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1.  TNFα is required for cholestasis-induced liver fibrosis in the mouse 
TNFα, a mediator of hepatotoxicity in several animal models, is elevated in acute and chronic liver diseases. Therefore, we investigated whether hepatic injury and fibrosis due to bile duct ligation (BDL) would be reduced in TNFα knockout mice (TNFα−/−). Survival after BDL was 60% in wild-type mice (TNFα+/+) and 90% in TNFα−/− mice. Body weight loss and liver to body weight ratios were reduced in TNFα−/− mice compared to TNFα+/+ mice. Following BDL, serum alanine transaminases (ALT) levels were elevated in TNFα+/+ mice (268.6 ± 28.2 U/L) compared to TNFα−/− mice (105.9 U/L ± 24.4). TNFα −/− mice revealed lower hepatic collagen expression and less liver fibrosis in the histology. Further, α-smooth muscle actin, an indicator for activated myofibroblasts, and TGF-β mRNA, a profibrogenic cytokine, were markedly reduced in TNFα−/− mice compared to TNFα+/+ mice. Thus, our data indicate that TNFα induces hepatotoxicity and promotes fibrogenesis in the BDL model.
doi:10.1016/j.bbrc.2008.10.155
PMCID: PMC5052129  PMID: 18996089
TNFα; Tumor necrosis factor α; Knockout mice; Bile duct ligation; BDL; Cholestasis; Liver fibrosis; TGF-β
2.  Identification of cytochrome CYP2E1 as critical mediator of synergistic effects of alcohol and cellular lipid accumulation in hepatocytes in vitro 
Oncotarget  2015;6(39):41464-41478.
Clinical studies propose a causative link between the consumption of alcohol and the development and progression of liver disease in obese individuals. However, it is incompletely understood how alcohol and obesity interact and whether the combined effects are additive or synergistic. In this study, we developed an in vitro model to address this question. Lipid accumulation in primary human hepatocytes was induced by incubation with oleic acid. Subsequently, steatotic and control hepatocytes were incubated with up to 50 mM alcohol. This alcohol concentration on its own revealed only minimal effects but significantly enhanced oleate-induced lipogenesis and cellular triglyceride content compared to control cells. Similarly, lipid peroxidation, oxidative stress and pro-inflammatory gene expression as well as CYP2E1 levels and activity were synergistically induced by alcohol and steatosis. CYP2E1 inhibition blunted these synergistic pathological effects. Notably, alcohol and cellular steatosis also induced autophagy in a synergistic manner, and also this was mediated via CYP2E1. Further induction of autophagy ameliorated the joint effects of alcohol and oleic acid on hepatocellular lipid accumulation and inflammatory gene expression while inhibition of autophagy further enhanced the dual pathological effects. Further analyses revealed that the joint synergistic effect of alcohol and steatosis on autophagy was mediated via activation of the JNK-pathway. In summary, our data indicate that alcohol induces not only pathological but also protective mechanisms in steatotic hepatocytes via CYP2E1. These findings may have important implications on the prognosis and treatment of alcoholic liver disease particularly in obese individuals.
PMCID: PMC4747167  PMID: 26497211
alcohol; steatosis; autophagy; CYP2E1; Pathology Section
3.  Glucose transporter isoform 1 expression enhances metastasis of malignant melanoma cells 
Oncotarget  2015;6(32):32748-32760.
The glucose transporter isoform 1 (GLUT1; SLC2A1) is a key rate-limiting factor in the transport of glucose into cancer cells. Enhanced GLUT1 expression and accelerated glycolysis have been found to promote aggressive growth in a range of tumor entities. However, it was unknown whether GLUT1 directly impacts metastasis. Here, we aimed at analyzing the expression and function of GLUT1 in malignant melanoma. Immunohistochemical analysis of 78 primary human melanomas on a tissue micro array showed that GLUT1 expression significantly correlated with the mitotic activity and a poor survival. To determine the functional role of GLUT1 in melanoma, we stably suppressed GLUT1 in the murine melanoma cell line B16 with shRNA. GLUT1 suppressed melanoma cells revealed significantly reduced proliferation, apoptosis resistance, migratory activity and matrix metalloproteinase 2 (MMP2) expression. In a syngeneic murine model of hepatic metastasis, GLUT1-suppressed cells formed significantly less metastases and showed increased apoptosis compared to metastases formed by control cells. Treatment of four different human melanoma cell lines with a pharmacological GLUT1 inhibitor caused a dose-dependent reduction of proliferation, apoptosis resistance, migratory activity and MMP2 expression. Analysis of MAPK signal pathways showed that GLUT1 inhibition significantly decreased JNK activation, which regulates a wide range of targets in the metastatic cascade. In summary, our study provides functional evidence that enhanced GLUT1 expression in melanoma cells favors their metastatic behavior. These findings specify GLUT1 as an attractive therapeutic target and prognostic marker for this highly aggressive tumor.
