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Role of liver sinusoidal endothelial cells in non-alcoholic fatty liver disease

  • Adel Hammoutene
    Affiliations
    Inserm, UMR-970, Paris Cardiovascular Research Center, PARCC, Paris, France

    University Paris Descartes, Paris, France
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  • Pierre-Emmanuel Rautou
    Correspondence
    Corresponding author. Address: Service d’hépatologie, Hôpital Beaujon, 100 boulevard du Général Leclerc, 92100 Clichy, France. Tel.: +33 1 40 87 52 83; fax: +33 1 40 87 55 30.
    Affiliations
    Inserm, UMR-970, Paris Cardiovascular Research Center, PARCC, Paris, France

    INSERM, UMR1149, Centre de Recherche sur l'Inflammation, Paris, France

    University Paris Diderot, Paris, France

    Service d'Hépatologie, Centre de Référence des Maladies Vasculaires du Foie, DHU Unity, Pôle des Maladies de l'Appareil Digestif, Hôpital Beaujon, AP-HP, Clichy, France
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Published:February 20, 2019DOI:https://doi.org/10.1016/j.jhep.2019.02.012

      Summary

      Non-alcoholic fatty liver disease (NAFLD) and its complications are an expanding health problem associated with the metabolic syndrome. Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells localized at the interface between the blood derived from the gut and the adipose tissue on the one side, and other liver cells on the other side. In physiological conditions, LSECs are gatekeepers of liver homeostasis. LSECs display anti-inflammatory and anti-fibrogenic properties by preventing Kupffer cell and hepatic stellate cell activation and regulating intrahepatic vascular resistance and portal pressure. This review focusses on changes occurring in LSECs in NAFLD and on their consequences on NAFLD progression and complications. Capillarization, namely the loss of LSEC fenestrae, and LSEC dysfunction, namely the loss of the ability of LSECs to generate vasodilator agents in response to increased shear stress both occur early in NAFLD. These LSEC changes favour steatosis development and set the stage for NAFLD progression. At the stage of non-alcoholic steatohepatitis, altered LSECs release inflammatory mediators and contribute to the recruitment of inflammatory cells, thus promoting liver injury and inflammation. Altered LSECs also fail to maintain hepatic stellate cell quiescence and release fibrogenic mediators, including Hedgehog signalling molecules, promoting liver fibrosis. Liver angiogenesis is increased in NAFLD and contributes to liver inflammation and fibrosis, but also to hepatocellular carcinoma development. Thus, improving LSEC health appears to be a promising approach to prevent NAFLD progression and complications.

      Keywords

      Introduction

      Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of conditions including simple steatosis and non-alcoholic steatohepatitis (NASH), defined as the association of steatosis, hepatocellular damage, inflammation and varying degrees of fibrosis.
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      The vascular endothelium, representing the interface between blood and other tissues of the body, is not only a physical barrier but is implicated in different physiological roles, such as haemostasis, metabolite transportation, inflammation, thrombosis, angiogenesis and vascular tone. The liver endothelium is mainly formed of liver sinusoidal endothelial cells (LSECs) which are highly specialized endothelial cells at the interface between blood derived from the visceral adipose tissue and the gut, on the one side, and hepatic stellate cells and hepatocytes, on the other side. LSECs have a unique phenotype in the human body as they lack a basement membrane and have a multitude of fenestrae organized into sieves, that regulate the transport of macromolecules, including lipids and lipoproteins, across the sinusoid.
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      LSECs and simple steatosis

      Role of LSECs in lipid transfer in the normal liver

      Dietary lipids present in the circulation have to be transported through the vascular endothelium to be metabolized by tissues. In physiological conditions, LSECs are major regulators of the bidirectional lipid exchange between the blood and the liver parenchyma. First, LSEC fenestrae allow for efficient transfer of lipoproteins, chylomicron remnants (small lipoproteins derived from chylomicrons generated by enterocytes from dietary lipids), and other macromolecules, from the sinusoidal blood to the space of Disse, where they are taken up by hepatocytes.
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      LSEC capillarization occurs early in NAFLD and promotes steatosis

      LSECs undergo morphological and functional changes during liver steatosis.
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      Triggers for sinusoidal capillarization are not fully identified, but we can speculate that excessive dietary macronutrients, including lipids, carbohydrates, and gut microbiota-derived products play a role.
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      The effect of FFA on fenestrae has also been tested in primary rat LSECs, but firm conclusions cannot be drawn as the authors did not test several concentrations of FFA but rather the presence vs. the absence of FFA, which does not adequately mimic in vivo conditions.
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      Moreover, it has been shown that a single injection of endotoxin in rats induces a decrease in both diameter and number of fenestrae suggesting that gut microbiota-derived products may contribute to LSEC capillarization, although caution is needed since the concentration of endotoxin used in that study was high.
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      In turn, capillarization favours liver steatosis (Fig. 1), as observed in mice deficient in plasmalemma vesicle-associated protein (PLVAP), an endothelial-specific integral membrane glycoprotein required for the formation of endothelial fenestrae.
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      Figure thumbnail gr1
      Fig. 1LSEC capillarization promotes steatosis. In physiological conditions, chylomicron remnants cross LSEC fenestrae and provide cholesterol and triglycerides for VLDL synthesis. VLDL are then released by hepatocytes and reach blood flow through fenestrae. In metabolic syndrome conditions, LSEC capillarization arises early in the course of NAFLD, possibly because of exposure of LSECs to dietary macronutrients. In turn, LSEC capillarization promotes steatosis, possibly because capillarization blocks the transfer of chylomicron remnants to hepatocytes, thus stimulating endogenous cholesterol and triglyceride synthesis, as a compensatory mechanism for the synthesis of VLDL, which reach blood flow through the lymphatic system. FFA, free fatty acids; LDL, low-density lipoprotein; LSECs, liver sinusoidal endothelial cells; NAFLD, non-alcoholic fatty liver disease; ox-LDL, oxidized low-density lipoprotein; VLDL; very low-density lipoprotein.
      A first hypothesis explaining this consequence of capillarization on steatosis could be that reduced LSEC permeability impairs the passage of hepatocyte-derived very low-density lipoprotein toward the sinusoidal lumen, thus inducing cholesterol and triglyceride retention in the liver. However, these lipoproteins may escape the liver through the lymphatic system.
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      However, there is no available data to ascertain this hypothesis. Similarly, Fraser and collaborators postulated that, following LSEC capillarization, chylomicron remnants and dietary cholesterol no longer cross the fenestrae to inhibit HMGCoA reductase, the rate limiting enzyme for hepatocyte cholesterol biosynthesis, consequently activating endogenous cholesterol synthesis in hepatocytes.
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      LSEC capillarization and dysfunction occur very early in NAFLD progression and contribute to dietary induced steatosis.

