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Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs’ communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Liver sinusoidal endothelial cells (LSECs) are ideally situated to sense stiffness and generate angiocrine programs that potentially regulate liver fibrosis and portal hypertension. We explored how specific focal adhesion (FA) proteins parlay LSEC mechanotransduction into stiffness-induced angiocrine signaling in vitro and in vivo.Primary human and murine LSECs were placed on gels with incremental stiffness (0.2 kPa vs. 32 kPa). Cell response was studied by FA isolation, actin polymerization assay, RNA-sequencing and electron microscopy. Glycolysis was assessed using radioactive tracers. Epigenetic regulation of stiffness-induced genes was analyzed by chromatin-immunoprecipitation (ChIP) analysis of histone activation marks, ChIP sequencing and circularized chromosome conformation capture (4C). Mice with LSEC-selective deletion of glycolytic enzymes (Hk2Glycolytic enzymes, particularly phosphofructokinase 1 isoform P (PFKP), are enriched in isolated FAs from LSECs on gels with incremental stiffness. Stiffness resulted in PFKP recruitment to FAs, which paralleled an increase in glycolysis. Glycolysis was associated with expansion of actin dynamics and was attenuated by inhibition of integrin β1. Inhibition of glycolysis attenuated a stiffness-induced CXCL1-dominant angiocrine program. Mechanistically, glycolysis promoted CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. Biochemically, this CXCL1 expression was mediated through spatial re-organization of nuclear chromatin resulting in formation of super-enhancers, histone acetylation and NF-kB interaction with the CXCL1 promoter. Hk2Glycolytic enzymes are involved in stiffness-induced angiocrine signaling in LSECs and represent druggable targets in early liver disease.Treatment options for liver fibrosis and portal hypertension still represent an unmet need. Herein, we uncovered a novel role for glycolytic enzymes in promoting stiffness-induced angiocrine signaling, which resulted in inflammation, fibrosis and portal hypertension. This work has revealed new targets that could be used in the prevention and treatment of liver fibrosis and portal hypertension.

With the epidemic of obesity, nonalcoholic fatty liver disease (NAFLD) has become the most common pediatric liver disease. The influence of a perinatal obesity‐inducing diet (OID) on the development and progression of NAFLD in offspring is important but incompletely studied. Hence, we fed breeding pairs of C57BL/6J mice during gestation and lactation (perinatally) either chow or an OID rich in fat, fructose, and cholesterol (FFC). The offspring were weaned to either chow or an FFC diet, generating four groups: perinatal (p)Chow‐Chow, pChow‐FFC, pFFC‐Chow, and pFFC‐FFC. Mice were sacrificed at 10 weeks of age. We examined the whole‐liver transcriptome by RNA sequencing (RNA‐seq) and whole‐liver genome methylation by reduced representation bisulfite sequencing (RRBS). Our results indicated that the pFFC‐FFC mice had a significant increase in hepatic steatosis, injury, inflammation, and fibrosis, as assessed histologically and biochemically. We identified 189 genes that were differentially expressed and methylated in the pFFC‐FFC mice versus the pChow‐FFC mice. Gene set enrichment analysis identified hepatic fibrosis/hepatic stellate cell activation as the top canonical pathway, suggesting that the differential DNA methylation events in the mice exposed to the FFC diet perinatally were associated with a profibrogenic transcriptome. To verify that this finding was consistent with perinatal nutritional reprogramming of the methylome, we exposed pFFC‐Chow mice to an FFC diet in adulthood. These mice developed significant hepatic steatosis, injury, inflammation, and more importantly fibrosis when compared to the appropriate controls. Conclusion: Perinatal exposure to an OID primes the immature liver for an accentuated fibrosing nonalcoholic steatohepatitis (NASH) phenotype, likely through nutritional reprogramming of the offspring methylome. These data have potential clinical implications for monitoring children of obese mothers and risk stratification of children with NAFLD.

