Control compounds for preclinical drug-induced liver injury assessment: Consensus-driven systematic review by the ProEuroDILI network.

BACKGROUND & AIMS: Idiosyncratic drug-induced liver injury (DILI) is a complex and unpredictable event caused by drugs, and herbal or dietary supplements. Early identification of human hepatotoxicity at preclinical stages remains a major challenge, in which the selection of validated in vitro systems and test drugs has a significant impact. In this systematic review, we analyzed the compounds used in hepatotoxicity assays and established a list of DILI-positive and -negative control drugs for validation of in vitro models of DILI, supported by literature and clinical evidence and endorsed by an expert committee from the COST Action ProEuroDILI Network (CA17112). METHODS: Following 2020 PRISMA guidelines, original research articles focusing on DILI which used in vitro human models and performed at least one hepatotoxicity assay with positive and negative control compounds, were included. Bias of the studies was assessed by a modified 'Toxicological Data Reliability Assessment Tool'. RESULTS: A total of 51 studies (out of 2,936) met the inclusion criteria, with 30 categorized as reliable without restrictions. Although there was a broad consensus on positive compounds, the selection of negative compounds lacked clarity. 2D monoculture, short exposure times and cytotoxicity endpoints were the most tested, although there was no consensus on drug concentrations. CONCLUSIONS: Extensive analysis highlighted the lack of agreement on control compounds for in vitro DILI assessment. Following comprehensive in vitro and clinical data analysis together with input from the expert committee, an evidence-based consensus-driven list of 10 positive and negative control drugs for validation of in vitro models of DILI is proposed. IMPACT AND IMPLICATIONS: Prediction of human toxicity early in the drug development process remains a major challenge, necessitating the development of more physiologically relevant liver models and careful selection of drug-induced liver injury (DILI)-positive and -negative control drugs to better predict the risk of DILI associated with new drug candidates. Thus, this systematic study has crucial implications for standardizing the validation of new in vitro models of DILI. By establishing a consensus-driven list of positive and negative control drugs, the study provides a scientifically justified framework for enhancing the consistency of preclinical testing, thereby addressing a significant challenge in early hepatotoxicity identification. Practically, these findings can guide researchers in evaluating safety profiles of new drugs, refining in vitro models, and informing regulatory agencies on potential improvements to regulatory guidelines, ensuring a more systematic and efficient approach to drug safety assessment.

Next-generation sequencing of bile cell-free DNA for the early detection of patients with malignant biliary strictures.

OBJECTIVE: Despite significant progresses in imaging and pathological evaluation, early differentiation between benign and malignant biliary strictures remains challenging. Endoscopic retrograde cholangiopancreatography (ERCP) is used to investigate biliary strictures, enabling the collection of bile. We tested the diagnostic potential of next-generation sequencing (NGS) mutational analysis of bile cell-free DNA (cfDNA). DESIGN: A prospective cohort of patients with suspicious biliary strictures (n=68) was studied. The performance of initial pathological diagnosis was compared with that of the mutational analysis of bile cfDNA collected at the time of first ERCP using an NGS panel open to clinical laboratory implementation, the Oncomine Pan-Cancer Cell-Free assay. RESULTS: An initial pathological diagnosis classified these strictures as of benign (n=26), indeterminate (n=9) or malignant (n=33) origin. Sensitivity and specificity of this diagnosis were 60% and 100%, respectively, as on follow-up 14 of the 26 and eight of the nine initially benign or indeterminate strictures resulted malignant. Sensitivity and specificity for malignancy of our NGS assay, herein named Bilemut, were 96.4% and 69.2%, respectively. Importantly, one of the four Bilemut false positives developed pancreatic cancer after extended follow-up. Remarkably, the sensitivity for malignancy of Bilemut was 100% in patients with an initial diagnosis of benign or indeterminate strictures. Analysis of 30 paired bile and tissue samples also demonstrated the superior performance of Bilemut. CONCLUSION: Implementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

Advanced preclinical models for evaluation of drug-induced liver injury - consensus statement by the European Drug-Induced Liver Injury Network [PRO-EURO-DILI-NET].

