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Heterogeneity of non-alcoholic fatty liver disease (NAFLD): Implication for cardiovascular risk stratification

      Highlights

      • Non-alcoholic fatty liver disease (NAFLD) associates with increased risk of clinical and subclinical cardiovascular disease (CVD).
      • NAFLD encompasses multiple phenotypes with different clinical outcomes.
      • Subjects with genetic NAFLD and without metabolic disturbances do not have increased CVD risk.
      • To recognize metabolic from genetic factors is fundamental to disentangle the relationship between NAFLD and atherosclerosis.

      Abstract

      NAFLD is currently considered the most common liver disease worldwide and mounting data support its strong link with atherosclerotic cardiovascular disease (ASCVD). This association is important as cardiovascular disease (CVD) is generally recognized as the leading cause of death in individuals with NAFLD. However, NAFLD represents a heterogeneous condition showing a wide spectrum of clinical and pathophysiological sub-phenotypes with different adverse outcomes ranging from ASCVD to liver damage progression.
      The contribution to NAFLD pathogenesis of different environmental, metabolic, and genetic factors underlies this heterogeneity. The more frequent phenotype of NAFLD patients is associated with metabolic dysfunctions such as obesity and insulin-resistant syndrome and this has been recently named as Metabolic Associated Fatty Liver disease (MAFLD). However, NAFLD is encountered also in subjects without insulin resistance and metabolic alterations and in whom genetic factors play a major role. It has been suggested that these individuals are at risk of liver disease progression but not of cardiovascular complications. Separating metabolic from genetic factors could be useful in disentangling the intricate relationship between NAFLD and atherosclerosis.
      In the present review, we aim to address the epidemic of NAFLD, its epidemiologically association with ASCVD complications and the overall mechanisms involved in the pathophysiology of atherosclerotic vascular damage in NAFLD patients. Finally, we will revise the potential role of genetics in identifying disease subtyping and predicting individualised CVD risk.

      Graphical abstract

      Keywords

      1. Introduction

      Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease and affects 17%–51% of the adult population worldwide [
      • Angulo P.
      Nonalcoholic fatty liver disease.
      ,
      • Rinella M.E.
      Nonalcoholic fatty liver disease: a systematic review.
      ,
      • Kim Y.S.
      • Jung E.S.
      • Hur W.
      • et al.
      Noninvasive predictors of nonalcoholic steatohepatitis in Korean patients with histologically proven nonalcoholic fatty liver disease.
      ]. Its prevalence is estimated to continuously increase in parallel to the growth of obesity and diabetes incidence [
      • Williams C.D.
      • Stengel J.
      • Asike M.I.
      • et al.
      Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study.
      ]. In 2016, 85.3 million people were estimated to have NAFLD in the United States, and this number is expected to reach 100.9 million in 2030. In parallel with the increase of NAFLD prevalence, its liver complications are estimated to increase by 63% for non-alcoholic steatohepatitis (NASH), 168% for decompensated cirrhosis and 137% for hepatocarcinoma (HCC) [
      • Estes C.
      • Anstee Q.M.
      • Arias-Loste M.T.
      • et al.
      Modeling NAFLD disease burden in China, France, Germany, Italy, Japan, Spain, United Kingdom, and United States for the period 2016-2030.
      ]. Finally, to date NAFLD is the second most common cause for liver transplantation, and it will probably become the first one soon [
      • Younossi Z.M.
      • Stepanova M.
      • Ong J.
      • et al.
      Nonalcoholic steatohepatitis is the most rapidly increasing indication for liver transplantation in the United States.
      ].
      Overall, atherosclerotic-related cardiovascular complications are the leading cause of morbidity and mortality in NAFLD patients being 6-fold more frequent than those related to liver causes (incident rate of 4.79 vs. 0.77 per 1000 person-years) [
      • Rinella M.E.
      Nonalcoholic fatty liver disease: a systematic review.
      ,
      • Younossi Z.M.
      • Koenig A.B.
      • Abdelatif D.
      • Fazel Y.
      • Henry L.
      • Wymer M.
      Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes.
      ,
      • Ong J.P.
      • Pitts A.
      • Younossi Z.M.
      Increased overall mortality and liver-related mortality in non-alcoholic fatty liver disease.
      ]. Moreover the annual incidence of ASCVD in NAFLD patients is around 1.1% [
      • Targher G.
      • Byrne C.D.
      • Lonardo A.
      • Zoppini G.
      • Barbui C.
      Non-alcoholic fatty liver disease and risk of incident cardiovascular disease: a meta-analysis.
      ] and considering the spread of the disease, the absolute number of these events is destined to grow significantly [
      • Del Ben M.
      • Baratta F.
      • Pastori D.
      • Angelico F.
      The challenge of cardiovascular prevention in NAFLD.
      ].
      Many studies have been published on the association between NAFLD and CVD risk and their results have been summarised in several meta-analyses, as shown in Table 1. In comparison to patients without NAFLD, those with NAFLD have an increased risk to develop acute myocardial infarction by 65%, stroke by 55% and CKD by 53% [
      • Cusi K.
      • Isaacs S.
      • Barb D.
      • et al.
      American association of clinical endocrinology clinical practice guideline for the diagnosis and management of nonalcoholic fatty liver disease in primary care and endocrinology clinical settings.
      ]. Not least, up to 80% of non-diabetic NAFLD patients will develop impairing fasting glucose or diabetes during their life [
      • Targher G.
      • Byrne C.D.
      Nonalcoholic fatty liver disease: a novel cardiometabolic risk factor for type 2 diabetes and its complications.
      ]. Notably, these associations were independent from the standard cardiovascular risk factors, such as age, gender, body mass index (BMI), waist circumference, smoking status, hypertension, or dyslipidemia [
      • Targher G.
      • Byrne C.D.
      • Lonardo A.
      • Zoppini G.
      • Barbui C.
      Non-alcoholic fatty liver disease and risk of incident cardiovascular disease: a meta-analysis.
      ,
      • Mantovani A.
      • Csermely A.
      • Petracca G.
      • et al.
      Non-alcoholic fatty liver disease and risk of fatal and non-fatal cardiovascular events: an updated systematic review and meta-analysis.
      ,
      • Wu S.
      • Wu F.
      • Ding Y.
      • Hou J.
      • Bi J.
      • Zhang Z.
      Association of non-alcoholic fatty liver disease with major adverse cardiovascular events: a systematic review and meta-analysis.
      ,
      • Zhou Y.Y.
      • Zhou X.D.
      • Wu S.J.
      • et al.
      Synergistic increase in cardiovascular risk in diabetes mellitus with nonalcoholic fatty liver disease: a meta-analysis.
      ]. Briefly, three meta-analyses including both diabetic and nondiabetic individuals indicated that NAFLD increases the relative risk of CVD, by 1.64 (95% CI: 1.26–2.13), 1.88 (95% CI: 1.68–2.01), and 1.81 (95% CI: 1.23–2.66), respectively [
      • Targher G.
      • Byrne C.D.
      • Lonardo A.
      • Zoppini G.
      • Barbui C.
      Non-alcoholic fatty liver disease and risk of incident cardiovascular disease: a meta-analysis.
      ,
      • Mantovani A.
      • Csermely A.
      • Petracca G.
      • et al.
      Non-alcoholic fatty liver disease and risk of fatal and non-fatal cardiovascular events: an updated systematic review and meta-analysis.
      ,
      • Wu S.
      • Wu F.
      • Ding Y.
      • Hou J.
      • Bi J.
      • Zhang Z.
      Association of non-alcoholic fatty liver disease with major adverse cardiovascular events: a systematic review and meta-analysis.
      ]. Zhou et al. also explored the correlation between NAFLD and CVD among adults with diabetes [
      • Zhou Y.Y.
      • Zhou X.D.
      • Wu S.J.
      • et al.
      Synergistic increase in cardiovascular risk in diabetes mellitus with nonalcoholic fatty liver disease: a meta-analysis.
      ]. Based on 11 observational studies, authors found that diabetic patients with NAFLD showed a two-fold increased risk for CVD compared with the non-NAFLD group concluding that NAFLD and type 2 diabetes (T2DM) might have a synergistic effect on CVD risk [
      • Zhou Y.Y.
      • Zhou X.D.
      • Wu S.J.
      • et al.
      Synergistic increase in cardiovascular risk in diabetes mellitus with nonalcoholic fatty liver disease: a meta-analysis.
      ]. Several lines of evidence also linked NAFLD with subclinical markers of atherosclerosis such as increased carotid artery intima-media thickness (IMT), arterial stiffness, coronary calcification, endothelial dysfunction, and impaired left ventricular diastolic function [
      • Zhou Y.Y.
      • Zhou X.D.
      • Wu S.J.
      • et al.
      Synergistic increase in cardiovascular risk in diabetes mellitus with nonalcoholic fatty liver disease: a meta-analysis.
      ]. In a second meta-analysis, Zhou et al. [
      • Zhou Y.Y.
      • Zhou X.D.
      • Wu S.J.
      • et al.
      Nonalcoholic fatty liver disease contributes to subclinical atherosclerosis: a systematic review and meta-analysis.
      ] found that NAFLD associated with increased IMT/plaques [OR 1.74; 95%CI (1.47–2.06)], increased arterial stiffness [OR 1.56; 95%CI (1.24–1.96)], coronary artery calcification [OR 1.40; 95%CI (1.22–1.60)] and endothelial cell dysfunction [OR 3.73; 95%CI (0.99–14.1)]. However, these results must be interpreted with caution due to the heterogeneity of the populations included in these meta-analyses and the observational nature of the studies, which fail to identify any causal or temporal relationships.
      Table 1Metanalyses on the association between NAFLD and CVD risk.
      ReferenceCharacteristicsOutcomeOR* or HR°
      [
      • Targher G.
      • Byrne C.D.
      • Lonardo A.
      • Zoppini G.
      • Barbui C.
      Non-alcoholic fatty liver disease and risk of incident cardiovascular disease: a meta-analysis.
      ]
      16 longitudinal studies

