SARS-CoV-2 receptor ACE2 is upregulated by fatty acids in human MASH.

Background & Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) results in steatosis, inflammation (steatohepatitis), and fibrosis. Patients with MASLD more likely develop liver injury in coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As viral RNA has been identified in liver tissues, we studied expression levels and cellular sources of the viral receptor angiotensin-converting enzyme 2 (ACE2) and coreceptors in MASLD and fibroinflammatory liver diseases. Methods: We built a transcriptomic MASLD meta-dataset (N = 243) to study SARS-CoV-2 receptor expression and verified results in 161 additional cases of fibroinflammatory liver diseases. We assessed the fibroinflammatory microenvironment by deconvoluting immune cell populations. We studied the cellular sources of ACE2 by multiplex immunohistochemistry followed by high-resolution confocal microscopy (N = 9 fatty livers; N = 7 controls), meta-analysis of two single-cell RNA sequencing datasets (N = 5 cirrhotic livers; N = 14 normal livers), and bulk transcriptomics from 745 primary cell samples. In vitro, we tested ACE2 mRNA expression in primary human hepatocytes treated with inflammatory cytokines, bacterial lipopolysaccharides, or long-chain fatty acids. Results: We detected ACE2 at the apical and basal poles of hepatocyte chords, in CLEC4M+ liver sinusoidal endothelial cells, the lumen of ABCC2+ bile canaliculi, HepPar-1+-TMPRSS2+ hepatocytes, cholangiocytes, and CD34+ capillary vessels. ACE2 steeply increased between 30 and 50 years of age; was related to liver fat area, inflammation, high immune reactivity, and fibrogenesis; and was upregulated in steatohepatitis. Although ACE2 mRNA was unmodified in alcoholic or viral hepatitis, it was upregulated in fibroinflammatory livers from overweight patients. In vitro, treatment of primary human hepatocytes with inflammatory cytokines alone downregulated but long chain fatty acids upregulated ACE2 mRNA expression. Conclusions: Lipid overload in fatty liver disease leads to an increased availability of ACE2 receptors. Impact and implications: COVID-19 can be a deadly disease in vulnerable individuals. Patients with fatty liver disease are at a higher risk of experiencing severe COVID-19 and liver injury. Recent studies have indicated that one of the reasons for this vulnerability is the presence of a key cell surface protein called ACE2, which serves as the main SARS-CoV-2 virus receptor. We describe the cellular sources of ACE2 in the liver. In patients with fatty liver disease, ACE2 levels increase with age, liver fat content, fibroinflammatory changes, enhanced positive immune checkpoint levels, and innate immune reactivity. Moreover, we show that long chain fatty acids can induce ACE2 expression in primary human hepatocytes. Understanding the cellular sources of ACE2 in the liver and the factors that influence its availability is crucial. This knowledge will guide further research and help protect potentially vulnerable patients through timely vaccination boosters, dietary adjustments, and improved hygiene practices.

Low concentrations of ethylene bisdithiocarbamate pesticides maneb and mancozeb impair manganese and zinc homeostasis to induce oxidative stress and caspase-dependent apoptosis in human hepatocytes.

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

Mitochondrial remodeling and energy metabolism adaptations in colonic crypts during spontaneous epithelial repair after colitis induction in mice.

Mucosal healing has emerged as a therapeutic goal to achieve lasting clinical remission in ulcerative colitis. Intestinal repair in response to inflammation presumably requires higher energy supplies for the restoration of intestinal barrier and physiological functions. However, epithelial energy metabolism during intestinal mucosal healing has been little studied, whereas inflammation-induced alterations have been reported in the main energy production site, the mitochondria. The aim of the present work was to assess the involvement of mitochondrial activity and the events influencing their function during spontaneous epithelial repair after colitis induction in mouse colonic crypts. The results obtained show adaptations of colonocyte metabolism during colitis to ensure maximal ATP production for supporting energetic demand by both oxidative phosphorylation and glycolysis in a context of decreased mitochondrial biogenesis and through mitochondrial function restoration during colon epithelial repair. In parallel, colitis-induced mitochondrial ROS production in colonic epithelial cells was rapidly associated with transient expression of GSH-related enzymes. Mitochondrial respiration in colonic crypts was markedly increased during both inflammatory and recovery phases despite decreased expression of several mitochondrial respiratory chain complex subunits after colitis induction. Rapid induction of mitochondrial fusion was associated with mitochondrial function restoration. Finally, in contrast with the kinetics expression of genes involved in mitochondrial oxidative metabolism and in glycolysis, the expression of glutaminase was markedly reduced in the colonic crypts both during colitis and repair phases. Overall, our data suggest that the epithelial repair after colitis induction is characterized by a rapid and transient increased capacity for mitochondrial ATP production in a context of apparent restoration of mitochondrial biogenesis and metabolic reorientation of energy production. The potential implication of energy production adaptations within colonic crypts to sustain mucosal healing in a context of altered fuel supply is discussed.

