Detoxification of xenobiotics including drugs is catalyzed by liver phase I and phase II enzymes. There are three main families of phase I cytochrome P450 (CYP450) monoxygenases that introduce polar groups on drugs. These phase I metabolites can then be further conjugated by transferases during phase II reaction. Liver biotransformation can also lead to toxic drug metabolites, the most common cause of drug failure during clinical investigation. CYP3A4 is considered to be the most important enzyme in drug metabolism. Drug development relies on the use of human liver cells in order to investigate drug metabolism and potential toxicity. With primary human liver cells as the gold standard several problems have to be solved, i.e. scarcity of functional human liver tissue, donor variation of CYP activity and rapid dedifferentiation processes during primary cell cultivation. These features make it difficult to use primary human liver cells as standard to measure CYP activity. To avoid problems with primary human liver cells, many attempts have been undertaken to establish liver carcinoma cell lines, non-transformed proliferating human liver cell systems and induced pluripotent stem cell-derived hepatocytes. Due to different problems with these surrogate systems, the one cell line that could be used as convenient standard cell system to benchmark CYP3A4 enzyme activity has not been established yet. Based on the widely used hepatocellular carcinoma cell line HepG2 and a lentiviral vector system, we generated cell clones for stable CYP3A4 overexpression. Here we present data on a new HepG2 cell clone (clone 9) showing higher than 10,000-fold overexpression of CYP3A4 compared to HepG2 parental cells. As measured by conversion of testosterone into 6␤-hydroxytestosterone, we found an enzyme activity of about 600 pmol per minute per mg total cellular protein, which ranges at the upper end reported for primary human liver cells. This enzyme activity appeared to be kept stable in clone 9 cells, because there was no influence detectable when cells were treated with 5-azacytidine, a drug that interferes with epigenetic silencing processes. Prototypic CYP3A4 inducer rifampicin led to significant increase of CYP3A4 testosterone hydroxylase activity in HepG2 clone 9 cells. Altogether, HepG2 clone 9 strongly and stably overexpressed CYP3A4 leading to a physiological enzyme activity, which apparently was unaffected by epigenetic processes. Thus, HepG2 clone 9 could be a useful reference cell clone for CYP3A4 enzyme activity.

Primary human hepatocytes are in great demand during drug development and in hepatology. However, both scarcity of tissue supply and donor variability of primary cells create a need for the development of alternative hepatocyte systems. By using a lentivirus vector system to transfer coding sequences of Upcyte® proliferation genes, we generated non-transformed stable hepatocyte cultures from human liver tissue samples. Here, we show data on newly generated proliferation-competent HepaFH3 cells investigated as conventional two-dimensional monolayer and as organotypical three-dimensional (3D) spheroid culture. In monolayer culture, HepaFH3 cells show typical cobblestone-like hepatocyte morphology and anchorage-dependent growth for at least 20 passages. Immunofluorescence staining revealed that characteristic hepatocyte marker proteins cytokeratin 8, human serum albumin, and cytochrome P450 (CYP) 3A4 were expressed. Quantitative real-time PCR analyses showed that expression levels of analyzed phase I CYP enzymes were at similar levels compared to those of cultured primary human hepatocytes and considerably higher than in the liver carcinoma cell line HepG2. Additionally, transcripts for phase II liver enzymes and transporter proteins OATP-C, MRP2, Oct1, and BSEP were present in HepaFH3. The cells produced urea and converted model compounds such as testosterone, diclofenac, and 7-OH-coumarin into phases I and II metabolites. Interestingly, phases I and II enzymes were expressed at about the same levels in convenient monolayer cultures and complex 3D spheroids. In conclusion, HepaFH3 cells and related primary-like hepatocyte lines seem to be promising tools for in vitro research of liver functions and as test system in drug development and toxicology analysis.

Atrial fibrillation (AF) is regularly accompanied by cardiac fibrosis and concomitant heart failure. Due to the heterogeneous nature and complexity of fibrosis, the knowledge about the underlying mechanisms is limited, which prevents effective pharmacotherapy. A deeper understanding of cardiac fibroblasts is essential to meet this need. We previously described phenotypic and functional differences between atrial fibroblasts from patients in sinus rhythm and with AF. Herein, we established and characterized a novel human atrial fibroblast line, which displays typical fibroblast morphology and function comparable to primary cells but with improved proliferation capacity and low spontaneous myofibroblast differentiation. These traits make our model suitable for the study of fibrosis mechanisms and for drug screening aimed at developing effective antifibrotic pharmacotherapy. Cardiovascular diseases (CVD) such as heart failure and arrhythmia represent the leading cause of death worldwide [1,2]. A well-recognized concomitant of CVD is cardiac fibrosis [1], which is the structural manifestation of an imbalance in extracellular matrix (ECM) homeostasis. With nearly 45% of all deaths in the Western world attributable to fibroproliferative disease, the clinical relevance of fibrotic remodeling is enormous [3,4]. This is particularly true in the case of atrial fibrosis, associated with a detrimental clinical outcome of highly abundant supraventricular arrhythmias like atrial fibrillation (AF) [5]. However, due to

