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Diabetes & Metabolic Syndrome: Clinical Research & Reviews 14 (2020) 1875e1887

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Diabetes & Metabolic Syndrome: Clinical Research & Reviews journal homepage: www.elsevier.com/locate/dsx

Pathophysiological mechanisms underlying MAFLD Mohammad Shafi Kuchay a, *, Narendra Singh Choudhary b, Sunil Kumar Mishra a a b

Division of Endocrinology and Metabolism, Medanta the Medicity Hospital, Gurugram, 122001, Haryana, India Institute of Digestive and Hepatobiliary Sciences, Medanta-The Medicity Hospital, Gurugram, 122001, Haryana, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 August 2020 Received in revised form 21 September 2020 Accepted 23 September 2020

Background and aims: The pathophysiology underlying metabolic associated fatty liver disease (MAFLD) involves a multitude of interlinked processes, including insulin resistance (IR) underlying the metabolic syndrome, lipotoxicity attributable to the accumulation of toxic lipid species, infiltration of proinflammatory cells causing hepatic injury and ultimately leading to hepatic stellate cell (HSC) activation and fibrogenesis. The proximal processes, such as IR, lipid overload and lipotoxicity are relatively well established, but the downstream molecular mechanisms, such as inflammatory processes, hepatocyte lipoapoptosis, and fibrogenesis are incompletely understood. Methods: A literature search was performed with Medline (PubMed), Scopus and Google Scholar electronic databases till June 2020, using relevant keywords (nonalcoholic fatty liver disease; metabolic associated fatty liver disease; nonalcoholic steatohepatitis; NASH pathogenesis) to extract relevant studies describing pathogenesis of MAFLD/MASH. Results: Several studies have reported new concepts underlying pathophysiology of MAFLD. Activation of HSCs is the common final pathway for diverse signals from damaged hepatocytes and proinflammatory cells. Activated HSCs then secrete excess extracellular matrix (ECM) which accumulates and impairs structure and function of the liver. TAZ (a transcriptional regulator), hedgehog (HH) ligands, transforming growth factor-b (TGF-b), bone morphogenetic protein 8B (BMP8B) and osteopontin play important roles in activating these HSCs. Dysfunctional gut microbiome, dysregulated bile acid metabolism, endogenous alcohol production, and intestinal fructose handling, modify individual susceptibility to MASH. Conclusions: Newer concepts of pathophysiology underlying MASH, such as TAZ/Ihh pathway, extracellular vesicles, microRNA, dysfunctional gut microbiome and intestinal fructose handling present promising targets for the development of therapeutic agents. © 2020 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Keywords: Metabolic associated fatty liver disease Metabolic associated steatohepatitis TAZ/IHH pathway Notch pathway Extracellular vesicles Fibroblast growth factor 19 Bone morphogenetic protein 8B Non-alcoholic fatty liver disease NASH

1. Introduction MAFLD comprises a spectrum of histological abnormalities, ranging from bland steatosis to steatohepatitis, hepatofibrosis and cirrhosis. It is the most common liver disease worldwide [1]. In around 25% of patients, MAFLD progresses to steatohepatitis (MASH), which increases the risk for end-stage liver disease [2]. In this review, we discuss the pathophysiology of MASH, mechanisms for development of fibrosis, including crosstalk among stressed hepatocytes; resident and bone marrow-derived proinflammatory cells; and HSCs. We also discuss relatively newer concepts in the pathogenesis of MASH, including the reactivation of developmental pathways, such as TAZ, hedgehog and Notch, dysfunctional gut microbiome, extracellular vesicles and microRNAs.

2. Use of the term ‘MAFLD’ for ‘non-alcoholic fatty liver disease (NAFLD)’ in our review An international panel of experts in a consensus statement recommended a change in name for non-alcoholic fatty liver disease (NAFLD) to metabolic-associated fatty liver disease (MAFLD) in order to reflect evolved understanding of the disease [3,4]. Several editorials have also commented on the importance of the move [5e10]. This change in nomenclature shifts the focus towards inclusionary diagnostic criteria, the presence of metabolic abnormalities, rather than the absence of excessive alcohol intake. It also avoids competition between a diagnosis of alcoholic liver disease and MAFLD, recognizing their synergistic effects on the liver. 3. MAFLD from South Asian perspective

* Corresponding author. . E-mail address: drshafi[email protected] (M.S. Kuchay). https://doi.org/10.1016/j.dsx.2020.09.026 1871-4021/© 2020 Diabetes India. Published by Elsevier Ltd. All rights reserved.

It has been suggested that South Asians are more prone to suffer from MAFLD, as they are metabolically more obese compared to


M.S. Kuchay, N.S. Choudhary and S.K. Mishra

associated with increased risk of MASH [38]. The role of other gene polymorphisms, such as GCKR, MBOAT7, in conferring susceptibility to MASH is incompletely understood.

other ethnic populations [11e14]. South Asians usually have higher percentage of visceral body fat, abdominal obesity, insulin resistance, and low muscle mass [15e19]. Furthermore, genetic susceptibility for development of components of metabolic syndrome has been observed in South Asians [20e23]. Genetic susceptibility, dysfunctional body adiposity, and insulin resistance underlying metabolic syndrome make South Asians more prone to development of MAFLD and its consequences.

