Human Liver Slices Research Articles

Kisspeptin Alleviates Human Hepatic Fibrogenesis by Inhibiting TGFβ Signaling in Hepatic Stellate Cells.

The peptide hormone kisspeptin attenuates liver steatosis, metabolic dysfunction-associated steatohepatitis (MASH), and fibrosis in mouse models by signaling via the kisspeptin 1 receptor (KISS1R). However, whether kisspeptin impacts fibrogenesis in the human liver is not known. We investigated the impact of a potent kisspeptin analog (KPA) on fibrogenesis using human precision-cut liver slices (hPCLS) from fibrotic livers from male patients, in human hepatic stellate cells (HSCs), LX-2, and in primary mouse HSCs. In hPCLS, 48 h and 72 h of KPA (3 nM, 100 nM) treatment decreased collagen secretion and lowered the expression of fibrogenic and inflammatory markers. Immunohistochemical studies revealed that KISS1R is expressed and localized to HSCs in MASH/fibrotic livers. In HSCs, KPA treatment reduced transforming growth factor b (TGFβ)-the induced expression of fibrogenic and inflammatory markers, in addition to decreasing TGFβ-induced collagen secretion, cell migration, proliferation, and colony formation. Mechanistically, KISS1R signaling downregulated TGFβ signaling by decreasing SMAD2/3 phosphorylation via the activation of protein phosphatases, PP2A, which dephosphorylates SMAD 2/3. This study revealed for the first time that kisspeptin reverses human hepatic fibrogenesis, thus identifying it as a new therapeutic target to treat hepatic fibrosis.

UCHL1-dependent control of hypoxia-inducible factor transcriptional activity during liver fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most types of chronic liver disease. At the cellular level, liver fibrosis is associated with the activation of hepatic stellate cells (HSCs) which transdifferentiate into a myofibroblast-like phenotype that is contractile, proliferative and profibrogenic. HSC transdifferentiation induces genome-wide changes in gene expression that enable the cell to adopt its profibrogenic functions. We have previously identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation; however, the cellular targets of its deubiquitinating activity are poorly defined. Here, we describe a role for UCHL1 in regulating the levels and activity of hypoxia-inducible factor 1 (HIF1), an oxygen-sensitive transcription factor, during HSC activation and liver fibrosis. HIF1 is elevated during HSC activation and promotes the expression of profibrotic mediator HIF target genes. Increased HIF1α expression correlated with induction of UCHL1 mRNA and protein with HSC activation. Genetic deletion or chemical inhibition of UCHL1 impaired HIF activity through reduction of HIF1α levels. Furthermore, our mechanistic studies have shown that UCHL1 elevates HIF activity through specific cleavage of degradative ubiquitin chains, elevates levels of pro-fibrotic gene expression and increases proliferation rates. As we also show that UCHL1 inhibition blunts fibrogenesis in a pre-clinical 3D human liver slice model of fibrosis, these results demonstrate how small molecule inhibitors of DUBs can exert therapeutic effects through modulation of HIF transcription factors in liver disease. Furthermore, inhibition of HIF activity using UCHL1 inhibitors may represent a therapeutic opportunity with other HIF-related pathologies.

Alcohol-Associated Liver Disease Outcomes: Critical Mechanisms of Liver Injury Progression.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Metabolic Dysfunction-Associated Steatotic Liver Disease in a Dish: Human Precision-Cut Liver Slices as a Platform for Drug Screening and Interventions.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing healthcare problem with limited therapeutic options. Progress in this field depends on the availability of reliable preclinical models. Human precision-cut liver slices (PCLSs) have been employed to replicate the initiation of MASLD, but a comprehensive investigation into MASLD progression is still missing. This study aimed to extend the current incubation time of human PCLSs to examine different stages in MASLD. Healthy human PCLSs were cultured for up to 96 h in a medium enriched with high sugar, high insulin, and high fatty acids to induce MASLD. PCLSs displayed hepatic steatosis, characterized by accumulated intracellular fat. The development of hepatic steatosis appeared to involve a time-dependent impact on lipid metabolism, with an initial increase in fatty acid uptake and storage, and a subsequent down-regulation of lipid oxidation and secretion. PCLSs also demonstrated liver inflammation, including increased pro-inflammatory gene expression and cytokine production. Additionally, liver fibrosis was also observed through the elevated production of pro-collagen 1a1 and tissue inhibitor of metalloproteinase-1 (TIMP1). RNA sequencing showed that the tumor necrosis factor alpha (TNFα) signaling pathway and transforming growth factor beta (TGFβ) signaling pathway were consistently activated, potentially contributing to the development of inflammation and fibrosis. In conclusion, the prolonged incubation of human PCLSs can establish a robust ex vivo model for MASLD, facilitating the identification and evaluation of potential therapeutic interventions.

