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.

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.

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.

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.

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.

The let-7 microRNA enhances heme oxygenase-1 by suppressing Bach1 and attenuates oxidant injury in human hepatocytes.

The let-7 microRNA (miRNA) plays important roles in human liver development and diseases such as hepatocellular carcinoma, liver fibrosis and hepatitis wherein oxidative stress accelerates the progression of these diseases. To date, the role of the let-7 miRNA family in modulation of heme oxygenase 1 (HMOX1), a key cytoprotective enzyme, remains unknown. Our aims were to determine whether let-7 miRNA directly regulates Bach1, a transcriptional repressor of the HMOX1 gene, and whether indirect up-regulation of HMOX1 by let-7 miRNA attenuates oxidant injury in human hepatocytes. The effects of let-7 miRNA on Bach1 and HMOX1 gene expression in Huh-7 and HepG2 cells were determined by real-time qRT-PCR, Western blot, and luciferase reporter assays. Dual luciferase reporter assays revealed that let-7b, let-7c, or miR-98 significantly decreased Bach1 3'-untranslated region (3'-UTR)-dependent luciferase activity but not mutant Bach1 3'-UTR-dependent luciferase activity, whereas mutant let-7 miRNA containing base complementarity with mutant Bach1 3'-UTR restored its effect on mutant reporter activity. let-7b, let-7c, or miR-98 down-regulated Bach1 protein levels by 50-70%, and subsequently up-regulated HMOX1 gene expression by 3-4 fold, compared with non-specific controls. Furthermore, Huh-7 cells transfected with let-7b, let-7c or miR-98 mimic showed increased resistance against oxidant injury induced by tert-butyl-hydroperoxide (tBuOOH), whereas the protection was abrogated by over-expression of Bach1. In conclusion, let-7 miRNA directly acts on the 3'-UTR of Bach1 and negatively regulates expression of this protein, and thereby up-regulates HMOX1 gene expression. Over-expression of the let-7 miRNA family members may represent a novel approach to protecting human hepatocytes from oxidant injury.

Chronic ethanol feeding accelerates hepatocellular carcinoma progression in a sex-dependent manner in a mouse model of hepatocarcinogenesis.

BACKGROUND: Chronic ethanol consumption increases the risk of hepatic cirrhosis and hepatocellular carcinoma (HCC). While sex differences exist in susceptibility to ethanol-induced liver damage/HCC development, little is known about the effects of ethanol on tumor progression. METHODS: Neonatal male and female mice were initiated with a single dose of diethylnitrosamine (DEN). Sixteen or 40 weeks later, animals were placed on a 10/20% (v/v) ethanol-drinking water (EtOH-DW; alternate days) regime for 8 weeks. At study end, liver tissue and serum were analyzed for liver pathology/function and cytokine expression. RESULTS: DEN reproducibly induced hepatic foci/tumors in male and female mice. Ethanol diminished hepatic function and increased liver damage, but ethanol alone did not induce hepatic foci/HCC formation. In DEN-initiated EtOH-DW animals, ethanol significantly increased tumor incidence and burden, but only in male mice. Male and female mice (±DEN) demonstrated comparable blood alcohol content at necropsy, yet increased hepatic damage and diminished hepatic function/antioxidant capacity were significantly greater in males. Analysis of liver mRNA for Th1, Th2, or T-regulatory factors demonstrated significantly elevated SMAD3 in male compared to female mice in response to EtOH, DEN initiation, and DEN + EtOH-DW. CONCLUSIONS: These data demonstrate male mice are more susceptible to HCC incidence and progression in the setting of chronic ethanol feeding than females. Differences in markers of hepatic immune response in male mice suggest that increased TGFß-SMAD3 signaling may enhance promotion in this model of HCC progression, effects modulated by chronic ethanol feeding.

Neonatal androgenization exacerbates alcohol-induced liver injury in adult rats, an effect abrogated by estrogen.

Alcoholic liver disease (ALD) affects millions of people worldwide and is a major cause of morbidity and mortality. However, fewer than 10% of heavy drinkers progress to later stages of injury, suggesting other factors in ALD development, including environmental exposures and genetics. Females display greater susceptibility to the early damaging effects of ethanol. Estrogen (E2) and ethanol metabolizing enzymes (cytochrome P450, CYP450) are implicated in sex differences of ALD. Sex steroid hormones are developmentally regulated by the hypothalamic-pituitary-gonadal (HPG) axis, which controls sex-specific cycling of gonadal steroid production and expression of hepatic enzymes. The aim of this study was to determine if early postnatal inhibition of adult cyclic E2 alters ethanol metabolizing enzyme expression contributing to the development of ALD in adulthood. An androgenized rat model was used to inhibit cyclic E2 production. Control females (Ctrl), androgenized females (Andro) and Andro females with E2 implants were administered either an ethanol or isocalorically-matched control Lieber-DeCarli diet for four weeks and liver injury and CYP450 expression assessed. Androgenization exacerbated the deleterious effects of ethanol demonstrated by increased steatosis, lipid peroxidation, profibrotic gene expression and decreased antioxidant defenses compared to Ctrl. Additionally, CYP2E1 expression was down-regulated in Andro animals on both diets. No change was observed in CYP1A2 protein expression. Further, continuous exogenous administration of E2 to Andro in adulthood attenuated these effects, suggesting that E2 has protective effects in the androgenized animal. Therefore, early postnatal inhibition of cyclic E2 modulates development and progression of ALD in adulthood.

Effects of a single dose of oral iron on hepcidin concentrations in human urine and serum analyzed by a robust LC-MS/MS method.

