We will extend our studies of the cellular and molecular mechanisms that are responsible for increased permeability of the lung vasculature. This increase in lung vascular permeability results in an array of pathological conditions including Adult Respiratory Distress Syndrome. The studies proposed here are based on the hypothesis that the VEcadherin- containing adherens junctions (AJs) of endothelial cells are critical determinants in both the increased permeability response of the endothelium and its time-dependent reversal. The dynamic AJ affects cell shape through the interaction of VE-cadherin with catenins that in turn associate with the actin cytoskeleton. The proposed studies will characterize the mechanisms by which AJs disassemble in response to thrombin and subsequently reassemble in a time-dependent manner. We will identify the phosphorylation and dephosphorylation """"""""switches"""""""", present in the VEcadherin/ eatenin complexes, which are responsible for regulating endothelial permeability. This project has the following Aims: (i) to identify the signaling mechanisms mediating the loss of homotypic VE-cadherin binding, the disassembly of AJs, and the resultant increased permeability of the endothelium, (ii), to characterize the signaling mechanisms responsible for re-assembly of AJs and reversal of this increased permeability response, and (iii) to characterize the mechanism of angiopoietin-1-induced AJ stabilization and its role in inhibiting the loss of endothelial barrier function. The studies will be carried out both in endothelial cells and in vivo with intact mice. We will exploit a variety of methodologies including cell imaging, expression of mutant constructs, gene transfer, and the evaluation of transgenic mouse models. By more fully understanding mechanisms of increased vascular permeability and the reversal of this process, we will be in a position to develop novel strategies directed against components of the AJ complex that will prevent vascular leakiness and the formation of pulmonary edema.
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