SMART MODE:

NORMAL GENOMIC

• Kampfer H, Pfeilschifter J, Frank S
• Expressional regulation of angiopoietin-1 and -2 and the tie-1 and -2 receptor tyrosine kinases during cutaneous wound healing: a comparative study of normal and impaired repair.
• Lab Invest. 2001; 81: 361-73
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• It has become evident that a closely regulated presence of vascular endothelial growth factor (VEGF) and angiopoietin (Ang) factors determines the fate of blood vessel formation during angiogenesis. As angiogenesis is central to a normal wound-healing process, we investigated the regulation of Ang-1 and -2 and the related tyrosine kinase with immunoglobulin and epidermal growth factor homology (Tie)-1 and -2 receptors during normal repair in Balb/c mice and diabetes-impaired wound healing conditions in genetically diabetic (db/db) mice. For both normal and impaired healing conditions, we observed a constitutive expression of Ang-1, which was paralleled by an increase of Ang-2 upon injury. Whereas the observed Ang-2 expression declines from Day 7 after injury in control mice, diabetic-impaired healing was characterized by still increasing amounts of Ang-2 at these time points. Furthermore, Tie-1 was strongly induced during repair with a prolonged expression in diabetic mice, whereas Tie-2 expression was constitutive during normal repair but completely absent in diabetes-impaired healing. The overexpression of Ang-2 in the presence of markedly reduced VEGF in wounds of diabetic mice was associated with a dramatic decrease in endothelial cell numbers compared with normal healing as assessed by analysis of the endothelium-specific markers CD31 and von Willebrand factor, whereas the lymphatic endothelium remained stable as determined by expression of VEGF receptor-3 (VEGFR-3/Flt-4).

• Voskas D, Kim M, Hurta RA
• Platelet-derived growth factor mediated altered expression and regulation of ornithine decarboxylase in H-ras-transformed cell lines.
• Cell Signal. 2001; 13: 401-9
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• This study demonstrates a novel link between alterations in platelet-derived growth factor (PDGF) regulation of ornithine decarboxylase (ODC) expression during malignant conversion. H-ras-transformed cell lines exhibited PDGF-mediated alterations in ODC gene expression. These alterations involved transcriptional, posttranscriptional, and cycloheximide-mediated events. PDGF-mediated alterations in ODC gene expression in NR3 cells (capable of only benign tumour formation) were ras-dependent, involved a tyrosine kinase activity and mitogen-activated protein (MAP) kinase-mediated signalling events, and were independent of both protein kinase C (PKC) events and pertussis toxin-sensitive (PTS) G-protein-mediated signalling. PDGF-mediated alterations in ODC gene expression in C2 cells [capable of malignant progression (metastasis formation)] were ras-dependent, required a tyrosine kinase activity, involved both MAP kinase-mediated events and phosphatidylinositol-3-kinase (PI-3-kinase)-mediated events, and were dependent upon PTS G-protein-mediated signalling but independent of PKC-mediated events. PDGF-mediated regulation of ODC gene expression changes in response to H-ras-mediated cellular transformation and malignant progression.

• Hamada T, Ui-Tei K, Imaki J, Miyata Y
• Molecular cloning of SCDGF-B, a novel growth factor homologous to SCDGF/PDGF-C/fallotein.
• Biochem Biophys Res Commun. 2001; 280: 733-7
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• Spinal cord-derived growth factor (SCDGF)/platelet-derived growth factor (PDGF)-C/fallotein has a unique two-domain structure, as it contains two regions homologousto CUB and PDGF/vascular endothelial growth factor (VEGF) domains. In this study, we isolateda novel gene homologous to SCDGF/PDGF-C/fallotein, and named SCDGF-B. The culture supernatant of CHO-K1 cells stably transfected with SCDGF-B showed mitogenic activity as SCDGF/PDGF-C/fallotein did. Although SCDGF-B and SCDGF/PDGF-C/fallotein might be the members of the PDGF/VEGF superfamily of growth factors, they were categorized into a new subfamily in addition to PDGF and VEGF subfamilies. Copyright 2001 Academic Press.

• Kim J, Wu H, Hawthorne L, Rafii S, Laurence J
• Endothelial cell apoptotic genes associated with the pathogenesis of thrombotic microangiopathies: an application of oligonucleotide genechip technology.
• Microvasc Res. 2001; 62: 83-93
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• Idiopathic thrombotic thrombocytopenic purpura (TTP) is a disease characterized by the apoptotic injury of all microvascular endothelial cells (MVEC) except those of pulmonary origin. It notably also spares EC of large vessel origin. It is fatal unless treated with plasma exchange. The EC lineage restriction of the apoptotic lesions in vivo is reproduced in vitro following exposure of primary human MVEC derived from various tissues to TTP plasma. Oligonucleotide genechip technology was used to identify genes that may contribute to the resistance of lung MVEC to apoptosis induced by TTP plasma and to explore the intrinsic genotypic heterogeneity between MVEC of TTP-sensitive (skin) versus resistant (lung) lineage. Exposure of cells to TTP or normal plasma yielded 157 genes that were differentially expressed in primary human lung MVEC. A global change in expression of pro- and anti-apoptotic genes was seen, including increases in caspase 1, Fas, and Bcl-xl, already shown by experimental means to be involved in TTP pathogenesis. Additional differences suggest the importance of pathways related to the death receptor ligand TRAIL, as well as a role for disruption of EC-extracellular matrix interactions in the initiation of apoptosis. Maintenance of specific prosurvival signals at baseline may be a feature of lung MVEC resistance in TTP as suggested by higher expression than skin EC of the TRAIL antagonist, osteoprotegerin, and the vascular endothelial growth factors, VEGF/VPF and VEGF-C, and their receptors, VEGFR-2 (KDR) and VEGFR-3 (Flt4). Copyright 2001 Academic Press.

• Baldwin ME et al.
• The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man.
• J Biol Chem. 2001; 276: 19166-71
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• Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where the VEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man.

• Makinen T et al.
• Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.
• Nat Med. 2001; 7: 199-205
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• The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.

• Ribatti D et al.
• Coordinate immunoreactivity to vascular endothelial growth factor receptor-2 and its ligand suggests a paracrine regulation during the development of the vascular system in the chick embryo bursa of Fabricius.
• Int J Mol Med. 2001; 7: 365-8
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• The bursa of Fabricius is a lymphoid organ of the chick which plays an important role in the development of the immune system. The role of angiogenic factors in the development of the vascular system of this organ has been poorly investigated. Vascular endothelial growth factor (VEGF) is a major regulator of endothelial cell proliferation, angiogenesis and vascular permeability, and its activities are mediated by two receptors, VEGFR-1 and VEGFR-2. In this study we have investigated by immunohistochemistry the VEGF and VEGFR-2 immunoreactivity in developing bursa of Fabricius. Starting from day 10 of incubation, the endodermal epithelium reacts with VEGF and gives rise to the lymphoid follicles, while the vascular endothelium reacts with VEGFR-2. These data support the view that VEGF acts as a paracrine stimulator of angiogenesis in the avian embryo and confirm the requirement of the endodermal layer for the normal formation of blood vessels by mesodermal cells.

• Dunk C, Ahmed A
• Expression of VEGF-C and activation of its receptors VEGFR-2 and VEGFR-3 in trophoblast.
• Histol Histopathol. 2001; 16: 359-75
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• Placental villous development requires the co-ordinated action of angiogenic factors on both endothelial and trophoblast cells. Like vascular endothelial growth factor (VEGF), VEGF-C increases vascular permeability, stimulates endothelial cell proliferation and migration. In the present study, we investigated the expression of VEGF-C and its receptors VEGFR-3 and VEGFR-2 in normal and intrauterine growth-restricted (IUGR) placenta. Immunolocalisation studies showed that like VEGF and VEGFR-1, VEGF-C, VEGFR-3 and VEGFR-2 co-localised to the syncytiotrophoblast, to cells in the maternal decidua, as well as to the endothelium of the large placental blood vessels. Western blot analysis demonstrated a significant decrease in placental VEGF-C and VEGFR-3 protein expression in severe IUGR as compared to gestationally-matched third trimester pregnancies. Conditioned medium from VEGF-C producing pancreatic carcinoma (Suit-2) and endometrial epithelial (Hec-1B) cell lines caused an increased association of the phosphorylated extracellular signal regulated kinase (ERK) in VEGFR-3 immunoprecipitates from spontaneously transformed first trimester trophoblast cells. VEGF121 caused dose-dependant phosphorylation of VEGFR-2 in trophoblast cells as well as stimulating DNA synthesis. In addition, premixing VEGF165 with heparin sulphate proteoglycan potentiated trophoblast proliferation and the association of phospho-ERK with the VEGFR-2 receptor. VEGF165-mediated DNA synthesis was inhibited by anti-VEGFR-2 neutralising antibody. The results demonstrate functional VEGFR-2 and VEGFR-3 receptors on trophoblast and suggest that the decreased expression of VEGF-C and VEGFR-3 may contribute to the abnormal villous development observed in IUGR placenta.

• Kadambi A et al.
• Vascular endothelial growth factor (VEGF)-C differentially affects tumor vascular function and leukocyte recruitment: role of VEGF-receptor 2 and host VEGF-A.
• Cancer Res. 2001; 61: 2404-8
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• Unlike vascular endothelial growth factor (VEGF)-A, the effect of VEGF-C on tumor angiogenesis, vascular permeability, and leukocyte recruitment is not known. To this end, we quantified in vivo growth and vascular function in tumors derived from two VEGF-C-overexpressing (VC+) and mock-transfected cell lines (T241 fibrosarcoma and VEGF-A-/- embryonic stem cells) grown in murine dorsal skinfold chambers. VC+ tumors grew more rapidly than mock-transfected tumors and exhibited parallel increases in tumor angiogenesis. Furthermore, VEGF-C overexpression elevated vascular permeability in T241 tumors, but not in VEGF-A-/- tumors. Surprisingly, unlike VEGF-A, VEGF-C did not increase leukocyte rolling or adhesion in tumor vessels. Administration of VEGF receptor (VEGFR)-2 neutralizing antibody DC101 reduced vascular density and permeability of both VC+ and mock-transduced T241 tumors. These data suggest that VEGFR-2 signaling is critical for tumor angiogenesis and vascular permeability and that VEGFR-3 signaling does not compensate for VEGFR-2 blockade. An alternate VEGFR, VEGFR-1 or neuropilin-1, may modulate adhesion of leukocytes to tumor vessels.

• Kajita T et al.
• The expression of vascular endothelial growth factor C and its receptors in non-small cell lung cancer.
• Br J Cancer. 2001; 85: 255-60
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• Expression of vascular endothelial growth factor (VEGF)-C and that of its receptors were assessed in non-small cell lung cancer. Immunohistochemistry revealed positive VEGF-C expression in 38.7% (24/62) of the patients studied. A significant positive correlation was found between VEGF-C in cancer cells and VEGF receptor-3 (VEGFR-3) in vascular endothelial cells, but not between VEGF-C in cancer cells and VEGFR-2 in endothelial cells. In this cohort of lung cancer patients, VEGF-C expression was significantly associated with lymph node metastasis, lymphatic vessel invasion, and worse outcomes after the operation. Although the independent prognostic impact of VEGF-C and VEGFR-3 was not clear, VEGFR-2 expression in endothelial cells retained the independency as the prognostic indicator. In light of these findings, we conclude that VEGF-C plays an important role in lymphatic invasion/metastasis and tumour progression in non-small cell lung cancer.

• Karkkainen MJ, Jussila L, Ferrell RE, Finegold DN, Alitalo K
• Molecular regulation of lymphangiogenesis and targets for tissue oedema.
• Trends Mol Med. 2001; 7: 18-22
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• New insight has recently been obtained into the molecular mechanisms regulating the function of lymphatic endothelial cells. Vascular endothelial growth factors-C and -D have been shown to stimulate lymphangiogenesis, and their receptor VEGFR-3 has been linked to human hereditary lymphoedema, although there is evidence that other genes are also involved. These data suggest that it may become possible to stimulate lymphatic growth and function and to treat tissue oedema involved in many diseases.

• Huang K, Andersson C, Roomans GM, Ito N, Claesson-Welsh L
• Signaling properties of VEGF receptor-1 and -2 homo- and heterodimers.
• Int J Biochem Cell Biol. 2001; 33: 315-24
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• Vascular endothelial growth factor (VEGF-A) exerts its effects through receptor tyrosine kinases VEGF receptor-1 (VEGFR-1) and VEGFR-2, which are expressed on most endothelial cell types in vitro and in vivo. We have examined VEGF-A-induced signal transduction in porcine aortic endothelial (PAE) cells individually expressing VEGFR-1 or VEGFR-2, and cells co-expressing both receptor types. We show that VEGF-A-stimulated PAE cells co-expressing VEGFR-1 and -2 contain receptor heterodimers. VEGF-A-stimulation of all three cell lines (expressing VEGFR-1, -2 and -1/2) resulted in signal transduction with different efficiencies. Thus, tyrosine phosphorylation of phospholipase Cgamma, and accumulation of inositol polyphosphates were efficiently transduced in the VEGFR-1/2 cells whereas cells expressing VEGFR-1 responded poorly in these assays. In contrast, VEGF-A-induced activation of phosphoinositide 3-kinase and induction of Ca2+ fluxes were transduced well by VEGFR-1 and VEGFR-2 homo- and heterodimers. The pattern of Ca2+ fluxes was unique for each type of VEGF receptor dimer. Our data show that signal transduction induced by VEGF-A is transduced in distinct manners by homo- and heterodimers of VEGF receptors.

• Hillman NJ, Whittles CE, Pocock TM, Williams B, Bates DO
• Differential effects of vascular endothelial growth factor-C and placental growth factor-1 on the hydraulic conductivity of frog mesenteric capillaries.
• J Vasc Res. 2001; 38: 176-86
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• Vascular endothelial growth factors (VEGFs) are known to increase vascular permeability. VEGF-A acts on two receptor tyrosine kinases, VEGF receptor-1 (VEGF-R1 or flt-1) and VEGF receptor-2 (VEGF-R2, flk-1 or KDR). VEGF-C acts only on VEGF-R2 on vascular endothelial cells, whereas placental growth factor-1 (PlGF-1) acts only on VEGF-R1. The effects of perfusion of these receptor-specific proteins on hydraulic conductivity (L(p)) was measured in frog mesenteric capillaries. The effect of PlGF on L(p) was not conclusive, and overall fluid flux did not increase during that time. VEGF-C acutely and transiently increased L(p) (4.5 +/- 0.9-fold), which was more obvious in a subset of vessels, in a similar manner to that reported for VEGF-A. In the subset of vessels in which VEGF-C significantly increased L(p) acutely, there was a sustained 12-fold increase in L(p) 20 min after perfusion, but this was not seen in those vessels which did not respond acutely to VEGF-C, or in vessels exposed to PlGF-1. L(p) was also increased 24 h after perfusion with VEGF-C, but not with PlGF-1. Western blot analysis showed that VEGF-R1 and VEGF-R2 are both present in frog tissue. These data show that the VEGFs that stimulate VEGF-R2 chronically increase L(p), but not those that stimulate VEGF-R1 only. This supports the hypothesis that chronic increases in microvascular permeability induced by VEGF are mediated via activation of VEGF-R2 rather than VEGF-R1. Copyright 2001 S. Karger AG, Basel

• Yu J, Moon A, Kim HR
• Both platelet-derived growth factor receptor (PDGFR)-alpha and PDGFR-beta promote murine fibroblast cell migration.
• Biochem Biophys Res Commun. 2001; 282: 697-700
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• Cell motility plays a critical role for many physiological and pathological processes including wound healing, fibrosis, angiogenesis, and tumor metastasis. Platelet-derived growth factor (PDGF) is among the most potent stimuli for mesenchymal cell migration. The PDGF B-chain homodimer PDGF BB activates both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR), and promotes cell migration in many cell types including fibroblasts and smooth muscle cells. PDGF-A chain homodimer PDGF AA activates alpha-PDGFR only, and its role for cell migration is still debatable. PDGF BB, but not PDGF AA, induces smooth muscle cell migration. Interestingly, alpha-PDGFR was shown to antagonize beta-PDGFR-induced smooth muscle cell migration. In the present study, we investigated the role of alpha-PDGFR and beta-PDGFR in PDGF-mediated cell migration of murine fibroblasts (NIH 3T3). Unlike smooth muscle cells, both PDGF AA and PDGF BB promoted NIH 3T3 cell migration. The effect of PDGF BB activation of beta-PDGFR alone for cell migration was examined using previously established NIH 3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. PDGF BB activation of beta-PDGFR alone was sufficient to induce cell migration, but the efficiency was significantly lower compared to PDGF activation of both receptors. These results showed that both alpha- and beta-PDGFRs promote fibroblast cell migration and their effects are additive. Taken together, we propose that cell-type specific alpha-PDGFR signaling is critical for regulation of mesenchymal cell migration in response to PDGF isoform, whereas beta-PDGFR mainly promotes cell migration. Copyright 2001 Academic Press.

• Ostman A, Heldin CH
• Involvement of platelet-derived growth factor in disease: development of specific antagonists.
• Adv Cancer Res. 2001; 80: 1-38
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• Platelet-derived growth factor (PDGF) is a family of dimeric isoforms that stimulates, e.g., growth, chemotaxis and cell shape changes of various connective tissue cell types and certain other cells. The cellular effects of PDGF isoforms are exerted through binding to two structurally related tyrosine kinase receptors. Ligand binding induces receptor dimerization and autophosphorylation. This enables a number of SH2 domain containing signal transduction molecules to bind to the receptors, thereby initiating various signaling pathways. PDGF isoforms have important roles during the embryonic development, particularly in the formation of connective tissue in various organs. In the adult, PDGF stimulates wound healing. Overactivity of PDGF has been implicated in certain disorders, including fibrotic conditions, atherosclerosis, and malignancies. Different kinds of PDGF antagonists are currently being developed and evaluated in different animal disease models, as well as in clinical trials.

• Gilbertson DG et al.
• Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor.
• J Biol Chem. 2001; 276: 27406-14
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• We have characterized platelet-derived growth factor (PDGF) C, a novel growth factor belonging to the PDGF family. PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1, and the C-terminal region consists of a growth factor domain (GFD) with homology to vascular endothelial growth factor (25%) and PDGF A-chain (23%). A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain. Competition binding and immunoprecipitation studies on cells bearing both PDGF alpha and beta receptors reveal a high affinity binding of recombinant GFD (PDGF-CC) to PDGF receptor-alpha homodimers and PDGF receptor-alpha/beta heterodimers. PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types. Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision. Together, these studies describe a third member of the PDGF family (PDGF-C) as a potent mitogen for cells of mesenchymal origin in in vitro and in vivo systems with a binding pattern similar to PDGF-AB.

• Yonemura Y et al.
• Lymphangiogenesis and the vascular endothelial growth factor receptor (VEGFR)-3 in gastric cancer.
• Eur J Cancer. 2001; 37: 918-23
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• Vascular endothelial growth factor C (VEGF-C) is the only factor known to cause lymphangiogenesis. We studied the correlation between VEGF-C and vascular endothelial growth factor receptor-3 (VEGFR-3) expression of 85 primary gastric cancers by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry, and the results were correlated with the number of lymphatic vessels, stained with anti-VEGFR-3 antibody. RT-PCR and immunohistology demonstrated that VEGF-C was mainly produced from cancer cells, but not from stromal elements. Morphologically, VEGFR-3 expression was detected in the endothelial cells of the stromal lymphatic vessels. There was a statistically positive correlation between the incidence of VEGF-C and VEGFR-3 mRNA expression in the primary tumours (P=0.0002). The number of VEGFR-3-positive lymphatic vessels in VEGF-C mRNA positive tumours was significantly larger than that in VEGF-C-negative tumours. The number of VEGFR-3-positive vessels in the tumour stroma was closely related to the grade of lymphatic invasion of gastric cancer. These results strongly indicate that VEGF-C may induce the proliferation of lymphatic vessels in the stroma of primary gastric cancer via activation of VEGFR-3, expressed on the endothelial cells of lymphatic vessels. In these circumstances, cancer cells can easily invade the lymphatic vessel, because of the increase of the contact points of cancer cells with the lymphatic vessels.

• Hughes DC
• Alternative splicing of the human VEGFGR-3/FLT4 gene as a consequence of an integrated human endogenous retrovirus.
• J Mol Evol. 2001; 53: 77-9
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• The vascular endothelial growth factor receptor 3 (VEGFR-3/FLT4) is a receptor tyrosine kinase that regulates angiogenesis and vasculogenesis in response to the binding of the ligands VEGF-C and VEGF-D. Mutations in VEGFR-3 have been identified in patients with primary lymphoedema. It has been noted previously that whilst in the mouse there is only a single Vegfr-3 transcript, in humans there are two transcripts of 5.8 and 4.5 kb, of which the shorter encodes a protein that lacks the C-terminal 65 amino acids. These two isoforms also differ in their biological activity. Analysis of the human VEGFR-3 cDNA and genomic sequence reveals that these two isoforms arise by alternative splicing of the terminal exons. The shorter transcript is generated by splicing into the long terminal repeat of a human endogenous retrovirus located between the last two exons, thus explaining the lack of the shorter transcript in the mouse. The retention of the retroviral sequences in the FLT4 locus suggests that this retrotransposition event has contributed significant additional function to this gene. This provides support for a role for integrated retroviruses in modulating gene activity and participating in evolutionary processes.

• Lamb DJ, Avades TY, Ferns GA
• Endogenous neutralizing antibodies against platelet-derived growth factor-aa inhibit atherogenesis in the cholesterol-fed rabbit.
• Arterioscler Thromb Vasc Biol. 2001; 21: 997-1003
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• Previous studies have shown that the B chain of platelet-derived growth factor (PDGF) has an important role in atherogenesis. In this study we have investigated the contribution of PDGF-A chain in cholesterol-induced atherogenesis in the New Zealand White rabbit. High titers of antibodies to PDGF-AA or to platelet cytosolic protein (PCP) were induced in these animals by immunization against recombinant human PDGF-AA or human PCP. Rabbits were then fed a 0.25% to 1% cholesterol-containing diet for 10 weeks to induce atherosclerotic lesions; the rabbits were then humanely killed and perfusion-fixed and their aortas were removed. The extent of atherosclerosis in the thoracic aortas was determined by quantitative morphometry after staining with oil red O. The intimal and medial areas in histological sections taken at the level of the first intercostal branch were quantified by image analysis. Immunization against PDGF-AA and PCP, but not against adjuvant alone, resulted in rising titers of antibodies within 2 weeks, the levels of which reached a plateau by 8 weeks. The antibodies to PDGF-AA were isoform-specific, recognized both human and rabbit PDGF-AA, and neutralized the biological activity of PDGF-AA in vitro. Integrated plasma cholesterol levels were similar in both groups. Compared with nonimmune rabbits (n=10), animals immunized against PDGF-AA (n=10) or PCP (n=10) had significantly smaller areas of the aorta covered by atherosclerotic lesions (24.6+/-5.1% and 18.7+/-4.2%, respectively, vs 34.4+/-4.3%; P<0.05). This was associated with a reduced aortic intimal-medial area ratio in PDGF-AA-immunized (0.009+/-0.006) and PCP-immunized (0.025+/-0.017) rabbits than in nonimmune animals (0.159+/-0.066; P<0.05). These data suggest that PDGF-AA is actively involved in cholesterol-induced atherosclerosis in the rabbit.

• Veikkola T et al.
• Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice.
• EMBO J. 2001; 20: 1223-31
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• Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.

• Backer MV, Backer JM
• Functionally active vegf fusion proteins.
• Protein Expr Purif. 2001; 23: 1-7
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• Angiogenesis is stimulated by vascular endothelial growth factor (VEGF) acting via endothelial cell-specific receptors, such as VEGFR-2, that are overexpressed at the sites of angiogenesis. If VEGF retains activity as a fusion protein with a large N-terminal extension, it would facilitate development of VEGF-based vehicles for receptor-mediated delivery of therapeutic and diagnostic agents to the sites of angiogenesis. We have constructed, expressed in Escherichia coli, and purified VEGF fusion proteins containing a 158-amino acid N-terminal extension fused to human VEGF(121), VEGF(165), and VEGF(189). We report here that VEGF fusion proteins induce tyrosine autophosphorylation of VEGFR-2 and its downstream targets, as well as cell contraction in cells overexpressing VEGFR-2. Although N-terminal extensions decrease the affinity of VEGF fusion proteins to VEGFR-2, at saturating concentrations these proteins are as efficient as correct size VEGF(165). We hypothesize that VEGF fusion proteins may be employed for targeting endothelial cells at the sites of angiogenesis.

• Tang RF et al.
• Overexpression of lymphangiogenic growth factor VEGF-C in human pancreatic cancer.
• Pancreas. 2001; 22: 285-92
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• Vascular endothelial growth factor C (VEGF-C) is a lymphangiogenic polypeptide that has been implicated in cancer growth. In this study, we characterized VEGF-C expression in cultured human pancreatic cancer cell lines and determined whether the presence of VEGF-C in human pancreatic cancers is associated with clinicopathologic characteristics. VEGF-C mRNA transcripts were present in all five tested cell lines (Capan-1, MIA-PaCa-2, PANC-1, COLO-357, and T3M4). Immunoblotting with a highly specific anti-VEGF-C antibody revealed the presence of VEGF-C protein in all the cell lines. Northern blot analysis of total RNA revealed an approximately 2.2-fold increase in VEGF-C mRNA transcript in the cancer samples compared with the normal pancreas. Immunohistochemical analysis confirmed the expression of VEGF-C and its receptor flt-4 in the cancer cells within the tumor mass. Immunohistochemical analysis of 51 pancreatic cancer tissues revealed the presence of strong VEGF-C immunoreactivity in the cancer cells in 80.4% of the cancer tissues. The presence of VEGF-C in these cells was associated with increased lymphatic vessels invasion and lymph node metastasis, but not with decreased patient survival. These findings indicate that VEGF-C and its receptor are commonly overexpressed in human pancreatic cancers and that this factor may contribute to the lymphangiogenic process and metastasis in this disorder.

• Makinen T et al.
• Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3.
• EMBO J. 2001; 20: 4762-73
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• Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4) binds two known members of the VEGF ligand family, VEGF-C and VEGF-D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR-3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. We show that VEGFR-3 stimulation alone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C-dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin-sensitive induction of Akt phosphorylation. These results define the critical role of VEGF-C/VEGFR-3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.

• Boldicke T et al.
• Anti-VEGFR-2 scFvs for cell isolation. Single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/flk-1) on the surface of primary endothelial cells and preselected CD34+ cells from cord blood.
• Stem Cells. 2001; 19: 24-36
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• Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells) as detected by surface immunofluorescence using confocal microscopy. In addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34+VEGFR-2+ hematopoietic immature cells within the population of enriched CD34+ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K(d) = 3.8 x 10(-9) M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.

• Enholm B et al.
• Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis in the skin.
• Circ Res. 2001; 88: 623-9
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• The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet-endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors.

• Betsholtz C, Karlsson L, Lindahl P
• Developmental roles of platelet-derived growth factors.
• Bioessays. 2001; 23: 494-507
• Display abstract

• Platelet-derived growth factor (PDGF) was originally identified in platelets and in serum as a mitogen for fibroblasts, smooth muscle cells (SMC) and glia cells in culture. PDGF has since expanded to a family of dimers of at least four gene products, whose biological actions are mediated through two receptor tyrosine kinases, PDGFRs. The present review summarizes and discusses the biological functions of PDGFs and PDGFRs in developmental processes, mainly as revealed through genetic analysis in mice. Such studies have demonstrated multiple critical roles of PDGFs and PDGFRs in embryonic and postnatal development. PDGFs seem to act upon specific populations of progenitor cells that give rise to several different cell types with distinct functions in a variety of developmental processes. Analogies are seen between the cell functions and the developmental processes controlled by PDGFs. This suggests that ancestral PDGF and PDGFR expression patterns and functions may have been iterated in related sets of morphogenetic processes in the course of evolution. Copyright 2001 John Wiley & Sons, Inc.

• LaRochelle WJ et al.
• PDGF-D, a new protease-activated growth factor.
• Nat Cell Biol. 2001; 3: 517-21
• Display abstract

• Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-alpha, whereas PDGF-B activates both PDGFR-alpha and PDGFR-beta. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-beta but not PDGFR-alpha. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-alpha activation may result from PDGFR-alpha/beta heterodimerization.

• Maehara K, Oh-Hashi K, Isobe KI
• Early growth-responsive-1-dependent manganese superoxide dismutase gene transcription mediated by platelet-derived growth factor.
• FASEB J. 2001; 15: 2025-6
• Display abstract

• Manganese superoxide dismutase Mn-SOD plays a major role in protecting mitochondria from oxidative damage. Overexpression of Mn-SOD maintains cell survival under conditions that lead to apoptotic death. In addition to the antioxidative enzyme, platelet-derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation by activating survival signaling pathways in various cells. Here we show that PDGF induced the expression of the Mn-SOD gene in NIH3T3 cells, and its induction was associated with early growth response-1 (Egr-1), a transcription factor. An electrophoretic mobility shift assay demonstrated that Egr-1 bound to the proximal promoter of the Mn-SOD gene in response to PDGF. The proximal promoter region of Mn-SOD was shown to be transcriptionally responsive to both basal and PDGF stimulation by transfection studies. Forced expression of Egr-1 in the cells activated Mn-SOD transcription in a dose-dependent manner. The pathway by which PDGF induced Egr-1 involved the mitogen-activated protein kinase kinase-1 (MEK1) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), because the effect of PDGF on the induction of Egr-1 was blocked by U0126, a specific MEK1 inhibitor. These findings indicate that the induction of Mn-SOD is part of the anti-apoptotic properties mediated by PDGF.

• Metheny-Barlow LJ, Flynn B, van Gijssel HE, Marrogi A, Gerwin BI
• Paradoxical effects of platelet-derived growth factor-A overexpression in malignant mesothelioma. Antiproliferative effects in vitro and tumorigenic stimulation in vivo.
• Am J Respir Cell Mol Biol. 2001; 24: 694-702
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• Malignant mesothelioma is associated with asbestos exposure and remains resistant to all therapeutic intervention. Previous studies have suggested an enhancing role for platelet-derived growth factor (PDGF) in mesothelial tumorigenicity, although the mechanism by which PDGF facilitates tumorigenicity is unknown. Here, we evaluate the contribution of PDGF-A expression to mesothelial tumorigenicity using ectopic modulation of PDGF-A expression. We find, in accordance with other reports, that the receptor for PDGF-A, although expressed at high levels in normal human mesothelial cells, is not easily detectable in mesothelioma. Further, we show that PDGF-A overexpression is responsible for autocrine downregulation of its receptor. Our data indicate, surprisingly, that for mesothelioma cells in vitro, high-level activation of a PDGF-A-PDGF receptor loop is antiproliferative whereas abrogation of PDGF-A expression stimulates growth. These data suggest that PDGF-A does not contribute to tumorigenicity by autocrine stimulation of proliferation. In contrast, increased PDGF-A expression in vivo increases tumor incidence and growth rate and decreases the latency period to tumor formation whereas abrogation of PDGF-A expression decreases tumor incidence and increases latency. Thus, the tumorigenic effect of PDGF-A must act through paracrine mechanisms relevant at early stages of tumor initiation.

• Foss B, Ulvestad E, Bruserud O
• Platelet-derived growth factor (PDGF) in human acute myelogenous leukemia: PDGF receptor expression, endogenous PDGF release and responsiveness to exogenous PDGF isoforms by in vitro cultured acute myelogenous leukemia blasts.
• Eur J Haematol. 2001; 66: 365-76
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• We investigated effects of Platelet-derived growth factor (PDGF) and Platelet factor 4 (PF-4) on the functional characteristics of native, human acute myelogenous leukemia (AML) blasts. AML blast expression of the PDGF-receptor alpha-chain was detected for a subset of patients (45%), whereas PDGF-receptor beta-chain expression was detected for most patients (90%). Constitutive AML blast release of the PDGF-AB isoform (the major form also derived from normal platelets) was detected for 43% of patients, whereas PDGF-BB release was not detected for any patient. The PDGF isoforms AA, AB and BB had dose-dependent and divergent effects on spontaneous and cytokine-dependent AML blast proliferation, whereas for constitutive cytokine secretion (IL-1beta, IL-6, TNF-alpha) inhibitory effects were rare and all three isoforms usually had no effect or enhanced the constitutive secretion. The PDGF effects were caused by a direct effect on the AML blasts and were not dependent on the presence of serum. The PDGF effects could also be detected after in vitro culture of AML cells in the presence of IL-4+granulocyte-macrophage colony stimulating factor. PF-4 had divergent effects on proliferation and cytokine secretion by native AML blasts. Our results suggest that exogenous (e.g. platelet-secreted) PDGF and PF-4 can function as regulators of leukemic hematopoiesis and possibly also modulate the function of residual AML cells in peripheral blood stem cell grafts. On the other hand, endogenous release of PDGF-AB by native blasts may modulate the function of normal cells in the bone marrow microenvironment (e.g. bone marrow stromal cells).

• Chiarugi P et al.
• Two vicinal cysteines confer a peculiar redox regulation to low molecular weight protein tyrosine phosphatase in response to platelet-derived growth factor receptor stimulation.
• J Biol Chem. 2001; 276: 33478-87
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• Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H(2)O(2) or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H(2)O(2), evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H(2)O(2) production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.

• Maeda Y, Solanky M, Menonna J, Chapin J, Li W, Dowling P
• Platelet-derived growth factor-alpha receptor-positive oligodendroglia are frequent in multiple sclerosis lesions.
• Ann Neurol. 2001; 49: 776-85
• Display abstract

• Platelet-derived growth factor (PDGF) ligand is a potent glial cell mitogen. When its cognate receptor (PDGF-alphaR) is expressed on oligodendroglial lineage cells, such cells are considered capable of division, and the receptor thus serves as a phenotypic marker for oligodendrocyte precursor cells. Here we identify using immunohistochemistry a considerably enlarged, PDGF-alphaR-expressing oligodendrocyte cell population within multiple sclerosis (MS) white matter lesions compared to control brains. Numerous PDGF-alphaR-positive oligodendroglia also colabel heavily with the nuclear cell proliferation marker antibody Ki-67. Our finding of large numbers of proliferating oligodendroglia in MS brains expressing up-regulated PDGF-alphaR suggests that these progenitor-like cells represent an important source of regenerating cells for the healing MS lesion.

• Mimura T, Amano S, Usui T, Kaji Y, Oshika T, Ishii Y
• Expression of vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 in corneal lymphangiogenesis.
• Exp Eye Res. 2001; 72: 71-8
• Display abstract

• Lymphangiogenesis has been reported in vascularized corneas. However, the molecular mechanisms of lymphangiogenesis in the cornea are still unclear. Since lymphatic vessels may contribute to a decreased success rate of keratoplasty in vascularized cornea by accelerating antigen recognition and graft rejection, elucidation of the mechanisms of corneal lymphangiogenesis will facilitate the inhibition of lymphatic vessels and may improve the outcome of keratoplasty. This study aimed to examine the expression of vascular endothelial growth factor-C (VEGF-C), which is the only endogenous lymphangiogenic factor reported so far, and one of its receptors, vascular endothelial growth factor receptor-3 (VEGFR-3), in corneal lymphangiogenesis. A rat model was used in which silver nitrate application resulted in corneal circumferential neovascularization. The presence of lymphatic vessels in the rat injured cornea was examined with electron microscope. Corneal VEGF-C and VEGFR-3 mRNA levels were quantified with competitive reverse transcription polymerase chain reaction (RT-PCR), and VEGF-C and VEGFR-3 proteins were studied in situ using immunohistochemical analysis. Electron microscopy revealed lymphatic vessels in the vascularized rat corneas. Competitive RT-PCR demonstrated that the expression of VEGF-C mRNA in the rat cornea was normally absent, and was dramatically up-regulated 3 days after the injury, which gradually decreased. The VEGFR-3 expression in the rat cornea was minimally detected before the injury and was up-regulated 3 and 7 days after the injury. It was also minimally detected 2 and 4 weeks after the injury. In immunohistochemical analysis of the rat cornea 3 days after the injury, VEGF-C was mainly detected in inflammatory cells, and VEGFR-3 was demonstrated in several new vessels in the corneal stroma. These data suggest that VEGF-C and VEGFR-3 are pathophysiologically relevant endogenous factors in corneal lymphangiogenesis.

• Rahimi N, Dayanir V, Lashkari K
• Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells.
• J Biol Chem. 2000; 275: 16986-92
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• Vascular endothelial growth factor (VEGF) provokes angiogenesis in vivo and stimulates growth and differentiation of endothelial cells in vitro. Although VEGF receptor-1 (VEGFR-1) and VEGFR-2 are known to be high affinity receptors for VEGF, it is not clear which of the VEGFRs are responsible for the transmission of the diverse biological responses of VEGF. For this purpose we have constructed a chimeric receptor for VEGFR-1 (CTR) and VEGFR-2 (CKR) in which the extracellular domain of each receptor was replaced with the extracellular domain of human colony-stimulating factor-1 receptor (CSF-1R), and these receptors were expressed in pig aortic endothelial (PAE) cells. We show that CKR individually expressed in PAE cells is readily tyrosine-phosphorylated in vivo, autophosphorylated in vitro, and stimulates cell proliferation in a CSF-1-dependent manner. In contrast, CTR individually expressed in PAE cells showed no significant in vivo, in vitro tyrosine phosphorylation and cell growth in response to CSF-1 stimulation. The kinase activity of CKR was essential for its biological activity, since mutation of lysine 866 to arginine abolished its in vivo, in vitro tyrosine phosphorylation and mitogenic signals. Remarkably, activation of CTR repressed CKR-mediated mitogen-activate protein kinase activation and cell proliferation. Similar effects were observed for VEGFR-2 co-expressed with VEGFR-1. Collectively, these findings demonstrate that VEGFR-2 activation plays a positive role in angiogenesis by promoting endothelial cell proliferation. In contrast, activation of VEGFR-1 plays a stationary role in angiogenesis by antagonizing VEGFR-2 responses.

• Hamada K et al.
• VEGF-C signaling pathways through VEGFR-2 and VEGFR-3 in vasculoangiogenesis and hematopoiesis.
• Blood. 2000; 96: 3793-800
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• Signaling by vascular endothelial growth factors (VEGFs) through VEGF receptors (VEGFRs) plays important roles in vascular development and hematopoiesis. The authors analyzed the function of VEGF-C signaling through both VEGFR-2 and VEGFR-3 in vasculoangiogenesis and hematopoiesis using a coculture of para-aortic splanchnopleural mesoderm (P-Sp) explants from mouse embryos with stromal cells (OP9). Vasculogenesis and angiogenesis were evaluated by the extent of vascular bed and network formation, respectively. Addition of VEGF-C to the P-Sp culture enhanced vascular bed formation and suppressed definitive hematopoiesis. Both vascular bed and network formations were completely suppressed by addition of soluble VEGFR-1-Fc competitor protein. Formation of vascular beds but not networks could be rescued by VEGF-C in the presence of the competitor, while both were rescued by VEGF-A. VEGFR-3-deficient embryos show the abnormal vasculature and severe anemia. Consistent with these in vivo findings, vascular bed formation in the P-Sp from the VEGFR-3-deficient embryos was enhanced to that in wild-type or heterozygous embryos, and hematopoiesis was severely suppressed. When VEGFR-3-Fc chimeric protein was added to trap endogenous VEGF-C in the P-Sp culture of the VEGFR-3-deficient embryos, vascular bed formation was suppressed and hematopoiesis was partially rescued. These results demonstrate that because VEGF-C signaling through VEGFR-2 works synergistically with VEGF-A, the binding of VEGF-C to VEGFR-3 consequently regulates VEGFR-2 signaling. In VEGFR-3-deficient embryos, an excess of VEGF-C signals through VEGFR-2 induced the disturbance of vasculogenesis and hematopoiesis during embryogenesis. This indicates that elaborated control through VEGFR-3 signaling is critical in vasculoangiogenesis and hematopoiesis. (Blood. 2000;96:3793-3800)

• Gille H et al.
• A repressor sequence in the juxtamembrane domain of Flt-1 (VEGFR-1) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 3'-kinase activation and endothelial cell migration.
• EMBO J. 2000; 19: 4064-73
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• Vascular endothelial growth factor (VEGF) has two highly homologous tyrosine kinase receptors: Flt-1 (VEGFR-1) and KDR (VEGFR-2). KDR is strongly phosphorylated on tyrosines and can transmit mitogenic and motogenic signals following VEGF binding, while Flt-1 is markedly less effective in mediating such functions. To dissect the regions that account for the differences between the two receptors, we generated a series of chimeric Flt-1-KDR molecules. We found that the juxtamembrane region of Flt-1 prevents key signaling functions. When the juxtamembrane region of Flt-1 is replaced by that of KDR, Flt-1 becomes competent to mediate endothelial cell migration and phosphatidylinositol 3'-kinase activation in response to VEGF. Further mutational analysis shows that a short divergent sequence is responsible for such repressor function. However, mutant Flt-1 receptors lacking this sequence do not transmit effective proliferative signals, suggesting that this receptor function is regulated separately. These results define a novel functional domain that serves to repress Flt-1 activity in endothelial cells.

• Langer I, Vertongen P, Perret J, Fontaine J, Atassi G, Robberecht P
• Expression of vascular endothelial growth factor (VEGF) and VEGF receptors in human neuroblastomas.
• Med Pediatr Oncol. 2000; 34: 386-93
• Display abstract

• BACKGROUND: Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen that stimulates angiogenesis and plays a crucial role in tumor growth. The aim of the present study was to evaluate the expression of VEGF and of its two high-affinity tyrosine kinase receptors (KDR and Flt-1) in neuroblastoma surgical samples and cell lines. PROCEDURE: The VEGF, KDR, and Flt-1 mRNA expression in neuroblastoma surgical samples and cell lines was studied by RT-PCR. The receptors were identified in [(125)I]VEGF binding and in functional studies (effect on cell growth). VEGF production by neuroblastomas was investigated by the ELISA method. RESULTS: It was possible to observe the mRNAs encoding for VEGF and its two receptors in some of the surgical specimens examined, including most of the high-grade tumors. It was also possible to demonstrate that the SK-N-BE cell line expressed VEGF, KDR, and Flt-1 mRNAs as well as biologically active receptors: The cells bound [(125)I]-VEGF, and their growth was stimulated by exogenous VEGF. Moreover, VEGF protein could be detected in their culture conditioned medium. CONCLUSIONS: These results suggest that, in addition to its effect on angiogenesis, VEGF may affect neuroblastoma cell growth directly and could be an autocrine growth factor.

• Zacour ME, Tolloczko B, Martin JG
• Calcium and growth responses of hyperresponsive airway smooth muscle to different isoforms of platelet-derived growth factor (PDGF).
• Can J Physiol Pharmacol. 2000; 78: 867-73
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• Airway smooth muscle (ASM) mass is likely to be an important determinant of airway responsiveness. Highly inbred Fisher rats model innate hyperresponsiveness, and also have more ASM in vivo than control Lewis rats. Platelet derived growth factor (PDGF) is an important endogenous growth factor for ASM, and partially purified PDGF-AB causes enhanced growth of Fisher rat ASM cells, compared to Lewis cells. The aim of the present study was to determine the mitogenic effects of all three recombinant PDGF isoforms on ASM cells, and investigate the mechanisms of enhanced Fisher ASM growth responses. The potential mechanisms assessed include PDGF receptor expression and activation (tyrosine phosphorylation), and intracellular calcium (Ca2+) responses to PDGF isoforms. Fisher ASM cells had a greater mitogenic response to PDGF-AB and -AA, and a greater Ca2+ response to -BB than Lewis ASM cells. A Ca2+ response was not necessary for a mitogenic response, and the effects of PDGF isoforms on Ca2+ were not associated with their effects on growth. Therefore, we suggest that enhanced Fisher mitogenic response to PDGF-AA and -AB is not mediated by differences in Ca2+ signalling. Western analysis of the PDGF receptor indicated a similar expression of beta-PDGF receptor in ASM cells from the two rat strains, but a greater expression of alpha-PDGF receptor in Fisher cells; however, phosphorylation of the PDGF receptor following growth stimulation did not differ between strains. This suggests a role for post-receptor signals, in addition to enhanced receptor expression, in the enhanced growth response of Fisher ASM cells to PDGF-AA and -AB.

• Clauss M
• Molecular biology of the VEGF and the VEGF receptor family.
• Semin Thromb Hemost. 2000; 26: 561-9
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• Vascular endothelial growth factor (VEGF) is the founding member of a still growing family of endothelial cell growth factors. The diverse functions of VEGF and its homologues (PIGF, VEGF-B, VEGF-C, VEGF-D, and VEGF-E) can be explained by their differential binding to the three signaling VEGF receptors. The VEGF family members PIGF and VEGF-B with exclusive binding capacities to the VEGFR-1 can influence monocyte activation and differentiation. The VEGFR-2 and VEGFR-3 binding VEGF homologues, VEGF-C and VEGF-D, are mitogens for both vascular and lymphatic endothelial cells. The orf virus encoded VEGF-E homologue binds and activates only the VEGFR-2 and thus may be the prototype of a vascular endothelial cell-specific growth factor. Further specific activities of VEGF and its homologues result from receptor-specific signaling and differential expression of ligands or receptors. A naturally occurring soluble form of the VEGFR-1 suggests a regulatory role for this receptor. Finally, the production and activation of factors involved in the coagulation/fibrinolytic system provide further evidence for the hypothesis that processes of hemostasis are involved in angiogenesis.

• Lu D et al.
• Identification of the residues in the extracellular region of KDR important for interaction with vascular endothelial growth factor and neutralizing anti-KDR antibodies.
• J Biol Chem. 2000; 275: 14321-30
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• The kinase domain receptor (KDR) of vascular endothelial growth factor (VEGF) is the main human receptor responsible for the angiogenic activity of VEGF. The extracellular region of KDR is comprised of seven immunoglobulin-like domains, of which the first three have been shown to be required for ligand binding. We have previously described antibodies directed against the extracellular region of KDR, including MAB383 and MAB664, which were shown to block the binding of VEGF to the receptor and to inhibit both VEGF-induced mitogenesis of human endothelial cells in vitro and tumor growth in vivo. Here we generated a series of KDR deletion mutants consisting of truncated extracellular regions and mapped out the domain(s) responsible for binding to VEGF and the neutralizing anti-KDR antibodies. All neutralizing antibodies were found to require domain 3 for efficient binding. Alanine-scanning mutagenesis of domain 3 identified two different sets of five residues, Ile(256), Asp(257), Glu(261), Leu(313), and Thr(315) and Tyr(262), Pro(263), Ser(264), Ser(265), and Lys(266), that were critical for binding to MAB383 and MAB664, respectively. Combination of alanine mutations affecting both MAB383 and MAB664 binding resulted in a variant that also lost binding to VEGF. These results suggest that the residues within this region of domain 3 are critical for VEGF binding. Our studies provide a basis for the mechanism of action of our anti-KDR antibodies and establish a functional foundation for the development of other classes of antagonists to the receptor.

• Larrivee B, Karsan A
• Signaling pathways induced by vascular endothelial growth factor (review).
• Int J Mol Med. 2000; 5: 447-56
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• Vasculogenesis and angiogenesis are the mechanisms responsible for the development of the blood vessels. Angiogenesis refers to the formation of capillaries from pre-existing vessels in the embryo and adult organism, while vasculogenesis is the development of new blood vessels from the differentiation of endothelial precursors (angioblasts) in situ. Vascular endothelial growth factor (VEGF) family members are major mediators of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to promote endothelial cell viability, mitogenesis, chemotaxis, and vascular permeability. It mediates its activity mainly via two tyrosine kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR), although other receptors, such as neuropilin-1 and -2, can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (flt-4) binds VEGF-C and VEGF-D and is more important in the development of lymphatic vessels. While the functional effects of VEGF on endothelial cells has been well studied, not as much is known about VEGF signaling. This review summarizes the different pathways known to be involved in VEGF signal transduction and the biological responses triggered by the VEGF signaling cascade.

• Achen MG et al.
• Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.
• Eur J Biochem. 2000; 267: 2505-15
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• Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.

• Hiltunen MO et al.
• Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta.
• Circulation. 2000; 102: 2262-8
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• BACKGROUND: Gene transfer to the vessel wall may provide new possibilities for the treatment of vascular disorders, such as postangioplasty restenosis. In this study, we analyzed the effects of adenovirus-mediated vascular endothelial growth factor (VEGF)-C gene transfer on neointima formation after endothelial denudation in rabbits. For comparison, a second group was treated with VEGF-A adenovirus and a third group with lacZ adenovirus. Clinical-grade adenoviruses were used for the study. METHODS AND RESULTS: Aortas of cholesterol-fed New Zealand White rabbits were balloon-denuded, and gene transfer was performed 3 days later. Animals were euthanized 2 and 4 weeks after the gene transfer, and intima/media ratio (I/M), histology, and cell proliferation were analyzed. Two weeks after the gene transfer, I/M in the lacZ-transfected control group was 0. 57+/-0.04. VEGF-C gene transfer reduced I/M to 0.38+/-0.02 (P:<0.05 versus lacZ group). I/M in VEGF-A-treated animals was 0.49+/-0.17 (P:=NS). The tendency that both VEGF groups had smaller I/M persisted at the 4-week time point, when the lacZ group had an I/M of 0.73+/-0.16, the VEGF-C group 0.44+/-0.14, and the VEGF-A group 0. 63+/-0.21 (P:=NS). Expression of VEGF receptors 1, 2, and 3 was detected in the vessel wall by immunocytochemistry and in situ hybridization. As an additional control, the effect of adenovirus on cell proliferation was analyzed by performing gene transfer to intact aorta without endothelial denudation. No differences were seen in smooth muscle cell proliferation or I/M between lacZ adenovirus and 0.9% saline-treated animals. CONCLUSIONS: Adenovirus-mediated VEGF-C gene transfer may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations.

• Gluzman-Poltorak Z, Cohen T, Herzog Y, Neufeld G
• Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected].
• J Biol Chem. 2000; 275: 18040-5
• Display abstract

• Neuropilin-1 (np-1) and neuropilin-2 (np-2) are receptors for axon guidance factors belonging to the class 3 semaphorins. np-1 also binds to the 165-amino acid heparin-binding form of VEGF (VEGF(165)) but not to the shorter VEGF(121) form, which lacks a heparin binding ability. We report that human umbilical vein-derived endothelial cells express the a17 and a22 splice forms of the np-2 receptor. Both np-2 forms bind VEGF(165) with high affinity in the presence of heparin (K(D) 1.3 x 10(-10) m) but not VEGF(121). np-2 also binds the heparin-binding form of placenta growth factor. These binding characteristics resemble those of np-1. VEGF(145) is a secreted heparin binding VEGF form that contains the peptide encoded by exon 6 of VEGF but not the peptide encoded by exon 7, which is present in VEGF(165). VEGF(145) binds to np-2 with high affinity (K(D) 7 x 10(-10) m). Surprisingly, VEGF(145) did not bind to np-1. Indeed, VEGF(145) does not bind to MDA-MB-231 breast cancer cells, which predominantly express np-1. By contrast, VEGF(145) binds to human umbilical vein-derived endothelial cells, which express both np-1 and np-2. The binding of VEGF(165) to porcine aortic endothelial cells expressing recombinant np-2 did not affect the proliferation or migration of the cells. Nevertheless, it is possible that VEGF-induced np-2-mediated signaling will take place only in the presence of other VEGF receptors such as VEGF receptor-1 or VEGF receptor-2.

• Scrofani SD, Fabri LJ, Xu P, Maccarone P, Nash AD
• Purification and refolding of vascular endothelial growth factor-B.
• Protein Sci. 2000; 9: 2018-25
• Display abstract

• Vascular endothelial growth factor (VEGF)-A interacts with the receptor tyrosine kinases VEGF-R1 and R2, and the importance of this interaction in endothelial cell (EC) function and blood vessel development has been well documented. Other ligands that interact differentially with these receptors and that are structurally related to VEGF-A include VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PLGF). Compared with VEGF-A, relatively little is known about the biological role of the VEGF-R1 specific ligand, VEGF-B. Two splice variant isoforms that differ at the COOH-terminus and which retain unique solubility characteristics are widely expressed throughout embryonic and postnatal development. Recent analysis of mice with a targeted deletion of the VEGF-B gene has revealed a defect in heart development and function consistent with an important role in vascularization of the myocardium (Bellomo D et al., 2000, Circ Res 86:E29-E35). To facilitate further characterization of VEGF-B, we have developed a protocol for expression and purification of refolded recombinant protein from Escherichia coli inclusion bodies (IBs). The approach developed resolves a number of significant issues associated with VEGF-B, including the ability to heterodimerize with endogenous VEGF-A when co-expressed in mammalian cells, a complex secondary structure incorporating inter- and intrachain disulfide bonds and hydrophobic characteristics that preclude the use of standard chromatographic resins. The resulting purified disulfide-linked homodimer was demonstrated to bind to VEGF-R1 and to compete with VEGF-A for binding to this receptor.

• Gunningham SP et al.
• The short form of the alternatively spliced flt-4 but not its ligand vascular endothelial growth factor C is related to lymph node metastasis in human breast cancers.
• Clin Cancer Res. 2000; 6: 4278-86
• Display abstract

• Angiogenesis is essential for tumor growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently, VEGF-C, a new VEGF family member, has been identified that binds to the tyrosine kinase receptors flt-4 [VEGF receptor (VEGFR) 3] and KDR (VEGFR2). Although the importance of VEGF has been shown in many human tumor types, the contribution of VEGF-C and its primary receptor flt-4 to tumor progression is less well understood. We have therefore measured the level of VEGF-C, flt-4, and KDR mRNA by RNase protection assay and the pattern of VEGF-C expression by immunohistochemistry in 11 normal breast tissue samples and 61 invasive breast cancers. No significant difference in VEGF-C expression was observed between normal and neoplastic breast tissues (P = 0.11). There was a significant correlation between VEGF-C and both flt-4 (P = 0.02) and KDR (P = 0.0002), but no association was seen between VEGF-C and either lymph node status (P = 0.66) or number of involved nodes (P = 0.88), patient age (P = 0.83), tumor size (P = 0.20), estrogen receptor status (P = 0.67), or tumor grade (P = 0.35). No significant relationship was present between VEGF-C and vascular invasion (P = 0.30), tumor vascularity (P = 0.21), VEGF-A (P = 0.62), or thymidine phosphorylase expression (P = 1.00). VEGF-C was expressed predominantly in the cytoplasm of tumor cells, although occasional stromal components including fibroblasts were also positive. We could demonstrate no association between lymph node metastasis and either VEGF-C (P = 0.66) or flt-4 (P = 0.4). However, we did observe a significant loss of the long but not the short isoform of flt-4 in tumors compared with normal tissues (P = 0.02 and P = 0.25, respectively), and this difference was largely accounted for by the reduction of long flt-4 in node-positive tumors. These findings strongly support a role for VEGF-C/flt-4 signaling in tumor growth by enhancement of angiogenesis and/or lymphangiogenesis and suggest that differential regulation of these processes may be controlled via flt-4 isoform transcription. They further suggest that the measurement of flt-4 isoform expression may identify a patient group that is likely to have node-positive disease and therefore benefit from additional treatment and also emphasize an additional ligand interaction that could be exploited by anti-VEGFR therapy.

• Li B et al.
• Receptor-selective variants of human vascular endothelial growth factor. Generation and characterization.
• J Biol Chem. 2000; 275: 29823-8
• Display abstract

• Vascular endothelial growth factor (VEGF) is a pleiotropic factor that exerts a multitude of biological effects through its interaction with two receptor tyrosine kinases, fms-like tyrosine kinase (Flt-1) or VEGF receptor 1 and kinase insert domain-containing receptor (KDR) or VEGF receptor 2. Whereas it is commonly accepted that KDR is responsible for the proliferative activities of VEGF, considerable controversy and uncertainty exist about the role of the individual receptors in eliciting many of the other effects. Based on a comprehensive mutational analysis of the receptor-binding site of VEGF, an Flt-1-selective variant was created containing four substitutions from the wild-type protein. This variant bound with wild-type affinity to Flt-1, was at least 470-fold reduced in binding to KDR, and had no activity in cell-based assays measuring autophosphorylation of KDR or proliferation of primary human vascular endothelial cells. Using a competitive phage display strategy, two KDR-selective variants were discovered with three and four changes from wild-type, respectively. Both variants had approximately wild-type affinity for KDR, were about 2000-fold reduced in binding to Flt-1, and showed activity comparable with the wild-type protein in KDR autophosphorylation and endothelial cell proliferation assays. These variants will serve as useful reagents in elucidating the roles of Flt-1 and KDR.

• Saaristo A et al.
• Vascular endothelial growth factor-C and its receptor VEGFR-3 in the nasal mucosa and in nasopharyngeal tumors.
• Am J Pathol. 2000; 157: 7-14
• Display abstract

• Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. Both blood and lymphatic vessels of the upper respiratory tract play important roles in pathological conditions, such as infections and tumors. Here we have studied the expression of VEGF-C and its receptor VEGFR-3 in the upper respiratory system by Northern blot analysis and immunohistochemistry of human tissues, and in situ mRNA hybridization of developing mouse embryos and beta-galactosidase staining of mouse embryos having a LacZ marker gene in the VEGFR-3 gene locus. The results demonstrate expression of VEGF-C and VEGFR-3 in the developing and adult nasal respiratory epithelium and in the nasal vascular plexus, respectively. Unlike in most other tissues, in the nasal mucosa VEGFR-3 is expressed in both blood and lymphatic vessels. Expression of VEGF-C was also detected in nasal and nasopharyngeal tumor islands, which were surrounded by VEGFR-3-positive angiogenic blood vessels. These results suggest that VEGF-C and VEGFR-3 have a role in the development of the nasal submucosal vascular plexus and in its normal function and that they are associated with angiogenesis in nasal and nasopharyngeal tumors.

• Karkkainen MJ, Petrova TV
• Vascular endothelial growth factor receptors in the regulation of angiogenesis and lymphangiogenesis.
• Oncogene. 2000; 19: 5598-605
• Display abstract

• VEGFR-1 (Flt-1), VEGFR-2 (KDR) and VEGFR-3 (Flt4) are endothelial specific receptor tyrosine kinases, regulated by members of the vascular endothelial growth factor family. VEGFRs are indispensable for embryonic vascular development, and are involved in the regulation of many aspects of physiological and pathological angiogenesis. VEGF-C and VEGF-D, as ligands for VEGFR-3 are also capable of stimulating lymphangiogenesis and at least VEGF-C can enhance lymphatic metastasis. Recent studies have shown that missense mutations within the VEGFR-3 tyrosine kinase domain are associated with human hereditary lymphedema, suggesting an important role for this receptor in the development of the lymphatic vasculature.

• Binetruy-Tournaire R et al.
• Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis.
• EMBO J. 2000; 19: 1525-33
• Display abstract

• Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

• Arbiser JL et al.
• Overexpression of VEGF 121 in immortalized endothelial cells causes conversion to slowly growing angiosarcoma and high level expression of the VEGF receptors VEGFR-1 and VEGFR-2 in vivo.
• Am J Pathol. 2000; 156: 1469-76
• Display abstract

• Vascular endothelial growth factor (VEGF or vascular permeability factor) is an important angiogenic factor that is up-regulated in numerous benign and malignant disorders, including angiosarcoma, hemangiomas, and solid tumors. To determine the functional role of VEGF in the development of endothelial tumors, we expressed primate VEGF 121 in an endothelial cell line, MS1, derived from primary murine cells by immortalization with a temperature-sensitive SV40 large T antigen. This cell line expresses the VEGFR-2 (Flk-1/Kdr) receptor for VEGF. Expression of VEGF 121 led to the development of slowly growing endothelial tumors, which were histologically well-differentiated angiosarcomas. The angiosarcomas generated from MS1 VEGF cells demonstrated up-regulation of the VEGF receptors VEGFR-2 and VEGFR-1 (Flt-1) in vivo compared with benign hemangiomas generated from MS1 cells. Treatment of these cells with the VEGFR-2 tyrosine kinase inhibitor SU 1498 led to decreased expression of ets-1, a transcription factor which has been shown to be stimulated by VEGF. These results suggest that high level expression of VEGF in endothelial cells may result in malignant transformation. This transformation process likely involves both autocrine and paracrine pathways.