      34,000 patients followed-up

      7 years
      Fatal/Non-fatal CV event1.64* [1.26–2.13]
      CV mortality for advanced liver disease3.28* [2.26–4.77]
      [
      • Wu S.
      • Wu F.
      • Ding Y.
      • Hou J.
      • Bi J.
      • Zhang Z.
      Association of non-alcoholic fatty liver disease with major adverse cardiovascular events: a systematic review and meta-analysis.
      ]
      13 prospective studies

      21 cross-sectional

      164,494 patients
      CV mortality1.10° [0.86–1.4]
      CV incidence1.37° [1.10–1.72]
      CAD incidence2.31° [1.46–3.65]
      [
      • Zhou Y.Y.
      • Zhou X.D.
      • Wu S.J.
      • et al.
      Synergistic increase in cardiovascular risk in diabetes mellitus with nonalcoholic fatty liver disease: a meta-analysis.
      ]
      11 studies

      8,342 NAFLD individuals
      CVD risk T2DM vs NGT NAFLD2.20* [1.67–2.90]
      Increased IMT1.74* [1.47–2.06]
      Increased arterial stiffness1.56* [1.24–1.96]
      Increased coronary artery calcification1.40* [1.22–1.60]
      Endothelial dysfunction3.73* [0.99–14.09]
      [
      • Mantovani A.
      • Csermely A.
      • Petracca G.
      • et al.
      Non-alcoholic fatty liver disease and risk of fatal and non-fatal cardiovascular events: an updated systematic review and meta-analysis.
      ]
      36 longitudinal studies

      5,802,226 individuals
      Fatal or non-fatal CV events1·45° [1·31–1·61]
      CV events in NAFLD at fibrosis stage2·50° [1·68–3·72]
      Indeed, despite the large amount of evidence linking NAFLD to ASCVD, there are several open issues on this topic. It is still debated whether this association derives from the overlap of common risk factors or aetiologies or if hepatic steatosis independently contributes to an increased atherosclerotic risk. Moreover, it is still highly unpredictable which NAFLD patient will develop ASCVD or severe liver complications. Interest is growing in identifying patients’ characteristics able to predict the different lines of disease progression.
      In the present review, we aim to provide evidence supporting the existence of different forms of NAFLD sub-phenotypes and the need to separate the metabolic from the genetic disease determinants to disentangle the intricate relationship between NAFLD and ASCVD.

      2. NAFLD: a spectrum of pathophysiological conditions

      NAFLD is a heterogeneous and complex disease, whose natural course may include cardiovascular, metabolic, neoplastic or liver-related complications. Both metabolic and genetic factors influence this heterogeneity [
      • Arrese M.
      • Arab J.P.
      • Barrera F.
      • Kaufmann B.
      • Valenti L.
      • Feldstein A.E.
      Insights into nonalcoholic fatty-liver disease heterogeneity.
      ]. The structural dissection of the differential pathways involved shall help us in identifying precise disease subtyping to predict individualised CVD risk.

      2.1 The intricate relationship between NAFLD and cardiometabolic disorders

      The most common NAFLD phenotype is associated with metabolic dysfunction. Recently, a consensus of international experts in fatty liver disease have agreed that the NAFLD term does not reflect the knowledge on the heterogeneity of the pathophysiological mechanisms associated with the disease. Metabolic-associated fatty liver disease (MAFLD), has been suggested as a more proper term [
      • Eslam M.
      • Newsome P.N.
      • Sarin S.K.
      • et al.
      A new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement.
      ] to emphasise the role of metabolic alterations on the pathogenesis of fatty liver disease. Based on this consensus, the major metabolic associated disorders leading to MAFLD are obesity and/or diabetes and/or at least two out of five features of metabolic syndrome (MetS) [
      • Eslam M.
      • Newsome P.N.
      • Sarin S.K.
      • et al.
      A new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement.
      ].
      The strong association between NAFLD and obesity was well demonstrated in the European DIONYSOS study cohort including 3000 participants. Notably, NAFLD was present in 25% of participants with a normal weight (BMI 20.0–24.9 kg/m2), 67% of overweight participants (BMI 25.0–29.9 kg/m2) and 94% of participants with obesity (BMI ∼30 kg/m2) [
      • Bellentani S.
      • Tiribelli C.
      • Saccoccio G.
      • et al.
      Prevalence of chronic liver disease in the general population of northern Italy: the Dionysos Study.
      ]. The worldwide prevalence of obesity in NAFLD and NASH patients have been reported to be 51% and 81%, respectively [
      • Younossi Z.M.
      • Koenig A.B.
      • Abdelatif D.
      • Fazel Y.
      • Henry L.
      • Wymer M.
      Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes.
      ]. Therefore, it is generally accepted that the initiating events in NAFLD are dependent on the development of obesity and insulin resistance [
      • Baratta F.
      • Pastori D.
      • Polimeni L.
      • et al.
      Adherence to mediterranean diet and non-alcoholic fatty liver disease: effect on insulin resistance.
      ,
      • Anstee Q.M.
      • Targher G.
      • Day C.P.
      Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis.
      ].
      Several studies consistently demonstrated a strong association between NAFLD and the features of metabolic syndrome (MetS) [
      • Marchesini G.
      • Brizi M.
      • Morselli-Labate A.M.
      • et al.
      Association of nonalcoholic fatty liver disease with insulin resistance.
      ,
      • Marchesini G.
      • Brizi M.
      • Blanchi G.
      • et al.
      Nonalcoholic fatty liver disease: a feature of the metabolic syndrome.
      ,
      • Hsiao P.J.
      • Kuo K.K.
      • Shin S.J.
      • et al.
      Significant correlations between severe fatty liver and risk factors for metabolic syndrome.
      ,
      • Jimba S.
      • Nakagami T.
      • Takahashi M.
      • et al.
      Prevalence of non-alcoholic fatty liver disease and its association with impaired glucose metabolism in Japanese adults.
      ] so that NAFLD is currently considered as the liver feature of MetS pushing in naming it as metabolic fatty liver disease, MAFLD [
      • Marchesini G.
      • Brizi M.
      • Blanchi G.
      • et al.
      Nonalcoholic fatty liver disease: a feature of the metabolic syndrome.
      ,
      • Hsiao P.J.
      • Kuo K.K.
      • Shin S.J.
      • et al.
      Significant correlations between severe fatty liver and risk factors for metabolic syndrome.
      ,
      • Chalasani N.
      • Younossi Z.
      • Lavine J.E.
      • et al.
      The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American association for the study of liver diseases, American college of gastroenterology, and the American gastroenterological association.
      ,
      • Nascimbeni F.
      • Pais R.
      • Bellentani S.
      • et al.
      From NAFLD in clinical practice to answers from guidelines.
      ]. Roughly, 42% of NAFLD subjects have MetS, 69% hyperlipidemia, 39% hypertension and 22% diabetes [
      • Younossi Z.M.
      • Koenig A.B.
      • Abdelatif D.
      • Fazel Y.
      • Henry L.
      • Wymer M.
      Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes.
      ]. High prevalence of NAFLD has been reported in patients with T2DM, ranging from 40% to 70% [
      • Targher G.
      • Bertolini L.
      • Padovani R.
      • et al.
      Prevalence of nonalcoholic fatty liver disease and its association with cardiovascular disease among type 2 diabetic patients.
      ,
      • Williamson R.M.
      • Price J.F.
      • Glancy S.
      • et al.
      Prevalence of and risk factors for hepatic steatosis and nonalcoholic Fatty liver disease in people with type 2 diabetes: the Edinburgh Type 2 Diabetes Study.
      ]. Conversely, the prevalence of NAFLD in patients with Type 1 diabetes mellitus is relatively low (8.8%) [
      • Cusi K.
      • Sanyal A.J.
      • Zhang S.
      • et al.
      Non-alcoholic fatty liver disease (NAFLD) prevalence and its metabolic associations in patients with type 1 diabetes and type 2 diabetes.
      ] thus further supporting the hypothesis that insulin resistance, which is typically present in obesity and T2DM, but rarely in T1DM, is the main contributor in the pathogenesis of MAFLD [
      • Kasper P.
      • Martin A.
      • Lang S.
      • et al.
      NAFLD and cardiovascular diseases: a clinical review.
      ].
      To this regard, it is worth to mention that the accumulation of visceral adipose tissue (VAT), frequently associated with hepatic steatosis, represents a strong determinant of complications usually seen in NAFLD patients such as atherogenic dyslipidaemia, insulin resistance, pro-inflammatory states, and elevated blood pressure [
      • Petta S.
      • Amato M.C.
      • Di Marco V.
      • et al.
      Visceral adiposity index is associated with significant fibrosis in patients with non-alcoholic fatty liver disease.
      ]. The portal/fatty acid flux theory proposes that visceral fat, through its increased lipolytic activity, releases toxic free fatty acids (FFA), which are carried directly by portal circulation into the liver in high concentrations leading to the development of hepatic insulin resistance through the accumulation of hepatic fat [
      • Yu S.J.
      • Kim W.
      • Kim D.
      • et al.
      Visceral obesity predicts significant fibrosis in patients with nonalcoholic fatty liver disease.
      ]. During this process visceral fat also releases inflammatory cytokines, contributing to the inflammatory milieu that represents a potent driver of promoting atherosclerosis process [
      • Nobarani S.
      • Alaei-Shahmiri F.
      • Aghili R.
      • et al.
      Visceral adipose tissue and non-alcoholic fatty liver disease in patients with type 2 diabetes.
      ,
      • Hanlon C.L.
      • Yuan L.
      Nonalcoholic fatty liver disease: the role of visceral adipose tissue.
      ]. At the same time, it is known that the serum concentrations of adipokines, derived from visceral fat as well as some proinflammatory cytokine in overweight patients, are closely linked with insulin resistance and NAFLD development [
      • Abenavoli L.
      • Luigiano C.
      • Guzzi P.H.
      • et al.
      Serum adipokine levels in overweight patients and their relationship with non-alcoholic fatty liver disease.
      ].
      Given the metabolic hallmark of the new definition of MAFLD, it could be speculated that an increased risk of cardiovascular complications should be expected in this subtype of NAFLD [
      • Lombardi R.
      • Cespiati A.
      • Francione P.
      • Cinque F.
      • Fargion S.
      • Fracanzani A.L.
      MAFLD and NAFLD: is there the need for redefining the risk of cardiovascular disease and mortality?.
      ]. Indeed, despite data about cardiovascular abnormalities in MAFLD are limited, it has been showed that individuals with MAFLD phenotype had a substantially increased ASCVD risk compared to those without MAFLD [
      • Han E.
      • Lee Y.
      • Lee J.S.
      • et al.
      Fibrotic burden determines cardiovascular risk among subjects with metabolic dysfunction-associated fatty liver disease.
      ]. However, while the new MAFLD definition better outlines the syndromic of NAFLD and encloses the large majority of fatty liver patients, it excludes those with metabolic-healthy NAFLD, that could be probably considered as the “pure” genetic ones [
      • Di Costanzo A.
      • D'Erasmo L.
      • Polimeni L.
      • et al.
      Non-alcoholic fatty liver disease and subclinical atherosclerosis: a comparison of metabolically- versus genetically-driven excess fat hepatic storage.
      ]. These patients might represent a useful human model to evaluate the potential individual association between fatty liver per se and ASCVD.

