SIRT5 rs12216101 T>G variant is associated with liver damage and mitochondrial dysfunction in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Sirtuin 5, encoded by the SIRT5 gene, is a NAD+-dependent deacylase that modulates mitochondrial metabolic processes through post-translational modifications. In this study, we aimed to examine the impact of the SIRT5 rs12216101 T>G non-coding single nucleotide polymorphism on disease severity in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: The rs12216101 variant was genotyped in 2,606 consecutive European patients with biopsy-proven NAFLD. Transcriptomic analysis, expression of mitochondrial complexes and oxidative stress levels were measured in liver samples from a subset of bariatric patients. Effects of SIRT5 pharmacological inhibition were evaluated in HepG2 cells exposed to excess free fatty acids. Mitochondrial energetics in vitro were investigated by high-performance liquid chromatography. RESULTS: In the whole cohort, the frequency distribution of SIRT5 rs12216101 TT, TG and GG genotypes was 47.0%, 42.3% and 10.7%, respectively. At multivariate logistic regression analysis adjusted for sex, age >50 years, diabetes, and PNPLA3 rs738409 status, the SIRT5 rs12216101 T>G variant was associated with the presence of non-alcoholic steatohepatitis (odds ratio 1.20, 95% CI 1.03-1.40) and F2-F4 fibrosis (odds ratio 1.18; 95% CI 1.00-1.37). Transcriptomic analysis showed that the SIRT5 rs12216101 T>G variant was associated with upregulation of transcripts involved in mitochondrial metabolic pathways, including the oxidative phosphorylation system. In patients carrying the G allele, western blot analysis confirmed an upregulation of oxidative phosphorylation complexes III, IV, V and consistently higher levels of reactive oxygen species, reactive nitrogen species and malondialdehyde, and lower ATP levels. Administration of a pharmacological SIRT5 inhibitor preserved mitochondrial energetic homeostasis in HepG2 cells, as evidenced by restored ATP/ADP, NAD+/NADH, NADP+/NADPH ratios and glutathione levels. CONCLUSIONS: The SIRT5 rs12216101 T>G variant, heightening SIRT5 activity, is associated with liver damage, mitochondrial dysfunction, and oxidative stress in patients with NAFLD. IMPACT AND IMPLICATIONS: In this study we discovered that the SIRT5 rs12216101 T>G variant is associated with higher disease severity in patients with non-alcoholic fatty liver disease (NAFLD). This risk variant leads to a SIRT5 gain-of-function, enhancing mitochondrial oxidative phosphorylation and thus leading to oxidative stress. SIRT5 may represent a novel disease modulator in NAFLD.

The PD-1/PD-L1 Axis in the Biology of MASLD.

Metabolic Dysfunction-Associated Steatotic Liver (MASL), previously named nonalcoholic fatty liver (NAFL), is a multifactorial disease in which metabolic, genetic, and environmental risk factors play a predominant role. Obesity and type 2 diabetes act as triggers of the inflammatory response, which contributes to the progression of MASL to Metabolic Dysfunction-Associated Steatohepatitis and the development of hepatocellular carcinoma. In the liver, several parenchymal, nonparenchymal, and immune cells maintain immunological homeostasis, and different regulatory pathways balance the activation of the innate and adaptative immune system. PD-1/PD-L1 signaling acts, in the maintenance of the balance between the immune responses and the tissue immune homeostasis, promoting self-tolerance through the modulation of activated T cells. Recently, PD-1 has received much attention for its roles in inducing an exhausted T cells phenotype, promoting the tumor escape from immune responses. Indeed, in MASLD, the excessive fat accumulation dysregulates the immune system, increasing cytotoxic lymphocytes and decreasing their cytolytic activity. In this context, T cells exacerbate liver damage and promote tumor progression. The aim of this review is to illustrate the main pathogenetic mechanisms by which the immune system promotes the progression of MASLD and the transition to HCC, as well as to discuss the possible therapeutic applications of PD-1/PD-L1 target therapy to activate T cells and reinvigorate immune surveillance against cancer.

Metabolic memory in diabetic foot syndrome (DFS): MICRO-RNAS, single nucleotide polymorphisms (SNPs) frequency and their relationship with indices of endothelial function and adipo-inflammatory dysfunction.

