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Non-alcoholic fatty liver disease and cardiovascular risk: Pathophysiological mechanisms and implications

  • Sven M. Francque
    Correspondence
    Corresponding author at: Department of Gastroenterology and Hepatology, Antwerp University Hospital, Wilrijkstraat 10, B-2650 Edegem, Belgium. Tel.: +32 3 821 45 72; fax: +32 3 821 44 78.
    Affiliations
    Laboratory of Experimental Medicine and Paediatrics (LEMP) – Gastroenterology & Hepatology, University of Antwerp, Wilrijk (Antwerp), Belgium

    Department of Gastroenterology and Hepatology, Antwerp University Hospital, Edegem (Antwerp), Belgium
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  • Denise van der Graaff
    Affiliations
    Laboratory of Experimental Medicine and Paediatrics (LEMP) – Gastroenterology & Hepatology, University of Antwerp, Wilrijk (Antwerp), Belgium
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  • Wilhelmus J. Kwanten
    Affiliations
    Laboratory of Experimental Medicine and Paediatrics (LEMP) – Gastroenterology & Hepatology, University of Antwerp, Wilrijk (Antwerp), Belgium

    Department of Gastroenterology and Hepatology, Antwerp University Hospital, Edegem (Antwerp), Belgium
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Published:April 15, 2016DOI:https://doi.org/10.1016/j.jhep.2016.04.005

      Summary

      Non-alcoholic fatty liver disease (NAFLD) has become one of the most frequent chronic liver diseases in the Western society and its prevalence is likely to rise even further. An increasing body of evidence shows that NAFLD is not only a potentially progressive liver disease, but also has systemic consequences. More specifically, evidence points out that NAFLD has to be considered as a significant independent risk factor for subclinical and clinical cardiovascular disease (CVD). Long-term follow-up studies demonstrate cardiovascular mortality to be the most important cause of death in NAFLD patients. Moreover, ample evidence associates NAFLD with endothelial dysfunction, increased pulse wave velocity, increased coronary arterial calcifications and increased carotid intima media thickness, all established markers for CVD.
      Despite of all this evidence, the mechanisms by which NAFLD causally contributes to CVD are not fully elucidated. Furthermore, an extensive overview of all potential pathophysiological mechanisms and the corresponding current data are lacking. In this review we summarise current knowledge, originating from fundamental and clinical research, that mechanistically links NAFLD to CVD. Subsequently, the impact of CVD on current clinical practice and future research in the area of NALFD are discussed.

      Abbreviations:

      NAFLD (non-alcoholic fatty liver disease), CV (cardiovascular), CVD (cardiovascular disease), MetS (metabolic syndrome), AT (adipose tissue), TG (triglycerides), DM (diabetes mellitus), NASH (non-alcoholic steatohepatitis), CVRF (cardiovascular risk factor), cIMT (carotid intima media thickness), LV (left ventricle), ADMA (asymmetric dimethyl arginine), LDL (low-density lipoproteins), HDL (high-density lipoproteins,), VLDL (very low-density lipoproteins), NAFL (non-alcoholic fatty liver (also known as simple steatosis)), VEGF (vascular endothelial growth factor), CAD (coronary artery disease), PAI-1 (plasminogen inhibitor activator 1), hsCRP (high sensitive C-reactive protein), FetA (fetuine A), FGF21 (fibroblast growth factor 21), SeP (selenoprotein P), ANGPTL (angiopoietin like protein), TMA (trimethylamine), TMAO (trimethylamine-n-oxide), PNPLA3 (patatin-like phospholipase domain containing protein 3), TM6SF2 (transmembrane 6 superfamily member 2), SNP (single nucleotide polymorphism)

      Keywords

      Introduction

      Non-alcoholic fatty liver disease (NAFLD) has become a major cause of chronic liver disease in Western societies and will become the main underlying cause for liver transplantation within 10 years [
      • Charlton M.R.
      • Burns J.M.
      • Pedersen R.A.
      • Watt K.D.
      • Heimbach J.K.
      • Dierkhising R.A.
      Frequency and outcomes of liver transplantation for nonalcoholic steatohepatitis in the United States.
      ]. Although awareness amongst physicians has increased and the importance is recognised, levels of screening and referral to hepatologists in suspected NAFLD is low in primary care and non-hepatology specialties [
      • Said A.
      • Gagovic V.
      • Malecki K.
      • Givens M.L.
      • Nieto F.J.
      Primary care practitioners survey of non-alcoholic fatty liver disease.
      ,
      • Bergqvist C.-J.
      • Skoien R.
      • Horsfall L.
      • Clouston A.D.
      • Jonsson J.R.
      • Powell E.E.
      Awareness and opinions of non-alcoholic fatty liver disease by hospital specialists.
      ]. As a result, NAFLD is relatively underdiagnosed and long-term outcomes of hepatic and extrahepatic manifestations of NAFLD are compromised. Indeed, NAFLD is not only associated with increased liver-related morbidity and mortality, but also with increased mortality due to cardiovascular disease (CVD) and cancer [
      • Ekstedt M.
      • Hagström H.
      • Nasr P.
      • Fredrikson M.
      • Stål P.
      • Kechagias S.
      • et al.
      Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up.
      ,
      • Satapathy S.K.
      • Sanyal A.J.
      Epidemiology and natural history of nonalcoholic fatty liver disease.
      ].
      Increased cardiovascular mortality and morbidity are observed in NAFLD.
      The role of NAFLD as an independent cardiovascular risk factor is still debated. Several studies demonstrated unequivocally an increased cardiovascular (CV) mortality in NAFLD [
      • Ekstedt M.
      • Hagström H.
      • Nasr P.
      • Fredrikson M.
      • Stål P.
      • Kechagias S.
      • et al.
      Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up.
      ,
      • Söderberg C.
      • Stål P.
      • Askling J.
      • Glaumann H.
      • Lindberg G.
      • Marmur J.
      • et al.
      Decreased survival of subjects with elevated liver function tests during a 28-year follow-up.
      ]. Nevertheless, some studies failed to confirm this association, including two large cohort studies with long-term follow-up [
      • Lazo M.
      • Hernaez R.
      • Bonekamp S.
      • Kamel I.R.
      • Brancati F.L.
      • Guallar E.
      • et al.
      Non-alcoholic fatty liver disease and mortality among US adults: prospective cohort study.
      ,
      • Stepanova M.
      • Younossi Z.M.
      Independent association between nonalcoholic fatty liver disease and cardiovascular disease in the US population.
      ]. However, the data should be interpreted with caution because of several methodological issues, including retrospective diagnosis based on recorded ultrasound imaging or on biochemistry [
      • Lazo M.
      • Hernaez R.
      • Bonekamp S.
      • Kamel I.R.
      • Brancati F.L.
      • Guallar E.
      • et al.
      Non-alcoholic fatty liver disease and mortality among US adults: prospective cohort study.
      ], which is known to poorly correlate with histological NAFLD features [
      • Verma S.
      • Jensen D.
      • Hart J.
      • Mohanty S.R.
      Predictive value of ALT levels for non-alcoholic steatohepatitis (NASH) and advanced fibrosis in non-alcoholic fatty liver disease (NAFLD).
      ]. Even in the absence of a significant relation with CV mortality, CVD was still undoubtedly increased in NALFD patients compared to controls [
      • Stepanova M.
      • Younossi Z.M.
      Independent association between nonalcoholic fatty liver disease and cardiovascular disease in the US population.
      ], supporting the many convincing data that NAFLD independently contributes to (sub)clinical CVD.
      Distillation of NAFLD as a separate risk factor is impeded by overlap with other well-established risk factors for CVD, as they are also risk factors for NAFLD itself [
      • Satapathy S.K.
      • Sanyal A.J.
      Epidemiology and natural history of nonalcoholic fatty liver disease.
      ]. Assuming that NAFLD is a contributor to CVD implies the need for knowledge on the underlying pathophysiological mechanisms that explain how NAFLD independently impacts on CVD. An extensive overview of potential mechanisms is currently lacking.
      In this review we summarise knowledge, originating from animal research as well as translational and clinical research, about the underlying pathophysiological mechanisms that might link NAFLD to CVD. We subsequently discuss the potential implications of these findings for clinical management of patients with NAFLD and future research goals.

      The cardiovascular risk associated with NAFLD

      General considerations

      The specific contribution of NAFLD to increased CVD risk is, especially in clinical studies, difficult to dissect from the combination of risk factors that are shared by both NAFLD and CVD. The population of NAFLD patients is furthermore probably heterogeneous, in some of whom NAFLD is just part of and victim of the global metabolic derangement whilst in others, the liver is particularly involved in the pathophysiology of the Metabolic Syndrome (MetS) itself, and in the emergence of CVD and other complications [
      • Kotronen A.
      • Yki-Järvinen H.
      Fatty liver: a novel component of the metabolic syndrome.
      ,
      • Lonardo A.
      • Ballestri S.
      • Marchesini G.
      • Angulo P.
      • Loria P.
      Nonalcoholic fatty liver disease: a precursor of the metabolic syndrome.
      ]. Many patients will be somewhere in between, with the liver being diseased because of some metabolic abnormalities, and, once diseased, also contributing significantly to disease progression in terms of MetS, CVD and malignancies. This concept is fundamental in our understanding of NAFLD as part of a systemic disease.
      The mechanisms by which the liver might contribute are also complex and heterogeneous. The liver plays a crucial role in lipid and glucose homeostasis and is hence in the center of cardiometabolic disease. There is a very complex interplay between the gut, visceral and subcutaneous adipose tissues (AT), muscle tissues, the cardiovascular system and the liver [
      • Tilg H.
      • Moschen A.R.
      Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis.
      ].
      One of the starting points is most probably an imbalance in calorie-intake and expenditure, exceeding the storing-capacity of AT leading to deposition of ectopic fat, including the liver [
      • Heilbronn L.
      • Smith S.R.
      • Ravussin E.
      Failure of fat cell proliferation, mitochondrial function and fat oxidation results in ectopic fat storage, insulin resistance and type II diabetes mellitus.
      ].
      Once these mechanisms are initiated, a vicious circle starts, after which interplay between the different players is so complex that simple cause-effect relations become extremely difficult to assess (Fig. 1). As the liver is centrally positioned between different players, it is surprising that the mechanisms contribution to diabetes mellitus (DM) and CVD have gained so little attention. If it can be demonstrated that non-alcoholic steatohepatitis (NASH) livers play a pivotal role in that vicious circle, targeting the liver becomes attractive to break through the circle and halt metabolic and CVD progression.
      NAFLD is not only associated with, but also contributes to the pathogenesis of cardiovascular diseases.
      Figure thumbnail gr1
      Fig. 1Complex interplay of NAFLD and cardiovascular disease. The liver is centrally positioned in the metabolic syndrome, where NAFLD can be considered as the consequence of mechanism driven by the other components of the metabolic syndrome. However, reciprocal crosstalk exists, wherein the liver may actually drive diabetes mellitus or cardiovascular disease. These synergetic effects become more complex and create a vicious circle. NAFLD, non-alcoholic fatty liver disease.