4.2. Substrate overload and generation of toxic lipid species The prominent proximal event of MAFLD is the accumulation of free fatty acids (FFAs) in the liver. The liver gets FFAs primarily from three sources. (A) FFA uptake from the circulation. Excessive mobilization of FFAs derived from the lipolysis of adipose tissues, which is driven by IR, is the main source of hepatic TGs (about 60%) [39,40]. (B) de novo lipogenesis (DNL) accounts for about 26% of stored hepatic TGs. Excessive carbohydrates are converted to FFAs in the liver by the process of DNL [39]. The rate of this process is tightly regulated by several nuclear transcription factors (TFs), the most important being sterol regulatory element binding protein-1c (SREBP-1c). This TF promotes DNL by increasing the transcription of lipogenic enzyme genes, such as acetyl Co-A carboxylase (ACC), fatty acyl synthase (FAS) and steroyl carboxy desaturase (SCDs). Hyperinsulinemia causes upregulation of SREBP-1c, thereby, causing unbridled DNL. (C) Dietary lipids constitute around 15% of TGs in the liver [39]. Furthermore, evidence indicates that saturated FAs are more hepatotoxic than unsaturated FAs (both MUFA and PUFA) [41]. Palmitate (16-C) and stearate (18-C) are major saturated FAs that accumulate and are associated with disease progression [42]. There are two mechanisms for FFA disposal in the liver. (A) boxidation, in which FFAs are oxidized mainly in mitochondria. (B) VLDL export, in which FFAs are re-esterified generating TGs. TGs are then assembled and secreted into the systemic circulation as a constituent of VLDLs. Hepatic steatosis occurs when the TG homeostasis is disrupted due to an increase in FA uptake and DNL, and a reduction in FFA oxidation and VLDL export. A body of evidence suggests that TGs contribute to steatosis but not to injury and fibrosis [43,44]. The protective role of TG-containing lipid droplets was demonstrated in mice lacking diacylglycerol acyltransferase 2 (DGAT-2) with inability to convert FFAs into inert TGs [43]. However, recent phase I and II trials demonstrated a significant reduction of liver fat content following inhibition of DGAT-2 for treatment of MAFLD, without any significant adverse effects on liver [45,46]. When these FFA disposal mechanisms are overwhelmed, ROS and toxic lipid species, such as LPCs, DAGs and ceramides, are generated triggering lipotoxicity (Fig. 1). Since substrate overload is at the centre of MASH pathogenesis, strategies targeted at decreasing substrate delivery to the liver or promoting disposal of FFAs from the liver represent promising therapeutic avenues. Some examples are given in Table 1.

4. Pathophysiology of MASH MASH is characterized by lipotoxicity, inflammation and fibrosis, ultimately leading to end-stage liver disease [1]. However, the pathophysiology of MASH is complex, multifactorial and incompletely understood. 4.1. Genetic susceptibility to MAFLD MAFLD is a heterogeneous and multifactorial disease. Genetic factors play a crucial role in the development and progression of MAFLD. Most of the genetic variation influencing MAFLD is driven by genes involved in lipid droplet biology, including patatin-like phospholipase domain-containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), 17b-Hydroxysteroid dehydrogenase type 13 (HSD17B13), membrane bound O-acyltransferase domain-containing 7 (MBOAT7) and glucokinase regulator (GCKR). However, the first two genes are consistently found to be associated with the disease, and knowledge about other genes and polymorphisms has been evolving. The most strongly associated genetic variant with MASH is a single-nucleotide polymorphism (SNP, I148M variant) in the PNPLA3 gene [24]. This gene encodes a lipid droplet protein which is involved at the lipolytic step. I148M variant PNPLA3 is resistant to degradation and accumulates on lipid droplets and impairs mobilization of TGs from lipid droplets. I148M PNPLA3 variant increases the risk for all the stages of MAFLD spectrum, from simple steatosis to liver cirrhosis and HCC [25,26]. This variant of PNPLA3 interacts with environmental factors, such as fructose-rich beverages and physical inactivity, to trigger hepatic fat accumulation and MAFLD progression [27]. At the molecular level, the PNPLA3 gene expression is under the direct transcriptional control of SREBP-1c (induced by insulin) and ChREBP (induced by glucose) [28]. Recently, PNPLA3 silencing with antisense oligonucleotides ameliorated MASH and fibrosis in PNPLA3 I143M knock-In mice, opening up new avenues for precision medicine [29]. Retinol is stored as retinyl palmitate in lipid droplets of HSCs, accounting for 50e80% of total vitamin A body stores in humans [30]. PNPLA3 is responsible for release of retinol from lipid droplets of HSCs. The I148 M variant has reduced activity of retinyl-palmitate lipase leading to reduced release of retinol into the circulation. Circulatory levels of retinol binding protein 4 (RBP4), a marker of retinol stores also secreted by hepatocytes, are also lower in patients carrying PNPLA3 I148M variant [31]. TM6SF2 E167K variant is associated with increased risk for progressive MASH but reduced risk of cardiovascular disease. The E167K variant leads to decreased expression of the gene and leads to its diminished function [32]. It is associated with increased fat accumulation in the liver, due to reduction in lipid secretion (via VLDL export). As this variant also reduces circulating lipids, it protects against the risk of developing cardiovascular disease [33]. HSD17B13 is a lipid droplet-associated retinol dehydrogenase protein mainly expressed in the liver [34]. It is a target gene for the insulin regulated SREBP-1c and is upregulated in the liver in human MAFLD [35]. A loss-of-function variant in HSD17B13 is associated with a reduced risk of chronic liver disease and of progression from simple steatosis to MASH [36,37]. A splice variant of HSD17B13 is

4.3. Activation of hepatic stress mechanisms Oxidative stress, ER stress and inflammasome formation are the key processes that contribute to development and progression of MASH. These processes are initiated by FFA overload and then perpetuated by feedback loops from several proinflammatory cells. 4.4. Oxidative stress and mitochondrial dysfunction Reactive oxygen species (ROS) interact with biological compounds (proteins, lipids, DNA) and alter their structure and function [47]. The main intracellular sources of ROS are mitochondria, endoplasmic reticulum (ER), peroxisomes, xanthine oxidase (XO) and cytochrome P450 metabolism [48]. Under physiological conditions, these ROS are neutralized by antioxidant mechanisms, such as superoxide dismutases (Cu/Zn SOD and Mn SOD)) and 1876



impair the synthesis of respiratory chain polypeptides [52]. ROS also interacts with polyunsaturated FAs (PUFAs) resulting in lipid peroxidation and formation of 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) [53]. These compounds can freely diffuse into the extracellular space to affect distant cells, therefore amplifying the effects of oxidative stress [54]. Almost all endogenous intracellular sources of ROS (mitochondria, peroxisomes, CYP2E1, XO, and ER) contribute to disease progression in MAFLD (Fig. 2). A breakdown in antioxidant defenses has been proven to play a significant role in oxidative stress associated with MASH, as evidenced by decreased hepatic glutathione (GSH) and diminished SOD, GPx, catalase, and glutathione transferase activities in correlation with disease severity [55]. Oxidative stress and mitochondrial dysfunction contribute to

glutathione peroxidase (GPx). Oxidative stress occurs as a result of either excessive production of ROS or reduced antioxidant defenses. In MASH, oxidative stress results from both increased production of oxidative species as well as breakdown of antioxidant defenses [48,49]. A substantial increase in b-oxidation (secondary to FFAs overload) is the main source of ROS in MAFLD [50]. Overexpression of CYP2E1 also contributes to ROS accumulation in MAFLD [51]. Once mitochondrial ROS formation increases in MASH, it triggers several vicious cycles. ROS directly damages respiratory chain polypeptides and thereby block electron flow in the respiratory chain. ROS also oxidizes unsaturated lipids and release lipid peroxidation products, which inactivate cytochrome c oxidase. Both ROS and lipid peroxidation products damage mitochondrial DNA (mtDNA) and further

