MECHANISMS OF RESPIRATORY ENDOTHELIAL INJURY BY XO

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Xanthine oxidase (XO) appears to participate in a variety of vascular injury syndromes including pulmonary, cardiac, renal, cerebral, and intestinal ischemia-reperfusion or hypoxia/reoxygenation. This possibility is consistent with (a) the high concentrations of XO in capillary endothelial cells, (b) the capacity of XO to generate toxic O2 metabolites and chemotaxins for neutrophils in vitro, and (c) the ability of XO inhibitors to decrease injury in animals. The problem is that the role of XO in human vascular disease is still unproven because XO has been implicated only by using non-specific or incomplete inhibitors of XO activity in animals rather than more accurate modern approaches in relevant human systems. Indeed, the mechanisms which govern regulation of expression of XO and its precursor, xanthine dehydrogenase (XD), the effects of this regulation on pathologic conditions, and the molecular basis for formation of XO and from XD following ischemia and other vascular insults are unclear. Our preliminary data indicate involvement of XO in many diseases. To address definitively the contribution of XO to inflammation and vascular damage, we chose to clone and sequence human XD/XO. Since our prior submission, we have 1) cloned and partially sequenced two candidate DNA probes specific for XO, 2) developed a monoclonal antibody specific for mammalian XO, 3) determined that our human liver cDNA library contains the human XO gene, 4) developed a yeast expression system for human XD, and 5) analyzed new sequence data which supports our hypothesis for XD to XO conversion. Our goal is to test definitively the premise that XO contributes to human disease and to explore the structural basis for XD to XO conversion. Our major objectives are: (a) to construct and test an XD/XO null mutant to produce definitive evidence for the hypothesis that XO dependent mechanisms contribute to cell injury and neutrophil adherence following exposure to hypoxia and reoxygenation; (b) to determine the structural difference between XD and XO activities and the basis for conversion of XD to XO in vitro; and (c) to determine if parameters of ischemia/reperfusion regulate XD/XO gene expression. These critical objectives require cloning and sequencing a human XD/XO cDNA clone. The significance of this project is that it will precisely define the role in ischemic vascular disease of XO in light of its native regulatory mechanisms. This approach will generate new understanding of fundamental mechanisms of tissue injury and inflammation.