Liposome-Mediated Gene Transfer in Differentiated HepaRG™ Cells: Expression of Liver Specific Functions and Application to the Cytochrome P450 2D6 Expression.

The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 and progenitor HepaRG™ human hepatoma cells. The lipophosphoramidate Syn1-based nanovector, which allowed the highest transfection efficiencies of progenitor HepaRG™ cells, was next used to transfect differentiated HepaRG™ cells. Lipofection of these cells using Syn1-based liposome was poorly efficient most likely because the differentiated HepaRG™ cells are highly quiescent. Thus, we engineered the differentiated HepaRG™ Mitogenic medium supplement (ADD1001) that triggered robust proliferation of differentiated cells. Importantly, we characterized the phenotypical changes occurring during proliferation of differentiated HepaRG™ cells and demonstrated that mitogenic stimulation induced a partial and transient decrease in the expression levels of some liver specific functions followed by a fast recovery of the full differentiation status upon removal of the mitogens. Taking advantage of the proliferation of HepaRG™ cells, we defined lipofection conditions using Syn1-based liposomes allowing transient expression of the cytochrome P450 2D6, a phase I enzyme poorly expressed in HepaRG cells, which opens new means for drug metabolism studies in HepaRG™ cells.

Transcriptional and Epigenetic Consequences of DMSO Treatment on HepaRG Cells.

Dimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPARα whereas genes not affected by this addition are regulated by HNF1α, HNF4α, and PPARα. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation.

The Role of Catecholamines in Pathophysiological Liver Processes.

Over the last few years, the number of research publications about the role of catecholamines (epinephrine, norepinephrine, and dopamine) in the development of liver diseases such as liver fibrosis, fatty liver diseases, or liver cancers is constantly increasing. However, the mechanisms involved in these effects are not well understood. In this review, we first recapitulate the way the liver is in contact with catecholamines and consider liver implications in their metabolism. A focus on the expression of the adrenergic and dopaminergic receptors by the liver cells is also discussed. Involvement of catecholamines in physiological (glucose metabolism, lipids metabolism, and liver regeneration) and pathophysiological (impact on drug-metabolizing enzymes expression, liver dysfunction during sepsis, fibrosis development, or liver fatty diseases and liver cancers) processes are then discussed. This review highlights the importance of understanding the mechanisms through which catecholamines influence liver functions in order to draw benefit from the adrenergic and dopaminergic antagonists currently marketed. Indeed, as these molecules are well-known drugs, their use as therapies or adjuvant treatments in several liver diseases could be facilitated.

Xenobiotic-Induced Aggravation of Metabolic-Associated Fatty Liver Disease.

Metabolic-associated fatty liver disease (MAFLD), which is often linked to obesity, encompasses a large spectrum of hepatic lesions, including simple fatty liver, steatohepatitis, cirrhosis and hepatocellular carcinoma. Besides nutritional and genetic factors, different xenobiotics such as pharmaceuticals and environmental toxicants are suspected to aggravate MAFLD in obese individuals. More specifically, pre-existing fatty liver or steatohepatitis may worsen, or fatty liver may progress faster to steatohepatitis in treated patients, or exposed individuals. The mechanisms whereby xenobiotics can aggravate MAFLD are still poorly understood and are currently under deep investigations. Nevertheless, previous studies pointed to the role of different metabolic pathways and cellular events such as activation of de novo lipogenesis and mitochondrial dysfunction, mostly associated with reactive oxygen species overproduction. This review presents the available data gathered with some prototypic compounds with a focus on corticosteroids and rosiglitazone for pharmaceuticals as well as bisphenol A and perfluorooctanoic acid for endocrine disruptors. Although not typically considered as a xenobiotic, ethanol is also discussed because its abuse has dire consequences on obese liver.

Well-differentiated liver cancers reveal the potential link between ACE2 dysfunction and metabolic breakdown.

