Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.     

Rapid transactivation of the vascular endothelial growth factor receptor KDR/Flk-1 by the bradykinin B2 receptor contributes to endothelial nitric-oxide synthase activation in cardiac capillary endothelial cells.

Bradykinin (BK) and vascular endothelial growth factor (VEGF)-165 stimulate vasodilatation, microvascular permeability, and angiogenesis via the activation of the B2-type and KDR/Flk-1 receptors. To delineate the signal transduction pathways distal to the receptor activation in microvascular permeability, we compared their effects on two downstream targets, i.e. endothelial nitric-oxide (NO) synthase (eNOS) and F-actin, in primary cultures of cardiac capillary endothelial cells. The two mediators induced a similar cytoskeletal reorganization and both the translocation and activation of eNOS, leading to NO release within the first minutes of cell exposure. At the same time, BK produced the tyrosine phosphorylation and internalization of KDR/Flk-1 as did VEGF itself. This transactivation was blocked by the selective inhibitor of VEGF receptor tyrosine kinase activity but not by inhibitors of epidermal growth factor receptor or protein kinase C activity. The selective inhibitor of VEGF receptor tyrosine kinase activity totally prevented the effects of VEGF but only partially inhibited NO release induced by BK without affecting the concomitant cytoskeletal reorganization. Thus, BK transactivated KDR/Flk-1 through an intrinsic kinase activity of KDR/Flk-1, resulting in a further eNOS activation in endothelial cells. This represents a novel mechanism whereby a G protein-coupled receptor activates a receptor tyrosine kinase to generate biological response.     

Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway.

The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pM) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated with L-N(G)-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition of S-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway.     

Mechanism of thrombin-induced angiogenesis

Clinical, laboratory, histopathological and pharmacological evidence support the notion that a systemic activation of blood coagulation is often present in cancer patients. Additionally, thrombin was shown to promote tumour progression and metastasis in animals, and epidemiological studies suggest an increased risk of cancer diagnosis after primary thromboembolism. We have proposed that the aforementioned results may be related to our finding that thrombin is a potent activator of angiogenesis. This is a thrombin receptor-mediated event (the receptor is referred to as protease-activate receptor) and is independent of fibrin formation. Many cellular effects of thrombin on endothelial cells can contribute to the angiogenic action of thrombin. (i) Exposure of endothelial cells to thrombin cause a time- and dose-dependent decrease in the attachment of these cells to basement membrane components, with a concomitant increase in matrix metalloproteinase 2 activation. (ii) Thrombin upregulates the expression of integrin alphavbeta3, the marker of the angiogenic phenotype of endothelial cells. (iii) Thrombin has chemotactic and aptotactic effects on endothelial cells and upregulates the expression of the vascular endothelial growth factor (VEGF) receptors (KDR and Flt1). Thus, thrombin synergizes with the key angiogenic factor VEGF in endothelial cell proliferation. Furthermore, thrombin enhances the secretion of VEGF and matrix metalloproteinase 9 of PC3 prostate cancer cells. These results can explain the angiogenic and tumour-promoting effect of thrombin and provide the basis for development of thrombin receptor mimetics or antagonists for therapeutic application.     

Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.     

Metabolic responses induced by thrombin in human umbilical vein endothelial cells

Metabolic responses induced by thrombin in human umbilical vein endothelial cells (HUVECs) were investigated by using the cytosensor technique. Thrombin increased the extracellular acidification rate of endothelial cells, measured as an index of metabolic activity with a cytosensor microphysiometer, in a concentration-dependent fashion with an EC(50) of 1.27+/-0.59 IU/ml, which was abolished by the MAP kinase inhibitor PD98059. When intracellular Ca(2+) was chelated or PKC was inactivated, PD98059 failed to abolish the thrombin-induced acidification rate response in HUVECs. In addition, the tyrosine kinase inhibitor genistein, PKC inhibitor calphostin C, and Na(+)/H(+)exchanger antagonist MIA also partly inhibited thrombin-induced acidification rate responses. It is suggested that thrombin stimulated rapid metabolic responses via MAP kinase in HUVECs, which are calcium- and PKC-dependent.     

Homeostatic modulation of cell surface KDR and Flt1 expression and expression of the vascular endothelial cell growth factor (VEGF) receptor mRNAs by VEGF

Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function.     

