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.     

Vascular permeability factor/vascular endothelial growth factor-mediated signaling in mouse mesentery vascular endothelium

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine and growth factor that has important roles in both pathological and physiological angiogenesis. VPF/VEGF induces vascular hyperpermeability, cell division, and other activities by interacting with two specific receptor tyrosine kinases, KDR/Flk-1 and Flt-1, that are selectively expressed on vascular endothelium. The signaling cascade that follows VPF/VEGF interaction with cultured endothelium is only partially understood but is known to result in increased intracellular calcium, activation of protein kinase C, and tyrosine phosphorylations of both receptors, phospholipase C-gamma (PLC-gamma) and phosphatidylinositol 3'-kinase. For many reasons, signaling events elicited in cultured endothelium may not mimic mediator effects on intact normal or tumor-induced microvessels in vivo. Therefore, we developed a system that would allow measurement of VPF/VEGF-induced signaling on intact microvessels. We used mouse mesentery, a tissue whose numerous microvessels are highly responsive to VPF/VEGF and that we found to express Flk-1 and Flt-1 selectively. At intervals after injecting VPF/VEGF i.p., mesenteries were harvested, extracted, and immunoprecipitated. Immunoblots confirmed that VPF/VEGF induced tyrosine phosphorylation of several proteins in mesenteric microvessels as in cultured endothelium: Flk-1; PLC-gamma; and mitogen-activated protein kinase. Similar phosphorylations were observed when mesentery was exposed to VPF/VEGF in vitro, or when mesenteries were harvested from mice bearing the mouse ovarian tumor ascites tumor, which itself secretes abundant VPF/VEGF. Other experiments further elucidated the VPF/VEGF signaling pathway, demonstrating phosphorylation of both PYK2 and focal adhesion kinase, activation of c-jun-NH2-kinase with phosphorylation of c-Jun, and an association between Flk-1 and PLC-gamma. In addition, we demonstrated translocation of mitogen-activated protein kinase to the cell nucleus in cultured endothelium. Taken together, these experiments describe a new model system with the potential for investigating signaling events in response to diverse mediators on intact microvessels in vivo and have further elucidated the VPF/VEGF signaling cascade.     

Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4 degrees C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (approximately 20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were paralleled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37 degrees C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125I-VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-labeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.     

Increased expression of vascular permeability factor (vascular endothelial growth factor) in bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the increased vascular permeability and angiogenesis associated with many malignant tumors. In addition, VPF/VEGF is strongly expressed by epidermal keratinocytes in wound healing and psoriasis, disorders that are also characterized by increased microvascular permeability and angiogenesis. In this study, we investigated the expression of VPF/VEGF in three bullous diseases with subepidermal blister formation that are characterized by hyperpermeable dermal microvessels and pronounced papillary dermal edema. The expression of VPF/VEGF mRNA was strongly up-regulated in the lesional epidermis of bullous pemphigoid (n = 3), erythema multiforme (n = 3), and dermatitis herpetiformis (n = 4) as detected by in situ hybridization. Epidermal labeling was particularly intense over blisters, but strong expression was also noted in areas of the epidermis adjacent to dermal inflammatory infiltrates at a distance from blisters. Moreover, the VPF/VEGF receptors, flt-1 and KDR, were up-regulated in endothelial cells in superficial dermal microvessels. High levels of VPF/VEGF (138-238 pM) were detected in blister fluids obtained from five patients with bullous pemphigoid. Addition of blister fluid to human dermal microvascular endothelial cells exerted a dose-dependent mitogenic effect that was suppressed after depletion of VPF/VEGF by immunoadsorption. These findings strongly suggest that VPF/VEGF plays an important role in the induction of increased microvascular permeability in bullous diseases, leading to papillary edema and fibrin deposition and contributing to the bulla formation characteristic of these disorders.     

