nvh – dutch liver retreat · nvh - dutch liver retreat 2020 page | 2 dear participants of the...

NVH - Dutch Liver Retreat 2020

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Index Program Page Welcome 2 Program day 1 3 Program day 2 5 Abstracts Session 1 6 Session 2 12 Session 3 17 Session 4 21 Session 5 26 Session 6 31 List of participants 35 Notes 38 Sponsors 39


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Dear Participants of the 10th DLR, Welcome to the 10th Dutch Liver Retreat! Please find the program below:

1. A main program with oral presentations with junior scientists appointed as session chairs. 2. A scientific speed-date session and a competition for the best liver research idea of DLR 2020. 3. A group session. More information will follow. 4. Ample opportunities to meet and discuss with your fellow liver scientists.

The DLR is organized by the Netherlands Association for the Study of the Liver (NASL or NVH). The DLR2020 offers members a strong discount on the registration fee. Furthermore, members are eligible for the NASL/NVH travel grants to go to international scientific meetings and funds for printing your PhD thesis. Moreover, you will receive our quarterly magazine “Lever” with all the news and views on Dutch Hepatology. We look forward to an exciting DLR2020! Ronit Sverdlov (MUMC), Stan van de Graaf (AUMC, loc. AMC), Bart (A.J.A.) van de Sluis (UMCG) and Floris Roos (Erasmus MC)


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Thursday February 6, 2020 9.45 – 10.50 Registration with coffee/tea 10.50 – 11.00 Welcome by organizing committee Full presentations (F) are 15 min with 5 min discussion Short Pitch (P) are 5 min with 5 min discussion 11.00 – 12.30 Session 1 Session chairs: A. Saeed & T. Houben F. Qinghong Li (UMCG) P. Turtushikh Damba (UMCG) F. Ronald Oude Elferink (AMC) P. Roni Kunst (AMC) F. Richell Booijink (UT) P. Sabrah Niesten (Erasmus MC) 12.30 – 13.30 Lunch

13.30 – 14.50 Session 2 Session chairs: J.C. Chang & W. van Zwol F. Martijn Rutten (UMCG) P. Andries Heida (UMCG) F. Eline Geervliet (UT) P. Lavinija Matakovic (UMCG) F. Valentina Gomez Mellado (AMC) 14.50 – 15.20 Coffee/Tea break 15.20 – 16.30 Session 3: Session chairs: D.Y. Vos & F.A. Aguilar F. Anna Palmiotti (UMCG) F. Tom Houben (MUMC) P. Begoña Porteiro (AMC) F. Ali Saeed (UMCG)


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16.30 – 17.00 Coffee/Tea break 17.00 – 18.00 Keynote speaker: The Age of Organoids: Liver Disease Modeling, Precision Medicine and Tissue Engineering Luc van der Laan, professor of regenerative medicine of the liver, Erasmus MC 18.00 – 18.30 Speed date session 18.30 – 20.00 Dinner 20.00 – 21.00 Group session 21.00 Bar, Drinks, Dance Prof. dr. Luc van der Laan is Professor in Liver Regenerative Medicine and Head of the Laboratory of Experimental Transplantation and Intestinal Surgery (LETIS) at the Erasmus MC, Dept. of Surgery. He started his career by studying biology at the University of Amsterdam (Uva) and obtained his PhD in the field of immunology at the Vrije Universiteit Amsterdam (VU Amsterdam) in 1998. Afterword’s he worked for 3 years as a Post-Doc in the Scripps Research Institute (San Diego, California) under supervision of prof. dr. Salomon. It is here, where his interest was raised in transplantation- and regenerative medicine (van der Laan LJW et al. Nature 2000). Returning to the Netherlands he became a staff researcher at The Erasmus MC to continue his reseach on transplantation and viral infections. Nowadays, his research focus has shifted towards tissue injury and regeneration in liver disease and liver transplantation. His research has a strongly translational character and aims to provide basic and applied knowledge to improve the outcome of patients with end-stage liver failure or liver cancer. Novel regenerative strategies to improve liver graft quality involve perfusion systems and applications of human hepatic mesenchymal stromal cells and epithelial stem cell-derived liver or bile duct organoids (key publications: Huch M, Van der Laan LJW, Clevers H, Cell 2014; Broutier L, Verstegen MMA, Van der Laan LJW, Huch M, Nat. Med. 2017).


