Src-mediated phosphorylation of Hsp90 in response to vascular endothelial growth factor (VEGF) is required for VEGF receptor-2 signaling to endothelial NO synthase

Nitric oxide (NO) release from endothelial cells, via endothelial NO synthase (eNOS) activation, is central to the proangiogenic actions of vascular endothelial growth factor (VEGF). VEGF signaling to eNOS is principally mediated by an Akt-dependent phosphorylation of eNOS and by increased association of eNOS to the molecular chaperone, heat-shock protein 90 kDa (Hsp90). Herein, we report that VEGFR-2 activation induces tyrosine phosphorylation of VEGF receptor 2 (VEGFR-2)-associated Hsp90beta. Tyrosine phosphorylation of Hsp90beta in response to VEGF is dependent on internalization of the VEGFR-2 and on Src kinase activation. Furthermore, we demonstrate that c-Src directly phosphorylates Hsp90 on tyrosine 300 residue and that this event is essential for VEGF-stimulated eNOS association to Hsp90 and thus NO release from endothelial cells. Our work identifies Y300 phosphorylation of Hsp90 as a novel regulated posttranslational modification of the chaperone and demonstrates its importance in the proangiogenic actions of VEGF, namely by regulating NO release from endothelial cells.     

Lipocortin V may function as a signaling protein for vascular endothelial growth factor receptor-2/Flk-1.

Binding of vascular endothelial growth factor (VEGF) to its receptor, VEGFR-2 (Flk-1/KDR), induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for signaling proteins that relay the signals for cell proliferation, migration, and permeability enhancement. We explored the VEGF/receptor signaling pathway by performing a two-hybrid screen of a rat lung cDNA library in yeast using the intracellular domain of rat VEGFR-2 as bait. Two clones encoding lipocortin V were isolated. Subsequent studies with the yeast two-hybrid assay showed that the complete intracellular domain of VEGFR-2 was required for the interaction. Co-immunoprecipitation of translated proteins confirmed the interaction between the VEGF receptor and lipocortin V. VEGF induced a rapid tyrosine phosphorylation of lipocortin V in human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with antisense oligodeoxyribonucleotide (ODN) for lipocortin V significantly inhibited VEGF-induced cell proliferation, which was accompanied by a decrease in protein synthesis and tyrosine phosphorylation of lipocortin V. Our results indicate that lipocortin V may function as a signaling protein for VEGFR-2 by directly interacting with the intracellular domain of the receptor and appears to be involved in regulation of vascular endothelial cell proliferation mediated by VEGFR-2.     

Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR.

Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitric-oxide synthase inhibitors diphenyleneiodonium and N(G)methyl-l-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-gamma-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies.     

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin,beta-catenin,and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.     

Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments

Receptor endocytosis is a fundamental step in controlling the magnitude, duration, and nature of cell signaling events. Confluent endothelial cells are contact inhibited in their growth and respond poorly to the proliferative signals of vascular endothelial growth factor (VEGF). In a previous study, we found that the association of vascular endothelial cadherin (VEC) with VEGF receptor (VEGFR) type 2 contributes to density-dependent growth inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. 2003. J. Cell Biol. 161:793-804). In the present study, we describe the mechanism through which VEC reduces VEGFR-2 signaling. We found that VEGF induces the clathrin-dependent internalization of VEGFR-2. When VEC is absent or not engaged at junctions, VEGFR-2 is internalized more rapidly and remains in endosomal compartments for a longer time. Internalization does not terminate its signaling; instead, the internalized receptor is phosphorylated, codistributes with active phospholipase C-gamma, and activates p44/42 mitogen-activated protein kinase phosphorylation and cell proliferation. Inhibition of VEGFR-2 internalization reestablishes the contact inhibition of cell growth, whereas silencing the junction-associated density-enhanced phosphatase-1/CD148 phosphatase restores VEGFR-2 internalization and signaling. Thus, VEC limits cell proliferation by retaining VEGFR-2 at the membrane and preventing its internalization into signaling compartments.     