BACKGROUND: The measurement of serum hepcidin, a peptide hormone that regulates iron metabolism, is clinically important to the understanding of iron homeostasis in health and disease. To date, the quantification of serum hepcidin levels by conventional immunological detection methods has proven problematic due to challenges in obtaining high quality antibodies which demonstrate good reproducibility. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been employed recently for more sensitive quantification of hepcidin; however, this method has high background levels and therefore less than optimal specificity. METHODS: In order to increase the specificity of the mass spectrometry based assay, we developed a robust, ultra-performance liquid-chromatography-tandem mass spectrometry (UPLC-MS/MS) protocol using multiple selected reaction monitoring (mSRM) for quantification of hepcidin levels in urine and serum of human subjects. With this assay, we assessed levels of hepcidin before and for up to 8 h after oral ingestion of ferrous sulfate in ten adult human subjects without known disease. RESULTS: The linear response of hepcidin quantitation on each instrument was measured, and the correlation coefficients of these calibrations were r(2)=0.9512±0.0202 (n=5) for urine and r(2)=0.9709±0.0291 (n=5) for serum [r(2)=mean±SD]. Compared to baseline, the levels of urinary hepcidin between 2-4 h and 4-8 h of both women and men showed significant increases with p<0.05 and p<0.001, respectively. The levels of serum hepcidin between 4 h and 8 h in both women and men showed significant increases, compared with baseline values, with both p<0.01. Interestingly, we also observed some degree of oscillation of levels, occurring at later time points. CONCLUSIONS: We have developed and validated a new method for measuring hepcidin concentrations in human serum and urine and used it to demonstrate early increases with iron supplement in both urinary and serum levels of hepcidin, which return to baseline levels, except in urine samples from men.

Targeting collagen expression in alcoholic liver disease.

Alcoholic liver disease (ALD) is a leading cause of liver disease and liver-related deaths globally, particularly in developed nations. Liver fibrosis is a consequence of ALD and other chronic liver insults, which can progress to cirrhosis and hepatocellular carcinoma if left untreated. Liver fibrosis is characterized by accumulation of excess extracellular matrix components, including type I collagen, which disrupts liver microcirculation and leads to injury. To date, there is no therapy for the treatment of liver fibrosis; thus treatments that either prevent the accumulation of type I collagen or hasten its degradation are desirable. The focus of this review is to examine the regulation of type I collagen in fibrogenic cells of the liver and to discuss current advances in therapeutics to eliminate excessive collagen deposition.

S-adenosyl-L-methionine inhibits collagen secretion in hepatic stellate cells via increased ubiquitination.

BACKGROUND: Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) components that disrupt normal liver microcirculation and lead to organ injury. Hepatic stellate cells (HSCs), following transdifferentiation, are the central mediators of hepatic fibrosis through increased secretion of ECM components, including type I collagen. AIMS: The mechanism(s) by which the antioxidant S-adenosyl-L-methionine (SAMe) acts to modulate type I collagen secretion in activated HSCs was examined. METHODS: Hepatic stellate cells were culture-activated for 13-15 days and treated with SAMe. Type I collagen, proteasomal activity and resident endoplasmic reticulum (ER) protein [78-kDa glucose-regulated protein (Grp78) and protein disulphide isomerase (PDI)] expression were measured. Nuclear factor-κB (NF-κB) activity, and its role in SAMe-mediated collagen inhibition, was determined. Type I collagen polyubiquitination was examined. RESULTS: S-adenosyl-L-methionine significantly inhibited type I collagen secretion without significant changes in type I collagen mRNA expression. SAMe also increased NF-κB activity, and blocking NF-κB activity using a dominant-negative IκBα abolished the SAMe-mediated type I collagen secretion. Examination of the post-transcriptional fate of procollagen demonstrated that SAMe treatment led to intracellular type I collagen polyubiquitination accompanied by diminution of proteasomal activity. Expression of Grp78 and PDI (resident ER proteins) were significantly decreased by SAMe treatment. CONCLUSIONS: S-adenosyl-L-methionine inhibits collagen processing leading to increased ubiquitination and decreased secretion. These findings represent a novel mechanism for modulating type I collagen expression in activated HSCs.

Altered expression and function of regulator of G-protein signaling-17 (RGS17) in hepatocellular carcinoma.

Guanine nucleotide regulatory proteins (G-proteins) are central to normal hepatocyte function and are implicated in hepatic disease initiation and progression. Regulators of G-protein signaling (RGS) are critical to defining G-protein-dependent signal fidelity, yet the role of RGS proteins in the liver is poorly defined. The aims of this study were to determine RGS17 expression in normal and transformed hepatic tissue and cells, and address the function of RGS17 in hepatic tumorgenicity. RGS17 expression was determined in human and rat HCC tissue and cell lines. Molecular approaches were used to alter RGS17 expression in HCC cells, effects on cell function measured, and RGS17 association with specific Gα-subunits determined. Using these approaches RGS17 mRNA, but not protein, was detectable in human and rat HCC tissue and cells. Conversely, RGS17 mRNA was not detected in normal tissue, isolated hepatocytes, or non-tumorigenic hepatic cells. Subsequent studies using transfected cells demonstrated that RGS17 proteins were not post-translationally modified in HCC cells, and RGS17 expression is governed by protein degradation and not via miRNAs. Notwithstanding inherently low RGS17 protein levels, altering RGS17 expression profoundly affected HCC cell mitogenesis and migration. Analysis of RGS17-G-protein interaction demonstrated RGS17 associates with both Giα- and Gqα-subunits in HCC cells of human and rat origin. In conclusion, these data demonstrate that, despite difficulties in measuring endogenous RGS protein expression, RGS17 is differentially expressed in HCC and plays a central role in regulating transformed hepatocyte tumorgenicity.