J023 Estrogen receptor-alpha mediated the endothelial no release triggered by delphinidin

Abstract

We previously reported that deletion of estrogen receptor-alpha (ER-alpha) abolishes endothelial response to wine polyphenols. The present study was designed to demonstrate that delphinidin, an anthocyanin that possess the same pharmacological profile than wine polyphenols in vascular protection, mediates its action via direct activation of ER-alpha receptor. Both 17-beta estradiol and the specific ER-alpha agonist, propyl pyrazole triol (PPT), induced endothelium-dependent relaxation in aorta from ovariectomized female ER-alpha wild type but not in ER-alpha knock out mice. In the following experiments, 17-beta estradiol, PPT and delphinidin were used at maximally active concentrations at which they produced endothelium-dependent relaxation. ER-apha deletion abrogated the endothelial relaxation to delphinidin. In human endothelial cell line, Eahy 926, both 17-beta estradiol (10 μm) and PPT (10 μm) were able to increase nitric oxide (NO) production evaluated by electron paramagnetic resonance. Interestingly, the specific ER-alpha antagonist, fulvestrant (30 nM), did not affect basal NO level but it completely prevented the response to 17-beta estradiol and PPT. Besides, the capacity of delphinidin (10 μg/ml) in increasing NO production was blunted when ER-alpha was inhibited either pharmacologically with fulvestrant or after depletion of ER-alpha receptor with siRNA. Western blot analysis showed that 17-beta estradiol, PPT and delphinidin increased interaction between ER-alpha, caveolin-1 (Cav-1) and c-Src, and enhanced phosphorylation of Cav-1, c-Src, P42/44 MAPkinase and endothelial NO synthase (eNOS) in endothelial cells. Antagonizing ER-alpha with fulvestrant or depletion of endogenous ER-alpha with siRNA completely abolished 17-beta estradiol-, PPT- and delphinidininduced phosphorylation of Cav-1, c-Src, P42/44 MAPkinase and eNOS. Finally, delphinidin induced 73 % inhibition of specific binding of ER-alpha agonist fluoligand using in vitro human ER-alpha receptor binding assay. Altogether, we provide direct evidence that delphinidin activates ER-alpha receptor and stimulates the interaction of this receptor with Cav-1, c-Src, P42/44 MAPkinase and eNOS to induce NO production in endothelial cells. Thus, ERalpha activation is probably involved in the therapeutic benefit of delphinidin in cardiovascular diseases associated with endothelial dysfunction.