Recruitment and activation of phospholipase Cgamma1 by vascular endothelial growth factor receptor-2 are required for tubulogenesis and differentiation of endothelial cells

Vascular endothelial growth factor-mediated angiogenic signal transduction relay is achieved by coordinated induction of endothelial cell proliferation, migration, and differentiation. These complex cellular processes are most likely controlled by activation of both cooperative and antagonistic signals by vascular endothelial growth factor receptors (VEGFRs). Here, we investigated the contribution of tyrosine-phosphorylated residues of VEGFR-2/fetal liver kinase-1 to endothelial cell proliferation and differentiation and activation of signaling proteins. Mutation of tyrosine 1006 of VEGFR-2 to phenylalanine severely impaired the ability of this receptor to stimulate endothelial cell differentiation and tubulogenesis. Paradoxically, the mutant receptor stimulated endothelial cell proliferation far better than the wild-type receptor. Further analysis showed that tyrosine 1006 is responsible for phospholipase Cgamma1 (PLCgamma1) activation and intracellular calcium release in endothelial cells. Activation of PLCgamma1 was selectively mediated by tyrosine 1006. Mutation of tyrosines 799, 820, 949, 994, 1080, 1173, and 1221 had no measurable effect on the ability of VEGFR-2 to stimulate PLCgamma1 activation. Association of VEGFR-2 with PLCgamma1 was mainly established between tyrosine 1006 and the C-terminal SH2 domain of PLCgamma1 in vitro and in vivo. Taken together, the results indicate that phosphorylation of tyrosine 1006 is essential for VEGFR-2-mediated PLCgamma1 activation, calcium flux, and cell differentiation. More importantly, VEGFR-2-mediated endothelial cell proliferation is inversely correlated with the ability of VEGFR-2 to associate with and activate PLCgamma1.     

Thematic review series: sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

Gangliosides are sialic acid-containing glycosphingolipids that have long been associated with tumor malignancy and metastasis. Mounting evidence suggests that gangliosides also modulate tumor angiogenesis. Tumor cells shed gangliosides into the microenvironment, which produces both autocrine and paracrine effects on tumor cells and tumor-associated host cells. In this study, we show that the simple monosialoganglioside GM3 counteracts the proangiogenic effects of vascular endothelial growth factor (VEGF) and of the complex disialoganglioside GD1a. GM3 suppressed the action of VEGF and GD1a on the proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited the migration of HUVECs toward VEGF as a chemoattractant. Enrichment of added GM3 in the HUVEC membrane also reduced the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream Akt. Moreover, GM3 reduced the proangiogenic effects of GD1a and growth factors in the in vivo Matrigel plug assay. Inhibition of GM3 biosynthesis with the glucosyl transferase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), increased HUVEC proliferation and the phosphorylation of VEGFR-2 and Akt. The effects of NB-DNJ on HUVECs were reversed with the addition of GM3. We conclude that GM3 has antiangiogenic action and may possess therapeutic potential for reducing tumor angiogenesis.     

Tyrosine residues 951 and 1059 of vascular endothelial growth factor receptor-2 (KDR) are essential for vascular permeability factor/vascular endothelial growth factor-induced endothelium migration and proliferation, respectively.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) exerts its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with two receptors intact, we, recently developed chimeric receptors (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR and Flt-1, respectively. With these fusion receptors, we have shown that KDR is solely responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration, whereas Flt-1 showed an inhibitory effect on KDR-mediated proliferation but not migration. To further characterize the VPF/VEGF-stimulated HUVEC proliferation and migration here, we have created several EGDR mutants by site-directed mutagenesis. We show that tyrosine residues 1059 and 951 of KDR are essential for VPF/VEGF-induced HUVEC proliferation and migration, respectively. Furthermore, the mutation of tyrosine 1059 to phenylanaline results in the complete loss of KDR/EGDR-mediated intracellular Ca(2+) mobilization and MAPK phosphorylation, but the mutation of tyrosine 951 to phenylanaline did not affect these events. Our results suggest that KDR mediates different signaling pathways for HUVEC proliferation and migration and, moreover, intracellular Ca(2+) mobilization and MAPK phosphorylation are not essential for VPF/VEGF-induced HUVEC migration.     

Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation

Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin’s actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin’s actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin’s effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.     

Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells

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

Differential regulation of vascular endothelial growth factor receptors (VEGFR) revealed by RNA interference: interactions of VEGFR-1 and VEGFR-2 in endothelial cell signaling

Vascular endothelial growth factor (VEGF) plays a central role in vascular homeostasis. VEGF receptors (VEGFRs) include several subtypes that may have a differential role in endothelial signal transduction, but interactions among these receptors are incompletely understood. In these studies, we designed small interfering RNA (siRNA) duplexes that targeted specific VEGFR subtypes in bovine aortic endothelial cells (BAEC). siRNA-mediated downregulation of VEGFR-2 by its cognate siRNA resulted in a significant attenuation of VEGF-mediated signaling. Compared to control siRNA-treated cells, VEGFR-2 siRNA markedly inhibited VEGF-mediated activation of PI3K/Akt/GSK3-beta as well as MAP kinase and PKC pathways. VEGFR-2 siRNA also blocked VEGF-stimulated phosphorylation and dephosphorylation of endothelial nitric oxide synthase (eNOS) at Ser(1179) and Ser(116), respectively. VEGFR-2-specific siRNA had no effect on the abundance of VEGFR-1 protein. By contrast, VEGFR-1-specific siRNA markedly not only downregulated the abundance of VEGFR-1 but also significantly reduced VEGFR-2 protein and mRNA abundance. VEGFR-1 siRNA had no effect on the stability of VEGFR-2 protein or mRNA. However, VEGFR-1 siRNA significantly inhibited VEGFR-2 promoter activity, as determined in luciferase assays using VEGFR-2 promoter fusion constructs in transfected BAEC. Deletion of either the 5' E box or the 3' E box and the GATA element in the VEGFR-2 promoter completely abolished the inhibition of VEGFR-2 promoter activity elicited by VEGFR-1 siRNA. Taken together, our data suggest that VEGFR-1 receptor is a critical determinant of VEGFR-2 abundance, while VEGFR-2 is the key receptor directly responsible for endothelial cell signaling stimulated by VEGF.     

The role of c-Fes in vascular endothelial growth factor-A-mediated signaling by endothelial cells

c-Fes plays pivotal roles in angiogenic cellular responses of endothelial cells. Here we examined the role of c-Fes in vascular endothelial growth factor-A (VEGF-A)-mediated signaling pathways in endothelial cells. We introduced either wild-type or kinase-inactive c-Fes in porcine aortic endothelial (PAE) cell lines, which endogenously express VEGF receptor (VEGFR)-1, and PAE cells ectopically expressing VEGFR-2 (denoted KDR/PAE cells) and generated stable cell lines. VEGF-A induced autophosphorylation of c-Fes only in KDR/PAE cells, suggesting that VEGFR-2 was required for its activation. Expression of kinase-inactive c-Fes failed to demonstrate dominant negative effect on VEGF-A-induced chemotaxis and capillary morphogenesis. Phosphoinositide 3-kinase (PI3-kinase) was activated in KDR/PAE cells and c-Fes contributed to this process in a kinase activity-dependent manner. However, VEGFR-2, insulin receptor substrate-1, and c-Src were also involved in VEGF-A-induced activation of PI3-kinase, resulting in the compensation in cells expressing kinase-inactive c-Fes. Interestingly, overexpression of wild-type c-Fes in PAE cells induced VEGF-A-independent capillary morphogenesis. Considered collectively, VEGF-A activated PI3-kinase partly through c-Fes and increase in c-Fes kinase activity enhanced capillary morphogenesis by yet unknown signaling pathways.     

Anti-tumor effects of vascular endothelial growth factor/vascular endothelial growth factor receptor binding domain-modified chimeric antigen receptor T cells

Background aimsThe vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) signaling pathway plays an important role in angiogenesis and lymphangiogenesis, which are closely related to tumor cell growth, survival, tissue infiltration and metastasis. Blocking/interfering with the interaction between VEGF and VEGFR to inhibit angiogenesis/lymphangiogenesis has become an important means of tumor therapy.MethodsHere the authors designed a novel chimeric antigen receptor (CAR) lentiviral vector expressing the VEGF-C domain targeting both VEGFR-2 and VEGFR-3 (VEGFR-2/3 CAR) and then transduced CD3-positive T cells with VEGFR-2/3 CAR lentivirus.ResultsAfter co-culturing with target cells, VEGFR-2/3 CAR T cells showed potent cytotoxicity against both VEGFR-2- and VEGFR-3-positive breast cancer cells, with increased simultaneous secretion of interferon gamma, tumor necrosis factor alpha and interleukin-2 cytokines. Moreover, CAR T cells were able to destroy the tubular structures formed by human umbilical vein endothelial cells and significantly inhibit the growth, infiltration and metastasis of orthotopic mammary xenograft tumors in a female BALB/c nude mice model.ConclusionsThe authors’ results indicate that VEGFR-2/3 CAR T cells targeting both VEGFR-2 and VEGFR-3 have significant anti-tumor activity, which expands the application of conventional CAR T-cell therapy.     