PMCID: PMC4741727  PMID: 26293674
GLUT1; melanoma; metastasis; glycolysis; JNK
4.  Effect of chronic alcohol consumption on the development and progression of non-alcoholic fatty liver disease (NAFLD) 
A number of epidemiologic studies show a protective effect of light to moderate daily alcohol consumption on the development of non-alcoholic fatty liver disease (NAFLD). Although these small amounts of ethanol may prevent fatty liver, they may also be a risk factor for other diseases such as breast and colon cancer. Those individuals who have underlying hepatic steatosis or non-alcoholic steatohepatitis (NASH) should not use ethanol chronically since the data available at present do not support a beneficial effect of alcohol in this situation. Especially overweight and obese individuals may be more susceptible towards alcohol even at moderate doses. Animal experiments show a negative effect of ethanol on liver histology in either dietary or genetic NASH models. In addition, patients with NASH reveal a significant increased risk for hepatocellular cancer (HCC) even with social alcohol consumption. Thus, subjects with underlying NASH should abstain from alcohol at any amounts.
doi:10.3978/j.issn.2304-3881.2014.12.01
PMCID: PMC4465604  PMID: 26151054
Alcohol; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH)
5.  Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts 
Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3’UTR binding site for miR-188-5p. COL1A1 and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA.
PMCID: PMC4525876  PMID: 26261542
Rheumatoid arthritis; synovial fibroblasts; miR-188-5p; KIAA1199
6.  Causal Modeling of Cancer-Stromal Communication Identifies PAPPA as a Novel Stroma-Secreted Factor Activating NFκB Signaling in Hepatocellular Carcinoma 
PLoS Computational Biology  2015;11(5):e1004293.
Inter-cellular communication with stromal cells is vital for cancer cells. Molecules involved in the communication are potential drug targets. To identify them systematically, we applied a systems level analysis that combined reverse network engineering with causal effect estimation. Using only observational transcriptome profiles we searched for paracrine factors sending messages from activated hepatic stellate cells (HSC) to hepatocellular carcinoma (HCC) cells. We condensed these messages to predict ten proteins that, acting in concert, cause the majority of the gene expression changes observed in HCC cells. Among the 10 paracrine factors were both known and unknown cancer promoting stromal factors, the former including Placental Growth Factor (PGF) and Periostin (POSTN), while Pregnancy-Associated Plasma Protein A (PAPPA) was among the latter. Further support for the predicted effect of PAPPA on HCC cells came from both in vitro studies that showed PAPPA to contribute to the activation of NFκB signaling, and clinical data, which linked higher expression levels of PAPPA to advanced stage HCC. In summary, this study demonstrates the potential of causal modeling in combination with a condensation step borrowed from gene set analysis [Model-based Gene Set Analysis (MGSA)] in the identification of stromal signaling molecules influencing the cancer phenotype.
Author Summary
All living cells rely on communication with other cells to ensure their function and survival. Molecular signals are sent among cells of the same cell type and from cells of one cell type to another. In cancer, not only the cancer cells themselves are responsible for the malignancy, but also stromal (non-cancerous) cells and the molecular signals they send to cancer cells are important factors that determine the severity and outcome of the disease. Therefore, the identification of stromal signals and their influence on cancer cells is important for the development of novel treatment strategies. With a computational systems biology model of stroma-cancer cell communication, we have compiled a set of ten proteins secreted by stromal cells that shape the cancer phenotype. Most importantly, our causal analysis uncovered Pregnancy-Associated Plasma Protein A (PAPPA) as a novel paracrine inducer of the pro-tumorigenic NFκB signaling pathway. In liver cancer patients, higher levels of PAPPA protein indicate a more progressed tumor stage, confirming its clinical relevance.
doi:10.1371/journal.pcbi.1004293
PMCID: PMC4447342  PMID: 26020769
7.  The hop constituent xanthohumol exhibits hepatoprotective effects and inhibits the activation of hepatic stellate cells at different levels 
Xanthohumol is the principal prenylated flavonoid of the female inflorescences of the hop plant. In recent years, various beneficial xanthohumol effects including anti-inflammatory, antioxidant, hypoglycemic activities, and anticancer effects have been revealed. This review summarizes present studies indicating that xanthohumol also inhibits several critical pathophysiological steps during the development and course of chronic liver disease, including the activation and pro-fibrogenic genotype of hepatic stellate cells. Also the various mechanism of action and molecular targets of the beneficial xanthohumol effects will be described. Furthermore, the potential use of xanthohumol or a xanthohumol-enriched hop extract as therapeutic agent to combat the progression of chronic liver disease will be discussed. It is notable that in addition to its hepatoprotective effects, xanthohumol also holds promise as a therapeutic agent for treating obesity, dysregulation of glucose metabolism and other components of the metabolic syndrome including hepatic steatosis. Thus, therapeutic xanthohumol application appears as a promising strategy, particularly in obese patients, to inhibit the development as well as the progression of non-alcoholic fatty liver disease.
doi:10.3389/fphys.2015.00140
PMCID: PMC4422013  PMID: 25999863
xanthohumol; hops; fibrosis; hepatic stellate cells; liver disease
8.  Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts 
Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3’UTR binding site for miR-188-5p. COL1A1and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA.
