Endothelin-1 enhances oxidative stress, cell proliferation and reduces apoptosis in human umbilical vein endothelial cells: role of ETB receptor, NADPH oxidase and caveolin-1.
1 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 microM) or the NADPH oxidase inhibitor apocynin (1 microM). 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, IkappaB, caveolin-1 and eNOS was evaluated by Western blot analysis. 2 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. 3 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.
Dong
Feng
F
Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071-3375, USA.
Zhang
Xiaochun
X
Wold
Loren E
LE
Ren
Qun
Q
Zhang
Zhaojie
Z
Ren
Jun
J
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D019255:NADPH Oxidases / Q000502:physiology*; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D044023:Receptor, Endothelin B / Q000819:agonists / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D019332:Endothelin-1; D044023:Receptor, Endothelin B; D019255:NADPH Oxidases