Vascular damage in patients with nonalcoholic fatty liver disease : Possible role of iron and ferritin

Non Alcoholic Fatty Liver Disease (NAFLD) is the most common chronic liver disease in Western countries. Recent data indicated that NAFLD is a risk factor by itself contributing to the development of cardiovascular disease independently of classical known risk factors. Hyperferritinemia and mild increased iron stores are frequently observed in patients with NAFLD and several mechanisms have been proposed to explain the role of iron, through oxidative stress and interaction with insulin metabolism, in the development of vascular damage. Moreover, iron depletion has been shown to decrease atherogenesis in experimental models and in humans. This review presents the recent evidence on epidemiology, pathogenesis, and the possible explanation of the role of iron and ferritin in the development of cardiovascular damage in patients with NAFLD, and discusses the possible interplay between metabolic disorders associated with NAFLD and iron in the development of cardiovascular disease

Mir-101-3p downregulation promotes fibrogenesis by facilitating hepatic stellate cell transdifferentiation during insulin resistance

Insulin resistance (IR) and microRNAs (miRNAs), which regulate cell-to-cell communication between hepatocytes and hepatic stellate cells (HSCs), may intertwine in nonalcoholic fatty liver disease (NAFLD) pathogenesis. The aim of this study was to evaluate whether epigenetics and environmental factors interact to promote progressive NAFLD during IR. We examined the miRNA signature in insulin receptor haploinsufficient (InsR+/ 12) and wild-type (wt) HSCs by RNAseq (n = 4 per group). Then, we evaluated their impact in an IR-NASH (nonalcoholic steatohepatitis) model (InsR+/ 12 mice fed standard or methionine choline deficient (MCD) diet, n = 10 per group) and in vitro. InsR+/ 12 HSCs displayed 36 differentially expressed miRNAs (p < 0.05 vs. wt), whose expression was then analyzed in the liver of InsR+/ 12 mice fed an MCD diet. We found that miR-101-3p negatively associated with both InsR+/ 12 genotype and MCD (p < 0.05) and the histological spectrum of liver damage (p < 0.01). miR-101-3p was reduced in InsR+/ 12 hepatocytes and HSCs and even more in InsR+/ 12 cells exposed to insulin (0.33 \ub5M) and fatty acids (0.25 mM), resembling the IR-NASH model. Conversely, insulin induced miR-101-3p expression in wt cells but not in InsR+/ 12 ones (p < 0.05). In conclusion, IR combined with diet-induced liver injury favors miR-101-3p downregulation, which may promote progressive NAFLD through HSC and hepatocyte transdifferentiation and proliferation

MiRNA signature in NAFLD: A turning point for a non-invasive diagnosis

Nonalcoholic fatty liver disease (NAFLD) defines a wide pathological spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) which may predispose to liver cirrhosis and hepatocellular carcinoma. It represents the leading cause of hepatic damage worldwide. Diagnosis of NASH still requires liver biopsy but due to the high prevalence of NAFLD, this procedure, which is invasive, is not practicable for mass screening. Thus, it is crucial to non-invasively identify NAFLD patients at higher risk of progression to NASH and fibrosis. It has been demonstrated that hepatic fat content and progressive liver damage have a strong heritable component. Therefore, genetic variants associated with NAFLD have been proposed as non-invasive markers to be used in clinical practice. However, genetic variability is not completely explained by these common variants and it is possible that many of the phenotypic differences result from gene-environment interactions. Indeed, NAFLD development and progression is also modulated by epigenetic factors, in particular microRNAs (miRNAs), which control at post-transcriptional level many complementary target mRNAs and whose dysregulation has been shown to have high prognostic and predictive value in NAFLD. The premise of the current review is to discuss the role of miRNAs as pathogenic factors, risk predictors and therapeutic targets in NAFLD

Cardiovascular risk after orthotopic liver transplantation, a review of the literature and preliminary results of a prospective study

