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

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

AMP-activated protein kinase mediates VEGF-stimulated endothelial NO production

Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell function. VEGF stimulates NO production, proposed to be a result of phosphorylation and activation of endothelial NO synthase (eNOS) at Ser1177. Phosphorylation of eNOS at this site also occurs after activation of AMP-activated protein kinase (AMPK) in cultured endothelial cells. We therefore determined whether AMPK mediates VEGF-stimulated NO synthesis in endothelial cells. VEGF caused a rapid, dose-dependent stimulation of AMPK activity, with a concomitant increase in phosphorylation of eNOS at Ser1177. Infection of endothelial cells with an adenovirus expressing a dominant negative mutant AMPK partially inhibited both VEGF-stimulated eNOS Ser1177 phosphorylation and NO production. VEGF-stimulated AMPK activity was completely inhibited by the Ca(2+)/calmodulin-dependent protein kinase kinase inhibitor, STO-609. Stimulation of AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase represents a novel signalling mechanism utilised by VEGF in endothelial cells that contributes to eNOS phosphorylation and NO production.     

Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells

Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2 h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells.     

Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway.

The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pM) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated with L-N(G)-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition of S-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway.     

Dephosphorylation of endothelial nitric-oxide synthase by vascular endothelial growth factor. Implications for the vascular responses to cyclosporin A

The endothelial isoform of nitric-oxide synthase (eNOS) is a key determinant of vascular tone. eNOS, a Ca(2+)/camodulin-dependent enzyme, is also regulated by a variety of agonist-activated protein kinases, but the role and regulation of the protein phosphatase pathways involved in eNOS dephosphorylation are much less well understood. Treatment of endothelial cells with vascular endothelial growth factor (VEGF), a potent eNOS agonist, leads to the activation of calcineurin, a Ca(2+)/camodulin-dependent protein phosphatase. In these studies, we used a phosphorylation state-specific antibody to show that VEGF promotes dephosphorylation of eNOS at serine residue 116 in cultured endothelial cells. Cyclosporin A, an inhibitor of calcineurin, completely blocks VEGF-induced eNOS dephosphorylation; under identical conditions, cyclosporin A also inhibits VEGF-induced eNOS activation. VEGF-induced eNOS dephosphorylation shows an EC(50) of 2 ng/ml and is maximal 30 min after agonist addition. eNOS phosphorylation at serine 116 is completely blocked by the protein kinase C inhibitor calphostin but is blocked by neither wortmannin (an inhibitor of phosphatidylinositide 3-kinase) nor the MAP kinase pathway inhibitor U0126. A phosphorylation-deficient mutant of eNOS in which serine 116 is changed to an alanine residue (S116A) shows significantly enhanced enzyme activity compared with the wild-type enzyme. Taken together, these findings indicated that VEGF-induced eNOS dephosphorylation at serine 116 leads to enzyme activation. Cyclosporin A is widely used as an immunosuppressive drug for which hypertension is an important dose-limiting side effect. Our results suggest that cyclosporin A-induced hypertension may involve, at least in part, the attenuation of endothelium-derived NO production through a calcineurin-sensitive pathway regulating eNOS dephosphorylation.     

Exogenous nitric oxide stimulates cell proliferation via activation of a mitogen-activated protein kinase pathway in ovine fetoplacental artery endothelial cells

Sodium nitroprusside (SNP), a nitric oxide (NO) donor and a nitrovasodilator drug used for patients with hypertensive crisis, has been shown to promote angiogenesis. However, direct evidence showing the involvement of NO in the SNP-induced angiogenesis is not available. Accordingly, we assessed whether NO generated from SNP-stimulated ovine fetoplacental artery endothelial (OFPAE) cell proliferation via activation of mitogen-activated protein kinase 3/1 (MAPK3/1, also termed ERK1/2). We observed that SNP dose dependently stimulated (P < 0.05) cell proliferation with a maximal effect at 1 microM and that SNP rapidly (相似文献   

Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI. Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation, but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERK activation and PI3K/Akt/eNOS/NO signaling.     

