Procyanidins from grape seeds protect endothelial cells from peroxynitrite damage and enhance endothelium-dependent relaxation in human artery: new evidences for cardio-protection

The peroxynitrite scavenging ability of Procyanidins from Vitis vinifera L. seeds was studied in homogeneous solution and in human umbilical endothelial cells (EA.hy926 cell line) using 3-morpholinosydnonimine (SIN-1) as peroxynitrite generator. In homogeneous phase procyanidins dose-dependently inhibited 2',7'-dichloro-dihydrofluorescein (DCFH) oxidation induced by SIN-1 with an IC50 value of 0.28 microM. When endothelial cells (EC) were exposed to 5 mM SIN-1, marked morphological alterations indicating a necrotic cell death (cell viability reduced to 16 +/- 2.5%) were observed. Cell damage was suppressed by procyanidins, with a minimal effective concentration of 1 microM (cell morphology and integrity completely recovered at 20 microM). Cellular localization of procyanidins in EC was confirmed using a new staining procedure and site-specific peroxyl radical inducers: AAPH and cumene hydroperoxide (CuOOH). Endothelial cells (EC) pre-incubated with procyanidins (20 microM) and exposed to FeCl3/K3Fe(CN)6 showed a characteristic blue staining, index of a site-specific binding of procyanidins to EC. Procyanidins dose-dependently inhibit the AAPH induced lipid oxidation and reverse the consequent loss of cell viability, but were ineffective when oxidation was driven at intracellular level (CuOOH). This demonstrates that the protective effect is due to their specific binding to the outer surface of EC thus to quench exogenous harmful radicals. Procyanidins dose-dependently relaxed human internal mammary aortic (IMA) rings (with intact endothelium) pre-contracted with norepinephrine (NE), showing a maximal vasorelaxant effect (85 +/- 9%) at 50 microM (catechin: 18 +/- 2% relaxation at 50 microM). This effect was completely abolished when IMA-rings were de-endothelized and when IMA-rings with intact endothelium were pretreated with L-NMMA or with the soluble guanylate cyclase inhibitor, ODQ. Pre-incubation with indomethacin reduces (by almost 50%) the vasodilating effect of procyanidins, indicating the involvement also of a COX-dependent mechanism. This was confirmed in another set of experiments, where procyanidins dose-dependently stimulate the prostacyclin (PGI2) release, reaching a plateau between 25 and 50 microM. Finally, pre-incubation of IMA-rings with procyanidins (from 6.25 to 25 microM) resulted in a dose-dependent prevention of the endothelin-1 (ET-1) vasoconstriction. The ability of procyanidins to prevent peroxynitrite attack to vascular cells, by layering on the surface of coronary EC, and to enhance endothelial NO-synthase-mediated relaxation in IMA rings provide further insight into the molecular mechanisms through which they exert cardioprotective activity in ischemia/reperfusion injury in vivo.