      Determinants of outcomes in NAFLD: subclinical and clinical CVD data

      NAFLD encompasses a spectrum of liver diseases, ranging from non-alcoholic fatty liver (NAFL, also known as simple steatosis) over NASH and might lead to advanced fibrosis or cirrhosis and hepatocellular carcinoma (HCC). NAFLD is characterized by excessive fat accumulation in the hepatocytes (steatosis). When steatosis is accompanied by both hepatocellular ballooning degeneration and lobular inflammation, a diagnosis of NASH is made [
      • Kleiner D.E.
      • Brunt E.M.
      Nonalcoholic fatty liver disease: pathologic patterns and biopsy evaluation in clinical research.
      ]. The natural history of NAFLD and its different subtypes is not so well described, in part because the gold standard for the accurate diagnosis of NASH is liver biopsy, and large long-term follow data with repeated biopsies are scarce and should be interpreted with caution because, amongst others, of potential selection bias. Nevertheless, NAFLD is generally considered to run a benign course, with a low (but not completely absent) risk of fibrosis progression, whereas NASH has a significantly higher risk of progressive liver disease [
      • Nascimbeni F.
      • Pais R.
      • Bellentani S.
      • Day C.P.
      • Ratziu V.
      • Loria P.
      • et al.
      From NAFLD in clinical practice to answers from guidelines.
      ].
      This dichotomous concept has recently been challenged [
      • Adams L.A.
      • Ratziu V.
      Non-alcoholic fatty liver - perhaps not so benign.
      ]. Singh et al. [
      • Singh S.
      • Allen A.M.
      • Wang Z.
      • Prokop L.J.
      • Murad M.H.
      • Loomba R.
      Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies.
      ] systematically reviewed and performed a meta-analysis on 11 paired biopsy cohort studies. Although the majority of the patients had stable disease, it was shown that fibrosis progression occurred in both patients with NASH as well as NAFL (annually increase 0.14 and 0.07 fibrosis stage respectively). Moreover, a subset of patients was identified with considerable rapid fibrosis progression. Of note, progressors with baseline NAFL frequently had mild lobular inflammation or ballooning compared to non-progressors and although insufficient for the diagnosis of NASH, these subtle differences might explain their progression and are still in line with the concept of necro-inflammation being the driving force of disease progression [
      • Singh S.
      • Allen A.M.
      • Wang Z.
      • Prokop L.J.
      • Murad M.H.
      • Loomba R.
      Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies.
      ]. Another recent paired biopsy study also showed that 44% of the patients with NAFL progressed to NASH, and 37% to fibrosis (including some with progression to stage 3 fibrosis). Of note, at baseline NAFL patients were significantly younger compared to NASH patients, and most of the progressors out of the NAFL group had NASH at follow-up, and frequently had mild lobular inflammation at baseline. Development of type 2 DM was also an important determinant of progression [
      • McPherson S.
      • Hardy T.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      • Anstee Q.M.
      Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: implications for prognosis and clinical management.
      ].
      NAFLD is unambiguously related to increased liver-related and all-cause mortality [
      • Ekstedt M.
      • Hagström H.
      • Nasr P.
      • Fredrikson M.
      • Stål P.
      • Kechagias S.
      • et al.
      Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up.
      ,
      • Söderberg C.
      • Stål P.
      • Askling J.
      • Glaumann H.
      • Lindberg G.
      • Marmur J.
      • et al.
      Decreased survival of subjects with elevated liver function tests during a 28-year follow-up.
      ]. Importantly, CVD is the main cause of death in NAFLD patients (38% of all causes [
      • Angulo P.
      • Kleiner D.E.
      • Dam-Larsen S.
      • Adams L.A.
      • Bjornsson E.S.
      • Charatcharoenwitthaya P.
      • et al.
      Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease.
      ]), with baseline fibrosis being the strongest predictor [
      • Ekstedt M.
      • Hagström H.
      • Nasr P.
      • Fredrikson M.
      • Stål P.
      • Kechagias S.
      • et al.
      Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up.
      ,
      • Angulo P.
      • Kleiner D.E.
      • Dam-Larsen S.
      • Adams L.A.
      • Bjornsson E.S.
      • Charatcharoenwitthaya P.
      • et al.
      Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease.
      ]. Earlier studies suggested that the risk was higher in patients with NASH compared to NAFLD [
      • Ekstedt M.
      • Franzén L.E.
      • Mathiesen U.L.
      • Thorelius L.
      • Holmqvist M.
      • Bodemar G.
      • et al.
      Long-term follow-up of patients with NAFLD and elevated liver enzymes.
      ]. In a meta-analysis of 2011 mortality did not differ between NAFL and NASH, though the same analysis was inconclusive on potential differences in incident CVD [
      • Musso G.
      • Gambino R.
      • Cassader M.
      • Pagano G.
      Meta-analysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity.
      ]. Whilst NASH category as such was significantly associated with long-term outcomes [
      • Angulo P.
      • Kleiner D.E.
      • Dam-Larsen S.
      • Adams L.A.
      • Bjornsson E.S.
      • Charatcharoenwitthaya P.
      • et al.
      Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease.
      ], fibrosis remained the only prognostic histological feature in subsequent analyses [
      • Ekstedt M.
      • Hagström H.
      • Nasr P.
      • Fredrikson M.
      • Stål P.
      • Kechagias S.
      • et al.
      Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up.
      ,
      • Angulo P.
      • Kleiner D.E.
      • Dam-Larsen S.
      • Adams L.A.
      • Bjornsson E.S.
      • Charatcharoenwitthaya P.
      • et al.
      Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease.
      ].
      Although valuable information comes from these recent data, several methodological issues make it still difficult to accurately answer the question of the natural history of NAFLD and what drives its progression both as a liver disease and concerning the extrahepatic consequences. NAFLD is a systemic trait with a complex and multidirectional interplay with CVD and the MetS, all dynamic conditions that may substantially fluctuate between 2 time points of evaluation. Fluctuations in life style, body weight and glycaemic control might substantially impact on disease progression, which is probably far from linear. The placebo effect observed in the clinical trials conducted so far (as well as the percentage of regressors in the paired biopsy studies) also illustrate the dynamic nature of the disease, especially in milder cases [
      • Neuschwander-Tetri B.A.
      • Loomba R.
      • Sanyal A.J.
      • Lavine J.E.
      • Van Natta M.L.
      • Abdelmalek M.F.
      • et al.
      Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial.
      ,
      • Ratziu V.
      • Harrison S.
      • Francque S.
      • Bedossa P.
      • Lehert P.
      • Serfaty L.
      • et al.
      Elafibranor, an Agonist of the Peroxisome Proliferator-Activated Receptor-α and -δ, Induces Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsening.
      ]. Another aspect that deserves to be considered, is the way necro-inflammatory changes are assessed: histological analysis with routine stains is still the reference method used in scoring systems and diagnostic decision trees, but presumably not so very accurate to assess subtle activation of inflammatory cascades, especially in milder cases. The role of fibrosis as a prognostic marker also needs to be further clarified, as it is not clear whether this is to be considered a reflection of a median disease activity in the period preceding the baseline biopsy and hence an indirect prognostic factor (but presumably stronger than the actual disease activity because the latter only reflects the current status at the time of biopsy) or whether it is a direct prognostic factor by itself. Furthermore, fibrosis is assessed as a static parameter that does not accurately reflect the dynamics of pro- and antifibrogenic processes. The concept that inflammation is the main driver of fibrogenesis and disease progression [
      • Argo C.K.
      • Northup P.G.
      • Al-Osaimi A.M.S.
      • Caldwell S.H.
      Systematic review of risk factors for fibrosis progression in non-alcoholic steatohepatitis.
      ] is hence still important in defining target populations for screening and treatment.
      Besides mortality, evidence linking clinical CVD to NAFLD is scarce. Elevated liver enzymes are related to stroke [
      • Ying I.
      • Saposnik G.
      • Vermeulen M.J.
      • Leung A.
      • Ray J.G.
      Nonalcoholic fatty liver disease and acute ischemic stroke.
      ] and the prevalence of NAFLD in ST-elevated myocardial infarction is high [
      • Boddi M.
      • Tarquini R.
      • Chiostri M.
      • Marra F.
      • Valente S.
      • Giglioli C.
      • et al.
      Nonalcoholic fatty liver in nondiabetic patients with acute coronary syndromes.
      ]. In contrast to hard clinical endpoints, ample evidence links NAFLD to subclinical CVD, often independently of other well-established CV risk factors (CVRFs). Patients with NAFLD exhibit endothelial dysfunction of conducting vessels [
      • Villanova N.
      • Moscatiello S.
      • Ramilli S.
      • Bugianesi E.
      • Magalotti D.
      • Vanni E.
      • et al.
      Endothelial dysfunction and cardiovascular risk profile in nonalcoholic fatty liver disease.
      ], as well of the microvasculature [
      • Long M.T.
      • Wang N.
      • Larson M.G.
      • Mitchell G.F.
      • Palmisano J.
      • Vasan R.S.
      • et al.
      Nonalcoholic fatty liver disease and vascular function: cross-sectional analysis in the Framingham heart study.
      ]. Arterial stiffness is a well-accepted marker of CVD preceding arterial hypertension [
      • Kaess B.M.
      • Rong J.
      • Larson M.G.
      • Hamburg N.M.
      • Vita J.A.
      • Levy D.
      • et al.
      Aortic stiffness, blood pressure progression, and incident hypertension.
      ] and NAFLD is independently associated with increased vascular stiffness [
      • Long M.T.
      • Wang N.
      • Larson M.G.
      • Mitchell G.F.
      • Palmisano J.
      • Vasan R.S.
      • et al.
      Nonalcoholic fatty liver disease and vascular function: cross-sectional analysis in the Framingham heart study.
      ]. The carotid intimal media thickness (cIMT), a marker for generalised atherosclerotic burden, or the effective carotid plaque burden is also associated with NAFLD [
      • Madan S.A.
      • John F.
      • Pyrsopoulos N.
      • Pitchumoni C.S.
      Nonalcoholic fatty liver disease and carotid artery atherosclerosis in children and adults: a meta-analysis.
      ]. Finally, significant associations are observed of NAFLD with left ventricular (LV) diastolic dysfunction and LV mass [
      • Bonci E.
      • Chiesa C.
      • Versacci P.
      • Anania C.
      • Silvestri L.
      • Pacifico L.
      Association of nonalcoholic fatty liver disease with subclinical cardiovascular changes: a systematic review and meta-analysis.
      ,
      • VanWagner L.B.
      • Wilcox J.E.
      • Colangelo L.A.
      • Lloyd-Jones D.M.
      • Carr J.J.
      • Lima J.A.
      • et al.
      Association of nonalcoholic fatty liver disease with subclinical myocardial remodeling and dysfunction: a population-based study.
      ]. For further details about the (sub)clinical data we refer to already published extensive reviews [
      • Bhatia L.S.
      • Curzen N.P.
      • Calder P.C.
      • Byrne C.D.
      Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor?.
      ,
      • Oni E.T.
      • Agatston A.S.
      • Blaha M.J.
      • Fialkow J.
      • Cury R.
      • Sposito A.
      • et al.
      A systematic review: burden and severity of subclinical cardiovascular disease among those with nonalcoholic fatty liver; should we care?.
      ,
      • Luo J.
      • Xu L.
      • Li J.
      • Zhao S.
      Nonalcoholic fatty liver disease as a potential risk factor of cardiovascular disease.
      ,
      • Ballestri S.
      • Lonardo A.
      • Bonapace S.
      • Byrne C.D.
      • Loria P.
      • Targher G.
      Risk of cardiovascular, cardiac and arrhythmic complications in patients with non-alcoholic fatty liver disease.
      ,
      • Byrne C.D.
      • Targher G.
      NAFLD: a multisystem disease.
      ], since this review focusses on the potential mechanisms underlying this association.
      The contribution of NAFLD to cardiovascular diseases is independent of shared risk factors, e.g. obesity or diabetes mellitus.
      Several potential pathophysiological mechanisms are involved, including endothelial dysfunction, systemic inflammation, systemic oxidative imbalance, dyslipidaemia, organokines and genetic factors.

      Potential pathophysiological mechanisms

      The mechanisms explaining development of CVD in NAFLD are not completely understood. NAFLD is part of a complex multisystem disease with multiple bidirectional relationships. Moreover, each individual patient might exhibit a unique combination of causal mechanisms. The next section summarises the different potential mechanisms by which NAFLD may contribute to CVD (Fig. 2; Table 1). These mechanisms are rather complex, incompletely known and often interrelated.
      Figure thumbnail gr2
      Fig. 2Summary of potential pathophysiological mechanism responsible for increased CVD in NAFLD. NAFLD drives multiple mechanisms that ultimately lead to cardiovascular disease. These mechanisms are summarised in this figure. Genetic background, adipose tissue and the gut all contribute, in part via the liver (direct effects also exists but are not within the scope of this review). The details about these mechanisms are described in the text. Structural alterations of the cardiovascular system are marked in red. ANGPTL, angiopoietin like proteins; FetA, fetuin-A; FGF21, fibroblast growth factor 21; GIP, gastric inhibitory peptide; GLP-1, glucagon-like peptide 1; HDL, high-density lipoproteins; HMGB-1, high mobility group box 1; hsCRP, high sensitivity C-reactive protein; IL-1β, interleukin 1β; IL-6, interleukin 6; M1/M2, macrophage phenotype 1/2 ratio; OxLDL, oxidized low-density lipoprotein; PAI-1, plasminogen activator inhibitor 1; PNPLA3, patatin-like phospholipase domain containing protein 3; sdLDL, small dense low-density lipoproteins; SeP, selenoprotein P; SNP, single nucleotide polymorphism; TG, triglycerides; TM6SF2, transmembrane 6 superfamily member 2; TMA, trimethylamine; TMAO, trimethylamine-N-Oxide; TNF-α, tumor necrosis factor α; VEGF, vascular endothelial growth factor; VLDL, very low-density lipoproteins.
      Table 1Potential pathophysiological mechanism responsible for increased CVD in NAFLD. Non-alcoholic fatty liver disease (NAFLD) drives multiple mechanisms that ultimately may lead to cardiovascular disease. This table summarises the potential mechanisms by which the liver can influence CVD. Only mechanisms directly involved are incorporated in the table, e.g., adipokines are not included. See the text for more details about the tabulated mechanisms.
      ADMA, asymmetric dimethyl arginine; ANGPTL, angiopoietin like proteins; CCL3, Chemokine (C-C motif) ligand 3; cIMT, carotid intima media thickness; CAD, coronary artery disease; CVD, cardiovascular disease; EPC, endothelial progenitor cells; FGF21, fibroblast growth factor 21; FMO, flavin monooxygenase; HDL, high-density lipoproteins; HMGB-1, high mobility group box 1; hsCRP, high sensitivity C-reactive protein; IL-1β, interleukin 1β; IL-6, interleukin 6; LDL, low-density lipoproteins; LV, left ventricle; M1/M2, macrophage phenotype 1/2 ratio; NAFLD, non-alcoholic fatty liver disease; NAFL, non-alcoholic fatty liver; NASH, non-alcoholic steatohepatitis; OxLDL, oxidized low-density lipoprotein; PAI-1, plasminogen activator inhibitor 1; PNPLA3, patatin-like phospholipase domain containing protein 3; PWV, pulse wave velocity; sdLDL, small dense low-density lipoproteins; SeP, selenoprotein P; sICAM, soluble intercellular adhesion molecule-1; TGF-β, transforming growth factor β; TM6SF2, transmembrane 6 superfamily member 2; TMA, trimethylamine; TMAO, trimethylamine-N-Oxide; TNF-α, tumor necrosis factor α; VEGF, vascular endothelial growth factor; VLDL, very low-density lipoproteins.

      Structural alterations

      Hepatic microvasculature shows important alterations in case of NAFLD, with distortion of the sinusoidal pattern, occurrence of sinusoidal blebs, compression of sinusoids by fat-laden hepatocytes and loss of fenestrae [
      • Francque S.
      • Laleman W.
      • Verbeke L.
      • Van Steenkiste C.
      • Casteleyn C.
      • Kwanten W.
      • et al.
      Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture.
      ,
      • Farrell G.C.
      • Teoh N.C.
      • McCuskey R.S.
      Hepatic microcirculation in fatty liver disease.
      ,
      • Coulon S.
      • Legry V.
      • Heindryckx F.
      • Van Steenkiste C.
      • Casteleyn C.
      • Olievier K.
      • et al.
      Role of vascular endothelial growth factor in the pathophysiology of nonalcoholic steatohepatitis in two rodent models.
      ]. These effects occurred prior to development of inflammation and fibrosis, indicating an early event [
      • Francque S.
      • Laleman W.
      • Verbeke L.
      • Van Steenkiste C.
      • Casteleyn C.
      • Kwanten W.
      • et al.
      Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture.
      ]. These structural alterations are in part held responsible for the observed increase in portal pressure in non-cirrhotic NAFLD both in animals and humans [
      • Francque S.
      • Laleman W.
      • Verbeke L.
      • Van Steenkiste C.
      • Casteleyn C.
      • Kwanten W.
      • et al.
      Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture.
      ,
      • Francque S.
      • Verrijken A.
      • Mertens I.
      • Hubens G.
      • Van Marck E.
      • Pelckmans P.
      • et al.
      Noncirrhotic human nonalcoholic fatty liver disease induces portal hypertension in relation to the histological degree of steatosis.
      ]. The exact underlying pathophysiological mechanisms behind these alterations aren’t known, but may be explained by alterations in angiogenic factors.
      The arterial wall exists out of three layers, the intima, media and adventitia. In case of increased arterial stiffness, associated with NAFLD and CVD, the media of the large arteries is structurally altered: its collagen content and crosslinking increases, whereas elastin fibres decrease and become fractioned [
      • Gkaliagkousi E.
      • Douma S.
      The pathogenesis of arterial stiffness and its prognostic value in essential hypertension and cardiovascular diseases.
      ]. Levels of metalloproteinases, as well serum elastase are correlated to arterial stiffness [
      • Gkaliagkousi E.
      • Douma S.
      The pathogenesis of arterial stiffness and its prognostic value in essential hypertension and cardiovascular diseases.
      ]. Interestingly, the latter was increased in NAFLD [
      • Zang S.
      • Ma X.
      • Zhuang Z.
      • Liu J.
      • Bian D.
      • Xun Y.
      • et al.
      Increased ratio of neutrophil elastase to α1-antitrypsin is closely associated with liver inflammation in patients with nonalcoholic steatohepatitis.
      ]. Others speculate a potential role of TGF-β [
      • Sunbul M.
      • Agirbasli M.
      • Durmus E.
      • Kivrak T.
      • Akin H.
      • Aydin Y.
      • et al.
      Arterial stiffness in patients with non-alcoholic fatty liver disease is related to fibrosis stage and epicardial adipose tissue thickness.
      ].

