The long term objective of this project is to determine the pathogenesis of stricture formation in Crohn's disease. Work to date has demonstrated that stricture formation results from an accumulation of collagen in the intestinal wall, and that the intestinal smooth muscle cell plays a major role in this exaggerated repair process by proliferating and depositing collagen in response to chronic inflammation. An in vitro model of human intestinal smooth muscle cells has been developed and characterized. The current proposal will utilize this model to answer the following questions and hence approach the long term objectives: 1. Do the cytokines IL-1beta and TGF-beta regulate procollagen expression through the early response genes c-fos, c-jun and jun b, how are these regulatory activities inter- connected, and how are they modified by corticosteroids? Is this regulation modified in cells isolated from inflamed bowel, and if so, how are the changes different in cells isolated from Crohn's disease and Ulcerative Colitis bowel? The effects of IL-1beta and TGF-beta and corticosteroids on the expression of c-fos, c-jun and jun b, procollagen, collagenase, and TGF-beta mRNA will be studied in cells from normal bowel and cells from Crohn's bowel and Ulcerative colitis bowel; 2. Does TGF- beta regulate the phenotype of human intestinal smooth muscle and how do these effects on phenotype correlate with effects on the expression of collagen and collagenase. The effect of exogenous TGF-beta on normal cell morphology and the expression of smooth muscle cytoskeletal markers will be studied and correlated with the expression of collagen and collagenase. In phenotypically modified cells from Crohn's bowel expressing increased TGFb, increased spreading, increased smooth muscle-specific cytoskeletal markers, and increased collagen, does TGF-beta antagonism induce a reversion of phenotype? The effect of TGF-beta antagonists, such as Decorin, TGF-beta antibody and retinoic acid will be studied on those cell lines; 3. Do the phenotypic changes in intestinal smooth muscle cells in Crohn's disease result in increased """"""""contracture"""""""" by those cells, and what factors are involved in this process? A model of HISM cell-populated collagen lattices will be utilized to compare """"""""contracture"""""""", by cells from normal, Crohn's and Ulcerative colitis bowel, and correlate this parameter with the other phenotypic changes under examination in specific aim #2; 4. How do mast cell proteases degrade human intestinal smooth muscle cell procollagen to collagen, and do the propeptide products of this cleavage regulate procollagen expression in human intestinal smooth muscle cells?
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