Impaired insulin-induced vasodilation in small coronary arteries of Zucker obese rats is mediated by reactive oxygen species

Insulin resistance (IR) and associated hyperinsulinemia are major risk factors for coronary artery disease. Mechanisms linking hyperinsulinemia to coronary vascular dysfunction in IR are unclear. We evaluated insulin-induced vasodilation in isolated small coronary arteries (SCA; approximately 225 microm) of Zucker obese (ZO) and control Zucker lean (ZL) rats. Vascular responses to insulin (0.1-100 ng/ml), ACh (10(-9)-10(-5) mol/l), and sodium nitroprusside (10(-8)-10(-4) mol/l) were assessed in SCA by measurement of intraluminal diameter using videomicroscopy. Insulin-induced dilation was decreased in ZO compared with ZL rats, whereas ACh and sodium nitroprusside elicited similar vasodilations. Pretreatment of arteries with SOD (200 U/ml), a scavenger of reactive oxygen species (ROS), restored the vasorelaxation response to insulin in ZO arteries, whereas ZL arteries were unaffected. Pretreatment of SCA with N-nitro-L-arginine methyl ester (100 micromol/l), an inhibitor of endothelial nitric oxide (NO) synthase (eNOS), elicited a vasoconstrictor response to insulin that was greater in ZO than in ZL rats. This vasoconstrictor response was reversed to vasodilation in ZO and ZL rats by cotreatment of the SCA with SOD or apocynin (10 micromol/l), a specific inhibitor of vascular NADPH oxidase. Lucigenin-enhanced chemiluminescence showed increased basal ROS levels as well as insulin (330 ng/ml)-stimulated production of ROS in ZO arteries that was sensitive to inhibition by apocynin. Western blot analysis revealed increased eNOS expression in ZO rats, whereas Mn SOD and Cu,Zn SOD expression were similar to ZL rats. Thus IR in ZO rats leads to decreased insulin-induced vasodilation, probably as a result of increased production of ROS by vascular NADPH oxidase, leading to decreased NO bioavailability, despite a compensatory increase in eNOS expression.     

Renal activity of Akt kinase in experimental Type 1 diabetes

Akt kinase regulates numerous cell functions including glucose metabolism, cell growth, survival, protein synthesis, and control of local hemodynamics. mTOR is one of down-stream effectors of Akt involved in the initiation of protein translation. However, renal Akt signaling in Type 1 diabetes (DM) in vivo, in particular under the conditions reflecting differences in metabolic control, has received less attention. Renal cortical activity and expression of Akt and mTOR (kinase assay, western blotting) were determined in streptozotocin-diabetic rats (D) with different levels of glycemic control (blood glucose 22.0+/-1.0, 13.4+/-1.5, 8.1+/-0.4 mmol/l, p<0.05 between the groups), achieved by varying insulin treatment (0, 4 and 12 IU/day), and in control rats with (C4) or without (C) chronic insulin administration. Renal Akt activity was reduced in D rats without insulin treatment and severe hyperglycemia (D-0, -62 %, p<0.01 vs. C), partially restored in moderately hyperglycemic rats (D-4, -30 %, p<0.05 vs. C), and normalized in D rats with intensive insulin and tight metabolic control (D-12). Expression of active mTOR paralleled Akt activity in D-0 (-51 %, p<0.01 vs. C), but not in D-4 and D-12 that demonstrated increases in active mTOR (+55 %, +80 % resp., p<0.05) as compared to C. Moreover, insulin activated renal Akt (+82 %, p<0.01), but not mTOR in C4. In conclusion, glycemic control and intensity of insulin treatment are important modulators of renal Akt and mTOR activity in diabetes. While Akt activity is reversible by tight metabolic control, combination of hyperglycemia and insulin treatment resulted in enhancement of mTOR activity. In addition to Akt, other signaling pathways likely contribute to regulation of renal mTOR activity in diabetes.     

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.     

