Genomics of liver fibrosis and cirrhosis

Hepatic fibrosis is the response to chronic injury from viral, toxic, metabolic, cholestatic, or autoimmune liver injury. However, only a minority of affected individuals develop advanced fibrosis or cirrhosis, suggesting that modifiers determine the individual risk. Aside from well-established progression factors including gender, duration of exposure to the disease, and ethnicity, unknown host genetic factors are likely to influence disease progression and prognosis. Potential genetic susceptibility loci are single nucleotide polymorphisms in fibrosis-associated genes, point mutations, microsatellites, and haplotype blocks composed of genes pivotal for fibrosis development. However, the study of complex polygenetic diseases poses numerous pitfalls in contrast to the elucidation of monogenetic (i.e., Mendelian) diseases. Many publications on the role of certain genetic variants in modulating the progression of hepatic fibrosis have been limited by inadequate study design and low statistical power. At present, powerful research strategies are being developed that allow the application of knowledge derived from the successful sequencing of the human genome that will help to translate our newly acquired insight into practical improvements for research activities and medical practice.

Integrin alphavbeta6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies

BACKGROUND/AIMS: The integrin alphavbeta6 promotes proliferation of specialized epithelia and acts as a receptor for the activation of latent TGFbeta1. We studied alphavbeta6 expression in experimental and human liver fibrosis and the potential of its pharmacological inhibition for treatment of hepatic fibrosis. METHODS: alphavbeta6 expression was studied by quantitative PCR and immunohistochemistry in rats with cirrhosis due to bile duct ligation (BDL), administration of thioacetamide (TAA), in Mdr2(Abcb4)(-/-) mice with spontaneous biliary fibrosis, and in livers of patients with chronic hepatitis C (n=79) and end-stage liver disease due to various etiologies (n=18). The effect of a selective alphavbeta6 inhibitor was evaluated in Mdr2(Abcb4)(-/-) mice with ongoing fibrogenesis. RESULTS: Integrin beta6 mRNA increased with fibrosis stage in hepatitis C and was upregulated between 25- and 100-fold in TAA- and BDL-induced cirrhosis, in Mdr2(Abcb4)(-/-) mice and in human end-stage liver disease. alphavbeta6 protein was absent in normal livers and expressed de novo on (activated) bile duct epithelia and transitional hepatocytes. A single dose of the alphavbeta6 inhibitor injected into Mdr2(Abcb4)(-/-) mice significantly induced profibrolytic matrix metalloproteinases (MMP)-8 and -9 after 3 h, with a corresponding increase in extracellular matrix-degrading activities. In parallel profibrogenic transcripts (procollagen alpha1(I), TGFbeta2, and MMP-2) showed a trend of downregulation. CONCLUSIONS: (1) Integrin alphavbeta6 is induced de novo in rodent and human liver fibrosis, where it is expressed on activated bile duct epithelia and (transitional) hepatocytes during fibrosis progression. (2) In vivo a single dose of a small molecule alphavbeta6 inhibitor induced antifibrogenic and profibrolytic genes and activities, suggesting alphavbeta6 is a unique target for treatment of liver fibrosis.

Alveolar echinococcosis: from a deadly disease to a well-controlled infection. Relative survival and economic analysis in Switzerland over the last 35 years

BACKGROUND/AIMS: Alveolar echinococcosis (AE) is a serious liver disease. The aim of this study was to explore the long-term prognosis of AE patients, the burden of this disease in Switzerland and the cost-effectiveness of treatment. METHODS: Relative survival analysis was undertaken using a national database with 329 patient records. 155 representative cases had sufficient details regarding treatment costs and patient outcome to estimate the financial implications and treatment costs of AE. RESULTS: For an average 54-year-old patient diagnosed with AE in 1970 the life expectancy was estimated to be reduced by 18.2 and 21.3 years for men and women, respectively. By 2005 this was reduced to approximately 3.5 and 2.6 years, respectively. Patients undergoing radical surgery had a better outcome, whereas the older patients had a poorer prognosis than the younger patients. Costs amount to approximately Euro108,762 per patient. Assuming the improved life expectancy of AE patients is due to modern treatment the cost per disability-adjusted life years (DALY) saved is approximately Euro6,032. CONCLUSIONS: Current treatments have substantially improved the prognosis of AE patients compared to the 1970s. The cost per DALY saved is low compared to the average national annual income. Hence, AE treatment is highly cost-effective in Switzerland.

