The overall objective of this renewal application is to study the signaling mechanisms regulating Ca2+ influx through transient receptor potential (TRP) channels in endothelial cells and their role in mediating increased ndothelial permeability and pulmonary edema. We have shown that thrombin activation of Protease- Activated Receptor-1 (PAR-1) expressed on the endothelial cell surface increases intracellular Ca2+, which is critical in the mechanism of increased endothelial permeability. Increased intracellular Ca2+ concentration is dependent on both Ca2+ store depletion and Ca2+ store depletion-mediated Ca2+ influx. Ca2+ influx due to store depletion is mediated by store-operated cation channels (SOCs). We have identified TRPC family channels expressed in endothelial cells that function as SOCs. We have also demonstrated that Ca2+ influx nduced by thrombin causing increased microvascular permeability was impaired in TRPC4 knockout (TRPC4''"""""""") mice. Our Supporting Data show that PAR-1 agonist-induced increase in lung vascular permeability was impaired in caveolin-1-null (Cav-1""""""""'"""""""") mice. Further, we showed that PAR-1 agonist- nduced Ca2+ influx was absent in Cav-1""""""""'"""""""" mouse lung endothelial cells (MLEC) and the Ca2+ influx response was rescued by re-expression of caveolin-1 in Cav-1 MLEC. These findings suggest that caveolin-1 expression is required for SOC function in endothelial cells. In addition, we show Supporting Data that in ubiquitin ligase Cbl-b-null (Cbl-b""""""""'"""""""") mice, PAR-1 agonist-induced Ca2+ influx and increased lung vascular permeability were abrogated;thus, Cbl-b signaling is important in regulating lung vascular permeability via Ca2+ influx. Based on these data, we propose to determine (i) the role of caveolin-1 in the regulation of Ca2+ entry through TRPC channels and thereby signaling increased endothelial permeability and (ii) the role of Cbl-b as a key mechanism regulating Ca2+ influx and increased endothelial permeability. The proposed studies will utilize cell and in vivo approaches utilizing gene deletion mouse models to address the role of TRPC channels in the mechanism of increased endothelial permeability and pulmonary edema formation. These studies are expected to provide novel molecular insights into the understanding of mechanisms of lung vascular injury and tissue inflammation. It is hoped that this work will lead to the development of new drug targeting acute lung injury associated with leaky lung microvessel and tissue edema formation.
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