Activated mesangial cells produce vascular permeability factor in early-stage mesangial proliferative glomerulonephritis

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine involved in angiogenesis, inflammation, and wound healing. Although VPF/VEGF has been reported to be produced only by glomerular podocytes in glomeruli, it was found that it is produced by human cultured mesangial cells (MC). Therefore, immunohistochemical analysis (using indirect immunofluorescence and in situ hybridization) of VPF/VEGF in normal kidneys (n = 7) and biopsy specimens taken from 83 patients with renal diseases, including mesangial proliferative glomerulonephritis (PGN) (n = 58), was performed to examine whether VPF/VEGF is produced by MC in human PGN. In all of the healthy subjects and all of the patients except those with PGN (disease control subjects), VPF/VEGF protein and mRNA were detected mainly in podocytes. However, in some PGN patients, VPF/VEGF protein was demonstrated clearly in MC as well as in podocytes, as some of the VPF/VEGF was colocalized with alpha-smooth muscle actin, a marker of activated MC, and VPF/VEGF mRNA was expressed by MC and podocytes. Mesangial VPF/VEGF expression level increased significantly in PGN patients with early lesions (P < 0.01 versus healthy subjects or disease control subjects, P < 0.05 versus PGN with later lesions). The time between biopsy and disease onset was significantly shorter in PGN patients with than in those without mesangial VPF/VEGF expression (P < 0.01). These findings provide the first evidence that activated MC are a source of VPF/VEGF in human PGN, and indicate that mesangial VPF/VEGF expression is characteristic of early lesions of PGN. Because VPF/ VEGF plays a pivotal role in tissue repair, MC-produced VPF/VEGF may play pathophysiologic roles, including promoting recovery from glomerular injuries, in early-stage PGN.     

Brain edema in meningiomas is associated with increased vascular endothelial growth factor expression

OBJECTIVE: Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-specific cytokine, induces proliferation of endothelial cells and increases vascular permeability dramatically. All gliomas secrete significant amounts of VEGF, whereas meningiomas are variable in expression. Thus, we sought to determine whether the extent of VPF/VEGF expression in meningiomas correlated with differences in brain edema associated with these tumors. METHODS: Meningioma tissue samples from 37 patients (15 men, average age 65 +/- 13 yr; 22 women, average age 60 +/- 10 yr) who underwent surgery at or were referred to the University of Alabama Hospital were examined retrospectively for the extent of expression of immunoreactive VPF/VEGF. Additionally, peritumoral edema was assessed on a blinded basis radiographically from preoperative magnetic resonance imaging scans. Selected specimens were examined by in situ hybridization to document the source of VPF/VEGF. RESULTS: The predominant meningioma subclassifications were transitional (57%) or meningothelial (27%) subtypes. VPF/VEGF immunoreactivity ranged from 0 to 3.5, with a median value of 2 on a subjective 5-point scale; magnetic resonance imaging-assessed edema ranged in extent from 0 to 4 (subjective 5-point scale), with a median value of 2.5. The correlation of determination (R2) of magnetic resonance imaging-assessed tumor edema rating and VPF/VEGF staining intensity rating was 0.6087 (r = 0.78; P = 0.0001). In situ hybridization localized VPF/VEGF messenger ribonucleic acid in meningioma cells and not in normal parenchymal brain cells. CONCLUSION: These data suggest that meningioma-associated edema may be a result of the capacity of meningioma cells to produce VPF/VEGF locally, leading to increased tumor neovascularization and enhanced vascular permeability.     

Phagocytic synovial lining cells regulate acute and chronic joint inflammation after antigenic exacerbation of smouldering experimental murine arthritis

Vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF) can both potently enhance vascular permeability and induce proliferation of vascular endothelial cells. We report here that mouse or human mast cells can produce and secrete VPF/VEGF. Mouse mast cells release VPF/VEGF upon stimulation through Fcepsilon receptor I (FcepsilonRI) or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore, A23187; such mast cells can rapidly release VPF/VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein. Notably, the Fc epsilonRI-dependent secretion of VPF/VEGF by either mouse or human mast cells can be significantly increased in cells which have undergone upregulation of Fc epsilonRI surface expression by a 4-d preincubation with immunoglobulin E. These findings establish that at least one cell type, the mast cell, can be stimulated to secrete VPF/VEGF upon immunologically specific activation via a member of the multichain immune recognition receptor family. Our observations also identify a new mechanism by which mast cells can contribute to enhanced vascular permeability and/or angiogenesis, in both allergic diseases and other settings.     

Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody

The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 micrograms/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/ VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression-i.e., blocking the interactions between VEFG/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.     

Vascular endothelial growth factor/vascular permeability factor produces nitric oxide-dependent hypotension. Evidence for a maintenance role in quiescent adult endothelium

In vitro studies suggest that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) may stimulate release of nitric oxide (NO) from endothelial cells. To investigate the hemodynamic consequences of recombinant VEGF/VPF administered in vivo, recombinant human VEGF/VPF was administered as a bolus dose of 500 micrograms to anesthetized (n = 6) or conscious (n = 5) New Zealand White rabbits, as well as anesthetized rabbits with diet-induced hypercholesterolemia (HC; n = 7). Anesthetized Yorkshire farm pigs (no specific dietary pretreatment) were studied before and after receiving 500 micrograms intravenous (IV; n = 5) or intracoronary (IC; n = 5) VEGF/VPF. In anesthetized, normal rabbits, mean arterial pressure (MAP) fell by 20.5 +/- 1.4% (P < .05 versus baseline) within 3 minutes after IV VEGF/VPF. Pretreatment with N omega-nitro-L-arginine caused a significant inhibition of VEGF/VPF-induced hypotension. In conscious, normal rabbits, VEGF/VPF produced a consistent though lesser reduction in MAP. The fall in MAP induced by VEGF/VPF in anesthetized, HC rabbits (21.5 +/- 2.5% from baseline) was no different from that observed in normal anesthetized rabbits. In pigs, both IV and IC administration of VEGF/VPF produced a prompt reduction in MAP. Heart rate increased, while cardiac output, stroke volume, left atrial pressure, and total peripheral resistance all declined to a similar, statistically significant degree in both IV and IC groups. Epicardial echocardiography disclosed neither global nor segmental wall motion abnormalities in response to VEGF/VPF. We conclude that (1) VEGF/VPF-stimulated release of NO, previously suggested in vitro, occurs in vivo; (2) this finding suggests that functional VEGF/VPF receptors are present on quiescent adult endothelium, consistent with a maintenance function for VEGF/VPF, which may include regulation of NO; and (3) the preserved response of HC rabbits suggests that endothelial cell receptors for VEGF/VPF are spared in the setting of hypercholesterolemia.     

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.     

Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.     

Putative control of angiogenesis in hemangioblastomas by the von Hippel-Lindau tumor suppressor gene

The hypoxia-inducible endothelial cell-specific mitogen vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is expressed in low amounts in adult human brain, but is highly upregulated in the perinecrotic palisading cells of glioblastomas. We observed high VEGF expression in cerebellar hemangioblastomas, which are highly vascular, nonnecrotic and presumably nonhypoxic tumors, and hypothesized that a mechanism other than hypoxia leads to VEGF upregulation. Because hemangioblastomas develop in patients with von Hippel-Lindau disease, and mutations of the von Hippel-Lindau tumor suppressor (VHL) gene have also been reported in sporadic hemangioblastomas, we investigated VHL expression in normal cerebellum and in hemangioblastomas and tested the hypothesis that mutations in the VHL gene lead to upregulation of VEGE We observed constitutive expression of VHL mRNA, but downregulation of VEGF mRNA in the postnatal cerebellum. In the adult cerebellum, VHL is predominantly expressed in neuronal cells. In hemangioblastomas, VHL expression appears to be restricted to stromal cells, suggesting that the neoplastic component is the stromal cell. VHL-deficient renal cell carcinoma cells (786-0) produced significantly higher levels of VEGF mRNA and protein compared with 786-0/ wt10 cells, which were stably transfected with the wild-type VHL gene. Our observations suggest that VHL mutations affect stromal cells in hemangioblastomas and that VEGF is upregulated in stromal cells as a consequence of mutations in the VHL gene.     

