Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway.

Endothelial nitric-oxide synthase (eNOS) is an important component of vascular homeostasis. During vascular disease, endothelial cells are exposed to excess reactive oxygen species that can alter cellular phenotype by inducing various signaling pathways. In the current study, we examined the implications of H(2)O(2)-induced signaling for eNOS phosphorylation status and activity in porcine aortic endothelial cells. We found that H(2)O(2) treatment enhanced eNOS activity and NO bioactivity as determined by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline and cellular cGMP content. Concomitant with eNOS activation, H(2)O(2) also activated Akt, increased eNOS phosphorylation at Ser-1177, and decreased eNOS phosphorylation at Thr-495. H(2)O(2)-induced promotion of eNOS activity and modulation of the eNOS phosphorylation status at Ser-1177 and Thr-495 were significantly attenuated by selective inhibitors of Src kinase, the ErbB receptor family, and phosphoinositide 3-kinase (PI 3-K). We found that Akt activation, eNOS Ser-1177 phosphorylation, and eNOS activation by H(2)O(2) were calcium-dependent, whereas eNOS dephosphorylation at Thr-495 was not, suggesting a branch point in the signaling cascade downstream from PI 3-K. Consistent with this, overexpression of a dominant negative isoform of Akt inhibited H(2)O(2)-induced phosphorylation of eNOS at Ser-1177 but not dephosphorylation of eNOS at Thr-495. Together, these data indicate that H(2)O(2) promotes calcium-dependent eNOS activity through a coordinated change in the phosphorylation status of the enzyme mediated by Src- and ErbB receptor-dependent PI 3-K activation. In turn, PI 3-K mediates eNOS Ser-1177 phosphorylation via a calcium- and Akt-dependent pathway, whereas eNOS Thr-495 dephosphorylation does not involve calcium or Akt. This response may represent an attempt by endothelial cells to maintain NO bioactivity under conditions of enhanced oxidative stress.     

Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.     

The effect of n-3 PUFA on eNOS activity and expression in Ea hy 926 cells

The aim of this study was to evaluate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on NO synthase (eNOS) activation in Ea hy 926 endothelial cells. EPA or DHA (0-80 microM), added to the culture medium during 24h, were dose-dependently incorporated into the cells. In control medium, eNOS activity (evaluated by the citrulline assay) and eNOS phosphorylation on Ser 1177 were correlated. They were increased by 10 microM histamine and prevented by 20 microM lysophosphatidylcholine (LPC). By contrast, EPA or DHA increased basal phosphorylation without affecting eNOS activity in non-stimulated cells, but dose-dependently decreased this activity in histamine-stimulated cells without modifying the phosphorylation level. Furthermore, EPA and DHA did not prevent the deleterious effects of LPC on histamine stimulation. In conclusion, incorporation of EPA and DHA could be deleterious for endothelial cells by deregulating the activation of eNOS and preventing NO liberation.     

Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells

Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled.     

The anandamide effect on NO/cGMP pathway in human platelets

In this study the effect of the endocannabinoid anandamide on platelet nitric oxide (NO)/cGMP pathway was investigated. Data report that anandamide in a dose-and time-dependent manner increased NO and cGMP levels and stimulated endothelial nitric oxide synthase (eNOS) activity. These parameters were significantly reduced by LY294002, selective inhibitor of PI3K and by MK2206, specific inhibitor of AKT. Moreover anandamide stimulated both eNOSser1177 and AKTser473 phosphorylation. Finally the anandamide effect on NO and cGMP levels, eNOS and AKT phosphorylation/activation were inhibited by SR141716, specific cannabinoid receptor 1 antagonist, supporting the involvement of anandamide binding to this receptor. Overall data of this report indicate that low concentrations of anandamide, through PI3K/AKT pathway activation, stimulates eNOS activity and increases NO levels in human platelets. In such way anandamide contributes to extend platelet survival.     

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.     

Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.     

Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.     

