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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 28, 2004 as doi:10.1096/fj.04-2146fje. |
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Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, Strasbourg, France
2Correspondence: UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, 74, route du Rhin, B.P. 60024, F-67401 Illkirch, France. E-mail: schini{at}aspirine.u-strasbg.fr
SPECIFIC AIMS
Enhanced endothelial formation of nitric oxide (NO) by red wine polyphenols has been involved in the protective effect of chronic intake of red wine on coronary diseases. The aim of the present study was to determine the mechanism(s) underlying the activation of endothelial NO synthase and the subsequent formation of NO in coronary arteries in response to red wine polyphenols.
PRINCIPAL FINDINGS
1. Red wine polyphenols induce the redox-sensitive, endothelium-dependent, NO-mediated relaxation of coronary arteries
In the presence of indomethacin and charybdotoxin + apamin to prevent the formation of prostanoids and endothelium-derived hyperpolarizing factor, respectively, red wine polyphenols caused pronounced endothelium-dependent relaxations in porcine coronary arteries. Relaxations to red wine polyphenols were abolished by N
-nitro-L-arginine (L-NA, a competitive inhibitor of NO synthase). These NO-mediated relaxations to red wine polyphenols were also abolished by the membrane-permeant analog of superoxide dismutase (SOD), MnTMPyP, and significantly reduced by polyethyleneglycol-SOD (PEG-SOD) and PEG-catalase but remained unaltered by native SOD and native catalase (Fig. 1
A–C). In contrast, bradykinin-induced, endothelium-dependent, NO-mediated relaxation was not affected by the SOD mimetic MnTMPyP (Fig. 1D)
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2. Role of redox-sensitive p38 MAPK, ERK1/2, and PI3-kinase/Akt pathways in the red wine polyphenol-induced, NO-mediated relaxations
Since ROS have important signaling function in vascular cells, experiments were performed to determine the role of several redox-sensitive protein kinases including p38 MAPK, ERK1/2, and PI3-kinase in red wine polyphenol-induced, NO-mediated relaxations. Relaxations to red wine polyphenols were significantly inhibited by wortmannin and LY294002, two PI3-kinase inhibitors, and not affected by SB203580, an inhibitor of p38 MAPK, or by PD98059, an inhibitor of ERK1/2 kinase kinase. In contrast to red wine polyphenols, bradykinin-induced relaxations were unaffected by wortmannin and LY294002.
3. Red wine polyphenols stimulate the redox-sensitive, PI3-kinase-dependent formation of NO in coronary endothelial cells
Exposure of cultured porcine coronary artery endothelial cells to red wine polyphenols caused an 
2-fold increase in formation of NO assessed by electron spin resonance spectroscopy. The stimulatory effect of red wine polyphenols was similar to that induced by bradykinin (2.5-fold at 1 µM). NO formation in response to red wine polyphenols and bradykinin was abolished by L-NA, indicating it was due to increased eNOS activity. The stimulatory effect of red wine polyphenols was markedly reduced by MnTMPyP and abolished by PEG-catalase and the antioxidant N-acetylcysteine, whereas that of bradykinin was not significantly affected by MnTMPyP. The kinetic of the red wine polyphenol-induced endothelial formation of NO, assessed indirectly by the cellular content of cyclic GMP, indicated that this response reached a plateau within 1 min and persisted for at least 10 min. The red wine polyphenol-induced formation of cyclic GMP was markedly reduced by inhibitors of PI3-kinase (wortmannin and LY294002). Exposure of cells to wortmannin alone reduced by 48% the cyclic GMP content; LY294002 had no such effect.
4. Red wine polyphenols cause redox-sensitive, PI3-kinase-dependent phosphorylation of Akt and eNOS in endothelial cells
Studies indicate that the PI3-kinase pathway mediates increased eNOS activation in response to hydrogen peroxide (H2O2), shear stress, estrogens, vascular endothelial growth factor through Akt-dependent phosphorylation of eNOS at Ser1177 and dephosphorylation of eNOS at Thr495. Therefore, the possibility that red wine polyphenols affect the phosphorylation level of Akt and eNOS was assessed by immunoblotting. Unstimulated endothelial cells had either no or only a low level of phosphorylated Akt and phosphorylated eNOS at Ser1177, but eNOS was phosphorylated at Thr495. Exposure of cells to red wine polyphenols caused the appearance within 3 min of a pronounced phosphorylation level of Akt and eNOS at Ser1177 whereas the phosphorylation level of eNOS at Thr495 was markedly reduced. These effects persisted for at least 30 min. Red wine polyphenol-induced phosphorylation of Akt and eNOS at Ser1177 was abolished by pretreatment of cells with the SOD mimetic MnTMPyP, wortmannin, or LY294002).