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF) and one of the leading indications for liver transplantation in Western societies. Given the wide use of both prescribed and over the counter drugs, DILI has become a major health issue for which there is a pressing need to find novel and effective therapies. Although significant progress has been made in understanding the molecular mechanisms underlying DILI, our incomplete knowledge of its pathogenesis and inability to predict DILI is largely due to both discordance between human and animal DILI in preclinical drug development and a lack of models that faithfully recapitulate complex pathophysiological features of human DILI. This is exemplified by the hepatotoxicity of acetaminophen (APAP) overdose, a major cause of ALF because of its extensive worldwide use as an analgesic. Despite intensive efforts utilising current animal and in vitro models, the mechanisms involved in the hepatotoxicity of APAP are still not fully understood. In this expert Consensus Statement, which is endorsed by the European Drug-Induced Liver Injury Network, we aim to facilitate and outline clinically impactful discoveries by detailing the requirements for more realistic human-based systems to assess hepatotoxicity and guide future drug safety testing. We present novel insights and discuss major players in APAP pathophysiology, and describe emerging in vitro and in vivo pre-clinical models, as well as advanced imaging and in silico technologies, which may improve prediction of clinical outcomes of DILI.

Protective role of c-Jun N-terminal kinase-2 (JNK2) in ibuprofen-induced acute liver injury.

Ibuprofen is a worldwide used non-steroidal anti-inflammatory drug which may cause acute liver injury (ALI) requiring liver transplantation. We aimed to unveil the molecular pathways involved in triggering ibuprofen-induced ALI, which, at present, remain elusive. First, we investigated activation of essential pathways in human liver sections of ibuprofen-induced ALI. Next, we assessed the cytotoxicity of ibuprofen in vitro and developed a novel murine model of ibuprofen intoxication. To assess the role of JNK, we used animals carrying constitutive deletion of c-Jun N-terminal kinase 1 (Jnk1-/- ) or Jnk2 (Jnk2-/- ) expression and included investigations using animals with hepatocyte-specific Jnk deletion either genetically (Jnk1Δhepa ) or by siRNA (siJnk2Δhepa ). We found in human and murine samples of ibuprofen-induced acute liver failure that JNK phosphorylation was increased in the cytoplasm of hepatocytes and other non-liver parenchymal cells (non-LPCs) compared with healthy tissue. In mice, ibuprofen intoxication resulted in a significantly stronger degree of liver injury compared with vehicle-treated controls as evidenced by serum transaminases, and hepatic histopathology. Next, we investigated molecular pathways. PKCα, AKT, JNK and RIPK1 were significantly increased 8 h after ibuprofen intoxication. Constitutive Jnk1-/- and Jnk2-/- deficient mice exhibited increased liver dysfunction compared to wild-type (WT) animals. Furthermore, siJnk2Δhepa animals showed a dramatic increase in biochemical markers of liver function, which correlated with significantly higher serum liver enzymes and worsened liver histology, and MAPK activation compared to Jnk1Δhepa or WT animals. In our study, cytoplasmic JNK activation in hepatocytes and other non-LPCs is a hallmark of human and murine ibuprofen-induced ALI. Functional in vivo analysis demonstrated a protective role of hepatocyte-specific Jnk2 during ibuprofen ALI. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Reclassifying Hepatic Cell Death during Liver Damage: Ferroptosis-A Novel Form of Non-Apoptotic Cell Death?

Ferroptosis has emerged as a new type of cell death in different pathological conditions, including neurological and kidney diseases and, especially, in different types of cancer. The hallmark of this regulated cell death is the presence of iron-driven lipid peroxidation; the activation of key genes related to this process such as glutathione peroxidase-4 (gpx4), acyl-CoA synthetase long-chain family member-4 (acsl4), carbonyl reductase [NADPH] 3 (cbr3), and prostaglandin peroxidase synthase-2 (ptgs2); and morphological changes including shrunken and electron-dense mitochondria. Iron overload in the liver has long been recognized as both a major trigger of liver damage in different diseases, and it is also associated with liver fibrosis. New evidence suggests that ferroptosis might be a novel type of non-apoptotic cell death in several liver diseases including non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), drug-induced liver injury (DILI), viral hepatitis, and hemochromatosis. The interaction between iron-related lipid peroxidation, cellular stress signals, and antioxidant systems plays a pivotal role in the development of this novel type of cell death. In addition, integrated responses from lipidic mediators together with free iron from iron-containing enzymes are essential to understanding this process. The presence of ferroptosis and the exact mechanisms leading to this non-apoptotic type of cell death in the liver remain scarcely elucidated. Recognizing ferroptosis as a novel type of cell death in the liver could lead to the understanding of the complex interaction between different types of cell death, their role in progression of liver fibrosis, the development of new biomarkers, as well as the use of modulators of ferroptosis, allowing improved theranostic approaches in the clinic.