Fig. 1. Free fatty acids (FFAs) are at the centre of MASH pathogenesis. The liver gets FFAs from expanded adipose tissue, under the influence of IR and from diet. Excess carbohydrates are converted into FFAs by de novo lipogenesis (DNL) with the help of upregulated transcription factors (e.g., SREBP-1c). These transcription factors subsequently upregulate all the enzymes necessary for DNL (e.g., ACC, FAS, SCDs). FFAs in the liver undergo b-oxidation by mitochondria or converted to triglycerides (TGs) and exported to the systemic circulation as constituents of VLDLs. When these two FFA disposal mechanisms are overwhelmed, TGs start accumulating as lipid droplets in the hepatocytes (steatosis). Excessive b-oxidation of FFAs results in production of ROS and cytotoxic lipid species (e.g., LPCs, DAGs and Ceramides). Misfolded proteins accumulate in the ER and activate unfolded protein response (UPR). Oxidative and ER stress then activate inflammasomes. These pathological processes lead to hepatocellular injury, inflammatory cell recruitment, and apoptosis/necroptosis to produce the histological phenotype of MASH. Overwhelming inflammatory processes then stimulate HSCs and activate fibrogenesis. SREBP1c, sterol response element binding protein-1c; ACC, acetyl-Coenzyme A carboxylase; FAS, fatty acid synthetase; SCD, steroyl-Coenzyme A desaturase; PNPLA3, patatin like phospholipase domain containing 3; VLDL, very low density lipoprotein; OSA, obstructive sleep apnea; HCC, hepatocellular carcinoma. 1877



Table 1 Therapeutic agents targeted predominantly at substrate delivery to the liver and/or disposal of FFAs from the liver in MASH. Target

Mechanism

Drug

PPAR-g agonist

Increase FFA oxidation Improve IR Anti-inflammatory Anti-fibrotic Increase FFA oxidation Improve IR Anti-inflammatory Anti-fibrotic Increase FFA oxidation Improve IR Anti-inflammatory Anti-fibrotic Increase FFA oxidation Improve IR Anti-inflammatory Anti-fibrotic Reduce glucose delivery to liver by diverting through urine

Pioglitazone

PPAR-a/d agonist

PPAR-a/g agonist

Pan-PPAR agonist

SGLT-2 inhibitors

GLP-1 receptor agonists

ACC 1/2 antagonists SCD 1 antagonist DGAT2 inhibitor Ketohexokinase inhibitor Thyroid hormone receptor b (THR-b) mimetic Sirtuin-1 agonist

Niacin-R agonist

FGF 19 agonist

Improve IR Reduce food intake Induce weight loss Reduce DNL Reduce DNL Reduce hepatic lipid levels Reduce VLDL TG secretion Reduce DNL Increase hepatic fat metabolism Decrease circulating lipids Reduce DNL Increase FA oxidation Anti-inflammatory Reduce lipolysis in adipose tissue Decrease TG synthesis Increase FFA oxidation Decrease bile acid synthesis Anti-inflammatory Anti-fibrotic

Elafibranor ZLY16

Saroglitazar

IVA337

Empagliflozin Dapagliflozin Ipragliflozin Liraglutide Dulaglutide Semaglutide PF-05221304 Firsocostat Aramchol IONIS-DGAT2 Rx PF-06835919 Resmetirom Resveratrol

Niacin

Aldafermin

MCJ expression enhances b-oxidation of FFAs, minimize lipid accumulation, which results in reduced hepatocyte damage and fibrosis [61].

the progression of MAFLD by inducing hepatic inflammatory cytokines. ROS along with products of lipid peroxidation result in increased release of several cytokines (TGF-b, TNF-a, IL-8, Fas ligand) which plays a key role in cell death, inflammation, and fibrosis [56]. The aldehyde end products of lipid peroxidation are well-documented proinflammatory mediators which activate HSCs, leading to increased collagen synthesis and development of liver fibrosis [57]. The inflammatory cytokines also play an important role in directing polymorpho- and mononuclear leukocytes into inflamed tissues [58]. Normally, hepatocytes express Fas (a membrane receptor), but not Fas ligand, thus preventing cell death. Expression of hepatocyte Fas is increased in MASH. Moreover, increased ROS also cause hepatocyte Fas ligand expression. This leads to Fas ligand on one hepatocyte interact with Fas on another hepatocyte to cause fratricidal apoptosis [59]. As already mentioned, mitochondria are major sources of ROS. Excessive ROS directly causes mitochondrial swelling and suppresses electron transport chain enzymes, ultimately leading to mitochondrial damage. Under physiologic conditions, damaged mitochondria are removed by mitochondrial autophagy or mitophagy. It has been demonstrated that impaired mitophagy machinery triggers NLRP3 inflammasome activation, which in turn causes progression of MASH [60]. Recently, a protein called methylation-controlled J protein (MCJ) was found to be an endogenous negative regulator of the respiratory chain Complex I that act to restrain mitochondrial respiration. MCJ levels in the liver of MAFLD patients are elevated. Decreasing

4.5. Endoplasmic reticulum stress Under stress conditions, such as FFA overload, the unfolded and misfolded proteins accumulate in the ER lumen. In order to reestablish ER homeostasis, a specific signalling pathway called the unfolded protein response (UPR), is activated [62]. In the setting of ER stress, the three transmembrane biosensors, namely protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring signalling protein 1 (IRE1) are activated. PERK activation induces the expression of the proapoptotic protein CCAAT/enhancer-binding homologous protein (CHOP), which in turn, mediates apoptosis through several mechanisms including generation of ROS. IRE-1a activates C-Jun N-terminal kinase-1 (JNK), which promotes apoptosis. IRE-1a also generates a spliced form of XBP (s-XBP) that promotes degradation of misfolded proteins. ATF6 contributes in CHOP induction, and heterodimerizes with XBP, enhancing protein degradation. The cumulative response of these three signalling pathways is to block the initiation of translation, in order to reduce the load upon ER, correct the misfolded proteins, and when the damage is beyond repair, induce apoptosis [63,64]. Suppression of the JNK1 can prevent the development of steatohepatitis in a mice model of MASH, explaining the importance of JNK signalling in the pathogenesis of 1878