Regulation of vascular endothelial growth factor receptor-2 activity by caveolin-1 and plasma membrane cholesterol

The stimulation of vascular endothelial growth factor receptor-2 (VEGFR-2) by tumor-derived VEGF represents a key event in the initiation of angiogenesis. In this work, we report that VEGFR-2 is localized in endothelial caveolae, associated with caveolin-1, and that this complex is rapidly dissociated upon stimulation with VEGF. The kinetics of caveolin-1 dissociation correlated with those of VEGF-dependent VEGFR-2 tyrosine phosphorylation, suggesting that caveolin-1 acts as a negative regulator of VEGF R-2 activity. Interestingly, we observed that in an overexpression system in which VEGFR-2 is constitutively active, caveolin-1 overexpression inhibits VEGFR-2 activity but allows VEGFR-2 to undergo VEGF-dependent activation, suggesting that caveolin-1 can confer ligand dependency to a receptor system. Removal of caveolin and VEGFR-2 from caveolae by cholesterol depletion resulted in an increase in both basal and VEGF-induced phosphorylation of VEGFR-2, but led to the inhibition of VEGF-induced ERK activation and endothelial cell migration, suggesting that localization of VEGFR-2 to these domains is crucial for VEGF-mediated signaling. Dissociation of the VEGFR-2/caveolin-1 complex by VEGF or cyclodextrin led to a PP2-sensitive phosphorylation of caveolin-1 on tyrosine 14, suggesting the participation of Src family kinases in this process. Overall, these results suggest that caveolin-1 plays multiple roles in the VEGF-induced signaling cascade.     

Identification of novel neutralizing single-chain antibodies against vascular endothelial growth factor receptor 2

Human vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2/kinase domain receptor [KDR]) play a crucial role in angiogenesis, which makes the VEGFR-2 signaling pathway a major target for therapeutic applications. In this study, a single-chain antibody phage display library was constructed from spleen cells of mice immunized with recombinant human soluble extracellular VEGFR-2/KDR consisting of all seven extracellular domains (sKDR D1-7) to obtain antibodies that block VEGF binding to VEGFR-2. Two specific single-chain antibodies (KDR1.3 and KDR2.6) that recognized human VEGFR-2 were selected; diversity analysis of the clones was performed by BstNI fingerprinting and nucleotide sequencing. The single-chain variable fragments (scFvs) were expressed in soluble form and specificity of interactions between affinity purified scFvs and VEGFR-2 was confirmed by ELISA. Binding of the recombinant antibodies for VEGFR-2 receptors was investigated by surface plasmon resonance spectroscopy. In vitro cell culture assays showed that KDR1.3 and KDR2.6 scFvs significantly suppressed the mitogenic response of human umbilical vein endothelial cells to recombinant human VEGF(165) in a dose-dependent manner, and reduced VEGF-dependent cell proliferation by 60% and 40%, respectively. In vivo analysis of these recombinant antibodies in a rat cornea angiogenesis model revealed that both antibodies suppressed the development of new corneal vessels (p < 0.05). Overall, in vitro and in vivo results disclose strong interactions of KDR1.3 and KDR2.6 scFvs with VEGFR-2. These findings indicate that KDR1.3 and KDR2.6 scFvs are promising antiangiogenic therapeutic agents.     

