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Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo -activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments. [ABSTRACT FROM AUTHOR]

Background & Aims: Chronic liver disease (CLD) remains a global health issue associated with a significant disease burden. Liver fibrosis, a hallmark of CLD, is characterised by the activation of hepatic stellate cells (HSCs) that gain profibrotic characteristics including increased production of extracellular matrix protein. Currently, no antifibrotic therapies are available clinically, in part because of the lack of HSC-specific drug targets. Here, we aimed to identify HSC-specific membrane proteins that can serve as targets for antifibrotic drug development., Methods: Small interfering RNA-mediated knockdown of GPR176 was used to assess the in vitro function of GPR176 in HSCs and in precision cut liver slices (PCLS). The in vivo role of GPR176 was assessed using the carbon tetrachloride (CCl 4 ) and common bile duct ligation (BDL) models in wild-type and GPR176 knockout mice. GPR176 in human CLD was assessed by immunohistochemistry of diseased human livers and RNA expression analysis in human primary HSCs and transcriptomic data sets., Results: We identified Gpr176 , an orphan G-protein coupled receptor, as an HSC-enriched activation associated gene. In vitr o, Gpr176 is strongly induced upon culture-induced and hepatocyte-damage-induced activation of primary HSCs. Knockdown of GPR176 in primary mouse HSCs or PCLS cultures resulted in reduced fibrogenic characteristics. Absence of GPR176 did not influence liver homeostasis, but Gpr176 -/- mice developed less severe fibrosis in CCl 4 and BDL fibrosis models. In humans, GPR176 expression was correlated with in vitro HSC activation and with fibrosis stage in patients with CLD., Conclusions: GPR176 is a functional protein during liver fibrosis and reducing its activity attenuates fibrogenesis. These results highlight the potential of GPR176 as an HSC-specific antifibrotic candidate to treat CLD., Impact and Implications: The lack of effective antifibrotic drugs is partly attributed to the insufficient knowledge about the mechanisms involved in the development of liver fibrosis. We demonstrate that the G-protein coupled receptor GPR176 contributes to fibrosis development. Since GPR176 is specifically expressed on the membrane of activated hepatic stellate cells and is linked with fibrosis progression in humans, it opens new avenues for the development of targeted interventions., Competing Interests: This work was partially funded by Gilead Sciences through a BeLux Fellowship 2020 grant (ID: 08309). Please refer to the accompanying ICMJE disclosure forms for further details., (© 2024 The Author(s).)

