MicroRNAs (miRNAs) are well-known regulators of disease pathogenesis and have great potential as biomarkers and therapeutic targets. We aimed at profiling miRNAs in alcoholic hepatitis (AH) and ...identifying miRNAs potentially involved in liver injury.
MiRNA profiling was performed in liver samples from patients with AH, alcohol liver disease, non-alcoholic steatohepatitis, HCV disease and normal liver tissue. Expression of miRNAs was assessed in liver and serum from patients with AH and animal models. Mimic and decoy miR-182 were used in vitro and in vivo to evaluate miR-182's biological functions.
MiRNA expression profile in liver was highly altered in AH and distinctive from alcohol-induced cirrhotic livers. Moreover, we identified a set of 18 miRNAs predominantly expressed in AH as compared with other chronic liver conditions. Integrative miRNA-mRNA functional analysis revealed the association of AH-altered miRNAs with nuclear receptors, IGF-1 signalling and cholestasis. Interestingly, miR-182 was the most highly expressed miRNA in AH, which correlated with degree of ductular reaction, disease severity and short-term mortality. MiR-182 mimic induced an upregulation of inflammatory mediators in biliary cells. At experimental level, miR-182 was increased in biliary cells in mice fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet but not upregulated by alcohol intake or fibrosis. Inhibition of miR-182 in DDC-fed mice reduced liver damage, bile acid accumulation and inflammatory response.
AH is characterised by a deregulated miRNA profile, including miR-182, which is associated with disease severity and liver injury. These results highlight the potential of miRNAs as therapeutic targets and biomarkers in AH.
Alcoholic hepatitis (AH) is a severe clinical condition that needs novel therapies. The identification of targets for therapy is hampered by the lack of animal models of advanced AH. The authors ...performed a translational study through a transcriptome analysis in patients with AH to identify new molecular targets.
Hepatic gene expression profiling was assessed by DNA microarray in patients with AH (n=15) and normal livers (n=7). Functional analysis was assessed by gene set enrichment analysis. Quantitative PCR was performed in patients with AH (n=40), hepatitis C (n=18), non-alcoholic steatohepatitis (n=20) and in mouse models of acute and chronic liver injury. Protein expression was assessed by immunohistochemistry and western blotting.
Gene expression analysis showed 207 genes >5-fold differentially expressed in patients with AH and revealed seven pathways differentially regulated including 'cytokine-cytokine receptor interaction'. Several tumour necrosis factor (TNF) superfamily receptors, but not ligands, were overexpressed in AH. Importantly, Fn14 was the only TNF superfamily receptor exclusively upregulated in AH compared with other liver diseases and correlated with both 90-day mortality and severity of portal hypertension. Fn14 protein expression was detected in areas of fibrogenesis and in a population of hepatocytes. Fn14 expression was increased in experimental models of liver injury and was detected in progenitor cells.
Translational research revealed that TNF superfamily receptors are overexpressed in AH. Fn14, the receptor for TNF-like weak inducer of apoptosis, is selectively upregulated in patients with AH. TNF superfamily receptors could represent a potential target for therapy.
Background/Aims Non-alcoholic fatty liver disease (NAFLD) pathogenesis remains unknown. Due to the emerging role of free cholesterol (FC) in NAFLD, our aim was to examine the correlation between FC ...accumulation in patients with NAFLD and the expression of enzymes that regulate cholesterol homeostasis. Methods Filipin staining, indicative of FC accumulation, and real-time PCR analyses were performed in 31 NAFLD patients and in seven controls. Results All NASH patients ( n = 14) and 4 out of 17 patients with steatosis exhibited filipin staining compared to controls (0 out of 7 subjects with normal liver histology and BMI). Sterol regulatory element-binding protein-2 (SREBP-2) mRNA levels were 7- and 3-fold higher in NASH and steatosis patients, respectively, compared to controls. Since hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the key enzyme in cholesterol synthesis and transcriptionally controlled by SREBP-2 we measured its mRNA levels, being 3- to 4-fold higher in NAFLD compared to controls, without any difference between NASH and steatosis patients. Fatty acid synthase (FAS) and SREBP-1c expression were not significantly induced in NAFLD, while ATP-binding cassette sub-family G member 1 (ABCG1), a transporter involved in cholesterol egress, and acyl-CoA-cholesterol acyltransferase mRNA levels were modestly increased (1.5- to 2.5-fold, p < 0.05), regardless of fibrosis. Interestingly, mRNA levels of steroidogenic acute regulatory protein (StAR), a mitochondrial-cholesterol transporting polypeptide, increased 7- and 15-fold in steatosis and NASH patients, respectively, compared to controls. Conclusions FC increases in NASH and correlates with SREBP-2 induction. Moreover, StAR overexpression in NASH suggests that mitochondrial FC may be a player in disease progression and a novel target for intervention.
Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease. Most studies have focused on short‐term prognosis, whereas factors associated with long‐term survival are largely unknown. ...The aims of our study were to (1) determine the impact of complete abstinence from alcohol on long‐term survival and (2) identify prognostic factors at admission capable of predicting abstinence during long‐term follow‐up in patients with AH. One hundred forty‐two patients with biopsy‐proven AH that survived the first episode were included. Demographic, psychiatric, and biochemical variables at admission and drinking status during follow‐up were obtained. Cox regression, logistic regression, and classification and regression trees (CART) analyses were used for statistical analysis. Overall mortality was 38% with a median follow‐up of 55 months. During follow‐up, complete abstinence was reported in 39% and was associated with better long‐term survival (hazard ratio, 0.53; P = 0.03). After adjustment for baseline prognostic scoring systems (Model for End‐Stage Liver Disease and age, bilirubin, international normalized ratio, creatinine scores), complete abstinence was independently associated with survival (P < 0.05). Age and lack of past alcoholism treatments were independently associated with complete abstinence (P < 0.001 and P = 0.02, respectively) during follow‐up. CART analysis generated a simple and practical algorithm based on the combination of past alcoholism treatments and age. Using CART analysis, we stratified 2 subgroups of patients with high (65%) and low (26%‐29%) rates of complete abstinence after an episode of AH. Conclusion: Complete abstinence after an episode of AH positively impacts long‐term survival. The combination of 2 variables easily obtained at admission might be useful to predict long‐term abstinence after an episode of AH. Strategies aimed at promoting alcohol abstinence in these patients are necessary. (Hepatology 2017;66:1842–1853)
Alcoholic hepatitis (AH) is a severe condition developed in patients with underlying alcoholic liver disease. Ductular reaction has been associated with chronic alcohol consumption but there is no ...information regarding the extent of liver progenitor cell (LPC) proliferation in AH. The aim of this study was to investigate LPC markers in AH and its correlation with disease severity. Fifty‐nine patients with clinical and histological diagnosis of AH were included in the study. LPC markers were assessed by real‐time polymerase chain reaction (PCR) and immunohistochemistry. Standard logistic regression analysis and classification and regression trees (CART) analysis were used for statistical analysis. A microarray analysis showed an up‐regulation of LPC markers in patients with AH. Real‐time PCR demonstrated that epithelial cell adhesion molecule (EpCAM), Prominin‐1, and Keratin7 were significantly increased in patients with AH compared with normal livers (P ≤ 0.01), chronic hepatitis C (P ≤ 0.01), and HCV‐induced cirrhosis (P ≤ 0.01). Immunohistochemistry scores generated for Keratin7 and EpCAM demonstrated a good correlation with gene expression. Keratin7 gene expression correlated with liver failure as assessed by model for endstage liver disease score (r = 0.41, P = 0.006) and Maddrey's discriminant function (r = 0.43, P = 0.004). Moreover, Keratin7 (OR1.14, P = 0.004) and Prominin‐1 (OR1.14, P = 0.002), but not EpCAM (OR1.16, P = 0.06), were identified as independent predictors of 90‐day mortality. CART analysis generated an algorithm based on the combination of Keratin7 and EpCAM gene expression that stratified three groups of patients with high, intermediate, and low short‐term mortality (89%, 33%, and 6%, respectively; area under the receiver operating curve 0.73, 95% confidence interval 0.60‐0.87). Keratin7 expression provided additional discrimination potential to the age, bilirubin, international normalization ratio, creatinine (ABIC) score. Conclusion: LPC markers correlate positively with severity of liver disease and short‐term mortality in AH patients. This study suggests that LPC proliferation may be an important feature of AH pathophysiology. (HEPATOLOGY 2012;55:1931–1941)
Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in most western countries. Current NAFLD diagnosis methods (e.g., liver biopsy analysis or imaging ...techniques) are poorly suited as tests for such a prevalent condition, from both a clinical and financial point of view. The present work aims to demonstrate the potential utility of serum metabolic profiling in defining phenotypic biomarkers that could be useful in NAFLD management. A parallel animal model/human NAFLD exploratory metabolomics approach was employed, using ultra performance liquid chromatography−mass spectrometry (UPLC−MS) to analyze 42 serum samples collected from nondiabetic, morbidly obese, biopsy-proven NAFLD patients, and 17 animals belonging to the glycine N-methyltransferase knockout (GNMT-KO) NAFLD mouse model. Multivariate statistical analysis of the data revealed a series of common biomarkers that were significantly altered in the NAFLD (GNMT-KO) subjects in comparison to their normal liver counterparts (WT). Many of the compounds observed could be associated with biochemical perturbations associated with liver dysfunction (e.g., reduced Creatine) and inflammation (e.g., eicosanoid signaling). This differential metabolic phenotyping approach may have a future role as a supplement for clinical decision making in NAFLD and in the adaption to more individualized treatment protocols.