Fig. 2. Basic model demonstrating excessive ROS production and mitochondrial dysfunction. IR leads to enhanced lipolysis and unbridled release of FFAs. In hepatocytes, FFAs saturate b-oxidation (both in mitochondria and peroxisomes) and results in excessive ROS production. Mitochondrial CYP2E1 is also a direct source of ROS. A reduction in antioxidant mechanisms (GPx and MnSOD) also contributes to ROS accumulation. Excessive ROS then results in lipid peroxidation, both processes further impair mitochondrial function (by ATP depletion, oxidative damage, mtDNA mutation), thereby starting a vicious circle. Lipid peroxidation leads to formation of highly reactive metabolites (4-HNE and MDA), which diffuse freely into the extracellular space and affect distant cells. Lipid peroxidation also activate Kupffer cells, upregulate proinflammatory cytokines (TNF-a, IL-6, IL-8) and promote neutrophil infiltration into the liver tissue. Incomplete b-oxidation of FFAs lead to accumulation of toxic lipid species, such as LPCs, DAGs and ceramides, which further enhance ROS production and act as inflammatory substances. ROS, reactive oxygen species; FFAs, free fatty acids; 4-HNE, 4-hydroxy-2-nonenal; MDA, malondialdehyde; LPCs, lysophosphatidylcholines; DAGs, diacylglycerols; CYP2E1, cytochrome P450 2E1; GPx, glutathione peroxidase; MnSOD, manganese superoxide dismutase.

development of MASH.

this disease [65]. Furthermore, the ER lumen is the main site of calcium storage. Saturated FAs may induce a disruption of ER calcium store resulting in an increased generation of ROS and induction of apoptosis [66]. Lastly, UPR causes decrease in antioxidant mechanism through inhibition of nuclear factor- (erythroid-derived 2-) like 2 (Nrf2). Nrf2 is a critical effector of cell survival, which activates transcription of antioxidant enzymes, playing a critical role in elimination of ROS. In a mouse model, deletion of Nrf2 was associated with a significant increase in oxidative stress as a result of decreased expression of antioxidative stress genes, resulting in rapid progression to MASH [67].

4.7. Recruitment and activation of proinflammatory cells Immune dysregulation plays a crucial role in the pathogenesis of MASH. The major immune cells that contribute to MASH are Kupffer cells, Ly6Chi monocytes, neutrophils, T-helper (Th), and cytotoxic CD8þ T cells. Activation of yolk-sac derived, liver-resident Kupffer cells release chemotactic and proinflammatory cytokines, such as CCL2, IL-1b, IL-6, and TNF-a [71]. These cytokines result in recruitment of bone-marrow derived Ly6Chi monocytes and neutrophils that further contribute to the inflammatory process [72]. Macrophage depletion or inhibition of macrophage recruitment via CCR2/CCR5 inhibition suppresses fibrogenesis in murine and human MASH [73,74]. Activated neutrophils enhance MASH by releasing MPO and ROS [72]. T-helper (Th) cells are main players of adaptive immune response. After immune activation, Th cells differentiate into Th1, Th2 and Th17 effector cells, depending on the cytokines in their environment. In MASH, there are excessive Th1-derived IFNg and Th17-derived IL-17; and deficiency of Th2-derived IL-4, IL-5, and IL13 [75]. Th17 cells producing IL-17 accumulate in the liver of humans with MASH and have been shown to aggravate inflammation and fibrosis through effects on macrophages and HSCs, respectively. Cytotoxic CD8þ T cells accumulate in the liver during MAFLD, and their inhibition results in decreased steatosis, IR, inflammation, and HSC activation [76]. Activation of these cytotoxic CD8þ T cells is

4.6. Inflammasome activation Inflammasomes are intracellular pattern recognition receptors (PRRs) that are responsible for the production of proinflammatory cytokines, such as IL-1b and IL-18 [68]. Inflammasome-mediated IL-1b secretion is initiated by activation of TLR as a priming signal and stimulated by a variety of second signals including pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs), and toxic lipids (palmitic acid, free cholesterol) [68]. The expression of NLRP3 inflammasome components are very low in healthy hepatocytes. However, the expression of NLRP3 inflammasome components was observed to be significantly increased both in murine and human MASH [69]. Moreover, pharmacological inhibition of these genes resulted in the alleviation of steatosis, inflammation and fibrogenesis [70]. These results suggest that NLRP3 inflammasome plays a critical role in the 1879



cells including stressed hepatocytes, Ly6Cþ monocytes, Kupffer cells, Th17 cells, and cytotoxic CD8þ T cells further promote HSC activation (Fig. 3).

supported by type I IFN responses and leads to the production of the proinflammatory cytokines, IFNg and TNFa [77]. Cytotoxic CD8þ T cells have also been shown to promote MASH development and subsequent transition to HCC in a process that requires crosstalk with natural killer T cells. Src homology region 2 domain-containing phosphatase 1 (SHP1, also called PTPN6) is a negative regulator of inflammation. Hepatocyte-specific PTPN6 knockout mice showed exacerbated hepatic steatosis, inflammation and fibrosis. Forced ectopic expression of SHP-1 significantly ameliorates MASH and inhibits proinflammatory cytokines, including TGF-b, IL-6, and TNF-a. SHP1 could be a potential therapeutic target for MASH [78]. Investigational therapeutic agents targeted at anti-inflammatory and antifibrotic mechanisms are listed in Table 2.