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Friday February 7, 2020 7.00 – 9.00 Breakfast 09.00 – 10.10 Session 4 Session chairs: M. Rutten & B.P. Porteiro F. Jung-Chin Chang (AMC) P. Yana Geng (UMCG) F. Dyonne Vos (UMCG) P. Willemien van Zwol (UMCG) P. Ana Ortiz Perez (UT) 10.10 – 10.40 Coffee/Tea break 10.40 – 12.10 Session 5 Session chairs: R. Bansal & T. Damba F. Willemien van Zwol (UMCG) F. Cong Liu (LUMC) P. Cristy Verzijl (UMCG) F. Simei Go (AMC)

12.10 – 13.00 Lunch 13.00 – 14.20 Session 6 Session chairs: S. Go & R.C. Verzijl F. Natalia Loaiza (UMCG) F. M. Zhang (UMCG) F. Hilde Nijland (UT) F. Enschen Zhou (LUMC) 14.20 – 14.40 Coffee/Tea break 14.40 – 15.40 Presentation of Breakout projects &

winner selection 15.40 – 15.55 Final Remarks & Closure

F. Fabio Aguilar (UMCG)


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Session 1 A molecular mechanism underlying genotype-specific intrahepatic cholestasis resulting from MYO5B mutations Q. Li, A. Overeem, Y. Qiu, F. Carton-Garciá, C. Leng, K. Klappe, J. Wang, D. Arango University Medical Center Groningen, Groningen, The Netherlands. Background and rationale for the study: Progressive familial intrahepatic cholestasis (PFIC)-type 6 has been associated with missense but not biallelic nonsense or frameshift mutations in MYO5B, encoding the motor protein myoVb. This genotype-phenotype correlation and the mechanism via which MYO5B mutations give rise to PFIC are not understood. The aim of this study was to determine whether the loss of myoVb or expression of patient-specific myoVb mutants can be causally related to defects in canalicular protein localization and, if so, via which mechanism. Main results: We demonstrate that the cholestasis-associated P660L mutation in myoVb caused the intracellular accumulation of bile canalicular proteins in vesicular compartments. Remarkably, the knockout of MYO5B in vitro and in vivo produced no canalicular localization defects. In contrast, the expression of myoVb mutants consisting of only the tail domain phenocopied the effects of the Myo5b-P660L mutation. Using additional myoVb and rab11a mutants, we demonstrate that motor domain-deficient myoVb inhibited the formation of specialized apical recycling endosomes, and that its disrupting effect on the localization of canalicular proteins was dependent on its interaction with active rab11a and occurred at the trans-Golgi Network/recycling endosome interface. Conclusions: Our results reveal a mechanism via which MYO5B motor domain mutations can cause the mislocalization of canalicular proteins in hepatocytes which, unexpectedly, does not involve myoVb loss-of-function but, as we propose, a rab11a-mediated gain-of-toxic function. The results explain why biallelic MYO5B mutations that affect the motor domain but not those that eliminate myoVb expression are associated with PFIC-type 6.


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Session 1 Inhibition of endogenous hydrogen sulfide production reduces activation of hepatic stellate cells via induction of cellular senescence T. Damba1, M.E.N.G.F Zhang1, M. Buist-Homan2, H.V. Goor3, A.J. Moshage2 1Dep. Gastroenterology and Hepatology, 2Dep. Gastroenterology and Hepatology, Dep. Laboratory Medicine, 3Dep. Pathology and Medical Biology, UMCG, Groningen, The Netherlands.

Background: The hepatic stellate cell (HSC) is the key regulatory cell during liver fibrosis. During fibrogenesis, non-proliferating quiescent stellate cells transdifferentiate to highly proliferative and fibrogenic myofibroblast-like cells. Recent studies indicate that the induction of senescence in HSCs leads to the resolution of liver fibrosis. Cellular senescence is characterized by an irreversible cell-cycle arrest, resulting in a reduction of cell proliferation and increased senescence-associated secretory phenotype (SASP). The gasotransmitter hydrogen sulfide (H2S) has anti-senescence properties and modulates different signal transduction pathways in pathophysiological conditions. Endogenous H2S is produced by the enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (MPST). Recently we reported that CTH/H2S expression increased during HSC activation and that the inhibition of endogenous H2S shows anti-fibrotic effects. However, little is known about the mechanism of the anti-fibrotic effect of endogenous H2S in HSCs and its relation to senescence. We hypothesize that the inhibition of endogenous H2S production induces cellular senescence. Methods: Rat HSCs were isolated and cultured for 7 days for complete activation and then treated with H2S slow-releasing donor GYY4137 and/or the CTH inhibitor DL- propargylglycine (DL-PAG), as well as the PI3K inhibitor LY294002. Results: CTH was significantly upregulated in fully activated HSCs compared to the quiescent state. Inhibition of CTH by DL-PAG reduced HSCs proliferation and expression of the fibrotic markers Col1a1 and Acta2, while simultaneously increasing the cell-cycle arrest markers Cdkn1a, p53, senescent associated (SA) secretory phenotype marker IL6 and SA-β-gal staining. In addition, exogenous H2S donor GYY4137 partially restored the proliferation of HSCs and repressed the increased expression level of senescence markers Cdkn1a, p53 and SA-β-gal staining induced by DL-PAG. . The induction of senescence by DL-PAG was accompanied by increased phosphorylation of Akt. The non-selective PI3K inhibitor LY294002 blocks Akt activity. Co-treatment of LY294002 partially reduced the senescence phenotype markers Cdkn1a, Tp53 and SA-β-gal activity of HSCs induced by DL-PAG. Conclusion: Inhibition of endogenous H2S production reduces hepatic stellate cell activation via induction of cellular senescence in a PI3K-Akt dependent manner. Our results suggest that H2S and/or induction of senescence may be a novel target for anti-fibrotic therapy.


