Date Published: January 1, 2010
Publisher: Public Library of Science
Author(s): Matthieu Chalopin, Angela Tesse, Maria Carmen Martínez, Didier Rognan, Jean-François Arnal, Ramaroson Andriantsitohaina, Carlo Polidori. http://doi.org/10.1371/journal.pone.0008554
Abstract: A greater reduction in cardiovascular risk and vascular protection associated with diet rich in polyphenols are generally accepted; however, the molecular targets for polyphenols effects remain unknown. Meanwhile evidences in the literature have enlightened, not only structural similarities between estrogens and polyphenols known as phytoestrogens, but also in their vascular effects. We hypothesized that alpha isoform of estrogen receptor (ERα) could be involved in the transduction of the vascular benefits of polyphenols.
Partial Text: Epidemiological studies have enlightened that women have lower cardiovascular risk than men, and this protection progressively disappears after menopause. These studies (protection in premenopausal women) suggest and experimental studies (prevention of atheroma development in animals) demonstrate a major atheroprotective action of 17-β-estradiol (E2) , . E2 actions are essentially mediated by two molecular targets: estrogen receptor alpha (ERα) and beta (ERβ), but the former appears to mediate most of the actions of E2 on the cardiovascular system , . Endothelium represents a well recognized target of E2, which elicits several beneficial actions as increased NO production – subsequent to activation of endothelial NO synthase (eNOS) via a G-protein , and ERK and phosphatidylinositol-3-kinase pathways.
The role of ERα in the endothelium-dependent relaxation to Provinols™ and to delphinidin was evaluated by using vessels taken from ERα WT and KO mice. First, we tested the ability of ERα agonists such as E2, which acts on both ERα and ERβ isoforms, and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), which is specific for ERα, to activate the endothelium. This was demonstrated by the capacity of the two ERα agonists to induce relaxation in aortas from ERα WT but not from KO mice in the presence of functional endothelium only (Figure 1A and 1B). The concentration of E2 to elicit maximal relaxation was in accordance with that reported by Li et al in the same vessels. As previously described by our group, red wine polyphenols and delphinidin are able to induce endothelium-dependent relaxation in mice aortas. Interestingly, the vasorelaxant effect of these two polyphenols was found in aortas from ERα WT mice (Figure 1C and 1D), but was completely abolished when ERα is deleted. In ERα deficient mice, a slight contraction to Provinols™ and delphinidin were even detected (Figure 1C and 1D).
The present study identifies ERα as the, or at least one of, key receptor transducing vascular effects exerted by red wine polyphenols, particularly delphinidin with respect to NO production. Indeed, E2 and PPT, as well as Provinols™ and delphinidin, are able to activate molecular pathways, involving Src, ERK1/2, eNOS and caveolin-1 phosphorylations, by a mechanism that required ERα activation, with subsequent increase of endothelial NO production and endothelium-dependent vascular relaxation. Moreover, using a binding assay and a docking, we showed that delphinidin fits on ERα’s activation site. Most importantly, evidence is provided that ERα triggers the in vivo effects of Provinols™ with respect to improvement in endothelial function given by the concomitant increase in NO and decrease in O2− superoxide anions releases in vessels. The later demonstrate for the first time the physiological relevance of this receptor in triggering the vascular protection induced by red wine polyphenols.
Provinols™ was obtained from Société Française des Distilleries (Vallon Pont d’Arc, France) and delphinidin was purchased from Extrasynthèse (Genay, France). The university of Angers ethical committee approved the present protocol. All animal studies were carried out using approved institutional protocols and were conformed the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Methods for vascular reactivity performed in mice ,  and endothelial cells extraction and culture were set up as previously described . Methods for RNA interference and transient transfection to silence ERα were adapted from Agouni et al.. NO and O2− spin trapping and electronic paramagnetic resonance (EPR) studies and Western blotting were conducted as previously described . Binding assay was performed by CEREP (Paris, France) using fluorescence polarization methods in human recombinant Sf9. Delphinidin was docked on ERα using default settings of the GOLD4.0 program . Additional details of the methods used are provided in the Supplemental Data file (Methods S1).