Received for publication November 9, 2007.
Revised March 17, 2008.
Accepted for publication March 18, 2008.

Mitochondrial effects of estrogen are mediated by ER in brain endothelial cells

Abstract

Mitochondrial reactive oxygen species (ROS) and endothelial dysfunction are key contributors to cerebrovascular pathophysiology. Previously, we found that 17-estradiol profoundly impacts mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress. To determine if estrogen specifically affects endothelial mitochondria through receptor mechanisms, we used cultured human brain microvascular endothelial cells (HBMEC). 17-estradiol treatment for 24 hours increased mitochondrial cytochrome c protein and mRNA; use of silencing RNA for estrogen receptors (ER) showed that this effect involved ER, but not ER. Mitochondrial ROS were determined by measuring the activity of aconitase, an enzyme with an iron-sulfur center inactivated by mitochondrial superoxide. 17-Estradiol increased mitochondrial aconitase activity in HBMEC, indicating a reduction in ROS. Direct measurement of mitochondrial superoxide with MitoSOX Red showed that 17-estradiol, but not 17-estradiol, significantly decreased mitochondrial superoxide production, an effect blocked by the ER antagonist, ICI-182,780. Selective ER agonists demonstrated that the decrease in mitochondrial superoxide was mediated by ER, not ER. The selective estrogen receptor modulators, raloxifene and 4-hydroxy tamoxifen, differentially affected mitochondrial superoxide production, with raloxifene acting as an agonist but 4-hydroxy-tamoxifen acting as an estrogen antagonist. Changes in superoxide by 17-estradiol could not be explained by changes in manganese superoxide dismutase. Instead, ER-mediated decreases in mitochondrial ROS may depend on the concomitant increase in mitochondrial cytochrome c, previously shown to act as an antioxidant. Mitochondrial protective effects of estrogen in cerebral endothelium may contribute to sex differences in the occurrence of stroke and other age-related neurodegenerative diseases.

Key words: Aconitase activity, Cerebral Circulation, Cytochrome c, Estrogen Receptors, Reactive Oxygen Species, Selective estrogen receptor modulators