Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.     

Stem cell-derived endothelial cells/progenitors migrate and pattern in the embryo using the VEGF signaling pathway

Endothelial precursor cells respond to molecular cues to migrate and assemble into embryonic blood vessels, but the signaling pathways involved in vascular patterning are not well understood. We recently showed that avian vascular patterning cues are recognized by mammalian angioblasts derived from somitic mesoderm through analysis of mouse-avian chimeras. To determine whether stem cell-derived endothelial cells/progenitors also recognize global patterning signals, murine ES cell-derived embryoid bodies (EBs) were grafted into avian hosts. ES cell-derived murine endothelial cells/progenitors migrated extensively and colonized the appropriate host vascular beds. They also formed mosaic vessels with avian endothelial cells. Unlike somite derived-endothelial cells, ES cell-derived endothelial cells/progenitors migrated across the host embryonic midline to the contralateral side. To determine the role of VEGF signaling in embryonic vascular patterning, EBs mutant for a VEGF receptor (flk-1(-/-)) or a signal (VEGF-A(-/-)) were grafted into quail hosts. Flk-1(-/-) EB grafts produced only rare endothelial cells that did not migrate or assemble into vessels. In contrast, VEGF-A(-/-) EB grafts produced endothelial cells that resembled wild-type and colonized host vascular beds, suggesting that host-derived signals can partially rescue mutant graft vascular patterning. VEGF-A(-/-) graft endothelial cells/progenitors crossed the host midline with much lower frequency than wild-type EB grafts, indicating that graft-derived VEGF compromised the midline barrier when present. Thus, ES cell-derived endothelial cells/progenitors respond appropriately to global vascular patterning cues, and they require the VEGF signaling pathway to pattern properly. Moreover, EB-avian chimeras provide an efficient way to screen mutations for vascular patterning defects.     

Heparan sulfate regulates VEGF165- and VEGF121-mediated vascular hyperpermeability

VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF(165) and VEGF(121). Because VEGF(121) does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF(165) and VEGF(121). Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF(165) and VEGF(121), suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.     

Paracrine VEGF/VE-cadherin action on ovarian cancer permeability

Ascites formation associated with neoplasms is most likely due to increased vascular permeability, a process in which vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) plays a pivotal role. We hypothesized that tumor-derived VEGF/VPF modulates ascites formation through a paracrine effect on both tumor and peritoneal vessels. We investigated human vascular endothelial permeability using a newly developed dual-chamber permeability assay by co-culturing human umbilical vein cells with and without ovarian cancer cell lines (OVCAR-3, Hey-A8, and OCC-1) in the presence or absence of a human VEGF monoclonal antibody and VE-cadherin function-blocking antibody. This method permits determination of mechanisms by which substances released from neoplasms and other sources of vascular endothelial cell secretagogues modulate vascular permeability and likely other pathologic states.     

Vascular endothelial cells: Targets for studying the activity of hair follicle cell-produced VEGF

Fetal bovine aortic endothelial cells (FBAEC) were exposed to purified fractions of conditioned medium from cultures of hair dermal papilla cells (DPC) to determine the existence of any vascular endothelial growth factor (VEGF)-like paracrine activity of the latter. Such fractions were tested for stimulation of growth and migration of cultured FBAEC. In addition, VEGF secretion by DPC was measured by radioassay of VEGF receptors using FBAEC as target cells. The results showed that stimulation of FBAEC proliferation and migration following exposure to purified conditioned medium was dose-dependent. Radioreceptor assays of recombinant VEGF and purified DPC-conditioned medium showed competitive VEGF binding in FBAEC.Abbreviations CM conditioned medium - DMEM Dulbecco's modified eagle's medium - DPC dermal papilla cells - EDTA ethylenediaminetetra-acetic acid - FBAEC fetal bovine aortic endothelial cells - FCS fetal calf serum - VEGF vascular endothelial growth factor     

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

AIDS-associated Kaposi's sarcoma cells in culture express vascular endothelial growth factor.

