Mechanotransduction Pathways of Endothelial Barrier Regulation

肺血管内皮细胞(EC)屏障的破坏是一种中枢病理生理学 炎症性肺损伤综合征中的事件和临床上显著的氧合 危重病人的精神错乱。项目2的目标是从机械上描述连杆机构 关键的外周EC结构之间(动态皮质肌动蛋白、脂筏、紧密连接、粘连连接) 以及对调节肺内皮细胞屏障功能起关键作用的细胞骨架元件。EC细胞骨架是一种 与调节EC屏障所需的细胞形状变化密切相关的复杂蛋白质阵列 功能。我们已经确定了多个细胞骨架连接物和效应器蛋白在 通过调节这些EC细胞骨架重排来调节血管通透性：MLCK， 皮质素、肌动蛋白、放射素、ZO-1。为了评估这些细胞骨架元素在心脏周围的重要性 细胞，特殊目标#1将快速定义动态皮质肌动蛋白细丝的结构和调节 在EC外围聚合，以响应多种屏障增强刺激。具体目标#2将 严格表征细胞骨架与富含胆固醇的膜相关脂筏的连接 质膜内的微区是浓缩跨膜的重要部位 并将它们的信号传递到细胞内。沿EC单层的外围细胞-细胞接触为 屏障维持的基本成分，作为与肌动蛋白细胞骨架的联系，提供两者 机械稳定性以及细胞外信号向细胞内的传递。在EC中，这些细胞间 接触主要由两种类型的连接复合体组成？紧密连接(具体目标3的重点) 并坚持连接(具体目标4的重点)，其细胞骨架连接将被精确定义。 该项目将利用生化、分子和细胞生物学方法、小鼠模型、翻译 基因组技术，以及高度新颖的原子力显微镜方法 表征细胞骨架连接物/效应器蛋白对关键外周结构的调节。通过 对关键的膜细胞骨架激活剂、关键的细胞骨架效应器和 在这些过程中受影响的结合点目标，我们期望能为制定有效的 肺血管通透性功能障碍和ALL的治疗干预。

Disruption of the endothelial cell (EC) barrier that lines the pulmonary vasculature is a central pathophysiologic event in inflammatory lung injury syndromes and results in clinically significant oxygenation derangements in critically ill patients. The goal of Project #2 is to mechanistically characterize linkage between key peripheral EC structures (dynamic cortical actin, lipid rafts, tight junctions, adherens junctions) and the cytoskeletal elements critical to the regulation of lung EC barrier function. The EC cytoskeleton is a complex array of proteins intimately involved in the cell shape changes necessary for regulation of EC barrier function. We have identified essential roles for multiple cytoskeletal linker and effector proteins in the regulation of vascular permeability through modulation of these EC cytoskeletal rearrangements: MLCK, cortactin, actinin, radixin, ZO-1. To assess the importance of these cytoskeletal elements at the periphery of the cell, Specific Aim #1 will define the structure and the regulation of dynamic cortical actin filaments rapidly polymerized at the EC periphery in response to multiple barrier enhancing stimuli. Specific Aim #2 will rigorously characterize the cytoskeletal linkage to membrane-associated lipid rafts, cholesterol-enriched microdomains within the plasma membrane that are important sites for concentrating transmembrane receptors and transducing their signals into the cell. Peripheral cell-cell contacts along the EC monolayer are essential components of barrier maintenance that serve as linkages to the actin cytoskeleton to provide both mechanical stability as well as transduction of extracellular signals into the cell. In the EC, these intercellular contacts consist primarily of two types of junctional complexes¿tight junctions (the focus of Specific Aim #3) and adherens junctions (the focus of Specific Aim #4),whose cytoskeletal linkages will be precisely defined. This project will utilize biochemical, molecular, and cell biology approaches, murine models, translational genomic techniques, and highly novel atomic force microscopy approaches to focus intensely on characterizing the regulation of key peripheral structures by cytoskeletal linker/effector proteins. By mechanistically characterizing key membrane cytoskeletal activators, key cytoskeletal effectors, and the junctional targets affected in these processes, we expect to provide a basis for development of effective therapeutic interventions for pulmonary vascular permeability dysfunction and All.