ANG II type I receptor antagonism improved nitric oxide production and enhanced eNOS and PKB/Akt expression in hearts from a rat model of insulin resistance

Exogenous insulin therapy improves endothelial function in insulin resistant patients, indirectly indicating that nitric oxide synthase activity and NO production may be impaired. Insulin stimulates production of NO by activating a signaling pathway including insulin receptor substrate-1, phosphatidylinositol-3-kinase and protein kinase B (PKB/Akt). Angiotensin II type I (AT1) receptor-evoked oxidative stress is implicated in the inactivation of NO, impairing endothelium-dependent vasodilatation. Blocking the actions of Angiotensin II with an AT1 receptor antagonist (Losartan), has beneficial effects in patients with insulin resistance or type 2 diabetes mellitus. This study investigated whether elevated Angiotensin II influences myocardial insulin resistance, insulin signaling and NO production in a rat model of diet-induced obesity (DIO) by antagonizing the actions of the AT1 receptor with Losartan. Isolated, perfused hearts, Western blotting and flow-cytometric methods were utilized to determine myocardial function, expression and phosphorylation of key proteins and NO production, respectively. Results showed that hearts from DIO rats are insulin resistant (higher serine phosphorylation of IRS-1, lower insulin-stimulated phosphorylation of PKB/Akt and eNOS, lower NO production) and had poorer functional recovery and larger infarct development after ischaemia/reperfusion. Losartan improved the impaired functional recovery, and NO production and enhanced eNOS expression and phosphorylation and reduced infarct size in hearts from the DIO animals. Data obtained from Losartan treatment also revealed that Angiotensin II signaling modulates myocardial PKB/Akt expression. We conclude that Angiotensin II signaling exacerbates inhibition of NO production in insulin resistance and that this can be improved by AT1 antagonism.     

Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser(1179)

Vasodilator actions of insulin are mediated by activation of endothelial nitric-oxide synthase (eNOS) and subsequent production of NO. Phosphatidylinositol 3-kinase and Akt play important roles in insulin-signaling pathways leading to production of NO in vascular endothelium. Here we dissected mechanisms whereby insulin activates eNOS by using the fluorescent dye DAF-2 to directly measure NO production in single cells. Insulin caused a rapid increase in intracellular NO in NIH-3T3(IR) cells transiently transfected with eNOS. The stimulation of NO production by lysophosphatidic acid (LPA) was abrogated by pretreatment of cells with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Remarkably, in the same cells, insulin-stimulated production of NO was unaffected. However, cells expressing the eNOS-S1179A mutant (disrupted Akt phosphorylation site) did not produce detectable NO in response to insulin, whereas the response to LPA was similar to that observed in cells expressing wild-type eNOS. Moreover, production of NO in response to insulin was blocked by coexpression of an inhibitory mutant of Akt, whereas the response to LPA was unaffected. Phosphorylation of eNOS at Ser(1179) was observed only in response to treatment with insulin, but not with LPA. Interestingly, platelet-derived growth factor treatment of cells activated Akt but not eNOS. Results from human vascular endothelial cells were qualitatively similar to those obtained in transfected NIH-3T3(IR) cells, although the magnitude of the responses was smaller. We conclude that insulin regulates eNOS activity using a Ca(2+)-independent mechanism requiring phosphorylation of eNOS by Akt. Importantly, phosphorylation-dependent mechanisms that enhance eNOS activity can operate independently from Ca(2+)-dependent mechanisms.     

Impaired endothelin-induced vasoconstriction in coronary arteries of Zucker obese rats is associated with uncoupling of [Ca2+]i signaling

Although insulin resistance (IR) is a major risk factor for coronary artery disease, little is known about the regulation of coronary vascular tone in IR by endothelin-1 (ET-1). We examined ET-1 and PGF(2alpha)-induced vasoconstriction in isolated small coronary arteries (SCAs; approximately 250 microM) of Zucker obese (ZO) rats and control Zucker lean (ZL) rats. ET-1 response was assessed in the absence and presence of endothelin type A (ET(A); BQ-123), type B (ET(B); BQ-788), or both receptor inhibitors. ZO arteries displayed reduced contraction to ET-1 compared with ZL arteries. In contrast, PGF(2alpha) elicited similar vasoconstriction in both groups. ET(A) inhibition diminished the ET-1 response in both groups. ET(B) inhibition alone or in combination with ET(A) blockade, however, restored the ET-1 response in ZO arteries to the level of ZL arteries. Similarly, inhibition of endothelial nitric oxide (NO) synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME) enhanced the contraction to ET-1 and abolished the difference between ZO and ZL arteries. In vascular smooth muscle cells from ZO, ET-1-induced elevation of myoplasmic intracellular free calcium concentration ([Ca2+]i) (measured by fluo-4 AM fluorescence), and maximal contractions were diminished compared with ZL, both in the presence and absence of l-NAME. However, increases in [Ca2+]i elicited similar contractions of the vascular smooth muscle cells in both groups. Analysis of protein and total RNA from SCA of ZO and ZL revealed equal expression of ET-1 and the ET(A) and ET(B) receptors. Thus coronary arteries from ZO rats exhibit reduced ET-1-induced vasoconstriction resulting from increased ET(B)-mediated generation of NO and diminished elevation of myoplasmic [Ca2+]i.     

