Disruption of the endothelial cell (EC) barrier that lines the pulmonary vasculature is a central pathophysiologicevent in inflammatory lung injury syndromes and results in clinically significant oxygenationderangements in critically ill patients. The goal of Project #2 is to mechanistically characterize linkagebetween key peripheral EC structures (dynamic cortical actin, lipid rafts, tight junctions, adherens junctions)and the cytoskeletal elements critical to the regulation of lung EC barrier function. The EC cytoskeleton is acomplex array of proteins intimately involved in the cell shape changes necessary for regulation of EC barrierfunction. We have identified essential roles for multiple cytoskeletal linker and effector proteins in theregulation of vascular permeability through modulation of these EC cytoskeletal rearrangements: MLCK,cortactin, actinin, radixin, ZO-1. To assess the importance of these cytoskeletal elements at the periphery ofthe cell, Specific Aim #1 will define the structure and the regulation of dynamic cortical actin filaments rapidlypolymerized at the EC periphery in response to multiple barrier enhancing stimuli.
Specific Aim #2 willrigorously characterize the cytoskeletal linkage to membrane-associated lipid rafts, cholesterol-enrichedmicrodomains within the plasma membrane that are important sites for concentrating transmembranereceptors and transducing their signals into the cell. Peripheral cell-cell contacts along the EC monolayer areessential components of barrier maintenance that serve as linkages to the actin cytoskeleton to provide bothmechanical stability as well as transduction of extracellular signals into the cell. In the EC, these intercellularcontacts consist primarily of two types of junctional complexes tight junctions (the focus of Specific Aim #3)and adherens junctions (the focus of Specific Aim #4),whose cytoskeletal linkages will be precisely defined.This project will utilize biochemical, molecular, and cell biology approaches, murine models, translationalgenomic techniques, and highly novel atomic force microscopy approaches to focus intensely oncharacterizing the regulation of key peripheral structures by cytoskeletal linker/effector proteins. Bymechanistically characterizing key membrane cytoskeletal activators, key cytoskeletal effectors, and thejunctional targets affected in these processes, we expect to provide a basis for development of effectivetherapeutic interventions for pulmonary vascular permeability dysfunction and All.
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