      2.2 NAFLD genetics

      Thanks to the recent discoveries on genetic factors predisposing to NAFLD, the opportunity to classify the disease has been enriched by the chance to cluster the ‘genetic’ sub-phenotypes of NAFLD.
      An increasing number of single-nucleotide polymorphisms (SNPs) have been discovered as associated with the presence and severity of NAFLD, namely, PNPLA3, transmembrane 6 superfamily member 2 (TM6SF2), glucokinase regulator (GCKR), membrane bound O-acyltransferase domain-containing 7 (MBOAT7) [
      • Di Costanzo A.
      • Belardinilli F.
      • Bailetti D.
      • et al.
      Evaluation of polygenic determinants of non-alcoholic fatty liver disease (NAFLD) by a candidate genes resequencing strategy.
      ,
      • Di Costanzo A.
      • Pacifico L.
      • Chiesa C.
      • et al.
      Genetic and metabolic predictors of hepatic fat content in a cohort of Italian children with obesity.
      ,
      • Dongiovanni P.
      • Stender S.
      • Pietrelli A.
      • et al.
      Causal relationship of hepatic fat with liver damage and insulin resistance in nonalcoholic fatty liver.
      ,
      • Grimaudo S.
      • Pipitone R.M.
      • Pennisi G.
      • et al.
      Association between PNPLA3 rs738409 C>G variant and liver-related outcomes in patients with nonalcoholic fatty liver disease.
      ,
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      ,
      • Sookoian S.
      • Pirola C.J.
      Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
      ,
      • Valenti L.
      • Al-Serri A.
      • Daly A.K.
      • et al.
      Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease.
      ,
      • Mancina R.M.
      • Dongiovanni P.
      • Petta S.
      • et al.
      The MBOAT7-TMC4 variant rs641738 increases risk of nonalcoholic fatty liver disease in individuals of European descent.
      ]. In addition, the hydroxysteroid 17β-dehydrogenase (HSD17B13) and the rs2642438 (p.A165T) variant in the mitochondrial amidoxime reducing component 1 (MARC1) have been found to be associated with protection from NAFLD progression and all-cause cirrhosis, respectively [
      • Dongiovanni P.
      • Meroni M.
      • Mancina R.M.
      • et al.
      Protein phosphatase 1 regulatory subunit 3B gene variation protects against hepatic fat accumulation and fibrosis in individuals at high risk of nonalcoholic fatty liver disease.
      ,
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.
      ,
      • Luukkonen P.K.
      • Juuti A.
      • Sammalkorpi H.
      • et al.
      MARC1 variant rs2642438 increases hepatic phosphatidylcholines and decreases severity of non-alcoholic fatty liver disease in humans.
      ]. Interestingly, most of these variants were found to have a plausible pathophysiological role in causing lipids alteration in hepatocytes, such as in droplets retention or very-low density lipoproteins (VLDLs) secretion, impaired synthesis or catabolism determining lipotoxicity [
      • Romeo S.
      • Sanyal A.
      • Valenti L.
      Leveraging human genetics to identify potential new treatments for fatty liver disease.
      ].
      Nowadays, the rs738409 polymorphism in PNPLA3 (I148 M) is the most potent gene variant predicting NAFLD risk [
      • Sookoian S.
      • Pirola C.J.
      Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
      ,
      • Valenti L.
      • Al-Serri A.
      • Daly A.K.
      • et al.
      Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease.
      ]. Interestingly, the variant associates with the full spectrum of NAFLD (from NASH to fibrosis, cirrhosis and HCC) [
      • Dongiovanni P.
      • Stender S.
      • Pietrelli A.
      • et al.
      Causal relationship of hepatic fat with liver damage and insulin resistance in nonalcoholic fatty liver.
      ,
      • Grimaudo S.
      • Pipitone R.M.
      • Pennisi G.
      • et al.
      Association between PNPLA3 rs738409 C>G variant and liver-related outcomes in patients with nonalcoholic fatty liver disease.
      ,
      • Valenti L.
      • Al-Serri A.
      • Daly A.K.
      • et al.
      Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease.
      ,
      • Liu Y.L.
      • Patman G.L.
      • Leathart J.B.S.
      • et al.
      Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.
      ,
      • Carpino G.
      • Pastori D.
      • Baratta F.
      • et al.
      PNPLA3 variant and portal/periportal histological pattern in patients with biopsy-proven non-alcoholic fatty liver disease: a possible role for oxidative stress.
      ], in absence of body weight alterations, insulin resistance or the MetS [
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      ]. Overall, these findings shed light on the hypothesis that liver fat accumulation per se is not a risk factor for atherosclerosis.
      Over the past years, in an attempt to separate patients in whom hepatic fat is due to metabolic alterations from those in whom it is due to genetic factors, we enrolled two groups of NAFLD patients. One group was carrying metabolic disorders and was wild-type for PNPLA3 variant (MetS-related NAFLD) the other included metabolic healthy NAFLD patients carrying the pro-steatogenic rs738409 PNPLA3 GG genotype [
      • Di Costanzo A.
      • D'Erasmo L.
      • Polimeni L.
      • et al.
      Non-alcoholic fatty liver disease and subclinical atherosclerosis: a comparison of metabolically- versus genetically-driven excess fat hepatic storage.
      ]. The latter group represents a small proportion of NAFLD patients expressing a “pure genetic NAFLD”, which should not be confused with lean NAFLD. It must be noted that ‘lean NAFLD’ definition has been often used to identify patients with low metabolic load to set against dysmetabolic ones. However, many lines of evidence have proved that these patients might have metabolic dysfunction (e.g.: insulin resistance, diabetes, hypertension [
      • Younossi Z.M.
      • Stepanova M.
      • Negro F.
      • et al.
      Nonalcoholic fatty liver disease in lean individuals in the United States.
      ]) and, based on these cardio-metabolic risk profiles, might be included in the new MAFLD definition [
      • Baratta F.
      • Ferro D.
      • Pastori D.
      • et al.
      Open issues in the transition from NAFLD to MAFLD: the experience of the Plinio study.
      ]. Not by chance, lean NAFLD patients have similar risk to develop cardiometabolic and liver complications as compared to obese ones [
      • Younes R.
      • Govaere O.
      • Petta S.
      • et al.
      Caucasian lean subjects with non-alcoholic fatty liver disease share long-term prognosis of non-lean: time for reappraisal of BMI-driven approach?.
      ].
      By identifying these two distinct forms of NAFLD, we demonstrated that the hepatic fat excess increased the burden of subclinical atherosclerosis, only when associated with metabolic disturbances [
      • Di Costanzo A.
      • D'Erasmo L.
      • Polimeni L.
      • et al.
      Non-alcoholic fatty liver disease and subclinical atherosclerosis: a comparison of metabolically- versus genetically-driven excess fat hepatic storage.
      ]. Moreover, we also identified an impairment in HDL functionality, namely reduced HDL-mediated cholesterol efflux capacity (CEC), in metabolically- but not in genetically- driven NAFLD [
      • Di Costanzo A.
      • Ronca A.
      • D'Erasmo L.
      • et al.
      HDL-mediated cholesterol efflux and plasma loading capacities are altered in subjects with metabolically- but not genetically driven non-alcoholic fatty liver disease (NAFLD).
      ]. As altered HDL-mediated cholesterol efflux is associated with increased risk of ASCVD, these findings further suggest the existence of pro-atherogenic pathways which are differently active in metabolically- and genetically- driven NAFLD. Several Mendelian randomization studies have confirmed these findings. Lauridsen B.K. et al. [
      • Lauridsen B.K.
      • Stender S.