BACKGROUND: Diabetic foot is a significant cause of morbidity in diabetic patients, with a rate that is approximately twice that of patients without foot ulcers. "Metabolic memory" represents the epigenetic changes induced by chronic hyperglycaemia, despite the correction of the glucose levels themselves. These epigenetic modifications appear to perpetuate the damage caused by persistently elevated glucose levels even in their absence, acting at various levels, mostly affecting the molecular processes of diabetic ulcer healing. METHODS: The aim of our cross-sectional study was to analyse a cohort of patients with diabetes with and without lower limb ulcers. We examined the effects of epigenetic changes on miRNA 126, 305, and 217 expression and the frequency of the SNPs of genes encoding inflammatory molecules (e.g., IL-6 and TNF-alpha) and their correlations with serum levels of proangiogenic molecules (e.g., ENOS, VEGF and HIF-1alpha) and several adipokines as well as with endothelial dysfunction, assessed noninvasively by reactive hyperaemia peripheral artery tonometry. Between March 2021 and June 2022, 110 patients were enrolled into the study: 50 diabetic patients with diabetic foot injuries, 40 diabetic patients without ulcerative complications and 20 nondiabetic patients as the control group. RESULTS: Diabetic subjects with lower limb ulcerative lesions exhibited higher levels of inflammatory cytokines, such as VEGF (191.40 ± 200 pg/mL vs. 98.27 ± 56.92 pg/mL vs. 71.01 ± 52.96 pg/mL; p = 0.22), HIF-1alpha (40.18 ± 10.80 ng/mL vs. 33.50 ± 6.16 ng/mL vs. 33.85 ± 6.84 ng/mL; p = 0.10), and Gremlin-1 (1.72 ± 0.512 ng/mL vs. 1.31 ± 0.21 ng/mL vs. 1.11 ± 0.19 ng/mL; p < 0.0005), than those without lower limb ulcers and healthy controls. Furthermore, we observed that miR-217-5p and miR-503-5p were 2.19-fold (p < 0.05) and 6.21-fold (p = 0.001) more highly expressed in diabetic foot patients than in healthy controls, respectively. Additionally, diabetic patients without lower limb ulcerative complications showed 2.41-fold (p = 0) and 2.24-fold (p = 0.029) higher expression of miR-217-5p and miR-503-5p, respectively, than healthy controls. Finally, diabetic patients with and without ulcerative complications of the lower limbs showed higher expression of the VEGFC2578A CC polymorphism (p = 0.001) and lower expression of the VEGFC2578A AC polymorphism (p < 0.005) than the healthy control population. We observed a significant increase in Gremlin-1 levels in patients with diabetic foot, suggesting that this inflammatory adipokine may serve as a predictive marker for the diagnosis of diabetic foot. CONCLUSIONS: Our results highlighted that patients with diabetic foot showed predominant expression of the VEGF C2578A CC polymorphism and reduced expression of the AC allele. Additionally, we found an overexpression of miR-217-5p and miR-503-5p in diabetic patients with and without diabetic foot syndrome compared with healthy controls. These results align with those reported in the literature, in which the overexpression of miR-217-5p and miR-503-5p in the context of diabetic foot is reported. The identification of these epigenetic modifications could therefore be helpful in the early diagnosis of diabetic foot and the treatment of risk factors. However, further studies are necessary to confirm this hypothesis.

Curcumin and Andrographolide Co-Administration Safely Prevent Steatosis Induction and ROS Production in HepG2 Cell Line.

Non-alcoholic fatty liver disease (NAFLD) is an emerging chronic liver disease worldwide. Curcumin and andrographolide are famous for improving hepatic functions, being able to reverse oxidative stress and release pro-inflammatory cytokines, and they are implicated in hepatic stellate cell activation and in liver fibrosis development. Thus, we tested curcumin and andrographolide separately and in combination to determine their effect on triglyceride accumulation and ROS production, identifying the differential expression of genes involved in fatty liver and oxidative stress development. In vitro steatosis was induced in HepG2 cells and the protective effect of curcumin, andrographolide, and their combination was observed evaluating cell viability, lipid and triglyceride content, ROS levels, and microarray differential gene expression. Curcumin, andrographolide, and their association were effective in reducing steatosis, triglyceride content, and ROS stress, downregulating the genes involved in lipid accumulation. Moreover, the treatments were able to protect the cytotoxic effect of steatosis, promoting the expression of survival and anti-inflammatory genes. The present study showed that the association of curcumin and andrographolide could be used as a therapeutic approach to counter high lipid content and ROS levels in steatosis liver, avoiding the possible hepatotoxic effect of curcumin. Furthermore, this study improved our understanding of the antisteatosis and hepatoprotective properties of a curcumin and andrographolide combination.