• Partanen TA et al.
• VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues.
• FASEB J. 2000; 14: 2087-96
• Display abstract

• Recently, vascular endothelial growth factor receptor 3 (VEGFR-3) has been shown to provide a specific marker for lymphatic endothelia in certain human tissues. In this study, we have investigated the expression of VEGFR-3 and its ligands VEGF-C and VEGF-D in fetal and adult tissues. VEGFR-3 was consistently detected in the endothelium of lymphatic vessels such as the thoracic duct, but fenestrated capillaries of several organs including the bone marrow, splenic and hepatic sinusoids, kidney glomeruli and endocrine glands also expressed this receptor. VEGF-C and VEGF-D, which bind both VEGFR-2 and VEGFR-3 were expressed in vascular smooth muscle cells. In addition, intense cytoplasmic staining for VEGF-C was observed in neuroendocrine cells such as the alpha cells of the islets of Langerhans, prolactin secreting cells of the anterior pituitary, adrenal medullary cells, and dispersed neuroendocrine cells of the gastrointestinal tract. VEGF-D was observed in the innermost zone of the adrenal cortex and in certain dispersed neuroendocrine cells. These results suggest that VEGF-C and VEGF-D have a paracrine function and perhaps a role in peptide release from secretory granules of certain neuroendocrine cells to surrounding capillaries.

• Karkkainen MJ et al.
• Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema.
• Nat Genet. 2000; 25: 153-9
• Display abstract

• Primary lymphoedema is a rare, autosomal dominant disorder that leads to a disabling and disfiguring swelling of the extremities and, when untreated, tends to worsen with time. Here we link primary human lymphoedema to the FLT4 locus, encoding vascular endothelial growth factor receptor-3 (VEGFR-3), in several families. All disease-associated alleles analysed had missense mutations and encoded proteins with an inactive tyrosine kinase, preventing downstream gene activation. Our study establishes that VEGFR-3 is important for normal lymphatic vascular function and that mutations interfering with VEGFR-3 signal transduction are a cause of primary lymphoedema.

• Cooper ME et al.
• Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes.
• Diabetes. 1999; 48: 2229-39
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• It has been suggested that the cytokine vascular endothelial growth factor (VEGF) has an important role in the pathogenesis of diabetic retinopathy, but its role in nephropathy has not been clearly demonstrated. Assessment of VEGF, 125I-VEGF binding, and vascular endothelial growth factor receptor-2 (VEGFR-2) in the kidney was performed after 3 and 32 weeks of streptozotocin-induced diabetes. Gene expression of both VEGF and VEGFR-2 was assessed by Northern blot analysis and the localization of the ligand and receptor was examined by in situ hybridization. VEGF and VEGFR-2 protein were also evaluated by immunohistochemistry. Binding of the radioligand 125I-VEGF was evaluated by in vitro and in vivo autoradiography. Diabetes was associated with increased renal VEGF gene expression. VEGF mRNA and protein were localized to the visceral epithelial cells of the glomerulus and to distal tubules and collecting ducts in both diabetic and nondiabetic rats. Renal VEGFR-2 mRNA was increased after 3 weeks of diabetes but not in long-term diabetes. In situ hybridization and immunohistochemical studies revealed that glomerular endothelial cells were the major site of VEGFR-2 expression. In addition, VEGFR-2 gene expression was detected in cortical and renomedullary interstitial cells and on endothelial cells of peritubular capillaries. There was an increase in 125I-VEGF binding sites after 3 but not 32 weeks of diabetes. The major VEGF binding sites were in the glomeruli. 125I-VEGF binding was also observed in medullary rays and in the renal papillae. These studies indicate an early and persistent increase in renal VEGF gene expression in association with experimental diabetes. In addition, an early and transient increase in renal VEGF receptors was also observed in diabetic rats. These findings are consistent with a role for VEGF in mediating some of the changes observed in the diabetic kidney.

• Wise LM et al.
• Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1.
• Proc Natl Acad Sci U S A. 1999; 96: 3071-6
• Display abstract

• Orf virus, a member of the poxvirus family, produces a pustular dermatitis in sheep, goats, and humans. The lesions induced after infection with orf virus show extensive proliferation of vascular endothelial cells, dilation of blood vessels and dermal swelling. An explanation for the nature of these lesions may lie in the discovery that orf virus encodes an apparent homolog of the mammalian vascular endothelial growth factor (VEGF) family of molecules. These molecules mediate endothelial cell proliferation, vascular permeability, angiogenesis, and lymphangiogenesis via the endothelial cell receptors VEGFR-1 (Flt1), VEGFR-2 (KDR/Flk1), and VEGFR-3 (Flt4). The VEGF-like protein of orf virus strain NZ2 (ORFV2-VEGF) is most closely related in primary structure to VEGF. In this study we examined the biological activities and receptor specificity of the ORFV2-VEGF protein. ORFV2-VEGF was found to be a disulfide-linked homodimer with a subunit of approximately 25 kDa. ORFV2-VEGF showed mitogenic activity on bovine aortic and human microvascular endothelial cells and induced vascular permeability. ORFV2-VEGF was found to bind and induce autophosphorylation of VEGFR-2 and was unable to bind or activate VEGFR-1 and VEGFR-3, but bound the newly identified VEGF165 receptor neuropilin-1. These results indicate that, from a functional viewpoint, ORFV2-VEGF is indeed a member of the VEGF family of molecules, but is unique, however, in that it utilizes only VEGFR-2 and neuropilin-1.

• Meyer M et al.
• A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases.
• EMBO J. 1999; 18: 363-74
• Display abstract

• The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.

• Skobe M et al.
• Vascular endothelial growth factor-C (VEGF-C) and its receptors KDR and flt-4 are expressed in AIDS-associated Kaposi's sarcoma.
• J Invest Dermatol. 1999; 113: 1047-53
• Display abstract

• Kaposi's sarcoma is characterized by clusters of spindle-shaped cells that are considered to be tumor cells and by prominent vasculature. Whereas spindle cells are most likely endothelial in origin, it remains controversial whether they are of lymphatic or blood vascular derivation. To test the hypothesis that the lymphangiogenesis factor vascular endothelial growth factor-C and its receptors, KDR and flt-4, are involved in the pathogenesis of Kaposi's sarcoma, we performed in situ hybridizations and immunofluorescent stainings on human immunodeficiency virus-associated Kaposi's sarcoma. Spindle-shaped tumor cells strongly expressed KDR and flt-4 mRNA. Immunofluorescent staining confirmed expression of the flt-4 receptor in Kaposi's sarcoma cells, and double labeling revealed its colocalization with the endothelial cell marker CD31. Vascular endothelial growth factor-C was strongly expressed in blood vessels associated with Kaposi's sarcoma. In vitro, human dermal microvascular endothelial cells also expressed vascular endothelial growth factor-C mRNA that was further upregulated by vascular permeability factor/vascular endothelial growth factor. Vascular endothelial growth factor-C potently stimulated the proliferation of Kaposi's sarcoma tumor cells in vitro. These results demonstrate important paracrine functions of vascular endothelial growth factor-C, produced by blood vessels, in the pathogenesis of cutaneous Kaposi's sarcoma, and suggest a lymphatic origin and/or differentiation of Kaposi's sarcoma tumor cells.

• Andrews A et al.
• Platelet-derived growth factor plays a key role in proliferative vitreoretinopathy.
• Invest Ophthalmol Vis Sci. 1999; 40: 2683-9
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• PURPOSE: The action of growth factors is thought to make a substantial contribution to the events leading to proliferative vitreoretinopathy (PVR). In this study, the importance of platelet-derived growth factor (PDGF) was tested in a rabbit model of PVR. METHODS: The approach was to compare the extent of PVR induced by cells that do or do not express the receptors for PDGF and therefore differ in their ability to respond to PDGF. RESULTS: Mouse embryo fibroblasts derived from PDGF receptor knock-out embryos that do not express either of the two PDGF receptors induced PVR poorly when injected into the eyes of rabbits that had previously undergone gas vitrectomy. Re-expression of the PDGF beta receptor in these cells did not improve the ability of the cells to cause PVR. In contrast, injection of cells expressing the PDGF alpha receptor resulted in stage 3 or higher PVR in 8 of 10 animals. CONCLUSIONS: These findings show that PDGF makes an important contribution to the development of PVR in this animal model. Furthermore, there is a marked difference between the two receptors for PDGF, and it is the PDGF alpha receptor that is capable of driving events that lead to PVR.

• Marconcini L et al.
• c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro.
• Proc Natl Acad Sci U S A. 1999; 96: 9671-6
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• c-fos-induced growth factor/vascular endothelial growth factor D (Figf/Vegf-D) is a secreted factor of the VEGF family that binds to the vessel and lymphatic receptors VEGFR-2 and VEGFR-3. Here we report that Figf/Vegf-D is a potent angiogenic factor in rabbit cornea in vivo in a dose-dependent manner. In vitro Figf/Vegf-D induces tyrosine phosphorylation of VEGFR-2 and VEGFR-3 in primary human umbilical cord vein endothelial cells (HUVECs) and in an immortal cell line derived from Kaposi's sarcoma lesion (KS-IMM). The treatment of HUVECs with Figf/Vegf-D induces dose-dependent cell growth. Figf/VEGF-D also induces HUVEC elongation and branching to form an extensive network of capillary-like cords in three-dimensional matrix. In KS-IMM cells Figf/Vegf-D treatment results in dose-dependent mitogenic and motogenic activities. Taken together with the previous observations that Figf/Vegf-D expression is under the control of the nuclear oncogene c-fos, our data uncover a link between a nuclear oncogene and angiogenesis, suggesting that Figf/Vegf-D may play a critical role in tumor cell growth and invasion.

• Marchio S et al.
• Vascular endothelial growth factor-C stimulates the migration and proliferation of Kaposi's sarcoma cells.
• J Biol Chem. 1999; 274: 27617-22
• Display abstract

• Recent evidence suggesting vascular endothelial growth factor-C (VEGF-C), which is a regulator of lymphatic and vascular endothelial development, raised the question whether this molecule could be involved in Kaposi's sarcoma (KS), a strongly angiogenic and inflammatory tumor often associated with infection by human immunodeficiency virus-1. This disease is characterized by the presence of a core constituted of three main populations of "spindle" cells, having the features of lymphatic/vascular endothelial cells, macrophagic/dendritic cells, and of a mixed macrophage-endothelial phenotype. In this study we evaluated the biological response of KS cells to VEGF-C, using an immortal cell line derived from a KS lesion (KS IMM), which retains most features of the parental tumor and can induce KS-like sarcomas when injected subcutaneously in nude mice. We show that VEGFR-3, the specific receptor for VEGF-C, is expressed by KS IMM cells grown in vitro and in vivo. In vitro, VEGF-C induces the tyrosine phosphorylation of VEGFR-2, a receptor also for VEGF-A, as well as that of VEGFR-3. The activation of these two receptors in KS IMM cells is followed by a dose-responsive mitogenic and motogenic response. The stimulation of KS IMM cells with a mutant VEGF-C unable to bind and activate VEFGR-2 resulted in no proliferative response and in a weak motogenic stimulation, suggesting that VEGFR-2 is essential in transducing a proliferative signal and cooperates with VEGFR-3 in inducing cell migration. Our data add new insights on the pathogenesis of KS, suggesting that the involvement of endothelial growth factors may not only determine KS-associated angiogenesis, but also play a critical role in controlling KS cell growth and/or migration and invasion.

• Stacker SA et al.
• A mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF receptor-2 activation retains the ability to induce vascular permeability.
• J Biol Chem. 1999; 274: 34884-92
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• Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.

• Eming SA, Yarmush ML, Krueger GG, Morgan JR
• Regulation of the spatial organization of mesenchymal connective tissue: effects of cell-associated versus released isoforms of platelet-derived growth factor.
• Am J Pathol. 1999; 154: 281-9
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• Platelet-derived growth factor (PDGF), a mitogen and chemoattractant for mesenchymal cells, occurs as cell-associated or released isoforms. To investigate their in vivo role, human keratinocytes, which normally synthesize both types of PDGF, were genetically modified to overexpress either wild-type PDGF-B (cell-associated) or the truncation mutant PDGF-B211 (released). Cells expressing the mutant isoform released 20 times more PDGF (145 ng/hour/10(7) cells) than cells expressing the wild-type isoform (6 ng/ hour/10(7) cells). When grafted as epithelial sheets onto athymic mice, modified cells formed a stratified epithelium and induced a connective tissue response that differed depending on the PDGF isoform expressed. Expression of PDGF-B211 induced a thick connective tissue with increased numbers of fibroblasts, mononuclear cells, and blood vessels evenly distributed throughout the connective tissue layer, whereas expression of PDGF-B induced a zone of fibroblasts and mononuclear cells localized to the interface of the epidermis and connective tissue, which often disrupted the continuity of the basement membrane. Immunostaining revealed that wild-type PDGF protein was deposited in the basement membrane region. These data suggest that the different binding properties of PDGF isoforms control the spatial organization of cellular events in regenerating mesenchymal tissue in vivo.

• Scheidegger P et al.
• Vascular endothelial growth factor (VEGF) and its receptors in tumor-bearing dogs.
• Biol Chem. 1999; 380: 1449-54
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• The molecular biology of the angiogenic growth factor, vascular endothelial growth factor (VEGF), has been studied in the dog. All major isoforms of VEGF are present in the dog. The amino acid sequences are identical between human and dog in the loop regions that are responsible for receptor binding. Accordingly, the VEGF receptors of dogs and humans are very similar and permit functional exchange of the growth factor. Here we show that canine VEGF activates human endothelial cells to the same extent as human VEGF. Similarly, the two proteins display identical cell binding properties. The VEGF receptor 1 (Flt-1) shows the same alternative splicing in humans and dogs and is overexpressed in the majority of tumors in both species. VEGF occurs also in canine tumors in similar relative quantities as in human malignancies. Based on the literature and our study we suggest that the molecular biology and the function of the VEGF signaling system are virtually identical in humans and canines and in healthy as well as in disease conditions.

• Mori S
• [PDGF and PDGF receptors]
• Nippon Ronen Igakkai Zasshi. 1999; 36: 457-65

• Rahimi N, Kazlauskas A
• A role for cadherin-5 in regulation of vascular endothelial growth factor receptor 2 activity in endothelial cells.
• Mol Biol Cell. 1999; 10: 3401-7
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• FLK-1/vascular endothelial growth factor receptor 2 (VEGFR-2) is one of the receptors for VEGF. In this study we examined the effect of cell density on activation of VEGFR-2. VEGF induces only very slight tyrosine phosphorylation of VEGFR-2 in confluent (95-100% confluent) pig aortic endothelial (PAE) cells. In contrast, robust VEGF-dependent tyrosine phosphorylation of VEGFR-2 was observed in cells plated in sparse culture conditions (60-65% confluent). A similar cell density-dependent phenomenon was observed in different endothelial cells but not in NIH-3T3 fibroblast cells expressing VEGFR-2. Stimulating cells with high concentrations of VEGF or replacing the extracellular domain of VEGFR-2 with that of the colony-stimulating factor 1 receptor did not alleviate the sensitivity of VEGFR-2 to cell density, indicating that the confluent cells were probably not secreting an antagonist to VEGF. Furthermore, in PAE cells, ectopically introduced platelet-derived growth factor alpha receptor could be activated at both high and low cell density conditions, indicating that the density effect was not universal for all receptor tyrosine kinases expressed in endothelial cells. In addition to lowering the density of cells, removing divalent cations from the medium of confluent cells potentiated VEGFR-2 phosphorylation in response to VEGF. These findings suggested that cell-cell contact may be playing a role in regulating the activation of VEGFR-2. To this end, pretreatment of confluent PAE cells with a neutralizing anti-cadherin-5 antibody potentiated the response of VEGFR-2 to VEGF. Our data demonstrate that endothelial cell density plays a critical role in regulating VEGFR-2 activity, and that the underlying mechanism appears to involve cadherin-5.

• Yonemura Y et al.
• Role of vascular endothelial growth factor C expression in the development of lymph node metastasis in gastric cancer.
• Clin Cancer Res. 1999; 5: 1823-9
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• Neogenesis of lymphatic vessel and lymphatic invasion is frequently found in the stroma of cancers, but the mechanisms of this phenomenon remain unclear. Vascular endothelial growth factor C (VEGF-C) is known to be the only growth factor for the lymphatic vascular system, and its receptor has been identified as Flt4. To clarify the mechanism of lymphatic invasion in cancer, we studied the expression of VEGF-C and flt4 genes in gastric cancer tissues. VEGF-C mRNA was mainly expressed in primary tumors (15 of 32; 47%), but the frequency of VEGF-C mRNA expression was low in normal mucosa (4 of 32; 13%). In primary tumors, there was a significant relationship between VEGF-C and flt4 mRNA expression. In contrast, Flt4 was mainly expressed on the lymphatic endothelial cells but not in cancer cells. A strong correlation was found between VEGF-C expression and lymph node status, lymphatic invasion, venous invasion, and tumor infiltrating patterns. Cancer cells in the lymphatic vessels frequently showed intracytoplasmic VEGF-C immunoreactivity. Furthermore, there was a close correlation between VEGF-C tissue status and the grade of lymph node metastasis. Patients with high expression of VEGF-C protein had a significantly poorer prognosis than did those in low VEGF-C expression group. By the Cox regression model, depth of wall invasion, lymph node metastasis, and VEGF-C tissue status emerged as independent prognostic parameters, and the VEGF-C tissue status was ranked third as an independent risk factor for death. These results strongly suggest that cancer cells producing VEGF-C may induce the proliferation and dilation of lymphatic vessels, resulting in the development of invasion of cancer cells into the lymphatic vessel and lymph node metastasis.

• Kranz A, Mayr U, Frank H, Waltenberger J
• The coronary endothelium: a target for vascular endothelial growth factor. Human coronary artery endothelial cells express functional receptors for vascular endothelial growth factor in vitro and in vivo.
• Lab Invest. 1999; 79: 985-91
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• Vascular endothelial growth factor (VEGF) is an angiogenic peptide that can stimulate endothelial cell proliferation and migration in vitro and collateral development in ischemic organs in vivo. Although postulated, the expression of functional VEGF receptors in the heart has not been demonstrated yet. To prove this hypothesis and to extend the molecular basis of myocardial angiogenesis, we have characterized the expression and function of VEGF receptors in human coronary artery endothelial cells (HCAEC) and in human heart tissue. VEGF strongly induces proliferation and migration of HCAEC. These cells express transcripts of the two VEGF receptors KDR and Flt-1. Their expression levels are higher in HCAEC as compared with human umbilical vein endothelial cells. In HCAEC, VEGF stimulates phosphorylation of KDR in a concentration-dependent manner proving that KDDR is a functional receptor tyrosine kinase. Scatchard analysis demonstrated the presence of the high affinity receptor Flt-1 in HCAEC with a kd of 8 pM. Flt-1 protein could be visualized as a single band corresponding to a size of 210 kd. In addition mature KDR protein could be detected in adult human heart. Taken together, HCAEC and human heart tissue express high levels of functional VEGF receptors. These results broaden the molecular basis for understanding and manipulating VEGF-induced endothelial function and angiogenesis in the coronary circulation.

• Olofsson B, Jeltsch M, Eriksson U, Alitalo K
• Current biology of VEGF-B and VEGF-C.
• Curr Opin Biotechnol. 1999; 10: 528-35
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• Endothelial growth factors and their receptors may provide important therapeutic tools for the treatment of pathological conditions characterised by defective or aberrant angiogenesis. Vascular endothelial growth factor (VEGF) is pivotal for vasculogenesis and for angiogenesis in normal and pathological conditions. VEGF-B and VEGF-C provide this gene family with additional functions, for example, VEGF-C also regulates lymphangiogenesis.

• Herold-Mende C et al.
• Expression and functional significance of vascular endothelial growth factor receptors in human tumor cells.
• Lab Invest. 1999; 79: 1573-82
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• Vascular endothelial growth factor (VEGF) is one of the key factors in tumor neoangiogenesis, acting through its receptors KDR (VEGFR-2) and fit-1 (VEGFR-1) expressed on endothelial cells. Our data demonstrate that VEGFR-1 and to a lesser extent VEGFR-2 are expressed in a number of human tumor tissues and derived cells in culture. VEGFR-1 protein is expressed in 26 of 42 glioma tissues, 22 of which show a coexpression of VEGFR-1 with VEGFR-2; 1 glioma tissue expresses exclusively VEGFR-2. In the derived glioma cell cultures, we found VEGFR-1 mRNA expression in 6 of 11 cultures, with one coexpressing VEGFR-1 and VEGFR-2. Of four established glioma cell lines, two expressed VEGFR-1. In addition VEGFR-1 protein expression was demonstrated in 30 of 37 tumor tissues of squamous cell carcinomas of the head and neck, with VEGFR-2 coexpression in 15 tissues and an expression of VEGFR-2 alone in 1 tissue. Derived tumor cell cultures showed mRNA expression of VEGFR-1 alone in seven of seven cases. Established melanoma cell lines expressed VEGFR-1 mRNA in four of five lines, with VEGFR-2 coexpression in two lines. Concerning the functional significance of VEGF receptor expression, VEGF treatment of VEGFR-1-expressing tumor cells induced the inhibition of cell proliferation by 25 to 55% and the inhibition of tumor cell migration by 29 to 55%. Thus our data indicate that the coexpression of VEGF and VEGFR-1 in tumor cells could have an inhibitory effect on tumor cell proliferation and migration, a mechanism possibly induced as a response to a deficiency in nutrient and oxygen supply.

• Abdiu A, Wingren S, Larsson SE, Wasteson A, Walz TM
• Effects of human platelet-derived growth factor-AB on sarcoma growth in vitro and in vivo.
• Cancer Lett. 1999; 141: 39-45
• Display abstract

• Platelet-derived growth factor (PDGF) has been proposed to play an important role in the growth of tumors. In order to study the effects of PDGF-AB on tumor growth in vivo, sarcoma-bearing mice were treated with PDGF-AB. The tumors, a malignant fibrous histiocytoma and an osteosarcoma, had functional PDGF receptors in vitro, as demonstrated by stimulation of PDGF-AB using a [3H]thymidine incorporation assay. Immunohistochemistry also revealed that both sarcoma xenografts expressed PDGF receptors. The tumor-bearing mice were given human PDGF-AB for 14 days, either continuously by an intraperitoneally placed mini-osmotic pump, or by daily injections. No effects on tumor growth in vivo were observed, as measured by tumor volume, autoradiography or cell cycle distribution. The histological appearance and ploidy of the tumors remained unaltered. The results indicate that, although the tumor cells are stimulated by PDGF-AB in vitro, the in vivo milieu or tumor growth pattern may render the tumors less susceptible to exogenously administered PDGF-AB in vivo.

• Whittle C, Gillespie K, Harrison R, Mathieson PW, Harper SJ
• Heterogeneous vascular endothelial growth factor (VEGF) isoform mRNA and receptor mRNA expression in human glomeruli, and the identification of VEGF148 mRNA, a novel truncated splice variant.
• Clin Sci (Colch). 1999; 97: 303-12
• Display abstract

• Vascular endothelial growth factor (VEGF) mediates increased vascular permeability and endothelial mitogenesis, and may orchestrate normal glomerular permselectivity and proteinuria. Distinct isoforms result from differential gene splicing. VEGF binds to two cell surface tyrosine-kinase receptors, KDR (kinase domain region) and Flt-1 (fms-like tyrosine kinase-1). The latter also exists in a soluble form (sFlt), which is inhibitory. We have studied patterns of VEGF-isoform and VEGF-receptor expression in isolated single normal human glomeruli. mRNA from 190 glomeruli (from 20 individuals) was harvested on to magnetic beads, and nested reverse transcription-PCR was performed using primers for the VEGF isoforms and VEGF receptors. Simultaneous nested reverse transcription-PCR for CD45 was conducted in order to exclude leucocyte contamination. Unexpected products were isolated, cloned and sequenced. Multiple patterns of glomerular VEGF mRNA isoform expression were identified. Most frequently (58%), all three common forms were expressed. VEGF(189) (i.e. 189-amino-acid form of VEGF) was expressed in 63%, VEGF(165) in 85% and VEGF(121) in 84% of glomeruli. Two unexpected PCR products were also identified: 18% of glomeruli expressed VEGF(145), and 27% of glomeruli expressed a new truncated VEGF splice variant, VEGF(148), lacking exon 6, the terminal part of exon 7 and exon 8. Multiple patterns of VEGF-receptor expression were also identified, the most common being expression of all three isoforms (28%). Overall, KDR was seen in 59% of glomeruli, Flt-1 in 45% and sFlt in 57%. Thus the expression of VEGF within normal glomeruli is complex and variable, with inter- and intra-individual variation. Furthermore, sFlt appears to be the co-dominant form of VEGF receptor expressed within glomeruli, suggesting that, in healthy individuals, a degree of VEGF autoregulation is the norm. The physiological importance of VEGF(148) remains to be confirmed.

• Lymboussaki A, Olofsson B, Eriksson U, Alitalo K
• Vascular endothelial growth factor (VEGF) and VEGF-C show overlapping binding sites in embryonic endothelia and distinct sites in differentiated adult endothelia.
• Circ Res. 1999; 85: 992-9
• Display abstract

• Vascular endothelial growth factor (VEGF) is a key modulator of angiogenesis during development and in adult tissues, whereas the related VEGF-C has been shown to induce both lymphangiogenesis and angiogenesis. To better understand the specific functions of these growth factors, we have here analyzed their binding to sections of mouse embryonic and adult tissues and compared the distribution of the bound growth factors with the expression patterns of the 3 known members of the VEGF receptor family as well as with neuropilin-1, a coreceptor for VEGF(165). Partially overlapping patterns of VEGF and VEGF-C binding were obtained in embryonic tissues, consistent with the expression of all known VEGF receptors by vascular endothelial cells. However, the most striking differences of binding were observed in the developing and adult heart, in which VEGF decorated all vessels, whereas strong VEGF-C signals were obtained only from epicardial vessels. In the lymph nodes, VEGF and VEGF-C showed distinct binding patterns in agreement with the differential location of their specific receptors. These results show that both VEGF-C and VEGF target embryonic blood vessels, whereas a more selective binding of VEGF-C occurs to its lymphatic vascular receptor in certain adult tissues. Our results suggest that VEGF and VEGF-C have both overlapping and distinct activities via their endothelial receptors.

• Heldin CH, Westermark B
• Mechanism of action and in vivo role of platelet-derived growth factor.
• Physiol Rev. 1999; 79: 1283-316
• Display abstract

• Platelet-derived growth factor (PDGF) is a major mitogen for connective tissue cells and certain other cell types. It is a dimeric molecule consisting of disulfide-bonded, structurally similar A- and B-polypeptide chains, which combine to homo- and heterodimers. The PDGF isoforms exert their cellular effects by binding to and activating two structurally related protein tyrosine kinase receptors, denoted the alpha-receptor and the beta-receptor. Activation of PDGF receptors leads to stimulation of cell growth, but also to changes in cell shape and motility; PDGF induces reorganization of the actin filament system and stimulates chemotaxis, i.e., a directed cell movement toward a gradient of PDGF. In vivo, PDGF has important roles during the embryonic development as well as during wound healing. Moreover, overactivity of PDGF has been implicated in several pathological conditions. The sis oncogene of simian sarcoma virus (SSV) is related to the B-chain of PDGF, and SSV transformation involves autocrine stimulation by a PDGF-like molecule. Similarly, overproduction of PDGF may be involved in autocrine and paracrine growth stimulation of human tumors. Overactivity of PDGF has, in addition, been implicated in nonmalignant conditions characterized by an increased cell proliferation, such as atherosclerosis and fibrotic conditions. This review discusses structural and functional properties of PDGF and PDGF receptors, the mechanism whereby PDGF exerts its cellular effects, and the role of PDGF in normal and diseased tissues.

• Stacker SA et al.
• Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers.
• J Biol Chem. 1999; 274: 32127-36
• Display abstract

• Vascular endothelial growth factor-D (VEGF-D) binds and activates the endothelial cell tyrosine kinase receptors VEGF receptor-2 (VEGFR-2) and VEGF receptor-3 (VEGFR-3), is mitogenic for endothelial cells, and shares structural homology and receptor specificity with VEGF-C. The primary translation product of VEGF-D has long N- and C-terminal polypeptide extensions in addition to a central VEGF homology domain (VHD). The VHD of VEGF-D is sufficient to bind and activate VEGFR-2 and VEGFR-3. Here we report that VEGF-D is proteolytically processed to release the VHD. Studies in 293EBNA cells demonstrated that VEGF-D undergoes N- and C-terminal cleavage events to produce numerous secreted polypeptides including a fully processed form of M(r) approximately 21,000 consisting only of the VHD, which is predominantly a non-covalent dimer. Biosensor analysis demonstrated that the VHD has approximately 290- and approximately 40-fold greater affinity for VEGFR-2 and VEGFR-3, respectively, compared with unprocessed VEGF-D. In situ hybridization demonstrated that embryonic lung is a major site of expression of the VEGF-D gene. Processed forms of VEGF-D were detected in embryonic lung indicating that VEGF-D is proteolytically processed in vivo.

• Piossek C, Schneider-Mergener J, Schirner M, Vakalopoulou E, Germeroth L, Thierauch KH
• Vascular endothelial growth factor (VEGF) receptor II-derived peptides inhibit VEGF.
• J Biol Chem. 1999; 274: 5612-9
• Display abstract

• Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor.

• Stacker SA, Achen MG
• The vascular endothelial growth factor family: signalling for vascular development.
• Growth Factors. 1999; 17: 1-11

• Zhu Z et al.
• Inhibition of vascular endothelial growth factor induced mitogenesis of human endothelial cells by a chimeric anti-kinase insert domain-containing receptor antibody.
• Cancer Lett. 1999; 136: 203-13
• Display abstract

• The kinase insert domain-containing receptor (KDR) is the human vascular endothelial growth factor (VEGF) receptor responsible for the mitogenic and angiogenic effects of VEGF. There is much experimental evidence to suggest that the VEGF/KDR pathway plays an important role in tumor angiogenesis, a process essential for tumor growth and metastasis. Here we produced a chimeric anti-KDR antibody (IgG1), c-p1C11, from a single chain (scFv) antibody isolated from a phage display library. C-p1C11 binds specifically to the extracellular domain of soluble as well as cell-surface expressed KDR. It effectively blocks VEGF-KDR interaction and inhibits VEGF-stimulated activation of KDR and MAP kinases p44/p42 of human endothelial cells. Furthermore, c-p1C11 efficiently neutralizes VEGF-induced mitogenesis of human endothelial cells. Our results suggest that antibodies against KDR have potential clinical applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.

• Mandriota SJ, Pepper MS
• [Lymphangiogenesis and biological activity ov vascular endothelial growth factor-C]
• J Soc Biol. 1999; 193: 159-63
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• Vascular endothelial growth factor (VEGF)-C is a new member of the VEGF family, a group of polypeptide growth factors which play key roles in the physiology and pathology of many aspects of the cardiovascular system, including vasculogenesis, hematopoiesis, angiogenesis and vascular permeability. VEGF signalling in endothelial cells occurs through three tyrosine kinase receptors (VEGFRs), expressed by endothelial cells and hematopoietic precursors. With respect to the first VEGF described, VEGF-A, which is an endothelial cell specific mitogen and key angiogenic factor, VEGF-C seems to play a major role in the development of the lymphatic system. This may reflect the different binding properties of VEGFs to VEGFRs, in that VEGF-A binds to VEGFR-1 and -2, whereas VEGF-C acts through VEGFR-3, whose expression becomes restricted to lymphatics and certain veins during development. However, the finding that VEGF-C also binds to and activates VEGFR-2 may explain why it induces angiogenesis under certain conditions, which makes it relevant to experimental or clinical settings in which one would wish to block or to stimulate angiogenesis. In this paper we briefly discuss current knowledge on the biological activity of VEGF-C, emphasizing that, as has already been shown for a number of other angiogenic factors, the biological effects of VEGF-C are strictly dependent on the activity of other angiogenic regulators present in the microenvironment of the responding endothelial cells.

• Cao Y et al.
• Vascular endothelial growth factor C induces angiogenesis in vivo.
• Proc Natl Acad Sci U S A. 1998; 95: 14389-94
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• Vascular endothelial growth factor C (VEGF-C) recently has been described to be a relatively specific growth factor for the lymphatic vascular system. Here we report that ectopic application of recombinant VEGF-C also has potent angiogenic effects in vivo. VEGF-C is sufficiently potent to stimulate neovascularization from limbal vessels in the mouse cornea. Similar to VEGF, the angiogenic response of corneas induced by VEGF-C is intensive, with a high density of new capillaries. However, the outgrowth of microvessels stimulated by VEGF-C was significantly longer than that induced by VEGF. In the developing embryo, VEGF-C was able to induce branch sprouts from the established blood vessels. VEGF-C also induced an elongated, spindle-like cell shape change and actin reorganization in both VEGF receptor (VEGFR)-2 and VEGFR-3-overexpressing endothelial cells, but not in VEGFR-1-expressing cells. Further, both VEGFR-2 and VEGFR-3 could mediate proliferative and chemotactic responses in endothelial cells on VEGF-C stimulation. Thus, VEGF-C may regulate physiological angiogenesis and participate in the development and progression of angiogenic diseases in addition to lymphangiogenesis.

• Witzenbichler B et al.
• Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia.
• Am J Pathol. 1998; 153: 381-94
• Display abstract

• Recently, vascular endothelial growth factor-C (VEGF-C or VEGF-2) was described as a specific ligand for the endothelial receptor tyrosine kinases VEGFR-2 and VEGFR-3. In vivo data, limited to constitutive overexpression in transgenic mice, have been interpreted as evidence that the growth-promoting effects of VEGF-C are restricted to development of the lymphatic vasculature. The current studies were designed to test the hypothesis that constitutive expression of VEGF-C in adult animals promotes angiogenesis. In vitro, VEGF-C exhibited a dose-dependent mitogenic and chemotactic effect on endothelial cells, particularly for microvascular endothelial cells (72% and 95% potency, respectively, compared with VEGF-A/VEGF-1). VEGF-C stimulated release of nitric oxide from endothelial cells and increased vascular permeability in the Miles assay; the latter effect was attenuated by pretreatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Both VEGFR-2 and VEGFR-3 receptors were shown to be expressed in human saphenous vein and internal mammary artery. The potential for VEGF-C to promote angiogenesis in vivo was then tested in a rabbit ischemic hindlimb model. Ten days after ligation of the external iliac artery, VEGF-C was administered as naked plasmid DNA (pcVEGF-C; 500 microg) from the polymer coating of an angioplasty balloon (n = 8 each) or as recombinant human protein (rhVEGF-C; 500 microg) by direct intra-arterial infusion. Physiological and anatomical assessments of angiogenesis 30 days later showed evidence of therapeutic angiogenesis for both pcVEGF-C and rhVEGF-C. Hindlimb blood pressure ratio (ischemic/normal) after pcVEGF-C increased to 0.83 +/- 0.03 after pcVEGF-C versus 0.59 +/- 0.04 (P < 0.005) in pGSVLacZ controls and to 0.76 +/- 0.04 after rhVEGF-C versus 0.58 +/- 0.03 (P < 0.01) in control rabbits receiving rabbit serum albumin. Doppler-derived iliac flow reserve was 2.7 +/- 0.1 versus 2.0 +/- 0.2 (P < 0.05) for pcVEGF-C versus LacZ controls and 2.9 +/- 0.3 versus 2.1 +/- 0.2 (P < 0.05) for rhVEGF-C versus albumin controls. Neovascularity was documented by angiography in vivo (angiographic scores: 0.85 +/- 0.05 versus 0.51 +/- 0.02 (P < 0.001) for plasmid DNA and 0.74 +/- 0.08 versus 0.53 +/- 0.03 (P < 0.05) for protein), and capillary density (per mm2) was measured at necropsy (252 +/- 12 versus 183 +/- 10 (P < 0.005) for plasmid DNA and 229 +/- 20 versus 164 +/- 20 (P < 0.05) for protein). In contrast to the results of gene targeting experiments, constitutive expression of VEGF-C in adult animals promotes angiogenesis in the setting of limb ischemia. VEGF-C and its receptors thus constitute an apparently redundant pathway for postnatal angiogenesis and may represent an alternative to VEGF-A for strategies of therapeutic angiogenesis in patients with limb and/or myocardial ischemia.

• Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M
• Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.
• Cell. 1998; 92: 735-45
• Display abstract

• Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis, binds to two receptor tyrosine kinases, KDR/Flk-1 and Flt-1. We now describe the purification and the expression cloning from tumor cells of a third VEGF receptor, one that binds VEGF165 but not VEGF121. This isoform-specific VEGF receptor (VEGF165R) is identical to human neuropilin-1, a receptor for the collapsin/semaphorin family that mediates neuronal cell guidance. When coexpressed in cells with KDR, neuropilin-1 enhances the binding of VEGF165 to KDR and VEGF165-mediated chemotaxis. Conversely, inhibition of VEGF165 binding to neuropilin-1 inhibits its binding to KDR and its mitogenic activity for endothelial cells. We propose that neuropilin-1 is a novel VEGF receptor that modulates VEGF binding to KDR and subsequent bioactivity and therefore may regulate VEGF-induced angiogenesis.

• Liu W, Ellis LM
• Regulation of vascular endothelial growth factor receptor KDR in vitro by a soluble factor in confluent endothelial cells.
• Pathobiology. 1998; 66: 247-52
• Display abstract

• Hypoxia regulates the expression of both vascular endothelial growth factor (VEGF) and its receptor (KDR). We have shown that cell density regulates VEGF expression in colon cancer and hypothesized that a similar mechanism regulates KDR in endothelial cells. Human umbilical vein endothelial cells were grown as sparse and confluent monolayers. Northern blot analysis revealed that KDR and VEGF mRNA expression in confluent cells was more than two-fold greater than in sparse cells. In contrast, flt-1 expression increased only slightly in cells grown to confluence. Cells were then plated at various concentrations and subjected to semi-quantitative PCR; KDR mRNA expression increased as cell density increased. Serum-free conditioned medium from cells grown to confluency for 48 h was added to sparsely plated cells, and KDR expression in the sparse cells increased twofold. We conclude that cell density regulates KDR endothelial cell expression via an unidentified soluble factor.

• Dumont DJ et al.
• Cardiovascular failure in mouse embryos deficient in VEGF receptor-3.
• Science. 1998; 282: 946-9
• Display abstract

• Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Unlike VEGF, the related VEGF-C stimulates the growth of lymphatic vessels through its specific lymphatic endothelial receptor VEGFR-3. Here it is shown that targeted inactivation of the gene encoding VEGFR-3 resulted in defective blood vessel development in early mouse embryos. Vasculogenesis and angiogenesis occurred, but large vessels became abnormally organized with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at embryonic day 9.5. Thus, VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of the lymphatic vessels.

• Fairbrother WJ et al.
• Novel peptides selected to bind vascular endothelial growth factor target the receptor-binding site.
• Biochemistry. 1998; 37: 17754-64
• Display abstract

• Peptides that inhibit binding of vascular endothelial growth factor (VEGF) to its receptors, KDR and Flt-1, have been produced using phage display. Libraries of short disulfide-constrained peptides yielded three distinct classes of peptides that bind to the receptor-binding domain of VEGF with micromolar affinities. The highest affinity peptide was also shown to antagonize VEGF-induced proliferation of primary human umbilical vascular endothelial cells. The peptides bind to a region of VEGF known to contain the contact surface for Flt-1 and the functional determinants for KDR binding. This suggests that the receptor-binding region of VEGF is a binding "hot spot" that is readily targeted by selected peptides and supports earlier assertions that phage-derived peptides frequently target protein-protein interaction sites. Such peptides may lead to the development of pharmacologically useful VEGF antagonists.

• Di Rocco F, Carroll RS, Zhang J, Black PM
• Platelet-derived growth factor and its receptor expression in human oligodendrogliomas.
• Neurosurgery. 1998; 42: 341-6
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• OBJECTIVE: Platelet-derived growth factor (PDGF) induces cellular proliferation and differentiation by activating intracellular signaling mechanisms via their cognate receptors. In previous studies, we demonstrated that human brain tumors such as meningiomas, astrocytomas, medulloblastomas, and ependymomas expressed the messenger ribonucleic acid for the PDGF subunits and their receptors. In the present study, we investigated the expression of the messenger ribonucleic acid PDGF A and B chains and the PDGF alpha and beta receptors in 17 cases of oligodendrogliomas. METHODS: Measurements of messenger ribonucleic acid levels were obtained using radioactive complementary deoxyribonucleic acid probes. Protein expression was analyzed with specific antibodies. RESULTS: Sixteen of 17 tumors expressed the PDGF A subunit and all the PDGF alpha receptors. Furthermore, all the tumors expressed PDGF B and PDGF beta receptor subunits. CONCLUSION: The results of this study suggest that oligodendrogliomas may have an autocrine loop stimulated by the interaction of PDGF and its receptor simultaneously produced by these tumors.

• Heldin CH, Ostman A, Ronnstrand L
• Signal transduction via platelet-derived growth factor receptors.
• Biochim Biophys Acta. 1998; 1378: 79113-79113
• Display abstract

• Platelet-derived growth factor (PDGF) exerts its stimulatory effects on cell growth and motility by binding to two related protein tyrosine kinase receptors. Ligand binding induces receptor dimerization and autophosphorylation, allowing binding and activation of cytoplasmic SH2-domain containing signal transduction molecules. Thereby, a number of different signaling pathways are initiated leading to cell growth, actin reorganization migration and differentiation. Recent observations suggest that extensive cross-talk occurs between different signaling pathways, and that stimulatory signals are modulated by inhibitory signals arising in parallel.

• Pepper MS, Mandriota SJ, Jeltsch M, Kumar V, Alitalo K
• Vascular endothelial growth factor (VEGF)-C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity.
• J Cell Physiol. 1998; 177: 439-52
• Display abstract

• Vascular endothelial growth factor-C (VEGF-C) is a recently characterized member of the VEGF family of angiogenic polypeptides. We demonstrate here that VEGF-C is angiogenic in vitro when added to bovine aortic or lymphatic endothelial (BAE and BLE) cells but has little or no effect on bovine microvascular endothelial (BME) cells. As reported previously for VEGF, VEGF-C and basic fibroblast growth factor (bFGF) induced a synergistic in vitro angiogenic response in all three cells lines. Unexpectedly, VEGF and VEGF-C also synergized in the in vitro angiogenic response when assessed on BAE cells. Characterization of VEGF receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express VEGFR-1 and -2, whereas of the three cell lines assessed, only BAE cells express VEGFR-3. We also demonstrate that VEGF-C increases plasminogen activator (PA) activity in the three bovine endothelial cell lines and that this is accompanied by a concomitant increase in PA inhibitor-1. Addition of alpha2-antiplasmin to BME cells co-treated with bFGF and VEGF-C partially inhibited collagen gel invasion. These results demonstrate, first, that by acting in concert with bFGF or VEGF, VEGF-C has a potent synergistic effect on the induction of angiogenesis in vitro and, second, that like VEGF and bFGF, VEGF-C is capable of altering endothelial cell extracellular proteolytic activity. These observations also highlight the notion of context, i.e., that the activity of an angiogenesis-regulating cytokine depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell.

• Robinson CJ, Stammers R, Gaines-Das R
• The international standard for platelet-derived growth factor-BB: comparison of candidate preparations by in vitro bioassays and immunoassays.
• Growth Factors. 1998; 16: 153-60
• Display abstract

• In an international collaborative study, four preparations of recombinant, human sequence, platelet-derived growth factor-BB (PDGF-BB), two glycosylated and two nonglycosylated, were evaluated, using in vitro bioassays and immunoassays, by seven laboratories in three countries, for their suitability to serve as the international standard (IS) for PDGF-BB. The study shows that interlaboratory variation in estimates of PDGF-BB potency is reduced by use of a common standard. The bioassays, using various types of fibroblast, detected PDGF-BB and PDGF-AB as equally potent, while immunoassays discriminated between the two isoforms. On the basis of the results reported here, the World Health Organization (WHO) established the preparation coded 94/728 as the first IS for PDGF-BB, with an assigned unitage of 3000 International Units per ampoule. The IS may be obtained by writing to NIBSC, PO Box 1193, Potters Bar, EN6 3QH, UK, or through web site http:??www.nibsc.ac.uk.

• Ergun S, Luttmer W, Fiedler W, Holstein AF
• Functional expression and localization of vascular endothelial growth factor and its receptors in the human epididymis.
• Biol Reprod. 1998; 58: 160-8
• Display abstract

• Blood supply is essential for the maintenance of epididymal function. Since there is no considerable neovascularization in the epididymis, this tissue could represent a suitable model to study the vascular endothelial growth factor (VEGF) effect for vascular permeability. We studied the expression and function of VEGF and its receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase (designated as kinase insert domain-containing receptor, KDR in the human) in the human epididymis. VEGF and VEGF receptors mRNA were detected in the human epididymal tissue. VEGF protein was localized in peritubular and in ciliated cells of efferent ducts as well as in peritubular and basal cells of the epididymal duct. Vascular endothelial cells did not express VEGF. Flt-1 protein was localized in ciliated cells of efferent ducts and in lymphatic vessels. Vascular endothelial cells were negative for Flt-1 but positive for KDR. In vitro VEGF165 treatment of epididymal tissue induced endothelial fenestrations and opening of interendothelial junctions. Additionally, we observed for the first time that VEGF could induce transendothelial gaps. We conclude that these gaps might be of importance not only for molecular transport but also for cell passage across the vessel wall, which may be significant for tumor metastasis. VEGF may act as a paracrine effector to influence the permeability of lymphatic vessels via Flt-1, and of blood vessels via KDR.

• Rossi E et al.
• Increased plasma levels of platelet-derived growth factor (PDGF-BB + PDGF-AB) in patients with never-treated mild essential hypertension.
• Am J Hypertens. 1998; 11: 1239-43
• Display abstract

• Platelet-derived growth factor (PDGF) could play a role in both vascular hypertrophy and atherosclerotic disease associated with hypertension. To assess whether plasma PDGF level is increased in mild essential hypertension, we measured plasma PDGF concentration in 25 never-treated patients with uncomplicated mild essential hypertension and in 22 normotensive healthy subjects. To evaluate the contribution of platelets to plasma PDGF in the two groups, we also measured plasma beta-thromboglobulin (BTG). Measurement of PDGF was carried out through an enzyme-linked immunoadsorbent assay, which detects two PDGF dimers, namely PDGF-BB and PDGF-AB. Both plasma PDGF and BTG were higher in the hypertensive than in the normotensive subjects. The ratio of PDGF to BTG was similar in the two groups. Plasma PDGF was weakly correlated with plasma BTG in the normotensive subjects, whereas this relationship was lost in the hypertensive patients. Our results suggest that the increase in plasma PDGF (PDGF-AB + PDGF-BB) in never-treated essential hypertension is mainly due to platelet activation. The increased circulating level of PDGF could play a role in the vascular structural changes associated with hypertension.

• Kaetzel DM, Reid JD 4th, Pedigo N, Zimmer SG, Boghaert ER
• A dominant-negative mutant of the platelet-derived growth factor A-chain increases survival of hamsters implanted intracerebrally with the highly invasive CxT24-neo3 glioblastoma cell.
• J Neurooncol. 1998; 39: 33-46
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• Evidence is accumulating to suggest a role for PDGF in stimulating malignant growth in astrocytoma, although it has been obtained using model systems (growth in 2-dimensional cell culture, athymic nude mice) that do not assess the complex interactions of these tumors with normal brain tissue. In the current study, the highly invasive hamster glioblastoma cell line CxT24-neo3 was used as a model to study the role of platelet-derived growth factor (PDGF) in mediating malignant growth both in vitro and in vivo when implanted directly into the right lateral ventricle of the brain. Co-expression of PDGF B-chain mRNA and PDGF alpha-receptors was detected in these cells, indicating potential for autocrine activation of their growth. CxT24-neo3 cells transfected with wild-type and receptor binding-deficient forms of the PDGF A- and B-chains displayed alterations in their abilities to grow as three-dimensional spheroids, with overexpression of wild-type B-chain resulting in increased spheroid formation, but a decreased rate of spheroid growth. Influence of these PDGF polypeptides on tumor invasion and survival time in vivo was evaluated following implantation of these spheroids in the brain. While all hamsters implanted with control spheroids died within 21 d (average 17 d), those implanted with cells expressing the receptor binding-deficient A-chain survived for much greater periods of time (average 80 d). Modest increases in survival were also seen in cells stably expressing wild-type A-chain (25 d) and mutant B-chain (26 d) proteins. The present study suggests an important role of PDGF in mediating the malignant growth of the CxT24-neo3 cell line in cerebral cortex, possibly via paracrine interactions with normal cortical cell types (i.e., glia, neurons).

• Siemeister G, Schirner M, Reusch P, Barleon B, Marme D, Martiny-Baron G
• An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells.
• Proc Natl Acad Sci U S A. 1998; 95: 4625-9
• Display abstract

• Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

• McCarthy MJ, Crowther M, Bell PR, Brindle NP
• The endothelial receptor tyrosine kinase tie-1 is upregulated by hypoxia and vascular endothelial growth factor.
• FEBS Lett. 1998; 423: 334-8
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• The receptor tyrosine kinase tie-1 is essential for angiogenesis where it appears to have a role in vessel maturation. Here we have examined the effects of hypoxia and vascular endothelial growth factor (VEGF) on the level of tie-1 protein expressed in bovine aortic endothelial cells. Both hypoxia (2% O2) and VEGF were found to increase tie-1 in a time-dependent manner. Hypoxic induction was direct and effects of hypoxia and VEGF were not additive. Experiments with actinomycin D indicate that these activators regulate tie-1 at the transcriptional level.

• Lindahl P, Betsholtz C
• Not all myofibroblasts are alike: revisiting the role of PDGF-A and PDGF-B using PDGF-targeted mice.
• Curr Opin Nephrol Hypertens. 1998; 7: 21-6
• Display abstract

• Analyses of platelet-derived growth factor (PDGF) and PDGF-receptor knockout mice show that the development of specific subsets of smooth muscle cells (SMCs) depend on PDGF. Pericytes and mesangial cells failed to develop in PDGF-B deficient embryos, whereas alveolar SMCs failed to develop in PDGF-A deficient embryos. The ontogeny of pericytes and alveolar SMCs suggests a common theme in the formation of PDGF-dependent SMCs, in which PDGF-receptor positive SMC progenitors spread from proximal to distal sites along PDGF-expressing epithelial, or endothelial, tubes.

• Kagami S et al.
• Collagen type I modulates the platelet-derived growth factor (PDGF) regulation of the growth and expression of beta1 integrins by rat mesangial cells.
• Biochem Biophys Res Commun. 1998; 252: 728-32
• Display abstract

• Mesangial cell (MC) proliferation and the deposition of collagen type I (collagen I) are the major pathological features in many types of glomerulonephritis (GN). Recent work suggested that beta-integrins play a critical role in the cell proliferation and extracellular matrix (ECM) remodeling observed in tissue repair after injury. To examine the involvement of beta-integrins in MC proliferation in association with the interaction of MCs with pathological collagen I, we investigated the effect of a prominent mitogen, platelet-derived growth factor-BB (PDGF-BB) on the growth and expression of beta-integrins by MCs cultured on plastic or in a three-dimensional collagen I gel. Immunoprecipitation using 35S-metabolic labeling, flow cytometry and a 3H-thymidine-uptake analysis demonstrated that PDGF-BB stimulated the cell mitogenicity and the expression of alpha5beta1 integrin (a fibronectin receptor), but not alpha1beta1 integrin (a collagen and laminin receptor) of MCs on plastic, in a dose-dependent manner. In contrast, MCs in the collagen I gels showed no significant changes in mitogenicity or alpha1beta1 and alpha5beta1 integrin expression, but increased alpha1beta1 integrin-mediated gel contraction was observed after PDGF-BB stimulation. Thus, the parallel up-regulation of MC-mitogenicity and alpha5beta1 integrin expression by PDGF-BB suggested that alpha5beta1 integrin is an important ECM receptor involved in the proliferative phenotype of MC. A spatial interaction between MCs and pathological collagen I in GN may influence the PDGF regulation of the MC phenotype regarding the cell growth and the expression of beta1 integrins.

• Eichmann A et al.
• Avian VEGF-C: cloning, embryonic expression pattern and stimulation of the differentiation of VEGFR2-expressing endothelial cell precursors.
• Development. 1998; 125: 743-52
• Display abstract

• VEGF-C is a recently discovered secreted polypeptide related to the angiogenic mitogen VEGF. We have isolated the quail VEGF-C cDNA and shown that its protein product is secreted from transfected cells and interacts with the avian VEGFR3 and VEGFR2. In situ hybridization shows that quail VEGF-C mRNA is strongly expressed in regions destined to be rich in lymphatic vessels, particularly the mesenteries, mesocardium and myotome, in the region surrounding the jugular veins, and in the kidney. These expression sites are similar to those observed in the mouse embryo (E. Kukk, A. Lymboussaki, S. Taira, A. Kaipainen, M. Jeltsch, V. Joukov and K. Alitalo, 1996, Development 122, 3829-3837). We have observed VEGFR3-positive endothelial cells in proximity to most of the VEGF-C-expressing sites, suggesting functional relationships between this receptor-ligand couple. The comparison of the VEGF and VEGFR2 knockout phenotypes had suggested the existence of another ligand for VEGFR2. We therefore investigated the effect of VEGF-C on VEGFR2-positive cells isolated from the posterior mesoderm of gastrulating embryos. We have recently shown that VEGF binding triggers endothelial differentiation of these cells, whereas hemopoietic differentiation appears to be mediated by binding of a so far unidentified VEGFR2 ligand. We show here that VEGF-C also triggers endothelial differentiation of these cells, presumably via VEGFR2. These results indicate that VEGF and VEGF-C can act in a redundant manner via VEGFR2. In conclusion, VEGF-C appears to act during two different developmental phases, one early in posterior mesodermal VEGFR2-positive endothelial cell precursors which are negative for VEGFR3 and one later in regions rich in lymphatic vessels at a time when endothelial cells express both VEGFR2 and VEGFR3.

• Ristimaki A, Narko K, Enholm B, Joukov V, Alitalo K
• Proinflammatory cytokines regulate expression of the lymphatic endothelial mitogen vascular endothelial growth factor-C.
• J Biol Chem. 1998; 273: 8413-8
• Display abstract

• Vascular endothelial growth factor (VEGF) is a prime regulator of normal and pathological angiogenesis. Three related endothelial cell growth factors, VEGF-B, VEGF-C, and VEGF-D were recently cloned. We have here studied the regulation of VEGF-C, a lymphatic endothelial growth factor, by angiogenic proinflammatory cytokines. Interleukin (IL)-1beta induced a concentration- and a time-dependent increase in VEGF-C, but not in VEGF-B, mRNA steady-state levels in human lung fibroblasts. The increase in VEGF-C mRNA levels was mainly due to increased transcription rather than elevated mRNA stability as detected by the nuclear run-on method and by following mRNA decay in the presence of an inhibitor of transcription, respectively. In contrast, angiopoietin-1 mRNA, encoding the ligand for the endothelial-specific Tek/Tie-2 receptor, was down-regulated by IL-1beta. Tumor necrosis factor-alpha and IL-1alpha also elevated VEGF-C mRNA steady-state levels, whereas the IL-1 receptor antagonist and dexamethasone inhibited the effect of IL-1beta. Experiments with cycloheximide indicated that the effect of IL-1beta was independent of protein synthesis. Hypoxia, which is an important inducer of VEGF expression, had no effect on VEGF-B or VEGF-C mRNA levels. IL-1beta and tumor necrosis factor-alpha also stimulated the production of VEGF-C protein by the fibroblasts. Cytokines and growth factors have previously been shown to down-regulate VEGF receptors in vascular endothelial cells. We found that the mRNA for the VEGF- and VEGF-C-binding VEGFR-2 (KDR/Flk-1) was stimulated by IL-1beta in human umbilical vein endothelial cells, whereas the mRNA levels of VEGFR-1 (Flt-1) and VEGFR-3 (Flt-4) were not altered. Our data suggest that in addition to VEGF, VEGF-C may also serve as an endothelial stimulus at sites of cytokine activation. In particular, these results raise the possibility that certain proinflammatory cytokines regulate the lymphatic vessels indirectly via VEGF-C.

• Fuh G, Li B, Crowley C, Cunningham B, Wells JA
• Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor.
• J Biol Chem. 1998; 273: 11197-204
• Display abstract

• Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2-3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind approximately 100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.

• Lymboussaki A et al.
• Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors.
• Am J Pathol. 1998; 153: 395-403
• Display abstract

• It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.

• Olofsson B et al.
• Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells.
• Proc Natl Acad Sci U S A. 1998; 95: 11709-14
• Display abstract

• The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp63, Asp64, and Glu67 to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B186 dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.

• Pepper MS, Mandriota SJ
• Regulation of vascular endothelial growth factor receptor-2 (Flk-1) expression in vascular endothelial cells.
• Exp Cell Res. 1998; 241: 414-25
• Display abstract

• We have previously reported the existence of a synergistic interaction between vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the induction of angiogenesis in vitro. Here we demonstrate that bFGF increases VEGF receptor-2 (VEGFR-2/Flk-1) expression: mRNA levels were increased by 4.5- to 8.0-fold and total protein by 2.0- to 3.5-fold, in bovine microvascular endothelial (BME), aortic endothelial (BAE), and transformed fetal aortic (GM7373) endothelial cells. VEGF itself did not affect VEGFR-2 expression, and neither bFGF nor VEGF altered expression of FGF receptor-1. We also show that synergism occurs at the level of proliferation when this is measured in a three-dimensional but not in a conventional two-dimensional assay. Differences in the level of VEGFR-2 expression were also observed when cells were grown on or within collagen gels under different conditions: mRNA levels were lowest under sparse conditions, increased 20- to 26-fold at confluence, and increased even further (57-fold) when cells were cultured in suspension in three-dimensional collagen gels. Finally, a synergistic increase was seen in the level of expression of urokinase and urokinase receptor mRNAs when cells were exposed to bFGF and VEGF for 4 days. These findings demonstrate that the level of VEGFR-2 expression can be modulated by environmental factors including cytokines and the geometry of the culture conditions and provide some insight into the mechanisms of synergism between bFGF and VEGF in the induction of angiogenesis in vitro.

• Zhu Z et al.
• Inhibition of vascular endothelial growth factor-induced receptor activation with anti-kinase insert domain-containing receptor single-chain antibodies from a phage display library.
• Cancer Res. 1998; 58: 3209-14
• Display abstract

• A single-chain antibody phage display library was constructed from spleen cells of mice immunized with a soluble form of a human vascular endothelial growth factor (VEGF) receptor, kinase insert domain-containing receptor (KDR). After two rounds of biopanning, >90% of the clones recovered were specifically reactive to KDR. Subsequent selection identified two clones that blocked VEGF binding to KDR. The clones were expressed in Escherichia coli and purified as soluble single-chain Fv (scFv) antibodies. The affinities of the scFv for binding to KDR were determined by BIAcore analysis (2.1 x 10(-9)-5.9 x 10(-9) M). One scFv, p1C11, was shown to inhibit VEGF-induced KDR phosphorylation and VEGF-stimulated DNA synthesis in human umbilical vein endothelial cells. There is much experimental evidence to suggest that the VEGF/KDR/Flk-1 pathway plays an important role in tumor angiogenesis, a process that is essential for tumor growth and metastasis. The antibodies discussed here, which block VEGF binding to KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved.

