The scaffolding adapter Gab1 mediates vascular endothelial growth factor signaling and is required for endothelial cell migration and capillary formation

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.     

Identification of novel angiogenesis inhibitors

Vascular endothelial growth factor (VEGF) is a key stimulant of angiogenesis, which is the process of generating new capillary blood vessels. Inhibition of the vascular endothelial growth factor receptor (VEGFR) kinase is known to result in blockage of angiogenesis. A pharmacophore was developed based on the binding of ATP to the hinge region of the kinase domain of VEGFR and a database search of 18,000 compounds was conducted. Selected hits were assessed for their ability to limit the induction of web-like network of capillary tubes by the human umbilical vascular endothelial cells. Two compounds (1 and 4) showed good inhibitory ability to prevent sprouting and closed polygon formation of the tubular networks, promising them to be lead compounds. Compound 4 showed 60% inhibition at 0.05 microM.     

Intrinsic tyrosine kinase activity is required for vascular endothelial growth factor receptor 2 ubiquitination, sorting and degradation in endothelial cells

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis co-ordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.     

Hypoxia differentially regulates VEGFR1 and VEGFR2 levels and alters intracellular signaling and cell migration in endothelial cells

The role of hypoxia on endothelial cell function and response to growth factors is unknown. Here, we tested the hypothesis that hypoxia re-programs endothelial function by modulating vascular endothelial growth factor receptor levels which in turn alter intracellular signaling and cell function. Hypoxia stimulated VEGF-A and VEGFR1 expression but decreased VEGFR2 levels in endothelial cells. During hypoxia, plasma membrane VEGFR1 levels were elevated whereas VEGFR2 levels were depleted. One functional consequence of hypoxia is a reduction in VEGF-A-stimulated and VEGFR2-regulated intracellular signaling including lowered endothelial nitric oxide synthase activation. Venous, arterial and capillary endothelial cells subjected to hypoxia all exhibited reduced cell migration in response to VEGF-A. A mechanistic explanation is that VEGFR1:VEGFR2 ratio is substantially increased during hypoxia to block VEGF-A-stimulated and VEGFR2-regulated endothelial responses to maximize cell viability and recovery.     

VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability

Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ～70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.     

Selective Inhibition of Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) Identifies a Central Role for VEGFR‐2 in Human Aortic Endothelial Cell Responses to VEGF

Abstract

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR‐family proteins consist of VEGFR‐1/Flt‐1, VEGFR‐2/KDR/Flk‐1, and VEGFR‐3/Flt‐4. Among these, VEGFR‐2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1–3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR‐specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR‐2 tyrosine kinase, ZM323881 (5‐[[7‐(benzyloxy) quinazolin‐4‐yl]amino]‐4‐fluoro‐2‐methylphenol), to explore the role of VEGFR‐2 in endothelial cell function. Consistent with its reported effects on VEGFR‐2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR‐2, but not of VEGFR‐1, epidermal growth factor receptor (EGFR), platelet‐derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR‐1 and VEGFR‐2, but not VEGFR‐3, in the absence or presence of ZM323881. Inhibition of VEGFR‐2 blocked activation of extracellular regulated‐kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR‐1‐specific ligand, placental growth factor (PlGF). Inhibition of VEGFR‐2 also perturbed VEGF‐induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor‐2 inhibition also reversed VEGF‐stimulated phosphorylation of CrkII and its Src homology 2 (SH2)‐binding protein p130^Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR‐2 thus blocked all VEGF‐induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR‐2 is critical for VEGF signaling and/or that VEGFR‐2 may function in a heterodimer with VEGFR‐1 in human vascular endothelial cells.     