Improved surgical techniques and greater efficacy of new anti-rejection drugs have significantly improved the survival of patients undergoing orthotopic liver transplantation (OLT). This has led to an increased incidence of metabolic disorders as well as cardiovascular and cerebrovascular diseases as causes of morbidity and mortality in OLT patients. In the last decade, several studies have examined which predisposing factors lead to increased cardiovascular risk (i.e., age, ethnicity, diabetes, NASH, atrial fibrillation, and some echocardiographic parameters) as well as which factors after OLT (i.e., weight gain, metabolic syndrome, immunosuppressive therapy, and renal failure) are linked to increased cardiovascular mortality. However, currently, there are no available data that evaluate the development of atherosclerotic damage after OLT. The awareness of high cardiovascular risk after OLT has not only lead to the definition of new but generally not accepted screening of high risk patients before transplantation, but also to the need for careful patient follow up and treatment to control metabolic and cardiovascular pathologies after transplant. Prospective studies are needed to better define the predisposing factors for recurrence and de novo occurrence of metabolic alterations responsible for cardiovascular damage after OLT. Moreover, such studies will help to identify the timing of disease progression and damage, which in turn may help to prevent morbidity and mortality for cardiovascular diseases. Our preliminary results show early occurrence of atherosclerotic damage, which is already present a few weeks following OLT, suggesting that specific, patient-tailored therapies should be started immediately post OLT

The A736V TMPRSS6 Polymorphism Influences Hepatic Iron Overload in Nonalcoholic Fatty Liver Disease

AIMS: Hepatic iron accumulation due to altered trafficking is frequent in patients with nonalcoholic fatty liver disease (NAFLD), and is associated with more severe liver damage and hepatocellular carcinoma. The p.Ala736Val TMPRSS6 variant influences iron metabolism regulating the transcription of the hepatic hormone hepcidin, but its role in the pathogenesis of iron overload disorders is controversial. Aim of this study was to evaluate the whether the TMPRSS6 p.Ala736Val variant influences hepatic iron accumulation in a well-characterized series of Italian patients with histological NAFLD. METHODS: 216 patients with histological NAFLD. TMPRSS6 and HFE variants were assessed by allele specific PCR, liver histology by the NAFLD activity score and Perls' staining for iron. RESULTS: Homozygosity for the p.736Val allele previously linked to higher hepcidin did not influence transferrin saturation (TS), but was associated with lower hepatic iron stores (p\u200a=\u200a0.01), and ferritin levels (median 223 IQR 102-449 vs. 308 IQR 141-618 ng/ml; p\u200a=\u200a0.01). Homozygosity for TMPRSS6 p.736Val was nearly associated with lower ballooning (p\u200a=\u200a0.05), reflecting hepatocellular damage related to oxidative stress. The influence of TMPRSS6 on hepatic iron accumulation was more marked in patients negative for HFE genotypes predisposing to iron overload (p.Cys282Tyr + and p.His63Asp +/+; p\u200a=\u200a0.01), and the p.736Val variant was negatively associated with hepatic iron accumulation independently of age, gender, HFE genotype, and beta-thalassemia trait (OR 0.59, 0.39-0.88). CONCLUSIONS: The p.Ala736Val TMPRSS6 variant influences secondary hepatic iron accumulation in patients with NAFLD

Patatin-like phospholipase domain containing-3 gene I148M polymorphism, steatosis, and liver damage in hereditary hemochromatosis

