Ectodysplasin-A mRNA in exosomes released from activated hepatic stellate cells stimulates macrophage response.

INTRODUCTION: The interaction between activated hepatic stellate cells (aHSCs) and macrophages is central to liver fibrosis development. The cargo contained within aHSC exosomes (aHSC-EXOs) and how aHSC-EXOs affect macrophage function is poorly understood. METHODS: RNA from aHSC-EXOs was separated into small (<200-basepairs) and large (≥200-basepairs) RNA species, transfected into macrophages, and macrophage IL-6 and TNFα mRNA expression and protein secretion measured. Next generation sequencing was performed on EXOs from rat quiescent and aHSCs and human aHSCs. aHSCs were transfected with siRNA against ectodysplasin-A (EDA), EXOs collected, and their effect on macrophage function analyzed. Human cirrhotic liver was analyzed for EDA mRNA expression and compared to non-tumor liver (NTL). RESULTS: Transfection with large RNA from aHSC-EXOs stimulated macrophage IL-6 and TNFα mRNA expression and protein secretion. EDA mRNA was highly expressed in aHSCs and transfection of aHSCs with EDA-siRNA decreased aHSC-EXO EDA mRNA and blunted the effect of aHSC-EXOs on macrophage function (IL-6/TNFα expression and macrophage migration). Human cirrhotic liver exhibited high EDA mRNA compared to NTL. CONCLUSIONS: HSC activation leads to altered EXO mRNA/miRNA profiles with aHSC-EXOs mRNAs exerting a dominant role in altering macrophage function. Ectodysplasin-A mRNA is an important component in aHSC-EXOs in regulating macrophage function.

Hepatic stellate cell-derived exosomes modulate macrophage inflammatory response.

BACKGROUND: Hepatic stellate cell (HSC) differentiation/activation is central to liver fibrosis and is innately linked to the immune response to liver injury. Exosomes (EXOs) are important means of communication between cell populations. This study sought to characterize EXO release from HSCs and the effect of HSC-EXOs on macrophage cytokine release/function. METHODS: Liver from a rat fibrosis model was analyzed for EXO expression and localization. Quiescent and culture-activated rat and mouse HSCs and activated human HSCs were analyzed for microRNA expression. Mouse, rat, and human HSCs were culture-activated and EXOs purified from culture medium prior to addition to macrophages, and interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) mRNA and protein measured. The effect of activated HSC-EXOs on macrophage migration was assayed. RESULTS: Activation of rat HSCs led to increased EXO production in vivo, an effect mirrored by in vitro rat HSC culture-activation. Culture activation of mouse and rat HSCs led to altered EXO microRNA profiles, with a similar microRNA profile detected in activated human HSCs. Addition of activated HSC-EXOs to macrophages stimulated IL-6 and TNFα mRNA expression and protein secretion in mouse and human macrophages, but not for rat HSC-EXO-macrophages. Addition of human EXOs to macrophages stimulated migration, effects mirrored by the direct addition of rhIL-6 and rhTNFα. CONCLUSIONS: HSC-EXOs associate with macrophages and stimulate cytokine synthesis-release and macrophage migration. Constructing a comprehensive understanding of EXO interactions between liver cell populations in the setting of inflammation/fibrosis increases the potential for developing new diagnostic/therapeutic approaches.

Nanoparticle mediated silencing of tenascin C in hepatic stellate cells: effect on inflammatory gene expression and cell migration.

Chronic liver dysfunction often begins with hepatic fibrosis. A pivotal event in the progression of liver fibrosis and cirrhosis is hepatic stellate cell (HSC) activation and secretion of extracellular matrix proteins, including tenascin-C (TnC). TnC is often chosen as a therapeutic target for treatment of liver disease. TnC is minimally detected in healthy tissue, but is transiently expressed during tissue injury, and plays a critical role in fibrogenesis and tumorigenesis. siRNA therapy is a promising alternative to knock-down proteins relevant for fibrosis therapy. This study describes the application of a functionalized mesoporous silica nanoparticles (MSNs) for the efficient transport and delivery of siTnC in HSCs. Silencing experiments in HSCs demonstrate the effective reduction of TnC mRNA and protein levels. In addition, attenuation of TnC expression due to the cellular uptake and release of siTnC from MSNs resulted in decreases of inflammatory cytokine levels and hepatocyte migration. We envision this siTnC-MSN platform as a promising alternative to evaluate siRNA therapy of chronic liver disease in preclinical trials.