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.     

A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension

Nitric oxide (NO) production by endothelial cell nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. We sought to understand the mechanism underlying defective eNOS function. Phosphorylation of the serine-threonine kinase Akt, which activates eNOS, was substantially reduced in sinusoidal endothelial cells from injured livers. Overexpression of Akt in vivo restored phosphorylation of Akt and production of NO and reduced portal pressure in portal hypertensive rats. We found that Akt physically interacts with G-protein-coupled receptor kinase-2 (GRK2), and that this interaction inhibits Akt activity. Furthermore, GRK2 expression increased in sinusoidal endothelial cells from portal hypertensive rats and knockdown of GRK2 restored Akt phosphorylation and NO production, and normalized portal pressure. Finally, after liver injury, GRK2-deficient mice developed less severe portal hypertension than control mice. Thus, an important mechanism underlying impaired activity of eNOS in injured sinusoidal endothelial cells is defective phosphorylation of Akt caused by overexpression of GRK2 after injury.     

Dissociation between metabolic and vascular insulin resistance in aging

Physiological actions of insulin via activation of the phosphatidylinositol 3-kinase/Akt pathway in the endothelium serve to couple regulation of hemodynamic and metabolic homeostasis. Insulin resistance, endothelial dysfunction, and hypertension increase in prevalence with aging. We investigated the metabolic and endothelial actions of insulin in 24- vs. 3-mo Sprague-Dawley rats. With the use of the hyperinsulinemic euglycemic clamp, the rate of glucose infusion necessary to maintain equivalent plasma glucose (5.5 mmol/l) was similar in 24- vs. 3-mo rats, as was fasting glucose (5.2 +/- 0.33 vs. 4.4 +/- 0.37 mmol/l; mean +/- SE) and insulin (0.862 +/- 0.193 vs. 1.307 +/- 0.230 mg/l). Systolic blood pressure was higher in 24-mo rats (133 +/- 5 vs. 110 +/- 4 mmHg; P = 0.005). Endothelial nitric oxide (NO)-dependent relaxation to insulin was impaired in aortas of 24- vs. 3-mo rats (maximal response 8.9 +/- 4.3 vs. 34.9 +/- 3.9%; P = 0.002); N(G)-nitro-l-arginine methyl ester abolished insulin-mediated relaxation in 3- but not 24-mo rats. Endothelium NO-dependent (acetylcholine) and -independent (sodium nitroprusside) relaxation, as well as NADPH oxidase activity, were similar in 3- and 24-mo rats. Insulin increased aortic serine phosphorylation of Akt in 3-mo rats by 120% over 24-mo rats (P < 0.05) and serine phosphorylation of endothelial NO synthase (eNOS) in 3-mo rats by 380% over 24-mo rats (P < 0.05). Aortic expression of phosphorylated c-Jun NH(2)-terminal kinase-1 and serine phosphorylated insulin receptor substrate-1, known mediators of metabolic insulin resistance, was similar in 3- and 24-mo rats. Expression of caveolin-1, a regulator of eNOS activity and insulin signaling, was 55% lower in 24- than 3-mo rats (P = 0.002). In summary, impaired vasorelaxation to insulin in aging was independent of metabolic insulin sensitivity and associated with impaired insulin-mediated activation of the Akt/eNOS pathway, but intact activation of the acetylcholine-mediated Ca(2+)-calmodulin/eNOS pathway. Vascular insulin resistance in aging may add to the increased susceptibility of this population to vascular injury induced by traditional cardiovascular risk factors.     

