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. 2008 Jan;134(1):145-55.
doi: 10.1053/j.gastro.2007.09.033.

Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition

Andreas Robinson  1 , Simon Keely, Jörn Karhausen, Mark E Gerich, Glenn T Furuta, Sean P Colgan
Affiliations

Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition

Andreas Robinson et al. Gastroenterology. 2008 Jan.

Abstract

Background & aims: A number of recent studies have implicated tissue hypoxia in both acute and chronic inflammatory diseases, particularly as they relate to mucosal surfaces lined by epithelial cells. In this context, a protective role for the transcriptional regulator hypoxia-inducible factor (HIF) was shown through conditional deletion of epithelial HIF-1alpha in a murine model of colitis. Here, we hypothesized that pharmacologic activation of HIF would similarly provide a protective adaptation to murine colitic disease.

Methods: For these purposes, we used a novel prolyl hydroxylase (PHD) inhibitor (FG-4497) that readily stabilizes HIF-1alpha and subsequently drives the expression downstream of HIF target genes (eg, erythropoietin).

Results: Our results show that the FG-4497-mediated induction of HIF-1alpha provides an overall beneficial influence on clinical symptoms [weight loss, colon length, tissue tumor necrosis factor-alpha (TNFalpha)] in murine trinitrobenzene sulfonic acid (TNBS) colitis, most likely because of their barrier protective function and wound healing during severe tissue hypoxia at the site of inflammation.

Conclusions: Taken together these findings emphasize the role of epithelial HIF-1alpha during inflammatory diseases in the colon and may provide the basis for a therapeutic use of PHD inhibitors in inflammatory mucosal disease.

