Novel role of ARF6 in vascular endothelial growth factor-induced signaling and angiogenesis

Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6(T27N) and mutant caveolin-1(Y14F), which cannot be phosphorylated, block VEGF-stimulated EC migration and proliferation. Moreover, ARF6 expression is markedly upregulated in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. Thus, ARF6 is involved in the temporal-spatial organization of caveolae/lipid rafts- and ROS-dependent VEGF signaling in ECs as well as in angiogenesis in vivo.     

Role of protein tyrosine phosphatase 1B in vascular endothelial growth factor signaling and cell-cell adhesions in endothelial cells

Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo.     

Redox signaling in angiogenesis: role of NADPH oxidase

Angiogenesis, a process of new blood vessel formation, is a key process involved in normal development and wound repair as well as in the various pathophysiologies such as ischemic heart and limb diseases and atherosclerosis. Reactive oxygen species (ROS) such as superoxide and H(2)O(2) function as signaling molecules in many aspects of growth factor-mediated responses including angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor and stimulates proliferation, migration, and tube formation of endothelial cells (ECs) primarily through the VEGF receptor type2 (VEGR2, KDR/Flk1). VEGF binding initiates autophosphorylation of VEGFR2, which results in activation of downstream signaling enzymes including ERK1/2, Akt, and eNOS in ECs, thereby stimulating angiogenesis. The major source of ROS in EC is a NADPH oxidase which consists of Nox1, Nox2 (gp91phox), Nox4, p22phox, p47phox, p67phox and the small G protein Rac1. The endothelial NADPH oxidase is activated by angiogenic factors including VEGF and angiopoietin-1. ROS derived from this enzyme stimulate diverse redox signaling pathways leading to angiogenesis-related gene induction as well as EC migration and proliferation, which may contribute to postnatal angiogenesis in vivo. The aim of this review is to provide an overview of the recent progress on the emerging area of the role of ROS derived from NADPH oxidase and redox signaling in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for treatment of angiogenesis-dependent cardiovascular diseases and for promoting angiogenesis in ischemic limb and heart diseases.     

Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis

Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac1 and gp91(phox) are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O2*- production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac1 (N17Rac1) and transfection of gp91(phox) antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N17Rac1 significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp91(phox) antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of sphingosine 1-phosphate (S1P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S1P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp91(phox-/-) mice, suggesting that ROS derived from gp91(phox)-containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.     

Control of endothelial cell proliferation and migration by VEGF signaling to histone deacetylase 7

VEGF has been shown to regulate endothelial cell (EC) proliferation and migration. However, the nuclear mediators of the actions of VEGF in ECs have not been fully defined. We show that VEGF induces the phosphorylation of three conserved serine residues in histone deacetylase 7 (HDAC7) via protein kinase D, which promotes nuclear export of HDAC7 and activation of VEGF-responsive genes in ECs. Expression of a signal-resistant HDAC7 mutant protein in ECs inhibits proliferation and migration in response to VEGF. These results demonstrate that phosphorylation of HDAC7 serves as a molecular switch to mediate VEGF signaling and endothelial function.     

A chemically defined culture of VEGFR2+ cells derived from embryonic stem cells reveals the role of VEGFR1 in tuning the threshold for VEGF in developing endothelial cells

Vascular endothelial growth factor (VEGF) is a major growth factor for developing endothelial cells (ECs). Embryonic lethality due to haploinsufficiency of VEGF in the mouse highlighted the strict dose dependency of VEGF on embryonic vascular development. Here we investigated the dose-dependent effects of VEGF on the differentiation of ES cell-derived fetal liver kinase 1 (Flk-1)/VEGF receptor 2(+) (VEGFR2(+)) mesodermal cells into ECs on type IV collagen under a chemically defined serum-free condition. These cells could grow even in the absence of VEGF, but differentiated mostly into mural cells positive for alpha-smooth muscle actin. VEGF supported in a dose-dependent manner the differentiation into ECs defined by the expression of VE-cadherin, platelet-endothelial cell adhesion molecule 1 (PECAM-1)/ CD31, CD34, and TIE2/TEK. VEGF requirement was greater at late than at early phase of culture during EC development, whereas response of VEGFR2(+) cells to VEGF-E, which is a virus-derived ligand for VEGFR2 but not for Flt-1/VEGFR1, was not dose sensitive even at late phase of culture. Delayed expression of VEGFR1 correlated with increased dose dependency of VEGF. These results suggested that greater requirement of VEGF in the maintenance than induction of ECs was due to the activity of VEGFR1 sequestering VEGF from VEGFR2 signal. The chemically defined serum-free culture system described here provides a new tool for assessing different factors for the proliferation and differentiation of VEGFR2(+) mesodermal cells.     