Liver-targeted nanoparticles delivering nitric oxide reduce portal hypertension in cirrhotic rats

Nitric oxide (NO) is a small vasodilator playing a key role in the pathogenesis of portal hypertension. Here, we assessed the potential therapeutic effect of a NO donor targeted to the liver by poly(beta-amino ester) nanoparticles (pBAE NPs) in experimental cirrhosis. Retinol-functionalized NO donor pBAE NPs (Ret pBAE NPs) were synthetized with the aim of actively targeting the liver. Administration of Ret pBAE NPs resulted in uptake and transfection by the liver and spleen. NPs were not found in other organs or the systemic circulation. Treatment with NO donor Ret pBAE NPs (30 mg/ kg body weight) significantly decreased aspartate aminotransferase, lactate dehydrogenase and portal pressure (9.75 ± 0.64 mmHg) compared to control NPs (13.4 ± 0.53 mmHg) in cirrhotic rats. There were no effects on mean arterial pressure and cardiac output. Liver-targeted NO donor NPs reduced collagen fibers and steatosis, activation of hepatic stellate cells and mRNA expression of profibrogenic and proinflammatory genes. Finally, Ret pBAE NPs displayed efficient transfection in human liver slices. Overall, liver-specific NO donor NPs effectively target the liver and mitigated inflammation and portal hypertension in cirrhotic rats. The use of Ret pBAE may prove to be an effective therapeutic strategy to treat advanced liver disease.

Human Precision-Cut Liver Slices: A Potential Platform to Study Alcohol-Related Liver Disease.

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

WED-407 - Characterization of extracellular vesicles derived from human precision-cut liver slices in non-alcoholic fatty liver disease

WED-407 - Characterization of extracellular vesicles derived from human precision-cut liver slices in non-alcoholic fatty liver disease

WED-538 - The effects of lipopolysaccharides on inducing non-alcoholic fatty liver disease in human precision-cut liver slices

WED-538 - The effects of lipopolysaccharides on inducing non-alcoholic fatty liver disease in human precision-cut liver slices

Transcriptomic profiling of induced steatosis in human and mouse precision-cut liver slices

There is a high need for predictive human ex vivo models for non-alcoholic fatty liver disease (NAFLD). About a decade ago, precision-cut liver slices (PCLSs) have been established as an ex vivo assay for humans and other organisms. In the present study, we use transcriptomics by RNASeq to profile a new human and mouse PCLSs based assay for steatosis in NAFLD. Steatosis as quantified by an increase of triglycerides after 48 h in culture, is induced by incremental supplementation of sugars (glucose and fructose), insulin, and fatty acids (palmitate, oleate). We mirrored the experimental design for human vs. mouse liver organ derived PCLSs and profiled each organ at eight different nutrient conditions after 24 h and 48 h time in culture. Thus, the provided data allows a comprehensive analysis of the donor, species, time, and nutrient factor specific regulation of gene expression in steatosis, despite the heterogeneity of the human tissue samples. Exemplified this is demonstrated by ranking homologous gene pairs by convergent or divergent expression pattern across nutrient conditions.