Angiotensin-converting enzyme 2 (ACE2) is the receptor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus disease 2019 (COVID-19). Transmembrane serine protease 2 (TMPRSS2) is a coreceptor. Abnormal hepatic function in COVID-19 suggests specific or bystander liver disease. Because liver cancer cells express the ACE2 viral receptor, they are widely used as models of SARS-CoV-2 infection in vitro. Therefore, the purpose of this study was to analyze ACE2 and TMPRSS2 expression and localization in human liver cancers and in non-tumor livers. We studied ACE2 and TMPRSS2 in transcriptomic datasets totaling 1503 liver cancers, followed by high-resolution confocal multiplex immunohistochemistry and quantitative image analysis of a 41-HCC tissue microarray. In cancers, we detected ACE2 and TMPRSS2 at the biliary pole of tumor hepatocytes. In whole mount sections of five normal liver samples, we identified ACE2 in hepatocyte's bile canaliculi, biliary epithelium, sinusoidal and capillary endothelial cells. Tumors carrying mutated ß-catenin showed ACE2 DNA hypomethylation and higher mRNA and protein expression, consistently with predicted ß-catenin response sites in the ACE2 promoter. Finally, ACE2 and TMPRSS2 co-expression networks highlighted hepatocyte-specific functions, oxidative stress and inflammation, suggesting a link between inflammation, ACE2 dysfunction and metabolic breakdown.

Loss of hepatocyte identity following aberrant YAP activation: A key mechanism in alcoholic hepatitis.

BACKGROUND & AIMS: Alcoholic hepatitis (AH) is a life-threatening disease with limited therapeutic options, as the molecular mechanisms leading to death are not well understood. This study evaluates the Hippo/Yes-associated protein (YAP) pathway which has been shown to play a role in liver regeneration. METHOD: The Hippo/YAP pathway was dissected in explants of patients transplanted for AH or alcohol-related cirrhosis and in control livers, using RNA-seq, real-time PCR, western blot, immunohistochemistry and transcriptome analysis after laser microdissection. We transfected primary human hepatocytes with constitutively active YAP (YAPS127A) and treated HepaRG cells and primary hepatocytes isolated from AH livers with a YAP inhibitor. We also used mouse models of ethanol exposure (Lieber de Carli) and liver regeneration (carbon tetrachloride) after hepatocyte transduction of YAPS127A. RESULTS: In AH samples, RNA-seq analysis and immunohistochemistry of total liver and microdissected hepatocytes revealed marked downregulation of the Hippo pathway, demonstrated by lower levels of active MST1 kinase and abnormal activation of YAP in hepatocytes. Overactivation of YAP in hepatocytes in vitro and in vivo led to biliary differentiation and loss of key biological functions such as regeneration capacity. Conversely, a YAP inhibitor restored the mature hepatocyte phenotype in abnormal hepatocytes taken from patients with AH. In ethanol-fed mice, YAP activation using YAPS127A resulted in a loss of hepatocyte differentiation. Hepatocyte proliferation was hampered by YAPS127A after carbon tetrachloride intoxication. CONCLUSION: Aberrant activation of YAP plays an important role in hepatocyte transdifferentiation in AH, through a loss of hepatocyte identity and impaired regeneration. Thus, targeting YAP is a promising strategy for the treatment of patients with AH. LAY SUMMARY: Alcoholic hepatitis is characterized by inflammation and a life-threatening alteration of liver regeneration, although the mechanisms behind this have not been identified. Herein, we show that liver samples from patients with alcoholic hepatitis are characterized by profound deregulation of the Hippo/YAP pathway with uncontrolled activation of YAP in hepatocytes. We used human cell and mouse models to show that inhibition of YAP reverts this hepatocyte defect and could be a novel therapeutic strategy for alcoholic hepatitis.

Cas9-targeted nanopore sequencing reveals epigenetic heterogeneity after de novo assembly of native full-length hepatitis B virus genomes.

Hepatitis B virus (HBV) contains a 3.2 kb DNA genome and causes acute and chronic hepatitis. HBV infection is a global health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). Methylation of HBV DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues as to why certain infections are cleared or persist with or without progression to cancer. The detection of 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. With Cas9-guided RNPs to specifically target the HBV genome, we enriched in HBV DNA from primary human hepatocytes (PHHs) infected with different HBV genotypes, as well as enriching in HBV from infected patient liver tissue, followed by sequencing with Oxford Nanopore Technologies MinION. Detection of 5mCpG by nanopore sequencing was benchmarked with bisulfite-quantitative methyl-specific qPCR (BS-qMSP). The 5mCpG levels in HBV determined by BS-qMSP and nanopore sequencing were highly correlated. Our nanopore sequencing approach achieved a coverage of ~2000× of HBV depending on infection efficiency, sufficient coverage to perform a de novo assembly and detect small fluctuations in HBV methylation, providing the first de novo assembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences. Moreover, by capturing entire HBV genomes, we explored the epigenetic heterogeneity of HBV in infected patients and identified four epigenetically distinct clusters based on methylation profiles. This method is a novel approach that enables the enrichment of viral DNA in a mixture of nucleic acid material from different species and will serve as a valuable tool for infectious disease monitoring.