Analysis of Plasma Tenascin-C in Post-HCV Cirrhosis: A Prospective Study.

BACKGROUND AND AIM: Hepatitis C virus (HCV)-related cirrhosis, one of the most common etiologies of liver cirrhosis in the Western world, is a risk factor for hepatocellular carcinoma. To confirm and improve current effectiveness of screening and prognosis of patients with established cirrhosis, a credible, simple plasma biomarker is needed. Hepatic stellate cell activation, a pivotal event in cirrhosis development, results in increased secretion of extracellular matrix proteins, including tenascin-C (TnC). Herein, we tested TnC as a simple biomarker to identify cirrhotic patients with active HCV infection from those with HCV eradication. METHODS: A prospective study of subjects with HCV-related cirrhosis, stratified into two groups, HCV or virologic cure, was conducted. Plasma TnC expression was measured by ELISA and Western blots. TnC values were correlated with markers of liver injury and ROC analyses performed between groups. RESULTS: The HCV cirrhotic cohort, consisting mostly of men (56%), Caucasians (76%), and genotype 1a or 1b (84%), was compared to healthy controls (HCs). Plasma TnC was significantly higher in HCV cirrhotic patients with active infection compared to HCs (P < 0.0001) and virologic cure (P < 0.0001). TnC concentrations in virologic cure subjects were not statistically different from HCs. TnC levels correlated with AST, platelets, MELD, APRI, FIB-4, and Child-Pugh score. TnC and AST together were significantly better indicators of cirrhosis in patients with active HCV infection than other markers tested. CONCLUSIONS: TnC and AST provided the best model for discriminating HCV cirrhotics with active infection from HC and virologic cure cohorts over current liver injury markers, suggesting TnC as a potential indicator of ongoing hepatic injury and inflammation.

Adeno-Associated Virus Serotype 2 Vector-Mediated Reintroduction of microRNA-19b Attenuates Hepatic Fibrosis.

Fibrotic liver injury is a significant healthcare burden in the United States. It represents a major cause of morbidity and mortality for which there are no effective Food and Drug Administration-approved treatment strategies. Fibrosis is considered a disruption of the normal wound healing responses mediated by fibroblastic cells, which are triggered and sustained by pro-fibrotic cytokines such as transforming growth factor beta 1 (TGF-ß1). TGF-ß1-mediated trans-differentiation of hepatic stellate cells (HSCs) from quiescent to activated myofibroblasts is a pivotal event in the development of fibrosis. Activation is accompanied by global changes in microRNA (miR) expression. It has been previously reported that miR19b is decreased in activated HSCs and contributes to increased expression of TGF-ß receptor II and connective tissue growth factor, both confirmed targets of miR19b. An adeno-associated virus serotype 2 vector (AAV2) with a miR19b transgene downstream of enhanced green fluorescent protein under the murine collage alpha 1(I) promoter was developed specifically to target HSCs. Male Sprague Dawley rats (250 g) underwent sham or bile-duct ligation (BDL) surgery. Directly after BDL, rats received AAV2-miR19b, AAV2-control, or vehicle normal saline (NS) by portal-vein injection. After 2 weeks, the animals were euthanized, and blood was collected for alanine and aspartate aminotransferase, total and direct bilirubin, and alkaline phosphatase. Tissue was collected for RNA and protein extraction and histology. Fibrosis and measures of hepatic injury were significantly reduced in AAV2-miR19b-treated rats in combination with significant improvements in total and direct bilirubin. Histological analysis of collagen by PicroSirius Red staining revealed a ∼50% reduction compared to AAV2-control or NS-injected animals. Pro-fibrotic markers, smooth-muscle alpha-actin, TGF-ß receptor II, and collagen alpha 2(I) mRNA and protein were significantly decreased compared to AAV2-control and NS groups. AAV2-mediated reintroduction of miR-19b, specifically expressed in HSCs, improved liver function, inhibited fibrosis, and improved measures of hepatic injury in a BDL model.

Dietary fructose augments ethanol-induced liver pathology.