      • Kristensen T.S.
      • et al.
      Liver fat content, non-alcoholic fatty liver disease, and ischaemic heart disease: mendelian randomization and meta-analysis of 279 013 individuals.
      ] found that PNPLA3 gene variant was not associated with increased risk of CHD in the general population, despite causing a high risk of the entire spectrum of NAFLD. Recently, Grimaudo et al. [
      • Grimaudo S.
      • Pipitone R.M.
      • Pennisi G.
      • et al.
      Association between PNPLA3 rs738409 C>G variant and liver-related outcomes in patients with nonalcoholic fatty liver disease.
      ] in the first prospective study on PNPLA3 gene variants carriers, found that this variant was independently associated with a higher risk of liver disease progression and liver-related death (HR, 3.64, 95% CI, 1.18–11.2; p = 0.02), but not with cardiovascular events [
      • Grimaudo S.
      • Pipitone R.M.
      • Pennisi G.
      • et al.
      Association between PNPLA3 rs738409 C>G variant and liver-related outcomes in patients with nonalcoholic fatty liver disease.
      ]. Overall, these results confirm the notion that the subset of metabolically healthy patients carrying the rs738409 PNPLA3 variant are potentially affected by genetic NAFLD without increased ASCVD risk [
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      ].
      Also the TM6SF2 C > T (E167K) rs58542926 variant was found to predispose patients to hepatic fat accumulation [
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      ]. This variant has been reported to interfere with the efflux of fat from hepatocytes, by reducing apolipoprotein B (apoB) lipidation and, thereby, VLDLs secretion [
      • Dongiovanni P.
      • Petta S.
      • Maglio C.
      • et al.
      Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease.
      ]. Interestingly, several studies have demonstrated that NAFLD risk and CVD risk do not coincide in carriers of the TM6SF2 167K pro-steatogenic allele [
      • Dongiovanni P.
      • Petta S.
      • Maglio C.
      • et al.
      Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease.
      ,
      • Holmen O.L.
      • Zhang H.
      • Fan Y.
      • et al.
      Systematic evaluation of coding variation identifies a candidate causal variant in TM6SF2 influencing total cholesterol and myocardial infarction risk.
      ]. Thus, both carriers of the rs738409 G allele in PNPLA3 and rs58592926 T allele in TM6SF2 were found to be at higher risk of NAFLD hepatic progression rather than cardiometabolic complications (see Table 2).
      Table 2Eeffects of PNPLA3 and TM6SF2 gene variants on liver disease severity and CVD risk.
      PNPLA3CohortSeverity of liver diseasesCardiometabolic risk
      NAFLDNASHFibrosisCirrhosisHCC
      [
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      ]
      NAFLD
      [
      • Sookoian S.
      • Castaño G.O.
      • Burgueño A.L.
      • Gianotti T.F.
      • Rosselli M.S.
      • Pirola C.J.
      A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity.
      ]
      NAFLD3.43
      [
      • Speliotes E.K.
      • Butler J.L.
      • Palmer C.D.
      • Voight B.F.
      • Hirschhorn J.N.
      PNPLA3 variants specifically confer increased risk for histologic nonalcoholic fatty liver disease but not metabolic disease.
      ]
      NAFLD

      NASH
      3.123.263.37↓BMI, T2DM, TG, WC
      [
      • Valenti L.
      • Al-Serri A.
      • Daly A.K.
      • et al.
      Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease.
      ]
      NAFLD1.351.51.54
      [
      • Sookoian S.
      • Pirola C.J.
      Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
      ] meta-analysis
      NAFLD3.263.263.25No association with BMI or HOMA-IR
      [
      • Krawczyk M.
      • Grünhage F.
      • Zimmer V.
      • Lammert F.
      Variant adiponutrin (PNPLA3) represents a common fibrosis risk gene: non-invasive elastography-based study in chronic liver disease.
      ]
      Mixed cohort1.86
      [
      • Burza M.A.
      • Pirazzi C.
      • Maglio C.
      • et al.
      PNPLA3 I148M (rs738409) genetic variant is associated with hepatocellular carcinoma in obese individuals.
      ]
      OBESITY7.42

      HR:5.88
      [
      • Petta S.
      • Valenti L.
      • Marchesini G.
      • et al.
      PNPLA3 GG genotype and carotid atherosclerosis in patients with non-alcoholic fatty liver disease.
      ]
      NAFLD↑IMT
      [
      • Liu Y.L.
      • Patman G.L.
      • Leathart J.B.S.
      • et al.
      Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.
      ]
      NAFLD-related HCC2.26
      [
      • Singal A.G.
      • Manjunath H.
      • Yopp A.C.
      • et al.
      The effect of PNPLA3 on fibrosis progression and development of hepatocellular carcinoma: a meta-analysis.
      ] meta-analysis
      Mixed1.351.321.40
      [
      • Shen J.H.
      • Li Y.L.
      • Li D.
      • Wang N.N.
      • Jing L.
      • Huang Y.H.
      The rs738409 (I148M) variant of the PNPLA3 gene and cirrhosis: a meta-analysis.
      ] meta-analysis
      Mixed1.86
      [
      • Zhang L.
      • You W.
      • Zhang H.
      • et al.
      PNPLA3 polymorphisms (rs738409) and non-alcoholic fatty liver disease risk and related phenotypes: a meta-analysis.
      ] meta-analysis
      NAFLD1.92
      [
      • Xu R.
      • Tao A.
      • Zhang S.
      • Deng Y.
      • Chen G.
      Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis.
      ] meta-analysis
      NAFLD3.414.443.11
      [
      • Dongiovanni P.
      • Petta S.
      • Maglio C.
      • et al.
      Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease.
      ]
      NAFLD NASH1.652.08
      [
      • Simons N.
      • Isaacs A.
      • Koek G.H.
      • Kuč S.
      • Schaper N.C.
      • Brouwers M.C.G.J.
      PNPLA3, TM6SF2, and MBOAT7 genotypes and coronary artery disease.
      ]
      CARDIoGRAMplusC4D cohort↓CAD
      [
      • Krawczyk M.
      • Rau M.
      • Schattenberg R.M.
      • et al.
      Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: a multicenter biopsy-based study.
      ]
      NAFLD2.421.67
      [
      • Lauridsen B.K.
      • Stender S.
      • Kristensen T.S.
      • et al.
      Liver fat content, non-alcoholic fatty liver disease, and ischaemic heart disease: mendelian randomization and meta-analysis of 279 013 individuals.
      ]
      Danish general population2.033.28↓IHD
      [
      • Di Costanzo A.
      • D'Erasmo L.
      • Polimeni L.
      • et al.
      Non-alcoholic fatty liver disease and subclinical atherosclerosis: a comparison of metabolically- versus genetically-driven excess fat hepatic storage.
      ]
      NAFLD↓IMT
      [
      • Yang J.
      • Trépo E.
      • Nahon P.
      • et al.
      PNPLA3 and TM6SF2 variants as risk factors of hepatocellular carcinoma across various etiologies and severity of underlying liver diseases.
      ]
      HCC discovery cohort1.67
      [
      • Grimaudo S.
      • Pipitone R.M.
      • Pennisi G.
      • et al.
      Association between PNPLA3 rs738409 C>G variant and liver-related outcomes in patients with nonalcoholic fatty liver disease.
      ]
      NAFLD or NAFLD-related cirrhosisHR:2.68no CVD events
      [
      • Wu J.T.
      • Liu S.S.
      • Xie X.J.
      • Liu Q.
      • Xin Y.N.
      • Xuan S.Y.
      Independent and joint correlation of PNPLA3 I148M and TM6SF2 E167K variants with the risk of coronary heart disease in patients with non-alcoholic fatty liver disease.
      ]
      NAFLD1.53↓CHD
      TM6SF2NAFLDNASHFibrosisCirrhosisHCC
      [
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      ]
      NAFLD↓LDL-C ↓TG
      [
      • Holmen O.L.
      • Zhang H.
      • Fan Y.
      • et al.
      Systematic evaluation of coding variation identifies a candidate causal variant in TM6SF2 influencing total cholesterol and myocardial infarction risk.
      ]
      Population-based cohort in Norway↓TC ↓TG