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Session 1 Identification of a pruritogen in plasma of cholestatic patients that may contribute to cholestatic itch. J. Langedijk1, W.D. Tolenaars1, M. van Weeghel2, D.R. de Waart1, K.F.J. van de Graaf1, C.C. Paulusma1, H.J. Verkade3, U.H. Beuers1, R.P. Oude Elferink1 1Dept. Gastroenterology and Hepatology, Amsterdam UMC, Amsterdam, The Netherlands. 2Metabolomics Facility, Amsterdam UMC, Amsterdam, The Netherlands. 3Dept. Pediatrics, UMC Groningen, Groningen, The Netherlands.

Itch is a frequent symptom in patients with chronic cholestasis. Biliary diversion often rapidly reduces cholestatic itch. We hypothesized that biliary diversion efficiently removes pruritogens, present in the enterohepatic circulation, which during cholestasis spill over to the peripheral circulation. A number of receptors, such as TRPV1, TRPA1 and MRGPR act as chemical sensors on itch neurons. In order to identify pruritogens we screened bile collected from diverted itchy patients (PBC and PSC) for ligands that activate these receptors. Methods: Bile collected during biliary diversion was fractionated by HPLC and fractions were screened for activation of cells overexpressing TRPA1 using a fluorescent Ca2+ assay. Receptor specificity was verified with inhibitors and non-transduced HEK cells. Active fractions were analyzed by mass spectrometry and compounds were identified by fragmentation patterns using Metfrag software. Results: Early diverted bile from a PBC patient activated TRPA1 cells at dilutions up to 2000-fold while bile collected after 6 days of diversion was 40-fold less potent. HPLC fractionation of the early bile sample revealed 4 TRPA1-activating peaks of which three coincided with the presence of bile salts. Indeed, pure conjugated bile salts also activated HEK-TRPA1 cells, but only at concentrations >100 uM. A fourth, more hydrophobic peak, not containing bile salt, most strongly activated TRPA1. In this fraction three compounds were detected by HPLC-MS, of which only one was also observed in diverted bile from other patients (PBC, PSC, Alagille and PFIC). MS/MS fragmentation identified this compound as 9,10-Epoxy-13-hydroxy-11-octadecenoic (9-EPHOME), an oxidized linoleic acid metabolite (OXLAM). We subsequently analyzed serum from cholestatic patients for OXLAMs and found a prominent peak that was identified as 9-hydroperoxyoctadeca-10,12-dienoic acid (9-HPODE) which is the a precursor molecule to 9-EPHOME. Concentrations of 9-HPODE in serum and 9-EPHOME in bile were significantly higher in cholestatic persons compared to controls. Administration of pure 9-HPODE to HEK-TRPA1 cells confirmed its potency as a TRPA1 agonist (EC50 = 1.5 uM). Intradermal injection of 9-HPODE in wild type mice induced significant scratch behavior, which was not observed in Trpa1-/- mice. Conclusion: 9-HPODE is a potent pruritogen, present in the plasma of patients with cholestatic itch.


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Session 1 Looking beyond TGR5: search for potentially new cell surface bile acid receptors R.F. Kunst1, S. Kooijman2, J.M. Donkers1, K.F.J. van de Graaf1 1Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands. 2Dept. of Medicine, Division of Endocrinology, LUMC, Leiden, The Netherlands. Background: The sodium-taurocholate cotransporting polypeptide (NTCP) transports bile salts from the portal vein back into the hepatocytes. Therefore, inhibition of NTCP prolongs the presence of elevated plasma bile acid concentrations and postprandially stimulates bile acid signaling. As a consequence, the NTCP knock-out mouse has reduced body weight gain, increased energy expenditure and upregulated brown adipose tissue activity compared to wild-type mice when fed a high fat diet (Donkers et al., JCI Insight 2019). The G protein-coupled bile acid receptor 1 (GPBAR1/TGR5) is a well-known regulator of body weight. However, NTCP-TGR5 double knock out mice have a similar reduction in body weight gain as the NTCP knock out mice upon high fat diet. Hypothesis We speculate that prolonged bile acid signaling causes cell surface membrane receptor activation other than TGR5, that can explain the observed metabolic changes in our in vivo model. Research Plan: First, we will use the G-protein coupled receptor (GPCR) PRESTO-TANGO screen (Kroeze et al., Nat Struct Mol Biology 2015) with 314 human orphan and non-orphan receptors to find receptors that are activated by bile salts. Thereafter, we aim to localize bile salt-responsive receptors in different tissues, and examine the effect of receptor activation in vitro. Furthermore, the metabolic profile of the NTCP-TGR5 double knock out mouse will be determined using calorimetric cages. Anticipated results: We expect an increase in energy expenditure of NTCP-TGR5 double knock out mice compared to wild type and TGR5 KO mice upon high fat diet. In addition, we expect to find GPCRs that are activated upon bile salt exposure, differentially expressed in various tissues such as brown adipose tissue, macrophages and the small intestine, explaining TGR5-independent effects of bile salts.