Certain dietary components when combined with alcohol exacerbate alcohol-induced liver injury (ALI). Here, we tested whether fructose, a major ingredient of the western diet, enhances the severity of ALI. We fed mice ethanol for 8 weeks in the following Lieber-DeCarli diets: (a) Regular (contains olive oil); (b) corn oil (contains corn oil); (c) fructose (contains fructose and olive oil) and (d) corn+fructose (contains fructose and corn oil). We compared indices of metabolic function and liver pathology among the different groups. Mice fed fructose-free and fructose-containing ethanol diets exhibited similar levels of blood alcohol, blood glucose and signs of disrupted hepatic insulin signaling. However, only mice given fructose-ethanol diets showed lower insulin levels than their respective controls. Compared with their respective pair-fed controls, all ethanol-fed mice exhibited elevated levels of serum ALT; the inflammatory cytokines TNF-α, MCP-1 and MIP-2; hepatic lipid peroxides and triglycerides. All the latter parameters were significantly higher in mice given fructose-ethanol diets than those fed fructose-free ethanol diets. Mice given fructose-free or fructose-containing ethanol diets each had higher levels of hepatic lipogenic enzymes than controls. However, the level of the lipogenic enzyme fatty acid synthase (FAS) was significantly higher in livers of mice given fructose control and fructose-ethanol diets than in all other groups. Our findings indicate that dietary fructose exacerbates ethanol-induced steatosis, oxidant stress, inflammation and liver injury, irrespective of the dietary fat source, to suggest that inclusion of fructose in or along with alcoholic beverages increases the risk of more severe ALI in heavy drinkers.

Alcohol, microbiome, life style influence alcohol and non-alcoholic organ damage.

This paper is based upon the "8th Charles Lieber's Satellite Symposium" organized by Manuela G. Neuman at the Research Society on Alcoholism Annual Meeting, on June 25, 2016 at New Orleans, Louisiana, USA. The integrative symposium investigated different aspects of alcohol-induced liver disease (ALD) as well as non-alcohol-induced liver disease (NAFLD) and possible repair. We revealed the basic aspects of alcohol metabolism that may be responsible for the development of liver disease as well as the factors that determine the amount, frequency and which type of alcohol misuse leads to liver and gastrointestinal diseases. We aimed to (1) describe the immuno-pathology of ALD, (2) examine the role of genetics in the development of alcoholic hepatitis (ASH) and NAFLD, (3) propose diagnostic markers of ASH and non-alcoholic steatohepatitis (NASH), (4) examine age and ethnic differences as well as analyze the validity of some models, (5) develop common research tools and biomarkers to study alcohol-induced effects, 6) examine the role of alcohol in oral health and colon and gastrointestinal cancer and (7) focus on factors that aggravate the severity of organ-damage. The present review includes pre-clinical, translational and clinical research that characterizes ALD and NAFLD. Strong clinical and experimental evidence lead to recognition of the key toxic role of alcohol in the pathogenesis of ALD with simple fatty infiltrations and chronic alcoholic hepatitis with hepatic fibrosis or cirrhosis. These latter stages may also be associated with a number of cellular and histological changes, including the presence of Mallory's hyaline, megamitochondria, or perivenular and perisinusoidal fibrosis. Genetic polymorphisms of ethanol metabolizing enzymes and cytochrome p450 (CYP) 2E1 activation may change the severity of ASH and NASH. Other risk factors such as its co-morbidities with chronic viral hepatitis in the presence or absence of human deficiency virus were discussed. Dysregulation of metabolism, as a result of ethanol exposure, in the intestine leads to colon carcinogenesis. The hepatotoxic effects of ethanol undermine the contribution of malnutrition to the liver injury. Dietary interventions such as micro and macronutrients, as well as changes to the microbiota have been suggested. The clinical aspects of NASH, as part of the metabolic syndrome in the aging population, have been presented. The symposium addressed mechanisms and biomarkers of alcohol induced damage to different organs, as well as the role of the microbiome in this dialog. The microbiota regulates and acts as a key element in harmonizing immune responses at intestinal mucosal surfaces. It is known that microbiota is an inducer of proinflammatory T helper 17 cells and regulatory T cells in the intestine. The signals at the sites of inflammation mediate recruitment and differentiation in order to remove inflammatory inducers and promote tissue homeostasis restoration. The change in the intestinal microbiota also influences the change in obesity and regresses the liver steatosis. Evidence on the positive role of moderate alcohol consumption on heart and metabolic diseases as well on reducing steatosis have been looked up. Moreover nutrition as a therapeutic intervention in alcoholic liver disease has been discussed. In addition to the original data, we searched the literature (2008-2016) for the latest publication on the described subjects. In order to obtain the updated data we used the usual engines (Pub Med and Google Scholar). The intention of the eighth symposia was to advance the international profile of the biological research on alcoholism. We also wish to further our mission of leading the forum to progress the science and practice of translational research in alcoholism.

