bs_bs_banner
Journal of Gastroenterology and Hepatology (2014) 29 (Suppl. 2)
Basic Science Liver Exercise slows growth of dysplastic hepatocytes by improving insulin sensitivity and enhancing DNA damage surveillance pathways in mice genetically predisposed to obesity and diabetes E ARFIANTI,1 V BARN,1 WG HAIGH,2 GN IOANNOU,2 N TEOH,1 G FARRELL1 1 Liver Research Group, ANU Medical School, The Canberra Hospital, ACT, 2Division of Gastroenterology, Veterans Affairs Puget Sound Health Care System and University of Washington, Seattle, WA, Australia Background: We previously reported that obesity and diabetes accelerate diethylnitrosamine (DEN)-induced hepatocarcinogenesis in Alms1 mutant (foz/foz) NOD.B10 mice, replicating the increased hepatocellular carcinoma (HCC) risk in obese, diabetic patients. Last AGW we reported an association between accelerated hepatocarcinogenesis with hyperinsulinemia/hyperglycemia-induced Akt/mTORC1 activation and Nrf1/2-mediated metabolic reprogramming.1 In the present study, we investigated whether exercise sufficient to reduce the rate of weight gain, reduces growth of dysplastic hepatocytes and HCC development in DENinjected foz/foz mice. Methods: Male foz/foz and wild-type (WT) littermates were injected with DEN (10 mg/kg i.p.) day 12–15 of age; controls were injected with vehicle (saline). They were then randomly assigned either to cages provided with an exercise wheel (from 4 wks of age, until 12 or 24 wks of age) or housed similarly without an exercise wheel. Dysplastic hepatocytes were identified by glutathione S-transferase pi (GST-pi) immunohistochemistry (IHC), protein and phospho-protein expression by immunoblotting and IHC, gene expression by semi-quantitative real-time PCR, glucose tolerance by i.p. glucose tolerance test, hepatic lipid content using HPLC, and oxidative status by reduced (GSH) and oxidized glutathione (GSSG). Results: Mice provided with in-cage exercise wheels ran (on average) 4 km/day, irrespective of whether they were WT or foz/foz. In association with such physical activity, foz/foz mice maintained weight gain similar to WT mice, at least until 12 wks of age. At 12 wks of age, GST-pi immunostaining showed a significant reduction in the number of dysplastic hepatocytes in exercising foz/foz mice compared to foz/foz littermates without exercise wheel provision (these mice are observed to be inactive). The exercise-associated reduction in number of pre-neoplastic hepatocytes correlated with prevention of excessive weight gain and adiposity, compared to their non-exercising littermates. Exercise also improved insulin sensitivity in foz/foz mice, as indicated by lower fasting blood glucose, reduced serum insulin and enhanced glucose tolerance. Improvement of insulin signaling was evident in livers of exercising mice by upregulation of insulin receptor substrate-2 (IRS-2) protein and attenuation of hepatic lipid accumulation, particularly decreased triglyceride and cholesterol ester levels. Interestingly, exercise increased rather than decreased GSSG levels in livers from foz/foz mice, suggesting that exercise increases generation of reactive oxygen species; such a link has been previously linked to the capacity of exercise to enhance insulin sensitivity.2 Despite the amelioration of insulin resistance by exercise in foz/foz mice (which usually develop obesity and diabetes), there was no difference in Akt/ mTORC1 or AMPK activation, and exercise had no effect on hepatic TNFα and MCP-1 expression. We previously observed in this model that obesity-promoted hepatocarcinogenesis is associated with Nrf1/2mediated shuttle of glucose and glutamine metabolism into purine synthesis.3 Nrf1/2 signaling was downregulated by exercise, inferring decreased metabolic activity to support hepatocellular proliferation. There was a parallel increase in activation of the Chk2/p53 cell cycle regulatory
pathway, associated with downregulation of cyclin E1. The resultant changes in cell cycle regulatory control likely contribute to the reduced number of dysplastic hepatocytes in exercising foz/foz mice compared to their overweight inert littermates. Analysis of another cohort of mice at 24 wks is underway to establish whether changes at 12 wks translate to reduction of HCC at 6 mth after DEN injection. Conclusions: Exercise prevents growth of dysplastic hepatocytes in the early (premalignant) stage of DEN-induced HCC in mice genetically predisposed to obesity and diabetes. This is associated with increased insulin sensitivity (including in the liver), reduced hepatic lipid content, suppression of cyclin E1 and enhancement Chk2/p53 cell cycle control. Whether this is sufficient to delay DEN hepatocarcinogenesis in foz/foz mice will be apparent by August 2014. References 1. Arfianti E, Lee SS, Heydet D, Larter C, Barn V, Teoh NC, Farrell GC. Enhanced growth of dysplastic hepatocytes is associated with activation of Akt/mTORC1 pathway in a murine model of hyperphagic-obesity. J Gastroenterol Hepatol 2013, 28(Suppl 2), 3. 2. Ristow M, Zarse K, Oberbach A, Klöting N, Birringer M, Kiehntopf M, Stumvoll M, Kahn CR, Blüher M. Antioxidants prevent healthpromoting effects of physical exercise in humans. Proc Natl Acad Sci U S A 2009, 106(21), 8665–8670. 3. Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, Yamamoto M, Motohashi H. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell 2012, 22(1), 66–79.
TLR9 may be a double-edged sword in the liver: is it pathogenic or protective in NASH? AR MRIDHA,1 F HACZEYNI,1 MM YEH,2 G HAIGH,3 V BARN,1 H AJAMIEH,1 JM HAMDORF,4 L ADAMS,5 NC TEOH,1 GC FARRELL1 1 Liver Research Group, Australian National University Medical School at the Canberra Hospital, Garran, ACT, Australia, 2Department of Pathology, University of Washington, Seattle, WA, USA, 3Department of Gastroenterology, University of Washington, Seattle, WA, USA, 4School of Surgery, University of Western Australia, Crawley, WA, Australia, 5School of Medicine and Pharmacology, University of Western Australia, Crawley, WA, Australia Background: Inflammation with macrophage recruitment and activation as characteristic features is a hallmark of non-alcoholic steatohepatitis (NASH) vs simple steatosis in NAFLD, while NF-κB and c-Jun/AP-1 are invariable pro-inflammatory signals. In addition to effects of lipids and pro-oxidants, such signaling could be triggered by cytokine or pattern recognition receptors, such as toll-like receptors (TLRs). TLR4 and TLR9 have both been implicated in nutritional depletion models of NAFLD, but TLR9 is a “master switch” of macrophage recruitment. Here we first measured TLR4 and 9 expression in human and mouse livers showing NASH vs simple steatosis, then studied the roles of TLR9 for inflammatory recruitment to fatty livers and for pathways to hepatocyte injury in NASH. Methods: Liver biopsies of patients (n = 7–9/grp) with NASH, simple steatosis or healthy controls were used to measure mRNA expression of TLR subtypes. Female wildtype (Wt), appetite-dysregulated Alms1 mutant (foz/foz) and Tlr9−/− mice were fed chow or an atherogenic (Ath) diet containing 0.2% cholesterol (n = 6–14/grp). Steatosis, liver inflammation and macrophage and neutrophil recruitment, fibrosis, NF-κB and c-Jun activation, cytokines/chemokines, circulating endotoxin, and markers of hepatocyte injury were assessed. We created bone marrow (BM) chimeric mice to examine the role of TLR9 on myeloid-derived vs parenchymal
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
1
Basic Science Liver
cells in Ath-induced NAFLD, studied effects of TLR9 deletion on susceptibility of primary hepatocytes to palmitic acid lipotoxicity and endotoxin, and BM macrophage cultures to determine the effects of CpG DNA (TLR9 ligand), necrotic media and LPS/IL-4 on M1/M2 polarization. Results: In human NASH, hepatic TLR4 variant 3 (but not variants 1 and 4) and TLR9 mRNA levels were higher than simple steatosis or control. In both Ath-fed Wt and foz/foz mice with NASH, Tlrs-4, 7, 9 transcripts increased, with similar pattern for TLR4 and 9 proteins. In Ath-fed Tlr9−/− mice, liver necro-inflammatory score and fibrosis markers were substantially diminished compared with Wt, despite similar steatosis and hepatic lipid levels. Likewise, Ath feeding failed to increase NF-κB and c-Jun activation, macrophage/neutrophil infiltration and pro-inflammatory Th1 cytokines in Tlr9−/− mice compared to major increases in Wt. Conversely, expression of anti-inflammatory Th2 cytokines (IL-4, IL-10) was not different. Despite less inflammation in Ath-fed Tlr9−/− vs Wt mice, hepatocyte damage (serum ALT, high mobility group box 1 [HMGB-1], CK-18, asialoglycoprotein [ASGPR] levels) and circulating endotoxin levels were higher. We interpret these changes as reflecting enhanced necrosis (and/or necroptosis) in response to endotoxemia; correspondingly, livers showed increased RIP3 (necrosis marker) and MLKL (necroptosis) expression. Further, Tlr9−/− hepatocytes were more susceptible to palmitic acid and endotoxin-induced injury than Wt. Using BM chimeras, we showed that TLR9 in BM-derived myeloid cells and not hepatocytes at least partially mediates Ath diet-induced hepatic injury. This is supported by our observation that, compared to Wt, Tlr9−/− BM-derived macrophages are resistant to activation by CpG DNA, necrotic mediators and LPS induced M1 polarization to produce pro-inflammatory cytokines. Conclusion: These novel data in human liver, in mice with metabolic syndrome-related NASH and with the atherogenic dietary model of NAFLD indicate that TLR9 activation is a critical pro-inflammatory trigger in NASH that is likely mediated via macrophages. In addition, however, TLR9 appears to confer hepatocyte protection in NASH as TLR9-deleted cells are more susceptible to lipotoxicity and endotoxin-induced necrosis. If this is correct, TLR9 blockade may not be an attractive therapeutic approach in NASH because, while it could dampen macrophage activation, it could also abrogate an intrinsic hepatoprotective pathway against lipotoxic molecules and gut-derived pathogen-associated molecular patterns.