• Joukov V et al.
• A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities.
• J Biol Chem. 1998; 273: 6599-602
• Display abstract

• The vascular endothelial growth factor (VEGF) and the VEGF-C promote growth of blood vessels and lymphatic vessels, respectively. VEGF activates the endothelial VEGF receptors (VEGFR) 1 and 2, and VEGF-C activates VEGFR-3 and VEGFR-2. Both VEGF and VEGF-C are also potent vascular permeability factors. Here we have analyzed the receptor binding and activating properties of several cysteine mutants of VEGF-C including those (Cys156 and Cys165), which in other platelet-derived growth factor/VEGF family members mediate interchain disulfide bonding. Surprisingly, we found that the recombinant mature VEGF-C in which Cys156 was replaced by a Ser residue is a selective agonist of VEGFR-3. This mutant, designated DeltaNDeltaC156S, binds and activates VEGFR-3 but neither binds VEGFR-2 nor activates its autophosphorylation or downstream signaling to the ERK/MAPK pathway. Unlike VEGF-C, DeltaNDeltaC156S neither induces vascular permeability in vivo nor stimulates migration of bovine capillary endothelial cells in culture. These data point out the critical role of VEGFR-2-mediated signal transduction for the vascular permeability activity of VEGF-C and strongly suggest that the redundant biological effects of VEGF and VEGF-C depend on binding and activation of VEGFR-2. The DeltaNDeltaC156S mutant may provide a valuable tool for the analysis of VEGF-C effects mediated selectively via VEGFR-3. The ability of DeltaNDeltaC156S to form homodimers also emphasizes differences in the structural requirements for VEGF and VEGF-C dimerization.

• Cleaver O, Tonissen KF, Saha MS, Krieg PA
• Neovascularization of the Xenopus embryo.
• Dev Dyn. 1997; 210: 66-77
• Display abstract

• The receptor tyrosine kinase, Flk-1 or VEGFR-2, and its ligand, vascular endothelial growth factor (VEGF) are required for the development of the embryonic vasculature. Targeted disruption of either gene in mice results in the failure of vascular system formation. The Xenopus homologues of flk-1 and VEGF have been cloned and their expression has been examined throughout early embryonic development. These studies indicate that flk-1 is expressed in groups of endothelial precursor cells which will form the major blood vessels of the embryo, including the posterior cardinal veins, the dorsal aorta, the vitelline veins, and the endocardium. VEGF expression is found in tissues adjacent to the mesenchyme containing the flk-1-expressing endothelial precursors. Expression of both flk-1 and VEGF is transient, appearing as the primary vascular plexus is forming and declining steadily after the onset of functional embryonic circulation. After establishment of the primary vascular structures, flk-1 expression is also observed in the intersegmental veins which form by an angiogenic mechanism. Overall, these results support a role for VEGF/flk-1 signaling in both vasculogenesis and angiogenesis in the Xenopus embryo. When VEGF is expressed ectopically in Xenopus embryos by microinjection of either plasmid DNA or synthetic mRNA, large, disorganized vascular structures are produced. This result indicates that ectopic VEGF is capable of altering the architecture of the developing vascular network.

• Chilov D et al.
• Genomic organization of human and mouse genes for vascular endothelial growth factor C.
• J Biol Chem. 1997; 272: 25176-83
• Display abstract

• We report here the cloning and characterization of human and mouse genes for vascular endothelial growth factor C (VEGF-C), a newly isolated member of the vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) family. Both VEGF-C genes comprise over 40 kilobase pairs of genomic DNA and consist of seven exons, all containing coding sequences. The VEGF homology domain of VEGF-C is encoded by exons 3 and 4. Exons 5 and 7 encode cysteine-rich motifs of the type C6C10CRC, and exon 6 encodes additional C10CXCXC motifs typical of a silk protein. A putative alternatively spliced rare RNA form lacking exon 4 was identified in human fibrosarcoma cells, and a major transcription start site was located in the human VEGF-C gene 523 base pairs upstream of the translation initiation codon. The upstream promoter sequences contain conserved putative binding sites for Sp-1, AP-2, and NF-kappaB transcription factors but no TATA box, and they show promoter activity when transfected into cells. The VEGF-C gene structure is thus assembled from exons encoding propeptides and distinct cysteine-rich domains in addition to the VEGF homology domain, and it shows both similarities and distinct differences in comparison with other members of the VEGF/PDGF gene family.

• Fournier E, Birnbaum D, Borg JP
• [Receptors for factors of the VEGF (Vascular Endothelial Growth Family)]
• Bull Cancer. 1997; 84: 397-405
• Display abstract

• Growth factors of the VEGF (vascular endothelial growth factor) family comprises 4 well characterized members that play a crucial role in the biology of blood vessels. They interact with 3 high affinity tyrosine kinase receptors (FLT1/VEGFR1, FLK1/KDR/VEGFR2, FLT4/VEGFR3). VEGF/VEGFR interactions have essential functions in blood vessel formation during development, specific phases of adult life, and in some pathological processes with neo-vascularization such as tumor growth.

• Joukov V et al.
• Proteolytic processing regulates receptor specificity and activity of VEGF-C.
• EMBO J. 1997; 16: 3898-911
• Display abstract

• The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor. Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C. The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2. Recombinant 'mature' VEGF-C made in yeast bound VEGFR-3 (K[D] = 135 pM) and VEGFR-2 (K[D] = 410 pM) and activated these receptors. Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure-function relationships in the PDGF/VEGF family.

• Dougher AM et al.
• Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor.
• Growth Factors. 1997; 14: 257-68
• Display abstract

• Vascular endothelial growth factor (VEGF), a potent and specific activator of endothelial cells, is expressed as multiple homodimeric forms resulting from alternative RNA splicing. VEGF121 does not bind heparin while the other three isoforms do, and it has been documented that the binding of VEGF165 to its receptor is dependent upon cell surface heparin sulfate proteoglycans. Little is known about the biochemical mechanism that allows for heparin regulation of growth factor binding. For example, it is not clear whether heparin interactions with growth factor or with cell surface receptors or both are essential for VEGF binding to its receptor. In this manuscript we provide results which are consistent with the hypothesis that an interaction between heparin and a site on the KDR receptor subtype is essential for VEGF165 binding. First, we demonstrate that expression of KDR into a CHO cell line deficient in heparan sulfate biosynthesis does not allow VEGF165 binding unless heparin is exogenously added during the binding assay. Secondly, we show that a ten amino acid synthetic peptide, corresponding to a sequence from the extracellular domain of the KDR, both inhibits VEGF165 binding to the receptor and also binds heparin with high avidity. Third, affinity purification of heparin molecules on a KDR-derived peptide affinity column, together with capillary electrophoresis and polyacrylamide electrophoresis analysis, was used to show that the KDR-derived peptide interacts with a specific subset of polysaccharide chains contained in the unfractionated heparin. Taken together, these results are consistent with the hypothesis that interactions between cell surface heparan sulfate proteoglycans and the VEGF receptor contribute to allowing maximal VEGF binding.

• Kirsch M, Wilson JC, Black P
• Platelet-derived growth factor in human brain tumors.
• J Neurooncol. 1997; 35: 289-301
• Display abstract

• This paper initially reviews ligand and receptor systems for the PDGF family and the signalling systems they use as well as their role in neural developments. It then describes the putative role of this family in astrocytoma, meningioma, and pituitary adenoma pathogenesis. Potential therapies with receptor antagonists or dominant negative mutants are discussed in the final sections.

• Muller YA, Christinger HW, Keyt BA, de Vos AM
• The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding.
• Structure. 1997; 5: 1325-38
• Display abstract

• BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays a role in pathogenic vascularization which is associated with a number of clinical disorders, including cancer and rheumatoid arthritis. The development of VEGF antagonists, which prevent the interaction of VEGF with its receptor, may be important for the treatment of such disorders. VEGF is a homodimeric member of the cystine knot growth factor superfamily, showing greatest similarity to platelet-derived growth factor (PDGF). VEGF binds to two different tyrosine kinase receptors, kinase domain receptor (KDR) and Fms-like tyrosine kinase 1 (Flt-1), and a number of VEGF homologs are known with distinct patterns of specificity for these same receptors. The structure of VEGF will help define the location of the receptor-binding site, and shed light on the differences in specificity and cross-reactivity among the VEGF homologs. RESULTS: We have determined the crystal structure of the receptor-binding domain of VEGF at 1.93 A resolution in a triclinic space group containing eight monomers in the asymmetric unit. Superposition of the eight copies of VEGF shows that the beta-sheet core regions of the monomers are very similar, with slightly greater differences in most loop regions. For one loop, the different copies represent different snapshots of a concerted motion. Mutagenesis mapping shows that this loop is part of the receptor-binding site of VEGF. CONCLUSIONS: A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.

• Hart CE, Clowes AW
• Platelet-derived growth factor and arterial response to injury.
• Circulation. 1997; 95: 555-6

• Valenzuela CF, Kazlauskas A, Weiner JL
• Roles of platelet-derived growth factor in the developing and mature nervous systems.
• Brain Res Brain Res Rev. 1997; 24: 77-89
• Display abstract

• In spite of its association by history and name to platelets, platelet-derived growth factor (PDGF) exerts important actions in a myriad of tissues, including the nervous system. PDGF and PDGF receptors are widely expressed in neuronal and glial cells of many regions of both the central and peripheral nervous systems. In this topical review, the roles played by PDGF in the development and maintenance of the nervous system are discussed. We also discuss the modulatory effects of PDGF on synaptic transmission, its role in neoplastic and non-neoplastic conditions of the central nervous system, and the neuroprotective effects of this growth factor.

• Walsh TP, Grant GH
• Computer modelling of the receptor-binding domains of VEGF and PIGF.
• Protein Eng. 1997; 10: 389-98
• Display abstract

• Models of the platelet-derived growth factor (PDGF)-like domains of vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) were built based on their homology to PDGF. These domains contain most of the determinants for receptor binding. The sequences of these proteins exhibit limited but significant homology to that of platelet-derived growth factor (PDGF), a member of the cystine knot growth factor family. The eight cysteine residues that are involved in intra- and interchain disulphide bonds are conserved. Two high affinity receptors for VEGF have been identified, only one of which binds PIGF. The models presented here are consistent with results that show that VEGF receptor binding is mediated by charged residues in the loops. A comparison of the models suggests that the difference in receptor-binding specificity between VEGF and PIGF may be due to differences in the distribution of positively charged residues and the exposure of hydrophobic residues in the loops.

• Fielder W et al.
• Expression of FLT4 and its ligand VEGF-C in acute myeloid leukemia.
• Leukemia. 1997; 11: 1234-7
• Display abstract

• FLT4 represents a recently cloned member of class III receptor tyrosine kinases which include receptors for the angiogenic growth factor VEGF, namely FLT1 and KDR. The ligand of FLT4 has been identified as VEGF-C which shares sequence homology with VEGF and P1GF. In the adult FLT4 shows a restricted expression pattern that is limited to lymphatic endothelia and endothelia of some high endothelial venules (HEV). FLT4 has also been detected in some tumor cell lines including the hematopoietic line HEL. We therefore investigated expression of FLT4 and its ligand VEGF-C in fresh samples from patients with AML. Using a sensitive PCR method we detected FLT4 m-RNA in 15 of 41 patients with de novo AML at diagnosis or relapse and in three of 12 patients with secondary AML. FLT4 expression was confirmed by immunocytochemistry in a subgroup of the studied patient population. FLT4 was also found in leukemic cell line U937, but not TF-1 and KG1a. VEGF-C expression was found in leukemic samples of four of seven FLT4-positive and four of six FLT4-negative patients. U937 cells also produced VEGF-C m-RNA. Interestingly, FLT4 expression was not detected in bone marrow samples of 15 normal volunteer donors or in CD34-positive cells from three additional donors. Possible autocrine and paracrine growth stimulation of leukemic blasts by VEGF-C is currently being investigated in our laboratory.

• Fitz LJ et al.
• Characterization of murine Flt4 ligand/VEGF-C.
• Oncogene. 1997; 15: 613-8
• Display abstract

• Flt4 is a receptor protein tyrosine kinase that is expressed in the adult lymphatic endothelium and high endothelial venules. We have used a BIAcore assay to identify rodent and human cell conditioned media containing the ligand of Flt4 (Flt4-L). Receptor-based affinity chromatography was used to purify this growth factor, followed by amino acid sequencing and molecular cloning of the murine cDNA, the orthologue of human vascular endothelial growth factor-C and vascular endothelial growth factor related protein. The murine flt4-L gene was localized to chromosome 8 and demonstrated to be widely expressed. Flt4-L was found to have a hydrophobic signal sequence and a pro-peptide-like sequence that is removed to generate the mature N-terminus. In addition, the C-terminal region of Flt4-L has four repeats of a cysteine-rich motif that is presumably also proteolytically processed to generate the 21000 Mr polypeptide subunit of the Flt4-L homodimer. Recombinant Flt4-L activated Flt4 as judged by induction of tyrosyl phosphorylation, and induced mitogenesis in vitro of lymphatic endothelial cells.

• Saji M, Taga M, Matsui H, Suyama K, Kurogi K, Minaguchi H
• Gene expression and specific binding of platelet-derived growth factor and its effect on DNA synthesis in human decidual cells.
• Mol Cell Endocrinol. 1997; 132: 73-80
• Display abstract

• To clarify the biological significance of platelet-derived growth factor (PDGF) in human decidual cell function, which is important for the maintenance of pregnancy, we investigated gene expression of the PDGF subunits, PDGF-A and PDGF-B, specific binding of the PDGF isoform, and the effect of PDGF dimers on DNA synthesis in human decidual cells. We detected in decidua from early pregnancy the expected DNA bands of PDGF-A and PDGF-B by reverse transcriptase-polymerase chain reaction (RT-PCR) as well as mRNAs of each PDGF subunit by Northern blot hybridization, demonstrating that both PDGF subunits exist in this tissue. Scatchard plot analysis showed that decidual cells had both PDGF-alpha and PDGF-beta receptors. PDGF-AA, -AB and -BB stimulated [3H]-thymidine incorporation in cultured decidual cells in a dose-dependent manner. These results indicate the importance of PDGF in human decidua.

• Yamada Y, Nezu J, Shimane M, Hirata Y
• Molecular cloning of a novel vascular endothelial growth factor, VEGF-D.
• Genomics. 1997; 42: 483-8
• Display abstract

• We have identified and characterized a novel vascular endothelial growth factor (VEGF), VEGF-D, which is structurally related to vascular endothelial growth factor C. A full-length cDNA for human VEGF-D was cloned following the identification of an EST obtained through a TFASTA search of public EST databases. The murine VEGF-D was subsequently isolated from a mouse lung cDNA library. The human VEGF-D gene was mapped to human chromosome Xp22.31. Both human and mouse VEGF-D are strongly expressed in lung and encode the eight cysteine residues that are highly conserved among the members of this family. The high level of conservation between mouse and human VEGF-D may emphasize the biological importance of this gene. Recently the murine gene, FIGF, which is identical to mouse VEGF-D, was reported.

• Jeltsch M et al.
• Hyperplasia of lymphatic vessels in VEGF-C transgenic mice.
• Science. 1997; 276: 1423-5
• Display abstract

• No growth factors specific for the lymphatic vascular system have yet been described. Vascular endothelial growth factor (VEGF) regulates vascular permeability and angiogenesis, but does not promote lymphangiogenesis. Overexpression of VEGF-C, a ligand of the VEGF receptors VEGFR-3 and VEGFR-2, in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement. Thus, VEGF-C induces selective hyperplasia of the lymphatic vasculature, which is involved in the draining of interstitial fluid and in immune function, inflammation, and tumor metastasis. VEGF-C may play a role in disorders involving the lymphatic system and may be of potential use in therapeutic lymphangiogenesis.

• Olofsson B et al.
• Vascular endothelial growth factor B, a novel growth factor for endothelial cells.
• Proc Natl Acad Sci U S A. 1996; 93: 2576-81
• Display abstract

• We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when coexpressed. Conditioned medium from transfected 293EBNA cells expressing VEGF-B stimulated DNA synthesis in endothelial cells. Our results suggest that VEGF-B has a role in angiogenesis and endothelial cell growth, particularly in muscle.

• Liu YC, Chen SC, Chang C, Leu CM, Hu CP
• Platelet-derived growth factor is an autocrine stimulator for the growth and survival of human esophageal carcinoma cell lines.
• Exp Cell Res. 1996; 228: 206-11
• Display abstract

• Platelet-derived growth factors (PDGF) are important mitogens for mesenchyme-derived cells. Neither PDGF nor PDGF receptors (PDGFR) are expressed in epithelial cells under normal physiological conditions. However, we have found that PDGF-BB induces c-jun expression and promotes the growth of the human esophageal carcinoma cell line CE48T/VGH. Scatchard analysis revealed the presence of 6 x 10(5) binding sites for PDGF-BB per cell, with a Kd of 9.7 nM. Furthermore, our data indicate that CE48T/VGH expresses beta type PDGFR (PDGFRbeta) with in vitro auto-kinase activity. We have also found that CE48T/VGH expresses the mRNA of the PDGF-A and PDGF-B chains and secretes PDGF molecules. Addition of anti-PDGF neutralizing antibody significantly decreased cell numbers of CE48T/VGH under serum-free conditions. The detached cells underwent apoptosis characterized by micronucleation. These results suggest that expression of the PDGF autocrine system may not only provide the growth advantage but also prevent the apoptosis for CE48T/VGH.

• Lee J, Gray A, Yuan J, Luoh SM, Avraham H, Wood WI
• Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4.
• Proc Natl Acad Sci U S A. 1996; 93: 1988-92
• Display abstract

• The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney.

• Eichmann A, Marcelle C, Breant C, Le Douarin NM
• Molecular cloning of Quek 1 and 2, two quail vascular endothelial growth factor (VEGF) receptor-like molecules.
• Gene. 1996; 174: 3-8
• Display abstract

• We have previously reported the cloning of two partial cDNAs corresponding to two quail (Coturnix coturnix japonica) receptor tyrosine kinases (RTKs), named Quek 1 and Quek 2, and their expression in endothelial cells of the early avian embryo. We here report the cloning of the full-size cDNAs for both molecules. Sequence comparison shows that Quek 1 and 2 share an overall amino acid (aa) identity of 49%. They both comprise seven extracellular immunoglobulin-like (Ig-like) domains, a single transmembrane domain, and an intracellular kinase domain split into two by a 70 aa insertion. These structural characteristics are shared by the members of the recently discovered VEGF receptor (VEGFR) family. We have compared the sequences of Quek 1 and 2 to the other VEGFRs. At the aa level, Quek 1 is most closely related to KDR/flk-1 (VEGFR 2) (aa identity of 69% and 71%, respectively). Quek 2 shows a similar degree of aa identity to fit-4 (VEGFR 3). Quek 1 and 2 display a lower homology to fit-1 (VEGFR 1) (about 45% aa identity). These data suggest that Quek 1 and 2 are the avian homologues of VEGFRs 2 and 3, respectively.

• Watanabe S et al.
• Platelet-derived growth factor accelerates gastric epithelial restoration in a rabbit cultured cell model.
• Gastroenterology. 1996; 110: 775-9
• Display abstract

• BACKGROUND & AIMS: Growth factors play an important role in gastric wo und repair. The aim of this study was to assess the role of platelet-derived growth factor (PDGF) in gastric epithelial restoration. METHODS: PDGF-BB (1-50 ng/mL) was added to confluent cultures of rabbit gastric epithelial cells after wounding. Regrowth of the epithelial cells was monitored for 48 hours. The speed of cell migration was measured, and cell proliferation was detected by using a 5-bromodeoxyuridine (BrdU) staining technique. The labeling index was calculated for a 0.05-mm(2) area around the wound. RESULTS: After wounding, cells at the wound edge formed lamellipodia and showed ruffling movements. The addition of PDGF-BB significantly accelerated cell migration and proliferation as well as gastric restoration. Migration speed was 21 microm/h in control cultures and 32 microm/h and 40 microm/h in cultures containing 10 ng/mL and 50 ng/mL of PDGF-BB, respectively. In control cultures, BrdU-positive cells were rarely detected in the initial 24 hours after wounding, and maximum labeling occurred at 36 hours (labeling index, 3.4%). Cultures containing PDGF-BB showed maximum labeling at 24 hours (labeling index, 6.9%). CONCLUSIONS: PDGF-BB dose-dependently accelerated the migration rate and proliferation of cultured gastric epithelial cells after wounding. Therefore, PDGF-BB may play a role in gastric epithelial cell restoration during healing of gastric mucosal lesions.

• Claesson-Welsh L
• Mechanism of action of platelet-derived growth factor.
• Int J Biochem Cell Biol. 1996; 28: 373-85
• Display abstract

• More than 20 years ago, platelet-derived growth factor (PDGF) was identified and later purified. Through recent years of intense research, a large body of information has been collected on how PDGF transduces its biological effects to responding cells. Two homologous receptors, the PDGF alpha- and beta-receptors, have been identified, which are receptor tyrosine kinases. Binding of PDGF leads to activation of the kinase and autophosphorylation. Particularly in the PDGF beta-receptor, a considerable number of autophosphorylation sites have been identified, which allow for physical interaction with signal transduction molecules. The signal transduction molecules are often enzymes, which undergo activity changes in conjunction with binding to the receptor. Other signal transduction molecules function as adaptors, which can couple to subunits equipped with catalytic activity. Through the activity changes of inherent or directly coupled catalytic activities, a signal is propagated, which ultimately results in a cellular response. PDGF is known to induce migration, proliferation and differentiation of different cells types. An array of signal transduction molecules has been shown to interact with the PDGF beta-receptor; several appear to contribute to the generation of the proliferative response, indicating the existence of parallel pathways for this response, which are utilized by many different growth factor receptors. Migration of cells towards PDGF appears to be more strictly dependent on activation of phosphatidylinositol 3' kinase. Interestingly, the PDGF alpha-receptor emits negative signals that inhibit simultaneous positive signals for migration induced by this receptor, or by other receptors, such as the PDGF beta-receptor. Virtually nothing is known about signal transduction initiated by PDGF, which generates differentiation responses. Since PDGF appears to play a role in different physiological and pathological processes, it is important to continue delineation of signal transduction pathways initiated through activation of the PDGF receptors.

• Kreysing J, Ostman A, van de Poll M, Backstrom G, Heldin CH
• Identification of three amino acid residues in the B-chain of platelet-derived growth factor with different importance for binding to PDGF alpha- and beta-receptors.
• FEBS Lett. 1996; 385: 181-4
• Display abstract

• The B-chain homodimer isoform of platelet-derived growth factor (PDGF) binds with high affinity both to alpha- and to beta-receptors. In order to localize amino acid residues in PDGF-BB of differential importance for the binding to the two receptors, PDGF-BB mutants were analyzed in which single amino acid residues were changed to alanine residues. We found that Phe-118 in loop 1 of the PDGF B-chain is crucial for binding to both receptors, and that the surrounding amino acids, Asn-117 and Leu-119, appear to be important primarily for binding to the beta-receptor. In contrast, Lys-161 in loop 3 was found to be more important for binding to alpha-receptors than beta-receptors. Previous studies have shown that the receptor binding epitope of PDGF-BB is composed mainly of loops 1 and 3; the findings of the present study show that the alpha- and beta-receptors interact with different amino acid residues in these regions.

• Hatva E, Bohling T, Jaaskelainen J, Persico MG, Haltia M, Alitalo K
• Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas.
• Am J Pathol. 1996; 148: 763-75
• Display abstract

• Capillary hemangioblastomas and hemangiopericytomas are highly vascular central nervous system tumors of controversial origin. Of interest in their pathogenesis are mechanisms regulating endothelial cell growth. The endothelial cell mitogen vascular endothelial growth factor (VEGF) stimulates angiogenesis, and together with its two receptor tyrosine kinases VEGFR-1(FLT1) and VEGFR-2(KDR), is up-regulated during the malignant progression of gliomas. We have analyzed the expression of VEGF and its receptors, the related placental growth factor (PlGF) and the endothelial receptors FLT4 and Tie by in situ hybridization in capillary hemangioblastomas and hemangiopericytomas. VEGF mRNA was up-regulated in all of the hemangiopericytomas studied and highly expressed in the stromal cells of hemangioblastomas. In addition, some hemangioblastoma tumor cells expressed high levels of PlGF. Significantly elevated levels of Tie mRNA, Tie protein, VEGFR-1, and VEGFR-2 but not FLT4 mRNAs were observed in the endothelia of both tumor types. In hemangioblastomas, however, the receptors were also highly expressed by a subpopulation of stromal cells. Consistent results were obtained for a human hemangioblastoma cell line in culture. Up-regulation of the endothelial growth factors and receptors may result in autocrine or paracrine stimulation of endothelial cells and their precursors involved in the genesis of these two vascular tumors.

• Kukk E et al.
• VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development.
• Development. 1996; 122: 3829-37
• Display abstract

• The vascular endothelial growth factor family has recently been expanded by the isolation of two new VEGF-related factors, VEGF-B and VEGF-C. The physiological functions of these factors are largely unknown. Here we report the cloning and characterization of mouse VEGF-C, which is produced as a disulfide-linked dimer of 415 amino acid residue polypeptides, sharing an 85% identity with the human VEGF-C amino acid sequence. The recombinant mouse VEGF-C protein was secreted from transfected cells as VEGFR-3 (Flt4) binding polypeptides of 30-32x10(3) Mr and 22-23x10(3) Mr which preferentially stimulated the autophosphorylation of VEGFR-3 in comparison with VEGFR-2 (KDR). In in situ hybridization, mouse VEGF-C mRNA expression was detected in mesenchymal cells of postimplantation mouse embryos, particularly in the regions where the lymphatic vessels undergo sprouting from embryonic veins, such as the perimetanephric, axillary and jugular regions. In addition, the developing mesenterium, which is rich in lymphatic vessels, showed strong VEGF-C expression. VEGF-C was also highly expressed in adult mouse lung, heart and kidney, where VEGFR-3 was also prominent. The pattern of expression of VEGF-C in relation to its major receptor VEGFR-3 during the sprouting of the lymphatic endothelium in embryos suggests a paracrine mode of action and that one of the functions of VEGF-C may be in the regulation of angiogenesis of the lymphatic vasculature.

• Osornio-Vargas AR et al.
• Platelet-derived growth factor (PDGF)-AA, -AB, and -BB induce differential chemotaxis of early-passage rat lung fibroblasts in vitro.
• Am J Respir Cell Mol Biol. 1995; 12: 33-40
• Display abstract

• Platelet-derived growth factor (PDGF) isoforms are chemoattractants and mitogens for cells of mesenchymal origin that could be important mediators of pulmonary fibrogenesis. We have previously reported that particle-activated alveolar macrophages secrete homologues of PDGF that are composed of all three PDGF isoforms (PDGF-AA, -AB, and -BB). This mixture of macrophage-derived PDGF, once dissociated from the PDGF-alpha-macroglobulin complex, induces chemotaxis of rat lung fibroblasts (RLF) in the nanomolar range. In addition, we have reported that PDGF isoforms induce differential proliferation of RLF (PDGF-BB > PDGF-AB > PDGF-AA). In the present study, we sought to determine the relative chemotactic potency of the three PDGF isoforms and correlate these responses to the relative abundance of the two types of PDGF cell-surface receptors: PDGF-alpha receptor (PDGF-R alpha) and PDGF-beta receptor (PDGF-R beta). We also investigated the chemotactic activity of combinations of two PDGF isoforms simultaneously. Isolates of early-passage RLF were assayed for chemotaxis in 48-microwell chambers. Swiss mouse 3T3 cells were assayed in parallel as a positive control cell line for PDGF-R alpha and PDGF-R beta expression. RLF responded differentially to the PDGF isoforms: PDGF-AB and PDGF-BB were potent chemoattractants and stimulated maximal chemotactic responses between 4 and 8 ng/ml PDGF, whereas PDGF-AA elicited a weak chemotactic response that was maximally 15% of that obtained with either B-chain isoform. PDGF-AB and PDGF-BB were also the most potent chemoattractants for Swiss 3T3 cells, and their response to these B-chain isoforms was approximately 40% greater than that obtained for RLF.(ABSTRACT TRUNCATED AT 250 WORDS)

• Kaipainen A et al.
• Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.
• Proc Natl Acad Sci U S A. 1995; 92: 3566-70
• Display abstract

• We have recently cloned the human fms-like tyrosine kinase 4 gene FLT4, whose protein product is related to two vascular endothelial growth factor receptors FLT1 and KDR/FLK1. Here the expression of FLT4 has been analyzed by in situ hybridization during mouse embryogenesis and in adult human tissues. The FLT4 mRNA signals first became detectable in the angioblasts of head mesenchyme, the cardinal vein, and extraembryonally in the allantois of 8.5-day postcoitus (p.c.) embryos. In 12.5-day p.c. embryos, the FLT4 signal decorated developing venous and presumptive lymphatic endothelia, but arterial endothelia were negative. During later stages of development, FLT4 mRNA became restricted to vascular plexuses devoid of red cells, representing developing lymphatic vessels. Only the lymphatic endothelia and some high endothelial venules expressed FLT4 mRNA in adult human tissues. Increased expression occurred in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. Our results suggest that FLT4 is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. They also support the theory on the venous origin of lymphatic vessels.

• Lindner V
• Expression of platelet-derived growth factor ligands and receptors by rat aortic endothelium in vivo.
• Pathobiology. 1995; 63: 257-64
• Display abstract

• In situ hybridization and immunostaining were used to study the time course of expression for platelet-derived growth factor (PDGF) ligands and receptors in endothelium of the rat aorta after injury. The PDGF-A and -B chains were expressed in endothelial cells at the wound edge within 4 h after injury, but no expression was detectable in uninjured endothelium. PDGF alpha-receptor was expressed in a pattern similar to the PDGF-A chain, while expression of PDGF beta-receptor was not detected at any time. Expression of the PDGF-B chain remained elevated in endothelial cells at the leading edge even at later measurements when these cells had stopped replicating. Smooth muscle cells (SMCs), which are absent from the intima of the normal aorta and are known to express PDGF beta-receptors, were predominantly found to migrate into the intima near the endothelial leading edge where PDGF-B was expressed. These data suggest a paracrine role for endothelial PDGF in SMC migration.

• Grinspan JB, Franceschini B
• Platelet-derived growth factor is a survival factor for PSA-NCAM+ oligodendrocyte pre-progenitor cells.
• J Neurosci Res. 1995; 41: 540-51
• Display abstract

• Mature oligodendroglia, which synthesize and express lipids and proteins characteristic of myelin, are generated from precursor cells which are formed in germinal matrix, then migrate widely through the neuraxis. We now demonstrate that these precursor cells can be recognized at a very early stage by their surface expression of polysialylated neural cell adhesion molecules (PSA-NCAM), and only later bind anti-ganglioside antibodies that had previously been used to recognize "O-2A" oligodendroglial precursor cells. PSA-NCAM expression by these cells is likely to be of functional significance, since a recent study demonstrated that O-2A cells become immobile when stripped of PSA-NCAM. Platelet-derived growth factor (PDGF) proved to be a survival factor for these PSA-NCAM+cells, and in a defined medium, PDGF was sufficient to ensure maturation of immunopurified PSA-NCAM+cells to oligodendroglia.

• Pietrogrande F, Caenazzo A, Dazzi F, Polato G, Girolami A
• A role for platelet-derived growth factor in drug-induced chronic ergotism? A case report.
• Angiology. 1995; 46: 633-6
• Display abstract

• Generalized vasoconstriction in chronic ergot poisoning is attributed both to the ergotamine activity on alpha-adrenergic receptors and to its direct action on vascular smooth muscle cells. The authors propose that endothelial wall, chronically damaged by ergot alkaloids, releases platelet-derived growth factor (PDGF), which contributes to vasoconstriction and promotes further arterial obstruction. Their hypothesis is supported by the increased PDGF activity found in plasma of a patient suffering from chronic ergotism.

• Andersson M, Ostman A, Kreysing J, Backstrom G, Van de Poll M, Heldin CH
• Involvement of loop 2 of platelet-derived growth factor-AA and -BB in receptor binding.
• Growth Factors. 1995; 12: 159-64
• Display abstract

• Platelet-derived growth factor (PDGF) is a disulfide-bonded antiparallel dimer of A- and B-polypeptide chains. Each subunit contains two loops (loops 1 and 3) which point in the same direction, and which are located close to a region (loop 2) from the other subunit of the dimer. Previous studies have shown that epitopes in loops 1 and 3 are important for binding to PDGF alpha- and beta-receptors. The aim of the present investigation was to determine the importance of loop 2 for receptor interactions. PDGF A- and B-chain cDNA:s were mutated in the loop 2 regions and transfected into COS cells. Analyses of conditioned media of such cell cultures revealed that PDGF B-chain mutated in the loop 2 region lost its ability to compete with 125I-PDGF for binding to PDGF beta-receptors, but retained 2-5% of its binding of alpha-receptors. The A-chain binds only to alpha-receptors; 2-5% of this binding was also retained after mutation of the loop 2 region. In conclusion, the loop 2 region of PDGF is important for receptor binding, but appears to be more important for binding to the PDGF beta-receptors than to the alpha-receptors.

• Battegay EJ, Raines EW, Colbert T, Ross R
• TNF-alpha stimulation of fibroblast proliferation. Dependence on platelet-derived growth factor (PDGF) secretion and alteration of PDGF receptor expression.
• J Immunol. 1995; 154: 6040-7
• Display abstract

• TNF-alpha stimulates DNA synthesis and proliferation of cultured human fibroblasts. Maximal DNA synthesis in response to TNF-alpha occurs approximately 28 h after addition of TNF-alpha to quiescent fibroblasts, a delay of about 12 to 14 h as compared with DNA synthesis elicited by platelet-derived growth factor (PDGF). TNF-alpha induces PDGF A chain gene expression with a maximum at 4 h. DNA synthesis is abrogated in response to TNF-alpha by a goat anti-PDGF IgG but not by nonimmune goat IgG, suggesting induction of an autocrine PDGF-AA loop by TNF-alpha. The response to PDGF-AA requires the presence of PDGF receptor alpha-receptors. TNF-alpha does not significantly affect PDGF alpha-receptor mRNA or protein expression, nor does it alter the proliferative response to externally added PDGF-AA. In contrast, TNF-alpha reduces the levels of PDGF beta-receptor mRNA, protein expression, and cell proliferation in response to PDGF-BB. Thus, DNA synthesis in response to TNF-alpha depends upon autocrinely induced PDGF-AA. At the same time, TNF-alpha may alter the response to PDGF-BB from exogenous sources.

• Munson L, Upadhyaya NB, Van Meter S
• Platelet-derived growth factor promotes endometrial epithelial cell proliferation.
• Am J Obstet Gynecol. 1995; 173: 1820-5
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• OBJECTIVE: Our purpose was to determine the effects of platelet-derived growth factor on the proliferation of endometrial epithelial cells. Platelet-derived growth factor and its receptors have been identified in the endometrium, and platelet-derived growth factor is a mitogen for endometrial stromal cells. Released from macrophages and platelets at sites of ectopic endometrial growth, platelet-derived growth factor could promote the progression of endometriosis and endometrial cancer. STUDY DESIGN: Endometrial epithelial cell lines were developed from proliferative-phase endometria from two patients without endometrial lesions. Cell lines were confirmed to be epithelial. Proliferation assays were conducted on both lines with recombinant human platelet-derived growth factor-AA, AB, and BB. Assays were also performed at different doses, times, and cell densities with platelet-derived growth factor. RESULTS: All isoforms of platelet-derived growth factor were potent mitogens for both endometrial epithelial cell lines. The greatest proliferative responses were achieved at 10 ng/ml and at 24 hours. Responses decreased significantly in confluent cultures. CONCLUSIONS: These results suggest that endometrial epithelial cells have functional platelet-derived growth factor-alpha receptors that signal cell replication. The greater activity of platelet-derived growth factor in subconfluent cultures may indicate that receptor numbers or affinity are up-regulated when cell-cell contact is disrupted. These data support a role for platelet-derived growth factor in normal endometrial proliferation and in pathologic proliferation such as endometriosis and endometrial cancer.

• Borg JP, deLapeyriere O, Noguchi T, Rottapel R, Dubreuil P, Birnbaum D
• Biochemical characterization of two isoforms of FLT4, a VEGF receptor-related tyrosine kinase.
• Oncogene. 1995; 10: 973-84
• Display abstract

• The FLT4 gene encodes a tyrosine kinase receptor related to the two identified receptors for vascular endothelial growth factor (VEGF), FLT1 and FLK1/KDR. Two isoforms of FLT4, differing by their C-terminal ends, have been identified. The long form has 65 additional amino acid residues. We have shown that FLT4 is a highly glycosylated, relatively stable, cell surface associated kinase of approximately 180 kDa. In order to study the signal transduction molecules associated with the FLT4 pathway, and in the absence of a known ligand, we constructed two chimeric molecules (FF4S and FF4L) made of the extracellular region of the CSF1 receptor (Fms gene product) and of the transmembrane and intracellular regions of either form of FLT4. These two chimeric forms were expressed in Rat 2 transfectants. We assayed the ligand-induced capacity of the FF4 short and long forms to sustain growth of Rat 2 cells in semisolid medium. In a soft agar assay, only the long form was able to induce the growth of Rat 2 cells upon ligand treatment. The two forms of FLT4 therefore have different functional capacities. We looked for association and/or phosphorylation of phospholipase C gamma (PLC gamma) and phosphatidylinositol-3'-phosphate (PI3K), after stimulation of the FF4 molecules by CSF1. Finally, we have studied the expression of the Flt4 gene in mouse embryos and in the adult by in situ hybridization. Flt4 transcripts were found at day 12.5 post-coitum and thereafter, including the adult mouse, predominantly in the pericardium, pleural membranes and in the lung.

• Sharma HS, Tang ZH, Gho BC, Verdouw PD
• Nucleotide sequence and expression of the porcine vascular endothelial growth factor.
• Biochim Biophys Acta. 1995; 1260: 235-8
• Display abstract

• We cloned and sequenced two cDNAs encoding the angiogenic, vascular endothelial growth factor (VEGF) from the porcine heart. Deduced amino acid sequence of the clone pPVE-18 and pPVE-5 predicted 164 (VEGF164), and 120 (VEGF120) residues of VEGF, respectively, with a putative N-terminal signal sequence of 26 amino acids. The porcine VEGF is shorter by one amino acid as compared to human VEGF, but a potential glycosylation site is present at Asn-74. PCR detection, and verification of the identity of the PCR products by Southern hybridization, confirmed wide expression of VEGF in different porcine tissues. Northern blot analysis with a radiolabeled porcine specific VEGF probe, showed one major (3.9 kb) and one minor (1.7 kb) mRNA species expressed in all four chambers of the heart.

• Eming SA, Lee J, Snow RG, Tompkins RG, Yarmush ML, Morgan JR
• Genetically modified human epidermis overexpressing PDGF-A directs the development of a cellular and vascular connective tissue stroma when transplanted to athymic mice--implications for the use of genetically modified keratinocytes to modulate dermal regeneration.
• J Invest Dermatol. 1995; 105: 756-63
• Display abstract

• We investigated the hypothesis that keratinocyte-produced platelet-derived growth factor-AA (PDGF-AA) is involved in epidermal-dermal interactions and that PDGF-AA is an important mediator of the temporal and spatial events of tissue repair. Retroviral-mediated gene transfer was used to introduce the gene encoding human PDGF-A into cultures of human diploid keratinocytes. Genetic modification boosted the endogenous in vitro level of PDGF-AA secretion by over 300 fold. When PDGF-secreting cells were transplanted as epithelial sheets to athymic mice, modified keratinocytes underwent terminal differentiation and generated a stratified epithelium comparable to unmodified cells. Seven days after grafting the newly synthesized connective tissue layer subjacent to the PDGF-A-modified grafts was significantly thicker, was rich in mononuclear cells and fibroblasts, and had increased numbers of blood vessels when compared to control grafts of unmodified cells. These results suggest that PDGF-AA secreted by the epidermis is an important mediator of epithelial-mesenchymal interactions and helps to promote growth and vascularization of the underlying dermal tissue. Further, these data demonstrate the feasibility of using genetically modified cells to modulate tissue regeneration.

• Spindler KP, Mayes CE, Miller RR, Imro AK, Davidson JM
• Regional mitogenic response of the meniscus to platelet-derived growth factor (PDGF-AB).
• J Orthop Res. 1995; 13: 201-7
• Display abstract

• Since meniscal healing is region-specific, we studied the regional (peripheral compared with central) response of meniscal explants to human, recombinant platelet-derived growth factor-AB. Meniscal explants from the hindlimbs of both knees of mature sheep were sectioned and were cultured with variable doses of human, recombinant platelet-derived growth factor-AB, and incorporation of [3H]-thymidine was measured. The mitogenic response was measured at different times in culture (48 or 96 hours) and by location (lateral or medial). In the absence of the growth factor, the peripheral third of both menisci incorporated 10-fold more [3H]-thymidine on a weight basis than did the central two-thirds. Cellularity was equivalent in the two regions. Doses of less than 100 ng/ml of growth factor produced either no stimulation or a variable response. A dose of 100 ng/ml resulted in consistent, significant (p < 0.05) stimulation in all groups in the peripheral region, and a dose of 200 ng/ml provided more than a 2.5-fold increase. Multiple-factor analysis of variance demonstrated that there were no significant differences between experiments, times in culture, or menisci. The central region did not respond to stimulation with the growth factor at any of the doses tested. These data suggest that regional differences (peripheral compared with central) in responsiveness to human, recombinant platelet-derived growth factor-AB may reflect a different level of signal transduction machinery for growth factor receptors and distinct fibrobchondrocyte populations. These findings are consistent with the variable healing capacity of the meniscal regions in vivo and suggest a pharmacological means to promote the repair of the peripheral meniscal region.

• Westermark B
• [Platelet-derived growth factor. A growth factor of great physiopathological importance]
• Lakartidningen. 1995; 92: 2397-401
• Display abstract

• Platelet-derived growth factor (PDGF) is a 30 kilodalton dimeric peptide comprising A and B chains. The two types of PDGF receptors (alpha and beta) that have been identified belong to the protein-tyrosine kinase family of growth factor receptors. PDGF is a potent mitogen for a variety of cell types, and in gene knock-out experiments has been shown to be required for the development of inter alia mesangial cells of renal glomeruli and smooth muscle cells of lung alveolar septa. PDGF is believed to fulfil important autocrine and paracrine growth-related functions, and its overproduction has been recognised in various malignant tumours and in non-neoplastic hyperproliferative disorders.

• Takanami I, Imamura T, Yamamoto Y, Kodaira S
• Usefulness of platelet-derived growth factor as a prognostic factor in pulmonary adenocarcinoma.
• J Surg Oncol. 1995; 58: 40-3
• Display abstract

• Platelet-derived growth factor (PDGF) in the resected pulmonary adenocarcinoma tissue of 88 patients was detected immunohistochemically by the avidin-biotin-peroxidase technique to determine whether or not it is a prognostic parameter. The 88 patients were divided into PDGF (-) and PDGF (+) groups according to the stainability of the factor. The PDGF (-) group included 39 patients and the PDGF (+) group 49. The 5-year survival rate was 53% for the PDGF (-) group and 17% for the PDGF (+) group (P < 0.01). These findings indicate that the stainability of PDGF can be a prognostic parameter for pulmonary adenocarcinoma.

• Pierce GF et al.
• Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and absence of PDGF in chronic nonhealing wounds.
• J Clin Invest. 1995; 96: 1336-50
• Display abstract

• Some human chronic dermal wounds treated with recombinant platelet-derived growth factor-BB (rPDGF-BB) show increased healing coupled with fibroblast activation and granulation tissue formation. To determine whether endogenous PDGF is associated with healing and nonhealing dermal ulcer phenotypes, we developed monoclonal antibodies capable of recognizing the three isoforms of PDGF, AA, AB, and BB dimers, and capable of discriminating between two alternatively spliced A chain transcripts. We detected little PDGF isoform expression in normal skin and in nonhealing dermal ulcers. In contrast, in surgically created acute wounds and chronic ulcers treated with rPDGF-BB, markedly upregulated levels of PDGF-AA (long form) were found. In both types of wounds, increased PDGF-AA was detected primarily in capillaries and fibroblasts, although in rPDGF-BB-treated chronic wounds, widespread expression of PDGF-AA was somewhat delayed. With continued treatment, the long form of PDGF-AA, which can preferentially bind extracellular matrix, was expressed only in capillaries, while fibroblasts began synthesizing the short form of PDGF-AA. Within capillaries, all endothelial cells and varying numbers of pericytes and smooth muscle cells contained PDGF-AA. In all wounds, macrophages and keratinocytes were not a major contributor. While PDGF-BB and PDGF-AB were present in a minority of healing wounds, they were usually present at lower levels than PDGF-AA. PDGF-beta receptors, which bind only PDGF-BB and not other isoforms, were found in normal skin and granulation tissue, providing a molecular basis for treating human chronic wounds with exogenous rPDGF-BB.

• Basheeruddin K, Li X, Rechtoris C, Mazzone T
• Platelet-derived growth factor enhances Sp1 binding to the LDL receptor gene.
• Arterioscler Thromb Vasc Biol. 1995; 15: 1248-54
• Display abstract

• We have previously demonstrated that growth activation of quiescent cells enhances LDL receptor gene transcription and that the proximal 5' flanking region of the LDL receptor gene could transduce a platelet-derived growth factor (PDGF) response. This portion of the LDL receptor gene encompasses a previously characterized sterol response element and an adjacent Sp1 binding site. By use of mobility shift analyses we show that PDGF activation of quiescent cells enhances binding of Sp1 to the LDL receptor gene. Transfection analyses indicated that the Sp1 site, but not the sterol response element binding protein site, could confer PDGF responsiveness to a heterologous promoter in quiescent cells. Furthermore, cotransfection of an LDL receptor reporter gene (containing -141 to +35 bp of the LDL receptor gene promoter) along with an expression construct coding for high-level constitutive expression of an Sp1 cDNA led to marked enhancement in expression of the LDL receptor reporter gene in quiescent cells. Increased Sp1 binding due to PDGF could be due to enhanced production of Sp1; alternatively, posttranslational activation of binding could be involved. Western blot analysis showed no difference in Sp1 abundance in quiescent cells versus PDGF-stimulated cells, suggesting a posttranslational mechanism for activation of Sp1 binding by growth induction. Our data demonstrate that PDGF stimulation of quiescent cells leads to enhanced Sp1 binding to the LDL receptor gene. This enhanced binding could participate in PDGF induction of LDL receptor gene transcription.

• Mustonen T, Alitalo K
• Endothelial receptor tyrosine kinases involved in angiogenesis.
• J Cell Biol. 1995; 129: 895-8

• Royce PM, Kato T, Ohsaki K, Miura A
• The enhancement of cellular infiltration and vascularisation of a collagenous dermal implant in the rat by platelet-derived growth factor BB.
• J Dermatol Sci. 1995; 10: 42-52
• Display abstract

• The implantation of collagen-based dermal substitutes offers one means of management of full-thickness skin lesions. We have examined the effect of the recombinant BB homodimer of platelet-derived growth factor (rPDGF-BB) on the extent of cellular infiltration and vascularisation of collagen sponges implanted into full-thickness excision wounds in rats. Histological examination of sponges excised 14 and 21 days post-implantation in dose-response studies in which 0-4 micrograms rPDGF-BB were applied to the undersurface of each sponge, immediately prior to its implantation, demonstrated a progressively increased infiltration of host cells, especially fibroblasts, and enhanced capillary formation. With 4 micrograms rPDGF-BB, an enhanced infiltration of fibroblasts into sponges was already apparent 3 days post-implantation, and enhanced capillary formation was noticeable after 7 days. This neovascularisation was noted to be associated with improved survival of autologous split-thickness skin grafts applied to the sponges immediately following their implantation.

• Andrew JG, Hoyland JA, Freemont AJ, Marsh DR
• Platelet-derived growth factor expression in normally healing human fractures.
• Bone. 1995; 16: 455-60
• Display abstract

• Platelet-derived growth factor (PDGF) has been shown to have effects on bone and cartilage cells in vitro, but its role in vivo in bone repair is not clear. We studied biopsy material from 16 normally healing fractures at a variety of times after injury, using immunohistochemistry for PDGF and in situ hybridization for PDGF A and B chains. PDGF A-chain gene was found to be expressed by many cell types over a prolonged period during fracture healing. These cells included endothelial and mesenchymal cells in granulation tissue and osteoblasts, chondrocytes, and osteoclasts later during fracture healing. PDGF B-chain gene expression was more restricted, being detected principally in osteoblasts at the stage of bone formation. PDGF was detected using immunohistochemistry in the cell types expressing PDGF A. These findings indicate that PDGF is expressed during normal human fracture repair, and the in vitro data also suggest that PDGF is likely to be an important local regulator in this process.

• Lepisto J, Peltonen J, Vaha-Kreula M, Niinikoski J, Laato M
• Platelet-derived growth factor isoforms PDGF-AA, -AB and -BB exert specific effects on collagen gene expression and mitotic activity of cultured human wound fibroblasts.
• Biochem Biophys Res Commun. 1995; 209: 393-9
• Display abstract

• This study was designed to clarify the action of three different isoforms of PDGF (PDGF-AA, PDGF-AB and PDGF-BB) on the proliferation rate and collagen synthesis of fibroblasts cultured from normal human wounds. PDGF-AA and PDGF-BB down-regulated both the steady-state levels of pro alpha 1 (I) and pro alpha 1 (III) collagen chain mRNAs and the production of collagen, both in a dose-dependent manner. Interestingly, low concentrations (1 ng/ml) of PDGF-AB up-regulated the expression of type I and III procollagen mRNAs by cultured wound fibroblasts, while under high concentrations (30 ng/ml) of PDGF-AB this effect changed to the opposite. The proliferation rate of wound fibroblasts was stimulated by PDGF-BB which elicited a dose-dependent (1-30 ng/ml) stimulation of cell proliferation, whereas PDGF-AB and -AA were less effective in this respect.

• Zhang G, el Nahas AM
• Platelet-derived growth factor in experimental glomerulonephritis.
• Nephrol Dial Transplant. 1995; 10: 787-95
• Display abstract

• Growth factors and in particular platelet-derived growth factor (PDGF) have been implicated in the pathogenesis of glomerulonephritis and glomerulosclerosis. We have studied the distribution of immunoreactive PDGF (iPDGF) within serial kidney biopsies (days 7, 15, 30, 90 and 120) of eight rats with an accelerated form of nephrotoxic serum nephritis (NTN). The course of NTN was mild in five rats and seere in three. Two patterns of immunostain for PDGF were noted. The first consisted of iPDGF-B chain in a glomerular segmental distribution similar to that of infiltrating monocytes (OX6+cells). At all stages of NTN the distribution of iPDGF-B chain correlated closely with the immunostain for monocytes. The second pattern of immunostain showed iPDGF-A chain in a diffuse distribution along the glomerular capillary lining and to a lesser extent in some mesangial cells. In severely affected rats the magnitude of the iPDGF-A increased along with glomerulosclerosis but disappeared later from areas of segmental and global glomerular obsolescence. By contrast, in rats with milder NTN glomerular iPDGF-A chain peaked early and decreased subsequently.(ABSTRACT TRUNCATED AT 250 WORDS)

• Guha A, Glowacka D, Carroll R, Dashner K, Black PM, Stiles CD
• Expression of platelet derived growth factor and platelet derived growth factor receptor mRNA in a glioblastoma from a patient with Li-Fraumeni syndrome.
• J Neurol Neurosurg Psychiatry. 1995; 58: 711-4
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• Expression of platelet derived growth factor (PDGF) and PDGF-receptor mRNA was examined from a glioblastoma taken from a patient with Li-Fraumeni syndrome. Northern blot analysis and in situ hybridisation showed very high concentrations of both PDGF-A and PDGF alpha-receptor mRNA in the tumour. The overall pattern of PDGF expression was similar to those found in sporadic glioblastomas. Mutations in p53 has been implicated as an early pathogenic event leading to sporadic low grade astrocytomas, and is the third most common tumour type in patients with Li-Fraumeni syndrome, where they are predisposed due to a germline mutation in the p53 tumour suppressor gene. This study suggests that progression towards a glioblastoma in both the general population and in patients with Li-Fraumeni syndrome may involve potential autocrine and paracrine stimulation by growth factors such as PDGF.

• Hu JC, Zhang C, Slavkin HC
• The role of platelet-derived growth factor in the development of mouse molars.
• Int J Dev Biol. 1995; 39: 939-45
• Display abstract

• Platelet-derived growth factor (PDGF) is a potent mitogen that functions in cytodifferentiation and wound healing. PDGF receptor-alpha (PDGFR-alpha) is required for the normal development of the dental ectomesenchyme (Stephenson et al., Proc. Natl. Acad. Sci. USA 88: 6-10, 1991). To investigate the regulatory potential of PDGF on tooth development, the expression of PDGF-AA was determined by reverse transcription-polymerase chain reaction (RT-PCR), the PDGF ligands and receptors were localized by immunohistochemistry. The growth-promoting effects of exogenous PDGF-AA on molar explants were determined by assaying for tritiated thymidine incorporation, the presence of type I collagen and amelogenin messenger RNA transcripts, and total DNA, RNA and protein accumulation. Based upon the temporal and spatial localization, PDGFs and their receptors are present in the enamel epithelia and pulpal mesenchyme of developing mouse molars. Exogenous PDGF-AA administration increased the total protein accumulation of mouse molar explants but produces no discernible effect on amelogenin and type I collagen expression.

• Othberg A, Odin P, Ballagi A, Ahgren A, Funa K, Lindvall O
• Specific effects of platelet derived growth factor (PDGF) on fetal rat and human dopaminergic neurons in vitro.
• Exp Brain Res. 1995; 105: 111-22
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• The neurotrophic effects of the BB isoform of platelet-derived growth factor (PDGF) on rat and human fetal mesencephalic dopaminergic neurons have been characterized in vitro. A dose-response analysis demonstrated maximal responses at 30 ng/ml of PDGF-BB. This concentration resulted in a marked increase in the survival and neurite outgrowth from rat and human tyrosine hydroxylase-(TH) positive, presumed dopaminergic neurons after 7 days in vitro. The effects of PDGF-BB on survival of TH-positive neurons were comparable to those of brain-derived neurotrophic factor (BDNF), whereas neurite outgrowth was more pronounced after addition of BDNF. The combination of BDNF and PDGF-BB yielded no additive effects. Double immunohistochemical staining of rat cultures demonstrated PDGF beta-receptors on about 90% of the TH-positive neurons. PDGF-BB treatment of rat mesencephalic cultures induced an upregulation of c-fos and TH mRNA with maximal levels after 0.5-2 h as assessed by quantitative PCR analysis. An increased number of Fos protein-positive cells was detected immunohistochemically after 4 h of PDGF-BB treatment. The present results provide further evidence for specific and direct effects of PDGF-BB on gene expression, survival and neurite outgrowth of mesencephalic dopaminergic neurons of rat and human origin.

• Mauro A, Di Sapio A, Mocellini C, Schiffer D
• Control of meningioma cell growth by platelet-derived growth factor (PDGF).
• J Neurol Sci. 1995; 131: 135-43
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• We have examined the possible involvement of PDGF and PDGF receptors in the growth control of five meningiomas, analyzing the biopsy specimens and the primary cultures derived from the same tumors. Light and electron microscopy demonstrated that MAbs against PDGF beta-receptors immunodecorate meningioma cells in vivo and in vitro, while those against alpha-receptors gave negative results. The effects of PDGF isoforms AA, AB, BB and of PDGF neutralizing antibodies on meningioma cultures were examined using [3H]thymidine incorporation analysis. Only with PDGF-AB and -BB a mitogenic effect was observed, while PDGF-neutralizing antibodies produced a reduction of [3H]thymidine incorporation. The production of PDGF-like growth factors by meningioma cells was tested analyzing the effects of meningioma culture-conditioned media on the growth of Swiss 3T3 cells. In all cases meningioma conditioned media stimulated the in vitro growth of 3T3 fibroblasts and this stimulatory effect was strongly reduced by PDGF-neutralizing antibodies. Furthermore, Northern blot analysis demonstrated expression of c-sis/PDGF-B and PDGF beta-receptors mRNA in all meningioma biopsies and in all the derived cultures. Our results provide strong evidence that PDGF-B chain and PDGF beta-receptors are involved in growth control mechanisms of human meningiomas through autocrine and/or paracrine mechanisms.

• Ross JA et al.
• Dietary modulation of serum platelet-derived growth factor-AB levels.
• Cancer Epidemiol Biomarkers Prev. 1995; 4: 485-9
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• Epidemiological studies have demonstrated that diets high in vegetables and fruit are associated with a decreased risk of cancer and, possibly cardiovascular disease. Certain constituents of vegetables and fruit inhibit the in vitro activity of platelet-derived growth factor (PDGF), a potent mitogen implicated in both cancer and cardiovascular disease. Few studies have measured PDGF in relationship to diet in vivo. Specifically, there are no data regarding the changes in PDGF levels of mitogenic activity after a dietary intervention. In this study, 19 young, healthy individuals consumed four (9-day) experimental diets in random order: (a) control diet alone; (b) control diet plus carotenoid-rich vegetables; (c) control diet plus cruciferous vegetables; and (d) control diet plus soy foods. Compared to the control diet, there was a significant elevation in PDGF-AB serum levels when the individuals were consuming the soy diet (P = 0.016). Increased PDGF-AB levels were also noted for the carotenoid diet. There was no change from baseline levels when individuals were consuming the cruciferous diet. Overall, mitogenic activity did not change on any of the experimental diets. This study suggests that high soy and carotenoid diets increase serum levels of PDGF-AB. This may represent an additional mechanism by which diet influences individual risk of cancer; further investigation into the role of diet and growth factors is warranted.

• Qu J et al.
• Lack of effect of recombinant platelet-derived growth factor on human neutrophil function.
• J Immunol. 1995; 154: 4133-41
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• Platelet-derived growth factor (PDGF) has been reported to induce chemotaxis, degranulation, and superoxide anion generation, and to increase the expression of CD11b/CD18 in human neutrophils; hence, it has been proposed as an important regulator of neutrophil function. Most of the studies on PDGF, however, have been complicated by the use of nonrecombinant PDGF or the use of mixed leukocyte cell preparations. Assessment of the effects of recombinant human PDGF-AB or -BB which display agonist activity against both PDGF receptor subtypes failed to demonstrate any effect of this peptide on neutrophil shape change, respiratory burst activity, CD11/CD18, or CD62-L expression, inositol 1,4,5-trisphosphate accumulation, or phosphorylation of mitogen-activated protein kinase. This apparent lack of effect of PDGF was consistent with our findings that neutrophils display no specific 125I-PDGF-AB or -BB binding and lack detectable mRNA for PDGF alpha-receptor and beta-receptors. These data indicate that human neutrophils do not possess functional PDGF receptors and question previous reports of a functional effect of this peptide in these cells.