AIM: To investigate whether the patatin-like phospholipase domain containing-3 gene (PNPLA3) I148M polymorphism is associated with steatosis, fibrosis stage, and cirrhosis in hereditary hemochromatosis (HH). METHODS: We studied 174 consecutive unrelated homozygous for the C282Y HFE mutation of HH (C282Y+/+ HH) patients from Northern Italy, for whom the presence of cirrhosis could be determined based on histological or clinical criteria, without excessive alcohol intake (< 30/20 g/d in males or females) or hepatitis B virus and hepatitis C virus viral hepatitis. Steatosis was evaluated in 123 patients by histology (n = 100) or ultrasound (n = 23). The PNPLA3 rs738409 single nucleotide polymorphism, encoding for the p.148M protein variant, was genotyped by a Taqman assay (assay on demand, Applied Biosystems). The association of the PNPLA3 I148M protein variant (p.I148M) with steatosis, fibrosis stage, and cirrhosis was evaluated by logistic regression analysis. RESULTS: PNPLA3 genotype was not associated with metabolic parameters, including body mass index (BMI), the presence of diabetes, and lipid levels, but the presence of the p.148M variant at risk was independently associated with steatosis [odds ratio (OR) 1.84 per p.148M allele, 95% confidence interval (CI): 1.05-3.31; P = 0.037], independently of BMI and alanine aminotransaminase (ALT) levels. The p.148M variant was also associated with higher aspartate aminotransferase (P = 0.0014) and ALT levels (P = 0.017) at diagnosis, independently of BMI and the severity of iron overload. In patients with liver biopsy, the 148M variant was independently associated with the severity (stage) of fibrosis (estimated coefficient 0.56 \ub1 0.27, P = 0.041). In the overall series of patients, the p.148M variant was associated with cirrhosis in lean (P = 0.049), but not in overweight patients (P = not significant). At logistic regression analysis, cirrhosis was associated with BMI 65 25 (OR 1.82, 95% CI: 1.02-3.55), ferritin > 1000 ng/mL at diagnosis (OR 19.3, 95% CI: 5.3-125), and with the G allele in patients with BMI < 25 (OR 3.26, 95% CI: 1.3-10.3). CONCLUSION: The PNPLA3 I148M polymorphism may represent a permissive factor for fibrosis progression in patients with C282Y+/+ HH

The i148m Pnpla3 polymorphism influences serum adiponectin in patients with fatty liver and healthy controls

BACKGROUND: Reduced adiponectin is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), and the I148M Patatin-like phospholipase domain-containing 3 (PNPLA3) polymorphism predisposes to NAFLD and liver damage progression in NASH and chronic hepatitis C (CHC) by still undefined mechanisms, possibly involving regulation of adipose tissue function. Aim of this study was to evaluate whether the I148M PNPLA3 polymorphism influences serum adiponectin in liver diseases and healthy controls. METHODS: To this end, we considered 144 consecutive Italian patients with NAFLD, 261 with CHC, 35 severely obese subjects, and 257 healthy controls with very low probability of steatosis, all with complete clinical and genetic characterization, including adiponectin (ADIPOQ) genotype. PNPLA3 rs738409 (I148M) and ADIPOQ genotypes were evaluated by Taqman assays, serum adiponectin by ELISA. Adiponectin mRNA levels were evaluated by quantitative real-time PCR in the visceral adipose tissue (VAT) of 35 obese subjects undergoing bariatric surgery. RESULTS: Adiponectin levels were independently associated with the risk of NAFLD and with the histological severity of the disease. Adiponectin levels decreased with the number of 148\u2009M PNPLA3 alleles at risk of NASH both in patients with NAFLD (p\u2009=\u20090.03), and in healthy subjects (p\u2009=\u20090.04). At multivariate analysis, PNPLA3 148\u2009M alleles were associated with low adiponectin levels (<6\u2009mg/ml, median value) independently of NAFLD diagnosis, age, gender, BMI, and ADIPOQ genotype (OR 1.67, 95% c.i. 1.07-2.1 for each 148\u2009M allele). The p.148\u2009M PNPLA3 variant was associated with decreased adiponectin mRNA levels in the VAT of obese patients (p\u2009<\u20090.05) even in the absence of NASH. In contrast, in CHC, characterized by adiponectin resistance, low adiponectin was associated with male gender and steatosis, but not with PNPLA3 and ADIPOQ genotypes and viral features. CONCLUSIONS: The I148M PNPLA3 variant is associated with adiponectin levels in patients with NAFLD and in healthy subjects, but in the presence of adiponectin resistance not in CHC patients. The I148M PNPLA3 genotype may represent a genetic determinant of serum adiponectin levels. Modulation of serum adiponectin might be involved in mediating the susceptibility to steatosis, NASH, and hepatocellular carcinoma in carriers of the 148\u2009M PNPLA3 variant without CHC, with potential therapeutic implications