Vascular endothelial growth factor (VEGF)-D and VEGF-A differentially regulate KDR-mediated signaling and biological function in vascular endothelial cells

Vascular endothelial growth factor (VEGF)-D binds to VEGF receptors (VEGFR) VEGFR2/KDR and VEGFR3/Flt4, but the signaling mechanisms mediating its biological activities in endothelial cells are poorly understood. Here we investigated the mechanism of action of VEGF-D, and we compared the signaling pathways and biological responses induced by VEGF-D and VEGF-A in endothelial cells. VEGF-D induced KDR and phospholipase C-gamma tyrosine phosphorylation more slowly and less effectively than VEGF-A at early times but had a more sustained effect and was as effective as VEGF-A after 60 min. VEGF-D activated extracellular signal-regulated protein kinases 1 and 2 with similar efficacy but slower kinetics compared with VEGF-A, and this effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase. In contrast to VEGF-A, VEGF-D weakly stimulated prostacyclin production and gene expression, had little effect on cell proliferation, and stimulated a smaller and more transient increase in intracellular [Ca(2+)]. VEGF-D induced strong but more transient phosphatidylinositol 3-kinase (PI3K)-mediated Akt activation and increased PI3K-dependent endothelial nitric-oxide synthase phosphorylation and cell survival more weakly. VEGF-D stimulated chemotaxis via a PI3K/Akt- and endothelial nitric-oxide synthase-dependent pathway, enhanced protein kinase C- and PI3K-dependent endothelial tubulogenesis, and stimulated angiogenesis in a mouse sponge implant model less effectively than VEGF-A. VEGF-D-induced signaling and biological effects were blocked by the KDR inhibitor SU5614. The finding that differential KDR activation by VEGF-A and VEGF-D has distinct consequences for endothelial signaling and function has important implications for understanding how multiple ligands for the same VEGF receptors can generate ligand-specific biological responses.     

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis

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.     

Receptor-selective variants of human vascular endothelial growth factor. Generation and characterization

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.     

Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells

Fibroblastic proliferation accompanies many angiogenesis-related retinal and systemic diseases. Since connective tissue growth factor (CTGF) is a potent mitogen for fibrosis, extracellular matrix production, and angiogenesis, we have studied the effects and mechanism by which vascular endothelial growth factor (VEGF) regulates CTGF gene expression in retinal capillary cells. In our study, VEGF increased CTGF mRNA levels in a time- and concentration-dependent manner in bovine retinal endothelial cells and pericytes, without the need of new protein synthesis and without altering mRNA stability. VEGF activated the tyrosine receptor phosphorylation of KDR and Flt1 and increased the binding of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit to KDR and Flt1, both of which could mediate CTGF gene induction. VEGF-induced CTGF expression was mediated primarily by PI3-kinase activation, whereas PKC and ERK pathways made only minimal contributions. Furthermore, overexpression of constitutive active Akt was sufficient to induce CTGF gene expression, and inhibition of Akt activation by overexpressing dominant negative mutant of Akt abolished the VEGF-induced CTGF expression. These data suggest that VEGF can increase CTGF gene expression in bovine retinal capillary cells via KDR or Flt receptors and the activation of PI3-kinase-Akt pathway independently of PKC or Ras-ERK pathway, possibly inducing the fibrosis observed in retinal neovascular diseases.     

Thrombin stimulation of vascular adhesion molecule-1 in endothelial cells is mediated by protein kinase C (PKC)-delta-NF-kappa B and PKC-zeta-GATA signaling pathways

We recently demonstrated that thrombin induces the expression of vascular adhesion molecule-1 (VCAM-1) in endothelial cells by an NF-kappaB- and GATA-dependent mechanism. In the present study, we describe the signaling pathways that mediate this response. Thrombin stimulation of the VCAM-1 gene and promoter in human umbilical vein endothelial cells was inhibited by preincubation with the phosphatidylinositol 3-kinase inhibitor, LY294002, the protein kinase C (PKC)-delta inhibitor, rottlerin, a PKC-zeta peptide inhibitor, or by overexpression of dominant negative (DN)-PKC-zeta. In electrophoretic mobility shift assays, thrombin-mediated induction of NF-kappaB p65 binding to two NF-kappaB motifs in the upstream promoter region of VCAM-1 was blocked by LY294002 and rottlerin, whereas the inducible binding of GATA-2 to a tandem GATA motif was inhibited by LY294002 and the PKC-zeta peptide inhibitor. In co-transfection assays, thrombin stimulation of a minimal promoter containing multimerized VCAM-1 NF-kappaB sites was inhibited by DN-PKC-delta but not DN-PKC-zeta. In contrast, thrombin-mediated transactivation of a minimal promoter containing tandem VCAM-1 GATA motifs was inhibited by DN-PKC-zeta but not DN-PKC-delta. Finally, thrombin failed to induce VCAM-1 expression in vascular smooth muscle cells. Taken together, these data suggest that the endothelial cell-specific effect of thrombin on VCAM-1 expression involves the coordinate activity of PKC-delta-NF-kappaB and PKC-zeta-GATA signaling pathways.     

