2
We examined methylation events in both early and advanced stage HCC, and further correlated these data with the simultaneous presence of hepatitis B and C (HBV and HCV) infection. Methods: We quantified methylation levels at 19 CpG loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC, CDH1, RUNX3, RIZ1, SFRP2, MINT31, COX2, MINT1, CACNA1G, RASSF2, MINT2, Reprimo, DCC) using combined bisulfite restriction analysis (COBRA). In total, 81 advanced tumors (well-differentiated HCCs >2 cm in size or moder- ately-poorly differentiated HCC), 12 early stage tumors (dysplastic nodules or well-differenti- ated HCC <2 cm size), and 77 non-cancerous liver tissues were analyzed. In addition, we also compared background methylation levels in 22 normal livers. Results: Based upon methylation profiles, we categorized the 19 loci into three groups. Normal liver tissues showed methylation primarily in Group-1 loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16 and APC). Non-cancerous livers demonstrated increased methylation in both Group- 1 and Group-2 loci (CDH1, RUNX3, RIZ1, SFRP2 and MINT31). Methylation of Group-3 loci (COX2, MINT1, CACNA1G, RASSF2A, MINT2, Reprimo and DCC) was rare in non- cancerous liver tissues, but was observed in early stage tumors. There was a gradual increase in methylation levels between early and advanced stage tumors, with well-differentiated HCC of > 2 cm size showing denser methylation compared with early tumors at these CpG loci. Additionally, HCV-related tumors tended to carry higher methylation levels at Group- 3 loci compared with HBV-related and virus-negative tumor, in both early and advanced tumors. Conclusions: Aberrant methylation is a frequent event in the liver and sequentially progresses from non-cancerous liver to early stage cancers and finally to advanced HCC. The cumulative differences in methylation levels among various liver tissues (precancerous livers, early and advanced tumors) suggest that aberrant methylation is an early event in HCC that progresses with the advancing stage. Lastly, simultaneous HCV infection may accelerate the carcinogenetic process in HCC. 58 Evidence for a Novel Role for Histamine Regulation of Cholangiocarcinoma Growth By An Autocrine Mechanism Heather Francis, Sharon DeMorrow, Julie Venter, Shelley Kopriva, Bradley T. Vaculin, Jennifer Savage, Gianfranco Alpini Histamine exerts its effects by interacting with multiple histamine receptors (HRH1, HRH2, HRH3 and HRH4). We have shown that activation of H3R/H4R decreases cholangiocyte growth, whereas stimulation with H1R and H2R agonists increases cholangiocyte prolifera- tion. The Aims of our study were to: (i) measure the levels of histamine and its receptors in cholangiocarcinoma (CCH) cell lines and tissue arrays; and (ii) evaluate if histamine regulates CCH growth by an autocrine mechanism. Methods: We used Mz-ChA-1 cells (extrahepatic human CCH line) and normal human cholangiocytes (H69) to measure: (i) the expression levels of the four histamine receptors, histidine decarboxylase (the enzyme that catalyzes histamine synthesis from histidine), and monoamine oxidase B (MAO B, an enzyme that catalyzes histamine catabolism) by real time PCR; and (ii) the effects of histamine (10 µM, 24-72 hr) on cholangiocarcinoma growth in the absence or presence of α-Methyl-DL- histidine dihydrochloride (histidine decarboxylase inhibitor, 10 µM), terfendadine (HRH1 antagonist, 10 µM), cimetidine (HRH2 antagonist, 25 µM), or thioperamide (HRH3/HRH4 antagonist, 25 µM); and (iii) histamine secretion by EIA. Immunohistochemistry for H1- H4 histamine receptors, histidine decarboxylase and MAO B were evaluated in a human CCH tissue array containing liver biopsy samples from 46 CCH patients and 4 healthy controls. Results: By real time PCR, we found elevated expression for all four receptor subtypes in CCH tissue arrays and CCH cells compared to H69. There was an ~90-fold increase in histidine decarboxylase expression and an ~70-fold decrease in MAO B levels in CCH mRNA compared to H69 cells. Tissue array for MAO B and histidine decarboxlyase confirmed the results from real time PCR. Consistent with the concept that histamine receptors differentially regulate cholangiocyte proliferation i.e., H1 and H2 agonists increase growth and H3/H4 agonists decrease proliferation, histamine alone did not change Mz-ChA- 1 cell growth. Stimulation with α-Methyl-DL-histidine dihydrochloride decreased Mz-ChA- 1 growth compared to basal. When Mz-ChA-1 cells were treated with H1R and H2R antagonists (in combination with histamine), there was reduced Mz-ChA-1 growth compared to histamine alone, whereas specific H3R/H4R antagonists (in combination with histamine) increased CCH growth. By EIA, secretion of histamine was greater in Mz-ChA-1 cells compared to H69. Conclusion: Histamine (coordinately interacting with HR subtypes) regu- lates cholangiocarcinoma growth. Inhibition of histamine synthesis or histamine receptors by specific antagonists may offer new avenues for cholangiocarcinoma therapy. 59 Mass Spectrometric Detection of Lipid Oxidation Products in Murine and Human Nonalcoholic Fatty Liver Disease Tamali Bhattacharyya, Renliang Zhang, Lisa M. Yerian, Arthur J. McCullough, Stanley L. Hazen, Ariel E. Feldstein Background: Nonalcoholic fatty liver disease (NAFLD) is a major public health concern. Oxidative stress has been long identified as a key component of the “second hit” responsible for disease progression.Development of non-invasive markers of oxidative stress have been limited due to the lack of sensitive markers and limited understanding of the relative importance of different oxidation pathways in the context of NAFLD. Thus the Aims of our study were to quantify the oxidized fatty acid (OxFA) profile in the plasma and liver tissue of different murine models of NAFLD/NASH; examine the role of free radical vs. stereoselective processes in formation of lipid oxidation products and,define the OxFA profile in the plasma of patients referred for liver biopsy with suspected NAFLD. Methods: Plasma and liver tissue were collected and stored under argon atmosphere with an antioxidant cocktail at -80C from 14-wks old male C57BL6 mice, on a methionine-choline deficient diet (MCD), a high fat diet (HF), or regular Chow diet (n=4 in each group) for 7 wks. Plasma was also collected and stored from 74 patients at the time of liver biopsy for suspected NAFLD. Histology was assessed by an experienced hepatopathologist. The OxFA profile was quantified using liquid chromatography with electrospray ionization mass spectrometry assay (LC/ESI/MS/MS) in mice and humans. The OxFA levels in mice on different diets were compared in both plasma and liver tissue. The OxFA profile in plasma of patients A-753 AASLD Abstracts with NASH was compared with