• Floege J, Johnson RJ
• Multiple roles for platelet-derived growth factor in renal disease.
• Miner Electrolyte Metab. 1995; 21: 271-82
• Display abstract

• Platelet-derived growth factor (PDGF) is a pleiotropic cytokine, that is synthesized by various resident renal cells and also by infiltrating cells. The best established role for PDGF in the kidney is the mediation of glomerular mesangial cell proliferation. There is also evidence to suggest an involvement of PDGF in the regulation of renal extracellular matrix turnover, the chemoattraction of mesangial cells and/or other cells to sites of injury, the regulation of glomerular hemodynamics, and lipoprotein uptake in the glomerulus. The first studies investigating the efficacy of anti-PDGF therapy in glomerular disorders point to a potentially novel approach to treat progressive renal disease.

• Zhang FX, Pan W, Hutchins JB
• Phosphorylation of F1F0 ATPase delta-subunit is regulated by platelet-derived growth factor in mouse cortical neurons in vitro.
• J Neurochem. 1995; 65: 2812-5
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• The delta subunit of F1F0 ATPase (ATP synthase complex) is part of the stalk connecting the F1 and F0 moieties. Studies in Escherichia coli suggest that the analogous bacterial subunit, called epsilon, is essential for the ATPase assembly energy coupling. Platelet-derived growth factor (PDGF) is an important growth factor for various cell types, including neurons of the CNS. Using two-dimensional gel electrophoresis, microsequencing, western blot analysis, and immunoprecipitation techniques, we have found that PDGF induces phosphorylation of the delta subunit or a closely related peptide in cultured mouse cortical neurons.

• Chiang MK, Flanagan JG
• Interactions between the Flk-1 receptor, vascular endothelial growth factor, and cell surface proteoglycan identified with a soluble receptor reagent.
• Growth Factors. 1995; 12: 1-10
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• Fetal liver kinase-1 (Flk-1) is a transmembrane tyrosine kinase that was identified in endothelial cells and populations of cells enriched in hematopoietic progenitors. To characterize the interaction of Flk-1 with potential ligands the receptor extracellular domain was genetically fused to an alkaline phosphatase (AP) tag. A soluble ligand for Flk-1 was identified in the supernatants of numerous mesenchymal cell lines by co-immunoprecipitation with the Flk1-AP fusion protein. This polypeptide was shown by N-terminal sequencing to be vascular endothelial growth factor (VEGF). Receptor-AP fusion proteins can thus be used to identify soluble ligands as well as transmembrane ligands, and this approach is therefore likely to be widely applicable to many types of orphan receptor. The Flk1-AP soluble receptor was also found to bind to cell surfaces, showing two apparent classes of binding site with different affinities. This interaction could be reconstructed by introducing a VEGF expression plasmid into cells. These results indicate that VEGF presented at the cell surface can bind to the Flk-1 receptor, and could mediate a direct cell-cell interaction. The Flk1-AP fusion protein was also found to bind heparin, implying that ligand binding by the Flk-1 receptor may involve a three way interaction between the Flk-1 receptor, VEGF, and heparin-like cell surface proteoglycans.

• Failli P et al.
• The mitogenic effect of platelet-derived growth factor in human hepatic stellate cells requires calcium influx.
• Am J Physiol. 1995; 269: 11339-11339
• Display abstract

• Platelet-derived growth factor (PDGF) is a key mitogen for hepatic stellate cells (HSC) and has been shown to be implicated in liver tissue repair and fibrogenesis. In this study the relationship between PDGF-induced intracellular Ca2+ concentration ([Ca2+]i) increase and mitogenesis in cultured human HSC was evaluated. In high-density cell cultures (80-90% subconfluence), PDGF induced a significant increase in [Ca2+]i, characterized by a short-lasting peak phase, which was followed by a long-lasting plateau phase. The plateau phase was abolished in the absence of extracellular Ca2+. However, in low-density cell cultures (30-40% subconfluence), the plateau phase was absent or markedly less pronounced. In parallel sets of experiments, PDGF was significantly less effective in inducing mitogenesis in low-density cell cultures than in high-density cell cultures and was totally ineffective in the absence of extracellular Ca2+. These results suggest that 1) spatial and time dynamics of PDGF-induced [Ca2+]i increase are dependent on cell density and 2) PDGF-induced mitogenesis requires extracellular Ca2+ influx.

• Bonner JC, Osornio-Vargas AR
• Differential binding and regulation of platelet-derived growth factor A and B chain isoforms by alpha 2-macroglobulin.
• J Biol Chem. 1995; 270: 16236-42
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• Alpha 2-Macroglobulin (alpha 2M) is a multifunctional secreted glycoprotein that serves as a ubiquitous proteinase inhibitor and as a binding protein for platelet-derived growth factor (PDGF) BB and homologues of PDGF-BB secreted in culture by macrophages. The interaction of alpha 2M with PDGF-A chain molecules has not been addressed. This is a potentially important issue because fibroblasts and smooth muscle cells produce PDGF-AA, whereas macrophages produce mainly PDGF-BB. Recombinant human 125I-PDGF-B chain molecules (AB and BB) bound to plasma-derived, native human, or bovine alpha 2M and trypsin-activated alpha 2M on Superose 6 fast protein liquid chromatography gel filtration and on nondenaturing polyacrylamide gel electrophoresis, whereas 125I-PDGF-AA did not. Similar results were obtained with 125I-PDGF isoforms binding to immobilized bovine alpha 2M and alpha 2M-methylamine. The same differential pattern of unlabeled PDGF isoforms binding to alpha 2M was observed by Western blotting of PDGF. Human lung fibroblasts secreted alpha 2M as measured by Western blotting, and fibroblast-derived alpha 2M possessed the same differential binding pattern for PDGF isoforms as did plasma-derived alpha 2M. The specific binding of PDGF-AB and -BB to these fibroblasts was inhibited by native bovine alpha 2M, although PDGF-AA binding was not affected. Native alpha 2M preferentially blocked fibroblast chemotaxis to the PDGF-B chain dimers. These data suggest that only PDGF-B chain dimers, such as those produced by macrophages or released from platelets, are regulated by alpha 2M and that PDGF-AA produced by fibroblasts and smooth muscle cells is not controlled by this cytokine-binding protein.

• Wang HL, Pappert TD, Castelli WA, Chiego DJ Jr, Shyr Y, Smith BA
• The effect of platelet-derived growth factor on the cellular response of the periodontium: an autoradiographic study on dogs.
• J Periodontol. 1994; 65: 429-36
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• Platelet-derived growth factor (PDGF) is a polypeptide growth factor considered to have a role in the proliferation and migration of fibroblasts at a wound healing site. The aim of this investigation was to determine if PDGF, when applied to root surfaces, would stimulate the proliferation of fibroblasts and further enhance regeneration. Six mongrel dogs with healthy periodontia were selected for this study. Using a closed wound surgical model, standardized 4 x 4 mm fenestration defects were created into dentin on the mid-facial of the mesial and distal roots of 4 mandibular posterior teeth in each quadrant. Each defect received either: 1) saline solution (C); 2) expanded polytetrafluoroethylene (ePTFE) membrane; 3) PDGF; or 4) ePTFE + PDGF. 3H-thymidine was administered 1 hour prior to animal sacrifice at 1, 3, and 7 days postsurgery. Each time period included 2 dogs with each dog undergoing the four different treatments. Slides were prepared for autoradiography. 3H-thymidine-labeled cells were counted and results were statistically analyzed using the Bonferroni (Dunn) t test on the SAS program. Results indicated PDGF enhanced fibroblast proliferation when compared to the groups without PDGF. Significant differences (P < 0.05) were noted at day 1 and 7 when PDGF and PDGF + GT were compared to C and GT groups. No significant differences were observed in labeled fibroblasts between the C and GT groups at any time period. These findings suggest that PDGF enhances fibroblast proliferation in early periodontal wound healing, whether used alone or in combination with the ePTFE membrane.

• Marra F, Choudhury GG, Pinzani M, Abboud HE
• Regulation of platelet-derived growth factor secretion and gene expression in human liver fat-storing cells.
• Gastroenterology. 1994; 107: 1110-7
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• BACKGROUND/AIMS: Liver fat-storing cells (FSCs) actively proliferate and secrete extracellular matrix during liver injury. Platelet-derived growth factor (PDGF) is a potent mitogen for cultured FSCs. In the present study, we investigated the regulation of PDGF gene expression and production in cultured human liver FSCs. METHODS: PDGF A-chain and B-chain expression was analyzed by Northern blotting and ribonuclease protection assay, respectively. Secretion of PDGF was evaluated by immunoprecipitation and immunoblotting of conditioned medium and metabolic labeling of FSC followed by immunoprecipitation. RESULTS: Three PDGF A-chain transcripts were detectable. Stimulation of FSC with phorbol myristate acetate (10(-7) mol/L) or PDGF BB (20 ng/mL) increased steady-state levels of PDGF A-chain and B-chain messenger RNA. PDGF AA had a small stimulatory effect on A-chain but not B-chain messenger RNA levels. FSCs secrete PDGF in the conditioned medium. The secreted protein is bioactive, because concentrated conditioned medium induced an increase in thymidine incorporation that was inhibited by anti-PDGF antibodies. CONCLUSIONS: This study shows that cultured FSCs express PDGF A- and B-chain genes and release bioactive PDGF in the culture medium. These data raise the possibility of an autocrine or short-loop paracrine effect of PDGF in FSCs as a mechanism contributing to the maintenance of the proliferative state during liver injury.

• Nash TJ, Howlett CR, Martin C, Steele J, Johnson KA, Hicklin DJ
• Effect of platelet-derived growth factor on tibial osteotomies in rabbits.
• Bone. 1994; 15: 203-8
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• The effect of exogenous platelet-derived growth factor (PDGF) BB on bone healing was tested in a pilot study using a unilateral tibial osteotomy in rabbits. Each osteotomy was injected with collagen or collagen containing 80 micrograms of PDGF. At 28 days, both tibiae from each rabbit were harvested and subjected to three-point bending to failure. The effect upon bone healing was tested by comparing the healing rates of PDGF-treated and -nontreated osteotomies with their respective normal contralateral bones. Three animals died before 28 days. The remaining 6 experimental and 5 control animals were available for assessment. Radiographically, at 2 weeks and 4 weeks, there was a clear increase in callus density and volume around the PDGF-treated osteotomies compared with the control rabbits' osteotomies. Osteotomies treated with PDGF were not statistically different in strength from their nonoperated contralateral bones. In the control group, however, the osteotomies were statistically weaker than their nonoperated (contralateral) bones. Microscopically, it was generally observed that PDGF-treated tibiae displayed a more florid and advanced state of osteogenic differentiation, both endosteally and periosteally, than the control osteotomies. Radiographic, mechanical, and histopathological data suggest that exogenous PDGF has a stimulatory effect on fracture healing.

• Rydziel S, Shaikh S, Canalis E
• Platelet-derived growth factor-AA and -BB (PDGF-AA and -BB) enhance the synthesis of PDGF-AA in bone cell cultures.
• Endocrinology. 1994; 134: 2541-6
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• Platelet-derived growth factor (PDGF), an agent with important mitogenic effects for bone cells, exists in three isoforms, PDGF-AA, -BB, and -AB. PDGF-AB and -BB are the prevalent circulating isoforms, whereas normal unstimulated cells of the osteoblast lineage synthesize primarily PDGF-AA. We examined the effects of PDGF-BB on PDGF-A mRNA expression and PDGF-AA polypeptide concentrations in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). In a selected number of experiments we compared the effects of PDGF-BB with those of PDGF-AA on PDGF-A mRNA levels. Steady state PDGF-A mRNA levels were determined by Northern blot analysis, and PDGF-AA concentrations were determined in acidified and fractionated culture medium by a specific RIA for PDGF-A chains. Treatment of Ob cells with PDGF-AA or -BB at 0.3-3.3 nM caused a dose-dependent increase in steady state PDGF-A mRNA, an effect that was initially observed after 2 h. Treatment with PDGF-BB at 1-3.3 nM for 24 h increased PDGF-AA polypeptide concentrations by 2- to 5-fold. The effects of PDGF on PDGF-A mRNA and polypeptide levels were prevented by the protein synthesis inhibitor cycloheximide at 3.6 microM. Phorbol 12-myristate 13-acetate at 1 microM increased PDGF-A mRNA after 2-6 h and PDGF-AA polypeptide levels after 24 h by 2-fold. However, the protein kinase-C inhibitor staurosporine at 50 nM did not modify basal PDGF-A mRNA levels and did not prevent the stimulatory effect of PDGF-AA or -BB on PDGF-A mRNA or PDGF-AA polypeptide levels. In conclusion, PDGF-BB and -AA increase skeletal PDGF-A synthesis, an effect that reveals autoinduction of PDGF in bone cells.

• Jellinek D, Green LS, Bell C, Janjic N
• Inhibition of receptor binding by high-affinity RNA ligands to vascular endothelial growth factor.
• Biochemistry. 1994; 33: 10450-6
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• The proliferation of new blood vessels (angiogenesis) is a process that accompanies many pathological conditions including rheumatoid arthritis and solid tumor growth. Among angiogenic cytokines that have been identified to date, vascular endothelial growth factor (VEGF) is one of the most potent. We used SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 505-510] to identify RNA ligands that bind to VEGF in a specific manner with affinities in the low nanomolar range. Ligands were selected from a starting pool of about 10(14) RNA molecules containing 30 randomized positions. Isolates from the affinity-enriched pool were grouped into six distinct families on the basis of primary and secondary structure similarities. Minimal sequence information required for high-affinity binding to VEGF is contained in 29-36-nucleotide motifs. Binding of truncated (minimal) high-affinity ligands to VEGF is competitive with that of other truncated ligands and heparin. Furthermore, truncated ligands from the six ligand families inhibit binding of [125I]VEGF to its cell-surface receptors. Oligonucleotide ligands described here represent an initial set of lead compounds in our ongoing effort toward the development of potent and specific VEGF antagonists.

• Oikawa T, Onozawa C, Sakaguchi M, Morita I, Murota S
• Three isoforms of platelet-derived growth factors all have the capability to induce angiogenesis in vivo.
• Biol Pharm Bull. 1994; 17: 1686-8
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• Three isoforms of platelet-derived growth factors (PDGFs) composing of AA, AB, and BB chains all exhibited angiogenic activity in a dose-dependent manner in an in vivo assay system involving the chorioallantoic membrane of chick embryo. The order of potency as BB > AB = AA. They, however, failed to stimulate proliferation of vascular endothelial cells, suggesting that their effects are indirect. These data suggest that possibility that three PDGF isoforms are indirect angiogenic factors.

• Haynes JH et al.
• Platelet-derived growth factor induces fetal wound fibrosis.
• J Pediatr Surg. 1994; 29: 1405-8
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• The fetal response to cutaneous injury differs markedly from that of the adult, proceeding with only minimal inflammation, minimal fibroblast proliferation, and only essential collagen deposition. Although the sequence of events in adult wound healing is well defined and thought to be controlled in part by potent polypeptide cytokines, relatively sparse information exists regarding growth factor involvement in fetal wound repair. Thus, the authors sought to examine the effect of platelet-derived growth factor (PDGF), a putative adult wound healing regulator, on the cellular and extracellular matrix events at a fetal wound site. SILASTIC wound implants containing 0, 1.0, 5.0, or 10.0 ng of human PDGF were placed subcutaneously on the backs of 24-day-gestation fetal rabbits (full term, 31 days) and then harvested after either 1, 3, or 5 days in utero. The specimens underwent standard histological processing and were evaluated in a blinded fashion. Compared with controls, PDGF-treated implants had a marked increase in acute inflammation, fibroblast recruitment, and collagen and hyaluronic acid deposition; these differences appeared to be largely time- and PDGF dose-dependent. Thus, the fetal system is responsive to an adult wound healing mediator, and these data suggest that fetal repair proceeds in the absence of PDGF.

• Pajusola K, Aprelikova O, Pelicci G, Weich H, Claesson-Welsh L, Alitalo K
• Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors.
• Oncogene. 1994; 9: 3545-55
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• The FLT4, FLT1 and KDR/FLK1 genes encode structurally similar endothelial cell receptor tyrosine kinases. Recently it has been shown that the FLT1 and KDR/FLK-1 proteins function as high-affinity receptors for vascular endothelial growth factor (VEGF). Here we show that FLT4 does not act as a receptor for VEGF, as VEGF did not show specific binding to the FLT4 tyrosine kinase or induce its autophosphorylation. Also, FLT4 did not interact with KDR in response to VEGF. However, when fused with the ligand binding domain of the colony stimulating factor-1 receptor (CSF-1R), the FLT4 tyrosine kinase was specifically activated by CSF-1. The activated FLT4 tyrosine kinase domain was found to interact with the Src homology 2 domains of the SHC and GRB2 adaptor proteins in vitro and with SHC in cells. CSF-1 stimulation of the CSF-1R/FLT4 receptor chimera induced thymidine incorporation in serum-starved NIH3T3 fibroblasts, but not in porcine aortic or murine lung capillary endothelial cells, although tyrosyl phosphorylation of the receptor and SHC occurred in these cells as well. These results suggest that the endothelial cell FLT4 receptor tyrosine kinase transmits signals for an as yet unidentified growth factor.

• Takimoto Y, Kimura A
• [Basic and clinical study of platelet-derived growth factor gene expression]
• Rinsho Ketsueki. 1994; 35: 364-9
• Display abstract

• PDGF is a glycoprotein composed of A and B chains, and induced the expression not only in platelets but also in many types of cells. By stimulation inducing cell proliferation, transcription of the A chain is activated, resulting in an increase in mRNA. By stimulation inducing cell differentiation, the A chain is activated at the post-transcriptional level, and the B chain at the transcriptional level, resulting in an increase in both mRNA. The 5' region of the A chain shows a mosaic pattern of positive and negative regulatory elements, suggesting complicated regulation. Also the fragments of the first intron are bond by 110Kd and 90Kd nuclear proteins and regulate the A chain expression. Mechanisms of regulation of the A and B chain gene expression differ markedly. The role of PDGF was then investigated on the mechanism of myelofibrosis frequently observed in myeloproliferative disorders (MPD). In the chronic phase of MPD PDGF was suggested to be leaked or released from megakaryocytes. In the accelerated and blast phase of CML with fibrosis, PDGF-AB or PDGF-BB was found to be secreated from myeloid blasts. These results suggest the importance of PDGF in the pathogenesis of myelofibrosis associated with MPD.

• Karlsson C, Paulsson Y
• Age related induction of platelet-derived growth factor A-chain mRNA in normal human fibroblasts.
• J Cell Physiol. 1994; 158: 256-62
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• We have previously found that stimulation of normal neonatal fibroblasts with PDGF or EGF leads to a transient induction of PDGF A-chain mRNA and the synthesis of PDGF-AA proteins. This finding may imply the existence of an autocrine feedback mechanism to amplify the mitogenic signal under certain conditions. We have now studied the PDGF-BB mediated PDGF A-chain induction in a set of fibroblasts from young and old donors to clarify if the levels of induction are correlated to the donor age and the replicative capacity of the cells. The PDGF A-chain induction was found to be reduced in cells from old donors compared with cells from embryonic and neonatal donors. The different cell strains were also characterized further with respect to PDGF receptor expression and PDGF binding properties. PDGF beta-receptors were found to be enhanced in old donor cell strains, whereas the PDGF alpha-receptors showed more variability in expression between the strains. The PDGF A-chain mRNA induction was also decreased or absent in late passage human fibroblasts (senescent cells) when compared with early passage cells. These data suggest that the PDGF A-chain mRNA induction is regulated by an age related mechanism in human fibroblasts.

• Tamm I, Kikuchi T, Kreutter D, Pledger WJ, Pfeffer LM
• Selective interferon-alpha/beta effects on platelet-derived growth factor-stimulated processes in quiescent BALB/c-3T3 fibroblasts.
• J Interferon Res. 1994; 14: 265-73
• Display abstract

• Interferon-alpha/beta (IFN-alpha/beta) suppresses cell cycle activation by platelet-derived growth factor (PDGF) as well as the induction of the 31-kD (pI) and the 35-kD (pII) proteins in density-arrested BALB/c-3T3 cells. We report that elevation of [Ca2+]i by ionomycin induces the synthesis of the 31-kD protein, but not that of the 35-kD protein. Since IFN blocks the PDGF-induced elevation of [Ca2+]i, these results suggest that IFN treatment may suppress pI induction by impairing this PDGF-activated signal transduction pathway. In contrast, because ionomycin did not induce the 35-kD protein, the suppression by IFN of PDGF-induced pII appears to be mediated via a pathway distinct from that operating in the suppression of pI. In BALB/c-3T3 cells, IFN-alpha/beta did not itself affect the turnover or de novo synthesis of inositol phospholipids and the cellular content of diacylglycerol, nor did IFN block the enhancement of these parameters by PDGF.

• Bonner JC
• Regulation of platelet-derived growth factor (PDGF) and alveolar macrophage-derived PDGF by alpha 2-macroglobulin.
• Ann N Y Acad Sci. 1994; 737: 324-38
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• In vitro findings suggest that alpha 2M is an important regulator of PDGF-stimulated fibroblast proliferation and chemotaxis. Native alpha 2M binds to PDGF and prevents PDGF from interacting with its receptor, but serves as an extracellular reservoir for the growth factor, which can be released over time in a controlled fashion to interact with the PDGF-alpha or -beta receptor. Methylamine-activated alpha 2M synergistically enhances PDGF-induced cell growth, whereas plasmin-activated alpha 2M inhibits PDGF-stimulated fibroblast proliferation. The reason for the difference in the effect of these two receptor-recognized alpha 2Ms is unknown. PDGF secreted by rat alveolar macrophages is bound to homologues of human alpha 2M and it has been suggested that PDGF action in the lung is tightly controlled during normal tissue remodeling. It is important to consider another regulator of PDGF termed SPARC (secreted protein, acidic and rich in cysteine), which inhibits the binding of PDGF-BB and -AB to cell-surface PDGF-beta receptors. SPARC could modulate PDGF activity during inflammation and tissue repair by limiting the availability of dimers containing the PDGF B chain. Future studies should address the relative importance of SPARC and alpha 2M in regulating PDGF-induced chemotaxis and proliferation. During inflammation or during the progression of fibroproliferative lung disease, the regulation of PDGF might be lost. For example, oxidative bursts from inflammatory cells (neutrophils and eosinophils) functionally inactivate alpha 2M. Thus, inhaled environmental insults (particles and oxidants) could perturb the normal growth regulatory signaling system between cells via the network that includes cytokines, alpha 2M, and proteinases.

• Boyan LA, Bhargava G, Nishimura F, Orman R, Price R, Terranova VP
• Mitogenic and chemotactic responses of human periodontal ligament cells to the different isoforms of platelet-derived growth factor.
• J Dent Res. 1994; 73: 1593-600
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• A major focus of studies that center on regeneration of the periodontium is to determine the efficacy of the use of polypeptide growth factors. Platelet-derived growth factor has been reported to be a possible agent for clinical use. PDGF has various isoforms. Therefore, we decided to study the mitogenic and chemotactic responses of human periodontal ligament (PDL) cells to recombinant human PDGF-AB, AA, and BB. Addition of each isoform of PDGF to in vitro mitogenesis assays induced PDL cell proliferation in a dose-dependent manner. The maximum mitogenic effect was evident at the concentration of 100 ng/mL. In these assays, PDGF-BB was found to be the most potent mitogen. PDGF-AB elicited an intermediate response, and PDGF-AA was the least effective. The results of chemotaxis assays closely parallel those of the mitogenesis assays. PDGF-BB exhibited the most potent chemotactic effect. The maximal effect was observed at 10 ng/mL. The findings of these experiments indicate that PDGF-BB is more effective than the other isoforms in promoting mitogenesis and chemotaxis of PDL cells in vitro, and may therefore be a suitable ethical pharmaceutical for use in periodontal regeneration procedures.

• Khouri RK et al.
• De novo generation of permanent neovascularized soft tissue appendages by platelet-derived growth factor.
• J Clin Invest. 1994; 94: 1757-63
• Display abstract

• Treatment of wounds with pharmacologic doses of the BB homodimeric form of recombinant PDGF (rPDGF-BB) induces the recruitment, activation, and proliferation of mesenchymal cells, resulting in the deposition of provisional, and subsequently collagen-containing extracellular matrix. In preliminary experiments with an in vitro growth chamber model in the rat consisting of a silicone shell containing a dissected femoral vascular bundle, we found that rPDGF-BB incorporated into a rapidly dissolving collagen type I film induces the generation of a marked, but transient amount of de novo tissue around the femoral vascular bundle. In the present studies, the new tissue generated around the femoral vascular bundle was wrapped with a full thickness syngeneic skin graft to determine if functional support of the graft would lead to sustained maintenance of the underlying generated tissue and create an epithelialized soft tissue appendage. The tissue generated after a single application of rPDGF-BB was skin grafted on the 10th day, exteriorized 20 d later, and observed for an additional month. This led to the formation of soft tissue appendages which demonstrated marked neovascularization, fibroblast migration and proliferation, and increased glycosaminoglycan, fibronectin, and collagen fibril deposition, now leading to preservation of the newly generated tissue. In contrast, minimal new tissue was generated in control-treated vascular bundles or bundles treated with inactive PDGF-BB, and grafting with skin failed to sustain the underlying tissue. Thus, rPDGF-BB coupled with skin grafting induced the formation of functional large soft tissue appendages which are potentially useful clinically to fill tissue defects or to serve as a cell delivery system for transfected genes.

• Koyama N, Watanabe S, Tezuka M, Morisaki N, Saito Y, Yoshida S
• Migratory and proliferative effect of platelet-derived growth factor in rabbit retinal endothelial cells: evidence of an autocrine pathway of platelet-derived growth factor.
• J Cell Physiol. 1994; 158: 1-6
• Display abstract

• Angiogenesis is a crucial event in the progression of diabetic retinopathy. Migration and proliferation of endothelial cells (EC) are important steps in angiogenesis and are caused by angiogenic factors such as basic fibroblast growth factor (bFGF). In this work, capillary EC were isolated from rabbit retinal tissues and rabbit retinal EC (RREC) were found to secrete a migration factor for RREC in conditioned medium (CM). The activity was inhibited by an anti-platelet-derived growth factor (PDGF) antibody, but not by an anti-bFGF antibody. We also found that RREC showed a migratory response to PDGF. The response was induced by PDGF-BB and PDGF-AB dose dependently, but not by PDGF-AA, indicating that it was mediated by PDGF-beta receptor-dependent pathways, and that the PDGF-like factor was PDGF-BB or -AB. In addition, PDGF-BB induced the proliferation of RREC as well as bFGF. These data indicate that RREC have an autocrine pathway of PDGF by the secretion of and the response to PDGF. PDGF may play significant parts in angiogenesis in the progression of diabetic retinopathy.

• Dill RE, Miller EK, Dyer BJ, Iacopino AM
• Synthesis of platelet-derived growth factor by cells of splenic red pulp in normal rats.
• Cell Tissue Res. 1994; 276: 209-12
• Display abstract

• A population of cells in the spleens of normal rats was found to contain platelet-derived growth factor (PDGF) B chain mRNA. These cells were found predominantly in the red pulp and nuclear morphology of some was consistent with that of macrophages. Similar cells were also shown by immunocytochemical staining to contain PDGF-AB/BB. These PDGF-positive cells were also found almost exclusively in the red pulp. It has been suggested by others that PDGF plays an important role in the function of the lymphohemopoietic microenvironment.

• Pekny M, Ostman A, Hermansson A, Nister M, Heldin CH, Westermark B
• Differences in binding to the solid substratum and extracellular matrix may explain isoform-specific paracrine effects of platelet-derived growth factor.
• Growth Factors. 1994; 10: 77-87
• Display abstract

• We have studied the paracrine response of fibroblasts to the two homodimeric isoforms of platelet-derived growth factor (PDGF-AA and -BB). CHO-cells stably transfected with a B-chain cDNA expression vector (CHO-PDGF-B cells), were found to elicit a marked paracrine response when seeded at clonal density on preformed monolayers of human or murine fibroblasts; no such response was elicited by CHO-PDGF-A cells. Immunofluorescence microscopy of CHO-PDGF-B cell cultures showed the presence of a pericellular deposit of material reacting with antibodies against PDGF-BB; no corresponding PDGF-AA immunoreactive material was found in the CHO-PDGF-A cultures. The pericellular material, deposited by CHO-PDGF-B cells, was shown to have a growth promoting effect on target cells. Furthermore, we could show that 125I-PDGF-BB binds more efficiently than 125I-PDGF-AA to extracellular matrix prepared from foreskin fibroblast cultures, as well as to defined extracellular matrix components (fibronectin, laminin and collagen III). The results reveal a marked difference in the paracrine activity of PDGF-AA and PDGF-BB; the latter has a strong local growth enhancing effect, that is mostly likely to be ascribed to its association with components of the extracellular matrix.

• Schneppe B, Eichner W, McCarthy JE
• Translational regulation of a recombinant operon containing human platelet-derived growth factor (PDGF)-encoding genes in Escherichia coli: genetic titration of the peptide chains of the heterodimer AB.
• Gene. 1994; 143: 201-9
• Display abstract

• A new strategy is described for the production of recombinant heteromultimeric proteins using Escherichia coli as host. A recombinant operon was constructed containing modified cDNA sequences encoding the mature A and B chains of human platelet-derived growth factor (PDGF). The relative expression rates of the PDGF genes were varied over a range equivalent to A:B ratios from 0.8 to 3.7 by means of translational regulation. This was achieved using two different translational initiation sequences (TIS) upstream from the respective coding regions, one derived from the E. coli atpE translational initiation region, and the other containing a sequence with extended complementarity to the 3' end of the 16S rRNA. The generation of mature PDGF A and B chains in different relative amounts in E. coli provided the basis for developing a novel procedure for the production of the biologically active PDGF heterodimer AB in large quantities. The general strategy is applicable to the preparation of a wide range of heteromultimeric complexes. Moreover, the described PDGF operon constitutes a compact and versatile model system for studies of the posttranscriptional regulation of gene expression.

• Abboud HE, Grandaliano G, Pinzani M, Knauss T, Pierce GF, Jaffer F
• Actions of platelet-derived growth factor isoforms in mesangial cells.
• J Cell Physiol. 1994; 158: 140-50
• Display abstract

• Platelet-derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF-BB, -AA, and -AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF-BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phospholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF-AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down-regulation of the PDGF-alpha receptor since preincubation of MC with suramin restored PDGF AA-induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase-C activation. The potent biologic effects of PDGF-AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation.

• Tsai LH et al.
• Expression of platelet-derived growth factor and its receptors by two pre-B acute lymphocytic leukemia cell lines.
• Blood. 1994; 83: 51-5
• Display abstract

• Platelet-derived growth factors (PDGF) are potent regulators of cell proliferation. The three isoforms of PDGF AA, AB, and BB are encoded by two genes: PDGF A and PDGF B. The v-sis oncogene is homologous to the PDGF-B gene. v-sis can transform cells that express the appropriate PDGF receptors. Two different types of receptors, PDGF-alpha and PDGF-beta, also encoded by two genes, have been identified. We show that two cell lines. SMS-SB and NALM-6, both derived from pre-B-cell acute lymphocytic leukemias, express the PDGF-A chain gene, and one of them, SMS-SB, releases PDGF-A chains into the media. The SMS-SB cells also express the PDGF-beta receptor, whereas NALM-6 cells express the PDGF-alpha receptor and bind PDGF. This extends the possible targets for PDGF to the B-cell lineage lymphocytes.

• Takayama S, Sasahara M, Iihara K, Handa J, Hazama F
• Platelet-derived growth factor B-chain-like immunoreactivity in injured rat brain.
• Brain Res. 1994; 653: 131-40
• Display abstract

• Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells and glial cells. For a better understanding of the role of PDGF B-chain in the brain, the expression of PDGF B-chain was examined immunohistochemically in penetrating injury to the rat brain. Shrunken neurons were distributed with enhanced PDGF B-chain-related immunoreactivity (PBRI) in the vicinity of the lesion during a period from day 1 to day 4 post injury. Platelet-derived growth factor B-chain-related immunoreactivity was transiently observed also in the cytoplasm of the numerous brain macrophages in the lesion on day 3 and day 4. These distributions of PBRI in the lesion were closely related to the neovascularization and astrogliosis there. The close time and spatial correlation between the expression of PBRI and cellular responses to injury seen in this study suggests PDGF B-chain has an important role in the healing process of cerebral wound.

• Takimoto Y, Kuramoto A
• Gene regulation by the 5'-untranslated region of the platelet-derived growth factor A-chain.
• Biochim Biophys Acta. 1994; 1222: 511-4
• Display abstract

• Platelet-derived growth factor (PDGF) A-chain gene contains a long 5'-untranslated region (5'-UTR) of 850 bp. We evaluated the role of the 5'-UTR by chloramphenicol acetyltransferase (CAT) assay. CAT activity appeared when the fragment +99 bp downstream from the initiation site (+1) was present but disappeared in the fragment to +184 bp. It appeared again at +338 bp but disappeared again to +609 bp. The fragment from +99 to +184 inhibited CAT activity by a post-transcriptional mechanism, as RNA of CAT was observed but CAT activity was not.

• Caniggia I et al.
• Fetal lung epithelial cells express receptors for platelet-derived growth factor.
• Am J Respir Cell Mol Biol. 1993; 9: 54-63
• Display abstract

• There is increasing evidence to suggest that platelet-derived growth factor (PDGF) or PDGF-like molecules play a role in fetal lung morphogenesis. Our previous studies demonstrated that fetal lung epithelial cells respond mitogenically to exogenous PDGF, while fetal lung fibroblasts respond with increased glycosaminoglycan synthesis. To further study the target cells of PDGF in fetal rat lung, we investigated the presence and nature of PDGF receptors in fetal lung cells. Functional PDGF receptors were expressed on normal epithelial cells of fetal rat lung. All three isoforms of PDGF (AA, AB, and BB) were mitogenic for quiescent epithelial cells. Northern blot and protein analysis demonstrated the presence of PDGF alpha-receptor and PDGF beta-receptor. All isoforms of PDGF enhanced tyrosine kinase activity and stimulated receptor autophosphorylation. In contrast, fetal lung fibroblasts expressed only the PDGF beta-receptor. PDGF-AB and PDGF-BB, but not PDGF-AA, stimulated tyrosine kinase activity. No PDGF isoform was mitogenic for quiescent fibroblasts. However, PDGF-BB stimulated fibroblast proliferation on a collagen type I substratum in the presence of transferrin. Binding experiments with [125I]PDGF-AA and [125I]-PDGF-BB to epithelial cells and fibroblasts confirmed these observations.

• Van Zoelen EJ, Van Rotterdam W, Van de Wetering RA, Heldin CH
• Differential effects of PDGF isoforms on proliferation of normal rat kidney cells.
• Growth Factors. 1993; 9: 329-39
• Display abstract

• The effects of the PDGF isoforms AA, AB and BB have been studied on the proliferation of normal rat kidney cells, a non-tumorigenic fibroblast cell line which contains both type alpha and type beta PDGF receptors. On monolayer cells made quiescent by serum deprivation, PDGF-AA is a relatively poor mitogen compared to PDGF-AB and PDGF-BB. When these cells are made density-arrested following continuous incubation with epidermal growth factor, however, they can be restimulated to proliferate by all three PDGF isoforms with similar activity when added at sufficiently high concentration, resulting in phenotypic cellular transformation. Binding of radiolabelled isoforms to confluent NRK monolayers obeys the predictions of an induced receptor dimerisation model, and increases in the order AA < AB < BB. Upon preincubation of the cells with PDGF-AA, the dose-response curve for mitogenic activity of PDGF-AB is shifted to higher concentrations, indicating that PDGF-AA can partly antagonize the growth stimulating activity of PDGF-AB, as has also been observed in ligand binding studies. This observation has subsequently been confirmed using fluorescence cytometric analysis. PDGF-AB is highly active in inducing anchorage-independent proliferation of NRK cells, but in all such assays PDGF-AA is at least as potent as PDGF-BB. Intriguingly, PDGF-BB is almost devoid of activity in inducing soft agar growth of these cells, in contrast to PDGF-AA. When compared to substrate-attached cells, enhanced expression of the type alpha PDGF receptor was observed under anchorage-independent conditions. These results show that the relative potency of the three PDGF isoforms to stimulate proliferation of NRK cells is different for quiescent cells in monolayer, density-arrested cells and anchorage-independent cells. Moreover it is shown that the biological activity of PDGFs can be impaired by the additional presence of other isoforms.

• Yablonka-Reuveni Z, Seifert RA
• Proliferation of chicken myoblasts is regulated by specific isoforms of platelet-derived growth factor: evidence for differences between myoblasts from mid and late stages of embryogenesis.
• Dev Biol. 1993; 156: 307-18
• Display abstract

• In the present study we measured the level of PDGF receptor expression by chicken myoblasts and the effect of the three different PDGF isoforms (AA, AB, BB) on DNA synthesis by myoblasts. We examined PDGF receptor expression and function on clonally derived myoblasts in order to eliminate contaminating fibroblasts which are present in myogenic cultures and which bind PDGF. Furthermore, since we have previously shown that fetal myoblasts are replaced with adult myoblasts during late chicken embryogenesis, we compared PDGF receptor expression and function on myoblasts from Embryonic Day 10 (E10, mid development) and from Embryonic Day 19 (E19, late development). We found that all myogenic clones from late embryos (E19) express many receptors for PDGF-BB, far fewer receptors for PDGF-AB, and even fewer, if any, receptors for PDGF-AA. Myoblast clones derived from E10 were more heterogeneous in their PDGF binding pattern ranging from clones similar to E19 clones to clones having very few PDGF binding sites. We also found that both PDGF-AB and PDGF-BB can promote DNA synthesis by clonally derived chicken myoblasts maintained in 2.5% fetal bovine serum whereas PDGF-AA has no detectable effect. Finally, we observed that primary myogenic cultures from E10 and E19 differ strikingly in levels of PDGF binding; E19 cultures bind much more PDGF than do E10 cultures. We conclude that PDGF can enhance the proliferation of chicken myoblasts and that myoblasts responsive to PDGF are more frequent in late than in mid stages of development. We propose that PDGF may be a modulator of myogenesis of adult but not fetal myoblasts.

• Abboud SL
• A bone marrow stromal cell line is a source and target for platelet-derived growth factor.
• Blood. 1993; 81: 2547-53
• Display abstract

• Platelet-derived growth factor (PDGF) stimulates multipotent and erythroid progenitors as well as stromal fibroblasts. Any of the three dimeric forms of PDGF (AA, AB, or BB) could potentially interact with these cells; however, the precise cellular origin of PDGF production in the bone marrow microenvironment is not known. In the present study, we found that medium conditioned by MBA-2, murine bone marrow-derived endothelial cells, contains PDGF activity that competes for [125I]PDGF binding to human foreskin fibroblasts and is mitogenic for these fibroblasts. Northern analysis of poly(A)+ RNA from MBA-2 shows the expression of both PDGF A-chain and B-chain mRNAs. Because cytokines such as transforming growth factor-beta (TGF-beta) regulate hematopoiesis and stimulate PDGF in certain mesenchymal cells, we determined whether TGF-beta influences PDGF secretion and gene expression in MBA-2. TGF-beta induced PDGF A-chain and B-chain mRNAs and the release of PDGF activity. Each of the three PDGF isoforms also stimulated DNA synthesis in MBA-2, but with different potency (BB > AB > AA). Ligand binding studies showed specific binding of labeled PDGF BB and, to a lesser extent, PDGF AA isoform, consistent with predominant expression of the PDGF-beta receptor in MBA-2. These data show that murine endothelial stromal cells release PDGF activity and respond to PDGF. Local production of PDGF in the marrow microenvironment may play an important role in regulating hematopoietic and stromal cell proliferation.

• Eichmann A, Marcelle C, Breant C, Le Douarin NM
• Two molecules related to the VEGF receptor are expressed in early endothelial cells during avian embryonic development.
• Mech Dev. 1993; 42: 33-48
• Display abstract

• We present the partial cloning and the expression patterns of two putative growth factor receptor molecules named Quek1 and Quek2 (for quail endothelial kinase) in chick and quail embryos from gastrulation to embryonic day 9 (E9). Quek1 and Quek2 show high homology to three interrelated murine and human genes, flk-1, KDR and flt. Flt was recently shown to be the receptor for the endothelial cell mitogen vascular endothelial growth factor (VEGF). In situ hybridization of Quek1 and Quek2 to sections of avian embryos showed that they are both expressed essentially by endothelial cells, that we identified with a monoclonal antibody (Mab) QH1 specific for endothelial and white blood cells of the quail. Quek1 is expressed in the mesoderm from the onset of gastrulation, whereas Quek2 message is first detected on QH1-expressing endothelial cells. The expression pattern of Quek1 suggests that it could identify the putative precursor of both endothelial and hematopoietic lineages, the hemangioblast. Quek1 and Quek2 are not expressed in all endothelial cells throughout life. At E9, after the initial phase of vasculogenesis, these genes are switched off in various compartments of the vascular network.

• Risbridger GP
• Discrete stimulatory effects of platelet-derived growth factor (PDGF-BB) on Leydig cell steroidogenesis.
• Mol Cell Endocrinol. 1993; 97: 125-8
• Display abstract

• The effect of platelet-derived growth factor-BB on adult rat Leydig cell steroidogenesis has been determined using culture conditions which maintain testosterone production and responsiveness to luteinising hormone for 3 days. A significant stimulation of testosterone levels was observed in acute response to a maximally stimulating dose (8 ng/ml) of rat luteinising hormone on the third day of culture, but was dependent on pre-exposure of the Leydig cells to PDGF-BB for 2 days, whilst maintained in the presence of a minimum dose (0.1 ng/ml) of rat luteinising hormone. These data demonstrate a very discrete action of PDGF-BB on adult rat Leydig cell steroidogenesis in vitro, but whether or not this constitutes a significant paracrine effect in vivo remains to be established.

• Fretto LJ et al.
• Mechanism of platelet-derived growth factor (PDGF) AA, AB, and BB binding to alpha and beta PDGF receptor.
• J Biol Chem. 1993; 268: 3625-31
• Display abstract

• The biological effects of platelet-derived growth factor (PDGF) are mediated by cell surface alpha and beta PDGF receptors, which, as a result of ligand binding, undergo dimerization in a manner consistent with PDGF being bivalent. In order to directly demonstrate PDGF bivalency and to define the binding of PDGF AB to isolated beta receptor, we developed solid-phase binding assays using purified recombinant extracellular domain of human PDGF receptors. PDGF AA, AB, and BB were prepared from the monomeric chains expressed in Escherichia coli, and each was purified to homogeneity; PDGF AB contained < 0.5% of either homodimer. The interactions of these isoforms with immobilized PDGF receptors were examined by several approaches. Scatchard analysis revealed high affinity binding (Kd = 0.5-1.0 nM) of radiolabeled PDGF AA and AB to alpha receptor and of PDGF BB to both receptor subtypes. Contrary to previous reports, PDGF AB also bound beta receptor with high affinity (Kd = 0.9 nM). When a B-chain-specific monoclonal antibody that recognizes the putative binding domain of PDGF BB was used for ligand detection, we found that PDGF AB binding to beta receptor occurred exclusively through the B-chain subunit, whereas binding to alpha receptor occurred through either subunit. In addition, site-directed mutagenesis was used to specifically inactivate the B chain of PDGF AB, which eliminated binding to the beta receptor without affecting alpha receptor binding. These results establish that PDGF is bivalent and that monovalent ligand retains high affinity receptor binding.

• Katoh O et al.
• Isolation of the complementary DNA encoding a mouse heparin-binding growth factor receptor with the use of a unique kinase insert sequence.
• Cancer Res. 1993; 53: 1136-41
• Display abstract

• With the use of reverse transcriptase-polymerase chain reaction techniques focused on a unique kinase insert sequence, the complementary DNA for a mouse tyrosine kinase receptor gene, designated sam3, was isolated from a mouse brain complementary DNA library as a member of the heparin-binding growth factor receptor family or fibroblast growth factor receptor family. The kinase insert region was selected as the probe synthesized by polymerase chain reaction techniques because it composes a unique structure in this receptor family. The sam3 protein, 800 amino acids long, has high homology to mouse K-sam/bek (67%) and N-sam/flg (63%), which we also cloned as the mouse counterparts of human K-sam/bek and N-sam/flg genes, other members of this family. The sam3 protein also has high homology to human FGFR3 (92%) and chicken cek2 (80%) proteins. The sam3 protein is most likely to be a mouse counterpart of human FGFR3 and chicken cek2 proteins. mRNAs of K-sam/bek, N-sam/flg, and sam3/FGFR3 genes were detected in mouse embryo through some adult tissues. The relative amounts of these mRNAs were different depending on the organs examined. Thus, these gene products may have different biological functions in organ development including the central nervous system.

• May M, Aaronson SA, LaRochelle WJ
• Platelet-derived growth factor AB heterodimer interchain interactions influence secretion as well as receptor binding and activation.
• Biochemistry. 1993; 32: 11734-40
• Display abstract

• Platelet-derived growth factor (PDGF) is a disulfide-linked dimer comprised of two related polypeptide chains. To investigate the effects of an inactivating lesion introduced into one chain of the nascent PDGF dimer, approaches were developed to optimize synthesis, assembly, secretion, and purification of heterodimers between normal PDGF A and wild-type or mutant PDGF B. PDGF AB heterodimers were released into culture fluids less efficiently than PDGF AA, but to a greater degree than the cell-associated PDGF BB. These results suggest that interactions between two chains influence PDGF secretion. Analysis of heterodimers between PDGF A and disabled PDGF B mutants on cells that express either alpha or beta PDGFRs demonstrated that the impaired biologic activity of the mutant PDGF B chain was ameliorated with respect to binding and triggering of alpha PDGFRs. In cells that expressed both receptor types, heterodimers of mutant PDGF B and wild-type PDGF A gained substantially in their ability to recruit and trigger alpha, but not beta, PDGFRs. Partial rescue of impaired PDGF B mutant chain function by dimer formation with a wild-type PDGF A chain implies that interchain interactions markedly affect PDGFR binding and activation.

• Seymour L, Dajee D, Bezwoda WR
• Tissue platelet derived-growth factor (PDGF) predicts for shortened survival and treatment failure in advanced breast cancer.
• Breast Cancer Res Treat. 1993; 26: 247-52
• Display abstract

• In a study of plasma and tissue platelet derived growth factor (PDGF) concentration in patients with breast cancer, elevated levels of plasma PDGF were found in a significant proportion, 11/37 (30%), of patients. Sixteen patients (43%) had tumors which expressed PDGF-AA and 6 patients had tumors which in addition expressed the BB isoform of PDGF. All patients with elevated plasma levels of platelet derived growth factor had tumors which expressed the growth factor on immunohistochemical staining of tumor cells. Furthermore there was a significant correlation between plasma levels of platelet derived growth factor and the intensity of tissue staining. Patients with stage four breast cancer with tumors which were positive for platelet derived growth factor had a significantly lower response rate to chemotherapy as well as significantly shorter duration of survival. In addition, patients with stage four breast cancer who had elevated plasma PDGF levels had a significantly shorter survival. These results indicate that elevated plasma levels of platelet derived growth factor in patients with breast cancer are derived from the tumor cells and suggest that platelet derived growth factor may play a significant role in control tumor cell growth.

• Clark JG, Madtes DK, Raghu G
• Effects of platelet-derived growth factor isoforms on human lung fibroblast proliferation and procollagen gene expression.
• Exp Lung Res. 1993; 19: 327-44
• Display abstract

• Platelet-derived growth factor (PDGF), a potent mitogen for fibroblasts, is a potentially important cytokine in the pathogenesis of fibroproliferative disorders of lung. Different isoforms of PDGF include a heterodimer composed of A and B chains and homodimers composed of A or B chains. The biological significance of the different isoforms is unknown, but they have been shown to differ in their mitogenic potency in some systems. Their effects on other fibroblast functions have not been fully examined. We undertook this study to determine the effect of PDGF isoforms on human lung fibroblast proliferation and procollagen synthesis. Cultured lung fibroblasts (IMR-90, WI-38, GeNA) were incubated in the presence of varying concentrations of highly purified PDGF-AB obtained from platelets or recombinant PDGF-AA or -BB homodimers. Incorporation of [3H]thymidine was determined as a measure of mitogenic activity. Fetal lung fibroblasts (IMR-90, WI-38) and an adult fibroblast strain (GeNA) responded similarly to the different isoforms, with maximum mitogenic activity observed at 5-10 ng/mL. Cell cycle analysis using three additional normal adult lung fibroblast strains indicated that all PDGF isoforms stimulated similar proportions of cells to cycle over a 7-day period. Fibroblast procollagen synthesis, measured after pulse labeling with [3H]proline, was not increased even at concentrations of the PDGF isoforms that were maximally mitogenic. Moreover, steady-state levels of alpha 1(I) and alpha 1(III) procollagen mRNA levels, determined by northern analysis and dot blot hybridization, were not changed after exposure to any of the PDGF isoforms. While all PDGF isoforms are potent mitogens for fetal and adult lung fibroblasts, it was concluded that they do not directly stimulate procollagen gene expression or procollagen synthesis in vitro. The results suggest that PDGF isoforms are potentially important mitogens for lung fibroblasts, but other factors are likely to be involved in the stimulation of fibroblast procollagen synthesis that is observed in fibroproliferative disorders of lung.

• Walsh J, Absher M, Kelley J
• Variable expression of platelet-derived growth factor family proteins in acute lung injury.
• Am J Respir Cell Mol Biol. 1993; 9: 637-44
• Display abstract

• During acute lung injury, there is an outpouring of growth factors into the alveolar space that drive local repair and fibrosis. During the remodeling that follows the instillation of bleomycin via the trachea into the adult rat, at least two platelet-derived growth factor (PDGF)-like peptides are released sequentially into lung lining fluid. Groups of four to five animals were killed at 3, 6, 15, and 26 days after exposure to bleomycin and lungs lavaged with isotonic saline. PDGF-like peptides in epithelial lining fluid (ELF) were partially purified by cation exchange chromatography and concentrated. Isolated peptides were analyzed by immunoblotting to determine their molecular weight and immunologic identity. Western blots were probed with polyclonal antibodies to PDGF-BB and PDGF-AA. PDGF-like peptides of two distinct size classes (38-40 kD and 29 kD) were present in alveolar fluid from all rats with lung injury induced by bleomycin. No PDGF-like peptides were found in comparably prepared ELF from control animals. The 38-40 kD peptide was detected only with anti-PDGF-BB antibody; the 29 kD peptide was detected only with anti-PDGF-AA antibody. The presence of these two peptides varied independently with time after exposure to bleomycin. The 38-40 kD peptide was at peak levels at 3 to 6 days. In contrast, the 29 kD peptide was present at all times following injury but with far less variation over time. In parallel with these immunoassays for PDGF-like molecules, there was abundant growth-promoting activity for fibroblasts present in concentrated ELF during the course of injury.(ABSTRACT TRUNCATED AT 250 WORDS)

• Herren B, Rooney B, Weyer KA, Iberg N, Schmid G, Pech M
• Dimerization of extracellular domains of platelet-derived growth factor receptors. A revised model of receptor-ligand interaction.
• J Biol Chem. 1993; 268: 15088-95
• Display abstract

• The alpha- and beta-subunits of the receptor for platelet-derived growth factor (PDGFR) were found to be autophosphorylated in the absence of ligands at high expression levels which suggests a propensity of PDGFRs to dimerize spontaneously. When the extracellular domains (ED) of both receptors were expressed and purified to homogeneity, they could be dimerized specifically by the different PDGF isoforms. PDGF-BB-induced dimerization was dependent on uncleaved loop I sequences present on both chains. Whereas, in solution, the EDs were weak competitors for PDGF binding to cellular PDGFRs, they formed high and low affinity complexes upon immobilization on solid phase. Cross-competition experiments defined two distinct binding sites on PDGFR alpha-ED. PDGF-AB bound only to the low affinity form of immobilized PDGFR beta-ED and could not dimerize PDGFR beta-ED. Cross-linking studies, however, revealed that both chains of PDGF-AB can interact with a PDGFR beta-ED monomer. Cross-linking of PDGF homodimers with EDs also yielded complexes which contained more than two ligand chains. These results led to a revised model of receptor-ligand interaction and indicate that monomeric PDGF should be able to dimerize PDGF receptors.

• Kaipainen A et al.
• The related FLT4, FLT1, and KDR receptor tyrosine kinases show distinct expression patterns in human fetal endothelial cells.
• J Exp Med. 1993; 178: 2077-88
• Display abstract

• The growth factor receptors expressed on endothelial cells are of special interest because of their potential to program endothelial cell growth and differentiation during development and neovascularization in various pathological states, such as wound healing and angiogenesis associated with tumorigenesis. Vascular endothelial growth factor ([VEGF] also known as vascular permeability factor) is a potent mitogen and permeability factor, which has been suggested to play a role in embryonic and tumor angiogenesis. The newly cloned FLT4 receptor tyrosine kinase gene encodes a protein related to the VEGF receptors FLT1 and KDR/FLK-1. We have here studied the expression of FLT4 and the other two members of this receptor family in human fetal tissues by Northern and in situ hybridization. These results were also compared with the sites of expression of VEGF and the related placenta growth factor (PlGF). Our results reveal FLT4 mRNA expression in vascular endothelial cells in developing vessels of several organs. A comparison of FLT4, FLT1 and KDR/FLK-1 receptor mRNA signals shows overlapping, but distinct expression patterns in the tissues studied. Certain endothelia lack one or two of the three receptor mRNAs. These data suggest that the receptor tyrosine kinases encoded by the FLT gene family may have distinct functions in the regulation of the growth/differentiation of blood vessels.

• Heldin CH, Westermark B
• Possible in vivo effect and clinical utility of platelet-derived growth factor and PDGF antagonists.
• Transplant Proc. 1993; 25: 2074-6

• Johnson RJ, Floege J, Couser WG, Alpers CE
• Role of platelet-derived growth factor in glomerular disease.
• J Am Soc Nephrol. 1993; 4: 119-28
• Display abstract

• An approach for establishing a role for a growth factor in glomerular disease is presented. Using platelet-derived growth factor (PDGF) as an example, there is strong evidence to support the hypothesis that PDGF is a mediator of mesangial cell proliferation in glomerulonephritis. This includes evidence that (1) PDGF is a mitogen for mesangial cells in culture; (2) PDGF is expressed in both experimental and human glomerulonephritis in which mesangial cell proliferation occurs; (3) infusion of PDGF into rats induces mesangial cell proliferation and a hypercellular lesion; and (4) inhibition of PDGF in a model of experimental nephritis significantly reduces the mesangial cell proliferation. However, these data do not answer the question of whether or not the inhibition of PDGF in human diseases would be beneficial in the long term, because some cell proliferation is likely required for normal healing and repair. Further studies will be necessary to resolve this issue.

• Gesualdo L, Ranieri E, Pannarale G, Di Paolo S, Schena FP
• Platelet-derived growth factor and proliferative glomerulonephritis.
• Kidney Int Suppl. 1993; 39: 869-869

• Allam M, Martinet N, Gallati H, Vaillant P, Hosang M, Martinet Y
• Platelet-derived growth factor AA and AB dimers are present in normal human epithelial lining fluid.
• Eur Respir J. 1993; 6: 1162-8
• Display abstract

• Normal lung architecture is related to the presence of mesenchymal cells (fibroblasts and smooth muscle cells), and to the production by these cells of extracellular matrix. The turnover of mesenchymal cells is under a fine regulation due, at least in part, to the local presence of different mediators acting on their cell cycle. Since normal human alveolar macrophages obtained by bronchoalveolar lavage (BAL) spontaneously release platelet-derived growth factor (PDGF), a cytokine with chemotactic and growth activity on mesenchymal cells, we evaluated normal human epithelial lining fluid (ELF) for the presence of PDGF. Active only as a dimer, PDGF is a glycoprotein composed of two chains (A and B) and, thus, can be present in three forms: AA, AB, and BB dimers. Interestingly, normal ELF contains PDGF AA dimers, and to a lesser extent AB dimers, while no significant level of BB dimers is detected. Furthermore, ELF PDGF is biologically active and responsible for a significant part of the chemotactic activity and the "competence" growth activity for mesenchymal cells present in normal ELF. These findings suggest that ELF PDGF has a role in normal lung structure maintenance and tissue repair.

• Ansel JC et al.
• Human keratinocytes are a major source of cutaneous platelet-derived growth factor.
• J Clin Invest. 1993; 92: 671-8
• Display abstract

• PDGF has been implicated as one of the principal mitogens involved in cutaneous wound healing. While it has been previously reported that both platelets and monocytes are a source of PDGF in human dermal wound repair, the production of PDGF by human keratinocytes has not yet been described. In this manuscript, we report the production of PDGF by cultured human keratinocytes. Both PDGF A and B chain mRNA can be detected in cultured cells. While only PDGF-AA polypeptide is found in significant levels in keratinocyte-conditioned culture media, all three PDGF isoforms (AA, AB, and BB) are present in detergent-solubilized cell extracts. No evidence of PDGF receptor expression was observed in cultured keratinocytes when analyzed for either mRNA levels or polypeptide expression, suggesting that PDGF does not play an autocrine role in keratinocyte growth. Analysis of cryosections of human cutaneous wounds by immunostaining for PDGF showed that both PDGF A and B chain is constitutively expressed in normal epidermis, as well as in newly reconstituted wound epidermis. No evidence for PDGF receptor polypeptide expression in the epidermis was detected by immunostaining of cryosections.

• Herren B, Weyer KA, Rouge M, Lotscher P, Pech M
• Conservation in sequence and affinity of human and rodent PDGF ligands and receptors.
• Biochim Biophys Acta. 1993; 1173: 294-302
• Display abstract

• Platelet-derived growth factor (PDGF) consists of two chains, PDGF-A and -B, which activate as homo- or heterodimers two receptors, alpha and beta. To test PDGF function in vivo we have generated neutralizing monoclonal antibodies. When analyzed with rat PDGFs only antibodies raised against human PDGF-AA showed cross-species activity. This correlated with complete amino acid sequence conservation of PDGF-A whereas rat PDGF-B differed in six positions when cloned rat PDGF cDNAs were compared with their human homologs within the receptor binding region. Extracellular domains of cloned rat PDGF alpha- and beta-receptor cDNAs did not reflect this difference in cross-species ligand conservation. When rat extracellular domains were expressed as soluble proteins they bound human PDGF-BB with high affinity after immobilization of the purified proteins on solid phase. Dissociation constants were identical to those of their human homologs. Thus, high affinity binding of human PDGF-BB to extracellular domains does not depend on species origin but only on receptor type.

• Takimoto Y, Kuramoto A
• Presence of a regulatory element within the first intron of the human platelet-derived growth factor-A chain gene.
• Jpn J Cancer Res. 1993; 84: 1268-72
• Display abstract

• We detected a suppressive element in the first intron of the human platelet-derived growth factor A chain (PDGF-A) gene. Two or more proteins, at least 110-kd and 90-kd proteins, were bound over a wide region of this fragment, and the fragment suppressed the expression of the PDGF-A chain via these proteins in vivo. Since the fragment also had suppressor activity on the promoter of the PDGF-B chain, it may be involved in a suppressive mechanism of gene expression common to PDGF-A and -B chains. Four tandem repeats of CCCCAT(CCCC) and three direct repeats of GGGGAG were observed in this region. The expression of the PDGF-A chain is considered to be regulated by a mechanism involving not only the 5' upstream region but also introns.

• Daniel TO, Kumjian DA
• Platelet-derived growth factor in renal development and disease.
• Semin Nephrol. 1993; 13: 87-95
• Display abstract

• In aggregate, the evidence reviewed here supports a very important role for PDGF expression and action at local glomerular and interstitial sites in human kidney development and disease. PDGF delivered by platelets, or produced by endogenous cells of the kidney is capable of stimulating responses including proliferation, matrix deposition, chemotaxis, and contraction in renal cells, particularly mesangial cells and interstitial fibroblasts. During kidney development, PDGF may mediate processes of cellular recruitment, extracellular matrix deposition, and proliferation with the constructive outcome of glomerulogenesis and vascularization. Proliferation and production of extracellular matrix components by these cells likely contribute to destructive proliferative and sclerotic responses attending proliferative and other glomerulopathies. As additional information accumulates, therapeutic targets within the PDGF system may provide opportunities to arrest destructive renal processes.

