Src Regulation of Lung Endothelial Barrier Function

内皮屏障功能的丧失是急性肺损伤（AL 1）的重要特征。白蛋白和其他大分子通过内皮小窝的跨细胞转运是促进内皮屏障功能的因素。我们已经确定了小窝蛋白-1（小窝蛋白），异源三聚体G蛋白Gi和Src激酶之间的特异性相互作用的小窝介导的内吞作用的机制。项目4的目标是确定 小窝蛋白-1作为质膜小泡运输和蛋白质-蛋白质相互作用所必需的信号转导级联的组织者和调节剂，通过转胞吞作用调节白蛋白渗透性。项目4的研究将涉及以下具体目标。具体目标1：确定异源三聚体G蛋白Gi在内皮细胞单层中信号传导小窝介导的内吞作用和跨内皮白蛋白渗透性中的作用;具体目标#2：确定GT3、发动蛋白-2的Src活化在信号传导小窝介导的内吞作用和跨内皮白蛋白渗透性中的作用;具体目标#3：为了解决凝血酶/蛋白酶激活受体-1的组分，诱导肺血管通透性增加，这是由于小窝的内化和跨细胞膜白蛋白转运所致。因此，项目4将鉴定激活Src的受体偶联信号，Src信号传导小窝分裂的磷酸化靶点（具体地，小窝蛋白-1和 发动蛋白-2），以及Src活化在调节跨细胞渗透性中的作用。为了说明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，还将尽可能在完整小鼠肺模型中进行实验。研究将采用两种成像方法（即，使用荧光探针和电子显微镜评估）和生理学（即，内皮通透性测定 在单层和小鼠肺模型中）以解决小窝介导的内吞作用在激活增加的白蛋白渗透性中的作用。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传导机制，从而有助于肺中白蛋白跨内皮渗透的机制。这些目标的实现将有助于阐明调节小窝介导的细胞凋亡的信号。 内吞作用及其在促进凝血酶诱导的肺血管通透性增加中的作用。随着新信号通路的鉴定，有可能开发出特异性靶向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.