黄芪甲苷Ⅳ通过影响β-actin对内皮型一氧化氮合酶的调节作用

目的：研究黄芪甲苷Ⅳ（AS-Ⅳ）对体外培养脐静脉内皮细胞中内皮型一氧化氮合酶（eNOS）的调节作用及可能的机制。方法：培养人脐静脉内皮细胞系EA-Hy926,用AS-Ⅳ进行干预,同时给予或不给予骨架蛋白β—actin聚合稳定剂phalloidin,用免疫共沉淀方法检测eNOS与单体8-actin结合状态的变化,用L-3H．精氨酸转化为L-SH-瓜氨酸的同位素法测定eNOS活性,I^125环-磷酸鸟苷（cGMP）放射免疫法检测细胞内cGMP水平,Westernblotting方法检测细胞中eNOS和蛋白激酶B（Akt）磷酸化水平,总蛋白水平。结果：（1）AS-IV作用10min后,细胞内单体β—actin与eNOS的结合明显增加（P〈0．05或P〈0．01）,预先给予phalloidin显著抑制了AS．IV引起的两者结合的增加（P〈0．01）。②AS—IV明显增加了eNOS活性（P〈0．05）、cGMP含量（P〈0．01）、eNOSSer-1177磷酸化水平（P〈0．01）、AktSer-473磷酸化水平（P〈0．001）,预先给予phalloidin明显降低了AS—IV引起的eNOS活性（P〈0．05）、cGMP含量（P〈0．01）和磷酸化水平的增加（P〈0．01）,但对Akt的磷酸化没有影响。结论：单体β—actin与eNOS的结合在AS-IV激活eNOS的过程中起着不可或缺作用,其主要是通过促进Akt对eNOSSer—1177的磷酸化来实现的。     

High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser1177 phosphorylation in human aortic endothelial cells

Recent studies have indicated that insulin activates endothelial nitric-oxide synthase (eNOS) by protein kinase B (PKB)-mediated phosphorylation at Ser1177 in endothelial cells. Because hyperglycemia contributes to endothelial dysfunction and decreased NO availability in types 1 and 2 diabetes mellitus, we have studied the effects of high glucose (25 mM, 48 h) on insulin signaling pathways that regulate NO production in human aortic endothelial cells. High glucose inhibited insulin-stimulated NO synthesis but was without effect on NO synthesis stimulated by increasing intracellular Ca2+ concentration. This was accompanied by reduced expression of IRS-2 and attenuated insulin-stimulated recruitment of PI3K to IRS-1 and IRS-2, yet insulin-stimulated PKB activity and phosphorylation of eNOS at Ser1177 were unaffected. Inhibition of insulin-stimulated NO synthesis by high glucose was unaffected by an inhibitor of PKC. Furthermore, high glucose down-regulated the expression of CAP and Cbl, and insulin-stimulated Cbl phosphorylation, components of an insulin signaling cascade previously characterized in adipocytes. These data suggest that high glucose specifically inhibits insulin-stimulated NO synthesis and down-regulates some aspects of insulin signaling, including the CAP-Cbl signaling pathway, yet this is not a result of reduced PKB-mediated eNOS phosphorylation at Ser1177. Therefore, we propose that phosphorylation of eNOS at Ser1177 is not sufficient to stimulate NO production in cells cultured at 25 mM glucose.     

(-)-Epicatechin induces calcium and translocation independent eNOS activation in arterial endothelial cells

The consumption of cacao-derived (i.e., cocoa) products provides beneficial cardiovascular effects in healthy subjects as well as individuals with endothelial dysfunction such as smokers, diabetics, and postmenopausal women. The vascular actions of cocoa are related to enhanced nitric oxide (NO) production. These actions can be reproduced by the administration of the cacao flavanol (-)-epicatechin (EPI). To further understand the mechanisms behind the vascular action of EPI, we investigated the effects of Ca(2+) depletion on endothelial nitric oxide (NO) synthase (eNOS) activation/phosphorylation and translocation. Human coronary artery endothelial cells were treated with EPI or with bradykinin (BK), a well-known Ca(2+)-dependent eNOS activator. Results demonstrate that both EPI and BK induce increases in intracellular calcium and NO levels. However, under Ca(2+)-free conditions, EPI (but not BK) is still capable of inducing NO production through eNOS phosphorylation at serine 615, 633, and 1177. Interestingly, EPI-induced translocation of eNOS from the plasmalemma was abolished upon Ca(2+) depletion. Thus, under Ca(2+)-free conditions, EPI can stimulate NO synthesis independent of calmodulin binding to eNOS and of its translocation into the cytoplasm. We also examined the effect of EPI on the NO/cGMP/vasodilator-stimulated phosphoprotein (VASP) pathway activation in isolated Ca(2+)-deprived canine mesenteric arteries. Results demonstrate that under these conditions, EPI induces the activation of this vasorelaxation-related pathway and that this effect is inhibited by pretreatment with nitro-L-arginine methyl ester, suggesting a functional relevance for this phenomenon.     