      LSECs dysfunction occurs early in NAFLD and promotes steatosis

      Liver steatosis is associated with an increased portal pressure and increased intrahepatic vascular resistance.
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      Contribution of cyclooxygenase end products and oxidative stress to intrahepatic endothelial dysfunction in early non-alcoholic fatty liver disease.
      Intrahepatic vascular resistance is increased even when steatosis is the only histological feature of NAFLD. This is due to the combination of a compression of sinusoids by fat-laden enlarged hepatocytes and of a dysfunction of LSECs due to reduced nitric oxide bioavailability.
      Several mechanisms could account for this liver endothelial dysfunction associated with steatosis (Fig. 2). First, LSECs dysfunction can be induced by overabundance of lipids during steatosis. In vitro experiments showed that stimulation of human primary LSECs with ox-LDL downregulates eNOS expression through the ox-LDL receptor, LOX1.
      • Zhang Q.
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      • Liu J.
      • Huang W.
      • Tian L.
      • Quan J.
      • et al.
      oxLDL induces injury and defenestration of human liver sinusoidal endothelial cells via LOX1.
      In addition, exposure of primary LSECs to palmitic acid also attenuates nitric oxide bioavailability through peroxynitrite production by NOX1, a nitric oxide consuming enzyme highly expressed in LSECs of mice fed a high-fat diet.
      • Matsumoto M.
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      • Jiang J.X.
      • Yamaguchi K.
      • et al.
      The NOX1 isoform of NADPH oxidase is involved in dysfunction of liver sinusoids in nonalcoholic fatty liver disease.
      Second, steatosis induces insulin resistance in LSECs, leading to impairment of insulin-dependent vasodilation.
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      • La Mura V.
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      • García-Calderó H.
      • Rodríguez-Vilarrupla A.
      • García-Pagán J.C.
      • et al.
      Sinusoidal endothelial dysfunction precedes inflammation and fibrosis in a model of NAFLD.
      • Pasarín M.
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      • La Mura V.
      • García-Pagán J.C.
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      Insulin resistance and liver microcirculation in a rat model of early NAFLD.
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      • La Mura V.
      Intrahepatic vascular changes in non-alcoholic fatty liver disease: Potential role of insulin-resistance and endothelial dysfunction.
      This effect is due, on the one hand, to the downregulation of eNOS activity,
      • 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.
      and on the other hand to the upregulation of iNOS (also called NOS2), the inducible form of NOS which can cause endothelial dysfunction through increased nitro-oxidative stress.
      • Pasarín M.
      • Abraldes J.G.
      • Liguori E.
      • Kok B.
      • La Mura V.
      Intrahepatic vascular changes in non-alcoholic fatty liver disease: Potential role of insulin-resistance and endothelial dysfunction.
      • Gunnett C.A.
      • Lund D.D.
      • Chu Y.
      • Brooks R.M.
      • Faraci F.M.
      • Heistad D.D.
      NO-dependent vasorelaxation is impaired after gene transfer of inducible NO-synthase.
      • Chauhan S.D.
      • Seggara G.
      • Vo P.A.
      • Macallister R.J.
      • Hobbs A.J.
      • Ahluwalia A.
      Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice.
      Interestingly, V-PYRRO/NO – a diazeniumdiolate ion metabolized in the liver that spontaneously decomposes to nitric oxide with a very short half-life at physiological pH and that triggers cyclic guanosine 3′,5′-monophosphate (cGMP) synthesis – improves hepatic microcirculation in mice with steatosis induced by a high-fat diet.
      • Kus E.
      • Jasiński K.
      • Skórka T.
      • Czyzynska-Cichon I.
      • Chlopicki S.
      Short-term treatment with hepatoselective NO donor V-PYRRO/NO improves blood flow in hepatic microcirculation in liver steatosis in mice.
      Third, the gut microbiota also seems to contribute to liver endothelial dysfunction. Indeed, Garcia-Lezana and colleagues demonstrated that restoration of a healthy microbiota via faecal transplantation normalizes portal hypertension by improving intrahepatic vascular resistance and endothelial dysfunction in rats.
      • García-Lezana T.
      • Raurell I.
      • Bravo M.
      • Torres-Arauz M.
      • Salcedo M.T.
      • Santiago A.
      • et al.
      Restoration of a healthy intestinal microbiota normalizes portal hypertension in a rat model of nonalcoholic steatohepatitis.
      Figure thumbnail gr2
      Fig. 2LSEC dysfunction promotes steatosis. In physiological conditions, LSECs release NO which regulates intrahepatic vascular tone on the one hand, and hepatic lipid metabolism on the other hand. NO limits hepatic lipid content by inhibiting hepatic de novo lipogenesis, through a limitation of citrate synthesis in mitochondria, an inhibition of ACC and of GPAT, and by promoting fatty acids beta-oxidation. In metabolic syndrome conditions, overabundance of lipids and insulin resistance lead to downregulation of eNOS activity and to upregulation of iNOS and of NOX1 (a nitric oxide consuming enzyme), causing nitro-oxidative stress through peroxynitrite production and eventually endothelial dysfunction. Reduced NO availability promotes steatosis. Liver steatosis is associated with an increased intrahepatic vascular resistance which has a mechanical component, due to the compression of the sinusoidal lumen by enlarged fat-laden hepatocytes, and a dynamic component, due to a liver endothelial dysfunction. ACC, acetyl CoA carboxylase; AMPK, AMP-activated protein kinase; eNOS, endothelial nitric oxide synthase; GPAT, glycerol-3-phosphate acyltransferase; LSECs, liver sinusoidal endothelial cells; IHRV; intrahepatic vascular resistance; iNOS, inducible nitric oxide synthase; NO, nitric oxide; NOX1, NADPH oxidase 1.
      In turn, LSEC dysfunction favours steatosis (Fig. 2). Indeed, deficiency in nitric oxide in eNos−/− mice results in massive fat droplet deposition and increases liver triglyceride content.
      • Tateya S.
      • Rizzo N.O.
      • Handa P.
      • Cheng A.M.
      • Morgan-Stevenson V.
      • Daum G.
      • et al.
      Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding.
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      Impairment of endothelial nitric oxide synthase causes abnormal fat and glycogen deposition in liver.
      Nitric oxide contributes to the regulation of hepatic lipid content by limiting citrate synthesis in mitochondria, which is involved in fatty acid production.
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      • Schulz C.
      • Gardemann A.
      • Keilhoff G.
      Impairment of endothelial nitric oxide synthase causes abnormal fat and glycogen deposition in liver.
      Nitric oxide also attenuates synthesis of fatty acids in isolated cultured rat hepatocytes by nitrosylating acetyl-CoA
      • Roediger W.E.
      • Hems R.
      • Wiggins D.
      • Gibbons G.F.
      Inhibition of hepatocyte lipogenesis by nitric oxide donor: could nitric oxide regulate lipid synthesis?.
      and activating AMP-activated protein kinase,
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      • Hardie D.G.
      Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise.
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      • Shi W.
      • Spencer T.E.
      • et al.
      Dietary L-arginine supplementation reduces fat mass in Zucker diabetic fatty rats.
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      • Shi C.-M.
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      • et al.
      Activation of 5’-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite.
      an inhibitor of glycerol-3-phosphate acyltransferase and thus of triacylglycerol synthesis.
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      • et al.
      Coordinate regulation of malonyl-CoA decarboxylase, sn-glycerol-3-phosphate acyltransferase, and acetyl-CoA carboxylase by AMP-activated protein kinase in rat tissues in response to exercise.
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      • Witters L.A.
      • Coleman R.A.
      AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target.
      In addition, nitric oxide also allows efficient fatty acid beta-oxidation through s-nitrosylation of very long-chain acyl-CoA dehydrogenase in hepatocytes.
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      • Tenopoulou M.
      • Greene J.L.
      • Raju K.
      • Ischiropoulos H.
      Nitric oxide regulates mitochondrial fatty acid metabolism through reversible protein S-nitrosylation.
      Interestingly, therapies augmenting nitric oxide availability in the liver ameliorate steatosis. The V-PYRRO/NO or the improvement of nitric oxide/cGMP signalling with the phosphodiesterase-5 inhibitor sildenafil protect against liver steatosis in mice fed a high-fat diet.
      • Tateya S.
      • Rizzo N.O.
      • Handa P.
      • Cheng A.M.
      • Morgan-Stevenson V.
      • Daum G.
      • et al.
      Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding.
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      • et al.
      The liver-selective NO donor, V-PYRRO/NO, protects against liver steatosis and improves postprandial glucose tolerance in mice fed high fat diet.
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      Hepatoselective nitric oxide (NO) donors, V-PYRRO/NO and V-PROLI/NO, in nonalcoholic fatty liver disease: a comparison of antisteatotic effects with the biotransformation and pharmacokinetics.
      Moreover, treatment with simvastatin, a drug able to increase expression and activity of eNOS expression in the liver, decreases steatosis induced by a high-fat diet in rats.
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      • Zhou J.
      • Zhen Z.
      • Wang Y.
      • Shen C.
      Simvastatin ameliorates liver fibrosis via mediating nitric oxide synthase in rats with non-alcoholic steatohepatitis-related liver fibrosis.
      To summarize, steatosis is associated with LSEC dysfunction which in turn worsens steatosis (Fig. 2).
      Molecular events associated with angiogenesis are initiated during simple steatosis, but angiogenesis itself is only detected in NASH.

      Angiogenesis and steatosis

      Angiogenesis, defined as the formation of new vessels from pre-existing vessels, is a key event in the progression of NAFLD.
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      Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease.
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      Role of vascular endothelial growth factor in the pathophysiology of nonalcoholic steatohepatitis in two rodent models.
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      Could inflammatory markers help diagnose nonalcoholic steatohepatitis?.
      VEGF is the master pro-angiogenic regulator of this process supported by activation of hypoxia inducible factors (HIFs) in hypoxic areas.
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      • Shah V.
      • Rockey D.C.
      Vascular pathobiology in chronic liver disease and cirrhosis - current status and future directions.
      Serum VEGF levels are higher in patients with biopsy-proven steatosis than in healthy individuals.
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      • et al.
      Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease.
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      • et al.
      Role of vascular endothelial growth factor in the pathophysiology of nonalcoholic steatohepatitis in two rodent models.
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      • et al.
      Could inflammatory markers help diagnose nonalcoholic steatohepatitis?.
      In animal models, liver expression of VEGF and CD105, an endothelial cell marker, increase after 3 days of methionine- and choline-deficient diet in obese and diabetic db/db transgenic mice and after 1 week of this diet in C57BL6/J mice, before NASH appears.
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      • et al.
      Role of vascular endothelial growth factor in the pathophysiology of nonalcoholic steatohepatitis in two rodent models.
      However, 3 studies reported that new vessels develop in the livers of patients with NASH, but not in individuals with simple steatosis or normal livers.
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      Neovascularization and oxidative stress in the progression of non-alcoholic steatohepatitis.
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      • et al.
      Crosstalk between angiogenesis, cytokeratin-18, and insulin resistance in the progression of non-alcoholic steatohepatitis.
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      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      This suggests that molecular events associated with upregulation of angiogenic factors start early in the course of NAFLD, while angiogenesis appears later, as detailed below.