patients with fatty liver and normal liver biopsy. SPSS statistical program (version 11) was used for all analyses. Results: Significantly increased levels of fatty acid oxidation products including 5, 8, 9, 11, 12, 15 hydroxyl-eicosatetraenoic acids (HETEs); 9 and 13 hydroxyl-octadecadenoic acids (HODEs) and Prostaglandin F2 isoprostane α (PGF2α) were found in the livers of mice on the MCD or HF diet as compared to those on a Chow diet (MCD>HF>Chow). The results suggested a free radical mediated oxidation process with significant difference in levels of 9-HETE and PGF2α between the groups. The mouse plasma levels of the lipid oxidation products mimicked the changes found in the liver tissue. Moreover, patients with NASH (n=39) had significantly higher levels of HETEs, HODEs and PGF2α compared to patients with steatosis (n=20) and normal biopsy (n=15) (NASH>steatosis>normal) replicating the results from mice to humans. Con- clusion: These results demonstrate that in NASH free radical mediated processes contribute to the generation of bioactive lipid oxidation products, and also suggest that OxFA profile may be a novel marker for NASH detection. 60 Transcriptomic Microarray Analysis of the Early Response to Treatment of Hepatitis C Virus (HCV) with Pegylated Interferon and Ribavirin Emma J. Devitt, Denise Sadlier, John A. Browne, John P. Crowe Background: Chronic Hepatitis C infection is associated with significant morbidity & mortal- ity. Standard treatment with pegylated interferon & ribavirin is suboptimal with up to 60% of patients failing to respond. Due to treatment toxicities & cost early prediction of response is crucial. Reasons for the diverse responses are not fully understood but there is strong evidence that individual molecular response to therapy influences ultimate virologic outcome. Aims: To characterise earliest transcriptomic response to treatment in HCV patients and identify a gene signature that predicts outcome. Methods: Patients with HCV infection commencing standard therapy were recruited. On the day of starting treatment blood was taken from each patient immediately before and 6, 12 & 24 hours after the first dose. Total RNA extracted from peripheral blood mononuclear cells (PBMCs) was analysed using Applied Biosystems Human Genome Survey Microarrays V2.0 (32,878 probes). Subsequent data analysis was performed using CARMAweb (https://carmaweb.genome.tugraz.at/carma/) along with R-Bioconductor (http://www.r-project.org/) and hierarchical cluster analysis. HCV PCR taken at 12 weeks was used to classify patients as treatment responders [R] (PCR negative) or non-responders [NR] (PCR positive). Results: In total 48 microarrays were performed on samples from 12 patients at time 0,6,12 & 24 hours (R=7 patients, NR= 5 patients). After applying strict statistical criteria only 11,333 probes were included in further transcriptomic analysis. Of these, more genes were significantly dysregulated in R compared to NR (18% vs.12%). For both R & NR, mRNA expression peaked at T12 compared to other timepoints (T6:R = 8%,NR = 6%, T12:R = 12%,NR = 9%, T24:R = 10%,NR = 8%) with the majority of dysregulated genes common to both R & NR (T6: 57%, T12: 58% and T24: 53%). Gene expression profiles in R vs. NR were analysed with cluster analysis identifying 3 major patterns :(1) Identical patterns of dysregulation (2)Similar expression profile but with more marked dysregulation in R vs. NR (3)Opposite patterns of expression- a cohort of genes demonstrated contrasting patterns of expression in R vs. NR. These included mRNA tran- scripts that are critical to immune response including CCL3, CCR3, TNFSF4 & FGF1 and mRNA transcripts not previously described in this setting such as ATF5 and TLE4. Conclu- sion: These early changes to the transcript profile demonstrate a rapid and significant response to treatment within the first 24 hours. Correlation of gene expression changes with ultimate virological response can identify an early transcriptomic signature predictive of virologic response. 107 Liver Fibrosis in Diet-Induced Steatohepatitis Is Attenuated By Deficiency in the Expression of Toll-Like Receptor 4 (TLR4), or the TLR Adapter, MyD88 Arumugam Velayudham, Istvan Hritz, Donna Catalano, Pranoti Mandrekar, Gyongyi Szabo Activation of inflammation-associated pathways is a major component in the progression of steatohepatitis to fibrosis and cirrhosis. Lipopolysaccharide (LPS) induced activation of TLR4 is a central event in the pathology of alcoholic liver disease, which shares many pathological features with non-alcoholic steatohepatitis (NASH). The role of LPS is yet to be determined in the progression of NASH to cirrhosis. Downstream signaling following recognition of LPS by TLR4 involves the common TLR adapter molecule, MyD88, and leads to the induction of inflammatory cytokines. The goal of this study was to evaluate the role of TLR4 and MyD88 in development of liver fibrosis in the setting of NASH induced by a methionone- choline deficient (MCD) diet in mice. Wild-type (WT) and knockout (KO) mice, deficient in the expression of TLR4 or MyD88, were fed MCD or methionine choline supplemented (MCS) diet for 8 weeks. MCD diet induced steatohepatitis, liver fibrosis, and elevated serum ALT (10-fold increase) in WT but to a significantly lesser degree in TLR4- or MyD88-KO mice. Sirius red staining revealed marked liver fibrosis in MCD diet-fed WT mice, but not in TLR4- or MyD88-KO mice. In addition, immunohistochemistry revealed significant expression of alpha smooth muscle actin (αSMA) in MCD diet-fed WT but not in TLR4- or MyD88-KO livers. Consistent with this, mRNA expression of procollagen I, TGFβ1, αSMA, MMP2 and TIMP1 in the liver was markedly upregulated by MCD diet in WT mice but to a significantly lower degree in TLR4- and MyD88-KO mice. Furthermore, TLR4 as well as MyD88 deficiency was protective from MCD diet-induced increase in liver triglyceride accumulation and TBARs, a marker of lipid peroxidation. This was also associated with an MCD-diet induced upregulation of mRNA of the subunits of the NADPH oxidase complex, namely p22phox, p47phox, p67phox and gp91phox, in WT livers but not in the absence of MyD88- and TLR4. Serum TNFα, a marker of inflammatory activation and a mediator of insulin resistance, was significantly lower in MCD diet-fed TLR4- and MyD88-KO mice compared to WT. Conversely, the hepatoprotective cytokine, IL-6, and the anti-inflammatory cytokine, IL-10, were significantly higher in the sera of TLR4- and MyD88-KO MCD-fed mice compared to WT. In summary, these results demonstrate a critical role for the TLR4 receptor complex and NADPH oxidase in the development of NASH-associated liver damage, steatosis, inflammation, and fibrosis. The absence of TLR4 or the adapter molecule, MyD88, AASLD Abstracts

