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.

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.

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.

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.

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.

Mer Tyrosine Kinase (MERTK) modulates liver fibrosis progression and hepatocellular carcinoma development.

MerTK is a tyrosine kinase receptor that belongs to the TAM (Tyro3/Axl/Mer) receptor family. It is involved in different processes including cellular proliferation/survival, cellular adhesion/migration, and release of the inflammatory/anti-inflammatory cytokines. Although it is reported that MERTK polymorphisms affect the severity of viral and metabolic liver diseases, being able to influence fibrosis progression and hepatocellular carcinoma development, the mechanisms remain unknown. METHODS: using a microarray approach, we evaluated the liver expression of genes involved in fibrogenesis and hepatocarcinogenesis in patient with chronic hepatitis C (CHC), stratified for MERTK genotype and MERTK expression. RESULTS: we found that the rs 4374383 AA homozygosity is associated with lower MERTK expression in CHC patients and that, depending on MERTK genotype, Matrix Metallopeptidase 9 (MMP9), Matrix Metallopeptidase 7 (MMP7), Secreted Frizzled Related Protein 1 (SFRP1) and WNT gene family 11(WNT11) show differential expression in patients with CHC with or without neoplastic progression. CONCLUSIONS: our results confirm that MERTK represents a genetic biomarker for progression of liver disease and are suggestive of translational relevance for the study of downstream pathways involved in fibrogenesis and hepatocarcinogenesis.

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.

A Genetic and Metabolic Staging System for Predicting the Outcome of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is an emerging cause of liver-related events (LREs). Here, we have assessed the ability of a composite score based on clinical features, metabolic comorbidities, and genetic variants to predict LREs. A total of 546 consecutive patients with NAFLD were recruited and stratified according to the fibrosis-4 (FIB-4) index. LREs were defined as occurrence of hepatocellular carcinoma or hepatic decompensation. Cox regression multivariate analysis was used to identify baseline variables associated with LREs. The UK Biobank was used as the validation cohort, and severe liver disease (incidence of cirrhosis, decompensated liver disease, hepatocellular carcinoma, and/or liver transplantation) was used as the outcome. LREs were experienced by 58 patients, only one of whom was in the cohort of patients with a FIB-4 score < 1.3. Multivariate Cox regression analysis of 229 patients with a FIB-4 score ≥ 1.3 highlighted clinical variables independently associated with the development of LREs, including older age, low platelet count, low albumin, low high-density lipoprotein cholesterol, certain genetic factors, and interactions between genetic factors and sex or diabetes. The area under the curve (AUC) for the model was 0.87 at 1, 3, and 5 years. Our novel Genetic and Metabolic Staging (GEMS) scoring system was derived from the Cox model linear predictor, ranked from 0 to 10, and categorized into five classes (0-5, 5-6, 6-7, 7-8, and 8-10). The risk of LREs increased from 4% in patients in the best class (GEMS score 0-5) to 91% in the worst (GEMS score 8-10). GEMS score was associated with incident severe liver disease in the study population (hazard ratio, 1.56; 95% confidence interval, 1.48-1.65; P < 0.001) as well as in the UK Biobank cohort where AUCs for prediction of severe liver disease at 1, 3, and 5 years were 0.70, 0.69, and 0.67, respectively. Conclusion: The novel GEMS scoring system has an adequate ability to predict the outcome of patients with NAFLD.

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.

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.

Effects of Pimozide Derivatives on pSTAT5 in K562 Cells.

STAT5 is a transcription factor, a member of the STAT family of signaling proteins. STAT5 is involved in many types of cancer, including chronic myelogenous leukemia (CML), in which this protein is found constitutively activated as a consequence of BCR-ABL expression. The neuroleptic drug pimozide was recently reported to act as an inhibitor of STAT5 phosphorylation and is capable of inducing apoptosis in CML cells in vitro. Our research group has synthesized simple derivatives of pimozide with cytotoxic activity and that are able to decrease the levels of phosphorylated STAT5. In this work we continued the search for novel STAT5 inhibitors, synthesizing compounds in which the benzoimidazolinone ring of pimozide is either maintained or modified, in order to obtain further structure-activity relationship information for this class of STAT5 inhibitors. Two compounds of the series showed potent cytotoxic activity against BCR-ABL-positive and pSTAT5-overexpressing K562 cells and were able to markedly decrease the levels of phosphorylated STAT5.