• Galland F et al.
• The FLT4 gene encodes a transmembrane tyrosine kinase related to the vascular endothelial growth factor receptor.
• Oncogene. 1993; 8: 1233-40
• Display abstract

• Three receptor tyrosine kinases, FLT1, FLK1 and FLT4, contain seven immunoglobin-like domains in their extracellular region and are strongly related by sequence similarities to each other and, to a lesser degree, to the class III receptors CSF1R/FMS, PDGFR, SLFR/KIT and FLT3/FLK2. They constitute a family of receptors putatively involved in the growth regulation of endothelial cells. We describe here the structure and pattern of expression of the human FLT4 gene. Two FLT4 transcripts of 5.8 and 4.5 kb are expressed in the human placenta and several hematopoietic cell lines. In mouse, a 5.8-kb transcript is expressed in a variety of tissues. A translational product 1298 amino acids in length is predicted to be encoded by the largest open reading frame. The FLT4 protein, when transiently expressed in Cos-7 cells and immunoprecipitated with a FLT4-specific rabbit immune serum, has an apparent molecular weight of 170 kDa.

• Calderon-Cacia M, Tekamp-Olson P, Allen J, George-Nascimento C
• Incomplete process of recombinant human platelet-derived growth factor produced in yeast and its effect on the biological activity.
• Biochem Biophys Res Commun. 1992; 187: 1193-9
• Display abstract

• Partially purified recombinant human Platelet-derived Growth Factor BB homodimer isolated from yeast culture media contains variable amounts of unprocessed PDGF-BB. This unprocessed PDGF-BB is found as a result of incomplete cleavage of the precursor to form the mature protein. Although the signal peptide is efficiently removed, a fraction of the PDGF secreted has an extended sequence corresponding to the truncated yeast alpha-factor leader. The data suggest that it is the amino acid chain from the truncated a-factor leader and not the sugar moiety attached to it that is responsible for the higher mitogenic activity found in this unprocessed molecule compared to highly purified PDGF-BB.

• Terman BI et al.
• Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.
• Biochem Biophys Res Commun. 1992; 187: 1579-86
• Display abstract

• Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.

• Safi A et al.
• Presence of elevated levels of platelet-derived growth factor (PDGF) in lung adenocarcinoma pleural effusions.
• Chest. 1992; 102: 204-7
• Display abstract

• Significant tumor stroma development is a specific feature of adenocarcinoma of the lung in comparison to small-cell lung cancer (SCLC). The fibrotic component of tumor stroma is thought to result from the migration and local replication of mesenchymal cells in response to the presence of cytokines. One of them, platelet-derived growth factor (PDGF), is a chemotactic and growth factor for mesenchymal cells. Since several lung adenocarcinoma cell lines, but not SCLC cell lines, have been shown in vitro to express PDGF genes, we evaluated pleural effusions for the presence of PDGF in patients with adenocarcinoma of the lung, SCLC, or nonmalignant pleural effusions. In adenocarcinoma of the lung, PDGF levels in pleural effusions were higher than in SCLC and in nonmalignant pleural effusions and were associated with the presence of a growth-promoting activity for fibroblasts due, in part, to the presence of PDGF. This observation suggests the role of PDGF in tumor stroma formation in adenocarcinoma of the lung.

• Johnson RJ et al.
• Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor.
• J Exp Med. 1992; 175: 1413-6
• Display abstract

• Platelet-derived growth factor (PDGF), a potent mitogen for mesenchymal cells in culture, is expressed in vivo in a variety of inflammatory conditions associated with cell proliferation, including atherosclerosis, wound repair, pulmonary fibrosis, and glomerulonephritis. However, it is not known if PDGF mediates the fibroproliferative responses that characterize these inflammatory disorders. We administered neutralizing anti-PDGF IgG or control IgG to rats with mesangial proliferative nephritis. Inhibition of PDGF resulted in a significant reduction in mesangial cell proliferation, and largely prevented the increased deposition of extracellular matrix associated with the disease. This suggests that PDGF may have a central role in proliferative glomerular disease.

• Allam M, Martinet N, Gallati H, Martinet Y
• Differential expression of PDGF A- and B-chain genes and production of AA and AB dimers by activated human blood monocytes.
• Biochimie. 1992; 74: 1097-101
• Display abstract

• Platelet-derived growth factor (PDGF) is composed of two chains (A and B) bound by disulfide bridges. Blood monocytes (BM) express the c-sis proto-oncogene, the gene coding for PDGF B-chain, and release PDGF. To evaluate PDGF release and A- and B-chain gene expression by BM, normal BM were cultured with LPS and specific transcripts for PDGF A- and B-chain genes were detected by Northern analysis and PDGF dimers by specific Elisas. Normal BM did not spontaneously express either A- or B-chain gene while, when activated, PDGF B-chain expression was precocious (maximum at 1 h) and decreased over 24 h; PDGF A-chain transcripts were present after 4 h and progressively increased over 24 h. Furthermore, activated BM released more AB dimers than AA dimers and almost no BB dimers. This observation confirms the concept of PDGF A- and B-chain separate gene regulation, and defines the specific molecular pattern of PDGF released by activated BM.

• Risau W et al.
• Platelet-derived growth factor is angiogenic in vivo.
• Growth Factors. 1992; 7: 261-6
• Display abstract

• PDGF receptors have recently been found to be expressed in microvascular endothelium in vivo under circumstances of endothelial cell activation and angiogenesis suggesting that PDGF may have a direct effect on endothelial cells. We have tested the angiogenic activity of PDGF-AA and -BB homodimers in the chick chorioallantoic membrane in vivo. PDGF-BB was found to consistently induce an angiogenic response whereas PDGF-AA was less active. Morphological analyses revealed that there was little inflammation associated with this response but an increase in vessel density suggested a direct effect of PDGF on embryonic chorioallantoic endothelial cells. In vitro, PDGF-BB was found to be more potent than PDGF-AA in stimulating the chemotaxis of rat brain capillary endothelial cells. This is consistent with a direct effect of PDGF on endothelial cells. Thus, this novel angiogenic activity of PDGF has implications for several developmental and pathological events in which PDGF, particularly the B-chain, is expressed.

• Brody AR et al.
• Interstitial pulmonary macrophages produce platelet-derived growth factor that stimulates rat lung fibroblast proliferation in vitro.
• J Leukoc Biol. 1992; 51: 640-8
• Display abstract

• Alveolar macrophages from humans and several animal species produce factors in vitro that modulate fibroblast growth and have been proposed as mediators of interstitial pulmonary fibrosis. Pulmonary interstitial macrophages (IMs) have not been studied previously in this regard. Pulmonary IMs were isolated from prelavaged rat lungs by enzymatic digestion of tissue and subsequent differential adherence of cells to culture dishes. The ability of IMs to release modulators of fibroblast growth into the culture medium was assessed by measuring [3H]thymidine incorporation into DNA and/or nuclear labeling of early-passage rat lung fibroblasts exposed to medium conditioned by IMs. The percentages of nuclei labeled in fibroblast cultures exposed to interstitial macrophage-conditioned medium (IMCM) alone did not significantly differ from that observed in controls, but fibroblasts exposed to IMCM supplemented with 2% platelet-poor plasma showed a 2.6-fold increase in labeling, indicating that IMCM contains predominantly "competence" growth factor activity. Similar results were obtained using purified human platelet-derived growth factor (PDGF). The level of growth factor activity released by IMs increased in cells that had phagocytized iron spheres during the culture period. In addition, fractionation of IMCM by high-performance liquid chromatography demonstrated most of the growth factor activity at a relative molecular mass of about 35 kd. Subsequent quantitative analysis of the fractions by an enzyme immunoassay for PDGF demonstrated that IMCM contains a homologue of human PDGF. These results show that IMs are capable of producing a PDGF-like growth factor for autologous fibroblasts and that release of this factor is enhanced by exposure to an insoluble inorganic particle. Because PDGF is a potent growth factor for fibroblasts and is released by IMs, it is essential to ask in future studies whether this or similar macrophage products play a significant role in mediating fibroblast proliferation in vivo.

• Ostman A, Thyberg J, Westermark B, Heldin CH
• PDGF-AA and PDGF-BB biosynthesis: proprotein processing in the Golgi complex and lysosomal degradation of PDGF-BB retained intracellularly.
• J Cell Biol. 1992; 118: 509-19
• Display abstract

• Platelet-derived growth factor is a potent mitogen for cells of mesenchymal origin. It is made up of two polypeptide chains (A and B) combined in three disulfide-linked dimeric forms (AA, AB, and BB). Here, the biosynthesis and proteolytic processing of the two homodimeric forms of PDGF (AA and BB) were studied in CHO cells stably transfected with A-chain (short splice version) or B-chain cDNA. PDGF-AA was processed to a 30-kD molecule which was secreted from the cells. In contrast, PDGF-BB formed two structurally distinct end products; a minor secreted 30-kD form and a major cell-associated 24-kD form. Immunocytochemical studies at light- and electron-microscopical levels revealed presence of PDGF in the Golgi complex, in lysosomes, and to a smaller extent in the ER. From analysis of cells treated with brefeldin A, an inhibitor of ER to Golgi transport, it was concluded that dimerization occurs in the ER, whereas the proteolytic processing of PDGF-AA and PDGF-BB precursors normally occurs in a compartment distal to the ER. Exposure of the cultures to the lysosomal inhibitor chloroquine led to an increased cellular accumulation of PDGF-BB, as determined both by metabolic labeling experiments and immunocytochemical methods, indicating that the retained form of PDGF-BB is normally degraded in lysosomes. Structural analysis of the two end products of PDGF-BB revealed that the secreted 30-kD form is a dimer of peptides processed as the B-chain of PDGF purified from human platelets, and that the retained 24-kD form is made up of subunits additionally processed in the NH2-terminus. Also, the 24-kD form was shown to be composed of proteolytic fragments held together by disulfide bridges. Taken together these findings suggest that the newly synthesized PDGF A- and B-chains are dimerized in the ER and thereafter transferred to the Golgi complex for proteolytic processing. From there, PDGF-AA is carried in vesicles to the cell surface for release extracellularly by exocytosis. A smaller part of PDGF-BB (the 30-kD form) is handled in a similar way, whereas the major part (the 24-kD form) is generated by additional proteolysis in the Golgi complex, from which it is slowly carried over to lysosomes for degradation.

• Caniggia I, Post M
• Differential effect of platelet-derived growth factor on glycosaminoglycan synthesis by fetal rat lung cells.
• Am J Physiol. 1992; 263: 495500-495500
• Display abstract

• Lung morphogenesis is in part regulated by extracellular matrix (ECM), and cytokines may indirectly control lung development via modulation of ECM. In the present study, we investigated the effect of different platelet-derived growth factor (PDGF) isoforms AA, AB, and BB on the synthesis of glycosaminoglycans (GAG) by fetal rat lung cells. Independent of gestational age, PDGF-BB, but not PDGF-AA or -AB, stimulated GAG synthesis of fetal lung fibroblasts. In contrast, GAG synthesis by epithelial cells was not affected by any of the PDGF molecules. The stimulatory effect of PDGF-BB on fibroblast GAG biosynthesis was dose (> 10 ng/ml) and time (> 8 h) dependent. The relative proportion of the individual GAG molecules was not altered by PDGF-BB exposure. Blockage of tyrosine kinase activity with staurosporine did abolish the effect of PDGF-BB on fibroblast GAG formation. Actinomycin D and cycloheximide did not abrogate the PDGF-BB effect, suggesting that no new RNA or protein synthesis is required. The proteoglycan synthesis blocker, beta-D-xyloside, also did not inhibit the PDGF-BB action on fibroblast GAG synthesis. These data suggest that the effect of PDGF on GAG synthesis is cell type and isoform specific and is most likely a direct effect on the GAG chain elongation enzymes.

• Nakamura T, Takeshita I, Inamura T, Fukui M
• The 56 kd platelet-derived growth factor (PDGF)-related protein is phosphorylated and the most stable form in human glioma cells.
• J Neurooncol. 1992; 13: 105-9
• Display abstract

• We report herein the presence of a 56 kd platelet derived growth factor (PDGF)-related protein as a phosphorylated form in human glioma cells. The phosphorylation of the 56 kd form was found to be the longest of all PDGF-related proteins. By Western blotting using a monoclonal anti-PDGF B-chain, the 80 kd, 56 kd, 40 kd, 28 kd and 17 kd PDGF-related proteins were detected, while after treatment among the nitrocellulose membrane transblotted cell extracts with alkaline phosphatase, 40 kd was the most densely observed while the 56 kd and 80 kd PDGF-related proteins were also detected. In a 32P flush labeling study, it was revealed that PDGF-related proteins incorporated with 32P were detected at 28, 32, 35, 40, 56 and 80 kd but the 17 kd monomer was not labeled. Among the labeled PDGF-related proteins, the 56 kd PDGF-related protein alone remained intracellularly for at least 16 hours. These results indicated that the PDGF-related proteins in human glioma cells are synthesized in a phosphorylated form and partly remain in a 56 kd phosphorylated form intracellularly. The 56 kd form may thus be the most stable form and likely has a substantial biological effect.

• Polet H
• Effects of platelet-derived growth factor on degradation, nuclear translocation of nonhistone proteins, and DNA synthesis.
• Proc Soc Exp Biol Med. 1992; 199: 417-23
• Display abstract

• Stimulation of resting normal rat kidney fibroblasts, prelabeled with [3H]leucine, by platelet-derived growth factor (PDGF) caused inhibition of cellular protein degradation and a parallel increased nuclear translocation of 3H-labeled nonhistone proteins (3H-NHP) and DNA synthesis. Nuclear translocation of these proteins was independent of protein synthesis. Fractionation of the nuclear 3H-NHP in a pH gradient of 2.5-6.5 showed that the protein fractions with a high degree of proteolysis in resting cells corresponded to the protein fractions with a high extent of translocation in stimulated cells, suggesting that degradation and translocation of these proteins may be related. PDGF inhibited cellular uptake of [3H]chloroquine, suggesting that PDGF inhibits NHP degradation via the lysosomal pathway. These observations support the hypothesis that PDGF induces NHP translocation to the nucleus by inhibiting lysosomal degradation of these proteins.

• Alexander DM, Hesson T, Mannarino A, Cable M, Dalie BL
• Isolation and purification of a biologically active human platelet-derived growth factor BB expressed in Escherichia coli.
• Protein Expr Purif. 1992; 3: 204-11
• Display abstract

• Human platelet-derived growth factor (PDGF) was expressed in Escherichia coli from a high-level cytoplasmic expression vector. A cDNA fragment encoding the mature form of the human PDGF B chain (hPDGF-B) was cloned into a plasmid under transcriptional control of the inducible E. coli Tac promoter. Expression of hPDGF-B from the final construct, pTacBIq, is regulated by the lactose repressor (LacIq). Upon induction, a polypeptide of approximately 14 kDa that had the same molecular mass and immunoreactivity as authentic hPDGF-B was produced. The production of recombinant hPDGF-B was significantly increased in an E. coli strain (CAG629) defective in expression of the lon protease. Expression of hPDGF-B in the CAG629 strain accounted for approximately 1% of total cell protein. In this system, hPDGF-B is expressed as an insoluble, intracellular protein and can readily be obtained in a partially purified form after differential centrifugation. Amino acid sequence determination of the purified protein has verified that the amino-terminal portion of the recombinant PDGF is correct. After renaturation into dimers, the purified recombinant hPDGF is fully functional in assays for receptor binding and mitogenesis.

• Daynes RA, Araneo BA
• Natural regulators of T-cell lymphokine production in vivo.
• J Immunother. 1992; 12: 174-9
• Display abstract

• The mammalian immune system possesses the intrinsic capacity to evoke a wide variety of functionally distinct effector mechanisms following stimulation by a particular antigenic substance. Such diversity in available responses is absolutely essential to the immunocompetent host, which must continually deal with a diverse set of potential pathogens within its ever-changing environment. The development of appropriate types of immune responses, therefore, represents a highly dynamic process that requires that an equivalent consideration be given to a large array of components, any one of which is capable of modulating the final outcome. While the nature and complexity of the antigen(s), plus the intracellular or extracellular mode of presentation, provide specificity and some selection to the developing process, the route of antigen entry, as well as the physiological status of the host at the time of antigen insult, also contribute significantly to the formation of any immune response. The overall objective of this article is to introduce the concept that platelet-derived growth factor (PDGF) (either preformed or synthesized in response to stimulation), plus a number of steroid hormones (some of which are end-organ metabolized at local tissue sites), can all play significant roles in the genesis of immunologic responses in vivo.

• Han RN, Mawdsley C, Souza P, Tanswell AK, Post M
• Platelet-derived growth factors and growth-related genes in rat lung. III. Immunolocalization during fetal development.
• Pediatr Res. 1992; 31: 323-9
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• To further study the role of platelet-derived growth factor (PDGF) in fetal lung development, the distribution of the PDGF homodimers PDGF-AA and PDGF-BB was examined by immunohistochemistry in embryonic and fetal rat lung from d 12 to 22 of gestation (term = 22 d). PDGF-AA and PDGF-BB were localized to airway epithelial cells as early as d 12 of gestation, 2 d before their appearance in mesenchymal cells. Both PDGF homodimer immunoreactivities increased until the late pseudoglandular stage of lung development, followed by fluctuations in reactivity during the canalicular stage. Only weak immunoreactivity to either PDGF homodimer was evident during the saccular stage of lung development. Immunodetection by Western blotting revealed that PDGF-AA and PDGF-BB homodimer protein concentrations were high during the embryonic and pseudoglandular stage of lung development and decreased with advancing gestation. We conclude that the presence of PDGF in both developing airway epithelial cells and mesenchymal cells, as well as gestation-dependent changes of PDGF homodimers, is compatible with a role for this growth factor during fetal lung development.

• Soma Y, Dvonch V, Grotendorst GR
• Platelet-derived growth factor AA homodimer is the predominant isoform in human platelets and acute human wound fluid.
• FASEB J. 1992; 6: 2996-3001
• Display abstract

• Platelet-derived growth factor (PDGF) is a dimeric protein composed of A- and B-chains (AA, AB, and BB). PDGF purified from human platelets has been shown to be composed primarily of the AB heterodimer. Immunoblots of total platelet extracts, cell-bound PDGF from the platelet extracts, and acute human wound fluid with PDGF A- and B-chain-specific antisera all demonstrate that the PDGF A-chain is the predominant peptide. Chemotactic and immunochemical assays of chromatographic fractions during PDGF isolation support these observations and demonstrate that PDGF AA can be separated from PDGF AB and BB by ion exchange chromatography. These studies indicate that the AA isoform constitutes the major PDGF dimer contained in human platelets and is the major form present at sites of injury during the acute phase of the wound repair response.

• Kaji K
• [Function, molecular structure and gene expression regulation of Platelet-derived growth factor]
• Nippon Rinsho. 1992; 50: 1902-9
• Display abstract

• Platelet-derived growth factor (PDGF) is a cationic glycoprotein of approximately 30 kDa, composed of two subunits. These subunit chains are termed A (18 kDa) and B (12-14 kDa) with high homology of the peptide sequences, including 8 cysteine residues at identical positions. Three isoforms of PDGF, AA, BB homodimers and AB heterodimer are distributed in the different tissues and cell lines suggesting that these isoforms have different functions. Two types of PDGF receptors alpha, and beta with Mr of 160-180 kDa are seen on the cell surface. PDGFR alpha can bind to both A and B subunits of the PDGD, while PDGFR beta, only B subunit. PDGF (AA) combines alpha alpha, PDGF (AB) makes dimers of alpha alpha and alpha beta, and PDGF (BB) can make three types of dimers, alpha alpha, alpha beta, and beta beta. These dimeric PDGFRs are active forms and phosphorylate its own domain and other neighbor specific proteins. The substrates of the receptor kinase are phospholipase C-gamma, GTPase activating protein (GAP), serine/threonine kinase Raf-1 and others. These molecules are thought to transfer information of the PDGFs on its receptors to the nucleus.

• Pollock RA, Richardson WD
• The alternative-splice isoforms of the PDGF A-chain differ in their ability to associate with the extracellular matrix and to bind heparin in vitro.
• Growth Factors. 1992; 7: 267-77
• Display abstract

• Platelet-derived growth factor (PDGF) consists of disulfide-linked homo- or heterodimers of A and B chains. mRNA encoding the A chain (PDGF-A) occurs in two versions that differ by the presence or absence of a single short exon. These alternatively-spliced mRNAs encode polypeptides that differ in length by fifteen amino acids. The longer isoform (PDGF-AL) possesses a highly basic carboxy-terminal extension that is responsible for retaining PDGF-AL homodomers at the cell surface after secretion, while homodimers of the shorter isoform (PDGF-AS) are released into the extracellular medium. We have investigated the mechanism by which PDGF-AL remains in association with the cells that produce it. We expressed epitope-tagged versions of PDGF-AL and PDGF-AS in Cos cells and compared their intra- and extracellular distributions by immunofluorescence microscopy. PDGF-AL, but not PDGF-AS, was detected on and around cells in a diffuse pattern suggesting associated with the extracellular matrix (ECM). Metabolically radiolabelled PDGF-AL, but not PDGF-AS, could be eluted from ECM preparations by washing in high salt. Moreover, PDGF-AL bound reversibly to heparin-Sepharose in vitro at physiological salt concentrations, eluting at a salt concentration around 0.5 M. PDGF-AS did not bind to heparin under the same conditions. Thus, PDGF dimers that contain PDGF-AL may remain immobilized near the cells that secrete them by virtue of binding to heparin-like constituents of the ECM.

• Okamura M et al.
• Platelet-derived growth factor gene expression in the kidney of malignant hypertension.
• Blood Press Suppl. 1992; 3: 17-21
• Display abstract

• To examine the pathogenetic role of platelet-derived growth factor (PDGF) in hypertensive kidney damage, we studied the gene expression of PDGF A-chain and B-chain in an animal model of malignant hypertension. Experimental malignant hypertension induced by unilateral nephrectomy combined with deoxycorticosterone and salt loading in the spontaneously hypertensive rat resulted in severely elevated blood pressure and renal histological damage, characterized by necrotizing vasculitis. Using reverse transcription-polymerase chain reaction analysis followed by Southern blot analysis, we observed that PDGF B-chain gene expression was increased in the kidney of experimental malignant hypertension and was correlated with the severity of glomerular damage, while PDGF A-chain gene expression was unaffected. Antihypertensive treatment with manidipine reduced glomerular damage and a decreased gene expression of PDGF B-chain. These results suggest that PDGF B-chain may have a role in mediating hypertensive kidney damage.

• Buwitt U, Koch C, Tatje D, Hoppe J, Gross G
• Platelet-derived growth factor isoforms AA, AB, and BB differentially activate poly r(I):r(C)-induced genes in human fibroblast FS4 cells.
• DNA Cell Biol. 1992; 11: 641-50
• Display abstract

• Polyribocytidylic-polyriboinosinic acid [poly r(I):r(C)]-inducible genes were isolated by a differential screening procedure from a human fibroblast cell (FS-4) cDNA bank. Among yet unidentified genes (gene 274), one codes for a protein with multiple finger motifs and has previously been detected in endothelial cells after tumor necrosis factor-alpha (TNF-alpha) treatment (A20; Opipari et al., 1990), the second one codes for a variant of the I kappa B family (Haskill et al., 1991), and a third one for the Ca2+ ATPase (isoform 1). Platelet-derived growth factor (PDGF) isoforms (AA, AB, and BB) stimulated the expression of these immediate-early genes. But the extent of the respective induction correlated neither with the number of the two receptors alpha or beta nor with the level of PDGF-stimulated receptor autophosphorylation on tyrosine. Although alpha-receptors were less abundant than beta-receptors (12,500 binding sites were estimated for PDGF-AA, KD 0.03 nM; 20,000 for PDGF-AB, KD 0.03 nM; 35,000 for PDGF-BB KD 0.16 nM) and tyrosine phosphorylation induced by PDGF-AA was significantly less than that evoked by PDGF-BB, some of the investigated genes were more strongly induced by PDGF-AA. We discuss how the differences in the biological potency of the PDGF isoforms may reside in different functions of the two receptors by activation of alternative signaling pathways.

• Natarajan R, Nadler J
• Platelet-derived growth factor is a potent inhibitor of angiotensin II-induced aldosterone synthesis.
• Mol Cell Endocrinol. 1992; 83: 57-63
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• Platelet-derived growth factor (PDGF) is a potent mitogen for several cell types. In addition, PDGF has vasoconstrictive action and shares some signal transduction mechanisms with angiotensin II (AII). In the present study, we have examined the effects of PDGF on basal and AII-induced aldosterone synthesis in freshly isolated rat adrenal glomerulosa cells. Recombinant human PDGF-BB caused a dose-dependent inhibition of AII-induced aldosterone synthesis being effective at concentrations as low as 10(-12) M. We also investigated possible mechanisms of action of PDGF. We have previously reported that the 12-lipoxygenase (LO) pathway of arachidonic acid plays a key role in AII-induced aldosterone synthesis. We thus examined whether PDGF action is mediated by changes in 12-LO activation. PDGF, at the same doses that blocked AII-induced synthesis also significantly inhibited AII-induced increases in the 12-LO product, 12-hydroxyeicosatetraenoic acid (12-HETE) formation. Further, the addition of 12-HETE completely restored the stimulatory effect of AII during inhibition by PDGF. These results suggest that PDGF could act, at least in part, by inhibition of AII-induced 12-HETE formation. We also examined the role of diacylglycerol (DG) formation since we have previously reported that DG is the source of arachidonic acid for 12-HETE formation. We observed that both AII and PDGF stimulated [3H]arachidonic acid-labeled DG formation. However, PDGF did not alter AII-induced DG formation suggesting that PDGF action is not mediated by affecting AII-induced increases in DG.(ABSTRACT TRUNCATED AT 250 WORDS)

• Abdennagy B, Hott M, Marie PJ
• Effects of platelet-derived growth factor on human and mouse osteoblastic cells isolated from the trabecular bone surface.
• Cell Biol Int Rep. 1992; 16: 235-47
• Display abstract

• We show here that purified platelet derived growth factor (PDGF) stimulates DNA synthesis in normal endosteal mouse and human osteoblastic cells isolated by selective migration from the trabecular bone surface. Maximum DNA synthesis as measured by (3H)-thymidine incorporation into DNA was increased at 50 ng/ml PDGF (48-72 hours). In both species, the effect of PDGF (25 ng/ml) was lower than the mitogenic effect of 10% FCS. We found that the mitogenic effect of PDGF on human trabecular cells decreased with the number of cell passages. DNA synthesis was increased about 4-fold by PDGF (25 ng/ml) in early passaged cells that expressed low basal growth rate and high osteocalcin production in basal conditions and in response to 1,25(OH)2 vitamin D, whereas DNA synthesis was increased 1.2 fold by PDGF in late passaged cells that showed high basal growth rate and low osteocalcin release in absence or presence of 1,25(OH)2D. PDGF alone had no effect on osteocalcin production. These results indicate that PDGF has mitogenic effect on normal mouse and human osteoblastic cells lining the trabecular bone surface and that the responsiveness to PDGF of human trabecular cells varies with the stage of differentiation.

• Antoniades HN
• Linking cellular injury to gene expression and human proliferative disorders: examples with the PDGF genes.
• Mol Carcinog. 1992; 6: 175-81

• Pahlman S, Johansson I, Westermark B, Nister M
• Platelet-derived growth factor potentiates phorbol ester-induced neuronal differentiation of human neuroblastoma cells.
• Cell Growth Differ. 1992; 3: 783-90
• Display abstract

• Previous studies have shown that platelet-derived growth factor (PDGF) and PDGF receptors are expressed in the mammalian central nervous system and that primary cultured neuroblasts from rat hindbrain have functional PDGF beta-receptors. Here, it is shown that cultured human neuroblastoma cells express PDGF alpha- and beta-receptors, but not PDGF-A and PDGF-B chain mRNA. In contrast to alpha-receptor expression, beta-receptor expression appears to be associated with a mature neuronal phenotype. Under serum-free growth conditions, PDGF-AA and -BB induce a trophic and weak mitogenic response in SH-SY5Y neuroblastoma cells, showing that the PDGF receptors in these cells are functional. In combination with 12-O-tetradecanoylphorbol-13-acetate, all three PDGF isoforms induce sympathetic neuronal differentiation of the SH-SY5Y cells, as shown by morphology and by increased expression of the genes coding for growth-associated protein 43 and neuropeptide tyrosine, respectively. This indicates a potential role for PDGF in the development of sympathetic neurons in particular and of the nervous system in general.

• Canalis E, Varghese S, McCarthy TL, Centrella M
• Role of platelet derived growth factor in bone cell function.
• Growth Regul. 1992; 2: 151-5
• Display abstract

• PDGF is a mitogen for cells of the osteoblastic lineage. PDGF is present in the systemic circulation and is locally synthesized by skeletal cells. The systemic form primarily contains PDGF B chains, which are intrinsically more active than PDGF A subunits, the forms secreted by normal bone cells. PDGF AA is regulated by other growth factors and cytokines, which modulate its binding to osteoblastic receptors and its synthesis by skeletal cells. The exact role of PDGF in bone remodelling is still uncertain and current information suggests that this factor has a function in the response to inflammation and wound healing.

• Dvonch VM, Murphey RJ, Matsuoka J, Grotendorst GR
• Changes in growth factor levels in human wound fluid.
• Surgery. 1992; 112: 18-23
• Display abstract

• BACKGROUND. It has been suggested that platelet-derived growth factor (PDGF) plays a central role in wound healing. Analysis of human wound fluid revealed the presence of PDGF AA (30 kd) and monocyte/macrophage-derived growth factor (MDGF) (12 to 14 kd) in the immediate postoperative period. METHODS. The amount of PDGF AA present was assayed by Western blot analysis. The chemotactic and mitogenic potential of purified wound fluid containing PDGF AA and MDGF was determined on a responsive cell line. The biologic activity of MDGF was assayed with a cell line that is unresponsive to the PDGF AA found in wound fluid. RESULTS. Both the concentration and the biologic activity were highest in the immediate postoperative period and declined to negligible levels by 24 hours after surgery. The chemotactic activity of MDGF was highest in the immediate postoperative period and declined during the first 24 hours in a manner similar to that of the combined PDGF AA and MDGF activity. CONCLUSIONS. These data demonstrate the changing levels of PDGF and MDGF in human wound fluid over time, supporting the cascade model of wound repair. By demonstrating that MDGF acts on cell lines unresponsive to the PDGF AA found in wound fluid, these data suggest that MDGF may also play an important role in wound healing.

• Ostman A, Andersson M, Betsholtz C, Westermark B, Heldin CH
• Identification of a cell retention signal in the B-chain of platelet-derived growth factor and in the long splice version of the A-chain.
• Cell Regul. 1991; 2: 503-12
• Display abstract

• The B-chain homodimer of platelet-derived growth factor (PDGF) is only very inefficiently secreted and remains largely associated with the producer cell; in contrast, the dimer of the short, and most common, splice variant of the A-chain is secreted. To identify the structural background to the differences in the secretory pattern between the different isoforms of PDGF, a set of chimeric PDGF A/B cDNAs was generated and expressed in COS cells. Analyses of the biosynthesis and processing of the corresponding products led to the identification of a determinant for cell association in the carboxy-terminal third of the PDGF B-chain precursor. Introduction of stop codons at various positions in the carboxy-terminal prosequence of the PDGF B-chain localized this determinant to an 11-amino-acid-long region (amino acids 219-229). This region contains an 8-amino-acid-long basic sequence that is homologous to a sequence present in an alternatively spliced longer version of the PDGF A-chain. In contrast to the short splice variant, the long splice A-chain version, like the B-chain, was found to remain predominantly cell associated. Thus, we have identified a conserved sequence that inhibits the secretion of some of the PDGF isoforms. Our data also suggest that switching of splicing patterns can be a mechanism to regulate the formation of secreted or cell-associated forms of PDGF-AA and possibly other growth factors.

• Miyazono K, Usuki K
• [Structure and function of platelet-derived endothelial cell growth factor]
• Seikagaku. 1991; 63: 290-3

• Circolo A, Welgus HG, Pierce GF, Kramer J, Strunk RC
• Differential regulation of the expression of proteinases/antiproteinases in fibroblasts. Effects of interleukin-1 and platelet-derived growth factor.
• J Biol Chem. 1991; 266: 12283-8
• Display abstract

• We have previously reported that polypeptide growth factors had an anti-inflammatory effect by decreasing the cytokine-enhanced expression of factor B (FB), an activator of the alternative complement pathway, in human fibroblasts. To further characterize the role of cytokines and growth factors in the inflammatory/repair continuum, we have studied the effects of interleukin-1 (IL-1) and platelet-derived growth factor (PDGF) on the expression of metalloproteinases/antiproteinases of the extracellular matrix in cultured human fibroblasts. Co-incubation of IL-1 and PDGF synergistically increased the expression of stromelysin and interstitial collagenase to 23-fold (for both proteins) over background, while PDGF decreased the IL-1-enhanced expression of FB by 82%. PDGF, but not IL-1, alone or in combination, increased the synthesis of tissue inhibitor of metalloproteinases. RNA blot analysis indicated that the changes in protein synthesis were regulated at a pretranslational level. Cycloheximide treatment indicated that the effects of PDGF on the metalloproteinases/antiproteinases were not protein-dependent, in contrast to results obtained for FB. The effect of the three dimeric forms of PDGF (AA, AB, and BB) on the synthesis of metalloproteinases and FB was also analyzed. The effects were qualitatively similar for each of the dimeric forms; however, the BB and AB isoforms had considerably greater effects than PDGF-AA. It has been reported that the PDGF receptors found in human fibroblasts have higher binding affinity for the BB and AB isoforms of the growth factor. The results presented in this paper are in accord with the possibility that differences in the biological activity of the three isoforms of PDGF are due to differences in the number or affinity of the binding sites of the target cells, rather than to different activation pathways of the receptor. Thus, PDGF increased cytokine effects on metalloproteinases, while decreasing cytokine effects or complement activator FB. The net effect of these changes may be to decrease inflammation and enhance remodeling early in repair and to enhance matrix stability later in the repair process.

• Zhang L, Leeman E, Carnes DC, Graves DT
• Human osteoblasts synthesize and respond to platelet-derived growth factor.
• Am J Physiol. 1991; 261: 34854-34854
• Display abstract

• Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin. We previously demonstrated that PDGF is produced by osteogenic sarcoma cells. We report here that normal human bone-derived cells produce PDGF and that these cells have an osteoblastic phenotype. This was demonstrated by use of a double immunofluorescent technique and by examining cloned human adult osteoblasts. Northern blot analysis indicates that PDGF production is accounted for by expression of the PDGF-A gene. PDGF-AA and PDGF-BB generally stimulated thymidine incorporation in normal human bone explants to a similar extent. All of the cloned human osteoblasts responded to PDGF-BB while the response to PDGF-AA varied. Similarly, five cloned osteoblastic cell populations were shown to produce PDGF while one did not. This result supports the hypothesis that there are different osteoblastic cell populations that differ in their growth factor responses or in the production of growth factors. Our results suggest that PDGF-BB has the potential to act as a paracrine factor for normal human osteoblasts because all of the osteoblastic cell populations responded to PDGF-BB. None of the osteoblastic cell populations expressed the PDGF-B gene, indicating that it would not act as an autocrine factor. Although not definitive, our results suggest that PDGF-AA has the potential to act as an autocrine factor because osteoblastic bone cell populations were shown to express the PDGF-A gene and respond to PDGF-AA.

• Tada M, Aida T, Hosokawa M, Kobayashi H, Sawamura Y, Abe H
• Antiproliferative effect of trapidil on PDGF-associated growth of human glioma cell lines in vitro.
• Neurol Med Chir (Tokyo). 1991; 31: 313-7
• Display abstract

• The effects of trapidil on platelet-derived growth factor (PDGF)-associated growth of glioblastoma cells were studied. The assessment using PDGF-dependent rat lung endothelium cells revealed secretion of a PDGF-like factor from SF-126 cell line but not from SF-188. Human recombinant PDGF stimulated proliferation of both these glioblastoma cell lines. The anti-PDGF monoclonal antibody inhibited the growth of SF-126 more than SF-188. The results suggest the presence of an autocrine growth mechanism in SF-126 cells mediated by PDGF. The growth of both SF-126 and SF-188 cells was suppressed by trapidil, a specific PDGF antagonist, at 10 and 50 micrograms/ml, respectively. The proliferative response to exogenous PDGF and the antagonistic effect of trapidil were greater in the SF-126 cell line. In addition, trapidil markedly reduced production of prostaglandin E2 in both glioblastoma cell lines. This anti-proliferative effect on malignant glioma cells suggests that trapidil might be a new therapeutic agent for malignant gliomas.

• Wolswijk G, Riddle PN, Noble M
• Platelet-derived growth factor is mitogenic for O-2Aadult progenitor cells.
• Glia. 1991; 4: 495-503
• Display abstract

• We report that platelet-derived growth factor (PDGF) is a potent mitogen for oligodendrocyte type-2 astrocyte (O-2A) progenitor cells derived from the optic nerves of adult rats. Moreover, O-2Aadult progenitors cultured in PDGF express the range of properties we have described previously for O-2Aadult progenitors cultured in the presence of type-1 astrocytes. Similarly, previous studies have demonstrated that PDGF is able to mimic the influence of type-1 astrocytes on O-2Aperinatal progenitors. Specifically, O-2Aadult progenitors and O-2Aperinatal progenitors exposed to PDGF express differences in average cell cycle time (59 +/- 5 h for O-2Aadult progenitors versus 20 +/- 6 h for O-2Aperinatal progenitors), average rate of migration (4.1 +/- 0.6 microns h-1 versus 24.6 +/- 5.4 microns h-1), morphology (unipolar versus bipolar), and antigenic phenotype (04+ vimentin- versus 04- vimentin+). Thus, our present results indicate that a single signalling molecule secreted by type-1 astrocytes produces markedly different cellular behaviours in two related O-2A progenitor populations.

• Pinzani M et al.
• Mitogenic signals for platelet-derived growth factor isoforms in liver fat-storing cells.
• Am J Physiol. 1991; 260: 48591-48591
• Display abstract

• Platelet-derived growth factor (PDGF), a key mitogen for liver fat-storing cells (FSC), is a dimeric molecule that occurs as homodimers or heterodimers of related polypeptide chains (PDGF-BB, -AB, and -AA). In chronic inflammation of the liver lobule, any of the three dimeric forms of PDGF derived from multiple sources could potentially interact with FSC. We explored the effects of the three different PDGF isoforms on DNA synthesis and early signal transduction pathways potentially related to PDGF mitogenicity in rat liver FSC. PDGF-BB homodimer and -AB heterodimer induced a marked increase in DNA synthesis, whereas the effect of PDGF-AA homodimer was considerably lower. Moreover, the mitogenicity of each isoform proportionally correlated with their effects on phosphoinositide turnover and intracellular Ca2+. Both the PDGF-BB and -AB dimers likely interact with the PDGF-beta-receptor, although PDGF-AB requires at least one alpha-receptor. The low responsiveness to PDGF-AA could not be accounted for by downregulation of the PDGF-alpha-receptor because FSC expressed very low levels of PDGF-A- and B-chain mRNAs and did not secrete detectable amounts of PDGF activity in the conditioned media. In addition, preincubation of FSC with suramin, a potent inhibitor of PDGF binding to its receptor, failed to increase PDGF-AA-induced DNA synthesis. These results are consistent with a predominant expression of PDGF-beta-receptor in liver FSC, that is linked to phospholipase C activation.

• Centrella M, McCarthy TL, Kusmik WF, Canalis E
• Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet-derived growth factor in osteoblast-enriched cultures from fetal rat bone.
• J Cell Physiol. 1991; 147: 420-6
• Display abstract

• Platelet-derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF-A or PDGF-B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF-BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systematically in vivo, has not been reported. Therefore recombinant human PDGF-BB, PDGF-AB, and PDGF-AA were evaluated in osteoblast-enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half-maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF-BB, PDGF-AB, and PDGF-AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF-BB being the most inhibitory. Binding studies with 125I-PDGF-BB or 125I-PDGF-AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I-PDGF-BB binding was preferentially displaced by PDGF-BB (Ki approximately 0.7 nM), less by PDGF-AB (Ki approximately 2.3 nM) and poorly by PDGF-AA. In contrast, 125I-PDGF-AA binding was measurably reduced by PDGF-AA (Ki approximately 4.0 nM), but was more effectively displaced by PDGF-BB or PDGF-AB (each with Ki approximately 0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF-B subunit binding preferentially mediate these results in osteoblast-enriched bone cell cultures.

• Deuel TF
• Structural and functional diversity of the platelet-derived growth factor.
• Curr Opin Biotechnol. 1991; 2: 802-6
• Display abstract

• The platelet derived growth factor is the most potent mitogen for cells of mesenchymal origin. Recent investigations have identified and characterized the structures and genes of the isoforms of PDGF, of the isoforms of its receptor, and of genes activated by it. It is now possible to understand in part the striking diversity of activities mediated by platelet derived growth factor and to identify new roles for it in normal and abnormal cell growth, in inflammation and repair, and in mammalian development.

• Bach MK, Brashler JR, Stout BK, Johnson HG, Sanders ME
• Platelet-derived growth factor can activate purified primate, phorbol myristate acetate-primed eosinophils.
• Int Arch Allergy Appl Immunol. 1991; 94: 167-8
• Display abstract

• We are interested in the physiologic mechanisms of eosinophil activation because of the presumed participation of activated eosinophils in the inflammatory sequelae of asthma. Suspecting that other formed elements of the blood may contribute to such an activation, we examined the capacity of platelet-derived growth factor (PDGF), a product of activated platelets, to activate eosinophils. We found that highly purified monkey and human eosinophils, but not guinea pig eosinophils, were activated by PDGF (superoxide anion production) in a dose-dependent fashion. Moreover, this activation was further dependent on a prior 'priming' of the cells by a brief exposure to subthreshold concentrations of phorbol ester. The response was specific for the BB homodimer of PDGF suggesting it is receptor-dependent.

• Pierce GF, Mustoe TA, Altrock BW, Deuel TF, Thomason A
• Role of platelet-derived growth factor in wound healing.
• J Cell Biochem. 1991; 45: 319-26
• Display abstract

• Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.

• Risto O, Wahlstrom O, Abdiu A, Walz T
• Effect of platelet derived growth factor on heterotopic bone formation in rats.
• Acta Orthop Scand. 1991; 62: 49-51
• Display abstract

• Platelet derived growth factor (PDGF) and induction of newly woven bone growth were studied in rats. PDGF (20 ng/mL) was administered continuously for 2 weeks via micro-osmotic pumps to 6-mm-long pieces of demineralized rat femur inserted into muscle pouches. Each rat had a control piece of demineralized bone inserted into the contralateral gluteal muscle. The samples were collected after 4 weeks, and wet and ash weight were recorded. Fourteen rats were evaluated. There were no differences as regards wet weights. PDGF increased the ash weights.

• Gesualdo L et al.
• Platelet-derived growth factor expression in mesangial proliferative glomerulonephritis.
• Lab Invest. 1991; 65: 160-7
• Display abstract

• Proliferation of mesangial cells and expansion of mesangial matrix are common histologic features of proliferative glomerular disease, a frequent cause of renal failure. Proliferation of glomerular mesangial cells occurs in response to platelet-derived growth factor (PDGF), and these cells release PDGF and express PDGF A and B chain mRNAs. However, all studies relating PDGF to potential changes in glomerular structure and function to date have been performed in vitro. To explore the role of PDGF in proliferative glomerulonephritides, we studied the expression of PDGF in vivo in two animal models of IgA nephropathy with different histologic patterns of glomerular injury: either predominant mesangial proliferation or expansion of mesangial matrix. Increased expression of PDGF and PDGF B-chain mRNA in whole kidneys from diseased mice was demonstrated by immunohistochemical techniques and by solution hybridization assay, respectively. Immunohistochemically, PDGF was localized primarily within the mesangial area of glomeruli and to a much lower extent in interstitium. The increased PDGF expression correlated with the degree of hypercellularity and clinical features of the disease. In addition, PDGF expression was increased in some forms of human glomerulonephritis, characterized by mesangial proliferation. These findings suggest that PDGF may be a major contributor to mesangial cell proliferation seen in proliferative glomerulonephritides.

• Leitzel K et al.
• Elevated plasma platelet-derived growth factor B-chain levels in cancer patients.
• Cancer Res. 1991; 51: 4149-54
• Display abstract

• Platelet-derived growth factor (PDGF) is produced by a variety of normal and tumor cells in vitro. We have developed an enzyme-linked immunosorbent assay for the detection of the B-chain of PDGF. This assay can reliably detect 0.1 ng/ml of homodimeric recombinant PDGF B-chain and does not cross-react with recombinant PDGF-AA, epidermal growth factor, basic fibroblast growth factor, or transforming growth factor-beta. Citrated plasma from 72 control individuals had a PDGF B-chain (PDGF-B) level of 0.32 +/- 0.14 ng/ml (mean +/- SD) with a range of 0.10-0.69 ng/ml. The plasma platelet factor 4 (PF4) level was 97 +/- 70 ng/ml, with a range of 34-363 ng/ml. Citrated plasma was obtained from 131 cancer patients, and plasma PDGF-B was elevated in 19 (15%) of the patients. Both PDGF-B and PF4 were elevated in 14 (11%) of these patients, consistent with a platelet source of PDGF-B. In 5 patients (4%), however, PDGF-B was elevated and PF4 was not elevated compared to the control group. This last group of patients may have a tumor-derived source of PDGF-B which could be important in autocrine or paracrine growth stimulation of the tumor cells.

• Bonner JC, Osornio-Vargas AR, Badgett A, Brody AR
• Differential proliferation of rat lung fibroblasts induced by the platelet-derived growth factor-AA, -AB, and -BB isoforms secreted by rat alveolar macrophages.
• Am J Respir Cell Mol Biol. 1991; 5: 539-47
• Display abstract

• Platelet-derived growth factor (PDGF)-like molecules secreted by alveolar macrophages have been postulated to be mediators of lung fibrogenesis since these cytokines stimulate the proliferation and chemotaxis of lung fibroblasts. We are studying the biology and biochemistry of rat macrophage-derived PDGF and have identified for the first time the specific isoforms of PDGF (-AA, -AB, and -BB) that these macrophages secreted in vitro following activation with either chrysotile asbestos or carbonyl iron spheres. Subsequently, the proliferative response of rat lung fibroblasts (RLF) to the different PDGF isoforms was established. Using several antibodies raised against the distinct isoforms, we established that two different PDGF-like factors with molecular masses of 30 to 34 kD and 16 to 18 kD were contained in alveolar macrophage-conditioned medium. Within each of these molecular mass regions was a mixture of all three PDGF isoforms. We estimated that the 30- to 34-kD PDGF was mainly PDGF-BB (approximately 50%), while the remaining consisted of PDGF-AA (approximately 13%) and PDGF-AB (approximately 37%). Purified recombinant PDGF isoforms were tested for their ability to stimulate the growth of early-passage RLF and Swiss 3T3 cells in a 3-day cell proliferation assay. PDGF-BB and PDGF-AB were the most potent inducers of RLF proliferation and stimulated growth half-maximally at approximately 1 ng/ml and approximately 7 ng/ml, respectively. While these two B-chain-containing dimers stimulated lung fibroblast growth by as much as 150% above control medium, the PDGF-AA homodimer stimulated lung fibroblast proliferation less than 25% above control medium at the highest concentrations tested (20 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

• Ambrus JL et al.
• Studies on tumor induced angiogenesis.
• J Med. 1991; 22: 355-69
• Display abstract

• Methods were developed to test the angiogenic response to human tumor implants and various biologic agents in the cornea of rabbits and non-human primates (Macaca arctoides). Human malignant melanoma tissue and crude platelet derived growth factor (PDGF) preparations had significant angiogenic effects. Purified, recombinant PDGF preparations were also effective initiators. Hemorheologic agents which also inhibit platelet aggregation [e.g. pentoxifylline (Px) (Trental) (also found to release PgI2 and tissue plasminogen activator (t-PA)] inhibited human tumor implant-induced angiogenesis. Sodium diethyldithiocarbamate (DDTC), a metal complexing agent with special affinity to copper and anti-thyroid as well as immune stimulating activity, was shown to be anti-angiogenic and to increase the effect of Px. The anti-fibrinolytic agents epsilon amino caproic acid (EACA) and tranexamic acid (t-AMCHA) were anti-angiogenic.

• Haraguchi T, Alexander DB, King DS, Edwards CP, Firestone GL
• Identification of the glucocorticoid suppressible mitogen from rat hepatoma cells as an angiogenic platelet-derived growth factor A-chain homodimer.
• J Biol Chem. 1991; 266: 18299-307
• Display abstract

• We have previously shown that BDS.1 rat hepatoma cells are hypersensitive to the antiproliferative effects of glucocorticoids, and secrete a glucocorticoid suppressible mitogenic activity (denoted GSM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that GSM purified to near homogeneity migrated as a 28-kDa protein under nonreducing conditions and as a single 15-kDa polypeptide in the presence of sulfhydryl reducing agents suggesting a homodimeric structure. Anti-platelet-derived growth factor (PDGF) A-chain specific antibodies selectively immunodepleted the mitogenic activity which can be extracted from nonreducing gels in the 26-30-kDa fraction and, in Western blots, recognized the 15-kDa reduced form of GSM. Western blot analysis further showed that dexamethasone suppressed the level of secreted PDGF A-chain protein in BDS.1 cells but not in glucocorticoid receptor-deficient hepatoma cells. Northern blots revealed that dexamethasone reduced expression of the PDGF A-chain 2.3- and 1.7-kilobase transcripts in proportion to the level of detectable PDGF-AA protein. Similarly to PDGF-AA, the hepatoma cell-derived GSM has a potent angiogenic activity. Taken together, our results demonstrate that the predominant glucocorticoid suppressible mitogen secreted from rat hepatoma cells is a PDGF A-chain homodimer and suggest that in vivo glucocorticoids may potentially regulate hepatoma growth by modulating PDGF-stimulated tumor vascularization.

• LaRochelle WJ, May-Siroff M, Robbins KC, Aaronson SA
• A novel mechanism regulating growth factor association with the cell surface: identification of a PDGF retention domain.
• Genes Dev. 1991; 5: 1191-9
• Display abstract

• Platelet-derived growth factor (PDGF) chimeras were used to map a domain responsible for either efficient secretion of PDGF-A or the tight cell association of PDGF-B to their carboxy-terminal domains. Introduction of stop codons within PDGF-A or PDGF-B further dissected their respective carboxy-terminal domains. Although successive deletions of the PDGF-A carboxyl terminus did not impair its secretion, incremental deletions from the carboxyl terminus of PDGF-B abrogated its membrane retention properties and promoted secretion. By this approach, PDGF-B retention properties could be localized to PDGF-B residues 212-226. A processed form of PDGF-B, which contained this domain, was expressed at the cell surface but not released. Comparison of PDGF-B with PDGF-A revealed an analogous sequence located at the PDGF-A carboxyl terminus. We demonstrated that this PDGF-A domain also acts as a retention sequence under conditions that inhibit its proteolytic cleavage. Thus, differences in PDGF-A and PDGF-B secretion relate to differential proteolytic processing of analogous retention domains. All of these findings establish a new mechanism for stable growth factor presentation at the cell surface.

• Nagaoka I, Trapnell BC, Crystal RG
• Upregulation of platelet-derived growth factor-A and -B gene expression in alveolar macrophages of individuals with idiopathic pulmonary fibrosis.
• J Clin Invest. 1990; 85: 2023-7
• Display abstract

• Idiopathic pulmonary fibrosis (IPF) is characterized by accumulation of alveolar macrophages spontaneously releasing exaggerated amounts of the potent mesenchymal cell growth factor platelet-derived growth factor (PDGF). To evaluate the relative contribution of the two PDGF genes to this process, PDGF-A and -B gene transcription rates and mRNA levels were examined in normal and IPF alveolar macrophages. While normal alveolar macrophages constitutively transcribe both PDGF-A and PDGF-B genes, LPS stimulation increases the transcription of both genes more than threefold. Importantly, IPF alveolar macrophages spontaneously transcribe both genes at a rate similar to that observed for normal macrophages after in vitro stimulation. Consistent with the transcription data, normal macrophages contain mRNA for both PDGF-A and -B, but PDGF-B mRNA is 10-fold more abundant. Strikingly, in IPF, both PDGF-A and -B mRNA levels were markedly increased, with persistence of the 10-fold dominance of PDGF-B mRNA. Thus, the exaggerated release of PDGF by IPF alveolar macrophages is likely modulated by upregulated PDGF gene transcription rates and concomitantly increased mRNA levels and the persistent 10-fold excess of B greater than A PDGF mRNA suggests that the PDGF released by alveolar macrophages is likely mostly of the potent B-chain homodimeric form.

• Liapi C, Raynaud F, Anderson WB, Evain-Brion D
• High chemotactic response to platelet-derived growth factor of a teratocarcinoma differentiated mesodermal cell line.
• In Vitro Cell Dev Biol. 1990; 26: 388-92
• Display abstract

• Evidence is presented that a differentiated mesodermal line (MES-1) from P19 EC cells express a high chemotactic response to platelet-derived growth factor (PDGF) as assayed in a blind-well modified Boyden chamber. Compared to the NIH 3T3 fibroblasts the chemotactic response of MES-1 is increased by 10-fold at 0.3 ng/ml of PDGF, 4-fold at 1.25 ng/ml of PDGF, 2-fold at 2.5 ng/ml of PDGF. In contrast, PDGF induces the same increase in [3H]thymidine incorporation in both cell lines, made quiescent under reduced serum concentration. This high chemotactic response to PDGF seems specific for these mesodermal cells. Among the different teratocarcinoma cells tested, including stem cells (F9, PC 13, PCC4) and endodermal derivatives (PYS, F9 with retinoic acid, PSA 5E), only the visceral endodermlike cells (PSA5E) are slightly attracted by PDGF. This chemotactic response to PDGF is not related to the presence or characteristics of the type B PDGF receptors, which are less numerous in MES-1 cells (10(5) receptors/cell, KDa 1,2 mM) compared to NIH 3T3 cells (64 X 10(4) receptors per cell, KDa 1,8 nM). The MES-1 cell line might be of interest for studying the chemotactic effect of PDGF. These results also suggest a role for this soluble factor in cell migration during early embryogenesis.

• Buck C, Montenarh M
• [Thrombocyte growth factor]
• Immun Infekt. 1990; 18: 132-5
• Display abstract

• PDGF is one of the principal mitogens for cultured cells of mesenchymal origin. Apart from aggregated platelets PDGF can be secreted by a number of cells. There is a striking homology between the amino-acid sequence of PDGF and that of a transforming protein of simian-sarcoma virus. PDGF is an important chemotactic protein for cells implicated in wound healing. Furthermore, recent publications suggest a possible role in the pathogenesis of several diseases. This article summarizes studies and recent findings on the structure, biology, and functional role of PDGF.

• Yablonka-Reuveni Z, Balestreri TM, Bowen-Pope DF
• Regulation of proliferation and differentiation of myoblasts derived from adult mouse skeletal muscle by specific isoforms of PDGF.
• J Cell Biol. 1990; 111: 1623-9
• Display abstract

• The expression of receptors and the mitogenic response to PDGF by C2 myoblasts, derived from adult mouse skeletal muscle, was investigated. Employing 125I-PDGF binding assays, we showed that the cells exhibit high level binding of PDGF-BB (approximately 165 x 10(3) molecules/cell at saturation) and much lower binding of the PDGF-AA and PDGF-AB (6-12 x 10(3) molecules/cell at saturation). This indicates that the C2 myoblasts express high levels of PDGF receptor beta-subunits and low levels of alpha-subunits. PDGF-BB enhances the proliferation of C2 cells maintained in 2% FCS by about fivefold. PDGF-AB had a moderate effect on cell proliferation (less than twofold) and PDGF-AA had no effect. Inverse effects of PDGF isoforms on the frequency of differentiated myoblasts were observed; the frequency of myosin-positive cells was reduced in the presence of PDGF-BB while PDGF-AA and PDGF-AB had no effect. PDGF may thus act to increase the number of myoblasts that participate in muscle regeneration following muscle trauma by stimulating the proliferation and by inhibiting the differentiation of myogenic cells.

• Knorr M, Steuhl KP, Dartsch P, Thiel HJ
• [Activation of phosphatidylinositol metabolism of corneal epithelial cells by platelet derived growth factor]
• Fortschr Ophthalmol. 1990; 87: 649-52
• Display abstract

• Numerous recent studies have found that the ubiquitous platelet-derived growth factor (PDGF) is central to the regulation of proliferative processes. PDGF's mitogenic effect in many cell types, for instance, is made possible by a receptor-mediated activation of phospholipase C, the key enzyme in the phosphatidylinositol (PI) turnover in membranes. Activation of the PI turnover then leads to an increase in the intracellular concentration of inositoltriphosphate (IP3), which functions as a second messenger and mobilizes cytosolic-free calcium from the endoplasmatic reticulum. The goal of the present study was to show that PDGF in corneal epithelial cells stimulates this transmembrane signal transmission system. Cell incubation was carried out on long-term cultures of rabbit corneal epithelial cells. IP3 was determined by ion-exchange chromatography, as described by Berridge et al. PDGF in nanomolar concentrations (5-50 ng/ml) was found to induce a dose-dependent rise in IP3, which peaked after 60 s. The maximum 300% increase in IP3 was accompanied by a pronounced rise in cytosolic-free calcium. These results show that PDGF can activate PI turnover in corneal epithelial cells.

• Ross R, Bowen-Pope DF, Raines EW
• Platelet-derived growth factor and its role in health and disease.
• Philos Trans R Soc Lond B Biol Sci. 1990; 327: 155-69
• Display abstract

• Platelet-derived growth factor (PDGF) was first discovered in platelets because they are the principal source of mitogenic activity in whole blood serum for mesenchymal cells in culture. PDGF is ubiquitous in that it can be formed by a large number of normal cells as well as many varieties of transformed cells. However, its expression and biological activity appear to be controlled at a number of different levels. The molecule consists of two peptide chains (termed 'A' and 'B') and is found as one of at least three possible isoforms, (AB, AA or BB). Each of these isoforms binds to a high-affinity cell-surface receptor that is composed of two different subunits, each of which has specificity for one or the other of the peptide chains of PDGF. The two receptor subunits are present in differing amounts on different cell types, and therefore the capacity of the different isoforms of PDGF to induce mitogenesis depends on the specific PDGF isoform and the relative numbers of receptor subunits present on the responding cell. In addition to inducing cell replication, PDGF elicits a number of intracellular signals related to mitogenesis, is chemotactic, is a vasoconstrictor, activates leukocytes, and modulates extracellular matrix turnover. This growth factor is probably involved in a number of biologically important events including wound repair, embryogenesis and development, and inflammation, leading to fibrosis, atherosclerosis and neoplasia.