A novel, quantitative model for study of endothelial cell migration and sprout formation within three-dimensional collagen matrices

Interactions between migratory endothelial cells (ECs) and surrounding extracellular matrix (ECM) are of central importance to vascular growth. Here, we present a new model of EC migration and morphogenesis within three-dimensional ECM termed "radial invasion of matrix by aggregated cells" (RIMAC). In the RIMAC model, single aggregates of defined numbers of bovine aortic ECs were embedded within small, lenticular gels of type I collagen supported by annuli of nylon mesh. Culture of the gels in nutrient media resulted in quantifiable, reproducible, radial migration of ECs into the collagen. The angiogenic proteins basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) each stimulated migration of ECs in a concentration-dependent manner. In combination, bFGF and VEGF stimulated migration synergistically. In contrast, transforming growth factor-beta1 inhibited migration of ECs. Low concentrations (0.1-0.5 ng/ml) of VEGF induced ECs to form multicellular sprouts, some of which possessed lumen-like spaces. Mitomycin C, an inhibitor of cell proliferation, did not affect the migration of ECs into collagen induced by 0.5 ng/ml VEGF but moderately inhibited migration induced by 5 ng/ml VEGF. Increasing the density (concentration) of the collagen gel inhibited the migration of single ECs and increased the branching and anastomosis of multicellular sprouts. We conclude that the RIMAC model is a highly efficacious assay for the screening of potentially angiogenic and angiostatic compounds and, moreover, is advantageous for mechanistic studies of vascular morphogenesis.     

Neuropilin-1 regulates vascular endothelial growth factor-mediated endothelial permeability

Neuropilin-1 (Npn-1) is a cell surface receptor that binds vascular endothelial growth factor (VEGF), a potent mediator of endothelial permeability, chemotaxis, and proliferation. In vitro, Npn-1 can complex with VEGF receptor-2 (VEGFR2) to enhance VEGFR2-mediated endothelial cell chemotaxis and proliferation. To determine the role of Npn-1/VEGFR2 complexes in VEGF-induced endothelial barrier dysfunction, endothelial cells were stably transfected with Npn1 or VEGFR2 alone (PAE/Npn and PAE/KDR, respectively), or VEGFR2 and Npn-1 (PAE/KDR/Npn-1). Permeability, estimated by measurement of transendothelial electrical resistance (TER), of PAE/Npn and PAE/KDR cell lines was not altered by VEGF165. In contrast, TER of PAE/KDR/Npn-1 cells decreased in dose-dependent fashion following VEGF165 (10 to 200 ng/mL). Activation of VEGFR2, and 2 downstream signaling intermediates (p38 and ERK1/2 MAPK) involved in VEGF-mediated permeability, also increased in PAE/KDR/Npn-1. Consistent with these data, inhibition of Npn-1, but not VEGFR2, attenuated VEGF165-mediated permeability of human pulmonary artery endothelial cells (HPAE), and VEGF121 (which cannot ligate Npn-1) did not alter TER of HPAE. Npn-1 inhibition also attenuated both VEGF165-mediated pulmonary vascular leak and activation of VEGFR2, p38, and ERK1/2 MAPK, in inducible lung-specific VEGF transgenic mice. These data support a critical role for Npn-1 in regulating endothelial barrier dysfunction in response to VEGF and suggest that activation of distinct receptor complexes may determine specificity of cellular response to VEGF.     