Signaling pathway of nitric oxide production induced by ginsenoside Rb1 in human aortic endothelial cells: a possible involvement of androgen receptor

Ginsenosides have been shown to stimulate nitric oxide (NO) production in aortic endothelial cells. However, the signaling pathways involved have not been well studied in human aortic endothelial cells. The present study was designed to examine whether purified ginsenoside Rb1, a major active component of ginseng could actually induce NO production and to clarify the signaling pathway in human aortic endothelial cells. NO production was rapidly increased by Rb1. The rapid increase in NO production was abrogated by treatment with nitric oxide synthetase inhibitor, L-NAME. Rb1 stimulated rapid phosphorylation of Akt (Ser473), ERK1/2 (Thr202/Thr204) and eNOS (Ser1177). Rapid phosphorylation of eNOS (Ser1177) was prevented by SH-5, an Akt inhibitor or wortmannin, PI3-kinase inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. Interestingly, NO production and eNOS phosphorylation at Ser1177 by Rb1 were abolished by androgen receptor antagonist, nilutamide. The results suggest that PI3kinase/Akt and MEK/ERK pathways and androgen receptor are involved in the regulation of acute eNOS activation by Rb1 in human aortic endothelial cells.     

Hydrogen peroxide restrains endothelium-derived nitric oxide bioactivity -- role for iron-dependent oxidative stress

Vascular diseases are characterized by impairment of endothelial-derived nitric oxide (NO) bioactivity and increased vascular levels of hydrogen peroxide (H(2)O(2)). Here we examined the implications of H(2)O(2) for agonist-stimulated endothelial NO bioactivity in rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC). Vessels pre-treated with H(2)O(2) exhibited impaired endothelial-dependent relaxation induced by acetylcholine or calcium ionophore. In contrast, H(2)O(2) had no effect on endothelium-independent relaxation induced by a NO donor, indicating a defect in endothelium-derived NO. This defect was not related to eNOS catalytic activity; treatment of PAEC with H(2)O(2) enhanced agonist-stimulated eNOS activity indicated by increased eNOS phosphorylation at Ser-1177 and de-phosphorylation at Thr-495 and enhanced conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline that was prevented by inhibitors of Src and phosphatidylinositol-3 kinases. Despite activating eNOS, H(2)O(2) impaired endothelial NO bioactivity indicated by attenuation of the increase in intracellular cGMP in PAEC stimulated with calcium ionophore or NO. The decrease in cGMP was not due to impaired guanylyl cyclase as H(2)O(2) treatment increased cGMP accumulation in response to BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase. At concentrations that impaired endothelial NO bioactivity H(2)O(2) increased intracellular oxidative stress and size of the labile iron pool in PAEC. The increase in oxidative stress was prevented by the free radical scavenger's tempol or tiron and the iron chelator desferrioxamine and these antioxidants reversed the H(2)O(2)-induced impairment of NO bioactivity in PAEC. This study shows that despite promoting eNOS activity, H(2)O(2) impairs endothelial NO bioactivity by promoting oxidative inactivation of synthesized NO. The study highlights another way in which oxidative stress may impair NO bioactivity during vascular disease.     