• Mercola M, Deininger PL, Shamah SM, Porter J, Wang CY, Stiles CD
• Dominant-negative mutants of a platelet-derived growth factor gene.
• Genes Dev. 1990; 4: 2333-41
• Display abstract

• Using site-directed mutagenesis of a PDGF-A cDNA clone, we identify two domains that are required to generate stable, mitogenically active PDGF-AA homodimers. Alteration of the tetra-basic amino acid sequence (Arg84-Arg-Lys-Arg to Arg-Ser-Asn-Gly) results in the formation of stable pro-PDGF-A homodimers that lack mitogenic activity. Substitution of serine for Cys129 destabilizes PDGF-A subunits within the cell. Genes incorporating either the processing lesion or the cysteine substitution suppress wild-type PDGF-A gene expression in a trans-dominant fashion. Suppression occurs because the mutant PDGF subunits dimerize with wild-type subunits to form inactive or unstable heterodimers. Suppression is exerted across phylogenetic boundaries; thus, the mouse PDGF-A chain mutants inhibit the activity of the wild-type Xenopus PDGF-A. The cysteine mutant gene suppresses expression of PDGF-B (c-sis), as well as PDGF-A. The processing mutant gene, however, suppresses only PDGF-A. Dominant-negative mutations of PDGF and other growth factors which, like PDGF, function as dimers may prove useful for creating animals models of growth factor deficiency disease states and for revealing the function of growth factors during early embryonic development.

• Kuratsu J, Ushio Y
• Antiproliferative effect of trapidil, a platelet-derived growth factor antagonist, on a glioma cell line in vitro.
• J Neurosurg. 1990; 73: 436-40
• Display abstract

• Platelet-derived growth factor (PDGF) is produced by glioma cells. However, there is heterogeneity among glioma cell lines in the production of PDGF. It has been demonstrated that U251MG cells produce a PDGF-like molecule while U105MG cells do not. Trapidil, a specific antagonist of PDGF, competes for receptor binding with PDGF. Therefore, the inhibitory effect of trapidil on the proliferation of glioma cells was investigated in vitro using two glioma cell lines. At 100 micrograms/ml, trapidil significantly inhibited the proliferation of U251MG cells (which produce the PDGF-like molecule). At the same trapidil concentration, the proliferation of U105MG cells (which do not produce the PDGF-like molecule) was not inhibited. The inhibitory effect of trapidil was remarkable on Days 3 and 4 of culture. After 4 days of incubation, the proliferation of U251MG cells was 46% of the control preparation. Trapidil enhanced the antitumor effect of 3-[4-amino-2-methyl-5-pyrimidinyl)ethyl)-1-(2-chloroethyl)-1-nitrosourea (ACNU) against U251MG cells. The enhancing effect was highest on Days 4 and 6 of culture. After 6 days of incubation in the presence of 100 micrograms/ml trapidil and 1 microgram/ml ACNU, the proliferation of U251MG cells was 18% of the control preparation. These findings suggest that trapidil interrupts the autocrine loop at the PDGF and PDGF-receptor level and that combination therapy with trapidil and ACNU may be useful in the treatment of glioma.

• Eichner W, Jager V, Herbst D, Hauser H, Hoppe J
• Large-scale preparation of recombinant platelet-derived growth factor AA secreted from recombinant baby hamster kidney cells.
• Eur J Biochem. 1989; 185: 135-40
• Display abstract

• The short isoform of platelet-derived growth factor A (PDGF-A) was expressed in a mammalian host (BHK-21 cell). A cell line was obtained that secreted up to 0.3 micrograms/10(6) cells recombinant PDGF-A chain homodimer/day into the medium. For large-scale production of supernatant, cells were grown either in roller bottles or in 2.5-1 stirred tank fermenters. A simple two-step procedure was developed to purify recombinant PDGF-AA (rPDGF-AA). The first step was adsorption onto porous glass and the final step was reversed-phase high-performance liquid chromatography. The yield was 0.2 mg/l supernatant. A total amount of 20-30 mg pure rPDGF-AA may be obtained from a single fermenter run. Sequence analysis showed the correct amino terminus and no internal proteolytic cleavages. The specific activity was 5 ng/ml for mouse AKR-2B cells. [125I]rPDGF-AA had an affinity constant of approximately 0.5 nM to these cells and 25,000 binding sites were estimated/cell.

• Bryckaert MC, Rendu F, Tobelem G, Wasteson A
• Collagen-induced binding to human platelets of platelet-derived growth factor leading to inhibition of P43 and P20 phosphorylation.
• J Biol Chem. 1989; 264: 4336-41
• Display abstract

• Platelet-derived growth factor (PDGF) is known to inhibit collagen-induced platelet aggregation. Collagen-induced binding of 125I-PDGF to human washed platelets was therefore investigated. It was found 1) to be time-dependent, reaching a plateau at 20 degrees C after 30 min, 2) collagen concentration-dependent, 3) specifically inhibited by unlabeled PDGF, and 4) saturable. Scatchard plot analysis showed a single class of sites with 3000 +/- 450 molecules bound/cell and an apparent KD of 1.2 +/- 0.2 10(-8) M. The effects of PDGF on collagen-induced phosphoinositide breakdown and protein phosphorylation were also investigated. At 50 ng/ml PDGF, a concentration which completely inhibited collagen-induced aggregation, the breakdown of [32P]phosphatidylinositol 4,5-biphosphate (PIP2) and [32P]phosphatidylinositol 4-phosphate (PIP) was observed, but the subsequent replenishment of [32P]PIP2 was inhibited. The same PDGF concentration totally inhibited collagen-induced phosphatidic acid formation. PDGF also completely prevented phosphorylation of P43 and P20, as a result of protein kinase C activation consecutive to phosphoinositide metabolism. These results suggest that (i) a specific PDGF receptor can be induced by collagen, and (ii) PDGF can effect the early events of collagen-induced platelet activation by inhibiting PIP2 resynthesis and P43 and P20 phosphorylation. It is concluded that PDGF might be involved in a negative feed-back control of platelet activation.

• Kaplow JM, Tong BD, Hurwitz DR, Jaye M
• Expression of mitogenically active human recombinant platelet-derived growth factor A-chain.
• Mol Biol Med. 1989; 6: 209-17
• Display abstract

• Expression vectors encoding cDNAs for the human platelet-derived growth factor (PDGF) A-chain (pJKl) and murine dihydrofolate reductase (pTKDHFR) were cotransfected into dihydrofolate reductase-deficient Chinese hamster ovary cells. Methotrexate-induced coamplification of clones, expressing PDGF A-chain resulted in enhanced levels of A-chain-specific DNA, RNA and protein. A 30,500 Mr protein was immunoprecipitated with PDGF antisera from the conditioned media of metabolically labeled cells. Reducing conditions resolved the A-chain-specific protein into two polypeptides of 16,500 and 17,000 Mr, confirming the homodimeric nature of the recombinant A-chain protein. The recombinant PDGF A-chain produced constitutively by these amplified clones proved to be mitogenically active. The secretion of a recombinant PDGF A-chain into conditioned media may provide a continuous and abundant source of PDGF A.A dimers, normally produced by specific tissues in only minute quantities. Future purification of the recombinant homodimeric A-chain will allow the assessment of its ability to function in clinical applications such as wound healing.

• Fine RM
• Platelet-derived growth factor and vascular proliferative lesions of the skin.
• Int J Dermatol. 1989; 28: 170-1

• Hosang M, Rouge M, Wipf B, Eggimann B, Kaufmann F, Hunziker W
• Both homodimeric isoforms of PDGF (AA and BB) have mitogenic and chemotactic activity and stimulate phosphoinositol turnover.
• J Cell Physiol. 1989; 140: 558-64
• Display abstract

• Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB, which were previously shown to bind to two receptors with different isoform-specificity, the A/B-type (binds all three isoforms) and the B-type (binds only PDGF-BB). Results from competition binding experiments with Swiss 3T3 cells suggest the existence of a third receptor type, which recognizes PDGF-AB and PDGF-BB. Furthermore, Swiss 3T3 cells and human dermal fibroblasts express different relative and absolute levels of these receptor types. In particular, Swiss 3T3 cells express 90,000 PDGF-AA binding sites (A/B-receptors) per cell, whereas human fibroblasts express only 20,000 A/B-receptors per cell. All three PDGF isoforms were tested in either cell type for their effect on DNA synthesis. PDGF-BB and PDGF-AA were also tested in Swiss 3T3 cells for their effect on inositol phospholipid metabolism and chemotaxis. Each isoform promoted all three processes dose-dependently, but there were differences in the maximum cellular responses elicited. These responses reflect the capacity of the cells to bind the individual isoforms. These results demonstrate that the previous distinctions in responsiveness to the different PDGF isoforms are primarily a consequence of the differences in the levels of surface expression of the various isoform-specific receptor types, rather than of the differences in the intrinsic activity of these isoforms. Furthermore, these results suggest that all types of PDGF receptors are capable of responding to their respective ligands by mediating phosphoinositide breakdown, chemotactic responses, and DNA synthesis. Whether they exhibit other functional differences remains to be seen.

• Garcia-Aguilar J, Brown GE, Lanser ME
• Coagulation increases neutrophil CR1 and CR3 expression: primary role for platelet-derived growth factor.
• J Lab Clin Med. 1989; 114: 312-20
• Display abstract

• Neutrophil receptors for C3b(CR1) and C3bi(CR3) mediate a number of functions important for infection control and tissue repair, such as adherence, aggregation, orientation in chemotactic gradients, and phagocytosis of opsonized particles. We studied the effect of the coagulation of whole blood on the induction of neutrophil complement receptor (CR) expression in vitro. Neutrophils incubated in serum for 1 hour at 37 degrees C increased the expression of CR1 3.43-fold and CR3 3.06-fold compared with incubation in buffer (p less than 0.001). In contrast, incubation in plasma did not induce such an increase. The serum factor responsible for this CR-inducing effect appeared to be a platelet constituent, because (1) serum derived from platelet-rich plasma, but not platelet-poor plasma, contained the CR-inducing factor; (2) pretreatment with aspirin inhibited the adenosine diphosphate-induced expression of this factor in platelet-rich plasma; (3) the CR-inducing factor was also contained in supernatants derived from frozen/thawed platelets; (4) pure platelet-derived growth factor (PDGF) induced CR expression to the same extent as did whole serum; and (5) the CR-inducing activity of serum and platelet supernatants was inhibited by incubation with antibody against PDGF but not by antibody against C5. Thus, a platelet component that is probably PDGF appears to be the major CR-inducing factor generated during in vitro coagulation and may play a vital role in mediating the neutrophil response to tissue injury and inflammation.

• Bonner JC, Hoffman M, Brody AR
• Alpha-macroglobulin secreted by alveolar macrophages serves as a binding protein for a macrophage-derived homologue of platelet-derived growth factor.
• Am J Respir Cell Mol Biol. 1989; 1: 171-9
• Display abstract

• Evidence is presented to support our hypothesis that an alpha-macroglobulin (alpha M) produced by lung macrophages serves as a specific binding protein for platelet-derived growth factor (PDGF) from these same macrophages. Culture medium "conditioned" by alveolar macrophages was fractionated by gel filtration according to molecular weight. Proteins larger than 200 kD were bound to greater than 50% of the macrophage-derived PDGF (MD-PDGF) that was extractable by 1 M acetic acid. Another approximately 25% was bound to fractions at approximately 150 kD, and approximately 20% remained unbound. The two high molecular weight fractions inhibited approximately 40% of specific [125I]PDGF binding to rat lung fibroblasts, whereas other fractions did not block PDGF binding to its receptor. Only the greater than 200 kD fractions inhibited the binding of PDGF antisera to purified human PDGF by 20% of control and exhibited specific complex formation and coelution on a gel filtration column with [125I]PDGF. The macrophage-derived alpha M (MD-alpha M) was separated from other macrophage-derived proteins by nickel-affinity chromatography and exhibited clear characteristics of alpha Ms, i.e., cross-reactivity with antibodies to human alpha 2-macroglobulin (alpha 2M) on immunoblots as well as gel migration corresponding to the electrophoretic mobility of the protease-bound "fast" and protease-unbound "slow" forms of human alpha 2M. Nickel-bound protein identified as an alpha M was bound to greater than 50% of the acid-extractable MD-PDGF in macrophage-conditioned medium, supporting the view that the greater than 200 kD protein separated by gel filtration is an alpha M.(ABSTRACT TRUNCATED AT 250 WORDS)

• Keck PJ et al.
• Vascular permeability factor, an endothelial cell mitogen related to PDGF.
• Science. 1989; 246: 1309-12
• Display abstract

• Vascular permeability factor (VPF) is a 40-kilodalton disulfide-linked dimeric glycoprotein that is active in increasing blood vessel permeability, endothelial cell growth, and angiogenesis. These properties suggest that the expression of VPF by tumor cells could contribute to the increased neovascularization and vessel permeability that are associated with tumor vasculature. The cDNA sequence of VPF from human U937 cells was shown to code for a 189-amino acid polypeptide that is similar in structure to the B chain of platelet-derived growth factor (PDGF-B) and other PDGF-B-related proteins. The overall identity with PDGF-B is 18%. However, all eight of the cysteines in PDGF-B were found to be conserved in human VPF, an indication that the folding of the two proteins is probably similar. Clusters of basic amino acids in the COOH-terminal halves of human VPF and PDGF-B are also prevalent. Thus, VPF appears to be related to the PDGF/v-sis family of proteins.

• Graves DT, Valentin-Opran A, Delgado R, Valente AJ, Mundy G, Piche J
• The potential role of platelet-derived growth factor as an autocrine or paracrine factor for human bone cells.
• Connect Tissue Res. 1989; 23: 209-18
• Display abstract

• Platelet-derived growth factor, PDGF, is a potent mitogen for cells of mesenchymal origin such as fibroblasts, smooth muscle cells and glial cells. PDGF is thought to have the potential to act as both a paracrine and an autocrine factor. Studies described here extend these observations to human bone-derived cells. Exogenous PDGF induces biologic activity in two human osteogenic sarcoma cell lines and in one of these, the two PDGF genes, PDGF-1 and PDGF-2/c-sis are expressed. In addition, PDGF stimulates proliferation of normal osteoblastic cells derived from adult human cancellous bone. The expression of the PDGF-1 gene but not the PDGF-2/c-sis gene is demonstrated in normal human adult bone-derived cells by Northern blot analysis and synthesis of PDGF is shown by immunoprecipitation with PDGF antisera. These studies indicate that PDGF has the potential to act as a paracrine or autocrine regulator of bone cells.

• Ostman A et al.
• Expression of three recombinant homodimeric isoforms of PDGF in Saccharomyces cerevisiae: evidence for difference in receptor binding and functional activities.
• Growth Factors. 1989; 1: 271-81
• Display abstract

• Three recombinant homodimeric isoforms of platelet-derived growth factor (PDGF) were produced and purified in milligram quantities by expression of PDGF A- and B-chains in yeast cells. Structural analysis of the purified short and long variants of PDGF-AA (PDGF-AAS and PDGF-AAL) and PDGF-BB showed that they had been properly processed and assembled into dimers. PDGF-AAS and PDGF-AAL were found to bind only to the PDGF A-type receptor on human fibroblasts, with affinities of 0.1 and 0.2 nM, respectively. PDGF-BB bound to cells with A- and B-type receptors and to cells with B-type receptor only with affinities of 0.6 nM in both cases. Each fibroblast appeared to express about 4-5 times more B-type receptors than A-type receptors. The maximal mitogenic response to PDGF-BB of human fibroblasts was almost 2-fold higher than that induced by either of the two PDGF-AA forms. The three isoforms of PDGF also stimulated growth in soft agar of human fibroblasts with PDGF-BB inducing a higher maximal response.

• Hoppe J, Weich HA, Eichner W
• Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli.
• Biochemistry. 1989; 28: 2956-60
• Display abstract

• Preparations of the mitogen platelet-derived growth factor (PDGF) from human platelets contain two related polypeptides termed A chain and B chain. PDGF-B is highly homologous to a portion of p28v-sis, the transforming protein of simian sarcoma virus. We have studied the mitogenic potential of a PDGF-BB-like homodimer by expressing the sequence coding for the mature part of PDGF-B in Escherichia coli. Expression was achieved as cro-beta-gal-PDGF-B fusion protein which was exclusively found in the "inclusion bodies". A monomeric PDGF-B fragment shortened by 12 amino acid residues from the NH2 terminus was excised from the fusion protein by CNBr cleavage. After protection of thiols by S-sulfonation, this fragment was purified by gel permeation chromatography and reversed-phase high-performance liquid chromatography. This monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active rPDGF-BB with an overall yield of approximately 0.7 mg of rPDGF-BB/L of culture. Escherichia coli rPDGF-BB stimulated [3H]thymidine incorporation into AKR2B fibroblast at concentrations of about 1 ng/mL.

• Tamura M, Iwamoto Y
• The effect of platelet-derived growth factor on phagocytosis of cultured human trabecular cells.
• Exp Eye Res. 1989; 48: 761-70
• Display abstract

• Platelet-derived growth factor (PDGF) enhanced the phagocytic activity of cultured human trabecular cells in a dose-dependent manner (5-40 ng ml-1). By transmission and scanning electron microscopy, microvilli on the cell surface were shown to make contact with latex particles. Subsequently the cell surface invaginated and engulfed them. By indirect immunofluorescence, actin was found to form stress fibers in PDGF-free cells. PDGF-treated cells were stained in thread-like patterns on the cell surface. In addition to this image, membrane ruffles and actin ribbons were also observed. These findings suggest that PDGF may serve to facilitate phagocytosis and migration of trabecular cells by modifying trabecular cell actin.

• Hammacher A, Mellstrom K, Heldin CH, Westermark B
• Isoform-specific induction of actin reorganization by platelet-derived growth factor suggests that the functionally active receptor is a dimer.
• EMBO J. 1989; 8: 2489-95
• Display abstract

• Human platelet-derived growth factor (PDGF) occurs as three isoforms which are made up of disulfide-bonded A and B chains. The isoforms bind with different affinities to two different but structurally related cell surface receptors. The A type receptor binds all three isoforms (PDGF-AA, PDGF-AB, PDGF-BB) with high affinity, whereas the B type receptor binds PDGF-BB with high affinity, PDGF-AB with lower affinity but does not appear to bind PDGF-AA. We have utilized the differential effects of the three isoforms on actin reorganization and membrane ruffling in human foreskin fibroblasts to probe the idea that ligand-induced receptor dimerization is associated with receptor activation. Actin reorganization was found to be induced only by PDGF-AB and PDGF-BB and is therefore likely to be mediated by the B type receptor. Simultaneous addition of PDGF-AA, or downregulation of the A type receptor blocked the effect of PDGF-AB but not that of PDGF-BB. This is compatible with a model by which PDGF-AB binds to and dimerizes one A and one B type receptor; PDGF-AB therefore requires A type receptors in order to be functionally active at physiological concentrations. In cells with down-regulated A type receptors, high concentrations of PDGF-AB inhibited the effect of PDGF-BB on actin reorganization. We believe that this is due to a monovalent binding of PDGF-AB to the B type receptors which prevents PDGF-BB from dimerizing the receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

• Guillausseau PJ et al.
• Platelet-derived growth factor (PDGF) in type 1 diabetes mellitus.
• Eur J Clin Invest. 1989; 19: 172-5
• Display abstract

• In 10 patients with type 1 diabetes mellitus, platelet-derived growth factor (PDGF) was assessed using an in vitro assay based on the stimulation of DNA synthesis in the 3T3 mouse fibroblast cell line. beta-Thromboglobulin (beta-TG) was used as an index for platelet alpha-granules content. Platelet beta-TG content and PDGF were markedly decreased in diabetic patients while plasma beta-TG was increased as compared with control subjects. In diabetic patients, a significant negative correlation was found between plasma beta-TG level and beta-TG total platelet content, associated with a significant positive correlation between platelet beta-TG content and PDGF. These results suggest that PDGF release might be increased in diabetic subjects. This may account in part for the cell proliferation observed in diabetic angiopathy.

• Westermark B, Heldin CH
• Platelet-derived growth factor: structural and functional aspects.
• J Intern Med. 1989; 225: 55-8

• Whelan HT, Nelson DB, Strother D, Przybylski C, Figge G, Mamandi A
• Medulloblastoma cell line secretes platelet-derived growth factor.
• Pediatr Neurol. 1989; 5: 347-52
• Display abstract

• Medulloblastoma is the most common malignant childhood brain tumor in which aggressive growth produces recurrence in approximately 50% of appropriately treated cases and metastases along the neuraxis in 30%. To date, no studies exist concerning the production of autocrine growth factors by this brain tumor type. Malignant brain tumors in adults often produce platelet-derived growth factor (PDGF). A medulloblastoma cell line, TE-671, has been used for many years in pediatric neuro-oncologic studies. We assayed this medulloblastoma cell line for the production of PDGF. PDGF is produced by medulloblastoma cells grown in monolayer tissue culture and stimulates PDGF-sensitive 3T3 fibroblasts to incorporate tritiated thymidine in a dose-dependent fashion. This biologic activity is blocked by PDGF antibodies in a dose-dependent relationship. We postulate that PDGF produced by medulloblastoma cells plays a role in the growth of this tumor by stimulating mitogenic activity.

• Pech M, Gazit A, Arnstein P, Aaronson SA
• Generation of fibrosarcomas in vivo by a retrovirus that expresses the normal B chain of platelet-derived growth factor and mimics the alternative splice pattern of the v-sis oncogene.
• Proc Natl Acad Sci U S A. 1989; 86: 2693-7
• Display abstract

• A retrovirus containing the entire human platelet-derived growth factor B-chain (PDGF-B) gene was constructed in order to investigate the in vivo biological activity of its encoded growth factor. When this virus was introduced into newborn mice, it reproducibly generated fibrosarcomas at the site of inoculation. Proviruses in each fibrosarcoma analyzed had lost 149 nucleotides downstream of the PDGF-B coding region. This deletion originated from an alternative or aberrant splice event that occurred within exon 7 of the PDGF-B gene and mimicked the v-sis oncogene. Thus, deletion of this region may be necessary for efficient retrovirus replication or for more potent transforming function. Evidence that the normal growth factor coding sequence was unaltered derived from RNase protection studies and immunoprecipitation analysis. Tumors were generally polyclonal but demonstrated clonal subpopulations. Moreover, tumor-derived cell lines became monoclonal within a few tissue culture passages and rapidly formed tumors in vivo. These findings argue that overexpression of the normal human PDGF-B gene product under retrovirus control can induce the fully malignant phenotype.

• Matsuoka J, Grotendorst GR
• Two peptides related to platelet-derived growth factor are present in human wound fluid.
• Proc Natl Acad Sci U S A. 1989; 86: 4416-20
• Display abstract

• Platelet-derived growth factor (PDGF) has been suggested to play an important role in wound healing. In this study wound fluid collected from patients after radical mastectomy was analyzed for PDGF-related factors. Analysis revealed the presence of two PDGF-related peptides of 16-17 kDa and 34-36 kDa. These PDGF-related peptides were purified by using anti-PDGF immunoaffinity techniques and found to possess both chemotactic and mitogenic activities for NIH 3T3 cells in vitro. Surprisingly, we could not detect authentic native PDGF (30 kDa) or PDGF A or B chain in any of the wound-fluid samples examined. These results indicate that PDGF-related peptides are present in normal human wound fluid, whereas PDGF A- and B-chain peptides are absent or only present in trace amounts relative to the total PDGF-related biological activity.

• Sturani E, Brambilla R, Morello L, Cattaneo MG, Zippel R, Alberghina L
• Effect of the different dimeric forms of the platelet-derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts.
• FEBS Lett. 1989; 255: 191-5
• Display abstract

• PDGF consists of two polypeptide chains, A and B, and all three possible dimers have been isolated from different sources. Human PDGF, essentially AB, porcine PDGF (BB) and recombinant PDGF-AA were tested on Swiss 3T3 fibroblasts for their ability to stimulate mitogenesis, phosphoinositide turnover and tyrosine phosphorylation of the PDGF receptor. When used in saturating amounts, the three isoforms were equally active in inducing mitogenesis. However, PDGF-AA was less active than AB and BB to induce the phosphorylation of the receptor and the turnover of phosphoinositides (30% and 50%, respectively). These findings suggest that, in Swiss 3T3 fibroblasts, PDGF receptors of the alpha-type are present in a slightly lower amount than beta-type. In addition, the two types of receptor appear to have similar physiological functions.

• LaRochelle WJ, Robbins KC, Aaronson SA
• Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.
• Mol Cell Biol. 1989; 9: 3538-42
• Display abstract

• A monoclonal antibody (mAb), sis 1, generated against human c-sis-encoded platelet-derived growth factor (PDGF) BB, was shown by enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis to recognize human PDGF BB and human platelet PDGF AB but not the human PDGF AA. This monoclonal antibody potently inhibited PDGF receptor-binding and mitogenic activities of both human PDGF BB and PDGF AB but had no effect on PDGF AA. Finally, we demonstrated that an immunoaffinity-purified anti-c-sis peptide antibody (anti-V4) which also blocked binding of PDGF BB to its cognate receptor and competed with mAb sis 1 for binding to PDGF BB. All of these results suggest that mAb sis 1 recognizes an epitope of the c-sis gene product, PDGF BB, that spatially overlaps the V4 surface domain of PDGF BB, immunochemically localizing a region of PDGF BB critical for PDGF receptor binding and activation.

• Ostman A et al.
• Synthesis and assembly of a functionally active recombinant platelet-derived growth factor AB heterodimer.
• J Biol Chem. 1988; 263: 16202-8
• Display abstract

• A Chinese hamster ovary cell line that stably expresses transfected human platelet-derived growth factor (PDGF) A and B chain precursors was established. All three dimeric combinations of PDGF chains were produced by this cell line; their biosynthesis, assembly, and processing were followed by pulse-chase analyses. PDGF-AA, PDGF-AB, and PDGF-BB were processed to Mr values of about 30,000 and were accumulated in these forms in the medium. In addition, PDGF-BB was further processed to a 24-kDa component, which remained cell-associated. The major secreted component was PDGF-AB, which was purified and shown to have structural and functional characteristics indistinguishable from PDGF-AB purified from human platelets.

• Armelin MC, Joazeiro CA, Rocha KM, Armelin HA
• Generation of myc/fos transfectant Balb-3T3 cell lines.
• Arch Biol Med Exp (Santiago). 1988; 21: 429-33
• Display abstract

• Early and transient expression of proto-oncogenes c-fos and c-myc is involved in the mitogenic response to PDGF (platelet-derived growth factor). We used DNA-mediated transfection to approach the role played by these genes in cell growth control by PDGF and in growth deregulation (neoplasia). Cloned pFBJ-2 (v-fos) and glucocorticoid-inducible mouse c-myc were co-transfected with a neo genetic marker to allow a neutral selection on the basis of resistance to the neomycin derivative geneticin G418. pFBJ-2 transfection was found to interfere with the number of G418-resistant (G418r) colonies. By using a v-fos-deleted pFBJ-2 construct, the deleterious effect was attributed to v-fos coding sequences. Cellular fos gene disruption, by homologous recombination with exogenous v-fos, is proposed as the basis for the deleterious effect. Co-transfection with MMTV-H3-c-myc effectively counteracts the negative effects of v-fos. Different from the parental line or single myc or fos transfectants, double myc/fos transfectants are morphologically transformed. Double transfectants still retain the PDGF requirement for growth in monolayer cultures.

• Hammacher A et al.
• A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain.
• J Biol Chem. 1988; 263: 16493-8
• Display abstract

• Platelet-derived growth factor, PDGF, purified from human platelets is a disulfide-bonded dimer consisting of two homologous polypeptide chains denoted A and B; it has not been known whether it is a heterodimer or a mixture of homodimers. We present here evidence that a major part of PDGF has a heterodimer structure. A highly homogeneous, 31-kDa PDGF was purified in the presence of protease inhibitors and shown to contain both chains by means of immunoprecipitations with peptide antisera specific for the A and B chains, respectively. The susceptibility of PDGF to mild acid treatment and its chromatographic behavior in reversed-phase high performance liquid chromatography and immobilized metal ion affinity chromatography, as compared to A and B chain homodimers, is consistent with a heterodimer structure. Analysis of PDGF purified according to our routine, large scale procedure revealed the major part to have a heterodimer structure. In addition, B chain homodimers were also found. With the demonstration that a major part of PDGF purified from human platelets occurs as a heterodimer, all three dimeric forms of PDGF have been identified. The following nomenclature to distinguish the various forms is suggested: PDGF-AA, a homodimer of A chains; PDGF-AB, a heterodimer; PDGF-BB, a homodimer of B chains; PDGF, any dimeric form of A or B chains.

• Heldin CH, Hammacher A, Nister M, Westermark B
• Structural and functional aspects of platelet-derived growth factor.
• Br J Cancer. 1988; 57: 591-3
• Display abstract

• The finding that PDGF production is common in normal as well as transformed cells indicate that PDGF has a function in autocrine and paracrine stimulation of cells in several physiological and pathological conditions. The expression of mRNA for the two chains of PDGF are independently regulated. The fact that the different dimeric forms of PDGF have different functional effects, indicate that the cellular response to PDGF may be more complex than previously thought, and may involve binding to more than one receptor. The cellular effects ascribed to PDGF, growth stimulation, ruffling and chemotaxis, seems to be mainly associated with B chain containing dimers. The function of PDGF-AA remains to be elucidated.

• Pencev D, Grotendorst GR
• Human peripheral blood monocytes secrete a unique form of PDGF.
• Oncogene Res. 1988; 3: 333-42
• Display abstract

• PDGF isolated from platelets and forms of PDGF produced by cells transformed with the v-sis gene (PDGF B chain) or U-20S osteosarcoma cells which express the PDGF A chain gene are known to be processed as disulfide-linked dimers of approximately 30 kd. Western blot analysis with anti human PDGF antibody of the PDGF-like factor synthesized and secreted by human blood monocytes (MDGF) on SDS gels indicates that it lacks interchain disulfide bridges and behaves as a 16-kd monomer under nonreducing conditions. Additionally, MDGF exhibits different sensitivities to either formic acid or CNBr cleavage compared to PDGF A or B chain molecules indicating it may have a different primary structure. These data suggest that MDGF represents a unique form of PDGF which lacks interchain disulfide bridges and may represent a new member of the PDGF family of growth factors.

• Bywater M et al.
• Expression of recombinant platelet-derived growth factor A- and B-chain homodimers in rat-1 cells and human fibroblasts reveals differences in protein processing and autocrine effects.
• Mol Cell Biol. 1988; 8: 2753-62
• Display abstract

• The autocrine effects of platelet-derived growth factor (PDGF) A- and B-chain homodimers (PDGF-AA and PDGF-BB) on rat-1 cells and human fibroblasts have been investigated by using human PDGF A- and B-chain cDNA clones expressed in a retroviral vector. Infection with replication-defective virus carrying the B-chain cDNA resulted in a phenotypical transformation resembling that induced by simian sarcoma virus. The resulting cells were focus forming in monolayer cultures, grew to high saturation densities, and formed large colonies in soft agar. The PDGF A-chain transfectants showed no transformed morphology and lacked focus-forming activity but grew to high saturation density in monolayer culture and formed small colonies in soft agar. A similar but weaker effect was obtained with an A-chain cDNA variant containing a 69-base-pair insertion in the 3' end of the protein-coding domain. A- and B-chain transfectants released PDGF receptor-competing activity into the medium, but only the medium conditioned by the B-chain transfectants possessed potent mitogenic activity on human fibroblasts. Both types of transfectants had downregulated levels of PDGF receptors; however, the B-chain transfectants were downregulated to significantly lower levels. Metabolic labeling and immunoprecipitations with PDGF antiserum showed that the PDGF B-chain protein was processed to a 24-kilodalton cell-associated and a 30-kilodalton secreted dimeric protein. The A-chain protein was rapidly secreted as a 31-kilodalton dimeric protein. The present study shows a marked difference in the autocrine effects of PDGF-AA and -BB expressed under the control of a retroviral promoter and suggests that different biological properties may be assigned to these two PDGF isoforms.

• Dupuy E, Rohrlich PS, Tobelem G
• Heparin stimulates fibroblasts growth induced by platelet derived growth factor.
• Cell Biol Int Rep. 1988; 12: 17-28
• Display abstract

• We have demonstrated that 125I unfractionated heparin binds to cultured human skin fibroblasts with a Kd of 1.16 10(-8) M and is internalized partly. A low molecular weight heparin fraction (PK 10169) competed (50%) with 125I unfractionated heparin, but to a lesser extent than cold unfractionated heparin (90%). When the cell proliferation was induced by pure PDGF, heparin markedly potentiated the fibroblast growth. Similar stimulation was observed when the growth was induced by FGF or EGF. Low molecular weight heparin enhanced the fibroblast proliferation induced by PDGF but to a lesser extent than unfractionated heparin. Chondroitin sulfate has no effect. PDGF did not modify the heparin binding on fibroblast cultures either at 4 degrees C or 37 degrees C and did not alter the process of heparin internalization. PDGF binding to the cultured fibroblast (Kd 10.1 +/- 3.4 10(-10) M) was not modified by the presence of heparin when studied at 4 degrees C or 37 degrees C.

• Kumar RK, Bennett RA, Brody AR
• A homologue of platelet-derived growth factor produced by rat alveolar macrophages.
• FASEB J. 1988; 2: 2272-7
• Display abstract

• Rat alveolar macrophages secrete a growth factor that renders rat lung fibroblasts competent to initiate DNA synthesis in vitro in the presence of platelet-poor plasma. This biological activity resembles that of platelet-derived growth factor (PDGF). After separation from putative associated binding proteins by chromatography under acidic conditions, the macrophage-derived factor exhibited a relative molecular weight similar to that of highly purified human PDGF. The factor bound to a monospecific antibody to human PDGF and thus could be quantitated in an enzyme immunoassay for PDGF. It competed with radiolabeled human PDGF for receptor sites for PDGF on rat lung fibroblasts, and binding to these receptor sites could be specifically inhibited by anti-PDGF. These data strongly support the view that the factor derived from rat alveolar macrophages is homologous to human PDGF and is similar to human macrophage-derived PDGF-like growth factor. Furthermore, we have demonstrated that the lung contains both an effector cell (pulmonary macrophage) and a potential target cell (interstitial fibroblast) for this cytokine. Therefore the rat appears to be an appropriate animal model in which to study macrophage-derived PDGF-like growth factors as mediators of proliferation in pulmonary fibrogenesis.

• Lanser ME, Brown GE, Garcia-Aguilar J
• Neutrophil CR3 induction by platelet supernatants is due to platelet-derived growth factor.
• Surgery. 1988; 104: 287-91
• Display abstract

• Recently, serum was shown to contain a factor that increased expression of the complement receptor CR3 on neutrophil membranes. This factor was localized to platelet granules and was released during coagulation. This study was undertaken to identify this factor in platelet granules. Platelet supernatants containing granule contents were incubated with neutrophils, and CR3 expression was determined by flow cytometry. Incubation with platelet supernatants induced more than a twofold increase in the amount of CR3 expressed on the neutrophil membrane (p = 0.05). Anti-platelet-derived growth factor (anti-PDGF) Fab, when preincubated with the platelet supernatants, completely inhibited this CR3-inducing activity. Pure PDGF induced a dose-response increase in CR3, whereas platelet factor 4 had no effect. PDGF was active in concentrations well within the physiologic range. These data indicate that PDGF is responsible for the CR3-inducing activity of platelet supernatants. PDGF may well be an important regulator of neutrophil adherence and phagocytic function in areas of tissue injury.

• Kimura A, Katoh O, Kuramoto A
• Marrow fibroblasts from patients with myeloproliferative disorders show increased sensitivity to human serum mitogens.
• Br J Haematol. 1988; 69: 153-6
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• The growth of marrow fibroblasts from patients with myeloproliferative disorders (MPD) was investigated using platelet derived growth factor (PDGF) and human serum as mitogens in the presence of human plasma derived serum. The proliferation of fibroblasts in MPD patients was increased compared to normal individuals, especially in patients with chronic myelocytic leukaemia and essential thrombocythaemia. This increment of proliferation might be due to higher sensitivity of the fibroblasts to plasma derived serum than to PDGF, because the ratio of proliferation with PDGF to that without PDGF, when compared between patients and normals, remained unchanged. These results suggest that MPD fibroblasts are more sensitive to some factor(s) in plasma, and this fact could partially explain the pathogenesis of myelofibrosis in MPD patients.

• Rorsman F, Bywater M, Knott TJ, Scott J, Betsholtz C
• Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: alternative exon usage predicts two different precursor proteins.
• Mol Cell Biol. 1988; 8: 571-7
• Display abstract

• The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.

• Sariban E, Kufe D
• Expression of the platelet-derived growth factor 1 and 2 genes in human myeloid cell lines and monocytes.
• Cancer Res. 1988; 48: 4498-502
• Display abstract

• Platelet-derived growth factor (PDGF), a potent mitogen for mesenchymal cells, consists of PDGF-1 and PDGF-2 polypeptide chains which are linked by disulfide bonds. Sequence analysis has revealed that: (a) the PDGF-2 chain is encoded by the c-sis protooncogene, the cellular counterpart of the simian sarcoma viral oncogene; (b) the PDGF-1 and PDGF-2 chains are related; and (c) the PDGF-1 gene has no known viral homologue. We have previously shown that the PDGF-2 gene is expressed during 12-O-tetradecanoylphorbol-13-acetate (TPA) induced monocytic differentiation of human HL-60 leukemia cells. In the present study, PDGF-1 and PDGF-2 gene expression was compared in HL-60 cells, human THP-1 monocytic leukemia cells, and human monocytes. Uninduced HL-60 cells, uninduced THP-1 cells, and resting monocytes had no detectable PDGF-1 or PDGF-2 mRNA. In contrast, both PDGF-1 and PDGF-2 transcripts were detected in HL-60 cells and monocytes induced with TPA, while only PDGF-1 mRNA was found in TPA-treated THP-1 cells. Moreover, neither of these transcripts were found during drug induced granulocytic differentiation of HL-60 cells. Cycloheximide, an inhibitor of protein synthesis: (a) failed to increase PDGF-1 and PDGF-2 mRNA levels in uninduced HL-60 cells; (b) increased PDGF-2, but not PDGF-1, mRNA in resting monocytes; and (c) increased levels of PDGF-1 and PDGF-2 mRNA in HL-60 cells and monocytes treated with TPA. This effect of cycloheximide was related in part to stabilization of both transcripts. Thus, PDGF-1 and PDGF-2 genes are differentially regulated in myeloid cells, although they share common control mechanisms at the post-transcriptional level. Differential regulation of PDGF gene expression would result in altered chain composition of the PDGF protein and possibly changes in biological activity.

• Ross R, Raines EW
• Platelet-derived growth factor--its role in health and disease.
• Adv Exp Med Biol. 1988; 234: 9-21

• Lowenberg BF, Pilliar RM, Aubin JE, Sodek J, Melcher AH
• Cell attachment of human gingival fibroblasts in vitro to porous-surfaced titanium alloy discs coated with collagen and platelet-derived growth factor.
• Biomaterials. 1988; 9: 302-9
• Display abstract

• The influence of biological coating, with or without the incorporation of growth factor, on the migration, attachment and orientation of human gingival fibroblasts in relation to porous-surfaced titanium alloy (Ti6AI4V) discs, was measured. Comparison was made between coating the discs with collagen and with collagen incorporating platelet-derived growth factor (PDGF); controls comprised porous-surfaced discs coated with agar or collagen containing bovine serum albumin (used as a carrier for the PDGF), uncoated porous-surfaced Ti6AI4V discs (with or without additional protein additives) exhibited significantly higher attachment indices (AI) and orientation indices (OI) compared with naked control discs (p less than 0.01); OI was also significantly higher than that of surface-demineralized root slices (p less than 0.001) on days 1, 2 and 3. Addition of PDGF to the collagen resulted in a further enhancement in OI on days 1 and 2 (p less than 0.01) over that shown by discs coated with collagen incorporating the bovine serum albumin vehicle. There was no cell attachment and consequently, no cell orientation, in relation to Ti alloy discs that had been coated with agar. These data suggest that attachment and orientation of cells following migration in relation to porous-surfaced Ti6AI4V discs can be modified by the application of biological molecules to the surface of the disc. This may have a useful application in clinical implantology.

• van den Eijnden-van Raaij AJ, van Maurik P, Boonstra J, van Zoelen EJ, de Laat SW
• Ultrastructural localization of platelet-derived growth factor and related factors in normal and transformed cells.
• Exp Cell Res. 1988; 178: 479-92
• Display abstract

• Visualization of platelet-derived growth factor (PDGF) and PDGF-like growth factors in cultured cells has been achieved by cryo-ultramicrotomy in combination with immunogold labeling. Immunogold staining of cryosections requires a mild chemical fixation in order to ensure preservation of antigenicity and ultrastructural details. Therefore the effect of several chemical fixatives on the antigenic properties of PDGF and PDGF-like growth factors was studied by indirect immunofluorescence using a polyclonal anti-PDGF antiserum. These studies demonstrated that formaldehyde has no effect on antigenicity, in contrast to glutaraldehyde or acrolein. For this reason formaldehyde was used as the only fixative for the visualization of PDGF in cryosections. PDGF was visualized in cryosections of normal human fibroblasts, preincubated with PDGF under various conditions. Preincubation at 4 degrees C with PDGF resulted in partial internalization of the growth factor. During subsequent warming of the cells to 37 degrees C PDGF was translocated to the nucleus. PDGF was also detected in the cytoplasm of tumor cells producing endogenous PDGF-like growth factors (neuroblastoma and simian sarcoma virus-transformed cells) but in these cases no significant amounts of these growth factors were present in the nucleus or at the extracellular surface of these cells. These results will be discussed in view of the intracellular routing of PDGF in normal responsive cells and of PDGF-like growth factors in factor-producing cells.

• Katoh O, Kimura A, Kuramoto A
• Platelet-derived growth factor is decreased in patients with myeloproliferative disorders.
• Am J Hematol. 1988; 27: 276-80
• Display abstract

• Platelet-derived growth factor (PDGF) has been suggested to play some role in the pathogenesis of myelofibrosis frequently encountered in patients with myeloproliferative disorders (MPD). In this study we measured PDGF activity and PF4 content in circulating platelets of patients with MPD. Both factors were lower than those of normal controls. PDGF activity in patients with myelofibrosis was slightly lower than in those without fibrosis. However, when adjusted to whole blood volume, there was a positive correlation between platelet count and PDGF activity per ml whole blood. Nevertheless, no correlation was found between activity and grade of bone marrow fibrosis. These results may support the idea that an abnormal release of PDGF occurs from platelets or megakaryocytes in the bone marrow environment, resulting in the stimulation of fibroblast proliferation, and hence, the occurrence of myelofibrosis in patients with MPD.

• Claesson-Welsh L et al.
• cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules.
• Mol Cell Biol. 1988; 8: 3476-86
• Display abstract

• The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.

• Mellstrom K, Heldin CH, Westermark B
• Induction of circular membrane ruffling on human fibroblasts by platelet-derived growth factor.
• Exp Cell Res. 1988; 177: 347-59
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• One of the earliest effects of platelet-derived growth factor (PDGF) on human fibroblasts in culture is an induction of membrane ruffling. The morphology of the ruffles induced by PDGF is unique in that they form circular arrangements on the dorsal side of the cells. Here we report that the induction of circular ruffle arrangements is an effect specific for PDGF, dose-dependent and inhibitable by anti-PDGF antibodies. We have attempted to utilize this effect to design a rapid and sensitive bioassay for PDGF. The "membrane ruffling assay" is compared with other methods to measure PDGF and its specificity with regard to the different dimeric forms of PDGF is discussed. Introduction of Ca2+ into the cells via the Ca2+ ionophore A23187 or the addition of the tumor-promor 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a stimulator of protein kinase C, does not induce circular ruffle formations on human fibroblasts, neither does the addition of the combination of these two agents. However, addition of TPA almost completely inhibits PDGF-induced circular ruffle formations. Further, we find a shift in the time-course of the PDGF-induced circular ruffle formations by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases. This may indicate the involvement of protein phosphorylation in the regulation of PDGF-induced membrane ruffling.

• Hammacher A, Nister M, Westermark B, Heldin CH
• A human glioma cell line secretes three structurally and functionally different dimeric forms of platelet-derived growth factor.
• Eur J Biochem. 1988; 176: 179-86
• Display abstract

• A human malignant glioma cell line, U-343 MGa Cl2:6, has previously been shown to secrete platelet-derived-growth-factor(PDGF)-like activity [Nister, M., Heldin, C.-H., Wasteson, A. and Westermark, B. (1984) Proc. Natl Acad. Sci. USA 81, 926-930]. We report here that this activity consists of three different molecules separable by reversed-phase chromatography and immobilized-metal-ion affinity chromatography. HPLC reversed-phase chromatography resolved two peaks of activity, which were denoted glioma-derived growth factor-I (GDGF-I) and GDGF-II. GDGF-I was purified to greater than 90% purity; in SDS gel electrophoresis, it appeared as a 31-kDa component which by reduction was converted to 17 kDa. Immunoprecipitation of radiolabeled, reduced and alkylated GDGF-I with antisera made against peptides from the A and B chains of PDGF, gave a specific signal only with antiserum against the A chain. Furthermore, when reduced and alkylated GDGF-I was analyzed by reversed-phase HPLC, it eluted at the position of PDGF A chains. We conclude that GDGF-I is a homodimer of a polypeptide similar to the A chain of PGDF. GDGF-II was found to have higher mitogenic activity than GDGF-I. Analysis by immunoprecipitation with PDGF-chain-specific antisera revealed that GDGF-II contained a polypeptide similar to the B chain of PDGF. Immobilized-metal-ion affinity chromatography revealed that 95% of the mitogenic activity of GDGF-II consisted of a heterodimer of one A and one B chain, whereas 5% consisted of a B-chain homodimer. Thus, U-343 MGa Cl 2:6 cells secrete all three possible dimeric forms of PDGF.

• Raff MC, Lillien LE, Richardson WD, Burne JF, Noble MD
• Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture.
• Nature. 1988; 333: 562-5
• Display abstract

• The various cell types in a multicellular animal differentiate on a predictable schedule but the mechanisms responsible for timing cell differentiation are largely unknown. We have studied a population of bipotential glial (O-2A) progenitor cells in the developing rat optic nerve that gives rise to oligodendrocytes beginning at birth and to type-2 astrocytes beginning in the second postnatal week. Whereas, in vivo, these O-2A progenitor cells proliferate and give rise to postimitotic oligodendrocytes over several weeks, in serum-free (or low-serum) culture they stop dividing prematurely and differentiate into oligodendrocytes within two or three days. The normal timing of oligodendrocyte development can be restored if embryonic optic-nerve cells are cultured in medium conditioned by type-1 astrocytes, the first glial cells to differentiate in the nerve: in this case the progenitor cells continue to proliferate, the first oligodendrocytes appear on the equivalent of the day of birth, and new oligodendrocytes continue to develop over several weeks, just as in vivo. Here we show that platelet-derived growth factor (PDGF) can replace type-1-astrocyte-conditioned medium in restoring the normal timing of oligodendrocyte differentiation in vitro and that anti-PDGF antibodies inhibit this property of the appropriately conditioned medium. We also show that PDGF is present in the developing optic nerve. These findings suggest that type-1-astrocyte-derived PDGF drives the clock that times oligodendrocyte development.

• Kimura A, Katoh O, Kuramoto A
• [Role of PDGF on myeloproliferative disorders]
• Rinsho Ketsueki. 1988; 29: 989-93

• Nister M et al.
• Evidence for progressional changes in the human malignant glioma line U-343 MGa: analysis of karyotype and expression of genes encoding the subunit chains of platelet-derived growth factor.
• Cancer Res. 1987; 47: 4953-60
• Display abstract

• Three cell samples in different passages of the line U-343 MGa, derived from a human malignant glioma biopsy, gave rise to clones with different amounts of platelet-derived growth factor (PDGF)-like activity secreted to extracellular medium, and of 125I-labeled PDGF binding. Sixteen clones were completely karyotyped with the G-banding technique. The unique markers 1p-q+, 16p- found in all clones, as well as in the parallel uncloned line, U-343 MG, provided evidence of their common origin. The deduced early, possibly partly primary, deviations had the formula 44, XY, 1p-q+, -14, 16p-, -22, where loss of one chromosome 22 is in accordance with previous reports on early chromosomal deviations in gliomas. Two clones, the hypodiploid 26L and 5H, represented early progressional changes. The other clones followed two patterns of late progressional changes, probably starting from the karyotype of 5H, with additional markers and doubling of the stemlines. In late progressional line I 12q+ and in II +7 were the most characteristic findings. Northern blot analysis using complementary DNA clones for the A and B chains of PDGF showed that both PDGF chains were expressed in 26L and 5H indicating that activation of the PDGF genes could have been an early event in the development of this glioma. Clones with late progression pattern II had been subjected to the highest selective pressure in vitro, and they secreted the highest amount of PDGF-like activity to the extracellular medium. Among them were the most rapidly and tightly growing cells and some clones with high 125I-labeled epidermal growth factor binding. Possibly these findings reflect progressional changes including defective regulation of the growth factor/growth factor receptor genes, selected for in vitro, without involving gross rearrangements or amplifications of the genes. The possible significance of extra chromosomes 7, with the PDGF A chain and epidermal growth factor receptor genes, and of the 12q+ marker, located near the gamma interferon gene is discussed.

• Westermark B, Heldin CH
• Structure and function of platelet-derived growth factor.
• Acta Med Scand Suppl. 1987; 715: 19-23

• Takehara K, Grotendorst GR, Silver R, LeRoy EC
• Dipyridamole decreases platelet-derived growth factor levels in human serum.
• Arteriosclerosis. 1987; 7: 152-8
• Display abstract

• Mitogenic activities on confluent fibroblasts by sera from patients with scleroderma and normal controls were studied. In experiments using eight different fibroblast strains (one human fetal lung fibroblast, one foreskin fibroblast, three adult skin fibroblast, and three scleroderma fibroblast), pooled scleroderma serum showed lower mitogenic activity by 3H-thymidine incorporation assay than pooled normal serum. Individual serum investigations revealed that 11 of 33 patients (33%) showed low mitogenic activities; all 11 patients were receiving dipyridamole. Of 15 patients, 11 (73%) receiving dipyridamole showed low mutagenic activities; none of 18 patients not receiving dipyridamole showed low mitogenic activities. Approximately 70% of this serum activity was inhibited by the addition of anti-platelet-derived growth factor (PDGF) antibody indicating that most of this serum activity seemed to be derived from PDGF. Western blot analysis of extracts of normal sera before and after administration of dipyridamole with antihuman PDGF antibody showed a large decrease in the amount of immunoreactive PDGF present. These data indicate that dipyridamole is an effective drug to lower release of PDGF during blood clotting.

• Tong BD et al.
• cDNA clones reveal differences between human glial and endothelial cell platelet-derived growth factor A-chains.
• Nature. 1987; 328: 619-21
• Display abstract

• Human platelet-derived growth factor (PDGF) is a potent mitogenic polypeptide which is believed to be a heterodimer of A- and B-chains stabilized by interchain disulphide bonds. The B-chain of PDGF is encoded by the c-sis gene, the normal cellular homologue of the transforming gene of the simian sarcoma virus (SSV). cDNA clones of the B-chain from both normal and transformed cells have mutually consistent DNA sequences. Recently, an A-chain cDNA clone (D-1) was isolated from a transformed human glial cell cDNA library. We report the complete sequence of an A-chain cDNA clone (BT-1) isolated from a normal human umbilical vein endothelial (HUVE) cell cDNA library. BT-1 differs from the sequence of the D-1 clone by a 69 base pair deletion containing the predicted carboxy terminus of the protein. The mRNA levels of the A- and B-chains of PDGF in HUVE cells were analysed and shown to respond differently to the endothelial cell growth factor (ECGF).

• Collins T, Bonthron DT, Orkin SH
• Alternative RNA splicing affects function of encoded platelet-derived growth factor A chain.
• Nature. 1987; 328: 621-4
• Display abstract

• Platelet-derived growth factor (PDGF) is a basic protein of relative molecular mass 30,000 (Mr 30K) composed of two polypeptide chains, designated PDGF A and PDGF B. The B-chain is encoded by the c-sis gene, the cellular counterpart of the simian sarcoma virus transforming gene v-sis. The PDGF A-chain cDNA clones recently isolated and sequenced from a transformed human clonal glioma cell line represent at least two alternatively spliced transcript species differing by 69 base pairs at the C-terminus. Here we demonstrate that the normal human umbilical vein endothelial cell (EC) A chain precursor lacks the 15 carboxy-terminal, highly basic amino acids encoded by the larger tumour cell cDNA. Surprisingly, culture media from monkey kidney cells (COS) transfected with the endothelial cDNA clone contained much less mitogenic activity than media from cells transfected with the longer tumour cell-derived A-chain cDNA. This functional difference appeared to be due to inefficient assembly or secretion of the recombinant endothelial-type growth factor. This suggests that some transformed cells may use alternative RNA splicing to modify normal growth factors and by so doing increase the efficiency of mitogen assembly or secretion.

• Nakamura T
• [Platelet-derived growth regulators and mechanism of liver regeneration]
• Tanpakushitsu Kakusan Koso. 1987; 32: 1113-25

• Papayannopoulou T, Raines E, Collins S, Nakamoto B, Tweeddale M, Ross R
• Constitutive and inducible secretion of platelet-derived growth factor analogs by human leukemic cell lines coexpressing erythroid and megakaryocytic markers.
• J Clin Invest. 1987; 79: 859-66
• Display abstract

• We have examined the constitutive and inducible secretion of platelet-derived growth factor (PDGF)-like proteins in a variety of human hemopoietic cell lines. The highest levels of secreted protein were noted in four human erythroleukemia lines which, in addition to erythroid lineage markers, express one or more megakaryocytic lineage markers. Induction of these lines by 12-O-tetradecanoylphorbol-13-acetate enhanced the expression of megakaryocytic markers and increased secretion of PDGF-like proteins several fold. In concert with these changes, there was significant induction of c-sis/PDGF-B messenger RNA (mRNA) expression in all lines, whereas one line showed significant concurrent induction of PDGF-A mRNA expression. Whether PDGF-like secretion is part of the stem cell-like phenotype displayed by these lines or is secondary to their leukemic transformation remains to be determined. Nevertheless, these lines provide new cellular models for studying the expression and function of PDGF analogs in hemopoietic cells.

• Dmoszynska-Giannopoulou A, Rupniewska ZM
• [The role of platelet-derived growth factor in the pathogenesis of various diseases. I. The role of platelet-derived growth factor in the inflammatory processes and arteriosclerosis]
• Pol Arch Med Wewn. 1987; 77: 234-7

• Fox PL, Chisolm GM, DiCorleto PE
• Lipoprotein-mediated inhibition of endothelial cell production of platelet-derived growth factor-like protein depends on free radical lipid peroxidation.
• J Biol Chem. 1987; 262: 6046-54
• Display abstract

• Cultured vascular endothelial cells produce several mitogens including a platelet-derived growth factor-like protein (PDGF-c). We previously reported that acetylated low density lipoprotein (acetyl-LDL) caused accumulation of cholesterol and specific inhibition of PDGF-c production by bovine aortic endothelial cells (Fox, P. L., and DiCorleto, P. E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4774-4778). We have now examined the role of cholesterol and other lipids on the inhibition of production of PDGF-c. Incubation of endothelial cells with free cholesterol/albumin complexes resulted in a large increase in cellular cholesterol content but did not inhibit PDGF-c production, demonstrating that cholesterol itself is not inhibitory. Involvement of lipid peroxides in the suppression of PDGF-c production was indicated by three observations. LDL modified in vitro by free radical lipid peroxidation quantitatively inhibited PDGF-c production. The inhibition was dependent on the level of LDL oxidation (as measured by thiobarbituric acid reactivity) and was specific since total protein synthesis was not affected. Inhibition of PDGF-c production by acetyl-LDL was also dependent on peroxidation. A lipid extract from oxidized LDL, but not from native LDL, specifically inhibited PDGF-c production. Chloroquine, monensin, and NH4Cl, inhibitors of lysosomal hydrolytic activity, did not prevent acetyl-LDL-mediated inhibition of PDGF-c production, indicating that cellular metabolism of the lipoprotein was not required for the inhibition. Furthermore, acetyl-LDL suppressed PDGF-c production by endothelial cells even in the presence of butylated hydroxytoluene, an inhibitor of lipid peroxidation, suggesting that cellular propagation of free radicals was not required for the inhibition. Finally, inhibition of PDGF-c production may be regulated at the post-transcriptional level since Northern blot analysis using a v-sis probe showed that the PDGF B-chain mRNA amounts were unaffected by oxidized or acetylated LDL. In summary, levels of an oxidized lipoprotein that have no effect on endothelial cell viability or protein synthetic rates can completely suppress production of a growth factor which may act as a paracrine mitogen in normal and pathological vascular processes.

• Besnard F, Perraud F, Sensenbrenner M, Labourdette G
• Platelet-derived growth factor is a mitogen for glial but not for neuronal rat brain cells in vitro.
• Neurosci Lett. 1987; 73: 287-92
• Display abstract

• The effects of platelet-derived growth factor (PDGF) on the proliferation of isolated rat neural cells grown in serum-free chemically defined media have been investigated. It was found that PDGF drastically stimulates the proliferation of astroblasts and oligodendroblasts, but has no effect on the proliferation of neuroblasts in primary culture. A role of PDGF in the reactive gliosis, occurring after brain injury, can be suggested.

• Fujii Y, Dohi K, Yamada H, Takai M, Ishikawa H
• [Relation of platelet-derived growth factor with vascular involvements in diabetes mellitus]
• Nippon Jinzo Gakkai Shi. 1987; 29: 469-76

• Sekido Y, Morishima Y, Ohya K
• Activity of platelet-derived growth factor (PDGF) in platelet concentrates and cryopreserved platelets determined by PDGF bioassay.
• Vox Sang. 1987; 52: 27-30
• Display abstract

• The bioassay of platelet-derived growth factor (PDGF) in platelets was established using the rat fibroblast cell line 3Y1. 3Y1 cells (5 X 10(3)/well) were cultured in 24-well microculture plates with RPMI 1640 medium using 1% of PDGF-free serum and 5% of platelet extracts for 4 days. Cell proliferation was measured by the increase of DNA content. This assay made it possible to measure the biological PDGF activity. PDGF activity of platelet concentrates kept the same level as that of fresh platelet samples during preservation for up to 120 h at 22 degrees C; cryopreserved platelets also retained PDGF activity well.

• Rollins BJ, Morrison ED, Stiles CD
• A cell-cycle constraint on the regulation of gene expression by platelet-derived growth factor.
• Science. 1987; 238: 1269-71
• Display abstract

• In density-arrested monolayer cultures of Balb/c 3T3 cells, platelet-derived growth factor (PDGF) stimulates expression of the c-myc and c-fos proto-oncogenes, as well as the functionally uncharacterized genes, JE, KC, and JB. These genes are not coordinately regulated. Under ordinary conditions, c-fos, JE, KC, and JB respond to PDGF only when the cells are in a state of G0 growth arrest at the time of PDGF addition. The c-myc gene is regulated in opposition to the other genes, responding best to PDGF in cycling cultures.

• Hanai K, Kato H, Matsuhashi S, Morita H, Raines EW, Ross R
• Platelet proteins, including platelet-derived growth factor, specifically depress a subset of the multiple components of the response elicited by glutathione in Hydra.
• J Cell Biol. 1987; 104: 1675-81
• Display abstract

• Human serum more strongly depressed the feeding response of Hydra (ball formation) elicited by S-methylglutathione than plasma. On the basis of the effect of several proteins released by platelets, at least five apparent components of the response (R1-R5) were suggested. Each of the platelet proteins examined specifically depressed a subset of these components. Among the platelet proteins examined, platelet-derived growth factor (PDGF) specifically depressed the R2 response (the concentration at which the depressing effect was 50% of the maximum [ED50] was 0.17 pM), and basic fibroblast growth factor depressed the R3 and R5 responses (ED50 0.50 aM) and the R2 response (ED50 0.55 pM). With respect to the depression of the R2 response by PDGF, addition of an anti-PDGF IgG or chemical reduction of PDGF, both of which prevent PDGF from binding to its cell surface receptor on responsive cells, eliminated the depressing effect of PDGF on the hydra response. The implications of these observations are discussed.