Monoacylglycerol lipase reprograms hepatocytes and macrophages to promote liver regeneration

Liver regeneration is a repair process in which metabolic reprogramming of parenchymal and inflammatory cells plays a major role. Monoacylglycerol lipase (MAGL) is an ubiquitous enzyme at the crossroad between lipid metabolism and inflammation. It converts monoacylglycerols into free fatty acids and metabolises 2-arachidonoylglycerol into arachidonic acid, being thus the major source of pro-inflammatory prostaglandins in the liver. In this study, we investigated the role of MAGL in liver regeneration. Hepatocyte proliferation was studied invitro in hepatoma cell lines and exvivo in precision-cut human liver slices. Liver regeneration was investigated in mice treated with a pharmacological MAGL inhibitor, MJN110, as well as in animals globally invalidated for MAGL (MAGL-/-) and specifically invalidated in hepatocytes (MAGLHep-/-) or myeloid cells (MAGLMye-/-). Two models of liver regeneration were used: acute toxic carbon tetrachloride injection and two-thirds partial hepatectomy. MAGLMye-/- liver macrophages profiling was analysed by RNA sequencing. A rescue experiment was performed by invivo administration of interferon receptor antibody in MAGLMye-/- mice. Precision-cut human liver slices from patients with chronic liver disease and human hepatocyte cell lines exposed to MJN110 showed reduced hepatocyte proliferation. Mice with global invalidation or mice treated with MJN110 showed blunted liver regeneration. Moreover, mice with specific deletion of MAGL in either hepatocytes or myeloid cells displayed delayed liver regeneration. Mechanistically, MAGLHep-/- mice showed reduced liver eicosanoid production, in particular prostaglandin E2 that negatively impacts on hepatocyte proliferation. MAGL inhibition in macrophages resulted in the induction of the type I interferon pathway. Importantly, neutralising the type I interferon pathway restored liver regeneration of MAGLMye-/- mice. Our data demonstrate that MAGL promotes liver regeneration by hepatocyte and macrophage reprogramming. By using human liver samples and mouse models of global or specific cell type invalidation, we show that the monoacylglycerol pathway plays an essential role in liver regeneration. We unveil the mechanisms by which MAGL expressed in both hepatocytes and macrophages impacts the liver regeneration process, via eicosanoid production by hepatocytes and the modulation of the macrophage interferon pathway profile that restrains hepatocyte proliferation.

MAIT cell inhibition promotes liver fibrosis regression via macrophage phenotype reprogramming

Recent data have shown that liver fibrosis can regress even at later stages of cirrhosis and shifting the immune response from pro-inflammatory towards a resolutive profile is considered as a promising option. The immune regulatory networks that govern the shift of the inflammatory phenotype and thus potential reversal of liver fibrosis are lesser known. Here we show that in precision-cut human liver slices obtained from patients with end-stage fibrosis and in mouse models, inhibiting Mucosal-Associated Invariant T (MAIT) cells using pharmacological or antibody-driven approaches, limits fibrosis progression and even regresses fibrosis, following chronic toxic- or non-alcoholic steatohepatitis (NASH)-induced liver injury. Mechanistic studies, combining RNA sequencing, in vivo functional studies (performed in male mice) and co-culture experiments indicate that disruption of the MAIT cell-monocyte/macrophage interaction results in resolution of fibrosis both by increasing the frequency of restorative Ly6Clo at the expenses of pro-fibrogenic Ly6Chi monocyte-derived macrophages and promoting an autophagic phenotype in both subsets. Thus, our data show that MAIT cell activation and the consequential phenotype shift of liver macrophages are important pathogenic features of liver fibrosis and could be targeted by anti-fibrogenic therapy.

IL-17A in Human Liver: Significant Source of Inflammation and Trigger of Liver Fibrosis Initiation.