      ↓ MI
      [
      • Liu Y.L.
      • Patman G.L.
      • Leathart J.B.S.
      • et al.
      Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.
      ]
      NAFLD2.94
      [
      • Dongiovanni P.
      • Petta S.
      • Maglio C.
      • et al.
      Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease.
      ]
      NAFLD NASH1.842.08↓ TC ↓ TG

      ↓ Carotid plaques
      [
      • Pirola C.J.
      • Sookoian S.
      The dual and opposite role of the TM6SF2-rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: a meta-analysis.
      ]
      NAFLD NASH1.37nono↓ CVD risk
      [
      • Krawczyk M.
      • Rau M.
      • Schattenberg R.M.
      • et al.
      Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: a multicenter biopsy-based study.
      ]
      NAFLD4.721.76
      [
      • Simons N.
      • Isaacs A.
      • Koek G.H.
      • Kuč S.
      • Schaper N.C.
      • Brouwers M.C.G.J.
      PNPLA3, TM6SF2, and MBOAT7 genotypes and coronary artery disease.
      ]
      CARDIoGRAMplusC4D cohort↓ CAD
      [
      • Yang J.
      • Trépo E.
      • Nahon P.
      • et al.
      PNPLA3 and TM6SF2 variants as risk factors of hepatocellular carcinoma across various etiologies and severity of underlying liver diseases.
      ]
      HCC discovery cohort1.45
      [
      • Parisinos C.A.
      • Wilman H.R.
      • Thomas E.L.
      • et al.
      Genome-wide and Mendelian randomisation studies of liver MRI yield insights into the pathogenesis of steatohepatitis.
      ]
      UK Biobank with MRS↓CAD
      The magnitude of the effect is expressed by odd ratios (OR) values.
      Studies exclusively focused on alcoholic fatty liver disease (ALD) or chronic hepatitis study populations were not considered. OR were extracted from those obtained by multivariate analysis in single articles and by overall Fixed term from meta-analysis.
      BMI, body mass index, CAD, coronary artery disease, CHD, coronary heart disease, CVD, cardiovascular disease, HOMA-IR, homeostasis model assessment-insulin resistance, HR, Hazard ratio, IHD, ischemic heart disease, IMT, intima-media thickness, LDL-C, low-density lipoprotein cholesterol, MI, myocardial infarction, TG, triglycerides, T2DM, type 2 diabetes, WC, waist circumference.
      Despite this, it must be noted that the “genetic NAFLD sub-phenotype” should not be considered as a fully separated entity. In fact, the genetic pro-steatogenic background interacts with the environment in generating its pathological effect. A previously published study highlighted the synergistic interaction between obesity and three NAFLD risk variants (PNPLA3 I148 M, TM6SF2 E167K and GCKR P446L) across the entire spectrum of NAFLD [
      • Stender S.
      • Kozlitina J.
      • Nordestgaard B.G.
      • Tybjærg-Hansen A.
      • Hobbs H.H.
      • Cohen J.C.
      Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci.
      ]. Specifically, the steatogenic effect of the M-allele in PNPLA3 was amplified by obesity (BMI ≥30 kg/m). A similar amplifying effect of BMI on hepatic triglycerides content (HTGC) was seen for the TM6SF2 risk variant (E167K), with a less significant interaction (p-interaction = 0.006), likely due to the lower frequency of the risk allele when compared to the GCKR risk allele. Thus, the interaction between genotypes and BMI on hepatic fat content is not unique to PNPLA3 [
      • Stender S.
      • Kozlitina J.
      • Nordestgaard B.G.
      • Tybjærg-Hansen A.
      • Hobbs H.H.
      • Cohen J.C.
      Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci.
      ]. Probably, these “mixed” patients have higher cardiometabolic risk in comparison to those “purely” genetics (pure genetic NAFLD group). Thus, based on the current knowledge, we could identify at least three clinical subtypes of NAFLD (Fig. 1).
      In conclusion, it could be reasonable to speculate that hepatic fat excess per se is unlikely to cause CVD and the reported epidemiological association is mostly mediated by the classical risk factors for atherosclerosis (dyslipidemia, diabetes mellitus, insulin resistance). However, the underlying mechanisms linking NAFLD to CVD are very complex and involve several different metabolic pathways (see below).

      3. Mechanisms linking NAFLD to ASCVD

      Causal mechanisms underlying the association between NAFLD, and CVD are not fully understood. However, several pathophysiological pathways including pro-atherogenic lipid alterations (e.g. atherogenic dyslipidaemia), insulin resistance, low-grade inflammation, de novo lipogenesis (DNL), endothelial dysfunction and oxidative stress and, more recently abnormal microbiota, have been described as links between NAFLD and ASCVD.

