Anne Sibille, Frederique Bustin, Luciano Carestia, Gaetan Catala, Christophe Comp ere, Kristof Cuppens , Benoit Colinet, Stephanie Coulon, Nele De Brucker, Lore Decoster, Lynn Decoster, Ingel Demedts, Sofie Derijcke, Koen Deschepper, Danny Galdermans, Annelies Janssens, Sebahat Ocak, Christel Oyen, Karin Pat, Thierry Pieters, Vincent Pruniau, Veerle Surmont, Saar Vandekeere and Johan Vansteenkiste Department of Pulmonology, University Hospital of Li ege, Li ege, Belgium; Department of Pulmonology, CHR de la Citadelle, Li ege, Belgium; Department of Pulmonology, Clinique Saint-Pierre Ottignies, Ottignies, Belgium; Department of Oncology, CH Jolimont, Haine-StPaul, La Louvi ere, Belgium; Department of Pulmonology, CCI (CHIREC Cancer Institute), CHIREC Hospital, Brussels, Belgium; Department of Pulmonology and Thoracic Oncology, Jessa Hospital, Hasselt, Belgium; Department of Pulmonology, Grand Hôpital de Charleroi, Gilly, Belgium; Medical Affairs Department, MSD Belgium, Brussels, Belgium; Department of Pneumonology, Imelda Hospital, Bonheiden, Belgium; Department of Medical Oncology, Oncologisch Centrum, Universitair Ziekenhuis Brussel (UZ Brussel), Vrije Universiteit Brussel (VUB), Brussels, Belgium; Department of Pulmonology, AZ Turnhout, Turnhout, Belgium; Department of Pulmonary Diseases, AZ Delta, Roeselare, Belgium; Department of Pulmonology, AZ Groeninge, Kortrijk, Belgium; Algemeen Ziekenhuis Nikolaas, Sint-Niklaas, Belgium; Department of Pulmonology, ZNA Middelheim, Antwerp, Belgium; MOCA Thoracic Oncology, Antwerp University Hospital, Edegem, Belgium; Division of Pulmonology, CHU UCL Namur, Godinne Site, Yvoir, Belgium; Pole of Pneumology, ENT, and Dermatology (PNEU), Institut de Recherche Exp erimentale et Clinique (IREC), Universit e Catholique de Louvain (UCLouvain), Brussels, Belgium; Respiratory Oncology Unit, University Hospitals KU Leuven, Leuven, Belgium; Department of Pulmonology, Cliniques Universitaires Saint-Luc, Brussels, Belgium; Department of Respiratory Medicine, Ghent University, Ghent, Belgium

Objective:To describe comorbidities and concomitant medications in patients initiating treatment for hepatitis C virus (HCV) infection with direct-acting antiviral (DAA) regimens in Belgium.Methods:This was a noninterventional, observational, multicenter study of data from patient charts. Adult patients with HCV infection receiving second-generation DAA therapy were included. Comorbidities were assessed at the time of HCV treatment initiation. Concomitant medications were recorded at the time of diagnosis and at treatment initiation. Potential clinically relevant drug-drug interactions (DDIs) were assessed based on information available at www.hep-druginteractions.org. The primary objective was to describe concomitant medication use ; secondary objectives were to describe modifications in concomitant therapies and comorbidities.Results:405 patients were included. A total of 956 comorbidities were reported by 362 patients (median, 2 ; range, 0-15). The most common comorbidities were hypertension (27.2%) ; HIV coinfection (22.5%), and type 2 diabetes mellitus (14.3%). Overall, 1455 concomitant medications were being taken by 365 patients (90.1% ; median, 3 ; range 0-16). The most common concomitant medications were psycholeptics (28.6%), antiviral agents (24.2%), and medications for acid-related disorders (21.0%) Overall, 74/365 (20.3%) patients receiving a concomitant medication required an adaptation to their concomitant medication. The medications that most frequently required change were drugs for acid-related disorders (n = 14) and antiviral drugs (n = 5) ; those that were most frequently stopped were lipid-modifying drugs (n = 25) and drugs for acid-related disorders (n = 13).Conclusion:Physicians are aware of the potential for DDIs with DAAs, but improved alignment between clinical practice and theoretical recommendations is required.