CONCLUSIONS AND SIGNIFICANCE
The present findings indicate that the red wine polyphenol-induced, endothelium-dependent, NO-mediated relaxation is a redox-sensitive event that is critically dependent on the intracellular formation of superoxide anions and H2O2. Indeed, membrane-permeable analogs of SOD or catalase were able to markedly reduce NO-mediated relaxation in the coronary artery and the formation and bioactivity of NO in endothelial cells in response to red wine polyphenols whereas extracellular SOD and catalase were inactive. However, red wine polyphenol-induced intracellular formation of reactive oxygen species must be a well-controlled and localized phenomenon since reactive oxygen species can directly interact with NO in a diffusion-limited fashion to cause its inactivation.
Several lines of evidence indicate that reactive oxygen species act as key intracellular mediators to activate signal transduction pathways leading to numerous biological responses in vascular cells, including growth, hypertrophy, survival, and apoptosis. H2O2 has been shown to cause vasodilatation partly by stimulating the endothelial formation of NO and in part by inducing potassium channel opening, resulting in vascular smooth muscle hyperpolarization. H2O2 promotes eNOS activity through the coordinated phosphorylation of eNOS at Ser1177 and dephosphorylation at Thr495. Although H2O2 is able to activate the mitogen-activated protein kinases p38, ERK1/2, and JNK, and PI3-kinase in endothelial cells, eNOS phosphorylation at Ser1177 is mediated predominantly by the PI3-kinase/Akt pathway. Therefore, the possibility that red wine polyphenols activate eNOS through the redox-sensitive PI3-kinase/Akt-dependent changes in the phosphorylation level of eNOS was examined.
The present findings indicate that red wine polyphenols induce eNOS activity by causing within minutes eNOS phosphorylation at Ser1177 and dephosphorylation at Thr495 in endothelial cells. Moreover, changes in the phosphorylation level of eNOS at Ser1177 and Thr495 were maintained for at least 30 min, indicating a long-lasting eNOS activation. A pronounced phosphorylation of eNOS at Ser1179 has been observed in bovine aortic endothelial cells in response to the green tea polyphenol epigallocatechin-3-gallate; this effect was maintained for at least 6 h. In contrast to red wine polyphenols and tea polyphenols, activation of G-protein-coupled receptors by bradykinin or thrombin caused only a transient formation of NO, which reached a peak value within 1 min, then vanished rapidly. The present findings indicate that red wine polyphenol-induced eNOS activity is mediated by the PI3-kinase/Akt pathway. Indeed, red wine polyphenols induced phosphorylation of Akt concomitant with changes in the phosphorylation level of eNOS at Ser1177 and Thr495; these responses were abolished by inhibitors of PI3-kinase. Inhibitors of PI3-kinase markedly reduced the endothelial formation of NO to red wine polyphenols. Moreover, since the membrane-permeant analog of SOD, MnTMPyP, abolished the phosphorylation of Akt and eNOS at Ser1177, it can be concluded that reactive oxygen species act as upstream mediators of the PI3-kinase/Akt pathway leading to eNOS activation by red wine polyphenols. The PI3-kinase pathway mediates the redox-sensitive NO formation in native endothelial cells since the red wine polyphenol-induced, NO-mediated relaxation of the coronary artery is markedly reduced by inhibition of the PI3-kinase pathway but not of the p38 MAPK and the MEK/ERK1/2 pathways.
In contrast to redox-sensitive NO formation in endothelial cells, the protective effect of red wine polyphenols on vascular smooth muscle function and platelet function is attributable to their antioxidant properties. Although polyphenols can exert their antioxidant properties by directly scavenging superoxide, the protective effect might be due to their ability to inhibit the expression and activity of pro-oxidant enzymes such as NADPH oxidase and xanthine oxidase and to increase that of antioxidant enzymes such as catalase.
The present findings indicate that reactive oxygen species are key intracellular mediators of the rapid and sustained formation of NO induced by red wine polyphenols in endothelial cells via the PI3-kinase/Akt pathway-dependent changes in eNOS phosphorylation. The dual ability of red wine polyphenols to cause a redox-sensitive formation of NO in endothelial cells and, on the other hand, to prevent platelet activation and expression of proatherogenic factors in vascular smooth muscle cells via their antioxidant properties, might help explain their beneficial effect on the cardiovascular system.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2146fje;
1 Present address: Department of Pharmacology and Therapeutics, University of Louvain Medical School, 53 Mounier, 1200 Brussels. 
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