      Endothelial dysfunction

      Impaired endothelial function is an early step in the process of atherosclerosis, ahead of development of fatty streaks or plaque inflammation [
      • Vanhoutte P.M.
      Endothelial dysfunction: the first step toward coronary arteriosclerosis.
      ] and hence crucial in CVD development.
      In cirrhosis, intrahepatic and mesenterial endothelial dysfunction are well known [
      • Iwakiri Y.
      • Shah V.
      • Rockey D.C.
      Vascular pathobiology in chronic liver disease and cirrhosis - current status and future directions.
      ]. Intrahepatic dysfunction is also described in NAFLD [
      • Maslak E.
      • Gregorius A.
      • Chlopicki S.
      Liver sinusoidal endothelial cells (LSECs) function and NAFLD; NO-based therapy targeted to the liver.
      ] but, intriguingly, in the absence of inflammation or fibrosis [
      • Francque S.
      • Laleman W.
      • Verbeke L.
      • Van Steenkiste C.
      • Casteleyn C.
      • Kwanten W.
      • et al.
      Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture.
      ,
      • Pasarín M.
      • Abraldes J.G.
      • Rodríguez-Vilarrupla A.
      • La Mura V.
      • García-Pagán J.C.
      • Bosch J.
      Insulin resistance and liver microcirculation in a rat model of early NAFLD.
      ,
      • Pasarín M.
      • La Mura V.
      • Gracia-Sancho J.
      • García-Calderó H.
      • Rodríguez-Vilarrupla A.
      • García-Pagán J.C.
      • et al.
      Sinusoidal endothelial dysfunction precedes inflammation and fibrosis in a model of NAFLD.
      ], indicating it’s an early event that might drive disease progression.
      Endothelial dysfunction of the systemic circulation was also observed in NAFLD, more pronounced in NASH [
      • Villanova N.
      • Moscatiello S.
      • Ramilli S.
      • Bugianesi E.
      • Magalotti D.
      • Vanni E.
      • et al.
      Endothelial dysfunction and cardiovascular risk profile in nonalcoholic fatty liver disease.
      ]. Asymmetric dimethyl arginine (ADMA) is an endogenous antagonist of nitric oxide synthase (NOS), positively linked to CVD. Reduced breakdown, in which the liver plays a dominant role [
      • Kasumov T.
      • Edmison J.M.
      • Dasarathy S.
      • Bennett C.
      • Lopez R.
      • Kalhan S.C.
      Plasma levels of asymmetric dimethylarginine in patients with biopsy-proven nonalcoholic fatty liver disease.
      ], is supposed to cause increased ADMA levels [
      • Deanfield J.E.
      • Halcox J.P.
      • Rabelink T.J.
      Endothelial function and dysfunction: testing and clinical relevance.
      ]. NAFLD patients exhibit increased levels of circulating ADMA, an association disappearing after correction for metabolic risk factors [
      • Kasumov T.
      • Edmison J.M.
      • Dasarathy S.
      • Bennett C.
      • Lopez R.
      • Kalhan S.C.
      Plasma levels of asymmetric dimethylarginine in patients with biopsy-proven nonalcoholic fatty liver disease.
      ,
      • Dogru T.
      • Genc H.
      • Tapan S.
      • Aslan F.
      • Ercin C.N.
      • Ors F.
      • et al.
      Plasma fetuin-A is associated with endothelial dysfunction and subclinical atherosclerosis in subjects with nonalcoholic fatty liver disease.
      ]. Other markers for endothelial dysfunction (e.g., endocan) were also increased in NAFLD [
      • Elsheikh E.
      • Younoszai Z.
      • Otgonsuren M.
      • Hunt S.
      • Raybuck B.
      • Younossi Z.M.
      Markers of endothelial dysfunction in patients with non-alcoholic fatty liver disease and coronary artery disease.
      ].
      An intact endothelial monolayer is important for normal vessel wall functioning. Disruption of this layer plays a role in atherogenesis and is characterized by increased levels of endothelial microparticles (EMPs), indicating endothelial disruption, and endothelial progenitor cells (EPCs), indicating endothelial repair [
      • Bruyndonckx L.
      • Hoymans V.Y.
      • Van Craenenbroeck A.H.
      • Vissers D.K.
      • Vrints C.J.
      • Ramet J.
      • et al.
      Assessment of endothelial dysfunction in childhood obesity and clinical use.
      ]. The levels of circulating EPCs were reduced in NAFLD and their adhesive function was attenuated [
      • Chiang C.-H.
      • Huang P.-H.
      • Chung F.-P.
      • Chen Z.-Y.
      • Leu H.-B.
      • Huang C.-C.
      • et al.
      Decreased circulating endothelial progenitor cell levels and function in patients with nonalcoholic fatty liver disease.
      ].

      Homocysteine and oxidative stress

      The liver is the main organ handling amino acids, including homocysteine. Elevated serum homocysteine is frequently reported in NAFLD [
      • Gulsen M.
      • Yesilova Z.
      • Bagci S.
      • Uygun A.
      • Ozcan A.
      • Ercin C.N.
      • et al.
      Elevated plasma homocysteine concentrations as a predictor of steatohepatitis in patients with non-alcoholic fatty liver disease.
      ,
      • Bravo E.
      • Palleschi S.
      • Aspichueta P.
      • Buqué X.
      • Rossi B.
      • Cano A.
      • et al.
      High fat diet-induced non alcoholic fatty liver disease in rats is associated with hyperhomocysteinemia caused by down regulation of the transsulphuration pathway.
      ,
      • de Carvalho S.C.R.
      • Muniz M.T.C.
      • Siqueira M.D.V.
      • Siqueira E.R.F.
      • Gomes A.V.
      • Silva K.A.
      • et al.
      Plasmatic higher levels of homocysteine in non-alcoholic fatty liver disease (NAFLD).
      ,
      • Franco Brochado M.J.
      • Domenici F.A.
      • Candolo Martinelli Ade L.
      • Zucoloto S.
      • de Carvalho da Cunha S.F.
      • Vannucchi H.
      Methylenetetrahydrofolate reductase gene polymorphism and serum homocysteine levels in nonalcoholic fatty liver disease.
      ,
      • Pastore A.
      • Alisi A.
      • di Giovamberardino G.
      • Crudele A.
      • Ceccarelli S.
      • Panera N.
      • et al.
      Plasma levels of homocysteine and cysteine increased in pediatric NAFLD and strongly correlated with severity of liver damage.
      ], though not consequently [
      • Hirsch S.
      • Poniachick J.
      • Avendaño M.
      • Csendes A.
      • Burdiles P.
      • Smok G.
      • et al.
      Serum folate and homocysteine levels in obese females with non-alcoholic fatty liver.
      ,
      • Kalhan S.C.
      • Edmison J.
      • Marczewski S.
      • Dasarathy S.
      • Gruca L.L.
      • Bennett C.
      • et al.
      Methionine and protein metabolism in non-alcoholic steatohepatitis: evidence for lower rate of transmethylation of methionine.
      ,
      • Polyzos S.A.
      • Kountouras J.
      • Patsiaoura K.
      • Katsiki E.
      • Zafeiriadou E.
      • Deretzi G.
      • et al.
      Serum homocysteine levels in patients with nonalcoholic fatty liver disease.
      ]. Levels of vitamin B12 or folic acid, elevating serum homocysteine when impaired, were not different. Interestingly, plasma homocysteine was lower in those with NASH compared to NAFL [
      • Pastore A.
      • Alisi A.
      • di Giovamberardino G.
      • Crudele A.
      • Ceccarelli S.
      • Panera N.
      • et al.
      Plasma levels of homocysteine and cysteine increased in pediatric NAFLD and strongly correlated with severity of liver damage.
      ,
      • Polyzos S.A.
      • Kountouras J.
      • Patsiaoura K.
      • Katsiki E.
      • Zafeiriadou E.
      • Deretzi G.
      • et al.
      Serum homocysteine levels in patients with nonalcoholic fatty liver disease.
      ]. Alterations of the homocysteine metabolism were reported, with reduced transsulfuration to cysteine and impaired remethylation to methionine [
      • Bravo E.
      • Palleschi S.
      • Aspichueta P.
      • Buqué X.
      • Rossi B.
      • Cano A.
      • et al.
      High fat diet-induced non alcoholic fatty liver disease in rats is associated with hyperhomocysteinemia caused by down regulation of the transsulphuration pathway.
      ,
      • Kalhan S.C.
      • Edmison J.
      • Marczewski S.
      • Dasarathy S.
      • Gruca L.L.
      • Bennett C.
      • et al.
      Methionine and protein metabolism in non-alcoholic steatohepatitis: evidence for lower rate of transmethylation of methionine.
      ,
      • Dahlhoff C.
      • Desmarchelier C.
      • Sailer M.
      • Fürst R.W.
      • Haag A.
      • Ulbrich S.E.
      • et al.
      Hepatic methionine homeostasis is conserved in C57BL/6N mice on high-fat diet despite major changes in hepatic one-carbon metabolism.
      ,
      • Pacana T.
      • Cazanave S.
      • Verdianelli A.
      • Patel V.
      • Min H.-K.
      • Mirshahi F.
      • et al.
      Dysregulated hepatic methionine metabolism drives homocysteine elevation in diet-induced nonalcoholic fatty liver disease.
      ] (although polymorphisms altering methylene tetrahydrofolate reductase (MTHFR) enzyme-function, part of the folate cycle that regulates remethylation of homocysteine, weren’t different [
      • de Carvalho S.C.R.
      • Muniz M.T.C.
      • Siqueira M.D.V.
      • Siqueira E.R.F.
      • Gomes A.V.
      • Silva K.A.
      • et al.
      Plasmatic higher levels of homocysteine in non-alcoholic fatty liver disease (NAFLD).
      ,
      • Franco Brochado M.J.
      • Domenici F.A.
      • Candolo Martinelli Ade L.
      • Zucoloto S.
      • de Carvalho da Cunha S.F.
      • Vannucchi H.
      Methylenetetrahydrofolate reductase gene polymorphism and serum homocysteine levels in nonalcoholic fatty liver disease.
      ]). As a result, oxidative stress within the liver increases (whilst faced with higher levels of beta-oxidation and reduced repletion of glutathione stores), contributing to NASH progression [
      • Bravo E.
      • Palleschi S.
      • Aspichueta P.
      • Buqué X.
      • Rossi B.
      • Cano A.
      • et al.
      High fat diet-induced non alcoholic fatty liver disease in rats is associated with hyperhomocysteinemia caused by down regulation of the transsulphuration pathway.
      ,
      • Kalhan S.C.
      • Edmison J.
      • Marczewski S.
      • Dasarathy S.
      • Gruca L.L.
      • Bennett C.
      • et al.
      Methionine and protein metabolism in non-alcoholic steatohepatitis: evidence for lower rate of transmethylation of methionine.
      ]. Intrahepatic vasculature is directly influenced by homocysteine, with impaired nitric oxide (NO) formation and increased intrahepatic vascular resistance [
      • Distrutti E.
      • Mencarelli A.
      • Santucci L.
      • Renga B.
      • Orlandi S.
      • Donini A.
      • et al.
      The methionine connection: homocysteine and hydrogen sulfide exert opposite effects on hepatic microcirculation in rats.
      ].
      Inborn hyperhomocysteinaemia is related with increased CV mortality, irrespective of the underlying genetic defect. Moreover, observational studies support plasma homocysteine levels as an independent CVRF [
      • Ganguly P.
      • Alam S.F.
      Role of homocysteine in the development of cardiovascular disease.
      ,
      • Santilli F.
      • Davì G.
      • Patrono C.
      Homocysteine, methylenetetrahydrofolate reductase, folate status and atherothrombosis: a mechanistic and clinical perspective.
      ]. Homocysteine causes oxidative stress, endothelial dysfunction, impairs redox status and enhances platelet activation, all contributing to CV effects [
      • Santilli F.
      • Davì G.
      • Patrono C.
      Homocysteine, methylenetetrahydrofolate reductase, folate status and atherothrombosis: a mechanistic and clinical perspective.
      ]. One study looking directly at associations of NAFLD with serum homocysteine levels and preclinical CVD showed an increased level of homocysteine, associated with elevated oxidative stress in NAFLD [
      • Leach N.V.
      • Dronca E.
      • Vesa S.C.
      • Sampelean D.P.
      • Craciun E.C.
      • Lupsor M.
      • et al.
      Serum homocysteine levels, oxidative stress and cardiovascular risk in non-alcoholic steatohepatitis.
      ]. The cIMT and plaque frequency increased in NAFLD. The cIMT was significantly correlated with reduced glutathione, though not directly with homocysteine. Intriguingly, the PIVENS trial evidenced lower levels of homocysteine after treatment [
      • Pacana T.
      • Cazanave S.
      • Verdianelli A.
      • Patel V.
      • Min H.-K.
      • Mirshahi F.
      • et al.
      Dysregulated hepatic methionine metabolism drives homocysteine elevation in diet-induced nonalcoholic fatty liver disease.
      ].
      Postprandial increase in serum oxidized low density lipoprotein (oxLDL) and large very low density lipoprotein (VLDL), with parallel decrease of total antioxidative status, is seen in NASH. This oxidative imbalance is related to liver fat content, liver injury and the degree of fibrosis [
      • Musso G.
      • Gambino R.
      • De Michieli F.
      • Biroli G.
      • Fagà E.
      • Pagano G.
      • et al.
      Association of liver disease with postprandial large intestinal triglyceride-rich lipoprotein accumulation and pro/antioxidant imbalance in normolipidemic non-alcoholic steatohepatitis.
      ]. Postprandial lipaemia is an established CVRF (see below) and an important source of oxidative stress [
      • Musso G.
      • Gambino R.
      • De Michieli F.
      • Biroli G.
      • Fagà E.
      • Pagano G.
      • et al.
      Association of liver disease with postprandial large intestinal triglyceride-rich lipoprotein accumulation and pro/antioxidant imbalance in normolipidemic non-alcoholic steatohepatitis.
      ]. Not only postprandial, but oxidative stress in general is increased in NASH [
      • Chalasani N.
      • Deeg M.A.
      • Crabb D.W.
      Systemic levels of lipid peroxidation and its metabolic and dietary correlates in patients with nonalcoholic steatohepatitis.
      ,
      • Madan K.
      • Bhardwaj P.
      • Thareja S.
      • Gupta S.D.
      • Saraya A.
      Oxidant stress and antioxidant status among patients with nonalcoholic fatty liver disease (NAFLD).
      ]. Oxidative stress is essential in CV pathophysiology [
      • Madamanchi N.R.
      • Vendrov A.
      • Runge M.S.
      Oxidative stress and vascular disease.
      ], thus all of the above may contribute to CVD development in NAFLD.