(H&E), sirius red, and immunofluorescence performed for T-cadherin, adiponectin, CD31, F4/80, and alpha smooth muscle actin (α-SMA). cDNA was generated from tissues and cells, and quantitative polymerase chain reaction (qPCR) undertaken for T-cadherin, AdipoR1, AdipoR2, F4/80, CD68, Tumor necrosis factor-α (TNF-α), α-SMA, Tissue metallopeptidase inhibitor 1 (TIMP1) and Type I collagen (Col1). Through western blots analyses protein levels of 5′ adenosine monophosphateactivated protein kinase (AMPK), phospho-AMPK, Protein Kinase B (Akt), phospho-Akt, mammalian target of rapamycin (mTOR), and phospho-mTOR were determined. Migration and proliferation assays were undertaken with primary HSCs treated with adiponectin and Tcad siRNA. Results: Significantly, we find that liver fibrosis was increased in AdipoKO, and reduced in Tcad KO and unchanged in dKO mice compared to WT control. Immunofluorescence shows that T-cadherin is present on HSCs and colocalizes with adiponectin. In fibrotic Adipo KO mice, there was a significant decrease in T-cad expression in the liver. In WT mice, compared to AdipoR1 and -R2, only T-cad expression was increased during fibrosis. In in vitro assays, the administration of adiponectin to HSCs reduced Col1 and α-SMA expression, and the knocking down of T-cad further reduced their expression. The treatment of HSCs with adiponectin in the absence of T-cad further increased TIMP1 expression levels. Adiponectin treatment reduced HSC proliferation and migration, and the silencing of T-cad with siRNA only reduced HSC proliferation. Western blot revealed that adiponectin treatment in the absence of T-cad coincided with increased phospho-AMPK and reduced phospho-mTOR and phospho-AKT expression. Conclusion: These data show that the interaction of adiponectin and T-cadherin is important in mediating liver fibrosis. The absence of T-cadherin leads to a significant reduction in fibrosis in the presence of adiponectin. The absence of both adiponectin and T-cadherin leads to fibrosis similar to that observed in WT mice. At the molecular level we find that T-cadherin absence in HSCs influences their proliferation and growth signals. We are now further investigating the mechanistic events leading to these changes.
The role of adiponectin and T-cadherin interactions in liver fibrosis B ALZAHRANI,1,2 J GEORGE,1,2 L HEBBARD1,2 1 Storr Liver Unit, Westmead Millennium Institute, PO Box 412, Darcy Road, Westmead, NSW 2145, Australia, 2 Sydney Medical School, University of Sydney, NSW, Australia
Hepatic microRNA expression is altered in a murine model of iron and fat co-mediated liver injury CJ MCDONALD,1 Y YUAN,2 N SANTRAMPURWALA,2,3 DHG CRAWFORD,2,3 VN SUBRAMANIAM1,2,3 1 The QIMR Berghofer Medical Research Institute, Brisbane, Australia, 2School of Medicine, The University of Queensland, Brisbane, Australia, 3Gallipoli Medical Research Centre, Greenslopes Private Hospital, Brisbane Australia
Introduction: Liver fibrosis is the scarring process that represents the liver’s response to injury. Adiponectin has been shown to have an important role in the regulation of fibrosis, as adiponectin null mice have greater levels after carbon tetrachloride (CCl4) treatment. Adiponectin binds to three receptors: AdipoR1, AdipoR2 and T-cadherin. Our unpublished studies suggest that AdipoR1 and AdipoR2 null mice have unchanged fibrosis after CCl4 treatment. The role of T-cadherin in hepatic biology is unknown. Thus, using mouse models we examined the role of adiponectin and T-cadherin interactions in liver fibrosis. Methods: We used four mouse models: wild-type (WT), adiponectin (Adipo), T-cadherin (T-cad) knock-out and adiponectin T-cadherin double knock-out (dKO) mice. Liver injury was promoted by twice weekly intraperitoneal injections of carbon tetrachloride (CCl4) for 12 weeks. Control mice were injected with corn oil alone. Primary rat hepatic stellate cells (HSC) were isolated, and treated with T-cadherin siRNA followed by subsequent treatment with full length recombinant adiponectin. Liver tissues were examined for pathology changes by haematoxylin and eosin
Introduction: HFE-hemochromatosis and non-alcoholic fatty liver disease are both common conditions in the developed world and often occur in conjunction. Co-occurrence of these disorders appears to potentiate greater liver injury than either alone; the underlying mechanisms are poorly understood. In this study we have investigated the regulation of hepatic microRNA (miRNA) expression in a mouse model of iron and fat co-mediated liver injury. Methods: Hfe-knockout mice were fed either control or high calorie diets. Mice were sacrificed after 20 weeks of treatment. Small RNA was extracted from liver tissue and profiled using next-generation miRNAsequencing and differentially expressed miRNAs identified. mRNA-seq was available from these same mice, and was utilized to perform combination analysis (which identifies which differentially expressed mRNAs are potential targets of differentially expressed miRNAs) and correlation analysis (which compares the level of expression of all identified miRNAs with the levels of expression of all of their potential target mRNAs) to identify miRNA-mRNA pairs of interest.
2
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Basic Science Liver
Results: 39 differentially expressed miRNAs were identified. Of these 10 have published links to relevant pathologies including miRNAs-190 and -199 which have both been linked to hepatic fibrosis, miRNA-223 which has been linked to hepatocellular carcinoma and obesity-associated adipose tissue inflammation, and miRNA-103 which has been linked to glucose homeostasis and insulin sensitivity. Combination analysis identified 49 miRNA-mRNA pairs in which both the miRNA and its target mRNA were differentially expressed including miRNA-199a-3p for which nine of its target mRNAs are differentially expressed. Correlation analysis identified 424 miRNA-mRNA pairs with a Pearson correlation coefficient > ±0.8 and p < 0.05. Conclusions: This study demonstrates a dysregulation in hepatic miRNA expression in the setting of combined iron overload and steatosis. Ten miRNAs are identified which are linked to relevant pathologies supporting the validity of this study, and a further 29 miRNAs which have not previously been liked to either hepatic or lipid pathology where also identified. Further, the integration of mRNA data has identified potential mechanisms of action for several of the differentially expressed miRNAs.
revealed accumulation of immature erythroblasts in the spleens of the Tmprss6−/−/Tfr2−/− mice, suggesting an important role for Tfr2 in erythroid differentiation that may be mediated by lower erythropoietin expression in the kidney. Conclusions: Our results indicate that matriptase-2 predominates over Hfe and Tfr2 in hepcidin regulation in the liver. These findings indicate that therapies aimed at inhibiting Mt-2 activity would be beneficial in treating iron overload in patients with HH caused by mutations in HFE and/or TFR2. Furthermore, we have also uncovered an important role for erythroid-expressed Tfr2 in the regulation of erythropoiesis that is separate from its accepted role as a regulator of iron homeostasis in the liver.
Investigation of mice deficient in matriptase-2, Hfe and transferrin receptor 2 reveals a novel non-hepatic role for Tfr2 in erythropoiesis DF WALLACE,1,2 ES SECONDES,1 G RISHI,1,2 L OSTINI,1 CJ MCDONALD,1 SW LANE,2,3 JD HOOPER,4 C LOPEZ-OTIN,5 VN SUBRAMANIAM1,2 1 Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia, 2School of Medicine, University of Queensland, Brisbane, Queensland, Australia, 3Translational Leukaemia Research Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia, 4Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia, 5 Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain
Introduction: Hepatic stellate cells (HSCs) are responsible for collagen deposition leading to fibrosis following liver injury/inflammation. Serum levels of the acute phase protein ferritin are elevated in inflammation and act as an indicator of disease severity in chronic liver disease. We have previously demonstrated that H-subunit ferritin actually contributes to this process as a pro-inflammatory mediator in HSC via an iron-independent, NFkappaB-regulated signaling pathway, inducing the expression of cytokines IL-1beta, IL-6 and RANTES. Aims: To decipher the molecular events at the level of the plasma membrane and the endocytic pathways that mediate the pro-inflammatory response of Ferritin in HSC. Methods: Primary rat HSCs were treated with 10 nM H-ferritin for 0–24 hrs. HSCs were pre-treated with inhibitors of microtubule formation (colchicine, nocodazole), lysosomal acidification (chloroquine) and intracellular protein transport (monensin), dynamin-2-dependent internalization (Dyngo-4), clathrin-coated pit (CCP) internalization (PitStop2), lipid raft/caveolae formation (beta-methyl cyclodextrin, beta-MCD) prior to ferritin stimulation. qPCR was used to determine relative expression of Ferritin-induced transcripts. Results: Colchicine or nocodazole had no significant effect on ferritininduced expression of IL-1beta suggesting that microtubule-dependent endocytosis is not necessary for signaling. However, inhibition of CCP endocytosis and dynamin-dependent endocytosis in HSC totally or partially abolished Ferritin-induced pro-inflammatory response, respectively. Conversely, betaMCD–induced disruption of lipid rafts/caveoolae exacerbated response to Ferritin. Moreover, monensin treatment resulted in a 75% reduction in ferritin-induced IL-1beta expression while chloroquine completely abolished IL-1beta expression. Finally, experiments in 2-deoxyglucose-treated HSC supported that Ferritin-mediated proinflammatory signaling depends on glycolysis. Conclusion: These results suggest that ferritin uptake and intracellular traffic, and therefore consequent pro-inflammatory signaling, are mediated by CCP but not via lipid rafts/caveolae. In addition, ferritin-induced HSC pro-inflammatory signaling is regulated by glycolysis linking ferritin to fibrosis in metabolic disorders. Further insights on the different cellular events that mediate ferritin-induced HSC pro-inflammatory signaling will contribute to better understanding of ferritin in the context of hepatic fibrogenesis.