      LSECs in NASH

      LSECs contribute to oxidative stress in NASH

      In response to lipotoxicity, hepatocytes generate reactive oxygen species (ROS) and initiate a robust inflammatory response that accentuates liver injury.
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      Mechanisms of NAFLD development and therapeutic strategies.
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      Lipotoxicity and the gut-liver axis in NASH pathogenesis.
      In addition to hepatocytes,
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      Mechanisms of NAFLD development and therapeutic strategies.
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      Lipotoxicity and the gut-liver axis in NASH pathogenesis.
      the lipotoxic response also occurs in LSECs contributing to ROS generation.
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      • Jiang J.X.
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      • et al.
      The NOX1 isoform of NADPH oxidase is involved in dysfunction of liver sinusoids in nonalcoholic fatty liver disease.
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      • Borradaile N.M.
      Non-parenchymal hepatic cell lipotoxicity and the coordinated progression of non-alcoholic fatty liver disease and atherosclerosis.
      Indeed, ROS have been detected not only in hepatocytes but also in sinusoidal cells in patients with NASH.
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      In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases.
      Circulating lipids seem to account for the oxidative stress in LSECs. Indeed, exposure of murine LSECs to palmitic acid upregulates NOX1 expression, an enzyme implicated in ROS production.
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      • et al.
      The NOX1 isoform of NADPH oxidase is involved in dysfunction of liver sinusoids in nonalcoholic fatty liver disease.
      In addition, stimulation of human primary LSECs with ox-LDL increases ROS generation after binding to LOX1.
      • Zhang Q.
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      • Huang W.
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      • et al.
      oxLDL induces injury and defenestration of human liver sinusoidal endothelial cells via LOX1.
      This oxidative stress in LSECs contributes to NASH. Indeed, mice with a global deficiency in NOX1, which is highly expressed in LSECs in NAFLD, had attenuated liver lesions when fed a high-fat diet, as shown by lower serum ALT level and lower hepatic cleaved caspase-3 expression.
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      • et al.
      The NOX1 isoform of NADPH oxidase is involved in dysfunction of liver sinusoids in nonalcoholic fatty liver disease.
      Therefore, in NASH, ROS production takes place not only in hepatocytes, but also to some extent in LSECs, and seems to contribute to hepatocyte injury.

      Anti-inflammatory role of LSECs at initial stages of NASH

      Progression of simple steatosis to steatohepatitis is accompanied by adhesion of leukocytes to the sinusoidal endothelium followed by infiltration of leukocytes within liver parenchyma to form inflammatory foci.
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      Moderate and resolved inflammatory responses are beneficial to the liver as they promote the re-establishment of homeostasis, contribute to tissue repair and exert hepatoprotective effects.
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      Decoding cell death signals in liver inflammation.
      However, chronic inflammation, as seen in NASH, leads to death of hepatocytes and causes damage to the liver parenchyma.
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      Decoding cell death signals in liver inflammation.
      In physiological conditions, LSECs constitute a barrier regulating the entry of circulating leukocytes within liver parenchyma and playing an anti-inflammatory role.
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      Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation.
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      • et al.
      TGF-β-dependent induction of CD4+CD25+Foxp3+ Tregs by liver sinusoidal endothelial cells.
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      • Groettrup M.
      • et al.
      Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance.
      • Berg M.
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      • Hämmerling G.
      • et al.
      Cross-presentation of antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-specific CD8+ T cell tolerance.
      At early stages of NAFLD progression, some evidence indicates that LSECs also exhibit anti-inflammatory functions.
      • Tateya S.
      • Rizzo N.O.
      • Handa P.
      • Cheng A.M.
      • Morgan-Stevenson V.
      • Daum G.
      • et al.
      Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding.
      • McMahan R.H.
      • Porsche C.E.
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      Free fatty acids differentially downregulate chemokines in liver sinusoidal endothelial cells: insights into non-alcoholic fatty liver disease.
      Indeed, Tateya and colleagues elegantly demonstrated that nitric oxide derived from LSECs inhibits Kupffer cell activation in mice fed a high-fat diet for a short period of time (8 weeks).
      • Tateya S.
      • Rizzo N.O.
      • Handa P.
      • Cheng A.M.
      • Morgan-Stevenson V.
      • Daum G.
      • et al.
      Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding.
      In vitro, both human and murine LSECs exposed to FFA for a short period (16 hours) exhibit a downregulation of pro-inflammatory chemokines involved in monocyte and macrophage recruitment, through a MAPK dependent pathway.
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      • Porsche C.E.
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      Free fatty acids differentially downregulate chemokines in liver sinusoidal endothelial cells: insights into non-alcoholic fatty liver disease.
      Lipotoxicity and inflammation induce endothelial inflammation. Activated LSECs release cytokines and chemokines and overexpress adhesion molecules, thus sustaining liver inflammation.

