Vascular Reactivity: Gender and Hormonal Influence

描述(申请人提供)：线粒体功能障碍被认为是与年龄相关的疾病的原因之一；然而，关于线粒体在血管疾病中的作用，我们知之甚少。我们最近发现雌激素(E)对脑血管线粒体有有益的作用，这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保护脑血管内皮细胞功能不受年龄的影响。在这项建议中，将在体外和体内确定脑血管线粒体E调节的机制和后果。培养的人脑内皮细胞将用于前两个目标。假设1：E可提高脑内皮细胞线粒体氧化磷酸化效率，降低氧化应激。在正常条件下和培养的缺血模型中，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。将使用选择性siRNA敲除、拮抗剂、激动剂和选择性ER调节剂(SERM)来测试E受体(ERa和ERbeta)的参与。假设2：E通过PGC-1转录共激活因子调节线粒体功能。我们将确定E和ERs对线粒体相关转录因子水平的影响，包括PGC-1家族。PGC-1？在E的线粒体效应中的具体参与将通过siRNA下调PGC-1？来测试。ER-PGC-1？ 相互作用将通过免疫沉淀进行测试。在目的3中，将使用转基因小鼠模型(MnSOD)来检验假设3：体内E保护脑血管线粒体，并随着年龄的增加而减少内皮功能障碍。脑血管将从完整的、去卵巢的和E处理过的不同年龄的女性身上分离出来，以比较线粒体酶、ATP和ROS的产生，以及对线粒体含铁/硫酶和线粒体DNA的损伤。将对分离的大脑中动脉加压节段的内皮功能进行评估。这个项目将描绘雌激素和雌激素相关疗法调节脑血管系统的新方法。这些研究可能对线粒体在脑血管功能障碍中的作用提供洞察力，脑血管功能障碍是中风和血管性痴呆等与年龄相关的疾病的诱因。

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.