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Session 1 Autotaxin as a novel therapeutic target in non-alcoholic fatty liver disease R.S. Booijink1, F. Salgado Polo2, B. Özturk Ackora1, C. Jamieson3, A. Perrakis4, R. Bansal1 1Dept. of Medical Cell BioPhysics, University of Twente, Enschede, The Netherlands. 2Division of Biochemistry, The Netherlands Cancer Institute, and Oncode Institute, Amsterdam, The Netherlands. 3Dept. of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United Kingdom. 4Division of Biochemistry, the Netherlands Cancer Institute, Amsterdam and Oncod, Amsterdam, The Netherlands.

Background: Non-alcoholic fatty liver disease (NAFLD) is a growing cause of mortality worldwide. Due to increasing incidences of diabetes and obesity, the prevalence of NAFLD is growing rapidly. 30% of affected people develop severe form of NAFLD, non-alcoholic steatohepatitis (NASH) that further progresses to cirrhosis and hepatocellular carcinoma. Currently, there is an unmet need for the effective therapy for the treatment of this disease. Recently, upregulated serum levels lysophosphatidic acid (LPA) and autotaxin (ATX, ENPP2), the enzyme that generates LPA in vivo have been identified in various chronic liver diseases (CLDs), including NASH suggesting the implication of the ATX-LPA axis in NASH progression. Therefore, we hypothesized that the inhibition of the ATX-LPA signaling pathway might be a promising approach to attenuate NASH. In this study, we investigated a novel small molecule type IV ATX inhibitor FP10.47, in vitro in hepatic stellate cells (HSCs) and inflammatory macrophages, and in vivo in a NASH mouse model. Methods: ATX gene and protein expression was examined in human disease livers and in vivo in mouse models. Thereafter, the in vitro effectivity of ATX inhibitor FP10.47 on fibrotic parameters, contractility and wound healing was evaluated in TGFβ-activated human HSCs. Furthermore, effects of FP10.47 on inflammatory parameters and migration was evaluated in M1-activated murine macrophages. Finally, FP10.47 was evaluated in a methionine-choline-deficient (MCD) induced NASH mouse model. Results: Upregulation of ATX mRNA levels were identified in CLD patients with different etiologies. Additionally, ATX protein expression was upregulated in murine NASH and alcoholic steatohepatitis (ASH). In vitro, FP10.47 inhibited TGFβ-induced HSCs activation, migration and contractility on both protein and gene expression level. M1-mediated macrophage activation and migration was furthermore reduced by FP10.47. In vivo, in established NASH mouse model, post-disease treatment with FP10.47 attenuated fibrosis, steatosis and inflammation, as observed histologically, as well as on gene expression profiles. Furthermore, FP10.47 significantly reduced ALT, AST, cholesterol and triglyceride plasma levels. Conclusion: The ATX-LPA pathway is a novel player in the pathogenesis of NASH, and inhibition of ATX is a promising therapeutic approach for the treatment of this disease.


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Session 1 Creating branching biliary organoids to study the intrahepatic biliary tree L.A. Muñoz Albarinos, F.J.M. Roos, S. Niesten, H.J. Metselaar, S. Darwish Murad, M. van Royen, J.N.M. IJzermans, M.M.A. Verstegen, L.J.W. van der Laan Dept. Gastroenterology and Hepatology, Erasmus MC, Rotterdam, The Netherlands. Introduction: The biliary tree is divided into intra- and extrahepatic bile ducts which are lined by cholangiocytes. Although very similar in their function, morphologically they differ. A number of diseases affect the biliary tree, and to model these cholangiopathies, novel models are needed. Recently, organoids cultured from extrahepatic cholangiocytes were established. However, intrahepatic branching biliary organoid models are still lacking. Thus, we set out to create biliary branching organoids as a model to study the intrahepatic biliary tree and the pathobiology of intrahepatic cholangiopathies. Methods: Organoids were initiated from liver biopsies obtained from healthy donors during transplantation and cultured according to our newly established protocol for branching organoids. Branching organoids were compared to their paired non-branching organoids (from the same donor) on gene expression (using qPCR and single cell RNA sequencing) as well as protein expression (using immunofluorescent staining and enzyme-linked immunosorbent assays). Phenotypic analyses and branching ability were assessed by time lapse confocal microscopy. Results: Branching organoids (ductoids) could be cultured from liver biopsies (7/10). The ductoids form self-organizing branching tubular-like structures, morphologically resembling the intrahepatic biliary tree. Ductoids can be cultured long-term without losing their branching morphology (>6 months, n=3). Single cell RNA sequencing analyses revealed a unique differential gene expression of the branching organoids compared to the non-branching organoids, with more intrahepatic differentiation features in the branching organoids. Immunofluorescence showed expression of mature cholangiocyte markers (cytokeratin 7 and 19) and revealed a ductal columnar organization of the cells. Furthermore, Rhodamine-123 assay showed an open lumen and functional activity of the typical cholangiocyte transporter Multi Drug Resistance Protein-1. Finally, confocal time imaging demonstrated how the organoids branch. EdU incorportation showed only incorporation (and thus proliferation) at the tips of the branches. Moreover, it showed that tips do not fuse together and thus do not create a shunt. Conclusions: With the establishment of the self-organizing branching biliary organoids we successfully created the first human adult cell-derived system that resembles intrahepatic bile ducts in vitro. Potentially, ductoids could serve as a model to study intrahepatic cholangiopathies, embryogenic bile duct development and could potentially be employed in regenerative medicine.