Intrarenal resistance index for the assessment of early renal function impairment in patients with liver cirrhosis

BACKGROUND: Renovascular vasoconstriction in patients with hepatorenal syndrome can be quantified by the renal arterial resistance index (RI). We investigated the value of RI measurement in detection of renal function impairment in patients with different stages of chronic liver disease. METHODS: Subjects were divided into 4 groups containing 21 patients with liver cirrhosis and ascites, 25 patients with liver cirrhosis without ascites, 35 patients with fatty liver disease and 78 control subjects. All patients underwent abdominal ultrasound examination with renal RI measurement and correlation with laboratory results for renal function. RESULTS: RI was significantly higher in ascitic patients compared to non-ascitic patients (0.74 vs. 0.67, p<0.01) and in non-ascitic patients with liver cirrhosis than in control subjects (0.67 vs. 0.62, p<0.01). 48% (19/40) of patients with liver cirrhosis and normal serum creatinine concentration showed elevated RI levels. There were no significant differences in RI levels between patients with fatty liver disease and controls (0.63 vs. 0.62). CONCLUSIONS: Intrarenal RI measurement is a predictor of renal vasoconstriction and serves to detect early renal function impairment in cirrhotic patients. The diagnosis of elevated RI may be taken into account in the clinical management of these patients.

Do common genetic variants in endotoxin signaling pathway contribute to predisposition to alcoholic liver cirrhosis?

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), produced by endotoxin-activated Kupffer cells, play a key role in the pathogenesis of alcoholic liver cirrhosis (ALC). Alleles TNFA -238A, IL1B -31T and variant IL1RN*2 of repeat polymorphism in the gene encoding the IL-1 receptor antagonist increase production of TNF-alpha and IL-1beta, respectively. Alleles CD14 -159T, TLR4 c.896G and TLR4 c.1196T modify activation of Kupffer cells by endotoxin. We confirmed the published associations between these common variants and genetic predisposition to ALC by means of a large case-control association study conducted on two Central European populations. METHODS: The study population comprised a Czech sample of 198 ALC patients and 370 controls (MONICA project), and a German sample of 173 ALC patients and 331 controls (KORA-Augsburg), and 109 heavy drinkers without liver disease. RESULTS: Single locus analysis revealed no significant difference between patients and controls in all tested loci. Diplotype [IL1RN 2/ 2; IL1B -31T+] was associated with increased risk of ALC in the pilot study, but not in the validation samples. CONCLUSIONS: Although cytokine mediated immune reactions play a role in the pathogenesis of ALC, hereditary susceptibility caused by variants in the corresponding genes is low in Central European populations.

Immune cell-mediated liver injury

Liver diseases represent an important cause of morbidity and mortality in the world. Death of hepatocytes and other hepatic cell types is a characteristic feature of several forms of liver injury such as cholestasis, viral hepatitis, drug- or toxin-induced injury, and alcohol-induced liver damage. Moreover, irrespectively of the reason, liver injury seems to be facilitated by similar immune effector mechanisms common to these various liver diseases. Indeed, common immune effector mechanisms may explain the high prevalence of cirrhosis and cancer development in most forms of liver disease. Improved understanding of the immune cell-mediated mechanisms involved in hepatocyte cell death could be beneficial for the development of common therapeutic strategies against different forms of liver diseases. In this review, we will discuss novel findings on the role of different immune cells in liver disease and immune cell-induced death executioner mechanisms involved in hepatocyte cell death.

Maternal cholestasis during pregnancy programs metabolic disease in offspring

The intrauterine environment is a major contributor to increased rates of metabolic disease in adults. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy that affects 0.5%-2% of pregnant women and is characterized by increased bile acid levels in the maternal serum. The influence of ICP on the metabolic health of offspring is unknown. We analyzed the Northern Finland birth cohort 1985-1986 database and found that 16-year-old children of mothers with ICP had altered lipid profiles. Males had increased BMI, and females exhibited increased waist and hip girth compared with the offspring of uncomplicated pregnancies. We further investigated the effect of maternal cholestasis on the metabolism of adult offspring in the mouse. Females from cholestatic mothers developed a severe obese, diabetic phenotype with hepatosteatosis following a Western diet, whereas matched mice not exposed to cholestasis in utero did not. Female littermates were susceptible to metabolic disease before dietary challenge. Human and mouse studies showed an accumulation of lipids in the fetoplacental unit and increased transplacental cholesterol transport in cholestatic pregnancy. We believe this is the first report showing that cholestatic pregnancy in the absence of altered maternal BMI or diabetes can program metabolic disease in the offspring.