107 Liver Fibrosis in Diet-Induced Steatohepatitis Is Attenuated By Deficiency in the Expression of Toll-Like Receptor 4 (TLR4), or the TLR Adapter, MyD88

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We examined methylation events in both early and advanced stage HCC, and furthercorrelated these data with the simultaneous presence of hepatitis B and C (HBV and HCV)infection. Methods: We quantified methylation levels at 19 CpG loci (HIC-1, CASP8, GSTP1,SOCS1, RASSF1A, p16, APC, CDH1, RUNX3, RIZ1, SFRP2, MINT31, COX2, MINT1,CACNA1G, RASSF2, MINT2, Reprimo, DCC) using combined bisulfite restriction analysis(COBRA). In total, 81 advanced tumors (well-differentiated HCCs >2 cm in size or moder-ately-poorly differentiated HCC), 12 early stage tumors (dysplastic nodules or well-differenti-ated HCC <2 cm size), and 77 non-cancerous liver tissues were analyzed. In addition, wealso compared background methylation levels in 22 normal livers. Results: Based uponmethylation profiles, we categorized the 19 loci into three groups. Normal liver tissuesshowed methylation primarily in Group-1 loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A,p16 and APC). Non-cancerous livers demonstrated increased methylation in both Group-1 and Group-2 loci (CDH1, RUNX3, RIZ1, SFRP2 and MINT31). Methylation of Group-3loci (COX2, MINT1, CACNA1G, RASSF2A, MINT2, Reprimo and DCC) was rare in non-cancerous liver tissues, but was observed in early stage tumors. There was a gradual increasein methylation levels between early and advanced stage tumors, with well-differentiatedHCC of > 2 cm size showing denser methylation compared with early tumors at these CpGloci. Additionally, HCV-related tumors tended to carry higher methylation levels at Group-3 loci compared with HBV-related and virus-negative tumor, in both early and advancedtumors. Conclusions: Aberrant methylation is a frequent event in the liver and sequentiallyprogresses from non-cancerous liver to early stage cancers and finally to advanced HCC.The cumulative differences in methylation levels among various liver tissues (precancerouslivers, early and advanced tumors) suggest that aberrant methylation is an early event inHCC that progresses with the advancing stage. Lastly, simultaneous HCV infection mayaccelerate the carcinogenetic process in HCC.

