One early aspect of the Adult Respiratory Distress syndrome is the adhesion of neutrophils to the vascular endothelium, indeed, at pathological examination the vasculature is often occluded with PMNs. Large numbers of PMNs also are found in the alveolar space of patients with ARDS. The activation of these cells with resultant secretion of proteases and toxic oxygen species is thought to be an important component of the pathogenesis of ARDS. We have studied the adhesion and activation signals produced by the endothelium in response to agonists such as thrombin, histamine, peptidoleukotrienes, and bradykinin. We have found that there are two important components, one of which is platelet-activating factor (PAF), a phospholipid with potent, diverse proinflammatory actions. PAF is removed by an enzyme, PAF acetylhydrolase, that is found in cells and plasma. The studies proposed here focus on this enzyme because it serves the essential role of shutting off the PAF signal. Additionally, in recent experiments we have examined the response of endothelial cells to oxidants. This is relevant to pathogenesis of ARDS since oxidants can be generated both by the activation of inflammatory cells and by exposure to high concentrations of oxygen. One result under these circumstances is the oxidation of polyunsaturated fatty acids, including those that are esterified in phospholipids. We found that these compounds have potent inflammatory actions - like PAF, and are produced when endothelial cells are exposed to oxidants. Thus, extremely potent inflammatory signals can be generated at the endothelial cell surface, or other places, by strictly chemical means -- without biochemical regulation of the synthesis. Under these circumstances the rapid removal of such lipids would be crucial. We found that the PAF acetylhydrolase hydrolyzes phospholipids with oxidatively fragmented fatty acids. Hypothesis: PAF and related lipids are key mediators of inflammation in ARDS; and the PAF acetylhydrolase suppresses inflammation and protects cells against oxidative damage. This pilot will test these hypotheses using several approaches. We will measure the amount of PAF and oxidized phospholipids in the alveolar space and the blood at different stages of ARDS. We also will characterize the PAF acetylhydrolase under the same conditions. Finally, we plan to obtain cDNA clones of the enzyme, overexpress it or inhibit its expression in an endothelial cell line, and then test the functional consequences on inflammatory responses by endothelial cells.
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