Protein kinase C-dependent protein kinase D activation modulates ERK signal pathway and endothelial cell proliferation by vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.     

VEGF induces proliferation, migration, and TGF-beta1 expression in mouse glomerular endothelial cells via mitogen-activated protein kinase and phosphatidylinositol 3-kinase

The role of glomerular endothelial cells in kidney fibrosis remains incompletely understood. While endothelia are indispensable for repair of acute damage, they can produce extracellular matrix proteins and profibrogenic cytokines that promote fibrogenesis. We used a murine cell line with all features of glomerular endothelial cells (glEND.2), which dissected the effects of vascular endothelial growth factor (VEGF) on cell migration, proliferation, and profibrogenic cytokine production. VEGF dose-dependently induced glEND.2 cell migration and proliferation, accompanied by up-regulation of VEGFR-2 phosphorylation and mRNA expression. VEGF induced a profibrogenic gene expression profile, including up-regulation of TGF-beta1 mRNA, enhanced TGF-beta1 secretion, and bioactivity. VEGF-induced endothelial cell migration and TGF-beta1 induction were mediated by the phosphatidyl-inositol-3 kinase pathway, while proliferation was dependent on the Erk1/2 MAP kinase pathway. This suggests that differential modulation of glomerular angiogenesis by selective inhibition of the two identified VEGF-induced signaling pathways could be a therapeutic approach to treat kidney fibrosis.     

Requirement of protein kinase D tyrosine phosphorylation for VEGF-A165-induced angiogenesis through its interaction and regulation of phospholipase Cgamma phosphorylation

Vascular endothelial cell growth factor-A(165) (VEGF-A(165)) is critical for angiogenesis. Although protein kinase C-mediated protein kinase D(PKD)activation was implicated in the response, the detailed mechanism remains unclear. In this study, we found that VEGF-A(165)-stimulated tyrosine phosphorylation of PKD and the dominant negative mutant of PKD, PKD(Y463F), inhibited VEGF-A(165)-induced human umbilical vein endothelial cell (HUVEC) proliferation. In addition, PKD(S738A/S742A) overexpression inhibited VEGF-induced HUVEC migration. Furthermore, knockdown of PKD by its specific small interfering RNA inhibited VEGF-induced HUVEC proliferation and migration. Moreover transfection of PKD(Y463F), PKD(S738A/S742A), or PKD-small interfering RNA blocked VEGF-induced angiogenesis in vivo. Our signaling experiments show that KDR not Flt-1 mediated PKD tyrosine phosphorylation and KDR tyrosine residues 951 and 1059 were required for VEGF-A(165)-stimulated PKD serine and tyrosine phosphorylation, respectively. Whereas G protein Gbetagamma subunits were required for both PKD serine phosphorylation and tyrosine phosphorylation, intracellular Ca(2+) mobilization was required for VEGF-A(165)-stimulated PKD tyrosine phosphorylation and phospholipase C (PLC) activity was required for PKD serine phosphorylation. Surprisingly, the PLC inhibitor did not inhibit PKD tyrosine phosphorylation. Instead, PKD tyrosine 463 was required for VEGF-A(165)-stimulated PLCgamma tyrosine phosphorylation. Moreover, PKD interacted with PLCgamma even in unstimulated cells, and PKD tyrosine 463 phosphorylation was not required for this interaction. Together, we demonstrate that PKD interacts with PLCgamma and becomes tyrosine phosphorylated upon VEGF stimulation, leading to PLCgamma activation and angiogenic response of VEGF-A(165).     

Distinct angiogenic mediators are required for basic fibroblast growth factor- and vascular endothelial growth factor-induced angiogenesis: the role of cytoplasmic tyrosine kinase c-Abl in tumor angiogenesis

Signaling pathways engaged by angiogenic factors bFGF and VEGF in tumor angiogenesis are not fully understood. The current study identifies cytoplasmic tyrosine kinase c-Abl as a key factor differentially mediating bFGF- and VEGF-induced angiogenesis in microvascular endothelial cells. STI571, a c-Abl kinase inhibitor, only inhibited bFGF- but not VEGF-induced angiogenesis. bFGF induced membrane receptor cooperation between integrin beta(3) and FGF receptor, and triggered a downstream cascade including FAK, c-Abl, and MAPK. This signaling pathway is different from one utilized by VEGF that includes integrin beta(5), VEGF receptor-2, Src, FAK, and MAPK. Ectopic expression of wild-type c-Abl sensitized angiogenic response to bFGF, but kinase dead mutant c-Abl abolished this activity. Furthermore, the wild-type c-Abl enhanced angiogenesis in both Matrigel implantation and tumor xenograft models. These data provide novel insights into c-Abl's differential functions in mediating bFGF- and VEGF-induced angiogenesis.     

BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced angiogenesis and proliferation in human umbilical vein endothelial cells

Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the present study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migration assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103), among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells (HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.     

VEGF binding to NRP1 is essential for VEGF stimulation of endothelial cell migration, complex formation between NRP1 and VEGFR2, and signaling via FAK Tyr407 phosphorylation

In endothelial cells, neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF)-A and is thought to act as a coreceptor for kinase insert domain-containing receptor (KDR) by associating with KDR and enhancing VEGF signaling. Here we report mutations in the NRP1 b1 domain (Y297A and D320A), which result in complete loss of VEGF binding. Overexpression of Y297A and D320A NRP1 in human umbilical vein endothelial cells reduced high-affinity VEGF binding and migration toward a VEGF gradient, and markedly inhibited VEGF-induced angiogenesis in a coculture cell model. The Y297A NRP1 mutant also disrupted complexation between NRP1 and KDR and decreased VEGF-dependent phosphorylation of focal adhesion kinase at Tyr407, but had little effect on other signaling pathways. Y297A NRP1, however, heterodimerized with wild-type NRP1 and NRP2 indicating that nonbinding NRP1 mutants can act in a dominant-negative manner through formation of NRP1 dimers with reduced binding affinity for VEGF. These findings indicate that VEGF binding to NRP1 has specific effects on endothelial cell signaling and is important for endothelial cell migration and angiogenesis mediated via complex formation between NRP1 and KDR and increased signaling to focal adhesions. Identification of key residues essential for VEGF binding and biological functions provides the basis for a rational design of antagonists of VEGF binding to NRP1.     

Activation of multiple signaling pathways is critical for fibroblast growth factor 2- and vascular endothelial growth factor-stimulated ovine fetoplacental endothelial cell proliferation

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) are two key regulators of placental angiogenesis. The potent vasodilator nitric oxide (NO) could also act as a key mediator of FGF2- and VEGF-induced angiogenesis. However, the postreceptor signaling pathways governing these FGF2- and VEGF-induced placental angiogenic responses are poorly understood. In this study, we assessed the role of endogenous NO, mitogen-activated protein kinase 3/1 (MAPK3/1), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) in FGF2- and VEGF-stimulated proliferation of ovine fetoplacental endothelial (OFPAE) cells. Both FGF2 and VEGF time-dependently stimulated (P < 0.05) NO production and activated AKT1. Both FGF2- and VEGF-stimulated cell proliferation was dose-dependently inhibited (P < 0.05) by N(G)-monomethyl-L-arginine (L-NMMA; an NO synthase inhibitor), PD98059 (a selective MAPK3/1 kinase 1 and 2 [MAP2K1/2] inhibitor), or LY294002 (a selective phosphatidylinositol 3 kinase [PI3K] inhibitor) but not by phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (PTIO, a potent extracellular NO scavenger). At the maximal inhibitory dose without cytotoxicity, PD98059 and LY294002 completely inhibited VEGF-induced cell proliferation but only partially attenuated (P < 0.05) FGF2-induced cell proliferation. PD98059 and LY294002 also inhibited (P < 0.05) FGF2- and VEGF-induced phosphorylation of MAPK3/1 and AKT1, respectively. L-NMMA did not significantly affect FGF2- and VEGF-induced phosphorylation of either MAPK3/1 or AKT1. Thus, in OFPAE cells, both FGF2- and VEGF-stimulated cell proliferation is partly mediated via NO as an intracellular and downstream signal of MAPK3/1 and AKT1 activation. Moreover, activation of both MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for FGF2-stimulated cell proliferation, whereas activation of either one pathway is sufficient for mediating the VEGF-induced maximal cell proliferation, indicating that these two kinase pathways differentially mediate the FGF2- and VEGF-stimulated OFPAE cell proliferation.     

VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2

Vascular endothelial growth factor (VEGF) signaling is critical for both normal and disease-associated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the Rho family of small GTPases. As a member of this family, Rac1 promotes VEGF-induced endothelial cell migration by stimulating the formation of lamellipodia and membrane ruffles. To form these membrane protrusions, Rac1 is activated by guanine nucleotide exchange factors (GEFs) that catalyze the exchange of GDP for GTP. The goal of this study was to identify the GEF responsible for activating Rac1 in response to VEGF stimulation. We have found that VEGF stimulates biphasic activation of Rac1 and for these studies we focused on the peak of activation that occurs at 30 min. Inhibition of VEGFR-2 signaling blocks VEGF-induced Rac1 activation. Using a Rac1 nucleotide-free mutant (G15ARac1), which has a high affinity for binding activated GEFs, we show that the Rac GEF Vav2 associates with G15ARac1 after VEGF stimulation. Additionally, we show that depleting endothelial cells of endogenous Vav2 with siRNA prevents VEGF-induced Rac1 activation. Moreover, Vav2 is tyrosine phosphorylated upon VEGF treatment, which temporally correlates with Rac1 activation and requires VEGFR-2 signaling and Src kinase activity. Finally, we show that depressing Vav2 expression by siRNA impairs VEGF-induced endothelial cell migration. Taken together, our results provide evidence that Vav2 acts downstream of VEGF to activate Rac1.     

An attempt to promote neo-vascularization by employing a newly synthesized inhibitor of protein tyrosine phosphatase

Vascular endothelial growth factor (VEGF) and its receptors play a key role in angiogenesis. VEGF receptor-2 (VEGFR-2) has a tyrosine kinase domain, and, once activated, induces the phosphorylation of cytoplasmic signaling proteins. The phosphorylated VEGFR-2 may be a substrate for intracellular protein tyrosine phosphatases (PTPs) which prevent VEGF signaling. We synthesized a series of alpha,alpha-difluoro(phenyl)methylphosphonic acids (DFPMPAs) which inhibit the action of PTP. In this study, we test their effects on VEGF-induced angiogenesis. DFPMPA-3, the most effective inhibitor of human PTP-1B, promoted tube formation by human umbilical vein endothelial cells (HUVEC) on Matrigel more effectively than any other DFPMPAs. The inhibitor promoted the VEGF-induced proliferation and migration of HUVEC by inhibiting the dephosphorylation of VEGFR-2. Its effectiveness was proven through neo-vascularization in mice. The present findings suggest that targeting PTP to promote therapeutic neo-vascularization may be a potential strategy.     

Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.     

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis

Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.     

Protein kinase D in vascular biology and angiogenesis

The Protein Kinase D (PKD) family comprises diacylglycerol stimulated serine/threonine protein kinases that participate in many key signaling pathways in a diverse range of cells. Recent studies show that PKD isoforms 1 and 2 play critical roles in vascular biology and angiogenesis and there has been considerable progress in determining some of the key angiogenic signaling pathways mediated by PKD in endothelial cells. Less is currently known regarding the specific roles of PKD isoforms in endothelial cells and the role of PKD in smooth muscle cells. PKD is also emerging as a potentially important mediator of tumor growth and tumor angiogenesis and there is growing interest in PKD as a novel therapeutic target in cancer.     

An inhibitory role of the phosphatidylinositol 3-kinase-signaling pathway in vascular endothelial growth factor-induced tissue factor expression.

Vascular endothelial growth factor (VEGF) is not only essential for vasculogenesis and angiogenesis but is also capable of inducing tissue factor, the prime initiator of coagulation, in endothelial cells. In this study we have analyzed the VEGF-elicited pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells. Using specific low molecular weight inhibitors we could demonstrate a crucial role of the p38 and Erk-1/2 mitogen-activated protein (MAP) kinases. In contrast, treatment with wortmannin or LY294002, inhibitors of phosphatidylinositol 3 (PI3)-kinase, resulted in a strong enhancement of the VEGF-induced tissue factor production, indicating a negative regulatory role of the PI3-kinase on tissue factor-inducing pathways. Accordingly, transduction with constitutively active Akt led to a reduction of VEGF-induced tissue factor production. Western blot analyses using antibodies specific for phosphorylated p38 showed an enhanced activation of this MAP kinase in human umbilical cord vein endothelial cells when stimulated with VEGF in the presence of wortmannin in comparison to either agent alone. Thus, the negative regulation of the PI3-kinase pathway on endothelial tissue factor activity can be explained at least in part by a suppression of this MAP kinase-signaling pathway. This is the first demonstration of a reciprocal relationship between procoagulant activity and the PI3-kinase-Akt signaling pathway, and it reveals a novel mechanism by which tissue factor expression can be controlled in endothelial cells.     

