Petra Windmolders

Obeticholic acid, a farnesoid X receptor agonist, improves portal hypertension by two distinct pathways in cirrhotic rats.

The farnesoid X receptor (FXR) is a nuclear bile acid receptor involved in bile acid homeostasis, hepatic and intestinal inflammation, liver fibrosis, and cardiovascular disease. We studied the effect of short‐term treatment with obeticholic acid (INT‐747), a potent selective FXR agonist, on intrahepatic hemodynamic dysfunction and signaling pathways in different rat models of cirrhotic portal hypertension (PHT). For this, thioacetamide (TAA)‐intoxicated and bile‐duct–ligated (BDL) rats were used as models. After gavage of two doses of 30 mg/kg of INT‐747 or vehicle within 24 hours, in vivo hemodynamics were assessed. Additionally, we evaluated the direct effect of INT‐747 on total intrahepatic vascular resistance (IHVR) and intrahepatic vascular tone (endothelial dysfunction and hyperresponsiveness to methoxamine) by means of an in situ liver perfusion system and on hepatic stellate cell contraction in vitro. FXR expression and involved intrahepatic vasoactive pathways (e.g., endothelial nitric oxide synthase [eNOS], Rho‐kinase, and dimethylarginine dimethylaminohydrolase [DDAH]) were analyzed by immunohistochemistry, reverse‐transcriptase polymerase chain reaction, or western blotting. In both cirrhotic models, FXR expression was decreased. Treatment with INT‐747 in TAA and BDL reactivated the FXR downstream signaling pathway and decreased portal pressure by lowering total IHVR without deleterious systemic hypotension. In the perfused TAA and BDL cirrhotic liver, INT‐747 improved endothelial vasorelaxation capacity, but not hyperresponsiveness. In both groups, this was associated with an increased eNOS activity, which, in TAA, related to down‐regulation of Rho‐kinase and in BDL to up‐regulation of DDAH‐2. Conclusion: FXR agonist INT‐747 improves PHT in two different rat models of cirrhosis by decreasing IHVR. This hemodynamic effect relates to increased intrahepatic eNOS activity by pathways that differ depending on the etiology of cirrhosis. (Hepatology 2014;59:2286–2298)

Long-term exposure to sorafenib of liver cancer cells induces resistance with epithelial-to-mesenchymal transition, increased invasion and risk of rebound growth.

Sorafenib leads to a survival benefit in patients with advanced hepatocellular carcinoma but its use is hampered by the occurrence of drug resistance. To investigate the molecular mechanisms involved we developed five resistant human liver cell lines in which we studied morphology, gene expression and invasive potential. The cells changed their appearance, lost E-cadherin and KRT19 and showed high expression of vimentin, indicating epithelial-to-mesenchymal transition. Resistant cells showed reduced adherent growth, became more invasive and lost liver-specific gene expression. Furthermore, following withdrawal of sorafenib, the resistant cells showed rebound growth, a phenomenon also found in patients. This cell model was further used to investigate strategies for restoration of sensitivity to sorafenib.

The FXR Agonist Obeticholic Acid Prevents Gut Barrier Dysfunction and Bacterial Translocation in Cholestatic Rats

Bacterial translocation (BTL) drives pathogenesis and complications of cirrhosis. Farnesoid X-activated receptor (FXR) is a key transcription regulator in hepatic and intestinal bile metabolism. We studied potential intestinal FXR dysfunction in a rat model of cholestatic liver injury and evaluated effects of obeticholic acid (INT-747), an FXR agonist, on gut permeability, inflammation, and BTL. Rats were gavaged with INT-747 or vehicle during 10 days after bile-duct ligation and then were assessed for changes in gut permeability, BTL, and tight-junction protein expression, immune cell recruitment, and cytokine expression in ileum, mesenteric lymph nodes, and spleen. Auxiliary in vitro BTL-mimicking experiments were performed with Transwell supports. Vehicle-treated bile duct-ligated rats exhibited decreased FXR pathway expression in both jejunum and ileum, in association with increased gut permeability through increased claudin-2 expression and related to local and systemic recruitment of natural killer cells resulting in increased interferon-γ expression and BTL. After INT-747 treatment, natural killer cells and interferon-γ expression markedly decreased, in association with normalized permeability selectively in ileum (up-regulated claudin-1 and occludin) and a significant reduction in BTL. In vitro, interferon-γ induced increased Escherichia coli translocation, which remained unaffected by INT-747. In experimental cholestasis, FXR agonism improved ileal barrier function by attenuating intestinal inflammation, leading to reduced BTL and thus demonstrating a crucial protective role for FXR in the gut-liver axis.