      3.1 Lipids

      As mentioned above, NAFLD is usually associated with MetS, insulin resistance, and T2DM, which are characterised by the presence of a typical lipid plasma alteration called atherogenic dyslipidemia (AD). AD is defined as the combination of elevated concentration of TG-rich lipoproteins (TRLs; ie, very low-density lipoproteins - VLDLs - plus chylomicrons and their remnants), lower concentration of high-density lipoprotein cholesterol (HDL-C), and an increased proportion of circulating small-dense particles of low density lipoprotein cholesterol (LDL-C) [
      • Brouwers M.C.G.J.
      • Simons N.
      • Stehouwer C.D.A.
      • Isaacs A.
      Non-alcoholic fatty liver disease and cardiovascular disease: assessing the evidence for causality.
      ,
      • Van Gaal L.F.
      • Mertens I.L.
      • De Block C.E.
      Mechanisms linking obesity with cardiovascular disease.
      ,
      • Adiels M.
      • Taskinen M.R.
      • Borén J.
      Fatty liver, insulin resistance, and dyslipidemia.
      ].
      As TRLs contain apolipoprotein B (apoB), plasma concentration of apoB is typically elevated in NAFLD, thus representing an helpful biochemical marker for the overall lipoprotein alterations as well as CVD risk in this condition [
      • Siddiqui M.S.
      • Fuchs M.
      • Idowu M.O.
      • et al.
      Severity of nonalcoholic fatty liver disease and progression to cirrhosis are associated with atherogenic lipoprotein profile.
      ].
      To this regard, it is worth to mention that the over secretion of apoB-TRLs particles together with their impaired clearance by the liver are the main causes of hepatic fat accumulation and central to the development of hyperlipidemia. Interestingly, this typical lipid profile might also depend on the reduced activity of lipoprotein lipase (LPL) due to an increased activity of ANGPTL3 and ANGPTL4, which represent the physiological circulating inhibitors of LPL [
      • Altun Ö.
      • Dikker O.
      • Arman Y.
      • et al.
      Serum Angiopoietin-like peptide 4 levels in patients with hepatic steatosis.
      ,
      • Bini S.
      • D'Erasmo L.
      • Di Costanzo A.
      • Minicocci I.
      • Pecce V.
      • Arca M.
      The interplay between angiopoietin-like proteins and adipose tissue: another piece of the relationship between adiposopathy and cardiometabolic diseases?.
      ,
      • Bini S.
      • Pecce V.
      • Di Costanzo A.
      • et al.
      The fibrinogen-like domain of ANGPTL3 facilitates lipolysis in 3T3-L1 cells by activating the intracellular erk pathway.
      ]. Notably, plasma levels of ANGPTL3 and ANGPTL4 were found to be increased in obesity and T2DM as well as in NAFLD/NASH patients [
      • Morinaga J.
      • Zhao J.
      • Endo M.
      • et al.
      Association of circulating ANGPTL 3,4, and 8 levels with medical status in a population undergoing routine medical checkups: a cross-sectional study.
      ].
      Elevated TGs are typically found in MAFLD [
      • Toledo F.G.S.
      • Sniderman A.D.
      • Kelley D.E.
      Influence of hepatic steatosis (fatty liver) on severity and composition of dyslipidemia in type 2 diabetes.
      ]. Notably, higher TGs, remnant lipoprotein cholesterol (RLP-C) and high TRLs have been observationally and genetically associated with an increased risk of development of atherosclerosis [
      • Kaltoft M.
      • Langsted A.
      • Nordestgaard B.G.
      Triglycerides and remnant cholesterol associated with risk of aortic valve stenosis: mendelian randomization in the Copenhagen General Population Study.
      ,
      • Cao Y.X.
      • Zhang H.W.
      • Jin J.L.
      • et al.
      Prognostic utility of triglyceride-rich lipoprotein-related markers in patients with coronary artery disease.
      ], thus representing another potential pathophysiological link between NAFLD and ASCVD. Recently, in the post-hoc analysis of the Plinio study, higher levels of RLP-C were found in NAFLD patients in comparison to those without. In addition, among NAFLD patients, those with higher baseline RLP-C had a more than doubled risk of developing cardiovascular events during follow up [
      • Pastori D.
      • Baratta F.
      • Novo M.
      • et al.
      Remnant lipoprotein cholesterol and cardiovascular and cerebrovascular events in patients with non-alcoholic fatty liver disease.
      ]. On the other hand, AD was found to be associated with advanced liver fibrosis in diabetic NAFLD patients [
      • Julián M.T.
      • Pera G.
      • Soldevila B.
      • et al.
      Atherogenic dyslipidemia, but not hyperglycemia, is an independent factor associated with liver fibrosis in subjects with type 2 diabetes and NAFLD: a population-based study.
      ].
      Altered levels of other apoB-containing lipoproteins, namely small-dense LDL, which are more atherogenic than normal-sized LDL, have been described in patients with NAFLD [
      • Siddiqui M.S.
      • Fuchs M.
      • Idowu M.O.
      • et al.
      Severity of nonalcoholic fatty liver disease and progression to cirrhosis are associated with atherogenic lipoprotein profile.
      ]. NAFLD patients may present low levels of larger LDL-1 and increased smaller LDL-3 and LDL-4 particles leading to a more atherogenic profile. Interestingly, in 2011, Athyros et al. [
      • Athyros V.G.
      • Giouleme O.
      • Ganotakis E.S.
      • et al.
      Safety and impact on cardiovascular events of long-term multifactorial treatment in patients with metabolic syndrome and abnormal liver function tests: a post hoc analysis of the randomised ATTEMPT study.
      ] demonstrated that treating LDL intensely with atorvastatin reduced both markers of liver damage and cardiovascular events incidence more than a more cautious treatment, suggesting a role of LDL as marker of both liver and cardiovascular risk. Moreover, in the Greece study, atorvastatin treatment improved liver tests and reduced cardiovascular morbidity in patients with mild-to-moderately abnormal liver tests that were potentially attributable to NAFLD [
      • Athyros V.G.
      • Tziomalos K.
      • Gossios T.D.
      • et al.
      Safety and efficacy of long-term statin treatment for cardiovascular events in patients with coronary heart disease and abnormal liver tests in the Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) Study: a post-hoc analysis.
      ].
      Another hallmark of MAFLD-associated dyslipidemia is the alteration of HDL profile. By evaluating HDL particle subfractions, Bril et al. [
      • Bril F.
      • Sninsky J.J.
      • Baca A.M.
      • et al.
      Hepatic steatosis and insulin resistance, but not steatohepatitis, promote atherogenic dyslipidemia in NAFLD.
      ] found smaller HDL particles among patients with NAFLD. Of note, it has been previously reported, that these small HDL particles have poor functionality, and that their clearance is increased, contributing to lower HDL cholesterol and apoAI seen in these patients [
      • Garvey W.T.
      • Kwon S.
      • Zheng D.
      • et al.
      Effects of insulin resistance and type 2 diabetes on lipoprotein subclass particle size and concentration determined by nuclear magnetic resonance.
      ]. It has been postulated that HDL exerts an important antioxidative function, among other antiatherogenic activities. Such antioxidative capacity can be crucial for the growth and complications of atherosclerotic plaques [
      • Heeren J.
      • Scheja L.
      Metabolic-associated fatty liver disease and lipoprotein metabolism.
      ]. Finally, increased liver fat content was found to be closely correlated with a more prolonged postprandial lipemia as estimated by measurements of chylomicrons, VLDL1 TG, and apoB-48 increases after an oral fat load [
      • Matikainen N.
      • Mänttäri S.
      • Westerbacka J.
      • et al.
      Postprandial lipemia associates with liver fat content.
      ]. In addition, NAFLD was also associated with increased levels of oxidised LDL after an oral fat load [
      • Ho C.M.
      • Ho S.L.
      • Jeng Y.M.
      • et al.
      Accumulation of free cholesterol and oxidized low-density lipoprotein is associated with portal inflammation and fibrosis in nonalcoholic fatty liver disease.
      ]. Postprandial lipid levels contribute substantially to the hepatic TG content in NAFLD and have been reported to be an independent cardio metabolic risk factor [
      • Matikainen N.
      • Mänttäri S.
      • Westerbacka J.
      • et al.
      Postprandial lipemia associates with liver fat content.
      ,
      • Pirillo A.
      • Norata G.D.
      • Catapano A.L.
      Postprandial lipemia as a cardiometabolic risk factor.
      ,
      • Cassader M.
      • Gambino R.
      • Musso G.
      • et al.
      Postprandial triglyceride-rich lipoprotein metabolism and insulin sensitivity in nonalcoholic steatohepatitis patients.
      ].

      3.2 Insulin resistance, glucose metabolism and de novo lipogenesis (DNL)

      Abnormal glucose metabolism and insulin resistance are the major hallmarks of NAFLD. Both are crucial in NAFLD and CVD pathogenesis [
      • Dongiovanni P.
      • Rametta R.
      • Meroni M.
      • Valenti L.
      The role of insulin resistance in nonalcoholic steatohepatitis and liver disease development--a potential therapeutic target?.
      ]. Disorders of glucose metabolism in NAFLD patients can be attributed to visceral obesity in conjunction with increased body weight which in turn is associated with insulin resistance.
      Insulin resistance is accompanied by a persistent hyperinsulinemia, that is of central importance for the induction and maintenance of an unfavourable metabolic condition characterised by increased free fatty acids (FFA) and glucose levels, which subsequently promotes the development of cardiometabolic disorders. Insulin resistance is regarded as a major driving force for atherogenic dyslipidaemia because it increases FFA release, which stimulates hepatic triacylglycerol output [
      • Postic C.
      • Girard J.
      Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice.
      ].
      Insulin resistance also drives hepatic de novo lipogenesis (DNL) in NAFLD [
      • Smith G.I.
      • Shankaran M.
      • Yoshino M.
      • et al.
      Insulin resistance drives hepatic de novo lipogenesis in nonalcoholic fatty liver disease.
      ]. DNL contribution to liver TG content is less than 5% in healthy control subjects but contributes to liver triglyceride content for 26% and to VLDL triglycerides for a total of 23% in hyperinsulinemic subjects with NAFLD [
      • Donnelly K.L.
      • Smith C.I.
      • Schwarzenberg S.J.
      • Jessurun J.
      • Boldt M.D.
      • Parks E.J.
      Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.
      ]. Interestingly, in the work by Donelly KL et al., the DNL was increased in fasted and fed states, indicating that conceivably the elevation of transcription factors and enzymes are involved in hepatic DNL [
      • Donnelly K.L.
      • Smith C.I.
      • Schwarzenberg S.J.
      • Jessurun J.
      • Boldt M.D.
      • Parks E.J.
      Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.
      ]. These include the transcription factor SREBP-1c, which is activated by insulin, and activates many important enzymes involved in lipogenesis. Carbohydrate response element–binding protein (ChREBP) also regulates DNL by activating several enzymes involved in the lipogenesis and pyruvate kinase, a key regulator of glycolysis [
      • Koo S.H.
      • Dutcher A.K.
      • Towle H.C.
      Glucose and insulin function through two distinct transcription factors to stimulate expression of lipogenic enzyme genes in liver.
      ]. Notably, the rs1260326 GCKR gene variant, encoding for the loss-of-function variant (P446L), results in a net increase in intracellular glucose phosphate, leading to increase DNL via the induction of glycolysis and the stimulation of the ChREBP [
      • Romeo S.
      • Sanyal A.
      • Valenti L.
      Leveraging human genetics to identify potential new treatments for fatty liver disease.
      ]. The variant was found to be associated with higher susceptibility to NAFLD and advanced liver disease as described above [
      • Raimondo A.
      • Rees M.G.
      • Gloyn A.L.
      Glucokinase regulatory protein: complexity at the crossroads of triglyceride and glucose metabolism.
      ].
      Between NAFLD and diabetes, as already discussed above, there is a tight interplay and diabetes is one of the most important atherosclerotic risk factors in patients with NAFLD [
      • Morrison A.E.
      • Zaccardi F.
      • Khunti K.
      • Davies M.J.
      Causality between non-alcoholic fatty liver disease and risk of cardiovascular disease and type 2 diabetes: a meta-analysis with bias analysis.
      ]. It is worth mentioning that some glucose-lowering drugs have shown potential in ameliorating NAFLD. Data from randomised control data have highlighted that pioglitazone seems to be of some help in non-cirrhotic, non-diabetic adults with biopsy-confirmed NASH. More recently, a novel class of drugs, the glucagon-like peptide-1 receptor agonist (GLP-1RA), have been found to be of potential interest in treating NAFLD in diabetic patients. A recent meta-analysis by Mantovani et al. [
      • Mantovani A.
      • Petracca G.
      • Beatrice G.
      • Csermely A.
      • Lonardo A.
      • Targher G.
      Glucagon-like peptide-1 receptor agonists for treatment of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: an updated meta-analysis of randomized controlled trials.
      ] have showed that GLP-1RAs treatment was able to reduce the absolute percentage of liver fat content (as assessed by magnetic resonance-based techniques), as well as to induce histological resolution of NASH without worsening liver fibrosis. Nevertheless, the use of these drugs is actually a matter of debate. While several other studies have confirmed the potential use of thiazolidinediones (TZDs) for selected NAFLD patients, some others did not support findings regarding the beneficial effect of GLP-1RA [
      • Mantovani A.
      • Petracca G.
      • Beatrice G.
      • Csermely A.
      • Lonardo A.
      • Targher G.
      Glucagon-like peptide-1 receptor agonists for treatment of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: an updated meta-analysis of randomized controlled trials.
      ]. Preliminary but equally promising results concern the sodium-glucose cotransporter-2 (SGTL-2). They markedly reduce the atherosclerotic risk [
      • Palmer S.C.
      • Tendal B.
      • Mustafa R.A.
      • et al.
      Sodium-glucose cotransporter protein-2 (SGLT-2) inhibitors and glucagon-like peptide-1 (GLP-1) receptor agonists for type 2 diabetes: systematic review and network meta-analysis of randomised controlled trials.
      ] and seem to be able to reduce liver-related risk in NAFLD patients [
      • Dougherty J.A.
      • Guirguis E.
      • Thornby K.A.
      A systematic review of newer antidiabetic agents in the treatment of nonalcoholic fatty liver disease.
      ].