58

Evidence for a Novel Role for Histamine Regulation of CholangiocarcinomaGrowth By An Autocrine MechanismHeather Francis, Sharon DeMorrow, Julie Venter, Shelley Kopriva, Bradley T. Vaculin,Jennifer Savage, Gianfranco Alpini

Histamine exerts its effects by interacting with multiple histamine receptors (HRH1, HRH2,HRH3 and HRH4). We have shown that activation of H3R/H4R decreases cholangiocytegrowth, whereas stimulation with H1R and H2R agonists increases cholangiocyte prolifera-tion. The Aims of our study were to: (i) measure the levels of histamine and its receptorsin cholangiocarcinoma (CCH) cell lines and tissue arrays; and (ii) evaluate if histamineregulates CCH growth by an autocrine mechanism. Methods: We used Mz-ChA-1 cells(extrahepatic human CCH line) and normal human cholangiocytes (H69) to measure: (i)the expression levels of the four histamine receptors, histidine decarboxylase (the enzymethat catalyzes histamine synthesis from histidine), and monoamine oxidase B (MAO B, anenzyme that catalyzes histamine catabolism) by real time PCR; and (ii) the effects of histamine(10 µM, 24-72 hr) on cholangiocarcinoma growth in the absence or presence ofα-Methyl-DL-histidine dihydrochloride (histidine decarboxylase inhibitor, 10 µM), terfendadine (HRH1antagonist, 10 µM), cimetidine (HRH2 antagonist, 25 µM), or thioperamide (HRH3/HRH4antagonist, 25 µM); and (iii) histamine secretion by EIA. Immunohistochemistry for H1-H4 histamine receptors, histidine decarboxylase and MAO B were evaluated in a humanCCH tissue array containing liver biopsy samples from 46 CCH patients and 4 healthycontrols. Results: By real time PCR, we found elevated expression for all four receptorsubtypes in CCH tissue arrays and CCH cells compared to H69. There was an ~90-foldincrease in histidine decarboxylase expression and an ~70-fold decrease in MAO B levelsin CCH mRNA compared to H69 cells. Tissue array for MAO B and histidine decarboxlyaseconfirmed the results from real time PCR. Consistent with the concept that histaminereceptors differentially regulate cholangiocyte proliferation i.e., H1 and H2 agonists increasegrowth and H3/H4 agonists decrease proliferation, histamine alone did not change Mz-ChA-1 cell growth. Stimulation with α-Methyl-DL-histidine dihydrochloride decreased Mz-ChA-1 growth compared to basal. When Mz-ChA-1 cells were treated with H1R and H2Rantagonists (in combination with histamine), there was reduced Mz-ChA-1 growth comparedto histamine alone, whereas specific H3R/H4R antagonists (in combination with histamine)increased CCH growth. By EIA, secretion of histamine was greater in Mz-ChA-1 cellscompared to H69. Conclusion: Histamine (coordinately interacting with HR subtypes) regu-lates cholangiocarcinoma growth. Inhibition of histamine synthesis or histamine receptorsby specific antagonists may offer new avenues for cholangiocarcinoma therapy.

59

Mass Spectrometric Detection of Lipid Oxidation Products in Murine andHuman Nonalcoholic Fatty Liver DiseaseTamali Bhattacharyya, Renliang Zhang, Lisa M. Yerian, Arthur J. McCullough, Stanley L.Hazen, Ariel E. Feldstein

Background: Nonalcoholic fatty liver disease (NAFLD) is a major public health concern.Oxidative stress has been long identified as a key component of the “second hit” responsiblefor disease progression.Development of non-invasive markers of oxidative stress have beenlimited due to the lack of sensitive markers and limited understanding of the relativeimportance of different oxidation pathways in the context of NAFLD. Thus the Aims of ourstudy were to quantify the oxidized fatty acid (OxFA) profile in the plasma and livertissue of different murine models of NAFLD/NASH; examine the role of free radical vs.stereoselective processes in formation of lipid oxidation products and,define the OxFA profilein the plasma of patients referred for liver biopsy with suspected NAFLD. Methods: Plasmaand liver tissue were collected and stored under argon atmosphere with an antioxidantcocktail at -80C from 14-wks old male C57BL6 mice, on a methionine-choline deficientdiet (MCD), a high fat diet (HF), or regular Chow diet (n=4 in each group) for 7 wks.Plasma was also collected and stored from 74 patients at the time of liver biopsy for suspectedNAFLD. Histology was assessed by an experienced hepatopathologist. The OxFA profilewas quantified using liquid chromatography with electrospray ionization mass spectrometryassay (LC/ESI/MS/MS) in mice and humans. The OxFA levels in mice on different dietswere compared in both plasma and liver tissue. The OxFA profile in plasma of patients