Background and Aims: The liver expressed proteins matriptase-2 (MT-2, encoded by TMPRSS6), HFE and Transferrin receptor 2 (TFR2) play important roles in systemic iron homeostasis by regulating the expression of the iron regulatory hormone hepcidin in the liver. Mutations in TMPRSS6 lead to iron refractory iron deficiency anaemia, whereas mutations in HFE and TFR2 lead to hereditary hemochromatosis. We generated mice lacking various combinations of Tmprss6, Hfe and Tfr2 to further elucidate the potentially competing roles of these proteins in hepcidin regulation, iron homeostasis and erythropoiesis. Methods: Tmprss6−/− and Hfe−/−/Tfr2−/− mice, both on the C57BL/6 background were bred to produce six different genotype groups: wild type, Tmprss6−/−, Hfe−/−/Tfr2−/−, Tmprss6−/−/Hfe−/−/Tfr2−/−, Tmprss6−/−/Hfe−/− and Tmprss6−/−/Tfr2−/−. Male mice (n = 6–8 per group) were sacrificed at 10 weeks of age and blood and tissues taken for analysis. Results: The Tmprss6−/−/Hfe−/−/Tfr2−/− and Tmprss6−/−/Tfr2−/− mice had iron deficiency anaemia and a more severe phenotype than the Tmprss6−/− and Tmprss6−/−/Hfe−/− mice, characterized by splenomegaly and extramedullary hematopoiesis (EMH) in the spleen. Iron deficiency in all mice lacking Tmprss6 was related to an increase in hepatic hepcidin expression. Analysis of gene expression in the spleen revealed a tight correlation between Tfr2 mRNA and markers of erythropoiesis, suggesting a function for Tfr2 in erythroid cells. Furthermore, analysis of the newly identified erythroid iron regulator, erythroferrone, showed increased levels in mice with EMH that did not appear to overcome the hepcidin over-expression mediated by loss of Mt-2 in the liver. Further analysis by flow cytometry
The role of receptor-mediated endocytosis of H-ferritin-induced proinflammatory signaling in hepatic stellate cells M FERNANDEZ-ROJO, A BURGESS, A GLANFIELD, D HOANG-LE, N SUBRAMANIAM, G RAMM QIMR Berghofer MRI
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
3
Basic Science Liver
The potential role of lipopolysaccharides in the development of biliary injury in an animal partial liver ischemia model J REILING,1,2,3,4 KR BRIDLE,1,2 N SANTRAMPURWALA,1,2 LJ BRITTON,1,2 DHG CRAWFORD,1,2 CHC DEJONG,4,5 J FAWCETT1,2,3,6 1 School of Medicine, The University of Queensland, Brisbane, Australia, 2Gallipoli Medical Research Foundation, Greenslopes Private Hospital, Brisbane, Australia, 3PA Research Foundation, Princess Alexandra Hospital, Brisbane, Australia, 4NUTRIM – School for Nutrition, Toxicology and Metabolism, Maastricht University, Maastricht, The Netherlands, 5Department of Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands, 6Queensland Liver Transplant Service, Princess Alexandra Hospital, Brisbane, Australia Introduction: Presently liver transplantation is considered to be the best treatment option for end-stage liver disease. To meet increasing demand, more livers donated after cardiac death (DCD livers) are being used for transplantation. Unfortunately the use of DCD livers is associated with the development of ischemic type biliary strictures in up to 40% of recipients, a complication with high morbidity and mortality for which few effective treatments are available. Bacterial endotoxins in the form of lipopolysaccharides (LPS) are released in the portal circulation upon gut ischemia, an unavoidable event during the donation after cardiac death process. It is however unknown if LPS play a role in the development of biliary injury and subsequent stricture formation. We examined the possible contribution of LPS to the development of biliary injury in a rat partial liver ischemia model. Methods: Male Sprague Dawley rats underwent either sham operation with vehicle administration (N = 8) or 70% partial liver ischemia for thirty minutes in combination with 1 mg/kg LPS (isch + LPS, N = 8). After one hour or six hours of reperfusion blood, bile, liver and bile duct tissue was collected. Blood biliary barrier permeability was assessed by intravenous injection of 1000 U horseradish peroxidase (a medium sized protein used to estimate tight junction dysfunction), and subsequent bile collection. Serum liver function tests were performed and bile was analyzed for composition and markers of biliary injury. qRT-PCR was used to assess mRNA expression of bile acid transporters. Results: Partial liver ischemia in combination with LPS induced hepatocellular injury evidenced by increased serum aspartate transaminase levels after one hour (sham: 92.39 ± 6.06; isch + LPS: 143.97 ± 20.68 U/L p = 0.02) and six hours of reperfusion (sham: 92.39 ± 6.06; isch + LPS: 143.97 ± 20.68 U/L p = 0.058). Lactate dehydrogenase in bile was used as a marker for biliary injury and this was only detectable in bile collected from animals undergoing liver ischemia and LPS administration after six hours (4.69 ± 1.39 U/L/gram wet liver weight). Horseradish peroxidase concentration in bile was increased at both time points after liver ischemia and LPS reflecting an increase in blood biliary barrier permeability (1 hour time point, sham: 203.47 ± 64.6; isch + LPS: 600.58 ± 366.32 and 6 hour time point, sham: 222.07 ± 34.46; isch + LPS: 842.48 ± 580.55 mU/L/ gram wet liver weight). Cyp7b1 mRNA expression was 5.5 and 7.7 fold higher in the isch + LPS group compared to sham at one and six hours respectively. Abcc2 and Slc10a1 were significantly down-regulated at six hours of reperfusion when comparing isch + LPS after one hour and six hours of reperfusion (p = 0.023 and 0.032 respectively). Conclusion: This pilot study suggests that 70% partial liver ischemia in combination with LPS causes biliary injury after six hours of reperfusion. Altered bile composition, as reflected by altered gene expression of bile acid transporters, in combination with increased blood biliary barrier permeability may play a key role in the development of this injury. Further
4
work needs to be conducted to validate these preliminary results. The results of this study will contribute to a better understanding of the pathophysiology of ischemic type biliary strictures and possible treatment options.
Angiotensin converting enzyme 2 (ACE2) gene therapy using a high efficiency and liver-specific adeno-associated viral vector attenuates experimental liver fibrosis in mice KY MAK,1 R CHIN,3 J TORRESI,3 S CUNNINGHAM,4 I ALEXANDER,4 PW ANGUS,2 CB HERATH1 1 Department of Medicine, The University of Melbourne, Melbourne, Australia, 2Liver Transplant Unit, Austin Health, Melbourne, Australia, 3Department of Infectious Disease, Austin Health, Melbourne, Australia, 4The Children’s Hospital at Westmead, University of Sydney, Sydney, Australia Background: Recent studies suggest that the alternate arm of the RAS consisting of ACE2, angiotensin 1-7 (Ang-1-7) and its receptor, Mas, is a potential therapeutic target in liver fibrosis.1,2,3 ACE2 appears to be a negative regulator of the RAS by degrading potentially deleterious vasoconstrictor and profibrotic actions angiotensin II (Ang II) to produce Ang(1-7), a peptide that has anti-fibrotic activity. We therefore investigated a long-term therapeutic effect of ACE2 in mice with experimental liver disease. Methods: A single injection of recombinant AAV2/8 carrying murine ACE2 (rAAV2/8-ACE2) with a liver-specific promoter was intraperitoneally administered to mice with liver disease induced by bile duct ligation (BDL), carbon tetrachloride (CCl4) injection and methionine and choline deficient (MCD) diet feeding. The mice were sacrificed 1 week (BDL) and 6 weeks (CCl4 and MCD) after ACE2 treatment. To determine hepatic fibrosis, gene and protein expressions of collagen and pro-fibrotic mediators, and effects on Ang II signaling pathways were analyzed. Results: Untreated mice showed extensive hepatic fibrosis at 2 weeks after BDL and 8 weeks after and CCl4 injections and MCD diet feeding. However, ACE2 therapy for 1 week (BDL) and 6 weeks (CCl4 and MCD) significantly reduced fibrosis, as reflected by marked reductions in liver hydroxyproline content and picrosirius red staining compared to controls. In both models gene expression of collagen 1 (COL1A1), alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-β) were significantly down-regulated in ACE2 treated mice. These changes were accompanied by increases in hepatic levels of the antifibrotic peptide Ang-(1-7) and reduced Ang II levels, with associated reductions in membrane translocation of the cytoplasmic p67phox NADPH oxidase subunit and activation of p38 MAP kinase. Conclusion: We conclude that rAAV2/8-ACE2 reduces fibrosis by changing the intrahepatic balance of Ang II and Ang-(1-7) production in the liver and may be an effective therapeutic option for the treatment of hepatic fibrosis. References 1. Grace JA., et al., Update on new aspects of the renin-angiotensin system in liver disease: clinical implications and new therapeutic options. Clin Sci (Lond). 2012 Aug 1;123(4):225–39. 2. Lubel JS., et al., Angiotensin-(1-7), an alternative metabolite of the renin-angiotensin system, is up-regulated in human liver disease and has antifibrotic activity in the bile-duct-ligated rat. Clin Sci (Lond). 2009 Sep 14;117(11):375–86. 3. Osterreicher CH., et al., Angiotensin-converting-enzyme 2 inhibits liver fibrosis in mice. Hepatology. 2009 Sep;50(3):929–38.
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Basic Science Liver
Jagged2 is a master regulator of liver cancer stem cells G WU, G WILSON, J GEORGE, L QIAO Storr Liver Unit, Westmead Millennium Institute, The University of Sydney, Westmead, NSW, Australia Introduction: Hepatocellular carcinoma (HCC) is an aggressive disease with poor clinical outcomes. Liver cancer stem cells (LCSCs) are thought to be the major mediators of HCC tumor progression, metastasis and treatment resistance. However, the mechanisms by which these LCSC populations are maintained are not well understood or characterized. Methods: LCSC and non-LCSC populations were generated based on the expression of the stem cell marker Oct4 using an exogenous human Oct-4 promoter GFP vector. The expression of Notch receptors, Notch ligands, and Notch downstream targets were determined by western blot and quantitative PCR (qPCR). LCSCs (Oct4+) and non-LCSCs (Oct4-) were treated with siRNA targeting Jagged2 and recombinant Jagged2 protein. BrDU cell proliferation assay, Annexin V apoptosis assay, sorafenib resistance assay and tumor sphere assays were undertaken on the cell populations with modulated Jagged2 activity. Results: Notch signaling components Jagged2 and Notch1 were found to be upregulated in the LCSC (Oct4+) population. Treatment of these LCSCs with siRNA targeting Jagged2 resulted in the inhibition of Notch signaling, reduced cell proliferation and increased apoptosis. Jagged2 knockdown also sensitized these LCSCs to sorafenib treatment. Conversely, activation of the Notch pathway with recombinant Jagged2 in the non-LCSC (Oct4-) population increased tumor sphere formation and increased the expression of SOX-2, a major transcription factor which induces stem-like characteristics and is also a novel predictor of poor prognosis in HCC. Conclusions: Jagged 2 is a critical mediator of Notch signaling in LCSCs. Jagged2 mediated Notch signaling is critical for the maintenance and treatment resistance of LCSCs in HCC and can increase stem-like characteristics of differentiated cells. Given these findings we believe Jagged2 is a potential therapeutic target for the treatment of HCC.