Glucose-6-phosphate dehydrogenase modulates vascular endothelial growth factor-mediated angiogenesis

Glucose-6-phosphate dehydrogenase (G6PD), the first enzyme of the pentose phosphate pathway, is the principal intracellular source of NADPH. NADPH is utilized as a cofactor by vascular endothelial cell nitric-oxide synthase (eNOS) to generate nitric oxide (NO*). To determine whether G6PD modulates NO*-mediated angiogenesis, we decreased G6PD expression in bovine aortic endothelial cells using an antisense oligodeoxynucleotide to G6PD or increased G6PD expression by adenoviral gene transfer, and we examined vascular endothelial growth factor (VEGF)-stimulated endothelial cell proliferation, migration, and capillary-like tube formation. Deficient G6PD activity was associated with a significant decrease in endothelial cell proliferation, migration, and tube formation, whereas increased G6PD activity promoted these processes. VEGF-stimulated eNOS activity and NO* production were decreased significantly in endothelial cells with deficient G6PD activity and enhanced in G6PD-overexpressing cells. In addition, G6PD-deficient cells demonstrated decreased tyrosine phosphorylation of the VEGF receptor Flk-1/KDR, Akt, and eNOS compared with cells with normal G6PD activity, whereas overexpression of G6PD enhanced phosphorylation of Flk-1/KDR, Akt, and eNOS. In the Pretsch mouse, a murine model of G6PD deficiency, vessel outgrowth from thoracic aorta segments was impaired compared with C3H wild-type mice. In an in vivo Matrigel angiogenesis assay, cell migration into the plugs was inhibited significantly in G6PD-deficient mice compared with wild-type mice, and gene transfer of G6PD restored the wild-type phenotype in G6PD-deficient mice. These findings demonstrate that G6PD modulates angiogenesis and may represent a novel angiogenic regulator.     

VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide

We tested the hypothesis that VEGF regulates endothelial hyperpermeability to macromolecules by activating the ERK-1/2 MAPK pathway. We also tested whether PKC and nitric oxide (NO) mediate VEGF-induced increases in permeability via the ERK-1/2 pathway. FITC-Dextran 70 flux across human umbilical vein endothelial cell monolayers served as an index of permeability, whereas Western blots assessed the phosphorylation of ERK-1/2. VEGF-induced hyperpermeability was inhibited by antisense DNA oligonucleotides directed against ERK-1/2 and by blockade of MEK and Raf-1 activities (20 microM PD-98059 and 5 microM GW-5074). These blocking agents also reduced ERK-1/2 phosphorylation. The PKC inhibitor bisindolylmaleimide I (10 microM) blocked both VEGF-induced ERK-1/2 activation and hyperpermeability. The NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (200 microM) and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidiazoline-1-oxyl-3-oxide (100 microM) abolished VEGF-induced hyperpermeability but did not block ERK-1/2 phosphorylation. These observations demonstrate VEGF-induced hyperpermeability involves activation of PKC and NOS as well as Raf-1, MEK, and ERK-1/2. Furthermore, our data suggest that ERK-1/2 and NOS are elements of different signaling pathways in VEGF-induced hyperpermeability.     

Angiotensin II elevates nitric oxide synthase 3 expression and nitric oxide production via a mitogen-activated protein kinase cascade in ovine fetoplacental artery endothelial cells

Normal pregnancy is associated with high angiotensin II (ANG II) concentrations in the maternal and fetal circulation. These high levels of ANG II may promote production vasodilators such as nitric oxide (NO). ANG II receptors are expressed in ovine fetoplacental artery endothelial (OFPAE) cells and mediate ANG II-stimulated OFPAE cell proliferation. Herein, we tested whether ANG II stimulated NO synthase 3 (NOS3, also known as eNOS) expression and total NO (NO(x)) production via activation of mitogen-activated protein kinase 3/1 (MAPK3/1, also known as ERK1/2) in OFPAE cells. ANG II elevated (P < 0.05) eNOS protein, but not mRNA levels with a maximum effect at 10 nM. ANG II also dose dependently increased (P < 0.05) NO(x) production with a maximal effect at doses of 1-100 nM. Activation of ERK1/2 by ANG II was determined by immunocytochemistry and Western blot analysis. ANG II rapidly induced positive staining for phosphorylated ERK1/2, appearing in cytosol after 1-5 min of ANG II treatment, accumulating in nuclei after 10 min, and disappearing at 15 min. ANG II increased (P < 0.05) phosphorylated ERK1/2 protein levels. Activation of ERK1/2 was confirmed by an immunocomplex kinase assay using ELK1 as a substrate. PD98059 significantly inhibited ANG II-induced ERK1/2 activation, and the ANG II-elevated eNOS protein levels but only partially reduced ANG II-increased NO(x) production. Thus, in OFPAE cells, the ANG II increased NO(x) production is associated with elevated eNOS protein expression, which is mediated at least in part via activation of the mitogen-activated protein kinase kinase1 and kinase2 (MAP2K1 and MAP2K2, known also as MEK1/2)/ERK1/2 cascade. Together with our previous observation that ANG II stimulates OFPAE cell proliferation, these data suggest that ANG II is a key regulator for both vasodilation and angiogenesis in the ovine fetoplacenta.     

MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor

Although the significance of vascular endothelial growth factor (VEGF) and its receptors in angiogenesis is well established, the signal transduction cascades activated by VEGF and their involvement in mediating the mitogenic response of endothelial cells to VEGF are incompletely characterized. Here we demonstrate that VEGF activates mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and p70 S6 kinase in human umbilical vein endothelial cells (HUVEC). The activation of these enzymes was assayed by kinase phosphorylation and by kinase activity towards substrates. Studies with PI 3-kinase inhibitors revealed that activation of p70 S6 kinase was mediated by PI 3-kinase. Selective inhibition of ERK, PI 3-kinase, and p70 S6 kinase with the inhibitors PD098059, LY294002, and rapamycin, respectively, inhibited VEGF-stimulated HUVEC proliferation. In marked contrast, the p38 MAP kinase inhibitor SB203580 not only failed to inhibit but actually enhanced HUVEC proliferation; this effect was associated with the phosphorylation of Rb protein. Rb phosphorylation resulted from a decrease in the level of the cdk inhibitor p27KiP1. These results indicate that the activities of ERK, PI 3-kinase, and p70 S6 kinase are essential for VEGF-induced HUVEC proliferation. p38 MAP kinase suppresses endothelial cell proliferation by regulating cell-cycle progression.     

VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, promotes endothelial cell survival and angiogenesis. We recently showed that VEGF can support the growth of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells in serum-free medium. Reasoning that VEGF might be modulating apoptotic signal transduction pathways, we examined mechanisms involved in the anti-apoptotic effect of VEGF on starvation- and ceramide-induced apoptosis in HDMEC. We observed that VEGF ameliorated the time-dependent increase in apoptosis, as demonstrated by morphologic observations, TUNEL assay, and DNA fragmentation. On the other hand, basic fibroblast growth factor only partially prevented apoptosis in serum-starved HDMEC; platelet-derived growth factor-BB was completely ineffective. VEGF activated the phosphorylation of extracellular signal regulated kinase (ERK)1 (p44 mitogen-activated protein kinase; MAPK) and ERK2 (p42 MAPK) in a time- and concentration-dependent manner. Both the VEGF-induced activation and its anti-apoptotic effect were prevented by the specific MAPK/ERK inhibitor PD98059. The presence of VEGF also inhibited the sustained activation of stress-activated protein kinase/c-jun-NH2-kinase (SAPK/JNK) caused by serum starvation and ceramide treatment. Activation of the MAPK pathway together with inhibition of SAPK/JNK activity by VEGF appears to be a key event in determining whether an endothelial cell survives or undergoes programmed cell death.     

Glomerular endothelial PI3 kinase-α couples to VEGFR2, but is not required for eNOS activation

Vascular endothelial growth factor (VEGF)-dependent signals are central to many endothelial cell (EC) functions, including survival and regulation of vascular tone. Akt and endothelial nitric oxide synthase (eNOS) activity are implicated to mediate these effects. Dysregulated signaling is characteristic of endothelial dysfunction that sensitizes the glomerular microvasculature to injury. Signaling intermediates that couple VEGF stimulation to eNOS activity remain unclear; hence, we examined the PI3 kinase isoforms implicated to regulate these enzymes. Using a combination of small molecule inhibitors and RNAi to study responses to VEGF in glomerular EC, we observed that the PI3 kinase p110α catalytic isoform is coupled to VEGFR2 and regulates the bulk of Akt activity. Coimmunoprecipitation experiments support a physical association of p110α with VEGFR2. Downstream, Akt-mediated FOXO1 phosphorylation in EC is regulated by p110α. The p110δ isoform contributes a minor amount of VEGF-stimulated Akt activation. However, we observe no effect of p110α or p110δ to regulate VEGF-stimulated eNOS activation via Akt-mediated phosphorylation on eNOS Ser1177, or NO-mediated vasodilation of the afferent arteriole ex vivo. VEGFR2-stimulated eNOS activation and NO production are inhibited by Compound C, an inhibitor of AMP-stimulated kinase, independent of PI3 kinase signaling. PI3 kinase-α/δ-mediated signaling downstream of VEGFR2 activation regulates Akt-dependent survival signals, but our data suggest it is not required to activate eNOS or to elicit NO production in glomerular EC.     

Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells

We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.     

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.     