PMCID: PMC4503060  PMID: 26191188
Rheumatoid arthritis; synovial fibroblasts; miR-188-5p; KIAA1199
9.  Formation of Fenestrae in Murine Liver Sinusoids Depends on Plasmalemma Vesicle-Associated Protein and Is Required for Lipoprotein Passage 
PLoS ONE  2014;9(12):e115005.
Liver sinusoidal endothelial cells (LSEC) are characterized by the presence of fenestrations that are not bridged by a diaphragm. The molecular mechanisms that control the formation of the fenestrations are largely unclear. Here we report that mice, which are deficient in plasmalemma vesicle-associated protein (PLVAP), develop a distinct phenotype that is caused by the lack of sinusoidal fenestrations. Fenestrations with a diaphragm were not observed in mouse LSEC at three weeks of age, but were present during embryonic life starting from embryonic day 12.5. PLVAP was expressed in LSEC of wild-type mice, but not in that of Plvap-deficient littermates. Plvap-/- LSEC showed a pronounced and highly significant reduction in the number of fenestrations, a finding, which was seen both by transmission and scanning electron microscopy. The lack of fenestrations was associated with an impaired passage of macromolecules such as FITC-dextran and quantum dot nanoparticles from the sinusoidal lumen into Disse's space. Plvap-deficient mice suffered from a pronounced hyperlipoproteinemia as evidenced by milky plasma and the presence of lipid granules that occluded kidney and liver capillaries. By NMR spectroscopy of plasma, the nature of hyperlipoproteinemia was identified as massive accumulation of chylomicron remnants. Plasma levels of low density lipoproteins (LDL) were also significantly increased as were those of cholesterol and triglycerides. In contrast, plasma levels of high density lipoproteins (HDL), albumin and total protein were reduced. At around three weeks of life, Plvap-deficient livers developed extensive multivesicular steatosis, steatohepatitis, and fibrosis. PLVAP is critically required for the formation of fenestrations in LSEC. Lack of fenestrations caused by PLVAP deficiency substantially impairs the passage of chylomicron remnants between liver sinusoids and hepatocytes, and finally leads to liver damage.
doi:10.1371/journal.pone.0115005
PMCID: PMC4277272  PMID: 25541982
10.  Downregulation of P-cadherin expression in hepatocellular carcinoma induces tumorigenicity 
P-cadherin is a major contributor to cell-cell adhesion in epithelial tissues, playing pivotal roles in important morphogenetic and differentiation processes and in maintaining tissue integrity and homeostasis. Alterations of P-cadherin expression have been observed during the progression of several carcinomas where it appears to act as tumor suppressive or oncogenic in a context-dependent manner. Here, we found a significant downregulation of P-cadherin in hepatocellular carcinoma (HCC) cell lines and tissues compared to primary human hepatocytes and non-malignant liver tissues. Combined immunohistochemical analysis of a tissue microarray containing matched pairs of HCC tissue and corresponding non-tumorous liver tissue of 69 patients confirmed reduced P-cadherin expression in more than half of the cases. In 35 human HCC tissues, the P-cadherin immunosignal was completely lost which correlated with tumor staging and proliferation. Also in vitro, P-cadherin suppression in HCC cells via siRNA induced proliferation compared to cells transfected with control-siRNA. In summary, downregulation of P-cadherin expression appears to induce tumorigenicity in HCC. Therefore, P-cadherin expression may serve as a prognostic marker and therapeutic target of this highly aggressive tumor.
PMCID: PMC4203231  PMID: 25337260
P-cadherin; hepatocellular carcinoma; tumor staging; proliferation
11.  Deficiency of intestinal mucin-2 ameliorates experimental alcoholic liver disease in mice 
Hepatology (Baltimore, Md.)  2013;58(1):108-119.