Exome-Wide Association Study on Alanine Aminotransferase Identifies Sequence Variants in the GPAM and APOE Associated With Fatty Liver Disease.

BACKGROUND & AIMS: Fatty liver disease (FLD) is a growing epidemic that is expected to be the leading cause of end-stage liver disease within the next decade. Both environmental and genetic factors contribute to the susceptibility of FLD. Several genetic variants contributing to FLD have been identified in exome-wide association studies. However, there is still a missing hereditability indicating that other genetic variants are yet to be discovered. METHODS: To find genes involved in FLD, we first examined the association of missense and nonsense variants with alanine aminotransferase at an exome-wide level in 425,671 participants from the UK Biobank. We then validated genetic variants with liver fat content in 8930 participants in whom liver fat measurement was available, and replicated 2 genetic variants in 3 independent cohorts comprising 2621 individuals with available liver biopsy. RESULTS: We identified 190 genetic variants independently associated with alanine aminotransferase after correcting for multiple testing with Bonferroni method. The majority of these variants were not previously associated with this trait. Among those associated, there was a striking enrichment of genetic variants influencing lipid metabolism. We identified the variants rs2792751 in GPAM/GPAT1, the gene encoding glycerol-3-phosphate acyltransferase, mitochondrial, and rs429358 in APOE, the gene encoding apolipoprotein E, as robustly associated with liver fat content and liver disease after adjusting for multiple testing. Both genes affect lipid metabolism in the liver. CONCLUSIONS: We identified 2 novel genetic variants in GPAM and APOE that are robustly associated with steatosis and liver damage. These findings may help to better elucidate the genetic susceptibility to FLD onset and progression.

A cholestatic pattern predicts major liver-related outcomes in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: NAFLD patients usually have an increase in AST/ALT levels, but cholestasis can also be observed. We aimed to assess in subjects with NAFLD the impact of the (cholestatic) C pattern on the likelihood of developing major liver-related outcomes (MALO). METHODS: Five hundred and eighty-two consecutive patients with biopsy-proven NAFLD or a clinical diagnosis of NAFLD-related compensated cirrhosis were classified as hepatocellular (H), C and mixed (M) patterns, by using the formula (ALT/ALT Upper Limit of Normal-ULN)/(ALP/ALP ULN). MALO were recorded during follow-up. An external cohort of 1281 biopsy-proven NAFLD patients was enrolled as validation set. RESULTS: H, M and C patterns were found in 153 (26.3%), 272 (46.7%) and 157 (27%) patients respectively. During a median follow-up of 78 months, only 1 (0.6%) patient with H pattern experienced MALO, whilst 15 (5.5%) and 38 (24.2%) patients in M and C groups had MALO. At multivariate Cox regression analysis, age >55 years (HR 2.55, 95% CI 1.17-5.54; p = .01), platelets <150 000/mmc (HR 0.14, 95% CI 0.06-0.32; p < .001), albumin <4 g/L(HR 0.62, 95% CI 0.35-1.08; p = .09), C versus M pattern (HR 7.86, 95% CI 1.03-60.1; p = .04), C versus H pattern(HR 12.1, 95% CI 1.61-90.9; p = .01) and fibrosis F3-F4(HR 35.8, 95% CI 4.65-275.2; p < .001) were independent risk factors for MALO occurrence. C versus M pattern(HR 14.3, 95% CI 1.90-105.6; p = .008) and C versus H pattern (HR 15.6, 95% CI 2.10-115.1; p = .0068) were confirmed independently associated with MALO occurrence in the validation set. The immunohistochemical analysis found a significantly higher prevalence of moderate-high-grade ductular metaplasia combined with low-grade ductular proliferation in C pattern when compared with the biochemical H pattern. Gene expression analysis showed a lower expression of NR1H3, RXRα and VCAM1 in patients with the C pattern. CONCLUSIONS: The presence of a cholestatic pattern in patients with NAFLD predicts a higher risk of MALO independently from other features of liver disease.

PNPLA3 rs738409 C>G Variant Predicts Fibrosis Progression by Noninvasive Tools in Nonalcoholic Fatty Liver Disease.

