Regulation of eNOS by Shear Stress in Intact Arteries

描述（由申请人提供）：本项目的目标是扩大我们对血管壁细胞如何抵抗物理力剪切应力的理解。剪切应力是由于血液流过管腔而施加在血管壁上的摩擦力，并且是控制血管直径的急性变化和慢性结构重塑的主要刺激。这些过程对于确保适当的血液流向组织至关重要，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切应力转化为扩张反应或结构重塑尚不清楚。因此，本项目的总体目标是揭示控制血管对剪切应力的反应的关键分子信号传导事件，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的目标来实现。目的1是通过检测内皮型一氧化氮合酶（eNOS）上关键的磷酸化调节位点来确定内皮型一氧化氮合酶在完整动脉中是如何被切应力急性调节的（Tyr 83，Ser 116，Thr 497，Ser 617，Ser 635，Tyr 657，Ser 1179），上游激酶的作用，蛋白质-蛋白质相互作用（小窝蛋白-1，Hsp 90），以及伴随的直径变化，使用分离的、插管的、灌注的小鼠动脉制备物和视频显微镜。动脉（肠系膜动脉和颈动脉）将在连续的时间点经受不同大小的剪切应力（有或没有特异性激酶抑制剂），并使用免疫印迹和免疫沉淀法分析磷酸化和蛋白质-蛋白质相互作用。这些实验将阐明这种酶在剪切应力诱导的扩张过程中是如何调节的。目的2是确定如何eNOS的调节过程中慢性升高的剪切应力在完整的动脉，通过检查eNOS磷酸化和蛋白质-蛋白质相互作用在重塑过程中。这将被衡量：1）在体外使用创新方法，其中将分离的、插管的小鼠动脉在慢性灌注系统中培养数天，和2）在体内使用手术结扎模型以慢性增加小鼠的颈外动脉和小肠系膜动脉中的流量。这些实验将提供深入了解eNOS何时以及如何在慢性剪切应力诱导的重塑过程中被激活，这是目前未知的。 公共卫生相关性：该项目将在分子水平上研究动脉如何对流经动脉的血液产生的物理力作出反应。这种力影响动脉的结构和健康。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，就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.