      LSECs promotes liver inflammation at more advanced stages of NASH

      As mentioned, LSEC alterations arise early in NAFLD progression, prior to liver inflammation.
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      Pivotal role of liver sinusoidal endothelial cells in NAFLD/NASH progression.
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      Sinusoidal endothelial dysfunction precedes inflammation and fibrosis in a model of NAFLD.
      Indeed, LSEC capillarization precedes Kupffer cell activation
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      Pivotal role of liver sinusoidal endothelial cells in NAFLD/NASH progression.
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      Sinusoidal endothelial dysfunction precedes inflammation and fibrosis in a model of NAFLD.
      and liver nitric oxide content falls before liver NF-kB activation and TNFα, IL-6 and ICAM-1 upregulation.
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      Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture.
      • Tateya S.
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      • Morgan-Stevenson V.
      • Daum G.
      • et al.
      Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding.
      • Pasarín M.
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      • García-Calderó H.
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      • García-Pagán J.C.
      • et al.
      Sinusoidal endothelial dysfunction precedes inflammation and fibrosis in a model of NAFLD.
      LSEC capillarization and dysfunction are permissive for establishment of liver inflammation. Indeed, mice deficient in eNOS exhibit an accelerated hepatic inflammatory response, while improving nitric oxide/cGMP signalling with the phosphodiesterase-5 inhibitor sildenafil or with simvastatin prevents liver inflammation in rodents fed a high-fat diet.
      • Tateya S.
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      • Daum G.
      • et al.
      Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding.
      • Wang W.
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      • Shen C.
      Simvastatin ameliorates liver fibrosis via mediating nitric oxide synthase in rats with non-alcoholic steatohepatitis-related liver fibrosis.
      During NALFD progression, LSECs then acquire a pro-inflammatory phenotype and functions (Fig. 3). LSECs pro-inflammatory phenotype during NASH is characterized by progressive overexpression of adhesion molecules including ICAM-1, VCAM-1 and VAP-1 (AOC3) at the surface of LSECs, as observed in mouse models of NASH.
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      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
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      Administration of the potent PPARalpha agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice.
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      Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis.
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      Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice.
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      Roles for cell-cell adhesion and contact in obesity-induced hepatic myeloid cell accumulation and glucose intolerance.
      LSECs also produce a number of pro-inflammatory mediators in NASH, including TNFα, IL-6, IL-1 and MCP1 (CCL2).
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      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      • Miyachi Y.
      • Tsuchiya K.
      • Komiya C.
      • Shiba K.
      • Shimazu N.
      • Yamaguchi S.
      • et al.
      Roles for cell-cell adhesion and contact in obesity-induced hepatic myeloid cell accumulation and glucose intolerance.
      • Wu J.
      • Meng Z.
      • Jiang M.
      • Zhang E.
      • Trippler M.
      • Broering R.
      • et al.
      Toll-like receptor-induced innate immune responses in non-parenchymal liver cells are cell type-specific.
      • Feder L.S.
      • Todaro J.A.
      • Laskin D.L.
      Characterization of interleukin-1 and interleukin-6 production by hepatic endothelial cells and macrophages.
      Figure thumbnail gr3
      Fig. 3LSECs acquire pro-inflammatory functions in NASH. Lipotoxicity, inflammation and gut microbiota-derived products induce LSECs’ inflammatory phenotype and function mediated by NF-kB activation, which orchestrates the release of pro-inflammatory mediators and the overexpression of adhesion molecules. Inflammatory mediators and LSEC dysfunction promote Kupffer cell activation and leukocyte chemoattraction. Adhesion molecule overexpression allows adhesion and transendothelial migration of the recruited leukocytes in the hepatic parenchyma. ICAM-1, intercellular adhesion molecule-1; IL-1, interleukin 1; IL-6, interleukin 6; LSECs, liver sinusoidal endothelial cells; MCP1, monocyte chemoattractant protein-1; NASH, non-alcoholic steatohepatitis; NF-kB, nuclear factor kappa B; NK, natural killer; NO, nitric oxide; TNFα, tumor necrosis factor alpha; VAP-1, vascular adhesion protein-1; VCAM-1, vascular cell adhesion molecule-1.
      This pro-inflammatory phenotype of LSECs in NASH is associated with pro-inflammatory functions (Fig. 3). First, dysfunctional LSECs fail to maintain Kupffer cell quiescence.
      • Tateya S.
      • Rizzo N.O.
      • Handa P.
      • Cheng A.M.
      • Morgan-Stevenson V.
      • Daum G.
      • et al.
      Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding.
      Second, the release of inflammatory mediators by LSECs contributes to the inflammatory response by activating neighbouring Kupffer cells, and by favouring recruitment, adhesion and transmigration of blood leukocytes.
      • Miyachi Y.
      • Tsuchiya K.
      • Komiya C.
      • Shiba K.
      • Shimazu N.
      • Yamaguchi S.
      • et al.
      Roles for cell-cell adhesion and contact in obesity-induced hepatic myeloid cell accumulation and glucose intolerance.
      • Marra F.
      • Tacke F.
      Roles for chemokines in liver disease.
      • Roh Y.-S.
      • Seki E.
      Chemokines and chemokine receptors in the development of NAFLD.
      The mechanisms of interaction between leukocytes and LSECs in NASH have been reviewed elsewhere in detail and are summarized in Fig. 3.
      • Weston C.J.
      • Shepherd E.L.
      • Claridge L.C.
      • Rantakari P.
      • Curbishley S.M.
      • Tomlinson J.W.
      • et al.
      Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis.
      • Shetty S.
      • Lalor P.F.
      • Adams D.H.
      Lymphocyte recruitment to the liver: molecular insights into the pathogenesis of liver injury and hepatitis.
      • Lalor P.F.
      • Shields P.
      • Grant A.
      • Adams D.H.
      Recruitment of lymphocytes to the human liver.
      • Lalor P.F.
      • Edwards S.
      • McNab G.
      • Salmi M.
      • Jalkanen S.
      • Adams D.H.
      Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells.
      LSECs’ expression of ICAM-1, VCAM-1 and VAP-1 is crucial for these interactions since in vivo and in vitro studies showed reduced leukocyte adhesion to hepatic sinusoids when these receptors are blocked or not functional.
      • Weston C.J.
      • Shepherd E.L.
      • Claridge L.C.
      • Rantakari P.
      • Curbishley S.M.
      • Tomlinson J.W.
      • et al.
      Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis.
      • Edwards S.
      • Lalor P.F.
      • Nash G.B.
      • Rainger G.E.
      • Adams D.H.
      Lymphocyte traffic through sinusoidal endothelial cells is regulated by hepatocytes.
      Moreover, inhibition of the VCAM-1 ligand, VLA-4 (or ITGA4), on monocytes using an anti-VLA-4 antibody inhibits adhesion and transendothelial migration of monocytes across LSECs – from wild-type mice fed a high-fat diet and from ob/ob obese mice – and improves liver inflammation.
      • Miyachi Y.
      • Tsuchiya K.
      • Komiya C.
      • Shiba K.
      • Shimazu N.
      • Yamaguchi S.
      • et al.
      Roles for cell-cell adhesion and contact in obesity-induced hepatic myeloid cell accumulation and glucose intolerance.
      Although the stimuli responsible for LSECs’ inflammatory phenotype and functions in NASH are not firmly identified, several mediators are potential candidates. This includes products derived from the visceral adipose tissue, such as ox-LDL, FFA and adipokines. Indeed, in vitro studies showed that stimulation of LSECs with ox-LDL and FFA (palmitate) activate NF-kB and TLR-4, respectively.
      • Zhang Q.
      • Liu J.
      • Liu J.
      • Huang W.
      • Tian L.
      • Quan J.
      • et al.
      oxLDL induces injury and defenestration of human liver sinusoidal endothelial cells via LOX1.
      • Matsumoto M.
      • Zhang J.
      • Zhang X.
      • Liu J.
      • Jiang J.X.
      • Yamaguchi K.
      • et al.
      The NOX1 isoform of NADPH oxidase is involved in dysfunction of liver sinusoids in nonalcoholic fatty liver disease.
      • Sutter A.G.
      • Palanisamy A.P.
      • Lench J.H.
      • Esckilsen S.
      • Geng T.
      • Lewin D.N.B.
      • et al.
      Dietary saturated fat promotes development of hepatic inflammation through toll-like receptor 4 in mice.
      Moreover, circulating concentrations of several adipokines, including TNFα and IL-6, are increased in the portal vein in the context of metabolic syndrome, and may contribute to LSECs inflammatory phenotype.
      • 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.
      The gut microbiota also has an emerging role in NASH as a source of inflammatory stimuli.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      • Marra F.
      • Svegliati-Baroni G.
      Lipotoxicity and the gut-liver axis in NASH pathogenesis.
      Increased intestinal permeability and elevated plasma concentrations of lipopolysaccharide (LPS)
      • Harte A.L.
      • da Silva N.F.
      • Creely S.J.
      • McGee K.C.
      • Billyard T.
      • Youssef-Elabd E.M.
      • et al.
      Elevated endotoxin levels in non-alcoholic fatty liver disease.
      • Brun P.
      • Castagliuolo I.
      • Di Leo V.
      • Buda A.
      • Pinzani M.
      • Palù G.
      • et al.
      Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis.
      observed in NASH may also contribute to LSECs’ pro-inflammatory function.
      • Wu J.
      • Meng Z.
      • Jiang M.
      • Zhang E.
      • Trippler M.
      • Broering R.
      • et al.
      Toll-like receptor-induced innate immune responses in non-parenchymal liver cells are cell type-specific.
      Besides mediators derived from the portal vein, hepatocytes and liver inflammatory cells also release inflammatory mediators in NASH that can activate LSECs.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      • Kazankov K.
      • Jørgensen S.M.D.
      • Thomsen K.L.
      • Møller H.J.
      • Vilstrup H.
      • George J.
      • et al.
      The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
      To summarize, while LSECs play an anti-inflammatory role in the initial stages of NAFLD, a switch towards pro-inflammatory functions occurs during the course of NAFLD development, paving the way for NASH progression.