Data on the effect of pro-fibrotic cytokine TGF-ß on hepatic stellate cell autophagy.

Our data describe autophagic flux in primary rat hepatic stellate cells (rHSCs) treated with pro-fibrotic growth factor, transforming growth factor beta (TGF-ß). An autophagy flux experiment determines the rate of synthesis and degradation of the autophagosome marker, LC3-II in the presence and absence of the lysosomal inhibitor bafilomcyin, which blocks LC3-II degradation in lysosomes. The effects of a test agent on LC3-II flux through the autophagic pathway is determined immunochemically by its relative amounts detected in lysates of cells treated with and without bafilomycin. This measurement helps to validate whether exposure to an agent affects the biogenesis or the degradation of autophagosomes during autophagy, a major macromolecular degrading mechanism in eukaryotic cells. ("Rev-erb Agonist and TGF-ß Similarly Affect Autophagy but Differentially Regulate Hepatic Stellate Cell Fibrogenic Phenotype" (Thomes et al., in press) [1].

Rev-erb agonist and TGF-ß similarly affect autophagy but differentially regulate hepatic stellate cell fibrogenic phenotype.

We demonstrated that ligand-activated nuclear receptor Rev-erbα mitigates CCl4-induced liver fibrosis. Rev-erbα is also a novel regulator of autophagy, a crucial eukaryotic catabolic system in which lysosomes degrade substrates for energy generation. In hepatic stellate cells (HSC) autophagy is reportedly required for this purpose to activate HSCs during fibrogenesis. Here, we examined whether pharmacological activation of Rev-erb with its synthetic ligand SR9009 or treatment with the pro-fibrotic cytokine, TGF-ß, each differentially modulate autophagy to regulate the HSC phenotype. We measured the effects of SR9009 on autophagy markers in a CCl4-induced liver fibrosis model. Using primary and immortalized HSCs in vitro, we quantified SR9009 and TGF-ß effects on autophagy flux. Compared with vehicle-treated controls, livers from CCl4-treated mice exhibited lower AMPK, higher P70S6K phosphorylation, elevated P62 and lower levels of ATG proteins, indicating a disruption of autophagosome (AV) formation. SR9009 treatment prevented CCl4-induced P70S6K phosphorylation but did not affect CCl4-induced changes in AMPK, ATG proteins or P62. Analysis of autophagy markers and autophagy flux in primary HSCs or an immortalized human HSC line (LX2), revealed that SR9009 exposure down-regulated AV biogenesis. These events were associated with lower levels of fibrogenic gene expression, P70S6K phosphorylation and HSC proliferation. However, HSC exposure to TGF-ß enhanced fibrogenic gene expression, P70S6K phosphorylation and HSC proliferation, while it simultaneously decelerated AV synthesis. The autophagy activator rapamycin and the autophagy inhibitor wortmannin each decreased HSC activation, P70S6K phosphorylation and HSC proliferation. Furthermore, knock-down of P70S6K using siRNA blocked basal and TGF-ß-induced cell proliferation in human activated LX2. We conclude that SR9009 and TGF-ß both similarly affected autophagy but, differentially regulated HSC fibrogenic phenotype through modulation of P70S6K, which is crucial for cell proliferation and fibrogenesis.

Processing of miR17-92 Cluster in Hepatic Stellate Cells Promotes Hepatic Fibrogenesis During Alcohol-Induced Injury.