Background & AimsHepatopulmonary syndrome is a complication of chronic liver disease resulting in increased morbidity and mortality. It is caused by intrapulmonary vascular dilations and arteriovenous connections with devastating influence on gas exchange. The pathogenesis is not completely understood but evidence mounts for angiogenesis. Aims of this study were to identify angiogenic factors in serum of patients with hepatopulmonary syndrome and to study the possibility to predict its presence by these factors.MethodsMultiplex assays were used to measure the concentration of angiogenic factors in patients with (n=30) and without hepatopulmonary syndrome (n=30). Diagnosis was based on the presence of gas exchange abnormality and intrapulmonary vasodilations according to published guidelines.ResultsPatients with and without hepatopulmonary syndrome had similar MELD scores (median: 11.2 vs. 11.6; P=0.7), Child-Pugh score (P=0.7) and PaCO2 values (median: 35 vs. 37; P=0.06). PaO2 and P(A-a) O-2 gradient were significantly different (respectively median of 80 vs. 86, P=0.02; and 24 vs. 16, P=0.004). Based on area under the curve (AUC) data and P-values, the best predictors were vascular cell adhesion molecule 1 (VCAM1) (AUC=0.932; P<0.001) and intercellular adhesion molecule 3 (ICAM3) (AUC=0.741; P=0.003). Combining these factors results in an AUC of 0.99 (after cross-validation still 0.99).ConclusionsVCAM1 and ICAM3 might be promising biomarkers for predicting hepatopulmonary syndrome. Combining these factors results in an AUC of 0.99 and a negative predictive value of 100%. Determining the concentration of these biomarkers might be a screening method to detect hepatopulmonary syndrome. The use of these biomarkers should be validated in larger groups of patients.

When cells are subjected to stress by changes in their extracellular environment, unfolded proteins accumulate in the endoplasmic reticulum (ER), causing ER stress. This initiates the unfolded protein response (UPR), a signal transduction cascade aiming at restoring cellular homeostasis. The UPR and angiogenesis are involved in the pathogenesis of many diseases such as cancer, pulmonary diseases and chronic liver diseases (CLDs) including alcoholic liver disease, non-alcoholic steatohepatitis and hepatitis B. This review summarizes the upcoming knowledge of the interaction between the UPR and angiogenesis in physiological angiogenesis and in different CLDs and other diseases.

UNLABELLED:The pathophysiology of nonalcoholic steatohepatitis (NASH) should be approached as a multifactorial process. In several stages of NASH, a link between disease progression and hepatic microvasculature changes can be made. In this study we investigated the role of angiogenesis in two mouse models for NASH, and the effect of a preventive and therapeutic antiangiogenic treatment in a diet-induced mouse model for NASH. Protein and RNA levels of angiogenic and inflammatory factors were significantly up-regulated in the liver of C56BL/6 and db/db mice with NASH at different timepoints. To examine the effect of angiogenic factors on the disease progression of NASH, a prevention and treatment study was set up, blocking the placental growth factor (PlGF) or vascular endothelial growth factor receptor 2 (VEGFR2). Our study showed that treatment prevents the progression of NASH by attenuating steatosis and inflammation, both in a preventive and therapeutic setting, thereby confirming the hypothesis that angiogenic factors play an early role in the disease progression from steatosis to NASH. Anti-PlGF (αPlGF) did not significantly improve liver histology. Vascular corrosion casting showed a more disrupted liver vasculature in mice with NASH compared to controls. Treatment with αVEGFR2 showed an improvement of the liver vasculature. Moreover, fat-laden primary hepatocytes treated with αVEGFR2 stored significantly less lipids. CONCLUSION:Our results demonstrate that there is an increased expression of angiogenic factors in the liver in different mouse models for NASH. We found that VEGFR2 blockage attenuates steatosis and inflammation in a diet-induced mouse model for NASH in a preventive and therapeutic setting. Our findings warrant further investigation of the role of angiogenesis in the pathophysiology in NASH.