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Figures

Figure 1
Figure 1. Influence of PHD inhibitors FG-4442 and FG-4497 on HIF activation
HeLa cells were transfected with HRE-reporter-gene construct, exposed with FG-4442 (A) or FG-4497 (B) for 24 hours in normoxic and hypoxia (pO2 20 torr) and then assayed for luciferase activity. Data are expressed as mean ± SD luciferase/protein, and are pooled from 3 independent experiments with 3 samples per condition, where * p<0.025 and # is p<0.05. (C) HeLa cells were exposed to indicated concentrations of FG-4497 for 6hr and probed for HIF-1α stabilization by western blot. Representative blot from n = 3.
Figure 2
Figure 2. Influence of PHD inhibitor FG-4497 on EPO and EPO endpoints in vivo
In panel A, Swiss Webster mice were administered FG4497 (one dose 60mg/kg) IV (black bars) or PO (open bars). Animals were sacrificed after 4 and 6 hours, respectively, and plasma EPO was measured by human ELISA kit. Data are expressed as mean ± SD EPO (mIU/ml) and are pooled from 3 animals in each condition where * p<0.025. In panels B and C, animals were administered FG-4497 orally at the indicated doses on Monday, Wednesday and Friday (day 1, 3 and 5) and, hemoglobin (panel B) and hematocrit (panel C) were determined on day 7. Data are expressed as mean ± SD are pooled from 6 animals in each condition where * p<0.025.
Figure 3
Figure 3. Screen of PHD isoform expression and influence of FG-4497 on intestinal permeability: role of HIF-1α
A: Screen of murine PHD isoform expression in wild-type mice. Total RNA was obtained from mucosal scrapings (enriched in epithelial cells) and assessed for expression of PHD-1, -2 and-3 by RT-PCR (30 cycles), relative to actin controls. B: Hif1a WT and conditional hif1a-null mice were administered FG-4497 (60mg/kg IP) or PBS (vehicle) for 20 h, gavaged with FITC-dextran for an additional 4h and and intestinal permeability was quantified as serum FITC-dextran. Data are expressed as mean ± SD relative FITC-dextran (relative to WT control) and are pooled from 4 animals in each condition. C: Quantitation of serum Texas Red-conjugated E. coli as a measure of intestinal permeability. Hif1a WT and conditional hif1a-null mice were administered FG-4497 (60mg/kg IP) or PBS (vehicle) for 20 h, gavaged with Texas Red-conjugated E. coli and subjected to room air (Nmx) or hypoxia (Hpx, 4h at 8% O2, 92% N2) as indicated, harvested and serum extracted to measure LPS levels. Data are expressed as mean ± SD serum LPS (ng/ml) and are pooled from 3−5 animals in each condition where * p<0.025 compared to vehicle controls and # p<0.05 compared to wild-type animals. Hif1a WT and conditional hif1a-null mice were subjected to induction of TNBS colitis at day 0. Controls received TNBS vehicle alone.
Figure 4
Figure 4. Documentation of tissue hypoxia in colitis and influence of conditional hif1a mutation on colitis outcomes
Panel A shows localization of EF5 staining in colonic section from vehicle control animal at day 3. Panel B shows corresponding H&E stain from the same block. Panel C reflects EF5 localization in sections from the distal colon in TNBS exposed animals demonstrating intense EF5 immunofluorescence overlying the area of inflammation. Panel C demonstrates H&E staining of from the same block displaying intense inflammatory infiltration. In panel E, body weight was monitored following induction of TNBS colitis (* p<0.025 by ANOVA). In panel F, colon length was measured at the time of sacrifice. Conditional hif1a-null mice displayed significant colon shortening compared to their WT littermates after TNBS administration (*p<0.025 mutant vs. littermate TNBS mice). Data are expressed as mean ± SD percent colon length change and are pooled from 3−5 animals in each condition.WT (n=4) and mutant (n=5).
Figure 5
Figure 5. Influence of FG-4497 on changes of body weight following induction of TNBS colitis
Wild-type mice received vehicle or FG-4497 at indicated concentrations at day −1, day 0 and day +1. Mice were subjected to induction of TNBS colitis at day 0. Controls received TNBS vehicle. Body weight was monitored following induction of TNBS colitis (* p<0.025 by ANOVA) in animals receiving FG-4497 at 40mg/kg (panel A) or 20mg/kg (panel B). Panel C represents a weight loss summary on day 3. Data are expressed as mean ± SD percent change in initial body weight and are pooled from 4−6 animals in each condition, where * p<0.025 in animals receiving vehicle as compared to FG-4497.
Figure 6
Figure 6. Influence of FG-4497 on changes in colon length and tissue TNFα and IFNγ
Wild-type mice received vehicle or FG-4497 at indicated concentrations at day −1, day 0 and day +1. Mice were subjected to induction of TNBS colitis at day 0. Controls received TNBS vehicle. Panel A depicts histological sections on day 3 post-TNBS from animals receiving vehicle or FG-4497 (40mg/kg). In panel B, colon length was measured at sacrifice and compared between animals receiving FG-4497 at 40mg/kg or 20mg/kg. Panel B represents TNFα analysis by real-time PCR in tissue derived from animals exposed to a combination of TNFα and FG-4497. Data are expressed as mean ± SD and are pooled from 4−6 animals in each condition, where * p<0.025 and **p<0.01.
Figure 7
Figure 7. Influence of FG4497 on collagen gel contraction
NIH 3T3 fibroblasts stably transfected with a luciferase-HRE (NIH3T3/HIF-luc) were incorporated into collagen gels with and without indicated concentrations of FG-4497. Panel A shows HRE-luciferase responses to PHD inhibitor FG-4497, where indicated * p<0.025 and ** p<0.01. Panel B shows images of contracted of gel matrices in response to FG4497 for 24h. Collagen gel matrices were incubated with (i) 0, (ii) 1 nM, (iii) 10 nM, (iv) 100 nM and (v) 1 μM FG4497. Gel surface area quantified in terms of total pixel number using ImageJ, where indicated * p<0.025 and ** p<0.01. Panel C depicts the correlation between FG-4497-induced HIF activity and collagen gel contraction.
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