      Lipid profile

      The liver is a central regulator of whole body lipid metabolism by the combined action of de novo lipogenesis and breakdown of lipids, as well uptake and secretion of serum lipoproteins [
      • Neuschwander-Tetri B.A.
      Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites.
      ].
      Serum lipid profile correlates significantly with NAFLD severity, with more pronounced disturbances in NASH. Increased levels of triglycerides (TG) and LDL and decrease of high density lipoprotein (HDL) results in more atherogenic lipid ratios [
      • Alkhouri N.
      • Tamimi T.A.-R.
      • Yerian L.
      • Lopez R.
      • Zein N.N.
      • Feldstein A.E.
      The inflamed liver and atherosclerosis: a link between histologic severity of nonalcoholic fatty liver disease and increased cardiovascular risk.
      ]. These differences were less clear in more recent studies, but advanced analyses still reveal pro-atherogenic profiles. Moreover particle composition, subclasses, surface apolipoproteins and phospholipids are of increasing importance with respect to CVD risk [
      • Ouweneel A.B.
      • Van Eck M.
      Lipoproteins as modulators of atherothrombosis: from endothelial function to primary and secondary coagulation.
      ]. Small dense LDL particles (LDL3 and LDL4) were increased, whilst the large LDL particle LDL1 was decreased in NASH compared with NAFL [
      • Sonmez A.
      • Nikolic D.
      • Dogru T.
      • Ercin C.N.
      • Genc H.
      • Cesur M.
      • et al.
      Low- and high-density lipoprotein subclasses in subjects with nonalcoholic fatty liver disease.
      ]. Siddiqui et al. confirmed the relationship with small dense LDL accompanied with increased VLDL particles and impaired maturation of HDL. Larger VLDL particles impair lipoprotein lipase-mediated clearance, causing higher triglyceride-rich circulating remnants. Concurrently, the activity of sterol regulatory element-binding protein 1 was increased, fuelling cholesterol synthesis [
      • Siddiqui M.S.
      • Fuchs M.
      • Idowu M.O.
      • Luketic V.A.
      • Boyett S.
      • Sargeant C.
      • et al.
      Severity of nonalcoholic fatty liver disease and progression to cirrhosis are associated with atherogenic lipoprotein profile.
      ].
      Postprandial lipid profile is compatible with a more atherogenic profile through increased chylomicron remnants, more LDL and less HDL particles [
      • Roche H.M.
      • Gibney M.J.
      The impact of postprandial lipemia in accelerating atherothrombosis.
      ]. In patients with NAFLD this postprandial mechanism is accentuated with higher levels of triglyceride-rich and enlarged VLDL particles [
      • Cassader M.
      • Gambino R.
      • Musso G.
      • Depetris N.
      • Mecca F.
      • Cavallo-Perin P.
      • et al.
      Postprandial triglyceride-rich lipoprotein metabolism and insulin sensitivity in nonalcoholic steatohepatitis patients.
      ,
      • Musso G.
      • Gambino R.
      • De Michieli F.
      • Durazzo M.
      • Pagano G.
      • Cassader M.
      Adiponectin gene polymorphisms modulate acute adiponectin response to dietary fat: possible pathogenetic role in NASH.
      ].

      Angiogenic factors

      Centrozonal arteries and microvessels are a common finding in NAFLD, even without advanced fibrosis. These alterations are indicative of active angiogenesis, as part of vascular remodelling, in early stages of NAFLD [
      • Gill R.M.
      • Belt P.
      • Wilson L.
      • Bass N.M.
      • Ferrell L.D.
      Centrizonal arteries and microvessels in nonalcoholic steatohepatitis.
      ]. In line, anti-VEGFR2 treatment was able to improve steatosis and inflammation in mouse models [
      • Coulon S.
      • Legry V.
      • Heindryckx F.
      • Van Steenkiste C.
      • Casteleyn C.
      • Olievier K.
      • et al.
      Role of vascular endothelial growth factor in the pathophysiology of nonalcoholic steatohepatitis in two rodent models.
      ].
      Coulon et al. demonstrated increased serum levels of VEGF in NAFL and NASH compared to controls [
      • Coulon S.
      • Francque S.
      • Colle I.
      • Verrijken A.
      • Blomme B.
      • Heindryckx F.
      • et al.
      Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease.
      ]. Other studies showed no differences [
      • Yilmaz Y.
      • Yonal O.
      • Kurt R.
      • Alahdab Y.O.
      • Ozdogan O.
      • Celikel C.A.
      • et al.
      Circulating levels of vascular endothelial growth factor A and its soluble receptor in patients with biopsy-proven nonalcoholic fatty liver disease.
      ] or only an increase in NASH [
      • Tarantino G.
      • Conca P.
      • Pasanisi F.
      • Ariello M.
      • Mastrolia M.
      • Arena A.
      • et al.
      Could inflammatory markers help diagnose nonalcoholic steatohepatitis?.
      ] compared to controls. Serum sVEGFR1 showed the same trend with comparable increase in NAFL and NASH [
      • Coulon S.
      • Francque S.
      • Colle I.
      • Verrijken A.
      • Blomme B.
      • Heindryckx F.
      • et al.
      Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease.
      ]. Others reported lowered serum levels of sVEGFR1 [
      • Yilmaz Y.
      • Yonal O.
      • Kurt R.
      • Alahdab Y.O.
      • Ozdogan O.
      • Celikel C.A.
      • et al.
      Circulating levels of vascular endothelial growth factor A and its soluble receptor in patients with biopsy-proven nonalcoholic fatty liver disease.
      ]. Finally, the levels of sVEGFR2 weren’t different amongst the groups [
      • Coulon S.
      • Francque S.
      • Colle I.
      • Verrijken A.
      • Blomme B.
      • Heindryckx F.
      • et al.
      Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease.
      ]. At a transcriptional level only NAFL and NASH could be compared, showing increased expression of VEGF and VEGFR2 in livers with NAFL compared to NASH [
      • Coulon S.
      • Francque S.
      • Colle I.
      • Verrijken A.
      • Blomme B.
      • Heindryckx F.
      • et al.
      Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease.
      ], but fold-changes in expression were rather small. Other studies using a threshold of ⩾2 times fold-changes failed to demonstrate significant differences [
      • Cayón A.
      • Crespo J.
      • Guerra A.R.
      • Pons-Romero F.
      Gene expression in obese patients with non-alcoholic steatohepatitis.
      ].
      VEGF-A and other VEGF-family members are also implied in CV pathophysiology. Significant associations indicate an active role in atherogenesis and plaque instability, thus contributing to plaque formation and vulnerability [
      • Khurana R.
      • Simons M.
      • Martin J.F.
      • Zachary I.C.
      Role of angiogenesis in cardiovascular disease: a critical appraisal.
      ,
      • Ylä-Herttuala S.
      • Rissanen T.T.
      • Vajanto I.
      • Hartikainen J.
      Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine.
      ]. The increased levels of VEGF in NAFLD can therefore link the 2 conditions. However, the role of VEGF in atherosclerogenesis is challenged lately, with some negative studies in animal and clinical settings. Local concentrations and not systemic concentrations might explain this discrepancy [
      • Ylä-Herttuala S.
      • Rissanen T.T.
      • Vajanto I.
      • Hartikainen J.
      Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine.
      ].
      High mobility group box 1 (HMGB-1) is a molecule with diverse functions, amongst which induction of angiogenesis [
      • Chen R.
      • Hou W.
      • Zhang Q.
      • Kang R.
      • Fan X.-G.
      • Tang D.
      Emerging role of high-mobility group box 1 (HMGB1) in liver diseases.
      ]. It showed to be capable of inducing liver damage and systemic inflammation [
      • Li L.
      • Chen L.
      • Hu L.
      • Liu Y.
      • Sun H.-Y.
      • Tang J.
      • et al.
      Nuclear factor high-mobility group box1 mediating the activation of Toll-like receptor 4 signaling in hepatocytes in the early stage of nonalcoholic fatty liver disease in mice.
      ]. Moreover, it’s associated with plaque burden in coronary artery disease (CAD) [
      • Andrassy M.
      • Volz H.C.
      • Maack B.
      • Schuessler A.
      • Gitsioudis G.
      • Hofmann N.
      • et al.
      HMGB1 is associated with atherosclerotic plaque composition and burden in patients with stable coronary artery disease.
      ]. Interestingly, in a small study HMGB1 levels in patients with NAFLD and CAD were actually reduced compared to those without CAD [
      • Elsheikh E.
      • Younoszai Z.
      • Otgonsuren M.
      • Hunt S.
      • Raybuck B.
      • Younossi Z.M.
      Markers of endothelial dysfunction in patients with non-alcoholic fatty liver disease and coronary artery disease.
      ].

      Haemostasis

      The liver is an important and sometimes exclusive source of both pro- and anticoagulant factors [
      • Tripodi A.
      • Mannucci P.M.
      The coagulopathy of chronic liver disease.
      ]. Alterations of these factors have previously been extensively studied in relation to cirrhosis, obesity and MetS, but less in NAFLD.
      Most of the studies have low patient numbers or lack histologic diagnosis. Nevertheless, they all point towards an increase in prothrombotic factors even after correction for other cardiometabolic risk factors. Hypercoagulability can be linked to atherosclerosis and CVD [
      • Loeffen R.
      • Spronk H.M.H.
      • ten Cate H.
      The impact of blood coagulability on atherosclerosis and cardiovascular disease.
      ]. The factors VIII, IX, XI and XII were positively correlated to hepatic fat content [
      • Kotronen A.
      • Joutsi-Korhonen L.
      • Sevastianova K.
      • Bergholm R.
      • Hakkarainen A.
      • Pietiläinen K.H.
      • et al.
      Increased coagulation factor VIII, IX, XI and XII activities in non-alcoholic fatty liver disease.
      ].
      The strongest evidence exists regarding plasminogen inhibitor activator 1 (PAI-1). Circulating levels were positively related to hepatic fat content or NASH [
      • Alessi M.-C.
      • Bastelica D.
      • Mavri A.
      • Morange P.
      • Berthet B.
      • Grino M.
      • et al.
      Plasma PAI-1 levels are more strongly related to liver steatosis than to adipose tissue accumulation.
      ,
      • Barbato A.
      • Iacone R.
      • Tarantino G.
      • Russo O.
      • Sorrentino P.
      • Avallone S.
      • et al.
      Relationships of PAI-1 levels to central obesity and liver steatosis in a sample of adult male population in southern Italy.
      ,
      • Sookoian S.
      • Castaño G.O.
      • Burgueño A.L.
      • Rosselli M.S.
      • Gianotti T.F.
      • Mallardi P.
      • et al.
      Circulating levels and hepatic expression of molecular mediators of atherosclerosis in nonalcoholic fatty liver disease.
      ,
      • Verrijken A.
      • Francque S.
      • Mertens I.
      • Prawitt J.
      • Caron S.
      • Hubens G.
      • et al.
      Prothrombotic factors in histologically proven nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
      ]. Verrijken et al. analysed a large cohort and proved that fibrinogen, factor VII, von Willebrand factor and PAI-1 were increased, whilst antithrombin III was decreased. However, only PAI-1 levels were independently related to NAFLD. Furthermore, PAI-1 serum levels as well as hepatic expression were significantly correlated with histological severity of NAFLD [
      • Verrijken A.
      • Francque S.
      • Mertens I.
      • Prawitt J.
      • Caron S.
      • Hubens G.
      • et al.
      Prothrombotic factors in histologically proven nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
      ]. The increase in PAI-1 expression in NASH strongly supports the concept of increased secretion of PAI-1 in NASH [
      • Sookoian S.
      • Castaño G.O.
      • Burgueño A.L.
      • Rosselli M.S.
      • Gianotti T.F.
      • Mallardi P.
      • et al.
      Circulating levels and hepatic expression of molecular mediators of atherosclerosis in nonalcoholic fatty liver disease.
      ,
      • Verrijken A.
      • Francque S.
      • Mertens I.
      • Prawitt J.
      • Caron S.
      • Hubens G.
      • et al.
      Prothrombotic factors in histologically proven nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
      ].
      At present, aside from many correlations of prothrombotic factors with NAFLD, there is scarce epidemiological evidence to connect NAFLD directly to thrombosis in absence of cirrhosis [
      • Northup P.G.
      • Argo C.K.
      • Shah N.
      • Caldwell S.H.
      Hypercoagulation and thrombophilia in nonalcoholic fatty liver disease: mechanisms, human evidence, therapeutic implications, and preventive implications.
      ]. Nonetheless, Tripodi et al. elegantly demonstrated a procoagulant imbalance in NAFLD, correlating with disease severity, using advanced coagulation assays. Decreased protein C activity and increased levels of factor VIII are supposed to be responsible [
      • Tripodi A.
      • Fracanzani A.L.
      • Primignani M.
      • Chantarangkul V.
      • Clerici M.
      • Mannucci P.M.
      • et al.
      Procoagulant imbalance in patients with non-alcoholic fatty liver disease.
      ].
      Whole blood viscosity and related haemorheological factors are related with increased CVD [
      • Toth A.
      • Papp J.
      • Rabai M.
      • Kenyeres P.
      • Marton Z.
      • Kesmarky G.
      • et al.
      The role of hemorheological factors in cardiovascular medicine.
      ], probably due to compromised delivery of substrates and oxygen to tissues. Increased blood viscosity, in part because of dyslipidaemia, was also linked to NAFLD [
      • Yu K.
      • Zhang M.
      • Li Y.
      • Wang R.
      Increased whole blood viscosity associated with arterial stiffness in patients with non-alcoholic fatty liver disease.
      ,
      • Zhao H.-Y.
      • Li J.
      • Xu M.
      • Wang T.-G.
      • Sun W.-W.
      • Chen Y.
      • et al.
      Elevated whole blood viscosity is associated with insulin resistance and non-alcoholic fatty liver.
      ] and provide an additional link to CVD.