The intestinal mucus layer protects the epithelium from noxious agents, viruses, and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin-2 (Muc2), secreted by goblet cells of the intestine. Experimental alcoholic liver disease requires translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. We investigated the roles of the intestinal mucus layer, and in particular Muc2, in development of experimental alcohol-associated liver disease in mice. We studied experimental alcohol-induced liver disease, induced by the Tsukamoto-French method (which involves continuous intragastric feeding of an isocaloric diet or alcohol) in wild-type and Muc2−/− mice. Muc2−/− mice showed less alcohol-induced liver injury and steatosis that developed in wild-type mice. Most notably, Muc2−/− mice had significantly lower plasma levels of lipopolysaccharide than wild-type mice after alcohol feeding. In contrast to wild-type mice, Muc2−/− mice were protected from alcohol-associated microbiome changes that are dependent on intestinal mucins. The anti-microbial proteins Reg3b and Reg3g were expressed at significantly higher levels in the jejunum of Muc2−/− mice fed the isocaloric diet or alcohol, compared with wild-type mice. Consequently, Muc2−/− mice showed increased killing of commensal bacteria and prevented intestinal bacterial overgrowth. Conclusion: Muc2−/− mice are protected from intestinal bacterial overgrowth and dysbiosis in response to alcohol feeding. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation, decreasing liver disease.
doi:10.1002/hep.26321
PMCID: PMC3695050  PMID: 23408358
microbiome; intestinal bacterial overgrowth; bacterial translocation; endotoxin; Reg3
12.  Expression and Function of Methylthioadenosine Phosphorylase in Chronic Liver Disease 
PLoS ONE  2013;8(12):e80703.
To study expression and function of methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, in chronic liver disease.
Design
MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chromatography-tandem mass spectrometry.
Results
MTAP was downregulated in hepatocytes in murine fibrosis models and in patients with chronic liver disease, leading to a concomitant increase in MTA levels. In contrast, activated hepatic stellate cells (HSCs) showed strong MTAP expression in cirrhotic livers. However, also MTA levels in activated HSCs were significantly higher than in hepatocytes, and there was a significant correlation between MTA levels and collagen expression in diseased human liver tissue indicating that activated HSCs significantly contribute to elevated MTA in diseased livers. MTAP suppression by siRNA resulted in increased MTA levels, NFκB activation and apoptosis resistance, while overexpression of MTAP caused the opposite effects in HSCs. The anti-apoptotic effect of low MTAP expression and high MTA levels, respectively, was mediated by induced expression of survivin, while inhibition of survivin abolished the anti-apoptotic effect of MTA on HSCs. Treatment with a DNA demethylating agent induced MTAP and reduced survivin expression, while oxidative stress reduced MTAP levels but enhanced survivin expression in HSCs.
Conclusion
MTAP mediated regulation of MTA links polyamine metabolism with NFκB activation and apoptosis in HSCs. MTAP and MTAP modulating mechanisms appear as promising prognostic markers and therapeutic targets for hepatic fibrosis.
doi:10.1371/journal.pone.0080703
PMCID: PMC3855635  PMID: 24324622
13.  Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME 
Godoy, Patricio | Hewitt, Nicola J. | Albrecht, Ute | Andersen, Melvin E. | Ansari, Nariman | Bhattacharya, Sudin | Bode, Johannes Georg | Bolleyn, Jennifer | Borner, Christoph | Böttger, Jan | Braeuning, Albert | Budinsky, Robert A. | Burkhardt, Britta | Cameron, Neil R. | Camussi, Giovanni | Cho, Chong-Su | Choi, Yun-Jaie | Craig Rowlands, J. | Dahmen, Uta | Damm, Georg | Dirsch, Olaf | Donato, María Teresa | Dong, Jian | Dooley, Steven | Drasdo, Dirk | Eakins, Rowena | Ferreira, Karine Sá | Fonsato, Valentina | Fraczek, Joanna | Gebhardt, Rolf | Gibson, Andrew | Glanemann, Matthias | Goldring, Chris E. P. | Gómez-Lechón, María José | Groothuis, Geny M. M. | Gustavsson, Lena | Guyot, Christelle | Hallifax, David | Hammad, Seddik | Hayward, Adam | Häussinger, Dieter | Hellerbrand, Claus | Hewitt, Philip | Hoehme, Stefan | Holzhütter, Hermann-Georg | Houston, J. Brian | Hrach, Jens | Ito, Kiyomi | Jaeschke, Hartmut | Keitel, Verena | Kelm, Jens M. | Kevin Park, B. | Kordes, Claus | Kullak-Ublick, Gerd A. | LeCluyse, Edward L. | Lu, Peng | Luebke-Wheeler, Jennifer | Lutz, Anna | Maltman, Daniel J. | Matz-Soja, Madlen | McMullen, Patrick | Merfort, Irmgard | Messner, Simon | Meyer, Christoph | Mwinyi, Jessica | Naisbitt, Dean J. | Nussler, Andreas K. | Olinga, Peter | Pampaloni, Francesco | Pi, Jingbo | Pluta, Linda | Przyborski, Stefan A. | Ramachandran, Anup | Rogiers, Vera | Rowe, Cliff | Schelcher, Celine | Schmich, Kathrin | Schwarz, Michael | Singh, Bijay | Stelzer, Ernst H. K. | Stieger, Bruno | Stöber, Regina | Sugiyama, Yuichi | Tetta, Ciro | Thasler, Wolfgang E. | Vanhaecke, Tamara | Vinken, Mathieu | Weiss, Thomas S. | Widera, Agata | Woods, Courtney G. | Xu, Jinghai James | Yarborough, Kathy M. | Hengstler, Jan G.