      3.3 Endothelial cell dysfunction

      In recent years, an increasing amount of evidence has shown that patients with NAFLD have endothelial dysfunction (ED) [
      • Targher G.
      • Bertolini L.
      • Rodella S.
      • et al.
      NASH predicts plasma inflammatory biomarkers independently of visceral fat in men.
      ], a marker of early atherosclerosis [
      • Widmer R.J.
      • Lerman A.
      Endothelial dysfunction and cardiovascular disease.
      ]. Endothelial cells are a crucial component of the normal vascular wall. When the endothelium is injured, it loses its specialised properties, resulting in ED which is a hallmark of vascular diseases [
      • Shireman P.K.
      • Pearce W.H.
      Endothelial cell function: biologic and physiologic functions in health and disease.
      ]. Activation and damage of the endothelial monolayer seem to trigger the development of the atherosclerotic lesions damaging. Once the integrity of endothelium is interrupted, lipid penetration and mononuclear cell adhesion might be initiated. Also, the expression of adhesion molecules (V-CAM in particular), the secretion of proinflammatory chemokines, the reduced production of nitric oxide and reduced endothelial-mediated vasodilation impairing the vascular response to physiologic and pharmacologic stimuli, have been described [
      • Theofilis P.
      • Sagris M.
      • Oikonomou E.
      • et al.
      Inflammatory mechanisms contributing to endothelial dysfunction.
      ]. Atherosclerosis risk factors, commonly found in NAFLD patients, such as hyperlipidemia, hypertension, diabetes mellitus, smoking, and infections can directly or indirectly stress the arterial endothelium, resulting in its dysfunction, damage, or both [
      • Stahl E.P.
      • Dhindsa D.S.
      • Lee S.K.
      • Sandesara P.B.
      • Chalasani N.P.
      • Sperling L.S.
      Nonalcoholic fatty liver disease and the heart: JACC state-of-the-art review.
      ]. Several pieces of evidence have shown that markers of endothelial dysfunction such as flow-mediated dilatation percentage (FMD%), are significantly impaired in patients with NAFLD [
      • Narayan J.
      • Das H.S.
      • Nath P.
      • et al.
      Endothelial dysfunction, a marker of atherosclerosis, is independent of metabolic syndrome in NAFLD patients.
      ,
      • Gentili A.
      • Daviddi G.
      • De Vuono S.
      • et al.
      Non-alcoholic fatty liver disease fibrosis score and preclinical vascular damage in morbidly obese patients.
      ] with a significant gradient according to liver disease severity [
      • Loffredo L.
      • Baratta F.
      • Ludovica P.
      • et al.
      Effects of dark chocolate on endothelial function in patients with non-alcoholic steatohepatitis.
      ]. In addition, FMD impairment was found to correlate with Framingham risk score in NAFLD patients [
      • Pastori D.
      • Loffredo L.
      • Perri L.
      • et al.
      Relation of nonalcoholic fatty liver disease and Framingham Risk Score to flow-mediated dilation in patients with cardiometabolic risk factors.
      ], enforcing the hypothesis of the role of endothelial dysfunction in the increased cardiovascular risk in NAFLD. Also, the average of IMT, used as a non-invasively marker of endothelial dysfunction and arteriopathy, was significantly increased in NAFLD cases as compared with non NAFLD [
      • Sert A.
      • Pirgon Ö.
      • Aypar E.
      • Yilmaz H.
      • Dündar B.
      Relationship between aspartate aminotransferase-to-platelet ratio index and carotid intima-media thickness in obese adolescents with non-alcoholic fatty liver disease.
      ,
      • Di Costanzo A.
      • Perla F.M.
      • D'Erasmo L.
      • Arca M.
      • Chiesa C.
      • Pacifico L.
      Elevated serum concentrations of remnant cholesterol associate with increased carotid intima-media thickness in children and adolescents.
      ]. In a recent meta-analysis, NAFLD patients had more carotid atherosclerosis than controls, with enlarged mean IMT and higher plaque prevalence [
      • Brea A.
      • Mosquera D.
      • Martín E.
      • Arizti A.
      • Cordero J.L.
      • Ros E.
      Nonalcoholic fatty liver disease is associated with carotid atherosclerosis: a case-control study.
      ].

      3.4 Oxidative stress

      Oxidative stress is associated with many chronic diseases, especially those characterised by low-grade inflammation, such as diabetes, metabolic syndrome, and obesity. Excessive reactive oxygen species (ROS) production may induce Kupfer cell and stellate cell activation in the liver thus promoting fibrosis and NAFLD progression to NASH and cirrhosis. Oxidative stress is also an important factor for the pathogenesis and progression of cardiovascular disease. It has been suggested that it may be a possible mechanism linking NAFLD to cardiovascular disease [
      • Polimeni L.
      • del Ben M.
      • Baratta F.
      • et al.
      Oxidative stress: new insights on the association of non-alcoholic fatty liver disease and atherosclerosis.
      ,
      • Ferro D.
      • Baratta F.
      • Pastori D.
      • et al.
      New insights into the pathogenesis of non-alcoholic fatty liver disease: gut-derived lipopolysaccharides and oxidative stress.
      ]. Oxidative stress and the accumulation of reactive oxygen species in plasma favours endothelial inflammation.
      Increased oxidative stress was documented in vivo in a large cohort of subjects with NAFLD by measuring the urinary levels of 8-iso-prostaglandin F2α (8-iso-PGF2α), which results from the non-enzymatic oxidation of arachidonic acid. This was measured together with the serum levels of the soluble Nox2-derived peptide (sNox2-dp), which represents an indicator of Nox2 activation, the main isoform of NADPH-oxidase responsible for ROS production [
      • Del Ben M.
      • Polimeni L.
      • Carnevale R.
      • et al.
      NOX2-generated oxidative stress is associated with severity of ultrasound liver steatosis in patients with non-alcoholic fatty liver disease.
      ]. Plasma accumulation of ROS, proportional to NAFLD/NASH severity, triggers endothelial inflammation and ultimately atherosclerosis progression [
      • Violi F.
      • Pignatelli P.
      • Pignata C.
      • et al.
      Reduced atherosclerotic burden in subjects with genetically determined low oxidative stress.
      ]. In addition, oxidative stress may be the consequence of the reduction of circulating levels of antioxidant systems. To this regard, in NAFLD patients, independently from the severity of liver disease, we observed a reduction of circulating levels of Vitamin E [
      • Pastori D.
      • Baratta F.
      • Carnevale R.
      • et al.
      Similar reduction of cholesterol-adjusted Vitamin E serum levels in simple steatosis and non-alcoholic steatohepatitis.
      ].