Interplay between Metabolism Reprogramming and Epithelial-to-Mesenchymal Transition in Cancer Stem Cells.

Tumor cells display important plasticity potential, which contributes to intratumoral heterogeneity. Notably, tumor cells have the ability to retrodifferentiate toward immature states under the influence of their microenvironment. Importantly, this phenotypical conversion is paralleled by a metabolic rewiring, and according to the metabostemness theory, metabolic reprogramming represents the first step of epithelial-to-mesenchymal transition (EMT) and acquisition of stemness features. Most cancer stem cells (CSC) adopt a glycolytic phenotype even though cells retain functional mitochondria. Such adaptation is suggested to reduce the production of reactive oxygen species (ROS), protecting CSC from detrimental effects of ROS. CSC may also rely on glutaminolysis or fatty acid metabolism to sustain their energy needs. Besides pro-inflammatory cytokines that are well-known to initiate the retrodifferentiation process, the release of catecholamines in the microenvironment of the tumor can modulate both EMT and metabolic changes in cancer cells through the activation of EMT transcription factors (ZEB1, Snail, or Slug (SNAI2)). Importantly, the acquisition of stem cell properties favors the resistance to standard care chemotherapies. Hence, a better understanding of this process could pave the way for the development of therapies targeting CSC metabolism, providing new strategies to eradicate the whole tumor mass in cancers with unmet needs.

Adding the oxygen carrier M101 to a cold-storage solution could be an alternative to HOPE for liver graft preservation.

BACKGROUND & AIMS: Hypothermic oxygenated machine perfusion (HOPE) is a promising technique for providing oxygen to the liver during graft preservation; however, because of associated logistical constraints, addition of an oxygen transporter to static cold-storage solutions (SCS) might be easier. M101 is marine worm haemoglobin that has been shown to improve kidney preservation in the clinic when added to SCS. This study evaluated the effects of the addition of M101 to SCS on the quality of pig liver graft preservation. METHODS: Pig liver grafts were preserved using SCS, HOPE, or SCS+M101, and the liver functions were compared during cold preservation and after orthotopic allotransplantation (OLT) in pigs. RESULTS: During preservation of the liver grafts, mitochondrial function, ATP synthesis, antioxidant capacities, and hepatocyte architecture were better preserved, and free radical production, antioxidant activities, and inflammatory mediators were lower, with HOPE or SCS+M101 than with SCS alone. However, after 1 h of preservation, liver functions with HOPE were superior to those with SCS+M101. After 6 h of preservation and OLT, blood levels of aspartate and alanine aminotransferases and lactate dehydrogenase increased with a peak effect at Day 1 post-transplant; values were similar with HOPE and SCS+M101, and were significantly lower than those in the SCS group. At Days 1 and 3, tumor necrosis factor α levels remained lower with HOPE and SCS+M101 vs. SCS. At Day 7, liver cell necrosis and inflammation were less marked in both oxygenated groups. CONCLUSIONS: When added to SCS, M101 effectively oxygenates liver grafts during preservation, preventing post-transplant injury; although graft performances are below those achieved with HOPE. LAY SUMMARY: When transported between donors and recipients, even cold-stored liver grafts need oxygen to maintain their viability. To provide them with oxygen, we added a marine worm super haemoglobin (M101) to the cold-storage solution UWCS. Using a pig liver transplant model, we revealed that livers cold stored with UWCS+M101 showed improved oxygenation compared with simple cold-storage solutions, but did not reach the oxygenation level achieved with machine perfusion.

Urinary TIMP-2 and MMP-2 are significantly associated with poor bladder compliance in adult patients with spina bifida.