Insulin-like growth factor 1 receptor (IGF-1R) is a cell membrane receptor widely expressed in tissues and involved in different cancers in humans. IGF-1R expression in human osteosarcoma has been associated with the development of tumour metastasis and with prognosis, and represents an attractive therapeutic target. The goal of this study was to investigate the expression of IGF-1R in canine osteosarcoma tissues and cell lines and assess its role and prognostic value. Samples from 34 dogs were examined by immunohistochemistry for IGF-1R expression. IGF-1R/AKT/MAPK signalling was evaluated by western blot and quantitative polymerase chain reaction in the cell lines. In addition, the in vitro inhibition of IGF-1R with pycropodophillin (PPP) was used to evaluate molecular and biological effects.Immunohistochemical data showed that IGF-1R was expressed in 71% of the analysed osteosarcoma samples and that dogs with higher levels of IGF-IR expression (47% of cases) had decreased survival (P < 0.05) when compared to dogs with lower IGF-IR expression. Molecular studies demonstrated that in canine osteosarcoma IGF-IR is activated by IGF-1 mostly in a paracrine or endocrine (rather than autocrine) manner, leading to activation of AKT/MAPK signalling. PPP caused p-IGF-1R dephosphorylation with partial blocking of p-MAPK and p-AKT, as well as apoptosis. It was concluded that IGF-1R is expressed and plays a role in canine osteosarcoma and that its expression is correlated with a poor prognosis. As in humans, IGF-1R may represent a good therapeutic target and a prognostic factor for canine osteosarcoma.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Background & Aims: The placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family known to stimulate endothelial cell growth, migration and survival, attract angiocompetent macrophages, and determine the metastatic niche. Unlike VEGF, genetic studies have shown that PIGF is specifically involved in pathologic angiogenesis, thus its inhibition would not affect healthy blood vessels, providing an attractive drug candidate with a good safety profile.Methods: We assess whether inhibition of PlGF could be used as a potential therapy against hepatocellular carcinoma (HCC), by using PIGF knockout mice and monoclonal anti-PlGF antibodies in a mouse model for HCC. In addition, the effect of PlGF antibodies is compared to that of sorafenib, as well as the combination of both therapies.Results: We have found that both in a transgenic knockout model and in a treatment model, targeting PlGF significantly decreases tumor burden. This was achieved not only by inhibiting neovascularisation, but also by decreasing hepatic macrophage recruitment and by normalising the remaining blood vessels, thereby decreasing hypoxia and reducing the prometastatic potential of HCC.Conclusions: Considering the favourable safety profile and its pleiotropic effect on vascularisation, metastasis and inflammation, PlGF inhibition could become a valuable therapeutic strategy against HCC. (C) 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Life in hypersaline environments is typically limited by bioenergetic constraints. Microbial activity at the thermodynamic edge, such as the anaerobic oxidation of methane (AOM) coupled to sulphate reduction (SR), is thus unlikely to thrive in these environments. In this study, carbon and sulphur cycling was investigated in the extremely hypersaline cold seep sediments of Mercator mud volcano. AOM activity was partially inhibited but still present at salinity levels of 292 g L(-1) (c. eightfold sea water concentration) with rates of 2.3 nmol cm(-3) day(-1) and was even detectable under saturated conditions. Methane and evaporite-derived sulphate comigrated in the ascending geofluids, which, in combination with a partial activity inhibition, resulted in AOM activity being spread over unusually wide depth intervals. Up to 79% of total cells in the AOM zone were identified by fluorescence in situ hybridization (FISH) as anaerobic methanotrophs of the ANME-1. Most ANME-1 cells formed monospecific chains without any attached partner. At all sites, AOM activity co-occurred with SR activity and sometimes significantly exceeded it. Possible causes of these unexpected results are discussed. This study demonstrates that in spite of a very low energy yield of AOM, microorganisms carrying this reaction can thrive in salinity up to halite saturation.

