Background & Aims: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor (TGF)-β is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). We investigated TGF-β signalling by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in Crohn's disease (CD) patients.
Methods: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGF-β blocking antibody or TGF-β1. TGF-β transcripts were analysed by quantitative RT-PCR. Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration.
Results: TGF-β transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGF-β transcripts, a greater pSmad2-3 response to TGF-β, increased TIMP-1, lower Smad7, increased collagen production, and reduced migration ability compared to myofibroblasts from mucosa overlying non-strictured gut. TGF-β blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared to strictured gut.
Conclusions: We identified changes in TGF-β signalling and MMP production in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.