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A-753 AASLD Abstracts

with NASH was compared with patients with fatty liver and normal liver biopsy. SPSSstatistical program (version 11) was used for all analyses. Results: Significantly increasedlevels of fatty acid oxidation products including 5, 8, 9, 11, 12, 15 hydroxyl-eicosatetraenoicacids (HETEs); 9 and 13 hydroxyl-octadecadenoic acids (HODEs) and Prostaglandin F2isoprostane α (PGF2α) were found in the livers of mice on the MCD or HF diet as comparedto those on a Chow diet (MCD>HF>Chow). The results suggested a free radical mediatedoxidation process with significant difference in levels of 9-HETE and PGF2α between thegroups. The mouse plasma levels of the lipid oxidation products mimicked the changesfound in the liver tissue. Moreover, patients with NASH (n=39) had significantly higherlevels of HETEs, HODEs and PGF2α compared to patients with steatosis (n=20) and normalbiopsy (n=15) (NASH>steatosis>normal) replicating the results from mice to humans. Con-clusion: These results demonstrate that in NASH free radical mediated processes contributeto the generation of bioactive lipid oxidation products, and also suggest that OxFA profilemay be a novel marker for NASH detection.

60

Transcriptomic Microarray Analysis of the Early Response to Treatment ofHepatitis C Virus (HCV) with Pegylated Interferon and RibavirinEmma J. Devitt, Denise Sadlier, John A. Browne, John P. Crowe

Background: Chronic Hepatitis C infection is associated with significant morbidity & mortal-ity. Standard treatment with pegylated interferon & ribavirin is suboptimal with up to 60%of patients failing to respond. Due to treatment toxicities & cost early prediction of responseis crucial. Reasons for the diverse responses are not fully understood but there is strongevidence that individual molecular response to therapy influences ultimate virologic outcome.Aims: To characterise earliest transcriptomic response to treatment in HCV patients andidentify a gene signature that predicts outcome. Methods: Patients with HCV infectioncommencing standard therapy were recruited. On the day of starting treatment blood wastaken from each patient immediately before and 6, 12 & 24 hours after the first dose. TotalRNA extracted from peripheral blood mononuclear cells (PBMCs) was analysed using AppliedBiosystems Human Genome Survey Microarrays V2.0 (32,878 probes). Subsequent dataanalysis was performed using CARMAweb (https://carmaweb.genome.tugraz.at/carma/) alongwith R-Bioconductor (http://www.r-project.org/) and hierarchical cluster analysis. HCV PCRtaken at 12 weeks was used to classify patients as treatment responders [R] (PCR negative)or non-responders [NR] (PCR positive). Results: In total 48 microarrays were performed onsamples from 12 patients at time 0,6,12 & 24 hours (R=7 patients, NR= 5 patients). Afterapplying strict statistical criteria only 11,333 probes were included in further transcriptomicanalysis. Of these, more genes were significantly dysregulated in R compared to NR (18%vs.12%). For both R & NR, mRNA expression peaked at T12 compared to other timepoints(T6:R = 8%,NR = 6%, T12:R = 12%,NR = 9%, T24:R = 10%,NR = 8%) with the majorityof dysregulated genes common to both R & NR (T6: 57%, T12: 58% and T24: 53%). Geneexpression profiles in R vs. NR were analysed with cluster analysis identifying 3 majorpatterns :(1) Identical patterns of dysregulation (2)Similar expression profile but with moremarked dysregulation in R vs. NR (3)Opposite patterns of expression- a cohort of genesdemonstrated contrasting patterns of expression in R vs. NR. These included mRNA tran-scripts that are critical to immune response including CCL3, CCR3, TNFSF4 & FGF1 andmRNA transcripts not previously described in this setting such as ATF5 and TLE4. Conclu-sion: These early changes to the transcript profile demonstrate a rapid and significant responseto treatment within the first 24 hours. Correlation of gene expression changes with ultimatevirological response can identify an early transcriptomic signature predictive of virologicresponse.

107

Liver Fibrosis in Diet-Induced Steatohepatitis Is Attenuated By Deficiency inthe Expression of Toll-Like Receptor 4 (TLR4), or the TLR Adapter, MyD88Arumugam Velayudham, Istvan Hritz, Donna Catalano, Pranoti Mandrekar, GyongyiSzabo