FXR agonist obeticholic acid reduces hepatic inflammation and fibrosis in a rat model of toxic cirrhosis

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

Roux-en-y gastric bypass attenuates hepatic mitochondrial dysfunction in mice with non-alcoholic steatohepatitis

Objective No therapy for non-alcoholic steatohepatitis (NASH) has been approved so far. Roux-en-y gastric bypass (RYGB) is emerging as a therapeutic option, although its effect on NASH and related hepatic molecular pathways is unclear from human studies. We studied the effect of RYGB on pre-existent NASH and hepatic mitochondrial dysfunction—a key player in NASH pathogenesis—in a novel diet-induced mouse model nicely mimicking human disease. Design C57BL/6J mice were fed a high-fat high-sucrose diet (HF-HSD). Results HF-HSD led to early obesity, insulin resistance and hypercholesterolaemia. HF-HSD consistently induced NASH (steatosis, hepatocyte ballooning and inflammation) with fibrosis already after 12-week feeding. NASH was accompanied by hepatic mitochondrial dysfunction, characterised by decreased mitochondrial respiratory chain (MRC) complex I and IV activity, ATP depletion, ultrastructural abnormalities, together with higher 4-hydroxynonenal (HNE) levels, increased uncoupling protein 2 (UCP2) and tumour necrosis factor-α (TNF-α) mRNA and free cholesterol accumulation. In our model of NASH and acquired mitochondrial dysfunction, RYGB induced sustained weight loss, improved insulin resistance and inhibited progression of NASH, with a marked reversal of fibrosis. In parallel, RYGB preserved hepatic MRC complex I activity, restored ATP levels, limited HNE production and decreased TNF-α mRNA. Conclusions Progression of NASH and NASH-related hepatic mitochondrial dysfunction can be prevented by RYGB. RYGB preserves respiratory chain complex activity, thereby restoring energy output, probably by limiting the amount of oxidative stress and TNF-α. These data suggest that modulation of hepatic mitochondrial function contributes to the favourable effect of RYBG on established NASH.

Cytotoxicity evaluation and antioxidant enzyme expression related to heavy metals found in tuna by-products meal: An in vitro study in human and rat liver cell lines

Heavy metals can accumulate in organisms via various pathways, including respiration, adsorption and ingestion. They are known to generate free radicals and induce oxidative and/or nitrosative stress with depletion of anti-oxidants. Tuna by-product meal (TBM) is rich in proteins and can, therefore, offer an attractive protein source for animals. This study was undertaken to assess the effects of metals present in TBM, namely cadmium (Cd), lead (Pb), and mercury (Hg), separately or in combination with oxidative stress, on cell viability. Three cell models: rat liver FTO2B, human hepatoma HepG2, and human hepatic WRL-68, were used. Cell viability was determined following exposure to various concentrations of the metals. Two antioxidant genes, catalase (CAT) and superoxide dismutase (SOD), were measured to obtain a better understanding of oxidative stress-associated gene expression. Among the metals present in TBM, only Cd at a concentration of 30μM was noted to exhibit cytotoxic effects. This cytotoxicity was even more pronounced after co-stimulation with H2O2, used to mimic systemic oxidative stress. At non-toxic concentrations, Hg and Pb were noted to aggravate oxidative stress toxicity. The results further revealed that exposure to Cd, Pb, and a co-stimulation of H2O2 with Hg resulted in the increased expression of antioxidant gene SOD. A risk assessment of toxic contaminants in TBM indicated that food safety objectives should consider the human health impacts of foods derived from animals fed on contaminated meal and that much care should be taken when TBM is used in animal diet.

Characterization of a cell culture model for clinically aggressive hepatocellular carcinoma induced by chronic hypoxia

We demonstrated in an in vitro model (human HepG2 liver cells) that chronic hypoxia induced gene expression is associated with an aggressive phenotype in patients with hepatocellular carcinoma (HCC). The aim of this study was to characterize this model further using gene expression microarray, real-time PCR and immunocytochemistry. Subsequently, pathway analysis software was used to identify relevant processes. After examination, we selected 2% O2 during 72 h as conditions to study chronic hypoxia. The most affected signaling is centered on TGF-β1 and PPARα/RXRα. Cells at 2% O2 showed a shift in expression of Epithelial-to-Mesenchymal-Transition (EMT) related genes. Furthermore, a downregulation of liver specific detoxification pathways including cytochrome P450s and glutathione-S-transferases was observed. Both up- and downregulation events within different signaling cascades indicated a cellular adaptation and the onset of a new equilibrium. The prominent role of TGF-β1- and PPARα/RXRα signaling and cell motility pathways warrants their further investigation for therapeutic targets in HCC.