Combination treatment of ledipasvir and sofosbuvir (LDV/SOF) is first-line treatment for patients with chronic hepatitis C genotype 1 in the United States, Europe, and Japan. However, the influence of LDV/SOF on the cardiovascular system is poorly characterized. A total of 470 chronic hepatitis C patients who started LDV/SOF treatment between September 2015 and February 2016 at nine hospitals in Japan were prospectively enrolled in this study. Corrected QT (QTc) prolongation was defined as a QTc interval ≥450 milliseconds. The sustained virologic response rate was 96.0% (451/470), and the discontinuance rate due to adverse effects was 0.9% (4/470). Among 395 patients whose electrocardiogram was evaluated over time and compared with baseline, the QTc interval was significantly prolonged during treatment and returned to baseline levels 12 weeks after the end of treatment. Twenty-four of 376 patients with baseline QTc intervals

Aim Several case reports have shown that hepatitis B virus (HBV) reactivation developed in hepatitis C patients with a current or previous HBV infection during direct-acting antiviral (DAA) treatment, which led to severe hepatitis or death in some cases. However, its precise frequency and risk factors are not entirely clear. We analyzed a prospective cohort. Methods We analyzed HBV reactivation in 461 consecutive hepatitis C patients who received 12 weeks of ledipasvir/sofosbuvir for genotype 1 or sofosbuvir plus ribavirin for genotype 2 at multiple centers. Results By the examination of the preserved sera at baseline, 159 patients (34%) were identified as seropositive for HBV core antibody (anti-HBc) and were included in the subsequent analysis; 4 patients were positive for HBV surface antigen (HBsAg), and the others were negative. Serum HBV DNA was undetectable or was detectable but

AIM Forkhead Box M1 (FoxM1) is a proliferation-specific transcription factor. In this study, we aimed to elucidate the clinicopathological and prognostic values of FoxM1 expression in human hepatocellular carcinoma (HCC) and correlate FoxM1 expression with various etiologies of liver diseases. We also investigated its therapeutic value in HCC. METHODS We investigated the expression of FoxM1 in tumor tissues and adjacent non-tumor tissues of 79 Japanese HCC patients by quantitative real-time reverse transcription-polymerase chain reaction analysis. Depletion by siRNA or specific inhibition by siomycin A were also used to investigate the effect of FoxM1 inhibition on stem-like features of human HCC cells. RESULTS Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that tumor tissues displayed an approximately 14-fold increase in FoxM1 expression compared with adjacent non-tumor tissues. Interestingly, the expression levels of FoxM1in tumor tissues did not depend on the etiology of liver disease. The expression of FoxM1 in tumor tissues was associated with serum α-fetoprotein level, maximum tumor size, histological grade, TNM staging, and portal involvement. Kaplan-Meier analysis indicated that the high FoxM1 expression (≥median) group had a poor prognosis compared with the low FoxM1 expression (

Background & Aims Hepatic recruitment of monocyte-derived macrophages (MoMFs) contributes to the inflammatory response in non-alcoholic steatohepatitis (NASH). However, how hepatocyte lipotoxicity promotes MoMF inflammation is unclear. Here we demonstrate that lipotoxic hepatocyte-derived extracellular vesicles (LPC-EVs) are enriched with active integrin β1 (ITGβ1), which promotes monocyte adhesion and liver inflammation in murine NASH. Methods Hepatocytes were treated with either vehicle or the toxic lipid mediator lysophosphatidylcholine (LPC); EVs were isolated from the conditioned media and subjected to proteomic analysis. C57BL/6J mice were fed a diet rich in fat, fructose, and cholesterol (FFC) to induce NASH. Mice were treated with anti-ITGβ1 neutralizing antibody (ITGβ1Ab) or control IgG isotype. Results Ingenuity® Pathway Analysis of the LPC-EV proteome indicated that ITG signaling is an overrepresented canonical pathway. Immunogold electron microscopy and nanoscale flow cytometry confirmed that LPC-EVs were enriched with activated ITGβ1. Furthermore, we showed that LPC treatment in hepatocytes activates ITGβ1 and mediates its endocytic trafficking and sorting into EVs. LPC-EVs enhanced monocyte adhesion to liver sinusoidal cells, as observed by shear stress adhesion assay. This adhesion was attenuated in the presence of ITGβ1Ab. FFC-fed, ITGβ1Ab-treated mice displayed reduced inflammation, defined by decreased hepatic infiltration and activation of proinflammatory MoMFs, as assessed by immunohistochemistry, mRNA expression, and flow cytometry. Likewise, mass cytometry by time-of-flight on intrahepatic leukocytes showed that ITGβ1Ab reduced levels of infiltrating proinflammatory monocytes. Furthermore, ITGβ1Ab treatment significantly ameliorated liver injury and fibrosis. Conclusions Lipotoxic EVs mediate monocyte adhesion to LSECs mainly through an ITGβ1-dependent mechanism. ITGβ1Ab ameliorates diet-induced NASH in mice by reducing MoMF-driven inflammation, suggesting that blocking ITGβ1 is a potential anti-inflammatory therapeutic strategy in human NASH. Lay summary Herein, we report that a cell adhesion molecule termed integrin β1 (ITGβ1) plays a key role in the progression of non-alcoholic steatohepatitis (NASH). ITGβ1 is released from hepatocytes under lipotoxic stress as a cargo of extracellular vesicles, and mediates monocyte adhesion to liver sinusoidal endothelial cells, which is an essential step in hepatic inflammation. In a mouse model of NASH, blocking ITGβ1 reduces liver inflammation, injury and fibrosis. Hence, ITGβ1 inhibition may serve as a new therapeutic strategy for NASH.

