European Journal of Clinical Investigation Vol 39 145 DOI: 10.1111/j.1365-2362.2008.02076.x Blackwell Publishing Ltd ORIGINAL ARTICLE Smad3 loss confers resistance to colorectal fibrosis ORIGINAL ARTICLE Smad3 loss confers resistance to the development of trinitrobenzene sulfonic acid–induced colorectal fibrosis G. Latella * , A. Vetuschi * , R. Sferra * , G. Zanninelli * , A. D’Angelo * , V. Catitti * , R. Caprilli † , K. C. Flanders ‡ and E. Gaudio † * University of L’Aquila, L’Aquila, Italy, † University of Roma ‘La Sapienza’, Rome, Italy, ‡ National Cancer Institute, Bethesda, MD, USA ABSTRACT Background Transforming growth factor-β (TGF-β)/Smad3 signalling plays a central role in tissue fibrogenesis, acting as a potent stimulus of extracellular matrix (ECM) protein accumulation. The aim of this study was to evaluate the potential role of Smad3 in the pathogenesis of colonic fibrosis induced by trinitrobenzene sulfonic acid (TNBS) in Smad3 null mice. Materials and methods Chronic colitis-associated fibrosis was induced in 15 Smad3 null and 13 wild-type mice by intra-rectal administration of TNBS. Each mouse received an incremental dose of TNBS (0·5–1·0 mg per week) over a 6-week period. The colon was excised for macroscopic examination and histological, morphometric and immunohistochemical analyses. For immunohistochemistry, alpha-smooth muscle actin (α-SMA), collagen types I–III, TGF-β1, connective tissue growth factor (CTGF), Smad3, Smad7, and CD3 antibodies were used. Results At macroscopic examination, the colon of Smad3 wild-type mice appeared significantly harder, thicker and shorter than that of the Smad3 null mice. Of the wild-type mice, 50% presented colonic adhesions and strictures. Histological and morphometric evaluation revealed a significantly higher degree of colonic fibrosis and accumulation of collagen in the Smad3 wild-type compared to null mice, whereas the degree of colonic inflammation did not differ between the two groups of mice. Immunohistochemical evaluation showed a marked increase in CTGF, collagen I–III, TGF-β and Smad3 staining in the colon of Smad3 wild-type compared to null mice, whereas Smad7 was increased only in null mice. Conclusions These results indicate that Smad3 loss confers resistance to the development of TNBS-induced colonic fibrosis. The reduced fibrotic response appears to be due to a reduction in fibrogenic mesenchymal cell activation and ECM production and accumulation. Smad3 could be a novel target for potential treatment of intestinal fibrosis, especially in inflammatory bowel disease. Keywords ECM, fibrosis, Smad proteins, TGF-β, TNBS-induced colitis. Eur J Clin Invest 2009; 39 (2): 145–156 Introduction Acute injury of the intestinal mucosa usually promotes a process of wound healing that restores normal tissue architecture and function. In inflammatory bowel disease (IBD), and particularly in Crohn’s disease (CD), as well as in other enteropathies, the chronic transmural inflammation and tissue damage elicits an excessive wound-healing response that leads to distortion of the tissue architecture, fibrosis, stricture, stenosis and obstruction [1,2]. Fibrosis and associated complications are major causes of surgery since, at present, there is no effective medical treatment [3]. The cellular and molecular mechanisms underlying wound healing and fibrosis still remain to be fully elucidated. Physiological fibrogenesis triggered by the onset of inflammation may lead either to tissue repair or fibrosis depending on the balance between extracellular matrix (ECM) synthesis and degradation. Fibrosis is a chronic and progressive process acting through complex cell/matrix/cytokine and growth factor interactions, but it is a reversible event [1–6]. Intestinal fibrosis is related to the abnormal accumulation of ECM proteins produced by activated intestinal mesenchymal cells [1,2,4–6]. The main intestinal mesenchymal cell types are smooth muscle cells, fibroblasts, and myofibroblasts, where the latter includes subepithelial myofibroblasts (SEMFs) and interstitial