Juan Caballería

Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma

Abstract Background/Aims: It has been known for at least 50 years that alterations in methionine metabolism occur in human liver cirrhosis. However, the molecular basis of this alteration is not completely understood. In order to gain more insight into the mechanisms behind this condition, mRNA levels of methionine adenosyltransferase ( MAT1A ), glycine methyltransferase ( GNMT ), methionine synthase ( MS ), betaine homocysteine methyltransferase ( BHMT ) and cystathionine β-synthase ( CBS ) were examined in 26 cirrhotic livers, five hepatocellular carcinoma (HCC) tissues and ten control livers. Methods: The expression of the above-mentioned genes was determined by quantitative RT-PCR analysis. Methylation of MAT1A promoter was assessed by methylation-sensitive restriction enzyme digestion of genomic DNA. Results: When compared to normal livers MAT1A , GNMT, BHMT, CBS and MS mRNA contents were significantly reduced in liver cirrhosis. Interestingly, MAT1A promoter was hypermethylated in the cirrhotic liver. HCC tissues also showed decreased mRNA levels of these enzymes. Conclusions: These findings establish that the abundance of the mRNA of the main genes involved in methionine metabolism is markedly reduced in human cirrhosis and HCC. Hypermethylation of MAT1A promoter could participate in its reduced expression in cirrhosis. These observations help to explain the hypermethioninemia, hyperhomocysteinemia and reduced hepatic glutathione content observed in cirrhosis.

Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH

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.

A Histologic Scoring System for Prognosis of Patients With Alcoholic Hepatitis

BACKGROUND & AIMS There is no histologic classification system to determine prognoses of patients with alcoholic hepatitis (AH). We identified histologic features associated with disease severity and created a histologic scoring system to predict short-term (90-day) mortality. METHODS We analyzed data from 121 patients admitted to the Liver Unit (Hospital Clinic, Barcelona, Spain) from January 2000 to January 2008 with features of AH and developed a histologic scoring system to determine the risk of death using logistic regression. The system was tested and updated in a test set of 96 patients from 5 academic centers in the United States and Europe, and a semiquantitative scoring system called the Alcoholic Hepatitis Histologic Score (AHHS) was developed. The system was validated in an independent set of 109 patients. Interobserver agreement was evaluated by weighted κ statistical analysis. RESULTS The degree of fibrosis, degree of neutrophil infiltration, type of bilirubinostasis, and presence of megamitochondria were independently associated with 90-day mortality. We used these 4 parameters to develop the AHHS to identify patients with a low (0-3 points), moderate (4-5 points), or high (6-9 points) risk of death within 90 days (3%, 19%, and 51%, respectively; P < .0001). The AHHS estimated 90-day mortality in the training and test sets with an area under the receiver operating characteristic value of 0.77 (95% confidence interval, 0.71-0.83). Interrater agreement values were 0.65 for fibrosis, 0.86 for bilirubinostasis, 0.60 for neutrophil infiltration, and 0.46 for megamitochondria. Interestingly, the type of bilirubinostasis predicted the development of bacterial infections. CONCLUSIONS We identified histologic features associated with the severity of AH and developed a patient classification system that might be used in clinical decision making.

Hepatic Expression of CXC Chemokines Predicts Portal Hypertension and Survival in Patients With Alcoholic Hepatitis

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-alpha also were determined by immunohistochemistry. Serum levels of IL-8 and Gro-alpha 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-alpha, 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-gamma, 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-alpha 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.

A randomized placebo controlled trial of vitamin E for alcoholic hepatitis

BACKGROUND/AIMS The effect of vitamin E administration on clinical and laboratory parameters of liver function and on markers of fibrogenesis was assessed in patients with mild to moderate alcoholic hepatitis in a double blind placebo controlled randomized trial. METHODS Twenty-five patients received 1000 I.U. of vitamin E per day, while 26 patients received placebo for 3 months. The patients were followed for 1 year after entry into the trial. RESULTS Vitamin E did not result in significant greater decreases in serum aminotransferases and serum bilirubin or in greater increases in serum albumin as compared with placebo. Prothrombin time did not change, while serum creatinine remained in the normal range. Monocyte nuclear nuclear factor-kappa B binding activity decreased in patients who remained abstinent, regardless of whether they received vitamin E. As regards markers of hepatic fibrogenesis, vitamin E treatment decreased serum hyaluronic acid (P<0.05) while serum aminoterminal peptide of type III procollagen did not change in either group. Four patients in the treatment group and five in the placebo group died during the 1-year study. CONCLUSIONS Vitamin E treatment improves serum hyaluronic acid but has no beneficial effects on tests of liver function in patients with mild to moderate alcoholic hepatitis.

Ghrelin attenuates hepatocellular injury and liver fibrogenesis in rodents and influences fibrosis progression in humans.