Grb2-associated binder 1 (Gab1) adaptor protein amplifies signals downstream of a broad range of growth factors/receptor tyrosine kinases. Although these signals are implicated in liver fibrogenesis, the role of Gab1 remains unclear. To elucidate the role of Gab1, liver fibrosis was examined in hepatocyte-specific Gab1-conditional knockout (Gab1CKO) mice upon bile duct ligation (BDL). Gab1CKO mice developed exacerbated liver fibrosis with activation of hepatic myofibroblasts after BDL compared with control mice. The antifibrotic role of hepatocyte Gab1 was further confirmed by another well-established mouse model of liver fibrosis using chronic injections of carbon tetrachloride. After BDL, Gab1CKO mice also displayed exacerbated liver injury, decreased hepatocyte proliferation, and enhanced liver inflammation. Furthermore, cDNA microarray analysis was used to investigate the potential molecular mechanisms of the Gab1-mediated signal in liver fibrosis, and the fibrosis-promoting factor chemokine (C-C motif) ligand 5 ( Ccl5) was identified as upregulated in the livers of Gab1CKO mice following BDL. Interestingly, in vitro studies using primary hepatocytes isolated from control and Gab1CKO mice revealed that the loss of Gab1 resulted in increased hepatocyte CCL5 synthesis upon lipopolysaccharide stimulation. Finally, pharmacological antagonism of CCL5 reduced BDL-induced liver fibrosis in Gab1CKO mice. In conclusion, our results demonstrate that hepatocyte Gab1 is required for liver fibrosis and that hepatocyte CCL5 could be an important contributor to this process. Thus, we present a novel antifibrotic function of hepatocyte Gab1 in liver fibrogenesis.

Background & Aims Obesity-related adipocytokine dysregulation is known to accelerate liver fibrosis progression. Recently, a natural Wnt5a inhibitor, secreted frizzled-related protein 5 (Sfrp5), was identified as a novel adipocytokine that has reduced expression in obese adipose tissue in both rodents and human. In addition, hepatic gene expression of Wnt5a and its receptor frizzled 2 (Fz2) is elevated during fibrosis progression. Therefore, Sfrp5 could have biological significance in liver fibrosis. Methods We first investigated the effects of Sfrp5 on primary cultured mouse hepatic stellate cells (HSCs) in vitro. Next, to elucidate the roles of Sfrp5 in liver fibrosis, we investigated a carbon-tetrachloride (CCl4)-induced liver fibrosis model using Sfrp5 knockout (KO) and wild type (WT) mice in vivo. Each mouse was injected intraperitoneally with CCl4 (0.5 ml/kg) or olive oil as a single dose (acute liver injury model), or twice a week for 6 weeks (liver fibrosis model). Results In in vitro studies, Wnt5a enhanced both proliferation and migration of HSCs, and these effects could be completely blocked by Sfrp5. Moreover, siRNA knockdown of Fz2 in HSCs could block the effects of Wnt5a on both HSC proliferation and migration. In in vivo studies, there were no differences in the CCl4-induced liver injury between KO and WT mice. Hepatic Wnt5a gene expression and plasma Wnt5a levels significantly increased after a single CCl4 injection in both mice. Sfrp5 knockout significantly enhanced CCl4-induced liver fibrosis. Conclusions Our findings demonstrate that Sfrp5 may ameliorate mouse liver fibrosis through inhibition of Wnt5a/Fz2 signalling.

