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.

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.

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.

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.

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.

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.

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.

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.

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.

Inhibitory effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis.

UNLABELLED: Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGF-ß). MicroRNAs (miRs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGF-ß signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Because miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGF-ß signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGF-ß signaling components demonstrated by decreased TGF-ß receptor II (TGF-ßRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3' untranslated region of TGF-ßRII was validated by luciferase reporter assay. Inhibition of TGF-ß signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGF-ß-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased smooth muscle α-actin expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared with normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared with normal human livers. CONCLUSION: miR 19b is a novel regulator of TGF-ß signaling in HSCs, suggesting a potential therapeutic approach for hepatic fibrosis.

Lon peptidase 1 (LONP1)-dependent breakdown of mitochondrial 5-aminolevulinic acid synthase protein by heme in human liver cells.

5-Aminolevulinic acid synthase (ALAS-1) is the first rate controlling enzyme that controls cellular heme biosynthesis. Negative feedback regulation of ALAS-1 by the end product heme is well documented and provides the foundation for heme treatment of acute porphyrias, a group of diseases caused by genetic defects in the heme biosynthesis pathway and exacerbated by controlled up-regulation of ALAS-1. Heme is known to affect ALAS-1 activity by repressing gene transcription, accelerating mRNA degradation, and impeding pre-ALAS-1 mitochondrial translocation. In the current study, we examined the effect of heme on the rate of mature ALAS-1 protein turnover in human cells and tissues and explored the mediator involved in this new regulatory mechanism. We found that heme and other metalloporphyrins such as CoPP and CrPP decreased mitochondrial ALAS-1 protein through proteolysis. This degradative effect cannot be emulated by iron or free protoporphyrin, two major chemical components of the heme ring, and is independent of oxidative stress. Down-regulating the activity of mitochondrial LONP1, an ATP-dependent protease that controls the selective turnover of mitochondrial matrix proteins, with potent inhibitors and specific siRNA diminished the negative effect of heme on mitochondrial ALAS-1. Therefore, our data support the existence of a conserved heme feedback regulatory mechanism that functions on the mature form of ALAS-1 protein through the activity of a mitochondrial proteolytic system.

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.

Altered aquaporin expression and role in apoptosis during hepatic stellate cell activation.

BACKGROUND: Hepatic stellate cells (HSCs) are effector cells of hepatic fibrosis contributing to excessive collagen deposition and scar matrix formation. Sustained HSC activation leads to hepatic cirrhosis, a leading cause of liver-related death. Reversal of hepatic fibrosis has been attributed to the induction of HSC apoptosis. Aquaporins (AQPs) are critical proteinacious channels that mediate cellular water loss during the initiation and progression of apoptosis. AIMS: This study examined AQP expression in quiescent and activated HSCs and determined the responsiveness to AQP-dependent apoptosis. METHODS: Aquaporin gene and protein expressions in quiescent and activated HSCs were determined by reverse transcription polymerase chain reaction and Western blot analyses. AQP function was determined by cell swelling and apoptotic assays in the absence and presence of HgCl(2) , a non-specific AQP inhibitor. RESULTS: In this study, we report that activated HSCs showed no detectable expression of AQP 1, 5, 8, 9 and 12 mRNAs but expression was observed in quiescent HSCs. Similarly, AQP 0, 1, 8 and 9 protein was not detected in activated HSCs but was measured in quiescent HSCs. Dual fluorescent immunohistochemistry confirmed that AQP expression is decreased in activated HSCs in a model of liver injury. Functional studies demonstrated that quiescent HSCs were highly susceptible to osmotic challenge and apoptotic stimulus, whereas activated HSCs were less responsive. Finally, apoptosis was abrogated by the inhibition of AQP-dependent water movement. CONCLUSIONS: These findings demonstrate that increased resistance to apoptosis in activated HSCs is due, at least in part, to the changes in AQP expression and function that occur following HSC activation.

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.

Inhibition of phosphatidylinositol 3-kinase signaling in hepatic stellate cells blocks the progression of hepatic fibrosis.

UNLABELLED: The hepatic stellate cell (HSC) is the primary cell type in the liver responsible for excess collagen deposition during fibrosis. Following a fibrogenic stimulus the cell changes from a quiescent vitamin A-storing cell to an activated cell type associated with increased extracellular matrix synthesis and increased cell proliferation. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been shown to regulate several aspects of HSC activation in vitro, including collagen synthesis and cell proliferation. Using a targeted approach to inhibit PI3K signaling specifically in HSCs, we investigated the role of PI3K in HSCs using a rodent model of hepatic fibrosis. An adenovirus expressing a dominant negative form of PI3K under control of the smooth muscle alpha-actin (alphaSMA) promoter was generated (Ad-SMAdnPI3K). Transducing HSCs with Ad-SMAdnPI3K resulted in decreased proliferation, migration, collagen expression, and several additional profibrogenic genes, while also promoting cell death. Inhibition of PI3K signaling was also associated with reduced activation of Akt, p70 S6 kinase, and extracellular regulated kinase signaling as well as reduced cyclin D1 expression. Administering Ad-SMAdnPI3K to mice following bile duct ligation resulted in reduced HSC activation and decreased extracellular matrix deposition, including collagen expression. A reduction in profibrogenic mediators, including transforming growth factor beta, tissue inhibitor of metalloproteinase 1, and connective tissue growth factor was also noted. However, liver damage, assessed by alanine aminotransferase levels, was not reduced. CONCLUSION: Inhibition of PI3K signaling in HSCs during active fibrogenesis inhibits extracellular matrix deposition, including synthesis of type I collagen, and reduces expression of profibrogenic factors. These data suggest that targeting PI3K signaling in HSCs may represent an effective therapeutic target for hepatic fibrosis.

Role of alcohol in liver carcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world and contributes significantly to cancer-related morbidity and mortality. Chronic alcohol consumption has long been associated with progressive liver disease toward the development of hepatic cirrhosis and the subsequent increased risk for developing HCC. In assessing the role of alcohol during hepatic disease, and as a carcinogen, many of the deleterious effects of alcohol can be attributed to alcohol metabolism in hepatocytes. In addition to the direct effects of alcohol/alcohol metabolism on hepatocyte transformation, increasing evidence indicates that other intrahepatic and systemic effects of alcohol are likely to play an equally significant role in the process of hepatic tumorigenesis.

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.

Tissue-specific expression of ALA synthase-1 and heme oxygenase-1 and their expression in livers of rats chronically exposed to ethanol.

5-Aminolevulinic acid synthase-1 (ALAS1) and heme oxygenase-1 (HO-1) are the rate-controlling enzymes for heme biosynthesis and degradation, respectively. Expression of these two genes showed tissue-specific expression pattern at both mRNA and protein levels in selected non-treated rat tissues. In the livers of rats receiving oral ethanol for 10 weeks, ALAS1 mRNA levels were increased by 65%, and the precursor and mature ALAS1 protein levels were increased by 1.8- and 2.3-fold, respectively, while no changes were observed in HO-1 mRNA and protein levels, compared with pair-fed controls. These results provide novel insights into the effects of chronic ethanol consumption on hepatic heme biosynthesis and porphyrias.