Role of NOX4 in Mitochondrial Oxidative Stress and Dysfunction: Protection by Estrogen.

摘要

This dissertation describes experiments that reveal a novel mechanism by which endothelial cells in blood vessels of the brain become damaged by oxidative stress: induction of NADPH oxidase (NOX) type 4 in the mitochondria. Moreover, I have shown that upregulation of NOX4 and mitochondrial superoxide can be suppressed by estrogen acting on estrogen receptor alpha (ER alpha). These results are important contributions to our understanding of underlying mechanisms of vascular disease and potential treatments. Superoxide production in mitochondria is thought to be a critical factor in aging and age-related disorders such as stroke, and reactive oxygen species (ROS) are key contributors to cardiovascular pathophysiology. The primary generators of endothelial cell ROS are mitochondria and NOXs; however possible interactions between these two sources have been unclear. I hypothesized that angiotensin II (ANGII), a promoter of vascular dysfunction, would increase mitochondrial ROS and do so via its known ability to activate NOX enzymes. I found that ANGII treatment of mouse brain endothelial cells in culture (bEND.3 cells) caused a significant increase in mitochondrial superoxide as measured using live cell fluorescence imaging with Mitosox RED fluorogenic dye. ANGII also decreased mitochondrial membrane potential assessed using the fluorescent indicator TMRM (tetramethylrhodamine methyl). Both effects of ANGII were suppressed by NOX inhibitors VAS2879 and diphenylene iodonium chloride. Furthermore, silencing the gene for NOX4, but not the NOX1 or NOX2 isoforms, suppressed the ability of ANGII to increase mitochondrial superoxide. ANGII increased protein levels of NOX4, but not NOX1/NOX2. NOX4 was localized to the mitochondria, and NOX4 protein levels measured in isolated mitochondria were increased following ANGII treatment. Taken together, these data indicate that mitochondrial-associated NOX4 is a major contributor of mitochondrial superoxide during prolonged stimulation with ANGII. Because our laboratory has shown previously that 17 beta-estradiol suppresses mitochondrial superoxide production in brain endothelial cells, I tested the hypothesis that 17 beta-estradiol acts by inhibiting NOX4. Indeed, 17 beta-estradiol, but not its inactive isomer 17alpha-estradiol, suppressed increases in NOX4 protein and mitochondrial superoxide following stimulation by either ANGII or endothelin-1 in brain endothelial cells. These effects were blocked by the ER antagonist, ICI 182,780. In addition, gene silencing of either ER alpha or ER beta revealed that the effects of 17 beta-estradiol were ER alpha-mediated. The ability of estrogen to mitigate increases in NOX4 and mitochondrial superoxide likely contributes to the known protective effects of estrogen in animal models of cerebrovascular disease, such as stroke.