Mitochondrial effects of estrogen are mediated by estrogen receptor α in brain endothelial cells

Mitochondrial reactive oxygen species (ROS) and endothelial dysfunction are key contributors to cerebrovascular pathophysiology. We previously found that 17 beta-estradiol profoundly affects mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress. To determine whether estrogen specifically affects endothelial mitochondria through receptor mechanisms, we used cultured human brain microvascular endothelial cells (HBMECs). 17 beta-Estradiol treatment for 24 h increased mitochondrial cytochrome c protein and mRNA; use of silencing RNA for estrogen receptors (ERs) showed that this effect involved ER alpha, but not ER beta. Mitochondrial ROS were determined by measuring the activity of aconitase, an enzyme with an iron-sulfur center inactivated by mitochondrial superoxide. 17 beta-Estradiol increased mitochondrial aconitase activity in HBMECs, indicating a reduction in ROS. Direct measurement of mitochondrial superoxide with MitoSOX Red showed that 17 beta-estradiol, but not 17 alpha-estradiol, significantly decreased mitochondrial superoxide production, an effect blocked by the ER antagonist, ICI-182,780 (fulvestrant). Selective ER agonists demonstrated that the decrease in mitochondrial superoxide was mediated by ER alpha, not ER beta. 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 beta-estradiol could not be explained by changes in manganese superoxide dismutase. Instead, ER alpha-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.