Archives of Toxicology  2013;87(8):1315-1530.
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
Electronic supplementary material
The online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s00204-013-1078-5
PMCID: PMC3753504  PMID: 23974980
Non-parenchymal cells; Mechanisms of gene regulation; DILI; 3D Models; Cryopreservation; Clearance; Mathematical modeling
14.  Effect of chronic psychosocial stress on nonalcoholic steatohepatitis in mice 
Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which may progress towards inflammation (nonalcoholic steatohepatitis (NASH)). NAFLD is regarded as a consequence of a sedentary, food-abundant lifestyle which, in the modern world, often coincides with chronically high levels of perceived psychosocial stress. Here, we aimed to characterize the effect of chronic psychosocial stress on the development of NAFLD/NASH in male mice either fed with standard chow or NASH-inducing high fat diet. Chronic psychosocial stress was induced by chronic subordinate colony housing (CSC), a pre-clinically validated paradigm relevant for human affective and somatic disorders. Single housed (SHC) mice served as controls. Under standard chow conditions CSC mice revealed lower hepatic triglyceride levels but higher hepatic TNFα, MCP-1 and HMOX mRNA expression, while serum transaminase levels did not significantly differ from SHC mice. Under the NASH-inducing high-fat diet CSC and SHC mice showed similar body weight-gain and serum levels of glucose and adiponectin. Moreover, liver histology as well as TNFα, MCP-1 and HMOX expression were similar in CSC and SHC mice fed with HFD. Surprisingly, CSC showed even significantly lower transaminase levels than SHC mice fed with the same NASH-inducing diet. Together, these data indicate that under normal dietary conditions the CSC model induces noticeable hepatic oxidative stress and inflammation without causing manifest hepatocellular injury. In contrast, CSC exhibited a protective effect on hepatocellular injury in a dietary NASH-model. Identification of the underlying mechanisms of this phenomenon may lead to novel therapeutic strategies to prevent progression of NAFLD.
PMCID: PMC3726974  PMID: 23923077
Chronic psychosocial stress; nonalcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH)
15.  Adiponectin upregulates hepatocyte CMKLR1 which is reduced in human fatty liver 
Chemokine-like receptor 1 (CMKLR1) ligands chemerin and resolvin E1 are suggested to have a role in non-alcoholic fatty liver disease (NAFLD). Here, expression of CMKLR1 in liver cells and NAFLD was studied. CMKLR1 was detected in primary human hepatocytes (PHH), Kupffer cells, bile-duct cells and hepatic stellate cells. In human and rodent fatty liver and in fibrotic liver of mice fed a methionine–choline deficient diet CMKLR1 was reduced. Hepatocytes are the major cells in the liver and effects of adipokines, cytokines and lipids on CMKLR1 in PHH were analyzed. Increased cellular triglyceride or cholesterol content, lipopolysaccharide, IL-6, TNF and leptin did not influence CMKLR1 levels in PHH whereas profibrotic TGFβ tended to reduce CMKLR1. Adiponectin strongly upregulated CMKLR1 mRNA and protein in PHH and hepatic CMKLR1 when injected into wild type mice. Further, CMKLR1 was suppressed in the liver of adiponectin deficient mice. These data indicate that low CMKLR1 in NAFLD may partly result from reduced adiponectin activity.
doi:10.1016/j.mce.2011.10.032
PMCID: PMC3670424  PMID: 22118966
Adipokine; Hepatic steatosis; Chemerin receptor; Liver
16.  Targeting Melanoma Metastasis and Immunosuppression with a New Mode of Melanoma Inhibitory Activity (MIA) Protein Inhibition 
PLoS ONE  2012;7(5):e37941.
Melanoma is the most aggressive form of skin cancer, with fast progression and early dissemination mediated by the melanoma inhibitory activity (MIA) protein. Here, we discovered that dimerization of MIA is required for functional activity through mutagenesis of MIA which showed the correlation between dimerization and functional activity. We subsequently identified the dodecapeptide AR71, which prevents MIA dimerization and thereby acts as a MIA inhibitor. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy demonstrated the binding of AR71 to the MIA dimerization domain, in agreement with in vitro and in vivo data revealing reduced cell migration, reduced formation of metastases and increased immune response after AR71 treatment. We believe AR71 is a lead structure for MIA inhibitors. More generally, inhibiting MIA dimerization is a novel therapeutic concept in melanoma therapy.
