Ischemia-reperfusion injury leads to the activation and deposition of serum complement on the vascular endothelium. Inhibition of complement activation or depletion of complement proteins prior to reperfusion has been shown to reduce tissue injury. However, the mechanisms regulating complement activation and deposition at the endothelial cell surface during ischemia (hypoxia) and reperfusion (reoxygenation) have not been fully elucidated. The general aim of this proposal is to investigate the mechanisms and effects of complement activation and deposition on human endothelial cells following hypoxia and reoxygenation. Recently, the existence of a third pathway of complement activation known as the lectin complement pathway has been demonstrated. This proposal will investigate whether: 1) reoxygenation of hypoxic human endothelial cells increases cell surface deposition of mannan-binding lectin leading to activation of the lectin complement pathway; 2) deposition of the terminal complement complex (C5b-9) on human endothelial cells decreases intracellular levels of cyclic guanosine monophosphate (cGMP) and increases cell surface expression of vascular cell adhesion molecules (VCAM-1) and intercellular adhesion molecules (ICAM-1); and 3) in vivo inhibition of the lectin complement pathway attenuates myocardial reperfusion injury. Experimental techniques to be used in this proposal include ELISA, flow cytometry, protein affinity chromatography, western blotting, and monoclonal antibody production. This award will enable the Applicant to further develop his investigative skills by attaining proficiency in both cellular and molecular techniques, and by expanding his knowledge of immunology, cell biology, biochemistry, and pathology. Moreover, the career development plan associated with this proposal will enable the Applicant to achieve his long-term goal of becoming an independent Clinician-Scientist capable of fostering the development of other future Clinician-Scientists. The findings to be gained from these experiments are likely to have significant clinical implications regarding complement-mediated vascular injury in humans. In addition to furthering our understanding of the basic mechanisms regulating complement activation, the findings of this proposal may also lead to the development of a novel therapeutic strategy against complement-mediated vascular injury in man. Finally, the results from this proposal may apply to other organ systems where complement activation is thought to play a significant role in mediating vascular injury, including the myocardial (myocardial ischemia), cerebral (stroke), gastrointestinal (inflammatory bowel disease), pulmonary (adult respiratory distress syndrome) and renal (glomerular nephritis) circulations.
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