Fibronectin Type III Domain-Containing Protein 5 rs3480 A>G Polymorphism, Irisin, and Liver Fibrosis in Patients With Nonalcoholic Fatty Liver Disease.

Context: Contrasting data have been reported on the role of irisin, a novel myokine encoded by the fibronectin type III domain-containing protein 5 (FNDC5) gene, in nonalcoholic fatty liver disease (NAFLD) pathogenesis. We tested in patients with suspected nonalcoholic steatohepatitis (NASH) the association of FNDC5 variants, hepatic expression, and circulating irisin with liver damage (F2 to F4 fibrosis as main outcome). We also investigated whether irisin modulates hepatocellular fat accumulation and stellate cell activation in experimental models. Methods: We considered 593 consecutive patients who underwent liver biopsy for suspected NASH and 192 patients with normal liver enzymes and without steatosis. FNDC5 rs3480 and rs726344 genotypes were assessed by 5' nuclease assays. Hepatic irisin expression was evaluated in mice fed a high-fat diet or treated with CCl4. The effect of irisin was evaluated in fat-laden HepG2 hepatocytes and in hepatic stellate cells (HSCs). Results: In patients at risk for NASH [odds ratio (OR) = 0.64, 95% confidence interval (CI), 0.47 to 0.87; P = 0.005], and more so in the high-risk subgroup of those with impaired fasting glucose/diabetes (OR = 0.44, 95% CI, 0.26 to 0.74; P = 0.002), the rs3480 A>G variant was independently associated with protection from F2 to F4 fibrosis. Irisin is expressed in human activated HSC, where it mediated fibrogenic actions and collagen synthesis, and is overexpressed in NAFLD patients with F2 to F4 fibrosis and CCl4-treated mice. However, Irisin does not affect fat accumulation in HepG2 and is not induced by high-fat-diet-inducing NAFLD. Conclusions: The FNDC5 rs3480 variant is associated with protection from clinically significant fibrosis in patients with NAFLD, while irisin expression is correlated with the severity of NAFLD and may be involved in extracellular matrix deposition. These data suggest that irisin is involved in regulation of hepatic fibrogenesis.

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.

Novel iodoacetamido benzoheterocyclic derivatives with potent antileukemic activity are inhibitors of STAT5 phosphorylation.

Signal Transducer and Activator of Transcription 5 (STAT5) protein, a component of the STAT family of signaling proteins, is considered to be an attractive therapeutic target because of its involvement in the progression of acute myeloid leukemia. In an effort to discover potent molecules able to inhibit the phosphorylation-activation of STAT5, twenty-two compounds were synthesized and evaluated on the basis of our knowledge of the activity of 2-(3',4',5'-trimethoxybenzoyl)-3-iodoacetamido-6-methoxy benzo[b]furan derivative 1 as a potent STAT5 inhibitor. Most of these molecules, structurally related to compound 1, were characterized by the presence of a common 3',4',5'-trimethoxybenzoyl moiety at the 2-position of different benzoheterocycles such as benzo[b]furan, benzo[b]thiophene, indole and N-methylindole. Effects on biological activity of the iodoacetamido group and of different moieties (methyl and methoxy) at the C-3 to C-7 positions were examined. In the series of benzo[b]furan derivatives, moving the iodoacetylamino group from the C-4 to the C-5 or C-6 positions did not significantly affect antiproliferative activity. Compounds 4, 15, 20 and 23 blocked STAT5 signals and induced apoptosis of K562 BCR-ABL positive cells. For compound 23, the trimethoxybenzoyl moiety at the 2-position of the benzo[b]furan core was not essential for potent inhibition of STAT5 activation.

Novel antiproliferative chimeric compounds with marked histone deacetylase inhibitory activity.