IL-17A is considered to guide liver inflammation and fibrosis. From twenty-two human liver samples of different fibrosis stages (F0 to F4), IL-17A, IL-22, and TGFβ1 protein expression in liver tissue lysates were analyzed. Ten paired samples of liver tissue (F0–F1 stage) and blood from the same patient were used to analyze intrahepatic and blood T-lymphoid IL-17A+ cells by flow cytometry. The analyses have been performed regardless of pathology, considering the stage of fibrosis. Human liver tissue was used for the primary human liver slice cultures, followed by subsequent cytokine stimulation and fibrotic markers’ analysis by ELISA. IL-17A production in human liver tissue was significantly higher in the early fibrotic stage compared with the advanced stage. Th17 T cells and, to a lesser extent, MAIT cells were the main sources of IL-17A in both compartments, the liver and the blood. Moreover, the presence of liver Th17IL-17A+INFγ+ cells was detected in the liver. IL-17A stimulation of human liver slice culture increased the expression of profibrotic and pro-inflammatory markers. IL-17A, secreted by Th17 and MAIT cells in the liver, triggered fibrosis by inducing the expression of IL-6 and profibrotic markers and could be a target for antifibrotic treatment. Further amplitude studies are needed to confirm the current results.

Response of Human Liver Tissue to Innate Immune Stimuli.

Precision-cut human liver slice cultures (PCLS) have become an important alternative immunological platform in preclinical testing. To further evaluate the capacity of PCLS, we investigated the innate immune response to TLR3 agonist (poly-I:C) and TLR4 agonist (LPS) using normal and diseased liver tissue. Pathological liver tissue was obtained from patients with active chronic HCV infection, and patients with former chronic HCV infection cured by recent Direct-Acting Antiviral (DAA) drug therapy. We found that hepatic innate immunity in response to TLR3 and TLR4 agonists was not suppressed but enhanced in the HCV-infected tissue, compared with the healthy controls. Furthermore, despite recent HCV elimination, DAA-cured liver tissue manifested ongoing abnormalities in liver immunity: sustained abnormal immune gene expression in DAA-cured samples was identified in direct ex vivo measurements and in TLR3 and TLR4 stimulation assays. Genes that were up-regulated in chronic HCV-infected liver tissue were mostly characteristic of the non-parenchymal cell compartment. These results demonstrated the utility of PCLS in studying both liver pathology and innate immunity.

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease

Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and exvivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. Invitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.

A Mammalian Target of Rapamycin‐Perilipin 3 (mTORC1‐Plin3) Pathway is essential to Activate Lipophagy and Protects Against Hepatosteatosis

Background and Aims NAFLD is the most common hepatic pathology in western countries and no treatment is currently available. NAFLD is characterized by the aberrant hepatocellular accumulation of fatty acids in the form of lipid droplets (LDs). Recently, it was shown that liver LD degradation occurs through a process termed lipophagy, a form of autophagy. However, the molecular mechanisms governing liver lipophagy are elusive. Here, we aimed to ascertain the key molecular players that regulate hepatic lipophagy and their importance in NAFLD. Approach and Results We analyzed the formation and degradation of LD in vitro (fibroblasts and primary mouse hepatocytes), in vivo and ex vivo (mouse and human liver slices) and focused on the role of the autophagy master regulator mammalian target of rapamycin complex (mTORC) 1 and the LD coating protein perilipin (Plin) 3 in these processes. We show that the autophagy machinery is recruited to the LD on hepatic overload of oleic acid in all experimental settings. This led to activation of lipophagy, a process that was abolished by Plin3 knockdown using RNA interference. Furthermore, Plin3 directly interacted with the autophagy proteins focal adhesion interaction protein 200 KDa and autophagy‐related 16L, suggesting that Plin3 functions as a docking protein or is involved in autophagosome formation to activate lipophagy. Finally, we show that mTORC1 phosphorylated Plin3 to promote LD degradation. Conclusions These results reveal that mTORC1 regulates liver lipophagy through a mechanism dependent on Plin3 phosphorylation. We propose that stimulating this pathway can enhance lipophagy in hepatocytes to help protect the liver from lipid‐mediated toxicity, thus offering a therapeutic strategy in NAFLD.

