Little is known about the development of fibrosis associated with chronic intestinal inflammation. Actions of Insulin-like growth factor I (IGF-I) suggest that this peptide mitogen may be relevant to the pathogenesis of intestinal fibrosis. IGF-I is a potent mitogen for fibroblasts and smooth muscle cells' and stimulates synthesis of several collagen types in vitro. We have shown that IGF-I is dramatically up-regulated in inflamed and strictured tissue from patients with Crohn's disease and in rats with peptidoglycan-polysaccharide (PG-PS) induced chronic enterocolitis, a model of Crohn's disease. In the rat model, IGF-I is over-expressed in smooth muscle-like cells in regions of intense fibrosis surrounding intestinal granulomas. The studies proposed in this application will use in vitro and in vivo models to study the role of IGF-I in the pathogenesis of intestinal fibrosis and determine the molecular mechanisms by which IGF-I induces collagen synthesis.
Our specific aims are: A) to determine if intestinal collagen types are differentially expressed in PG-PS injected rats compared with controls. Quantitation of collagen types in tissue from PG-PS injected and control rats will be performed by protein analysis of solubilized collagens using polyacrylamide gel electrophoresis and by Northern analysis using collagen specific cDNA probes. B) to determine the cellular phenotype of cells that synthesize collagen and to determine the relationship between the sites of collagen synthesis and sites of IGF-I, IGF receptor and IGF binding protein expression. Localization of collagen alpha chain mRNAs will be performed by in situ hybridization. The phenotype of cells that synthesize collagen will be determined by combined in situ hybridization for collagen mRNAs and immunohistochemistry for alpha smooth muscle actin and desmin. Localization of collagen will be compared to localization of IGF-I, the IGF type I receptor and IGF-I binding protein mRNAs. C) to determine the role of IGF-I and IGF binding protein 5 (IGFBP-5) in mediating collagen synthesis and cellular proliferation in vitro. We will use two culture systems to test the in vitro role of IGF-I and IGFBP-5 in enhancing cellular proliferation and collagen synthesis: isolated rat intestinal smooth muscle cells and isolated granulomas from rats with PG-PS induced enterocolitis. These studies will allow us to directly test the hypothesis that IGF-I mediates collagen synthesis in intestinally derived cells and will provide a simple system to study the molecular mechanisms by which IGF-I and IGFBP-5 mediate intestinal collagen synthesis. Study of IGFBP-5 is based on data showing that IGFBP-5 enhances the fibrogenic actions of IGF-I by associating with the extracellular matrix and accumulating IGF-I near its receptor. IGFBP-5 mRNA is up-regulated in inflamed intestinal tissue in rats with PG-PS enterocolitis. D) to determine if infusion of a monoclonal antibody to IGF-l affects chronic intestinal inflammation and fibrosis in vivo. We will modify IGF-I action by in vivo administration of an IGF-I monoclonal antibody in PG-PS injected rats. We will determine the changes in intestinal inflammation and fibrosis resulting from modulation of IGF-I to lend insight into the in vivo roles of IGF-I in mediating intestinal fibrosis.
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