FC; conditioned media was collected and stored at −80°C. Primary (resting) Kupffer cell cultures were prepared by selective centrifugation using Percoll gradients (50/25) and cultured in RPMI before addition of conditioned media. Highly purified cholesterol crystals were prepared in acetone, as reported (Duewell et al 2010). Pathways of inflammasome activation were determined by semiquantitative real time PC, by IL-1β secretion into media (ELISA), and use of the potent (Ki50∼10 nM) NLRP3specific inhibitor, CRID3. Results: At 40 μM LDL, hepatocytes secreted HMGB1 into culture media and underwent liver injury (as LDH leakage) with cell death by apoptosis and necrosis. Addition of HMGB1-enriched culture medium from FC-loaded hepatocytes activated resting KCs, as assessed by nuclear translocation of NF-κB, release of IL-1β and TNF-α, and ultrastructural changes. To expose Kupffer cells directly to cholesterol crystals, we coated culture vessels, using purified FC crystals solubilized in 1:100 acetone:ethanol solvent. Solvent was allowed to evaporate, leaving cholesterol crystals at 15, 20, and 60 mM concentrations per culture vessel. After 24 h culture on crystal-coated coverslips, Kupffer cells showed significant induction of NLRP3, caspase 1, ASC, IL-18 and IL-1β mRNA, as well as a 50% increase in IL-1β secretion. KCs from Tlr4−/− mice were refractory to NLRP3 activation by cholesterol crystals. Even at 5 nM, the NLRP3 inhibitor CRID3 totally (100%) abrogated IL-1β secretion from Kupffer cells activated by cholesterol crystals directly. Conclusions: These highly novel findings reveal direct links (via HMGB1 and likely other DAMPs) between cholesterol lipotoxicity and engagement of Kupffer cell activation, in which cholesterol crystals may play an additional direct pathogenic role. Identification of TLR4 and the NLRP3 inflammasome, and particularly the impressive efficacy of CRID3 as an NLRP3 inhibitor, has implications for both the pathogenesis and treatment of NASH. References 1. Ioannou GN, Haigh WG, Thorning D et al. Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis. J Lipid Res 2013; 54: 1326–1334 2. Duewell P, Hajime K, Rayner KJ et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 2010; 464: 1357–1361
What causes Kupffer cell activation in NAFLD? HMGB1, TLR4, crystals and NLRP3 inflammasome link free cholesterol to inhibitable pro-inflammatory pathways in NASH LT GAN,1 DM VAN ROOYEN,1 M COOPER,2 A ROBERTSON,2 S MASTERS,3 N TEOH,1 G FARRELL1 1 Liver Research Group, ANU Medical School at The Canberra Hospital, Garran, ACT, 2Institute of Molecular Biology, University of Queensland, QLD, 3Walter and Eliza Hall Institute, Parkville, VIC
Manipulation of dietary advanced glycation end-product content influences the progression of non-alcoholic fatty liver disease (NAFLD) C LEUNG,1,2 CB HERATH,1,2 J ZHIYUAN,1,2 T LEONG,2 JM FORBES,1,3 PW ANGUS1,2 1 The University of Melbourne, Victoria, 2Liver Unit, Austin Hospital, Heidelberg, Victoria, 3Mater Medical Research Institute, South Brisbane, Queensland
Introduction: Increasing evidence implicates free cholesterol (FC) in pathogenesis of non-alcoholic steatohepatitis (NASH). We recently showed that loading primary murine hepatocytes with FC by incubation with human LDL causes JNK1-dependent cell death with release of high mobility group box 1 (HMGB1), inhibition of which blocks further injury in FC-loaded hepatocytes. Further, TLR4-deleted hepatocytes are refractory to FC lipotoxicity. HMGB1 is an archetypical danger associated molecular pattern (DAMP) which may facilitates interactions between other DAMPs and pattern recognition receptors, such as TLRs. Cholesterol crystals have recently been identified in human NASH (Ioannou et al 2013), and are abundant in livers from our metabolic syndrome mouse model. We tested whether conditioned media from lipotoxic hepatocytes and/or cholesterol crystals activates Kupffer cells via the NLRP3 inflammasome, which in this cell type is activated via TLR4. Materials and Methods: Primary murine hepatocytes from C57B6/J wild type (WT) mice were incubated with 40 μM LDL for 24 h to load with
Introduction: Advanced glycation end-products (AGEs) content is high in western diets and may contribute to tissue injury via RAGE (receptor for AGEs). Here, we determined if manipulation of dietary AGE intake affects NAFLD progression and whether these effects are mediated via RAGE. Methods: Male C57B6 mice were fed a high fat, high fructose, high cholesterol (HFHC) diet for 33 weeks and compared with animals on normal chow. A third group were given a HFHC diet that was high in AGEs through baking. Another group was given a HFHC diet that was marinated in vinegar to prevent the formation of AGEs. In a second experiment, RAGE KO animals were fed a HFHC diet or a high AGE HFHC diet and compared with WT controls. Hepatic biochemistry, histology, picro-sirius red morphometry and hepatic mRNA were determined. We also determined the effects of AGEs on primary Kupffer cells (KCs). Results: The long term HFHC diet model generated significant steatohepatitis and fibrosis. Hepatic 4-hydroxynonenal content (a marker
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
5
Basic Science Liver
of chronic oxidative stress) and hepatocyte ballooning (a marker of cellular injury) were significantly increased with a HFHC diet and further increased with a high AGE HFHC diet and abrogated by vinegar marination. Similarly, the high AGE HFHC diet significantly increased picrosirius red staining, α-smooth muscle actin and collagen type 1A gene expression compared with HFHC alone and this was reduced by vinegar marination. The increased oxidative stress, hepatocyte ballooning and fibrosis associated with a high AGE HFHC diet was significantly reduced in corresponding high AGE HFHC RAGE KO animals. We found KCs express RAGE and take up AGEs. AGEs increase ROS generation and proliferation (as measured by BrDU uptake) in these cells. Similar results were achieved with primary hepatic stellate cells (HSCs). Conclusions: In the HFHC model of NAFLD, manipulation of dietary AGEs modulates liver injury, inflammation, and liver fibrosis via a RAGE dependent pathway. Our cell work suggests that these proinflammatory and profibrotic effects are mediated via direct effects on KCs and HSCs via RAGE. This suggests that pharmacological and dietary strategies targeting the AGE/RAGE pathway could slow the progression of NAFLD. Our results also have important implications for diabetes associated NAFLD, a condition in which endogenous AGE production and RAGE expression is increased.
The protease fibroblast activation protein (FAP) in liver disease MD GORRELL,1,3 KH WILLIAMS,2,3 AJ VIERA DE RIBEIRO,1,3 S CHOWDHURY,1,3 E HAMSON,1,3 O SCHILLING,4 E PRAKOSO,1,3 NA SHACKEL,1,3 SV MCLENNAN,2,3 WW BACHOVCHIN,5 FM KEANE,1,3 GW MCCAUGHAN,1,3 A ZEKRY,6 SM TWIGG2,3 1 Centenary Institute, 2Bosch Institute, 3The University of Sydney Medical School, Australia, 4Albert-Ludwigs University of Freiburg, Germany, 5Tufts University, Boston, USA, 6The Inflammation and Infection Research Centre, University of New South Wales School of Medical Sciences, Australia More than 400 human genes encode proteases, many of which have potential medical application. The main advantages of exploiting proteases are that both therapeutics and assays can use specific chemical compounds that are far less expensive than antibodies. FAP is predominantly associated with disease states, including liver and lung fibrosis, solid tumors, arthritis and atherosclerosis. Substrates of this protease include α-2-antiplasmin, collagen I and Neuropeptide Y. In a diet-induced obesity model, we have found that FAP gene knockout (gko) mice have improved glucose tolerance and liver histopathology, and less insulin resistance and fatty liver, compared to wild type mice on this diet. FAP gko mice resist liver fibrosis. Using our recently published novel FAP activity assay (1), we observed that serum levels of FAP enzyme activity co-segregate with liver stiffness as a measure of fibrosis in two adult cohorts with NAFLD. Cohort 1 contained 108 patients with type 2 diabetes who had transient elastography and Cohort 2 contained 148 patients with morbid obesity with liver biopsies. In Cohort 1, serum FAP was an independent risk factor for median liver stiffness ≥ 10.3 kPa. There was an 8-fold increased odds ratio of having a median liver stiffness of ≥10.3 kPa for those in the highest FAP tertile, compared with subjects in the lowest tertile (p = 0.01). A serum FAP level below 730 pmol AMC/min/mL had a negative predictive value for significant fibrosis of 95%. In Cohort 2, the FAP level was added to the NAFLD fibrosis score (NFS) to correctly reclassify 49% of patients as low risk of severe fibrosis who by NFS had been classified as intermediate risk. Measuring FAP in serum is rapid and should thus become an inexpensive supplement to the NFS to avoid patients being sent for unnecessary further tests.
6
Cell lines derived from FAP gko mice were engineered to express functional FAP enzyme (FAPe+) vs inactive FAP (FAPe-). Proteomic analyses of these cells showed FAP-specific cleavage of many bioactive peptides. In vitro ‘wound healing’ found that cells with FAP activity exhibited greater cell migration but comparable proliferation and apoptosis. Conclusions: (1) FAP has an important role in glucose and lipid metabolism and in fibrosis progression. (2) Adding a FAP serum measurement to the existing clinical NFS algorithm may correctly diagnose as non-fibrotic about half of the patients who would otherwise receive an uncertain diagnosis and require further testing. (3) FAP enzyme activity causes increased cell migration and so may have roles in wound healing. Reference 1. Keane FM, et al. Quantitation of fibroblast activation protein (FAP)specific protease activity in mouse, baboon and human fluids and organs. FEBS open bio 2014;4:43–54.
Serum-free hepatocytic transdifferentiation of pancreatic progenitor cells facilitated by extracellular matrix proteins FD GRATTE,1 JK OLYNYK,1,2,3 GCT YEOH,4 D TOSH,5 DR COOMBE,1 JEE TIRNITZ-PARKER1,2 1 School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, Australia, 2 School of Medicine and Pharmacology, University of Western Australia, Fremantle, Australia, 3Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Australia, 4Harry Perkins Institute of Medical Research, Perth, Australia, 5Centre for Regenerative Medicine, University of Bath, Bath, UK Background: Rising incidences of chronic liver disease and organ shortage for orthotopic liver transplantation have prompted interest into the development of alternative sources of liver tissue. Previous studies have highlighted the potential of cell-based technologies for the in vitro production of hepatocytes for transplantation, including the use of pancreatic progenitor cells (PPCs).1 Pancreatic progenitor cells are able to generate hepatocyte-like cells via pancreas-to-liver transdifferentiation after stimulation with the glucocorticoid dexamethasone in conjunction with other liver-promoting growth factors and cell culture supplements. Traditional methods utilize fetal bovine serum, an undefined concoction of growth factors and extracellular matrix (ECM) components, which is unsuitable for use in human treatments. Therefore the development of novel methods using defined levels of growth factors and ECM proteins in a serum-free environment is necessary for future cell-based therapies. Methods: The clonal pancreatic cell lineAR42J-B13 was cultured in basal medium (control group) or under differentiation-inducing conditions, on fibronectin or laminin, with and without serum, for five days. Cells were continuously assessed for morphological changes and subjected to transcriptome or immunofluorescent analyses on days 3 and 5 of the transdifferentiation protocol. Changes in pancreatic (amylase) and hepatocytic (hepatocyte nuclear factor 4α, albumin, tyrosine aminotransferase and transthyretin) gene and/or protein expression were evaluated. To test for hepatocyte functionality, periodic acid-Schiff staining for glycogen storage and indocyanine green uptake and release assays were performed. Results: Undifferentiated AR42J-B13 cells grew in grape-like collections of small, amylase-expressing cells and displayed little or no expression of hepatocytic markers. All groups subjected to differentiationinducing conditions quickly formed monolayer cultures, showed rapid morphological changes including significant enlargement of all cells and bi- or multinucleation (hallmark of hepatocytes) in a subpopulation of cells. Correspondingly, cells changed their gene and protein expression pattern from a pancreatic to a hepatocytic phenotype. In addition,
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Basic Science Liver
transdifferentiated AR42J-B13 cells were demonstrated to have hepatocyte functions by glycogen storage and indocyanine release assays. Discussion: Utilization of fibronectin or laminin facilitated the successful transdifferentiation of AR42J-B13 cells into functional hepatocyte-like cells, importantly without the presence of fetal bovine serum in the culture medium. These results may help to improve current differentiation protocols and move approaches towards a more applicable stage for use in future cell-based therapies to treat liver-based metabolic disorders. Reference 1. Shen et al. Nature Cell Biology 2000.