• Shinkai Y, Cameron JS
• Trial of platelet-derived growth factor antagonist, trapidil, in accelerated nephrotoxic nephritis in the rabbit.
• Br J Exp Pathol. 1987; 68: 847-52
• Display abstract

• The platelet-derived growth factor (PDGF) antagonist, trapidil, which also blocks the thromboxane and/or PG-endoperoxide receptor and is an inhibitor of thromboxane synthetase, was administered during rabbit accelerated nephrotoxic nephritis; the clinical and histological evolution was studied as well as urinary immunoreactive thromboxane (i-TXB2) and immunoreactive prostaglandin E2 (i-PGE2) excretion. Although the dose we used has been shown to be effective in vivo, and it inhibited the urinary i-TXB2 excretion on days 5 and 10, it neither inhibited the enhanced production of i-TXB2 on day 1, nor prevented the glomerular influx of monocytes on days 5 and 10. All clinical and histological data tend to be worse rather than better in trapidil-treated animals on days 5 and 10.

• Dmoszynska-Giannopoulou A, Rupniewska ZM
• [The role of platelet-derived growth factor in the pathogenesis of various diseases. II. Platelet-derived growth factor and the development of neoplasms]
• Pol Arch Med Wewn. 1987; 77: 238-42

• Wilson E, Laster SM, Gooding LR, Lambeth JD
• Platelet-derived growth factor stimulates phagocytosis and blocks agonist-induced activation of the neutrophil oxidative burst: a possible cellular mechanism to protect against oxygen radical damage.
• Proc Natl Acad Sci U S A. 1987; 84: 2213-7
• Display abstract

• The effect of platelet-derived growth factor (PDGF) on agonist-induced activation of the superoxide-generating oxidative burst in human neutrophils was tested. PDGF had no effect on the resting level of superoxide generation but inhibited both the rate and the extent of fMet-Leu-Phe-stimulated superoxide production in a dose-dependent manner. The concentration required to inhibit the response by 50% was 95 +/- 26 pM (n = 10). PDGF also blocked activation by other receptor-mediated agonists such as the complement protein C5a and opsonized zymosan, but not by phorbol myristate acetate or arachidonate, both of which may act at postreceptor sites. The growth factor, however, had no effect on the binding of fMet-Leu-Phe to its receptor. PDGF in concentrations that blocked the oxidative burst stimulated phagocytosis of opsonized latex particles. Thus, PDGF functions as a heterologous "down-regulator" of receptor-mediated activation of the neutrophil oxidative burst and an activator of phagocytosis. A model for a feedback regulatory loop between platelets and neutrophils is proposed.

• Narczewska B, Czyrski JA
• Partial purification of porcine platelet-derived growth factor (PDGF).
• Arch Immunol Ther Exp (Warsz). 1987; 35: 831-7
• Display abstract

• Fresh porcine blood platelets, subjected to ethanol acid extraction and purification on DEAE-Sephadex A-50 and CM-Sephadex C-50, were used to obtain partially purified, cationic PDGF (pI 8.5-10) of Mr 38,000 and biological activity 10(4) U/ml. Reduction of porcine PDGF with 2-mercaptoethanol revealed the presence of two polypeptides with Mr 18,000-22,000.

• Savage K, Siebert E, Swann D
• The effect of platelet-derived growth factor on cell division and glycosaminoglycan synthesis by human skin and scar fibroblasts.
• J Invest Dermatol. 1987; 89: 93-9
• Display abstract

• The effect of platelet-derived growth factor (PDGF) on cell division and glycosaminoglycan (GAG) synthesis by fibroblasts isolated from skin and scar was measured. We found that PDGF stimulates cell division more efficiently in normal skin fibroblasts than in scar fibroblasts and decreases GAG synthesis in skin and scar fibroblasts. Using a 4-h pulse label with [3H]thymidine ([3H]Thd) following a 20-h incubation of confluent monolayer cultures with 0-5 units PDGF/ml Dulbecco's modified Eagle's medium, we found a concentration-dependent increase in [3H]Thd incorporation. After incubation of fibroblasts with [3H]glucosamine and 35SO4 in the presence or absence of PDGF, labeled constituents were isolated from the extracellular, pericellular, and cellular fractions by pronase digestion and column chromatography on Sepharose CL4B or DEAE-cellulose and analyzed by cellulose acetate electrophoresis. The presence of PDGF decreased the total amount of 35S incorporated into macromolecules by skin and scar fibroblasts and resulted in an altered distribution of labeled GAGs. Dermal fibroblasts exposed to PDGF for 24 h incorporated a greater percentage of radiolabeled 35S into dermatan sulfate prime (DS') and less into dermatan sulfate (DS) in the extracellular fractions and a greater percentage of 35S into heparan sulfate (HS) in the pericellular fractions than did parallel cultures grown in the absence of PDGF. It is thought than PDGF may have an effect on scar formation by increasing the fibroblast population in the wound tissue and by affecting the total amount and types of matrix components synthesized.

• Singh JP
• A radioreceptor assay for platelet-derived growth factor.
• Methods Enzymol. 1987; 147: 13-22

• Martinet Y, Rom WN, Grotendorst GR, Martin GR, Crystal RG
• Exaggerated spontaneous release of platelet-derived growth factor by alveolar macrophages from patients with idiopathic pulmonary fibrosis.
• N Engl J Med. 1987; 317: 202-9
• Display abstract

• Idiopathic pulmonary fibrosis is a fibrotic lung disease characterized by an increased number of mesenchymal cells in the alveolar walls. Alveolar macrophages constitutively express low levels of c-sis, the protooncogene coding for the B chain of platelet-derived growth factor, a protein with chemotactic and mitogenic activity toward mesenchymal cells. We therefore hypothesized that alveolar macrophages in patients with idiopathic pulmonary fibrosis may release increased amounts of platelet-derived growth factor, which might help to explain the accumulation of mesenchymal cells and the fibrosis of the lower respiratory tract in the disease. Evaluation of alveolar macrophages recovered from the lungs of patients with idiopathic pulmonary fibrosis demonstrated that these cells spontaneously released four times more platelet-derived growth factor than did alveolar macrophages recovered from normal persons (P less than 0.01). That the platelet-derived growth factor molecules were potentially active was shown by their chemotactic activity for smooth-muscle cells and their ability to act as a "competence" factor for fibroblast growth. These observations suggest the possibility that the accumulation of mesenchymal cells within the alveolar walls in patients with idiopathic pulmonary fibrosis may result partly from the exaggerated release of the potent mitogen platelet-derived growth factor by mononuclear phagocytes in the lower respiratory tract.

• Masuda H, Yokota J, Battifora H, Cline M
• Events on different chromosomes alter the genes for the two peptides of platelet-derived growth factor in an osteogenic sarcoma.
• Cancer Genet Cytogenet. 1987; 29: 303-9
• Display abstract

• Platelet derived growth factor consists of dimers of partially homologous peptides (A and B) encoded by separate genes on different chromosomes. A clinically aggressive osteogenic sarcoma had both an amplified A-chain gene on chromosome #7 and a reduplication of all or part of chromosome #22 containing the B-chain (c-sis) gene. Other oncogenes on chromosomes #1, #2, #5, #6, #7, #8, #11, #12, #14, and #15 were not reduplicated or amplified. These observations are consistent with the hypothesis of autocrine stimulation of growth of some sarcomas in humans, and indicate that amplification and reduplication of related genes on different chromosomes can occur in tumors in vivo.

• Katz FE, Michalevicz R, Lam G, Hoffbrand AV, Goldman JM
• Effect of platelet-derived growth factor on enriched populations of haemopoietic progenitors from patients with chronic myeloid leukaemia.
• Leuk Res. 1987; 11: 339-44
• Display abstract

• The effect of pure platelet-derived growth factor and fresh serum on the in-vitro growth of purified haemopoietic progenitors from the peripheral blood of 12 patients with CML was studied. Purified haemopoietic progenitors were prepared using Percoll separation followed by cell sorting with the monoclonal antibody BI.3C5. Both pure PDGF at a concentration of 20 ng/ml and fresh serum significantly increased the numbers of BFU-E (p less than 0.01) and CFU-GEMM (p less than 0.014), but not the CFU-GM. That the PDGF effect was not mediated to any significant extent via prostaglandins, was shown by the lack of inhibitory effect of indomethacin on the growth of purified progenitor cells in the presence of fresh serum. Increased amounts of pure PDGF were required to give maximal stimulation of purified CML peripheral blood progenitors compared to normal bone marrow progenitors. These results show that CML progenitors are capable of responding to PDGF. Whether the quantitative difference in response is due to a reduced proportion of mesenchymal cells in CML peripheral blood compared to normal marrow, or whether CML progenitors are most likely already stimulated by autocrime PDGF or other growth factors remains to be elucidated.

• Teplizky H, Dvilansky A
• [Does platelet derived growth factor induce myelofibrosis in agnogenic myeloid metaplasia?]
• Harefuah. 1987; 112: 102-4

• Romano M, Poggi A
• Myelofibrosis: a role for platelet derived growth factor (PDGF)?
• Haematologica. 1986; 71: 359-61

• Newmark P
• Growth factor A-chain back in train.
• Nature. 1986; 320: 683-683

• Frantz CN, Doherty K, Gelsomino N, Cervoni M, Rust L
• Phosphorylation of a 75,000 molecular weight cytosol protein induced by platelet derived growth factor and tumor promoter in BALB/c-3T3 cells.
• J Cyclic Nucleotide Protein Phosphor Res. 1986; 11: 217-31
• Display abstract

• In order to identify common mechanisms of action by which both the platelet-derived growth factor (PDGF) and the tumor promoter tetradecanoyl phorbol acetate (TPA) initiate cell growth, the effects of PDGF and TPA on phosphorylation of cellular proteins were examined in density-inhibited Balb/c-3T3 cells. Cultures were incubated with 32Pi and growth factor, and 32P-labeled cellular proteins were examined after separation by SDS-polyacrylamide gel electrophoresis and autoradiography. TPA and PDGF each induced phosphorylation of a major cytosol protein of approximately 75,000 molecular weight (pp75). Phosphorylation of this protein was not induced by either epidermal growth factor or insulin, neither of which initiate 3T3 cell growth but enhance growth later in the 3T3 cell cycle. pp75 was a single band under reduced and non-reduced conditions, and a single spot was seen on two-dimensional gels. Phosphorylation did not occur at 4 degrees C. Phosphorylation of the protein was observed within 3 min and reached a maximum in 10-30 min. Submitogenic doses of TPA and PDGF induced submaximal phosphorylation. The phosphoprotein was labeled only on serine. Cell free phosphorylation of pp75 occurred at 4 degrees C in the presence of Mg++ and Ca2+. Homogenates from cultures pretreated with TPA phosphorylated pp75 in the presence or absence of Ca2+. Phosphorylation of this protein may possibly be related to activation of the Ca2+-dependent, phospholipid sensitive protein kinase C.

• Michalevicz R, Katz F, Stroobant P, Janossy G, Tindle RW, Hoffbrand AV
• Platelet-derived growth factor stimulates growth of highly enriched multipotent haemopoietic progenitors.
• Br J Haematol. 1986; 63: 591-8
• Display abstract

• Platelet-derived growth factor (PDGF) has been shown to stimulate growth of normal and malignant fibroblasts, glial cells and smooth muscle cells. A growth promoting effect on human haemopoietic precursors has also been described, but the interpretation of this haemopoietic proliferative response to PDGF has been hampered by the lack of purity of the target population. In this study we show that PDGF promotes growth of early bone marrow haemopoietic progenitors depleted of either monocytes or T lymphocytes which are known to influence haemopoiesis. Moreover, the action of PDGF is even increased on a highly enriched BI-3C5 early bone marrow population. BI-3C5 is a novel monoclonal antibody which recognizes an antigen present on all multilineage colony-forming cells (CFU-mix) (Tindle et al. 1985). BI-3C5 positively and negatively sorted fractions were obtained by fluorescence activated cell sorting (FACS) and PDGF was found to stimulate growth of CFU-mix in the BI-3C5-positive fraction (consisting of only 4-6% of the marrow population), the effect being more marked than that on unsorted bone marrow. The results suggest that the product of the cellular proto-oncogene c-sis (the putative structural gene for the beta chain of PDGF) may play a regulatory role in the in vivo proliferation of multipotent haemopoietic progenitors.

• Heldin CH, Johnsson A, Wennergren S, Wernstedt C, Betsholtz C, Westermark B
• A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A-chains.
• Nature. 1986; 319: 511-4
• Display abstract

• Platelet-derived growth factor (PDGF), as purified from fresh human platelets, is a protein of relative molecular mass (Mr) 30,000 composed of two disulphide-linked subunit chains of similar size, named A and B (ref. 1). The dimer structure of PDGRF seems to be important for its biological effects, as reduction irreversibly inactivates the factor; it is not known, however, whether PDGF exists as a heterodimer or as a mixture of homodimers. Amino-acid sequence analysis has revealed that the A- and B-chains of human PDGF are related to each other, and that the B-chain is almost identical to part of the v-sis gene product of simian sarcoma virus (SSV). There is experimental evidence that a PDGF-like protein is indeed operational in SSV-induced transformation and the biologically active v-sis product is probably structurally similar to a putative dimer of PDGF B-chains. PDGF-like growth factors and/or a 4.2-kilobase (kb) c-sis transcript are present in several transformed mammalian cell lines and in certain nontransformed cells; cloned c-sis complementary DNA from human T cells transformed with human T-lymphotropic virus (HTLV) or from human endothelial cells contains the coding sequence for a putative PDGF B-chain precursor, but apparently lacks PDGF A-chain sequences. We have previously partially purified and characterized a PDGF-like growth factor from U-2 OS cells (osteosarcoma-derived growth factor, ODGF) and shown that this factor has structural, functional and immunological characteristics in common with PDGF. We describe here a procedure for the preparation of homogeneous ODGF, and provide evidence that this factor, which binds to the PDGF receptor, has a structure similar to a homodimer of PDGF A-chains.

• Bernabei PA, Arcangeli A, Casini M, Grossi A, Padovani R, Rossi Ferrini P
• Platelet-derived growth factor(s) mitogenic activity in patients with myeloproliferative disease.
• Br J Haematol. 1986; 63: 353-7
• Display abstract

• Platelet-derived growth factor has been invoked in the pathogenesis of medullary fibrosis during myeloproliferative disorders. In this study we compared the mitogenic activity of heat-stable platelet-growth factor(s) from 13 patients suffering from myeloproliferative disorders with that of a normal group. The test was carried out on Go growth arrested Balb/c 3T3 fibroblasts incubated with various concentrations of platelet extracts, determining the entrance into the S phase by means of [14C]thymidine uptake. The incorporation curves of [14C]thymidine by the fibroblast culture, under the effect of pathological extracts, were consistently lower than the control curve, indicating a lower level of PDGF(s) in platelets from patients. The greatest depression of this activity was found to be associated with highest degree of medullary fibrosis (agnogenic myeloid metaplasia patient group), in agreement with the hypothesis that fibroblast activation within bone marrow during myeloproliferative disorders might be correlated with a PDGF(s) release in the bone marrow environment.

• Westermark B, Heldin CH
• Platelet-derived growth factor as a mediator of normal and neoplastic cell proliferation.
• Med Oncol Tumor Pharmacother. 1986; 3: 177-83
• Display abstract

• Human platelet-derived growth factor is the major mitogen in serum for connective-tissue-derived cells in culture. The factor is 30,000 mol. wt protein composed of two disulphide-linked polypeptide chains, named A and B. The B-chain is virtually identical to part of the transforming protein of simian sarcoma virus (SSV), implying that SSV-transformation is mediated by a PDGF-like growth factor. This notion is supported by the finding that specific as well as nonspecific inhibitors of PDGF-action (PDGF antibodies and suramin, respectively) are efficient inhibitors of SSV-transformation and revert the transformed phenotype of SSV-transformed cells. Expression of the genes encoding the PDGF subunits and production of PDGF-like growth factors is a common feature of human sarcoma cell lines, suggesting a role of PDGF in the pathogenesis of sarcomas, although direct support in favor of this notion is lacking. An involvement of PDGF in autocrine and paracrine stimulation of normal cell growth is suggested by the finding that responsive (arterial smooth muscle cells and placental cytotrophoblasts) as well as nonresponsive (endothelial cells and macrophages) cells produce PDGF-like growth factors. In conclusion, PDGF-like growth factors may be widely implicated in normal as well as neoplastic growth processes.

• Takamiya Y, Kohsaka S, Toya S, Otani M, Mikoshiba K, Tsukada Y
• Possible association of platelet-derived growth factor (PDGF) with the appearance of reactive astrocytes following brain injury in situ.
• Brain Res. 1986; 383: 305-9
• Display abstract

• The association of platelet-derived growth factor (PDGF) with the appearance of reactive astrocytes following injury was investigated by using a specific antagonist of PDGF, Trapidil. The cerebral cortex of 4-week-old male rats was unilaterally injured with a 22-gauge needle. Immunohistochemical staining with antiserum to glial fibrillary acidic protein revealed that reactive astrocytes had increased in number around the wound by 2 days following the injury and had spread to the ipsilateral areas distant from the wound by 3 days. The appearance of reactive astrocytes in areas distant from the wound was dramatically suppressed by the administration of Trapidil. This finding indicates that PDGF might play a role in gliosis following injury.

• Hanks CT, Kim JS, Edwards CA
• Growth control of cultured rat calvarium cells by platelet-derived growth factor.
• J Oral Pathol. 1986; 15: 476-83
• Display abstract

• Platelet-derived growth factor (PDGF), which is released during the "clotting cascade", has a mitogenic effect on a variety of mesenchymal cells including those of the periosteum. Cell culture provides a method of studying these effects. In the present study, PDGF was studied for its ability to stimulate quiescent calvarium periosteal cells to enter G1, indicated by new protein synthesis. After addition of platelet poor plasma, these cells later entered S phase, indicated by uptake of 3H-thymidine. Three hours after PDGF stimulation, there were quantitative increases of at least 18 protein bands as compared to cells left in serum starvation medium. Two of these new protein bands (31K and 45K) required transcription. These data suggest that PDGF in extravasated blood may stimulate appositional bone formation beneath intact periosteum at the site of injury.

• Campochiaro PA, Glaser BM
• Platelet-derived growth factor is chemotactic for human retinal pigment epithelial cells.
• Arch Ophthalmol. 1985; 103: 576-9
• Display abstract

• The presence of blood or serum in the vitreous cavity has been associated with the formation of cellular membranes in proliferative vitreoretinopathy and after penetrating ocular trauma. Retinal pigment epithelial (RPE) cells are an important component of these membranes. For RPE cells to effectively spread throughout the vitreous cavity and form contractile membranes, cell migration must occur. Serum has been shown to initiate RPE cell migration. Fibronectin (FN), a glycoprotein found in serum, stimulates RPE cell migration but accounts for only part of the stimulatory effect of serum. We report that another serum component, platelet-derived growth factor (PDGF), also stimulates RPE cell migration. Furthermore, the effect of PDGF and FN are additive and together probably account for a large part of the chemotactic activity found in serum.

• Collins T, Ginsburg D, Boss JM, Orkin SH, Pober JS
• Cultured human endothelial cells express platelet-derived growth factor B chain: cDNA cloning and structural analysis.
• Nature. 1985; 316: 748-50
• Display abstract

• Vascular endothelial cells have a central role in various pathophysiological responses such as acute inflammation, wound healing and atherogenesis. The anatomical position of endothelial cells between blood leukocytes and the surrounding vascular smooth muscle cells or stromal fibroblasts may intensify and focus the effects of released endothelial cell products. Endothelial cells in culture produce a platelet-derived growth factor (PDGF)-like mitogen. PDGF purified from platelets is a basic protein with an apparent relative molecular mass (Mr) of approximately 30,000 (reviewed in refs 2, 3) and is believed to comprise two polypeptide chains, PDGF-A and PDGF-B (also referred to as PDGF-1 and PDGF-2; refs 5, 6). Sequence analysis of PDGF B chain has revealed a striking homology with the predicted sequence of p28sis, the transforming protein of simian sarcoma virus. sis-Homologous transcripts have been detected by Northern blot analysis of RNA from cultured endothelial cells. However, there are no structural data available on either the protein product or the messenger RNA to establish the identity of the endothelial-derived mitogen with either chain of PDGF. Here we report the isolation and complete sequence analysis of a sis-homologous complementary DNA clone from human endothelial cells, providing an opportunity to study the structure of sis as transcribed by a normal (untransformed) cell. Our results establish that normal human endothelial cells in culture express the B chain of PDGF, and that endothelial-derived PDGF B chain is synthesized as a predicted precursor polypeptide of Mr 27,281.

• Sinzinger H, Firbas W
• Irradiation depresses prostacyclin generation upon stimulation with the platelet-derived growth factor.
• Br J Radiol. 1985; 58: 1023-6
• Display abstract

• Experimental and clinical findings demonstrated rather severe arterial lesions occurring in irradiated arteries. It is suggested that a local haemostatic imbalance might be one causative factor. Liberation of PDGF causes smooth-muscle cell proliferation and PGI2 formation by the arterial wall cells, inhibiting in turn further release of PDGF from the alpha granules of the platelets. A decreased ability of irradiated vascular segments to generate PGI2 upon PDGF stimulation described here might cause haemostatic imbalance and subsequent radiation-induced lesions.

• Stroobant P, Gullick WJ, Waterfield MD, Rozengurt E
• Highly purified fibroblast-derived growth factor, an SV40-transformed fibroblast-secreted mitogen, is closely related to platelet-derived growth factor.
• EMBO J. 1985; 4: 1945-9
• Display abstract

• Fibroblast-derived growth factor (FDGF), a polypeptide secreted by an SV40-transformed baby hamster kidney cell line (SV28), was purified approximately 1000-fold from SV28-conditioned medium. FDGF, which gave a single band on SDS-polyacrylamide gel electrophoresis, is a hydrophobic and cationic protein of apparent mol. wt. 31 000 containing disulphide-linked polypeptides. This factor is positive in Western blots using human platelet-derived growth factor (PDGF) anti-serum. FDGF is a potent mitogen for Swiss 3T3 cells; half-maximal stimulation of DNA synthesis was achieved at a concentration of approximately 1 nM, comparable with those for human and porcine PDGF. FDGF inhibits EGF binding to Swiss 3T3 cells, as does PDGF. The coincidence of the physical, biological and immunological characteristics of FDGF and PDGF strongly suggests that they are closely related in structure.

• Pantazis P, Pelicci PG, Dalla-Favera R, Antoniades HN
• Synthesis and secretion of proteins resembling platelet-derived growth factor by human glioblastoma and fibrosarcoma cells in culture.
• Proc Natl Acad Sci U S A. 1985; 82: 2404-8
• Display abstract

• Immunoprecipitation of proteins extracted from metabolically labeled human glioblastoma and fibrosarcoma cells with antiserum to platelet-derived growth factor (PDGF) showed that these cells express and secrete proteins that are recognized specifically by the antiserum. The molecular masses of immunoprecipitated proteins in the lysates of the malignant cells ranged from 16 kDa to 140 kDa. Both cell lines secreted a 31-kDa polypeptide with structural, immunological, and biological properties similar to those of human PDGF. These cell lines were shown to synthesize a 4.4-kb mRNA that contained sequences from all the six currently identified exons of the human c-sis gene. These data suggest that the PDGF-like proteins in the two mesenchyme-derived transformed cells are encoded at least in part by the c-sis locus.

• Tzeng DY, Deuel TF, Huang JS, Baehner RL
• Platelet-derived growth factor promotes human peripheral monocyte activation.
• Blood. 1985; 66: 179-83
• Display abstract

• Like in the polymorphonuclear leukocyte (PMN), the platelet-derived growth factor (PDGF) purified to homogeneity is capable of inducing monocyte activation responses as evaluated by generation of superoxide anion (O-.2) from membrane-associated oxidase system, release of granule enzymes, and enhanced cell adherence and cell aggregation. Superoxide anion release was maximized at 10 ng/mL PDGF and was comparable to that induced by 10(-7) mol/L formyl-methionyl-leucyl-phenylalanine. The potency of PDGF to induce this response in monocytes was of the same magnitude as that observed in PMNs. Similarly, lysozyme release and monocyte adherence were also increased in a dose-dependent manner and achieved maximal responses at 40 ng/mL concentration of PDGF. The PDGF concentration required to achieve maximal monocyte aggregation was two-fold (60 ng/mL) of that found for PMNs. In contrast to PMNs, a positive correlation (gamma = .93; P less than .01) was observed between the increases of PDGF concentration and beta-glucuronidase release. These findings indicate that PDGF can induce the full sequence of cell activation events in human monocytes similar to human PMNs.

• Narczewska B, Czyrski JA, Inglot AD
• Properties of purified bovine platelet-derived growth factor stimulating proliferation of human and mouse fibroblasts.
• Can J Biochem Cell Biol. 1985; 63: 187-94
• Display abstract

• Platelet-derived growth factor (PDGF) was isolated from platelets obtained from fresh bovine blood. The platelet lysates were extracted with acid-ethanol, according to the classic procedure that has been applied to the preparation of various protein hormones and transforming growth factors, but not until now to PDGF isolation. Bovine PDGF was further purified by ion-exchange chromatography on DEAE-Sephadex, CM-Sephadex, and finally by molecular sieving on Sephadex G-100. Bovine PDGF has an isoelectric point of 9.45-10.6 and a molecular weight ranging from 28 000 to 31 000. Reduction of bovine PDGF with 2-mercaptoethanol revealed the presence of two polypeptides with molecular weights of approximately 14 000 and 15 000. A simple biologic microassay of cell multiplication-stimulating activity has been developed in our laboratory. Purified bovine PDGF stimulated the proliferation of BALB/c 3T3 or human fibroblasts at minimal concentrations of 0.36-1.5 ng/mL.

• Scher CD, Whipple AP, Singh JP, Pledger WJ
• Modulation of the platelet-derived growth factor induced replicative response.
• J Cell Physiol. 1985; 123: 10-6
• Display abstract

• Quiescent cultures of density arrested BALB/c-3T3 cells have been sensitized to the growth stimulatory action of the platelet-derived growth factor (PDGF). Sensitization was achieved by depriving the cultures of PDGF prior to growth stimulation and was noted after transfer of cultures from medium supplemented with 10% serum to medium containing either an equivalent concentration of platelet-poor plasma or a low concentration (0.5%) of serum. Sensitized cultures required less pure PDGF for growth stimulation than nonsensitized ones. In addition such cultures required less mitogen to synthesize a PDGF modulated major excreted protein (MEP). The mechanism of sensitization was investigated. Sensitized cultures did not bind more PDGF than non-sensitized ones. Rather, sensitization appeared to result from the loss of cells that occurred when cultures were deprived of PDGF. Such a loss increased the amount of PDGF available per cell, causing a higher percentage of cells to enter the S phase. Similarly, the amount of PDGF per cell regulated MEP synthesis. Furthermore, in non-sensitized cultures (containing the same number of cells), the absolute quantity rather than the concentration of PDGF regulated DNA synthesis. It appears that the amount of PDGF per cell modulates mitogenesis.

• Hosang M
• Suramin binds to platelet-derived growth factor and inhibits its biological activity.
• J Cell Biochem. 1985; 29: 265-73
• Display abstract

• The polyanion suramin was recently found to inhibit binding of 125I-PDGF (platelet-derived growth factor) to Balb/c 3T3 cell membranes. Cultured Swiss 3T3 cells were used to investigate the mode of action of suramin and to monitor its effect on the biological activity of PDGF. Evidence is presented that suramin inhibits cellular binding of PDGF by binding to PDGF itself, thereby preventing it from binding to its cell surface receptor: First, while suramin inhibited 125I-PDGF binding with a half maximum inhibition concentration of approximately 60 microM or 90 micrograms/ml in a simultaneous competition assay, it was inactive in a sequential radioreceptor assay, in which an inhibitor is expected to be active if it interacts with the receptor (even with relatively low affinity) but to be inactive if it interacts with PDGF. Second, suramin prevented immunoprecipitation of 125I-PDGF in a dose-dependent manner, with a half maximum effective concentration of approximately 50 microM. Furthermore, suramin efficiently dissociated 125I-PDGF bound to its cell surface receptor, whereas unlabeled PDGF even in large excess was virtually inactive. This is also in line with the proposed direct interaction between PDGF and suramin, since such an interaction can be envisaged to induce a conformational change in the PDGF-receptor complex, resulting in an increased off-rate of the complex. Reduced 125I-PDGF binding in the presence of suramin correlated directly with a suramin dose-dependent inhibition of PDGF-induced incorporation of 3H-thymidine into quiescent Swiss 3T3 cells and of the proliferation of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

• Tominaga S, Lengyel P
• beta-Interferon alters the pattern of proteins secreted from quiescent and platelet-derived growth factor-treated BALB/c-3T3 cells.
• J Biol Chem. 1985; 260: 1975-8
• Display abstract

• Mouse beta-interferon (at a concentration of 100 units/ml or higher) inhibited the platelet-derived growth factor (PDGF)-induced replication of quiescent BALB/c-3T3 cells. The interferon treatment did not inhibit, but slightly enhanced, the accumulation of the following three PDGF-induced RNAs: myc RNA, JE RNA, and KC RNA. The treatment with interferon changed the pattern of secreted proteins from quiescent cells and from cells treated with partially purified PDGF; it inhibited the accumulation of the PDGF-induced proteins (including proteins of 63 and 32 kDa) and it induced the accumulation of several other proteins (including proteins of 89, 31.5, 30, and 10.5 kDa) in both quiescent and also in PDGF-treated cells.

• Hasegawa-Sasaki H
• Early changes in inositol lipids and their metabolites induced by platelet-derived growth factor in quiescent Swiss mouse 3T3 cells.
• Biochem J. 1985; 232: 99-109
• Display abstract

• Inositol lipid turnover was studied in quiescent Swiss mouse 3T3 cells stimulated by platelet-derived growth factor (PDGF). Stimulation of the cells by PDGF for 10 min at 37 degrees C induced the following changes in lipids: in cells prelabelled with [32P]Pi, a 28% decrease in [32P]phosphatidylinositol 4,5-bisphosphate, a 41% decrease in [32P]phosphatidylinositol 4-phosphate and a 1.7-fold increase in the 32P-labelling of phosphatidic acid; in cells prelabelled with [3H8]arachidonic acid, a 17.9-fold increase in [3H]phosphatidic acid, a 20% decrease in [3H]phosphatidylinositol (PtdIns), an 8.6-fold increase in [3H]arachidonic acid released into the medium, a 57-fold increase in [3H]prostaglandin E2 in the medium, and a 5.3-fold increase in [3H]monoacylglycerol released into the medium (the last was identified as the 2-acyl derivative); in cells prelabelled with [2-3H]glycerol, a 1.7-fold increase in [3H]diacylglycerol, a 6.7-fold increase in [3H]phosphatidic acid, a 1.6-fold increase in [3H]lysophosphatidylcholine (lysoPtdCho), a 9% decrease in [3H]PtdIns, and a 1.6-fold increase in [3H]monoacylglycerol released into the medium. PDGF stimulated the formation of inositol tris-, bis- and mono-phosphates in the cells prelabelled with myo-[2-3H]inositol. These results indicate that, in Swiss 3T3 cells stimulated by PDGF, diacylglycerol produced by the hydrolysis of inositol lipids is partly degraded to 2-acylglycerol and partly converted into phosphatidic acid. The increase in lysoPtdCho indicates that a portion of arachidonic acid released from the stimulated cells is formed by the hydrolysis of PtdCho with a phospholipase A2. Different values of half-maximal doses of the partially purified PDGF used in this study were found for the various responses of quiescent Swiss 3T3 cells to PDGF. The values for half-maximal doses suggest that activation of a fraction of the cell-surface receptor for PDGF is sufficient for mitogenesis and for an increase in the cytoplasmic free Ca2+ concentration, and that the PGDF-stimulated lipid metabolism is probably proportional to the number of receptor sites activated by PDGF.

• hang JY, Tian JS
• [Advance in the research of platelet-derived growth factor]
• Sheng Li Ke Xue Jin Zhan. 1985; 16: 270-2

• Wu QY
• [Platelet-derived growth factor]
• Sheng Li Ke Xue Jin Zhan. 1985; 16: 356-8

• Habenicht AJ et al.
• Human platelet-derived growth factor stimulates prostaglandin synthesis by activation and by rapid de novo synthesis of cyclooxygenase.
• J Clin Invest. 1985; 75: 1381-7
• Display abstract

• Human platelet-derived growth factor (PDGF) stimulated prostaglandin (PG) E2 synthesis in the cell cycle of Swiss 3T3 cells at two distinct time intervals, with a first plateau within 10 min and a second plateau within 2-4 h after addition of PDGF. At 4 h, the concentration of PGE2 in PDGF-stimulated cultures exceeded the quiescent control cells by a factor of 10-15. Quiescent cells incubated with up to 16 microM exogenous arachidonic acid (AA) synthesized only small amounts of PGE2. In contrast, 4 h after addition of PDGF, the concentration of PGE2 synthesized from exogenous AA exceeded that in quiescent cultures by a factor of 28. The effect of PDGF stimulation on PG synthesis from exogenous AA could not be explained by growth factor-mediated increase in the cellular free AA pool as shown in experiments using [14C]AA. PDGF also stimulated synthesis of PGI2 (prostacyclin), thromboxane, and PGF2 alpha from exogenous AA. While inhibition of protein synthesis by 10 micrograms/ml cycloheximide had no effect on the early increase in PGE2 synthesis, the second increase was completely prevented. Additionally, cycloheximide treatment at 6 h after PDGF stimulation resulted in rapid decline of PGE2 synthesis from exogenous AA. Quiescent cultures pretreated with 100 microM aspirin and stimulated by PDGF thereafter recovered from cyclooxygenase inhibition within 180 min. Our results suggest that phospholipase activation and resultant AA release is not sufficient to induce the burst of PG synthesis observed in PDGF-stimulated cells. Instead, PDGF stimulates PG synthesis by direct effects on the PG-synthesizing enzyme system, one involving a protein synthesis-independent mechanism and another that requires rapid translation of cyclooxygenase.

• Heldin CH, Westermark B, Wasteson A
• [Platelet-derived growth factor in normal and malignant cell growth]
• Nord Med. 1984; 99: 319-22

• Ross R
• Multiple responses of connective tissue cells to mitogenic stimulation with platelet-derived growth factor.
• Prog Clin Biol Res. 1984; 154: 125-31
• Display abstract

• A number of important properties are held by mitogenic substances such as platelet derived growth factor for cells that can respond to such a mitogen. These include responses that occur early after exposure of the cells to the mitogens. Such responses include increases in endocytosis, binding of low density lipoprotein to high affinity cell surface receptors and increased uptake and degradation of LDL, increases in phospholipid metabolism, sodium and potassium ion flux, increases in protein and RNA synthesis, and chemotaxis. It is not yet clear whether some of these properties can be separated from the stimulus to enter DNA synthesis and mitosis, or whether they are tightly coupled to the latter. Nevertheless, they all precede DNA synthesis by many hours before the cells enter into S and go on to divide. It is highly probable that a number of other biological events will be found to be stimulated by exposing cells to mitogens, such as the platelet derived growth factor. Elucidation of these events and determination of the interrelation of each of these biological responses to the subsequent multiplication of the cells in a given set of circumstances will help to provide a basis for understanding how these mitogens work, and potentially how it may be possible to prevent some of the proliferative responses that represent such an inherent part of many disease processes.

• Fiedel BA, Vallve C, Izzi JM
• Enhanced skin reactivity to platelet-derived permeability factor (PDPF) and exogenous histamine in an acute phase rabbit model.
• Agents Actions. 1984; 14: 738-42
• Display abstract

• Rabbits made acute phase by sub-cutaneous trauma with 2% croton oil (in mineral oil) were tested by intradermal (ID) injection with platelet-granule extracts containing platelet-derived permeability factor (PDPF). Compared with controls, skin reactivity to PDPF was enhanced in acute phase animals 3-7 days post-trauma, a period of acute inflammation as reflected by the occurrence in the circulation of C-reactive protein; maximal skin responses were observed 3-4 days post-trauma. Individual skin sites reached maximum intensity 15 min-1 hour post-ID injection of PDPF and were sensitive to chlorpheniramine maleate, suggesting a major role for histamine. Intradermal injection of histamine revealed that acute phase animals yielded an initially more intense skin reaction, and were markedly less capable of recovering from the effects of histamine. These data suggest that in the acute phase, there exists a heightened and prolonged sensitivity to the action of histamine which can be exploited by pro-inflammatory agents such as PDPF.

• Czyrski JA, Narczewska B, Inglot AD
• New procedure for purification of human platelet-derived growth factor.
• Arch Immunol Ther Exp (Warsz). 1984; 32: 589-98
• Display abstract

• A new simple procedure was developed for purification of human platelet-derived growth factor (PDGF). PDGF was isolated and concentrated by acid/ethanol extraction of platelet lysate. Purification of this extract was accomplished by initial ion-exchange chromatography on DEAE-Sephadex and subsequent separation of "cationic" unadsorbed fraction on CM-Sephadex and controlled-pore glass--CPG-10. The product migrated as two biologically active homogeneous fractions, PDGF I (Mr = 34 000) and PDGF II (Mr = 31 000) in analytical gel electrophoresis in the presence of SDS. PDGF had isoelectric point ranging from 9.5-10.6. Multiplication-stimulating activity of PDGF was estimated using the clone of Balb/c 3T3 cells propagated in microplates in Eagle's minimal essential medium supplemented with 2% of platelet poor plasma serum. Purified PDGF was active in stimulating 3T3 cell proliferation at 0.4 ng/ml.

• Bockus BJ, Scher CD, Stiles CD
• Use of defined media for analysis of the action of platelet-derived growth factor on fibroblasts.
• Prog Clin Biol Res. 1984; 154: 133-41

• Almazov VA, Blagosklonnyi MV, Zaritskii AI
• [Platelet-derived growth factor: mechanism of action and role in the development of diseases]
• Ter Arkh. 1984; 56: 144-7

• Dresow B, Delbruck A
• The isolation and activity of growth-stimulating factors from human platelets.
• J Clin Chem Clin Biochem. 1984; 22: 527-33
• Display abstract

• Optimal conditions for determining the mitogenic activity of platelet growth factor in a homologous system (human palmar fascia fibroblasts/human platelet growth factor) were established. With the aid of this system it was possible to test the active fraction from crude platelet extract following affinity chromatography on heparin-Sepharose, chromatofocusing and molecular sieve filtration. This fraction has an isoelectric point of pH greater than 9 with a molecular mass of about 12000 daltons and reacts with beta-thromboglobulin antiserum. The addition of protease inhibitors proved essential for the isolation of the active growth factor, in order to prevent proteolytic degradation to a biologically inactive substance that is immunologically identical to beta-thromboglobulin. In addition to stimulation of DNA synthesis, the isolated growth factor induces enhanced synthesis of collagen and glycosaminoglycans in fibroblasts in vitro.

• Mondschein JS, Schomberg DW
• Effects of partially and more highly purified platelet-derived growth factor preparations on luteinizing hormone receptor induction in granulosa cell cultures.
• Biol Reprod. 1984; 30: 603-8
• Display abstract

• The effects of partially and more highly purified platelet-derived growth factor (PDGF) preparations on luteinizing hormone (LH) receptor induction by follicle-stimulating hormone (FSH) and cholera toxin (CTX) were studied in cultured granulosa cells from immature, diethylstilbestrol-primed rats. Partially purified PDGF, prepared by carboxymethyl Sephadex C-50 chromatography (CMS-PDGF), and more highly purified PDGF, further purified by Cibacron Blue Sepharose chromatography (BS-PDGF), enhance FSH-dependent LH receptor induction in serum-free and in serum-containing medium. BS-PDGF is more potent than CMS-PDGF, and is relatively more efficacious in serum-free and less efficacious in serum-containing medium than CMS-PDGF. CMS-PDGF and BS-PDGF enhance LH receptor induction by CTX in serum-containing medium, but levels achieved are significantly less than those attained with FSH and CMS- or BS-PDGF. BS-PDGF does not enhance induction by CTX in serum-free medium. The results suggest that the action of PDGF to enhance LH receptor induction is complex and may represent actions of several components of PDGF preparations. These findings may also provide indirect evidence for a component of FSH action which is independent of cAMP.

• Fairbanks KP, Witte LD, Goodman DS
• Relationship between mevalonate and mitogenesis in human fibroblasts stimulated with platelet-derived growth factor.
• J Biol Chem. 1984; 259: 1546-51
• Display abstract

• Relationships between mevalonate and DNA synthesis were explored in quiescent human fibroblasts stimulated with human platelet-derived growth factor (PDGF). Studies of others have indicated that mevalonate, or a product of mevalonate other than cholesterol, is essential for DNA replication. The present studies were designed to determine whether there was a critical time in the cell cycle when mevalonate was necessary for later DNA synthesis to occur. Compactin and mevinolin, inhibitors of hydroxymethylglutaryl CoA reductase, were employed to block both the synthesis of mevalonate and of DNA. Compactin inhibited the sharp peak of DNA synthesis which occurs in cells 24 h after PDGF addition in a concentration-dependent manner. This suppression of DNA synthesis was not prevented by low density lipoprotein but was fully reversed by mevalonate. Compactin inhibited DNA synthesis when the inhibitor was present during the time interval 10-20 h after PDGF addition. Its presence only in the interval before 10 h or after 20 h had no effect. Conversely, mevalonate could fully overcome the compactin block in DNA synthesis when present during the period of from 10-20 h after PDGF addition. Mevalonate present only before 10 h or after 20 h had no effect. When mevalonate was added to mevinolin-blocked cells for the interval 10-15 h after PDGF, the mevinolin block of DNA synthesis was 68% overcome; in contrast, only 20% of the reversal of the mevinolin block was seen when mevalonate was added from 15-20 h. Addition of mevalonate for only the 2-h interval of from 10-12 h after PDGF overcame the mevinolin block of DNA synthesis (assayed at 24 h) by 50%. The results show that there is a critical time period, several h before S phase, when PDGF-stimulated cells require mevalonate in order for DNA synthesis to proceed at 24 h. This critical period comprised the interval of approximately 10-20 h after PDGF addition and especially the early part of this interval.

• Owen AJ, Pantazis P, Antoniades HN
• Simian sarcoma virus--transformed cells secrete a mitogen identical to platelet-derived growth factor.
• Science. 1984; 225: 54-6
• Display abstract

• Normal rat kidney (NRK) cells transformed by simian sarcoma virus (SSV) release into the culture medium a biologically active mitogen with properties identical to those of human platelet-derived growth factor (PDGF). Like PDGF, the growth factor derived from SSV-NRK cells was shown to be stable to heat and sensitive to reducing agents. It was capable of inhibiting binding of labeled PDGF to the receptor on human fibroblasts. It also stimulated the phosphorylation of the same membrane protein (185 kilodaltons) in isolated plasma membranes from human fibroblasts. Immunoprecipitation of metabolically labeled proteins released by SSV-NRK cells showed that a 34-kilodalton protein was specifically precipitated by antiserum to PDGF. Upon reduction, this protein had a molecular size of 17 kilodaltons. PDGF has been shown to consist of two 14- to 18-kilodalton proteins linked by disulfide bonds.

• Fox PL, DiCorleto PE
• Regulation of production of a platelet-derived growth factor-like protein by cultured bovine aortic endothelial cells.
• J Cell Physiol. 1984; 121: 298-308
• Display abstract

• Platelet-derived growth factor (PDGF) is a potent mitogen for cultured cells of mesenchymal origin. Known sources of PDGF or PDGF-like protein are blood platelets, several transformed cell lines, and cultured endothelial cells (EC). We have examined the regulation of production of a PDGF-like protein in cultures of bovine aortic EC using a specific radioreceptor assay for PDGF. EC constitutively secreted PDGF-like protein into serum-containing or serum-free medium. The rate of production of PDGF-like protein was constant for at least 3 weeks and was not due to release of an internal store, since cell lysis by repeated freeze/thaw cycles did not release significant amounts of the protein. Synthesis of PDGF-like protein was sensitive to changes in the pH of the media and was maximal at pH 8.5. Production of PDGF-like protein was independent of EC growth rate: rapidly dividing cells and confluent, quiescent cells produced equal amounts per cell. However, sparse, quiescent EC produced more PDGF-like protein per cell than did confluent, quiescent cells. Several phorbol esters stimulated production of PDGF-like protein. At a concentration of 10(-6) M, a twofold stimulation was observed upon addition of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and nearly a fourfold stimulation upon addition of the nonpromoting analog, methyl TPA. Incubation of EC with endotoxin (10 micrograms/ml) resulted in a twofold stimulation of PDGF-like protein production. In all experiments with endotoxin and phorbol esters, an increase in the production of PDGF-like protein was accompanied by morphological changes in the EC cultures. The cells appeared elongated and fibroblastic and exhibited low viability. A mathematical model was developed in which PDGF-like protein production was shown to consist of two separate components--production at a constant rate by healthy cells and a large burst of synthesis and secretion by dying cells. These results suggest that injurious agents may be capable of stimulating production of a growth factor by the endothelium.

• Deschamps JF, Caen JP
• Abnormal megakaryocyte release and myelofibrosis.
• Lancet. 1984; 1: 567-8

• Niman HL, Houghten RA, Bowen-Pope DF
• Detection of high molecular weight forms of platelet-derived growth factor by sequence-specific antisera.
• Science. 1984; 226: 701-3
• Display abstract

• Antisera to synthetic peptides representing sequences of both chains of platelet-derived growth factor (PDGF) were used to structurally analyze PDGF isolated from outdated human platelets and PDGF-like proteins in normal and transformed cells. Most PDGF isolated from platelets did not contain the carboxyl portion of PDGF-2 in contrast to p20sis, the major form of p28sis detected in simian sarcoma virus-transformed cells. In addition, higher molecular weight forms of molecules containing PDGF-1 and PDGF-2 sequences were detected in all cell lines tested. These lines were heterogeneous with respect to species, cell type, and transforming agent.

• Syms AJ, Norris JS, Smith RG
• Autocrine regulation of growth: I. Glucocorticoid inhibition is overcome by exogenous platelet derived growth factor.
• Biochem Biophys Res Commun. 1984; 122: 68-74
• Display abstract

• The ductus deferens smooth muscle tumor cell line (DDT1MF-2) expresses c-sis protooncogene mRNA transcripts which encode at least one subunit of the potent mitogenic agent, platelet derived growth factor (PDGF). These cells also synthesize and secrete a protein which is immunologically identical to this growth factor. Therefore, PDGF is implicated in the autocrine regulation of DDT1MF-2 cell proliferation. While androgens also stimulate proliferation and induce an augmentation in androgen receptor levels in DDT1MF-2 cells, glucocorticoids inhibit both events and arrest cells in the G1 phase of the cell cycle. Addition of PDGF overcomes the glucocorticoid cell cycle arrest, but does not diminish the suppressive action on androgen receptor concentration. These findings are consistent with a mechanism by which glucocorticoids regulate DDT1MF-2 cell proliferation through modulation of PDGF expression which is independent of the glucocorticoid effects on androgen receptor concentrations.

• Poggi A, Rucinski B, James P, Holt JC, Niewiarowski S
• Partial purification and characterization of porcine platelet-derived growth factor (PDGF).
• Exp Cell Res. 1984; 150: 436-41
• Display abstract

• Platelet derived growth factor (PDGF) has been partially purified from porcine platelets. Purification steps included heparin-agarose chromatography of the material released by thrombin-stimulated washed porcine platelets and Blue-Sepharose chromatography. Preparative isoelectric focusing showed that isoelectric point of porcine PDGF is at pH 10.0-11.0 and elution experiments from sodium dodecyl sulfate (SDS) polyacrylamide gels indicated that its molecular weight is close to 30 kD. The immunoglobulin fraction prepared from anti-human PDGF serum inhibited the mitogenic activity of porcine PDGF. These experiments suggest a homology of porcine and human PDGF. Porcine platelet factor 4 and porcine platelet basic protein were devoid of significant mitogenic activity.

• Raines EW, Bowen-Pope DF, Ross R
• Plasma binding proteins for platelet-derived growth factor that inhibit its binding to cell-surface receptors.
• Proc Natl Acad Sci U S A. 1984; 81: 3424-8
• Display abstract

• Evidence is presented that the binding of platelet-derived growth factor (PDGF) to plasma constituents inhibits the binding of PDGF to its cell-surface mitogen receptor. Approximately equivalent amounts of PDGF-binding activity were found in plasma from a number of different species known by radioreceptor assay to contain PDGF homologues in their clotted blood. Activation of the coagulation cascade did not significantly alter the PDGF-binding activity of the plasma components. Three molecular weight classes of plasma fractions that inhibit PDGF binding to its cell-surface receptor were defined by gel filtration: approximately equal to 40,000, 150,000, and greater than 500,000. Specific binding of 125I-labeled PDGF to the highest molecular weight plasma fraction could also be demonstrated by gel filtration. The binding of PDGF to these plasma components was reversible under conditions of low pH or with guanidine X HCl, and active PDGF could be recovered from the higher molecular weight fractions. Immunologic and functional evidence is presented that the highest molecular weight plasma fraction may be alpha 2-macroglobulin. A model is proposed in which the activity of PDGF released in vivo may be regulated by association with these plasma binding components and by high-affinity binding to cell-surface PDGF receptors.

• Stroobant P, Waterfield MD
• Purification and properties of porcine platelet-derived growth factor.
• EMBO J. 1984; 3: 2963-7
• Display abstract

• The purification to homogeneity of a potent growth factor from porcine platelets is described. This cationic mitogen is named porcine platelet-derived growth factor (PDGF) on the basis of close structural, functional and immunological similarities to human PDGF. Porcine PDGF, like its human homologue, is a hydrophobic, disulphide cross-linked protein, which is stable to heat, acid, sodium dodecyl sulphate (SDS), and guanidine. The purified protein has an apparent mol. wt. on SDS-polyacrylamide gels of 38 000, similar to those reported for human PDGF (27 500-35 000). Amino terminal sequence analysis of native porcine PDGF gave a single 15 amino acid residue sequence, of which 11 residues were identical to the amino terminal sequence of the B chain of human PDGF. Gel permeation h.p.l.c. in guanidine solutions of the reduced protein revealed a single species of mol. wt. 17 000 suggesting that native porcine PDGF may be a homodimer of a 17 000 mol. wt. chain. Since porcine PDGF can be purified at low cost from large quantities of fresh platelets, it provides an alternative source of PDGF for structural and functional studies, and could be of use in preparing defined media for cell culture.

• Hill DJ, Milner RD
• Platelet-derived growth factor and multiplication-stimulating activity II, but not multiplication-stimulating activity III-2, stimulate [3H]thymidine and [35S]sulphate incorporation by fetal rat costal cartilage in vitro.
• J Endocrinol. 1984; 103: 195-203
• Display abstract

• The actions of partially purified porcine platelet-derived growth factor (PDGF) and highly purified multiplication-stimulating activity (MSA) II and MSA III-2, which are somatomedins, were investigated on the incorporation of [3H]thymidine and [35S]sulphate by fetal rat costal cartilage in vitro. This was compared with their effects in the presence of 1% fetal calf serum (FCS) on the uptake of thymidine by growth-arrested fetal rat fibroblasts. Platelet-derived growth factor at concentrations of 0.21-21 micrograms/l enhanced the incorporation of both isotopes by fetal cartilage in the presence of 1% FCS, but had an inconsistent action on thymidine uptake and no significant action on sulphate uptake in serum-free medium. Platelet-derived growth factor promoted thymidine uptake by growth-arrested, isolated fetal rat fibroblasts. Multiplication-stimulating activity II (10-100 micrograms/l) stimulated the uptake of thymidine and sulphate by fetal cartilage in medium containing 1% FCS but had no consistent action in serum-free medium, although MSA II and PDGF had a synergistic effect on thymidine uptake in the absence of serum. Multiplication-stimulating activity III-2 had no consistent action on thymidine or sulphate incorporation by fetal cartilage in either serum-free or serum-supplemented medium. However, the same preparation of MSA III-2 stimulated the uptake of [3H]thymidine into fetal rat fibroblasts with a half-maximal response at a concentration of 5-10 micrograms/l. The results identify PDGF as a possible mitogenic agent for fetal rat connective tissues in vitro and show a differential sensitivity of fetal cartilage to MSA peptides.

• Huang JS, Huang SS, Deuel TF
• Specific covalent binding of platelet-derived growth factor to human plasma alpha 2-macroglobulin.
• Proc Natl Acad Sci U S A. 1984; 81: 342-6
• Display abstract

• Attempts to measure the platelet-derived growth factor (PDGF) in human plasma resulted in the discovery of a specific plasma binding protein. The 125I-labeled PDGF (125I-PDGF)-plasma binding protein complex retained mitogenic activity but lost reactivity against rabbit anti-PDGF antiserum. Copurification of the plasma binding protein and alpha 2-macroglobulin (alpha 2M) in human plasma, the formation of a complex between 125I-PDGF and purified alpha 2M, and the comigration of the 125I-PDGF-plasma binding protein complex and the 125I-PDGF-alpha 2M complex in NaDodSO4/polyacrylamide gel electrophoresis and in pore-limiting polyacrylamide gel electrophoresis strongly suggested that alpha 2M is the plasma binding protein for 125I-PDGF. Immunoprecipitation of 125I-PDGF-alpha 2M and 125I-PDGF-plasma binding protein complexes by anti-human alpha 2M antiserum further established that alpha 2M and the plasma binding protein are the same molecule. Approximately 20% of 125I-PDGF is complexed by alpha 2M; further 125I-PDGF is complexed if the remaining 125I-PDGF is incubated with additional alpha 2M. Complex formation of 125I-PDGF with plasma or with alpha 2M was completely inhibited by 0.2 mM p-chloromercuric benzoate or 0.2 mM N-ethylmaleimide. The 125I-PDGF-alpha 2M complex or 125I-PDGF-plasma binding protein complex was not dissociated by 8 M urea, 1 M acetic acid, 0.1 M NaOH, or 1% NaDodSO4 but was dissociated by 2-mercaptoethanol, suggesting that the covalent binding of 125I-PDGF to alpha 2M occurs through a disulfide/sulfhydryl exchange reaction. The 125I-PDGF-alpha 2M complex (780,000 daltons) appears to contain two molecules of 125I-PDGF and two dimers of alpha 2M. The precise physiological role of the 125I-PDGF-alpha 2M interaction is unknown. alpha 2M may serve to limit PDGF released locally at sites of blood vessel injury. Alternatively, because of the nearly complete homology between the partial amino acid sequence of PDGF and the predicted amino acid sequence of the transforming protein of the simian sarcoma virus, p28sis, alpha 2M may play an important role in limiting the activity of a PDGF-like activity expressed by virus-transformed cells.

• Tzeng DY, Deuel TF, Huang JS, Senior RM, Boxer LA, Baehner RL
• Platelet-derived growth factor promotes polymorphonuclear leukocyte activation.
• Blood. 1984; 64: 1123-8
• Display abstract

• The platelet-derived growth factor (PDGF) has several well defined important biologic activities. Platelet-derived growth factor is the major mitogen in human serum for cells of mesenchymal origins; it is a potent chemoattractant protein for human monocytes, neutrophils, fibroblasts, and smooth muscle cells; and has been implicated in transformation by simian sarcoma virus and perhaps in transformation by other agents as well. In this article, PDGF has been shown to stimulate activation of human peripheral blood neutrophils defined by loss of membrane associated calcium as reflected by loss of chlortetracycline fluorescence, release of superoxide anion and specific granule enzymes, and enhanced neutrophil adherence and aggregation. These responses occurred in a dose-dependent fashion at concentrations of PDGF between 10 ng/mL (0.4 nmol/L) and 40 ng/mL (1.5 nmol/L) and were comparable to effects obtained with optimal concentrations of fMLP and C5a. Degranulation induced by PDGF was selective for secondary (specific) granules and not primary (azurophil) granules. Platelet-derived growth factor thus is ideally suited for a pivotal role in attracting inflammatory cells locally and initiating neutrophil activation at sites of blood vessel injury. Platelet-derived growth factor or a closely related protein also may play an important role in attracting and activating neutrophils in association with inflammatory tumors.

• Andersen HA, Flodgaard H, Klenow H, Leick V
• Platelet-derived growth factor stimulates chemotaxis and nucleic acid synthesis in the protozoan Tetrahymena.
• Biochim Biophys Acta. 1984; 782: 437-40
• Display abstract

• Platelet-derived growth factor (PDGF) is in concentrations of a few nanograms per ml a very active chemoattractant for the free-living ciliated protozoan Tetrahymena; at the same time it induces a rapid increase in incorporation of radioactive nucleic acid precursors into RNA and DNA. We find it remarkable that this lower eukaryote responds to platelet-derived growth factor in very much the same way as fibroblastic cells.

• Deuel TF, Huang JS
• Platelet-derived growth factor. Structure, function, and roles in normal and transformed cells.
• J Clin Invest. 1984; 74: 669-76

• Ek B, Heldin CH
• Use of an antiserum against phosphotyrosine for the identification of phosphorylated components in human fibroblasts stimulated by platelet-derived growth factor.
• J Biol Chem. 1984; 259: 11145-52
• Display abstract

• In search for possible intracellular mediators of the mitogenic signal induced by platelet-derived growth factor (PDGF), we have investigated tyrosine-specific phosphorylation stimulated by PDGF in intact human fibroblasts. Cells were metabolically labeled, either with [32P] orthophosphoric acid or with [35S]methionine, and thereafter treated with PDGF for various times. Lysates from the cell cultures were then immunoprecipitated with an antiserum specifically recognizing phosphotyrosine. Analysis of the precipitated radioactivity by sodium dodecyl sulfate-gel electrophoresis and autoradiography or fluorography showed the appearance of a 185-kDa protein in cells stimulated with PDGF; maximum yield was at about 5 min after the addition of PDGF. This component was found to have several characteristics in common with the PDGF receptor, including similar Mr, binding to immobilized wheat germ agglutinin, and incorporation of phosphate on tyrosine residues after exposure to PDGF. We conclude that the 185-kDa component probably represents the PDGF receptor proper. Phosphoamino acid analysis of the 185-kDa protein/PDGF receptor, precipitated with the antiphosphotyrosine immune serum, revealed that it, in addition to phosphotyrosine, also contained phosphoserine. PDGF also consistently stimulated the phosphorylation of components of Mr values of 300,000 to 200,000, 115,000, 72,000, 54,000, 45,000, and 35,000. Some of these components may be involved in the intracellular transmission of the PDGF-induced mitogenic signal.

• Johnsson A et al.
• The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.
• EMBO J. 1984; 3: 921-8
• Display abstract

• The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.

• Josephs SF, Ratner L, Clarke MF, Westin EH, Reitz MS, Wong-Staal F
• Transforming potential of human c-sis nucleotide sequences encoding platelet-derived growth factor.
• Science. 1984; 225: 636-9
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• The nucleotide sequence of a transforming human c-sis complementary DNA shows an open reading frame 723 base pairs in length located downstream from an in-phase terminator thymine-guanine-adenine codon. Sequences within this region were identical to those previously determined for the exons of the normal human c-sis gene. Thus, the predicted transforming product, a protein of 27,281 daltons, may be the actual precursor for normal human platelet-derived growth factor chain A.