      Angiogenesis in liver inflammation in NASH

      Inflammation promotes angiogenesis that in turn worsens liver inflammation as demonstrated by the anti-inflammatory effect of anti-angiogenic therapies.
      Pathologic angiogenesis increases with NASH
      • Coulon S.
      • Heindryckx F.
      • Geerts A.
      • Van Steenkiste C.
      • Colle I.
      • Van Vlierberghe H.
      Angiogenesis in chronic liver disease and its complications.
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      • Tamaki Y.
      • Nakade Y.
      • Yamauchi T.
      • Makino Y.
      • Yokohama S.
      • Okada M.
      • et al.
      Angiotensin II type 1 receptor antagonist prevents hepatic carcinoma in rats with nonalcoholic steatohepatitis.
      • Yoshiji H.
      • Kuriyama S.
      • Noguchi R.
      • Ikenaka Y.
      • Kitade M.
      • Kaji K.
      • et al.
      Angiotensin-II and vascular endothelial growth factor interaction plays an important role in rat liver fibrosis development.
      (Fig. 4). Indeed, several studies reported the formation of new vessels in the liver of patients with NASH. Moreover, serum VEGF and sVEGFR1 levels are higher in patients with steatosis and biopsy-proven NASH than in healthy individuals.
      • 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.
      • Kitade M.
      • Yoshiji H.
      • Noguchi R.
      • Ikenaka Y.
      • Kaji K.
      • Shirai Y.
      • et al.
      Crosstalk between angiogenesis, cytokeratin-18, and insulin resistance in the progression of non-alcoholic steatohepatitis.
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      • Cayón A.
      • Crespo J.
      • Guerra A.R.
      • Pons-Romero F.
      Gene expression in obese patients with non-alcoholic steatohepatitis.
      Similarly, in animal models of NASH, liver vasculature is disrupted and hepatic expression of VEGF and CD105 is increased.
      • 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.
      Figure thumbnail gr4
      Fig. 4Angiogenesis in NASH. Hypoxia, liver injury, lipids, oxidative stress, inflammation and fibrosis induce the release of pro-angiogenic factors, such as hepatocyte-derived microvesicles, VEGF and Angiopoietin-2, from parenchymal and non-parenchymal cells including LSECs, promoting pathologic angiogenesis. Adipokines, such as leptin, also exhibit pro-angiogenic activity contributing to pathologic angiogenesis in NASH. In turn, angiogenesis promotes liver inflammation and fibrosis as shown by anti-angiogenic therapies which prevent liver inflammation and fibrosis in experimental models of NASH. LSECs, liver sinusoidal endothelial cells; NASH, non-alcoholic steatohepatitis; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2.
      Several mechanisms trigger angiogenesis during NASH. First, chronic inflammation promotes angiogenesis. Indeed, chronic inflammation sustains tissue hypoxia and induces transcription of angiogenic genes modulated by HIF-1α.
      • Coulon S.
      • Heindryckx F.
      • Geerts A.
      • Van Steenkiste C.
      • Colle I.
      • Van Vlierberghe H.
      Angiogenesis in chronic liver disease and its complications.
      • Kietzmann T.
      • Görlach A.
      Reactive oxygen species in the control of hypoxia-inducible factor-mediated gene expression.
      Pro-inflammatory mediators also elicit a direct angiogenic response through the induction of HIF-1α transcriptional activity and VEGF production.
      • Coulon S.
      • Heindryckx F.
      • Geerts A.
      • Van Steenkiste C.
      • Colle I.
      • Van Vlierberghe H.
      Angiogenesis in chronic liver disease and its complications.
      Moreover, cytokines and ROS released during NASH can activate the MAPK/ERK pathway, a signalling pathway involved in cell migration and angiogenesis.
      • Coulon S.
      • Heindryckx F.
      • Geerts A.
      • Van Steenkiste C.
      • Colle I.
      • Van Vlierberghe H.
      Angiogenesis in chronic liver disease and its complications.
      Second, hepatocyte-derived microvesicles link lipotoxicity with angiogenesis. Indeed, hepatocytes exposed in vitro to excessive amounts of saturated FFA, that mimics steatosis, release microvesicles with a pro-angiogenic activity.
      • Povero D.
      • Eguchi A.
      • Niesman I.R.
      • Andronikou N.
      • de Mollerat du Jeu X.
      • Mulya A.
      • et al.
      Lipid-induced toxicity stimulates hepatocytes to release angiogenic microparticles that require Vanin-1 for uptake by endothelial cells.
      Likewise, mice fed a methionine- and choline-deficient diet have high circulating levels of hepatocyte-derived microvesicles able to induce angiogenesis. Third, angiopoietin-2 is another mechanism of liver angiogenesis in NASH. Angiopoietins are key regulators of angiogenesis. Although angiopoietins-1 and 2 contribute to vascular stability and quiescence in physiological conditions, angiopoietin-2 promotes pathological angiogenesis in inflammatory conditions.
      • Kim M.
      • Allen B.
      • Korhonen E.A.
      • Nitschké M.
      • Yang H.W.
      • Baluk P.
      • et al.
      Opposing actions of angiopoietin-2 on Tie2 signaling and FOXO1 activation.
      Lefere and coworkers recently showed that serum angiopoietin-2 levels are increased in patients with NASH and correlate with liver steatosis, inflammation and hepatocyte ballooning, but not with liver fibrosis.
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      Similar findings were observed with 2 murine models of NASH, namely mice fed a methionine- and choline-deficient diet and mice with neonatal injection of streptozotocin followed by 16 weeks of western diet.
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      The main source of hepatic angiopoietin-2 was LSECs.
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      Inhibiting angiopoietin-2 levels using the angiopoietin-2/Tie2 receptor inhibiting peptibody L1-10 reduced hepatic angiogenesis and normalized vascular microarchitecture.
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      In turn, angiogenesis promotes inflammation since various strategies of inhibition of angiogenesis all improve liver inflammation (Fig. 4). Coulon and colleagues showed in a mouse model of NASH that treatment with anti-VEGFR2 antibody improves liver vasculature and decreases liver inflammatory gene expression, both using preventive and therapeutic approaches.
      • 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.
      Lefere and colleagues showed that blocking angiopoietin-2/Tie2 interaction with the L1-10 peptibody also alleviates liver injury and inflammation in mice fed a methionine- and choline-deficient diet.
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      Importantly, this effect of L1-10 therapy is at least partly mediated by an effect on LSECs since L1-10 treatment downregulates VCAM-1, ICAM-1 and MCP1 expression in liver endothelial cells isolated from mice fed a methionine- and choline-deficient diet.
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      This anti-inflammatory effect of anti-angiogenic treatment is not specific for NASH, as it is observed in most models of chronic liver disease, namely carbon tetrachloride, and bile duct ligation.
      • Tugues S.
      • Fernandez-Varo G.
      • Muñoz-Luque J.
      • Ros J.
      • Arroyo V.
      • Rodés J.
      • et al.
      Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats.
      • Van Steenkiste C.
      • Ribera J.
      • Geerts A.
      • Pauta M.
      • Tugues S.
      • Casteleyn C.
      • et al.
      Inhibition of placental growth factor activity reduces the severity of fibrosis, inflammation, and portal hypertension in cirrhotic mice.
      • Thabut D.
      • Routray C.
      • Lomberk G.
      • Shergill U.
      • Glaser K.
      • Huebert R.
      • et al.
      Complementary vascular and matrix regulatory pathways underlie the beneficial mechanism of action of sorafenib in liver fibrosis.
      • Öztürk Akcora B.
      • Storm G.
      • Prakash J.
      • Bansal R.
      Tyrosine kinase inhibitor BIBF1120 ameliorates inflammation, angiogenesis and fibrosis in CCl4-induced liver fibrogenesis mouse model.
      • Yang L.
      • Kwon J.
      • Popov Y.
      • Gajdos G.B.
      • Ordog T.
      • Brekken R.A.
      • et al.
      Vascular endothelial growth factor promotes fibrosis resolution and repair in mice.
      To summarize, inflammation stimulates angiogenesis that in turn worsens inflammation, as shown by the anti-inflammatory effect of anti-angiogenic therapies (Fig. 4).

      LSECs in NASH-related liver fibrosis

      Liver fibrosis is defined as the excessive deposition of extracellular matrix in liver parenchyma. The main mechanism leading to liver fibrosis is a long-standing wound healing process caused by hepatocellular injury and inflammation and mediated by hepatic stellate cell activation.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      • Tsuchida T.
      • Friedman S.L.
      Mechanisms of hepatic stellate cell activation.
      Hepatic stellate cells are nonparenchymal cells close to LSECs, in the space of Disse, which store retinoids in physiological conditions and shift their phenotype to an activated myofibroblastic state during liver injury and inflammation, wherein they secrete large amounts of extracellular matrix compounds.
      • Tsuchida T.
      • Friedman S.L.
      Mechanisms of hepatic stellate cell activation.
      As detailed above, LSECs are major effectors of liver inflammation in NASH, and consequently also promote hepatic fibrosis. For example, LSECs overexpress VAP-1 during inflammation which, in addition to its pro-inflammatory functions in NASH, is directly involved in hepatic stellate cell activation.
      • Weston C.J.
      • Shepherd E.L.
      • Claridge L.C.
      • Rantakari P.
      • Curbishley S.M.
      • Tomlinson J.W.
      • et al.
      Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis.
      Inhibition or deficiency in VAP-1 in mice fed a methionine- and choline-deficient diet or a high-fat diet attenuates liver fibrosis.
      • Weston C.J.
      • Shepherd E.L.
      • Claridge L.C.
      • Rantakari P.
      • Curbishley S.M.
      • Tomlinson J.W.
      • et al.
      Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis.
      LSECs also contribute to liver fibrosis through capillarization and endothelial dysfunction, as detailed in the following sections.

