Microvascular hyperpermeability represents an end-point cellular process underlying the development of ischemic injury, diabetes, atherosclerosis, and various inflammatory diseases. The abnormality is largely attributed to neutrophil (PMN) activation and secretion of inflammatory mediators that are capable of altering the structural and functional integrity of the endothelial barrier. Currently our knowledge of the molecular control of endothelial barrier function is limited. Even less is known about PMN-induced endothelial changes in intact exchange microvessels. Among the reasons given for the lack of such information is the paucity of suitable experimental models that directly link subcellular events to physiological function. In this regard, one of our accomplishments over the prior funding period is the development of new techniques that facilitate correlative analyses of molecular reactions and microvascular function. Furthermore, the studies have produced a large body of experimental data that forms a signaling paradigm for venular hyperpermeability in inflammation. Three important endothelial structures, namely the contractile cytoskeleton, adherens junction, and focal adhesion, have been found to mediate barrier dysfunction. The current proposal is to extend these original investigations to a detailed mechanism of endothelial response to PMN activation. Our working hypothesis centers on biochemical and conformational modulations characterized by myosin light chain phosphorylation-dependent endothelial cell contraction, VE-cadherin/b-catenin sequestration-induced junctional weakening, and focal adhesion kinase-signaled focal adhesion rearrangement. The hypothesis will be tested by an approach that integrates physiological experiments with biochemical, biophysical, and molecular biology analyses at both the intact microvascular and subcellular levels. The newly developed microvessel transfection technique will be incorporated into a comprehensive evaluation of the cause-effect relationship between molecular reactions and venular barrier dysfunction in PMN activation. The study should provide novel insights into the molecular basis of microvascular leakage in inflammation. Identification and characterization of the key molecules that serve as ultimate effectors in the hyperpermeability reaction is fundamental to the development of therapeutic strategies directed against the injury.
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