stearoyl-CoA desaturase-1 (Scd1), fatty acid binding protein (Fabp) and carnitine palmitoyltransferase-1 (Cpt1) was determined with qRT-PCR. Results: Serum levels of AST and ALT were significantly higher in MCD fed mice treated with anti-VEGFR2 compared to MCD fed mice treated with NaCl (respectively p =0.009;p =0.007). Liver histology showed a decrease of steatosis (p < 0.001) and fewer inflammatory foci (p = 0.015) in the liver of anti-VEGFR2 treated mice in comparison to MCD fed mice treated with NaCl. There was no difference in fibrosis. Expression of endoglin in the liver of MCD fed mice treated with anti-VEGFR2 was significant decreased compared to MCD fed mice treated with NaCl (p = 0.005). To investigate the molecular mechanisms, we looked into genes involved in fatty acid metabolism. Scd1 was significantly higher in MCD fed mice treated with anti-VEGFR2 compared to MCD fed mice treated with NaCl (p < 0.001). Low Scd1 expression results in excess fat deposition in the liver. FABP and Cpt1 expression remained the same in both groups. Conclusion: Our results demonstrate that inhibition of VEGFR2 is protective against the development of steatosis and inflammation in a diet-induced mouse model for NASH. The effect of anti-VEGFR2 might be found in the fatty acid metabolism. These findings might give rise to new therapeutic options for NASH.

Objectives Hepatocellular carcinoma and cholangiocarcinoma form the majority of primary hepatic tumours and are the third most common cause of cancer-related deaths. These liver tumours rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors and triggering the angiogenic switch. Therefore, inhibiting angiogenesis has proven to be a valuable therapeutic strategy in hepatocellular carcinoma, yet less is known about its use in cholangiocarcinoma. In this study, we assess whether inhibiting the placental growth factor (PlGF) could offer a therapeutic option in mice with hepatocellular carcinoma and cholangiocarcinoma. PlGF is a homologue of the vascular endothelial growth factor, which is only involved in pathological angiogenesis, therefore, its inhibition does not induce adverse effects. Methods We have used a chemically induced transgenic mouse model in which both hepatocellular carcinoma and cholangiocarcinoma develop after 25 weeks and are treated with murine monoclonal antibodies targeting PlGF. Results This study has shown for the first time that inhibiting PlGF decreases the burden of cholangiocarcinoma, by affecting both angiogenesis and inflammation. Conclusion The use of monoclonal antibodies targeting PlGF could thus offer a potential systemic treatment for patients who suffer from primary liver tumours.

The liver is a major target of injury in obese patients. Non-alcoholic fatty liver disease (NAFLD) is present in 60-90% of obese Americans and can range from simple steatosis to the more severe non-alcoholic steatohepatitis (NASH). The onset of a chronic inflammatory reaction marks the progression from simple steatosis to NASH and the expansion of adipose tissue is strongly associated with angiogenesis. Therefore, we determined the serum concentration of inflammatory [tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL6)] and angiogenic [vascular endothelial growth factor A (VEGF)] cytokines and soluble VEGF receptors 1 and 2 (sVEGFR1, sVEGFR2) in the serum of an obese population with simple steatosis and NASH compared to healthy controls. Moreover, we determined the TNF alpha, IL6, VEGF, VEGFR1 and VEGFR2 gene expression in the liver of these simple steatosis and NASH patients. The population consisted of 30 obese patients, which were diagnosed with simple steatosis and 32 patients with NASH and compared to 30 age-and-sex matched healthy controls. Mean serum TNF alpha levels were elevated in the serum of simple steatosis and NASH patients compared to healthy controls, reaching significance in NASH patients. IL6 was significantly increased in simple steatosis and NASH patients compared to the healthy controls. VEGF levels were significantly elevated in patients with simple steatosis and borderline significantly elevated in NASH patients compared to the serum levels of healthy control subjects. The concentration of sVEGFR1 was significantly increased in serum of simple steatosis and NASH patients compared to controls. sVEGFR2 concentration was not significantly different in the three groups. TNF alpha mRNA expression was higher in NASH patients compared to simple steatosis patients. Hepatic gene expression of VEGF, VEGFR1 and VEGFR2 were slightly decreased in NASH patients compared to simple steatosis patients.These data indicate the involvement of inflammatory (TNF alpha and IL6), angiogenic (VEGF) cytokines and sVEGFR1 in the pathophysiology of NAFLD. (C) 2012 Elsevier Ltd. All rights reserved.

