Ayako Suzuki

Increased fructose consumption is associated with fibrosis severity in patients with nonalcoholic fatty liver disease

The rising incidence of obesity and diabetes coincides with a marked increase in fructose consumption. Fructose consumption is higher in individuals with nonalcoholic fatty liver disease (NAFLD) than in age‐matched and body mass index (BMI)‐matched controls. Because fructose elicits metabolic perturbations that may be hepatotoxic, we investigated the relationship between fructose consumption and disease severity in NAFLD. We studied 427 adults enrolled in the NASH Clinical Research Network for whom Block food questionnaire data were collected within 3 months of a liver biopsy. Fructose consumption was estimated based on reporting (frequency × amount) of Kool‐aid, fruit juices, and nondietary soda intake, expressed as servings per week, and classified into none, minimum to moderate (<7 servings/week), and daily (≥7 servings/week). The association of fructose intake with metabolic and histological features of NAFLD was analyzed using multiple linear and ordinal logistic regression analyses with and without controlling for other confounding factors. Increased fructose consumption was univariately associated with decreased age (P < 0.0001), male sex (P < 0.0001), hypertriglyceridemia (P < 0.04), low high‐density lipoprotein (HDL) cholesterol (<0.0001), decreased serum glucose (P < 0.001), increased calorie intake (P < 0.0001), and hyperuricemia (P < 0.0001). After controlling for age, sex, BMI, and total calorie intake, daily fructose consumption was associated with lower steatosis grade and higher fibrosis stage (P < 0.05 for each). In older adults (age ≥ 48 years), daily fructose consumption was associated with increased hepatic inflammation (P < 0.05) and hepatocyte ballooning (P = 0.05). Conclusion: In patients with NAFLD, daily fructose ingestion is associated with reduced hepatic steatosis but increased fibrosis. These results identify a readily modifiable environmental risk factor that may ameliorate disease progression in patients with NAFLD. HEPATOLOGY 2010

The use of liver biopsy evaluation in discrimination of idiopathic autoimmune hepatitis versus drug-induced liver injury.

Distinguishing drug‐induced liver injury (DILI) from idiopathic autoimmune hepatitis (AIH) can be challenging. We performed a standardized histologic evaluation to explore potential hallmarks to differentiate AIH versus DILI. Biopsies from patients with clinically well‐characterized DILI [n = 35, including 19 hepatocellular injury (HC) and 16 cholestatic/mixed injury (CS)] and AIH (n = 28) were evaluated for Ishak scores, prominent inflammatory cell types in portal and intra‐acinar areas, the presence or absence of emperipolesis, rosette formation, and cholestasis in a blinded fashion by four experienced hepatopathologists. Histologic diagnosis was concordant with clinical diagnosis in 65% of cases; but agreement on final diagnosis among the four pathologists was complete in only 46% of cases. Interface hepatitis, focal necrosis, and portal inflammation were present in all evaluated cases, but were more severe in AIH (P < 0.05) than DILI (HC). Portal and intra‐acinar plasma cells, rosette formation, and emperiopolesis were features that favored AIH (P < 0.02). A model combining portal inflammation, portal plasma cells, intra‐acinar lymphocytes and eosinophils, rosette formation, and canalicular cholestasis yielded an area under the receiver operating characteristic curve (AUROC) of 0.90 in predicting DILI (HC) versus AIH. All Ishak inflammation scores were more severe in AIH than DILI (CS) (P ≤ 0.05). The four AIH‐favoring features listed above were consistently more prevalent in AIH, whereas portal neutrophils and intracellular (hepatocellular) cholestasis were more prevalent in DILI (CS) (P < 0.02). The combination of portal inflammation, fibrosis, portal neutrophils and plasma cells, and intracellular (hepatocellular) cholestasis yielded an AUC of 0.91 in predicting DILI (CS) versus AIH. Conclusion: Although an overlap of histologic findings exists for AIH and DILI, sufficient differences exist so that pathologists can use the pattern of injury to suggest the correct diagnosis. (Hepatology 2011;)

