The present goal of the Principal Investigator is to continue to develop the intellectual, technical and analytical skills to become a successfully funded independent investigator. A program of additional didactic and laboratory training in molecular biology and mentoring by senior professors with expertise in mucosal immunology, molecular and cell biology, and nitric oxide chemistry and biology will be used. The long-term research objective is to elucidate the role of nitric oxide (NO), particularly that produced by epithelial cells, in intestinal inflammation and injury. The purpose is to improve understanding of the pathogenesis of inflammatory bowel diseases (IBD) and the treatment and management of these diseases. The long-term career goal of the PI is to establish himself as a nationally recognized investigator in intestinal NO biology and IBD research. Despite intensive investigation, mechanisms of intestinal inflammation remain incompletely understood. Evidence is rapidly mounting that the free radical gas and pleiotrophic signaling molecule NO may be a key piece in the puzzle. With stimulation, as in mucosal immune activation, a high output NO pathway can be activated by transcription of the inducible enzyme (iNOS). Our data indicate that iNOS expression is increased in the intestinal mucosa in endotoxemia and colitis and that iNOS can be abundantly expressed in intestinal epithelial cells (IECs). NO can be cytotoxic, induce apoptosis, and modulate cyclooxygenase (COX)-2 and cytokine expression; whether such effects are induced by NO in GI inflammation is unknown. We hypothesize that in the presence of inciting stimuli, the intestinal epithelium is a regulatable source of high output NO production by iNOS, and that NO modulates fundamental aspects of epithelial integrity that can contribute to intestinal inflammation.
Our Specific Aims are to: (1) demonstrate that high output NO is produced by epithelial cells in intestinal inflammation and that this is involved in injury; (2) assess IEC iNOS regulation by EGF and TGF-beta; (3) determine the effects of NO on IEC proliferation, apoptosis, wound repair, COX-2 expression, and pro-inflammatory cytokine expression in vitro; and (4) demonstrate that iNOS expression has similar effects in situ. For these studies we will use intestinal epithelial cell lines transfected with iNOS, stimulated with cytokines, or exposed to NO-donating compounds; rat models of colitis; and human IBD tissues. The role of NO in experimental colitis and the effect of TGF-beta on iNOS expression will be further assessed in iNOS and TGF-beta knock- out mice, respectively. Our studies should provide insight into the role of NO and IECs in intestinal inflammation and into potential novel targets of pharmacologic intervention in IBD.
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