Proteomic profiling of cellular steatosis with concomitant oxidative stress in vitro.

BACKGROUND: Nutrient excess underpins the development of nonalcoholic fatty liver disease (NAFLD). The ensuing metabolic derangement is characterised by increased cellular respiration, oxidative stress and mitochondrial impairment. We have previously recapitulated these events in an in vitro cellular steatosis model. Here, we examined the distinct patterns of protein expression involved using a proteomics approach. METHODS: Human hepatoblastoma C3A cells were treated with a combination of energy substrates; lactate (L), pyruvate (P), octanoate (O) and ammonia (N). Proteins extracts were trypsinized and analyzed on a capillary HPLC OrbitrapXL mass spectrometer. Proteins were quantified using a label-free intensity based approach. Functional enrichment analysis was performed using ToppCluster via Gene Ontology (GO) database. RESULTS: Of the 1327 proteins identified, 104 were differentially expressed between LPON and untreated cells (defined as: ≥2 peptides; fold change ≥1.5; p-value <0.05). Seventy of these were upregulated with LPON. Functional enrichment analysis revealed enhanced protein biosynthesis accompanied by downregulation of histones H2A type 1-A, H1.2, H1.5 and H1.0I in LPON cells. Lipid binding annotations were also enriched as well as proteins involved in cholesterol synthesis, uptake and efflux. Increased expression of aldo-keto reductase family 1, member C1 and C3 suggests enhanced sterol metabolism and increased ROS-mediated lipid peroxidation. CONCLUSIONS: The surge of energy substrates diverts free fatty acid metabolism towards pathways that can mitigate lipotoxicity. The histones depletion may represent an adaptation to increased protein synthesis. However, this can also expose DNA to oxidative stress thus should be explored further in the context of NAFLD progression.

Fibrinogen production is enhanced in an in-vitro model of non-alcoholic fatty liver disease: an isolated risk factor for cardiovascular events?

BACKGROUND: Cardiovascular disease (CVD) remains the major cause of excess mortality in patients with non-alcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the individual contribution of NAFLD to CVD risk factors in the absence of pathogenic influences from other comorbidities often found in NAFLD patients, by using an established in-vitro model of hepatic steatosis. METHODS: Histopathological events in non-alcoholic fatty liver disease were recapitulated by focused metabolic nutrient overload of hepatoblastoma C3A cells, using oleate-treated-cells and untreated controls for comparison. Microarray and proteomic data from cell culture experiments were integrated into a custom-built systems biology database and proteogenomics analysis performed. Candidate genes with significant dysregulation and concomitant changes in protein abundance were identified and STRING association and enrichment analysis performed to identify putative pathogenic pathways. RESULTS: The search strategy yielded 3 candidate genes that were specifically and significantly up-regulated in nutrient-overloaded cells compared to untreated controls: fibrinogen alpha chain (2.2 fold), fibrinogen beta chain (2.3 fold) and fibrinogen gamma chain (2.1 fold) (all rank products pfp <0.05). Fibrinogen alpha and gamma chain also demonstrated significant concomitant increases in protein abundance (3.8-fold and 2.0-fold, respectively, p <0.05). CONCLUSIONS: In-vitro modelling of NAFLD and reactive oxygen species formation in nutrient overloaded C3A cells, in the absence of pathogenic influences from other comorbidities, suggests that NAFLD is an isolated determinant of CVD. Nutrient overload-induced up-regulation of all three fibrinogen component subunits of the coagulation cascade provides a possible mechanism to explain the excess CVD mortality observed in NAFLD patients.

Non-invasive methods of monitoring Fe3O4 magnetic nanoparticle toxicity in human liver HepaRG cells using impedance biosensing and Coherent anti-Stokes Raman spectroscopic (CARS) microscopy.