doi:10.1371/journal.pone.0037941
PMCID: PMC3362532  PMID: 22666418
17.  Zinc finger protein 267 is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation and migration 
Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulates diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 mRNA is up-regulated in liver cirrhosis, which is the main risk factor for hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in human HCC cells and tissue specimens and found a significant up-regulation compared to primary human hepatocytes and corresponding non-tumorous liver tissue. Over-expression of the transcription factor Ets-1 further enhanced ZNF267 expression, and reporter gene assays revealed that mutation of the Ets-1 binding site to the ZNF267 promotor markedly inhibited ZNF267 promotor activity. Hypoxic conditions induced Ets-1 in HCC cells via HIF1alpha activation, and hypoxia induced ZNF267 expression while HIF1alpha inhibition significantly reduced both hypoxia-induced as well as basal ZNF267 expression in HCC cells. It is known that hypoxic conditions in tumorous tissues induce the formation of reactive oxygen species (ROS), and ROS have been identified as important factor in the regulation of Ets-1 expression in tumor cells. Here, we found that ROS induction induced and ROS scavenging reduced ZNF267 expression in HCC cells, respectively. Loss and gain of function analysis applying siRNA directed against ZNF267 or transient transfection revealed that ZNF267 promotes proliferation and migration of HCC cells in vitro. These findings indicate Ets-1 and HIF1alpha as critical regulators of basal and hypoxia- or ROS-induced ZNF267 expression in HCC, and further suggest that the pro-tumorigenic effect of these factors is at least in part mediated via increased ZNF267 expression in HCC. Since ZNF267 is already elevated in cirrhosis, ZNF267 appears as promising target for both prevention as well as treatment of HCC in patients with chronic liver disease.
doi:10.1016/j.yexmp.2011.07.006
PMCID: PMC3342774  PMID: 21840307
Hepatocellular carcinoma; ZNF267; Kruppel-like factor
18.  Micro-RNA Profiling in Human Serum Reveals Compartment-Specific Roles of miR-571 and miR-652 in Liver Cirrhosis 
PLoS ONE  2012;7(3):e32999.
Background and Aims
Micro-RNAs (miRNAs) have recently emerged as crucial modulators of molecular processes involved in chronic liver diseases. The few miRNAs with previously proposed roles in liver cirrhosis were identified in screening approaches on liver parenchyma, mostly in rodent models. Therefore, in the present study we performed a systematic screening approach in order to identify miRNAs with altered levels in the serum of patients with chronic liver disease and liver cirrhosis.
Methods
We performed a systematic, array-based miRNA expression analysis on serum samples from patients with liver cirrhosis. In functional experiments we evaluated the relationship between alterations of miRNA serum levels and their role in distinct cellular compartments involved in hepatic cirrhosis.
Results
The array analysis and the subsequent confirmation by qPCR in a larger patient cohort identified significant alterations in serum levels of miR-513-3p, miR-571 and miR-652, three previously uncharacterized miRNAs, in patients with alcoholic or hepatitis C induced liver cirrhosis. Of these, miR-571 serum levels closely correlated with disease stages, thus revealing potential as a novel biomarker for hepatic cirrhosis. Further analysis revealed that up-regulation of miR-571 in serum reflected a concordant regulation in cirrhotic liver tissue. In isolated primary human liver cells, miR-571 was up-regulated in human hepatocytes and hepatic stellate cells in response to the pro-fibrogenic cytokine TGF-β. In contrast, alterations in serum levels of miR-652 were stage-independent, reflecting a concordant down-regulation of this miRNA in circulating monocytes of patients with liver cirrhosis, which was inducible by proinflammatory stimuli like bacterial lipopolysaccharide.
Conclusion
Alterations of miR571 and miR-652 serum levels in patients with chronic liver disease reflect their putative roles in the mediation of fibrogenic and inflammatory processes in distinct cellular compartments involved in the pathogenesis of liver cirrhosis.
doi:10.1371/journal.pone.0032999
PMCID: PMC3296762  PMID: 22412969
19.  Protective effect of xanthohumol on toxin-induced liver inflammation and fibrosis 
Xanthohumol, the major prenylated chalcone found in hops, is known for its anti-inflammatory properties. We have recently shown that xanthohumol inhibits hepatic inflammation and fibrosis in a murine model of nonalcoholic steatohepatitis. The aim of this study was to investigate the effect of xanthohumol in an acute model of liver injury. Carbon tetrachloride (CCl4), an industrial solvent, is a hepatotoxic agent and its administration is widely used as an animal model of toxin-induced liver injury. Xanthohumol was applied orally at a dose of 1 mg/g body weight 2 days prior as well as during and after exposure to CCl4. 72 h after a single CCl4 application histomorphology and serum levels of transaminases revealed considerable hepatocellular necrosis, which was accompanied by significantly enhanced hepatic expression of pro-inflammatory cytokines. Furthermore, elevated hepatic alpha-smooth muscle actin expression indicated activation of hepatic stellate cells, and in accordance, we detected enhanced hepatic expression levels of TGF-β and collagen type I reflecting a marked fibrogenic response to CCl4 exposure. While the degree of hepatocellular damage in response to CCl4 was similar in mice which received xanthohumol and the control group, pro-inflammatory and profibrogenic hepatic gene expression were almost completely blunted in xanthohumol fed mice. Furthermore, xanthohumol fed mice revealed decreased hepatic NFκB activity. These results suggest that the protective effects of xanthohumol in this toxic liver injury model involves direct mechanisms related to its ability to block both hepatic inflammation and the activation of hepatic stellate cells, presumable at least in part via decreasing NFκB activity. Thus, this study further indicates the potential of xanthohumol application to prevent or ameliorate the development and progression of liver fibrosis in response to hepatic injury.
