1. Volume 153, Issue 4, Pages 1054-1067 (October 2017)
Treatment of Intestinal Fibrosis in Experimental Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase Inhibitor 
Tom Holvoet, Sarah Devriese, Karolien Castermans, Sandro Boland, Dirk Leysen, Yves-Paul Vandewynckel, Lindsey Devisscher, Lien Van den Bossche, Sophie Van Welden, Melissa Dullaers, Roosmarijn E. Vandenbroucke, Riet De Rycke, Karel Geboes, Arnaud Bourin, Olivier Defert, Pieter Hindryckx, Martine De Vos, Debby Laukens 
Gastroenterology 
Volume 153, Issue 4, Pages (October 2017)
DOI: /j.gastro
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2. Figure 1
ROCK activity is elevated in inflamed and fibrotic intestinal tissue. (A) ROCK activity was measured by immunoassay in biopsies of healthy ileum (n=4), inflamed ileum of CD patients (CD-I, n=5), ileum from UC patients with backwash ileitis (UC, n=3), and the mucosa of the resected ileum of CD patients with fibrostenotic disease, collected from non-stenotic (CD-NS, n=5) and stenotic regions (CD-S, n=5). (B) Immunohistochemical staining for ROCK in the stenotic ileum of a CD patient showing immunopositivity in (1) epithelial cells, (2) subepithelial fibroblasts, (3) submucosal fibroblasts, (4) endothelial cells, and (5) muscle cells. (C) Representative Masson’s trichrome images of the distal colon in different stages of the development of chronic colitis induced by DSS 6, 9, and 12 weeks (wks) after the first DSS administration and after adoptive T cell transfer (n=8 in each group, bar=200 μmol/L). (D) Colonic inflammation and (E) fibrosis scores. (F) ROCK activity in full-thickness distal colonic tissue during different stages of chronic DSS and adoptive T cell transfer. Data are the mean ± SEM. *P < .05; $P < .05, $$P < .01, $$$P < .001 compared with the group not treated with DSS.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Local ROCK inhibition attenuates the development of fibrosis in chronic DSS-induced colitis. C57BL/6J mice were subjected to 3 cycles of 2.5% DSS for 1 week followed by 2 weeks of recuperation. Mice were treated preventively by oral gavage with 3 mg/kg per day AMA0825 from the start of DSS administration (n=8 in each group). (A) Colonic weight/length ratio. (B) Qualitative and quantitative fibrosis scores, and representative Masson's trichrome images of the distal colon (bar=200 μmol/L). (C) Colonic transcript levels of collagen I (Col1a1) and Ctgf measured by quantitative real-time polymerase chain reaction (qRT-PCR), and colonic protein concentrations of TGFβ1 and IL6. (D) MMP-2, -3 and -9 measured by Luminex bead technology. (E) Quantification of alpha smooth muscle actin (αSMA) by immunohistochemical staining of the distal colon (200×). Data are the mean ± SEM. NRQ, normalized relative quantities. *P < .05, **P < .01, ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Anti-TNF combined with AMA0825 prevents fibrosis, but anti-TNF alone does not. CD4+CD25-CD62L+ naive T cells were injected IP in CB-17 SCID mice on day 0 (adoptive transfer, AT). Mice developed symptoms of colitis starting on week 2, at which time therapy was initiated. Mice were treated with 25 mg/kg anti-TNF IP 3 times per week, with 3 mg/kg per day AMA0825 by oral gavage, or with a combination of both (n=10 in each group). Placebo-treated mice received 25 mg/kg IgG1 IP 3 times per week and oral gavage of vehicle. (A) Changes in body weight, (B) histologic inflammation, (C) colonic cytokine concentration, (D) Masson's trichrome-based fibrosis, and (E) colonic TGFb1 and IL6 concentrations. Data are the mean ± SEM. NS, not significant; *P < .05; **P < .01; ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Local inhibition of ROCK by AMA0825 reverses end-stage fibrosis. C57BL/6J mice were subjected to 3 cycles of 2.5% DSS for 1 week followed by 2 weeks of recuperation. (A) Treatment was initiated on week 6, after completion of 2 cycles of DSS treatment, when intestinal fibrosis was already present. Treatment consisted of 3 or 10 mg/kg of AMA0825 per day, or a placebo, for 3 or 6 weeks (n=7 in each group). (B) Colonic fibrosis assessed by Masson’s trichrome staining. (C) Distal colonic protein concentrations of IL6, TGFβ1 and (D) MMP-2, - 3, -8, -9, and -12 measured using Luminex bead technology. (F) Quantification of alpha smooth muscle actin (αSMA) by immunohistochemical staining of the distal colon. Data are the mean ± SEM.*P < .05, **P < .01; $P < .05, $$P < . 01 compared with the group not treated with DSS.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