Cyclophilin-mediated pathways in the effect of cyclosporin A on endothelial cells: role of vascular endothelial growth factor

The relative importance of cyclophilin (CyP) versus calcineurin (Cn)-mediated mechanisms in the effect of cyclosporin A (CsA) on endothelial cells (ECs) is largely unknown. In cultured ECs, CsA was cytotoxic/proapoptotic or cytoprotective/antiapoptotic at high or low concentrations, respectively. CsA analogs (MeVal-4-CsA and MeIle-4-CsA), which bind to CyP but do not inhibit Cn, closely reproduced the CsA effects. Based on our previous data, the role of vascular endothelial growth factor (VEGF) as a mediator of CsA-induced cytoprotection was further analyzed. The actions of CsA and CsA analogs were shifted from a protective to a cell-damaging pattern in the presence of a specific anti-VEGF monoclonal antibody (mAb). This positive interaction was further supported by a transient increase in cytosolic free calcium concentration ([Ca(2+)](i)) by VEGF after pretreatment with either CsA or MeVal-4-CsA and an increase in the expression and synthesis of VEGF receptor 2 (VEGFR2). Of functional importance, blockade of the interaction between VEGF and VEGFR2 by a VEGFR2 mAb abolished the cytoprotective effect of CsA. In addition, preconditioning with low concentrations of CsA or CsA analogs increased both cytoprotection and VEGFR2 mRNA expression when EC were exposed to higher concentrations of CsA. In summary, our results reveal that (1) the biphasic responses to CsA in EC are related to the interaction of CsA with CyP rather than with Cn and (2) VEGF is a critical factor in the cytoprotective effect of CsA, by a mechanism that involves VEGFR2.     

Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin αvβ₃

Vascular endothelial growth factor (VEGF) acting through VEGF receptor 2 (VEGFR2) on endothelial cells (ECs) is a key regulator of angiogenesis, a process essential for wound healing and tumor metastasis. Rap1a and Rap1b, 2 highly homologous small G proteins, are both required for angiogenesis in vivo and for normal EC responses to VEGF. Here we sought to determine the mechanism through which Rap1 promotes VEGF-mediated angiogenesis. Using lineage-restricted Rap1-knockout mice we show that Rap1-deficiency in endothelium leads to defective angiogenesis in vivo, in a dose-dependent manner. Using ECs obtained from Rap1-deficient mice we demonstrate that Rap1b promotes VEGF-VEGFR2 kinase activation and regulates integrin activation. Importantly, the Rap1b-dependent VEGF-VEGFR2 activation is in part mediated via integrin α(v)β(3). Furthermore, in an in vivo model of zebrafish angiogenesis, we demonstrate that Rap1b is essential for the sprouting of intersomitic vessels, a process known to be dependent on VEGF signaling. Using 2 distinct pharmacologic VEGFR2 inhibitors we show that Rap1b and VEGFR2 act additively to control angiogenesis in vivo. We conclude that Rap1b promotes VEGF-mediated angiogenesis by promoting VEGFR2 activation in ECs via integrin α(v)β(3). These results provide a novel insight into the role of Rap1 in VEGF signaling in ECs.     

Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family

The central role of vascular endothelial growth factor (VEGF) in angiogenesis in health and disease makes it attractive both as a therapeutic target for anti-angiogenic drugs and as a pro-angiogenic cytokine for the treatment of ischaemic heart disease. While VEGF binds to two receptor protein tyrosine kinases, VEGFR1 (Flt-1) and VEGFR2 (KDR), most biological functions of VEGF are mediated via VEGFR2, and the role of VEGFR1 is currently unknown. Neuropilin-1, a non-tyrosine kinase transmembrane molecule, may function as a co-receptor for VEGFR2. Considerable progress has recently been made towards delineating the signal transduction pathways distal to activation of VEGFR2. Activation of the mitogen-activated protein kinase, protein kinase C and Akt pathways are all strongly implicated in mediating diverse cellular biological functions of VEGF, including cell survival, proliferation, the generation of nitric oxide and prostacyclin and angiogenesis. Upregulation of metalloproteinases, activation of focal adhesion kinase and interactions between VEGF receptors and integrins are strongly implicated in VEGF-induced endothelial cell migration. Recent findings suggest important roles for the vasodilators nitric oxide and prostacyclin, in linking post-receptor signaling networks to downstream biological effects and in mediating some in vivo endothelial functions of VEGF.     