Pro-inflammatory cytokines but not endotoxin-related parameters associate with disease severity in patients with NAFLD

Intestinal dysbiosis and elevated lipopolysaccharides (LPS) levels have been implicated in the development of obesity, insulin resistance and non-alcoholic steatohepatitis (NASH). In order to determine if LPS levels are elevated in patients with NASH compared to patients with non-alcoholic fatty liver (NAFL) and, if elevated LPS levels correlated with histological severity of non-alcoholic fatty liver disease (NAFLD) we compared LPS, markers of LPS bioactivity and pro-inflammatory cytokines/chemokines in patients undergoing bariatric surgery. At the time of surgery a liver biopsy was taken allowing the stratification into well-delineated subgroups including: No NAFL/NAFL; NASH; NASH with fibrosis and NASH cirrhotics, using the NAFLD Activity Score (NAS). Anthropometric data and plasma were collected for assessment of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14), intestinal-type fatty acid binding protein (iFABP), Toll-like receptors 2 and 4 (TLR2, 4) and a panel of cytokines/chemokines. Similar analysis was performed on plasma from a cohort of healthy controls. Our data indicate elevated levels of LPS, LBP, sCD14, iFABP and TLR2,4 in obese patients compared to healthy controls, however, these parameters remained unaltered within patients with limited liver disease (NAFL) compared to NASH/NASH with fibrosis subgroups. Hierarchic cluster analysis using endotoxin-related parameters failed to discriminate between lean controls, NAFLD. While similar cluster analysis implementing inflammation-related parameters clearly distinguished lean controls, NALFD subgroups and NASH cirrhotics. In addition, LPS levels was not associated with disease severity while TNFα, IL8, and CCL3 featured a clear correlation with transaminase levels and the histological severity of NALFD. In conclusion our data indicate a stronger correlation for circulating inflammatory- rather than endotoxin-related parameters in progression of NAFLD and highlights the need for additional larger studies in unravelling further mechanistic insights.

Acriflavine Inhibits Acquired Drug Resistance by Blocking the Epithelial-to-Mesenchymal Transition and the Unfolded Protein Response

Epithelial-to-mesenchymal transition (EMT) is linked to tumor invasion, drug resistance and aggressive disease and this is largely dependent on the cells microenvironment. Acriflavine (ACF) is an old antibacterial drug recently also suggested as anticancer agent and HIF inhibitor. We wanted to study the effect of acriflavine on EMT in different human cancer models. Pancreatic cancer cells (Panc-1) were exposed to TGF-β1 or cobalt chloride (to mimick severe hypoxia) to induce EMT. For our third model we exposed HepG2 liver cancer cells to sorafenib which resulted in development of acquired drug resistance with strong features of EMT and aggressive behavior. These models were morphologically and functionally (invasion assay) characterized. Markers of EMT were determined using qRT-PCR and Western blotting. Transcriptome analysis was performed following gene expression determination and combining the iRegulon tool and Gene Set Enrichment Analysis (GSEA). We made the following observations: (1) acriflavine inhibited EMT based on changes in cell morphology, invasive capacities and markers of EMT (at protein and gene expression level). (2) Transcriptome analysis revealed potent inhibition of ATF4 target genes and of the unfolded protein response. We showed that acriflavine blocked eIF2a phosphorylation and reduced ATF4 translation thereby inhibiting the PERK/eIF2a/ATF4 UPR pathway. (3) ACF restored drug sensitivity of cells that obtained acquired resistance. Conclusions: We identified acriflavine as a potent inhibitor of EMT and the UPR, thereby re-sensitizing the cancer cells to antineoplastic drugs.

The plant decapeptide OSIP108 can alleviate mitochondrial dysfunction induced by cisplatin in human cells.

We investigated the effect of the Arabidopsis thaliana-derived decapeptide OSIP108 on human cell tolerance to the chemotherapeutic agent cisplatin (Cp), which induces apoptosis and mitochondrial dysfunction. We found that OSIP108 increases the tolerance of HepG2 cells to Cp and prevents Cp-induced changes in basic cellular metabolism. More specifically, we demonstrate that OSIP108 reduces Cp-induced inhibition of respiration, decreases glycolysis and prevents Cp-uptake in HepG2 cells. Apart from its protective action against Cp in human cells, OSIP108 also increases the yeast Saccharomyces cerevisiae tolerance to Cp. A limited yeast-based study of OSIP108 analogs showed that cyclization does not severely affect its activity, which was further confirmed in HepG2 cells. Furthermore, the similarity in the activity of the d-stereoisomer (mirror image) form of OSIP108 with the l-stereoisomer suggests that its mode of action does not involve binding to a stereospecific receptor. In addition, as OSIP108 decreases Cp uptake in HepG2 cells and the anti-Cp activity of OSIP108 analogs without free cysteine is reduced, OSIP108 seems to protect against Cp-induced toxicity only partly via complexation. Taken together, our data indicate that OSIP108 and its cyclic derivatives can protect against Cp-induced toxicity and, thus, show potential as treatment options for mitochondrial dysfunction- and apoptosis-related conditions.