eNOS activation induced by a polyphenol-rich grape skin extract in porcine coronary arteries.

BACKGROUND/AIMS: Drinking red wine is associated with a decreased mortality from coronary heart diseases. This study examined whether polyphenols contained in a grape skin extract (GSE) triggered the endothelial formation of nitric oxide (NO) and investigated the underlying mechanism. METHODS: Vascular reactivity was assessed in organ chambers using porcine coronary artery rings in the presence of indomethacin (a cyclooxygenase inhibitor) and charybdotoxin plus apamin (inhibitors of endothelium-derived hyperpolarizing factor-mediated responses). The phosphorylation level of Src, Akt and endothelial NO synthase (eNOS) were assessed by Western blot analysis, and the formation of reactive oxygen species (ROS) was investigated using dihydroethidine and dichlorodihydrofluorescein. RESULTS: GSE-induced endothelium-dependent relaxations were abolished by N(G)-nitro-L-arginine (an eNOS inhibitor) and ODQ (a soluble guanylyl cyclase inhibitor), and they were reduced by MnTMPyP, polyethyleneglycol catalase, PP2 (an inhibitor of Src kinase) and wortmannin (an inhibitor of phosphoinositide 3-kinase). GSE caused phosphorylation of Src, which was prevented by MnTMPyP. It also caused phosphorylation of Akt and eNOS, which were prevented by MnTMPyP, polyethyleneglycol catalase, PP2, wortmannin and LY294002. GSE elicited the formation of ROS in native and cultured endothelial cells, which was prevented by MnTMPyP. CONCLUSIONS: GSE causes endothelium-dependent NO-mediated relaxations of coronary arteries. This effect involves the intracellular formation of ROS in endothelial cells leading to the Src kinase/phosphoinositide 3-kinase/Akt-dependent phosphorylation of eNOS.

Angiotensin II-induced hypertension is associated with a selective inhibition of endothelium-derived hyperpolarizing factor-mediated responses in the rat mesenteric artery.

Hypertension has been shown to be associated with impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated arterial relaxation and hyperpolarization. Treatments of hypertensive rats with inhibitors of the renin-angiotensin system have been shown to restore both EDHF-mediated responses and the expression of connexins involved in the intercellular transfer of the hyperpolarization in mesenteric arteries. The present study was designed to determine whether chronic treatment of rats with angiotensin II impairs EDHF-mediated responses and the expression of connexins in the mesenteric arterial wall. Male Wistar rats were treated with angiotensin II (0.4 mg/kg/day) for 21 days using osmotic minipumps. Arterial pressure was measured by tail-cuff plethysmography. Contractile responses and membrane potential were measured in isolated mesenteric arteries. The expression of the three connexins (Cxs), Cx37, Cx40, and Cx43, was quantified in segments of mesenteric arteries by immunohistochemistry and quantitative real-time reverse transcriptase-polymerase chain reaction. Angiotensin II administration increased the mean systolic blood pressure. EDHF-mediated relaxation and hyperpolarization to acetylcholine and red wine polyphenols were significantly impaired in mesenteric arteries from angiotensin II-treated rats in comparison with control animals, whereas nitric oxide-mediated relaxation was unaltered. The expression of connexins Cx37, Cx40, and Cx43 was significantly decreased in the mesenteric artery from angiotensin II-treated rats. These findings indicate that angiotensin II-induced hypertension is associated with a selective impairment of EDHF-mediated relaxation and hyperpolarization in the rat mesenteric artery. The inhibition of EDHF-mediated responses is due, at least in part, to a decreased expression of connexins Cx37, Cx40, and Cx43 in the arterial wall.

Targeted and persistent effects of NO mediated by S-nitrosation of tissue thiols in arteries with endothelial dysfunction.