Functional proteomic analysis of a three-tier PKCepsilon-Akt-eNOS signaling module in cardiac protection

Cardiac protective signaling networks have been shown to involve PKCepsilon. However, the molecular mechanisms by which PKCepsilon interacts with other members of these networks to form task-specific modules remain unknown. Among 93 different PKCepsilon-associated proteins that have been identified, Akt and endothelial nitric oxide (NO) synthase (eNOS) are of importance because of their independent abilities to promote cell survival and prevent cell death. The simultaneous association of PKCepsilon, Akt, and eNOS has not been examined, and, in particular, the formation of a module containing these three proteins and the role of such a module in the regulation of NO production and cardiac protection are unknown. The present study was undertaken to determine whether these molecules form a signaling module and, thereby, play a collective role in cardiac signaling. Using recombinant proteins in vitro and PKCepsilon transgenic mouse hearts, we demonstrate the following: 1) PKCepsilon, Akt, and eNOS interact and form signaling modules in vitro and in the mouse heart. Activation of either PKCepsilon or Akt enhances the formation of PKCepsilon-Akt-eNOS signaling modules. 2) PKCepsilon directly phosphorylates and enhances activation of Akt in vitro, and PKCepsilon activation increases phosphorylation and activation of Akt in PKCepsilon transgenic mouse hearts. 3) PKCepsilon directly phosphorylates eNOS in vitro, and this phosphorylation enhances eNOS activity. Activation of PKCepsilon in vivo increased phosphorylation of eNOS at Ser(1177), indicating eNOS activation. This study characterizes, for the first time, the physical, as well as functional, coupling of PKCepsilon, Akt, and eNOS in the heart and implicates these PKCepsilon-Akt-eNOS signaling modules as critical signaling elements during PKCepsilon-induced cardiac protection.     

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.     

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.     

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.     

Changes in eNOS phosphorylation contribute to increased arteriolar NO release during juvenile growth

Nitric oxide (NO) mediates a major portion of arteriolar endothelium-dependent dilation in adults, but indirect evidence has suggested that NO contributes minimally to these responses in the young. Isolated segments of arterioles were studied in vitro to verify this age-related increase in NO release and investigate the mechanism by which it occurs. Directly measured NO release induced by ACh or the Ca(2+) ionophore A-23187 was five- to sixfold higher in gracilis muscle arterioles from 42- to 46-day-old (juvenile) rats than in those from 25- to 28-day-old (weanling) rats. There were no differences between groups in arteriolar endothelial NO synthase (eNOS) expression or tetrahydrobiopterin levels, and arteriolar l-arginine levels were lower in juvenile vessels than in weanling vessels (104 ± 6 vs.126 ± 3 pmol/mg). In contrast, agonist-induced eNOS Thr(495) dephosphorylation and eNOS Ser(1177) phosphorylation (events required for maximal activity) were up to 30% and 65% greater, respectively, in juvenile vessels. Juvenile vessels did not show increased expression of enzymes that mediate these events [protein phosphatases 1 and 2A and PKA and PKB (Akt)] or heat shock protein 90, which facilitates Ser(1177) phosphorylation. However, agonist-induced colocalization of heat shock protein 90 with eNOS was 34-66% greater in juvenile vessels than in weanling vessels, and abolition of this difference with geldanamycin also abolished the difference in Ser(1177) phosphorylation between groups. These findings suggest that growth-related increases in arteriolar NO bioavailability may be due at least partially to changes in the regulation of eNOS phosphorylation and increased signaling activity, with no change in the abundance of eNOS signaling proteins.     

Vascular Endothelial Growth Factor Receptor-2 Activates ADP-ribosylation Factor 1 to Promote Endothelial Nitric-oxide Synthase Activation and Nitric Oxide Release from Endothelial Cells

Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates endothelial function via production and release of nitric oxide (NO), an important signaling molecule. The molecular basis leading to NO production involves phosphatidylinositiol-3 kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) activation. In this study, we have examined whether small GTP-binding proteins of the ADP-ribosylation factor (ARF) family act as molecular switches to regulate signaling cascades activated by VEGF in endothelial cells. Our results show that this growth factor can promote the rapid and transient activation of ARF1. In endothelial cells, this GTPase is present on dynamic plasma membrane ruffles. Inhibition of ARF1 expression, using RNA interference, markedly impaired VEGF-dependent eNOS phosphorylation and NO production by preventing the activation of the PI3K/Akt signaling axis. Furthermore, our data indicate that phosphorylation of Tyr⁸⁰¹, on VEGF receptor 2, is essential for activating Src- and ARF1-dependent signaling events leading to NO release from endothelial cells. Lastly, this mediator is known to regulate a broad variety of endothelial cell functions. Depletion of ARF1 markedly inhibits VEGF-dependent increase of vascular permeability as well as capillary tubule formation, a process important for angiogenesis. Taken together, our data indicate that ARF1 is a novel modulator of VEGF-stimulated NO release and signaling in endothelial cells.     

