A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain

Vascular endothelial growth factor (VEGF) mediates endothelial cell proliferation, angiogenesis, and vascular permeability via the endothelial cell receptors, KDR/Flk-1 and Flt-1. Recently, a gene encoding a polypeptide with about 25% amino acid identity to mammalian VEGF was identified in the genome of Orf virus (OV), a parapoxvirus that affects sheep and goats and occasionally, humans, to generate lesions with angiogenesis. In this study, we examined the biological activities and receptor of OV-derived NZ-7 VEGF (VEGF-E). VEGF-E was found to be a dimer of about 20 kDa with no basic domain nor affinity for heparin column, similar to VEGF121 subtype. VEGF121 has 10-100-fold less endothelial cell mitotic activity than VEGF165 due to lack of a heparin-binding basic region. Interestingly, however, VEGF-E showed almost equal levels of mitotic activity on primary endothelial cells and vascular permeability activity as VEGF165. Furthermore, VEGF-E bound KDR/Flk-1 (VEGFR-2) and induced its autophosphorylation to almost the same extent as VEGF165, but did not bind Flt-1 (VEGFR-1) nor induce autophosphorylation of Flt-1. These results indicate that VEGF-E is a novel type of endothelial growth factor, utilizing only one of the VEGF receptors, and carrying a potent mitogenic activity without affinity to heparin.     

Up-regulation of flk-1/vascular endothelial growth factor receptor 2 by its ligand in a cerebral slice culture system

Vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR) are key mediators of physiological and pathological angiogenesis. They are expressed in most tissues during embryonic development but are down-regulated in the adult, when angiogenesis ceases. Up-regulation of VEGFR-2 and of VEGF are observed in many pathological conditions under which angiogenesis is reinduced. A major regulator of VEGF expression is hypoxia. Although the temporal expression pattern of VEGFR-2 parallels VEGF expression to a high extent, little is known about its regulation. Here, we show that VEGFR-2 is highly expressed in early postnatal mouse brain but is down-regulated commencing at postnatal day 15 (P15) of mouse brain development and is hardly detectable in P30 mouse brain. Using P30 mouse brain slices, we observed that hypoxia up-regulates VEGFR-2 in the slices but not in human umbilical vein endothelial cells, suggesting the presence of a hypoxia-inducible factor in the murine neuroectoderm that up-regulates VEGFR-2. To identify the factors involved, normoxic P30 cerebral slices were cultured with growth factors that are either hypoxia-inducible (e.g., PDGF-BB, erythropoietin, and VEGF) and/or are known to act on endothelial cells (e.g., PDGF-BB, VEGF, and PIGF). Exogenously added recombinant VEGF led to an up-regulation of VEGFR-2 expression, which could be inhibited by preincubation with a neutralizing anti-VEGF antibody. Addition of PDGF-BB, PIGF, and erythropoietin had no effect on VEGFR-2 expression. Our results suggest a differential but synergistic regulation by hypoxia of VEGF and VEGFR-2: a direct induction of VEGF that subsequently up-regulates VEGFR-2 in endothelial cells. This autoenhancing system may represent an important mechanism of tumor angiogenesis.     