      3.5 Gut microbiota

      Gut microbiota influences different metabolic pathways, and its alterations are widely recognized as a significant pathophysiological mechanism in metabolic disorders onset [
      • Fan Y.
      • Pedersen O.
      Gut microbiota in human metabolic health and disease.
      ]. In addition to obesity and T2DM, an association between microbiota dysregulation (i.e. dysbiosis) and cardiovascular disease was found [
      • Fan Y.
      • Pedersen O.
      Gut microbiota in human metabolic health and disease.
      ]. Patients developing cardiovascular disease show less abundance of Bacteroides, Alistipes and Oscillibacter spp. and of Faecalibacterium prausnitzii while show more abundance of Ruminococcus, Acinetobacter, Escherichia coli, Enterobacter aerogenes, Klebsiella spp, Veillonella spp [
      • Jie Z.
      • Xia H.
      • Zhong S.L.
      • et al.
      The gut microbiome in atherosclerotic cardiovascular disease.
      ,
      • Sokol H.
      • Pigneur B.
      • Watterlot L.
      • et al.
      Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients.
      ,
      • Cui X.
      • Ye L.
      • Li J.
      • et al.
      Metagenomic and metabolomic analyses unveil dysbiosis of gut microbiota in chronic heart failure patients.
      ]. A recent meta-analysis [
      • Mohammad N.S.
      • Nazli R.
      • Zafar H.
      • Fatima S.
      Effects of lipid based Multiple Micronutrients Supplement on the birth outcome of underweight pre-eclamptic women: a randomized clinical trial.
      ] summarised findings on gut microbiota composition in NAFLD, reporting the increase of Streptococcus, Prevotella and Escherichia genera, and the decrease of Faecalibacterium, Coprococcus and Ruminococcus genera. Authors suggested that changes of Faecalibacterium and Prevotella abundance were associated with BMI, while the change of Streptococcus and Faecalibacterium quantity were associated with inflammatory status. Some of these results overlap with those observed in patients at high risk for CVD (e.g changes in Escherichia coli abundance) thus partially explaining the link between NAFLD and CVD. In patients with atherosclerosis, gut microbiota produces high levels of trimethylamine (TMA) from dietary l-carnitine, phosphatidylcholine and lecithin. TMA is oxidised in trimethylamine N-oxide (TMAO) in the liver [
      • Wang Z.
      • Klipfell E.
      • Bennett B.J.
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      ]. Notably, patients with high plasma levels of TMAO show increased risk for major cardiovascular events and all-cause mortality [
      • Guasti L.
      • Galliazzo S.
      • Molaro M.
      • et al.
      TMAO as a biomarker of cardiovascular events: a systematic review and meta-analysis.
      ]. The link between TMAO and cardiovascular disease is due to the association between TMAO, increased oxidative stress [
      • Violi F.
      • Loffredo L.
      • Carnevale R.
      • Pignatelli P.
      • Pastori D.
      Atherothrombosis and oxidative stress: mechanisms and management in elderly.
      ], platelet activation [
      • Zhu W.
      • Wang Z.
      • Tang W.H.W.
      • Hazen S.L.
      Gut microbe-generated trimethylamine N-oxide from dietary choline is prothrombotic in subjects.
      ] and lipid macrophage uptake, leading to foam cells formation [
      • Wang Z.
      • Klipfell E.
      • Bennett B.J.
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      ]. Recent studies demonstrated that circulating levels of TMAO are associated with NASH diagnosis and circulating levels of bile acids [
      • León-Mimila P.
      • Villamil-Ramírez H.
      • Li X.S.
      • et al.
      Trimethylamine N-oxide levels are associated with NASH in obese subjects with type 2 diabetes.
      ] probably via the inhibition of hepatic farnesoid X receptor (FXR) activation [
      • Tan X.
      • Liu Y.
      • Long J.
      • et al.
      Trimethylamine N-oxide aggravates liver steatosis through modulation of bile acid metabolism and inhibition of farnesoid X receptor signaling in nonalcoholic fatty liver disease.
      ]. Another mechanism by which gut microbiota dysregulation affects cardiovascular system health is represented by the lipopolysaccharides (LPS) translocation in blood stream [
      • Violi F.
      • Loffredo L.
      • Carnevale R.
      • Pignatelli P.
      • Pastori D.
      Atherothrombosis and oxidative stress: mechanisms and management in elderly.
      ]. LPS is a component of the bacterial lipid membrane in Gram-negative and a potent stimulator of innate immunity. LPS activates toll-like receptors (TLRs), in particular on the endothelial surface, leading to the inflammatory response and endothelial dysfunction [
      • Soares J.B.
      • Pimentel-Nunes P.
      • Roncon-Albuquerque R.
      • Leite-Moreira A.
      The role of lipopolysaccharide/toll-like receptor 4 signaling in chronic liver diseases.
      ] and on platelets surface inducing oxidative stress and platelet activation [
      • Barillà F.
      • Cammisotto V.
      • Bartimoccia S.
      • et al.
      Toll-like receptor 4 activation in platelets from myocardial infarction patients.
      ].
      Finally, it was recently proved that LPS from Escherichia coli localises in human plaque and may contribute to atherosclerotic damage via TLR4-induced oxidative stress [
      • Carnevale R.
      • Nocella C.
      • Petrozza V.
      • et al.
      Localization of lipopolysaccharide from Escherichia Coli into human atherosclerotic plaque.
      ]. Notably, patients with NAFLD showed an increase of 38–40% of their LPS serum levels in comparison to dysmetabolic patients without NAFLD [
      • Ferro D.
      • Baratta F.
      • Pastori D.
      • et al.
      New insights into the pathogenesis of non-alcoholic fatty liver disease: gut-derived lipopolysaccharides and oxidative stress.
      ]. This increase in circulating LPS may be the consequence of several factors. The overgrowth of intestinal Gram-negative bacteria and the increased intestinal permeability have been found [
      • Ferro D.
      • Baratta F.
      • Pastori D.
      • et al.
      New insights into the pathogenesis of non-alcoholic fatty liver disease: gut-derived lipopolysaccharides and oxidative stress.
      ]. LPS localises in NASH patients’ liver, particularly in hepatocytes and induces nuclear factor-κB activation [
      • Carpino G.
      • Del Ben M.
      • Pastori D.
      • et al.
      Increased liver localization of lipopolysaccharides in human and experimental NAFLD.
      ]. In addition, LPS may promote macrophages-platelets (TLR4+) hepatic colocalization, suggesting a LPS-induced platelet activation in the liver [
      • Carpino G.
      • Del Ben M.
      • Pastori D.
      • et al.
      Increased liver localization of lipopolysaccharides in human and experimental NAFLD.
      ].

      4. Conclusions and future perspectives

      Prevalence of NAFLD suggests that it is the liver disease of our century. Since patients with NAFLD present an increased CVD incidence, to predict what proportion of subjects will develop cardiovascular complications represents a public health issue.
      To date, differently from other clinical conditions [
      • Pastori D.
      • Menichelli D.
      • Del Sole F.
      • et al.
      Long-term risk of major adverse cardiac events in atrial fibrillation patients on direct oral anticoagulants.
      ], a cardiovascular risk score specifically validated in NAFLD patients is missing. Identifying a tool for this purpose could represent one of the major challenges for clinical research on NAFLD and could address one of the unmet needs in the clinical management of these patients. Meanwhile, waiting to know if the incoming drugs resolving NAFLD will also reduce the cardiovascular risk of these patients, by treating cardiometabolic risk factors in those with NAFLD, we should consider fatty liver as a “compelling indication” in choosing the best drugs.
      In the future, the recognition of the heterogeneous drivers responsible for the different disease subtypes of NAFLD, shall improve the development of integrated risk model of NAFLD – CVD progression based on genetic, molecular, histology and “omics” based data (transcriptome, metabolite, proteome, microbiome) toward a precision medicine approaches [
      • Sanyal A.J.
      Past, present and future perspectives in nonalcoholic fatty liver disease.
      ]. This would help in improving the design of clinical trials by pooling patients' subgroups with specific phenotypes and in applying customised therapeutic strategies able to prevent CV events occurrence.

      Author contributions

      A.D.C, F.B and L.D researched data for the article, contributed substantially to discussion of the content, wrote the article, and reviewed and/or edited the manuscript before submission. S.B, D.P, F.A, M.D.B and M.A contributed substantially to discussion of the content and reviewed and/or edited the manuscript before submission.

      Declaration of competing interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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