Stimulatory effect of IGF-I and VEGF on eNOS message, protein expression, eNOS phosphorylation and nitric oxide production in rat glomeruli, and the involvement of PI3-K signaling pathway.

Nitric oxide (NO) is reported to be involved in the pathogenesis of renal hyperfiltration in the early stage of diabetic nephropathy. We set out to determine whether IGF-I and/or VEGF165 directly stimulate NO production in rat glomeruli and whether the expression of NO synthase (NOS) isoforms as well as eNOS phosphorylation contribute to NO generation by IGF-I and VEGF. Long-term exposure to IGF-I and/or VEGF165 augments NO production through increased eNOS mRNA, protein expression and phosphatidylinositol 3-kinase (PI3-K) signaling pathway plays a major role in this process; short-term exposure to IGF-I and/or VEGF(165) activates eNOS activity via phosphorylation by a PI3-K/Akt dependent pathway. Our data suggest the great possibility that increased endogenous IGF-I and VEGF may be responsible for the up-regulation of eNOS expression and NO production which contributes to glomerular hyperfiltration in early diabetic kidneys. IGF-I is a newly described growth factor that up-regulates eNOS expression and PI3-K plays a major role in this process.     

Dysfunction of endothelium-dependent relaxation to insulin via PKC-mediated GRK2/Akt activation in aortas of ob/ob mice

In diabetic states, hyperinsulinemia may negatively regulate Akt/endothelial nitric oxide synthase (eNOS) activation. Our main aim was to investigate whether and how insulin might negatively regulate Akt/eNOS activities via G protein-coupled receptor kinase 2 (GRK2) in aortas from ob/ob mice. Endothelium-dependent relaxation was measured in aortic rings from ob/ob mice (a type 2 diabetes model). GRK2, β-arrestin2, and Akt/eNOS signaling-pathway protein levels and activities were mainly assayed by Western blotting. Plasma insulin was significantly elevated in ob/ob mice. Insulin-induced relaxation was significantly decreased in the ob/ob aortas [vs. age-matched control (lean) ones]. The response in ob/ob aortas was enhanced by PKC inhibitor or GRK2 inhibitor. Akt (at Thr(308)) phosphorylation and eNOS (at Ser(1177)) phosphorylation, and also the β-arrestin2 protein level, were markedly decreased in the membrane fraction of insulin-stimulated ob/ob aortas (vs. insulin-stimulated lean ones). These membrane-fraction expressions were enhanced by GRK2 inhibitor and by PKC inhibitor in the ob/ob group but not in the lean group. PKC activity was much greater in ob/ob than in lean aortas. GRK2 protein and activity levels were increased in ob/ob and were greatly reduced by GRK2 inhibitor or PKC inhibitor pretreatment. These results suggest that in the aorta in diabetic mice with hyperinsulinemia an upregulation of GRK2 and a decrease in β-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.     

Epigallocatechin gallate, a green tea polyphenol, mediates NO-dependent vasodilation using signaling pathways in vascular endothelium requiring reactive oxygen species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.     

Mechanisms of shear stress-induced endothelial nitric-oxide synthase phosphorylation and expression in ovine fetoplacental artery endothelial cells

Placental blood flow, nitric-oxide (NO) levels, and endothelial NO synthase (eNOS) expression increase during human and ovine pregnancy. Shear stress stimulates NO production and eNOS expression in ovine fetoplacental artery endothelial (OFPAE) cells. Because eNOS is the rate-limiting enzyme essential for NO synthesis, its activity and expression are both closely regulated. We investigated signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by OFPAE cells. The OFPAE cells were cultured at 3 dynes/cm2 shear stress, then exposed to 15 dynes/cm2 shear stress. Western blot analysis for phosphorylated ERK1/2, Akt, p38 mitogen activated protein kinase (MAPK), and eNOS showed that shear stress rapidly increased phosphorylation of ERK1/2 and Akt but not of p38 MAPK. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by the phosphatidyl inositol-3-kinase (PI-3K)-inhibitors wortmannin and LY294002 but not by the mitogen activated protein kinase kinase (MEK)-inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels otherwise induced by the 15 dynes/cm2 shear stress. Blockade of either signaling pathway changed the shear stress-induced increase in eNOS protein levels. In conclusion, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibition of MEK. Prolonged shear stress-stimulated increases in eNOS protein were not affected by inhibition of MEK- or PI-3K-mediated pathways.