Alterations in vascular permeability are defining feature of diverse processes including inflammation, ischemia/reperfusion and ventilator-induced lung injury, and may increase alveolar flooding, leukocyte infiltration and hypoxemia leading to increased morbidity and mortality. Oxidative stress accompanying these pathologies results in increased levels of oxidized phospholipids (OxPL) in the pulmonary circulation. We have previously characterized a group of barrier-protective OxPLs in vitro and in vivo and described enhancement of basal pulmonary endothelial (EC) barrier function and attenuation of permeability increase caused by inflammatory agonists. Barrier protective effects of OxPLs have been linked to enhancement of peripheral actin cytoskeleton, increased interactions between cell-cell and cell-substrate junctions mediated by small GTPases Rac and Cdc42, and Rac-mediated attenuation of Rho-dependent pathways of endothelial hyper-permeability. Although importance of endothelial cytoskeleton in dynamic regulation of EC permeability is well recognized, the role of EC adhesive complexes in the EC barrier responses is less understood. Our preliminary studies strongly suggest that barrier protective effects of OxPLs in the models of lung EC barrier dysfunction induced by inflammatory agonists are mediated by cell-cell adhesive structures, which are regulated by small GTPases Rac and Rap1. Our studies indicate that Rac- and Rap1-dependent signaling is also involved in the OxPL- mediated inhibition of Rho pathway. We hypothesize that bioactive components of OxPLs promote the lung vascular barrier integrity via activation of Rap1-dependent engagement of adherens junctions and tight junctions. We also hypothesize that OxPL-induced enhancement of adherens junctions may inhibit Rho GTPase via its negative regulator p190RhoGAP controlled by adherens junctions and lipid rafts.
Specific Aim #1 will test a novel upstream mechanism of OxPL-induced Rac activation via engagement of lipid rafts and activation of GRP78 receptor.
Specific Aim #2 will evaluate involvement of Rap1 and its cell adhesion effector afadin in the OxPLs-induced adherens junction and tight junction enhancement.
Specific Aim #3 will explore a role of p190RhoGAP in the Rac- and Rap1-dependent mechanisms of OxPL-induced Rho inhibition. We believe that these studies will significantly impact our understanding of the role of oxidized phospholipids in the EC barrier regulation and will help to identify novel protein targets and propose new therapies for prevention of pulmonary vascular barrier dysfunction associated with acute lung inflammation and injury.
The acute phase of lung injury is characterized by increased endothelial permeability and compromise of the blood-gas barrier, which allows an influx of protein-rich fluid into the air spaces, causing pulmonary edema. However, despite recent advances in ventilation strategies and a better understanding of the pathophysiology of ALI, there remain few effective treatments for this devastating illness. This application will investigate molecular mechanisms underlying protective effects of oxidized phospholipids against pulmonary vascular endothelial leak induced by edemagenic agonists and pathologic mechanical strain associated with acute lung injury and will characterize novel groups of barrier protective oxidized phospholipid compounds.
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