Relationship Between Methylome and Transcriptome in Patients With Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMSnCirrhosis and liver cancer are potential outcomes of advanced nonalcoholic fatty liver disease (NAFLD). It is not clear what factors determine whether patients will develop advanced or mild NAFLD, limiting noninvasive diagnosis and treatment before clinical sequelae emerge. We investigated whether DNA methylation profiles can distinguish patients with mild disease from those with advanced NAFLD, and how these patterns are functionally related to hepatic gene expression.nnnMETHODSnWe collected frozen liver biopsies and clinical data from patients with biopsy-proven NAFLD (56 in the discovery cohort and 34 in the replication cohort). Samples were divided into groups based on histologic severity of fibrosis: F0-1 (mild) and F3-4 (advanced). DNA methylation profiles were determined and coupled with gene expression data from the same biopsies; differential methylation was validated in subsets of the discovery and replication cohorts. We then analyzed interactions between the methylome and transcriptome.nnnRESULTSnClinical features did not differ between patients known to have mild or advanced fibrosis based on biopsy analysis. There were 69,247 differentially methylated CpG sites (76% hypomethylated, 24% hypermethylated) in patients with advanced vs mild NAFLD (P < .05). Methylation at fibroblast growth factor receptor 2, methionine adenosyl methyltransferase 1A, and caspase 1 was validated by bisulfite pyrosequencing and the findings were reproduced in the replication cohort. Methylation correlated with gene transcript levels for 7% of differentially methylated CpG sites, indicating that differential methylation contributes to differences in expression. In samples with advanced NAFLD, many tissue repair genes were hypomethylated and overexpressed, and genes in certain metabolic pathways, including 1-carbon metabolism, were hypermethylated and underexpressed.nnnCONCLUSIONSnFunctionally relevant differences in methylation can distinguish patients with advanced vs mild NAFLD. Altered methylation of genes that regulate processes such as steatohepatitis, fibrosis, and carcinogenesis indicate the role of DNA methylation in progression of NAFLD.

Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation

In bile duct-ligated (BDL) rodents, as in humans with chronic cholangiopathies, biliary obstruction triggers proliferation of bile ductular cells that are surrounded by fibrosis produced by adjacent myofibroblastic cells in the hepatic mesenchyme. The proximity of the myofibroblasts and cholangiocytes suggests that mesenchymal–epithelial crosstalk promotes the fibroproliferative response to cholestatic liver injury. Studying BDL mice, we found that bile duct obstruction induces activity of the Hedgehog (Hh) pathway, a system that regulates the viability and differentiation of various progenitors during embryogenesis. After BDL, many bile ductular cells and fibroblastic-appearing cells in the portal stroma express Hh ligands, receptor and/or target genes. Transwell cocultures of an immature cholangiocyte line that expresses the Hh receptor, Patched (Ptc), with liver myofibroblastic cells demonstrated that both cell types produced Hh ligands that enhanced each others viability and proliferation. Further support for the concept that Hh signaling modulates the response to BDL was generated by studying PtcLacZ mice, which have an impaired ability to constrain Hh signaling due to a heterozygous deficiency of Ptc. After BDL, PtcLacZ mice upregulated fibrosis gene expression earlier than wild-type controls and manifested an unusually intense ductular reaction, more expanded fibrotic portal areas, and a greater number of lobular necrotic foci. Our findings reveal that adult livers resurrect developmental signaling systems, such as the Hh pathway, to guide remodeling of the biliary epithelia and stroma after cholestatic injury.

Drugs Associated with Hepatotoxicity and their Reporting Frequency of Liver Adverse Events in VigiBase Unified List Based on International Collaborative Work

Background: Challenges exist in the clinical diagnosis of drug-induced liver injury (DILI) and in obtaining information on hepatotoxicity in humans.Objective: (i) To develop a unified list that combines drugs incriminated in well vetted or adjudicated DILI cases from many recognized sources and drugs that have been subjected to serious regulatory actions due to hepatotoxicity; and (ii) to supplement the drug list with data on reporting frequencies of liver events in the WHO individual case safety report database (VigiBase™).Data Sources and Extraction: (i) Drugs identified as causes of DILI at three major DILI registries; (ii) drugs identified as causes of drug-induced acute liver failure (ALF) in six different data sources, including major ALF registries and previously published ALF studies; and (iii) drugs identified as being subjected to serious governmental regulatory actions due to their hepatotoxicity in Europe or the US were collected. The reporting frequency of adverse events was determined using VigiBase™, computed as Empirical Bayes Geometric Mean (EBGM) with 90% confidence interval for two customized terms, ‘overall liver injury’ and ‘ALF’. EBGM of ≥2 was considered a disproportional increase in reporting frequency. The identified drugs were then characterized in terms of regional divergence, published case reports, serious regulatory actions, and reporting frequency of ‘overall liver injury’ and ‘ALF’ calculated from VigiBase™.Data Synthesis: After excluding herbs, supplements and alternative medicines, a total of 385 individual drugs were identified; 319 drugs were identified in the three DILI registries, 107 from the six ALF registries (or studies) and 47 drugs that were subjected to suspension or withdrawal in the US or Europe due to their hepatotoxicity. The identified drugs varied significantly between Spain, the US and Sweden. Of the 319 drugs identified in the DILI registries of adjudicated cases, 93.4% were found in published case reports, 1.9% were suspended or withdrawn due to hepatotoxicity and 25.7% were also identified in the ALF registries/studies. In VigiBase™, 30.4% of the 319 drugs were associated with disproportionally higher reporting frequency of ‘overall liver injury’ and 83.1% were associated with at least one reported case of ALF.Conclusions: This newly developed list of drugs associated with hepatotoxicity and the multifaceted analysis on hepatotoxicity will aid in causality assessment and clinical diagnosis of DILI and will provide a basis for further characterization of hepatotoxicity.

