Regulation of eNOS by Shear Stress in Intact Arteries

描述(申请人提供)：这个项目的目标是扩大我们对血管壁细胞如何传递物理力剪应力的理解。切应力是血液流过管腔而施加在血管壁上的摩擦力，是导致血管内径急剧变化和慢性结构重建的主要刺激因素。这些过程对于确保适当的血液流向组织是至关重要的，这对人类健康至关重要。值得注意的是，血管壁中的细胞如何将剪应力转化为扩张反应或结构重塑尚不清楚。因此，该项目的总体目标是揭示控制血管对剪切力的反应的关键分子信号事件，重点是内皮型一氧化氮合酶(ENOS)。ENOS是血管扩张和血管重塑的关键酶。揭示其在剪切应力过程中的调节机制将通过两个具体目标实现。目的1是通过研究eNOS(Tyr83，Ser116，Thr497，Ser617，Ser635，Tyr657，Ser1179)上的关键调节磷酸化位点，上游激酶的作用，蛋白质-蛋白质相互作用(小窝蛋白-1，热休克蛋白90)，以及伴随的直径变化，通过分离、插管、灌流的小鼠动脉制备和视频显微镜，确定eNOS是如何在完整的动脉中受到剪切力的调节的。动脉(肠系膜动脉和颈动脉)将在连续的时间点受到不同程度的剪应力(有或没有特定的激酶抑制剂)，并使用免疫印迹和免疫沉淀分析磷酸化和蛋白质相互作用。这些实验将阐明这种酶在剪应力诱导的扩张过程中是如何调节的。目的2是通过检测eNOS的磷酸化和重塑过程中的蛋白质-蛋白质相互作用，确定eNOS在正常动脉慢性剪应力升高过程中是如何被调节的。这将被测量：1)在体外使用一种创新的方法，在慢性灌流系统中培养几天分离的插管的小鼠动脉，以及2)在体内使用手术结扎模型来慢性增加小鼠的颈外动脉和肠系膜小动脉的流量。这些实验将深入了解eNOS在慢性剪应力诱导的重塑过程中何时以及如何被激活，这一点目前尚不清楚。 与公共卫生相关：该项目将在分子水平上研究动脉如何对血液流经动脉所产生的体力做出反应。这种力量既影响动脉的结构，也影响动脉的健康。健康的动脉对人类的整体健康至关重要，因为当它们不健康时，会导致动脉粥样硬化和心脏病。

DESCRIPTION (provided by applicant): The goal of this project is to expand our understanding of how cells in the blood vessel wall transduce the physical force shear stress. Shear stress is the frictional force exerted on the vessel wall due to the flow of blood through the lumen, and is the primary stimulus that governs both acute changes in blood vessel diameter and chronic structural remodeling. These processes are critically important for ensuring appropriate blood flow to tissues, which is essential for human health. Remarkably, how the cells in the vessel wall transduce the shear stress into a dilation response or structural remodeling is not clearly understood. Therefore, the overall objective of this project is to uncover key molecular signaling events that control the vascular response to shear stress, with a focus on endothelial nitric oxide synthase (eNOS). eNOS is a key enzyme critical to blood vessel dilation and vascular remodeling. Uncovering its regulatory mechanisms during shear stress will be achieved through two specific aims. Aim 1 is to determine how eNOS is regulated acutely by shear stress in intact arteries, through examining key regulatory phosphorylation sites on eNOS (Tyr83, Ser116, Thr497, Ser617, Ser635, Tyr657, Ser1179), the role of upstream kinases, protein-protein interactions (caveolin-1, Hsp90), and concomitant diameter changes, using an isolated, cannulated, perfused mouse artery preparation and video microscopy. Arteries (mesenteric and carotid arteries) will be subjected to various magnitudes of shear stress (with or without specific kinase inhibitors), at sequential time-points, and analyzed for phosphorylation and protein-protein interaction using immunoblotting and immunoprecipitation. These experiments will illuminate how this enzyme is regulated during shear stress-induced dilation. Aim 2 is to determine how eNOS is regulated during chronically elevated shear stress in intact arteries, by examining eNOS phosphorylation and protein-protein interactions during the remodeling process. This will be measured: 1) in vitro using an innovative method in which isolated, cannulated mouse arteries are cultured for several days in a chronic perfusion system, and 2) in vivo using a surgical ligation model to chronically increase flow in the external carotid artery and the small mesenteric arteries of a mouse. These experiments will provide insight into when and how eNOS is activated during chronic shear stress-induced remodeling, which is currently unknown. PUBLIC HEALTH RELEVANCE: This project will examine, at the molecular level, how arteries respond to the physical force produced by blood flowing through the artery. This force influences both the structure and the health of the artery. Healthy arteries are critical to overall human health because when they are unhealthy, atherosclerosis and heart disease results.