Hypoxic vasospasm and/or thrombosis of the major coronary arteries may be instrumental in the development of transient myocardial ischemia and fatal infarction. A rapidly expanding literature indicates that the endothelium is an important modulator of vascular smooth muscle (VSM) tone by virtue of its ability to secrete vasoactive second messengers in response to luminal stimuli. The overall goal of this project is to investigate the interrelationships between hypoxic coronary vasospasm and thrombosis with emphasis on the role of the endothelial cell (EC) in modulating the interactions. During the past project period, we have isolated, purified and partially characterized a polypeptide from ECs in culture, which is now referred to as the endothelium-derived constricting factor (EDCF). We have examined species specificity, its effect in isolated VSM cells, beating heart preparations and in whole animal vasculatures. We have also established a working model of its potential mechanism of action and have shown that hypoxia induces a change in EC-morphology that is associated with an enhanced release of EDCF. Other investigators have shown that EDCF activity can be expressed by the intact endothelial lining and that anoxia enhances the release of a diffusable, polypeptide vasoconstrictor from intact vessels in an EC-dependent fashion. These data obtained in """"""""intact"""""""" vessels provide validation of EC culture as a model system and appear to be focused on the same EDCF molecule whose release under conditions of 02-deprivation may be instrumental in the pathophysiology of coronary vasospasm. ECs cultured in a hypoxic or acidotic environment secrete significantly less of the clot-dissolving activator protein (plasminogen activator) -- a condition which could favor thrombosis. Thus, the EC may logically represent the critical link or intermediary between thrombosis and coronary artery vasospasm and forms the rationale for our proposal. The requested renewal period will focus on: 1) scaling-up of our purification of EDCF and production of a monoclonal antibody for use in specifically identifying, quantifying and further purifying the EDCF molecule; 2) completing two remaining specific aims left over from the current year; 3) continuing and expanding studies of the effect of hypoxia (and a potential serum-derived cofactor) on EDCF production; 4) continuing studies on the mechanism of action of EDCF on VSM; and 5) beginning studies on how immune cell-products affect the role of the EC in modulation of VSM tone and thrombosis. All of the protocols will utilize established cell cultures or co-culture models. The project will provide new and meaningful information highly relevant to our understanding and treatment of coronary artery disease.
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