Liver fibrosis is the main predictor of events in patients with nonalcoholic fatty liver disease (NAFLD),1 and its evolution is characterized by a nonlinear trend2,3 mostly affected by metabolic risk factors, severity of liver inflammation and steatosis, and weight loss.3 The rs738409 C>G common variant in PNPLA3 gene has been associated with severity of fibrosis and risk of liver-related events in NAFLD.4,5 Noninvasive tests as Fibrosis-4 (FIB-4) and liver stiffness measurement (LSM) are useful to rule-out advanced fibrosis and they could be reliable to predict fibrosis progression.2,6 We aimed to evaluate in patients with NAFLD whether PNPLA3 rs738409 C>G variant impacts on fibrosis progression, noninvasively assessed by FIB-4 and LSM.

NR1H4 rs35724 G>C variant modulates liver damage in nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Farnesoid X receptor (FXR) plays a key role in bile acid and lipid homeostasis. Experimental evidence suggests that it can modulate liver damage related to nonalcoholic fatty liver disease (NAFLD). We examined the impact of the NR1H4 rs35724 G>C, encoding for FXR, on liver damage in a large cohort of patients at risk of steatohepatitis. METHODS: We considered 2,660 consecutive individuals at risk of steatohepatitis with liver histology. The rs35724 G>C polymorphisms were genotyped by TaqMan assays. Gene expression was evaluated by RNASeq in a subset of patients (n = 124). RESULTS: The NR1H4 rs35724 CC genotype, after adjusting for clinic-metabolic and genetic confounders and for enrolling centre, was protective against severity of steatosis (GG vs CC OR 0.77, 95% CI 0.62-0.95; P = .01), steatohepatitis (GG vs CC OR 0.62, 95% CI 0.47-0.83; P = .001) and severity of fibrosis (GG vs CC OR 0.83, 95% CI 0.67-0.98; P = .04). The C allele was associated with higher total circulating cholesterol (P = .01). Patients carrying the NR1H4 rs35724 C allele had significantly higher hepatic mRNA levels of FXR and were associated with higher hepatic FGFR4 and Cyp39A1 that are in turn involved in bile acid synthesis. CONCLUSIONS: Increased hepatic FXR expression due to the NR1H4 rs35724 C allele is linked to higher serum cholesterol but protects against steatosis, steatohepatitis and liver fibrosis. The translational relevance of these results for patient risk stratification and FXR-targeted therapy warrants further investigation.

Association Between PNPLA3 rs738409 C>G Variant and Liver-Related Outcomes in Patients With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Patients with nonalcoholic fatty liver disease (NAFLD) have an increased risk for liver-related complications, such as decompensation, hepatocellular carcinoma (HCC), and death; the severity of liver fibrosis and metabolic comorbidities are the main risk factors. A single nucleotide polymorphism in patatin-like phospholipase domain-containing-3 (PNPLA3) gene is associated with higher prevalence of liver damage and HCC, but there are no data from prospective studies of outcomes of patients with this polymorphism. We investigated whether the common rs738409 variant in PNPLA3 gene associates with the occurrence of liver-related events and death in a large cohort of patients with NAFLD. METHODS: We followed 471 consecutive individuals at a hospital in Italy with a diagnosis of NAFLD based on histologic factors or a diagnosis of compensated NAFLD-related cirrhosis based on clinical factors for at least 6 months, from March 2004 through December 2018. We collected data on the occurrence of hepatic and extrahepatic outcomes, including decompensation and HCC, cardiovascular events and extrahepatic cancers, and overall and liver-related death. We detected the rs738409 G>C polymorphism in DNA from patient blood samples using the TaqMan assay. RESULTS: During a median follow-up time of 64.6 months (range 6.1-175 months) 26 cases of decompensation, 13 HCCs, and 16 deaths (12 liver-related) were recorded. All liver-related events, including liver-related death, occurred in patients with F3 fibrosis or cirrhosis. The prevalence of PNPLA3 rs738409 GG, GT, and TT genotypes was 31.8%, 45.6%, and 22.6%, respectively. After adjusting for clinical, metabolic, and histologic risk factors, PNPLA3 C>G variant was associated with a higher risk of decompensation (hazard ratio [HR], 2.10; 95% CI, 1.03-4.29; P = .04), HCC (HR, 2.68; 95% CI, 1.01-7.26; P = .04), and liver-related death (HR, 3.64; 95% CI, 1.18-11.2; P = .02) by multivariate Cox regression analysis. In the subgroup of 162 patients with F3 fibrosis or cirrhosis, we confirmed the independent association between the PNPLA3 variant and decompensation (HR, 2.00; 95% CI, 1.01-3.97; P = .04), HCC (HR, 2.66; 95% CI, 1.02-7.13; P = .04), and liver-related death (HR, 3.64, 95% CI, 1.18-11.2; P = .02). We found no association between PNPLA3 genotype and cardiovascular events, extrahepatic cancers, or overall mortality. CONCLUSIONS: Patients with NAFLD carrying PNPLA3 rs738409 G>C variant are at higher risk of liver-related events and death.