Heat shock protein 47 is a potential therapeutic target for the depletion of cancer-promoting stromal cells in pancreatic cancer J YOUKHANA,1* J MCCARROLL,2* G SHARBEEN,1 M ERKAN,3 J LIU,1 D GOLDSTEIN,1 PA PHILLIPS1 1 Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia, 2Children’s Cancer Institute Australia, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia, 3Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany Introduction: Pancreatic cancer (PC) is a lethal disease with a 5-year survival rate
Journal of Gastroenterology and Hepatology (2014) 29 (Suppl. 2)
Basic Science Liver Exercise slows growth of dysplastic hepatocytes by improving insulin sensitivity and enhancing DNA damage surveillance pathways in mice genetically predisposed to obesity and diabetes E ARFIANTI,1 V BARN,1 WG HAIGH,2 GN IOANNOU,2 N TEOH,1 G FARRELL1 1 Liver Research Group, ANU Medical School, The Canberra Hospital, ACT, 2Division of Gastroenterology, Veterans Affairs Puget Sound Health Care System and University of Washington, Seattle, WA, Australia Background: We previously reported that obesity and diabetes accelerate diethylnitrosamine (DEN)-induced hepatocarcinogenesis in Alms1 mutant (foz/foz) NOD.B10 mice, replicating the increased hepatocellular carcinoma (HCC) risk in obese, diabetic patients. Last AGW we reported an association between accelerated hepatocarcinogenesis with hyperinsulinemia/hyperglycemia-induced Akt/mTORC1 activation and Nrf1/2-mediated metabolic reprogramming.1 In the present study, we investigated whether exercise sufficient to reduce the rate of weight gain, reduces growth of dysplastic hepatocytes and HCC development in DENinjected foz/foz mice. Methods: Male foz/foz and wild-type (WT) littermates were injected with DEN (10 mg/kg i.p.) day 12–15 of age; controls were injected with vehicle (saline). They were then randomly assigned either to cages provided with an exercise wheel (from 4 wks of age, until 12 or 24 wks of age) or housed similarly without an exercise wheel. Dysplastic hepatocytes were identified by glutathione S-transferase pi (GST-pi) immunohistochemistry (IHC), protein and phospho-protein expression by immunoblotting and IHC, gene expression by semi-quantitative real-time PCR, glucose tolerance by i.p. glucose tolerance test, hepatic lipid content using HPLC, and oxidative status by reduced (GSH) and oxidized glutathione (GSSG). Results: Mice provided with in-cage exercise wheels ran (on average) 4 km/day, irrespective of whether they were WT or foz/foz. In association with such physical activity, foz/foz mice maintained weight gain similar to WT mice, at least until 12 wks of age. At 12 wks of age, GST-pi immunostaining showed a significant reduction in the number of dysplastic hepatocytes in exercising foz/foz mice compared to foz/foz littermates without exercise wheel provision (these mice are observed to be inactive). The exercise-associated reduction in number of pre-neoplastic hepatocytes correlated with prevention of excessive weight gain and adiposity, compared to their non-exercising littermates. Exercise also improved insulin sensitivity in foz/foz mice, as indicated by lower fasting blood glucose, reduced serum insulin and enhanced glucose tolerance. Improvement of insulin signaling was evident in livers of exercising mice by upregulation of insulin receptor substrate-2 (IRS-2) protein and attenuation of hepatic lipid accumulation, particularly decreased triglyceride and cholesterol ester levels. Interestingly, exercise increased rather than decreased GSSG levels in livers from foz/foz mice, suggesting that exercise increases generation of reactive oxygen species; such a link has been previously linked to the capacity of exercise to enhance insulin sensitivity.2 Despite the amelioration of insulin resistance by exercise in foz/foz mice (which usually develop obesity and diabetes), there was no difference in Akt/ mTORC1 or AMPK activation, and exercise had no effect on hepatic TNFα and MCP-1 expression. We previously observed in this model that obesity-promoted hepatocarcinogenesis is associated with Nrf1/2mediated shuttle of glucose and glutamine metabolism into purine synthesis.3 Nrf1/2 signaling was downregulated by exercise, inferring decreased metabolic activity to support hepatocellular proliferation. There was a parallel increase in activation of the Chk2/p53 cell cycle regulatory
pathway, associated with downregulation of cyclin E1. The resultant changes in cell cycle regulatory control likely contribute to the reduced number of dysplastic hepatocytes in exercising foz/foz mice compared to their overweight inert littermates. Analysis of another cohort of mice at 24 wks is underway to establish whether changes at 12 wks translate to reduction of HCC at 6 mth after DEN injection. Conclusions: Exercise prevents growth of dysplastic hepatocytes in the early (premalignant) stage of DEN-induced HCC in mice genetically predisposed to obesity and diabetes. This is associated with increased insulin sensitivity (including in the liver), reduced hepatic lipid content, suppression of cyclin E1 and enhancement Chk2/p53 cell cycle control. Whether this is sufficient to delay DEN hepatocarcinogenesis in foz/foz mice will be apparent by August 2014. References 1. Arfianti E, Lee SS, Heydet D, Larter C, Barn V, Teoh NC, Farrell GC. Enhanced growth of dysplastic hepatocytes is associated with activation of Akt/mTORC1 pathway in a murine model of hyperphagic-obesity. J Gastroenterol Hepatol 2013, 28(Suppl 2), 3. 2. Ristow M, Zarse K, Oberbach A, Klöting N, Birringer M, Kiehntopf M, Stumvoll M, Kahn CR, Blüher M. Antioxidants prevent healthpromoting effects of physical exercise in humans. Proc Natl Acad Sci U S A 2009, 106(21), 8665–8670. 3. Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, Yamamoto M, Motohashi H. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell 2012, 22(1), 66–79.
TLR9 may be a double-edged sword in the liver: is it pathogenic or protective in NASH? AR MRIDHA,1 F HACZEYNI,1 MM YEH,2 G HAIGH,3 V BARN,1 H AJAMIEH,1 JM HAMDORF,4 L ADAMS,5 NC TEOH,1 GC FARRELL1 1 Liver Research Group, Australian National University Medical School at the Canberra Hospital, Garran, ACT, Australia, 2Department of Pathology, University of Washington, Seattle, WA, USA, 3Department of Gastroenterology, University of Washington, Seattle, WA, USA, 4School of Surgery, University of Western Australia, Crawley, WA, Australia, 5School of Medicine and Pharmacology, University of Western Australia, Crawley, WA, Australia Background: Inflammation with macrophage recruitment and activation as characteristic features is a hallmark of non-alcoholic steatohepatitis (NASH) vs simple steatosis in NAFLD, while NF-κB and c-Jun/AP-1 are invariable pro-inflammatory signals. In addition to effects of lipids and pro-oxidants, such signaling could be triggered by cytokine or pattern recognition receptors, such as toll-like receptors (TLRs). TLR4 and TLR9 have both been implicated in nutritional depletion models of NAFLD, but TLR9 is a “master switch” of macrophage recruitment. Here we first measured TLR4 and 9 expression in human and mouse livers showing NASH vs simple steatosis, then studied the roles of TLR9 for inflammatory recruitment to fatty livers and for pathways to hepatocyte injury in NASH. Methods: Liver biopsies of patients (n = 7–9/grp) with NASH, simple steatosis or healthy controls were used to measure mRNA expression of TLR subtypes. Female wildtype (Wt), appetite-dysregulated Alms1 mutant (foz/foz) and Tlr9−/− mice were fed chow or an atherogenic (Ath) diet containing 0.2% cholesterol (n = 6–14/grp). Steatosis, liver inflammation and macrophage and neutrophil recruitment, fibrosis, NF-κB and c-Jun activation, cytokines/chemokines, circulating endotoxin, and markers of hepatocyte injury were assessed. We created bone marrow (BM) chimeric mice to examine the role of TLR9 on myeloid-derived vs parenchymal
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
1
Basic Science Liver
cells in Ath-induced NAFLD, studied effects of TLR9 deletion on susceptibility of primary hepatocytes to palmitic acid lipotoxicity and endotoxin, and BM macrophage cultures to determine the effects of CpG DNA (TLR9 ligand), necrotic media and LPS/IL-4 on M1/M2 polarization. Results: In human NASH, hepatic TLR4 variant 3 (but not variants 1 and 4) and TLR9 mRNA levels were higher than simple steatosis or control. In both Ath-fed Wt and foz/foz mice with NASH, Tlrs-4, 7, 9 transcripts increased, with similar pattern for TLR4 and 9 proteins. In Ath-fed Tlr9−/− mice, liver necro-inflammatory score and fibrosis markers were substantially diminished compared with Wt, despite similar steatosis and hepatic lipid levels. Likewise, Ath feeding failed to increase NF-κB and c-Jun activation, macrophage/neutrophil infiltration and pro-inflammatory Th1 cytokines in Tlr9−/− mice compared to major increases in Wt. Conversely, expression of anti-inflammatory Th2 cytokines (IL-4, IL-10) was not different. Despite less inflammation in Ath-fed Tlr9−/− vs Wt mice, hepatocyte damage (serum ALT, high mobility group box 1 [HMGB-1], CK-18, asialoglycoprotein [ASGPR] levels) and circulating endotoxin levels were higher. We interpret these changes as reflecting enhanced necrosis (and/or necroptosis) in response to endotoxemia; correspondingly, livers showed increased RIP3 (necrosis marker) and MLKL (necroptosis) expression. Further, Tlr9−/− hepatocytes were more susceptible to palmitic acid and endotoxin-induced injury than Wt. Using BM chimeras, we showed that TLR9 in BM-derived myeloid cells and not hepatocytes at least partially mediates Ath diet-induced hepatic injury. This is supported by our observation that, compared to Wt, Tlr9−/− BM-derived macrophages are resistant to activation by CpG DNA, necrotic mediators and LPS induced M1 polarization to produce pro-inflammatory cytokines. Conclusion: These novel data in human liver, in mice with metabolic syndrome-related NASH and with the atherogenic dietary model of NAFLD indicate that TLR9 activation is a critical pro-inflammatory trigger in NASH that is likely mediated via macrophages. In addition, however, TLR9 appears to confer hepatocyte protection in NASH as TLR9-deleted cells are more susceptible to lipotoxicity and endotoxin-induced necrosis. If this is correct, TLR9 blockade may not be an attractive therapeutic approach in NASH because, while it could dampen macrophage activation, it could also abrogate an intrinsic hepatoprotective pathway against lipotoxic molecules and gut-derived pathogen-associated molecular patterns.
(H&E), sirius red, and immunofluorescence performed for T-cadherin, adiponectin, CD31, F4/80, and alpha smooth muscle actin (α-SMA). cDNA was generated from tissues and cells, and quantitative polymerase chain reaction (qPCR) undertaken for T-cadherin, AdipoR1, AdipoR2, F4/80, CD68, Tumor necrosis factor-α (TNF-α), α-SMA, Tissue metallopeptidase inhibitor 1 (TIMP1) and Type I collagen (Col1). Through western blots analyses protein levels of 5′ adenosine monophosphateactivated protein kinase (AMPK), phospho-AMPK, Protein Kinase B (Akt), phospho-Akt, mammalian target of rapamycin (mTOR), and phospho-mTOR were determined. Migration and proliferation assays were undertaken with primary HSCs treated with adiponectin and Tcad siRNA. Results: Significantly, we find that liver fibrosis was increased in AdipoKO, and reduced in Tcad KO and unchanged in dKO mice compared to WT control. Immunofluorescence shows that T-cadherin is present on HSCs and colocalizes with adiponectin. In fibrotic Adipo KO mice, there was a significant decrease in T-cad expression in the liver. In WT mice, compared to AdipoR1 and -R2, only T-cad expression was increased during fibrosis. In in vitro assays, the administration of adiponectin to HSCs reduced Col1 and α-SMA expression, and the knocking down of T-cad further reduced their expression. The treatment of HSCs with adiponectin in the absence of T-cad further increased TIMP1 expression levels. Adiponectin treatment reduced HSC proliferation and migration, and the silencing of T-cad with siRNA only reduced HSC proliferation. Western blot revealed that adiponectin treatment in the absence of T-cad coincided with increased phospho-AMPK and reduced phospho-mTOR and phospho-AKT expression. Conclusion: These data show that the interaction of adiponectin and T-cadherin is important in mediating liver fibrosis. The absence of T-cadherin leads to a significant reduction in fibrosis in the presence of adiponectin. The absence of both adiponectin and T-cadherin leads to fibrosis similar to that observed in WT mice. At the molecular level we find that T-cadherin absence in HSCs influences their proliferation and growth signals. We are now further investigating the mechanistic events leading to these changes.