PMCID: PMC3267483  PMID: 22295144
Xanthohumol; carbon tetrachloride; fibrosis; inflammation; acute liver injury
20.  DNA methylation is associated with downregulation of the organic cation transporter OCT1 (SLC22A1) in human hepatocellular carcinoma 
Genome Medicine  2011;3(12):82.
Background
Organic cation transporters (OCTs) determine not only physiological processes but are also involved in the cellular uptake of anticancer agents. Based on microarray analyses in hepatocellular carcinoma (HCC), SLC22A1/OCT1 mRNA seems to be downregulated, but systematic protein expression data are currently missing. Moreover, the underlying molecular mechanisms responsible for altered SLC22A1 expression in HCC are not fully understood. Therefore, we investigated the role of DNA methylation in the transcriptional regulation of the family members SLC22A1/OCT1, SLC22A2/OCT2 and SLC22A3/OCT3 in HCC.
Methods
Semiquantitative immunohistochemistry of SLC22A1 protein expression was performed in paired HCC and histological normal adjacent liver tissues (n = 71) using tissue microarray analyses, and the results were correlated with clinicopathological features. DNA methylation, quantified by MALDI-TOF mass spectrometry and gene expression of SLC22A1, SLC22A2 and SLC22A3 were investigated using fresh-frozen HCC (n = 22) and non-tumor adjacent liver tissues as well as histologically normal liver samples (n = 120) from a large-scale liverbank.
Results
Based on tissue microarray analyses, we observed a significant downregulation of SLC22A1 protein expression in HCC compared to normal adjacent tissue (P < 0.0001). SLC22A1 expression was significantly inverse correlated with expression of the proliferation marker MIB1/Ki-67 (rs = -0.464, P < 0.0001). DNA methylation of SLC22A1 was significantly higher in HCC compared with non-tumor adjacent liver tissue and was lowest in histologically normal liver tissue. Methylation levels for SLC22A1 in combination with RASSF1A resulted in a specificity of > 90% and a sensitivity of 82% for discriminating HCC and tumor-free liver tissue.
Conclusions
DNA methylation of SLC22A1 is associated with downregulation of SLC22A1 in HCC and might be a new biomarker for HCC diagnosis and prognosis. Moreover, targeting SLC22A1 methylation by demethylating agents may offer a novel strategy for anticancer therapy of HCC.
doi:10.1186/gm298
PMCID: PMC3334547  PMID: 22196450
21.  FGF receptors 1 and 2 control chemically-induced injury and compound detoxification in regenerating livers of mice 
Gastroenterology  2010;139(4):1385-1396.e8.
Background & Aims
Fibroblast growth factor receptor (FGFR) 4 controls bile acid metabolism and protects the liver from fibrosis, but the roles of FGFR1 and FGFR2 in the adult liver are largely unknown. We investigated the functions and mechanisms of action of these receptors in liver homeostasis, regeneration, and fibrosis.
Methods
We generated mice with hepatocytes that lack FGFR1 and FGFR2 and subjected them to acute and chronic carbon tetrachloride-induced liver injury and partial hepatectomy; mice were also injected with FGF7. We performed histology, histomorphometry, real-time reverse transcription PCR, and immunoblot analyses.
Results
In hepatocytes, loss of FGFR1 and FGFR2 eliminated responsiveness to FGF7 and related FGF family members, but did not affect toxin-induced liver injury and fibrosis. However, mortality after partial hepatectomy increased because of severe hepatocyte necrosis. These effects appeared to be mediated by a failure of hepatocyes to induce the expression of the transcriptional regulators Dbp and Tef upon liver surgery; this affected expression of their target genes, which encode detoxifying cytochrome P450 enzymes. We found that Dbp and Tef expression was directly controlled by FGFR signalling in hepatocytes. As a consequence of the reduced expression of genes that control detoxification, the liver tissue that remained after partial hepatectomy failed to efficiently metabolize endogenous compounds and the drugs applied for anaesthesia/analgesia.