Aim Direct and indirect effects of leptin on hepatic stellate cells (HSCs) have been documented in the literature, whereas little is known about leptin’s actions on hepatocytes. Leptin mediates its profibrogenic and proinflammatory effects on HSCs in part through the production of intracellular reactive oxygen species (ROS). In this study, we focus our analysis on leptin-induced ROS production in hepatocytes. Methods The expression of leptin receptor isoforms on primary mouse liver cells was examined by real-time quantitative-PCR and western blotting. Cultures were exposed to leptin in combination with inhibitors for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, MAP kinase/ERK kinase 1 (MEK1) or janus kinase 2 (JAK2). ROS levels were quantified by measuring fluorescence. The effects of leptin on hepatocyte functions and programmed cell death were evaluated by fluorescent or luminescent assays. Results Leptin induced ROS production in primary hepatocytes by 150–450%, compared with a 20–30% increase in HSCs and liver sinusoidal endothelial cells (LSECs). This ROS production could be inhibited by NADPH oxidase, MEK1 and JAK2 inhibitors. Western blotting indicated that mouse HSCs and LSECs mainly express short leptin receptor isoforms, whereas hepatocytes appeared to express both short and long isoform(s). Leptin-induced ROS production in db/db hepatocytes did not differ from wild-type mice. Finally, leptin had no negative influence on primary hepatocyte functions. Conclusion Leptin induced higher ROS levels in primary hepatocytes than in LSECs and HSCs, depending on NADPH oxidase, MEK1 and JAK2 signalling but not on the long leptin receptor isoform. Furthermore, leptin exposure did not influence primary hepatocyte functionality negatively.

Background & Aims: The two major primary liver cancers in adults are hepatocellular carcinoma and cholangiocarcinoma. These tumors rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors. Recently, interest has grown in the regulators of these factors. Several reports have been published describing the role of prolyl hydroxylase domains - the key oxygen sensor responsible for the degradation of hypoxia inducible factors - in tumor progression and vascularisation. The effect of prolyl hydroxylase domain 2 on the pathogenesis of liver cancer has never been studied.Methods: A diethylnitrosamine-induced mouse model was used in this study, allowing primary hepatic tumors to occur as a result of chronic liver damage. Several parameters of prolyl hydroxylase domain 2-haplodeficient mice were compared to those of wild type mice, thereby focussing on the expression of angiogenic factors and on the hepatic progenitor cell activation and differentiation.Results: This study shows that inhibiting prolyl hydroxylase domain 2 increases the hepatocarcinogenesis and stimulates the development of cholangiocarcinoma. Furthermore, PHD2 deficiency and the accompanying continuous HIF activation, selected for a more metastatic tumor phenotype.Conclusions: The effect of prolyl hydroxylase domain 2 deficiency on hepatocarcinogenesis hold a great potential for therapeutic intervention, since hypoxia and the selection for a more aggressive cholangiocarcinoma phenotype might also have a repercussion on patients receiving long-term treatment with anti-angiogenic compounds. (C) 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.