In vitro models of liver fibrosis have evolved from mono-cultures of primary rodent hepatic stellate cells and stellate cell lines, to more complex co-cultures of primary or stem cell-derived liver cells. Great progress has been made in the development of stem cell-derived liver cultures; however, the liver cells obtained from stem cells do not yet fully recapitulate the phenotype of their in vivo counterparts. Freshly isolated rodent cells remain the most representative cell type to use for in vitro culture. To study liver injury-induced fibrosis, co-cultures of hepatocytes and stellate cells are an informative minimal model. Here, we describe a robust protocol to isolate hepatocytes and hepatic stellate cells from one mouse and a method for the subsequent seeding and culture as free-floating spheroids., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Chronic liver disease can lead to liver fibrosis and ultimately cirrhosis, which is a significant health burden and a major cause of death worldwide. Reliable in vitro models are lacking and thus mono-cultures of cell lines are still used to study liver disease and evaluate candidate anti-fibrotic drugs. We established functional multicellular liver spheroid (MCLS) cultures using primary mouse hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells and Kupffer cells. Cell-aggregation and spheroid formation was enhanced with 96-well U-bottom plates generating over ±700 spheroids from one mouse. Extensive characterization showed that MCLS cultures contain functional hepatocytes, quiescent stellate cells, fenestrated sinusoidal endothelium and responsive Kupffer cells that can be maintained for 17 days. MCLS cultures display a fibrotic response upon chronic exposure to acetaminophen, and present steatosis and fibrosis when challenged with free fatty acid and lipopolysaccharides, reminiscent of non-alcoholic fatty liver disease (NAFLD) stages. Treatment of MCLS cultures with potential anti-NAFLD drugs such as Elafibranor, Lanifibranor, Pioglitazone and Obeticholic acid shows that all can inhibit steatosis, but only Elafibranor and especially Lanifibranor inhibit fibrosis. Therefore, primary mouse MCLS cultures can be used to model acute and chronic liver disease and are suitable for the assessment of anti-NAFLD drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aims: Hepatic capillaries are lined with specialized liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialization is co-determined by transcription factors. The zinc-finger E-box-binding homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis., Methods and Results: To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called 'capillarization'. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC-HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarization, HSC activation, and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis., Conclusion: Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background & Aims: Liver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis., Methods: Intracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl 4 induced liver fibrosis model., Results: In our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets' expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl 4 -treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease., Conclusions: We provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Activated hepatic stellate cells (aHSC) are the main source of extra cellular matrix in liver fibrosis. Activation is classically divided in two phases: initiation and perpetuation. Currently, HSC-based therapeutic candidates largely focus on targeting the aHSCs in the perpetuation phase. However, the importance of HSC initiation during chronic liver disease (CLD) remains unclear. Here, we identified transcriptional programs of initiating and activated HSCs by RNA sequencing, using in vitro and in vivo mouse models of fibrosis. Importantly, we show that both programs are active in HSCs during murine and human CLD. In human cirrhotic livers, scar associated mesenchymal cells employ both transcriptional programs at the single cell level. Our results indicate that the transcriptional programs that drive the initiation of HSCs are still active in humans suffering from CLD. We conclude that molecules involved in the initiation of HSC activation, or in the maintenance of aHSCs can be considered equally important in the search for druggable targets of chronic liver disease., (© 2021. The Author(s).)

Primary and secondary liver cancer are the third cause of death in the world, and as the incidence is increasing, liver cancer represents a global health burden. Current treatment strategies are insufficient to permanently cure patients from this devastating disease, and therefore other approaches are under investigation. The importance of cancer-associated fibroblasts (CAFs) in the tumour microenvironment is evident, and many pre-clinical studies have shown increased tumour aggressiveness in the presence of CAFs. However, it remains unclear how hepatic stellate cells are triggered by the tumour to become CAFs and how the recently described CAF subtypes originate and orchestrate pro-tumoural effects. Specialized in vitro systems will be needed to address these questions. In this review, we present the currently used in vitro models to study CAFs in primary and secondary liver cancer and highlight the trend from using oversimplified 2D culture systems to more complex 3D models. Relatively few studies report on the impact of cancer (sub)types on CAFs and the tumour microenvironment, and most studies investigated the impact of secreted factors due to the nature of the models.

Liver sinusoidal endothelial cells have a gatekeeper function in liver homeostasis by permitting substrates from the bloodstream into the space of Disse and regulating hepatic stellate cell activation status. Maintenance of LSEC's highly specialized phenotype is crucial for liver homeostasis. During liver fibrosis and cirrhosis, LSEC phenotype and functions are lost by processes known as capillarization and LSEC dysfunction. LSEC capillarization can be demonstrated by the loss of fenestrae (cytoplasmic pores) and the manifestation of a basement membrane. Currently, no protein or genetic markers can clearly distinguish healthy from damaged LSECs in acute or chronic liver disease. Single cell (sc)RNA sequencing efforts have identified several LSEC populations in mouse models for liver disease and in human cirrhotic livers. Still, there are no clearly defined genesets that can identify LSECs or dysfunctional LSEC populations in transcriptome data. Here, we developed genesets that are enriched in healthy and damaged LSECs which correlated very strongly with healthy and early stage- vs. advanced human liver diseases. A damaged LSEC signature comprised of Fabp4/5 and Vwf/a1 was established which could efficiently identify damaged endothelial cells in single cell RNAseq data sets. In LSECs from an acute CCl 4 liver injury mouse model, Fabp4/5 and Vwf/a1 expression is induced within 1-3 days while in cirrhotic human livers these 4 genes are highly enriched in damaged LSECs. In conclusion, our newly developed gene signature of damaged LSECs can be applicable to a wide range of liver disease etiologies, implicating a common transcriptional alteration mechanism in LSEC damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Verhulst, van Os, De Smet, Eysackers, Mannaerts and van Grunsven.)