Conclusions
We identified a new, cytoprotective effect of FGFR1 and FGFR2 in the regenerating liver and suggest the use of recombinant FGF7 to increase survival of patients after surgical resection of large amounts of liver tissue.
doi:10.1053/j.gastro.2010.06.069
PMCID: PMC2949525  PMID: 20603121
liver disease; cirrhosis; drug toxicity; cytoprotection
22.  Increased expression of Zinc finger protein 267 in non-alcoholic fatty liver disease 
Hepatocellular lipid accumulation is a hallmark of non-alcoholic
fatty liver disease (NAFLD), which encompasses a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and ultimately cirrhosis. Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulate diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 expression is up-regulated in liver cirrhosis and is further increased in hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in tissue specimens of NAFLD patients and found a significant up-regulation compared to normal liver tissue. Noteworthy, ZNF267 mRNA was already significantly increased in steatotic liver tissue without inflammation. In line with this, incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation and corresponding dose-dependent ZNF267 induction in vitro. Furthermore, hepatocellular lipid accumulation induced formation of reactive oxygen species (ROS), and also chemically induced ROS formation increased ZNF267 mRNA expression. In summary with previous findings, which revealed ZNF267 as pro-fibrogenic and pro-cancerogenic factor in chronic liver disease, the present study further suggests ZNF267 as promising therapeutic target particularly for NAFLD patients. In addition, it further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign.
PMCID: PMC3209606  PMID: 22076166
Non-alcoholic fatty liver disease; Kruppel-like factor; ZNF267
23.  Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition 
BMC Cancer  2010;10:668.
Background
Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer.
Methods
Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression.
Results
The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer.
Conclusion
In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor.
doi:10.1186/1471-2407-10-668
PMCID: PMC3003660  PMID: 21129190
24.  Hepatic steatosis causes induction of the chemokine RANTES in the absence of significant hepatic inflammation 
Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to cirrhosis. Hepatocellular lipid accumulation is a hallmark of both nonalcoholic steatosis and steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine CCL5/RANTES plays an important role in the progression of hepatic inflammation and fibrosis. We here aimed to investigate its expression in NAFLD. Incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation, and corresponding dose-dependent RANTES induction in vitro. Furthermore, we observed significantly elevated hepatic RANTES expression in a dietary model of NAFLD, in which mice were fed a high-fat diet for 12 weeks. This diet induced significant hepatic steatosis but only minimal inflammation. In contrast to the liver, RANTES expression was not induced in visceral adipose tissue of the group fed with high-fat diet. Finally, RANTES serum levels were elevated in patients with ultrasound-diagnosed NAFLD. In conclusion, our data indicate hepatocytes as cellular source of elevated hepatic as well as circulating RANTES levels in response to hepatic steatosis. Noteworthy, upregulation of RANTES in response to lipid accumulation occurs in the absence of relevant inflammation, which further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign.
PMCID: PMC2933387  PMID: 20830238
Hepatic steatosis; liver cirrhosis; nonalcoholic steatosis; steatohepatitis; chemokine CCL5; RANTES; inflammation; nonalcoholic fatty liver disease (NAFLD)
25.  Prevalence of ultrasound-diagnosed non-alcoholic fatty liver disease in a hospital cohort and its association with anthropometric, biochemical and sonographic characteristics 
Non-alcoholic fatty liver disease (NAFLD) is considered as the most common liver disease in Western countries with still rising prevalence due to a lifestyle favoring the development of the metabolic syndrome. Aim: To investigate the prevalence of ultrasound-diagnosed NAFLD in patients with referral for sonographic examination of the abdomen, and to determine risk factors. Methods: After exclusion of patients with known liver disease or risk factors for secondary NAFLD, a total of 155 arbitrarily selected patients (mean age 53.6±17.4 years; 52.6% male) from the interdisciplinary ultrasound department of a German University Hospital were included in this prospective study. Each patient underwent a standardized ultrasound, anthropometric and biochemical examination. Results: The prevalence of ultrasound-diagnosed NAFLD was 40.0%. NAFLD-patients had significantly higher body mass index (BMI) and waist-to-hip ratio, higher rates of reported hypertension and diabetes mellitus, and lower HDL cholesterol serum levels. Furthermore, NAFLD-patients revealed significantly higher serum ALT levels (23.2±22.1 U/l vs. 15.0±8.2 U/l; p=0.001), lower AST/ALT ratio (1.76±0.79 vs. 2.11±0.94; p=0.019), and notably, decreased flow in the portal vein (22.9±6.3 cm/s vs. 26.7±10.5 cm/s; p=0.011). Multivariate analysis revealed BMI (odds ratio (OR): 14.05; 95% Confidence interval (CI): 3.3-59.8), AST/ALT ratio (OR: 0.39; CI: 0.18-0.82), and HDL-C (OR: 4.33; CI: 1.6-11.9) as independent risk factors. Conclusions: Ultrasound-diagnosed NAFLD is frequent in patients with referral for ultrasound examination of the abdomen, and our findings further support that NAFLD is the hepatic manifestation of the metabolic syndrome with obesity being the most important risk factor.
PMCID: PMC2929946  PMID: 20827318
Non-alcoholic fatty liver disease (NAFLD); ultrasound; diagnosis; prospective study; obesity; risk factor

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