The inflammatory process, stimulated by reperfusion following tissue ischemia, leads to local injury. With sufficiently large regions and times of ischemia, sizable quantities of mediators gain entry into the vasculature and lead to remote organ failure accompanied by prominent leukosequestration. This study of reperfusion injury will test the postulates that; (1) in the ischemic region, endothelial phenotype changes with the appearance of neoantigens, causing the deposition of immune complexes and activation of the classical complement pathway; (2) local injury via the terminal C5b-9 complex is permitted by loss of membrane complement regulatory proteins as well as the appearance of a scaffolding to strengthen complement binding to the cell membrane; (3) the scaffolding is thought to be provided by the adhesion glycoprotein P selectin which contains 9 short consensus repeats homologous with other complement binding proteins; (4) remote pulmonary injury is dependent upon binding of C3 components to an altered endothelium. This complement fragment works in synergy with neutrophils. Reoxygenation and reperfusion experiments will be conducted in endothelial cell cultures, ex-vivo blood vessels as well as in intact mice, rats, human volunteers, patients undergoing thoracoabdominal aneurysmectomy and femoral-popliteal bypass surgery. Assays will include the detection of membrane bound immune complexes, complement fragments, complement regulatory proteins as well as cell bound and soluble P-selectin. A variety of reagents will be employed to study mechanisms of injury, including monoclonal antibodies to proteins as well as genetically engineered materials: peptides homologous with selectin complementarily determining regions; soluble P-selectin, soluble counter receptor of P-selectin including sialyl Lewis and P-selectin glycoprotein ligand-1. Measurement techniques include immunohistochemistry, flow cytometry as well as standard methods for muscle and lung permeability using 125I-albumin, myeloperoxidase for neutrophil sequestration and the hemolytic red cell assay for classical and alternative complement activation. The data generated should provide an understanding of the putative functions of P selectin in leukocyte-endothelial adhesion as well as in complement binding.
Showing the most recent 10 out of 34 publications