Inhibition of mini-TyrRS-induced angiogenesis response in endothelial cells by VE-cadherin-dependent mini-TrpRS

To clarify whether a VE-cadherin-dependent pathway allows mini-TrpRS to inhibit mini-TrpRS-induced new blood vessel formation in endothelial cells (ECs), the inhibitory effects of mutant mini-TrpRS and VE-cadherin on mini-TrpRS-induced angiogenesis were investigated. The effects of mini-TyrRS and mini-TrpRS on EC proliferation were evaluated using an MTT colorimetric assay. Cell migration was assayed using a modified Boyden chamber technique. The angiogenic activity in vitro was evaluated by transwell migration assay and matrigel-induced capillary tube formation. It was found that mini-TrpRS does not inhibit the mini-TyrRS-induced proliferation and migration of EC under the condition of VE-cadherin knockout. While wild-type mini-TrpRS inhibited mini-TyrRS-induced angiogenesis, this activity vanished for the mutant protein. Also, the promotion of angiogenesis by mini-TyrRS and the inhibition of angiogenesis by mini-TrpRS were VEGFR2 dependent but not VEGF dependent. Mini-TyrRS was able to increase the protein expression of VEGFR-2 in the presence of VE-cadherin, while no stimulatory effect of mini-TyrRS was detected when VE-cadherin was not present. Angiogenesis is therefore stimulated by mini-TyrRS and inhibited by mini-TrpRS, raising the possibility that mini-TyrRS and mini-TrpRS stimulate a common downstream signaling event: VE-cadherin. Thus, naturally occurring fragments of the two proteins involved in translation, TyrRS and TrpRS, have opposing activities on angiogenesis. The opposing activities of the two tRNA synthetases suggest tight regulation of the balance between pro- and antiangiogenic stimuli.     

Vascular endothelial growth factor-induced endothelial cell migration and proliferation depend on a nitric oxide-mediated decrease in protein kinase Cdelta activity.

Vascular endothelial growth factor (VEGF) promotes angiogenesis and endothelial cell (EC) migration and proliferation by affecting intracellular mediators, only some of which are known, distal to its receptors. Protein kinase C (PKC) participates in the function of VEGF, but the role of individual PKC isoenzymes is unknown. In this study, we tested the importance of the activity of specific PKC isoenzymes in human EC migration and proliferation in response to VEGF. PKCdelta specific activity was depressed by the addition of VEGF (by 41+/-8% [P<0.05] at 24 hours) in human umbilical vein ECs (HUVECs) and in a HUVEC-derived EC line, ECV, without changing the total amount of either protein or mRNA encoding PKCdelta. Neither basic fibroblast growth factor (FGF-2) nor serum altered PKCdelta specific activity. The VEGF-induced decrease of PKCdelta activity, which began at 8 hours after stimulation, was strongly blocked by pretreatment with the nitric oxide (NO) synthase inhibitor N(G)-monomethyl-L-arginine in HUVECs; NO release peaked within 2 hours after stimulation. An exogenous NO donor, sodium nitroprusside, also decreased PKCdelta activity. The inhibition by N(G)-monomethyl-L-arginine of VEGF-induced HUVEC migration and proliferation, but not that induced by FGF-2 or serum, suggested that the decrease in PKCdelta via NO pathway is required for VEGF-induced EC migration and proliferation. Overexpression of PKCdelta in ECV cells specifically prevented EC response to VEGF but not to FGF-2 or serum. Thus, we conclude that suppression of PKCdelta activity via a NO synthase mechanism is required for VEGF-induced EC migration and proliferation, but not for that induced by FGF-2 or serum.     

