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Background and purpose: Red wine polyphenols (RWPs) inhibit the expression of vascular endothelial growth factor (VEGF), a major pro-angiogenic and pro-atherosclerotic factor, in vascular smooth muscle cells (VSMCs). The aim of this study was to identify which red wine polyphenols were inhibitory and to determine the mechanism underlying the inhibitory effects. Experimental approach: Release of VEGF stimulated by platelet derived growth factor AB (PDGF AB ) , from human aortic VSMCs was measured by immunoassay and phosphorylation of kinases by Western blot analysis. The direct antioxidant properties of polyphenols were determined by electron paramagnetic resonance and the cellular formation of reactive oxygen species (ROS) by dichlorofluorescein. Key results: The inhibitory effect of RWPs on PDGF AB -induced release of VEGF was mimicked by delphinidin but not by quercetin, catechins, resveratrol, gallic acid or caffeic acid. In the anthocyanin class, not only delphinidin but also cyanidin prevented VEGF release whereas malvidin and peonidin were without effect. RWPs, delphinidin and cyanidin directly scavenged ROS and prevented the PDGF AB -induced formation of ROS in VSMCs. Malvidin and peonidin did not scavenge ROS but prevented the cellular formation of ROS. Although the p38 MAPK, ERK1/2 and JNK pathways have been involved in the PDGF AB -induced expression of VEGF, in our experiments, only phosphorylation of p38 MAPK and JNK was inhibited by RWPs, delphinidin and cyanidin. Conclusions and implications: Anthocyanins presenting a hydroxyl residue at position 3 0 are able to inhibit PDGF AB -induced VEGF expression by preventing activation of p38 MAPK and JNK in VSMCs.

The possible involvement of the endothelium in the vasodilator action of eugenol was investigated in the mesenteric vascular bed (MVB) of the rat. Bolus injections of eugenol (0.2, 2 and 20 micromol) and acetylcholine (ACh; 10, 30 and 100 pmol) induced dose-dependent vasodilator responses in noradrenaline-precontracted beds that were partially inhibited by pretreatment of the MVB with deoxycholate (1 mg mL(-1)) to remove the endothelium (approximately 14% and approximately 30% of the control response remaining at the lowest doses of ACh and eugenol, respectively). The vasodilator effect of glyceryl trinitrate (1 micromol) was unaltered by deoxycholate. In the presence of either N(omega)-nitro-L-arginine methyl ester (300 microM) or tetraethylammonium (1 mM)the response to ACh was partially reduced, whereas eugenol-induced vasodilation was unaffected. Similarly the vasodilator effect of eugenol was not inhibited by indometacin (3 microM). Under calcium-free conditions the vasoconstrictor response elicited by bolus injections of noradrenaline (10 nmol) was dose-dependently and completely inhibited by eugenol (0.1-1 mM). Additionally, the pressor effects of bolus injections of calcium chloride in potassium-depolarized MVBs were greatly reduced in the presence of eugenol (0.1 mM), with a maximal reduction of approximately 71% of the control response. Our data showed that eugenol induced dose-dependent, reversible vasodilator responses in the rat MVB, that were partially dependent on the endothelium, although apparently independent of nitric oxide, endothelium-derived hyperpolarizing factor or prostacyclin. Furthermore, an endothelium-independent intracellular site of action seemed likely to participate in its smooth muscle relaxant properties.

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.

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