Hepatitis C virus eradication by direct antiviral agents abates oxidative stress in patients with advanced liver fibrosis.

BACKGROUND AND AIMS: HCV eradication improves non-hepatic outcomes such as cardiovascular diseases, although without clearly defined mechanisms. In this study we aimed to assess whether improvement of carotid atherosclerosis may be linked to a reduction in systemic oxidative stress after viral clearance. METHODS: We studied a retrospective cohort of 105 patients (age 62.4 ± 11.2 years; 62 men) with F3/F4 fibrosis, characterized by carotid ultrasonography at baseline and at sustained virologic response (SVR) follow-up. Levels of 8-iso-prostaglandin F2α (F2 -isoprostanes) and other oxidative stress markers were measured on frozen sera. Association between change (denoted as Δ) in oxidative stress markers (exposures) and change in carotid intima-media thickness (cIMT) (outcome) was examined using multiple linear regression. RESULTS: Subclinical atherosclerosis, defined as the presence of carotid plaque and/or cIMT ≥ 0.9, was present in 72% of the cohort. All patients achieved SVR that led to reduction in cIMT (0.92 ± 0.20 vs 0.83 ± 0.21 mm, P < .001). HCV eradication markedly decreased serum levels of F2 -isoprostanes (620.5 [143.2; 1904.1] vs 119.51 [63.2; 400.6] pg/mL, P < .0001), lipid hydroperoxides (13.8 [6.3; 20.7] vs 4.9 [2.3; 9.6] nmol/µl, P < .0001) and 8-hydroxy-2'-deoxyguanosine (558.9 [321.0; 6301.2] vs 294.51 [215.31; 408.95] pg/mL, P < .0001), whereas increased serum GPx activity (10.44 [4.6; 16.3] vs 13.75 [9.42; 20.63] nmol/min/mL, P = .001). By multiple linear regression analysis ΔcIMT was independently associated with ΔF2 -isoprostanes (ß: 1.746 [0.948; 2.543]; P < .0001) after adjustment for age, baseline F2 -isoprostanes and baseline IMT. CONCLUSIONS: Besides association of lipid peroxidation with severity of liver disease, the reduction in F2 -isoprostanes may be involved in the improvement of atherosclerosis after HCV eradication.

Hepatitis C virus eradication by direct-acting antiviral agents improves carotid atherosclerosis in patients with severe liver fibrosis.

BACKGROUND AND AIMS: Recent studies suggest an association between hepatitis C virus (HCV) infection and cardiovascular damage, including carotid atherosclerosis, with a possible effect of HCV clearance on cardiovascular outcomes. We aimed to examine whether HCV eradication by direct-acting antiviral agents (DAA) improves carotid atherosclerosis in HCV-infected patients with advanced fibrosis/compensated cirrhosis. MATERIALS AND METHODS: One hundred eighty-two consecutive patients with HCV and advanced fibrosis or compensated cirrhosis were evaluated. All patients underwent DAA-based antiviral therapy according to AISF/EASL guidelines. Intima-media thickness (IMT), carotid thickening (IMT ≥1 mm) and carotid plaques, defined as focal thickening of ≥1.5 mm at the level of the common carotid, were evaluated by ultrasonography (US) at baseline and 9-12 months after the end of therapy. Fifty-six percent of patients were male, mean age 63.1 ±â€¯10.4 years, and 65.9% had compensated cirrhosis. One in five had diabetes, 14.3% were obese, 41.8% had arterial hypertension and 35.2% were smokers. At baseline, mean IMT was 0.94 ±â€¯0.29 mm, 42.8% had IMT ≥1 mm, and 42.8% had carotid plaques. RESULTS: All patients achieved a 12-week sustained virological response. IMT significantly decreased from baseline to follow-up (0.94 ±â€¯0.29 mm vs. 0.81 ±â€¯0.27, p <0.001). Consistently, a significant reduction in the prevalence of patients with carotid thickening from baseline to follow-up was observed (42.8% vs. 17%, p <0.001), while no changes were reported for carotid plaques (42.8% vs. 47.8%, p = 0.34). These results were confirmed in subgroups of patients stratified for cardiovascular risk factors and liver disease severity. CONCLUSION: HCV eradication by DAA improves carotid atherosclerosis in patients with severe fibrosis with or without additional metabolic risk factors. The impact of this improvement in the atherosclerotic burden in terms of reduction of major cardiovascular outcomes is worth investigating in the long term. LAY SUMMARY: Hepatitis C virus eradication by direct-acting antiviral agents improves carotid atherosclerosis in patients with advanced fibrosis/compensated cirrhosis. The improvement in intima-media thickness and carotid thickening was confirmed after stratification for severity of liver disease and cardiovascular risk factors. Hepatitis C virus eradication by direct-acting antiviral agents also lead to improvement in glucose homeostasis and increased cholesterol levels.

