Microvascular barrier injury represents a significant problem associated with trauma and inflammatory disease. In patients with major burns, edema occurs not only locally but also in tissues remote from the wound, leading to hypovolemic shock and dysfunction of multiple organs including the gut and lungs. The end-organ effects of systemic inflammation have not been well characterized at the cellular or molecular level. The goal of this long- standing project is to elucidate the endothelial-specific mechanisms of microvascular hyperpermeability following burns. Our work during the previous funding period has led to the development of unique experimental models and imaging techniques that enable quantitative analyses of microvascular function in rodent models of thermal injury. The studies also revealed burn-elicited signaling and structural changes in the endothelial barrier characterized by cytoskeleton contraction and cell-cell junction opening. In this competing renewal application, we plan to extend the investigation to a more in-depth examination of endothelial barrier molecules, focusing on our newly discovered pathway of palmitoylation in mediating paracellular permeability.
Three specific aims are proposed: 1) to establish the pathophysiological importance and functional role of palmitoylation in burn-induced microvascular injury; 2) to characterize the molecular basis of palmitoylating enzyme (DHHC PAT)-mediated permeability responses in vivo and in vitro; and 3) to identify molecular targets of palmitoylation in endothelial cell-cell adherens junction barrier that regulate microvascular permeability. The central hypothesis to be tested is that endothelial palmitoylation activated following burn injury contributes to the pathogenesis of microvascular barrier failure by promoting ?atenin sequestration from cell-cell junctions, an end-point cellular response attenuated by pharmacological or molecular inhibition of palmitoylation. This novel and mechanistic pathway will be evaluated in a series of complementary studies integrating in vivo microcirculation analyses with isolated microvessel experiments and cultured endothelial cell assays. Innovative experimental models and molecular tools will be developed and tested. Data derived from this project may lead to a new theory about the pathophysiological regulation of microvascular function in trauma. The study also has the potential to identify novel therapeutic or diagnostic targets for inflammatory diseases.
Thermal injury affects more than 2 million Americans each year. Despite the remarkable improvement in critical care and wound management, inflammation-induced multiple organ dysfunction remains a major cause of mortality and morbidity in patients with severe burns. Attempts to block individual inflammatory mediators as therapeutic means have met with limited success, owing to our incomplete understanding of the endpoint cellular processes underlying tissue injury. The objective of this study is to elucidate the endothelial-specific mechanisms of microvascular dysfunction during thermal injury. We propose an in-depth analysis of novel molecular pathways and therapies by targeting the palmitoylation process of endothelial proteins that serve as endpoint effectors in barrier injury. Data derived from this project will lead to a new theory regarding the pathophysiological regulation of microvascular function in trauma. The study also has the potential to identify novel therapeutic or diagnostic targets for effective treatment of traumatic injury or inflammatory disease.
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