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Session 2 CRISPR-based disease modeling for GSD Ia: clinical spectrum and gene-gene interactions M.G.S. Rutten1, N.C.A. Huijkman1, T. Bos1, N.J. Kloosterhuis1, R.E. Thomas2, A. de Bruin2, B. van de Sluis1, M.H. Oosterveer1 1UMCG, Groningen, The Netherlands. 2Dutch Molecular Pathology Center, Faculty of Veterinary Medicine, Utrecht, The Netherlands. Background: Glycogen storage disease type 1 (GSD Ia) is an inborn error of metabolism caused by a defect in glucose-6-phosphatase (G6PC) activity and primarily results in fasting hypoglycemia. Evidence is accumulating that glycemic control in GSD Ia patients affects the risk of long-term complications such as liver tumor development. Moreover, heterogeneity in glycemic control as well as other symptoms and complications is observed between individual patients. Because the available mouse models do not cover the spectrum of glycaemia and biochemical phenotypes in GSD Ia patients, we employed CRISPR/Cas9 gene editing technology to generate hepatocyte-specific GSD Ia mice with variable G6PC activities in the liver. In addition, we used this approach to investigate the interaction between G6PC and Carbohydrate Response Element Binding Protein (ChREBP) to liver disease development in GSD Ia. Methods: To study the spectrum of GSD Ia disease phenotypes, male hepatocyte-specific Cas9-expressing mice were injected with various titers of adenovirus particles expressing three sgRNAs targeting G6pc and sacrificed 4 weeks after virus injection. As proof-of-concept to study gene-gene interactions, male hepatocyte-specific Cas9-expressing mice were injected with sgRNAs targeting G6pc, with/without sgRNAs targeting either Chrebpα or Chrebpα/β. These animals were sacrificed 2 weeks after virus injection. Results: sgRNA-G6pc injection in Cas9-expressing mice resulted in dose-dependent reductions in hepatic G6PC expression, residual G6Pase activities, and fasting blood glucose levels. Furthermore, it dose-dependently increased hepatomegaly and hepatocyte vacuolation, hepatic glycogen, glucose-6-phosphate, and lipid contents, as well as plasma triglyceride levels. Simultaneous editing of hepatic G6pc and Chrebp exacerbated GSD Ia hepatomegaly, as liver weight and hepatic content of glucose-6-phosphate, glycogen, and lipids showed additional increases compared to mice deficient for hepatic G6pc only. Discussion: CRISPR/Cas9-mediated inactivation of hepatic G6pc elicits the biochemical symptoms of GSD Ia in a dose-dependent fashion. Hence, this novel approach can be employed to induce a spectrum of disease phenotypes, thereby enabling mechanistic investigation of the relationship between glycemic control and long-term complications in GSD Ia. Moreover, this system allows editing of multiple genes simultaneously and thus enables to systematically study gene-gene interactions in the pathophysiology of GSD Ia.


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Session 2 Is the liver-specific Hep-lncRNA a gatekeeper of hepatic differentiation? A. Heida1, B. Atanasovska1, M. Rutten1, N. Huijkman1, N. Kloosterhuis1, A. de Bruin3, M. Oosterveer1, J. Yang-Fu1,2, B. van de Sluis1 1Dept. of Pediatrics, UMCG, Groningen, The Netherlands. 2Dept. of Genetics, UMCG, Groningen, The Netherlands. 3Dutch Molecular Pathology Center, UU, Utrecht, The Netherlands. Background: Non-alcoholic fatty liver disease (NAFLD) is characterized by fat accumulation in the liver and can further progress to non-alcoholic steatohepatitis (NASH). NASH is characterized by steatosis, lobular inflammation and signs of liver damage. NASH livers are predisposed to other complications, such as fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Although the mechanism by which NAFLD progresses to NASH is not well understood accumulating data implicate a role for long non-coding RNAs (lncRNAs) in lipogenesis, inflammation and hepatic proliferation and thus might contribute to the disease progression of NAFLD. Recently, we have identified a liver-specific lncRNA (Hep-lncRNA) whose expression is negatively correlated with NASH grade in humans and mice. In this study, we aim to elucidate the contribution of Hep-lncRNA to NASH development. Methods: Using somatic CRISPR/Cas9 gene editing, Hep-lncRNA was specifically targeted in mouse livers. Wildtype and hepatic deficient Hep-lncRNA mice were fed either a chow or a high-fat high cholesterol diet (HFC), subsequently, hepatic inflammation and lipid accumulation were determined. In addition, the expression of Hep-lncRNA was determined in other preclinical models for liver diseases. Results: Hepatic ablation of Hep-lncRNA did not lead to changes in body weight or liver weight compared to wild-type mice. Histological and biochemical analyses suggest that hepatic loss of Hep-lncRNA does not affect the development of simple steatosis. However, the expression of several inflammatory markers, such as TNF-α and CD68, were slightly increased upon Hep-lncRNA ablation. Interestingly, in other preclinical liver disease models, we found that Hep-lncRNA expression was positively correlated with markers for hepatic differentiation, and negatively correlated with hepatic proliferation. Furthermore, preliminary data show that the lncRNA is highly upregulated during the acute phase response after partial hepatectomy (PH) but strongly down-regulated during the hepatic proliferating phase. Conclusion: Although we did not find a strong evidence for a role of lncRNA in NASH development, we hypothesize that Hep-lncRNA is essential to maintain hepatic differentiation, and currently, additional experiments are being performed to test our hypothesis.