Interferon lambda 4 rs368234815 TT>δG variant is associated with liver damage in patients with nonalcoholic fatty liver disease.

The interferon (IFN) lambda 3/4 (IFNL3/4) locus, influencing innate immunity regulation, has been associated with the severity of hepatitis and fibrosis progression during chronic hepatitis C infection, while contrasting results were reported in nonalcoholic fatty liver disease. In this study, we examined whether rs12979860 and the linked causal rs368234815 variant encoding for the alternative IFNL4 protein variant are associated with liver fibrosis and damage in a large multicenter cohort of patients at risk of nonalcoholic steatohepatitis. To clarify the mechanism, we also evaluated the impact on IFN-stimulated gene hepatic expression in a subset of patients. We considered 946 consecutive Italian individuals at risk of nonalcoholic steatohepatitis with liver histology evaluated according to Kleiner. The rs368234815 TT>δG, rs12979860 C>T, and patatin-like phospholipase-3 rs738409 C>G polymorphisms were genotyped; and IFN-stimulated gene hepatic expression (n = 16) was tested by TaqMan assays. We found that the rs368234815 TT allele was independently associated with severe F3-F4 fibrosis (odds ratio, 1.53; 95% confidence interval, 1.15-2.31; P = 0.005) and with severe (grade 2-3) lobular necroinflammation (odds ratio, 1.47; 95% confidence interval, 1.14-1.88; P = 0.002). The impact of rs368234815 on liver damage was generally more marked in nonobese individuals, where association with severe fibrosis, necroinflammation, and nonalcoholic steatohepatitis was observed (P < 0.05). IFN-stimulated genes were hypo-expressed in the liver of patients carrying the IFNL4 rs368234815 TT/TT genotype (P < 0.05). Similar results were observed when considering the rs12979860 polymorphism, which was in high linkage disequilibrium with rs368234815 (R2 = 0.87). CONCLUSION: The IFNL4 genotype is associated with severity of fibrosis in nonalcoholic fatty liver disease patients of European ancestry, likely by modulating the activation of innate immunity and necroinflammation. (Hepatology 2017;66:1885-1893).

Prevalence and severity of nonalcoholic fatty liver disease by transient elastography: Genetic and metabolic risk factors in a general population.

BACKGROUND & AIMS: The worldwide spread of obesity is leading to a dramatic increase in the prevalence of nonalcoholic fatty liver disease (NAFLD) and its complications. We aimed to evaluate both prevalence and factors associated with NAFLD in a general population in a Mediterranean area. METHODS: We considered 890 consecutive individuals included in the community-based ABCD (Alimentazione, Benessere Cardiovascolare e Diabete) study (ISRCTN15840340). Controlled attenuation parameter (CAP) and liver stiffness measurement (LSM) were measured with FibroScan. Participants were genotyped for PNPLA3 rs738409 and TM6SF2 rs58542926 variants. RESULTS: The prevalence of NAFLD in the cohort was 48%. NAFLD participants exhibited elevated LSM values, suggesting advanced fibrosis (6.5% of cases). Both NAFLD and advanced fibrosis were independently associated with traditional risk factors (NAFLD: age >50 years, obesity, hypertension, elevated ALT and low HDL-cholesterol serum concentrations. Advanced fibrosis: IFG/diabetes, elevated ALT serum concentrations). A high prevalence (>60%) of NAFLD was found in obese people, while it varied widely in non-obese people according to the presence of metabolic risk factors. PNPLA3 G variant (OR = 1.33, 95% C.I. = 1.01-1.8; P < .05) was independently associated with NAFLD. Prevalence of advanced fibrosis (high LSM values) ranged from 3.4% (no risk factors) to 60% (presence of all risk factors). TM6SF2 T variant (OR = 3.06, 95% C.I. = 1.08-8.65, P < .05) was independently associated with advanced fibrosis (high LSM values). CONCLUSIONS: In a cohort of a general population, the prevalence of NAFLD was very high, and among NAFLD patients a significant proportion had advanced fibrosis (high LSM values). Apart from traditional risk factors, genetic factors may have a significant role that needs to be further investigated.

