Mitochondrial Biogenesis and Endothelial Cell Phenotype

说明(申请人提供)：内皮是正常血管稳态的重要组成部分，内皮功能障碍是血管疾病及其临床表现(如心脏病发作和中风)发展的前奏。内皮功能障碍被认为部分是由于已有的血管疾病危险因素如高胆固醇血症、高血压和糖尿病的损害行为所致。抵抗内皮功能障碍和损伤是对抗血管疾病的重要保护机制，因为内皮功能保留的患者不容易发生临床血管事件。然而，决定内皮功能障碍易感性或抵抗力的确切分子事件尚不清楚。本申请中提供的初步数据表明，代谢应激反应的诱导(通过AMP激酶激活)刺激线粒体的生物生成，并保护内皮免受损伤性刺激。此外，我们的数据将这种保护作用与抑制c-jun氨基末端激酶激活联系在一起--c-jun氨基末端激酶激活是环境应激反应的关键组成部分。因此，我们的中心假设是代谢应激反应和由此产生的线粒体生物发生的刺激是内皮细胞表型和对损伤性刺激抵抗力的关键决定因素。因此，这项应用的目的是确定代谢应激和线粒体生物发生调节内皮功能的分子机制(S)，并检验增加内皮细胞对功能障碍的抵抗力将改善血管疾病发展的假设。为了实现这一目标，我们将首先确定保护内皮免受损伤性刺激所需的代谢应激反应和线粒体生物发生的组成部分(S)。我们将使用药理学(AICAR，2-脱氧葡萄糖)和分子(PGC-11，RIP140)方法模拟代谢应激，并量化线粒体的生物发生。然后，我们将使用药理学(氯霉素)和分子(TFAM)工具将代谢应激反应与线粒体生物发生分离，并确定其对内皮保护的影响。然后我们将探讨已知的代谢靶点，如eNOS、FOXO和SIRT1在内皮应激抵抗中的作用。接下来，我们将确定代谢应激反应和线粒体生物发生减弱JNK激活的机制(S)。代谢应激和线粒体生物发生将被操纵，我们将通过研究重要的上游(MAP3K和MAP2K)激酶以及通过化学遗传学方法涉及的特定JNK亚型来研究JNK激活的意义。然后，我们将研究JNK失活的重要决定因素，如ROS和MAP激酶磷酸酶。最后，使用化学遗传学方法，我们将确定JNK调控的时间方面。最后，我们将在体内确定操纵内皮细胞线粒体生物发生对内皮功能障碍和血管疾病的影响。我们已经开发出工具来操纵内皮细胞PGC11，作为体内线粒体生物发生和质量的模型。利用这些动物，我们将确定内皮细胞PGC11对线粒体生物发生以及与高血压和动脉粥样硬化相关的功能障碍的质量和内皮抵抗的影响。上述实验应该为我们提供坚实的工作知识，了解线粒体的生物发生和增加的线粒体质量如何有助于控制内皮表型，以及这如何转化为体内的动态平衡反应。有了这些信息，我们应该有必要的洞察力来设计新的工具，以调节血管氧化还原状态和表型，着眼于血管疾病的治疗。

DESCRIPTION (provided by applicant): The endothelium is an important component of normal vascular homeostasis and endothelial dysfunction is a prelude to the development of vascular disease and its clinical manifestations such as heart attack and stroke. Endothelial dysfunction is thought to result, in part, from the injurious actions of established vascular disease risk factors such as hypercholesterolemia, hypertension, and diabetes. Resistance to endothelial dysfunction and injury is an important protective mechanism against vascular disease, as patients with preserved endothelial function are not predisposed to clinical vascular events. However, the precise molecular events that determine susceptibility or resistance to endothelial dysfunction are not known. Preliminary data presented in this application indicate that induction of a metabolic stress response (via AMP kinase activation) stimulates mitochondrial biogenesis and protects the endothelium from injurious stimuli. Furthermore, our data link this protective effect to suppression of c-Jun N-terminal kinase activation - a key component of the environmental stress response. Therefore, our central hypothesis is that the metabolic stress response and resultant stimulation of mitochondrial biogenesis are key determinants of endothelial cell phenotype and resistance to injurious stimuli. The objective of this application, therefore, is to determine the molecular mechanism(s) whereby metabolic stress and mitochondrial biogenesis modulate endothelial function and test the hypothesis that increased endothelial cell resistance to dysfunction will ameliorate the development of vascular disease. In order to achieve this objective, we will first determine the component(s) of the metabolic stress response and mitochondrial biogenesis required to protect the endothelium from injurious stimuli. We will model metabolic stress using pharmacologic (AICAR, 2-deoxyglucose) and molecular (PGC-11, RIP140) means and quantify mitochondrial biogenesis. We will then dissociate the metabolic stress response from mitochondrial biogenesis using pharmacologic (chloramphenicol) and molecular (Tfam) tools and determine the implications for endothelial protection. We will then probe the involvement of known metabolic targets such as eNOS, FOXOs, and SIRT1in endothelial stress resistance. Next we will determine the mechanism(s) whereby the metabolic stress response and mitochondrial biogenesis attenuates JNK activation. Metabolic stress and mitochondrial biogenesis will be manipulated and we will examine the implications for JNK activation by investigating important upstream (MAP3K and MAP2K) kinases as well as the specific JNK isoforms involved via a chemical genetic approach. We will then examine important determinants of JNK inactivation such as ROS and MAP kinase phosphatases. Finally, using a chemical genetic approach, we will determine temporal aspects of JNK regulation. Finally, we will determine the implications of manipulating endothelial cell mitochondrial biogenesis on endothelial dysfunction and vascular disease in vivo. We have developed tools to manipulate endothelial cell PGC11 as a model of mitochondrial biogenesis and mass in vivo. Using these animals, we will determine the implications of endothelial cell PGC11 on mitochondrial biogenesis and mass and endothelial resistance to the dysfunction associated with hypertension and atherosclerosis. The experiments outlined above should provide us with a solid working knowledge of how mitochondrial biogenesis and increased mitochondrial mass contributes to the control of endothelial phenotype and how this translates into homeostatic responses in vivo. With this information in hand, we should have the requisite insight to design new tools directed at modulating vascular redox status and phenotype with an eye toward the treatment of vascular disease.