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Session 2 pH-responsive polymersomes for matrix metalloproteinase-1 delivery as a novel therapeutic approach for the treatment of liver fibrosis E.K. Geervliet1, D. Appelhans2, S. Moreno Pinilla2, R. Bansal1 1University of Twente, Enschede, The Netherlands. 2Leibniz Institute for polymer research Dresden e.v., Dresden, Germany.

Background: Liver fibrosis is a growing health problem affecting millions of people worldwide. Chronic liver injury leads to the formation of scar tissue due to the excessive accumulation of extracellular matrix, mainly collagen-I and -III produced by activated hepatic stellate cells (HSCs). Currently, there are no therapies available and liver transplantation is the only option, however has limitations. Matrix metalloproteinase-1 (MMP1) is an enzyme that degrades the scar tissue by degrading collagen-I and -III favoring liver regeneration. We hypothesized that liver-specific delivery of MMP1 as a promising approach for the treatment of liver cirrhosis. Using state-of-the-art technologies, we synthesized innovative pH-responsive smart MMP1-polymersomes with the MMP1 decorated on the surface of polymersomes. Finally, we investigated the therapeutic efficacy of MMP1 and MMP1-polymersomes on human HSCs in vitro and on liver fibrosis mouse models in vivo, and ex vivo on fibrotic mouse livers. Methods: Polymersomes were fabricated using the pH switch method and MMP1 post-loading at pH 5-6 was performed to increase the interaction between MMP1 and the polymersome membrane. Physiochemical characterization and enzymatic assays were performed to characterize the attachment and functionalization of enzyme on polymersomes. In vitro studies were performed on TGF-beta-activated human HSCs to evaluate the efficacy of MMP1 and polymersome-MMP1 on the cell viability, wound healing, gene and protein expression of collagen and HSCs activation marker alpha-SMA. Finally, the efficacy of MMP1 and polymersome-MMP1 was evaluated in vivo and ex vivo on CCl4-induced liver fibrosis mouse models, and investigated for toxicity and therapeutic efficacy Results: Decoration of MMP1 on the surface of the polymersome was successfully established with an affinity of 30%, without inhibition of function. Synthesized MMP1-polymersome hybrid structures showed favorable size and charge. MMP1 and more significantly polymersome-MMP1 showed dose-dependent inhibition of collagen-I, -III and alpha-SMA gene expression, and collagen-I protein expression in TGF-beta-activated human HSCs with no significant effects on cell viability. The studies on CCl4-induced liver fibrosis mouse models have been performed, and are currently under investigation. Conclusions: In conclusion, we present an innovative approach of MMP1 delivery for the treatment of liver fibrosis.


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Session 2 Protein therapy for hereditary thrombophilia and haemophilia L.M. Matakovic, K.K. Klappe, R.M. Mulder, K.M. Meijer, S.C.D. van IJzendoorn University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. Background: The blood-clotting disorders haemophilia C, due to factor XI deficiency and thrombophilia due to protein S deficiency are rare diseases which can be caused by nonsense mutations in the FXI and PROS1 gene, respectively. These proteins are produced by hepatocytes in the liver and secreted into the blood. Current therapies based on the administration of anticoagulant or coagulation factors, are expensive and associated with increased risk of overdose. Therefore, new alternative treatment would be to rescue the nonsense mutation by drug-induced read-through. Methods: To test if the treatment with aminoglycosides (gentamicin and geneticin) would promote read-through of specific nonsense mutant forms of PROS1 and FXI, the nonsense mutations generating truncated forms of PROS1 and FXI will be expressed in transiently transfected HEK-293 cells. Furthermore, the patient-specific iPSC lines from carefully selected patients are generated and will be used for differentiation into functional human-induced hepatocytes (hiHEPS) and assessing the effectiveness of most promising “readthrough compounds”. Conclusion: However, this does not rule out the possibility of nonsense-mediated mRNA decay affecting any or all of these nonsense mutations in vivo. This is the first step towards a new personalized “first-in-class” preventive treatment of hereditary coagulation diseases.