Hypoglycemia is associated with increased mortality in patients with acute decompensated liver cirrhosis

PRINCIPALS The liver plays an important role in glucose metabolism, in terms of glucolysis and gluconeogenesis. Several studies have shown that hyperglycemia in patients with liver cirrhosis is associated with progression of the liver disease and increased mortality. However, no study has ever targeted the influence of hypoglycemia. The aim of this study was to assess the association of glucose disturbances with outcome in patients presenting to the emergency department with acute decompensated liver cirrhosis. METHODS Our retrospective data analysis comprised adult (≥16 years) patients admitted to our emergency department between January 1, 2002, and December 31, 2012, with the primary diagnosis of decompensated liver cirrhosis. RESULTS A total of 312 patients were eligible for study inclusion. Two hundred thirty-one (74.0%) patients were male; 81 (26.0%) were female. The median age was 57 years (range, 51-65 years). Overall, 89 (28.5%) of our patients had acute glucose disturbances; 49 (15.7%) of our patients were hypoglycemic and 40 (12.8%) were hyperglycemic. Patients with hypoglycemia were significantly more often admitted to the intensive care unit than hyperglycemic patients (20.4% vs 10.8%, P < .015) or than normoglycemic patients (20.4% vs 10.3%, P < .011), and they significantly more often died in the hospital (28.6% hypoglycemic vs 7.5% hyperglycemic, P < .024; 28.6% hypoglycemic vs 10.3% normoglycemic P < .049). Survival analysis showed a significantly lower estimated survival for hypoglycemic patients (36 days) than for normoglycemic patients (54 days) or hyperglycemic patients (45 days; hypoglycemic vs hyperglycemic, P < .019; hypoglycemic vs normoglycemic, P < .007; hyperglycemic vs normoglycemic, P < .477). CONCLUSION Hypoglycemia is associated with increased mortality in patients with acute decompensated liver cirrhosis. It is not yet clear whether hypoglycemia is jointly responsible for the increased short-term mortality of patients with acute decompensated liver cirrhosis or is only a consequence of the severity of the disease or the complications.

Health-related quality of life and survival in liver transplant candidates.

Health-related quality of life (HRQOL) is an important measure of the effects of chronic liver disease in affected patients that helps guide interventions to improve well-being. However, the relationship between HRQOL and survival in liver transplant candidates remains unclear. We examined whether the Physical Component Summary (PCS) and Mental Component Summary (MCS) scores from the Short Form 36 (SF-36) Health Survey were associated with survival in liver transplant candidates. We administered the SF-36 questionnaire (version 2.0) to patients in the Pulmonary Vascular Complications of Liver Disease study, a multicenter prospective cohort of patients evaluated for liver transplantation in 7 academic centers in the United States between 2003 and 2006. Cox proportional hazards models were used with death as the primary outcome and adjustment for liver transplantation as a time-varying covariate. The mean age of the 252 participants was 54 +/- 10 years, 64% were male, and 94% were white. During the 422 person years of follow-up, 147 patients (58%) were listed, 75 patients (30%) underwent transplantation, 49 patients (19%) died, and 3 patients were lost to follow-up. Lower baseline PCS scores were associated with an increased mortality rate despite adjustments for age, gender, Model for End-Stage Liver Disease score, and liver transplantation (P for the trend = 0.0001). The MCS score was not associated with mortality (P for the trend = 0.53). In conclusion, PCS significantly predicts survival in liver transplant candidates, and interventions directed toward improving the physical status may be helpful in improving outcomes in liver transplant candidates.

The metabolomic window into hepatobiliary disease

The emergent discipline of metabolomics has attracted considerable research effort in hepatology. Here we review the metabolomic data for non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), alcoholic liver disease (ALD), hepatitis B and C, cholecystitis, cholestasis, liver transplantation, and acute hepatotoxicity in animal models. A metabolomic window has permitted a view into the changing biochemistry occurring in the transitional phases between a healthy liver and hepatocellular carcinoma or cholangiocarcinoma. Whether provoked by obesity and diabetes, alcohol use or oncogenic viruses, the liver develops a core metabolomic phenotype (CMP) that involves dysregulation of bile acid and phospholipid homeostasis. The CMP commences at the transition between the healthy liver (Phase 0) and NAFLD/NASH, ALD or viral hepatitis (Phase 1). This CMP is maintained in the presence or absence of cirrhosis (Phase 2) and whether or not either HCC or CCA (Phase 3) develops. Inflammatory signalling in the liver triggers the appearance of the CMP. Many other metabolomic markers distinguish between Phases 0, 1, 2 and 3. A metabolic remodelling in HCC has been described but metabolomic data from all four Phases demonstrate that the Warburg shift from mitochondrial respiration to cytosolic glycolysis foreshadows HCC and may occur as early as Phase 1. The metabolic remodelling also involves an upregulation of fatty acid β-oxidation, also beginning in Phase 1. The storage of triglycerides in fatty liver provides high energy-yielding substrates for Phases 2 and 3 of liver pathology. The metabolomic window into hepatobiliary disease sheds new light on the systems pathology of the liver.

