Directly or indirectly, via IGF binding proteins, IGF-I causes increased smooth muscle cell hyperplasia, increased collagen II production and fibrosis in TNBS-induced colitis

Directly or indirectly, via IGF binding proteins, IGF-I causes increased smooth muscle cell hyperplasia, increased collagen II production and fibrosis in TNBS-induced colitis. or wildtype C57BL/6J mice by rectal administration of TNBS or ethanol vehicle. After 7 days colonic smooth muscle cells were isolated and used to prepare RNA or protein lysates. Transcript levels of IGF-IEa, IGF binding protein (IGFBP)-3, IGFBP-5, TGF-1 and collagen II were measured by quantitative RT-PCR. Corresponding protein levels were measured by western blot or ELISA. Fibrosis was measured using digital image analysis of Massons-trichrome stained histologic sections. == Results == In IGF-I(+/) mice, which express significantly lower levels of IGF-I than wildtype, the response to TNBS-induced colitis: upregulation of IGF-I, IGFBP-3, IGFBP-5, and collagen II expression, the resulting collagen deposition, and fibrosis are all significantly diminished compared to C57BL/6J wildtype controls. TGF-1 expression and its increase following TNBS administration are not altered in IGF-I(+/) mice compared to wildtype. == Conclusions == The findings indicate IGF-I is a key regulator in intestinal smooth muscle of smooth hyperplasia and excess collagen production that leads to fibrosis and longterm to stricture formation. Keywords:Stricture, Colitis, Crohns disease, smooth muscle == INTRODUCTION == Trinitrobenzene sulfonic acid (TNBS) induces chronic inflammation in murine colon that all portions of the wall, including muscularis propria, and is utilized as a model of Crohns disease1. The response of smooth muscle cells of muscularis propria to inflammation, whether from TNBS or Crohns Disease, cellular hyperplasia and hypertrophy and increased extracellular matrix production, including collagen I, results in fibrosis2,3. In Crohns disease, expression of Insulin-like Growth Factor-I (IGF-I) is increased in the intestine including muscle cells of the muscularis propria3,4. Similar events occur in animal models of inflammation: TNBS-induced and dextran sulfate-induced colitis5,6and peptidoglycan-polysaccharide-induced ileocolitis7. IGF-I is an endogenous growth factor playing a central role in the Cisplatin growth and development of visceral and vascular smooth muscle. In human intestine, IGF-I endogenous to smooth muscle cells regulates muscle cell growth of the muscularis propria in normal intestine by simultaneously stimulating proliferation and inhibiting apoptosis, and also stimulates collagen-I production8,9. While increased XE169 expression of IGF-I in the muscularis propria in Crohns Disease and in animal models of colitis is conjectured to contribute to the resulting muscle hyperplasia and fibrosis that contributes to stricture formation, this paper is the first to demonstrate this directly3,4,7,10,11. IGF-I is produced by the liver, with the main circulating form encoded by the IGF-IEa splice variant in humans, rats and mice, and also by target tissues including intestinal smooth muscle8,12. Two lines of investigation suggest that the effects of IGF-I on the smooth muscle cells of the intestine are largely Cisplatin governed by endogenous IGF-I production. First, in mice with hepatic-deleted IGF-I, intestinal muscle develops normally13. Second, mice over-expressing IGF-I develop hyperplasia of smooth muscle tissues including the intestinal muscularis propria14,15. The effects of endogenous IGF-I in intestinal smooth muscle is mediated by the cognate IGF-I receptor tyrosine kinase coupled to activation of Erk1/2 and p70S6 kinase that jointly stimulate proliferation, and collagen I gene expression, and GSK-3 that inhibits apoptosis,9,16,17. IGF-I stimulates collagen I expression via Erk1/2 in rat colon18. We recently showed that V3 integrin (the vitronectin receptor) and its ligands, vitronectin and fibronectin, Cisplatin are expressed by intestinal smooth muscle cells in humans and upregulated in Crohns disease strictures19. Occupancy of V3 integrin by these ligands increases the intensity and duration of IGF-I stimulated IGF-I receptor activation and mediates excess smooth muscle growth in patients with stricturing Crohns disease4. This study examines the role of endogenous IGF-I in the response of the smooth muscle cells of the murine muscularis propria to TNBS-induced colitis. Deletion of IGF-I or of its receptor is a lethal mutation20, thus the current study was performed in IGF(+/) heterozygous mice which have a ~70% reduction in IGF-I, and in C57BL/6J littermate controls. The results show that the effects of TNBS-induced colitis on the smooth muscle cells of the muscularis propria, upregulation of gene expression and protein levels of IGF-I, IGFBP-5 and IGFBP-3 and collagen II, smooth muscle cell growth.