Activation of inflammation-associated pathways is a major component in the progression ofsteatohepatitis to fibrosis and cirrhosis. Lipopolysaccharide (LPS) induced activation of TLR4is a central event in the pathology of alcoholic liver disease, which shares many pathologicalfeatures with non-alcoholic steatohepatitis (NASH). The role of LPS is yet to be determinedin the progression of NASH to cirrhosis. Downstream signaling following recognition of LPSby TLR4 involves the common TLR adapter molecule, MyD88, and leads to the inductionof inflammatory cytokines. The goal of this study was to evaluate the role of TLR4 andMyD88 in development of liver fibrosis in the setting of NASH induced by a methionone-choline deficient (MCD) diet in mice. Wild-type (WT) and knockout (KO) mice, deficientin the expression of TLR4 or MyD88, were fed MCD or methionine choline supplemented(MCS) diet for 8 weeks. MCD diet induced steatohepatitis, liver fibrosis, and elevated serumALT (10-fold increase) in WT but to a significantly lesser degree in TLR4- or MyD88-KOmice. Sirius red staining revealed marked liver fibrosis in MCD diet-fed WT mice, butnot in TLR4- or MyD88-KO mice. In addition, immunohistochemistry revealed significantexpression of alpha smooth muscle actin (αSMA) in MCD diet-fed WT but not in TLR4-or MyD88-KO livers. Consistent with this, mRNA expression of procollagen I, TGFβ1,αSMA, MMP2 and TIMP1 in the liver was markedly upregulated by MCD diet in WT micebut to a significantly lower degree in TLR4- and MyD88-KO mice. Furthermore, TLR4 aswell as MyD88 deficiency was protective from MCD diet-induced increase in liver triglycerideaccumulation and TBARs, a marker of lipid peroxidation. This was also associated with anMCD-diet induced upregulation of mRNA of the subunits of the NADPH oxidase complex,namely p22phox, p47phox, p67phox and gp91phox, in WT livers but not in the absenceof MyD88- and TLR4. Serum TNFα, a marker of inflammatory activation and a mediatorof insulin resistance, was significantly lower in MCD diet-fed TLR4- and MyD88-KO micecompared to WT. Conversely, the hepatoprotective cytokine, IL-6, and the anti-inflammatorycytokine, IL-10, were significantly higher in the sera of TLR4- and MyD88-KO MCD-fedmice compared to WT. In summary, these results demonstrate a critical role for the TLR4receptor complex and NADPH oxidase in the development of NASH-associated liver damage,steatosis, inflammation, and fibrosis. The absence of TLR4 or the adapter molecule, MyD88,

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swas equally protective suggesting a role for the MyD88-dependent TLR4 pathway activationin the pathogenesis of NASH and associated liver fibrosis in this model.

108

NRF2 Prevents Alcohol Induced Fulminant Liver InjuryJutta Lamlé, Silke Marhenke, Jürgen Borlak, Masayuki Yamamoto, Michael P. Manns,Arndt Vogel

The transcription factor Nrf2 is essential for protecting cells against xenobiotic and oxidativestress. To clarify the role of Nrf2 in ethanol induced liver injury, WT and Nrf2-/- mice weretreated with an alcohol-containing liquid diet. With doses of ethanol that were tolerated byWT mice, Nrf2-/- mice died of liver failure. Livers of critically sick Nrf2-/- mice exhibitedsevere macrovesicular steatosis, multiple Tunel-positive hepatocytes and focal inflammation.Following ingestion ethanol is oxidized by cytosolic alcohol dehydrogenases to acetaldehyde,which is oxidized by aldehyde dehydrogenases to produce non-toxic acetate. Loss of Nrf2did not impair the expression of genes involved in the oxidation of alcohol to acetaldehyde.However, Nrf2-/- mice displayed a reduced expression of Aldh enzymes leading to signific-antly lower Aldh activity in Nrf2-/- mice. After ethanol administration, toxic acetaldehydelevels were accordingly significantly higher in livers of Nrf2-/- mice that likely contributeto the increased mortality of Nrf2-/- mice. Ethanol feeding induced a severe steatosis inNrf2-/- mice, which is reflected by a gene expression profile that governs lipid homeostasisin the liver. Pathway analysis identified Srebp-1 as a likely candidate contributing to theearly adaptations in Nrf2-/- mice. Furthermore, ethanol consumption led to a progressivedepletion of total and mitochondrial GSH, which was associated with more pronouncedstructural and functional changes to mitochondria of Nrf2-/- mice. Progression of alcoholicliver disease beyond steatosis does not occur in the absence of a second hit that promotesinflammation and cell death. Accumulating evidence indicates that ethanol increases thesusceptibility to toxic effects of endotoxins released from the gut. Here, ethanol treatmentelicits an aggravated inflammatory response in Nrf2-/- mice. High Tnf-α/ Il-6 levels occurredsimultaneously with the onset of the severe steatosis shortly before Nrf2-/- mice died,suggesting that the overwhelming immune response mediated by Kupffer cells is directlyrelated to the increased mortality of ethanol-fed Nrf2-/- mice. Taken together, our dataestablish a central role for Nrf2 in the protection against ethanol-induced liver injury.Detoxification of acetaldehyde was significantly impaired in Nrf2-/- mice, leading to anaccumulation of toxic acetaldehyde. Disruption of Nrf2 further aggravated ethanol-inducedliver damage by eliciting an aggravated inflammatory response in ethanol-fed Nrf2-/- mice.Together this changes lead to a vicious cycle of accumulating hepatocellular damage, ulti-mately leading to liver failure and death of Nrf2-/- mice.