Regulation of distinct steps of angiogenesis by different angiogenic molecules

This study was designed to determine the relative activity of basic fibroblast growth factor (bFGF), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor (PD-ECGF), hepatocyte growth factor (HGF), and interleukin-8 (IL-8) in regulating endothelial cell division, migration, degradation of the extracellular matrix (ECM), morphogenesis, and survival. Human umbilical vein endothelial cells (HUVEC) were treated with different concentrations of the six cytokines. bFGF was the most potent mitogen followed by VEGF/VPF and PD-ECGF. VEGF/VPF and bFGF also enhanced the survival of the endothelial cells in serum-free medium. Interstitial collagenase (MMP-1) and urokinase plasminogen activator (uPA) were significantly upregulated only by bFGF. HGF, bFGF, and VEGF/VPF induced chemotactic migration of the endothelial cells, but only HGF (scatter factor) enhanced nondirectional motility. The organization of endothelial cells to form tubes on Matrigel was induced by bFGF and, to a lesser extent, by VEGF/VPF and IL-8. Permeability across endothelial cell monolayers was induced only by VEGF/VPF. These data demonstrate that different angiogenic molecules differentially regulate distinct steps in the process of angiogenesis, suggesting that any given molecule may be necessary but in itself insufficient for establishment of a viable vasculature.     

Female choice for spot asymmetry in the Trinidadian guppy

The overexpression in tumor cells of (proto)-oncogenic receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) or ErbB2/neu (also known as HER-2) is generally thought to contribute to the development of solid tumors primarily through their effects on promoting uncontrolled cell proliferation. However, agents that antagonize the function of the protein products encoded by these (proto)-oncogenes are known to behave in vivo in a cytotoxic-like manner. This implies that such oncogenes may regulate critical cell survival functions, including angiogenesis. The latter could occur as a consequence of regulation of relevant growth factors by such oncogenes. We therefore sought to determine whether EGFR or ErbB2/neu may contribute to tumor angiogenesis by examining their effects on the expression of vascular endothelial cell growth factor (VEGF)/vascular permeability factor (VPF), one of the most important of all known inducers of tumor angiogenesis. We found that in vitro treatment of EGFR-positive A431 human epidermoid carcinoma cells, which are known to be heavily dependent on VEGF/VPF in vivo as an angiogenesis growth factor, with the C225 anti-EGFR neutralizing antibody caused a dose-dependent inhibition of VEGF protein expression. Prominent suppression of VEGF/VPF expression in vivo, as well as a significant reduction in tumor blood vessel counts, were also observed in established A431 tumors shortly after injection of the antibody as few as four times into nude mice. Transformation of NIH 3T3 fibroblasts with mutant ErbB2/neu, another EGFR-like oncogenic tyrosine kinase, resulted in a significant induction of VEGF/VPF, and the magnitude of this effect was further elevated by hypoxia. Moreover, treatment of ErbB2/neu-positive SKBR-3 human breast cancer cells in vitro with a specific neutralizing anti-ErbB2/neu monoclonal antibody (4D5) resulted in a dose-dependent reduction of VEGF/VPF protein expression. Taken together, the results suggest that oncogenic properties of EGFR and ErbB2/neu may, at least in part, be mediated by stimulation of tumor angiogenesis by up-regulating potent angiogenesis growth factors such as VEGF/VPF. These genetic changes may cooperate with epigenetic/environmental effects such as hypoxia to maximally stimulate VEGF/VPF expression. Therapeutic disruption of EGFR or ErbB2/neu protein function in vivo may therefore result in partial suppression of angiogenesis, a feature that could enhance the therapeutic index of such agents in vivo and endow them with anti-tumor effects, the magnitude of which may be out of proportion with their observed cytostatic effects in monolayer tissue culture.