Knockdown of VEGFR2 Inhibits Proliferation and Induces Apoptosis in Hemangioma-Derived Endothelial Cells

Angiogenesis is a process of development and growth of new capillary blood vessels from pre-existing vessels. Angiogenic growth factors play important roles in the development and maintenance of some malignancies, of which vascular endothelial growth factor (VEGF)/VEGFR2 interactions are involved in proliferation, migration, and survival of many cancer cells. The aim of this study was to investigate the function of VEGFR2 in human hemangiomas (HAs). Using immunohistochemistry assay, we examined the expression levels of VEGF, VEGFR2, Ki-67, glucose transporter-1 (Glut-1), phosphorylated protein kinase B (p-AKT) and p-ERK in different phases of human HAs. Positive expression of VEGF, VEGFR2, Ki-67, Glut-1, p-AKT and p-ERK was significantly increased in proliferating phase HAs, while decreased in involuting phase HAs (P=0.001; P=0.003). In contrast, cell apoptotic indexes were decreased in proliferating phase HAs, but increased in involuting phase HAs (P<0.01). Furthermore, we used small hairpin RNA (shRNA)-mediated VEGFR2 knockdown in primary HA-derived endothelial cells (HemECs) to understand the role of VEGF/VEGFR2 signaling. Knockdown of VEGFR2 by Lv-shVEGFR2 inhibited cell viability and induced apoptosis in primary HemECs companied with decreased expression of p-AKT, p-ERK, p-p38MAPK and Ki-67 and increased expression of caspase-3 (CAS-3); Overexpression of VEGFR2 promoted cell viability and blocked apoptosis in Lv-VEGFR2-transfected HemECs. Taken together, our findings demonstrate that, increased expression of VEGFR2 is involved in the development of primary HemECs possibly through regulation of the AKT and ERK pathways, suggesting that VEGFR2 may be a potential therapeutic target for HAs.Key words: vascular endothelial growth factor receptor 2, hemangioma, proliferation, apoptosis     

Ligand-Stimulated VEGFR2 Signaling is Regulated by Co-Ordinated Trafficking and Proteolysis

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.     

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.     

A Heat-Shock Protein Axis Regulates VEGFR2 Proteolysis,Blood Vessel Development and Repair

Vascular endothelial growth factor A (VEGF-A) binds to the VEGFR2 receptor tyrosine kinase, regulating endothelial function, vascular physiology and angiogenesis. However, the mechanism underlying VEGFR2 turnover and degradation in this response is unclear. Here, we tested a role for heat-shock proteins in regulating the presentation of VEGFR2 to a degradative pathway. Pharmacological inhibition of HSP90 stimulated VEGFR2 degradation in primary endothelial cells and blocked VEGF-A-stimulated intracellular signaling via VEGFR2. HSP90 inhibition stimulated the formation of a VEGFR2-HSP70 complex. Clathrin-mediated VEGFR2 endocytosis is required for this HSP-linked degradative pathway for targeting VEGFR2 to the endosome-lysosome system. HSP90 perturbation selectively inhibited VEGF-A-stimulated human endothelial cell migration in vitro. A mouse femoral artery model showed that HSP90 inhibition also blocked blood vessel repair in vivo consistent with decreased endothelial regeneration. Depletion of either HSP70 or HSP90 caused defects in blood vessel formation in a transgenic zebrafish model. We conclude that perturbation of the HSP70-HSP90 heat-shock protein axis stimulates degradation of endothelial VEGFR2 and modulates VEGF-A-stimulated intracellular signaling, endothelial cell migration, blood vessel development and repair.     

Membrane fixation of vascular endothelial growth factor receptor 1 ligand-binding domain is important for vasculogenesis and angiogenesis in mice

Vascular endothelial growth factor (VEGF) regulates vasculogenesis and angiogenesis by using two tyrosine kinase receptors, VEGFR1 and VEGFR2. VEGFR1 null mutant mice die on embryonic day 8.5 (E8.5) to E9.0 due to an overgrowth of endothelial cells and vascular disorganization, suggesting that VEGFR1 plays a negative role in angiogenesis. We previously showed that the tyrosine kinase (TK) domain of VEGFR1 is dispensable for embryogenesis, since VEGFR1 TK-deficient mice survived and were basically healthy. However, the molecular basis for this is not yet clearly understood. To test the hypothesis that the specific role of VEGFR1 during early embryogenesis is to recruit its ligand to the cell membrane, we deleted the transmembrane (TM) domain in TK-deficient VEGFR1 mice. Surprisingly, about half of the VEGFR1(TM-TK)-deficient mice succumbed to embryonic lethality due to a poor development of blood vessels, whereas other mice were healthy. In VEGFR1(TM-TK)(-/-) mice with growth arrest, membrane-targeted VEGF was reduced, resulting in the suppression of VEGFR2 phosphorylation. Furthermore, the embryonic lethality in VEGFR1(TM-TK)(-/-) mice was significantly increased to 80 to 90% when the genotype of VEGFR2 was changed from homozygous (+/+) to heterozygous (+/-) in 129/C57BL6 mice. These results strongly suggest that the membrane-fixed ligand-binding region of VEGFR1 traps VEGF for the appropriate regulation of VEGF signaling in vascular endothelial cells during early embryogenesis.     