ROCK inhibition abrogates the fibroblast to myofibroblast transition and their subsequent activation. Primary HIFs were stimulated for 48 hours with 1 ng/mL TGFβ1 and a dose-range of AMA0825 and (A) ROCK activity (left panel) and IL6 release (right panel) was measured (n=3 per condition). Next, cells were stimulated with 1 ng/mL TGFβ1, a combination of 1 ng/mL TGFβ1 and 1 μmol/L AMA0825, or the vehicle control (n=3 per condition). (B) Representative images and quantification of F-actin and vimentin staining. (C) Transcript levels of COL1A1, TGFβ1, and CTGF analyzed by qRT-PCR. (D) Representative western blot images of CTGF expression. (E) MMP-2 and MMP-12 protein concentrations in supernatant samples measured using Luminex bead technology. (F) The former experiment was repeated using an experimental set-up (left panel) to determine the reversibility of myofibroblast formation by exposing fibroblasts to 1 ng/mL TGFβ1 or vehicle for 48 hours, after which the supernatant was discarded and the cells were incubated further for 24 hours with vehicle, TGFβ1 alone, or TGFβ1 combined with 1 μmol/L AMA0825. IL6 protein concentrations (right panel) in supernatant measured using Luminex bead technology. Data are the mean ± SEM. NRQ, normalized relative quantities. *P < .05, **P < .01, ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
ROCK inhibition by AMA0825 amplifies the autophagic response in fibroblasts and controls collagen, IL6, and MMP expression. (A) Autophagic flux (LC3B-II/I ratio quantified by western blot analysis) in fibroblasts isolated from C57/BL6J mice undergoing repeated cycles of DSS treatment with or without oral treatment with 3 mg/kg per day AMA0825 (n=6 per group). (B) Representative electron microscopy images (5000×) of primary HIFs treated for 48 hours with 1 ng/mL TGFβ1, a combination of 1 ng/mL TGFβ1 and 1 μmol/L AMA825 or the vehicle control. Autophagosomes (arrows) were counted in 10 cells per condition in 4 images per cell. (C) Primary HIFs were treated for 48 hours with 1 ng/mL TGFβ1 or a combination of TGFβ1 and 1 μmol/L AMA0825 in the presence or absence of 10 nmol/L bafilomycin A1 (n=3 per condition). The expression of p62 and collagen I was quantified by western blot analysis. (D) Collagen I and IL6 secretion was measured using western blot and Luminex bead technology, respectively. (E) Transcript levels of COL1A1, IL6, and MMP2 analyzed by qRT-PCR. (F) Protein concentration of IL6 in the supernatant mouse intestinal fibroblasts isolated from Atg16l1 hypomorphic (ATG16L1HM/HM) and wild-type mice (n=4 per group) treated for 48 hours with 1 ng/mL TGFβ1, a combination of 1 ng/mL TGFβ1 and 1 μmol/L AMA0825, or the vehicle control. Data are the mean ± SEM. NRQ, normalized relative quantities; NS, not significant. *P < .05, **P < .01; $P < .05, $$P < .01 compared with the control group.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
AMA0825 reduces pro-fibrotic protein secretion from human ileal stenotic biopsies and from primary ileal fibroblasts. (A) Ileal biopsies obtained from stenotic regions of CD patients during colonoscopy (n=8) were incubated for 24 hours with 5 μmol/L AMA0825 or vehicle. Levels of MMP-2, -3,-9, -12, TGFβ, and IL6 were determined in the supernatant using Luminex bead technology (n=3 per condition). (B) Fibroblasts were isolated from resection specimens of healthy ileum (n=2) and from non-stenotic (CD-NS) and stenotic (CD-S) resection specimens from CD patients (n=6). Cells were stimulated for 24 hours with 1 ng/mL TGFβ1 in the presence of 1 μmol/L AMA0825 or the vehicle. The protein concentration of IL6 was determined in the supernatant using Luminex bead technology. Data are the mean ± SEM. NS, not significant. *P < .05, **P < .01, ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
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9. Supplementary Figure 2
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10. Supplementary Figure 3
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11. Supplementary Figure 4
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12. Supplementary Figure 5
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13. Supplementary Figure 6
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14. Supplementary Figure 7
Gastroenterology  , DOI: ( /j.gastro )
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15. Supplementary Figure 8
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16. Supplementary Figure 9
Gastroenterology  , DOI: ( /j.gastro )
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