Endothelin-1 enhances oxidative stress, cell proliferation and reduces apoptosis in human umbilical vein endothelial cells: Role of ET B receptor, NADPH oxidase and caveolin-1

Abstract

Endothelin-1 (ET-1), an endothelium-derived vasoactive peptide, participates in the regulation of endothelial function through mechanisms that are not fully elucidated. This study examined the impact of ET-1 on oxidative stress, apoptosis and cell proliferation in human umbilical vein endothelial cells (HUVEC). HUVECs were challenged for 24 h with ET-1 (10 pM-10 nM) in the absence or presence of the ET B receptor antagonist BQ788 (1 μM) or the NADPH oxidase inhibitor apocynin (1 μM). Reactive oxygen species (ROS) were detected using chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated with 4′,6′-diamidino-2′- phenylindoladihydrochloride staining and by the caspase-3 assay. Cell proliferation was measured by a colorimetric assay. Expression of NADPH oxidase, Akt, pAkt, Bcl-2, Bax, IκB, caveolin-1 and eNOS was evaluated by Western blot analysis. ET-1 significantly enhanced ROS generation and cell proliferation following 24-h incubation, both of which were prevented by BQ788 or apocynin, consistent with the ability of ET-1 to directly upregulate NADPH oxidase. ET-1 itself did not affect apoptosis but attenuated homocysteine-induced apoptosis through an ET B receptor-mediated mechanism. Western blot analysis indicated that ET-1 alleviated homocysteine (Hcy)-induced apoptosis, likely acting by antagonizing the Hcy-induced decreases in Akt, pAkt, pAkt-to-Akt, Bcl-2-to-Bax ratios and increases in Bax and caveolin-1 expression. Furthermore, ET-1 downregulated expression of caveolin-1 and eNOS, which was attenuated by BQ788 or apocynin. In summary, our results suggest that ET-1 affects oxidative stress, proliferation and apoptosis possibly through ET B, NADPH oxidase, Akt, Bax and caveolin-1-mediated mechanisms. © 2005 Nature Publishing Group. All rights reserved.