AIMS: To assess the predictive values of six urinary markers (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], matrix metalloproteinase 2 [MMP-2], tissue inhibitor metalloproteinase 2 [TIMP-2], transformation growth factor ß-1 [TGF-B1], and prostaglandin 2 [PGE2]) for adverse urodynamic features and for upper urinary tract damage in adult patients with spina bifida. MATERIALS AND METHODS: A single-center prospective trial was conducted from March 2015 to March 2017 including all consecutive adult patients with spina bifida seen for urodynamic testing. The urine was collected and stored at -80°C. A urodynamic and an upper urinary tract were systematically performed. At the end of the inclusion period, urines were defrosted and urinary nerve growth factor, BDNF, TIMP-2, and TGF-B1 were assessed using validated ELISA kits. The urinary markers levels were adjusted on the urinary creatinine level. Urinary MMP-2 levels were assessed by zymography. RESULTS: Fourty patients were included. Only TIMP-2 and MMP-2 were significantly associated with poor bladder compliance (P = .043 and P = .039, respectively). TIMP-2 was also the only urinary marker significantly associated with upper urinary tract damage on imaging (OR = 19.81; P = .02). Of all urodynamic parameters, bladder compliance and maximum detrusor pressure were the only ones associated with upper urinary tract damage on imaging (P = .01 and P = .02), The diagnostic performances of urinary TIMP-2 for upper urinary tract damage were slightly superior to PdetMax and bladder compliance with an area under the curve of 0.72. CONCLUSION: Urinary TIMP-2 and MMP-2 were significantly associated with poor bladder compliance and urinary TIMP-2 was significantly associated with upper urinary tract damage. These findings support a pathophysiological role of extracellular matrix remodeling in poor bladder compliance of adult patients with spina bifida.

Cytochrome P450 1A1/2, 2B6 and 3A4 HepaRG Cell-Based Biosensors to Monitor Hepatocyte Differentiation, Drug Metabolism and Toxicity.

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.

Retrodifferentiation of Human Tumor Hepatocytes to Stem Cells Leads to Metabolic Reprogramming and Chemoresistance.

Human hepatocellular carcinoma (HCC) heterogeneity promotes recurrence and therapeutic resistance. We recently demonstrated that inflammation favors hepatocyte retrodifferentiation into progenitor cells. Here, we identify the molecular effectors that induce metabolic reprogramming, chemoresistance, and invasiveness of retrodifferentiated HCC stem cells. Spheroid cultures of human HepaRG progenitors (HepaRG-Spheres), HBG-BC2, HepG2, and HuH7 cells and isolation of side population (SP) from HepaRG cells (HepaRG-SP) were analyzed by transcriptomics, signaling pathway analysis, and evaluation of chemotherapies. Gene expression profiling of HepaRG-SP and HepaRG-Spheres revealed enriched signatures related to cancer stem cells, metastasis, and recurrence and showed that HepaRG progenitors could retrodifferentiate into an immature state. The transcriptome from these stem cells matched that of proliferative bad outcome HCCs in a cohort of 457 patients. These HCC stem cells expressed high levels of cytokines triggering retrodifferentiation and displayed high migration and invasion potential. They also showed changes in mitochondrial activity with reduced membrane potential, low ATP production, and high lactate production. These changes were, in part, related to angiopoietin-like 4 (ANGPTL4)-induced upregulation of pyruvate dehydrogenase kinase 4 (PDK4), an inhibitor of mitochondrial pyruvate dehydrogenase. Upregulation of ANGPTL4 and PDK4 paralleled that of stem cells markers in human HCC specimens. Moreover, the PDK4 inhibitor dichloroacetate reversed chemoresistance to sorafenib or cisplatin in HCC stem cells derived from four HCC cell lines. In conclusion, retrodifferentiated cancer cells develop enhanced invasion and therapeutic resistance through ANGPTL4 and PDK4. Therefore, restoration of mitochondrial activity in combination with chemotherapy represents an attractive therapeutic approach in HCC. SIGNIFICANCE: Restoring mitochondrial function in human hepatocellular carcinomas overcomes cancer resistance.

Large-Scale Modeling Approach Reveals Functional Metabolic Shifts during Hepatic Differentiation.

Being able to explore the metabolism of broad metabolizing cells is of critical importance in many research fields. This article presents an original modeling solution combining metabolic network and omics data to identify modulated metabolic pathways and changes in metabolic functions occurring during differentiation of a human hepatic cell line (HepaRG). Our results confirm the activation of hepato-specific functionalities and newly evidence modulation of other metabolic pathways, which could not be evidenced from transcriptomic data alone. Our method takes advantage of the network structure to detect changes in metabolic pathways that do not have gene annotations and exploits flux analyses techniques to identify activated metabolic functions. Compared to the usual cell-specific metabolic network reconstruction approaches, it limits false predictions by considering several possible network configurations to represent one phenotype rather than one arbitrarily selected network. Our approach significantly enhances the comprehensive and functional assessment of cell metabolism, opening further perspectives to investigate metabolic shifts occurring within various biological contexts.