The role of adiponectin and T-cadherin interactions in liver fibrosis B ALZAHRANI,1,2 J GEORGE,1,2 L HEBBARD1,2 1 Storr Liver Unit, Westmead Millennium Institute, PO Box 412, Darcy Road, Westmead, NSW 2145, Australia, 2 Sydney Medical School, University of Sydney, NSW, Australia
Hepatic microRNA expression is altered in a murine model of iron and fat co-mediated liver injury CJ MCDONALD,1 Y YUAN,2 N SANTRAMPURWALA,2,3 DHG CRAWFORD,2,3 VN SUBRAMANIAM1,2,3 1 The QIMR Berghofer Medical Research Institute, Brisbane, Australia, 2School of Medicine, The University of Queensland, Brisbane, Australia, 3Gallipoli Medical Research Centre, Greenslopes Private Hospital, Brisbane Australia
Introduction: Liver fibrosis is the scarring process that represents the liver’s response to injury. Adiponectin has been shown to have an important role in the regulation of fibrosis, as adiponectin null mice have greater levels after carbon tetrachloride (CCl4) treatment. Adiponectin binds to three receptors: AdipoR1, AdipoR2 and T-cadherin. Our unpublished studies suggest that AdipoR1 and AdipoR2 null mice have unchanged fibrosis after CCl4 treatment. The role of T-cadherin in hepatic biology is unknown. Thus, using mouse models we examined the role of adiponectin and T-cadherin interactions in liver fibrosis. Methods: We used four mouse models: wild-type (WT), adiponectin (Adipo), T-cadherin (T-cad) knock-out and adiponectin T-cadherin double knock-out (dKO) mice. Liver injury was promoted by twice weekly intraperitoneal injections of carbon tetrachloride (CCl4) for 12 weeks. Control mice were injected with corn oil alone. Primary rat hepatic stellate cells (HSC) were isolated, and treated with T-cadherin siRNA followed by subsequent treatment with full length recombinant adiponectin. Liver tissues were examined for pathology changes by haematoxylin and eosin
Introduction: HFE-hemochromatosis and non-alcoholic fatty liver disease are both common conditions in the developed world and often occur in conjunction. Co-occurrence of these disorders appears to potentiate greater liver injury than either alone; the underlying mechanisms are poorly understood. In this study we have investigated the regulation of hepatic microRNA (miRNA) expression in a mouse model of iron and fat co-mediated liver injury. Methods: Hfe-knockout mice were fed either control or high calorie diets. Mice were sacrificed after 20 weeks of treatment. Small RNA was extracted from liver tissue and profiled using next-generation miRNAsequencing and differentially expressed miRNAs identified. mRNA-seq was available from these same mice, and was utilized to perform combination analysis (which identifies which differentially expressed mRNAs are potential targets of differentially expressed miRNAs) and correlation analysis (which compares the level of expression of all identified miRNAs with the levels of expression of all of their potential target mRNAs) to identify miRNA-mRNA pairs of interest.
2
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Basic Science Liver
Results: 39 differentially expressed miRNAs were identified. Of these 10 have published links to relevant pathologies including miRNAs-190 and -199 which have both been linked to hepatic fibrosis, miRNA-223 which has been linked to hepatocellular carcinoma and obesity-associated adipose tissue inflammation, and miRNA-103 which has been linked to glucose homeostasis and insulin sensitivity. Combination analysis identified 49 miRNA-mRNA pairs in which both the miRNA and its target mRNA were differentially expressed including miRNA-199a-3p for which nine of its target mRNAs are differentially expressed. Correlation analysis identified 424 miRNA-mRNA pairs with a Pearson correlation coefficient > ±0.8 and p < 0.05. Conclusions: This study demonstrates a dysregulation in hepatic miRNA expression in the setting of combined iron overload and steatosis. Ten miRNAs are identified which are linked to relevant pathologies supporting the validity of this study, and a further 29 miRNAs which have not previously been liked to either hepatic or lipid pathology where also identified. Further, the integration of mRNA data has identified potential mechanisms of action for several of the differentially expressed miRNAs.
revealed accumulation of immature erythroblasts in the spleens of the Tmprss6−/−/Tfr2−/− mice, suggesting an important role for Tfr2 in erythroid differentiation that may be mediated by lower erythropoietin expression in the kidney. Conclusions: Our results indicate that matriptase-2 predominates over Hfe and Tfr2 in hepcidin regulation in the liver. These findings indicate that therapies aimed at inhibiting Mt-2 activity would be beneficial in treating iron overload in patients with HH caused by mutations in HFE and/or TFR2. Furthermore, we have also uncovered an important role for erythroid-expressed Tfr2 in the regulation of erythropoiesis that is separate from its accepted role as a regulator of iron homeostasis in the liver.
Investigation of mice deficient in matriptase-2, Hfe and transferrin receptor 2 reveals a novel non-hepatic role for Tfr2 in erythropoiesis DF WALLACE,1,2 ES SECONDES,1 G RISHI,1,2 L OSTINI,1 CJ MCDONALD,1 SW LANE,2,3 JD HOOPER,4 C LOPEZ-OTIN,5 VN SUBRAMANIAM1,2 1 Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia, 2School of Medicine, University of Queensland, Brisbane, Queensland, Australia, 3Translational Leukaemia Research Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia, 4Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia, 5 Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain
Introduction: Hepatic stellate cells (HSCs) are responsible for collagen deposition leading to fibrosis following liver injury/inflammation. Serum levels of the acute phase protein ferritin are elevated in inflammation and act as an indicator of disease severity in chronic liver disease. We have previously demonstrated that H-subunit ferritin actually contributes to this process as a pro-inflammatory mediator in HSC via an iron-independent, NFkappaB-regulated signaling pathway, inducing the expression of cytokines IL-1beta, IL-6 and RANTES. Aims: To decipher the molecular events at the level of the plasma membrane and the endocytic pathways that mediate the pro-inflammatory response of Ferritin in HSC. Methods: Primary rat HSCs were treated with 10 nM H-ferritin for 0–24 hrs. HSCs were pre-treated with inhibitors of microtubule formation (colchicine, nocodazole), lysosomal acidification (chloroquine) and intracellular protein transport (monensin), dynamin-2-dependent internalization (Dyngo-4), clathrin-coated pit (CCP) internalization (PitStop2), lipid raft/caveolae formation (beta-methyl cyclodextrin, beta-MCD) prior to ferritin stimulation. qPCR was used to determine relative expression of Ferritin-induced transcripts. Results: Colchicine or nocodazole had no significant effect on ferritininduced expression of IL-1beta suggesting that microtubule-dependent endocytosis is not necessary for signaling. However, inhibition of CCP endocytosis and dynamin-dependent endocytosis in HSC totally or partially abolished Ferritin-induced pro-inflammatory response, respectively. Conversely, betaMCD–induced disruption of lipid rafts/caveoolae exacerbated response to Ferritin. Moreover, monensin treatment resulted in a 75% reduction in ferritin-induced IL-1beta expression while chloroquine completely abolished IL-1beta expression. Finally, experiments in 2-deoxyglucose-treated HSC supported that Ferritin-mediated proinflammatory signaling depends on glycolysis. Conclusion: These results suggest that ferritin uptake and intracellular traffic, and therefore consequent pro-inflammatory signaling, are mediated by CCP but not via lipid rafts/caveolae. In addition, ferritin-induced HSC pro-inflammatory signaling is regulated by glycolysis linking ferritin to fibrosis in metabolic disorders. Further insights on the different cellular events that mediate ferritin-induced HSC pro-inflammatory signaling will contribute to better understanding of ferritin in the context of hepatic fibrogenesis.