      Inflammation and cytokines

      The liver contains the largest number of residential macrophages and contains high numbers of immune cells [
      • Racanelli V.
      • Rehermann B.
      The liver as an immunological organ.
      ]. It’s conceivable that cytokines secreted by the diseased liver drain into the systemic circulation and cause secondary CV effects. Systemic inflammation and circulating cyto- and chemokines are associated with CVD [
      • Stoner L.
      • Lucero A.A.
      • Palmer B.R.
      • Jones L.M.
      • Young J.M.
      • Faulkner J.
      Inflammatory biomarkers for predicting cardiovascular disease.
      ]. Inflammation fuels CVD via endothelial dysfunction, altered vascular tone, enhanced plaque formation and coagulation [
      • Kofler S.
      • Nickel T.
      • Weis M.
      Role of cytokines in cardiovascular diseases: a focus on endothelial responses to inflammation.
      ]. Interestingly, 18 genes were significantly differently expressed in NASH compared to NAFL, all of which were linkable to inflammation and/or plaque formation [
      • Sookoian S.
      • Gianotti T.F.
      • Rosselli M.S.
      • Burgueño A.L.
      • Castaño G.O.
      • Pirola C.J.
      Liver transcriptional profile of atherosclerosis-related genes in human nonalcoholic fatty liver disease.
      ].
      The observed increased hepaticovenous pressure gradient in non-cirrhotic NAFLD was positively associated with IL-1β [
      • Vonghia L.
      • Magrone T.
      • Verrijken A.
      • Michielsen P.
      • Van Gaal L.
      • Jirillo E.
      • et al.
      Peripheral and hepatic vein cytokine levels in correlation with non-alcoholic fatty liver disease (NAFLD)-related metabolic, histological, and haemodynamic features.
      ]. In NAFLD, hepatic venous blood (representing the outflow tract of the liver) showed a higher M1/2 macrophage ratio (hence a more inflammatory pattern) than systemic blood, correlated with a higher hepaticovenous pressure gradient and hence affected vascular function, at least within the liver. Hepaticovenous levels of several cytokines (IL-6, IL1β, TNFα, IL10/IL17 ratio) also correlated with more disturbed parameters of glucose metabolism [
      • Vonghia L.
      • Magrone T.
      • Verrijken A.
      • Michielsen P.
      • Van Gaal L.
      • Jirillo E.
      • et al.
      Peripheral and hepatic vein cytokine levels in correlation with non-alcoholic fatty liver disease (NAFLD)-related metabolic, histological, and haemodynamic features.
      ].
      Increased circulating markers for systemic inflammation are associated with NAFLD. This link is most pronounced in NASH. Levels of IL-6 were increased in line with histological severity [
      • Coulon S.
      • Legry V.
      • Heindryckx F.
      • Van Steenkiste C.
      • Casteleyn C.
      • Olievier K.
      • et al.
      Role of vascular endothelial growth factor in the pathophysiology of nonalcoholic steatohepatitis in two rodent models.
      ,
      • Wieckowska A.
      • Papouchado B.G.
      • Li Z.
      • Lopez R.
      • Zein N.N.
      • Feldstein A.E.
      Increased hepatic and circulating interleukin-6 levels in human nonalcoholic steatohepatitis.
      ,
      • Hamirani Y.S.
      • Katz R.
      • Nasir K.
      • Zeb I.
      • Blaha M.J.
      • Blumenthal R.S.
      • et al.
      Association between inflammatory markers and liver fat: The Multi-Ethnic Study of Atherosclerosis.
      ], though not consequently [
      • du Plessis J.
      • van Pelt J.
      • Korf H.
      • Mathieu C.
      • van der Schueren B.
      • Lannoo M.
      • et al.
      Association of adipose tissue inflammation with histologic severity of nonalcoholic fatty liver disease.
      ]. Hepatic expression of IL-6 was also related to NAFLD, though lost significance when adding metabolic risk factors [
      • Wieckowska A.
      • Papouchado B.G.
      • Li Z.
      • Lopez R.
      • Zein N.N.
      • Feldstein A.E.
      Increased hepatic and circulating interleukin-6 levels in human nonalcoholic steatohepatitis.
      ]. Similarly, serum levels and expression of hsCRP were elevated in relation with disease severity [
      • Hamirani Y.S.
      • Katz R.
      • Nasir K.
      • Zeb I.
      • Blaha M.J.
      • Blumenthal R.S.
      • et al.
      Association between inflammatory markers and liver fat: The Multi-Ethnic Study of Atherosclerosis.
      ,
      • Yoneda M.
      • Mawatari H.
      • Fujita K.
      • Iida H.
      • Yonemitsu K.
      • Kato S.
      • et al.
      High-sensitivity C-reactive protein is an independent clinical feature of nonalcoholic steatohepatitis (NASH) and also of the severity of fibrosis in NASH.
      ]. Finally, serum levels of TNFα, CCL3 and sICAM-1 were positively related with NAFLD [
      • Sookoian S.
      • Castaño G.O.
      • Burgueño A.L.
      • Rosselli M.S.
      • Gianotti T.F.
      • Mallardi P.
      • et al.
      Circulating levels and hepatic expression of molecular mediators of atherosclerosis in nonalcoholic fatty liver disease.
      ,
      • du Plessis J.
      • van Pelt J.
      • Korf H.
      • Mathieu C.
      • van der Schueren B.
      • Lannoo M.
      • et al.
      Association of adipose tissue inflammation with histologic severity of nonalcoholic fatty liver disease.
      ]. Hepatic expression of TNF, CXCL10 and IL1RN were congruently elevated [
      • Wolfs M.G.M.
      • Gruben N.
      • Rensen S.S.
      • Verdam F.J.
      • Greve J.W.
      • Driessen A.
      • et al.
      Determining the association between adipokine expression in multiple tissues and phenotypic features of non-alcoholic fatty liver disease in obesity.
      ].
      Taking together, these data provide evidence that the liver is indeed directly contributing to the observed systemic inflammation that affects the CV system at distance.

      Hepatokines

      Hepatokines are a type of organokines (proteins exclusively/predominantly produced by and secreted from a specific tissue) that are less well known than adipokines. Organokines are not only simple markers of their tissue of origin, but can exert active para- and endocrine effects. Several hepatokines have been described and the majority influences insulin sensitivity, although some of them showed to have CV effects [
      • Stefan N.
      • Häring H.-U.
      The role of hepatokines in metabolism.
      ].
      Fetuin-A (FetA) is a hepatokine that inhibits the insulin receptor tyrosine kinase in liver and skeletal muscle causing insulin resistance [
      • Stefan N.
      • Häring H.-U.
      The role of hepatokines in metabolism.
      ] and it is capable of inducing the expression of inflammatory cytokines and inhibits adiponectin [
      • Hennige A.M.
      • Staiger H.
      • Wicke C.
      • Machicao F.
      • Fritsche A.
      • Häring H.-U.
      • et al.
      Fetuin-A induces cytokine expression and suppresses adiponectin production.
      ]. The expression of FetA is linked with that of key enzymes of lipid and glucose metabolism. NAFLD is related with higher expression, transcription and serum levels of FetA [
      • Haukeland J.W.
      • Dahl T.B.
      • Yndestad A.
      • Gladhaug I.P.
      • Løberg E.M.
      • Haaland T.
      • et al.
      Fetuin A in nonalcoholic fatty liver disease: in vivo and in vitro studies.
      ,
      • Kahraman A.
      • Sowa J.-P.
      • Schlattjan M.
      • Sydor S.
      • Pronadl M.
      • Wree A.
      • et al.
      Fetuin-A mRNA expression is elevated in NASH compared with NAFL patients.
      ], and levels in NASH are the highest [
      • Kahraman A.
      • Sowa J.-P.
      • Schlattjan M.
      • Sydor S.
      • Pronadl M.
      • Wree A.
      • et al.
      Fetuin-A mRNA expression is elevated in NASH compared with NAFL patients.
      ]. Since myocardial infarction and stroke are also related with FetA [
      • Weikert C.
      • Stefan N.
      • Schulze M.B.
      • Pischon T.
      • Berger K.
      • Joost H.-G.
      • et al.
      Plasma fetuin-a levels and the risk of myocardial infarction and ischemic stroke.
      ], FetA can be a causative link between NASH and CVD.
      Sato et al. showed cIMT and endothelial dysfunction to be positively related to FetA levels. In contrast with others, FetA levels were, however, not different between controls and NAFLD. Contrariwise, cIMT was even negatively correlated with FetA levels in NAFLD, as was the degree of liver fibrosis [
      • Sato M.
      • Kamada Y.
      • Takeda Y.
      • Kida S.
      • Ohara Y.
      • Fujii H.
      • et al.
      Fetuin-A negatively correlates with liver and vascular fibrosis in nonalcoholic fatty liver disease subjects.
      ], which could be explained by the inhibitory effects of FetA on TGF-β1 signalling. The authors suggest a dual role of FetA, with a kind of self-defensive mechanism with respect to fibrosis, whilst a positive trend between FetA and steatohepatitis was seen. This inverse relation with fibrosis wasn’t reconfirmed in a biopsy-proven series, a discrepancy attributed to the outweighing of inflammation in NASH in case of lower degrees of fibrosis [
      • Kamada Y.
      • Miyoshi E.
      Value of fetuin-A as a predictor of liver fibrosis in patients with nonalcoholic fatty liver disease.
      ]. Finally, and in line with a dual role of FetA, NAFLD could be independently associated with FetA, whilst the risk of coronarographically diagnosed CAD decreased [
      • Ballestri S.
      • Meschiari E.
      • Baldelli E.
      • Musumeci F.E.
      • Romagnoli D.
      • Trenti T.
      • et al.
      Relationship of serum fetuin-A levels with coronary atherosclerotic burden and NAFLD in patients undergoing elective coronary angiography.
      ]. More recent analyses confirm the conflicting CV protective and harmful role of FetA, potentially explained by the balance between promoting a CVD risk profile and the capability to decrease vascular calcifications [
      • Jensen M.K.
      • Bartz T.M.
      • Mukamal K.J.
      • Djoussé L.
      • Kizer J.R.
      • Tracy R.P.
      • et al.
      Fetuin-A, type 2 diabetes, and risk of cardiovascular disease in older adults: the cardiovascular health study.
      ].
      Fibroblast growth factor 21 (FGF21) has beneficial effects on insulin sensitivity and cholesterol. Despite its favourable effects, increased levels are strongly associated with increased CVD, potentially reflecting an adaptive hypersecretion to FGF21 resistance [
      • Domouzoglou E.M.
      • Naka K.K.
      • Vlahos A.P.
      • Papafaklis M.I.
      • Michalis L.K.
      • Tsatsoulis A.
      • et al.
      Fibroblast growth factors in cardiovascular disease: The emerging role of FGF21.
      ]. In NAFLD both increased serum levels and hepatic expression of FGF21 were seen [
      • Li H.
      • Fang Q.
      • Gao F.
      • Fan J.
      • Zhou J.
      • Wang X.
      • et al.
      Fibroblast growth factor 21 levels are increased in nonalcoholic fatty liver disease patients and are correlated with hepatic triglyceride.
      ,
      • Dushay J.
      • Chui P.C.
      • Gopalakrishnan G.S.
      • Varela-Rey M.
      • Crawley M.
      • Fisher F.M.
      • et al.
      Increased fibroblast growth factor 21 in obesity and nonalcoholic fatty liver disease.
      ], correlating with the degree of steatosis [
      • Li H.
      • Fang Q.
      • Gao F.
      • Fan J.
      • Zhou J.
      • Wang X.
      • et al.
      Fibroblast growth factor 21 levels are increased in nonalcoholic fatty liver disease patients and are correlated with hepatic triglyceride.
      ]. In a coronarographic study both NAFLD and CAD were positively associated with FGF21, albeit this association was independent of NAFLD [
      • Shen Y.
      • Ma X.
      • Zhou J.
      • Pan X.
      • Hao Y.
      • Zhou M.
      • et al.
      Additive relationship between serum fibroblast growth factor 21 level and coronary artery disease.
      ].
      Selenoprotein P (SeP) is increased in NAFLD, and the risk of NAFL increases 7.5 times in the highest plasma level tertile in non-diabetics. Moreover, SeP is positively correlated with insulin resistance, hsCRP and arterial stiffness [
      • Choi H.Y.
      • Hwang S.Y.
      • Lee C.H.
      • Hong H.C.
      • Yang S.J.
      • Yoo H.J.
      • et al.
      Increased selenoprotein p levels in subjects with visceral obesity and nonalcoholic Fatty liver disease.
      ] or cIMT [
      • Yang S.J.
      • Hwang S.Y.
      • Choi H.Y.
      • Yoo H.J.
      • Seo J.A.
      • Kim S.G.
      • et al.
      Serum selenoprotein P levels in patients with type 2 diabetes and prediabetes: implications for insulin resistance, inflammation, and atherosclerosis.
      ]. The close relationship with glucose handling and associated inflammation might explain the effects on CVD. A longitudinal follow-up after bariatric surgery in 10 patients unexpectedly showed decrease of ANGPTL6 (see below) and increase of SeP after 9 months, making the role of SeP more complex. The simultaneous antioxidative properties of SeP and potential U-shaped relationship between SeP and DM may explain this paradox [
      • Lim J.
      • Park H.S.
      • Lee S.K.
      • Jang Y.J.
      • Lee Y.J.
      • Heo Y.
      Longitudinal changes in serum levels of angiopoietin-like protein 6 and selenoprotein P after gastric bypass surgery.
      ].
      Angiopoietin like proteins (ANGPTL) are considered orphan ligands, because they don’t bind to receptors targeted by angiopoietins. They have pleiotropic effects, including angiogenic properties and influence on glucose and lipid metabolism. Some of these ANGPTLs are mainly produced by the liver [
      • Katagiri H.
      • Yamada T.
      • Oka Y.
      Adiposity and cardiovascular disorders: disturbance of the regulatory system consisting of humoral and neuronal signals.
      ,
      • Santulli G.
      Angiopoietin-like proteins: a comprehensive look.
      ]. Besides metabolic linkage, ANGTPL’s are associated with subclinical CVD [
      • Hatsuda S.
      • Shoji T.
      • Shinohara K.
      • Kimoto E.
      • Mori K.
      • Fukumoto S.
      • et al.
      Association between plasma angiopoietin-like protein 3 and arterial wall thickness in healthy subjects.
      ,
      • Adachi H.
      • Fujiwara Y.
      • Kondo T.
      • Nishikawa T.
      • Ogawa R.
      • Matsumura T.
      • et al.
      Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis.
      ,
      • Jung C.H.
      • Lee W.J.
      • Lee M.J.
      • Kang Y.M.
      • Jang J.E.
      • Leem J.
      • et al.
      Association of serum angiopoietin-like protein 2 with carotid intima-media thickness in subjects with type 2 diabetes.
      ]. Interestingly, patients with NASH, not NAFL, have higher levels of ANGTPL3 [
      • Yilmaz Y.
      • Ulukaya E.
      • Atug O.
      • Dolar E.
      Serum concentrations of human angiopoietin-like protein 3 in patients with nonalcoholic fatty liver disease: association with insulin resistance.
      ].