Hepatic Gene Expression Profiles Differentiate Presymptomatic Patients With Mild Versus Severe Nonalcoholic Fatty Liver Disease

Clinicians rely upon the severity of liver fibrosis to segregate patients with well‐compensated nonalcoholic fatty liver disease (NAFLD) into subpopulations at high‐ versus low‐risk for eventual liver‐related morbidity and mortality. We compared hepatic gene expression profiles in high‐ and low‐risk NAFLD patients to identify processes that distinguish the two groups and hence might be novel biomarkers or treatment targets. Microarray analysis was used to characterize gene expression in percutaneous liver biopsies from low‐risk, “mild” NAFLD patients (fibrosis stage 0‐1; nu2009=u200940) and high‐risk, “severe” NAFLD patients (fibrosis stage 3‐4; nu2009=u200932). Findings were validated in a second, independent cohort and confirmed by real‐time polymerase chain reaction and immunohistochemistry (IHC). As a group, patients at risk for bad NAFLD outcomes had significantly worse liver injury and more advanced fibrosis (severe NAFLD) than clinically indistinguishable NAFLD patients with a good prognosis (mild NAFLD). A 64‐gene profile reproducibly differentiated severe NAFLD from mild NAFLD, and a 20‐gene subset within this profile correlated with NAFLD severity, independent of other factors known to influence NAFLD progression. Multiple genes involved with tissue repair/regeneration and certain metabolism‐related genes were induced in severe NAFLD. Ingenuity Pathway Analysis and IHC confirmed deregulation of metabolic and regenerative pathways in severe NAFLD and revealed overlap among the gene expression patterns of severe NAFLD, cardiovascular disease, and cancer. Conclusion: By demonstrating specific metabolic and repair pathways that are differentially activated in livers with severe NAFLD, gene profiling identified novel targets that can be exploited to improve diagnosis and treatment of patients who are at greatest risk for NAFLD‐related morbidity and mortality. (Hepatology 2014;59:471–482)

Hedgehog pathway activation parallels histologic severity of injury and fibrosis in human nonalcoholic fatty liver disease

The Hedgehog (HH)‐signaling pathway mediates several processes that are deregulated in patients with metabolic syndrome (e.g., fat mass regulation, vascular/endothelial remodeling, liver injury and repair, and carcinogenesis). The severity of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome generally correlate. Therefore, we hypothesized that the level of HH‐pathway activation would increase in parallel with the severity of liver damage in NAFLD. To assess potential correlations between known histologic and clinical predictors of advanced liver disease and HH‐pathway activation, immunohistochemistry was performed on liver biopsies from a large, well‐characterized cohort of NAFLD patients (n = 90) enrolled in the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) Database 1 study. Increased HH activity (evidenced by accumulation of HH‐ligand–producing cells and HH‐responsive target cells) strongly correlated with portal inflammation, ballooning, and fibrosis stage (each P < 0.0001), supporting a relationship between HH‐pathway activation and liver damage. Pathway activity also correlated significantly with markers of liver repair, including numbers of hepatic progenitors and myofibroblastic cells (both P < 0.03). In addition, various clinical parameters that have been linked to histologically advanced NAFLD, including increased patient age (P < 0.005), body mass index (P < 0.002), waist circumference (P < 0.0007), homeostatic model assessment of insulin resistance (P < 0.0001), and hypertension (P < 0.02), correlated with hepatic HH activity. Conclusion: In NAFLD patients, the level of hepatic HH‐pathway activity is highly correlated with the severity of liver damage and with metabolic syndrome parameters that are known to be predictive of advanced liver disease. Hence, deregulation of the HH‐signaling network may contribute to the pathogenesis and sequelae of liver damage that develops with metabolic syndrome. (HEPATOLOGY 2012;55:1711–1721)