Hepatic stellate cells (HSCs) are nonparenchymal liver cells responsible for extracellular matrix homeostasis and are the main cells involved in the development of liver fibrosis following injury. The lack of reliable sources of HSCs has hence limited the development of complex in vitro systems to model liver diseases and toxicity. Here we describe a protocol to differentiate human induced pluripotent stem cells (iPSCs) into hepatic stellate cells (iPSC-HSCs). The protocol is based on the addition of several growth factors important for liver development sequentially over 12 d. iPSC-HSCs present phenotypic and functional characteristics of primary HSCs and can be expanded or frozen and used to perform high-throughput in vitro studies. We also describe how to coculture iPSC-HSCs with hepatocytes, which self-assemble into three-dimensional (3D) hepatic spheroids. This protocol enables the generation of HSC-like cells for in vitro modeling and drug screening studies.

A major obstacle in the development of efficient therapies for progressive liver fibrosis is the lack of representative in vitro models of liver fibrosis to aid in understanding the mechanisms of the disease and to promote the development of pharmaceuticals. Our aim was to develop a relevant in vitro mouse liver fibrosis model, based on the central hypothesis that liver fibrosis in vitro cannot be studied using only hepatic stellate cells (HSCs)-the main producer of scar tissue during fibrosis-, but requires cultures in which at least hepatocytes are integrated. We established robust methods to generate co-culture spheroids from freshly isolated mouse hepatocytes and HSCs. Characteristics and functionality of these spheroids were analyzed by qPCR of cell-type specific markers, CYP induction and immunohistochemistry. Compound toxicity was determined by ATP-assays. Hepatocytes and HSCs maintained their cell-type specific marker expression over a 15-day culture period without major hepatocyte dedifferentiation or HSC activation. Exposure of spheroids to TGFβ can directly activate HSCs, while acetaminophen exposure mounts a hepatocyte damage dependent activation of HSCs. Pharmaceuticals with known anti-fibrotic properties, such as Valproic acid and Verteporfin, reduce HSC activation in response to hepatocyte damage in these cultures. A comparison between the fibrotic response of the spheroid co-cultures and in vivo activated HSCs showed that these 3D co-cultures are more representative than the commonly used 2D HSC monocultures. Finally, we showed that the 3D cultures can be integrated in microfluidic chips. We conclude that our hepatocyte-stellate cell-spheroid cultures are a robust in vitro model of liver fibrosis. This model could be used to further unravel the mechanism of HSC activation and facilitate the discovery of, or testing for novel anti-fibrotic compounds, as these spheroids better reproduce HSC in vivo activation compared to the more traditional 2D mono-culture models., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Hippo signaling pathway and its two downstream effectors, the YAP and TAZ transcriptional coactivators, are drivers of tumor growth in experimental models. Studying mouse models, we show that YAP and TAZ can also exert a tumor-suppressive function. We found that normal hepatocytes surrounding liver tumors displayed activation of YAP and TAZ and that deletion of Yap and Taz in these peritumoral hepatocytes accelerated tumor growth. Conversely, experimental hyperactivation of YAP in peritumoral hepatocytes triggered regression of primary liver tumors and melanoma-derived liver metastases. Furthermore, whereas tumor cells growing in wild-type livers required YAP and TAZ for their survival, those surrounded by Yap - and Taz -deficient hepatocytes were not dependent on YAP and TAZ. Tumor cell survival thus depends on the relative activity of YAP and TAZ in tumor cells and their surrounding tissue, suggesting that YAP and TAZ act through a mechanism of cell competition to eliminate tumor cells., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Platelet Derived Growth Factor Receptor beta (PDGFRβ) has been associated to hepatic stellate cell activation and has been the target of multiple therapeutic studies. However, little is known concerning its use as a diagnostic agent., Methods: Circulating PDGFRβ levels were analysed in a cohort of patients with liver fibrosis/cirrhosis due to chronic alcohol abuse, viral hepatitis, or non-alcoholic fatty liver disease (NAFLD). The diagnostic performance of PDGFRβ as individual blood parameter, or in combination with other metabolic factors was evaluated., Findings: sPDGFRβ levels are progressively increased with increasing fibrosis stage and the largest difference was observed in patients with significant fibrosis, compared to no or mild fibrosis. The accuracy of sPDGFRβ-levels predicting fibrosis could be increased by combining it with albumin levels and platelet counts into a novel diagnostic algorithm, the PRTA-score, generating a predictive value superior to Fib-4, APRI, and AST/ALT. The sPDGFRβ levels and the PRTA-score are independent of liver disease aetiology, thus overcoming one of the major weaknesses of current non-invasive clinical and experimental scores. Finally, we confirmed the diagnostic value of sPDGFRβ levels and the PRTA-score in an independent patient cohort with NAFLD which was staged for fibrosis by liver biopsy., Interpretation: The PRTA-score is an accurate tool for detecting significant liver fibrosis in a broad range of liver disease aetiologies. FUND: Vrije Universiteit Brussel, the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Flanders) (HILIM-3D; SBO140045), and the Fund of Scientific Research Flanders (FWO)., (Copyright © 2019. Published by Elsevier B.V.)