Neuropilin-1 is a placenta growth factor-2 receptor

Placenta growth factor (PlGF) belongs to the family of vascular endothelial growth factors (VEGFs). It binds to the flt-1 VEGF receptor but not to the KDR/flk-1 receptor which is thought to mediate most of the angiogenic and proliferative effects of VEGF. Three PlGF isoforms are produced by alternative splicing. PlGF-1 and PlGF-3 differ from PlGF-2 since they lack the exon 6 encoded peptide which bestows upon PlGF-2 its heparin binding properties. Cross-linking experiments revealed that 125I-PlGF-2 binds to two endothelial cell surface receptors in a heparin dependent fashion. The binding of 125I-PlGF-2 to these receptors was inhibited by an excess of PlGF-2 and by the 165-amino acid form of VEGF (VEGF165), but not at all by VEGF121 and very marginally if at all by PlGF-1. The apparent molecular weight and the binding characteristics of these receptors correspond to those of the recently identified VEGF165 specific receptor neuropilin-1, and we therefore conclude that neuropilin-1 is a receptor for PlGF-2. The binding of 125I-PlGF-2 as well as the binding of 125I-VEGF165 to these receptors was inhibited by a synthetic peptide derived from exon 6 of PlGF. Furthermore, the binding of 125I-PlGF-2, but not that of 125I-VEGF165, was also inhibited by a synthetic peptide derived from exon 7 of PlGF. These observations indicate that the peptides encoded by these exons probably participate in the formation of the domain which mediates the binding of PlGF-2 to these receptors. We have also determined, using chemically modified heparin species, that the presence of sulfate moieties on the glucosamine-O-6 and on the iduronic acid-O-2 groups of heparin was required for the potentiation of 125I-PlGF-2 binding to these receptors. To determine if PlGF-2 is able to induce biological responses that are not induced by PlGF-1, we compared the effects of PlGF-1 and PlGF-2 on the migration and proliferation of endothelial cells. Both PlGF forms induced migration of endothelial cells. However, there was no quantitative difference between the response to PlGF-2 and the response to PlGF-1. Furthermore, neither PlGF-1 nor PlGF-2 had any effect upon the proliferation of the endothelial cells.     

A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities

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.     

Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders

Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis associated with tumors and other pathological conditions, including proliferative diabetic retinopathy and age-related macular degeneration. The murine anti-human VEGF monoclonal antibody (muMAb VEGF) A.4.6.1 has been shown to potently suppress angiogenesis and growth in a variety of human tumor cells lines transplanted in nude mice and also to inhibit neovascularization in a primate model of ischemic retinal disease. In this report, we describe the humanization of muMAb VEGF A.4.6.1. by site-directed mutagenesis of a human framework. Not only the residues involved in the six complementarity-determining regions but also several framework residues were changed from human to murine. Humanized anti-VEGF F(ab) and IgG1 variants bind VEGF with affinity very similar to that of the original murine antibody. Furthermore, recombinant humanized MAb VEGF inhibits VEGF-induced proliferation of endothelial cells in vitro and tumor growth in vivo with potency and efficacy very similar to those of muMAb VEGF A.4.6.1. Therefore, recombinant humanized MAb VEGF is suitable to test the hypothesis that inhibition of VEGF-induced angiogenesis is a valid strategy for the treatment of solid tumors and other disorders in humans.     

Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.     

Differential mobilization of fatty acids from adipose tissue

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, and the permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two known receptors Flt1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2). The Flt4 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. In this study, we have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human prostatic carcinoma cells. While VEGF-C is homologous to other members of the VEGF/platelet derived growth factor (PDGF) family, its C-terminal half contains extra cysteine-rich motifs characteristic of a protein component of silk produced by the larval salivary glands of the midge, Chironomus tentans. VEGF-C is proteolytically processed, binds Flt4, which we rename as VEGFR-3 and induces tyrosine autophosphorylation of VEGFR-3 and VEGFR-2. In addition, VEGF-C stimulated the migration of bovine capillary endothelial cells in collagen gel. VEGF-C is thus a novel regulator of endothelia, and its effects may extend beyond the lymphatic system, where Flt4 is expressed.     

Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate

Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the sflt-1 gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis in vivo, since it did not affect tumor cell mitogenesis in vitro. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that sflt-1 gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth.     

Vascular endothelial growth factor C induces angiogenesis in vivo

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.     

Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis

FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2-induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-Mr FGF-2 (22-24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF.     

2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain

Vascular endothelial growth factor (VEGF) has been implicated in the pathological induction of new blood vessel growth in a variety of proliferative disorders. Using the SELEX process (systematic evolution of ligands by exponential enrichment), we have isolated 2'-F-pyrimidine RNA oligonucleotide ligands (aptamers) to human VEGF165. Representative aptamers from three distinct sequence families were truncated to the minimal sequence capable of high affinity binding to VEGF (23-29 nucleotides) and were further modified by replacement of 2'-O-methyl for 2'-OH at all ribopurine positions where the substitution was tolerated. Equilibrium dissociation constants for the interaction of VEGF with the truncated, 2'-O-methyl-modified aptamers range between 49 and 130 pM. These aptamers bind equally well to murine VEGF164, do not bind to VEGF121 or the smaller isoform of placenta growth factor (PlGF129), and show reduced, but significant affinity for the VEGF165/PlGF129 heterodimer. Cysteine 137 in the exon 7-encoded domain of VEGF165 forms a photo-inducible cross-link to a single uridine residue in each of the three aptamers. The aptamers potently inhibit the binding of VEGF to the human VEGF receptors, KDR and Flt-1, expressed by transfected porcine aortic endothelial cells. Furthermore, one of the aptamers is able to significantly reduce intradermal VEGF-induced vascular permeability in vivo.     

