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. 2015 Jul;64(7):1120-31.
doi: 10.1136/gutjnl-2013-306484. Epub 2014 Jun 5.

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice

J D Coombes  1 , M Swiderska-Syn  2 , L Dollé  3 , D Reid  4 , B Eksteen  4 , L Claridge  5 , M A Briones-Orta  1 , S Shetty  5 , Y H Oo  5 , A Riva  6 , S Chokshi  6 , S Papa  7 , Z Mi  8 , P C Kuo  8 , R Williams  1 , A Canbay  9 , D H Adams  5 , A M Diehl  2 , L A van Grunsven  3 , S S Choi  10 , W K Syn  11
Affiliations

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice

J D Coombes et al. Gut. 2015 Jul.

Abstract

Background: Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis.

Methods: Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease.

Results: OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-β signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis.

Conclusions: OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.

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Conflict of interest statement

Disclosures / Competing Interests

On behalf of authors, I declare no Competing Interests

Figures

Figure 1
Figure 1. Osteopontin (OPN) is upregulated in human CLD and is expressed by Sox9+ liver progenitors
Liver sections from patients with NASH-cirrhosis (n = 5), ALD-cirrhosis (n = 5), PBC (n = 5), and excess normal donor livers (n = 3), were stained for OPN, and Sox9. OPN-staining was analyzed by morphometry; the number of Sox9+ cells (stained nuclei) was counted in 20 randomly-chosen high-power fields (HPF)/section. Representative photomicrographs are shown. (A) OPN staining and OPN morphometry. (B) Sox9 staining and Sox9 morphometry. (C) Sox9 (Brown) and OPN (Green)–double-immunostaining. (D) High magnification of Sox9 / OPN–double-immunostaining in NASH-cirrhosis (black arrows) (x400). Sox9 / OPN quantification by cell counting; number of double-positive cells per HPF. Results are expressed as fold change relative to normal liver and graphed as mean ± SEM. *p<0.05 vs. normal liver
Figure 2
Figure 2. OPN promotes 603B-LPC viability
603B-LPC were analyzed for OPN, Sox9 and K19 by western blot, and 603B-conditioned medium (CM) assayed using OPN ELISA. In separate experiments, LPC were treated with sham or OPN-aptamers, in the presence or absence of TGF-β. Viability/proliferation was assessed using the CCK8 assay, and apoptotic activity evaluated by caspase-3/7 activity and by sub-G1 analysis (A) OPN, Sox9, and K19 protein expression; OPN levels in 603B-CM. (B) Cell numbers under basal (non-TGF-β) conditions; 24–72 h. (C) Cell numbers under TGF-β conditions; 24–72 h (solid line: sham-aptamer-treated; dashed line: OPN-aptamer-treated). Mean ± SEM (O.D) are graphed. (D) Caspase-3/7 under TGF-β conditions; 72 h. Mean ± SEM (RFU) are graphed. (E) Sub-G1 analysis under TGF-β conditions; 72 h. % of cells in sub-G1 are graphed (F) Representative sub-G1 histograms from sham or OPN-aptamer treated 603B at 72 h. All experiments were performed in triplicate. *p<0.05 vs. respective baseline
Figure 3
Figure 3. OPN enhances 603B progenitor-associated wound healing responses
603B-LPC were treated with sham or OPN-aptamers, in the presence or absence of TGF-β for 48 h. Cells were analyzed for EMT markers. (A) Snail and Collagen 1αI mRNA. (B) E-cadherin and Id2 mRNA. Mean ± SEM were graphed. Wound-healing and transmigration studies were performed using conditions described (C–D). (C) Wound-healing under TGF-β conditions at time 0 (time of scratch) and 12 h later; migration was quantified by measuring the distance dividing the two sides of the monolayer. Mean (% wound closure) ± SEM were graphed. (D) Transmigration under TGF-β conditions was evaluated 24 h after seeding of cells, by counting crystal violet-stained cells on the underside membrane in 15 random HPF. Mean cell numbers ± SEM were graphed. *p<0.05 vs sham-aptamer. To determine if OPN modulated TGF-β signaling, 603B were treated as above, and protein analyzed. (E) Representative western blot: phospho-Smad 2/3, SnoN, Ski, Smad 7, and beta-actin. Quantitative data for these studies are shown in Supplemental Fig 5.
Figure 4
Figure 4. OPN-aptamers ameliorate fibrogenesis in CCL4 and MCD diet-treated mice
