W1675 A Protective Role for HSP70 Against Indomethacin-Induced Small Intestinal Lesions

Background: Crohn's disease is complicated by intestinal fibrosis. The RAAS has been implicated in the process of fibrosis of the heart and kidney. In addition to the mineralocorticoid receptor (MR), spironolactone can affect other (FXR, PXR) nuclear receptors. We investigated whether inhibitors of the RAAS (spironolactone, canrenone, enalprilat, losartan, aliskiren) affect fibrosis in In Vitro models of intestinal fibrosis. Aims: 1) To determine whether RAAS inhibitors inhibit TGFβ-induced fibrosis in colonicmyofibroblasts and primary myofibroblasts; 2) To determine if RAAS inhibitors block TGFβ transcription; and 3) To elucidate the role of nuclear receptors FXR and PXR in spironolactone's inhibition of fibrogenesis. Methods: Human colonic myofibroblast lines (CCD-18co) or primary myofibroblasts were serum-starved for 24 hours prior to stimulation with 1 nM TGFβ or co-treatment with TGFβ and RAAS inhibitors, or TGFβ and inhibitors of FXR or PXR. Fibrogenic response was measured by QRT-PCR analysis of αSMA, CTGF, and collagen I mRNA expression in a minimum of 7 independent experiments. The fibrogenic response was confirmed by Western blotting for αSMA and collagen I protein expression in 3 independent experiments. Results: Stimulation of CCD-18co human colonic myofibroblasts with TGFβ produces a fibrogenic phenotype with a > 50-fold induction of αSMA, a 9.5-fold increase in CTGF, and a 2.5-fold increase in collagen I transcription. Spironolactone and canrenone reduce the transcription of αSMA and CTGF to untreated levels and collagen by ~7-fold. Aliskiren, enalprilat, losartan, and eplerenone significantly reduce TGFβ-induced αSMA transcription by >50%. Similar inhibition is seen with spironolactone and canrenone in primary intestinal and lung myofibroblasts. Inhibitors of FXR or PXR did not alter the fibrogenic gene activation. Treatment of CCD-18co myofibroblasts with TGFβ induces MR transcription and TGFβ transcription by ~3-fold (p <0.0001). Though spironolactone and other RAAS inhibitors block the downstream effects of TGFβ-induced fibrosis, neither spironolactone nor other RAAS inhibitors affect this TGFβ positive feedback transcriptional loop.Conclusions: Spironolactone and its metabolite, canrenone, are potent inhibitors of fibrosis In Vitro. Other RAAS pathway inhibitors reduce TGFβ-induced fibrosis to a lesser extent. TGFβ induces a positive feedback transcriptional loop in myofibroblasts. Spironolactone and other RAAS inhibitors inhibit TGFβ-induced fibrosis by a mechanism independent of TGFβ transcription and independent of the PXR and FXR nuclear receptors. The RAAS pathway may play a role in intestinal fibrosis.