      Adipokines

      The adipokines are produced by white AT, which is nowadays recognised as an active metabolic player and no longer as a simple storage body compartment. The presence of alterations in circulating adipokines (decreased adiponectin, increased leptin) in MetS or NAFLD is hence not surprising. Adipokines are implicated in both NAFLD and CVD pathogenesis. Nonetheless, since the liver isn’t directly involved in their production and since systemic levels are rather similar or lower than portal levels [
      • Fontana L.
      • Eagon J.C.
      • Trujillo M.E.
      • Scherer P.E.
      • Klein S.
      Visceral fat adipokine secretion is associated with systemic inflammation in obese humans.
      ,
      • Faber D.R.
      • Moll F.L.
      • Vink A.
      • van der Waal C.
      • Kalkhoven E.
      • Schipper H.S.
      • et al.
      Adipose tissue quantity and composition contribute to adipokine concentrations in the subclavian vein and the inferior mesenteric vein.
      ,
      • Karbaschian Z.
      • Hosseinzadeh-Attar M.J.
      • Giahi L.
      • Golpaie A.
      • Masoudkabir F.
      • Talebpour M.
      • et al.
      Portal and systemic levels of visfatin in morbidly obese subjects undergoing bariatric surgery.
      ], the role of adipokines in the link between NAFLD and CVD seems minor. Publications on other adipokines, e.g., visfatin or resistin, are in light of CVD in NAFLD limited. Similarly, there is limited knowledge of the effects of brown AT on CVD.

      Gut-liver axis

      Intestinal dysbiosis is related to the aetiology of NAFLD and progression to NASH [
      • Zhu L.
      • Baker S.S.
      • Gill C.
      • Liu W.
      • Alkhouri R.
      • Baker R.D.
      • et al.
      Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH.
      ,
      • Wong V.W.-S.
      • Tse C.-H.
      • Lam T.T.-Y.
      • Wong G.L.-H.
      • Chim A.M.-L.
      • Chu W.C.-W.
      • et al.
      Molecular characterization of the fecal microbiota in patients with nonalcoholic steatohepatitis--a longitudinal study.
      ,
      • Abu-Shanab A.
      • Quigley E.M.M.
      The role of the gut microbiota in nonalcoholic fatty liver disease.
      ]. Likewise intestinal dysbiosis can be linked to atherogenesis, myocardial infarction and heart failure [
      • Aron-Wisnewsky J.
      • Clément K.
      The gut microbiome, diet, and links to cardiometabolic and chronic disorders.
      ,
      • Brown J.M.
      • Hazen S.L.
      The gut microbial endocrine organ: bacterially derived signals driving cardiometabolic diseases.
      ]. Within plaques the presence of intestinal DNA was even demonstrated and associated with CV events [
      • Aron-Wisnewsky J.
      • Clément K.
      The gut microbiome, diet, and links to cardiometabolic and chronic disorders.
      ].
      The gut microbiome can secrete different molecules that, via the intestinal wall, end up into the blood stream. The most well-known are the secondary bile acids, but also trimethylamine (TMA) and short chain fatty acids are amongst them. These molecules can modulate energy balance, insulin sensitivity and may indirectly influence NAFLD and CVD [
      • Brown J.M.
      • Hazen S.L.
      The gut microbial endocrine organ: bacterially derived signals driving cardiometabolic diseases.
      ]. More direct evidence of a link with CVD is found with trimethylamine-N-Oxide (TMAO), considered as a pro-atherogenic compound. Impact on cholesterol metabolism and the promotion of foam cells are assumed to be responsible [
      • Brown J.M.
      • Hazen S.L.
      The gut microbial endocrine organ: bacterially derived signals driving cardiometabolic diseases.
      ].
      TMAO is formed out of TMA by flavin monoxygenase (FMO) in the liver. Genetic analysis of inbred mouse strains couldn’t show a positive relation of FMO3, the most active isoform, with hepatic TG content, but FMO5 instead was upregulated [
      • Hui S.T.
      • Parks B.W.
      • Org E.
      • Norheim F.
      • Che N.
      • Pan C.
      • et al.
      The genetic architecture of NAFLD among inbred strains of mice.
      ]. The main substrates for TMA formation are dietary choline, phosphatidylcholine and L-carnitine. Clinically, and in line with many animal models of NASH [
      • Hebbard L.
      • George J.
      Animal models of nonalcoholic fatty liver disease.
      ], choline-deficiency and corresponding microbiome alterations are related to NAFLD [
      • Spencer M.D.
      • Hamp T.J.
      • Reid R.W.
      • Fischer L.M.
      • Zeisel S.H.
      • Fodor A.A.
      Association between composition of the human gastrointestinal microbiome and development of fatty liver with choline deficiency.
      ], which seems to imply that this should rather be beneficial towards CVD. The intestinal microbiome activity may contribute to both steatosis, due to prompt a relative choline-deficiency, and to increased levels of produced TMA out of this choline [
      • Dumas M.-E.
      • Barton R.H.
      • Toye A.
      • Cloarec O.
      • Blancher C.
      • Rothwell A.
      • et al.
      Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice.
      ].
      Incretins are gastrointestinal peptide hormones regulating postprandial glucose metabolism. Glucose-induced release of glucagon-like peptide 1 (GLP-1), nor gastric inhibitory protein (GIP), was impaired in non-diabetic patients with NAFLD [
      • Bernsmeier C.
      • Meyer-Gerspach A.C.
      • Blaser L.S.
      • Jeker L.
      • Steinert R.E.
      • Heim M.H.
      • et al.
      Glucose-induced glucagon-like Peptide 1 secretion is deficient in patients with non-alcoholic fatty liver disease.
      ]. Treatment with GLP-1 receptor agonists or DPP-4 inhibitors improve liver histology [
      • Carbone F.
      • Montecucco F.
      • Mach F.
      • Pontremoli R.
      • Viazzi F.
      The liver and the kidney: two critical organs influencing the atherothrombotic risk in metabolic syndrome.
      ] and seem promising. GLP-1 is known to have multiple cardioprotective effects [
      • Kim J.
      • Samson S.L.
      Cardiovascular effects of incretin therapy in diabetes care.
      ]. Furthermore, exendine-4 (a GLP-1 receptor agonist) improved NASH, vessel inflammation and plaque size via the inhibition of macrophage recruitment and activation [
      • Wang Y.
      • Parlevliet E.T.
      • Geerling J.J.
      • van der Tuin S.J.L.
      • Zhang H.
      • Bieghs V.
      • et al.
      Exendin-4 decreases liver inflammation and atherosclerosis development simultaneously by reducing macrophage infiltration.
      ].
      Of note, the role of the gut is also particular interest in light of the upcoming treatments, including obeticholic acid, which exerts its action through agonism on the farnesoid X receptor (a receptor of secondary bile acids), located in intestinal epithelial cells and hepatocytes and a key regulator of bile acid homeostasis but possesses important crosstalk with glucose and lipid metabolism [
      • Neuschwander-Tetri B.A.
      • Loomba R.
      • Sanyal A.J.
      • Lavine J.E.
      • Van Natta M.L.
      • Abdelmalek M.F.
      • et al.
      Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial.
      ].

      Genetics

      There are increasing arguments for a role of genetic modifiers of NAFLD. When restricting to those genes with robust validation or the use of transmission disequilibrium testing, only a handful of genes currently seems to be of interest [
      • Anstee Q.M.
      • Day C.P.
      The genetics of nonalcoholic fatty liver disease: spotlight on PNPLA3 and TM6SF2.
      ], of which patatin-like phospholipase domain containing protein 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2) are most documented.
      Familial clustering, ethnic differences and twin studies give arguments for the presence of heritable components. These studies estimate that 18–50% can be attributed to genetic factors [
      • Anstee Q.M.
      • Day C.P.
      The genetics of nonalcoholic fatty liver disease: spotlight on PNPLA3 and TM6SF2.
      ,
      • Tarnoki A.D.
      • Tarnoki D.L.
      • Bata P.
      • Littvay L.
      • Osztovits J.
      • Jermendy G.
      • et al.
      Heritability of non-alcoholic fatty liver disease and association with abnormal vascular parameters: a twin study.
      ,
      • Loomba R.
      • Schork N.
      • Chen C.-H.
      • Bettencourt R.
      • Bhatt A.
      • Ang B.
      • et al.
      Heritability of hepatic fibrosis and steatosis based on a prospective twin study.
      ]. In a recent twin study CV risk and NAFLD was specifically studied [
      • Tarnoki A.D.
      • Tarnoki D.L.
      • Bata P.
      • Littvay L.
      • Osztovits J.
      • Jermendy G.
      • et al.
      Heritability of non-alcoholic fatty liver disease and association with abnormal vascular parameters: a twin study.
      ]. Although CV parameters were more frequently abnormal in NAFLD, they failed to establish a role of genetic components in acquiring NAFLD (in contrast to [
      • Loomba R.
      • Schork N.
      • Chen C.-H.
      • Bettencourt R.
      • Bhatt A.
      • Ang B.
      • et al.
      Heritability of hepatic fibrosis and steatosis based on a prospective twin study.
      ]) or the associated CVRFs [
      • Tarnoki A.D.
      • Tarnoki D.L.
      • Bata P.
      • Littvay L.
      • Osztovits J.
      • Jermendy G.
      • et al.
      Heritability of non-alcoholic fatty liver disease and association with abnormal vascular parameters: a twin study.
      ].
      Robust data now associate the PNPLA3 rs738409 mutation with NAFLD severity [
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • Pertsemlidis A.
      • Cox D.
      • Pennacchio L.A.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      ,
      • Speliotes E.K.
      • Yerges-Armstrong L.M.
      • Wu J.
      • Hernaez R.
      • Kim L.J.
      • Palmer C.D.
      • et al.
      Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.
      ]. The function of the gene seems to be directly related to TG metabolism, whereas mutations have not been associated with insulin resistance or glucose intolerance. The PNPLA3 mutation is increasingly recognised as a modifier of NAFLD [
      • Anstee Q.M.
      • Day C.P.
      The genetics of nonalcoholic fatty liver disease: spotlight on PNPLA3 and TM6SF2.
      ] in terms of disease severity and the risk on NAFLD-related HCC [
      • Liu Y.-L.
      • Patman G.L.
      • Leathart J.B.S.
      • Piguet A.-C.
      • Burt A.D.
      • Dufour J.-F.
      • et al.
      Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.
      ], whilst the link of this mutation with CVD currently remains elusive.
      Another non-synonymous SNP, TM6SF2 has also been associated with NAFLD and fibrosis [
      • Liu Y.-L.
      • Reeves H.L.
      • Burt A.D.
      • Tiniakos D.
      • McPherson S.
      • Leathart J.B.S.
      • et al.
      TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease.
      ]. Mutation of this gene probably results in retention of TG and cholesterol in the liver [
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • Nordestgaard B.G.
      • Zhou H.H.
      • Tybjærg-Hansen A.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      ], predisposing to NAFLD and fibrosis. Paradoxically, this mutation results in a reduction of VLDL secretion and improvement of serum TG, whilst insulin sensitivity was unaltered [
      • Holmen O.L.
      • Zhang H.
      • Fan Y.
      • Hovelson D.H.
      • Schmidt E.M.
      • Zhou W.
      • et al.
      Systematic evaluation of coding variation identifies a candidate causal variant in TM6SF2 influencing total cholesterol and myocardial infarction risk.
      ,
      • Zhou Y.
      • Llauradó G.
      • Orešič M.
      • Hyötyläinen T.
      • Orho-Melander M.
      • Yki-Järvinen H.
      Circulating triacylglycerol signatures and insulin sensitivity in NAFLD associated with the E167K variant in TM6SF2.
      ]. Therefor this mutation would be cardioprotective rather than deleterious for the CV system, recently called the “Catch-22” paradigm [
      • Kahali B.
      • Liu Y.-L.
      • Daly A.K.
      • Day C.P.
      • Anstee Q.M.
      • Speliotes E.K.
      TM6SF2: catch-22 in the fight against nonalcoholic fatty liver disease and cardiovascular disease?.
      ].
      Other SNP’s have been associated with NAFLD [
      • Naik A.
      • Košir R.
      • Rozman D.
      Genomic aspects of NAFLD pathogenesis.
      ] and whilst biologically plausible, their association still warrants further validation [
      • Anstee Q.M.
      • Day C.P.
      The genetics of nonalcoholic fatty liver disease: spotlight on PNPLA3 and TM6SF2.
      ] and the link with CVD is largely unknown.