Hepatic stellate cells activate upon liver injury and help at restoring damaged tissue by producing extracellular matrix proteins. A drastic increase in matrix proteins results in liver fibrosis and we hypothesize that this sudden increase leads to accumulation of proteins in the endoplasmic reticulum and its compensatory mechanism, the unfolded protein response. We indeed observe a very early, but transient induction of unfolded protein response genes during activation of primary mouse hepatic stellate cells in vitro and in vivo, prior to induction of classical stellate cell activation genes. This unfolded protein response does not seem sufficient to drive stellate cell activation on its own, as chemical induction of endoplasmic reticulum stress with tunicamycin in 3D cultured, quiescent stellate cells is not able to induce stellate cell activation. Inhibition of Jnk is important for the transduction of the unfolded protein response. Stellate cells isolated from Jnk knockout mice do not activate as much as their wild-type counterparts and do not have an induced expression of unfolded protein response genes. A timely termination of the unfolded protein response is essential to prevent endoplasmic reticulum stress-related apoptosis. A pathway known to be involved in this termination is the non-sense-mediated decay pathway. Non-sense-mediated decay inhibitors influence the unfolded protein response at early time points during stellate cell activation. Our data suggest that UPR in HSCs is differentially regulated between acute and chronic stages of the activation process. In conclusion, our data demonstrates that the unfolded protein response is a JNK1-dependent early event during hepatic stellate cell activation, which is counteracted by non-sense-mediated decay and is not sufficient to drive the stellate cell activation process. Therapeutic strategies based on UPR or NMD modulation might interfere with fibrosis, but will remain challenging because of the feedback mechanisms between the stress pathways.

P311 was first identified by the group of Studler et al. (1993) in the developing brain. In healthy, but mainly in pathological tissues, P311 is implicated in cell migration and proliferation. Furthermore, evidence in models of tissue fibrosis points to the colocalization with and the stimulation of transforming growth factor β1 by P311. This review provides a comprehensive overview on P311 and discusses its potential as an anti-fibrotic target.

