Gemma Odena

Alcoholic liver disease: Pathogenesis, management, and novel targets for therapy

Alcohol use is a leading cause of preventable morbidity and mortality worldwide, with much of its negative impact as the result of alcoholic liver disease (ALD). ALD is a broad term that encompasses a spectrum of phenotypes ranging from simple steatosis to steatohepatitis, progressive fibrosis, cirrhosis, and hepatocellular carcinoma. The mechanisms underlying the development of these different disease stages are incompletely understood. Standard treatment of ALD, which includes abstinence, nutritional support, and corticosteroids, has not changed in the last 40 years despite continued poor outcomes. Novel therapies are therefore urgently needed. The development of such therapies has been hindered by inadequate resources for research and unsuitable animal models. However, recent developments in translational research have allowed for identification of new potential targets for therapy. These targets include: (i) CXC chemokines, (ii) IL-22/STAT3, (iii) TNF receptor superfamily, (iv) osteopontin, (v) gut microbiota and lipopolysaccharide (LPS), (vi) endocannabinoids, and (vii) inflammasomes. We review the natural history, risk factors, pathogenesis, and current treatments for ALD. We further discuss the findings of recent translational studies and potential therapeutic targets.

Deletion of SIRT1 from Hepatocytes in Mice Disrupts Lipin-1 Signaling and Aggravates Alcoholic Fatty liver

BACKGROUND & AIMS Sirtuin (SIRT1) is a nicotinamide adenine dinucleotide-dependent protein deacetylase that regulates hepatic lipid metabolism by modifying histones and transcription factors. Ethanol exposure disrupts SIRT1 activity and contributes to alcoholic liver disease in rodents, but the exact pathogenic mechanism is not clear. We compared mice with liver-specific deletion of Sirt1 (Sirt1LKO) mice with their LOX littermates (controls). METHODS We induced alcoholic liver injury in male Sirt1LKO and control mice, placing them on Lieber-DeCarli ethanol-containing diets for 10 days and then administering a single dose of ethanol (5 g/kg body weight) via gavage. Liver and serum samples were collected. We also measured messenger RNA levels of SIRT1, SFRS10, and lipin-1β and lipin-1α in liver samples from patients with alcoholic hepatitis and individuals without alcoholic hepatitis (controls). RESULTS On the ethanol-containing diet, livers of Sirt1LKO mice accumulated larger amounts of hepatic lipid and expressed higher levels of inflammatory cytokines than control mice; serum of Sirt1LKO mice had increased levels of alanine aminotransferase and aspartate aminotransferase. Hepatic deletion of SIRT1 exacerbated ethanol-mediated defects in lipid metabolism, mainly by altering the function of lipin-1, a transcriptional regulator of lipid metabolism. In cultured mouse AML-12 hepatocytes, transgenic expression of SIRT1 prevented fat accumulation in response to ethanol exposure, largely by reversing the aberrations in lipin-1 signaling induced by ethanol. Liver samples from patients with alcoholic hepatitis had reduced levels of SIRT1 and a higher ratio of Lpin1β/α messenger RNAs than controls. CONCLUSIONS In mice, hepatic deletion of Sirt1 promotes steatosis, inflammation, and fibrosis in response to ethanol challenge. Ethanol-mediated impairment of hepatic SIRT1 signaling via lipin-1 contributes to development of alcoholic steatosis and inflammation. Reagents designed to increase SIRT1 regulation of lipin-1 can be developed to treat patients with alcoholic fatty liver disease.

Fat-Specific Protein 27/CIDEC Promotes Development of Alcoholic Steatohepatitis in Mice and Humans