Risk of obstructive sleep apnea with daytime sleepiness is associated with liver damage in non-morbidly obese patients with nonalcoholic Fatty liver disease

BACKGROUND: A high prevalence of obstructive sleep apnea syndrome (OSAS) has been reported in severely obese patients with nonalcoholic fatty liver disease (NAFLD), but few studies have evaluated OSAS in non-morbidly obese NAFLD patients. AIMS: To determine the prevalence of risk for OSAS with or without daytime sleepiness in non-morbidly obese patients with NAFLD and evaluate the association with the severity of liver damage. METHODS: We considered 159 consecutive patients with histological NAFLD and body mass index (BMI) <35 Kg/m2, and 80 controls without ultrasonographic steatosis matched for age, sex, and BMI. OSAS risk was determined by positivity for Berlin questionnaire (BQ), and daytime sleepiness by the Sleepness Epworth Scale (ESS). Liver damage was evaluated according to the NAFLD activity score. RESULTS: In NAFLD patients, BQ alone was positive in 39 (25%), ESS in 8 (5%), and both in 13 (8%, OSAS with sleepines); p\u200a=\u200ans vs. controls without steatosis. In NAFLD patients at risk for OSAS with (but not in those without) sleepiness, we observed a higher prevalence of nonalcoholic steatohepatitis (NASH; 11/13, 85% vs. 72/146, 49%; p\u200a=\u200a0.018), and of clinically significant fibrosis (stage>1; 9/13, 69% vs. 39/146, 27%; p\u200a=\u200a0.003). At multivariate logistic regression analysis, OSAS with sleepiness was strongly associated with NASH and fibrosis>1 independently of known clinical risk factors such as age, gender, BMI, diabetes, and ALT levels (OR 7.1, 95% c.i. 1.7-51, p\u200a=\u200a0.005 and OR 14.0, 95% c.i. 3.5-70, p\u200a=\u200a0.0002, respectively). CONCLUSIONS: A proportion of NAFLD patients without severe obesity is at risk for OSAS with daytime sleepiness, which is associated with the severity of liver damage independently of body mass and other cofactors

CYBRD1 as a modifier gene that modulates iron phenotype in HFE p.C282Y homozygous patients

Background. Most patients with Hereditary Hemochromatosis are homozygous for the p.C282Y mutation in the HFE gene in Caucasian population. Penetrance and expression of Hemochromatosis largely differ in p.C282Y homozygous cases. Besides environmental factors, genetic factors might be implicated. Design and Methods. In the present study, we analysed 50 candidate genes involved in iron metabolism and evaluated the association between 214 single nucleotide polymorphisms in these genes and three phenotypic outcomes of iron overload (serum ferritin, iron removed and transferrin saturation) in a large group of 296 Italian p.C282Y homozygous cases. Polymorphisms were tested for genetic association with each single outcome using linear regression models adjusted for age, sex and alcohol consumption. Results. We found a series of 17 genetic variants located in different genes with possible additive effect on the studied outcomes. In order to evaluate if the selected polymorphisms could provide a predictive signature for adverse phenotype, we re-evaluated data by dividing patients in two extreme phenotype classes based on the three phenotypic outcomes. We found that only a small improvement in prediction can be achieved adding genetic information to clinical data. Among the selected polymorphisms, a significant association between rs3806562, located in the 5\u2019UTR of CYBRD1, and transferrin saturation was observed. This variant belongs to the same haplotype block which contains the CYBRD1 polymorphism rs884409, found to be associated with serum ferritin in another population of p.C282Y homozygotes, and able to modulate promoter activity. Luciferase assay indicates that rs3806562 has not a significant functional role, suggesting that it is a genetic marker linked to the putative genetic modifier rs884409. Conclusions. While our results support the hypothesis that polymorphisms in genes regulating iron metabolism may modulate penetrance of HFE-HH, with emphasis on CYBRD1, they strengthen the notion that none of these polymorphisms alone is a major modifier of HH phenotype