Hydroxylase inhibition reduces transforming growthfactor-beta 1 induced fibrosis associated with colitis

Mario Manresa, Murtaza Tambuwala, Praveen Radhakrishnan, Jonathan Harnoss, Miguel Cavadas, Ciara Keogh, Alex Cheong, Eoin Cummins, Kim Barrett, Martin Schneider, Cormac Taylor

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ntestinal fibrosis is a severe complication of inflammatory bowel disease (IBD) that often leads to surgical interventions, highlighting an urgent need for alternative therapies. The inhibition of key oxygen‐sensing hydroxylases that regulate the response to hypoxia ameliorates inflammation in mouse models of colitis. Here we investigated the impact of hydroxylase inhibition on intestinal fibrosis. Intestinal fibrosis was induced in mice secondary to colitis through exposure to dextran sodium sulfate (DSS) in drinking water. Cultures of human colonic (CCD‐18Co) and mouse embryonic (MEF) fibroblasts were used to investigate the actions of hydroxylase inhibitors on the response to transforming growth factor‐β1 (TGF‐β1). Mice allowed to recover for 8 or 14 days after exposure to DSS developed fibrosis with profound alterations in the pattern of collagen deposition and infiltration of α‐smooth muscle actin (α‐SMA)‐positive fibroblasts. Treatment with the pan‐hydroxylase inhibitor, dimethyloxalylglycine (DMOG) during the recovery period reduced signs of fibrosis. In contrast, exposure of prolyl hydroxylase‐2+/− (PHD‐2+/−) mice to DSS resulted in severe intestinal fibrosis suggesting that the effects of DMOG are independent of the PHD‐2‐HIF axis. In vitro, hydroxylase inhibitors reduced TGF‐β1 mediated production of key fibrosis markers such as α‐SMA, Connective Tissue Growth Factor (CTGF) and collagen‐1‐α, in both CCD‐18Co and MEF, strongly suggesting an effect on TGF‐β1 signalling. Furthermore, these antifibrotic effects were present in PHD‐2+/− fibroblasts even though these cells showed enhanced responsiveness to TGF‐β1. In order to investigate the molecular mechanisms of the anti‐TGF effects of hydroxylase inhibitors, we analyzed their impact on the activation of TGF‐β1 signalling pathways. While no major effects were detected on the ability of TGF‐β1 to activate the Smad pathway, hydroxylase inhibition strongly inactivated TGF‐ERK signalling. Furthermore, areas of the intestinal mucosa of mice exposed to DSS where α‐SMA positive fibroblasts were concentrated, exhibited abundant positive phosphoERK nuclear staining. This observation highlighted the importance of the activation of the ERK pathway in fibrotic colons. Taken together, our studies showed that hydroxylase inhibition ameliorates intestinal fibrosis, likely via effects on TGF‐ERK signalling. We propose hydroxylase inhibition as a potential new therapeutic approach for the treatment of intestinal fibros
Original languageEnglish
Publication statusPublished - 1 Apr 2016

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