NEUTROPHIL DEPENDENT REGULATION OF VENULAR PERMEABILIT

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Coronary microvascular exchange plays an essential role in the maintenance of normal cardiac function. In acute inflammation and myocardial ischemia-reperfusion, neutrophil adhesion to postcapillary venular endothelium followed by an increase in venular permeability comprises a common pathophysiological consequence in the development of myocardial dysfunction. Although much work has been dedicated to the clarification of neutrophil-derived agonists and second messengers, little is known about their molecular targets. Within this context, whether activated neutrophils affect microvascular barrier function by altering the structural and functional integrity of the endothelium remains elusive. Therefore, the overall goal of this study is to identify at molecular levels the signaling mechanism that controls the endothelial hyperpermeability response to activated neutrophils. Corresponding to this goal is the major hypothesis that neutrophil- induced barrier dysfunction occurs through a chain of events in the endothelium from cytoskeletal reorganization to junctional disorganization, which ultimately facilitate transendothelial flux of fluid and proteins. Specifically, three hypotheses will be tested: (1) activated neutrophils trigger the cytoskeletal contractile process through myosin light chain phosphorylation, (2) activated neutrophils downregulate the endothelial adherence junction through phosphorylation and dissociation of VE-cadherin/beta-catenin complex, and (3) activated neutrophils promote anchorage-dependent cell contraction through the formation and redistribution of focal adhesions. To test these hypotheses, several advanced experimental approaches will be employed, including the intact perfused coronary venule preparation, the cultured venular endothelial monolayer model, western blot analysis, fluorescence immunocytochemistry, DNA and protein transfection, and dominant negative inhibition approaches. The study will provide a new insight into the molecular pathobiology of inflammatory and ischemic heart diseases. Identification of specific target proteins of activated neutrophils will enhance the development of therapeutic strategies to effectively inhibit the deleterious process of edema without suppressing the beneficial effects of inflammatory responses.