HSP90 and Akt modulate Ang-1-induced angiogenesis via NO in coronary artery endothelium.

This study examines the notion that heat shock protein (HSP) 90 binding to nitric oxide (NO), endothelial NO synthase (eNOS), and PI3K-Akt regulate angiopoietin (Ang)-1-induced angiogenesis in porcine coronary artery endothelial cells (PCAEC). Exposure to Ang-1 (250 ng/ml) for periods up to 2 h resulted in a time-dependent increase in eNOS phosphorylation at Ser 1177 that occurred by 5 min and peaked at 60 min. This was accompanied by a gradual increase in NO release. Ang-1 also led to stimulation of HSP90 binding to eNOS and a significant increase in Akt phosphorylation. Thirty minutes of pretreatment of cells with either 1 microg/ml geldanamycin (a specific inhibitor of HSP90) or 500 nM wortmannin [a specific phosphatidylinositol 3 (PI3)-kinase (PI3K) inhibitor] significantly attenuated Ang-1-stimulated eNOS phosphorylation and NO production. Exposure to Ang-1 caused an increase in endothelial cell migration, tube formation, and sprouting from PCAEC spheroids, and pharmacological blockage of HSP90 function or inhibition of PI3K-Akt pathway completely abolished these effects. Inhibition of nitric oxide synthase by NG-nitro-l-arginine methyl ester (2.5 mM) also resulted in a significant decrease in Ang-1-induced angiogenesis. We conclude that stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to Ang-1-induced eNOS phosphorylation, NO production, and angiogenesis in PCAEC.     

Synergistic activation of endothelial nitric-oxide synthase (eNOS) by HSP90 and Akt: calcium-independent eNOS activation involves formation of an HSP90-Akt-CaM-bound eNOS complex

Endothelial nitric-oxide synthase (eNOS), which generates the endogenous vasodilator, nitric oxide (NO), is highly regulated by post-translational modifications and protein interactions. We recently used purified proteins to characterize the mechanisms by which heat shock protein 90 (HSP90) increases eNOS activity at low and high Ca2+ levels (Takahashi, S. and Mendelsohn, M. E. (2003) J. Biol. Chem. 278, 9339-9344). Here we extend these studies to explore interactions between HSP90, Akt, and eNOS. In studies with purified proteins, HSP90 increased the initial rate and maximal extent of Akt-mediated eNOS phosphorylation and activation at low Ca2+ levels. Akt was not observed in the eNOS complex in the absence of HSP90, but both active and inactive Akt associated with eNOS in the presence of HSP90. Direct binding of Akt to HSP90 was observed even in the absence of eNOS. HSP90 also facilitated CaM binding to eNOS irrespective of Akt presence. Geldanamycin (GA) disrupted HSP90-eNOS binding, reduced HSP90-stimulated CaM binding, and blocked both recruitment of Akt to the eNOS complex and phosphorylation of eNOS at Ser-1179. Akt phosphorylated only CaM-bound eNOS, in an HSP90-independent manner. HSP90 and active Akt together increased eNOS activity synergistically, which was reversed by GA. In bovine aortic endothelial cells (BAECs), the effects of vascular endothelial growth factor (VEGF) and insulin on eNOS-HSP90-Akt complex formation and eNOS activation were compared. BAPTA-AM inhibited VEGF- but not insulin-induced eNOS-HSP90-Akt complex formation and eNOS phosphorylation. Insulin caused rapid, transient increase in eNOS activity correlated temporally with the formation of eNOS-HSP90-Akt complex. GA prevented insulin-induced association of HSP90, Akt and CaM with eNOS and inhibited eNOS activation in BAECs. Both platelet-derived growth factor (PDGF) and insulin induced activation of Akt in BAECs, but only insulin caused HSP90-Akt-eNOS association and eNOS phosphorylation. These results demonstrate that HSP90 and Akt synergistically activate eNOS and suggest that this synergy contributes to Ca2+-independent eNOS activation in response to insulin.     

Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells

Adiponectin is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Here, we investigated whether adiponectin regulates angiogenic processes in vitro and in vivo. Adiponectin stimulated the differentiation of human umbilical vein endothelium cells (HUVECs) into capillary-like structures in vitro and functioned as a chemoattractant in migration assays. Adiponectin promoted the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase Akt/protein kinase B, and endothelial nitric oxide synthesis (eNOS) in HUVECs. Transduction with either dominant-negative AMPK or dominant-negative Akt abolished adiponectin-induced eNOS phosphorylation as well as adiponectin-stimulated HUVEC migration and differentiation. Dominant-negative AMPK also inhibited adiponectin-induced Akt phosphorylation, suggesting that AMPK is upstream of Akt. Dominant-negative Akt or the phosphatidylinositol 3-kinase inhibitor LY294002 blocked adiponectin-stimulated Akt and eNOS phosphorylation, migration, and differentiation without altering AMPK phosphorylation. Finally, adiponectin stimulated blood vessel growth in vivo in mouse Matrigel plug implantation and rabbit corneal models of angiogenesis. These data indicate that adiponectin can function to stimulate the new blood vessel growth by promoting cross-talk between AMP-activated protein kinase and Akt signaling within endothelial cells.     

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.     

Up-regulation of the association between heat shock protein 90 and endothelial nitric oxide synthase prevents high glucose-induced apoptosis in human endothelial cells

Hyperglycemia is the hallmark of diabetes mellitus. Poor glycemic control is correlated with increased cardiovascular morbidity and mortality. High glucose can trigger endothelial cell apoptosis by de-activation of endothelial nitric oxide synthase (eNOS). eNOS was recently demonstrated to be extensively regulated by Akt and heat shock protein 90 (HSP90). Yet, little is known about the molecular mechanisms that regulate eNOS activity during high glucose exposure. The present study was designed to determine the involvement of protein interactions between eNOS and HSP90 in high glucose-induced endothelial cell apoptosis. The protein interaction of eNOS/HSP90 and eNOS/Akt were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to either control-level (5.5 mM) or high-level (33 mM) glucose for different durations (2, 4, 6, and 24 h). The results showed that the protein interactions between eNOS and HSP90 and between eNOS and Akt and the phosphorylation of eNOS were up-regulated by high glucose exposure for 2-4 h. With longer exposures, these effects decreased gradually. During early hours of exposure, the protein interactions of eNOS/HSP90 and eNOS/Akt and the phosphorylation of eNOS were all inhibited by geldanamycin, an HSP90 inhibitor. High glucose-induced endothelial cell apoptosis was also enhanced by geldanamycin and was reversed by NO donors. LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor, inhibited the association of eNOS/Akt and the phosphorylation of eNOS but had no effect on the interaction between eNOS and HSP90 during early hours of exposure. From our results we propose that, in HUVECs, during early phase of high glucose exposure, apoptosis can be prevented by enhancement of eNOS activity through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt. With longer exposure, dysregulation of eNOS activity would result in apoptosis. The present study provides a molecular basis for the effects of eNOS in the prevention of endothelial cells apoptosis during early phase of high glucose exposure. These observations may contribute to the understanding of the pathogenesis of vascular complications in diabetes mellitus.     

Fractalkine stimulates angiogenesis by activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways

Fractalkine (FKN) has been implicated in modulation of angiogenesis and vascular inflammation, but the underlying mechanism has not been elucidated. We have investigated the molecular mechanism by which FKN regulates angiogenesis. We found that recombinant FKN increases in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells and stimulates in vivo angiogenesis. FKN-induced angiogenesis was accompanied by phosphorylation of ERK, Akt, and endothelial nitric oxide (NO) synthase (eNOS), as well as an increase in NO production. These biochemical events and angiogenesis were completely inhibited by the G protein-coupled receptor inhibitor pertussis toxin. Inhibitors of Raf-1, MEK, phosphatidylinositol 3-kinase (PI3K), and eNOS or transfection with dominant-negative forms of ERK and Akt significantly suppressed the angiogenic activity of FKN. However, inhibitors of Raf-1 and MEK or a dominant-negative ERK mutant blocked FKN-induced ERK, but not Akt and eNOS, phosphorylation. The PI3K inhibitor and a dominant-negative mutant of Akt suppressed Akt and eNOS phosphorylation and NO production. Our results demonstrated that FKN stimulated angiogenesis by activating the Raf-1/MEK/ERK and PI3K/Akt/eNOS/NO signal pathways via the G protein-coupled receptor CX3CR1, indicating that two pathways are required for full angiogenic activity of FKN. This study suggests that FKN may play an important role in the pathophysiological process of inflammatory angiogenesis.     

Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells

Sphingosine 1-phosphate (SPP) binds to members of the endothelial differentiation gene family (EDG) of receptors and leads to diverse signaling events including cell survival, growth, migration and differentiation. However, the mechanisms of how SPP activates these proangiogenic pathways are poorly understood. Here we show that SPP signals through the EDG-1 receptor to the heterotrimeric G protein G(i), leading to activation of the serine/threonine kinase Akt and phosphorylation of the Akt substrate, endothelial nitric-oxide synthase (eNOS). Inhibition of G(i) signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP also stimulates eNOS phosphorylation and NO release and these effects are also attenuated by inhibition of G(i) signaling, PI 3-kinase, and Akt. However, inhibition of NO production did not influence SPP-induced chemotaxis but effectively blocked the chemotactic actions of vascular endothelial growth factor. Thus, SPP signals through G(i) and PI 3-kinase leading to Akt activation and eNOS phosphorylation.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen

17beta-Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17beta-estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17beta-estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17beta-estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17beta-estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17beta-estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17beta-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.     

Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric-oxide synthase

We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS) on basal and stimulated NO release, cooperative phosphorylation, and co-association with hsp90 and Akt. Mutation of the serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site, demonstrating cooperation between multiple phosphorylation events, whereas mutation of serine 635 to alanine did not cause compensation. Mutation of serines 116 and 617 to alanine promoted a greater protein-protein interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation. More importantly, using alanine substitutions, Ser-116 is important for agonist, but not basal NO release, Ser-635 is important for basal, but not stimulated, Ser-617 negatively regulates basal and stimulated NO release, and Ser-1179 phosphorylation is stimulatory for both basal and agonist-mediated NO release. Using putative "gain of function" mutants (serine to aspartate) serines 635 and 1179 are important positive regulators of basal and stimulated NO release. S635D eNOS is the most efficacious, yielding 5-fold increases in basal and 2-fold increases in stimulated NO release from cells. However, S617A and S617D eNOS both increased NO release with opposite actions in NOS activity assays. Thus, multiple serine phosphorylation events regulate basal and stimulate NO release with Ser-635 and Ser-1179 being important positive regulatory sites and Ser-116 as a negative regulatory. Ser-617 may not be important for directly regulating NO release but is important as a modulator of phosphorylation at other sites and protein-protein interactions.     

A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation

Epidemiological studies suggest that tea catechins may reduce the risk of cardiovascular disease, but the mechanisms of benefit have not been determined. The objective of the present study was to investigate the effects of epigallocatechin-3-gallate (EGCG), the major constituent of green tea, on vasorelaxation and on eNOS expression and activity in endothelial cells. EGCG (1-50 microm) induced dose-dependent vasodilation in rat aortic rings. Vasodilation was abolished by pretreatment with Ng-nitro L-arginine methyl ester. In bovine aortic endothelial cells, EGCG increased endothelial nitric oxide (eNOS) activity dose-dependently after 15 min. Treatment with EGCG induced a sustained activation of Akt, ERK1/2, and eNOS Ser1179 phosphorylation. Inhibition of extracellular signal-regulated kinase (ERK)1/2 had no influence on eNOS activity or Ser1179 phosphorylation. Simultaneous treatment of cells with selective inhibitors for cAMP-dependent protein kinase (PKA) and Akt completely prevented the increase in eNOS activity by EGCG after 15 min, indicating that both kinases act in concert. Specific phosphatidylinositol-3-OH-kinase inhibitors yielded identical results. Akt inhibition prevented eNOS Ser1179 phosphorylation, whereas inhibition of PKA did not influence Akt and eNOS Ser1179 phosphorylation. Pretreatment of endothelial cells with EGCG for 4 h markedly enhanced the increase in eNOS activity stimulated by Ca-ionomycin, suggesting that Akt accounts for prolonged eNOS activation. Treatment of cells for 72 h with EGCG did not change eNOS protein levels. Our results indicate that EGCG-induced endothelium-dependent vasodilation is primarily based on rapid activation of eNOS by a phosphatidylinositol 3-kinase-, PKA-, and Akt-dependent increase in eNOS activity, independently of an altered eNOS protein content.     

Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity

Plasma adiponectin level is significantly reduced in patients with metabolic syndrome, and vascular dysfunction is an important pathological event in these patients. However, whether adiponectin may protect endothelial cells and attenuate endothelial dysfunction caused by metabolic disorders remains largely unknown. Adult rats were fed with a regular or a high-fat diet for 14 wk. The aorta was isolated, and vascular segments were incubated with vehicle or the globular domain of adiponectin (gAd; 2 mug/ml) for 4 h. The effect of gAd on endothelial function, nitric oxide (NO) and superoxide production, nitrotyrosine formation, gp91(phox) expression, and endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) activity/expression was determined. Severe endothelial dysfunction (maximal vasorelaxation in response to ACh: 70.3 +/- 3.3 vs. 95.2 +/- 2.5% in control, P < 0.01) was observed in hyperlipidemic aortic segments, and treatment with gAd significantly improved endothelial function (P < 0.01). Paradoxically, total NO production was significantly increased in hyperlipidemic vessels, and treatment with gAd slightly reduced, rather than increased, total NO production in these vessels. Treatment with gAd reduced (-78%, P < 0.01) superoxide production and peroxynitrite formation in hyperlipidemic vascular segments. Moreover, a moderate attenuation (-30%, P < 0.05) in gp91(phox) and iNOS overexpression in hyperlipidemic vessels was observed after gAd incubation. Treatment with gAd had no effect on eNOS expression but significantly increased eNOS phosphorylation (P < 0.01). Most noticeably, treatment with gAd significantly enhanced eNOS (+83%) but reduced iNOS (-70%, P < 0.01) activity in hyperlipidemic vessels. Collectively, these results demonstrated that adiponectin protects the endothelium against hyperlipidemic injury by multiple mechanisms, including promoting eNOS activity, inhibiting iNOS activity, preserving bioactive NO, and attenuating oxidative/nitrative stress.     

Adiponectin stimulates production of nitric oxide in vascular endothelial cells

Adiponectin is secreted by adipose cells and mimics many metabolic actions of insulin. However, mechanisms by which adiponectin acts are poorly understood. The vascular action of insulin to stimulate endothelial production of nitric oxide (NO), leading to vasodilation and increased blood flow is an important component of insulin-stimulated whole body glucose utilization. Therefore, we hypothesized that adiponectin may also stimulate production of NO in endothelium. Bovine aortic endothelial cells in primary culture loaded with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA) were treated with lysophosphatidic acid (LPA) (a calcium-releasing agonist) or adiponectin (10 microg/ml bacterially produced full-length adiponectin). LPA treatment increased production of NO by approximately 4-fold. Interestingly, adiponectin treatment significantly increased production of NO by approximately 3-fold. Preincubation of cells with wortmannin (phosphatidylinositol 3-kinase inhibitor) blocked only adiponectin- but not LPA-mediated production of NO. Using phospho-specific antibodies, we observed that either adiponectin or insulin treatment (but not LPA treatment) caused phosphorylation of both Akt at Ser473 and endothelial nitric-oxide synthase (eNOS) at Ser1179 that was inhibitable by wortmannin. We next transfected bovine aortic endothelial cells with dominant-inhibitory mutants of Akt (Akt-AAA) or AMP-activated protein kinase (AMPK) (AMPKK45R). Neither mutant affected production of NO in response to LPA treatment. Importantly, only AMPKK45R, but not Akt-AAA, caused a significant partial inhibition of NO production in response to adiponectin. Moreover, AMPK-K45R inhibited phosphorylation of eNOS at Ser1179 in response to adiponectin but not in response to insulin. We conclude that adiponectin has novel vascular actions to directly stimulate production of NO in endothelial cells using phosphatidylinositol 3-kinase-dependent pathways involving phosphorylation of eNOS at Ser1179 by AMPK. Thus, the effects of adiponectin to augment metabolic actions of insulin in vivo may be due, in part, to vasodilator actions of adiponectin.     