Increased production of sonic hedgehog by ballooned hepatocytes

Ballooned hepatocytes distinguish non‐alcoholic steatohepatitis (NASH) from steatosis. Such cells contain dilated endoplasmic reticulum and ubiquitin aggregates, characteristics of endoplasmic reticulum stress. Hepatocyte ballooning increases the risk for fibrosis in NASH, suggesting that ballooned hepatocytes release pro‐fibrogenic factors. Hedgehog ligands function as pro‐fibrogenic factors in liver diseases, but mechanisms for hedgehog ligand production remain poorly understood. We evaluated the hypothesis that endoplasmic reticulum stress induces hepatocyte production of hedgehog ligands that provide paracrine pro‐fibrogenic signals to neighbouring cells. In livers from NASH patients, keratin 8/18 and ubiquitin staining demonstrated enlarged, keratin 8/18‐negative/ubiquitin‐positive hepatocytes (ballooned hepatocytes) that were positive for Sonic hedgehog. In order to model endoplasmic reticulum stress in vitro, primary mouse hepatocytes were treated with tunicamycin. Compared to vehicle, tunicamycin significantly increased Sonic hedgehog and Indian hedgehog expression. Furthermore, conditioned medium from tunicamycin‐treated hepatocytes increased Gli‐luciferase reporter activity 14‐fold more than conditioned medium from vehicle‐treated hepatocytes. Cyclopamine (hedgehog signalling inhibitor) abrogated the effect of conditioned medium from tunicamycin‐treated hepatocytes, verifying that soluble hepatocyte‐derived factors activate hedgehog signalling. Ballooned hepatocytes in NASH patients did not express the hedgehog target gene, Gli2, α‐smooth muscle actin or vimentin, but were surrounded by Gli2‐positive stromal cells expressing these myofibroblast markers. Trichrome staining demonstrated the accumulation of ballooned hepatocytes in areas of matrix deposition, and numbers of Sonic hedgehog‐positive hepatocytes correlated with the degree of ballooning and fibrosis stage. Hepatocytes undergoing endoplasmic reticiulum stress generate hedgehog ligands which act as paracrine pro‐fibrogenic factors for hedgehog‐responsive stromal cells. These results help to explain why fibrosis stage correlates with hepatocyte ballooning in NASH. Copyright

Gender and Menopause Impact Severity of Fibrosis among Patients with Nonalcoholic Steatohepatitis

Estrogens inhibit stellate cell activation and fibrogenesis. Thus, gender and reproductive states may influence the degree of fibrosis in patients with nonalcoholic steatohepatitis (NASH). To investigate the association between gender, menopause, and the severity of liver fibrosis in patients with NASH, we analyzed 541 adult patients enrolled from our Duke Liver Clinics (nu2009=u2009338) and the Duke Metabolic and Weight Loss Surgery Program (nu2009=u2009203) who had a histologic diagnosis of NASH. Multiple ordinal logistic regression models were used to assess the association between gender, menopause, and severity of liver fibrosis. Overall, men, premenopausal, and postmenopausal women composed 35.1%, 28.4%, and 36.5% of the population, respectively. The mean age was 48 years and 22% had advanced fibrosis. After adjusting for covariates (enrolling site, grades of portal inflammation, and hepatocyte ballooning) and potential confounders (race, body mass index, diabetes/prediabetes, hypertension), adjusted cumulative odd ratio (ACOR) and 95% confidence interval (CI) for greater fibrosis severity was 1.4 (0.9, 2.1) (Pu2009=u20090.17) for postmenopausal women and 1.6 (1.0, 2.5) (Pu2009=u20090.03) for men, having premenopausal women as a reference. There was borderline interaction between gender and age group divided by age 50, the average age at menopause in the U.S. (Pu2009=u20090.08): ACOR and 95% CI of having greater fibrosis severity in men compared to women was 1.8 (1.1, 2.9) for patients with age <50 years (Pu2009=u20090.02) and 1.2 (0.7, 2.1) for patients with age ≥50 years (Pu2009=u20090.59). Conclusion: Men are at a higher risk of having more severe fibrosis compared to women before menopause, while postmenopausal women have a similar severity of liver fibrosis compared to men. These findings may be explained by the protective effects of estrogen against fibrogenesis. (Hepatology 2014;59:1406‐1414)

Nonalcoholic fatty liver disease in women

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in developed countries and is increasing in prevalence with the rise of diabetes and obesity. In addition to obesity and age, gender may also influence the prevalence and severity of NAFLD. However, mechanisms underlying gender-based differences in NAFLD have not been clearly defined. Furthermore, alterations in body composition, fat distribution and/or hormonal or metabolic changes that occur following menopause and in the setting of polycystic ovary syndrome may influence the development and progression of NAFLD. In this article, we will summarize known gender differences as well as the proposed mechanisms for gender differences in NAFLD, review two women-specific issues that may influence the prevalence and severity of NAFLD, menopause and polycystic ovary syndrome, and discuss potential therapeutic options for women with NAFLD who are postmenopausal or have polycystic ovary syndrome.