BACKGROUND & AIMS Alcoholic steatohepatitis (ASH) is the progressive form of alcoholic liver disease and may lead to cirrhosis and hepatocellular carcinoma. We studied mouse models and human tissues to identify molecules associated with ASH progression and focused on the mouse fat-specific protein 27 (FSP-27)/human cell death-inducing DFF45-like effector C (CIDEC) protein, which is expressed in white adipose tissues and promotes formation of fat droplets. METHODS C57BL/6N mice or mice with hepatocyte-specific disruption of Fsp27 (Fsp27(Hep-/-) mice) were fed the Lieber-Decarli ethanol liquid diet (5% ethanol) for 10 days to 12 weeks, followed by 1 or multiple binges of ethanol (5 or 6 g/kg) during the chronic feeding. Some mice were given an inhibitor (GW9662) of peroxisome proliferator-activated receptor γ (PPARG). Adenoviral vectors were used to express transgenes or small hairpin (sh) RNAs in cultured hepatocytes and in mice. Liver tissue samples were collected from ethanol-fed mice or from 31 patients with alcoholic hepatitis (AH) with biopsy-proved ASH and analyzed histologically and immunohistochemically and by transcriptome, immunoblotting, and real-time PCR analyses. RESULTS Chronic-plus-binge ethanol feeding of mice, which mimics the drinking pattern of patients with AH, produced severe ASH and mild fibrosis. Microarray analyses revealed similar alterations in expression of many hepatic genes in ethanol-fed mice and humans with ASH, including up-regulation of mouse Fsp27 (also called Cidec) and human CIDEC. Fsp27(Hep-/-) mice and mice given injections of adenovirus-Fsp27shRNA had markedly reduced ASH following chronic-plus-binge ethanol feeding. Inhibition of PPARG and cyclic AMP-responsive element binding protein H (CREBH) prevented the increases in Fsp27α and FSP27β mRNAs, respectively, and reduced liver injury in this chronic-plus-binge ethanol feeding model. Overexpression of FSP27 and ethanol exposure had synergistic effects in inducing production of mitochondrial reactive oxygen species and damage to hepatocytes in mice. Hepatic CIDEC mRNA expression was increased in patients with AH and correlated with the degree of hepatic steatosis and disease severity including mortality. CONCLUSIONS In mice, chronic-plus-binge ethanol feeding induces ASH that mimics some histological and molecular features observed in patients with AH. Hepatic expression of FSP27/CIDEC is highly up-regulated in mice following chronic-plus-binge ethanol feeding and in patients with AH; this up-regulation contributes to alcohol-induced liver damage.

Rifaximin, but not growth factor 1, reduces brain edema in cirrhotic rats

AIM To compare rifaximin and insulin-like growth factor (IGF)-1 treatment of hyperammonemia and brain edema in cirrhotic rats with portal occlusion. METHODS Rats with CCl₄-induced cirrhosis with ascites plus portal vein occlusion and controls were randomized into six groups: Cirrhosis; Cirrhosis + IGF-1; Cirrhosis + rifaximin; Controls; Controls + IGF-1; and Controls + rifaximin. An oral glutamine-challenge test was performed, and plasma and cerebral ammonia, glucose, bilirubin, transaminases, endotoxemia, brain water content and ileocecal cultures were measured and liver histology was assessed. RESULTS Rifaximin treatment significantly reduced bacterial overgrowth and endotoxemia compared with cirrhosis groups, and improved some liver function parameters (bilirubin, alanine aminotransferase and aspartate aminotransferase). These effects were associated with a significant reduction in cerebral water content. Blood and cerebral ammonia levels, and area-under-the-curve values for oral glutamine-challenge tests were similar in rifaximin-treated cirrhotic rats and control group animals. By contrast, IGF-1 administration failed to improve most alterations observed in cirrhosis. CONCLUSION By reducing gut bacterial overgrowth, only rifaximin was capable of normalizing plasma and brain ammonia and thereby abolishing low-grade brain edema, alterations associated with hepatic encephalopathy.

LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development.

Determination of the ideal preparation for colonoscopy in a rat model.

Background: Quality bowel preparation is essential to examine the entire colon adequately. No trials comparing different purgative regimens are available in animal models. The aim was to compare 5 methods for bowel cleansing to develop a rat model to study therapeutic colonoscopy. Methods: Twenty-five rats were assigned to one of 5 regimens: (1) high-volume polyethylene glycol electrolyte solution (HV-PEG-ES, 40 mL); (2) low-volume PEG-ES (LV-PEG-ES, 20 mL); (3) high-volume PEG-ES+ascorbic acid (HV-PEG-ES+AA, 20 mL); (4) LV-PEG-ES+AA, 10 mL; (5) rectal enema with saline solution (RE-SS). Bowel preparation quality was rated by total colonoscopy. Results: RE-SS is the best regime for left colon cleansing, whereas HV-PEG-ES and HV-PEG-ES+AA solutions resulted in significantly better cleansing in the whole colon. HV-PEG-ES+AA regimen needed less volume, and administration was easier. Conclusions: A total of 20 mL of PEG-ES+AA before colonoscopy is the best regimen to explore the whole colon, whereas to explore the left colon RE-SS is adequate.

Colonoscopy in rats: An endoscopic, histological and tomographic study.

