Razmara Ali, Sunday Lorraine, Stirone Chris, Wang Xiao Bo, Krause Diana N, Duckles Sue P, Procaccio Vincent
Department of Pharmacology, School of Medicine, University of California, Irvine, CA 92697-4625, USA.
J Pharmacol Exp Ther. 2008 Jun;325(3):782-90. doi: 10.1124/jpet.107.134072. Epub 2008 Mar 19.
Mitochondrial reactive oxygen species (ROS) and endothelial dysfunction are key contributors to cerebrovascular pathophysiology. We previously found that 17beta-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). 17beta-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 ERalpha, but not ERbeta. Mitochondrial ROS were determined by measuring the activity of aconitase, an enzyme with an iron-sulfur center inactivated by mitochondrial superoxide. 17beta-Estradiol increased mitochondrial aconitase activity in HBMECs, indicating a reduction in ROS. Direct measurement of mitochondrial superoxide with MitoSOX Red showed that 17beta-estradiol, but not 17alpha-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 ERalpha, not ERbeta. 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 17beta-estradiol could not be explained by changes in manganese superoxide dismutase. Instead, ERalpha-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.
线粒体活性氧（ROS）和内皮功能障碍是脑血管病理生理学的关键因素。我们之前发现，17β-雌二醇对脑血管中的线粒体功能有深远影响，可提高能量产生效率并抑制线粒体氧化应激。为了确定雌激素是否通过受体机制特异性影响内皮线粒体，我们使用了培养的人脑微血管内皮细胞（HBMECs）。17β-雌二醇处理24小时可增加线粒体细胞色素c蛋白和mRNA；使用雌激素受体（ERs）的沉默RNA表明，这种作用涉及ERα，而非ERβ。通过测量乌头酸酶的活性来测定线粒体ROS，乌头酸酶是一种具有铁硫中心的酶，会被线粒体超氧化物灭活。17β-雌二醇可增加HBMECs中线粒体乌头酸酶的活性，表明ROS减少。用MitoSOX Red直接测量线粒体超氧化物表明，17β-雌二醇而非17α-雌二醇可显著降低线粒体超氧化物的产生，ER拮抗剂ICI-182,780（氟维司群）可阻断这一作用。选择性ER激动剂表明，线粒体超氧化物的减少是由ERα介导的，而非ERβ。选择性雌激素受体调节剂雷洛昔芬和4-羟基他莫昔芬对线粒体超氧化物的产生有不同影响，雷洛昔芬起激动剂作用，而4-羟基他莫昔芬起雌激素拮抗剂作用。17β-雌二醇引起的超氧化物变化无法用锰超氧化物歧化酶的变化来解释。相反，ERα介导的线粒体ROS减少可能取决于线粒体细胞色素c的同时增加，之前已证明细胞色素c可作为抗氧化剂。雌激素对脑内皮的线粒体保护作用可能导致中风和其他与年龄相关的神经退行性疾病发生中的性别差异。