Effects of Eradicating Hepatitis C Virus Infection in Patients With Cirrhosis Differ With Stage of Portal Hypertension.

BACKGROUND & AIMS: Clearance of hepatitis C virus (HCV) via antiviral treatment changes the course of liver disease. We evaluated the benefit of sustained virologic response (SVR) in patients with HCV and cirrhosis without (stage 1) and with (stage 2) esophageal varices (EV). METHODS: We performed a prospective cohort study of 444 patients with HCV and compensated cirrhosis (218 with stage 1 and 226 with stage 2 disease) treated with peg-interferon and ribavirin from June 2001 through December 2009 at the University of Palermo, Italy and followed for a median of 7.6 years (range, 1-12.6 years). We used Cox regression analysis to identify variables associated with appearance or progression of EVs, development of hepatocellular carcinoma (HCC), liver decompensation, and overall survival. RESULTS: In the intention-to-treat analysis, 67 patients with stage 1 disease (30.7%) and 41 patients with stage 2 disease (18.1%) achieved an SVR (P = .003). Patients with stage 1 disease and an SVR were less likely to develop EVs than stage 1 patients without an SVR (hazard ratio [HR], 0.23; 95% confidence interval [CI], 0.11-0.48; P < .001). However, SVR did not affect whether patients with stage 2 disease developed further EVs (HR, 1.58; 95% CI, 0.33-1.03; P = .07, by log-rank test). An SVR was associated with lower risk for HCC (HR, 0.25; 95% CI, 0.12-0.55; P < .001). Patients with stage 2 disease, regardless of SVR, were at greater risk than patients with stage 1 disease for liver decompensation (HR, 2.82; 95% CI, 1.73-4.59; P < .001) or death (HR, 1.77; 95% CI, 1.12-2.80; P = .015). A lower proportion of patients with stage 1 disease and an SVR died from HCC (2.9%), compared with those without an SVR (11.9%) (P = .03) or developed liver decompensation (none vs 7.1% without an SVR; P = .009). A lower proportion of patients with stage 2 disease and an SVR died from causes secondary to HCC (2.0%) compared with those without an SVR (18.4%) (P = .003). Death from causes secondary to liver decompensation did not differ significantly between patients with stage 2 disease with or without an SVR (12.1% vs 25.4%; P = .15). CONCLUSIONS: In a prospective study of 444 patients with HCV and compensated cirrhosis, HCV eradication reduced risk for liver decompensation, HCC, and death, regardless of whether the patients had EVs.

The MBOAT7-TMC4 Variant rs641738 Increases Risk of Nonalcoholic Fatty Liver Disease in Individuals of European Descent.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver damage and is characterized by steatosis. Genetic factors increase risk for progressive NAFLD. A genome-wide association study showed that the rs641738 C>T variant in the locus that contains the membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7, also called LPIAT1) and transmembrane channel-like 4 gene (TMC4) increased the risk for cirrhosis in alcohol abusers. We investigated whether the MBOAT7-TMC4 is a susceptibility locus for the development and progression of NAFLD. METHODS: We genotyped rs641738 in DNA collected from 3854 participants from the Dallas Heart Study (a multi-ethnic population-based probability sample of Dallas County residents) and 1149 European individuals from the Liver Biopsy Cross-Sectional Cohort. Clinical and anthropometric data were collected, and biochemical and lipidomics were measured in plasma samples from participants. A total of 2736 participants from the Dallas Heart Study also underwent proton magnetic resonance spectroscopy to measure hepatic triglyceride content. In the Liver Biopsy Cross-Sectional Cohort, a total of 1149 individuals underwent liver biopsy to diagnose liver disease and disease severity. RESULTS: The genotype rs641738 at the MBOAT7-TMC4 locus associated with increased hepatic fat content in the 2 cohorts, and with more severe liver damage and increased risk of fibrosis compared with subjects without the variant. MBOAT7, but not TMC4, was found to be highly expressed in the liver. The MBOAT7 rs641738 T allele was associated with lower protein expression in the liver and changes in plasma phosphatidylinositol species consistent with decreased MBOAT7 function. CONCLUSIONS: We provide evidence for an association between the MBOAT7 rs641738 variant and the development and severity of NAFLD in individuals of European descent. This association seems to be mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling.