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Rationale: Fibrosis promotes the maintenance of atrial fibrillation (AF), making it resistant to therapy. Improved understanding of the molecular mechanisms leading to atrial fibrosis will open new pathways towards effective antifibrotic therapies. Objective: This study aims to decipher the mechanistic interplay between polo-like kinase 2 (PLK2) and the pro-fibrotic cytokine osteopontin (OPN) in the pathogenesis of atrial fibrosis and atrial fibrillation. Methods and Results: Atrial PLK2 mRNA expression was 10-fold higher in human fibroblasts than in cardiomyocytes. Compared to sinus rhythm (SR), right atrial appendages and isolated right atrial fibroblasts from AF patients showed downregulation of PLK2 mRNA and protein, along with increased PLK2 promotor methylation. Genetic deletion as well as pharmacological inhibition of PLK2 induced pro-fibrotic phenotype conversion in cardiac fibroblasts and led to a striking de novo secretion of OPN. Accordingly, PLK2-deficient (PLK2 KO) mice showed cardiac fibrosis and were prone to experimentally induced AF. In line with these findings, OPN plasma levels were significantly higher only in AF patients with atrial low-voltage zones (surrogates of fibrosis) compared to SR controls. Mechanistically, we identified ERK1/2 as the relevant downstream mediator of PLK2 leading to increased OPN expression. Finally, oral treatment with the clinically-available drug mesalazine, known to inhibit ERK1/2, prevented cardiac OPN overexpression and reversed the pathological PLK2 KO phenotype in PLK2 KO-mice. Conclusions: In summary, abnormal PLK2/ERK1/2/OPN axis function critically contributes to AF-related atrial fibrosis, suggesting reinforcing PLK2 activity and/or OPN inhibition as innovative targets to prevent fibrosis progression in AF. Mesalazine derivatives may be used as lead compounds for the development of novel anti-AF agents targeting fibrosis.

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Abstract. Chronic alcohol abuse is the leading cause of liver cirrhosis in western countries. Ethanol, even at lower concentrations, can cause pleiotropic effects at the cellular level such as the formation of reactive oxygen species (ROS) and DNA damage. Ethanol is oxidized to acetaldehyde by liver alcohol dehydrogenase and by a microsomal ethanol-oxidizing system which is dependent on CYP2E1. There are numerous reports on CYP2E1-mediated formation of ROS. These lower the levels of intracellular glutathione, an efficient antioxidant. We were interested to investigate ethanol effects on glutathione levels independent of CYP2E1. We chose human hepatoma cell line HepG2 as a model known to lack physiological CYP2E1 expression. We found that 40 mM ethanol, a dosage comparable to blood ethanol concentration after heavy alcohol drinking, reduced intracellular HepG2 glutathione levels only by 18%. Within 24 hours, this effect could be normalized by the glutathione regeneration system. When HepG2 cells were exposed to 40 mM ethanol for one week, the cells gradually lost their ability to regenerate intracellular glutathione stores. We conclude that chronic ethanol exposure has a substantial effect on the glutathione regeneration capacity in liver cells which might contribute to alcohol-induced liver disease.

Fibrosis and inflammation promote atrial fibrillation (AF) and worsen its clinical outcome. The underlying molecular mechanisms, that are relevant for effective antifibrotic drug development, are still under debate. This study deciphers a novel mechanistic interplay between polo-like kinase 2 (PLK2) and the pro-inflammatory cytokine osteopontin (OPN) in the pathogenesis of atrial fibrosis. Compared to sinus rhythm (SR) controls, right atrial appendages and isolated right atrial fibroblasts from AF patients showed downregulation of PLK2 mRNA and protein levels, which were accompanied by remarkable hypoxia-sensitive DNA-methylation of the PLK2 promotor. In an experimental setting, both, genetic deletion and pharmacological inhibition of PLK2 induced myofibroblast differentiation and reduced fibroblast proliferation.Notably, proteomics from PLK2-deleted fibroblasts revealed de novo secretion of OPN.