LDL attenuates VEGF-induced angiogenesis via mechanisms involving VEGFR2 internalization and degradation following endosome-trans-Golgi network trafficking

Considerable efforts have been made to amplify angiogenesis under conditions of hypoxia and ischemia by vascular endothelial growth factor (VEGF) delivery, so far with limited success. Ischemic vascular diseases are often associated with hypercholesterolemia. To elucidate whether the exposure to blood lipids influences VEGF responses of microvessels, we characterized effects of low density lipoprotein (LDL) exposure on the proliferation, migration and tube formation of human umbilical vein endothelial cells. By examining the expression, phosphorylation and downstream signals of VEGF’s receptor VEGFR2, we characterized mechanisms controlling angiogenic responses following LDL exposure. LDL attenuated endothelial proliferation, migration and tube formation in a dose-dependent way. Reduced abundance of VEGFR2 and VEGFR1 were noticed in LDL-exposed endothelial cells. In subcellular localization studies that we combined with pharmacological experiments, we showed that the loss of VEGFR2 resulted from its internalization and degradation, the latter of which required syntaxin-16-dependent endosome-trans-Golgi network trafficking. As a consequence, VEGFR2 phosphorylation and downstream signals -specifically Akt and ERK1/2 phosphorylation- were attenuated in response to VEGF treatment. VEGF only partly reversed the effects of LDL on angiogenesis under conditions of normoxia and hypoxia. Our results suggest that angiogenic responses to VEGF are compromised in hypercholesterolemia as a consequence of endosomal VEGFR2 degradation.     

Lysophosphatidylcholine inhibits endothelial cell migration and proliferation via inhibition of the extracellular signal-regulated kinase pathway

Lysophosphatidylcholine (lysoPC), a major lipid component of oxidized low density lipoprotein, inhibits endothelial cell (EC) migration and proliferation, which are critical processes during angiogenesis and the repair of injured vessels. However, the mechanism(s) of lysoPC-induced inhibition of EC migration and proliferation has not been clarified. In this report, we demonstrate the critical role of extracellular signal-regulated kinase (ERK) in growth factor-stimulated EC migration and proliferation as well as their inhibition by lysoPC. EC migration and proliferation stimulated by basic fibroblast growth factor (FGF-2) were blocked by inhibition of ERK activity by both the specific mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 and the overexpression of a dominant-negative mutant of MEK1. Conversely, overexpression of a constitutively active mutant of MEK1 increased EC migration and proliferation, which were comparable to those of ECs stimulated with FGF-2. LysoPC inhibited FGF-2-induced ERK activation via prevention of Ras activation without inhibiting tyrosine phosphorylation of phospholipase C-gamma. Taken together, our data demonstrate that ERK activity is required for FGF-2-induced EC migration and proliferation and suggest that inhibition of the Ras/ERK pathway by lysoPC contributes to the reduced EC migration and proliferation.     

Xanthine oxidase interaction with vascular endothelial growth factor in human endothelial cell angiogenesis

OBJECTIVES: Reduced capillary density occurs early in cardiovascular diseases. Oxidant stress is implicated in endothelial apoptosis. We investigated the effects of xanthine oxidase (XO) on endothelial survival signaling: protein kinase B/Akt, its cross-talk with p38 MAPK and apoptosis pathways, and its effect on vascular tube formation in vascular endothelial growth factor (VEGF)-simulated human umbilical vein cells. METHODS: We studied primary cultured human endothelial cells from the umbilical cord. Reactive oxygen species (ROS) production was detected by dihydroethidium staining, cell-signaling pathways by western blots, cell survival by western blots, and nuclear chromatin and angiogenesis response by MTT proliferation assay and three-dimensional Matrigel cultures. RESULTS: Exogenous XO increased cellular ROS production and caused superoxide-dependent inhibition of Akt phosphorylation and enhancement of p38 MAPK phosphorylation in a time-and dose-dependent manner. In contrast, application of the XO inhibitor oxypurinol or allopurinol inhibited VEGF-stimulated Akt phosphorylation, indicating that endogenous XO promotes VEGF-induced endothelial cell (EC) survival signaling. Exogenous XO induced activation of caspase-3 and reduced expression of the anti-apoptosis protein Bcl-2. Exogenous XO also reduced EC viability, proliferation, and vascular tube formation by p38 MAPK-dependent, phosphoinositide 3-kinase (PI3-K) reversible mechanisms; whereas VEGF promoted EC survival by PI3-K-dependent, p38 MAPK-independent effects. CONCLUSIONS: Exogenous XO activity is an important contributor to endothelial mechanisms for microvascular rarefaction, by modulation of cell survival signaling pathways; however, endogenous XO is necessary for maintaining EC survival.     

