The aim of this proposal is to explore the regulation of the enzyme xanthine dehydrogenase/xanthine oxidase (XDH/XO) by oxygen and cytokines, and the role of XDH/XO in the development of acute lung injury through its interaction with inducible nitric oxide synthase (iNOS). XDH/XO and iNOS are upregulated by hypoxia and proinflammatory cytokines (e.g., interleukins 1 and 6, IFNg and TNF). The products of XO and iNOS, superoxide and NO, interact to form the highly toxic oxidant peroxynitrite. We have previously shown that XHD/XO is transciptionally upregulated by hypoxia in pulmonary artery endothelial cells (EC). Preliminary studies, using transient transfection of EC, indicate that specific regulatory elements within the XDH/XO promoter might be responsible for the transcriptional regulation of this enzyme by hypoxia and IL-1beta. Furthermore, using an in vivo model of acute lung injury, we have demonstrated an increase in lung XDH/XO activity and gene expression in response to hypoxia, endotoxin, and IL-1beta treatment. Pulmonary edema was prevented by pharmacological inhibition of XDH/XO, supporting a role for this enzyme in the pathogenesis of acute lung injury. Preliminary studies indicating upregulation of lung iNOS by hypoxia, endotoxin, and IL-1beta treatment, as well as the formation of peroxynitrite in this model, support a role for iNOS in the pathogenesis of acute lung injury through interaction of NO with XO-derived superoxide. We hypothesize that: 1) induction of IL-1 by hypoxia contributes to upregulation of lung XDH/XO and iNOS in vivo; 2) transcriptional upregulation of XDH/XO by hypoxia and cytokines (e.g., IL-1 and 6) involves a HIF-1 like element, as well as NF-kb, AP-1 and C/EBP binding sites in the 5'-flanking region of the promoter; and 3) lung XDH/XO and iNOS contribute to increased capillary permeability, in an animal model of acute lung injury, through peroxynitrite formation.
The Specific Aims of the project are: 1) to examine the expression of IL-1 in response to hypoxia, as well as the effect of IL-1 receptor antagonist and IL-1 polyclonal antibody on the upregulation of XDH/XO and iNOS expression by hypoxia, in pulmonary microvascular EC and rat lung; 2) to characterize the role of cis DNA elements of the 5'-flanking XDH/XO promoter, to assess the role of XDH/XO and iNOS in the development of pulmonary edema and oxidative stress in this model, using inhibitors as well as antisense oligonucleotides targeted at these enzymes.
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