Ongoing investigations in our laboratory have examined in vitro mechanisms of the endothelial damage in dialysis-induced pulmonary leukostasis and extended these findings to suggest the pathophysiology underlying shock (""""""""shock lung"""""""") and vasculitis. In addition to neutrophil-mediated lytic and denuding endothelial injury, variety of sublytic endothelial metabolic and permeability alterations can occur as an endothelial """"""""activation"""""""" response to neutrophil-derived oxidants or proteolytic stimuli; these alterations may play a crucial role in vessel wall physiology. This proposal focuses upon functional changes of the endothelium in response to inflammatory stimuli such as proteases, endotoxin, tumor necrosis factor, and neutrophils and their releasable products, which lead to the recruitment and priming of neutrophils. Platelet activating factor (PAF), a cell-derived phospholipid, will be examined as a model molecule, which can be synthesized rapidly by endothelium in response inflammatory mediators. We have demonstrated that minuscule quantities (10-12M) of platelet activating factor can prime neutrophil responses, that is, prior exposure of neutrophils to fluid-phase PAF can augment their responses to a subsequently provided, but sub-optimal, stimulus. Neutrophils primed with PAF can cause more endothelial damage. This proposal will further investigate the ability of inflammatory mediators to """"""""activate"""""""" endothelium and thereby enhance neutrophil-mediated endothelial damage by priming neutrophil functions. Neutrophils, themselves, by elaborating specific products, can """"""""activate"""""""" endothelium. The specific neutrophil product, activating endothelium will be investigated in an attempt to answer whether """"""""neutrophil-activated endothelium"""""""" feeds back and primes neutrophil functions. Neutrophils can alter endothelial cell proteoglycans by the release of heparanases, proteases, or oxidants. Loss of heparin sulfate from endothelium would decrease its anticoagulant capacity by diminishing anti-thrombin III binding. This proposal will address whether neutrophils """"""""primed"""""""" on activated endothelium are especially efficient in removing endothelial cell heparin sulfate proteoglycans. The cellular pathways involved in endothelial cell-neutrophil """"""""cross-talk"""""""" will also be studied. Specifically, the role of neutrophil adhesion and the interaction between endothelial membrane-associated PAF and neutrophils in priming will be examined. Finally, attempts will be made to validate insights arrived from the in vitro priming studies using an explanted rat-lung model. The proposed studies describe a novel paracrine trigger for granulocyte- mediated endothelial injury, which may sere to amplify vascular injury in inflammatory sites where coagulation processes are activated and multiple mediators are signalling the endothelium.
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