The mechanisms linking hepatocellular death, hepatic stellate cell (HSC) activation, and liver fibrosis are largely unknown. Here, we investigate whether acidic sphingomyelinase (ASMase), a known ...regulator of death receptor and stress-induced hepatocyte apoptosis, plays a role in liver fibrogenesis. We show that selective stimulation of ASMase (up to sixfold), but not neutral sphingomyelinase, occurs during the transdifferentiation/activation of primary mouse HSCs into myofibroblast-like cells, coinciding with cathepsin B (CtsB) and D (CtsD) processing. ASMase inhibition or genetic down-regulation by small interfering RNA blunted CtsB/D processing, preventing the activation and proliferation of mouse and human HSCs (LX2 cells). In accordance, HSCs from heterozygous ASMase mice exhibited decreased CtsB/D processing, as well as lower levels of α-smooth muscle actin expression and proliferation. Moreover, pharmacological CtsB inhibition reproduced the antagonism of ASMase in preventing the fibrogenic properties of HSCs, without affecting ASMase activity. Interestingly, liver fibrosis induced by bile duct ligation or carbon tetrachloride administration was reduced in heterozygous ASMase mice compared with that in wild-type animals, regardless of their sensitivity to liver injury in either model. To provide further evidence for the ASMase-CtsB pathway in hepatic fibrosis, liver samples from patients with nonalcoholic steatohepatitis were studied. CtsB and ASMase mRNA levels increased eight- and threefold, respectively, in patients compared with healthy controls. These findings illustrate a novel role of ASMase in HSC biology and liver fibrogenesis by regulating its downstream effectors CtsB/D.
Background & Aims Alcoholic hepatitis (AH) is characterized by hepatocellular damage, inflammation, and fibrosis. We performed a prospective study to associate hepatic expression of the CXC subfamily ...of chemokines with histology findings and prognosis of patients with AH. Methods Liver biopsy samples from 105 patients with AH and 5 normal liver samples (controls) were evaluated for steatosis, inflammation, fibrosis, and cholestasis. Computer-based morphometric analysis assessed the numbers of infiltrating CD3 + T cells and CD15 + cells (neutrophils); terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining was used to quantify apoptosis. Expression of CXC and CC chemokines and selected signaling components were assessed by quantitative reverse-transcription polymerase chain reaction; protein levels of interleukin (IL)-8 and Gro-α also were determined by immunohistochemistry. Serum levels of IL-8 and Gro-α were measured by enzyme-linked immunosorbent assay. The Cox regression model identified variables associated with mortality. Results Most patients (75%) had severe AH; their 90-day mortality rate was 21.9%. In AH liver samples, expression of the CXC subfamily members IL-8, Gro-α, CXCL5, CXCL6, CXCL10, and platelet factor 4 was up-regulated and compared with controls. The CC chemokine CCL2, but not CCL5, also was up-regulated. Higher expression levels of IL- 8, CXCL5, Gro-γ, and CXCL6 were associated with worse prognosis. Expression of CXC components correlated with neutrophil infiltration and the severity of portal hypertension. In the multivariate analysis, IL-8 protein levels were an independent predictor of 90-day mortality. IL-8 and Gro-α serum levels did not correlate with prognosis. Conclusions Hepatic expression of CXC components correlates with prognosis of patients with AH. Reagents that target CXC chemokines might be developed as therapeutics.
Prohibitin‐1 (PHB1) is an evolutionarily conserved pleiotropic protein that participates in diverse processes depending on its subcellular localization and interactome. Recent data have indicated a ...diverse role for PHB1 in the pathogenesis of obesity, cancer, and inflammatory bowel disease, among others. Data presented here suggest that PHB1 is also linked to cholestatic liver disease. Expression of PHB1 is markedly reduced in patients with primary biliary cirrhosis and biliary atresia or with Alagille syndrome, two major pediatric cholestatic conditions. In the experimental model of bile duct ligation, silencing of PHB1 induced liver fibrosis, reduced animal survival, and induced bile duct proliferation. Importantly, the modulatory effect of PHB1 is not dependent on its known mitochondrial function. Also, PHB1 interacts with histone deacetylase 4 (HDAC4) in the presence of bile acids. Hence, PHB1 depletion leads to increased nuclear HDAC4 content and its associated epigenetic changes. Remarkably, HDAC4 silencing and the administration of the HDAC inhibitor parthenolide during obstructive cholestasis in vivo promote genomic reprogramming, leading to regression of the fibrotic phenotype in liver‐specific Phb1 knockout mice. Conclusion: PHB1 is an important mediator of cholestatic liver injury that regulates the activity of HDAC4, which controls specific epigenetic markers; these results identify potential novel strategies to treat liver injury and fibrosis, particularly as a consequence of chronic cholestasis. (Hepatology 2015;62:1237‐1248)
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