109

Primary Alterations in Hepatic Lipid Metabolism Do Not Affect LiverRegeneration Following Partial HepatectomyElizabeth P. Newberry, Susan Kennedy, Yan Xie, Jianyang Luo, Nicholas O. Davidson

A marked, but transient hepatic steatosis occurs after 70% partial hepatectomy (PH) in mice,with hepatic triglyceride (TG) levels peaking 18-24h after PH, returning to baseline after~72h. Studies have shown that genetic or pharmacologic modifications that alter wholebody lipid metabolism prevent PH-induced hepatic lipid accumulation and abrogate liverregeneration, leading to the suggestion that hepatic steatosis is required for normal liverregeneration. We have reexamined this issue using a range of genetically manipulated micewith primary alterations in hepatic lipid metabolism in order to modulate hepatic TGaccumulation following PH and then examined liver regeneration. L-Fabp-/- mice exhibitreduced (50-80%) hepatic TG content compared to WT mice at 6, 12, 24, and 48h afterPH, with no effect on liver regeneration or hepatocyte proliferation. This phenotype correlateswith the absence of L-Fabp in liver, since WT mice treated with L-Fabp antisense oligonucleo-tide (ASO), which manifest decreased hepatic but not intestinal L-Fabp mRNA and protein,had a 50% reduction in hepatic TG 24h after PH compared to control ASO mice. BrdUincorporation 48h after PH is similar in control and L-Fabp ASO treated mice (27.6 ± 6.7vs 32.2 ± 13.0% positive nuclei, respectively). Mice with intestine-specific deletion ofmicrosomal TG transfer protein (MTP-IKO) exhibit virtually complete fat malabsorption,resulting in a lack of peripheral fat and manifest a compensatory induction of hepaticlipogenesis. Hepatic TG accumulation following PH is reduced 2-4x in MTP-IKO mice,likely due to decreased availability of FA, yet there is no decrease in hepatocyte proliferation.Mice lacking PPARα exhibit ~4x elevated hepatic TG compared to WT mice 48h after PH,with no difference in liver regeneration (PPARα-/-, 36.2 ± 5.7; C57BL/6, 39.1 ± 6.2% BrdUpositive nuclei). This temporary inability to mobilize TG stores likely reflects decreasedinduction of hepatic MTP expression, which is markedly attenuated in PPARα null mice(increased 70% 24h after PH in both WT and L-Fabp-/- mice vs 0% in PPARα-/- mice) andreduced ketogenesis following PH. Double knockout (L-Fabp-/-, PPARα-/-) mice demonstratedreversal of hepatic TG accumulation at 48h following PH, but again there was no defect inhepatic regeneration. Together these data indicate that hepatic TG accumulation is regulatedthrough MTP-dependent defects in lipid mobilization but is not required for liver regenera-tion.

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Over Twenty Five Years of Follow Up for a Non-Alcoholic Fatty Liver Disease(NAFLD) CohortNila Rafiq, Chunhong Bai, Yun Fang, Manirath K. Srishord, Arthur J. McCullough, ZobairM. Younossi

NAFLD is one of the most common causes of chronic liver disease. From the spectrum ofNAFLD, patients with non-alcoholic steatohepatitis (NASH) have a potentially progressivecourse. Aim: To assess the long term mortality outcomes of patients with NAFLD and itssubtypes. Design: From our existing databases, patients with biopsy proven NAFLD with aminimum of 5 years of follow up were included in the study. All other causes of liver disease

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A-754AASLD Abstracts

(viral hepatitis, excessive alcohol use etc.) have been excluded and liver biopsies read in asystematic fashion for NAFLD (age at biopsy 50.1±14.5, male 39.7%, Caucasians 80.4%,type II Diabetes 28.7% and hyperlipidemia 48.5%). NAFLD patients were categorized asNASH, Simple Steatosis (SS) or steatosis with non-specific changes [Non-NASH NAFLD(NNN)]. Mortality data and causes of death were obtained from National Death Index Plus.Univariate and multivariate analyses were performed comparing different NAFLD subtypesand to identify predictors of mortality. Results: A total of 174 NAFLD patients were included(NASH= 66, SS= 75, NNN= 32). The median follow up from the time of biopsy to the timeof death or the last NDI follow up was 11.13 years with longest follow up being 28.55years. Over this period, 78 NAFLD patients had died with the most common causes ofdeath being coronary artery disease (13%), malignancy (8%) and liver death (7%). Althoughoverall mortality was not different between the NAFLD subtypes, liver related mortality washigher in NASH (16%) as compared to SS (2%) or NNN (0%), p-values<0.05. Kaplan Meirsurvival estimates confirmed the increase in liver-related mortality in NASH as comparedto SS and NNN (p=0.0043). In multivariate analysis, independent predictors of liver relatedmortality included histologic NASH [Hazard Ratio (HR) of 8.647, p=0.0056], presence ofdiabetes (HR of 5.137, p=0.0108) and age of biopsy (HR of 1.1 per year, p=0.0037).Conclusions: This long term follow up of patients with NAFLD confirms that NASH patientshave higher liver related mortality than non-NASH patients with NAFLD. Additionally,patients with NAFLD and diabetes are especially at risk for liver related mortality.