Ghrelin has novel vascular actions that mimic PI 3-kinase-dependent actions of insulin to stimulate production of NO from endothelial cells

Ghrelin is an orexigenic peptide hormone secreted by the stomach. In patients with metabolic syndrome and low ghrelin levels, intra-arterial ghrelin administration acutely improves their endothelial dysfunction. Therefore, we hypothesized that ghrelin activates endothelial nitric oxide synthase (eNOS) in vascular endothelium, resulting in increased production of nitric oxide (NO) using signaling pathways shared in common with the insulin receptor. Similar to insulin, ghrelin acutely stimulated increased production of NO in bovine aortic endothelial cells (BAEC) in primary culture (assessed using NO-specific fluorescent dye 4,5-diaminofluorescein) in a time- and dose-dependent manner. Production of NO in response to ghrelin (100 nM, 10 min) in human aortic endothelial cells was blocked by pretreatment of cells with NG-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor), wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor], or (D-Lys3)-GHRP-6 (selective antagonist of ghrelin receptor GHSR-1a), as well as by knockdown of GHSR-1a using small-interfering (si) RNA (but not by mitogen/extracellular signal-regulated kinase inhibitor PD-98059). Moreover, ghrelin stimulated increased phosphorylation of Akt (Ser473) and eNOS (Akt phosphorylation site Ser1179) that was inhibitable by knockdown of GHSR-1a using siRNA or by pretreatment of cells with wortmannin but not with PD-98059. Ghrelin also stimulated phosphorylation of mitogen-activated protein (MAP) kinase in BAEC. However, unlike insulin, ghrelin did not stimulate MAP kinase-dependent secretion of the vasoconstrictor endothelin-1 from BAEC. We conclude that ghrelin has novel vascular actions to acutely stimulate production of NO in endothelium using a signaling pathway that involves GHSR-1a, PI 3-kinase, Akt, and eNOS. Our findings may be relevant to developing novel therapeutic strategies to treat diabetes and related diseases characterized by reciprocal relationships between endothelial dysfunction and insulin resistance.     

2-Methoxyestradiol Induces Vasodilation by Stimulating NO Release via PPARγ/PI3K/Akt Pathway

The endogenous estradiol metabolite 2-methoxyestradiol (2-ME) reduces atherosclerotic lesion formation, while the underlying mechanisms remain obscure. In this work, we investigated the vasodilatory effect of 2-ME and the role of nitric oxide (NO) involved. In vivo studies using noninvasive tail-cuff methods showed that 2-ME decreased blood pressure in Sprague Dawley rats. Furthermore, in vitro studies showed that cumulative addition of 2-ME to the aorta caused a dose- and endothelium-dependent vasodilation. This effect was unaffected by the pretreatment with the pure estrogen receptor antagonist ICI 182,780, but was largely impaired by endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or by phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (WM). Moreover, 2-ME（10^{−7 ∼}10⁻⁵ M）enhanced phosphorylation of Akt and eNOS and promoted NO release from cultured human umbilical endothelial cells (HUVECs). These effects were blocked by PI3K inhibitor WM, or by the transfection with Akt specific siRNA, indicating that endothelial Akt/eNOS/NO cascade plays a crucial role in 2-ME-induced vasodilation. The peroxisome proliferator-activated receptor γ (PPARγ) mRNA and protein expression were detected in HUVECs and the antagonist GW9662 or the transfection with specific PPARγ siRNA inhibited 2-ME-induced eNOS and Akt phosphorylation, leading to the impairment of NO production and vasodilation. In conclusion, 2-ME induces vasodilation by stimulating NO release. These actions may be mediated by PPARγ and the subsequent activation of Akt/eNOS cascade in vascular endothelial cells.     