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Session 2 ATP8B1 Deficiency Affects Lipid Metabolism in Human Macrophages V.E. Gómez Mellado, K.S. Ho-Mok, R.P.J. Oude Elferink, C.C. Paulusma Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands. Background: ATP8B1 deficiency causes cholestatic liver disease characterized by a continuous disease spectrum, from intermittent in Benign Recurrent Intrahepatic Cholestasis type 1 (BRIC1) to progressive in Progressive Familial Intrahepatic Cholestasis type 1 (PFIC1). PFIC1 usually associates with dyslipidemia, which is manifested by low serum HDL, high oxidized LDL (oxLDL) and hypertriglyceridemia, while serum total cholesterol levels are low to normal. The etiology of dyslipidemia is not understood and is subject of our present research. Here we studied the role of ATP8B1 in lipid metabolism in macrophages. Methods: Experiments were performed in human ATP8B1-deficient THP-1 macrophages. Fluorescently-labeled native low-density lipoprotein (LDL) uptake and accumulation was measured by FACS analysis. Cholesterol and lipid distribution was studied by filipin and Nile red stainings. Proteins and mRNAs relevant for lipid metabolism were studied by Western blot analysis and quantitative RT-PCR, respectively, w/wo activation of LXR by T0901317. Lipidomic analysis was performed on ATP8B1-depleted THP-1 cells. Bone marrow-derived macrophages were isolated from wild type and ATP8B1 mutant mice. Results: ATP8B1 knockdown THP-1 macrophages showed increased accumulation of fluorescence after incubation with fluorescently-labeled LDL. Filipin staining showed affected distribution of cholesterol. Quantitative PCR showed downregulation of transcription factors and targets involved in cholesterol and fatty acid metabolism, including LXRα, SREBP1, HMG-CoA reductase, FAS, SCD1 and ABCA1. Downregulation coincided with reduced abundance of several triglyceride species as demonstrated by lipodomic analysis. ABCA1 protein was strongly decreased under both lipoprotein-supplemented and -deficient conditions, however recovered completely upon incubation with T0901317 as shown by surface biotinylation experiments. All experiments are currently being conducted in wild type and ATP8B1-deficient murine bone marrow-derived macrophages and results will be presen ted. Conclusion: ATP8B1 plays an important role in cholesterol and fatty acid metabolism in human macrophages and its deficiency may contribute to dyslipidemia in PFIC1 disease.


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Session 3 Cholangiopathy and biliary fibrosis in Cyp2c70-deficient mice with a human-like bile acid pool are fully reversed by ursodeoxycholic acid A. Palmiotti1, R. Li1, H.D. de Vries2, N.L. Mulder1, M.V. Hovingh1, M. Koehorst2, V.W. Bloks1, B. van de Sluis1, J.F. de Boer3, F. Kuipers3 1Dept. of Pediatrics, 2Dept. of Laboratory Medicine, 3Dept. of Pediatrics and Laboratory Medicine, UMCG, Groningen, The Netherlands. Background: Bile acids (BA) are natural detergents that aid intestinal absorption of fat-soluble nutrients and exert hormone-like functions by signaling via nuclear (e.g., FXR) and membrane-bound (e.g., TGR5) receptors. The latter have been identified as bona fide drug targets for human diseases. The various BA species that are present in the BA pool possess different detergent properties and signaling characteristics. Consequently, differences in BA composition between humans and mice, particularly the presence of hydrophilic muricholic acids (MCA) in the latter, hamper translation of preclinical outcomes to the human situation. We have recently shown that acute knock-down of hepatic Cyp2c70 in mice strongly reduces the contribution of MCA in the BA pool and alters responses to pharmacological FXR activation. Now, we have generated a Cyp2c70-deficient mouse model to study the impact of a human-like BA pool during early development that can be used to evaluate treatment strategies for pediatric cholestatic syndromes and for metabolic diseases that target BA signaling pathways. Methods: BA-related parameters, gene expression and liver morphology were assessed in male and female Cyp2c70-/- and wild-type mice at different ages. Results: Plasma and bile of Cyp2c70-/- mice were completely devoid of MCA and showed high abundances of hydrophobic chenodeoxycholic (~55%) and lithocholic (~6%) acids. BA pool size was unaffected in male but reduced by 40% in female Cyp2c70-/- mice. Bile flow and biliary BA secretion were normal in adult Cyp2c70-/- mice, indicating absence of cholestasis. Nonetheless, plasma BA concentrations were higher and transaminases were ~4-fold increased. The hydrophobic BA pool of Cyp2c70-/- mice was associated with proliferation of cholangiocytes and mild portal fibrosis. Interestingly, plasma BA and transaminases were highest in Cyp2c70-/- mice at weaning and spontaneously decreased with age. Surprisingly, aged female Cyp2c70-/- mice, but not the males, developed severe bridging fibrosis in the absence of steatosis. Addition of UDCA to the diet of Cyp2c70-/- mice completely normalized all hepatic functions and fully restored liver morphology. Conclusion: Cyp2c70-/- mice with a hydrophobic human-like BA pool show transient neonatal cholestasis and develop a Primary Biliary Cholangitis-like phenotype that is fully restored by early treatment with UDCA.


