Aims. We recently reported that hyperosmolar sucrose enhances lung microvascular barrier properties by activating the protein, focal adhesion kinase (FAK), thereby blocking pulmonary edema in acute lung injury (ALI). Here, our overall objective is to understand relevant endothelial cell (EC) mechanisms. The three specific aims will test the hypotheses that (1) FAK signals through (-actinin and vinculin to enhance E- cadherin and barrier function;(2) EC uptake of a purified form of constitutively activated FAK (FAKP) enhances E-cadherin and barrier function;(3) intravascular infusions of hyperosmolar sucrose and FAKP protect against microvascular hyperpermeability and pulmonary edema. Procedures.
In Specific Aim 1, studies will be developed through immunoblotting, immunoprecipitation, and real-time live cell imaging in monolayers of rat lung microvascular EC (RLMEC). Emphasis will be placed on mutational approaches to determine protein-protein interactions in the signaling pathway.
In Specific Aim 2, we will purify constitutively active FAK (FAKP). Then to enable EC uptake, we will infuse FAKP with the Chariot reagent. We will determine functional effects in terms of barrier properties and E-cadherin expression in optically imaged microvessels.
In Specific Aim 3 we will determine microvascular filtration coefficient and blood-free extravascular lung water in acid- and lipolysaccharide-models of ALI (Aim 3). We will determine protective effects of hyperosmolar sucrose and FAKP. Significance. Despite high mortality and morbidity in ALI, specific barrier enhancement therapy is lacking. In ALI, critical pathology results from EC barrier deterioration that causes pulmonary edema. Hence, barrier enhancement will be protective. We will define novel protein-protein interactions underlying signaling that causes barrier enhancement. Understanding of this cell signaling will reveal new mechanisms that will facilitate development of barrier enhancement therapy. The FAKP studies are outstandingly novel. They will directly test the critical role of FAK in causing barrier enhancement, and reveal FAK therapy as a novel therapeutic possibility for ALI. PROJECT NARRATIVE. This project is to determine fundamental mechanisms underlying enhancement of the lung endothelial barrier by focal adhesion kinase. The findings of this research are likely to impact understanding of mechanisms of acute lung injury and the development of relevant therapy.
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