Derangement in the intestinal mucosal barrier may play an important role m the pathogenesis of systemic infection critically ill patients. Perturbations in the host defense mechanisms that result in sustained upregulation of inducible nitric oxide synthase (iNOS) in the gut may lead to profound alterations in intestinal mucosal barrier function. Evidence suggests that peroxynitrite (ONOO-), a potent oxidant formed by the reaction of NO with mperoxide, may be a key reactive nitrogen intermediate responsible for the eytopathic effects of NO in inflammatory conditions such as endotoxemia, inflammatory bowel disease (IBD), or necrotizing enterocolitis. Our objective is to determine the mechanisms by which overproduction of NO or ONOO- may promote tissue injury enterocyte apoptosis or necrosis) and inhibit tissue repair mechanisms (epithelial restitution via enterocyte migration and proliferation), thereby leading to gut barrier failure. We propose two specific aims.
Aim I : To elucidate the potential mechanisms by which ONOO induces enterocyte apoptosis. We will examine the cytopathic effect of ONOO- in various enterocytic cell lines and the biochemical pathways that may be involved (mitochondrial dysregulation, caspases, and PARS activation).
Aim II : To investigate the mechanisms by which NO or ONOO- inhibits tissue repair mechanisms, epithelial restitution and proliferation. We will examine how ONOO- affects epithelial restitution by enterocyte migration, the critical phase that precedes the proliferative response to repair the mucosal injury. Migration may be regulated in part by Rho-GTPases, which modify the actin cytoskeleton. We will determine whether Rho is required for enterocyte migration and stress fiber formation in vitro. We will test the hypothesis that ONOO can inhibit migration and proliferation by nitrating critical tyrosine residues of key members of the Src family of tyrosine kinases: Src; focal adhesion kinase (FAK); and P13K. We will attempt to modulate Rho, Src kinase, or mitochondrial signaling pathways with various cytoprotective agents to enhance intestinal barrier function in conditions associated with excessive NO/ONOO- production (endotoxemia IBD) in vivo.
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