Dysregulation of cell metabolism is a hallmark of cancer. The mevalonate pathway in lipid metabolism has been implicated as a potential target of cancer therapy for hepatocellular carcinoma (HCC). The role of the Forkhead Box M1 (FoxM1) transcription factor in HCC development has been well documented, however, its involvement in cancer metabolism of HCC has not been fully determined. Here, we hypothesized that FoxM1 is involved in the mevalonate pathway of cholesterol biosynthesis in HCC. Inhibition of the mevalonate pathway by statins, inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), resulted in reduced expression of FoxM1 and increased cell death in human hepatoma cells. Re-exposure of mevalonate, a product of HMGCR, restored these effects. Likewise, knockdown of HMGCR reduced FoxM1 expression, indicating that FoxM1 expression was regulated by the mevalonate pathway in HCC. Mechanistically, protein geranylgeranylation was found to be responsible for FoxM1 expression and geranylgeranylated proteins, including RhoA, Rac1 or Cdc42, were shown to be involved in this process. In surgically resected human HCC tissues, the gene expression of FoxM1 had a positive correlation with that of the mevalonate pathway-related genes, such as HMGCR or sterol regulatory element-binding protein 2 (SREBP2). Furthermore, the gene expression of FoxM1 along with that of HMGCR or SREBP2 defined prognosis of HCC patients, suggesting the clinical significance of the mevalonate-FoxM1 pathway in human HCC. Our data indicate that FoxM1 links the mevalonate pathway to oncogenic signals in HCC. Thus, we propose a novel therapeutic approach to inhibit FoxM1 by targeting the mevalonate pathway for HCC.

Background and aim Impaired fatty liver regeneration has already been reported in many genetic modification models. However, in diet-induced simple hepatic steatosis, which showed similar phenotype with clinical pathology, whether liver regeneration is impaired or not remains unclear. In this study, we evaluated liver regeneration in mice with diet-induced simple hepatic steatosis, and focused on excess lipid accumulation occurring during liver regeneration. Methods Mice were fed high fat diet (HFD) or control diet for 9–10 weeks. We analyzed intrahepatic lipid accumulation, DNA replication, and various signaling pathways including cell proliferation and ER stress during liver regeneration after partial hepatectomy. In addition, some of mice were pretreated with tauroursodeoxycholic acid (TUDCA), a chemical chaperone which alleviates ER stress, and then we estimated TUDCA effects on liver regeneration. Results The peak of hepatocyte BrdU incorporation, the expression of proliferation cell nuclear antigen (PCNA) protein, and the expressions of cell cycle-related genes were observed in delayed time in HFD mice. The expression of phosphorylated Erk1/2 was also delayed in HFD mice. The amounts of liver triglyceride were at least twofold higher in HFD mice at each time point. Intrahepatic palmitic acid was increased especially in HFD mice. ER stress induced during liver regeneration was significantly higher in HFD mice. In HFD mice, pretreatment with TUDCA reduced ER stress and resulted in improvement of delayed liver regeneration. Conclusion In simple hepatic steatosis, lipid overloading occurring during liver regeneration might be caused ER stress and results in delayed hepatocyte DNA replication.

Forkhead Box M1 (FoxM1) is a proliferation-specific transcription factor. In this study, we aimed to elucidate the clinicopathological and prognostic values of FoxM1 expression in human hepatocellular carcinoma (HCC) and correlate FoxM1 expression with various etiologies of liver diseases. We also investigated its therapeutic value in HCC.We investigated the expression of FoxM1 in tumor tissues and adjacent non-tumor tissues of 79 Japanese HCC patients by quantitative real-time reverse transcription-polymerase chain reaction analysis. Depletion by siRNA or specific inhibition by siomycin A were also used to investigate the effect of FoxM1 inhibition on stem-like features of human HCC cells.Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that tumor tissues displayed an approximately 14-fold increase in FoxM1 expression compared with adjacent non-tumor tissues. Interestingly, the expression levels of FoxM1in tumor tissues did not depend on the etiology of liver disease. The expression of FoxM1 in tumor tissues was associated with serum α-fetoprotein level, maximum tumor size, histological grade, TNM staging, and portal involvement. Kaplan-Meier analysis indicated that the high FoxM1 expression (≥median) group had a poor prognosis compared with the low FoxM1 expression (median) group. Using multivariate analysis, the expression of FoxM1 in tumor tissues was shown to be an independent prognostic factor that affected overall survival and disease-free survival. Furthermore, FoxM1 inhibition by siRNA or siomycin A reduced spheroid colony formation of HCC cells in vitro.Our data suggest that FoxM1 might be a prognostic biomarker and a promising therapeutic target for HCC.