Endothelin-1 activates endothelial cell nitric-oxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein jinase B/Akt

Endothelin-1 has dual vasoactive effects, mediating vasoconstriction via ETA receptor activation of vascular smooth muscle cells and vasorelaxation via ETB receptor activation of endothelial cells. Although it is commonly accepted that endothelin-1 binding to endothelial cell ETB receptors stimulates nitric oxide (NO) synthesis and subsequent smooth muscle relaxation, the signaling pathways downstream of ETB receptor activation are unknown. Here, using a model in which we have utilized isolated primary endothelial cells, we demonstrate that ET-1 binding to sinusoidal endothelial cell ETB receptors led to increased protein kinase B/Akt phosphorylation, endothelial cell nitric-oxide synthase (eNOS) phosphorylation, and NO synthesis. Furthermore, eNOS activation was not dependent on tyrosine phosphorylation, and pretreatment of endothelial cells with pertussis toxin as well as overexpression of a dominant negative G-protein-coupled receptor kinase construct that sequesters betagamma subunits inhibited Akt phosphorylation and NO synthesis. Taken together, the data elucidate a G-protein-coupled receptor signaling pathway for ETB receptor-mediated NO production and call attention to the absolute requirement for heterotrimeric G-protein betagamma subunits in this cascade.     

Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased nitric oxide (NO) release and impaired pulmonary vasodilation. We investigated the hypothesis that decreased association of heat shock protein 90 (HSP90) with endothelial NO synthase (eNOS) impairs NO release and vasodilation in PPHN. The responses to the NOS agonist ATP were investigated in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus, and in sham ligation controls. ATP caused dose-dependent vasodilation in control pulmonary resistance arteries, and this response was attenuated in PPHN vessels. The response of control pulmonary arteries to ATP was attenuated by NG-nitro-l-arginine methyl ester (l-NAME), a NOS antagonist, and geldanamycin, an inhibitor of HSP90-eNOS interaction. The attenuated response to ATP observed in PPHN was improved by pretreatment of vessels with l-NAME or 4,5-dihydroxy-1,3-benzene-disulfonate, a superoxide scavenger. Pulmonary arteries from PPHN lambs had decreased basal levels of HSP90 in association with eNOS. Association of HSP90 with eNOS and NO release increased in response to ATP in control pulmonary artery endothelial cells, but not in cells from PPHN lambs. Decreased HSP90-eNOS interactions may contribute to the impaired NO release and vasodilation observed in the ductal ligation model of PPHN.     

High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases

High density lipoprotein (HDL) activates endothelial nitric-oxide synthase (eNOS), leading to increased production of the antiatherogenic molecule NO. A variety of stimuli regulate eNOS activity through signaling pathways involving Akt kinase and/or mitogen-activated protein (MAP) kinase. In the present study, we investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I. HDL (10-50 microg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. Phosphoinositide 3-kinase (PI3 kinase) inhibition or dominant negative PI3 kinase also blocked the phosphorylation and activation of eNOS by HDL. Studies with genistein and PP2 showed that the nonreceptor tyrosine kinase, Src, is an upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition fully attenuated eNOS stimulation by HDL without affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt did not alter HDL-induced MAP kinase activation. These results indicate that HDL stimulates eNOS through common upstream, Src-mediated signaling, which leads to parallel activation of Akt and MAP kinases and their resultant independent modulation of the enzyme.     

Involvement of androgen receptor in nitric oxide production induced by icariin in human umbilical vein endothelial cells

Icariin, a flavonoid isolated from Epimedii herba, stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177, Akt (Ser473) and ERK1/2 (Thr202/Tyr204). The icariin-induced eNOS phosphorylation was abolished by an androgen receptor (AR) antagonist, nilutamide in human umbilical vein endothelial cells (HUVECs). Furthermore, it was also reduced in the cells transfected with small interfering RNA in which the expression of AR was broken down. The icariin-induced eNOS phosphorylation was inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. These data suggest that icariin stimulates release of NO by AR-dependent activation of eNOS in HUVECs. PI3K/Akt and MAPK-ERK kinase (MEK)/ERK1/2 pathways were involved in the phosphorylation of eNOS by icariin.     

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.