We have previously demonstrated that in endothelium-denuded arteries, S-nitrosation of cysteine residues is a mechanism of formation of releasable nitric oxide (NO) stores, accounting for the long-lasting relaxation induced by S-nitrosating agents like S-nitrosoglutathione (GSNO). Here, we have investigated whether such effects could also be obtained in arteries exhibiting oxidative stress-associated endothelial dysfunction. Rats were implanted or not with a minipump delivering saline or angiotensin II for 14 days. As expected, aorta from angiotensin II-infused rats exhibited increased level of superoxide anions (as evaluated with dihydroethidine as fluorescent probe) and a reduced relaxation to acetylcholine in comparison to saline group. Unlike aortic rings with endothelium from controls, those from angiotensin II-infused rats exhibited persistent hyporesponsiveness to phenylephrine after pre-exposure to GSNO, as well as relaxation upon addition of N-acetylcysteine (NAC, which can displace NO from cysteine-NO residues) or HgCl(2) (which cleaves S-NO bonds). In aorta from angiotensin II-infused rats, GSNO also induced a persistent increase in cysteine-NO residues (as determined using anti-cysteine-NO antiserum), which was blunted by NAC and HgCl(2). These data indicate that (i) the vasorelaxant influence of releasable NO stores is unmasked by endothelial dysfunction (ii) S-nitrosation of cysteine residues remains an effective mechanism of formation of releasable NO stores in arteries exhibited oxidative stress-associated endothelial dysfunction. Thus, formation of releasable NO stores by S-nitrosating agents allows targeted vasculoprotective effects of NO at sites of endothelial dysfunction.

Grape juice causes endothelium-dependent relaxation via a redox-sensitive Src- and Akt-dependent activation of eNOS.

OBJECTIVES: An enhanced endothelial formation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), is thought to contribute to the protective effect of moderate consumption of red wine on coronary diseases. The present study has characterized endothelium-dependent relaxations to Concord grape juice (CGJ), a non-alcoholic rich source of grape-derived polyphenols, in the coronary artery. METHODS: Porcine coronary artery rings were suspended in organ chambers for the measurement of changes in isometric tension in the presence of indomethacin. NO formation was assessed by electron spin resonance spectroscopy, and the phosphorylation of Src, Akt and endothelial NO synthase (eNOS) by Western blot analysis in cultured endothelial cells. RESULTS: Endothelium-dependent relaxations to CGJ were slightly but significantly reduced by L-NA, not affected by charybdotoxin (CTX) plus apamin (APA, two inhibitors of EDHF-mediated responses) whereas the combination of L-NA, CTX plus APA reduced maximal relaxation to about 50%. In the presence of CTX plus APA, relaxations to CGJ were markedly reduced by the membrane permeant mimetic of superoxide dismutase (SOD), MnTMPyP, the membrane permeant analogue of catalase polyethyleneglycol-catalase (PEG-catalase), PP2, an inhibitor of Src kinase, and by wortmannin, an inhibitor of the PI3-kinase. CGJ stimulated the formation of reactive oxygen species and the N(omega)-nitro-L-arginine-, PP2- and wortmannin-sensitive formation of NO in endothelial cells. The formation of NO was associated with a redox-sensitive and time-dependent phosphorylation of Src, Akt and eNOS. CONCLUSIONS: CGJ induces endothelium-dependent relaxations of coronary arteries, which involve a NO-mediated component and also, to a minor extent, an EDHF-mediated component. In addition, CGJ-induced NO formation is due to the redox-sensitive activation of Src kinase with the subsequent PI3-kinase/Akt-dependent phosphorylation of eNOS.

Progestins overcome inhibition of platelet aggregation by endothelial cells by down-regulating endothelial NO synthase via glucocorticoid receptors.

Hormone replacement therapy with estroprogestin preparations is associated with an increased risk of venous and arterial thromboembolic events in postmenopausal women. This study examined whether progestins affect the formation of NO in endothelial cells, and, if so, to determine the underlying mechanism. Experiments were performed with human umbilical vein endothelial cells. Endothelial nitric oxide synthase (eNOS) expression was assessed by real-time polymerase chain reaction (PCR) and Western blot analysis, NO formation by electron spin resonance spectroscopy, nuclear translocation of the glucocorticoid receptor by immunofluorescence microscopy, and platelet aggregation by an aggregometer. Medroxyprogesterone acetate (MPA) and progesterone markedly decreased the eNOS mRNA and protein levels, whereas levonorgestrel and nomegestrol acetate had only small effects. This effect was associated with a decreased NO formation leading to a reduced ability of endothelial cells to prevent platelet aggregation and was prevented by knockdown of the glucocorticoid receptor using siRNA. MPA and progesterone, but not levonorgestrel and nomegestrol acetate, caused nuclear translocation of the glucocorticoid receptor. The present findings indicate that certain progestins, including MPA, reduce the antiaggregatory effect of endothelial cells by decreasing the expression of eNOS and the formation of NO in endothelial cells, an effect that is mediated via activation of glucocorticoid receptors.