4.10. Changes in HSC cellular metabolism Reprogramming of quiescent HSCs into active HSCs depends upon the induction of aerobic glycolysis. This is accomplished through the activation of hedgehog (Hh) pathway, which upregulates hypoxia-inducible transcription factor-1a (HIF-1a), a key modulator of the activity of glycolytic enzymes [85]. By contrast, inhibition of Hh signalling, HIF-1a expression, glycolysis or lactate accumulation results in the reversal of active HSCs to quiescent HSCs phenotype. These findings indicate that cellular metabolism plays a vital role in the fibrogenic response. Another change in the cellular metabolism that happens in active HSC is induction of glutaminolysis (conversion of glutamine to a-ketoglutarate), which then fuels the Krebs cycle to meet the enhanced demands of bioenergetic and biosynthetic pathways needed for maintaining the active HSC phenotype. Similar to aerobic glycolysis, glutaminolysis is also mediated through HH pathway [86,87]. Succinate, an intermediate in the Krebs cycle, functions as a paracrine signal between hepatocytes and HSCs, through activation of its cognate G protein-coupled receptor 91 (GPR91), which results in fibrogenesis [88]. Sirtuin 3 (SIRT3), a protein deacetylase, is a key regulator of SDH activity [89,90]. The SIRT3-SDH-GPR91 axis modulates HSC activation. Repression of succinate-GPR91 signalling by an analogue of FGF21 inhibits HSC activation [91].

4.8. Adipose-liver axis Adipose tissue dysfunction is closely associated with MAFLD in humans [79]. Adipose tissue dysfunction involves its failure to expand in presence of positive energy balance and store excess energy. This leads to enhanced lipolysis and secretion of FFAs [80]. As already mentioned, lipolysis of adipose tissue is the main source of FFAs for steatosis. Adipose tissue as an endocrine organ secretes several adipokines, such as leptin and adiponectin, with systemic effects. Leptin and adiponectin influence MAFLD through regulation of food intake, insulin sensitivity, and inflammation [81]. In people with MASH, there are reduced adiponectin levels and increased leptin levels. In addition, excessive production of proinflammatory cytokines by adipose tissue macrophages is critical in development of obesity-associated low grade inflammation [81]. Activated adipose tissue macrophages secrete cytokines, including TNFa, IL-1b, IL-6, and CCL2, which cause local IR resulting in dysregulated lipid metabolism and can also reach the circulation leading to systemic IR [82,83].

4.11. Free cholesterol mediates HSC activation FC accumulation increases TLR4 levels in HSCs and thereby sensitizes the cells to TGF-b induced activation [92]. Along with HSC activation, subsequent upregulation of both SREBP2 and MiR-33a leads to further FC accumulation and exaggerates liver fibrosis in a positive feedforward loop [93,94].

4.9. Activation of HSCs Hepatic stellate cells (HSCs) play a crucial role in MASH progression. Activation of HSC involves the transition from a quiescent vitamin A-storing cells to a proliferative migratory and fibrogenic phenotype, which is characteristic of liver fibrogenesis [84]. Activation of HSCs involve upregulation of various genes, including asmooth muscle actin (a-SMA), collagen-1a1, tissue inhibitor of metalloproteinases (TIMP-1 and 2), and transforming growth factor-b (TGF-b). A multitude of metabolic pathways and molecular signals that contribute to HSC activation have been identified. In addition, extracellular/paracrine signals from various inflammatory

4.12. Hepatocytes mediate HSC activation MASH-induced hepatocyte damage and cell death are the prominent drivers of fibrosis in MASH [95,96]. Hepatocytes contribute to HSC activation through at least four mechanisms. Firstly, hepatocyte stress and death promote inflammation, resulting in recruitment of macrophages. Macrophages in turn secrete profibrogenic mediators such as TGFb, thus putting the hepatocyte-macrophage-HSC axis at the centre of fibrogenic response in MASH. Secondly, stressed and death hepatocytes interact directly with HSCs and causing their activation, without macrophage mediation. This may be through the release of profibrogenic damage-associated molecular patterns (DAMPs) or other mediators such as Hh ligands and osteopontin [95,97]. Thirdly, hepatocyte apoptotic bodies may directly act on HSCs and lead to their activation and fibrogenesis [98]. Fourthly, injured but living hepatocytes (ballooned hepatocytes) secrete sonic hedgehog (SHH) which promotes HSC activation and fibrinogenesis [99] (Fig. 3).

Table 2 Investigational therapeutic agents directed at downstream processes of MASH. Target

Mechanism

Drug

CCR2/CCR5 dual antagonist

Anti-inflammatory Anti-fibrotic Improve insulin sensitivity Anti-inflammatory Anti-fibrotic Anti-inflammatory Anti-fibrotic Anti-inflammatory Anti-fibrotic Anti-inflammatory Anti-inflammatory Anti-fibrotic Anti-inflammatory

Cenicriviroc

FXR agonists

Galectin-3 protein inhibitor ASK1 inhibitor Inflammasome inhibitor TLR4 antagonist Anti-LOXL2 inhibitor Caspase inhibitor

Obeticholic acid GS-9674 LMB763 GR-MD-02

5. Evolving concepts for progression of simple steatosis to fibrosis

Selonsertib* SGM-1019 JKB-121 Simtuzumab* Emricasan

Fibrogenesis is a dynamic process. Tissue homeostasis is maintained by constant synthesis and degradation of ECM. When there is an excessive and prolonged injurious stimuli, such as lipotoxic species, profibrogenic processes predominate (HSC activation, expression of TIMPs) and fibrous tissue accumulate in the liver [96].

ASK-1, apoptosis signal-regulating kinase-1; TLR, Toll-like receptor; *found to be ineffective; **. 1880