HCPTPA, a protein tyrosine phosphatase that regulates vascular endothelial growth factor receptor-mediated signal transduction and biological activity

Angiogenesis is a tightly controlled process in which signaling by the receptors for vascular endothelial growth factor (VEGF) plays a key role. In order to define signaling pathways downstream of VEGF receptors (VEGFR), the kinase domain of VEGFR2 (Flk-1) was used as a bait to screen a human fetal heart library in the yeast two-hybrid system. One of the signaling molecules identified in this effort was HCPTPA, a low molecular weight, cytoplasmic protein tyrosine phosphatase. Although HCPTPA possesses no identifiable phosphotyrosine binding domains (i.e. SH2 or phosphotyrosine binding domains), it bound specifically to active, autophosphorylated VEGFR2 but not to a mutated, kinase-inactive VEGFR2. Recombinant VEGFR2 and endogenous VEGFR2 were substrates for recombinant HCPTPA, and HCPTPA was co-expressed with VEGFR2 in endothelial cell lines, suggesting that HCPTPA may be a negative regulator of VEGFR2 signal transduction. To pursue this possibility, an adenovirus directing the expression of HCPTPA was constructed. When used to infect cultured endothelial cells, this adenovirus directed high level expression of HCPTPA that resulted in impairment of VEGF-mediated VEGFR2 autophosphorylation and mitogen-activated protein kinase activation. Adenovirus-mediated overexpression of HCPTPA also inhibited VEGF-induced cellular responses (endothelial cell migration and proliferation) and inhibited angiogenesis in the rat aortic ring assay. Taken together, these findings indicate that HCPTPA may be an important regulator of VEGF-mediated signaling and biological activity. Potential interactions with other signaling pathways and possible therapeutic implications are discussed.     

Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived VEGFR2(+) mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras(-/-) mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2(+) progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2(+) progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of VEGFR2(+) vascular progenitor cells.     

Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis

Vascular endothelial growth factor (VEGF)-A, a major regulator for angiogenesis, binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). These receptors regulate physiological as well as pathological angiogenesis. VEGFR2 has strong tyrosine kinase activity, and transduces the major signals for angiogenesis. However, unlike other representative tyrosine kinase receptors which use the Ras pathway, VEGFR2 mostly uses the Phospholipase-Cgamma-Protein kinase-C pathway to activate MAP-kinase and DNA synthesis. VEGFR2 is a direct signal transducer for pathological angiogenesis including cancer and diabetic retinopathy, thus, VEGFR2 itself and the signaling appear to be critical targets for the suppression of these diseases. VEGFR1 plays dual role, a negative role in angiogenesis in the embryo most likely by trapping VEGF-A, and a positive role in adulthood in a tyrosine kinase-dependent manner. VEGFR1 is expressed not only in endothelial cells but also in macrophage-lineage cells, and promotes tumor growth, metastasis, and inflammation. Furthermore, a soluble form of VEGFR1 was found to be present at abnormally high levels in the serum of preeclampsia patients, and induces proteinurea and renal dysfunction. Therefore, VEGFR1 is also an important target in the treatment of human diseases. Recently, the VEGFR2-specific ligand VEGF-E (Orf-VEGF) was extensively characterized. Interestingly, the activation of VEGFR2 via VEGF-E in vivo results in a strong angiogenic response in mice with minor side effects such as inflammation compared with VEGF-A, suggesting VEGF-E to be a novel material for pro-angiogenic therapy.     

