The major objective of this proposal is to elucidate the mechanism of the endothelial dysfunction occurring after reperfusion of a previously ischemic coronary or splanchnic vasculature or after induction of soft tissue trauma. The major manifestation of the endothelial dysfunction is a marked reduction in the basal release and agonist-stimulated release of nitric oxide (NO). The primary mechanisms responsible for the reduced release of NO are (a) increased superoxide radical formation which inactivates NO, (b)polymorphonuclear (PMN) leukocyte adherence to endothelial cells (EC), and (c) inhibition of constitutive nitric oxide synthase. The previous proposal was directed toward studying the time course of the endothelial dysfunction and the interactions between NO and oxygen derived free radicals. The major thrust of the present proposal is to investigate the adhesion molecules responsible for PMN-EC interaction and to relate these interactions to tissue injury, since PMNs play a significant role in reperfusion injury even beyond adherence to the endothelium. In order to achieve these goals, we will study the effect of monoclonal antibodies (MAb) directed against L-selectin and CD18 on the neutrophil cell membrane as well as MAbs directed against ICAM-1, PECAM-1, P-selectin and L-selectin on endothelial cells. Additional studies will be conducted using anti-selectin oligosaccharides (i.e., sialyl Lewis[X] analogs). Utilizing flow cytometry and immunohistochemistry, we will determine the time course of activation-inactivation of these adhesion molecules, their interaction among each other and their significance to reperfusion- and trauma-induced endothelial dysfunction. Employing intravital microscopy, we will directly visualize leukocyte-EC interactions at the level of the microvasculature (i.e., mesenteric venules). Further work will be directed toward studying cellular events in cultured ECs, and in investigating mechanisms of attenuation of reperfusion-induced increased adhesiveness of ECs and reduced NO generation. These findings should provide important basic insight into the pathophysiology and therapeutics of myocardial and splanchnic ischemia/reperfusion and traumatic shock. All of these are serious life threatening disorders, and knowledge of their pathophysiology provides important information to the clinician treating these conditions.
Showing the most recent 10 out of 24 publications