BACKGROUND: Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through hepatic stellate cell (HSC) activation, leading to global changes in mRNA and microRNA (miR) expression. miRs are expressed in cells or shuttled to exosomes which can be detected in tissue culture media (TCM) and biological fluids. The mechanisms and function underlying the differential expression and processing of miRs and their downstream effects during hepatic injury remain poorly understood. METHODS: Expression of primary (pri)-miR17-92. and individual members of this cluster, miR17a, 18a, 19a, 20a, 19b, and 92, were examined in primary HSCs and human LX2 cells exposed to alcohol-conditioned media (CM), liver tissue from a rodent model of alcoholic injury, and in exosomes from TCM and plasma of rodent models and patients with alcoholic liver disease (ALD). miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter. RESULTS: Profibrotic markers were enhanced in primary HSCs and LX2 cells exposed to alcohol-CM, concomitant with decreased miR19b expression and a significant increase in pri-miR17-92. Increased pri-miR17-92 was confirmed in a rodent model of alcohol-induced liver injury. Individual members of the cluster were inversely proportionate in cells and exosomes. AAV2-mediated miR19b overexpression inhibited miR17-92 and altered expression of individual cluster members in cells and exosomes. Expression of individual miR17-92 cluster members in plasma exosomes isolated from patients with ALD was similar to that seen in a rodent model of alcoholic injury and in vitro. CONCLUSIONS: Reintroduction of miR19b inhibits HSC activation and modulates expression of pri-miR17-92 and the inverse expression of individual cluster members in cells and exosomes. Better understanding of miR17-92 processing may provide mechanistic insights into the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.

Mitigation of myocardial fibrosis by molecular cardiac surgery-mediated gene overexpression.

OBJECTIVE: Heart failure is accompanied by up-regulation of transforming growth factor beta signaling, accumulation of collagen and dysregulation of sarcoplasmic reticulum calcium adenosine triphosphatase cardiac isoform 2a (SERCA2a). We examined the fibrotic response in small and large myocardial infarct, and the effect of overexpression of the SERCA2a gene. METHODS: Ischemic cardiomyopathy was induced via creation of large or small infarct in 26 sheep. Animals were divided into 4 groups: small infarct; large infarct with heart failure; gene-treated (large infarct with heart failure followed by adeno-associated viral vector, serotype 1.SERCA2a gene construct transfer by molecular cardiac surgery with recirculating delivery); and control. RESULTS: Heart failure was significantly less pronounced in the gene-treated and small-infarct groups than in the large-infarct group. Expression of transforming growth factor beta signaling components was significantly higher in the large-infarct group, compared with the small-infarct and gene-treated groups. Both the angiotensin II type 1 receptor and angiotensin II were significantly elevated in the small- and large-infarct groups, whereas gene treatment diminished this effect. Active fibrosis with de novo collagen synthesis was evident in the large-infarct group; the small-infarct and gene-treated groups showed less fibrosis, with a lower ratio of de novo to mature collagen. CONCLUSIONS: The data presented provide evidence that progression of fibrosis is mediated through increased transforming growth factor beta and angiotensin II signaling, which is mitigated by increased SERCA2a gene expression.

Diet-Induced Obesity Enhances Progression of Hepatocellular Carcinoma through Tenascin-C/Toll-Like Receptor 4 Signaling.

Obesity is an independent risk factor for the development of liver fibrosis/cirrhosis and hepatocellular carcinoma (HCC). Tenascin-C (TnC), an extracellular matrix protein, is transiently expressed during tissue injury and plays a role in fibrogenesis and tumorigenesis. However, the mechanistic role of TnC signaling in the development of HCC remains unknown. We developed a diet-induced obesity HCC mouse model and examined TnC expression and liver injury. To determine the cellular mechanism of TnC signaling in promoting inflammation and hepatocyte epithelial-mesenchymal transition and migration, we used primary hepatocytes and hepatoma and macrophage cell lines. Further, to determine whether elevated TnC expression correlated with obesity-associated HCC, we measured plasma TnC in obese patients with various levels of liver injury. Increased tissue inflammation accompanied with elevated hepatic stellate cell-derived TnC and Toll-like receptor 4 expression was observed in the diet-induced obesity HCC animal model. In vitro studies found enhanced Toll-like receptor 4 signaling activated by TnC, promoting an increased inflammatory response, hepatocyte transformation, and migration. Further, obese patients with cirrhosis alone and in combination with HCC showed significant increases in plasma TnC compared with healthy volunteers and patients with less severe liver injury. Overall, these studies suggest TnC/Toll-like receptor 4 signaling as an important regulator in HCC; inhibiting this signaling axis may be a viable therapeutic target for impeding HCC.