fibrosis in mice.81 Pharmacologic inhibition of TGF-b partially reduces MASH-induced fibrosis [103]. 5.2. Hepatocyte TAZ-IHH pathway and HSC activation Hepatic fibrosis is a key feature of MASH that distinguishes it from simple steatosis and determines long term liver-related morbidity and mortality [104]. Progression of simple steatosis to MASH requires overexpression of the transcription regulator TAZ, also called WWTR1 (Fig. 4). TAZ is a key component of the HIPPOYAP/TAZ TEAD signalling cascade. TAZ levels are markedly elevated in livers of humans with MASH-related fibrosis and in the livers of several mouse models of MASH. Genetically induced TAZ expression in hepatocytes promotes MASH in a mouse model of steatosis. Hepatocyte TAZ promotes fibrosis by inducing Indian Hedgehog (Ihh), which is a secretory factor that activates fibrogenic genes in HSCs. It has been demonstrated that hepatocyte TAZ silencing in mice steatotic liver prevents inflammation, apoptosis and fibrosis [105]. 5.3. Hepatocyte notch signalling and HSC activation In the liver, during embryonic development, Notch converts bipotential hepatoblasts to the biliary lineage, whereas Notch suppressed hepatoblasts commit to hepatocyte lineage [106]. Notch activity is absent in hepatocytes of adult healthy liver, but is reactivated upon liver injury and regulates the complex crosstalk among the distinct cellular types involved in the repair process [107]. Notch signalling is mildly elevated in bland steatosis and markedly increased in murine and human MASH [107,108]. Hepatocyte-specific Notch loss-of-function rodent models showed attenuated MASH-associated liver fibrosis without affecting cell death and inflammation [109]. Notch-activated hepatocytes promote secretion of osteopontin, which is responsible for the majority of the profibrogenic effects of Notch activation (Fig. 4). A Notch inhibitor (Nicastrin, antisense oligonucleotide) reduced fibrosis in NASH mice, opening up new vistas for targeting maladaptive Notch pathway for treatment of MASH [109]. It must be noted that Notch activation also increases FoxO 1 activation at gluconeogenic promoters, leading to glucose intolerance, which may partially explain the association between type 2 diabetes and accelerated MASH pathology [110].

Fig. 3. Overview of MASH from altered environmental factors (bad diet and physical inactivity) to hepatic stellate cell activation. Crosstalk between stressed hepatocytes, bone marrow-derived monocytes, Kupffer cells, T cells and HSCs are highlighted.

5.4. Bone morphogenetic protein 8B If the initial injurious stimuli are reversed, the degrading processes (expression of MMPs and inhibition of TIMPs) predominate and cause clearance of extra fibrous tissue (Fig. 2). In the liver, HSCs constitute the main source of ECM-producing myofibroblasts, contributing around 80%e95% of collagen-producing myofibroblasts. The various signalling pathways that activate these HSCs are discussed below.

BMP8B represents a novel regulatory pathway that contributes to the progression of MASH. BMP8B, a member of TGFb/BMP superfamily, is almost absent in the healthy and simple steatotic liver. However, hepatic expression of BMP8B increases proportionally to disease stage in people with MASH [111]. BMP8B promotes HSC activation and the hepatic wound healing responses causing MASH progression. It should be noted that chronic activation of the hepatic wound-healing response is crucial for MASH progression. Absence of BMP8B prevents HSC activation, reduces inflammation and affects the wound-healing responses, thereby attenuating MASH progression. Since there is near absence of BMP8B in healthy livers, inhibition of BMP8B might be a promising therapeutic avenue for MASH treatment [112].

5.1. Transforming growth factor-b (TGFb) and HSC activation TGF-b signalling is the main profibrogenic signal that activates HSCs [100]. TGF-b is released by several type of liver cells (e.g., Kupffer cells) in its latent form and is locally activated by HSCs expressing integrin aV [100]. Profibrogenic effects of TGFb are mediated by SMAD-dependent pathways, and by mitogenactivated protein kinase (MAPK) pathway [101]. Vitamin D nuclear receptor (VDR) plays a role in modulation of TGF-b Smad signalling. Activation of VDR antagonizes Smad binding to the promotor region of fibrogenic genes in HSCs [102]. Accordingly, VDR-deficiency promotes and vitamin D treatment attenuates liver

5.5. Extracellular vesicles (EVs) The intercellular communication via secreted EVs has been established as an important biological process, carrying cellspecific cargo from one cell to another cell [113]. EVs are released in a highly regulated manner from different cell types including 1881



Fig. 4. Fibrogenesis in MASH: Fibrogenesis requires a phenotypic switch from quiescent to activated HSCs. Metabolic insults activate HSC and produce fibrosis in the liver. MMPs are inhibited (via upregulated TIMPs), which normally degrade fibrosis, thereby increasing fibrosis burden in the liver. Metabolic insults also promote TAZ and NOTCH expression in hepatocytes. TAZ expression stimulates secretion of IHH, which in turn activate fibrogenic genes in the activated HSCs. NOTCH-activated hepatocytes promote secretion of osteopontin (OPN), which mediates majority of fibrogenic effects of NOTCH activation.

macrophage chemoattractant [120,121]. CXCL10 expression is increased in the liver of patients with NASH. CXCL10-enriched hepatic EVs recruit macrophages and also activate Kupffer cells during MASH progression. 4, Lipotoxicity also induces release of TNF-associated apoptosis inducing ligand (TRAIL)-bearing EVs from hepatocytes, which are death receptor 5 (DR5), caspase and Rho associated protein kinase 1 (ROCK1)-dependent. These EVs activate macrophages via a DR5 signalling. Furthermore, ROCK1 inhibition prevents lipotoxicityinduced EV release from hepatocytes and thus attenuates macrophage activation and improves MASH in a rodent model [122]. 5, Toll-like receptor 9 (TLR9) is an endosomal pattern recognition receptor (PRR) for which bacterial DNA and mammalian selfDNA are ligands [123]. There is an increase in hepatic EVs containing liver mitochondrial DNA (mtDNA) and oxidized DNA in MASH, and these molecules mediate macrophage activation through TLR-9 pathway [124]. In brief, hepatic EVs play crucial roles in modulation of a variety of cells in the liver, including hepatic macrophages and HSCs, resulting in the acceleration of MASH progression. Furthermore, proinflammatory EVs could be an attractive target for development of therapeutic agents.