Vascular endothelial growth factor (VEGF) induces rapid prourokinase (pro-uPA) activation on the surface of endothelial cells

Vascular endothelial growth factor (VEGF) is the pivotal angiogenic growth factor activating endothelial cells to migrate, proliferate, and form capillary tubes. For an ordered endothelial cell migration, tissue invasion, and degradation of the extracellular matrix, proteolytic machinery is indispensable. Such machinery, suitable for localized proteolysis, is provided by the prourokinase-urokinase-plasmin system. Prourokinase (pro-uPA), the initial component of this system, is, however, synthesized in its inactive precursor form and as such bound to its cellular receptor uPAR. Here we identify a mechanism via which VEGF(165) interacting with its receptor VEGFR-2 rapidly induces prourokinase activation that is dependent on a change in integrin affinity, activation of matrix metalloproteinase 2 (MMP-2), and pro-uPA being bound to its surface receptor uPAR. This VEGF-induced pro-uPA activation on endothelial cells is responsible for VEGF-dependent local fibrinolytic activity and might be one of the initial steps in the angiogenic process.     

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

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.     

Hyperplasia of lymphatic vessels in VEGF-C transgenic mice

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.     

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

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.     

Overexpression of VEGF in testis and epididymis causes infertility in transgenic mice: evidence for nonendothelial targets for VEGF

Vascular endothelial growth factor (VEGF) is a key regulator of endothelial growth and permeability. However, VEGF may also target nonendothelial cells, as VEGF receptors and responsiveness have been detected for example in monocytes, and high concentrations of VEGF have been reported in human semen. In this work we present evidence that overexpression of VEGF in the testis and epididymis of transgenic mice under the mouse mammary tumor virus (MMTV) LTR promoter causes infertility. The testes of the transgenic mice exhibited spermatogenic arrest and increased capillary density. The ductus epididymidis was dilated, containing areas of epithelial hyperplasia. The number of subepithelial capillaries in the epididymis was also increased and these vessels were highly permeable as judged by the detection of extravasated fibrinogen products. Intriguingly, the expression of VEGF receptor-1 (VEGFR-1) was detected in certain spermatogenic cells in addition to vascular endothelium, and both VEGFR-1 and VEGFR-2 were also found in the Leydig cells of the testis. The infertility of the MMTV-VEGF male mice could thus result from VEGF acting on both endothelial and nonendothelial cells of the male genital tract. Taken together, these findings suggest that the VEGF transgene has nonendothelial target cells in the testis and that VEGF may regulate male fertility.     

The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.     

The von Hippel-Lindau gene product inhibits vascular permeability factor/vascular endothelial growth factor expression in renal cell carcinoma by blocking protein kinase C pathways

Mutation or loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is regularly found in sporadic renal cell carcinomas (RCC), well vascularized malignant tumors that characteristically overexpress vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). The wild-type VHL (wt-VHL) gene product acts to suppress VPF/VEGF expression, which is overexpressed when wt-VHL is inactive. The present study investigated the pathways by which VHL regulates VPF/VEGF expression. We found that inhibition of protein kinase C (PKC) represses VPF/VEGF expression in RCC cells that regularly overexpress VPF/VEGF. The wt-VHL expressed by stably transfected RCC cells forms cytoplasmic complexes with two specific PKC isoforms, zeta and delta, and prevents their translocation to the cell membrane where they otherwise would engage in signaling steps that lead to VPF/VEGF overexpression. Other experiments implicated mitogen-activated protein kinase (MAPK) phosphorylation as a downstream step in PKC regulation of VPF/VEGF expression. Taken together, these data demonstrate that wt-VHL, by neutralizing PKC isoforms zeta and delta and thereby inhibiting MAPK activation, plays an important role in preventing aberrant VPF/VEGF overexpression and the angiogenesis that results from such overexpression.