Role of alcohol in the development and progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a significant cause of cancer-related morbidity and mortality. Chronic, heavy ethanol consumption is a major risk for developing the worsening liver pathologies that culminate in hepatic cirrhosis, the leading risk factor for developing HCC. A significant body of work reports the biochemical and pathological consequences of ethanol consumption and metabolism during hepatocarcinogeneis. The systemic effects of ethanol means organ system interactions are equally important in understanding the initiation and progression of HCC within the alcoholic liver. This review aims to summarize the effects of ethanol-ethanol metabolism during the pathogenesis of alcoholic liver disease, the progression toward HCC and the importance of ethanol as a comorbid factor for HCC development.

The effects of over expressing aquaporins on the cryopreservation of hepatocytes.

During cryopreservation, aquaporins are critical in regulating water transport across cellular membranes and preventing osmotic damages. Hepatocytes express aquaporin (AQP) 0, 8, 9, 11, and 12; this study investigates whether increasing the localization of AQP8 on the cellular membrane would improve cell viability by increasing water transport during cryopreservation. Primary rat hepatocytes were cultured and treated with dibutyryl cAMP (Bt(2)cAMP) or glucagon to increase the expression of AQP8 at the cellular membrane via translocation. This phenomenon is verified through two experiments - confocal immunofluorescence microscopy and cell shrinkage analysis. The immunofluorescence results showed increase in AQP8 on the cellular membrane of treated cells, and cell shrinkage analysis showed an increase in water transport of treated cells compared to controls. Primary rat hepatocytes were treated with Bt(2)cAMP or glucagon and cryopreserved using standard protocols in a controlled rate freezer. This resulted in a significant increase in the cell viability on warming. These results indicate that Bt(2)cAMP or glucagon treated hepatocytes had increased expression of aquaporin in the cellular membrane, increased water transport during cryopreservation, and increased post-thaw viability.

MiR-122 decreases HCV entry into hepatocytes through binding to the 3' UTR of OCLN mRNA.

BACKGROUND & AIMS: Analysis in silico suggests that occludin (OCLN), a key receptor for HCV, is a candidate target of miR-122; the most abundant hepatic micro RNA. We aimed to determine if miR-122 can decrease HCV entry through binding to the 3' UTR of OCLN mRNA. DESIGN: Huh7.5 cells were cotransfected with luciferase construct containing 3' UTR of OCLN (pLuc-OCLN) and with selected miRNAs (0-50 nM) and luciferase activity was measured. Huh7.5 cells were also infected by viral particles containing lenti-miR122 genome or control virus. After 48 h, the cells were infected with HCV pseudo-particles (HCVpp) and VSV pseudo-particles (VSVpp). After 72 h of infection, luciferase activity was measured and HCVpp activity was normalized to VSVpp activity. RESULTS: miR-122 binds to the 3'-UTR of OCLN and down-regulates its expression; cotransfection of miR-122 mimic with pLuc-OCLN resulted in a significant decrease in luciferase activity [by 55% (P < 0.01)], while a non-specific miRNA and a mutant miR-122 did not have any effect. miR-122 mimic significantly down-regulated [by 80% (P < 0.01)] OCLN protein in Huh7.5 cells. Accordingly, patients with chronic hepatitis C and higher levels of hepatic miR-122 have lower hepatic expression of OCLN. Immuno-fluorescence imaging showed a decrease in colocalization of OCLN and CLDN following miR-122 over-expression in HCV infected cells. Huh7.5 cells transiently expressing Lenti-miR122 system showed 42% (P < 0.01) decrease in HCV entry. CONCLUSION: This study uncovers a novel antiviral effect of miR-122 on human liver cells and shows that over-expression of miR-122 can decrease HCV entry into hepatocytes through down-regulation of OCLN.

Novel role of nuclear receptor Rev-erbα in hepatic stellate cell activation: potential therapeutic target for liver injury.