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Hepatic Lipid Partitioning and Liver Damage in NAFLD: Role of Stearoyl-CoADesaturaseZhengzheng Li, Michael P. Berk, Ariel E. Feldstein

Hepatic lipid overloading mainly in the form of triglycerides (TG) is considered a prerequisitefor the development of NAFLD. However, TG accumulation in the liver in response to lipidoverflow may represent a protective mechanism against lipotoxicity. Our aims were to assessthe fundamental cellular mechanisms that link lipid partitioning to liver injury by usingboth In Vivo dietary models of NAFLD and In Vitro cell models of lipid overloading.METHODS: Primary mouse hepatocytes, HepG2 and McNtcp.24 cells were treated withvaried concentrations of free fatty acids (FFA) with different degrees of saturation, alone orin combination for up to 24 hrs. Apoptosis was assessed morphologically and biochemically.TG accumulation was assessed by Oil Red O staining. Activity of Stearoyl-CoA Desaturase(SCD)-1, the enzyme that catalysis the rate limiting step in TG formation, was suppressedby genetic (siRNA) or pharmacological approaches. SCD-1 knock out and wildtype micewere placed on either a high fat(HSAT), a methionine-choline deficient (MCD), or a controlchow diet (n = 6 in each group) for 6 wks. Liver injury was assessed by TUNEL assay,active caspase 3 staining, WB for cytosolic cytochrome c, histopathology, and serum ALT.Hepatic lipids were quantified by capillary gas chromatography. SCD-1 expression wasassessed by WB analysis. RESULTS: Exposure of liver cells to unsaturated FFA (oleate)resulted in significant accumulation of TG without changes in cell viability. In contrast, cellsincubated with saturated FFA (palmitate or stearate) showed significant decrease in cellviability as a result of increase apoptotic cell death in conjunction with absence of TGaccumulation above the baseline levels. Moreover, blocking TG synthesis by either geneticor pharmacological inhibition of SCD-1 resulted in a significant increased sensitivity to FFAinduced apoptotic cell death. Hepatic SCD-1 expression was increased in HSAT fed anddecreased in MCD fed wildtype animals compared to those mice on the control diet. SCD-1-/- mice showed decreased in hepatic TG content on both HSAT and MCD diet comparedto the SCD-1+/+ on the respective diets. However SCD-/- mice on the MCD diet showedsignificant increase in hepatic saturated FFA levels, and marked increased in hepatocellularapoptosis, inflammation and liver injury. CONCLUSIONS: These data strongly suggest thatlipid partitioning in the liver and in particular excessive accumulation of saturated FFA mayplay an important role in disease progression from fatty liver to steatohepatitis. This concepthas important implications for the development of novel treatment strategies for patientswith this condition.

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18β-Glycyrrhetinic Acid Prevents Free Fatty Acids/High Fat Diet-InducedHepatic Toxicity By Inhibiting Cathepsin B Expression and Enzyme ActivitiesXudong Wu, Luyong Zhang, Jing Shang, Emily C. Gurley, Elaine Studer, Xuan Wang,Phillip Hylemon, William M. Pandak, Arun J. Sanyal, Huiping Zhou

Free fatty acids (FFAs)-induced lipotoxicity plays a pivotal role in the pathogenesis ofnonalcoholic fatty liver disease NAFLD. Inhibition of FFAs-associated hepatic toxicity repres-ents a potential therapeutic strategy. Glycyrrhizin (GL), the major bioactive component oflicorice root extract, has been used to treat hepatitis to reduce liver inflammation and hepaticinjury. However, the underlying mechanism remains unknown. 18β-glycyrrhetinic acid (GA)is the biologically active metabolite of GL. In the present study, we examined whether GAis able to prevent FFAs-induced lipotoxicity both in In Vitro and In Vivo NAFLD models.Methods: HepG2 cells were treated with 1 mM FFAs (oleic acid /palmitic acid, 2;1) in thepresence of GA (0 ~ 30 µM) for various time periods. High fat diet (HFD)-induced ratNAFLD models were used for In Vivo studies. Rats were fed HFD and gavaged with GA (25and 50 mg/kg) for 8-weeks. The apoptotic cells were detected by Annexin V-FITC/PI stainingfollowed by flow cytometry. The lipid accumulation was detected by nile red staining.The lysosomal stability was assessed using the acridine orange (AO)-uptake method. Theexpression of cathepsin B was detected by western blot analysis and immunofluorescentstaining. Serum lipids and liver function enzymes were measured using standard enzymatictechniques. Cathepsin B enzyme activities were measured using specific substrate Z-Arg-Arg-7-amido-4-methylcourmarin hydrochloride. Liver injury was examined by HE staining.The histopathological score of rat liver was analyzed by Mann-Whitney Rank Sum Test.Results: GA not only significantly inhibited FFAs-induced apoptosis in HepG2 cells, butalso protected the HFD-induced lipid accumulation and liver injury in rats. In HepG2 cells,GA (10 µM) markedly inhibited FFAs-induced cathepsin B expression and enzyme activitiesby 50% and 26%, respectively. In rat NAFLD models, GA (50 mg/kg) also significantlyinhibited HFD-induced cathepsin B expression and enzyme activity by 70% and 34%,