Aim Many studies indicate an accelerated progression of non-alcoholic steatohepatitis (NASH) in postmenopausal women. Very recently, we reported that estrogen deficiency enhanced the progression of steatohepatitis in mice fed a high fat and high cholesterol (HFHC) diet. Hypercholesterolemia is often observed in postmenopausal women, and recent studies indicate it to be an important risk factor for the progression of NASH. Statins can slow NASH progression in the estrogen-deficient state but the precise mechanisms of their effects are still unclear. Methods We investigated the effects of pitavastatin on steatohepatitis progression using ovariectomized (OVX) mice fed a HFHC diet or HFHC + pitava diet (containing 5 p.p.m. pitavastatin) for 6 weeks. Results Serum alanine aminotransferase and cholesterol levels significantly decreased in mice fed the HFHC + pitava diet compared with mice fed the HFHC diet. Real-time reverse transcription polymerase chain reaction representing hepatic inflammatory gene expressions significantly decreased in mice fed the HFHC + pitava diet compared with the HFHC-fed mice. Pitavastatin treatment also decreased both hepatic macrophage infiltration and hepatocyte chemokine (C-C motif) ligand 2 expression and improved the liver fibrosis condition when compared with the mice fed the HFHC diet. In addition, the enhanced spleen monocyte chemokine (C-C motif) receptor 2 expression in ovariectomized mice fed the HFHC diet was also decreased by pitavastatin administration. Conclusion Our study demonstrated that the exacerbated steatohepatitis progression in OVX mice fed a HFHC diet could be attenuated by pitavastatin treatment at least through inhibition of hepatic macrophage infiltration. We concluded that statins should be useful for treating NASH in postmenopausal women.

Aim: We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced in response to several liver injuries. Because the HB-EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver-specific HB-EGF conditional knockout mice using the interferon-inducible Mx-1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. Methods: We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl4) in HB-EGF KO mice and wild-type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB-EGF-dependent anti-apoptosis and wound-healing process of the liver in vitro. Results: HB-EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl4 injection. We also demonstrated that HB-EGF treatment inhibited tumor necrosis factor-α-induced apoptosis of AML12 mouse hepatocytes and promoted the wound-healing response of these cells. Conclusion: This study showed that HB-EGF plays a protective role during acute liver injury.

Aim: Central obesity, insulin resistance and alcohol consumption are thought to be major risk factors for fatty liver formation. Adiponectin (APN) prevents fatty liver formation, and its serum levels are lower in subjects with central obesity and/or insulin resistance. The aim of this study was to explore the association among serum APN levels, central obesity, insulin resistance and liver dysfunction with or without fatty liver classified by alcohol consumption in healthy subjects. Methods: A total of 5588 Japanese male subjects who underwent a health check-up were classified into three groups according to alcohol consumption: non- or light drinkers (15 g/day ≥ ethanol); mild drinkers (15 g/day

Acetaminophen (APAP) overdose is the leading cause of drug-induced acute liver failure. In APAP-induced acute liver failure, hepatocyte death and subsequent liver regeneration determines the prognosis of patients, making it necessary to identify suitable therapeutic targets based on detailed molecular mechanisms. Grb2-associated binder 1 (Gab1) adaptor protein plays a crucial role in transmitting signals from growth factor and cytokine receptors to downstream effectors. In this study, we hypothesized that Gab1 is involved in APAP-induced acute liver failure. Hepatocyte-specific Gab1 conditional knockout (Gab1CKO) and control mice were treated with 250 mg/kg of APAP. After APAP treatment, Gab1CKO mice had significantly higher mortality and elevated serum alanine aminotransferase levels compared to control mice. Gab1CKO mice had increased hepatocyte death and increased serum levels of high mobility group box 1, a marker of hepatocyte necrosis. In addition, Gab1CKO mice had reduced hepatocyte proliferation. The enhanced hepatotoxicity in Gab1CKO mice was associated with increased activation of stress-related c-Jun N-terminal kinase (JNK) and reduced activation of extracellular signal-regulated kinase and AKT. Furthermore, Gab1CKO mice showed enhanced mitochondrial translocation of JNK accompanied by an increase in the release of mitochondrial enzymes into the cytosol, which is indicative of increased mitochondrial dysfunction and subsequent nuclear DNA fragmentation. Finally, in vitro experiments showed that Gab1-deficient hepatocytes were more susceptible to APAP-induced mitochondrial dysfunction and cell death, suggesting that hepatocyte Gab1 is a direct target of APAP-induced hepatotoxicity. Conclusion: Our current data demonstrate that hepatocyte Gab1 plays a critical role in controlling the balance between hepatocyte death and compensatory hepatocyte proliferation during APAP-induced liver injury. (Hepatology 2016;63:1340–1355)

Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced in response to several liver injuries. Because the HB-EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver-specific HB-EGF conditional knockout mice using the interferon-inducible Mx-1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl4 ) in HB-EGF KO mice and wild-type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB-EGF-dependent anti-apoptosis and wound-healing process of the liver in vitro. HB-EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl4 injection. We also demonstrated that HB-EGF treatment inhibited tumor necrosis factor-α-induced apoptosis of AML12 mouse hepatocytes and promoted the wound-healing response of these cells. This study showed that HB-EGF plays a protective role during acute liver injury.

Central obesity, insulin resistance and alcohol consumption are thought to be major risk factors for fatty liver formation. Adiponectin (APN) prevents fatty liver formation, and its serum levels are lower in subjects with central obesity and/or insulin resistance. The aim of this study was to explore the association among serum APN levels, central obesity, insulin resistance and liver dysfunction with or without fatty liver classified by alcohol consumption in healthy subjects. A total of 5588 Japanese male subjects who underwent a health check-up were classified into three groups according to alcohol consumption: non- or light drinkers (15 g/day ≥ ethanol); mild drinkers (15 g/day ethanol ≤ 30 g/day); and moderate- or heavy drinkers (30 g/day ethanol). Central obesity and insulin resistance were assessed by waist circumference (WC) and Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR), respectively. WC was significantly increased, while HOMA-IR was significantly decreased according to the extent of alcohol consumption. Serum alanine aminotransferase levels were significantly lower and serum APN levels were significantly higher in mild drinkers than in the other two groups. Multiple linear regression analysis showed that serum APN level served as the significant and independent determinant for liver dysfunction in the subjects with fatty liver, irrespective of alcohol consumption. However, WC became a non-significant determinant of liver dysfunction as alcohol consumption increased. Hypoadiponectinemia is a significant determinant for steatotic dysfunction for all levels of alcohol consumption, but central obesity was not a significant determinant for alcoholic fatty liver-induced liver dysfunction.

Many studies indicate an accelerated progression of non-alcoholic steatohepatitis (NASH) in postmenopausal women. Very recently, we reported that estrogen deficiency enhanced the progression of steatohepatitis in mice fed a high fat and high cholesterol (HFHC) diet. Hypercholesterolemia is often observed in postmenopausal women, and recent studies indicate it to be an important risk factor for the progression of NASH. Statins can slow NASH progression in the estrogen-deficient state but the precise mechanisms of their effects are still unclear.We investigated the effects of pitavastatin on steatohepatitis progression using ovariectomized (OVX) mice fed a HFHC diet or HFHC + pitava diet (containing 5 p.p.m. pitavastatin) for 6 weeks.Serum alanine aminotransferase and cholesterol levels significantly decreased in mice fed the HFHC + pitava diet compared with mice fed the HFHC diet. Real-time reverse transcription polymerase chain reaction representing hepatic inflammatory gene expressions significantly decreased in mice fed the HFHC + pitava diet compared with the HFHC-fed mice. Pitavastatin treatment also decreased both hepatic macrophage infiltration and hepatocyte chemokine (C-C motif) ligand 2 expression and improved the liver fibrosis condition when compared with the mice fed the HFHC diet. In addition, the enhanced spleen monocyte chemokine (C-C motif) receptor 2 expression in ovariectomized mice fed the HFHC diet was also decreased by pitavastatin administration.Our study demonstrated that the exacerbated steatohepatitis progression in OVX mice fed a HFHC diet could be attenuated by pitavastatin treatment at least through inhibition of hepatic macrophage infiltration. We concluded that statins should be useful for treating NASH in postmenopausal women.