      LSEC capillarization promotes liver fibrosis

      Capillarization is observed in patients and animal models of NASH, preceding fibrosis,
      • Miyao M.
      • Kotani H.
      • Ishida T.
      • Kawai C.
      • Manabe S.
      • Abiru H.
      • et al.
      Pivotal role of liver sinusoidal endothelial cells in NAFLD/NASH progression.
      • 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.
      • Schaffner F.
      • Poper H.
      Capillarization of hepatic sinusoids in man.
      • Xu B.
      • Broome U.
      • Uzunel M.
      • Nava S.
      • Ge X.
      • Kumagai-Braesch M.
      • et al.
      Capillarization of hepatic sinusoid by liver endothelial cell-reactive autoantibodies in patients with cirrhosis and chronic hepatitis.
      • Sørensen K.K.
      • Simon-Santamaria J.
      • McCuskey R.S.
      • Smedsrød B.
      Liver sinusoidal endothelial cells.
      • DeLeve L.D.
      • Wang X.
      • Kanel G.C.
      • Atkinson R.D.
      • McCuskey R.S.
      Prevention of hepatic fibrosis in a murine model of metabolic syndrome with nonalcoholic steatohepatitis.
      • DeLeve L.D.
      Liver sinusoidal endothelial cells in hepatic fibrosis.
      but also promoting its development (Box 1). Indeed, PLVAP deficient mice, displaying a pronounced reduction in the number of LSEC fenestrae, spontaneously develop perisinusoidal liver fibrosis.
      • Herrnberger L.
      • Hennig R.
      • Kremer W.
      • Hellerbrand C.
      • Goepferich A.
      • Kalbitzer H.R.
      • et al.
      Formation of fenestrae in murine liver sinusoids depends on plasmalemma vesicle-associated protein and is required for lipoprotein passage.
      LSEC capillarization and dysfunction precede liver fibrosis and are permissive for it, through the loss of the ability of LSECs to maintain quiescence of hepatic stellate cells.
      Experiments performed using cultured LSECs and hepatic stellate cells highlighted the importance of cross-talk between these cells types in regulating each other’s phenotype. While healthy LSECs maintain hepatic stellate cell quiescence, capillarized LSECs lose this ability
      • DeLeve L.D.
      Liver sinusoidal endothelial cells in hepatic fibrosis.
      • Maslak E.
      • Gregorius A.
      • Chlopicki S.
      Liver sinusoidal endothelial cells (LSECs) function and NAFLD; NO-based therapy targeted to the liver.
      (Fig. 5). A vicious cycle between LSEC capillarization and hepatic stellate cell activation then occurs during the fibrotic process.
      Figure thumbnail gr5
      Fig. 5LSECs in NASH-related fibrosis. Healthy LSECs are gatekeepers of liver fibrosis by maintaining HSCs quiescence through NO, while altered LSECs (following capillarization and LSEC dysfunction) lose this ability. In addition, altered LSECs release profibrogenic molecules such as TGF-β, Hedgehog molecules, laminin and fibronectin, which activate HSCs. Activated HSCs produces Hedgehog molecules reinforcing their own activation and LSEC capillarization. Activated HSCs then produce large amounts of extracellular matrix thus inducing liver fibrosis. ECM, extracellular matrix; Hh, Hedgehog; HSCs, hepatic stellate cells; LSECs, liver sinusoidal endothelial cell; NO, nitric oxide; TGF-β, transforming growth factor-β.
      In NASH, ballooned hepatocytes produce Hedgehog molecules.
      • Rangwala F.
      • Guy C.D.
      • Lu J.
      • Suzuki A.
      • Burchette J.L.
      • Abdelmalek M.F.
      • et al.
      Increased production of sonic hedgehog by ballooned hepatocytes.
      The Hedgehog pathway regulates capillarization, as inhibition of Hedgehog signalling prevents capillarization and partially reverts the phenotype of LSECs from a dedifferentiated state to their differentiated state.
      • Xie G.
      • Choi S.S.
      • Syn W.-K.
      • Michelotti G.A.
      • Swiderska M.
      • Karaca G.
      • et al.
      Hedgehog signalling regulates liver sinusoidal endothelial cell capillarisation.
      LSECs are thus Hedgehog-sensitive cells, but they are also Hedgehog producing cells. Similarly, quiescent hepatic stellate cells are Hedgehog-sensitive cells, while activated hepatic stellate cells become Hedgehog-producing cells, which are also able to release macrovesicles loaded with Hedgehog signalling molecules that interact with LSECs.
      • Matz-Soja M.
      • Gebhardt R.
      The many faces of Hedgehog signalling in the liver: recent progress reveals striking cellular diversity and the importance of microenvironments.
      • Witek R.P.
      • Yang L.
      • Liu R.
      • Jung Y.
      • Omenetti A.
      • Syn W.-K.
      • et al.
      Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.
      It is thus tempting to speculate that during NASH, Hedgehog ligands are released by hepatocytes and LSECs, thus activating LSECs themselves, as well as quiescent hepatic stellate cells, by autocrine and paracrine effects. Activated hepatic stellate cells can then secrete Hedgehog molecules, promoting LSEC capillarization which in turn favours hepatic stellate cell activation, promoting the fibrogenic process.

      LSECs dysfunction promotes liver fibrosis

      Endothelial dysfunction appears very early in the course of NAFLD and precedes fibrosis in animal models of NASH.
      • 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.
      • Tateya S.
      • Rizzo N.O.
      • Handa P.
      • Cheng A.M.
      • Morgan-Stevenson V.
      • Daum G.
      • et al.
      Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding.
      • 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.
      Several lines of evidence suggest that liver endothelial dysfunction contributes to liver fibrosis. First, in rats fed a high-fat diet, simvastatin increases liver eNOS expression and ameliorates liver fibrosis.
      • Wang W.
      • Zhao C.
      • Zhou J.
      • Zhen Z.
      • Wang Y.
      • Shen C.
      Simvastatin ameliorates liver fibrosis via mediating nitric oxide synthase in rats with non-alcoholic steatohepatitis-related liver fibrosis.
      Second, eNOS inhibition using L-NAME blocks the ability of healthy LSECs to keep hepatic stellate cells quiescent.
      • Deleve L.D.
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      Sinusoidal endothelial cells prevent rat stellate cell activation and promote reversion to quiescence.
      • Marrone G.
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      • et al.
      The transcription factor KLF2 mediates hepatic endothelial protection and paracrine endothelial-stellate cell deactivation induced by statins.
      Third, an activator of soluble guanylate cyclase, a receptor for nitric oxide, can recapitulate the effect of healthy LSECs and reverse hepatic stellate cell activation.
      • Xie G.
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      • et al.
      Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats.
      However, a limitation of these studies is that most approaches might not only act on endothelial function but also on LSEC capillarization.
      • DeLeve L.D.
      Liver sinusoidal endothelial cells in hepatic fibrosis.
      • Deleve L.D.
      • Wang X.
      • Guo Y.
      Sinusoidal endothelial cells prevent rat stellate cell activation and promote reversion to quiescence.
      Indeed, although L-NAME blocks LSEC-induced hepatic stellate cell quiescence, addition of a nitric oxide donor does not directly reverse hepatic stellate cell activation in vitro,
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      suggesting that additional LSEC-derived factors could be responsible for the reversion of activated hepatic stellate cells to quiescence.
      To summarize, these data demonstrate that capillarization and LSEC dysfunction not only precede liver fibrosis, but also promote it (Fig. 5). In their differentiated state, LSECs are able to maintain hepatic stellate cell quiescence, making differentiated LSECs gatekeepers of fibrosis in NASH, as in other chronic liver diseases.

      Liver endothelial-to-mesenchymal transition: a process promoting liver fibrosis?

      Another important process that links endothelial cells to organ fibrosis is endothelial-to-mesenchymal transition, i.e. the mechanism by which endothelial cells convert into myofibroblasts and contribute to extracellular matrix deposition.
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      Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases.
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      Endothelial-to-mesenchymal transition occurs in various fibrotic cardiovascular and pulmonary diseases.
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      Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases.
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      Endothelial to mesenchymal transition (EndoMT) in the pathogenesis of human fibrotic diseases.
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      In vitro studies showed that healthy LSECs produce a modest amount of collagen and fibronectin.
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      Cell biology of liver endothelial and Kupffer cells.
      Capillarized LSECs secrete fibrogenic factors, such as TGF-β1, and extracellular matrix proteins, such as fibronectin and laminin, that may be considered as an endothelial-to-mesenchymal transition, as well as stimulating activation of neighbouring hepatic stellate cells.
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      Exosome adherence and internalization by hepatic stellate cells triggers sphingosine 1-phosphate-dependent migration.
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      Cellular sources of extracellular matrix in hepatic fibrosis.
      In the liver disease field, only 1 study has described endothelial-to-mesenchymal transition in vivo, in patients with alcohol- or hepatitis C virus-related cirrhosis and in mice treated with carbon tetrachloride.
      • Ribera J.
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      • Bosch A.
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      • et al.
      A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.
      This process might also occur during liver fibrosis in NASH, but further studies are required.

