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.
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