Endotoxin regulates matrix genes increasing reactive oxygen species generation by intercellular communication between palmitate-treated hepatocyte and stellate cell.

Previous studies have shown that gut-derived bacterial endotoxins contribute in the progression of simple steatosis to steatohepatitis, although the mechanism(s) remains inaccurate to date. As hepatic stellate cells (HSC) play a pivotal role in the accumulation of excessive extracellular matrix (ECM), leading to collagen deposition, fibrosis, and perpetuation of inflammatory response, an in vitro model was developed to investigate the crosstalk between HSC and hepatocytes (human hepatoma cell) pretreated with palmitate. Bacterial lipopolysaccharide (LPS) stimulated HSC with phosphorylation of the p38 mitogen-activated protein kinase/NF-κB pathway, while several important pro-inflammatory cytokines were upregulated in the presence of hepatocyte-HSC. Concurrently, fibrosis-related genes were regulated by palmitate and the inflammatory effect of endotoxin where cells were more exposed or sensitive to reactive oxygen species (ROS). This interaction was accompanied by increased expression of the mitochondrial master regulator, proliferator-activated receptor gamma coactivator alpha, and a cytoprotective effect of the agent N-acetylcysteine suppressing ROS production, transforming growth factor-ß1, and tissue inhibitor of metalloproteinase-1. In summary, our results demonstrate that pro-inflammatory mediators LPS-induced promote ECM rearrangement in hepatic cells transcriptionally committed to the regulation of genes encoding enzymes for fatty acid metabolism in light of differences that might require an alternative therapeutic approach targeting ROS regulation.

Thymoquinone protects rat liver after partial hepatectomy under ischaemia/reperfusion through oxidative stress and endoplasmic reticulum stress prevention.

Ischaemia reperfusion (I/R) is associated with liver injury and impaired regeneration during partial hepatectomy (PH). The aim of this study was to investigate the effect of thymoquinone (TQ), the active compound of essential oil obtained from Nigella sativa seeds, on rat liver after PH. Male Wistar rats were divided equally into four groups (n = 6) receiving an oral administration of either vehicle solution (sham and PH groups) or TQ at 30 mg/kg (TQ and TQ + PH groups) for 10 consecutive days. Then, rats underwent PH (70%) with 60 minutes of ischaemia followed by 24 hours of reperfusion (PH and TQ + PH groups). Alanine aminotransferase (ALT) activity and histopathological damage were determined. Also, antioxidant parameters, liver regeneration index, hepatic adenosine triphosphate (ATP) content, endoplasmic reticulum (ER) stress and apoptosis were assessed. In response to PH under I/R, liver damage was significantly alleviated by TQ treatment as evidenced by the decrease in ALT activity (P < .01) and histological findings (P < .001). In parallel, TQ preconditioning increased hepatic antioxidant capacities. Moreover, TQ improved mitochondrial function (ATP, P < .05), attenuated ER stress parameters and repressed the expression of apoptotic effectors. Taken together, our results suggest that TQ preconditioning could be an effective strategy to reduce liver injury after PH under I/R. The protective effects were mediated by the increase of antioxidant capacities and the decrease of ER stress and apoptosis.

A practical multi-step synthesis of ethyl N-functionalized [Formula: see text]-amino benzimidazole acrylate derivatives as promising cytotoxic agents.

A series of 16 new ethyl [Formula: see text]-amino benzimidazole acrylate derivatives 12(a-p) with a (2E)-s-cis/trans conformation and bearing two points of diversity was designed and synthesized by using a multi-step strategy (reductive amination, deprotection in acidic media and transamination) in moderate to good yields from ethyl 3-dimethylamino-2-(1H-benzimidazol-2-yl)acrylate (5) and monosubstituted N-Boc diamines (7a,7b) as starting building blocks. Products 12 were evaluated for their in vitro cytotoxic potential against six selected human cell lines (Huh7-D12, Caco2, MDA-MB231, HCT116, PC3 and NCI-H727). Compounds 12a, 12e and 12l exhibited selective and micromolar antitumor activities against Huh7-D12 and Caco2 cell lines.