      Angiogenesis in NASH-related liver fibrosis

      Liver angiogenesis correlates with the severity of liver fibrosis and promotes its development. Once established, fibrosis stimulates angiogenesis by increasing tissue hypoxia. Blocking pathologic angiogenesis prevents liver fibrosis.
      Liver angiogenesis correlates with liver fibrosis in patients with NASH.
      • Kitade M.
      • Yoshiji H.
      • Kojima H.
      • Ikenaka Y.
      • Noguchi R.
      • Kaji K.
      • et al.
      Neovascularization and oxidative stress in the progression of non-alcoholic steatohepatitis.
      • Kitade M.
      • Yoshiji H.
      • Noguchi R.
      • Ikenaka Y.
      • Kaji K.
      • Shirai Y.
      • et al.
      Crosstalk between angiogenesis, cytokeratin-18, and insulin resistance in the progression of non-alcoholic steatohepatitis.
      The mechanisms leading to liver angiogenesis in NASH-related fibrosis include those mentioned above, namely tissue hypoxia,
      • Coulon S.
      • Heindryckx F.
      • Geerts A.
      • Van Steenkiste C.
      • Colle I.
      • Van Vlierberghe H.
      Angiogenesis in chronic liver disease and its complications.
      hepatocyte-derived microvesicles
      • Povero D.
      • Eguchi A.
      • Niesman I.R.
      • Andronikou N.
      • de Mollerat du Jeu X.
      • Mulya A.
      • et al.
      Lipid-induced toxicity stimulates hepatocytes to release angiogenic microparticles that require Vanin-1 for uptake by endothelial cells.
      • Lemoinne S.
      • Thabut D.
      • Housset C.
      • Moreau R.
      • Valla D.
      • Boulanger C.M.
      • et al.
      The emerging roles of microvesicles in liver diseases.
      and angiopoietin-2,
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      but also leptin. Leptin concentrations are increased in the serum of patients with NAFLD.
      • Huang X.-D.
      • Fan Y.
      • Zhang H.
      • Wang P.
      • Yuan J.-P.
      • Li M.-J.
      • et al.
      Serum leptin and soluble leptin receptor in non-alcoholic fatty liver disease.
      This adipocytokine has both pro-angiogenic effects,
      • Kitade M.
      • Yoshiji H.
      • Kojima H.
      • Ikenaka Y.
      • Noguchi R.
      • Kaji K.
      • et al.
      Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats.
      and direct pro-fibrogenic effects, through the upregulation of TGF‐β in LSECs and Kupffer cells.
      • Marra F.
      • Bertolani C.
      Adipokines in liver diseases.
      In turn, angiogenesis promotes liver fibrosis since several approaches inhibiting liver angiogenesis prevent NASH-related fibrosis (Fig. 4). First, in the study by Kitade and colleagues, neither angiogenesis nor fibrosis were observed in the absence of leptin signalling in a rat model of NASH.
      • Kitade M.
      • Yoshiji H.
      • Kojima H.
      • Ikenaka Y.
      • Noguchi R.
      • Kaji K.
      • et al.
      Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats.
      Second, blocking the release of pro-angiogenic microvesicles from fat laden-hepatocytes or inhibiting their binding to target cells protects mice from steatohepatitis-induced pathologic angiogenesis and results in a reduction in liver fibrosis.
      • Povero D.
      • Eguchi A.
      • Niesman I.R.
      • Andronikou N.
      • de Mollerat du Jeu X.
      • Mulya A.
      • et al.
      Lipid-induced toxicity stimulates hepatocytes to release angiogenic microparticles that require Vanin-1 for uptake by endothelial cells.
      Third, Zhou and coworkers recently showed that a specific deletion of the physiological inhibitor of angiogenesis, prolyl-hydroxylase-2, in endothelial cells results in an overexpression of angiopoietin-2 and TGF‐β1 in the liver and promotes dietary-induced liver fibrosis in mice.
      • Zhou L.-Y.
      • Zeng H.
      • Wang S.
      • Chen J.-X.
      Regulatory role of endothelial PHD2 in the hepatic steatosis.
      • Ozer A.
      • Bruick R.K.
      Regulation of HIF by prolyl hydroxylases: recruitment of the candidate tumor suppressor protein ING4.
      Whether this pro-fibrotic effect of endothelial prolyl-hydroxylase-2 deficiency is directly induced by promoting angiogenesis remains to be demonstrated. Fourth, 2 studies reported that inhibiting angiotensin-II receptor using telmisartan or candesartan inhibits liver angiogenesis and fibrosis in rats fed a choline-deficient, L-amino acid-defined diet.
      • Tamaki Y.
      • Nakade Y.
      • Yamauchi T.
      • Makino Y.
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      • et al.
      Angiotensin II type 1 receptor antagonist prevents hepatic carcinoma in rats with nonalcoholic steatohepatitis.
      • Yoshiji H.
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      • Ikenaka Y.
      • Kitade M.
      • Kaji K.
      • et al.
      Angiotensin-II and vascular endothelial growth factor interaction plays an important role in rat liver fibrosis development.
      Finally, Lefere and colleagues demonstrated that blocking angiogenesis by inhibiting the interaction between angiopoietin-2/Tie2 using the L1-10 peptibody improves liver fibrosis in preventive and therapeutic strategies in mice fed a methionine- and choline-deficient diet. Therapeutic application of L1-10 peptibody also prevents liver fibrosis in diabetic mice with NASH (streptozotocin and western diet model).
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      It should be noted that an anti-fibrotic effect of anti-angiogenic treatments has also been observed in models of chronic liver disease without NASH.
      • Tugues S.
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      Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats.
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      Inhibition of placental growth factor activity reduces the severity of fibrosis, inflammation, and portal hypertension in cirrhotic mice.
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      Complementary vascular and matrix regulatory pathways underlie the beneficial mechanism of action of sorafenib in liver fibrosis.
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      Tyrosine kinase inhibitor BIBF1120 ameliorates inflammation, angiogenesis and fibrosis in CCl4-induced liver fibrogenesis mouse model.
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      Vascular endothelial growth factor promotes fibrosis resolution and repair in mice.
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      Resolution of liver fibrosis requires myeloid cell-driven sinusoidal angiogenesis.
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      Hepatic stellate cells secrete angiopoietin 1 that induces angiogenesis in liver fibrosis.
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      Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis.
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      Tetramethylpyrazine attenuates sinusoidal angiogenesis via inhibition of hedgehog signaling in liver fibrosis.
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      Disruption of negative feedback loop between vasohibin-1 and vascular endothelial growth factor decreases portal pressure, angiogenesis, and fibrosis in cirrhotic rats.
      • Chatterjee S.
      Reversal of vasohibin-driven negative feedback loop of vascular endothelial growth factor/angiogenesis axis promises a novel antifibrotic therapeutic strategy for liver diseases.