Omentin, a novel adipocytokine inhibits TNF-induced vascular inflammation in human endothelial cells

Omentin is a recently identified adipocytokine with insulin-sensitizing effect. While lack of omentin may be related to the pathogenesis of obesity-related cardiovascular diseases, its effect in vasculature is largely unknown. We examined effects of omentin on vascular endothelial inflammatory states. Western blotting was performed to analyze inflammatory signal transduction in cultured vascular endothelia cells. The cyclic guanosine monophosphate (cGMP) content was measured using enzyme immunoassay. Treatment of human umbilical vein endothelial cells with omentin (300 ng/ml, 20 min) induced phosphorylation of 5′-AMP-activated protein kinase (AMPK) (Thr 172) and endothelial nitric oxide (NO) synthase (eNOS) (Ser 1177). Consistently, omentin increased the cGMP level. Pretreatment with omentin (300 ng/ml, 30 min) significantly inhibited the phosphorylation of JNK as well as expression of cyclooxygenase (COX)-2 by TNF-α (5 ng/ml, 20 min–24 h). An inhibitor of JNK significantly inhibited the TNF-α-induced COX-2 expression. Inhibitory effect of omentin on TNF-α-induced COX-2 was reversed by a NOS inhibitor. The present results demonstrate for the first time that omentin plays an anti-inflammatory role by preventing the TNF-α-induced COX-2 expression in vascular endothelial cells. Omentin inhibits COX-2 induction via preventing the JNK activation presumably through activation of AMPK/eNOS/NO pathways.     

Protein kinase Cdelta regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

In this study, we explore the roles of the delta isoform of PKC (PKCdelta) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCdelta with either rottlerin or with the peptide, deltaV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCdelta inhibition using either rottlerin or the overexpression of a dominant negative PKCdelta mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCdelta inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCdelta is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCdelta-mediated Akt activation and NO generation in maintaining eNOS expression.     

Endothelium-derived microparticles inhibit angiogenesis in the heart and enhance the inhibitory effects of hypercholesterolemia on angiogenesis

Therapeutic angiogenesis remains unsuccessful in coronary artery disease. It is known that plasma endothelium-derived microparticles (EMPs) are increased in coronary artery disease and that hypercholesterolemia can inhibit angiogenesis. We evaluated the relationship between EMPs and hypercholesterolemia in the impairment of angiogenesis. EMPs isolated from human umbilical vein endothelial cells were injected into low-density lipoprotein receptor-null (LDLr(-/-)) mice fed a Western diet for 2 wk and C57BL6 mice for 6 h or were directly added to the tissue culture media. Hearts isolated from mice were sectioned and cultured, and endothelial tube formation was measured. The expression and phosphorylation of endothelial NO synthase (eNOS) and the generation of NO in the hearts were determined. Angiogenesis was inhibited by pathophysiological concentrations of EMPs but not physiological concentrations of EMPs in hearts from C57BL6 mice. However, angiogenesis was inhibited by EMPs at both physiological and pathophysiological concentrations of EMPs in hearts from hypercholesterolemic LDLr(-/-) mice. Pathophysiological concentrations of EMPs decreased eNOS phosphorylation at Ser(1177) and NO generation without altering eNOS expression in hearts from C57BL6 mice. Both physiological and pathophysiological concentrations of EMPs decreased not only eNOS phosphorylation at Ser(1177) and NO generation, but eNOS expression in hypercholesterolemic hearts from LDLr(-/-) mice. These data demonstrated that pathophysiological concentrations of EMPs could inhibit angiogenesis in hearts by decreasing eNOS activity. EMPs and hypercholesterolemia mutually enhanced their inhibitory effect of angiogenesis by inducing eNOS dysfunction. Our findings suggest a novel mechanism by which hypercholesterolemia impairs angiogenesis.     

Antihypertensive therapy increases tetrahydrobiopterin levels and NO/cGMP signaling in small arteries of angiotensin II-infused hypertensive rats