Red wine polyphenols prevent angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase.

OBJECTIVE: Chronic administration of moderate amounts of red wine has been associated with a protective effect on the cardiovascular system. This study examined whether red wine polyphenols prevent the angiotensin II (Ang II)-induced hypertension and endothelial dysfunction in rats, and, if so, to elucidate the underlying mechanism. METHODS: Hypertensive rats were obtained by a 14-day infusion of Ang II. Red wine polyphenols were administered in the drinking water one week before and during the Ang II infusion. Arterial pressure was measured in conscious rats. Ex vivo vascular relaxation was assessed in organ chambers, vascular superoxide anion production by dihydroethidine and vascular NADPH oxidase expression by immunohistochemistry. RESULTS: Ang II-induced hypertension was associated with decreased relaxation to acetylcholine but not to red wine polyphenols. The Ang II treatment also increased vascular superoxide anion production and expression of nox1 and p22phox NADPH oxidase subunits. Intake of red wine polyphenols prevented the Ang II-induced hypertension and endothelial dysfunction and normalized vascular superoxide anion production and NADPH oxidase subunit expression. Red wine polyphenol treatment alone did not affect blood pressure. CONCLUSION: Intake of red wine polyphenols prevents Ang II-induced hypertension and endothelial dysfunction. Prevention of vascular NADPH oxidase induction and preservation of arterial nitric oxide availability during Ang II administration likely contribute to this effect.

Red wine polyphenol-induced, endothelium-dependent NO-mediated relaxation is due to the redox-sensitive PI3-kinase/Akt-dependent phosphorylation of endothelial NO-synthase in the isolated porcine coronary artery.

An enhanced endothelial formation of nitric oxide (NO) by red wine polyphenolic compounds (RWPs) has been involved in the protective effect of chronic intake of red wine on coronary diseases. However, the mechanism underlying the activation of endothelial NO synthase (eNOS) remains unclear. In the presence of indomethacin and charybdotoxin plus apamin to prevent the formation of prostanoids and endothelium-derived hyperpolarizing factor, respectively, RWPs caused pronounced endothelium-dependent relaxations in porcine coronary arteries. Relaxations to RWPs were abolished by N(omega)-nitro-L-arginine (L-NA, a competitive inhibitor of NO synthase) and the membrane permeant analog of superoxide dismutase (SOD), MnTMPyP, and reduced by polyethylene glycol-SOD (PEG-SOD), PEG-catalase and inhibitors of PI3-kinase (wortmannin and LY294002). RWPs caused the L-NA-sensitive formation of NO, as assessed by electron spin resonance spectroscopy and the formation of cyclic guanosine monophosphate in coronary artery endothelial cells; these responses were reduced by MnTMPyP, PEG-catalase, and inhibitors of PI3-kinase. RWPs caused the sustained phosphorylation of Akt and eNOS at Ser1177 in endothelial cells, which were abolished by MnTMPyP and inhibitors of PI3-kinase. These data demonstrate that RWPs induce the redox-sensitive activation of the PI3-kinase/Akt pathway in endothelial cells which, in turn, causes phosphorylation of eNOS, resulting in an increased formation of NO.

Vascular protection by dietary polyphenols.

Consumption of polyphenol-rich foods, such as fruits and vegetables, and beverages derived from plants, such as cocoa, red wine and tea, may represent a beneficial diet in terms of cardiovascular protection. Indeed, epidemiological studies demonstrate a significant inverse correlation between polyphenol consumption and cardiovascular risk. Among the numerous plausible mechanisms by which polyphenols may confer cardiovascular protection, improvement of the endothelial function and inhibition of angiogenesis and cell migration and proliferation in blood vessels have been the focus of recent studies. These studies have indicated that, in addition to and independently from their antioxidant effects, plant polyphenols (1) enhance the production of vasodilating factors [nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostacyclin] and inhibit the synthesis of vasoconstrictor endothelin-1 in endothelial cells; and (2) inhibit the expression of two major pro-angiogenic factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in smooth muscle cells. The mechanisms of these effects involve: (1) in endothelial cells, increased Ca(2+) level and redox-sensitive activation of the phosphoinositide 3 (PI3)-kinase/Akt pathway (leading to rapid and sustained activation of nitric oxide synthase and formation of EDHF) and enhanced expression of nitric oxide synthase; and (2) in smooth muscle cells, both redox-sensitive inhibition of the p38 mitogen-activated protein kinase (p38 MAPK) pathway activation (leading to inhibition of platelet-derived growth factor (PDGF)-induced VEGF gene expression) and redox-insensitive mechanisms (leading to inhibition of thrombin-induced MMP-2 formation). The current evidence suggests that all these mechanisms are triggered by polyphenols with specific structures, although the structural requirements may be different from one effect to the other, and that they all contribute to the vasoprotective, anti-angiogenic, anti-atherogenic, vasorelaxant and anti-hypertensive effects of acute or chronic administration of plant polyphenols found in vivo in animals and in patients.

