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Background Stricture formation occurs in ≈30% of patients with Crohn’s disease (CD) and is a significant cause of morbidity. Strictures are characterized by intestinal smooth muscle cell hyperplasia, smooth muscle cell hypertrophy, and fibrosis due to excess net extracellular matrix production, including collagen. Transforming growth factor-β1 (TGF-β1) has profibrotic effects in many tissues due to its ability to regulate collagen expression and extracellular matrix dynamics. We previously showed that both insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) and TGF-β1 are expressed by normal human intestinal smooth muscle cells, bind to, and activate TGF-βRII/I receptors in these cells. Methods Smooth muscle cells isolated from the muscularis propria of patients were used to prepare RNA, protein lysates, or placed into primary culture. IGFBP-3, TGF-β1, and collagen IαI expression was measured with quantitative reverse-transcription polymerase chain reaction (RT-PCR) and protein levels by enzyme-linked immunosorbent assay (ELISA) or immunoblot. Results Expression and production of IGFBP-3, TGF-β1, and collagen IαI were significantly increased specifically in smooth muscle cells isolated from regions of strictured intestine in CD compared to nonstrictured histologically normal resection margin. IGFBP-3 and TGF-β1 regulated collagen IαI expression and production via a TGF-βRII/I-dependent and Smad2/3-dependent mechanism. Upregulated (excess) collagen IαI expression and production in smooth muscle cells of strictures and basal collagen IαI in smooth muscle cells of normal margin were inhibited by immunoneutralization of IGFBP-3 or TGF-β1. Conclusions The findings indicate that upregulated endogenous IGFBP-3 and TGF-β1 expression regulates excess collagen IαI production and contributes to fibrosis and stricture formation in CD.

Background Strictures develop in >30% of Crohn’s disease. No available medication prevents stricture development in susceptible patients. In Crohn’s strictures, but not adjacent normal intestine, TGF-β1 increases in muscularis smooth muscle, increasing collagen I production and strictures. Muscle cells express αVβ3 integrin containing an RGD binding domain. The aim was to determine whether increased TGF-β1 levels in strictures was the result of latent TGF-β1, which contains an RGD sequence , binding to and activation by αVβ3; and whether cilengitide, which is an RGD-containing αVβ3 integrin inhibitor, decreases TGF-β1 activation and development of fibrosis in chronic TNBS-induced colitis Design Muscle cells were isolated from Crohn’s disease strictures and normal resection margin and from colon of rats after 42 d of chronic TNBS-induced colitis were used to prepare RNA, protein lysates and initiate primary cultures. The mechanisms leading to increased TGF-β1 activation, collagen I production and fibrosis were examined in human muscle and in rats. Human cultured cells in vitro and rats in vivo were treated with cilengitide to determine it efficacy to decrease TGF-β1-activation, collagen production and decrease the development of fibrosis. Results Latent TGF-β1 is activated by the αVβ3 RGD domain in human and rat intestinal smooth muscle. Increased activation of TGF-β1 in Crohn’s disease and in TNBS-induced colitis causes increased collagen production, and fibrosis that could be inhibited by cilengitide. Conclusions Cilengitide, a αVβ3 integrin RGD inhibitor, could be a novel treatment to diminish excess TGF-β1 activation, collagen I production and development of fibrosis in Crohn’s disease.

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