Mechanotransduction Pathways of Endothelial Barrier Regulation

内衬肺血管系统的内皮细胞（EC）屏障的破坏是一种中心病理生理学 炎性肺损伤综合征事件，并导致临床显著氧合 危重病人的精神错乱项目#2的目标是机械地表征链接 关键的外周EC结构（动态皮质肌动蛋白、脂筏、紧密连接、粘附连接）之间 以及对调节肺EC屏障功能至关重要的细胞骨架元件。EC细胞骨架是一种 一系列复杂的蛋白质，密切参与调节EC屏障所必需的细胞形状变化 功能我们已经确定了多个细胞骨架连接蛋白和效应蛋白在细胞凋亡中的重要作用。 通过调节这些EC细胞骨架重排来调节血管通透性：MLCK， 角质蛋白辅肌动蛋白根蛋白ZO-1为了评估这些细胞骨架元素在外周的重要性， 细胞，具体目标#1将快速定义动态皮质肌动蛋白丝的结构和调节 响应于多种屏障增强刺激而在EC周边聚合。具体目标#2 严格表征细胞骨架与膜相关脂筏的连接，富含胆固醇的 质膜内的微区，其是跨膜浓缩的重要位点 受体并将其信号转导到细胞中。外周细胞-细胞接触沿着EC单层是 屏障维持的基本组成部分，作为连接肌动蛋白细胞骨架， 机械稳定性以及将细胞外信号转导到细胞中。在EC中，这些细胞间 接触主要由两种类型的连接复合物组成：紧密连接（具体目标#3的重点） 和粘附连接（具体目标#4的重点），其细胞骨架连接将被精确定义。 该项目将利用生物化学，分子和细胞生物学方法，小鼠模型，翻译 基因组技术和高度新颖的原子力显微镜方法， 表征通过细胞骨架接头/效应蛋白调节关键外周结构。通过 机械表征关键膜细胞骨架激活剂，关键细胞骨架效应物， 在这些过程中受影响的连接靶点，我们希望为开发有效的 肺血管通透性功能障碍和急性淋巴细胞白血病的治疗干预。

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.