We previously reported that small mesenteric arteries from hypertensive rats have increased NOS-derived H(2)O(2) and reduced NO/cGMP signaling. We hypothesized that antihypertensive therapy lowers blood pressure through a tetrahydrobiopterin (BH(4))-dependent mechanism restoring NO/cGMP signaling and endothelial NOS (NOS3; eNOS) phosphorylation in small arteries. To test this hypothesis, small mesenteric arteries from normotensive rats (NORM), angiotensin II-infused rats (ANG), ANG rats with triple therapy (reserperine, hydrochlorothiazide, and hydralazine), or ANG rats with oral BH(4) therapy were studied. Both triple therapy and oral BH(4) therapy attenuated the rise in systolic blood pressure in ANG rats and restored NO/cGMP signaling in small arteries similarly. Triple therapy significantly increased vascular BH(4) levels and BH(4)-to-BH(2) ratio similar to ANG rats with BH(4) supplementation. Furthermore, triple therapy (but not oral BH(4) therapy) significantly increased GTP cyclohydrolase I (GTPCH I) activity in small arteries without a change in expression. NOS3 phosphorylation at Ser1177 was reduced in small arteries from ANG compared with NORM, while NOS3 phosphorylation at Ser633 and Thr495 were similar in ANG and NORM. NOS3 phosphorylation at Ser1177 was restored with triple therapy or oral BH(4) in ANG rats. In conclusion, antihypertensive therapy regulates NO/cGMP signaling in small arteries through increasing BH(4) levels and NOS3 phosphorylation at Ser1177.     

Diminished NO release in chronic hypoxic human endothelial cells

The present study addressed whether chronic hypoxia is associated with reduced nitric oxide (NO) release due to decreased activation of endothelial NO synthase (eNOS). Primary cultures of endothelial cells from human umbilical veins (HUVECs) were used and exposed to different oxygen levels for 24 h, after which NO release, intracellular calcium, and eNOS activity and phosphorylation were measured after 24 h. Direct measurements using a NO microsensor showed that in contrast to 1-h exposure to 5% and 1% oxygen (acute hypoxia), histamine-evoked (10 microM) NO release from endothelial cells exposed to 5% and 1% oxygen for 24 h (chronic hypoxia) was reduced by, respectively, 58% and 40%. Furthermore, chronic hypoxia also lowered the amount and activity of eNOS enzyme. The decrease in activity could be accounted for by reduced intracellular calcium and altered eNOS phosphorylation. eNOS Ser(1177) and eNOS Thr(495) phosphorylations were reduced and increased, respectively, consistent with lowered enzyme activity. Akt kinase, which can phosphorylate eNOS Ser(1177), was also decreased by hypoxia, regarding both total protein content and the phosphorylated (active) form. Moreover, the protein content of beta- actin, which is known to influence the activity of eNOS, was almost halved by hypoxia, further supporting the fall in eNOS activity. In conclusion, chronic hypoxia in HUVECs reduces histamine-induced NO release as well as eNOS expression and activity. The decreased activity is most likely due to changed eNOS phosphorylation, which is supported by decreases in Akt expression and phosphorylation. By reducing NO, chronic hypoxia may accentuate endothelial dysfunction in cardiovascular disease.     

Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration

Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration. The protein kinase Akt activates the endothelial NO synthase (eNOS) by phosphorylation of Ser-1177. Therefore, we investigated the contribution of Akt-mediated eNOS phosphorylation to VEGF-induced EC migration. Inhibition of NO synthase or overexpression of a dominant negative Akt abrogated VEGF-induced cell migration. In contrast, overexpression of constitutively active Akt was sufficient to induce cell migration. Moreover, transfection of an Akt site phospho-mimetic eNOS (S1177D) potently stimulated EC migration, whereas a non-phosphorylatable mutant (S1177A) inhibited VEGF-induced EC migration. Our data indicate that eNOS activation via phosphorylation of Ser-1177 by Akt is necessary and sufficient for VEGF-mediated EC migration.     

Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), which is involved in various physiological functions of the cardiovascular system. eNOS is activated by dephosphorylation at Thr495 and phosphorylation at Ser1177. Inhibition of Rho-kinase, an effector of the small GTPase RhoA, leads to activation of Akt/PKB, which phosphorylates eNOS at Ser1177 and thereby promotes NO production. However, little is known about the effects of Rho-kinase on phosphorylation of Thr495. We here found that the constitutively active form of Rho-kinase phosphorylated eNOS at Thr495 in vitro. Expression of the constitutively active form of RhoA or Rho-kinase increased this phosphorylation in COS-7 cells. Addition of thrombin to cultured human umbilical vein endothelial cells induced phosphorylation of eNOS at Thr495. Treatment with Y27632, a Rho-kinase inhibitor, suppressed thrombin-induced phosphorylation at Thr495. These results indicate that Rho-kinase can directly phosphorylate eNOS at Thr495 to suppress NO production in endothelium.