The development of complex in vitro hepatic systems and artificial liver devices has been hampered by the lack of reliable sources for relevant cell types, such as hepatic stellate cells (HSCs). Here we report efficient differentiation of human pluripotent stem cells into HSC-like cells (iPSC-HSCs). iPSC-HSCs closely resemble primary human HSCs at the transcriptional, cellular, and functional levels and possess a gene expression profile intermediate between that of quiescent and activated HSCs. Functional analyses revealed that iPSC-HSCs accumulate retinyl esters in lipid droplets and are activated in response to mediators of wound healing, similar to their in vivo counterparts. When maintained as 3D spheroids with HepaRG hepatocytes, iPSC-HSCs exhibit a quiescent phenotype but mount a fibrogenic response and secrete pro-collagen in response to known stimuli and hepatocyte toxicity. Thus, this protocol provides a robust in vitro system for studying HSC development, modeling liver fibrosis, and drug toxicity screening., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Liver fibrosis is the result of persistent liver injury, and is characterized by sustained scar formation and disruption of the normal liver architecture. The extent of fibrosis is considered as an important prognostic factor for the patient outcome, as an absence of (early) treatment can lead to the development of liver cirrhosis and hepatocellular carcinoma. Till date, the most sensitive and specific way for the diagnosis and staging of liver fibrosis remains liver biopsy, an invasive diagnostic tool, which is associated with high costs and discomfort for the patient. Over time, non-invasive scoring systems have been developed, of which the measurements of serum markers and liver stiffness are validated for use in the clinic. These tools lack however the sensitivity and specificity to detect small changes in the progression or regression of both early and late stages of fibrosis. Novel non-invasive diagnostic markers with the potential to overcome these limitations have been developed, but often lack validation in large patient cohorts. In this review, we will summarize novel trends in non-invasive markers of liver fibrosis development and will discuss their (dis-)advantages for use in the clinic., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Pannexins are transmembrane proteins that form communication channels connecting the cytosol of an individual cell with its extracellular environment. A number of studies have documented the presence of pannexin1 in liver as well as its involvement in inflammatory responses. In this study, it was investigated whether pannexin1 plays a role in acute liver failure and non-alcoholic steatohepatitis, being prototypical acute and chronic liver pathologies, respectively, both featured by liver damage, oxidative stress and inflammation. To this end, wild-type and pannexin1 -/- mice were overdosed with acetaminophen for 1, 6, 24 or 48h or were fed a choline-deficient high-fat diet for 8weeks. Evaluation of the effects of genetic pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, lipid accumulation, protein adduct formation, oxidative stress and inflammation. In parallel, in order to elucidate molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. The results of this study show that pannexin1 -/- diseased mice present less liver damage and oxidative stress, while inflammation was only decreased in pannexin1 -/- mice in which non-alcoholic steatohepatitis was induced. A multitude of genes related to inflammation, oxidative stress and xenobiotic metabolism were differentially modulated in both liver disease models in wild-type and in pannexin1 -/- mice. Overall, the results of this study suggest that pannexin1 may play a role in the pathogenesis of liver disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Chronic hepatitis B (HBV) and C (HCV) virus infection is associated with the activation of hepatic stellate cells (HSCs) toward a myofibroblastic phenotype, resulting in excessive deposition of extracellular matrix, the development of liver fibrosis, and its progression toward cirrhosis. The gold standard for the detection and staging of liver fibrosis remains the liver biopsy, which is, however, associated with some mild and severe drawbacks. Other non-invasive techniques evade these drawbacks, but lack inter-stage specificity and are unable to detect early stages of fibrosis. We investigated whether circulating vesicle-associated miRNAs can be used in the diagnosis and staging of liver fibrosis in HBV and HCV patients. Methods : Plasma samples were obtained from 14 healthy individuals and 39 early stage fibrotic patients (F0-F2) with chronic HBV or HCV infection who underwent transient elastography (Fibroscan). Extracellular vesicles were extracted from the plasma and the level of miRNA-122, -150, -192, -21, -200b, and -92a was analyzed by qRT-PCR in total plasma and circulating vesicles. Finally, these same miRNAs were also quantified in vesicles extracted from in vitro activating primary HSCs. Results : In total plasma samples, only miRNA-200b (HBV: p = 0.0384; HCV: p = 0.0069) and miRNA-122 (HBV: p < 0.0001; HCV: p = 0.0007) were significantly up-regulated during early fibrosis. In circulating vesicles, miRNA-192 (HBV: p < 0.0001; HCV: p < 0.0001), -200b (HBV: p < 0.0001; HCV: p < 0.0001), -92a (HBV: p < 0.0001; HCV: p < 0.0001), and -150 (HBV: p = 0.0016; HCV: p = 0.004) displayed a significant down-regulation in both HBV and HCV patients. MiRNA expression profiles in vesicles isolated from in vitro activating primary mouse HSCs resembled the miRNA expression profile in circulating vesicles. Conclusion : Our analysis revealed a distinct miRNA expression pattern in total plasma and its circulating vesicles. The expression profile of miRNAs in circulating vesicles of fibrotic patients suggests the potential use of these vesicle-associated miRNAs as markers for early stages of liver fibrosis.

