RESPIRATORY ENDOTHELIAL INJURY BY XANTHINE OXIDASE

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We recently cloned two human genes (AOX1 and AOX2) that encode the obligatory oxygen radical generating enzyme, aldehyde oxidase (AO) and discovered unanticipated genetic complexity in this molybdenum hydroxylase family which includes xanthine oxidase (XO). It also appears that AO could contribute to acute lung leak: First, AO, but not XO, was expressed in human lung. Second, treatment with benzoquinone or SKF-525 (AO inhibitors), but not allopurinol (an XO inhibitor), decreased lung leak in rats given interleukin-1 (IL-1)-intratracheally. Third, lung slices given AO substrate made H2O2, and more H2O2 was observed using cerium chloride staining in electron micrographs of lungs given IL-1 than in control or IL-1 and AO inhibitor treated rats Our specific hypothesis is that AO generated O2 radicals contribute to acute lung lead. Our specific aims are: 1. To characterize human and rat AOX1 and AOX2 genes by DNA sequence analysis, functional expression, antigenic mapping, and amino acid sequence analysis. 2. To determine the levels and cellular localization of AO activity and characterize AO antigen and mRNA expression in human and rat lung by in situ hybridization, immunochemistry, and histochemistry. 3. To determine the contribution of AO to the development of acute lung leak in vivo. The primary justification for the proposed approach is that: (a) The expression of AO,but not XO, in the human lung suggests a heretofore unrecognized pathway for oxygen radical generation in acute lung leak (the so called acute respiratory distress syndrome, ARDS). (b) The structures, expression and regulation of AO and XO and their genes needs to be determined to understand their specific and possibly different functional contributions to oxygen radical mediated lung leak. (c) Combined analysis of the rat and human AO genes will enable us to determine the significance of AO and to assess the potential for manipulating AO to prevent acute lung leak (ARDS) in humans.