Ischemia/reperfusion (I/R) vascular disorders appear to depend on oxygen free-radicals (02*) produced by the molybdenum hydroxylase, xanthine oxidase (XO). Involvement of XO has been proposed because: (1) XO is abundant in vascular endothelial cells, (2) XO produces toxic O2* and neutrophil chemotaxins in vitro, (3) inhibitors of XO decrease I/R injury in animal models, and (4) XO is increased in patients with certain vascular disease. The problem is that the involvement of XO in human vascular disease is equivocal. Extrapolation from animal models to human disease has been unreliable, and data produced in humans has been inconsistent. We propose that several unrecognized mechanisms determine O2* synthesis by molybdenum hydroxylases and consequent vascular cell injury in humans. Differences in level and distribution of rodent and human XO, the existence of cell specific expression, unknown modes of regulation, XO secretion, and the unrecognized contribution of aldehyde oxidase have all contributed to this uncertainty. Our preliminary genetic data have supported this argument by revealing three highly similar genes encoding molybdenum hydroxylases in humans. mRNAs homologous to each gene exhibit tissue specific expression that differs from the apparent distribution of enzyme activity and is consistent with different endothelial and epithelial expression. Unexpectedly, two of these genes were linked genetically to important but unrelated O2* mediated human disease, including juvenile ALS and cancer. Our goal is to test the hypothesis that tissue and cell specific expression of each gene may determine the contribution of the molybdenum hydroxylases to human vascular disease. Our major objectives are: (1) to define precisely the coding difference between the three genes and confirm possible secretion of XO; (2) to determine the pattern of expression and modes of regulation of the molybdenum hydroxylase genes in human endothelial and epithelial cells; and (3) to determine the contribution of each gene to O2* synthesis and vascular cell injury. The significance of this project is that several uncertainties concerning the role of molybdenum hydroxylases in human vascular disease will be resolved in human cells. Our work will determine whether specific endothelial and epithelial gene expression affects the capacity of cells to produce cytotoxic O2*, and secrete XO. Also, we will generate vital information useful in evaluating genetic susceptibility or resistance to vascular disease.