Design,synthesis, and evaluation of 5-methyl-4-phenoxy-5H-pyrrolo[3,2-d]pyrimidine derivatives: Novel VEGFR2 kinase inhibitors binding to inactive kinase conformation

We synthesized a series of pyrrolo[3,2-d]pyrimidine derivatives and evaluated their application as type-II inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) kinase. Incorporation of a diphenylurea moiety at the C4-position of the pyrrolo[3,2-d]pyrimidine core via an oxygen linker resulted in compounds that were potent inhibitors of VEGFR2 kinase. Of these derivatives, compound 20d showed the strongest inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVEC). The co-crystal structure of 20d and VEGFR2 revealed that 20d binds to the inactive form of VEGFR2. Further studies indicated that 20d inhibited VEGFR2 kinase with slow dissociation kinetics and also inhibited PDGFR and Tie-2 kinases. Oral administration of the hydrochloride salt of 20d at 3 mg/kg/day showed potent inhibition of tumor growth in a DU145 human prostate cancer cell xenograft nude mouse model.     

VEGFR1 receptor tyrosine kinase localization to the Golgi apparatus is calcium-dependent

Vascular endothelial growth factor receptor 1 (VEGFR1) is an essential receptor tyrosine kinase that regulates mammalian vascular development and embryogenesis but its function is not well understood. Herein, we present evidence whereby endothelial VEGFR1 is largely resident within the Golgi apparatus but translocates to the plasma membrane via a calcium-regulated process. Primary human endothelial cells reveal differing VEGFR1 and VEGFR2 intracellular distribution and dynamics. The major proportion of the full-length VEGFR1 membrane protein was resident within the Golgi apparatus in primary endothelial cells. Whereas VEGFR2 displayed down-regulation in response to VEGF-A, VEGFR1 was not significantly affected arguing for a significant intracellular pool that was inaccessible to extracellular VEGF-A. This intracellular VEGFR1 pool showed significant co-distribution with key Golgi residents. Brefeldin A caused VEGFR1 Golgi fragmentation consistent with redistribution to the endoplasmic reticulum. Metabolic labeling experiments and microscopy using domain-specific VEGFR1 antibodies indicated that the mature processed VEGFR1 species and an integral membrane protein was resident within Golgi apparatus. Cytosolic calcium ions play a key role in VEGFR1 trafficking as treatment with either VEGF-A, histamine, thrombin, thapsigargin or A23187 ionophore caused VEGFR1 redistribution from the Golgi apparatus to small punctate vesicles and plasma membrane. We thus propose a model whereby the balance of VEGFR1 and VEGFR2 plasma membrane levels dictate either negative or positive endothelial signaling to influence vascular physiology.     

The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells

Identifying prosurvival mechanisms in stressed neuronal cells would provide protective strategies to hinder neurodegeneration. Recent evidence shows that vascular endothelial growth factor (VEGF), a well-established mitogen in endothelial cells, can mediate neuroprotection against damaging insults through the activation of its cognate receptor VEGFR2. In addition, growth factor receptor signaling pathways have been shown to crosstalk with cAMP-dependent Protein Kinase A (PKA) to protect neuronal cells from harmful stimuli. Whether a relationship exists between VEGFR2 and PKA in mediating neuroprotection under stressful conditions is unknown. Using SK-N-SH neuronal cells as a model system, we show that serum deprivation induces an upregulation in VEGF and VEGFR2 that concomitantly serves as a prosurvival signaling pathway. Inhibitor studies revealed that PKA functioned concurrently with VEGFR2 pathway to signal the activation of the extracellular signal-regulated protein kinases (ERK1/2) as protection against caspase-3/7 activation and a subsequent cell death. The loss in cell viability induced by VEGFR2 and PKA inhibition was prevented by caspase inhibition or overexpression of ERK1. Overexpression of the antiapoptotic protein Bcl-xL also promoted survival when VEGFR2 function was blocked. However, the protection elicited by all three treatments were prevented by the inclusion of a selective inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase that activates ERK1/2. Taken together, these findings suggested that PKA and VEGFR2 converge at the MEK/ERK1/2 pathway to protect serum starved neuronal cells from a caspase-dependent cell death. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.