Phospholipase Cgamma-Erk Axis in vascular endothelial growth factor-induced eukaryotic initiation factor 4E phosphorylation and protein synthesis in renal epithelial cells

Vascular endothelial growth factor (VEGF) increases protein synthesis and induces hypertrophy in renal tubular epithelial cells (Senthil, D., Choudhury, G. G., McLaurin, C., and Kasinath, B. S. (2003) Kidney Int. 64, 468-479). We examined the role of Erk1/2 MAP kinase in protein synthesis induced by VEGF. VEGF stimulated Erk phosphorylation that was required for induction of protein synthesis. VEGF-induced Erk activation was not dependent on phosphoinositide (PI) 3-kinase activation but required sequential phosphorylation of type 2 VEGF receptor, PLCgamma and c-Src, as demonstrated by inhibitors SU1498, U73122, and PP1, respectively. c-Src phosphorylation was inhibited by U73122, indicating it was downstream of phospholipase (PL)Cgamma. Studies with PP1/2 showed that phosphorylation of c-Src was required for tyrosine phosphorylation of Raf-1, an upstream regulator of Erk. VEGF also stimulated phosphorylation of Pyk-2; VEGF-induced phosphorylation of Pyk2, c-Src and Raf-1 could be abolished by BAPTA/AM, demonstrating requirement for induction of intracellular calcium currents. We examined the downstream events following the phosphorylation of Erk. VEGF stimulated phosphorylation of Mnk1 and eIF4E and induced Mnk1 to shift from the cytoplasm to the nucleus upon phosphorylation. VEGF-induced phosphorylation of Mnk1 and eIF4E required phosphorylation of PLCgamma, c-Src, and Erk. Expression of dominant negative Mnk1 abrogated eIF4E phosphorylation and protein synthesis induced by VEGF. VEGF-stimulated protein synthesis could be blocked by inhibition of PLCgamma by a chemical inhibitor or expression of a dominant negative construct. Our data demonstrate that VEGF-stimulated protein synthesis is Erk-dependent and requires the activation of VEGF receptor 2, PLCgamma, c-Src, Raf, and Erk pathway. VEGF also stimulates Erk-dependent phosphorylation of Mnk1 and eIF4E, crucial events in the initiation phase of protein translation.     

eNOS function is developmentally regulated: uncoupling of eNOS occurs postnatally

At birth, the transition to gas breathing requires the function of endothelial vasoactive agents. We investigated the function of endothelial nitric oxide synthase (eNOS) in pulmonary artery (PA) vessels and endothelial cells isolated from fetal and young (4-wk) sheep. We found greater relaxations to the NOS activator A-23187 in 4-wk-old compared with fetal vessels and that the NOS inhibitor nitro-L-arginine blocked relaxations in both groups. Relaxations in 4-wk vessels were not blocked by an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, but were partially blocked by catalase. We therefore hypothesized that activation of eNOS produced reactive oxygen species in 4-wk but not fetal PA. To address this question, we studied NO and superoxide production by endothelial cells at baseline and following NOS stimulation with A-23187, VEGF, and laminar shear stress. Stimulation of NOS induced phosphorylation at serine 1177, and this event correlated with an increase in NO production in both ages. Upon stimulation of eNOS, fetal PA endothelial cells (PAEC) produced only NO. In contrast 4-wk-old PAEC produced superoxide in addition to NO. Superoxide production was blocked by L-NAME but not by apocynin (an NADPH oxidase inhibitor). L-Arginine increased NO production in both cell types but did not block superoxide production. Heat shock protein 90/eNOS association increased upon stimulation and did not change with developmental age. Cellular levels of total and reduced biopterin were higher in fetal vs. 4-wk cells. Sepiapterin [a tetrahydrobiopterin (BH4) precursor] increased basal and stimulated NO levels and completely blocked superoxide production. We conclude that the normal function of eNOS becomes uncoupled after birth, leading to a developmental adaptation of the pulmonary vascular system to produce oxygen species other than NO. We speculate this may be related to cellular production and/or maintenance of BH4 levels.     

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.     

Vascular endothelial growth factor-dependent down-regulation of Flk-1/KDR involves Cbl-mediated ubiquitination. Consequences on nitric oxide production from endothelial cells

Ligand-stimulated degradation of receptor tyrosine kinase (RTK) is an important regulatory step of signal transduction. The vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of Flk-1 signaling in endothelial cells have not been investigated. Here we show that Flk-1 is rapidly down-regulated following VEGF stimulation of bovine aortic endothelial cells (BAECs). Consequently, VEGF pretreatment of endothelial cells prevents any further stimulation of Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with Flk-1 results in the phosphorylation of the ubiquitin ligase Cbl, the enhanced association of Cbl with Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of Flk-1, whereas a Cbl mutant lacking the ubiquitin ligase RING finger domain, 70Z/3-Cbl, does not. Moreover, expression of Cbl in contrast to 70Z/3-Cbl inhibits the Flk-1-dependent activation of eNOS and, thus, NO release. In BAEC overexpressing Cbl, the degradation of Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of Flk-1 signaling to eNOS. Cbl thus plays a role in the regulation of VEGF signaling by mediating the stimulated ubiquitination and, consequently, degradation of Flk-1 in endothelial cells.