Vascular Reactivity: Gender and Hormonal Influence

描述（由申请人提供）：线粒体功能障碍已被认为是与年龄相关的疾病的原因；然而，关于线粒体在血管疾病中的作用所知甚少。我们最近发现雌激素(E)对脑血管线粒体有有益作用，可能有助于血管保护。因此，我们的总体假设是：E对线粒体功能的影响可以防止随年龄增长的脑血管内皮功能障碍。在这一建议中，E调节脑血管线粒体的机制和后果将在体外和体内被确定。培养的人脑内皮细胞将用于前两个目的。假设1:E提高脑内皮线粒体氧化磷酸化效率，降低氧化应激。在正常条件下和缺血培养模型中，E处理后将测量线粒体功能、线粒体酶水平和活性、线粒体生物发生和活性氧（ROS）产生的关键指标。E受体（erα和erβ）的参与将使用选择性siRNA敲除、拮抗剂、激动剂和选择性ER调节剂（serm）进行测试。假设2:E通过PGC-1转录辅激活因子调控线粒体功能。我们将确定E对线粒体相关转录因子（包括PGC-1家族）水平的影响以及er的作用。PGC-1¿在线粒体E效应中的具体参与将使用PGC-1¿的siRNA下调进行测试。ER-PGC-1害怕

DESCRIPTION (provided by applicant): Mitochondrial dysfunction has been implicated as a cause of age-related disorders; however little is known regarding the role of mitochondria in vascular disease. We recently found that estrogen (E) has beneficial effects on cerebrovascular mitochondria that likely contribute to vasoprotection. Thus, our overall hypothesis is: Effects of E on mitochondrial function protect against cerebrovascular endothelial dysfunction with age. In this proposal, mechanisms and consequences of E modulation of cerebrovascular mitochondria will be determined both in vitro and in vivo. Cultured human brain endothelial cells will be used in the first two aims. HYPOTHESIS 1: E increases the efficiency of oxidative phosphorylation and decreases oxidative stress in cerebral endothelial mitochondria. Key indices of mitochondrial function, mitochondrial enzyme levels and activities, mitochondrial biogenesis, and production of reactive oxygen species (ROS) will be measured after E treatment in normal conditions and in culture models of ischemia. The involvement of E receptors (ERalpha and ERbeta) will be tested using selective siRNA knockdown, antagonists, agonists and selective ER modulators (SERMs). HYPOTHESIS 2: E regulates mitochondrial function via PGC-1 transcriptional coactivators. We will determine effects of E, and the role of ERs, on levels of mitochondria-related transcription factors, including the PGC-1 family. Specific involvement of PGC-1¿ in mitochondrial effects of E will be tested using siRNA downregulation of PGC-1¿. ER-PGC-1¿ interactions will be tested by immunoprecipitation. In aim 3, a transgenic mouse model (MnSOD) will be used to test HYPOTHESIS 3: In vivo E protects cerebrovascular mitochondria and decreases endothelial dysfunction with advancing age. Cerebral blood vessels will be isolated from intact, ovariectomized, and E-treated females of different ages to compare mitochondrial enzymes, ATP and ROS production, and damage to mitochondrial iron/sulphur- containing enzymes and mtDNA. Endothelial function will be assessed in pressurized segments of isolated middle cerebral arteries. This project will delineate novel ways in which estrogen and estrogen-related therapies modulate the cerebral vascular system. These studies may provide insight into the role of mitochondria in cerebrovascular dysfunction, a contributor to age-related disorders such as stroke and vascular dementia.