Introduction Connective tissue growth factor (CTGF) has been implicated in the development of fibrosis in different organs, including stricture formation in Crohn's disease. As a profibrotic mediator, CTGF acts downstream and together with transforming growth factor β (TGF-β). Expression of TGF-β has previously been shown to be altered in myofibroblasts isolated from mucosal samples affected by inflammatory bowel disease. Rho-kinase is a downstream mediator of RhoA, a GTP-binding protein that is believed to regulate a number of myofibroblast functions. The aims of this study were to investigate (i) the expression of CTGF by human intestinal myofibroblasts isolated from mucosal samples obtained from different individuals and (ii) the effect of the Rho-kinase inhibitor Y-27632 on CTGF expression by intestinal myofibroblasts.
Methods Myofibroblasts were isolated from normal colonic mucosal samples (obtained from operation resection specimens) and established in pure culture. Myofibroblasts were cultured in medium containing 0.1% or 10% fetal calf serum (FCS). CTGF transcript expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of protein (in cell lysates) by Western blot analysis. Following incubation with Y-27632 (final concentration 100 μM), changes in myofibroblast cell morphology, expression of CTGF and programmed cell death (analysis of the sub-G1 region of the cell cycle by flow cytometry) were studied.
Results Studies by RT-PCR showed that when grown in 10% FCS, intestinal myofibroblast cultures from 5 mucosal tissue donors expressed CTGF transcript. Expression of CTGF protein was confirmed by western blot analysis of intestinal myofibroblasts grown in 10% FCS (n=7 donors). By contrast, when cultured in 0.1% FCS, only 2 out 5 donors expressed CTGF protein (0.1% FCS vs 10% FCS: p<0.03). Exposure to Y-27632 led to change in morphology to spindle-shaped cells, with all the myofibroblasts affected by 24 h. When cultured for 24 h with 10% FCS, Y-27632 led to a reduction in expression of CTGF protein by intestinal myofibroblasts isolated from 6 donors. However, there was no significant difference in the number of myofibroblasts undergoing programmed cell death between those cultured in medium only, compared to cells exposed to Y-27632 (events in sub-G1 region of cells cycle: 203.8 (SEM 51.62) vs 393.0 (64.91), p=0.10).
Conclusion 1. Myofibroblasts isolated from normal human colonic mucosal samples are capable of expressing CTGF.
2. The Rho-kinase inhibitor Y-27632 reduces expression of CTGF protein by colonic myofibroblasts.
3. The Rho-kinase pathway may be a suitable therapeutic target in stricturing Crohn's disease.
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