There are no effective antifibrotic therapies for patients with liver diseases. We performed an experimental and translational study to investigate whether ghrelin, an orexigenic hormone with pleiotropic properties, modulates liver fibrogenesis. Recombinant ghrelin was administered to rats with chronic (bile duct ligation) and acute (carbon tetrachloride) liver injury. Hepatic gene expression was analyzed by way of microarray analysis and quantitative polymerase chain reaction. The hepatic response to chronic injury was also evaluated in wild‐type and ghrelin‐deficient mice. Primary human hepatic stellate cells were used to study the effects of ghrelin in vitro. Ghrelin hepatic gene expression and serum levels were assessed in patients with chronic liver diseases. Ghrelin gene polymorphisms were analyzed in patients with chronic hepatitis C. Recombinant ghrelin treatment reduced the fibrogenic response, decreased liver injury and myofibroblast accumulation, and attenuated the altered gene expression profile in bile duct–ligated rats. Moreover, ghrelin reduced the fibrogenic properties of hepatic stellate cells. Ghrelin also protected rats from acute liver injury and reduced the extent of oxidative stress and inflammation. Ghrelin‐deficient mice developed exacerbated hepatic fibrosis and liver damage after chronic injury. In patients with chronic liver diseases, ghrelin serum levels decreased in those with advanced fibrosis, and ghrelin gene hepatic expression correlated with expression of fibrogenic genes. In patients with chronic hepatitis C, polymorphisms of the ghrelin gene (−994CT and −604GA) influenced the progression of liver fibrosis. Conclusion: Ghrelin exerts antifibrotic effects in the liver and may represent a novel antifibrotic therapy. (HEPATOLOGY 2010;51:974–985.)

Systemic inflammatory response and serum lipopolysaccharide levels predict multiple organ failure and death in alcoholic hepatitis.

Alcoholic hepatitis (AH) frequently progresses to multiple organ failure (MOF) and death. However, the driving factors are largely unknown. At admission, patients with AH often show criteria of systemic inflammatory response syndrome (SIRS) even in the absence of an infection. We hypothesize that the presence of SIRS may predispose to MOF and death. To test this hypothesis, we studied a cohort including 162 patients with biopsy‐proven AH. The presence of SIRS and infections was assessed in all patients, and multivariate analyses identified variables independently associated with MOF and 90‐day mortality. At admission, 32 (19.8%) patients were diagnosed with a bacterial infection, while 75 (46.3%) fulfilled SIRS criteria; 58 patients (35.8%) developed MOF during hospitalization. Short‐term mortality was significantly higher among patients who developed MOF (62.1% versus 3.8%, P < 0.001). The presence of SIRS was a major predictor of MOF (odds ratio = 2.69, P = 0.025) and strongly correlated with mortality. Importantly, the course of patients with SIRS with and without infection was similar in terms of MOF development and short‐term mortality. Finally, we sought to identify serum markers that differentiate SIRS with and without infection. We studied serum levels of high‐sensitivity C‐reactive protein, procalcitonin, and lipopolysaccharide at admission. All of them predicted mortality. Procalcitonin, but not high‐sensitivity C‐reactive protein, serum levels identified those patients with SIRS and infection. Lipopolysaccharide serum levels predicted MOF and the response to prednisolone. Conclusion: In the presence or absence of infections, SIRS is a major determinant of MOF and mortality in AH, and the mechanisms involved in the development of SIRS should be investigated; procalcitonin serum levels can help to identify patients with infection, and lipopolysaccharide levels may help to predict mortality and the response to steroids. (Hepatology 2015;62:762–772)

Transcriptome analysis identifies TNF superfamily receptors as potential therapeutic targets in alcoholic hepatitis

Objective 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. Design 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. Results 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. Conclusion 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.

Effects of Ethanol Consumption on Hepatic Hemodynamics in Patients With Alcoholic Cirrhosis

BACKGROUND & AIMS Increased portal blood flow represents a compensatory mechanism preventing hepatic hypoxia after ethanol consumption. In addition, alcohol increases hepatic vascular resistance. Thus, ethanol consumption, by increasing hepatic vascular resistance and portal flow, may worsen portal hypertension in patients with cirrhosis. The aim of this study was to investigate the effects of ethanol consumption on hepatic hemodynamics in patients with alcohol-induced cirrhosis. METHODS Measurements of hepatic venous pressure gradient (HVPG), azygos blood flow, hepatic blood flow, heart rate, and arterial pressure were obtained in 16 patients with alcohol-induced cirrhosis and portal hypertension before and after random administration of a noncaloric fruit drink (250 mL, n = 7) or of an identical beverage plus 0.5 g/kg of ethanol (n = 9). RESULTS Vehicle caused no effects. By contrast, ethanol increased HVPG (P < 0.0001). The increase in HVPG was maximum at 15 minutes and remained significant at 45 minutes (P < 0.05 vs. vehicle). Ethanol increased azygos blood flow (P < 0.05) without changes in hepatic blood flow. Heart rate and arterial pressure slightly increased. CONCLUSIONS Oral ethanol consumption increases portal pressure and portocollateral blood flow in patients with alcohol-induced cirrhosis. These findings suggest that even moderate alcohol consumption worsens the portal-hypertensive syndrome and, therefore, may increase the risk of variceal bleeding in patients with alcohol-induced cirrhosis.

Liver progenitor cell markers correlate with liver damage and predict short-term mortality in patients with alcoholic hepatitis.

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 Maddreys 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)