Soluble TIM3 and Its Ligands Galectin-9 and CEACAM1 Are in Disequilibrium During Alcohol-Related Liver Disease and Promote Impairment of Anti-bacterial Immunity.

Background and AimsImmunoregulatory checkpoint receptors (CR) contribute to the profound immunoparesis observed in alcohol-related liver disease (ALD) and in vitro neutralization of inhibitory-CRs TIM3/PD1 on anti-bacterial T-cells can rescue innate and adaptive anti-bacterial immunity. Recently described soluble-CR forms can modulate immunity in inflammatory conditions, but the contributions of soluble-TIM3 and soluble-PD1 and other soluble-CRs to immune derangements in ALD remain unclear.MethodsIn Alcoholic Hepatitis (AH; n = 19), alcohol-related cirrhosis (ARC; n = 53) and healthy control (HC; n = 27) subjects, we measured by Luminex technology (i) plasma levels of 16 soluble-CRs, 12 pro/anti-inflammatory cytokines and markers of gut bacterial translocation; (ii) pre-hepatic, post-hepatic and non-hepatic soluble-CR plasma levels in ARC patients undergoing TIPS; (iii) soluble-CRs production from ethanol-treated immunocompetent precision cut human liver slices (PCLS); (iv) whole-blood soluble-CR expression upon bacterial challenge. By FACS, we assessed the relationship between soluble-TIM3 and membrane-TIM3 and rescue of immunity in bacterial-challenged PBMCs.ResultsSoluble-TIM3 was the dominant plasma soluble-CR in ALD vs. HC (p = 0.00002) and multivariate analysis identified it as the main driver of differences between groups. Soluble-CRs were strongly correlated with pro-inflammatory cytokines, gut bacterial translocation markers and clinical indices of disease severity. Ethanol exposure or bacterial challenge did not induce soluble-TIM3 production from PCLS nor from whole-blood. Bacterial challenge prompted membrane-TIM3 hyperexpression on PBMCs from ALD patient’s vs. HC (p < 0.002) and was inversely correlated with plasma soluble-TIM3 levels in matched patients. TIM3 ligands soluble-Galectin-9 and soluble-CEACAM1 were elevated in ALD plasma (AH > ARC; p < 0.002). In vitro neutralization of Galectin-9 and soluble-CEACAM1 improved the defective anti-bacterial and anti-inflammatory cytokine production from E. coli-challenged PBMCs in ALD patients.ConclusionsAlcohol-related liver disease patients exhibit supra-physiological plasma levels of soluble-TIM3, particularly those with greater disease severity. This is also associated with increased levels of soluble TIM3-ligands and membrane-TIM3 expression on immune cells. Soluble-TIM3 can block the TIM3-ligand synapse and improve anti-bacterial immunity; however, the increased levels of soluble TIM3-binding ligands in patients with ALD negate any potential immunostimulatory effects. We believe that anti-TIM3 neutralizing antibodies currently in Phase I clinical trials or soluble-TIM3 should be investigated further for their ability to enhance anti-bacterial immunity. These agents could potentially represent an innovative immune-based supportive approach to rescue anti-bacterial defenses in ALD patients.

Adult human liver slice cultures: Modelling of liver fibrosis and evaluation of new anti-fibrotic drugs.