hepatocytes, HSCs and immune cells in normal and pathological conditions. EVs are efficiently internalized into target cells, and the transferring of their cargo is the key mechanism by which EVs modulate cell signalling in target cells [114]. Here, we focus on EVs involved in the pathophysiology of MASH. During the MAFLD progression, one of the key events is the hepatocyte injury by lipotoxicity [115]. These damaged hepatocytes then release large quantities of EVs that contribute to key processes involved in MAFLD pathogenesis, including macrophage recruitment, inflammation and HSC activation [116,117]. Several studies have provided key insights regarding the mechanism by which EVs modulate pathogenesis of MASH. 1, EVs released by hepatocytes during lipotoxicity carry a variety of different bioactive molecules including miRNAs. These hepatic EVs are efficiently internalized by HSCs. A specific miRNA, called miR-128e3p inhibits peroxisome proliferator-activated receptor-g (PPAR-g) in HSCs. Inhibition of PPAR-g then lead to activation of HSCs and induce fibrosis in the liver [117]. 2, Palmitate induces the release of EVs from hepatocytes. These EVs are enriched in C16:0 ceramide and are released in IRE1a dependent manner. These EVs activate macrophage chemotaxis via formation of sphingosine-1 phosphate (SIP-1) using C16:0 ceramide. Thus, the ceramide metabolite activates macrophage chemotaxis and this chemotaxis is blocked by sphingosine kinase inhibitors and SIP receptor inhibitors [118,119]. 3, Lipotoxicity promote release of EVs from hepatocytes by Mixed linage kinase 3 (MLK3) dependent pathway. These hepatic EVs carry chemokine (C-X-C motif) ligand 10 (CXCL10), which is a

5.6. MicroRNA (miRNA) MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level [125]. Here, we summarize the current knowledge on the role of miRNAs 1882



through mechanisms involving small intestinal bacterial overgrowth (SIBO), increased intestinal permeability, tight junction alteration and bacterial translocation [145,146]. Recently, a sile acid sequestrant, sevelamer, was found to prevented hepatic steatosis, macrophage infiltration and fibrosis in mice. Sevelamer bound to LPS in the intestinal lumen and promoted its faecal excretion. Consequently, sevelamer treatment restored the tight intestinal junction proteins and reduced the portal LPS levels, leading to the suppression of hepatic TLR4 signalling [147].

in the pathogenesis, diagnosis and therapeutic targets of MAFLD/ MASH. 1, MicroRNA-21 (miR-21) expression is elevated in patients with MASH but not in simple steatosis. MiR-21 overexpression lead to inhibition of PPAR-a, which contributes to the key features of MASH, including cell injury, inflammation and fibrosis [126,127]. MiR-21 inhibition decreases liver injury, inflammation and fibrosis in patients with MASH, by restoring PPAR-a expression [127]. MiR21 is a druggable target and antagomir-21 might be a possible therapeutic strategy for treatment of MASH [128]. 2, MicroRNA-223 (miR-223) modulates macrophage polarization and inflammasome activation, the two processes that are crucial for MASH progression [129]. MiR-223 switches proinflammatory macrophage phenotype (M1) toward the antiinflammatory phenotype (M2) [130]. Overexpression of miR-223 prevents assembly of NLRP3 inflammasome and inhibits IL-1b production [131]. In a mouse model of MASH, administration of miR-223 alleviates MASH, by suppressing hepatic NLRP3 inflammasome activation and IL-1b release [132]. 3, Recently, a study aimed at evaluating whether epigenetics and environmental factors interact to promote progressive MAFLD during insulin resistance. They concluded that IR combined with diet-induced liver injury favoured miR-101e3p downregulation, which might promote progressive MAFLD through HSC and hepatocyte transdifferentiation and proliferation [133]. A significantly higher levels of MiR-101e3p was also found in the plasma of HCC patients in comparison with healthy and cirrhotic controls [134]. 4, MiR-122 is highly expressed in liver, accounting for 70% of liver miRNAs [135]. This miRNA plays a key role in regulating lipid and cholesterol biosynthesis in the liver [136]. Preliminary studies suggest miR-122 being an important biomarker of early MASH [137]. 5, MiR-34a is the second most important regulatory miRNA in the liver physiology. MiR-34a is involved in MAFLD progression through induction of p53-mediated hepatocyte apoptosis [138]. Mir-34a represses the SIRT1 expression thereby increasing p53 acetylation, which leads to the induction of proapoptotic genes and finally cell death [139]. Lack of miR-34a expression results in protection against apoptosis induced by P53 [140]. In brief, epigenetic modifiers, such as miRNA can represent promising molecular indicators which can determine not only the early risk assessment but also the disease progression and prognosis.

6.2. Dysregulated bile acid metabolism Abnormalities in bile acid metabolism is emerging as a novel mechanism contributing to MAFLD progression. Patients with MASH have been found to have reduced circulating fibroblast growth factor 19 (FGF19) levels and elevated bile acid levels [148e150]. It has been postulated that supraphysiologic bile acid levels may contribute to MASH-related liver injury and progressive fibrogenesis. FGF19 is a hormone in the gut-liver axis that inhibits de novo bile acid synthesis from cholesterol via cytochrome P450 7A1 (CYP7A1), and inhibits insulin-induced hepatic lipogenesis [151]. This makes FGF19 agonism a promising therapeutic target for treatment of MASH. Recently, an FGF19 analogue (NGM282) has been found to reduce liver fat [152] and improve histological features of MASH [153,154]. FGF19 functions through its receptor FGFR4 and requires a coreceptor b-klotho. Farnesoid X receptors (FXRs) are abundantly expressed in body tissues involved in bile acid metabolism, such as liver, and intestines. Bile acids are natural ligands of the FXRs and regulate expression of the gene encoding for CYP7A1, the rate limiting enzyme in bile acid synthesis [155]. In addition to its principle effect on bile acid homeostasis, FXR signalling has several pleiotropic functions on various metabolic pathways. It has been demonstrated that FXR activation lowers plasma glucose (repress gluconeogenesis), free fatty acids (enhance b oxidation of FFA via PPAR-a), TGs (repress TG synthesis and promotes TG clearance via Apo-CII) and improves insulin sensitivity [156,157] (Fig. 5). FXR deficiency results in peripheral insulin resistance, an important feature of MASH [158]. Hepatocyte inflammation and ER stress downregulate FXR which contributes to MASH. Inflammation activates Yin Yang 1 (YY1) protein, which then downregulates FXR. Furthermore, activated hepatic ER stress represses FXR expression via the transcriptional activity of hepatocyte nuclear factor 1a (HNF-1a). In addition to their direct role, FXRs indirectly regulate carbohydrate and lipid metabolism and insulin sensitivity through increasing the secretion of FGF19 into the small intestine [159]. Obeticholic acid, a selective FXR agonist has not only reduced liver enzymes and liver fat, but also improved markers of liver inflammation and fibrosis in patients with MASH (Table 1) [160].