Functionalized nanoparticles have been developed for use in nanomedicines for treating life threatening diseases including various cancers. To ensure safe use of these new nanoscale reagents, various assays for biocompatibility or cytotoxicity in vitro using cell lines often serve as preliminary assessments prior to in vivo animal testing. However, many of these assays were designed for soluble, colourless materials and may not be suitable for coloured, non-transparent nanoparticles. Moreover, cell lines are not always representative of mammalian organs in vivo. In this work, we use non-invasive impedance sensing methods with organotypic human liver HepaRG cells as a model to test the toxicity of PEG-Fe3O4 magnetic nanoparticles. We also use Coherent anti-Stokes Raman Spectroscopic (CARS) microscopy to monitor the formation of lipid droplets as a parameter to the adverse effect on the HepaRG cell model. The results were also compared with two commercial testing kits (PrestoBlue and ATP) for cytotoxicity. The results suggested that the HepaRG cell model can be a more realistic model than commercial cell lines while use of impedance monitoring of Fe3O4 nanoparticles circumventing the uncertainties due to colour assays. These methods can play important roles for scientists driving towards the 3Rs principle - Replacement, Reduction and Refinement.

Lack of Cyclin E1 in hepatocytes aggravates ethanol-induced liver injury and hepatic steatosis in experimental murine model of acute and chronic alcohol-associated liver disease.

BACKGROUND: Cyclin E1 is the regulatory subunit of cyclin-dependent kinase 2 (Cdk2) and one of the central players in cell cycle progression. We recently showed its crucial role for initiation of liver fibrosis and hepatocarcinogenesis. In the present study, we investigated the role of Cyclin E1 in the development of alcohol-associated liver disease (ALD). METHODS: Mice with constitutive (E1-/-), hepatocyte-specific (Cyclin E1Δhepa), or intestinal-epithelial-cell-specific (Cyclin E1ΔIEC) inactivation of Cyclin E1 and corresponding wild type littermate controls (WT) were administered either a Lieber-DeCarli ethanol diet (LDE) for 3 weeks or acute ethanol binges (6 g/kg) through oral gavage. Serum parameters of liver functionality were measured; hepatic tissues were collected for biochemical and histological analyses. RESULTS: The administration of acute EtOH binge and chronic LDE diet to E1-/- mice enhanced hepatic steatosis, worsened liver damage and triggered body weight loss. Similarly, in the acute EtOH binge model, Cyclin E1Δhepa mice revealed a significantly worsened liver phenotype. In contrast, inactivation of Cyclin E1 only in intestinal epithelial cell (IECs)did not lead to any significant changes in comparison to WT mice after acute EtOH challenge. Remarkably, both acute and chronic EtOH administration in E1-/- animals resulted in increased levels of ADH and decreased expression of ALDH1/2. The additional application of a pan-Cdk inhibitor (S-CR8) further promoted liver damage in EtOH-treated WT mice. CONCLUSION: Our data point to a novel unexpected role of Cyclin E1 in hepatocytes for alcohol metabolism, which seems to be independent of the canonical Cyclin E1/Cdk2 function as a cell cycle regulator.

Genetic and pharmacological inhibition of XBP1 protects against APAP hepatotoxicity through the activation of autophagy.

Acetaminophen (APAP) hepatotoxicity induces endoplasmic reticulum (ER) stress which triggers the unfolded protein response (UPR) in hepatocytes. However, the mechanisms underlying ER stress remain poorly understood, thus reducing the options for exploring new pharmacological therapies for patients with hyperacute liver injury. Eight-to-twelve-week-old C57BL/6J Xbp1-floxed (Xbp1f/f) and hepatocyte-specific knockout Xbp1 mice (Xbp1∆hepa) were challenged with either high dose APAP [500 mg/kg] and sacrificed at early (1-2 h) and late (24 h) stages of hepatotoxicity. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Pharmacological inhibition of XBP1 using pre-treatment with STF-083010 [STF, 75 mg/kg] and autophagy induction with Rapamycin [RAPA, 8 mg/kg] or blockade with Chloroquine [CQ, 60 mg/kg] was also undertaken in vivo. Cytoplasmic expression of XBP1 coincided with severity of human and murine hyperacute liver injury. Transcriptional and translational activation of the UPR and sustained activation of JNK1/2 were major events in APAP hepatotoxicity, both in a human hepatocytic cell line and in a preclinical model. Xbp1∆hepa livers showed decreased UPR and JNK1/2 activation but enhanced autophagy in response to high dose APAP. Additionally, blockade of XBP1 splicing by STF, mitigated APAP-induced liver injury and without non-specific off-target effects (e.g., CYP2E1 activity). Furthermore, enhanced autophagy might be responsible for modulating CYP2E1 activity in Xbp1∆hepa animals. Genetic and pharmacological inhibition of Xbp1 specifically in hepatocytes ameliorated APAP-induced liver injury by enhancing autophagy and decreasing CYP2E1 expression. These findings provide the basis for the therapeutic restoration of ER stress and/or induction of autophagy in patients with hyperacute liver injury.