Hepatic inflammation drives hepatic stellate cells (HSC), resulting in liver fibrosis. The Farnesoid-X receptor (FXR) antagonizes inflammation through NF-κB inhibition. We investigated preventive and therapeutic effects of FXR agonist obeticholic acid (OCA) on hepatic inflammation and fibrosis in toxic cirrhotic rats. Cirrhosis was induced by thioacetamide (TAA) intoxication. OCA was given during or after intoxication with vehicle-treated rats as controls. At sacrifice, fibrosis, hemodynamic and biochemical parameters were assessed. HSC activation, cell turn-over, hepatic NF-κB activation, pro-inflammatory and pro-fibrotic cytokines were determined. The effect of OCA was further evaluated in isolated HSC, Kupffer cells, hepatocytes and liver sinusoidal endothelial cells (LSEC). OCA decreased hepatic inflammation and fibrogenesis during TAA-administration and reversed fibrosis in established cirrhosis. Portal pressure decreased through reduced intrahepatic vascular resistance. This was paralleled by decreased expression of pro-fibrotic cytokines (transforming growth-factor β, connective tissue growth factor, platelet-derived growth factor β-receptor) as well as markers of hepatic cell turn-over, by blunting effects of pro-inflammatory cytokines (e.g. monocyte chemo-attractant protein-1). In vitro, OCA inhibited both LSEC and Kupffer cell activation; while HSC remained unaffected. This related to NF-κB inhibition via up-regulated IκBα. In conclusion, OCA inhibits hepatic inflammation in toxic cirrhotic rats resulting in decreased HSC activation and fibrosis.

Current models for in vitro fibrosis consist of simple mono-layer cultures of rodent hepatic stellate cells (HSC), ignoring the role of hepatocyte injury. We aimed to develop a method allowing the detection of hepatocyte-mediated and drug-induced liver fibrosis. We used HepaRG (Hep) and primary human HSCs cultured as 3D spheroids in 96-well plates. These resulting scaffold-free organoids were characterized for CYP induction, albumin secretion, and hepatocyte and HSC-specific gene expression by qPCR. The metabolic competence of the organoid over 21 days allows activation of HSCs in the organoid in a drug- and hepatocyte-dependent manner. After a single dose or repeated exposure for 14 days to the pro-fibrotic compounds Allyl alcohol and Methotrexate, hepatic organoids display fibrotic features such as HSC activation, collagen secretion and deposition. Acetaminophen was identified by these organoids as an inducer of hepatotoxic-mediated HSC activation which was confirmed in vivo in mice. This novel hepatic organoid culture model is the first that can detect hepatocyte-dependent and compound-induced HSC activation, thereby representing an important step forward towards in vitro compound testing for drug-induced liver fibrosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)