Volume 134, Issue 1, Pages 156-165.e1 (January 2008) The Hydroxylase Inhibitor Dimethyloxalylglycine Is Protective in a Murine Model of Colitis  Eoin P. Cummins, Fergal Seeballuck, Stephen J. Keely, Niamh E. Mangan, John J. Callanan, Padraic G. Fallon, Cormac T. Taylor  Gastroenterology  Volume 134, Issue 1, Pages 156-165.e1 (January 2008) DOI: 10.1053/j.gastro.2007.10.012 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Intestinal epithelial cell responses to hypoxia and DMOG. (A) Treatment of cultured Caco-2 cells with ambient hypoxia induces a time-dependent nuclear accumulation of the p65 subunit of NF-κB detectable by 3 hours and maximal by 18 hours. (B) Exposure to hypoxia results in a rapid-onset activation of NF-κB signaling as measured by phosphorylation of IKKα/β, detectable and maximal within 1 hour. (C) Exposure of Caco-2 cells to either ambient hypoxia or DMOG (24 hours) activates HIF-1α. (D) Treatment with DMOG results in a rapid-onset activation of NF-κB signaling as measured by phosphorylation of IKKα/β, detectable and maximal within 15 minutes of treatment. (E) Mice were treated with PBS or DMOG for 2 days and lysates from colonic scrapings were prepared. Western blot analysis of pooled lysates shows enhanced HIF-1α stabilization in DMOG-treated animals. Positive control (ctrl) is a whole-cell extract from Hela cells treated with 1 mmol/L DMOG for 8 hours to stabilize HIF-1α. Representative Western blots are shown with accompanying pooled densitometric analysis (n = 3). (F) Mice were treated with PBS or DMOG for 5 days and whole-colon homogenates were prepared. Homogenates were examined by a specific TransAM p65 DNA binding enzyme-linked immunosorbent assay and there is a trend toward enhanced p65 NF-κB binding to a consensus NF-κB binding site in the DMOG-treated animals (P = .0518). Values reflect mean corrected absorbance at 450 nm ± SEM for n = 5 animals in each group. Gastroenterology 2008 134, 156-165.e1DOI: (10.1053/j.gastro.2007.10.012) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 DMOG treatment is protective in acute DSS colitis. Mice were exposed to 2.5% DSS in drinking water for 5 days to induce colitis with and without co-administration of DMOG. The effects of DMOG on (A) DSS-induced weight loss, and (B) DAI were measured. (C) Gross appearance of the colonic anatomy illustrates the effect of DMOG on DSS-induced colon shortening (dashed lines, DSS-treated mouse; straight lines, untreated mouse) and the formation of pelleted stools (red arrows). (D) Colon length was measured at autopsy. (E) Histologic analysis shows the effect of DMOG treatment on colonic cross-sectional histology (H&E stained). (F) Histology sections were scored blindly. N = 5–6 mice per group. Gastroenterology 2008 134, 156-165.e1DOI: (10.1053/j.gastro.2007.10.012) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 DMOG treatment prevents DSS-induced increases in inflammatory markers in the colon. (A) MPO and (B) cytokine (IL-1β, TNF-α, IL-6, and IL-12) levels were measured in colons from control, DMOG-, DSS-, and DSS/DMOG-treated mice. MPO and cytokines are presented as Units of MPO or pg of cytokine per mg of colon protein. N = 5–6 mice per group. □, Untreated; ■, DSS treated. Gastroenterology 2008 134, 156-165.e1DOI: (10.1053/j.gastro.2007.10.012) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 DMOG accelerates recovery after DSS-induced colitis. Mice were exposed to 3% DSS in drinking water for 5 days (with cotreatment with DMOG or PBS) and returned to normal drinking water for 3 more days to allow recovery to occur. DMOG or PBS cotreatment began 1 day before DSS treatment and was maintained throughout the experiment. (A) The effects of cotreatment with DMOG on clinical signs of disease (DAI) for the duration of treatment were measured. After the completion of treatment, the effects of DMOG treatment on (B) fecal occult blood and (C) histopathology were measured. Epithelial cell restitution is clearly evident in DMOG-treated mice, with no repair seen in control mice (red arrows). (D) Histopathology scoring of attenuated colon damage in mice cotreated with DMOG and DSS relative to PBS-treated mice. N = 5–6 mice per group. (A) Black triangle, PBS; red triangle, DMOG; black circle, DSS-PBS; red circle, DSS-DMOG. Gastroenterology 2008 134, 156-165.e1DOI: (10.1053/j.gastro.2007.10.012) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 DMOG prevents intestinal epithelial apoptosis in vivo. Mice were treated with PBS or 2.5% DSS with or without DMOG for 5 days and colons were removed on day 5. (A) Apoptosis of colonic epithelial cells was detected by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining and cell morphology. N = 5–6 mice per group. □, Untreated; ■, DSS. (B) Western blot analysis of pooled colonic protein extract expression of cIAP-2, Bcl-xl, and cyclooxygenase-2. , Vehicle; ■, DMOG; , vehicle + DSS; □, DMOG + DSS. (C) T84 colonic epithelial cells pre-exposed to DMOG (1 mmol/L, 24 h) showed resistance to TRAIL/cyclohexamide-induced apoptosis. ■, -DMOG; □, +DMOG. Gastroenterology 2008 134, 156-165.e1DOI: (10.1053/j.gastro.2007.10.012) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 1 Alterations in weight and colon length in DMOG- and DSS-treated mice. Mice were exposed to 3% DMOG for 5 days to induce severe pathology and returned to normal drinking water for 3 more days. (A) Animal weight and (B) colon length were measured. (C) Twenty-four–hour DMOG treatment activates HIF in mouse intestinal tissue as shown by Western blotting of 4 independent animals. Black triangle, PBS; red triangle, DMOG; black circle, DSS-PBS; red circle, DSS-DMOG. Gastroenterology 2008 134, 156-165.e1DOI: (10.1053/j.gastro.2007.10.012) Copyright © 2008 AGA Institute Terms and Conditions