PCR cloning and cDNA sequencing have been used to identify mRNAs of two splice products of the vascular endothelial growth factor (VEGF) gene, VEGF121 and VEGF165, in cells isolated from Kaposi's sarcomas (KS) of AIDS patients (AIDS-KS). As demonstrated by Northern blot analysis, AIDS-KS cells as well as tumor cells show a high expression level of the VEGF gene as compared to primary human vascular cells like smooth muscle cells or endothelial cells. In addition to the lower expression of the gene, vascular cells express a 3.9 kb band together with a 3.2 kb band instead of a 3.9 kb and a 4.3 kb band in AIDS-KS cells. Our data suggest that the angiogenic properties of AIDS-KS cells might be mediated by the secretion of this growth factor and that this factor alone or in combination with other endothelial mitogens may be involved in endothelial proliferation associated with Kaposi's sarcoma.     

Intrauterine hypertension decreases lung VEGF expression and VEGF inhibition causes pulmonary hypertension in the ovine fetus

Although vascular endothelial growth factor (VEGF) plays a vital role in lung vascular growth in the embryo, its role in maintaining endothelial function and modulating vascular structure during late fetal life has not been studied. We hypothesized that impaired lung VEGF signaling causes pulmonary hypertension, endothelial dysfunction, and structural remodeling before birth. To determine whether lung VEGF expression is decreased in an experimental model of persistent pulmonary hypertension of the newborn (PPHN), we measured lung VEGF and VEGF receptor protein content from fetal lambs 7-10 days after ductus arteriosus ligation (132-140 days gestation; term = 147 days). In contrast with the surge in lung VEGF expression during late gestation in controls, chronic intrauterine pulmonary hypertension reduced lung VEGF expression by 78%. To determine whether VEGF inhibition during late gestation causes pulmonary hypertension, we treated fetal lambs with EYE001, an aptamer that specifically inhibits VEGF(165). Compared with vehicle controls, EYE001 treatment elevated pulmonary artery pressure and pulmonary vascular resistance by 22 and 50%, respectively, caused right ventricular hypertrophy, and increased wall thickness of small pulmonary arteries. EYE001 treatment reduced lung endothelial nitric oxide synthase protein content by 50% and preferentially impaired the pulmonary vasodilator response to ACh, an endothelium-dependent agent. We conclude that chronic intrauterine pulmonary hypertension markedly decreases lung VEGF expression and that selective inhibition of VEGF(165) mimics the structural and physiological changes of experimental PPHN. We speculate that hypertension downregulates VEGF expression in the developing lung and that impaired VEGF signaling may contribute to the pathogenesis of PPHN.     

Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 x 10(-6) mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 x 10(-5) mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 microM) and the selective COX-2 inhibitor NS-398 (5 microM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 microM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 x 10(-3) mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.     

低氧大鼠肺动脉内皮细胞VEGF变化与PKC活性关系的探讨

目的：探讨低氧培养大鼠肺动脉血管内皮细胞ＶＥＧＦ的表达变化与ＰＫＣ活性的关系。方法：培养大鼠肺动脉血管内皮细胞,观察低氧（１％Ｏ２）培养不同时间大鼠肺动脉血管内皮细胞浆、膜ＰＫＣ活性和培养液中ＶＥＧＦ水平变化;加入ＰＫＣ抑制剂（ｓｔａｕｒｏｓｐｏｒｉｎｅ）后,测定低氧、常氧培养不同时间二者的变化。结果：低氧时膜ＰＫＣ活性和培养液中ＶＥＧＦ水平明显升高（Ｐ＜０．０１）。而加入ＰＫＣ抑制剂后,常氧和低     

Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells

Vascular endothelial growth factor (VEGF) is a newly identified growth and permeability factor with a unique specificity for endothelial cells. Recently the flt-encoded tyrosine kinase was characterized as a receptor for VEGF. A novel tyrosine kinase receptor encoded by the KDR gene was also found to bind VEGF with high affinity when expressed in CMT-3 cells. Screening for flt and KDR expression in a variety of species and tissue-derived endothelial cells demonstrates that flt is predominantly expressed in human placenta and human vascular endothelial cells. Placenta growth factor (PIGF), a growth factor significantly related to VEGF, is coexpressed with flt in placenta and human vascular endothelial cells. KDR is more widely distributed and expressed in all vessel-derived endothelial cells. These data demonstrate that cultured human endothelial cells isolated from different tissues express both VEGF receptors in relative high levels and, additionally, that all investigated nonhuman endothelial cells in culture are also positive for KDR gene expression.     