Sphingosine kinase mediates vascular endothelial growth factor-induced activation of ras and mitogen-activated protein kinases

Vascular endothelial growth factor (VEGF) signaling is critical to the processes of angiogenesis and tumor growth. Here, evidence is presented for VEGF stimulation of sphingosine kinase (SPK) that affects not only endothelial cell signaling but also tumor cells expressing VEGF receptors. VEGF or phorbol 12-myristate 13-acetate treatment of the T24 bladder tumor cell line resulted in a time- and dose-dependent stimulation of SPK activity. In T24 cells, VEGF treatment reduced cellular sphingosine levels while raising that of sphingosine-1-phosphate. VEGF stimulation of T24 cells caused a slow and sustained accumulation of Ras-GTP and phosphorylated extracellular signal-regulated kinase (phospho-ERK) compared with that after EGF treatment. Small interfering RNA (siRNA) that targets SPK1, but not SPK2, blocks VEGF-induced accumulation of Ras-GTP and phospho-ERK in T24 cells. In contrast to EGF stimulation, VEGF stimulation of ERK1/2 phosphorylation was unaffected by dominant-negative Ras-N17. Raf kinase inhibition blocked both VEGF- and EGF-stimulated accumulation of phospho-ERK1/2. Inhibition of SPK by pharmacological inhibitors, a dominant-negative SPK mutant, or siRNA that targets SPK blocked VEGF, but not EGF, induction of phospho-ERK1/2. We conclude that VEGF induces DNA synthesis in a pathway which sequentially involves protein kinase C (PKC), SPK, Ras, Raf, and ERK1/2. These data highlight a novel mechanism by which SPK mediates signaling from PKC to Ras in a manner independent of Ras-guanine nucleotide exchange factor.     

RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.     

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor, and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-β to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.     

Vascular endothelial growth factor stimulates differential signaling pathways in in vivo microcirculation

Vascular endothelial growth factor (VEGF) induces mild vasodilation and strong increases in microvascular permeability. Using intravital microscopy and digital integrated optical intensity image analysis, we tested, in the hamster cheek pouch microcirculation, the hypothesis that differential signaling pathways in arterioles and venules represent an in vivo regulatory mechanism in the control of vascular diameter and permeability. The experimental design involved blocking specific signaling molecules and simultaneously assessing VEGF-induced changes in arteriolar diameter and microvascular transport of FITC-Dextran 150. Inhibition of Akt [indirectly via phosphatidylinositol 3-kinase with LY-294002 or wortmannin] or PKC (with bisindolylmaleimide) reduced VEGF-induced hyperpermeability. However, phosphatidylinositol 3-kinase/Akt inhibition enhanced the early phase and attenuated the late phase of VEGF-induced vasodilation, whereas blocking PKC had no effect. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 (with PD-98059 or AG-126) also reduced VEGF-induced hyperpermeability but did not block VEGF-induced vasodilation. Blockade of endothelial nitric oxide synthase (with N(omega)-monomethyl-l-arginine) inhibited VEGF-induced changes in both permeability and diameter. Furthermore, immunofluorescence studies with human umbilical vein endothelial cells revealed that bisindolylmaleimide, PD-98059, and l-NMMA attenuate VEGF-induced reorganization of vascular endothelial cadherin. Our data demonstrate that 1) endothelial nitric oxide synthase is a common convergence pathway for VEGF-induced changes in arteriolar diameter and microvascular permeability; 2) PKC and ERK-1/2 do not play a major role in VEGF-induced vasodilation in the hamster cheek pouch microcirculation; and 3) Akt, PKC, and ERK-1/2 are elements of the signaling cascade that regulates VEGF-stimulated microvascular hyperpermeability. Our data provide evidence for differential signaling as a regulatory step in VEGF-stimulated microvascular dynamics.     

Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen that promotes angiogenesis, vascular hyperpermeability, and vasodilation by autocrine mechanisms involving nitric oxide (NO) and prostacyclin (PGI(2)) production. These experiments used immunoprecipitation and immunoassay procedures to characterize the signaling pathways by which VEGF induces NO and PGI(2) formation in cultured endothelial cells. The data showed that VEGF stimulates complex formation of the flk-1/kinase-insert domain-containing receptor (KDR) VEGF receptor with c-Src and that Src activation is required for VEGF induction of phospholipase C gamma1 activation and inositol 1,4,5-trisphosphate formation. Reporter cell assays showed that VEGF promotes a approximately 50-fold increase in NO formation, which peaks at 5-20 min. This effect is mediated by a signaling cascade initiated by flk-1/KDR activation of c-Src, leading to phospholipase C gamma1 activation, inositol 1,4,5-trisphosphate formation, release of [Ca(2+)](i) and nitric oxide synthase activation. Immunoassays of VEGF-induced 6-keto prostaglandin F(1alpha) formation as an indicator of PGI(2) production revealed a 3-4-fold increase that peaked at 45-60 min. The PGI(2) signaling pathway follows the NO pathway through release of [Ca(2+)](i), but diverges prior to NOS activation and also requires activation of mitogen-activated protein kinase. These results suggest that NO and PGI(2) function in parallel in mediating the effects of VEGF.