New molecular mechanisms in cholangiocarcinoma: signals triggering interleukin-6 production in tumor cells and KRAS co-opted epigenetic mediators driving metabolic reprogramming.

BACKGROUND: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. METHODS: Cholangiocarcinogenesis was induced in rats (TAA) and mice (JnkΔhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. RESULTS: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. CONCLUSIONS: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.

Medical liability and health care reform.

We examine the impact of the Affordable Care Act (ACA) on medical liability and the controversy over whether federal medical reform including a damages cap could make a useful contribution to health care reform. By providing guaranteed access to health care insurance at community rates, the ACA could reduce the problem of under-compensation resulting from damages caps. However, it may also exacerbate the problem of under-claiming in the malpractice system, thereby reducing incentives to invest in loss prevention activities. Shifting losses from liability insurers to health insurers could further undermine the already weak deterrent effect of the medical liability system. Republicans in Congress and physician groups both pushed for the adoption of a federal damages cap as part of health care reform. Physician support for damages caps could be explained by concerns about the insurance cycle and the consequent instability of the market. Our own study presented here suggests that there is greater insurance market stability in states with caps on non-economic damages. Republicans in Congress argued that the enactment of damages caps would reduce aggregate health care costs. The Congressional Budget Office included savings from reduced health care utilization in its estimates of cost savings that would result from the enactment of a federal damages cap. But notwithstanding recent opinions offered by the CBO, it is not clear that caps will significantly reduce health care costs or that any savings will be passed on to consumers. The ACA included funding for state level demonstration projects for promising reforms such as offer and disclosure and health courts, but at this time the benefits of these reforms are also uncertain. There is a need for further studies on these issues.

Fibrotic Events in the Progression of Cholestatic Liver Disease.

Cholestatic liver diseases including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are associated with active hepatic fibrogenesis, which can ultimately lead to the development of cirrhosis. However, the exact relationship between the development of liver fibrosis and the progression of cholestatic liver disease remains elusive. Periductular fibroblasts located around the bile ducts seem biologically different from hepatic stellate cells (HSCs). The fibrotic events in these clinical conditions appear to be related to complex crosstalk between immune/inflammatory mechanisms, cytokine signalling, and perturbed homeostasis between cholangiocytes and mesenchymal cells. Several animal models including bile duct ligation (BDL) and the Mdr2-knockout mice have improved our understanding of mechanisms underlying chronic cholestasis. In the present review, we aim to elucidate the mechanisms of fibrosis in order to help to identify potential diagnostic and therapeutic targets.

Activation of the Unfolded Protein Response (UPR) Is Associated with Cholangiocellular Injury, Fibrosis and Carcinogenesis in an Experimental Model of Fibropolycystic Liver Disease.

Fibropolycystic liver disease is characterized by hyperproliferation of the biliary epithelium and the formation of multiple dilated cysts, a process associated with unfolded protein response (UPR). In the present study, we aimed to understand the mechanisms of cyst formation and UPR activation in hepatocytic c-Jun N-terminal kinase 1/2 (Jnk1/2) knockout mice. Floxed JNK1/2 (Jnkf/f) and Jnk∆hepa animals were sacrificed at different time points during progression of liver disease. Histological examination of specimens evidenced the presence of collagen fiber deposition, increased α-smooth muscle actin (αSMA), infiltration of CD45, CD11b and F4/80 cells and proinflammatory cytokines (Tnf, Tgfß1) and liver injury (e.g., ALT, apoptosis and Ki67-positive cells) in Jnk∆hepa compared with Jnkf/f livers from 32 weeks of age. This was associated with activation of effectors of the UPR, including BiP/GRP78, CHOP and spliced XBP1. Tunicamycin (TM) challenge strongly induced ER stress and fibrosis in Jnk∆hepa animals compared with Jnkf/f littermates. Finally, thioacetamide (TAA) administration to Jnk∆hepa mice induced UPR activation, peribiliary fibrosis, liver injury and markers of biliary proliferation and cholangiocarcinoma (CCA). Orthoallografts of DEN/CCl4-treated Jnk∆hepa liver tissue triggered malignant CCA. Altogether, these results suggest that activation of the UPR in conjunction with fibrogenesis might trigger hepatic cystogenesis and early stages of CCA.

