O219	Role of endothelial NO synthase (eNOS) in the effect of 17β-estradiol (E2) in reendothelialisation and in the rapid plasma membrane-associated signalisation.
1A.Billon, 2S.Lehoux, 1V.Fontaine, 1C.Filipe, 1A.Schambourg, 1H.Laurell, 1P.Gourdy, 3A-P.Gadeau, 1J-F.Arnal
1INSERM U589, Toulouse, FR; 2INSERM U541, Paris, FR; 3INSERM U441, Pessac, FR.

Even though many cell types are E2 targets in the cardiovascular system, the main part of the vasculo-protective effect of E2 seems to occur at the level of the endothelium. We have developed an injury model of the mouse carotid artery. Using this model, we have shown that the reendothelialisation is accelerated by E2 and that this effect is dependent on the estrogen receptor alpha (ERα). Recently, the accelerating effect of E2 was found to be abolished in eNOS-deficient mice (eNOS-/-). We evaluated the influence of NOS inhibition (L-NAME) in this effect. Surprisingly, the pharmacological inhibition of eNOS did not interfere with action of E2 on reendothelialisation. These discordant results led us to hypothesize that eNOS could act as a docking-protein independently of its enzymatic activity, and that this may be crucial for eNOS implication in E2 signalling. ERα is not only a nuclear receptor with transactivating capacities, but can also mediates rapid membrane-associated effects at the level of caveolae, leading for instance to the activation of MAPK and PI3K-AKT. In a model of isolated and perfused carotid artery, we have found that E2 stimulates the phosphorylation of MAPK in wild type mice and that this effect is abolished in carotid artery from eNOS-deficient mice. In conclusion, this work suggests the existence of an original and unexpected function of the eNOS protein in estrogen signalling. Altogether, eNOS could act as a docking-protein in caveolae and mediate an ERα-dependent phosphorylation of MAPK. We are currently further testing this hypothesis in cultured endothelial cells by an siRNA-mediated approach.

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