Focal Adhesion Kinase Regulation of Lung Vascular Permeability and Edemagenesis

描述（由申请方提供）：肺血管通透性增加导致急性呼吸窘迫综合征（ARDS）相关的发病率和死亡率。由一系列复杂的蛋白质维持的内皮粘附连接和细胞-基质粘附的动态组织对于建立内皮屏障功能至关重要。作为对炎症介质如凝血酶的响应，粘附连接和细胞-基质粘附由于RhoA诱导的细胞收缩而解体，从而产生内皮渗透性的快速增加。炎症介质引起的内皮通透性增加通常是一种可逆事件，是粘附连接和细胞-基质粘附重组的结果。然而，促进内皮屏障功能恢复的机制仍然知之甚少。粘着斑激酶（FAK）调节粘着斑部位的细胞-基质粘附接触。我们已经证明，抑制FAK导致凝血酶刺激后内皮通透性不可逆增加。重要的是，我们的支持数据表明，内皮通透性的不可逆增加是RhoA激活的结果。这些发现表明FAK在下调RhoA活性和重新退火粘附连接中的新作用，这是逆转水肿剂后内皮通透性增加所需的。在初步数据中，我们还确定了蛋白质p190 RhoGAP和N-WASP的关键功能，它们在被FAK激活后可以抑制RhoA并诱导粘附连接的重新组装。因此，我们将测试的假设，即FAK激活后，增加内皮通透性组装一个信号复合物，恢复内皮通透性。我们的具体目标是：（i）研究FAK在通过负调节RhoA活性并由此促进粘附连接和粘着斑的重新组装而重建内皮屏障功能中的作用，（ii）确定异源三聚体G蛋白的Gpy二聚体在调节FAK活化并由此恢复内皮屏障功能中的作用，（iii）鉴定抑制由FAK诱导的RhoA活性的机制及其在逆转增加的内皮渗透性反应中的功能作用，以及（iv）阐明FAK诱导的粘附连接再退火的机制及其在恢复内皮屏障功能中的作用。研究将在内皮细胞和从几种遗传小鼠模型分离的完整肺中进行，包括一种携带内皮细胞中FAK基因的条件性缺失的小鼠模型。我们将使用最先进的细胞成像技术，突变体结构的表达和siRNA诱导的基因下调来分析信号分子的作用。这些研究对于阐明内皮屏障功能的恢复机制和确定ARDS药物治疗的潜在新分子靶点具有重要意义。

DESCRIPTION (provided by applicant): Increased lung vascular permeability contributes to the morbidity and mortality associated with Acute Respiratory Distress Syndrome (ARDS). Dynamic organization of endothelial adherens junctions and cell- matrix adhesions maintained by a complex array of proteins is crucial for establishing endothelial barrier function. In response to inflammatory mediators such as thrombin adherens junctions and cell-matrix adhesions disassemble as a result of RhoA-induced cell contraction producing a rapid increase in endothelial permeability. This increase in endothelial permeability in response to inflammatory mediators is normally a reversible event occurring as a result of re-organization of adherens junctions and cell-matrix adhesions. However mechanisms, which promote the recovery of endothelial barrier function remain poorly understood. Focal adhesion kinase (FAK) regulates the cell-matrix adhesive contact at focal adhesion sites. We have demonstrated that inhibition of FAK resulted in an irreversible increase in endothelial permeability following thrombin challenge. Importantly, our supporting data show that the irreversible increase in endothelial permeability is the result of activation of RhoA. These findings indicate a novel role of FAK in down-modulating RhoA activity and re-annealing adherens junctions, required for reversing the increase in endothelial permeability after edemagenic agents. In Preliminary Data, we have also identified a crucial function of proteins, p190RhoGAP and N-WASP, which upon activation by FAK may inactivate RhoA and induce re-assembly of adherens junctions. Thus, we will test the hypothesis that FAK activation subsequent to increased endothelial permeability assembles a signaling complex, which restore endothelial permeability. Our Specific Aims are: (i) to investigate the role of FAK in re-establishing the endothelial barrier function by negatively regulating RhoA activity and thereby promoting reassembly of adherens junctions and focal adhesions, (ii) to determine the role of the Gpy dimer of the heterotrimeric G proteins in regulating FAK activation and thereby restoring endothelial barrier function, (iii) to identify the mechanisms of inhibition of RhoA activity induced by FAK and its functional role in reversing the increased endothelial permeability response, and (iv) to address the mechanisms of FAK-induced re-annealing of adherens junctions and its role in restoring endothelial barrier function. Studies will be carried out both in endothelial cells and in intact lungs isolated from several genetic mouse models, including one carrying a conditional deletion of the FAK gene in endothelial cells. We will analyze the role of the signaling molecules using state-of-the-art cell imaging techniques, expression of mutant constructs, and siRNA-induced down regulation of genes. These studies will be critical for defining the mechanism of restoration of endothelial barrier function and identifying potential new molecular targets in the pharmacotherapy of ARDS.