Low-shear modelled microgravity environment maintains morphology and differentiated functionality of primary porcine hepatocyte cultures.

Hepatocytes cultured in conventional static culture rapidly lose polarity and differentiated function. This could be explained by gravity-induced sedimentation, which prevents formation of complete three-dimensional (3D) cell-cell/cell-matrix interactions and disrupts integrin-mediated signals (including the most abundant hepatic integrin alpha(5)beta(1)), important for cellular polarity and differentiation. Cell culture in a low fluid shear modelled microgravity (about 10(-2) g) environment promotes spatial colocation/self-aggregation of dissociated cells and induction of 3D differentiated liver morphology. Previously, we demonstrated the utility of a NASA rotary bioreactor in maintaining key metabolic functions and 3D aggregate formation of high-density primary porcine hepatocyte cultures over 21 days. Using serum-free chemically defined medium, without confounding interactions of exogenous bioscaffolding or bioenhancing surface materials, we investigated features of hepatic cellular polarity and differentiated functionality, including expression of hepatic integrin alpha(5), as markers of functional morphology. We report here that in the absence of exogenous biomatrix scaffolding, hepatocytes cultured in serum-free chemically defined medium in a microgravity environment rapidly (<24 h) form macroscopic (2-5 mm), compacted 3D hepatospheroid structures consisting of a shell of glycogen-positive viable cells circumscribing a core of eosinophilic cells. The spheroid shell layers exhibited ultrastructural, morphological and functional features of differentiated, polarized hepatic tissue including strong expression of the integrin alpha(5) subunit, functional bile canaliculi, albumin synthesis, and fine ultrastructure reminiscent of in vivo hepatic tissue. The low fluid shear microgravity environment may promote tissue-like self-organization of dissociated cells, and offer advantages over spheroids cultured in conventional formats to delineate optimal conditions for enhanced directed tissue self-assembly.

Development of an invasively monitored porcine model of acetaminophen-induced acute liver failure.

BACKGROUND: The development of effective therapies for acute liver failure (ALF) is limited by our knowledge of the pathophysiology of this condition, and the lack of suitable large animal models of acetaminophen toxicity. Our aim was to develop a reproducible invasively-monitored porcine model of acetaminophen-induced ALF. METHOD: 35kg pigs were maintained under general anaesthesia and invasively monitored. Control pigs received a saline infusion, whereas ALF pigs received acetaminophen intravenously for 12 hours to maintain blood concentrations between 200-300 mg/l. Animals surviving 28 hours were euthanased. RESULTS: Cytochrome p450 levels in phenobarbital pre-treated animals were significantly higher than non pre-treated animals (300 vs 100 pmol/mg protein). Control pigs (n = 4) survived 28-hour anaesthesia without incident. Of nine pigs that received acetaminophen, four survived 20 hours and two survived 28 hours. Injured animals developed hypotension (mean arterial pressure; 40.8 +/- 5.9 vs 59 +/- 2.0 mmHg), increased cardiac output (7.26 +/- 1.86 vs 3.30 +/- 0.40 l/min) and decreased systemic vascular resistance (8.48 +/- 2.75 vs 16.2 +/- 1.76 mPa/s/m3). Dyspnoea developed as liver injury progressed and the increased pulmonary vascular resistance (636 +/- 95 vs 301 +/- 26.9 mPa/s/m3) observed may reflect the development of respiratory distress syndrome.Liver damage was confirmed by deterioration in pH (7.23 +/- 0.05 vs 7.45 +/- 0.02) and prothrombin time (36 +/- 2 vs 8.9 +/- 0.3 seconds) compared with controls. Factor V and VII levels were reduced to 9.3 and 15.5% of starting values in injured animals. A marked increase in serum AST (471.5 +/- 210 vs 42 +/- 8.14) coincided with a marked reduction in serum albumin (11.5 +/- 1.71 vs 25 +/- 1 g/dL) in injured animals. Animals displayed evidence of renal impairment; mean creatinine levels 280.2 +/- 36.5 vs 131.6 +/- 9.33 mumol/l. Liver histology revealed evidence of severe centrilobular necrosis with coagulative necrosis. Marked renal tubular necrosis was also seen. Methaemoglobin levels did not rise >5%. Intracranial hypertension was not seen (ICP monitoring), but there was biochemical evidence of encephalopathy by the reduction of Fischer's ratio from 5.6 +/- 1.1 to 0.45 +/- 0.06. CONCLUSION: We have developed a reproducible large animal model of acetaminophen-induced liver failure, which allows in-depth investigation of the pathophysiological basis of this condition. Furthermore, this represents an important large animal model for testing artificial liver support systems.

