Focal Adhesion Kinase Regulation of Lung Vascular Permeability and Edemagenesis

描述(申请人提供)：肺血管通透性增加导致与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细胞黏附连接和细胞-基质黏附的动态组织对于建立内皮屏障功能至关重要。在炎症介质如凝血酶黏附连接和细胞-基质黏附的反应中，RhoA诱导的细胞收缩导致解体，导致内皮通透性迅速增加。炎症介质引起的内皮通透性增加通常是由于黏附连接和细胞-基质黏附的重新组织而发生的可逆事件。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)调节着粘着斑处细胞-基质的粘着接触。我们已经证明，抑制FAK导致凝血酶攻击后内皮细胞通透性不可逆转地增加。重要的是，我们的支持数据表明，内皮通透性的不可逆转增加是RhoA激活的结果。这些发现表明，FAK在下调RhoA活性和重新退火粘连连接方面发挥了新的作用，这是逆转水肿剂后内皮通透性增加所必需的。在初步数据中，我们还确定了蛋白质p190RhoGAP和N-WASP的关键功能，它们在被FAK激活时可能会使RhoA失活并诱导粘连连接的重新组装。因此，我们将检验这样的假设，即内皮通透性增加后FAK的激活组装了一个信号复合体，从而恢复了内皮通透性。我们的具体目标是：(I)研究FAK通过负调控RhoA活性从而促进黏附连接和焦点黏附的重组来重建内皮屏障功能的作用；(Ii)确定异源三聚体G蛋白的GPY二聚体在调节FAK激活从而恢复内皮屏障功能中的作用；(Iii)确定FAK抑制RhoA活性的机制及其在逆转内皮通透性增加的反应中的作用；以及(Iv)研究FAK诱导的黏附连接重新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个遗传小鼠模型中分离出来的完整肺中进行，其中一个模型携带内皮细胞中FAK基因的有条件缺失。我们将使用最先进的细胞成像技术、突变结构的表达以及siRNA诱导的基因下调来分析信号分子的作用。这些研究对于明确内皮屏障功能恢复的机制以及在ARDS的药物治疗中寻找潜在的新的分子靶点将是至关重要的。

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.