• Bowen-Pope DF, Vogel A, Ross R
• Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor receptors accompany transformation by a wide spectrum of agents.
• Proc Natl Acad Sci U S A. 1984; 81: 2396-400
• Display abstract

• A series of nontransformed human and murine cells and derivative cell lines transformed by methylcholanthrene; by simian virus 40, Kirsten and Moloney murine sarcoma viruses, simian sarcoma virus, and adenovirus; and by a "spontaneous" event in culture were examined for the expression of receptors for the platelet-derived growth factor (PDGF) and for production of substances able to compete with 125I-labeled PDGF for binding to the cell-surface PDGF receptor. In each case, transformation resulted in a 50-100% decrease in available PDGF receptors. All transformed cells except the methylcholanthrene-transformed mouse cells produce a PDGF competitor into the conditioned medium. Levels of PDGF competitor in conditioned medium at the end of a 48-hr collection were as high as 2 ng/ml--high enough to be measured by radioreceptor assay diluted 1:30 and to maximally stimulate [3H]thymidine incorporation by human fibroblasts. The PDGF competitor activity detected in a radioreceptor assay does not reflect irreversible (e.g., proteolytic) damage to the receptor of test cells since its effects are reversed by acetic acid dissociation. Antiserum against human PDGF neutralizes 20-80% of the PDGF competitor found in conditioned medium from different transformed human cells and 100% of the activity from normal human endothelial cells. The possibility that induction of expression of the cellular PDGF gene may be involved in the mechanism of transformation of PDGF-responsive mesenchymal cells is discussed.

• Lopez-Rivas A, Stroobant P, Waterfield MD, Rozengurt E
• Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells.
• EMBO J. 1984; 3: 939-44
• Display abstract

• Addition of porcine platelet-derived growth factor (PDGF) to quiescent cultures of Swiss 3T3 cells caused a marked, dose-dependent stimulation of Na+ influx and Na-K pump-mediated 86Rb+ uptake. Porcine PDGF (a single component in SDS polyacrylamide gels) stimulated ion fluxes to the same maximal extent as partially purified preparations, and exhibited half-maximal effect at 6 ng/ml (2 X 10(-10) M). Maximal effect was achieved at 30 ng/ml (10(-9) M). In the presence of insulin, PDGF elicited mitogenesis at comparable concentrations. PDGF stimulated ion uptake in a time-dependent fashion; maximal effect was obtained after 5 min of exposure to the growth factor. PDGF stimulates Na+ influx via an amiloride-sensitive pathway, suggesting that PDGF enhances the activity of a Na+/H+ antiport system. In accordance with this possibility, the mitogen caused an increase of intracellular pH by 0.15 pH units, as judged by the steady-state distribution of labelled 5,5-dimethyloxazolidine-2,4-dione (DMO). Porcine PDGF stimulated E-type prostaglandin synthesis and cAMP accumulation but these events could be dissociated from the stimulation of the ionic fluxes, which was detected within minutes and was not blocked by indomethacin. It is suggested that PDGF elicits multiple signals to stimulate cell proliferation in 3T3 cells.

• Thiel HJ, Hafenrichter R
• Simian sarcoma virus transformation-specific glycopeptide: immunological relationship to human platelet-derived growth factor.
• Virology. 1984; 136: 414-24
• Display abstract

• The simian sarcoma virus transformation-specific glycopeptide (SSV-TrSgp) represents a proteoglycan which is released from SSV-transformed cells and can be detected by an autologous goat serum against SSV nonproducer cells (SSV-NP serum) (H.-J. Thiel, R. Hafenrichter, and B. Gregor, 1984, Virology 134, 138-147). This antiserum has now been shown to react also with human platelet-derived growth factor (PDGF). Antiserum to PDGF precipitated a glycosylated molecule from the tissue culture supernatant of SSV-NP cells. The respective antigen was identified as the SSV-TrSgp (after immunoprecipitation including enzymatic treatment with chondroitinases). The anti-SSV-TrSgp reactivity of both the anti-PDGF serum and the SSV-NP serum could be absorbed by pure PDGF. Therefore, the SSV-TrSgp is apparently immunologically related to human PDGF. Additional studies indicated that the SSV-TrSgp protein backbone and PDGF have very similar molecular weights.

• Niman HL
• Antisera to a synthetic peptide of the sis viral oncogene product recognize human platelet-derived growth factor.
• Nature. 1984; 307: 180-3
• Display abstract

• It has recently been reported that the sequences of the sis oncogene of simian sarcoma virus (SSV) and of human platelet-derived growth factor (PDGF) are very similar, establishing the most solid link yet between the mitogenic actions of growth factors and the transforming proteins of retroviruses. To investigate molecular mechanisms of transformation I have produced antisera against synthetic peptides corresponding to segments of the protein sequences predicted by the nucleotide sequences of viral oncogenes. Applying this approach to the case of sis and PDGF, I report here the results of probing outdated human platelets with an antiserum directed against a synthetic peptide representing residues 139-155 of the predicted sequence of the SSV transforming protein, p28sis (ref. 3). I detected peptides of apparent molecular weights (MWs) 30,000 to 31,000 (30-31K) and 16-18K, which correspond to the apparent molecular weights of nonreduced and reduced PDGF. In addition, a peptide of MW 21,000 was detected in platelets and a protein of MW 56,000 was detected in SSV-infected marmoset cells.

• Kellermayer M, Prestayko AW, Hazlewood CF
• Platelet-derived growth factor (PDGF) induces intranuclear protein accumulation in 3T3 fibroblasts.
• Exp Cell Res. 1984; 152: 255-9
• Display abstract

• Quiescent 3T3 fibroblasts were grown for a short time in the presence of [3H]amino acids then treated with PDGF, and the nucleo-cytoplasmic distribution of the 3H-labelled proteins was analysed by autoradiography. There was no difference in the total amount of 3H-labelled proteins in PDGF-treated and untreated cells but PDGF induced a significant increase in intranuclear protein accumulation.

• Pinkerton OD, Hokama Y, Shigemura LA
• Immunologic basis for the pathogenesis of pterygium.
• Am J Ophthalmol. 1984; 98: 225-8
• Display abstract

• We examined surgically excised pterygial tissues from 26 patients for the various immunoglobulin classes by direct immunofluorescence. Of the 26 patients, 19 (73.1%) showed positive staining with goat anti-human IgG fluorescein-labeled antibody. Direct immunofluorescence with goat anti-human IgE fluorescein-labeled antibody was found in all 26 samples, varying in fluorescent intensity from 1+ to 4+. Routine histologic staining disclosed an infiltration of small lymphocytes and plasma cells into the pterygium. These results suggest that an immunologic mechanism, possibly Type 1 hypersensitivity, may contribute to the pathogenesis of pterygium.

• Chiu IM, Reddy EP, Givol D, Robbins KC, Tronick SR, Aaronson SA
• Nucleotide sequence analysis identifies the human c-sis proto-oncogene as a structural gene for platelet-derived growth factor.
• Cell. 1984; 37: 123-9
• Display abstract

• The simian sarcoma virus transforming gene, v-sis, encodes a protein, p28sis , that is closely related to human platelet-derived growth factor (PDGF). The human locus related to v-sis was cloned and shown to contain at least five exons corresponding to the v-sis coding region. Nucleotide sequence analysis of these exons revealed that the predicted amino acid sequence of human c-sis differed by 6% from that of the woolly monkey-derived v-sis. These findings imply that the sis proto-oncogene has been well conserved during primate evolution. By comparison of the known amino acid sequences of PDGF peptides with the predicted human c-sis protein, it was possible to demonstrate that this human proto-oncogene is the structural gene encoding one of the two major polypeptides of this potent mitogen for connective tissue cells.

• Seits IF
• [A further step in the discovery of the secret of cancer (the platelet-derived growth factor (PDGF) in the context of the oncogen concept)]
• Vopr Onkol. 1984; 30: 106-16

• Straus DS, Coppock DL
• Growth control variant cell line having increased serum requirement and decreased response to platelet-derived growth factor: reversion by 5-azacytidine.
• J Cell Biol. 1984; 99: 1838-47
• Display abstract

• Variants of the mouse embryo fibroblast X melanoma hybrid clone 100A have been isolated by a procedure that selects against cells that are able to grow in medium containing low concentrations of serum plus insulin. Three variant clones derived from this selection were found to have a much higher serum requirement than the parental clone 100A cells, as evidenced by a very low rate of DNA synthesis and growth in medium containing low concentrations of serum. Two of the variants had approximately double the number of chromosomes as the parental cell line, while one had approximately the same number of chromosomes as the parental cells. One of the variants was very strongly reverted by 5-azacytidine but not by ethyl methanesulfonate, suggesting that it reverted by a nonmutational mechanism such as a stable change in DNA methylation. Analysis of the growth requirements in hormone-supplemented serum-free media of the 100A parent, the INS 471 variant, and revertants of the variant indicated that the variant had a specific deficiency in its growth response to platelet-derived growth factor (PDGF). PDGF dose-response curves obtained with the variant cells were shifted approximately an order of magnitude toward higher PDGF concentrations relative to PDGF dose-response curves obtained with the parental 100A cells. This quantitative increase in PDGF requirement of the INS 471 variant appears to explain the increased serum requirement of this variant. Equilibrium binding experiments performed with 125I-PDGF suggest that the variant does not have a decreased number of PDGF receptors.

• Bowen-Pope DF, Malpass TW, Foster DM, Ross R
• Platelet-derived growth factor in vivo: levels, activity, and rate of clearance.
• Blood. 1984; 64: 458-69
• Display abstract

• Platelet-derived growth factor (PDGF) is a potent mitogen for many cultured connective tissue cells. It is present in concentrated form within the platelet alpha-granules and is believed to be released during platelet degranulation at sites of vascular injury. We have used a sensitive radioreceptor assay to measure PDGF levels in whole blood serum from normal humans [17.5 +/- 3.1 (SD) ng/mL] and baboons (2.7 +/- 1.2 ng/mL). PDGF was not detected in plasma from either species. In addition, plasma was found to substantially reduce the ability of added purified PDGF to bind to the cell surface PDGF receptor on cultured cells, suggesting that plasma may contain a PDGF-binding protein that would serve to inactivate PDGF released into plasma. Calculations of PDGF concentrations in serum have been corrected for the effects of the binding protein. 125I-PDGF injected intravenously into normal baboons was cleared rapidly from the plasma (t1/2 = two minutes). The rapid clearance of 125I-PDGF did not result from iodination damage, as purified unlabeled PDGF was cleared with comparable kinetics. The rapid clearance of purified and iodinated PDGF did not result from changes in PDGF structure during purification or from removal of PDGF-associated proteins during purification, as PDGF present in freeze-thaw lysates of fresh platelets was cleared equally rapidly. We conclude that release of PDGF at sites of vascular injury would greatly increase the local concentration of PDGF and that PDGF not localized to the site of injury would be rapidly cleared from the circulation.

• Huang JS, Huang SS, Deuel TF
• Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma-binding protein.
• J Cell Biol. 1983; 97: 383-8
• Display abstract

• The platelet-derived growth factor (PDGF) is the principal mitogen in serum for cultured cells of mesenchymal origin. PDGF also is a potent chemotactic protein for inflammatory cells and for cells required for wound repair. Because activity levels of PDGF in biological fluids are difficult to measure, we attempted to develop a radioimmunoassay for PDGF. Rabbits were immunized with purified PDGF; the antiserum obtained was monospecific for PDGF in immunodiffusion analysis against concentrated platelet lysates, serum, and plasma. A radioimmunoassay for PDGF was developed with a sensitivity of congruent to 0.2 ng/ml. Levels of PDGF in plasma/serum were measured and compared with PDGF levels determined by a receptor-competition assay and by a standard biological assay measuring incorporation of [3H]thymidine into 3T3 cells. Radioimmunoassay showed apparent PDGF levels of 50 ng/ml in human plasma and 103 ng/ml in serum. The 50 ng/ml PDGF in plasma was unexpected because the plasma samples contained little or no platelet release products as determined by very low levels of platelet factor 4. We therefore sought an immunologically reactive PDGF molecule in human plasma. No immunologically reactive protein was detected by immunodiffusion analysis or when plasma was treated with an immunoaffinity gel. Subsequently, a 125I-PDGF-binding protein was identified; the 125I-PDGF-plasma-binding protein complex was not reactive with anti-PDGF immunoglobulin. Correction for 125I-PDGF bound by the plasma-binding protein established serum levels of PDGF of congruent to 50 ng/ml; congruent to 50 ng/ml PDGF was found in serum by radioreceptor-competition assays and by mitogenic assays as well. The plasma-binding protein may serve to clear PDGF released in the circulation, thereby limiting PDGF activity to its local interactions at the site of blood-vessel injury.

• Nishimura J, Deuel TF
• Platelet-derived growth factor stimulates the phosphorylation of ribosomal protein S6.
• FEBS Lett. 1983; 156: 130-4
• Display abstract

• The human platelet derived-growth factor (PDGF) is both a potent mitogen and a strong chemoattractant protein for cells involved in inflammation and repair. In seeking mechanisms by which PDGF might initiate specific activities in target cells, it was found that highly purified PDGF stimulates the phosphorylation of an Mr approximately 33000 protein in confluent Swiss mouse 3T3 cells [Biochem. Biophys. Res. Commun. (1981) 103, 355-361]. The Mr approximately 33000 protein has now been recovered in polysomes by differential centrifugation and identified as ribosomal protein S6 by two-dimensional polyacrylamide gel electrophoresis.

• Burns CP, Rozengurt E
• Serum, platelet-derived growth factor, vasopressin and phorbol esters increase intracellular pH in Swiss 3T3 cells.
• Biochem Biophys Res Commun. 1983; 116: 931-8
• Display abstract

• Addition of the growth-promoting agents phorbol esters, vasopressin, platelet-derived growth factor or fresh serum to quiescent cultures of Swiss 3T3 cells causes a significant increase in the uptake of 5,5-dimethyl oxazolidine-2-4-dione (DMO), a sensitive measure of intracellular pH. The results further indicate that cytoplasmic alkalinization is an early event associated with the action of a variety of mitogenic compounds.

• Graves DT, Owen AJ, Antoniades HN
• Evidence that a human osteosarcoma cell line which secretes a mitogen similar to platelet-derived growth factor requires growth factors present in platelet-poor plasma.
• Cancer Res. 1983; 43: 83-7
• Display abstract

• A human osteosarcoma-derived cell line, 2T, grows almost as well in medium supplemented with platelet-poor plasma (PPP) as it does in medium containing fetal bovine serum. Human diploid fibroblasts, in contrast, will not grow in medium containing PPP unless human platelet-derived growth factor (PDGF) is added. PPP treated with carboxymethyl-Sephadex at pH 7.4 was able to support 2T cell proliferation, although at a reduced rate compared to untreated PPP. Addition of PDGF to carboxymethyl-Sephadex-treated PPP did not restore the growth rate. However, insulin-like growth factor isolated from human plasma did partially restore the activity of carboxy-methyl-Sephadex-treated PPP. Medium conditioned by 2T cells was mitogenic for quiescent BALB/c3T3 cells and human diploid fibroblasts. Antiserum to human PDGF blocked the mitogenic activity of the conditioned medium. Partial characterization confirmed the biochemical similarity to PDGF. The data are consistent with the hypothesis that these osteosarcoma-derived cells have growth factor requirements similar to those of normal mesenchymal cells but are able to overcome the normal growth limitations by autocrine secretion of PDGF-like mitogens.

• Chesterman CN, Walker T, Grego B, Chamberlain K, Hearn MT, Morgan FJ
• Comparison of platelet-derived growth factor prepared from release products of fresh platelets and from outdated platelet concentrates.
• Biochem Biophys Res Commun. 1983; 116: 809-16
• Display abstract

• Platelet-derived growth factor was isolated from the release products of washed, human platelets and from freeze-thawed outdated platelet concentrates. On the basis of sodium dodecyl sulphate polyacrylamide gel electrophoresis and amino acid sequence determination we conclude that platelet derived growth factor released from platelets by the agonist thrombin (EC 3.4.4.13) is structurally similar to that isolated from lysed platelets and from platelet concentrates stored for more than 72 hr at room temperature.

• Antoniades HN, Hunkapiller MW
• Human platelet-derived growth factor (PDGF): amino-terminal amino acid sequence.
• Science. 1983; 220: 963-5
• Display abstract

• Human platelet-derived growth factor (PDGF) obtained from outdated human platelets was subjected to amino-terminal amino acid sequence analysis by automated Edman degradation. Despite the apparent presence of limited proteolytic degradation of the protein derived from this method, the sequence analysis reveals two primary peptide sequences and suggests that active PDGF is composed of two, possibly homologous, peptides linked by a disulfide bond or bonds.

• Merz B
• Publishing flurry attends oncogene-growth factor link.
• JAMA. 1983; 250: 11271131-11271131

• Lechner JF, McClendon IA, LaVeck MA, Shamsuddin AM, Harris CC
• Differential control by platelet factors of squamous differentiation in normal and malignant human bronchial epithelial cells.
• Cancer Res. 1983; 43: 5915-21
• Display abstract

• Recently, we developed a nutritionally optimal medium for rapid clonal growth (greater than 1 population doubling/day) of normal human bronchial epithelial (NHBE) cells. Adding fetal bovine or adult human blood-derived serum to this medium depresses the clonal growth rate of NHBE cells in a dose-dependent fashion. In contrast, 10 representative lines of human lung carcinomas either replicate poorly or fail to grow at all when inoculated at clonal density in serum-free medium, and their rates of multiplication increase in direct proportion to the amount of blood-divided serum added to the optimized medium. Thus, the growth factor requirements of these lung carcinoma cell lines are significantly different from those of their normal counterparts. Blood-divided serum reduces the clonal growth rate of NHBE cells by specifically inducing the normal cells, but not lung carcinoma cells, to undergo squamous differentiation. The differentiation-inducing activity was found in platelet lysates. In addition, a growth-inhibiting activity that did not induce squamous differentiation of NHBE cells was also identified in partially purified commercial preparations of platelet-derived growth factor. This observation was in marked contrast to results using human bronchial fibroblasts and human lung carcinoma cell lines; the growth rate of the former was significantly stimulated by commercial preparations of platelet-derived growth factor, whereas the growth rates of the tumor cell lines were unaffected. These results indicate that an aberration in the cellular differentiation as assayed in vitro is positively correlated with cancer and suggests that decreased responsiveness to inducer(s) of differentiation may be a major aspect of bronchial cell carcinogenesis.

• Mellstroom K, Hoglund AS, Nister M, Heldin CH, Westermark B, Lindberg U
• The effect of platelet-derived growth factor on morphology and motility of human glial cells.
• J Muscle Res Cell Motil. 1983; 4: 589-609
• Display abstract

• Platelet-derived growth factor (PDGF) is a mitogen for several cell types in culture. It is documented in this work that one of the earliest effects of PDGF on serum-starved glial cells is an induction of intensive motile activity. Within the first minute after the addition of PDGF thin membrane lamellae grow out around almost all of the cell circumference. Later, circular arrangements of small ruffles appear on the dorsal surface of the cells. These rings of ruffles vary in size and some encircle almost the whole cell. The organization of the peripheral weave of microfilaments in the PDGF-induced advancing lamellae was closely similar to that of normally growing cells. In the regions of the circular arrangements of ruffles there was an extensive reorganization of the surface actin with unusual arrangements of microfilament bundles and polygonal networks. There was also a general intensification of the translocation of membrane ruffles and spikes from the cell periphery towards the centre of the cell, increased micropinocytotic activity and shuttling of intracellular particles.

• Hendrickson SL, Scher CD
• Platelet-derived growth factor-modulated translatable mRNAs.
• Mol Cell Biol. 1983; 3: 1478-87
• Display abstract

• The treatment of density-arrested BALB/c 3T3 cells with electrophoretically homogeneous or highly purified preparations of the platelet-derived growth factor (PDGF) stimulated the rapid and selective accumulation of several species of abundant mRNA identified by cell-free translation. These translatable mRNAs appeared long before entry into the S phase. Less PDGF was required for selective mRNA accumulation than for PDGF-modulated DNA synthesis. The translatable mRNAs also accumulated after addition of the epidermal growth factor but not after addition of insulin or platelet-poor plasma. Their selective accumulation was blocked by addition of actinomycin D. Three classes of PDGF-modulated mRNAs were defined. An early (primary) RNA appeared within 30 to 60 min of PDGF addition; its accumulation was not blocked by cycloheximide. Another early mRNA also appeared within 60 min, but treatment with both PDGF and cycloheximide was required for optimal accumulation. A third class, secondary RNAs, began to accumulate later at 90 to 120 min; the appearance of this class was inhibited by cycloheximide. One- and two-dimensional gel electrophoresis of translation products demonstrated that a spontaneously transformed BALB/c 3T3 (ST2-3T3) cell line, which does not require PDGF or epidermal growth factor for growth, constitutively accumulated the secondary growth factor-regulated mRNAs. The accumulation of these translatable mRNAs may be required for PDGF-modulated DNA synthesis.

• Kakishita E
• Role of the platelet in maintaining the integrity of the vascular endothelial cell.
• Nippon Ketsueki Gakkai Zasshi. 1983; 46: 1551-7

• Dainiak N, Davies G, Kalmanti M, Lawler J, Kulkarni V
• Platelet-derived growth factor promotes proliferation of erythropoietic progenitor cells in vitro.
• J Clin Invest. 1983; 71: 1206-14
• Display abstract

• To investigate serum requirements for optimal erythropoiesis in vitro, we studied the response of erythroid progenitor cell proliferation in culture to platelet-derived growth factor (PDGF). Human bone marrow cells cultured with platelet-poor plasma-derived serum (PDS) form fewer erythroid colonies than do cells cultured with human whole blood serum or fetal calf serum (P less than 0.05). Treatment of washed platelets with thrombin releases a low molecular weight (less than 100,000) factor that enhances colony growth. This secreted factor appears to be PDGF, based upon the ability of partially purified and electrophoretically pure PDGF to restore colony-forming capacity of PDS-containing cultures to 70-96% of the level found in control cultures with whole blood serum or fetal calf serum. Enhancement of colony growth by PDGF was noted only in marrow cultures supplemented with erythropoietin and PDS. Presence of bioactive erythropoietin in PDGF preparations was excluded by assay in hypertransfused, polycythemic mice, and in fasted rats. Although PDGF stimulates erythroid burst formation in marrow cultures containing optimal concentrations of burst-promoting activity (BPA), it does not influence proliferation of circulating erythroid bursts, regardless of BPA concentration added to culture. We conclude that PDGF is a serum determinant of optimal erythroid progenitor cell proliferation in marrow culture. The activity of PDGF is distinct from that of the apparent erythroid specific growth factors erythropoietin and BPA.

• Williams LT, Antoniades HN, Goetzl EJ
• Platelet-derived growth factor stimulates mouse 3T3 cell mitogenesis and leukocyte chemotaxis through different structural determinants.
• J Clin Invest. 1983; 72: 1759-63
• Display abstract

• Platelet-derived growth factor (PDGF) stimulates both proliferation of fibroblasts and chemotaxis of leukocytes. In this study we compared the mitogenic and chemotactic activities of native PDGF and reduced PDGF. Reduction of PDGF (Mr = 32,000) to its constituent polypeptides (Mr = 14,000 and 17,000) caused a loss of the ability to stimulate proliferation of Balb/c 3T3 cells. However, reduced PDGF retained virtually all of its activity as a chemotactic agent for human neutrophils and monocytes. A half-maximal chemotactic response to both native and reduced PDGF occurred at a concentration of approximately 0.08 nM for neutrophils and 0.1 nM for monocytes. The maximal chemotactic response to reduced PDGF was at least as great as the maximal response to native PDGF. Both native and reduced PDGF stimulated the release of the lysosomal enzyme, beta-glucosaminidase, from neutrophils with a half-maximal response at less than 0.1 nM. However, the net maximum release of this enzyme by PDGF (and reduced PDGF) was significantly less than that stimulated by a maximal concentration of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. These results indicate that different structural determinants are required for the proliferative response of 3T3 cells to PDGF and for the chemotactic response of leukocytes to PDGF.

• Inglot AD, Inglot O
• Hormonal-like modulation of growth of moloney virus-induced tumors by interferon and platelet-derived growth factor.
• Arch Immunol Ther Exp (Warsz). 1983; 31: 243-8
• Display abstract

• Continued intramuscular (i.m.) administration of highly purified preparations of mouse interferon (MuIFN) and/or bovine platelet-derived growth factor (PDGF) to the Moloney sarcoma virus (MSV) infected Balb/c mice resulted in a hormonal-like modulation of growth of tumors. Large dose MuIFN treatment (10(5) units per mouse daily, for 14 days) inhibited the MSV-induced tumors. On the other hand, small-dose MuIFN treatment (100 units per mouse daily, for 14 days or 10(4) units per mouse, every second day in 3-4 doses) enhanced the tumor growth. The simultaneous administration of large doses of MuIFN and PDGF significantly diminished the antitumor effect of IFN. The results suggest that the negative and positive interaction between IFN and PDGF takes place in vivo.

• Deuel TF, Huang JS, Huang SS, Stroobant P, Waterfield MD
• Expression of a platelet-derived growth factor-like protein in simian sarcoma virus transformed cells.
• Science. 1983; 221: 1348-50
• Display abstract

• The near identity of the partial amino acid sequence of human platelet-derived growth factor (PDGF) and that predicted for p28sis, the putative transforming protein of the simian sarcoma virus (SSV), suggests expression of a growth factor activity may be central for transformation by SSV. It is now reported that SSV-transformed cells but not control cells contain a growth factor activity that is identical to PDGF in immunoassay, in mitogenic dose response, and in specific mitogenic activity. The protein immunoprecipitated by antiserum to human PDGF has an apparent molecular weight of 20,000, identical to that of p20sis, the putative intracellular degradation product of p28sis. The results support the concept that expression of a PDGF-like molecule, which appears to be the product of the viral-sis gene, is responsible for the abnormal regulation of growth is SSV-transformed cells.

• Cassel D, Rothenberg P, Zhuang YX, Deuel TF, Glaser L
• Platelet-derived growth factor stimulates Na+/H+ exchange and induces cytoplasmic alkalinization in NR6 cells.
• Proc Natl Acad Sci U S A. 1983; 80: 6224-8
• Display abstract

• Stimulation of Na+/H+ exchange by growth factors has been implicated as a mechanism allowing quiescent cells to resume growth because of a predicted elevation of intracellular pH(pHi). We tested this prediction in NR6 cells by using a further development of our technique for pHi measurement, based on introduction of the fluorescent pH indicator 4',5'-dimethylfluorescein (pKa = 6.75) coupled to dextran into the cytoplasm. Addition of the potent mitogens platelet-derived growth factor (PDGF) or serum to NR6 cells stimulates an amiloride-sensitive 22Na+ uptake and causes an elevation of pHi. The PDGF-dependent pHi increase follows a lag period of approximately equal to 2 min, reaches a maximal level within 10 min (delta pHi approximately equal to 0.1 at an external pH of 7.18), and remains at this level for at least 1 hr. Serum addition initially produces a large elevation of pHi, which later declines to a level similar to that obtained with PDGF. The effects of PDGF and serum are partially additive (delta pHi approximately equal to 0.14). The magnitude of pHi elevation by PDGF decreases with increasing extracellular pH. Serum- and PDGF-dependent elevations of pHi are inhibited by amiloride and by eliminating Na+ from the medium. Under conditions in which Na+/H+ exchange is inhibited, PDGF and serum induce an initial cytoplasmic acidification that does not show a lag period. The results show that a single purified growth factor, as well as serum, can promote a sustained elevation of pHi by stimulating Na+/H+ exchange. The extent of pHi elevation may be modulated by the concomitant stimulation by the growth factor of a process generating H+ within the cell.

• Kellermayer M
• [Cell-growth factor derived from human thrombocytes]
• Orv Hetil. 1983; 124: 1485-8

• Klenow H, Flodgaard H
• Both hypoxanthine and adenosine stimulate DNA synthesis independently in serum-starved L cells treated with platelet protein.
• Proc Natl Acad Sci U S A. 1983; 80: 7420-3
• Display abstract

• The effect of human platelet factors and purine derivatives on DNA synthesis has been investigated in mouse fibroblast-like L cells whose growth was arrested by serum starvation. When such cells were exposed to diluted platelet extract (e.g., 35 micrograms of protein per ml), a stimulatory effect on net DNA synthesis was observed. This effect was almost abolished by dialysis of the extract. The stimulation was, however, recovered by supplementing the diluted and dialyzed extract with hypoxanthine or adenosine. Similar phenomena were observed in pulse-labeling experiments performed with [3H]thymidine. In this case, however, there was a marginal stimulatory effect of adenosine or hypoxanthine alone. When the cells were treated with saturating concentrations of pure platelet-derived growth factor (PDGF), a stimulatory effect on pulse labeling was again obtained by the simultaneous presence of hypoxanthine or adenosine. In serum-starved cells of a mutant line of L cells deficient in hypoxanthine phosphoribosyltransferase, there was, however, no stimulatory effect on pulse labeling by hypoxanthine when it was added alone or together with either PDGF or diluted dialyzed platelet extract. It is suggested that the stimulation of DNA synthesis by the purine derivatives in the presence of a certain type of platelet proteins, probably involving PDGF, may be explained by their function as precursors for a purine ribonucleotide pool that is specifically related to DNA synthesis. Treatment of serum-starved L cells with high concentrations of dialyzed platelet extract (e.g., 240 micrograms of protein per ml) showed that platelets contain an additional type of factor that may substitute for the requirement of adenosine or hypoxanthine for DNA synthesis to take place. It is suggested that the effect of this type of factor may be to activate the catabolic activity of the purine salvage pathway.

• Wettenhall RE, Chesterman CN, Walker T, Morgan FJ
• Phosphorylation sites for ribosomal S6 protein kinases in mouse 3T3 fibroblasts stimulated with platelet-derived growth factor.
• FEBS Lett. 1983; 162: 171-6
• Display abstract

• Platelet release products and purified platelet-derived growth factor stimulated the phosphorylation of ribosomal protein S6 in cultured mouse Balb/c 3T3 fibroblasts. The post-nuclear fraction of the stimulated cells was enriched in S6 kinase activity specific for sites resembling those phosphorylated within intact cells in response to PDGF as determined by tryptic peptide mapping. 3T3-S6 sites closely resembled those phosphorylated in S6 of rat hepatocytes stimulated with insulin and included sites for both cAMP-dependent and independent kinases.

• Robbins KC, Antoniades HN, Devare SG, Hunkapiller MW, Aaronson SA
• Structural and immunological similarities between simian sarcoma virus gene product(s) and human platelet-derived growth factor.
• Nature. 1983; 305: 605-8
• Display abstract

• The predicted amino acid sequence of the simian sarcoma virus (SSV) transforming gene product, p28sis, closely corresponds to that of human platelet-derived growth factor (PDGF). We demonstrate that p28sis rapidly undergoes a series of discrete processing steps including dimer formation and proteolytic digestion to yield molecules structurally and immunologically resembling biologically active PDGF.

• Nilsson J, Thyberg J, Heldin CH, Westermark B, Wasteson A
• Surface binding and internalization of platelet-derived growth factor in human fibroblasts.
• Proc Natl Acad Sci U S A. 1983; 80: 5592-6
• Display abstract

• Surface binding and uptake of platelet-derived growth factor (PDGF) in human fibroblasts cultivated in vitro were studied by quantitative electron microscopic autoradiography using 125I-labeled PDGF and by indirect immunofluorescence using PDGF antibodies. After 120 min at 12 degrees C PDGF was found preferentially in coated regions of the plasma membrane. Warming the cells to 37 degrees C initiated rapid ingestion of the factor via small vesicles, usually lacking a membrane coat. After 10 min PDGF started to appear in lysosomes, and it showed maximal concentration within these organelles after 30 min. There were also signs of passage of PDGF through the Golgi complex, but only after 60 min. Treatment of the cells with chloroquine, a weak base that inhibits intralysosomal degradation, prevented disappearance of tracer from the lysosomes. The observations indicate that PDGF was internalized via coated regions of the plasma membrane and carried to lysosomes for degradation. Subsequent appearance of tracer within the Golgi complex could reflect receptor-ligand complexes that escaped degradation and were recirculated back to the cell surface.

• Marx JL
• Cooperation between oncogenes. Investigators focus on cooperation between two or more oncogenes to help explain the multistep development of human cancers.
• Science. 1983; 222: 602-3

• Singh JP, Chaikin MA, Pledger WJ, Scher CD, Stiles CD
• Persistence of the mitogenic response to platelet-derived growth factor (competence) does not reflect a long-term interaction between the growth factor and the target cell.
• J Cell Biol. 1983; 96: 1497-502
• Display abstract

• Quiescent BALB/c-3T3 cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA even if PDGF is removed from cell culture medium prior to the onset of DNA synthesis. We have suggested that persistence of the PDGF-induced competent state reflects a rapidly induced and relatively stable biochemical change within the target cells. Others suggest that the phenomenon reflects a long-term association between PDGF and its target cells or perhaps between PDGF and the cell culture dish. This controversy has been addressed (a) by examining the effect of anti-PDGF antibodies on PDGF-induced competence and (b) by studying the chemical fate of 125I-labeled PDGF. Anti-PDGF antibodies inactive both soluble and surface-bound PDGF. However, if quiescent 3T3 cells are exposed to PDGF for as little as 30 min, subsequent addition of these antibodies to the culture medium does not prevent the mitogenic response. Under conditions where the PDGF-induced competent state decays stochastically with a t1/2 of 18-20 h, cell-associated 125I-PDGF decays with a t1/2 of approximately 50 min. These data do not support the concept that persistence of the PDGF-induced competent state reflects a long-term association between PDGF and the target cells or between PDGF and the culture dish.

• Cochran BH, Reffel AC, Stiles CD
• Molecular cloning of gene sequences regulated by platelet-derived growth factor.
• Cell. 1983; 33: 939-47
• Display abstract

• We have screened a cDNA library for gene sequences that are regulated by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells. Of 8000 clones screened, less than 14 independent PDGF-inducible sequences were found. Two of these (KC and JE) were studied in detail. By hybrid-selection and translation the KC and JE mRNAs encode 10,000 and 19,000 dalton polypeptides, respectively. In the absence of PDGF, the JE and KC sequences correspond to low abundance mRNAs. One hour after addition of PDGF their abundance level can be increased 10- to 20-fold. Within 4 hr, a 60-fold induction of JE can be attained. Nanogram per ml quantities of pure PDGF regulate these sequences whereas microgram/ml quantities of chemically unrelated mitogens (EGF, insulin, or platelet-poor plasma) have either a weak or an undetectable effect. Inhibitors of protein synthesis block the progression of quiescent 3T3 cells through G1 into S phase; however these drugs do not block the induction of KC and JE by PDGF. This result indicates that these sequences correspond to "early genes" which are not induced as a consequence of cell growth, but rather are directly regulated by PDGF.

• Olashaw NE, Pledger WJ
• Association of platelet-derived growth factor-induced protein with nuclear material.
• Nature. 1983; 306: 272-4
• Display abstract

• Platelet-derived growth factor (PDGF) has been proposed to initiate the cell-cycle traverse of density-arrested BALB/c-3T3 cells by rendering quiescent cells 'competent' to respond to 'progression' factors contained in platelet-poor plasma (PPP). PDGF-treated cells remain competent for many hours following PDGF removal; subsequent addition of PPP triggers G0-G1 traversal and entry into S phase. Numerous observations suggest that the competent state reflects the existence of a stable PDGF-induced 'second signal' as opposed to a persistent association of PDGF with cells. Several unique proteins have been shown to be synthesized in response to PDGF; of these, the dose-dependent production of 'pI' (molecular weight 29,000) was found to correlate closely with the induction of competence. We report here the further characterization of pI in terms of its time-dependent synthesis, intracellular location and stability. Electrophoretic analysis of nuclear and non-nuclear extracts of PDGF-treated BALB/c-3T3 cells demonstrated that pI is synthesized during the first 6 h of G0-G1, is associated with nuclear material and is stable for 9-12 h. These findings are consistent with the proposed role of pI as the cellular mediator of PDGF.

• Burke JM
• Cultured retinal glial cells are insensitive to platelet-derived growth factor.
• Exp Eye Res. 1982; 35: 663-9

• Johnsson A, Heldin CH, Westermark B, Wasteson A
• Platelet-derived growth factor: identification of constituent polypeptide chains.
• Biochem Biophys Res Commun. 1982; 104: 66-74

• Katoh Y, Takayama S
• Conversion of platelet-derived growth factor-dependent cells to growth factor-independent cells during chemical carcinogenesis in vitro.
• Carcinogenesis. 1982; 3: 867-73
• Display abstract

• The growth responses of hamster dermal fibroblasts (HDF cells) to platelet-derived growth factor (PDGF) during chemical carcinogenesis in vitro were investigated. Normal HDF cell grew in medium with whole blood serum (WBS), but not in medium with plasma-derived serum (PDS). However, they grew in PDS medium when PDGF was added. In contrast to control cultures which finally stopped proliferating, 7 out of 8 cell strains treated with 4-nitroquinoline-1-oxide (4NQO) or N-methyl-N'-nitro-nitrosoguanidine changed to cell lines. Later, 5 of these 7 cell lines became able to grow in PDS medium in association with ability to grow in soft agar. When clonal cell lines were isolated at early stages of carcinogenesis while parental cell lines were still PDGF-dependent, most of them gradually became PDGF-independent as well. Dialysed cell lysates of transformed HDF cells showed strong growth stimulating activity on normal HDF cells in PDS medium. Thus, this conversion of PDGF-dependent cells to PDGF-independent cells was correlated with the appearance of a growth factor(s) produced specifically by transformed HDF cells.

• Seppa H, Grotendorst G, Seppa S, Schiffmann E, Martin GR
• Platelet-derived growth factor in chemotactic for fibroblasts.
• J Cell Biol. 1982; 92: 584-8
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• Chemotaxis assays in modified Boyden chambers were used to detect fibroblast chemoattractants in materials released from early-stage inflammatory cells, namely, mast cells, platelets, and neutrophils. Strong attractant activity was found in substances released from platelets. This activity was accounted for mainly by the platelet-derived growth factor (PDGF), which is released from the platelets and which was active as a chemoattractant at 0.5-1.0 mitogenic units/ml. The mitogenic activity of purified PDGF, measured by [3H]thymidine incorporation, occurs at a similar concentration range. By varying the gradient of PDGF, we demonstrated that PDGF stimulates chemotaxis rather than random motility. Preincubation of suspensions of fibroblasts in the presence of PDGF decreased the subsequent migration of cells to a gradient of PDGF as well as to a gradient of fibronectin, which is also in attractant for fibroblasts. The chemotactic response of fibroblasts to PDGF was not inhibited by hydroxyurea or azidocytidine but was inhibited by actinomycin D and cycloheximide, suggesting that synthesis of RNA and proteins but not of DNA is required for the chemotactic response to occur. Fibroblast growth factor, epidermal growth factor, nerve growth factor, and insulin were not chemotactic for human skin fibroblasts, suggesting that the chemoattractant activity of PDGF for fibroblasts is not a general property of growth factors and mitogens. These results suggest that PDGF could have two functions in wound healing: to attract fibroblasts to migrate into the clot and then to induce their proliferation.

• Raines EW, Ross R
• Platelet-derived growth factor. I. High yield purification and evidence for multiple forms.
• J Biol Chem. 1982; 257: 5154-60
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• Platelet-derived growth factor has been purified from human outdated platelet-rich plasma with a 21% overall yield. This five-step procedure represents a 500,000-fold purification over serum. 6.4 ng/ml (2 X 10(-10) M) of purified platelet-derived growth factor stimulated DNA synthesis in quiescent, density arrested cultures of Swiss 3T3 cells to a level equivalent to that produced by 5% calf serum. The growth-promoting activity and specific binding to 3T3 cells has been shown to be associated with four entities of molecular weights: 31,000, 29,000, 28,500, and 27,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. In the presence of 2-mercaptoethanol, the four molecular species are converted to three inactive chains of lower molecular weights: 17,500, 16,000, and 14,400. Other methods of chemically cleaving the disulfide bonds were investigated: reductive cleavage by S-sulfonation, reductive cleavage with dithiothreitol, and performic acid oxidation. In all cases, mitogenic activity was reduced by 80-100%. Attempts to restore mitogenic activity after reduction were unsuccessful. Two-dimensional 125I-peptide mapping of the nonreduced and reduced multiple forms was also investigated. The four nonreduced moieties gave basically identical maps. The maps of the reduced 17,500- and 16,000-dalton chains were also essentially identical, but the map of the reduced 14,400-dalton chain was significantly different. We propose that platelet-derived growth factor consists of two polypeptide chains: a 14,400-dalton chain and either a 17,500- or a 16,000-dalton chain.

• Nister M, Heldin CH, Wasteson A, Westermark B
• A platelet-derived growth factor analog produced by a human clonal glioma cell line.
• Ann N Y Acad Sci. 1982; 397: 25-33

• Leslie CC, Antoniades HN, Geyer RP
• Stimulatin of phospholipid and cholesterol ester synthesis by platelet-derived growth factor in normal and homozygous familial hypercholesterolemia human skin fibroblasts.
• Biochim Biophys Acta. 1982; 711: 290-304
• Display abstract

• Pure human platelet-derived growth factor at nanogram levels stimulates cholesterol ester, phospholipid and DNA synthesis in normal and familial hypercholesterolemia mutant human skin fibroblasts. Stimulation of DNA synthesis did not begin until 15-24 h after addition of platelet-derived growth factor to quiescent normal and FH mutant fibroblasts, In contrast, stimulation of [3H]oleic acid incorporation into cholesterol ester and phospholipid was evident 3-6 h after the addition of platelet-derived growth factor. In the normal cells, the rate of cholesterol ester synthesis was maximal at 24 h, then rapidly declined. Compared to the normal cells, cholesterol esterification was much lower in the FH cells; however, platelet-derived growth factor stimulated the rate of [3H]oleic acid incorporation into cholesterol ester by 5-fold, 31 h after addition of the growth factor. The stimulation of [3H]oleic acid incorporation into cholesterol ester by platelet-derived growth factor was inhibited in both normal and FH mutant skin fibroblasts by progesterone, an inhibitor of acyl-CoA: cholesterol acyltransferase. The rate of cholesterol ester synthesis in the normal cells increased as the concentration of platelet-poor plasma or low density lipoprotein (LDL) was increased, especially in the presence of platelet-derived growth factor. Linearization of the LDL dose-response curve indicated that platelet-derived growth factor increased the rate rather than the affinity of the overall cholesterol esterification system. The rate of cholesterol esterification in the FH mutant cells was highest in the absence of LDL or at low levels of platelet-poor plasma. Consequently, platelet-derived growth factor can stimulate cholesterol ester synthesis by LDL- and non-LDL-mediated processes.

• Owen AJ 3rd, Geyer RP, Antoniades HN
• Human platelet-derived growth factor stimulates amino acid transport and protein synthesis by human diploid fibroblasts in plasma-free media.
• Proc Natl Acad Sci U S A. 1982; 79: 3203-7
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• Purified human platelet-derived growth factor (PDGF) induces an increase in amino acid uptake via system A in quiescent human diploid fibroblasts. Cells must be exposed to PDGF for 45 min to obtain maximum transport stimulation. Transport stimulation requires protein synthesis. Transient exposure to PDGF, alone, in the absence of plasma components can stimulate transport. Acid-insoluble [3H]leucine incorporation is also stimulated by PDGF treatment, and this event also does not require the presence of plasma components. Finally, antiserum to PDGF that blocks PDGF-stimulated DNA synthesis in these cells also blocks PDGF-stimulated amino acid uptake and protein synthesis. Increased amino acid uptake and protein synthesis that occur soon after addition of fresh serum to quiescent cells can be attributed to the action of PDGF acting alone and should be useful as markers for the investigation of early cellular events caused by PDGF.

• Drouet L
• [PDGF (platelet derived growth factor): a regulator of connective tissue proliferation or an artefact?]
• Nouv Rev Fr Hematol. 1982; 24: 153-61
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• Platelets carry, and release after stimulation, mitotic activities for connective tissue cells in culture. One of these activities has been studied carefully: the PDGF, it is a peptide, molecular weight 30,000, pHi 9.8-10, heat stable. Beside this mitotic activity, PDGF bears migrative, chemotactic and metabolic functions. Hypothetical theories have implicated PDGF in different pathological conditions (atherosclerosis, myelofibrosis, ...) but there is no direct proof that PDGF is active in vivo. Recently there has been experimental data suggesting that the mitotic activity of PDGF in vitro could be a technical artefact.

• Deuel TF, Senior RM, Huang JS, Griffin GL
• Chemotaxis of monocytes and neutrophils to platelet-derived growth factor.
• J Clin Invest. 1982; 69: 1046-9
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• The platelet-derived growth factor (PDGF) is shown to be chemotactic for monocytes and neutrophils. Maximum monocyte chemotaxis to PDGF is fully equal to that achieved with C5a and occurs at congruent to 20 ng/ml (congruent to 0.7 nM). Maximum neutrophil chemotaxis is congruent to 60% that achieved with C5A but occurs at congruent to 1 ng/ml (congruent to 32 pM). The chemotactic activity of PDGF is blocked by specific antisera to PDGF and by protamine sulfate, a competitive inhibitor of PDGF binding to cell surfaces. In contrast to PDGF, epidermal growth factor shows no chemotactic activity for inflammatory cells at 0.5-100 ng/ml. The high level of chemotactic activity of PDGF suggests that in addition to its role as a mitogen for smooth muscle cells and fibroblasts, PDGF may be involved in attracting inflammatory cells to sites of platelet release.

• Singh JP, Chaikin MA, Stiles CD
• Phylogenetic analysis of platelet-derived growth factor by radio-receptor assay.
• J Cell Biol. 1982; 95: 667-71
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• Competition between 125I-labeled platelet-derived growth factor (PDGF) and unlabeled PDGF forms the basis of a specific "radio-receptor assay" for quantifying PDGF in clotted blood serum. Human clotted blood serum contains 15 ng/ml of PDGF by radio-receptor assay; this corresponds to a PDGF content of approximately 7.5 x 10(-5) pg per circulating platelet, a figure which is corroborated by purification data. Clotted blood sera from mammals, lower vertebrates and marine invertebrates were screened for homologues of human PDGF by radio-receptor assay. All tested specimens from phylum Chordata contain a mitogenic agent that competes with human PDGF for receptor binding. Sera from tunicates down on the chordate line of evolution and sera from all tested animals on the arthropod line of development were negative. The phylogenetic distribution of PDGF homologue does not correlate with platelet distribution since platelets and their precursor cell--the bone marrow megacaryocyte--are unique to the mammalian hematopoietic system. One anatomical feature appearing coordinately with PDGF on the vertebrate line of development is a pressurized circulatory system. The coincidental appearance of these features may lend support to the hypothesis that PDGF plays a role in maintenance and repair of the vascular lining in vivo.

• Currie GA
• Promotion of fibrosarcoma cell growth by products of syngeneic host macrophages.
• Br J Cancer. 1981; 44: 506-13
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• Cells from a C57BL/cbi chemically induced fibrosarcoma (FS6) require exogenous platelet-derived growth factor (PDGF) for in vitro proliferation (as do normal "untransformed" fibroblasts) whereas cells obtained from the FS6M1 tumour, a spontaneous metastasizing subline, show autonomy from PDGF in vitro. Furthermore, the FS6 cells exhibit very low colony formation in an anchorage-independent growth assay. In vivo, this tumour is immunogenic, rarely metastasizes and is heavily infiltrated by host macrophages. Studies of in vitro cell proliferation and anchorage-independent growth show that syngeneic host macrophages from the peritoneal cavity or from the growing tumour release a diffusible factor(s) which has (1) growth-stimulating activity on FS6 cells in monolayer cultures in PDGF-poor medium and (2) potent colony-stimulating activity on FS6 cell cultured in methyl-cellulose-containing medium. These macrophage supernatants stimulate proliferation of quiescent normal fibroblasts in monolayer culture as well as FS6 sarcoma cells, but do not stimulate anchorage-independent growth of normal cells. Supernatants from BCG-elicited macrophages were shown to contain abundant arginase, and were cytolytic to FS6 cells but not to normal cells. Heat inactivation abrogated the arginase and cytotoxicity, revealing heat-stable mitogenicity for FS6 cells and normal fibroblasts. The stimulatory effect of macrophages on FS6 sarcoma cells can be mimicked by the addition of the tumour promoter 12-tetradecanoyl-phorbol-13-acetate (TPA) and supports the hypothesis that macrophages could play a significant role in multistage carcinogenesis by providing a source of endogenous promoter.

• Cochran BH, Lillquist JS, Stiles CD
• Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma.
• J Cell Physiol. 1981; 109: 429-38
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• Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.

• Antoniades HN
• Human platelet-derived growth factor (PDGF): purification of PDGF-I and PDGF-II and separation of their reduced subunits.
• Proc Natl Acad Sci U S A. 1981; 78: 7314-7
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• Human platelet-derived growth factor (PDGF) was purified from lysates of clinically outdated human platelets by a direct procedure that allowed the recovery of active PDGF from stained sodium dodecyl sulfate/polyacrylamide gels. This technique enabled the identification and purification to homogeneity of two active PDGF polypeptides, and with a molecular weight of about 35,000 (PDGF-I) and the other with a molecular weight of about 32,000 (PDGF-II). Reduced PDGF-I produced two inactive subunits, with molecular weights of about 15,000 and 18,000. Reduced PDGF-II also produced two inactive subunits, with molecular weights of about 15,000 and 16,000. It is possible that PDGF-II derives from proteolytic cleavage of PDGF-I.

• Currie GA
• Platelet-derived growth-factor requirements for in vitro proliferation of normal and malignant mesenchymal cells.
• Br J Cancer. 1981; 43: 335-43
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• Serum obtained by clotting whole blood contains a potent mitogen with apparent specificity for mesenchymal cells. This peptide wound-healing hormone, derived from platelets, is known as platelet-derived growth factor (PDGF). Serum obtained by clotting plasma contains no detectable growth-promoting activity for fibroblasts, and is therefore a valuable additive to culture medium for an examination of the autonomy of cells from exogenous PDGF. Fibroblasts from man, mouse and hamster remain mitotically quiescent in plasma-derived serum and proliferate only when a source of PDGF is added. Normal human kidney epithelial cells and human T-cells proliferate normally in plasma-derived serum, and are unaffected by the addition of PDGF. A range of virally transformed cells and malignant cells from chemically induced rodent sarcomas was tested for their proliferative capacity in plasma-derived serum and their response to exogenous PDGF. A complete spectrum of PDGF-dependence was revealed. Polyoma-transformed BHK21 cells and SV40-transformed 3T3 cells showed complete PDGF independence. Cells from 7 chemically induced rat or mouse sarcomas provided results which ranged from the FS6 (a C57BL Cbi mouse sarcoma which was completely PDGF dependent) to MC28 (a hooded rat sarcoma) which was completely PDGF independent. The dependence of proliferation of these cells on PDGF showed a close correlation with several features of their in vivo behaviour. Tumours which were non-immunogenic in syngeneic hosts, contained few host macrophages and produced a high incidence of spontaneous distant metastases provided PDGF-independent cells. Cells from highly immunogenic, macrophage-rich "non-metastasizing" tumours were on the other hand PDGF dependent and tumours of intermediate "malignancy" provided cells with partial autonomy from PDGF. An assay for anchorage-independent growth provided data which also correlated with autonomy from PDGF. However, daily addition of large amounts of PDGF to BHK21 C13 cells induced reversible anchorage independent growth. The value of plasma-derived serum for the investigation of the proliferative autonomy of malignant cells is emphasized.

• Heldin CH, Westermark B, Wasteson A
• Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia.
• Proc Natl Acad Sci U S A. 1981; 78: 3664-8
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• A cellular receptor for platelet-derived growth factor (PDGF) was demonstrated by incubation of 125I-labeled PDGF with human foreskin fibroblast cultures followed by liberation of cell-bound radioactivity with Triton X-100. The cellular binding of labeled PDGF in the presence of increasing amounts of unlabeled PDGF showed saturation; Scatchard analysis of binding data indicated a single class of receptors having kd = 1 X 10(-9) M. The number of PDGF binding sites was approximately 3 X 10(5)/cell. Labeled PDGF binding reached an apparent equilibrium after 3 hr at 4 degrees C. At 37 degrees C, it passed a maximum after 30 min and then decreased with time due to degradation of the tracer. A large excess of unlabeled PDGF reduced labeled PDGF binding by more than 90% whereas similar doses of epidermal growth factor, fibroblast growth factor, or insulin had no effect. It was concluded that PDGF did not share receptors with these factors. PDGF receptors were found on skin fibroblasts, normal and malignant glial cells, smooth muscle cells, and 3T3 cells but not on epithelial-derived cells, neuroblastoma cells, endothelial cells, or peripheral lymphocytes.l As only the receptor-positive cells--i.e., the connective tissue- and glia-derived cells--are responsive to stimulation with PDGF, these findings imply a functional significance of the PDGF receptor.

• Smith JC, Stiles CD
• Cytoplasmic transfer of the mitogenic response to platelet-derived growth factor.
• Proc Natl Acad Sci U S A. 1981; 78: 4363-7
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• BALB/c 3T3 mouse cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA and divide. When cells are treated with PDGF and then fused to untreated cells, the resulting heterokaryons become competent to replicate their DNA. Cytoplasts derived from PDGF-treated cells are also able to transfer the growth response to untreated cells. After cytoplasmic transfer to another cell, the strength of the PDGF-induced mitogenic signal is attenuated by a factor roughly proportional to the increase in total cytoplasmic volume. When RNA synthesis is blocked during PDGF treatment, cells do not acquire the capacity to transfer the PDGF growth signal to untreated cells. By contrast, exposure to cycloheximide during PDGF treatment has no effect. These observations suggest that cytoplasmic transfer of the growth response to PDGF (competence) is mediated by a PDGF-induced stable RNA rather than by PDGF itself or a PDGF--receptor complex. The onset of DNA synthesis in PDGF--control heterokaryons occurs a minimum of 11 hr after cell fusion. Thus the substance that is transferred in these cell fusions is not directly involved in DNA synthesis; rather, it seems to trigger a sequence of events culminating in DNA synthesis.

• Nishimura J, Deuel TF
• Stimulation of protein phosphorylation in Swiss mouse 3T3 cells by human platelet derived growth factor.
• Biochem Biophys Res Commun. 1981; 103: 355-61

• Hiyama Y, Mahmud I, Karimi-Tari F, Fujita S, Fukui N, Miura Y
• Platelet-derived growth factor and thromboxane are necessary for liver regeneration.
• Cell Mol Biol. 1981; 27: 593-9

• Heldin CH, Westermark B, Wasteson A
• Platelet-derived growth factor. Isolation by a large-scale procedure and analysis of subunit composition.
• Biochem J. 1981; 193: 907-13
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• Platelet-derived growth factor was purified from fresh platelets by a large-scale procedure not involving the use of SDS (sodium dodecyl sulphate). The product, 0.5 mg of platelet-derived growth factor, obtained from about 3 x 10(13) platelets migrated as a single component in analytical gel electrophoresis in the presence of SDS and showed no inhomogeneity on sedimentation-equilibrium analysis in the ultracentrifuge. It had a high specific activity, 2 ng of platelet-derived growth factor/ml (70pM) being equivalent to 1% (v/v) human serum in an assay for multiplication-stimulating activity. Amino acid analysis revealed that platelet-derived growth factor contains all the common amino acids, except tryptophan, but no hexosamine. The molecular weight of platelet-derived growth factor, as determined by sedimentation-equilibrium analysis, was about 33 000. A similar value was obtained by gel electrophoresis in SDS under non-reducing conditions. In the presence of reducing agents the factor molecule was converted into two distinct components of lower molecular weight (17 000 and 14 000 respectively), as demonstrated by protein staining. The molecular model implicated by these findings is that platelet-derived growth factor consists of two different polypeptides chains, linked by disulphide bridges.

• Deuel TF, Huang JS, Proffitt RT, Baenziger JU, Chang D, Kennedy BB
• Human platelet-derived growth factor. Purification and resolution into two active protein fractions.
• J Biol Chem. 1981; 256: 8896-9
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• Human platelets secrete a factor that stimulates cultured human cells to initiate DNA synthesis and to divide. This human platelet-derived growth factor (PDGF) has been purified congruent to 100,000-fold into two equally active homogeneous fractions, PDGF I (Mr congruent to 31,000) and PDGF II (Mr congruent to 28,000). The amino acid compositions of each are similar, highly basic, and show 18 half-cystine residues. Both PDGF I and II are glycoproteins, but differ in their carbohydrate compositions. The data suggest that PDGF II may be a proteolytic cleavage product of PDGF I but do not rule out that the proteins may be separate but very similar gene products. Purified PDGF is active in stimulating DNA synthesis at 0.2 ng/ml.

• Inglot AD, Inglot O
• Platelet-derived growth factor (PDGF) enhances the growth of Moloney sarcoma virus-induced tumors in mice.
• Arch Immunol Ther Exp (Warsz). 1981; 29: 431-7
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• The intramuscular (i.m.) administration of bovine or human platelet derived growth factor (PDGF) had a marked potentiating effect on the growth of Moloney sarcoma virus (MSV)-induced tumors in Balb/c mice. PDGF had the tumor-promoting-activity when administered i.m. on the side of inoculation of MSV two days after the virus, and subsequently every second day for 2-3 weeks. The extent of MSV-induced tumor promoting activity of PDGF was age and sex-related. In view of the recently described antagonism in action between growth factors and interferons it is suggested that both families of hormones play an important role in controlling the neoplastic proliferation.

• Heldin CH, Westermark B, Wasteson A
• Chemical and biological properties of a growth factor from human-cultured osteosarcoma cells: resemblance with platelet-derived growth factor.
• J Cell Physiol. 1980; 105: 235-46
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• A human osteosarcoma cell line, U-2 OS, cultured under serum-free conditions, was shown to produce a growth factor (osteosarcoma-derived growth factor, ODGF) for human-cultured glial cells, fibroblasts, and other cells. ODGF, collected from the spent medium of 2 OS cultures, was purified by a sequence involving heparin-Sepharose chromatography, hydrophobic chromatography, gel chromatography, and preparative gel electrophresis in SDS. Purified ODGF, at a concentration of 3 ng/ml, elicited a mitogenic response in human glial cells equivalent to 50% of that afforded by human serum at a final concentration of 1%. The preparation was estimated to be > 50% pure. The biological activity of ODGF resided in a cationic, relatively heat-resistant, reduction-susceptible protein with a molecular weight of 30,000 (by gel chromatography and SDS-gel electrophoresis). The electrophoretic behaviour of radioiodinated ODGF suggested that the protein was composed of two different polypeptide chains (about 13,000-14,00 and 16,000-17,000 daltons, respectively) linked via disulphide bonds. The molecular makeup of ODGF was thus similar to that of platelet-derived growth factor. 125I-ODGF could be precipitated by an antibody to platelet-derived growth factor, indicating that the two factors were immunologically related. Resemblance with platelet-derived growth factor was also indicated by the finding that the latter (but not, e.g., fibroblast growth factor or epidermal growth factor) competed with 125I-ODGF for binding to human-cultured glial cells.

• Chernoff A, Levine RF, Goodman DS
• Origin of platelet-derived growth factor in megakaryocytes in guinea pigs.
• J Clin Invest. 1980; 65: 926-30
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• Growth factor activity, as determined by the stimulation of [3H]thymidine incorporation into the DNA of quiescent 3T3 cells in culture, was found in lysates of guinea pig platelets and megakaryocytes. Quantitative dilution studies demonstrated that, of the cells present in the guinea pig bone marrow, only the megakaryocyte possessed quantitatively significant growth factor activity. The amount of activity present in one megakaryocyte was equivalent to that present in 1,000-5,000 platelets, a value approximately comparable to the number of platelets shed from a single megakaryocyte. It is suggested that guinea pig platelet-derived growth factor has its origin in the megakaryocyte.

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