Given our interest in finding potential antitumor agents and in view of the multifactorial mechanistic nature of cancer, in the present work, taking advantage of the multifunctional ligands approach, new chimeric molecules were designed and synthesized by combining in single chemical entities structural features of SAHA, targeting histone deacetylases (HDACs), with substituted stilbene or terphenyl derivatives previously obtained by us and endowed with antiproliferative and pro-apoptotic activity. The new chimeric derivatives were characterized with respect to their cytotoxic activity and their effects on cell cycle progression on different tumor cell lines, as well as their HDACs inhibition. Among the other, trans -6 showed the most interesting biological profile, as it exhibited a strong pro-apoptotic activity in tumor cell lines in comparison with both of its parent compounds and a marked HDAC inhibition.

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.

Glucokinase regulatory protein gene polymorphism affects liver fibrosis in non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Variant in glucokinase regulatory protein (GCKR), associated with lipid and glucose traits, has been suggested to affect fatty liver infiltration. We aimed to assess whether GCKR rs780094 C→T SNP influences the expression of steatosis, lobular inflammation and fibrosis in NAFLD patients, after correction for PNPLA3 genotype. METHODS: In 366 consecutive NAFLD patients (197 from Sicily, and 169 from center/northern Italy), we assessed anthropometric, biochemical and metabolic features; liver biopsy was scored according to Kleiner. PNPLA3 rs738409 C>G and GCKR rs780094 C>T single nucleotide polymorphisms were also assessed. RESULTS: At multivariate logistic regression analysis in the entire NAFLD cohort, the presence of significant liver fibrosis (>F1) was independently linked to high HOMA (OR 1.12, 95% CI 1.01-1.23, p = 0.02), NAFLD activity score ≥ 5 (OR 4.09, 95% CI 2.45-6.81, p<0.001), and GCKR C>T SNP (OR 2.06, 95% CI 1.43-2.98, p<0.001). Similar results were observed considering separately the two different NAFLD cohorts. GCKR C>T SNP was also associated with higher serum triglycerides (ANOVA, p = 0.02) in the entire cohort. CONCLUSIONS: In patients with NAFLD, GCKR rs780094 C>T is associated with the severity of liver fibrosis and with higher serum triglyceride levels.

PNPLA3 GG genotype and carotid atherosclerosis in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIM: To evaluate if the presence of carotid atherosclerosis in patients with NAFLD, could be related to gene variants influencing hepatic fat accumulation and the severity of liver damage. METHODS: We recorded anthropometric, metabolic and histological data(Kleiner score) of 162 consecutive, biopsy-proven Sicilian NAFLD patients. Intima-media thickness(IMT), IMT thickening(IMT≥1 mm) and carotid plaques(focal thickening of >1.3 mm at the level of common carotid artery) were evaluated using ultrasonography. IL28B rs12979860 C>T, PNPLA3 rs738409 C>G, GCKR rs780094 C>T, LYPLAL1 rs12137855 C>T, and NCAN rs2228603 C>T single nucleotide polymorphisms were also assessed. The results were validated in a cohort of 267 subjects with clinical or histological diagnosis of NAFLD from Northern Italy, 63 of whom had follow-up examinations. RESULTS: Carotid plaques, IMT thickening and mean maximum IMT were similar in the two cohorts, whereas the prevalence of diabetes, obesity, NASH, and PNPLA3 GG polymorphism(21%vs.13%, p = 0.02) were significantly higher in the Sicilian cohort. In this cohort, the prevalence of carotid plaques and IMT thickening was higher in PNPLA3 GG compared to CC/CG genotype(53%vs.32%, p = 0.02; 62%vs.28%, p<0.001, respectively). These associations were confirmed at multivariate analyses (OR2.94;95%C.I. 1.12-7.71, p = 0.02, and OR4.11;95%C.I. 1.69-9.96, p = 0.002, respectively), although have been observed only in patients <50years. Also in the validation cohort, PNPLA3 GG genotype was independently associated with IMT thickening in younger patients only (OR: 6.00,95%C.I. 1.36-29, p = 0.01), and to IMT progression (p = 0.05) in patients with follow-up examinations. CONCLUSION: PNPLA3 GG genotype is associated with higher severity of carotid atherosclerosis in younger patients with NAFLD. Mechanisms underlying this association, and its clinical relevance need further investigations.