AIM To describe colon anatomy with colonoscopy and computed tomography (CT) to develop a rat model for future studies of therapeutic colonoscopy. METHODS Eighteen male Sprague-Dawley rats, on average 400-420 g, underwent total colonoscopy, CT and histological examination. Colonoscopy was performed after bowel preparation with a baby upper gastrointestinal endoscopy with an outer diameter of 6.7 mm. CT obtained a 3D image of total colon after a rectal enema with radiological contrast. Macroscopic and microscopic examinations were examined with a conventional technique (hematoxylin and eosin). Colonic wall thickness, length and diameter measurements were taken from the anus, 3, 7, 14 and 20 cm from the anal margin. RESULTS The median colonoscope depth was 24 cm (range 20-28 cm). Endoscopic and tomographic study of colon morphology showed an easy access with tubular morphology in the entire left colon (proximal left colon and rectum). Transverse colon was unapparent on colonoscopy. Right colon, proximal to the splenic flexure, was the largest part of the colon and assumed saccular morphology with tangential trabecula. Radiological measurements of the colonic length and diameter substantiate a subdivision of the right colon into two parts, the cecum and distal right colon. In addition, histological measurement of the colonic wall thickness confirmed a progressive decrease from rectum to cecum. The muscular layer was thinner in the proximal left colon. CONCLUSION The combination of colonoscopy, tomography and histology leads to a better characterization of the entire colon. These data are important for deciding when to perform endoscopic resections or when to induce perforations to apply endoscopic treatments.

Rat CCl4‐induced cirrhosis plus total portal vein ligation: a new model for the study of hyperammonaemia and brain oedema

Introduction: Animal models used to study hyperammonaemic disorders related to chronic liver disease are unsatisfactory. These animals only develop hyperammonaemia and brain oedema when fed with diets supplemented with amonium acetate.

Fibrogénesis hepática: fisiopatología

Liver fibrogenesis is the result of excessive tissue repair of chronic liver damage. This entity consists of the progressive extracellular matrix deposition in the liver parenchyma that is observed in most chronic liver diseases and which precedes the development of cirrhosis. In the last few years, several studies have identified activated stellate cells, portal fibroblasts, and myofibroblasts from distinct cell populations as the main collagen-producing cells in the damaged liver. Likewise, the main cytokines and molecules involved in liver fibrogenesis have been identified. The finding that advanced liver fibrogenesis can be reversed has stimulated research into possible antifibrogenic therapies. Nevertheless, the only effective treatment is elimination of the causal agent. The present article summarizes the progress made in the study of the pathogenesis of liver fibrogenesis and discusses the possible therapeutic targets for the development of antifibrogenic agents.

Hepatocyte-derived macrophage migration inhibitory factor mediates alcohol-induced liver injury in mice and patients

BACKGROUND & AIMS Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that contributes to the inflammatory response to injury. MIF is expressed by multiple cell types; however, the cellular source and actions of MIF in alcoholic liver disease (ALD) are not well known. Here we tested the hypothesis that non-myeloid cells, specifically hepatocytes, are an important cellular source of MIF in ALD. METHODS MIF expression was measured in HuH7 and differentiated THP-1 cells in response to ethanol. Ethanol-induced liver injury was assessed in C57BL/6 (WT) and Mif-/- bone marrow chimeras. MIF was measured in peripheral and suprahepatic serum, as well as visualized by immunohistochemistry in liver biopsies, from patients with alcoholic hepatitis (AH). RESULTS HuH7 hepatocytes, but not THP-1 macrophages, released MIF in response to ethanol challenge in culture. In chimeric mice expressing MIF in non-myeloid cells (Mif-/-→WT), chronic ethanol feeding increased ALT/AST, hepatic steatosis, and expression of cytokine/chemokine mRNA. In contrast, chimeric mice not expressing MIF in non-myeloid cells (WT→Mif-/-) were protected from ethanol-induced liver injury. Immunohistochemical staining of liver biopsies from patients with AH revealed a predominant localization of MIF to hepatocytes. Interestingly, the concentration of MIF in suprahepatic serum, but not peripheral serum, was positively correlated with clinical indicators of disease severity and with an increased risk of mortality in patients with AH. CONCLUSIONS Taken together, these data provide evidence that hepatocyte-derived MIF is critical in the pathogenesis of ALD in mice and likely contributes to liver injury in patients with AH. Lay summary: Alcoholic liver disease is a major cause of preventable mortality worldwide, and lacks specific pharmacological therapies. Recent studies have recognized that macrophage migration inhibitor factor (MIF) has a critical role in the inflammatory response to liver damage. However, the cells that produce this protein are still unknown. Our present findings reveal that hepatocytes, the main cell type in the liver, are primarily responsible for MIF production in response to alcohol, which promotes liver injury. Our study suggests that drugs inhibiting MIF production could be beneficial in treating patients with liver disease due to excessive alcohol consumption.