      Other potential mechanisms

      Sarcopenia (loss of muscle mass and muscle performance) [
      • Cooper C.
      • Fielding R.
      • Visser M.
      • van Loon L.J.
      • Rolland Y.
      • Orwoll E.
      • et al.
      Tools in the assessment of sarcopenia.
      ] and sarcopenic obesity, which links sarcopenia with obesity [
      • Prado C.M.M.
      • Wells J.C.K.
      • Smith S.R.
      • Stephan B.C.M.
      • Siervo M.
      Sarcopenic obesity: a critical appraisal of the current evidence.
      ] are associated with increased CVD [
      • Byeon C.-H.
      • Kang K.-Y.
      • Kang S.-H.
      • Bae E.-J.
      Sarcopenia is associated with Framingham risk score in the Korean population: Korean National Health and Nutrition Examination Survey (KNHANES) 2010–2011.
      ,
      • Atkins J.L.
      • Wannamethee S.G.
      The effect of sarcopenic obesity on cardiovascular disease and all-cause mortality in older people.
      ]. The pathophysiology is rather complex with involvement of joint risk factors/mechanisms [
      • Prado C.M.M.
      • Wells J.C.K.
      • Smith S.R.
      • Stephan B.C.M.
      • Siervo M.
      Sarcopenic obesity: a critical appraisal of the current evidence.
      ,
      • Stenholm S.
      • Harris T.B.
      • Rantanen T.
      • Visser M.
      • Kritchevsky S.B.
      • Ferrucci L.
      Sarcopenic obesity: definition, cause and consequences.
      ]. Importantly, also NAFLD could be associated with sarcopenia in three recent papers [
      • Moon J.S.
      • Yoon J.S.
      • Won K.C.
      • Lee H.W.
      The role of skeletal muscle in development of nonalcoholic Fatty liver disease.
      ,
      • Lee Y.-H.
      • Jung K.S.
      • Kim S.U.
      • Yoon H.-J.
      • Yun Y.J.
      • Lee B.-W.
      • et al.
      Sarcopaenia is associated with NAFLD independently of obesity and insulin resistance: Nationwide surveys (KNHANES 2008–2011).
      ,
      • Hong H.C.
      • Hwang S.Y.
      • Choi H.Y.
      • Yoo H.J.
      • Seo J.A.
      • Kim S.G.
      • et al.
      Relationship between sarcopenia and nonalcoholic fatty liver disease: the Korean Sarcopenic Obesity Study.
      ]. One of them observed a direct association with sarcopenia and increased pulse wave velocity, a parameter for subclinical CVD [
      • Hong H.C.
      • Hwang S.Y.
      • Choi H.Y.
      • Yoo H.J.
      • Seo J.A.
      • Kim S.G.
      • et al.
      Relationship between sarcopenia and nonalcoholic fatty liver disease: the Korean Sarcopenic Obesity Study.
      ].
      NAFLD and vitamin D deficiency are often associated [
      • Kwok R.M.
      • Torres D.M.
      • Harrison S.A.
      Vitamin D and nonalcoholic fatty liver disease (NAFLD): is it more than just an association?.
      ]. Vitamin D deficiency has proven to be a causal and independent CVRF [
      • Weyland P.G.
      • Grant W.B.
      • Howie-Esquivel J.
      Does sufficient evidence exist to support a causal association between vitamin D status and cardiovascular disease risk? An assessment using Hill’s criteria for causality.
      ]. Whether NAFLD causes hypovitaminosis D and so contributes to CVD has to be proven. We estimate that it’s most probably an association rather than a causal link.
      Decreased levels of serum albumin are classically seen in advanced liver disease (i.e., high grade fibrosis). Albumin has known inotropic effects [
      • Bortoluzzi A.
      • Ceolotto G.
      • Gola E.
      • Sticca A.
      • Bova S.
      • Morando F.
      • et al.
      Positive cardiac inotropic effect of albumin infusion in rodents with cirrhosis and ascites: molecular mechanisms.
      ] and low levels of albumin are linked to higher CVD and mortality [
      • Schalk B.W.M.
      • Visser M.
      • Bremmer M.A.
      • Penninx B.W.J.H.
      • Bouter L.M.
      • Deeg D.J.H.
      Change of serum albumin and risk of cardiovascular disease and all-cause mortality: longitudinal aging study Amsterdam.
      ]. However long-term analysis failed to show decrease in serum albumin levels in patients with NAFLD [
      • Charatcharoenwitthaya P.
      • Lindor K.D.
      • Angulo P.
      The spontaneous course of liver enzymes and its correlation in nonalcoholic fatty liver disease.
      ]. As a result, albumin doesn’t seem to mediate CVD in patients with NAFLD without advanced liver disease.
      Microvesicles are small vesicles formed out of cellular plasma by budding, excretion or after apoptosis and may contain lipids, proteins, RNAs and microRNAs. Initially regarded as cellular debris, they are now recognised as paracrine signals [
      • Lemoinne S.
      • Thabut D.
      • Housset C.
      • Moreau R.
      • Valla D.
      • Boulanger C.M.
      • et al.
      The emerging roles of microvesicles in liver diseases.
      ]. Circulating microvesicles are increased in liver diseases. These microvesicles can originate from other organs and cause liver damage. The liver itself can, however, also be a source of microvesicles, illustrated by the presence of liver-derived procoagulant microvesicles reported in acute liver failure [
      • Lemoinne S.
      • Thabut D.
      • Housset C.
      • Moreau R.
      • Valla D.
      • Boulanger C.M.
      • et al.
      The emerging roles of microvesicles in liver diseases.
      ]. Potential effects of liver-related microvesicles on the CV system and CVD have been reported in a mouse model with NASH and atherosclerosis [
      • Baron M.
      • Leroyer A.S.
      • Majd Z.
      • Lalloyer F.
      • Vallez E.
      • Bantubungi K.
      • et al.
      PPARα activation differently affects microparticle content in atherosclerotic lesions and liver of a mouse model of atherosclerosis and NASH.
      ]. In line with this potential role of microvesicles, NAFLD has very recently shown to have a unique profile of circulating microRNAs. This profile was associated with increased CV risk when the alterations were compared to known disease pathways in in silico analysis [
      • Pirola C.J.
      • Fernández Gianotti T.
      • Castaño G.O.
      • Mallardi P.
      • San Martino J.
      • Mora Gonzalez Lopez Ledesma M.
      • et al.
      Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis.
      ].

      Clinical implications

      Current guidelines

      In the current published recommendations, both on the clinical management of the patients with NAFLD and a fortiori on the design of therapeutic trials for NAFLD, the link with CVD disease is not a major issue [
      • Ratziu V.
      • Bellentani S.
      • Cortez-Pinto H.
      • Day C.
      • Marchesini G.
      A position statement on NAFLD/NASH based on the EASL 2009 special conference.
      ,
      • Chalasani N.
      • Younossi Z.
      • Lavine J.E.
      • Diehl A.M.
      • Brunt E.M.
      • Cusi K.
      • 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.
      ,
      • Sanyal A.J.
      • Friedman S.L.
      • McCullough A.J.
      • Dimick-Santos L.
      American Association for the Study of Liver Diseases, United States Food and Drug Administration. Challenges and opportunities in drug and biomarker development for nonalcoholic steatohepatitis: findings and recommendations from an American Association for the Study of Liver Diseases-U.S. Food and Drug Administration Joint Workshop.
      ]. In the clinical guidelines, the overt link of NAFLD with the MetS translates into instructions to screen NAFLD patients for associated CVRFs and to treat these according to their proper guidelines [
      • Nascimbeni F.
      • Pais R.
      • Bellentani S.
      • Day C.P.
      • Ratziu V.
      • Loria P.
      • et al.
      From NAFLD in clinical practice to answers from guidelines.
      ]. In the joined AASLD-AGA guidelines, these instructions are only found in the section for the treatment of associated dyslipidaemias, so screening for CVRFs is not even a specific topic [
      • Chalasani N.
      • Younossi Z.
      • Lavine J.E.
      • Diehl A.M.
      • Brunt E.M.
      • Cusi K.
      • 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.
      ]. In the European association for the study of the liver (EASL) position paper of 2010, CVD is recognised as an extrahepatic manifestation of NAFLD. Screening for CVRFs is a separate recommendation and is hence considered a standard element in the approach of a NAFLD patient [
      • Ratziu V.
      • Bellentani S.
      • Cortez-Pinto H.
      • Day C.
      • Marchesini G.
      A position statement on NAFLD/NASH based on the EASL 2009 special conference.
      ]. New EASL guidelines are expected to be presented in 2016. The subsequent sections propose and discuss a pragmatic approach based on the current knowledge.
      Subclinical cardiovascular alterations can be detected easily in NAFLD and screening is potentially cost-effective in selected patient-groups.

      Screening

      Screening for NAFLD in patients with CVRFs and CVD

      As NAFLD is contributing to the development of CVD, one might consider screening for NAFLD in patients with CVRFs, in patients with subclinical CVD, or in patients with clinical CVD. The first question is what to screen for? As discussed previously, data suggesting that the risk of CVD, and especially clinical CVD, seems to be confined to patients with NASH, have recently been challenged by data suggesting that fibrosis is the most important predictor of future CV events in both NAFL and NASH patients [
      • Ekstedt M.
      • Hagström H.
      • Nasr P.
      • Fredrikson M.
      • Stål P.
      • Kechagias S.
      • et al.
      Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up.
      ,
      • Angulo P.
      • Kleiner D.E.
      • Dam-Larsen S.
      • Adams L.A.
      • Bjornsson E.S.
      • Charatcharoenwitthaya P.
      • et al.
      Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease.
      ]. Nevertheless, detailed analysis of the data, including recent paired biopsy studies [
      • Singh S.
      • Allen A.M.
      • Wang Z.
      • Prokop L.J.
      • Murad M.H.
      • Loomba R.
      Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies.
      ,
      • McPherson S.
      • Hardy T.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      • Anstee Q.M.
      Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: implications for prognosis and clinical management.
      ] and the aforementioned consideration concerning methodology and the dynamic nature of both NAFLD and its related conditions like CVD and MetS, still support the concept of necro-inflammation as the driver of disease progression. This also explains why patients with NASH and some degree of fibrosis are currently the target population in most of the clinical trials [
      • Sanyal A.J.
      • Friedman S.L.
      • McCullough A.J.
      • Dimick-Santos L.
      American Association for the Study of Liver Diseases, United States Food and Drug Administration. Challenges and opportunities in drug and biomarker development for nonalcoholic steatohepatitis: findings and recommendations from an American Association for the Study of Liver Diseases-U.S. Food and Drug Administration Joint Workshop.
      ]. Therefore, screening should ideally address both the aspects of steatohepatitis and fibrosis
      The second question is which screening technique to use. For the diagnosis of steatosis, ultrasound is an ideal screening tool because it is safe and cheap and has a high accuracy if steatosis >30% [
      • Ryan C.K.
      • Johnson L.A.
      • Germin B.I.
      • Marcos A.
      One hundred consecutive hepatic biopsies in the workup of living donors for right lobe liver transplantation.
      ]. Liver enzymes are cheap, but lack sensitivity and specificity [
      • Verma S.
      • Jensen D.
      • Hart J.
      • Mohanty S.R.
      Predictive value of ALT levels for non-alcoholic steatohepatitis (NASH) and advanced fibrosis in non-alcoholic fatty liver disease (NAFLD).
      ,
      • Charatcharoenwitthaya P.
      • Lindor K.D.
      • Angulo P.
      The spontaneous course of liver enzymes and its correlation in nonalcoholic fatty liver disease.
      ]. Other diagnostic techniques are more expensive, less easily accessible and/or invasive, so currently not suitable for screening purposes. Biomarker research hasn’t provided a sufficiently accurate and validated alternative [
      • Castera L.
      Noninvasive evaluation of nonalcoholic fatty liver disease.
      ]. Screening must therefore currently rely on a combination of clinical (metabolic) assessment, serum liver enzymes and ultrasound. Screening for fibrosis can be done by liver stiffness measurement, clinicobiochemical parameters and scores, or by a combination of both [
      • Castera L.
      Noninvasive evaluation of nonalcoholic fatty liver disease.
      ] but these approaches still lack validation in large populations at risk.
      Thirdly, the group of patients to screen for NAFLD should be defined. Patients that already present with overt CVD represent a well-delineated group. Screening for NAFLD will hardly add to the overall cost of their treatment. In several countries, some high-risk patients, especially diabetics, undergo regular medical check-ups including screening for CVD [
      • Rydén L.
      • Grant P.J.
      • Anker S.D.
      • Berne C.
      • Cosentino F.
      • et al.
      Authors/Task Force Members
      ESC Guidelines on diabetes, pre-diabetes, and cardiovascular diseases developed in collaboration with the EASD: the Task Force on diabetes, pre-diabetes, and cardiovascular diseases of the European Society of Cardiology (ESC) and developed in collaboratio.
      ]. Also in this population, adding screening for NAFLD/NASH by liver enzymes and ultrasound would only minimally impact on overall cost. For other NAFLD risk groups, especially patients with obesity or MetS, baseline-screening guidelines exist (but stringent follow-up programs are less systematically organised) and frequently include liver enzymes and ultrasound. The third category of patients, in whom risk factors for CVD have been identified (e.g., smoking, positive familial history, dyslipidaemia) constitute a large group, in whom liver tests are frequently checked, but abdominal ultrasound isn’t part of their routine work-up.
      This leads to another problem that hampers screening for NAFLD, namely the unawareness of the relevance of NASH and related conditions (CVD, T2DM, MetS) in the physicians taking care of these patients, with, as a consequence, underdiagnosis of NAFLD [
      • Said A.
      • Gagovic V.
      • Malecki K.
      • Givens M.L.
      • Nieto F.J.
      Primary care practitioners survey of non-alcoholic fatty liver disease.
      ,
      • Bergqvist C.-J.
      • Skoien R.
      • Horsfall L.
      • Clouston A.D.
      • Jonsson J.R.
      • Powell E.E.
      Awareness and opinions of non-alcoholic fatty liver disease by hospital specialists.
      ]. Awareness of physicians for the presence of NASH and a correct interpretation of tests that are already performed would probably result in a considerably higher detection rate without any additional cost. Moreover, a Canadian cost-effectiveness study supports cost-effectiveness of non-invasive screening for NASH and advanced fibrosis in a high-risk obese or diabetic population [
      • Zhang E.
      • Wartelle-Bladou C.
      • Lepanto L.
      • Lachaine J.
      • Cloutier G.
      • Tang A.
      Cost-utility analysis of nonalcoholic steatohepatitis screening.
      ].
      Formal recommendation cannot be given based on the current evidence, but we suggest to screen for NAFLD and NASH by ultrasound and liver enzymes in patients with overt CVD or in patients with increased CV risk (e.g., SCORE or HeartSCORE estimated 10 year risk ⩾5% [
      • Perk J.
      • De Backer G.
      • Gohlke H.
      • Graham I.
      • Reiner Z.
      • Verschuren M.
      • et al.
      European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts)..
      ]) and if positive, to subsequently assess fibrosis by a combination of a clinicobiochemical score and a liver stiffness measurement [
      • Castera L.
      Noninvasive evaluation of nonalcoholic fatty liver disease.
      ].
      The need for screening, although defendable, is further challenged by the limited treatment options for NASH. Lifestyle modification improves NASH as well CV risk profile [
      • Nguyen V.
      • George J.
      Nonalcoholic fatty liver disease management: dietary and lifestyle modifications.
      ,
      • Eckel R.H.
      • Jakicic J.M.
      • Ard J.D.
      • de Jesus J.M.
      • Houston Miller N.
      • Hubbard V.S.
      • et al.
      2013 AHA/ACC guideline on lifestyle management to reduce cardiovascular risk: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines.
      ], but whether both are just associated because of a beneficial impact on the different metabolic risk factors that are shared by both conditions, or whether the improvement in NASH severity contributes to an improvement of the risk of a true CVD event, is an unresolved issue. The fact that NASH contributes to CVD logically leads to the hypothesis that improvement in NASH specifically contributes to an improvement in CVD. This, however, remains to be proven, which isn’t easy, as similar to pathophysiology, the specific contribution of NASH improvement to the overall improvement in CV risk is difficult to dissect from the beneficial contribution of other factors. The answer to this question remains nevertheless crucial and properly designed studied are highly warranted.