Red wine polyphenols induce EDHF-mediated relaxations in porcine coronary arteries through the redox-sensitive activation of the PI3-kinase/Akt pathway.

Red wine polyphenolic compounds (RWPCs) are potent inducers of endothelium-dependent relaxations of coronary arteries, which involve both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF). The EDHF-mediated relaxation to RWPCs is critically dependent on the formation of reactive oxygen species by a flavin-dependent enzyme. The aim of the present study was to determine the role of redox-sensitive protein kinases including p38 MAPK, ERK1/2 and PI3-kinase/Akt in RWPCs-induced EDHF-mediated relaxation. Porcine coronary artery rings were suspended in organ chambers for measurement of changes in isometric tension. Confluent cultures of porcine coronary artery endothelial cells were used to determine the phosphorylation level of p38 MAPK, ERK1/2 and Akt by Western blot analysis. All experiments were performed in the presence of indomethacin and Nomega-nitro-L-arginine. RWPCs caused pronounced endothelium-dependent relaxations, which were significantly reduced by wortmannin and LY294002, two inhibitors of PI3-kinase, and not affected by PD98059 (an inhibitor of ERK1/2 kinase kinase) and SB203580 (an inhibitor of p38 MAPK). In contrast, wortmannin did not affect relaxations to bradykinin or levcromakalim. RWPCs elicited within minutes a sustained and concentration-dependent phosphorylation of p38 MAPK, ERK1/2 and Akt in endothelial cells. The phosphorylation of Akt in response to RWPCs was abolished by wortmannin and LY294002, and by the membrane-permeant analogue of superoxide dismutase Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin. The present findings demonstrate that RWPCs cause EDHF-mediated relaxations of coronary arteries; these responses are critically dependent on the redox-sensitive activation of the PI3-kinase/Akt pathway in endothelial cells.

Chronic treatment with progesterone but not medroxyprogesterone acetate restores the endothelial control of vascular tone in the mesenteric artery of ovariectomized rats.

OBJECTIVE: To examine whether chronic administration of the natural hormone progesterone or a synthetic progestogen, medroxyprogesterone acetate, to ovariectomized rats affects the endothelial control of arterial tone in the isolated mesenteric artery. DESIGN: Sham-operated rats received a daily subcutaneous injection of solvent (sesame oil), whereas ovariectomized rats received either sesame oil, progesterone (22 mg kg/day), or medroxyprogesterone acetate (22 mg kg/day) for 4 weeks, according to their respective group. RESULTS: Phenylephrine-induced contractions were significantly increased (about 200% at 10 microM) by N-nitro-L-arginine, a nitric oxide synthase inhibitor, in intact mesenteric arterial rings from the sham-operated but not from the ovariectomized group. The progesterone but not the medroxyprogesterone treatment restored the potentiating effect of N-nitro-L-arginine on phenylephrine-induced contraction (about 180% at 10 microM). Contractions to phenylephrine were not affected by the combination of charybdotoxin plus apamin, two inhibitors of endothelium-derived hyperpolarizing factor-mediated responses, in all groups. Acetylcholine induced endothelium-dependent relaxations, which were partially inhibited by N-nitro-L-arginine and abolished by the combination of N-nitro-L-arginine plus charybdotoxin and apamin, in all groups. Acetylcholine induced similar charybdotoxin and apamin-sensitive hyperpolarizations in intact mesenteric artery segments from all groups. CONCLUSIONS: Chronic administration of progesterone, but not medroxyprogesterone, to ovarictomized rats restores the endothelium-dependent attenuation of contractile responses to phenylephrine in mesenteric arterial rings through the endothelial formation of nitric oxide. Thus, an enhancement of the protective effect of endothelial cells on the arterial wall might contribute to the beneficial effect of certain progestogen-containing preparations during hormonal treatment.