Neuropilin-1-mediated vascular permeability factor/vascular endothelial growth factor-dependent endothelial cell migration

Neuropilin-1 (NRP-1) has been found to be expressed by endothelial cells and tumor cells as an isoform-specific receptor for vascular permeability factor/vascular endothelial growth factor (VEGF). Previous studies were mainly focused on the extracellular domain of NRP-1 that can bind to VEGF165 and, thus, enables NRP-1 to act as a co-receptor for VEGF165, which enhances its binding to VEGFR-2 and its bioactivity. However, the exact functional roles and related signaling mechanisms of NRP-1 in angiogenesis are not well understood. In this study we constructed a chimeric receptor, EGNP-1, by fusing the extracellular domain of epidermal growth factor receptor to the transmembrane and intracellular domains of NRP-1 and transduced it into HUVECs with a retroviral expression vector. We observed that NRP-1/EGNP-1 mediates ligand-stimulated migration of human umbilical vein endothelial cells (HUVECs) but not proliferation. Our results show that NRP-1 alone can mediate HUVEC migration through its intracellular domain, and its C-terminal three amino acids (SEA-COOH) are essential for the process. We demonstrate that phosphatidylinositol 3-kinase inhibitor Ly294002 and the p85 dominant negative mutant can block NRP-1-mediated HUVEC migration. NRP-1-mediated migration can be significantly reduced by overexpression of the dominant negative mutant of RhoA (RhoA-19N). In addition, Gq family proteins and Gbetagamma subunits are also required for NRP-1-mediated HUVEC migration. These results show for the first time that NRP-1 can independently promote cell signaling in endothelial cells and also demonstrate the importance of last three amino acids of NRP-1 for its function.     

Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.     

A novel snake venom vascular endothelial growth factor (VEGF) predominantly induces vascular permeability through preferential signaling via VEGF receptor-1

Vascular endothelial growth factor (VEGF)/vascular permeability factor induces both angiogenesis and vascular permeability mainly through VEGF receptor (VEGFR)-2 activation. VEGF binds VEGFR-1 as well, but the importance of VEGFR-1 signaling in vascular permeability has been largely neglected. Here, we report the purification and characterization of a novel VEGF-like protein from Trimeresurus flavoviridis Habu snake venom. The Habu snake has a venom-specific VEGF-like molecule, T. flavoviridis snake venom VEGF (TfsvVEGF), in addition to VEGF-A. TfsvVEGF has almost 10-fold less mitotic activity than VEGF(165), a predominant isoform of human VEGF-A, but a similar effect on vascular permeability. TfsvVEGF bound VEGFR-1 and induced its autophosphorylation to almost the same extent as VEGF(165), but bound VEGFR-2 weakly and induced its autophosphorylation almost 10-fold less effectively than VEGF(165). This unique binding affinity for VEGFR-1 and VEGFR-2 leads to the vascular permeability-dominant activity of TfsvVEGF. These results suggest that Habu snakes have acquired a highly purposive molecule for a toxin, which enhances the toxicity in envenomation without inducing effective angiogenesis and the following regeneration of damaged tissues, taking advantage of the difference in signaling properties involving VEGFR-1 and VEGFR-2 between vascular permeability and angiogenesis. TfsvVEGF is thus a potent inducing factor selective for vascular permeability through preferential signaling via VEGFR-1. These data strongly indicate the importance of VEGFR-1 signaling in vascular permeability.     

The vascular endothelial growth factor VEGF165 induces perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular endothelial cells

A member of the vascular endothelial growth factor (VEGF) family, VEGF165, regulates vascular endothelial cell functions in autocrine and paracrine fashions in microvessels. Proteoglycans are highly glycosylated poly-anionic macromolecules that influence cellular behaviors such as proliferation and migration by interacting with cytokines/growth factors. In the present study, we investigated the regulation of proteoglycan synthesis by VEGF165 in cultured human brain microvascular endothelial cells. The cells were exposed to recombinant human VEGF165, and the proteoglycans were then characterized using biochemical techniques. VEGF165 treatment increased the accumulation of proteoglycans 1.4- and 1.6-fold in the cell layer and conditioned medium, respectively. This effect resulted from the activation of VEGFR-2, and was mimicked by vammin, a VEGFR-2 ligand from snake venom but not placenta growth factor, which binds specifically to VEGFR-1. VEGF165 stimulated the production and secretion of perlecan, substituted with shorter heparan sulfate side chains, but with unaltered sulfated disaccharide composition. The perlecan secreted by VEGF165-stimulated endothelial cells may be involved in the regulation of cellular behavior during angiogenesis, in diseases of the brain microvessels, and in the maintenance of the endothelial cell monolayer.