6. Gut-liver axis The liver constantly communicates with gut through nutrients, gut microbiome derived products, and bile acids via entero-hepatic circulation. Both alterations in the gut microbiome as well as abnormalities in the bile acid metabolism contribute to the pathogenesis of MASH.

6.3. Endogenous alcohol production 6.1. Dysfunctional gut microbiome Histologically, MASH is indistinguishable from alcoholic liver disease. It was, therefore, long believed that alcohol might play a role in MASH. The increased abundance of alcohol-producing bacteria in MASH microbiomes, elevated blood-ethanol levels in MASH patients, and the well-established role of alcohol metabolism in oxidative stress and liver inflammation suggest a role for alcoholproducing microbiota in the pathogenesis of MASH [161]. Recently, a high-alcohol-producing Klebsiella pneumoniae was found to occur in a large percentage of individuals with MAFLD in a Chinese cohort. Transfer of this bacterial strain into mice resulted in MAFLD, suggesting increased levels of this bacterial strain causing MAFLD in some individuals [162].

Various studies have demonstrated that gut microflora may play a pivotal role in the pathogenesis of NAFLD through LPS-TLR4 signalling [141]. Many studies in animals and humans suggest the involvement of gut microbiota in the development of obesity, IR and risk factors associated with NAFLD pathogenesis [142]. In genetically obese mice, an increased hepatic susceptibility to endotoxin-mediated damage has been observed [143]. Dysbiosis may lead to translocation of bacterial products into the systemic circulation, further flaming the inflammatory processes by activating macrophages and Kupffer cells [144]. In human studies, MAFLD has been associated with increased plasma LPS levels, 1883



Fig. 5. Farnesoid X receptor (FXR) functions: Liver receives bile acids (BAs) through enterohepatic circulation. These BAs act as endogenous ligands for FXR. FXR activation results in repression of gluconeogenesis, TG synthesis and VLDL export via small heterodimer partner (SHP) signalling cascade. FXR activation also increases FA oxidation via PPAR-a and enhance TG clearance by inducing Apo-C11 expression. Inflammation inhibits FXR signalling via Yin Yang 1 (YY1) protein and ER stress represses FXR expression via the inhibition of the transcriptional activity of hepatocyte nuclear factor 1 alpha (HNF-1a).

progression of MASH. In general, proximal processes of MASH, such as IR, lipid overload, and lipotoxicity are well established to some extent, but the downstream mechanisms of MASH, such as inflammatory, apoptotic, and fibrogenic processes are incompletely understood, and are rapidly evolving. New downstream molecular mechanisms are being discovered that influence progression of MASH. New targets such as EVs, miRNAs, and intestinal fructose handling are promising for development of therapeutic agents.

6.4. Intestinal fructose catabolism Epidemiological studies suggest that excessive fructose consumption is associated with hyperlipidemia, MAFLD, obesity and type 2 diabetes [163]. Dietary fructose is mostly cleared by the small intestine. Clearance requires the fructose-phosphorylating enzyme ketohexokinase (KHK). Low doses of fructose are ~90% cleared by the intestine, with only trace fructose but extensive fructose-derived glucose, lactate, and glycerate found in the portal blood. High doses of fructose (1 g/kg) overwhelm intestinal fructose absorption and clearance, resulting in fructose reaching both the liver and colonic microbiota. Fructose metabolism begins with its phosphorylation by the enzyme ketohexokinase (KHK), which exists in two forms, fructokinase C and A [164]. The more active isozyme, KHKeC, is expressed most strongly in the liver, but also substantially in the small intestine [165], where it drives dietary fructose absorption and conversion into other metabolites before fructose reaches the liver [166,167]. Intestinal fructose catabolism mitigates fructose-induced lipogenesis. In mice, intestine-specific KHKeC deletion increases dietary fructose transit to the liver and gut microbiota and sensitizes mice to fructose’s hyperlipidaemic effects and hepatic steatosis [168]. In contrast, intestine-specific KHKeC overexpression promotes intestinal fructose clearance and decreases fructose-induced lipogenesis [168]. Thus, intestinal fructose clearance capacity controls the rate at which fructose can be safely ingested. Collectively, these data demonstrate that fructose induces lipogenesis when its dietary intake rate exceeds the intestinal clearance capacity. These observations are important in several aspects. Firstly, single nucleotide polymorphism (SNPs) of KFK-c may explain why some individuals are more prone to fructose induced liver injury. Secondly, pharmacological overexpression of this enzyme may lead to better tolerance of fructose and prevention of fructose induced MASH.

Author contributions M.S.K. original idea for the review, search strategies, writing. N.S.C. search strategies, quality assessment, writing and revision of the manuscript. S.K.M. quality assessment, revision of the manuscript. All authors provided input throughout the entire review protocol, read and approved the final manuscript. PPAR, peroxisome proliferator activated receptor; SGLT2, sodium-glucose cotransporter 2; GLP-1, glucagon like peptide; FXR, farnesoid X receptor; ACC, Acetyl-CoA carboxylase; SCD, steroyl CoA desaturase; FGF, fibroblast growth factor; DGAT2, diacylglycerol acyltransferase; FFA, free fatty acid; IR, insulin resistance; DNL, de novo lipogenesis. Declaration of competing interest The authors declare no conflicts of interest. References [1] Younossi Z, Anstee QM, Marietti M, et al. Global burden of NAFLD and NASH: Trends, predictions, risk factors and prevention. Nat Clin Pract Gastroenterol Hepatol 2018;15(1):11e20. [2] Dulai PS, Singh S, Patel J, et al. Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Systematic review and meta-analysis. Hepatology 2017;65(5):1557e65. [3] Eslam M, Sanyal AJ, George J. International consensus panel. MAFLD: A consensus-driven proposed nomenclature for metabolic associated fatty liver disease. Gastroenterology 2020;158(7):1999e2014. e1. [4] Eslam M, Newsome PN, Anstee QM, Targher G, Gomez MR, Zelber-Sagi S, et al. A new definition for metabolic associated fatty liver disease: An international expert consensus statement. J Hepatol 2020;73:202e9. [5] Fouad Y, Waked I, Bollipo S, Gomaa A, Ajlouni Y, Attia D. What’s in a name?

7. Conclusions Over the last few year, there has been an increasing knowledge regarding pathogenesis of MASH. This review highlighted current understanding about molecular mechanisms for development and 1884


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