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 influences hepatic response and presents 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.

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). 16 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/anti-oxidant capacity was 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 increased TGFβ-SMAD3 signaling may enhance promotion in this model of HCC progression, effects modulated by chronic ethanol feeding.

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-erba, a transcriptional repressor involved in metabolic and circadian regulation known to promote adipogenesis in preadipocytes, in HSC transdifferentiation. We discovered that Rev-erba protein was upregulated 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-erba, which demonstrated distributions similar to myosin, the major cellular motor protein. Cells overexpressing a cytoplasm-localized Rev-erba exhibited enhanced contractility. Ectopically expressed Rev-erba responded to both adipogenic ligand and fibrogenic transforming growth factor beta treatment. Rev-erb ligand SR6452 down-regulated cytoplasmic expression of Rev-erba, decreased expression of fibrogenic markers and the activated phenotype in HSCs, and ameliorated fibrosis and PH in rodent models. Conclusions: Up-regulation of Rev-erba is an intrinsic fibrogenic response characterized by cytoplasmic accumulation of the protein in activated HSCs. Cytoplasmic expression of Rev-erba promotes a contractile phenotype. Rev-erba 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-erba. Our studies identify Rev-erba as a novel regulator of HSC transdifferentiation and offers exciting new insights on the therapeutic potential of Rev-erb ligands. (HEPATOLOGY 2014;59:2383-2396

Background Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through 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 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 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 tissue culture media and plasma of rodent models and patients with ALD. miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter. Results Pro-fibrotic 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 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 were similar to those 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 to the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.

Background Alcohol is a significant risk factor for development of hepatocellular carcinoma (HCC). To date, no rodent model has demonstrated formation of hepatic neoplasia in the setting of chronic alcohol consumption alone. Methods We investigated whether rats selectively bred for high alcohol preference (P rats), allowed free access to water, or water and 10% (v/v) alcohol for 6, 12 or 18 months, develop hepatic neoplasia. Results At necropsy, liver tumor incidence and multiplicity were significantly increased in 18-month alcohol-consuming versus water-consuming P rats. These data were confirmed histologically by glutathione-S-transferase pi-class (GSTp) staining. Phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK) staining was also increased in the sinusoidal lining cells within livers of alcohol-consuming versus water only P rats. In addition, cytochrome p450IIE1 (CYP2E1) mRNA, protein expression/activity, and intrahepatic oxidative stress were significantly increased in alcohol-consuming P rat livers versus water only. In contrast, acetaldehyde dehydrogenase expression decreased in alcohol-consuming versus water only P rats. No significant difference in alcohol dehydrogenase expression was detected. Conclusions These data demonstrate that chronic alcohol consumption is associated with hepatic neoplasia, MAPK/ERK activation, increased CYP2E1 activity, and intrahepatic oxidative stress in P rats. Since these rats are well-characterized as a model of alcoholism, these findings identify a novel rodent model of alcohol or “alcoholism”-induced liver neoplasia.