MERTK rs4374383 polymorphism affects the severity of fibrosis in non-alcoholic fatty liver disease.

BACKGROUND & AIM: Homozygosity for a common non-coding rs4374383 G>A polymorphism in MERTK (myeloid-epithelial-reproductive tyrosine kinase) has been associated with the protection against fibrosis progression in chronic hepatitis C. The main study objective was to assess whether MERTK AA genotype influences liver fibrosis, and secondarily MERTK expression in patients with non-alcoholic fatty liver disease (NAFLD). We also investigated whether MERTK is expressed in human hepatic stellate cells (HSC) and in murine models of fibrogenesis. METHODS: We considered 533 consecutive patients who underwent liver biopsy for suspected non-alcoholic steatohepatitis (NASH) without severe obesity from two Italian cohorts. As controls, we evaluated 158 patients with normal liver enzymes and without metabolic disturbances. MERTK rs4374383 genotype was assessed by 5'-nuclease assays. MERTK expression was analysed in mouse models of fibrosis, and the effect of the MERTK ligand GAS6 were investigated in human HSC. RESULTS: Clinically significant fibrosis (stage F2-F4) was observed in 19% of patients with MERTK AA compared to 30% in those with MERTK GG/GA (OR 0.43, CI 0.21-0.88, p=0.02; adjusted for centre, and genetic, clinical-metabolic and histological variables). The protective rs4374383 AA genotype was associated with lower MERTK hepatic expression. MERTK was overexpressed in the liver of NAFLD patients with F2-F4 fibrosis and in in vivo models of fibrogenesis. Furthermore, exposure of cultured human HSC to the MERTK ligand GAS6, increased cell migration and induced procollagen expression. These effects were counteracted by inhibition of MERTK activity, which also resulted in apoptotic death of HSC. CONCLUSIONS: The rs4374383 AA genotype, associated with lower intrahepatic expression of MERTK, is protective against F2-F4 fibrosis in patients with NAFLD. The mechanism may involve modulation of HSC activation.

Inhibition of activated STAT5 in Bcr/Abl expressing leukemia cells with new pimozide derivatives.

STATs are transcription factors acting as intracellular signaling after stimulation with cytokines, growth factors and hormones. STAT5 is also constitutively active in many forms of cancers, including chronic myelogenous leukemia, acute lymphoblastic leukemia and Hodgkin's lymphoma. Recently, literature reported that the neuroleptic drug pimozide inhibits STAT5 phosphorylation inducing apoptosis in CML cells. We undertook an investigation from pimozide structure, obtaining simple derivatives with cytotoxic and STAT5-inhibitory activity, two of them markedly more potent than pimozide.

Correction to "Novel Antiproliferative Chimeric Compounds with Marked Histone Deacetylase Inhibitory Activity".

[This corrects the article DOI: 10.1021/ml5000959.].

MAFLD: a multisystem disease.

Nonalcoholic fatty liver disease (NAFLD), affecting about 25% of general population and more than 50% of dysmetabolic patients, is an emerging cause of chronic liver disease and its complications. Recently, an international consensus of experts proposed to rename this disease as 'Metabolic dysfunction-Associated Fatty Liver Disease' (MAFLD) to focus on the bidirectional interplay between fatty liver and metabolic alterations and to stress the need of assessing fatty liver independently from alcohol consumption and other coexisting causes of liver disease. The peculiarity of NAFLD/MAFLD lies in the presence of a higher risk of not only - as expected - liver-related events but also of extrahepatic events, mostly cardiovascular and cancers. Available evidence suggests that these associations are not only the expression of sharing the same risk factors but shed light about the ability of NAFLD/MAFLD and particularly of its progressive form - nonalcoholic/metabolic dysfunction-associated steatohepatitis - to act as an independent risk factor via promotion of atherogenic dyslipidemia and a proinflammatory, profibrogenic, and procoagulant systemic environment. The present review summarizes available epidemiological and clinical evidence supporting the concept of NAFLD/MAFLD as a multisystemic disease, and highlights potential explanatory mechanisms underlying the association between NAFLD/MAFLD and extrahepatic disorders.