Oxygen Plasma Substrate and Specific Nanopattern Promote Early Differentiation of HepaRG Progenitors.

Fully differentiated HepaRG™ cells are the hepatic cell line of choice for in vitro study in toxicology and drug trials. They are derived from a hepatoblast-like progenitor (HepaRG-P) that differentiates into a coculture of hepatocyte-like and cholangiocyte-like cells. This process that requires 2 weeks of proliferation followed by 2 weeks of differentiation using dimethyl sulfoxide (DMSO) can be time consuming and costly. Identifying a method to accelerate HepaRG-Ps toward a mature lineage would save both time and money. The ability to do this in the absence of DMSO would remove the possibility of confounding toxicology results caused by DMSO induction of CYP pathways. It has been shown that tissue culture substrates play an important role in the development and maturity of a cell line, and this is particularly important for progenitor cells, which retain some form of plasticity. Oxygen plasma treatment is used extensively to modify cell culture substrates. There is also evidence that patterned rather than planar surfaces have a positive effect on proliferation and differentiation. In this study, we compared the effect of standard tissue culture plastic (TCP), oxygen plasma coated (OPC), and nanopatterned substrates (NPS) on early differentiation and function of HepaRG-P cells. Since NPS were OPC we initially compared the effect of TCP and OPC to enable comparison between all three culture surfaces using OPC as control to asses if patterning further enhanced early differentiation and functionality. The results show that HepaRG-P's grown on OPC substrate exhibited earlier differentiation, proliferation, and function compared with TCP. Culturing HepaRG-P's on OPC with the addition of NPS did not confer any additional advantage. In conclusion, OPC surface appeared to enhance hepatic differentiation and functionality and could replace traditional methods of differentiating HepaRG-P cells into fully differentiated and functional HepaRGs earlier than standard methods. Impact statement We show significantly earlier differentiation and function of HepaRG progenitor cells when grown in dimethyl sulfoxide-free medium on oxygen plasma substrates versus standard tissue culture plastic. Further investigation showed that nanopatterning of oxygen plasma substrates did not confer any additional advantage over smooth oxygen plasma, although one pattern (DSQ120) showed comparable early differentiation and function.

Mitogen-Activated Protein Kinases (MAPKs) and Cholangiocarcinoma: The Missing Link.

In recent years, the incidence of both liver and biliary tract cancer has increased. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common types of hepatic malignancies. Whereas HCC is the fifth most common malignant tumor in Western countries, the prevalence of CCA has taken an alarming increase from 0.3 to 2.1 cases per 100,000 people. The lack of specific biomarkers makes diagnosis very difficult in the early stages of this fatal cancer. Thus, the prognosis of CCA is dismal and surgery is the only effective treatment, whilst recurrence after resection is common. Even though chemotherapy and radiotherapy may prolong survival in patients with CCA, the 5-year survival rate is still very low-a significant global problem in clinical diagnosis and therapy. The mitogen-activated protein kinase (MAPK) pathway plays an important role in signal transduction by converting extracellular stimuli into a wide range of cellular responses including inflammatory response, stress response, differentiation, survival, and tumorigenesis. Dysregulation of the MAPK cascade involves key signaling components and phosphorylation events that play an important role in tumorigenesis. In this review, we discuss the pathophysiological role of MAPK, current therapeutic options, and the current situation of MAPK-targeted therapies in CCA.