PKC-delta/c-Src-mediated EGF receptor transactivation regulates thrombin-induced COX-2 expression and PGE(2) production in rat vascular smooth muscle cells

The thrombin/proteinase-activated receptors (PARs) have been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. Thrombin up-regulates expression of several proteins including cyclooxygenase (COX)-2 in vascular smooth muscle cells (VSMCs) and contributes to vascular diseases. However, the mechanisms underlying thrombin-regulated COX-2 expression in VSMCs remain unclear. Western blotting, RT-PCR, and EIA kit analyses showed that thrombin induced the expression of COX-2 mRNA and protein and PGE(2) release in a time-dependent manner, which was attenuated by inhibitors of PKC (GF109203X and rottlerin), c-Src (PP1), EGF receptor (EGFR; AG1478) and MEK1/2 (U0126), or transfection with dominant negative mutants of PKC-delta, c-Src or extracellular regulated kinase (ERK) and ERK1 short hairpin RNA interference (shRNA). These results suggest that transactivation of EGFR participates in COX-2 expression induced by thrombin in VSMCs. Accordingly, thrombin stimulated phosphorylation of ERK1/2 which was attenuated by GF109203X, rottlerin, PP1, GM6001, CRM197, AG1478, or U0126, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by selective inhibitors of AP-1 and NF-kappaB, curcumin and helenalin, respectively. Moreover, thrombin-stimulated activation of NF-kappaB, AP-1, and COX-2 promoter activity was blocked by the inhibitors of c-Src, PKC, EGFR, MEK1/2, AP-1 and NF-kappaB, suggesting that thrombin induces COX-2 promoter activity mediated through PKC(delta)/c-Src-dependent EGFR transactivation, MEK-ERK1/2, AP-1, and NF-kappaB. These results demonstrate that in VSMCs, activation of ERK1/2, AP-1 and NF-kappaB pathways was essential for thrombin-induced COX-2 gene expression. Understanding the regulation of COX-2 expression and PGE(2) release by thrombin/PARs system on VSMCs may provide potential therapeutic targets of vascular inflammatory disorders including arteriosclerosis.     

Endothelial thrombomodulin induces Ca2+ signals and nitric oxide synthesis through epidermal growth factor receptor kinase and calmodulin kinase II

Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLC(beta3) and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cgamma1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.     

Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line

Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.     

Platelet factor 4 disrupts the intracellular signalling cascade induced by vascular endothelial growth factor by both KDR dependent and independent mechanisms.

The mechanism by which the CXC chemokine platelet factor 4 (PF-4) inhibits endothelial cell proliferation is unclear. The heparin-binding domains of PF-4 have been reported to prevent vascular endothelial growth factor 165 (VEGF(165)) and fibroblast growth factor 2 (FGF2) from interacting with their receptors. However, other studies have suggested that PF-4 acts via heparin-binding independent interactions. Here, we compared the effects of PF-4 on the signalling events involved in the proliferation induced by VEGF(165), which binds heparin, and by VEGF(121), which does not. Activation of the VEGF receptor, KDR, and phospholipase Cgamma (PLCgamma) was unaffected in conditions in which PF-4 inhibited VEGF(121)-induced DNA synthesis. In contrast, VEGF(165)-induced phosphorylation of KDR and PLCgamma was partially inhibited by PF-4. These observations are consistent with PF-4 affecting the binding of VEGF(165), but not that of VEGF(121), to KDR. PF-4 also strongly inhibited the VEGF(165)- and VEGF(121)-induced mitogen-activated protein (MAP) kinase signalling pathways comprising Raf1, MEK1/2 and ERK1/2: for VEGF(165) it interacts directly or upstream from Raf1; for VEGF(121), it acts downstream from PLCgamma. Finally, the mechanism by which PF-4 may inhibit the endothelial cell proliferation induced by both VEGF(121) and VEGF(165), involving disruption of the MAP kinase signalling pathway downstream from KDR did not seem to involve CXCR3B activation.     

Thrombin stimulates c-sis gene expression in microvascular endothelial cells

We have determined whether expression of the c-sis gene product, platelet-derived growth factor (PDGF), is regulated in cultured renal microvascular endothelial cells by factors to which vascular endothelial cells may be exposed at sites of perivascular cellular proliferation. Thrombin exposure increased endothelial cell levels of c-sis message by 3-5-fold over a time course that peaked at 4 h after exposure. Similarly, thrombin-exposed microvascular endothelial cells released increased amounts of PDGF activity into their media. The thrombin effect was not mediated through the proteolytic activity of thrombin, as proteolytically inactive thrombin stimulated the c-sis expression as well as native thrombin. This stimulation was mimicked by exposure of cells to biologically active phorbol esters, suggesting that thrombin action may be mediated through activation of kinase C (Ca2+/phospholipid-dependent enzyme). Thus, thrombin regulates the expression and release of PDGF activity from endothelial cells in culture and may act in vivo to stimulate mitogen release from endothelial cells, thereby inducing proliferation of perivascular cells.