Functional roles of angiogenic factors and receptors on non‐endothelial cells in the oropharyngeal cavity of the chukar partridge (Alectoris chukar)

The vascular endothelial growth factor (VEGF) family belong to the platelet‐derived growth factor supergene family and is involved in angiogenesis and mitogenesis. The VEGF–VEGFR system regulates endothelial cell proliferation, migration, vascular permeability, secretion and other non‐endothelial cells functions. To clarify the possible role of endothelial and non‐endothelial cells, VEGF and its receptors, vascular endothelial cell growth inhibitor (VEGI) were immunohistochemically examined in oropharyngeal organs. Ten adult partridges were used in this study and the pharynx and larynx were dissected together with the palate and tongue. VEGI, VEGF and its receptor were highly expressed in luminal epithelial and stromal cells, when compared to glandular epithelial and muscle cells (P < 0.05). Moreover, VEGF, its receptors and VEGI were expressed rather strongly in the endothelial cells of the blood capillaries and in both the endothelial and smooth muscle cells of the large and small blood vessels. In conclusion, VEGF and its receptors (flt1/fms, flk1/KDR and flt4) and VEGI were expressed by various cell groups at varying intensity in the oropharyngeal organs. This demonstrates that they play a critical role in the regulation and maintenance of the functions in cells different from endothelial ones as well as in cell proliferation, differentiation, apoptosis and angiogenesis.     

逆转录病毒介导人VEGF在兔皮肤成纤维细胞中的表达

A replication-deficient recombinant retrovirus containing the cDNA coding for human vascular endothelial growth factor (VEGF) was generated, and then infected rabbit primary skin fibroblasts. After selection with G418, the transduced colonies have the ability of producing VEGF. The integration and expression of VEGF in transduced cells were confirmed by Southern blot, PCR, Northern blot and RT-PCR assay. The VEGF secreted by transduced cells has strong bioactivity when assayed by endothelial proliferation and Miles vascular permeability assay. Thus, this study pave the way for future study of biological and physiological effect of VEGF in vivo.     

逆转录病毒介导人VEGF在兔皮肤成纤维细胞中的表达

本文以人VEGFcDNA为目的基因,构建成逆转录病毒质粒载体,并进一步包装成重组缺陷型逆转录病毒,以此感染原代兔皮肤成纤维细胞,经G418筛选,获得了具有分泌VEGF能力的抗性细胞克隆,经Southern、PCR、Northern、RT-PCR等检测,证实了外源基因已整合至原代兔皮肤成纤维细胞基因组中,无重排和产生野生型病毒之患。整合的VEGF可在修饰细胞中转录,经内皮细胞增殖实验和血管通透性实验证实其表达产物具有强大的生物活性。为进一步研究VEGF的生物学功能和生理效应提供有用的工具。     

Nitric oxide mediates the mitogenic effects of insulin and vascular endothelial growth factor but not of leptin in endothelial cells

The regulation of vascular wall homeostasis by nitric oxide (NO) generated by endothelium is being intensively studied. In the present paper, the involvement of NO in the vascular endothelial growth factor (VEGF), insulin or leptin-stimulated proliferation of human endothelial cells (HUVEC) was measured by [3H]thymidine or bromodeoxyuridine incorporation. VEGF and insulin, but not leptin, increased NO generation in HUVEC, as detected with ISO-NO electrode. Proliferation of HUVEC induced by leptin was not changed or was higher in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) a nitric oxide synthase (NOS) inhibitor. In contrast, L-NAME blunted the proproliferative effect of VEGF and insulin. Furthermore, we demonstrated that, in human arterial smooth muscle cells (hASMC) transfected with endothelial NOS (eNOS) gene, the generation of biologically active VEGF protein was NO-dependent. Inhibition of NO generation by L-NAME decreased the synthesis of VEGF protein and attenuated HUVEC proliferation induced by conditioned media from transfected hASMC. Endothelium-derived NO seems to participate in VEGF and insulin, but not leptin, mitogenic activity. Additionally, the small amounts of NO released from endothelial cells, as mimicked by eNOS transfection into hASMC, may activate generation of VEGF in sub-endothelial smooth muscle cells, leading to increased synthesis of VEGF protein necessary for turnover and restitution of endothelial cells.     

Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1–4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix–binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases.     

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-M_r 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.     

Hantaviruses direct endothelial cell permeability by sensitizing cells to the vascular permeability factor VEGF, while angiopoietin 1 and sphingosine 1-phosphate inhibit hantavirus-directed permeability

Hantaviruses infect human endothelial cells and cause two vascular permeability-based diseases: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Hantavirus infection alone does not permeabilize endothelial cell monolayers. However, pathogenic hantaviruses inhibit the function of alphav beta3 integrins on endothelial cells, and hemorrhagic disease and vascular permeability deficits are consequences of dysfunctional beta3 integrins that normally regulate permeabilizing vascular endothelial growth factor (VEGF) responses. Here we show that pathogenic Hantaan, Andes, and New York-1 hantaviruses dramatically enhance the permeability of endothelial cells in response to VEGF, while the nonpathogenic hantaviruses Prospect Hill and Tula have no effect on endothelial cell permeability. Pathogenic hantaviruses directed endothelial cell permeability 2 to 3 days postinfection, coincident with pathogenic hantavirus inhibition of alphav beta3 integrin functions, and hantavirus-directed permeability was inhibited by antibodies to VEGF receptor 2 (VEGFR2). These studies demonstrate that pathogenic hantaviruses, similar to alphav beta3 integrin-deficient cells, specifically enhance VEGF-directed permeabilizing responses. Using the hantavirus permeability assay we further demonstrate that the endothelial-cell-specific growth factor angiopoietin 1 (Ang-1) and the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) inhibit hantavirus directed endothelial cell permeability at physiologic concentrations. These results demonstrate the utility of a hantavirus permeability assay and rationalize the testing of Ang-1, S1P, and antibodies to VEGFR2 as potential hantavirus therapeutics. The central importance of beta3 integrins and VEGF responses in vascular leak and hemorrhagic disease further suggest that altering beta3 or VEGF responses may be a common feature of additional viral hemorrhagic diseases. As a result, our findings provide a potential mechanism for vascular leakage after infection by pathogenic hantaviruses and the means to inhibit hantavirus-directed endothelial cell permeability that may be applicable to additional vascular leak syndromes.     

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 role of vascular endothelial cadherin in modulating c-Src activation and downstream signaling of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and vascular permeability, in which c-Src tyrosine kinase plays an essential role. However, the mechanisms by which VEGF stimulates c-Src activation have remained unclear. Here, we demonstrate that vascular endothelial cadherin (VE-cadherin) plays a critical role in regulating c-Src activation in response to VEGF. In vascular endothelial cells, VE-cadherin was basally associated with c-Src and Csk (C-terminal Src kinase), a negative regulator of Src activation. VEGF stimulated Csk release from VE-cadherin by recruiting the protein tyrosine phosphatase SHP2 to VE-cadherin signaling complex, leading to an increase in c-Src activation. Silencing VE-cadherin with small interference RNA significantly reduced VEGF-stimulated c-Src activation. Disrupting the association of VE-cadherin and Csk through the reconstitution of Csk binding-defective mutant of VE-cadherin also diminished Src activation. Moreover, inhibiting SHP2 by small interference RNA and adenovirus-mediated expression of a catalytically inactive mutant of SHP2 attenuated c-Src activation by blocking the disassociation of Csk from VE-cadherin. Furthermore, VE-cadherin and SHP2 differentially regulates VEGF downstream signaling. The inhibition of c-Src, VE-cadherin, and SHP2 diminished VEGF-mediated activation of Akt and endothelial nitric-oxide synthase. In contrast, inhibiting VE-cadherin and SHP2 enhanced ERK1/2 activation in response to VEGF. These findings reveal a novel role for VE-cadherin in modulating c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function.