BACKGROUNDLiver fibrosis can result in end-stage liver failure and death.AIMTo examine human liver fibrogenesis and anti-fibrotic therapies, we evaluated the three dimensional ex vivo liver slice (LS) model.METHODSFibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected from patients after liver resection. Human liver slices (HLS) were cultivated for up to 21 days. Hepatitis C virus (HCV) infection, alcohol (ethanol stimulation) and steatosis (palmitate stimulation) were examined in fibrotic (F2 to F4) liver slices infected (or not) with HCV. F0-F1 HLS were used as controls. At day 0, either ursodeoxycholic acid (choleretic and hepatoprotective properties) and/or α-tocopherol (antioxidant properties) were added to standard of care on HLS and fibrotic liver slices, infected (or not) with HCV. Expression of the biomarkers of fibrosis and the triglyceride production were checked by quantitative reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay.RESULTSThe cultures were viable in vitro for 21 days allowing to study fibrosis inducers and to estimate the effect of anti-fibrotic drugs. Expression of the biomarkers of fibrosis and the progression to steatosis (estimated by triglycerides production) was increased with the addition of HCV and /or ethanol or palmitate. From day 15 of the follow-up studies, a significant decrease of both transforming growth factor β-1 and Procol1A1 expression and triglycerides production was observed when a combined anti-fibrotic treatment was applied on HCV infected F2-F4 LS cultures.CONCLUSIONThese results show that the human three dimensional ex vivo model effectively reflects the in vivo processes in damaged human liver (viral, alcoholic, nonalcoholic steatohepatitis liver diseases) and provides the proof of concept that the LS examined model permits a rapid evaluation of new anti-fibrotic therapies when used alone or in combination.

Liver Abnormalities after Elimination of HCV Infection: Persistent Epigenetic and Immunological Perturbations Post-Cure.

Chronic hepatitis C (CHC) is a major cause of hepatocellular carcinoma (HCC) worldwide. While directly acting antiviral (DAA) drugs are now able to cure virtually all hepatitis C virus (HCV) infections, even in subjects with advanced liver disease, what happens to the liver and progression of the disease after DAA-induced cure of viremia is only beginning to emerge. Several large-scale clinical studies in different patient populations have shown that patients with advanced liver disease maintain a risk for developing HCC even when the original instigator, the virus, is eliminated by DAAs. Here we review emerging studies derived from multiple, complementary experimental systems involving patient liver tissues, human liver cell cultures, human liver slice cultures, and animal models, showing that HCV infection induces epigenetic, signaling, and gene expression changes in the liver associated with altered hepatic innate immunity and liver cancer risk. Of critical importance is the fact that these virus-induced abnormalities persist after DAA cure of HCV. These nascent findings portend the discovery of pathways involved in post-HCV immunopathogenesis, which may be clinically actionable targets for more comprehensive care of DAA-cured individuals.

Epigenetic mechanisms and metabolic reprogramming in fibrogenesis: dual targeting of G9a and DNMT1 for the inhibition of liver fibrosis

ObjectiveHepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and...

Design of a Gene Panel to Expose the Versatile Role of Hepatic Stellate Cells in Human Liver Fibrosis.

The pivotal cell involved in the pathogenesis of liver fibrosis, i.e., the activated hepatic stellate cell (HSC), has a wide range of activities during the initiation, progression and even regression of the disease. These HSC-related activities encompass cellular activation, matrix synthesis and degradation, proliferation, contraction, chemotaxis and inflammatory signaling. When determining the in vitro and in vivo effectivity of novel antifibrotic therapies, the readout is currently mainly based on gene and protein levels of α-smooth muscle actin (α-SMA) and the fibrillar collagens (type I and III). We advocate for a more comprehensive approach in addition to these markers when screening potential antifibrotic drugs that interfere with HSCs. Therefore, we aimed to develop a gene panel for human in vitro and ex vivo drug screening models, addressing each of the HSC-activities with at least one gene, comprising, in total, 16 genes. We determined the gene expression in various human stellate cells, ranging from primary cells to cell lines with an HSC-origin, and human liver slices and stimulated them with two key profibrotic factors, i.e., transforming growth factor β (TGFβ) or platelet-derived growth factor BB (PDGF-BB). We demonstrated that freshly isolated HSCs showed the strongest and highest variety of responses to these profibrotic stimuli, in particular following PDGF-BB stimulation, while cell lines were limited in their responses. Moreover, we verified these gene expression profiles in human precision-cut liver slices and showed similarities with the TGFβ- and PDGF-BB-related fibrotic responses, as observed in the primary HSCs. With this study, we encourage researchers to get off the beaten track when testing antifibrotic compounds by including more HSC-related markers in their future work. This way, potential compounds will be screened more extensively, which might increase the likelihood of developing effective antifibrotic drugs.