Src Regulation of Lung Endothelial Barrier Function

内皮屏障功能丧失是急性肺损伤(ALL)的重要特征。白蛋白和其他大分子通过内皮细胞小窝的跨细胞运输是促进内皮屏障功能的一个因素。我们已经确定了小窝蛋白-1(一种小窝蛋白)、异源三聚体G蛋白Gi和Src激酶之间在小窝介导的内吞作用机制中的特定相互作用。项目4的目标是定义 Caveolin-1作为信号转导通路的组织者和调节者，对质膜囊泡运输和通过跨细胞作用调节白蛋白通透性的蛋白质-蛋白质相互作用是必不可少的。项目4中的研究将涉及以下具体目标。具体目的#1：确定异三聚体G蛋白Gi在血管内皮细胞单层介导的内吞和跨内皮白蛋白通透性信号中的作用；特殊目的#2：确定GTPase的Src激活在信号转导小窝介导的内吞和跨内皮白蛋白通透性中的作用；特殊目的#3：研究凝血酶/蛋白酶激活的受体-1诱导的肺血管通透性增加的成分，这是由于小窝内化和跨细胞白蛋白运输引起的。因此，项目4将确定激活Src的受体偶联信号，Src信号小窝分裂的磷酸化目标(具体地说，小窝-1和 Dynamin-2)，以及Src激活在调节跨细胞通透性中的作用。为了解决这些研究在肺微血管内皮细胞中的体内相关性和功能意义，还将尽可能在完整的小鼠肺模型中进行实验。研究将在成像(即使用荧光探针和电子显微镜评估)和生理学(即确定内皮通透性)两个方面采用方法 在单层和小鼠肺模型中)，以探讨小窝介导的内吞作用在激活增加的白蛋白通透性中的作用。因此，这些研究将阐明调节小窝内化和质膜小泡运输的信号机制，从而有助于肺内皮细胞白蛋白通透性的机制。这些目标的实现将导致阐明调节小窝介导的信号。 内吞作用及其在凝血酶诱导的肺血管通透性增加中的作用。随着新的信号通路的识别，有可能开发专门针对导致肺血管通透性不适当增加的信号的治疗策略。

Loss of endothelial barrier function is an important characteristic of Acute Lung Injury (ALl). The transcellular transport of albumin and other macromolecules via endothelial caveolae is a factor contributing to endothelial barrier function. We have identified specific interactions between caveolin-1 (a caveolar protein), the heterotrimeric G protein Gi, and Src kinase in the mechanism of caveolae-mediated endocytosis. The goals of Project 4 are to define the role of caveolin-1 as an organizer and regulator of signal transduction cascades essential for plasmatemmal vesicle trafficking and the protein-protein interactions that regulate albumin permeability via transcytosis. The studies in Project 4 will address the following specific aims. Specific Aim #1: To determine the role of the heterotrimeric G protein, Gi, in signaling ceaveolae-mediated endocytosis and transendothelial albumin permeability in endothelial monolayers; Specific Aim #2: To determine the role of Src activation of the GTPase, dynamin-2, in signaling caveolae-mediated endocytosis and transendothelial albumin permeability; Specific Aim #3:To address the component of thrombin/Protease Activated Receptor-1-induced increase in lung vascular permeability resulting from internalization of caveolae and transcelinlar albumin transport. Thus, Project 4 will identify the receptor-coupled signals activating Src, the phosphorylation targets of Src signaling caveolae fission (specifically, caveolin-1 and dynamin-2), and the role of Src activation in regulating transcellular permeability. To address the in vivo relevance and functional significance of these studies in pulmonary microvascular endothelial cells, experiments will also be made, wherever possible, in intact mouse lung models. Studies will employ approaches in both imaging (i.e., using fluorescent probes and electron microscopic assessment) and physiology (i.e., determination of endothelial permeability in monoayers and mouse lung models) to address the role of caveolae-mediated endocytosis in activating increased albumin permeability. Thus, these studies will elucidate the signaling mechanisms that regulate caveolae internalization and plasmalemmal vesicle trafficking, and thus contribute to the mechanism of transendothelial albumin permeability in lungs. The achievement of these objectives will lead to tthe elucidation of the signals regulating caveolae-mediated endocytosis and its role in contributing to the thrombin-induced increase in lung vascular permeability. With the identification of novel signaling pathways, it may be possible to develop therapeutic strategies that specifically target signals leading to inappropriate increase in lung vascular permeability.