Background and Aims: The liver expressed proteins matriptase-2 (MT-2, encoded by TMPRSS6), HFE and Transferrin receptor 2 (TFR2) play important roles in systemic iron homeostasis by regulating the expression of the iron regulatory hormone hepcidin in the liver. Mutations in TMPRSS6 lead to iron refractory iron deficiency anaemia, whereas mutations in HFE and TFR2 lead to hereditary hemochromatosis. We generated mice lacking various combinations of Tmprss6, Hfe and Tfr2 to further elucidate the potentially competing roles of these proteins in hepcidin regulation, iron homeostasis and erythropoiesis. Methods: Tmprss6−/− and Hfe−/−/Tfr2−/− mice, both on the C57BL/6 background were bred to produce six different genotype groups: wild type, Tmprss6−/−, Hfe−/−/Tfr2−/−, Tmprss6−/−/Hfe−/−/Tfr2−/−, Tmprss6−/−/Hfe−/− and Tmprss6−/−/Tfr2−/−. Male mice (n = 6–8 per group) were sacrificed at 10 weeks of age and blood and tissues taken for analysis. Results: The Tmprss6−/−/Hfe−/−/Tfr2−/− and Tmprss6−/−/Tfr2−/− mice had iron deficiency anaemia and a more severe phenotype than the Tmprss6−/− and Tmprss6−/−/Hfe−/− mice, characterized by splenomegaly and extramedullary hematopoiesis (EMH) in the spleen. Iron deficiency in all mice lacking Tmprss6 was related to an increase in hepatic hepcidin expression. Analysis of gene expression in the spleen revealed a tight correlation between Tfr2 mRNA and markers of erythropoiesis, suggesting a function for Tfr2 in erythroid cells. Furthermore, analysis of the newly identified erythroid iron regulator, erythroferrone, showed increased levels in mice with EMH that did not appear to overcome the hepcidin over-expression mediated by loss of Mt-2 in the liver. Further analysis by flow cytometry
The role of receptor-mediated endocytosis of H-ferritin-induced proinflammatory signaling in hepatic stellate cells M FERNANDEZ-ROJO, A BURGESS, A GLANFIELD, D HOANG-LE, N SUBRAMANIAM, G RAMM QIMR Berghofer MRI
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
3
Basic Science Liver
The potential role of lipopolysaccharides in the development of biliary injury in an animal partial liver ischemia model J REILING,1,2,3,4 KR BRIDLE,1,2 N SANTRAMPURWALA,1,2 LJ BRITTON,1,2 DHG CRAWFORD,1,2 CHC DEJONG,4,5 J FAWCETT1,2,3,6 1 School of Medicine, The University of Queensland, Brisbane, Australia, 2Gallipoli Medical Research Foundation, Greenslopes Private Hospital, Brisbane, Australia, 3PA Research Foundation, Princess Alexandra Hospital, Brisbane, Australia, 4NUTRIM – School for Nutrition, Toxicology and Metabolism, Maastricht University, Maastricht, The Netherlands, 5Department of Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands, 6Queensland Liver Transplant Service, Princess Alexandra Hospital, Brisbane, Australia Introduction: Presently liver transplantation is considered to be the best treatment option for end-stage liver disease. To meet increasing demand, more livers donated after cardiac death (DCD livers) are being used for transplantation. Unfortunately the use of DCD livers is associated with the development of ischemic type biliary strictures in up to 40% of recipients, a complication with high morbidity and mortality for which few effective treatments are available. Bacterial endotoxins in the form of lipopolysaccharides (LPS) are released in the portal circulation upon gut ischemia, an unavoidable event during the donation after cardiac death process. It is however unknown if LPS play a role in the development of biliary injury and subsequent stricture formation. We examined the possible contribution of LPS to the development of biliary injury in a rat partial liver ischemia model. Methods: Male Sprague Dawley rats underwent either sham operation with vehicle administration (N = 8) or 70% partial liver ischemia for thirty minutes in combination with 1 mg/kg LPS (isch + LPS, N = 8). After one hour or six hours of reperfusion blood, bile, liver and bile duct tissue was collected. Blood biliary barrier permeability was assessed by intravenous injection of 1000 U horseradish peroxidase (a medium sized protein used to estimate tight junction dysfunction), and subsequent bile collection. Serum liver function tests were performed and bile was analyzed for composition and markers of biliary injury. qRT-PCR was used to assess mRNA expression of bile acid transporters. Results: Partial liver ischemia in combination with LPS induced hepatocellular injury evidenced by increased serum aspartate transaminase levels after one hour (sham: 92.39 ± 6.06; isch + LPS: 143.97 ± 20.68 U/L p = 0.02) and six hours of reperfusion (sham: 92.39 ± 6.06; isch + LPS: 143.97 ± 20.68 U/L p = 0.058). Lactate dehydrogenase in bile was used as a marker for biliary injury and this was only detectable in bile collected from animals undergoing liver ischemia and LPS administration after six hours (4.69 ± 1.39 U/L/gram wet liver weight). Horseradish peroxidase concentration in bile was increased at both time points after liver ischemia and LPS reflecting an increase in blood biliary barrier permeability (1 hour time point, sham: 203.47 ± 64.6; isch + LPS: 600.58 ± 366.32 and 6 hour time point, sham: 222.07 ± 34.46; isch + LPS: 842.48 ± 580.55 mU/L/ gram wet liver weight). Cyp7b1 mRNA expression was 5.5 and 7.7 fold higher in the isch + LPS group compared to sham at one and six hours respectively. Abcc2 and Slc10a1 were significantly down-regulated at six hours of reperfusion when comparing isch + LPS after one hour and six hours of reperfusion (p = 0.023 and 0.032 respectively). Conclusion: This pilot study suggests that 70% partial liver ischemia in combination with LPS causes biliary injury after six hours of reperfusion. Altered bile composition, as reflected by altered gene expression of bile acid transporters, in combination with increased blood biliary barrier permeability may play a key role in the development of this injury. Further
4
work needs to be conducted to validate these preliminary results. The results of this study will contribute to a better understanding of the pathophysiology of ischemic type biliary strictures and possible treatment options.
Angiotensin converting enzyme 2 (ACE2) gene therapy using a high efficiency and liver-specific adeno-associated viral vector attenuates experimental liver fibrosis in mice KY MAK,1 R CHIN,3 J TORRESI,3 S CUNNINGHAM,4 I ALEXANDER,4 PW ANGUS,2 CB HERATH1 1 Department of Medicine, The University of Melbourne, Melbourne, Australia, 2Liver Transplant Unit, Austin Health, Melbourne, Australia, 3Department of Infectious Disease, Austin Health, Melbourne, Australia, 4The Children’s Hospital at Westmead, University of Sydney, Sydney, Australia Background: Recent studies suggest that the alternate arm of the RAS consisting of ACE2, angiotensin 1-7 (Ang-1-7) and its receptor, Mas, is a potential therapeutic target in liver fibrosis.1,2,3 ACE2 appears to be a negative regulator of the RAS by degrading potentially deleterious vasoconstrictor and profibrotic actions angiotensin II (Ang II) to produce Ang(1-7), a peptide that has anti-fibrotic activity. We therefore investigated a long-term therapeutic effect of ACE2 in mice with experimental liver disease. Methods: A single injection of recombinant AAV2/8 carrying murine ACE2 (rAAV2/8-ACE2) with a liver-specific promoter was intraperitoneally administered to mice with liver disease induced by bile duct ligation (BDL), carbon tetrachloride (CCl4) injection and methionine and choline deficient (MCD) diet feeding. The mice were sacrificed 1 week (BDL) and 6 weeks (CCl4 and MCD) after ACE2 treatment. To determine hepatic fibrosis, gene and protein expressions of collagen and pro-fibrotic mediators, and effects on Ang II signaling pathways were analyzed. Results: Untreated mice showed extensive hepatic fibrosis at 2 weeks after BDL and 8 weeks after and CCl4 injections and MCD diet feeding. However, ACE2 therapy for 1 week (BDL) and 6 weeks (CCl4 and MCD) significantly reduced fibrosis, as reflected by marked reductions in liver hydroxyproline content and picrosirius red staining compared to controls. In both models gene expression of collagen 1 (COL1A1), alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-β) were significantly down-regulated in ACE2 treated mice. These changes were accompanied by increases in hepatic levels of the antifibrotic peptide Ang-(1-7) and reduced Ang II levels, with associated reductions in membrane translocation of the cytoplasmic p67phox NADPH oxidase subunit and activation of p38 MAP kinase. Conclusion: We conclude that rAAV2/8-ACE2 reduces fibrosis by changing the intrahepatic balance of Ang II and Ang-(1-7) production in the liver and may be an effective therapeutic option for the treatment of hepatic fibrosis. References 1. Grace JA., et al., Update on new aspects of the renin-angiotensin system in liver disease: clinical implications and new therapeutic options. Clin Sci (Lond). 2012 Aug 1;123(4):225–39. 2. Lubel JS., et al., Angiotensin-(1-7), an alternative metabolite of the renin-angiotensin system, is up-regulated in human liver disease and has antifibrotic activity in the bile-duct-ligated rat. Clin Sci (Lond). 2009 Sep 14;117(11):375–86. 3. Osterreicher CH., et al., Angiotensin-converting-enzyme 2 inhibits liver fibrosis in mice. Hepatology. 2009 Sep;50(3):929–38.
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Basic Science Liver
Jagged2 is a master regulator of liver cancer stem cells G WU, G WILSON, J GEORGE, L QIAO Storr Liver Unit, Westmead Millennium Institute, The University of Sydney, Westmead, NSW, Australia Introduction: Hepatocellular carcinoma (HCC) is an aggressive disease with poor clinical outcomes. Liver cancer stem cells (LCSCs) are thought to be the major mediators of HCC tumor progression, metastasis and treatment resistance. However, the mechanisms by which these LCSC populations are maintained are not well understood or characterized. Methods: LCSC and non-LCSC populations were generated based on the expression of the stem cell marker Oct4 using an exogenous human Oct-4 promoter GFP vector. The expression of Notch receptors, Notch ligands, and Notch downstream targets were determined by western blot and quantitative PCR (qPCR). LCSCs (Oct4+) and non-LCSCs (Oct4-) were treated with siRNA targeting Jagged2 and recombinant Jagged2 protein. BrDU cell proliferation assay, Annexin V apoptosis assay, sorafenib resistance assay and tumor sphere assays were undertaken on the cell populations with modulated Jagged2 activity. Results: Notch signaling components Jagged2 and Notch1 were found to be upregulated in the LCSC (Oct4+) population. Treatment of these LCSCs with siRNA targeting Jagged2 resulted in the inhibition of Notch signaling, reduced cell proliferation and increased apoptosis. Jagged2 knockdown also sensitized these LCSCs to sorafenib treatment. Conversely, activation of the Notch pathway with recombinant Jagged2 in the non-LCSC (Oct4-) population increased tumor sphere formation and increased the expression of SOX-2, a major transcription factor which induces stem-like characteristics and is also a novel predictor of poor prognosis in HCC. Conclusions: Jagged 2 is a critical mediator of Notch signaling in LCSCs. Jagged2 mediated Notch signaling is critical for the maintenance and treatment resistance of LCSCs in HCC and can increase stem-like characteristics of differentiated cells. Given these findings we believe Jagged2 is a potential therapeutic target for the treatment of HCC.