      LSECs in NASH-induced HCC

      In most cases, HCC develops on a background of chronic liver disease (70–90% of all patients). The role of liver endothelial cells in HCC development, outside the NAFLD setting, has been reviewed elsewhere and is summarized in Fig. 6.
      • Poisson J.
      • Lemoinne S.
      • Boulanger C.
      • Durand F.
      • Moreau R.
      • Valla D.
      • et al.
      Liver sinusoidal endothelial cells: physiology and role in liver diseases.
      • Berg M.
      • Wingender G.
      • Djandji D.
      • Hegenbarth S.
      • Momburg F.
      • Hämmerling G.
      • et al.
      Cross-presentation of antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-specific CD8+ T cell tolerance.
      • Yang Z.F.
      • Poon R.T.P.
      Vascular changes in hepatocellular carcinoma.
      • Géraud C.
      • Mogler C.
      • Runge A.
      • Evdokimov K.
      • Lu S.
      • Schledzewski K.
      • et al.
      Endothelial transdifferentiation in hepatocellular carcinoma: loss of Stabilin-2 expression in peri-tumourous liver correlates with increased survival.
      • Wu L.Q.
      • Zhang W.J.
      • Niu J.X.
      • Ye L.Y.
      • Yang Z.H.
      • Grau G.E.
      • et al.
      Phenotypic and functional differences between human liver cancer endothelial cells and liver sinusoidal endothelial cells.
      • Höchst B.
      • Schildberg F.A.
      • Böttcher J.
      • Metzger C.
      • Huss S.
      • Türler A.
      • et al.
      Liver sinusoidal endothelial cells contribute to CD8 T cell tolerance toward circulating carcinoembryonic antigen in mice.
      Figure thumbnail gr6
      Fig. 6Role of liver endothelial cells in HCC development in chronic liver diseases (not specific for NAFLD). During HCC progression, endothelial cells sequentially lose their specific markers including stabilin-1, stabilin-2, LYVE-1 and CD32b (SE-1). Conversely, endothelial expression of integrins increases, facilitating adhesion of liver cancer cells. In parallel, endothelial expression of ICAM-1 decreases, leading to a lower ability of leukocyte to adhere and infiltrate HCC. Endothelial cells within HCC can also alter tumour-associated immune responses via their ability to confer T cell tolerance towards cancer-associated antigens and to create an immunosuppressive environment. ICAM-1, intercellular adhesion molecule-1; LYVE-1, lymphatic vessel endothelial hyaluronic acid receptor 1; HCC, hepatocellular carcinoma; NAFLD, non-alcoholic fatty liver disease.
      Patients with metabolic syndrome and NAFLD also develop HCC in the absence of underlying cirrhosis, suggesting oncogenic pathways specific for NAFLD.
      • Paradis V.
      • Zalinski S.
      • Chelbi E.
      • Guedj N.
      • Degos F.
      • Vilgrain V.
      • et al.
      Hepatocellular carcinomas in patients with metabolic syndrome often develop without significant liver fibrosis: a pathological analysis.
      • Kanwal F.
      • Kramer J.R.
      • Mapakshi S.
      • Natarajan Y.
      • Chayanupatkul M.
      • Richardson P.A.
      • et al.
      Risk of hepatocellular cancer in patients with non-alcoholic fatty liver disease.
      Adipokines and angiogenesis associated with NAFLD seem to account – at least partly – for this specific link between NAFLD and HCC.
      Adipokines contribute to HCC development in NAFLD.
      Circulating concentrations of the adipokine FABP4 are increased in patients with NAFLD without HCC and correlate with liver inflammation and fibrosis.
      • Milner K.-L.
      • van der Poorten D.
      • Xu A.
      • Bugianesi E.
      • Kench J.G.
      • Lam K.S.L.
      • et al.
      Adipocyte fatty acid binding protein levels relate to inflammation and fibrosis in nonalcoholic fatty liver disease.
      Interestingly, Laouirem and colleagues recently demonstrated that LSECs exposed to conditions mimicking NAFLD – namely high concentrations of glucose, insulin, or VEGFA – release FABP4. They also observed that FABP4 released by LSECs exerts pro-oncogenic effects, since it induces hepatocyte proliferation. In mice fed a high-fat diet, specific inhibition of FABP4 reduces HCC growth.
      • Laouirem S.
      • Sannier A.
      • Norkowski E.
      • Cauchy F.
      • Doblas S.
      • Rautou P.E.
      • et al.
      Endothelial fatty liver binding protein 4: a new targetable mediator in hepatocellular carcinoma related to metabolic syndrome.
      We can speculate that FABP4 from LSECs not only contributes to HCC progression but also to HCC development in a NAFLD setting (Fig. 7).
      Figure thumbnail gr7
      Fig. 7Role of liver endothelial cells in hepatocellular carcinoma development in the NAFLD setting. Circulating concentrations of angiocrine factors, such as VEGF and angiopoeitin-2, and adipokines, such as leptin are increased in NAFLD. These mediators induce angiogenesis which promotes HCC growth. The adipokine FABP4 is released by adipose tissue and endothelial cells and contributes to HCC development and progression. FABP4, fatty acid binding protein 4; HCC, hepatocellular carcinoma; NAFLD, non-alcoholic fatty liver disease.
      Figure thumbnail gr8
      Box 1LSEC changes are not specific for certain animal models of NASH. LSEC, liver sinusoidal endothelial cells; NASH, non-alcoholic steatohepatitis.
      In NAFLD, angiogenesis is highly stimulated and promotes NAFLD-associated HCC, since various inhibitors of angiogenesis all prevent HCC development. First, leptin-mediated angiogenesis has been demonstrated to be involved in HCC development as neither angiogenesis nor HCC develop in the absence of leptin signalling in Zucker rats fed a choline-deficient, L-amino acid-defined diet.
      • Kitade M.
      • Yoshiji H.
      • Kojima H.
      • Ikenaka Y.
      • Noguchi R.
      • Kaji K.
      • et al.
      Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats.
      Second, Yoshiji and colleagues showed that a conventional anti-angiogenic treatment with sorafenib inhibits the appearance of preneoplastic lesions in rats fed a choline-deficient, l-amino acid-defined diet.
      • Yoshiji H.
      • Noguchi R.
      • Namisaki T.
      • Moriya K.
      • Kitade M.
      • Aihara Y.
      • et al.
      Combination of sorafenib and angiotensin-II receptor blocker attenuates preneoplastic lesion development in a non-diabetic rat model of steatohepatitis.
      In this study, authors also demonstrated that a treatment combining low doses of sorafenib with the angiotensin-II receptor inhibitor losartan also successfully inhibited preneoplastic lesions.
      • Yoshiji H.
      • Noguchi R.
      • Namisaki T.
      • Moriya K.
      • Kitade M.
      • Aihara Y.
      • et al.
      Combination of sorafenib and angiotensin-II receptor blocker attenuates preneoplastic lesion development in a non-diabetic rat model of steatohepatitis.
      Third, Tamaki and colleagues demonstrated that inhibition of angiotensin-II receptor with telmisartan inhibits HIF-1α activity and VEGF expression and prevents HCC development in the liver of rats fed a choline-deficient, L-amino acid-defined diet for 48 weeks.
      • Tamaki Y.
      • Nakade Y.
      • Yamauchi T.
      • Makino Y.
      • Yokohama S.
      • Okada M.
      • et al.
      Angiotensin II type 1 receptor antagonist prevents hepatic carcinoma in rats with nonalcoholic steatohepatitis.
      Finally, Lefere and coworkers recently showed that therapeutic inhibition of angiopoietin-2 alleviates steatohepatitis and prevents NASH-associated HCC progression in mice.
      • Lefere S.
      • Van de Velde F.
      • Hoorens A.
      • Raevens S.
      • Van Campenhout S.
      • Vandierendonck A.
      • et al.
      Angiopoietin-2 promotes pathological angiogenesis and is a novel therapeutic target in murine non-alcoholic fatty liver disease.
      NALFD associated angiogenesis promotes HCC. Blocking pathologic angiogenesis prevents HCC development and progression.

      Gaps in knowledge and future directions

      Even if our understanding of the role of LSECs in NAFLD has progressed over the last years, several aspects remain elusive. First, triggers responsible for LSEC alterations in NAFLD are mostly unknown. It has been suggested that mediators derived from the visceral adipose tissue and the gut are responsible, but this has not been convincingly established. Indeed, available in vitro studies considered each mediator individually and not in combination, as in vivo in the portal venous blood.
      • Zhang Q.
      • Liu J.
      • Liu J.
      • Huang W.
      • Tian L.
      • Quan J.
      • et al.
      oxLDL induces injury and defenestration of human liver sinusoidal endothelial cells via LOX1.
      • Matsumoto M.
      • Zhang J.
      • Zhang X.
      • Liu J.
      • Jiang J.X.
      • Yamaguchi K.
      • et al.
      The NOX1 isoform of NADPH oxidase is involved in dysfunction of liver sinusoids in nonalcoholic fatty liver disease.
      • McMahan R.H.
      • Porsche C.E.
      • Edwards M.G.
      • Rosen H.R.
      Free fatty acids differentially downregulate chemokines in liver sinusoidal endothelial cells: insights into non-alcoholic fatty liver disease.
      Second, mechanisms underlying endothelial changes in NAFLD, including capillarization, need to be defined which might provide new therapeutic targets for NAFLD. Third, the role of LSECs in NASH-related cirrhosis has not been specifically investigated. Whether LSEC function and phenotype differ in cirrhosis related to NASH from cirrhosis related to other causes remains to be assessed.
      • Gracia-Sancho J.
      • Marrone G.
      • Fernández-Iglesias A.
      Hepatic microcirculation and mechanisms of portal hypertension.
      Fourth, although NAFLD is well recognized as favouring HCC development, we are still at a very early stage of understanding how LSEC changes might favour HCC development.

      Conclusion

      LSECs are gatekeepers of liver homeostasis in physiological conditions. In NAFLD, sinusoidal endothelial alterations, including capillarization and LSEC dysfunction, occur early in disease progression, at the stage of simple steatosis. These initial changes, triggered by lipotoxicity, adipokines, inflammation and gut microbiota-derived products are associated with a loss of the ability of LSECs to prevent liver inflammation and fibrosis associated with NASH. Indeed, altered LSECs fail to maintain Kupffer cells and hepatic stellate cells in a quiescent state. At the stage of NASH, altered LSECs contribute to liver angiogenesis, inflammation, fibrosis and HCC. Improving LSEC health has great therapeutic potential for NAFLD. The current challenge is the identification of strategies to specifically target LSECs in order to modulate their activity.

      Financial support

      This work was supported by the “ Institut National de la Santé et de la Recherche Médicale ” (ATIP AVENIR), Paris Descartes University , the “Agence Nationale pour la Recherche” ( ANR-14-CE12-0011 , ANR-14-CE35-0022 , ANR-18-CE14-0006-01 ) and by the “ Association Française pour l’Etude du foie ” ( AFEF 2014 ). A.H. was supported by the “ Ministère de l’Enseignement Supérieur et de la Recherche ”.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      A.H. and P-E.R wrote the manuscript.

      Supplementary data

      The following are the Supplementary data to this article:

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