CCL4: In one group, mice (n =5/group) received twice-weekly injections of olive oil (control) or CCl4 for 6 weeks. In another group, mice receiving CCL4 were administered sham or OPN-aptamers (n =5/group) during the final week of CCL4. MCD: one group of mice (n=5/group) was fed control-chow or the methionine-choline deficient (MCD) diet for 5 weeks. Another group fed the MCD diet was treated with sham or OPN-aptamers (n =5/group) in the final week of dietary-challenge. Mice were sacrificed 24 h after final dose of aptamers, and livers analyzed. Representative staining are shown. (A–B) Sirius-red staining with morphometry, and liver hydroxyproline measurements in CCL4-treated (A), and MCD-fed (B) mice. (C–D) αSMA immunoreactivity (black arrows) and morphometry (20x fields for analysis) in CCL4-treated (C), and MCD-fed (D) mice. Results were expressed as fold change relative to control mice and graphed as mean ± SEM. *p<0.05 vs. control mice
Figure 5
Figure 5. OPN-aptamers attenuate LPC response in CCL4-treated mice
Mice were treated as described in Figure legend 4. Livers were harvested for IHC. Representative staining displayed. (A) OPN-staining and OPN-quantification (B) Sox9 staining and Sox9-quantification; % of positive cells per HPF. (C) Double immuno-staining for Sox9 (Brown) and OPN (Green). (Bottom left panel) Sox9 / OPN double staining in control mice. (Top left panel) Sox9 / OPN double staining (accumulation of Sox9 / OPN double staining) in CCL4-treated mice receiving sham-aptamers. (Top right panel) Sox9 / OPN double staining (loss of OPN staining) in CCL-treated mice receiving OPN-aptamers. (Bottom right) Sox9 / OPN quantification by cell counting; number of double-positive cells per HPF. Results are expressed as fold change relative to control mice and graphed as mean ± SEM. *p<0.05 vs. control mice. Black arrows indicate Sox9 positive cells which co-express (or should co-express) OPN.
Figure 6
Figure 6. OPN-aptamers attenuate LPC response in MCD diet-fed mice
Mice were fed control chow or the MCD diet for 5 weeks, in the presence of sham or OPN-aptamers, as described in Figure legend 4. Livers were harvested for IHC. Representative staining are shown. (A) OPN staining and OPN quantification by morphometric analysis. (B) Sox9 staining and Sox9 quantification by cell counting; % of positive cells per HPF. (C) Sox9 (Brown) and OPN (Green) – double immunostaining (magnification x400). (Left panel) Accumulation of Sox9 / OPN double positive cells in MCD-fed mice receiving sham-aptamers; (Right panel) Fewer Sox9 / OPN double positive cells in MCD-fed mice receiving OPN-aptamers. Graph shows Sox9 / OPN quantification by cell counting; number of double-positive cells per HPF. Results are expressed as fold change relative to control mice and graphed as mean ± SEM. *p<0.05 vs. control mice. Black arrows indicate Sox9 positive cells which co-express (or should co-express) OPN.
Figure 7
Figure 7. OPN-aptamers upregulate epithelial and repress mesenchymal genes in CCL4-treated mice
Mice were treated as described in Figure legend 4. (A) OPN mRNA. (B) Snail mRNA. (C) E-Cadherin mRNA. (D) Id2 mRNA. Results were expressed as fold change relative to control-treated mice and graphed as mean ± SEM. Livers were stained for Sox9 (Brown) and E-Cadherin (Green) (E-F). (E) (Top left) Sox9/E-Cadherin double-staining in control mice; (Top right) Loss of E-Cadherin staining in CCL4 mice with sham-aptamers; (Bottom left) Re-expression of E-Cadherin in Sox9+ cells in OPN-aptamer-treated mice; (Bottom right) Re-expression of membranous-E-Cadherin in OPN-aptamer-treated mice. (F) Sox9/E-Cadherin quantification by cell counting; number of double-positive cells/HPF. Results are expressed as fold change relative to control mice and graphed as mean ± SEM. *p<0.05 vs. control mice. Black arrows indicate Sox9 positive cells which co-express (or should co-express) E-Cadherin. White arrows indicate membranous-expression of E-Cadherin.
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References

    1. Friedman SL, Sheppard D, Duffield JS, et al. Therapy for fibrotic diseases: nearing the starting line. Sci Transl Med. 2013;5:167sr161. - PubMed
    1. Schuppan D, Kim YO. Evolving therapies for liver fibrosis. J Clin Invest. 2013;123:1887–1901. - PMC - PubMed
    1. Schuppan D, Afdhal NH. Liver cirrhosis. Lancet. 2008;371:838–851. - PMC - PubMed
    1. Rahimi RS, Rockey DC. End-stage liver disease complications. Curr Opin Gastroenterol. 2013;29:257–263. - PubMed
    1. Loomba R, Sanyal AJ. The global NAFLD epidemic. Nat Rev Gastroenterol Hepatol. 2013 Sep 17; doi: 10.1038/nrgastro.2013.171. Epub ahead of print. - DOI - PubMed

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