The liver may act as a firewall mediating mutualism between the host and its gut commensal microbiota.

A prerequisite for establishment of mutualism between the host and the microbial community that inhabits the large intestine is the stringent mucosal compartmentalization of microorganisms. Microbe-loaded dendritic cells trafficking through lymphatics are arrested at the mesenteric lymph nodes, which constitute the firewall of the intestinal lymphatic circulation. We show in different mouse models that the liver, which receives the intestinal venous blood circulation, forms a vascular firewall that captures gut commensal bacteria entering the bloodstream during intestinal pathology. Phagocytic Kupffer cells in the liver of mice clear commensals from the systemic vasculature independently of the spleen through the liver's own arterial supply. Damage to the liver firewall in mice impairs functional clearance of commensals from blood, despite heightened innate immunity, resulting in spontaneous priming of nonmucosal immune responses through increased systemic exposure to gut commensals. Systemic immune responses consistent with increased extraintestinal commensal exposure were found in humans with liver disease (nonalcoholic steatohepatitis). The liver may act as a functional vascular firewall that clears commensals that have penetrated either intestinal or systemic vascular circuits.

Evaluation of genome-wide loci of iron metabolism in hereditary hemochromatosis identifies PCSK7 as a host risk factor of liver cirrhosis

Genome-wide association studies (GWAS) have revealed genetic determinants of iron metabolism, but correlation of these with clinical phenotypes is pending. Homozygosity for HFE C282Y is the predominant genetic risk factor for hereditary hemochromatosis (HH) and may cause liver cirrhosis. However, this genotype has a low penetrance. Thus, detection of yet unknown genetic markers that identify patients at risk of developing severe liver disease is necessary for better prevention. Genetic loci associated with iron metabolism (TF, TMPRSS6, PCSK7, TFR2 and Chr2p14) in recent GWAS and liver fibrosis (PNPLA3) in recent meta-analysis were analyzed for association with either liver cirrhosis or advanced fibrosis in 148 German HFE C282Y homozygotes. Replication of associations was sought in additional 499 Austrian/Swiss and 112 HFE C282Y homozygotes from Sweden. Only variant rs236918 in the PCSK7 gene (proprotein convertase subtilisin/kexin type 7) was associated with cirrhosis or advanced fibrosis (P = 1.02 × 10(-5)) in the German cohort with genotypic odds ratios of 3.56 (95% CI 1.29-9.77) for CG heterozygotes and 5.38 (95% CI 2.39-12.10) for C allele carriers. Association between rs236918 and cirrhosis was confirmed in Austrian/Swiss HFE C282Y homozygotes (P = 0.014; ORallelic = 1.82 (95% CI 1.12-2.95) but not in Swedish patients. Post hoc combined analyses of German/Swiss/Austrian patients with available liver histology (N = 244, P = 0.00014, ORallelic = 2.84) and of males only (N = 431, P = 2.17 × 10(-5), ORallelic = 2.54) were consistent with the premier finding. Association between rs236918 and cirrhosis was not confirmed in alcoholic cirrhotics, suggesting specificity of this genetic risk factor for HH. PCSK7 variant rs236918 is a risk factor for cirrhosis in HH patients homozygous for the HFE C282Y mutation.