Red wine polyphenolic compounds inhibit vascular endothelial growth factor expression in vascular smooth muscle cells by preventing the activation of the p38 mitogen-activated protein kinase pathway.

OBJECTIVE: Moderate consumption of red wine has a beneficial effect on the cardiovascular system. This study examines whether red wine polyphenolic compounds (RWPCs) affect vascular endothelial growth factor (VEGF) expression, a major angiogenic and proatherosclerotic factor in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: VEGF mRNA expression was assessed by Northern blot analysis and the release of VEGF by immunoassay in cultured VSMCs. Short-term and long-term exposure of VSMCs to RWPCs inhibited VEGF mRNA expression and release of VEGF in response to platelet-derived growth factor AB (PDGFAB), transforming growth factor-beta1, or thrombin. The PDGFAB-induced expression of VEGF was markedly reduced by SB203580 (inhibitor of p38 mitogen-activated protein kinase [MAPK]), antioxidants, and diphenylene iodonium (inhibitor of flavin-dependent enzymes), slightly reduced by PD98059 (inhibitor of MEK), and not significantly affected by wortmannin (inhibitor of PI-3-kinase) and L-JNKI (inhibitor of JNK). Short-term and long-term treatment of VSMCs with RWPCs markedly reduced PDGFAB-induced production of reactive oxygen species and phosphorylation of p38 MAPK. CONCLUSIONS: These data indicate that RWPCs strongly inhibit growth factor-induced VEGF expression in VSMCs by preventing the redox-sensitive activation of the p38 MAPK pathway. The potential antiangiogenic and antiatherosclerotic properties of RWPCs are likely to contribute to cardiovascular protection by preventing the development of atherosclerotic lesions.

Cutaneous venous dysfunction studied in vivo in the LPS-treated rabbit: implication of NO in saphenous vein hyporeactivity.

Superficial vein pathology involves both mechanical (hyperpressure and distension) and inflammatory mechanisms. Conflicting results exist about the role of NO in the venous hyporeactivity induced by inflammation. In order to clarify this point, we aimed to investigate the effects of sepsis on cutaneous vein responsiveness in vivo and the possible contributions of constitutive and inducible NOS to the changes of venous contractility. Saphenous vein diameter was recorded by an ultrasonic echo-tracking device in pentobarbital-anaesthetised rabbits. Bacterial lipopolysaccharide (LPS) was administered i.v. at 20 mg/kg/15 min, inducing a progressive fall in mean arterial blood pressure after 2-3 h. The effects of LPS on saphenous vein responsiveness to noradrenaline (2 microg/kg i.v.) were measured simultaneously. In some rabbits, veins were removed for immunochemistry to detect iNOS staining. The venoconstriction to noradrenaline was already significantly reduced at 30 min after LPS (6+/-1% instead of 19+/-1% before LPS) and was completely abolished 3 h after LPS. A reduction of the venoconstriction induced by sumatriptan, a 5-HT(1B/D) agonist, (100 microg/kg, 11+/-1% after saline n=5) was also observed 180 min after LPS infusion (3+/-1%, n=4). The venodilatations induced by acetylcholine or sodium nitroprusside injected locally into the vein were not altered by LPS. When administered 90 min after LPS infusion, the NOS inhibitor L-NAME but not the selective iNOS inhibitor L-NIL (10 mg/kg) induced a recovery of the venoconstriction. Preventive perfusion with L-NAME (10 mg/kg/2 h) reduced the initial hyporeactivity to noradrenaline (30 to 60 min), but accelerated the lethal fall in MAP. L-NIL (10 mg/kg/2 h), to a lesser extent than L-NAME, also reduced the initial hyporeactivity to noradrenaline; in contrast to L-NAME, L-NIL also delayed the complete loss of noradrenaline constriction and improved animal survival. In control animals, neither L-NAME nor L-NIL modified the venoconstriction induced by noradrenaline. iNOS staining was observed in the saphenous vein endothelium after LPS. The experimental model developed in these experiments allows the study of venous responsiveness during sepsis in vivo. Our results show that LPS administration reduces saphenous vein contractility to both adrenergic and serotoninergic constrictor agents. The data suggest that both endothelial and inducible NO are involved in the loss of venous reactivity but these enzymes exert contrasting effects on blood pressure changes.