FC; conditioned media was collected and stored at −80°C. Primary (resting) Kupffer cell cultures were prepared by selective centrifugation using Percoll gradients (50/25) and cultured in RPMI before addition of conditioned media. Highly purified cholesterol crystals were prepared in acetone, as reported (Duewell et al 2010). Pathways of inflammasome activation were determined by semiquantitative real time PC, by IL-1β secretion into media (ELISA), and use of the potent (Ki50∼10 nM) NLRP3specific inhibitor, CRID3. Results: At 40 μM LDL, hepatocytes secreted HMGB1 into culture media and underwent liver injury (as LDH leakage) with cell death by apoptosis and necrosis. Addition of HMGB1-enriched culture medium from FC-loaded hepatocytes activated resting KCs, as assessed by nuclear translocation of NF-κB, release of IL-1β and TNF-α, and ultrastructural changes. To expose Kupffer cells directly to cholesterol crystals, we coated culture vessels, using purified FC crystals solubilized in 1:100 acetone:ethanol solvent. Solvent was allowed to evaporate, leaving cholesterol crystals at 15, 20, and 60 mM concentrations per culture vessel. After 24 h culture on crystal-coated coverslips, Kupffer cells showed significant induction of NLRP3, caspase 1, ASC, IL-18 and IL-1β mRNA, as well as a 50% increase in IL-1β secretion. KCs from Tlr4−/− mice were refractory to NLRP3 activation by cholesterol crystals. Even at 5 nM, the NLRP3 inhibitor CRID3 totally (100%) abrogated IL-1β secretion from Kupffer cells activated by cholesterol crystals directly. Conclusions: These highly novel findings reveal direct links (via HMGB1 and likely other DAMPs) between cholesterol lipotoxicity and engagement of Kupffer cell activation, in which cholesterol crystals may play an additional direct pathogenic role. Identification of TLR4 and the NLRP3 inflammasome, and particularly the impressive efficacy of CRID3 as an NLRP3 inhibitor, has implications for both the pathogenesis and treatment of NASH. References 1. Ioannou GN, Haigh WG, Thorning D et al. Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis. J Lipid Res 2013; 54: 1326–1334 2. Duewell P, Hajime K, Rayner KJ et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 2010; 464: 1357–1361
What causes Kupffer cell activation in NAFLD? HMGB1, TLR4, crystals and NLRP3 inflammasome link free cholesterol to inhibitable pro-inflammatory pathways in NASH LT GAN,1 DM VAN ROOYEN,1 M COOPER,2 A ROBERTSON,2 S MASTERS,3 N TEOH,1 G FARRELL1 1 Liver Research Group, ANU Medical School at The Canberra Hospital, Garran, ACT, 2Institute of Molecular Biology, University of Queensland, QLD, 3Walter and Eliza Hall Institute, Parkville, VIC
Manipulation of dietary advanced glycation end-product content influences the progression of non-alcoholic fatty liver disease (NAFLD) C LEUNG,1,2 CB HERATH,1,2 J ZHIYUAN,1,2 T LEONG,2 JM FORBES,1,3 PW ANGUS1,2 1 The University of Melbourne, Victoria, 2Liver Unit, Austin Hospital, Heidelberg, Victoria, 3Mater Medical Research Institute, South Brisbane, Queensland
Introduction: Increasing evidence implicates free cholesterol (FC) in pathogenesis of non-alcoholic steatohepatitis (NASH). We recently showed that loading primary murine hepatocytes with FC by incubation with human LDL causes JNK1-dependent cell death with release of high mobility group box 1 (HMGB1), inhibition of which blocks further injury in FC-loaded hepatocytes. Further, TLR4-deleted hepatocytes are refractory to FC lipotoxicity. HMGB1 is an archetypical danger associated molecular pattern (DAMP) which may facilitates interactions between other DAMPs and pattern recognition receptors, such as TLRs. Cholesterol crystals have recently been identified in human NASH (Ioannou et al 2013), and are abundant in livers from our metabolic syndrome mouse model. We tested whether conditioned media from lipotoxic hepatocytes and/or cholesterol crystals activates Kupffer cells via the NLRP3 inflammasome, which in this cell type is activated via TLR4. Materials and Methods: Primary murine hepatocytes from C57B6/J wild type (WT) mice were incubated with 40 μM LDL for 24 h to load with
Introduction: Advanced glycation end-products (AGEs) content is high in western diets and may contribute to tissue injury via RAGE (receptor for AGEs). Here, we determined if manipulation of dietary AGE intake affects NAFLD progression and whether these effects are mediated via RAGE. Methods: Male C57B6 mice were fed a high fat, high fructose, high cholesterol (HFHC) diet for 33 weeks and compared with animals on normal chow. A third group were given a HFHC diet that was high in AGEs through baking. Another group was given a HFHC diet that was marinated in vinegar to prevent the formation of AGEs. In a second experiment, RAGE KO animals were fed a HFHC diet or a high AGE HFHC diet and compared with WT controls. Hepatic biochemistry, histology, picro-sirius red morphometry and hepatic mRNA were determined. We also determined the effects of AGEs on primary Kupffer cells (KCs). Results: The long term HFHC diet model generated significant steatohepatitis and fibrosis. Hepatic 4-hydroxynonenal content (a marker
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
5
Basic Science Liver
of chronic oxidative stress) and hepatocyte ballooning (a marker of cellular injury) were significantly increased with a HFHC diet and further increased with a high AGE HFHC diet and abrogated by vinegar marination. Similarly, the high AGE HFHC diet significantly increased picrosirius red staining, α-smooth muscle actin and collagen type 1A gene expression compared with HFHC alone and this was reduced by vinegar marination. The increased oxidative stress, hepatocyte ballooning and fibrosis associated with a high AGE HFHC diet was significantly reduced in corresponding high AGE HFHC RAGE KO animals. We found KCs express RAGE and take up AGEs. AGEs increase ROS generation and proliferation (as measured by BrDU uptake) in these cells. Similar results were achieved with primary hepatic stellate cells (HSCs). Conclusions: In the HFHC model of NAFLD, manipulation of dietary AGEs modulates liver injury, inflammation, and liver fibrosis via a RAGE dependent pathway. Our cell work suggests that these proinflammatory and profibrotic effects are mediated via direct effects on KCs and HSCs via RAGE. This suggests that pharmacological and dietary strategies targeting the AGE/RAGE pathway could slow the progression of NAFLD. Our results also have important implications for diabetes associated NAFLD, a condition in which endogenous AGE production and RAGE expression is increased.
The protease fibroblast activation protein (FAP) in liver disease MD GORRELL,1,3 KH WILLIAMS,2,3 AJ VIERA DE RIBEIRO,1,3 S CHOWDHURY,1,3 E HAMSON,1,3 O SCHILLING,4 E PRAKOSO,1,3 NA SHACKEL,1,3 SV MCLENNAN,2,3 WW BACHOVCHIN,5 FM KEANE,1,3 GW MCCAUGHAN,1,3 A ZEKRY,6 SM TWIGG2,3 1 Centenary Institute, 2Bosch Institute, 3The University of Sydney Medical School, Australia, 4Albert-Ludwigs University of Freiburg, Germany, 5Tufts University, Boston, USA, 6The Inflammation and Infection Research Centre, University of New South Wales School of Medical Sciences, Australia More than 400 human genes encode proteases, many of which have potential medical application. The main advantages of exploiting proteases are that both therapeutics and assays can use specific chemical compounds that are far less expensive than antibodies. FAP is predominantly associated with disease states, including liver and lung fibrosis, solid tumors, arthritis and atherosclerosis. Substrates of this protease include α-2-antiplasmin, collagen I and Neuropeptide Y. In a diet-induced obesity model, we have found that FAP gene knockout (gko) mice have improved glucose tolerance and liver histopathology, and less insulin resistance and fatty liver, compared to wild type mice on this diet. FAP gko mice resist liver fibrosis. Using our recently published novel FAP activity assay (1), we observed that serum levels of FAP enzyme activity co-segregate with liver stiffness as a measure of fibrosis in two adult cohorts with NAFLD. Cohort 1 contained 108 patients with type 2 diabetes who had transient elastography and Cohort 2 contained 148 patients with morbid obesity with liver biopsies. In Cohort 1, serum FAP was an independent risk factor for median liver stiffness ≥ 10.3 kPa. There was an 8-fold increased odds ratio of having a median liver stiffness of ≥10.3 kPa for those in the highest FAP tertile, compared with subjects in the lowest tertile (p = 0.01). A serum FAP level below 730 pmol AMC/min/mL had a negative predictive value for significant fibrosis of 95%. In Cohort 2, the FAP level was added to the NAFLD fibrosis score (NFS) to correctly reclassify 49% of patients as low risk of severe fibrosis who by NFS had been classified as intermediate risk. Measuring FAP in serum is rapid and should thus become an inexpensive supplement to the NFS to avoid patients being sent for unnecessary further tests.
6
Cell lines derived from FAP gko mice were engineered to express functional FAP enzyme (FAPe+) vs inactive FAP (FAPe-). Proteomic analyses of these cells showed FAP-specific cleavage of many bioactive peptides. In vitro ‘wound healing’ found that cells with FAP activity exhibited greater cell migration but comparable proliferation and apoptosis. Conclusions: (1) FAP has an important role in glucose and lipid metabolism and in fibrosis progression. (2) Adding a FAP serum measurement to the existing clinical NFS algorithm may correctly diagnose as non-fibrotic about half of the patients who would otherwise receive an uncertain diagnosis and require further testing. (3) FAP enzyme activity causes increased cell migration and so may have roles in wound healing. Reference 1. Keane FM, et al. Quantitation of fibroblast activation protein (FAP)specific protease activity in mouse, baboon and human fluids and organs. FEBS open bio 2014;4:43–54.
Serum-free hepatocytic transdifferentiation of pancreatic progenitor cells facilitated by extracellular matrix proteins FD GRATTE,1 JK OLYNYK,1,2,3 GCT YEOH,4 D TOSH,5 DR COOMBE,1 JEE TIRNITZ-PARKER1,2 1 School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, Australia, 2 School of Medicine and Pharmacology, University of Western Australia, Fremantle, Australia, 3Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Australia, 4Harry Perkins Institute of Medical Research, Perth, Australia, 5Centre for Regenerative Medicine, University of Bath, Bath, UK Background: Rising incidences of chronic liver disease and organ shortage for orthotopic liver transplantation have prompted interest into the development of alternative sources of liver tissue. Previous studies have highlighted the potential of cell-based technologies for the in vitro production of hepatocytes for transplantation, including the use of pancreatic progenitor cells (PPCs).1 Pancreatic progenitor cells are able to generate hepatocyte-like cells via pancreas-to-liver transdifferentiation after stimulation with the glucocorticoid dexamethasone in conjunction with other liver-promoting growth factors and cell culture supplements. Traditional methods utilize fetal bovine serum, an undefined concoction of growth factors and extracellular matrix (ECM) components, which is unsuitable for use in human treatments. Therefore the development of novel methods using defined levels of growth factors and ECM proteins in a serum-free environment is necessary for future cell-based therapies. Methods: The clonal pancreatic cell lineAR42J-B13 was cultured in basal medium (control group) or under differentiation-inducing conditions, on fibronectin or laminin, with and without serum, for five days. Cells were continuously assessed for morphological changes and subjected to transcriptome or immunofluorescent analyses on days 3 and 5 of the transdifferentiation protocol. Changes in pancreatic (amylase) and hepatocytic (hepatocyte nuclear factor 4α, albumin, tyrosine aminotransferase and transthyretin) gene and/or protein expression were evaluated. To test for hepatocyte functionality, periodic acid-Schiff staining for glycogen storage and indocyanine green uptake and release assays were performed. Results: Undifferentiated AR42J-B13 cells grew in grape-like collections of small, amylase-expressing cells and displayed little or no expression of hepatocytic markers. All groups subjected to differentiationinducing conditions quickly formed monolayer cultures, showed rapid morphological changes including significant enlargement of all cells and bi- or multinucleation (hallmark of hepatocytes) in a subpopulation of cells. Correspondingly, cells changed their gene and protein expression pattern from a pancreatic to a hepatocytic phenotype. In addition,
Journal of Gastroenterology and Hepatology 2014; 29 (Suppl. 2): 1–9 © 2014 The Authors. Journal of Gastroenterology and Hepatology © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Basic Science Liver
transdifferentiated AR42J-B13 cells were demonstrated to have hepatocyte functions by glycogen storage and indocyanine release assays. Discussion: Utilization of fibronectin or laminin facilitated the successful transdifferentiation of AR42J-B13 cells into functional hepatocyte-like cells, importantly without the presence of fetal bovine serum in the culture medium. These results may help to improve current differentiation protocols and move approaches towards a more applicable stage for use in future cell-based therapies to treat liver-based metabolic disorders. Reference 1. Shen et al. Nature Cell Biology 2000.
Heat shock protein 47 is a potential therapeutic target for the depletion of cancer-promoting stromal cells in pancreatic cancer J YOUKHANA,1* J MCCARROLL,2* G SHARBEEN,1 M ERKAN,3 J LIU,1 D GOLDSTEIN,1 PA PHILLIPS1 1 Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia, 2Children’s Cancer Institute Australia, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia, 3Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany Introduction: Pancreatic cancer (PC) is a lethal disease with a 5-year survival rate