      Screening for CVD in patients with NAFLD

      The inverse screening question is if we need to screen for CVD in patients with NAFLD. Current recommendation just recommend to screen for classical CVRFs [
      • Nascimbeni F.
      • Pais R.
      • Bellentani S.
      • Day C.P.
      • Ratziu V.
      • Loria P.
      • et al.
      From NAFLD in clinical practice to answers from guidelines.
      ]. As presence of subclinical CVD is a well-established risk factor for subsequent clinical CVD [
      • Halcox J.P.J.
      • Schenke W.H.
      • Zalos G.
      • Mincemoyer R.
      • Prasad A.
      • Waclawiw M.A.
      • et al.
      Prognostic value of coronary vascular endothelial dysfunction.
      ] and NASH impacts on atherosclerosis, the question rises whether a NASH patient shouldn’t be screened for the presence of subclinical CVD. If present, the consequence could be that the patient should undergo more sophisticated examinations to look for clinically significant CV abnormalities and a more aggressive treatment of the CVRFs and lesions. The tool to screen for subclinical CVD is to be discussed and depends on local availability and expertise. Carotid ultrasound including cIMT measurement is one of the most widely used and validated [
      • Perk J.
      • De Backer G.
      • Gohlke H.
      • Graham I.
      • Reiner Z.
      • Verschuren M.
      • et al.
      European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts)..
      ,
      • O’Leary D.H.
      • Polak J.F.
      • Kronmal R.A.
      • Manolio T.A.
      • Burke G.L.
      • Wolfson S.K.
      Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. Cardiovascular Health Study Collaborative Research Group.
      ]. Coronary artery calcium score, ankle-brachial index and flow-mediated vasodilatation are valuable alternatives [
      • Perk J.
      • De Backer G.
      • Gohlke H.
      • Graham I.
      • Reiner Z.
      • Verschuren M.
      • et al.
      European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts)..
      ]. Besides, the population to be screened isn’t defined, but as the risk seems to be low in NAFL, limiting the screening to NASH seems appropriate. Anyhow, no data on cost-effectiveness are available to date to support this strategy but studies should be encouraged given the potential clinical relevance. A pragmatic approach to screen for CVD in NAFLD is proposed in Fig. 3.
      Figure thumbnail gr3
      Fig. 3Proposed algorithm to assess CVD in patients with NAFLD. Since patients with non-alcoholic fatty liver disease (NAFLD) are at high-risk to develop cardiovascular disease (CVD), there is a rationale to screen for CVD. Screening seems to be more appropriate and cost-effective in patients with NASH or clinical significant fibrosis, though must not be restrained to them solely. In case of sufficient clinical arguments, amongst which the presence of diabetes mellitus is of major importance, additional screening can be advocated. Negative assessments do not waive adequate follow-up and lifestyle interventions. Re-assessments can be proposed at 2–3 year intervals or based on symptomatic CVD. CACS, coronary artery calcium score; cIMT, carotid intima media thickness; CVD, cardiovascular disease; ECG, electrocardiogram; FMD, flow-mediated dilatation; NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PWV, pulse wave velocity.

      Pharmacological treatment

      Treatment of NAFLD should include assessment and treatment of extra-hepatic manifestations of NAFLD.
      Treatment of NASH might also result in improvement of cardiovascular disease, though this remains to be proven.
      The question is becoming even more relevant, as drugs specifically aiming at treating NAFLD/NASH are currently being developed, some of them entering phase 3 clinical trials. In the current recommendations for NASH trial design, CVD is poorly touched upon, except for the recommendation that, as most NASH patients die from CVD and treatment is presumably long-term or even life-long, the cardiac safety profile of the drug should be thoroughly monitored [
      • Sanyal A.J.
      • Friedman S.L.
      • McCullough A.J.
      • Dimick-Santos L.
      American Association for the Study of Liver Diseases, United States Food and Drug Administration. Challenges and opportunities in drug and biomarker development for nonalcoholic steatohepatitis: findings and recommendations from an American Association for the Study of Liver Diseases-U.S. Food and Drug Administration Joint Workshop.
      ]. There is hence, besides the monitoring of CVRFs and registration of CVD events, no specific assessment of the impact of the treatment of NASH on the CV outcome of the patients.
      Cardiovascular safety is of course an issue in all studies, and most include also impact on metabolic and CVRFs as one of the secondary endpoints. Effects on hard clinical CV endpoints would take many years and appropriately powered studies to show a benefit of the drug compared to placebo and is hence not realistic in this setting. One wonders, however, why a test for subclinical CVD has not been incorporated in the design of these studies as a secondary endpoint. As well as NASH histology is an accepted surrogate endpoint for clinical benefit in terms of liver disease, measurement of the impact on markers of subclinical CVD (such as cIMT, fibromuscular dysplasia) are accepted surrogate endpoints for clinical benefit in terms of CVD [
      • Halcox J.P.J.
      • Schenke W.H.
      • Zalos G.
      • Mincemoyer R.
      • Prasad A.
      • Waclawiw M.A.
      • et al.
      Prognostic value of coronary vascular endothelial dysfunction.
      ].
      Markers of subclinical cardiovascular disease should become co-primary or secondary endpoints in clinical trials as surrogate for long-term CV benefit.
      If a drug improving steatohepatitis could also show to have a beneficial effect on a parameter of subclinical CVD (and hence predictive of future CV clinical events), this would greatly enhance its utility in clinical practice as this substantially increases the likelihood that the drug results in a significant survival benefit. We would therefore strongly recommend to add the impact of a candidate drug on a marker of subclinical CVD as a co-primary or secondary endpoint in clinical trials for NASH and to ascertain that studies are sufficiently powered to assess this aspect of potential treatment benefit.
      Another aspect, besides demonstrating beneficial effects on cardiometabolic risk profile, or on a parameter of subclinical disease, is the treatment indication. The goal of treatment is to improve NASH rather than just improving fibrosis. Since NASH is believed to be the motor of disease progression, resolution of NASH without worsening of fibrosis is the preferred primary endpoint [
      • Sanyal A.J.
      • Friedman S.L.
      • McCullough A.J.
      • Dimick-Santos L.
      American Association for the Study of Liver Diseases, United States Food and Drug Administration. Challenges and opportunities in drug and biomarker development for nonalcoholic steatohepatitis: findings and recommendations from an American Association for the Study of Liver Diseases-U.S. Food and Drug Administration Joint Workshop.
      ,
      • Ratziu V.
      • Goodman Z.
      • Sanyal A.
      Current efforts and trends in the treatment of NASH.
      ]. The target population consists of patients with moderate-severe steatohepatitis and significant fibrosis (⩾F2).
      Studies on the impact of glycaemic control on long-term outcome, however, failed to show a reduction in CV mortality despite good glycaemic control. This has been attributed to the so-called “metabolic memory”, denoting the persistence of endothelial dysfunction despite correction of glucose levels. Only patients with short duration of DM, low baseline HbA1c and no history of a CVD event benefitted (in terms of improved survival) from good glycaemic control, suggesting that early intervention is needed to improve survival and once vascular damage has been installed, improved metabolic control does hardly impact on long-term survival.
      This concept challenges the restriction of treatment indication to patients with significant fibrosis, as it suggests that NASH patients should be treated early and hence regardless of fibrosis degree. The shift from a purely liver-centered approach to a holistic approach would therefore substantially impact on the number of patients to be treated, but also on the potential overall survival benefit that could be obtained.
      A final consideration in this ongoing debate is the relationship between the improvement in NASH and the improvement in cardiometabolic profile and endothelial dysfunction. The causal role of NASH in both the development of DM and CVD doesn’t necessarily mean that improvement of NAFLD automatically results in improvement of DM and CVD. A unilateral relationship is difficult to demonstrate in a clinical setting, where several processes are interrelated and patients are heterogeneous. Sub-analyses of the current and future trials, looking into the kinetics of changes in liver and cardiometabolic parameters, into differences between responders and non-responders and into relationships between the magnitude of the improvement of NASH and the observed changes in cardiometabolic risk, might help answering this question.
      Interestingly, although beyond the scope of this review, pharmacological treatment of CVRFs and CVD can also positively impact on NAFLD. Statins have no proven benefit on liver histology in randomized controlled trials, but were shown to reduce progression to cirrhosis and cirrhosis decompensation [
      • Mohanty A.
      • Tate J.P.
      • Garcia-Tsao G.
      Statins are associated with a decreased risk of decompensation and death in veterans with hepatitis c-related compensated cirrhosis.
      ], to reduce incident HCC [
      • Zhou Y.-Y.
      • Zhu G.-Q.
      • Wang Y.
      • Zheng J.-N.
      • Ruan L.-Y.
      • Cheng Z.
      • et al.
      Systematic review with network meta-analysis: statins and risk of hepatocellular carcinoma.
      ] and to improve hepatic endothelial function [
      • Pasarín M.
      • La Mura V.
      • Gracia-Sancho J.
      • García-Calderó H.
      • Rodríguez-Vilarrupla A.
      • García-Pagán J.C.
      • et al.
      Sinusoidal endothelial dysfunction precedes inflammation and fibrosis in a model of NAFLD.
      ], fibrogenesis [
      • Chong L.-W.
      • Hsu Y.-C.
      • Lee T.-F.
      • Lin Y.
      • Chiu Y.-T.
      • Yang K.-C.
      • et al.
      Fluvastatin attenuates hepatic steatosis-induced fibrogenesis in rats through inhibiting paracrine effect of hepatocyte on hepatic stellate cells.
      ] and angiogenesis [
      • Chang C.-C.
      • Wang S.-S.
      • Hsieh H.-G.
      • Lee W.-S.
      • Chuang C.-L.
      • Lin H.-C.
      • et al.
      Rosuvastatin improves hepatopulmonary syndrome through inhibition of inflammatory angiogenesis of lung.
      ]. Statins reduce fibrosis in animal models [
      • Shirai Y.
      • Yoshiji H.
      • Noguchi R.
      • Kaji K.
      • Aihara Y.
      • Douhara A.
      • et al.
      Cross talk between toll-like receptor-4 signaling and angiotensin-II in liver fibrosis development in the rat model of non-alcoholic steatohepatitis.
      ] and aspirin use was associated with lower indices of fibrosis in the US adult population [
      • Jiang Z.G.
      • Feldbrügge L.
      • Tapper E.B.
      • Popov Y.
      • Ghaziani T.
      • Afdhal N.
      • et al.
      Aspirin use is associated with lower indices of liver fibrosis among adults in the United States.
      ] and reduced steatohepatitis in an animal model [
      • Madrigal-Perez V.M.
      • García-Rivera A.
      • Rodriguez-Hernandez A.
      • Ceja-Espiritu G.
      • Briseño-Gomez X.G.
      • Galvan-Salazar H.R.
      • et al.
      Preclinical analysis of nonsteroidal anti-inflammatory drug usefulness for the simultaneous prevention of steatohepatitis, atherosclerosis and hyperlipidemia.
      ]. These and other data further highlight the complex reciprocal interactions of NAFLD and CVD and the need for a multidisciplinary approach.

      Conclusions

      The liver and the cardiovascular system are inextricably linked to each other: the hepato-cardiovascular axis.
      CVD remains the most important cause of death in patients with NAFLD. Clinical evidence for a hepato-cardiovascular axis, in which NAFLD is an independent risk factor for subclinical and clinical CVD is supported by evidence from fundamental and clinical research; unravelling the mechanisms by which NAFLD causally influences endothelial dysfunction and the development of atherosclerosis as well as other CV lesions. The current liver-centered approach of NAFLD should therefore shift to a more holistic approach, including specific assessment of CVD that shouldn’t be limited to the treatment of concomitant CVRFs, but should ideally include parameters of subclinical CVD. This not only applies to routine clinical practice, but a fortiori to clinical trials, as the ultimate goal of therapy is to improve patient’s survival. As metabolic memory negatively impacts on potential treatment-induced survival benefit, early treatment of NASH might be warranted, challenging the currently defined target population for clinical trials in NASH.

      Financial support

      WK and SF received funding from the fund for scientific research (FWO) flanders (11j9513n, 1802154n). The funders had no role in the preparation of the manuscript.

      Conflict of interest

      The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Authors’ contributions

      All authors contributed to the draft of the text. Furthermore, the intellectual content was subject to critical review by SF and WK. DG created the figures.

      Supplementary data

      References

      Author names in bold designate shared co-first authorshipThe authors wish to apologise to all researchers who contributed significantly to the knowledge of NAFLD and CVD, but whose work couldn’t be cited due to space limitation.

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