Endocrine Abstracts (2006) 11 P364

Anti-oxidative effect of 17beta-estradiol in human endothelial cells: the role of Bcl-2

W Zhong & SL Atkin


University of Hull, Hull, North Yorkshire, United Kingdom.


Aim: To determine if the endogenous estrogen 17beta-estradiol protected against oxidative stress-induced endothelial cell damage and the mechanism of this potential effect.

Methods: Human umbilical vein endothelial cells were isolated and cultured in phenol red free media with 2% charcoal-stripped serum. The following were undertaken (1) Cells were exposed to 0.1 to 1 nM 17beta-estradiol immediately prior to 100 μM hydrogen peroxide added for 24 hours, with and without the anti-estrogen ICI182,780. Cell proliferation, apoptotic DNA fragmentation and expression of Bcl-2 and Bax were subsequently measured by tritiated thymidine incorporation assay, Cell Death ELISA and Taqman quantitative PCR, respectively. (2) Cell Bcl-2 expression in response to 17beta-estradiol treatment was determined by Taqman quantitative PCR and western blotting. (3) Cells with or without Bcl-2 siRNA transfection (Bcl-2 knockdown) were exposed to 17beta-estradiol and hydrogen peroxide, and cell DNA fragmentation was then detected. The outcome of the Bcl-2 gene knockdown was confirmed by Taqman quantitative PCR and western blotting.

Results: Hydrogen peroxide significantly reduced cell proliferation, increased cell apoptotic DNA fragmentation level and caused reduced mRNA expression of Bcl-2 but elevated Bax. All of these were significantly protected against by 17beta-estradiol but reversed by the anti-estrogen ICI182780. 17beta-estradiol dramatically increased Bcl-2 expression at both mRNA and protein levels. Bcl-2 expression was silenced by the siRNA transfection, and within these cells, the protective effect of 17beta-estradiol on hydrogen peroxide induced apoptosis was completely lost.

Conclusion: 17beta-estradiol had protective effect against oxidative stress induced cell proliferation inhibition and cell apoptosis in human endothelial cells in vitro, mediated through a Bcl-2 dependent pathway.

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