CELLULAR MECHANISMS OF PULMONARY ENDOTHELIAL INJURY

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Pulmonary endothelial injury typifies the adult respiratory distress syndrome (ARDS). Gram negative bacterial sepsis is the most frequent clinical setting in which ARDS occurs, presumably a result of endotoxin- induced lung injury. In this renewal application, we propose to extend our observations of effects of endotoxin on lung endothelial cells in culture to a molecular level. We propose to test the hypothesis that endotoxin exposure results in increased oxidant stress within endothelial cells which causes increased production of interleukin-1 (IL-1) resulting in increased production of the antioxidant enzyme, manganous superoxide dismutase (MnSOD) (we have demonstrated that the first and last step in this sequence occur). Further, we propose that endotoxin stimulation of prostanoid production by endothelial cells is also oxidant mediated and that prostaglandin E2 (PGE2), a major product of microvascular endothelial cells, modulates IL-1 and thus MnSOD induction. We propose that differences in response of endothelial cells from different organs and from different sites within the lung can be explained by differences in constitutive production of MnSOD and PGE2 and that genetic engineering of endothelial cells to produce increased amounts of MnSOD will render the cells resistant to endotoxin-induced injury. To test these hypotheses we will conduct studies in cultured endothelial cells from large and small vessels in the lungs and from human umbilical veins. We will 1) elucidate the time course of induction of alteration in IL-1 and MnSOD gene expression following exposure of the cells to endotoxin and determine whether the responses are inhibited by antioxidants which access the cell interior, 2) determine whether the endotoxin effect are mimicked by the cytokine TNFalpha (which, like endotoxin, stimulates IL-1 production by endothelial cells) or exogenous IL-1, 3) determine effects of exogenous PGE2 on IL-1 and MnSOD gene expression, 4) compare these responses to endotoxin exposure in cells from different species, organs and sites, 5) determine whether endothelial cells genetically engineered to produce increased amounts of MnSOD are resistant to endotoxin effect and 6) initiate studies of the molecular mechanisms of endotoxin induction of MnSOD gene expression in endothelial cells. These studies follow logically the work supported by this grant over the past five years and promise a new level of understanding of how endotoxin affects endothelial cells and how the deleterious effects of endotoxin can be prevented. Such information will contribute to the development of novel interventions which may find eventual application in the clinical setting.