Long-term outcomes of patients with Wilson disease in a large Austrian cohort

BACKGROUND & AIMS Wilson disease is an autosomal recessive disorder that affects copper metabolism, leading to copper accumulation in liver, central nervous system, and kidneys. There are few data on long-term outcomes and survival from large cohorts; we studied these features in a well-characterized Austrian cohort of patients with Wilson disease. METHODS We analyzed data from 229 patients diagnosed with Wilson disease from 1961 through 2013; 175 regularly attended a Wilson disease outpatient clinic and/or their physicians were contacted for information on disease and treatment status and outcomes. For 53 patients lost during the follow-up period, those that died and reasons for their death were identified from the Austrian death registry. RESULTS The mean observation period was 14.8 ± 11.4 years (range, 0.5-52.0 years), resulting in 3116 patient-years. Of the patients, 61% presented with hepatic disease, 27% with neurologic symptoms, and 10% were diagnosed by family screening at presymptomatic stages. Patients with a hepatic presentation were diagnosed younger (21.2 ± 12.0 years) than patients with neurologic disease (28.8 ± 12.0; P < .001). In 2% of patients, neither symptoms nor onset of symptoms could be determined with certainty. Most patients stabilized (35%) or improved on chelation therapy (26% fully recovered, 24% improved), but 15% deteriorated; 8% required a liver transplant, and 7.4% died within the observation period (71% of deaths were related to Wilson disease). A lower proportion of patients with Wilson disease survived for 20 years (92%) than healthy Austrians (97%), adjusted for age and sex (P = .03). Cirrhosis at diagnosis was the best predictor of death (odds ratio, 6.8; 95% confidence interval, 1.5-31.03; P = .013) and need for a liver transplant (odds ratio, 07; 95% confidence interval, 0.016-0.307; P < .001). Only 84% of patients with cirrhosis survived 20 years after diagnosis (compared with healthy Austrians, P =.008). CONCLUSION Overall, patients who receive adequate care for Wilson disease have a good long-term prognosis. However, cirrhosis increases the risk of death and liver disease. Early diagnosis, at a precirrhotic stage, might increase survival times and reduce the need for a liver transplant.

Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis.

BACKGROUND & AIMS Unhealthy lifestyles predispose to non-alcoholic steatohepatitis (NASH), which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity, it is unknown whether regular exercise reduces the risk of developing HCC. Therefore, we studied the effect of regular exercise on the development of HCC in male hepatocyte-specific PTEN-deficient mice (AlbCrePten(flox/flox)), which develop steatohepatitis and HCC spontaneously. METHODS Mice were fed a standardized 10% fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 minutes/day, 5 days/week during 32 weeks. RESULTS After 32 weeks of regular exercise, 71% of exercised mice developed nodules larger than 15 mm(3) vs 100% of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise, as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the Non-alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically, exercise stimulated the phosphorylation of AMPK and its substrate raptor, which decreased the kinase activity of mTOR. CONCLUSIONS These data show a benefit of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC.

Progression of Liver Fibrosis in HIV/HCV Co-Infection: A Comparison between Non-Invasive Assessment Methods and Liver Biopsy.

OBJECTIVES To evaluate the diagnostic performance of seven non-invasive tests (NITs) of liver fibrosis and to assess fibrosis progression over time in HIV/HCV co-infected patients. METHODS Transient elastography (TE) and six blood tests were compared to histopathological fibrosis stage (METAVIR). Participants were followed over three years with NITs at yearly intervals. RESULTS Area under the receiver operating characteristic curve (AUROC) for significant fibrosis (> = F2) in 105 participants was highest for TE (0.85), followed by FIB-4 (0.77), ELF-Test (0.77), APRI (0.76), Fibrotest (0.75), hyaluronic acid (0.70), and Hepascore (0.68). AUROC for cirrhosis (F4) was 0.97 for TE followed by FIB-4 (0.91), APRI (0.89), Fibrotest (0.84), Hepascore (0.82), ELF-Test (0.82), and hyaluronic acid (0.79). A three year follow-up was completed by 87 participants, all on antiretroviral therapy and in 20 patients who completed HCV treatment (9 with sustained virologic response). TE, APRI and Fibrotest did not significantly change during follow-up. There was weak evidence for an increase of FIB-4 (mean increase: 0.22, p = 0.07). 42 participants had a second liver biopsy: Among 38 participants with F0-F3 at baseline, 10 were progessors (1-stage increase in fibrosis, 8 participants; 2-stage, 1; 3-stage, 1). Among progressors, mean increase in TE was 3.35 kPa, in APRI 0.36, and in FIB-4 0.75. Fibrotest results did not change over 3 years. CONCLUSION TE was the best NIT for liver fibrosis staging in HIV/HCV co-infected patients. APRI-Score, FIB-4 Index, Fibrotest, and ELF-Test were less reliable. Routinely available APRI and FIB-4 performed as good as more expensive tests. NITs did not change significantly during a follow-up of three years, suggesting slow liver disease progression in a majority of HIV/HCV co-infected persons on antiretroviral therapy.