Cytosolic and calcium-independent phospholipase A(2) mediate glioma-enhanced proangiogenic activity of brain endothelial cells

Glioma is characterized by an active production of proangiogenic molecules. We observed that conditioned medium (CM) from C6 glioma significantly enhanced proliferation and migration of immortalized rat brain GP8.3 endothelial cells (ECs) and primary bovine brain microvascular ECs. The glioma CM effect was significantly reduced by cytosolic (cPLA₂) and Ca⁺⁺-independent (iPLA₂) phospholipase A₂, cyclooxygenase-2, and protein kinase inhibitors. In GP8.3 ECs, cPLA₂ and iPLA₂ enzyme activities and phosphorylation of cPLA₂, significantly stimulated after 24 h CM co-incubation, were attenuated by PLA₂, PI3-K, MEK-1, and ERK1/2 inhibitors. By confocal microscopy, in glioma CM-stimulated ECs, enhancement of fluorescence signals for phospho-cPLA₂, phospho-ERK1/2, phospho-PKCα, COX-2, and iPLA₂ was in parallel observed. Electroporation of anti-iPLA₂ and cPLA₂ antibodies and siRNAs directed against iPLA₂ and cPLA₂ significantly inhibited cell proliferation and migration. Incubation of CM- or VEGF peptide-stimulated ECs with antibodies against VEGF or VEGFR-1/-2 receptors strongly reduced mitotic rate, cell migration, and phospho-cPLA₂ and iPLA₂ protein levels. The findings suggest that PLA₂ activities are involved in stimulating EC migration and proliferation in the presence of glioma CM and that cPLA₂ is positively regulated upstream by PI3-K, PKCα, and ERK1/2 signal cascades. Our work provides new insights in understanding EC metabolism and signaling during tumor angiogenesis.     

Down syndrome candidate region 1,a downstream target of VEGF, participates in endothelial cell migration and angiogenesis

Vascular endothelial growth factor (VEGF) is a principal stimulator of angiogenesis. However, the downstream targets of VEGF in endothelial cells (ECs) are not entirely clarified. Survey of downstream targets of VEGF in human ECs identified a number of genes, including Down syndrome candidate region 1 (DSCR1). Here, we confirmed the inducible expression of DSCR1 in ECs by Northern and Western blottings. Moreover, VEGF-stimulated induction of DSCR1 was blocked by anti-VEGF receptor-2 monoclonal antibody (mAb), or the specific calcineurin inhibitors cyclosporin A and FK506. The expression of DSCR1 in ECs of neovessels was further shown by immunohistochemical analysis. We therefore examined whether DSCR1 played any roles in angiogenesis. The specific downregulation of DSCR1 expression by antisense oligonucleotide (AS-ODN) inhibited VEGF-stimulated migration of ECs as well as angiogenesis in vivo. AS-ODN inhibited the spreading of ECs on vitronectin, as well as on the immobilized anti-alphavbeta3 mAb, but not on anti-alphavbeta5 mAb. Moreover, AS-ODN inhibited tyrosine phosphorylation of focal adhesion kinase when ECs were plated on a vitronectin-coated dish. Immunoprecipitation followed by Western blotting showed the coimmunoprecipitation of DSCR1 and integrin alphavbeta3. These results suggest that DSCR1 is involved in angiogenesis by regulating adhesion and migration of ECs via the interaction with integrin alphavbeta3.