UNLABELLED: Hepatic stellate cell (HSC) transdifferentiation from a quiescent, adipocyte-like cell to a highly secretory and contractile myofibroblast-like phenotype contributes to negative pathological consequences, including fibrosis/cirrhosis with portal hypertension (PH). Antiadipogenic mechanisms have been shown to underlie activation of HSCs. We examined the role of heme-sensing nuclear receptor Rev-erbα, a transcriptional repressor involved in metabolic and circadian regulation known to promote adipogenesis in preadipocytes, in HSC transdifferentiation. We discovered that Rev-erbα protein was up-regulated in activated HSCs and injured livers; however, transcriptional repressor activity was not affected by fibrogenic treatments. Surprisingly, increased protein expression was accompanied with increased cytoplasmic accumulation of Rev-erbα, which demonstrated distributions similar to myosin, the major cellular motor protein. Cells overexpressing a cytoplasm-localized Rev-erbα exhibited enhanced contractility. Ectopically expressed Rev-erbα responded to both adipogenic ligand and fibrogenic transforming growth factor beta treatment. Rev-erb ligand SR6452 down-regulated cytoplasmic expression of Rev-erbα, decreased expression of fibrogenic markers and the activated phenotype in HSCs, and ameliorated fibrosis and PH in rodent models. CONCLUSIONS: Up-regulation of Rev-erbα is an intrinsic fibrogenic response characterized by cytoplasmic accumulation of the protein in activated HSCs. Cytoplasmic expression of Rev-erbα promotes a contractile phenotype. Rev-erbα acts as a bifunctional regulator promoting either anti- or profibrogenic response, depending on milieu. Rev-erb ligand SR6452 functions by a previously undescribed mechanism, targeting both nuclear activity and cytoplasmic expression of Rev-erbα. Our studies identify Rev-erbα as a novel regulator of HSC transdifferentiation and offers exciting new insights on the therapeutic potential of Rev-erb ligands.

Cytoskeletal proteins: shaping progression of hepatitis C virus-induced liver disease.

Hepatitis C virus (HCV) infection, which results in chronic hepatitis C (CHC) in most patients (70-85%), is a major cause of liver disease and remains a major therapeutic challenge. The mechanisms determining liver damage and the key factors that lead to a high rate of CHC remain imperfectly understood. The precise role of cytoskeletal (CS) proteins in HCV infection remains to be determined. Some studies including our recent study have demonstrated that changes occur in the expression of CS proteins in HCV-infected hepatocytes. A variety of host proteins interact with HCV proteins. Association between CS and HCV proteins may have implications in future design of CS protein-targeted therapy for the treatment for HCV infection. This chapter will focus on the interaction between host CS and viral proteins to signify the importance of this event in HCV entry, replication and transportation.

Rodent models of alcoholic liver disease: of mice and men.

Alcoholic liver disease (ALD) is a major cause of acute and chronic liver disease worldwide. The progressive nature of ALD is well described; however, the complex interactions under which these pathologies evolve remain to be fully elucidated. Clinically there are no clear biomarkers or universally accepted, effective treatment strategies for ALD. Experimental models of ALD are an important component in identifying underlying mechanisms of alcohol-induced injury to develop better diagnostic markers, predictors of disease progression, and therapeutic targets to manage, halt, or reverse disease progression. Rodents remain the most accessible model for studying ALD pathology. Effective rodent models must mimic the natural history of ALD while allowing examination of complex interactions between multiple hepatic, and non-hepatic, cell types in the setting of altered metabolic or oxidative/nitrosative stress, inflammatory responses, and sensitivity to cytotoxic stress. Additionally, mode and duration of alcohol delivery influence hepatic response and present unique challenges in understanding disease pathology. This review provides an overview of rodent models of ALD, their strengths and weaknesses relative to human disease states, and provides insight of the potential to develop novel rodent models to simulate the course of human ALD.

Silibinin (Milk Thistle) potentiates ethanol-dependent hepatocellular carcinoma progression in male mice.

Hepatocellular carcinoma (HCC) is a global health burden with limited treatment options and poor prognosis. Silibinin, an antioxidant derived from the Milk Thistle plant (Silybum marianum), is reported to exert hepatoprotective and antitumorigenic effects in vitro and in vivo by suppressing oxidative stress and proliferation. Using a DEN-initiated mouse model of HCC, this study examined the effects of dietary silibinin supplementation alone, or in combination with chronic ethanol consumption on HCC progression. Our data demonstrate silibinin exerted marginal hepatoprotective effects in early stages of hepatocarcinogenesis but, when co-administered with ethanol, exacerbated the promotional effects of ethanol in HCC bearing mice, but only in males.