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Session 3 Sex-opposed hepatic inflammatory effects of 27-hydroxycholesterol are mediated via differences in estrogen signaling T. Houben, A.V. Bitorina, Y. Oligschlaeger, M. Jeurissen, S. Rensen, E. Köhler, M. Westerterp, D. Lütjohann, J. Theys, A. Romano, J. Plat, R. Shiri-Sverdlov Maastricht University, Maastricht, The Netherlands. Background: Despite the increasing amount of patients suffering from non-alcoholic steatohepatitis (NASH), the lack of mechanistic understanding towards the progression of the disease still encompasses the largest hurdle for developing effective therapies. We previously demonstrated that the cholesterol derivative, 27-hydroxycholesterol (27HC), reduced cholesterol accumulation in hepatic macrophages thereby reducing hepatic inflammation in female hyperlipidemic mice. However, 27HC paradoxically showed pro-inflammatory responses in the context of atherosclerosis in a comparable male hyperlipidemic mouse model. Due to the differences in sex between these experiments, we investigated whether sex plays a role in the hepatic inflammatory effect of 27HC in the context of NASH. Methods: To explore whether the sex-opposed hepatic inflammatory effects of 27HC translated to humans, plasma 27HC levels were measured and correlated with hepatic inflammatory parameters in obese individuals. Further, to investigate if 27HC exerts sex-opposed effects on inflammation, we injected 27HC to female and male Niemann-Pick disease type C1 mice (Npc1nih), used as a model for cholesterol-induced hepatic inflammation. Finally, the role of estrogen signaling in this mechanism was studied using bone marrow-derived macrophages (BMDMs) that were treated with 27HC and 17ß-estradiol (E2). Results: Plasma 27HC levels showed opposite correlations with hepatic inflammatory indicators between female and male obese individuals. Likewise, female Npc1nih mice showed reduced hepatic inflammation after 27HC injections in contrast to male Npc1nih mice. Similarly, hepatic 27HC levels oppositely correlated with female and male Npc1nih mice. Furthermore, 27HC administration also oppositely affected inflammation in female and male BMDMs cultured in E2-enriched medium. Interestingly, female BMDMs showed higher ERα expression compared to male BMDMs. Conclusions: Our findings identify that 27HC exerts sex-opposed effects on cholesterol-induced hepatic inflammatory responses that are related to differences in estrogen signaling between females and males. Hence, the individual’s sex needs to be taken into account when 27HC is employed as therapeutic tool.


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Session 3

Role of bile acid accumulation in the phenotype of FXR-deficient mice: focus on diet-induced obesity and liver carcinogenesis B.P. Porteiro, R.F. Kunst, J.M. Donkers, K.F.J. van de Graaf Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands. Background: The nuclear bile acid receptor farnesoid X receptor (FXR) is a master regulator of many metabolic processes, including the regulation of bile acid, lipid and glucose homeostasis. Activation of the nuclear receptor for bile acids using obeticholic acid decreases the nonalcoholic fatty liver disease activity score [NAS] by ≥2, with no worsening of fibrosis in almost half of the treated patients in te FLINT trial (placebo 21%). Remarkably, also the reverse modulation, genetic inactivation of FXR, leads to an improved metabolic phenotype on a high-fat diet, albeit with significant hepatic steatosis. Furthermore, FXR-/- mice display high levels of the proinflammatory cytokine IL-1β, proto-oncogene β-catenin, and the β-catenin target gene c-Myc at 3 months of age, and spontaneously develop hepatocellular carcinoma (HCC) after 12 months. We have recently identified inhibition of NTCP (Na+-Taurocholate Cotransporting Polypeptide) as a pharmacological strategy to dampen inflammation and lower hepatic steatosis. Hypothesis: We hypothesized that inhibition of hepatic NTCP transporter can have beneficial effects in the prevention or development of hepatocellular carcinoma. Experimental design: To study the potential benefits of prolonged bile acid signaling by NTCP inhibition for HCC, two models will be combined: a pharmacological approach using dimethylnitrosamine (DEN) and the genetic one, using FXR -/- mice. (Anticipated) results NTCP/FXR double knockout mice display reduced hepatosteatosis compared to FXR single knockout mice. Interestingly, the absence of NTCP dampened the strong body weight reduction of FXR KO mice suggesting this phenotype is related to bile salt toxicity. We anticipate an improvement in hepatic fibrosis, cellular proliferation and HCC formation in terms of tumor number and size and reduced expression of several proliferative and inflammatory markers upon genetic NTCP depletion.


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Session 3 Farnesoid X receptor and bile acids regulate vitamin A storage A. Saeed1, J. Yang2, J. Heegsma2, A.K. Groen3, S.W.C. van Mil4, C.C. Paulusma5, L. Zhou6, B. Wang6, K.N. Faber2 1Universitair Medisch Centrum Groningen, Groningen, The Netherlands. 2Dept. Gastroenterology & Hepatology, University Medical Center Groningen, Groningen, The Netherlands. 3Dept. of Pediatrics, University Medical Center Groningen,, Groningen, The Netherlands. 4Dept. of Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands. 5Amsterdam UMC, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands. 6Dept. of Gastroenterology and Hepatology, Tianjin Medical University, Tianjin, China. Abstract: The nuclear receptor Farnesoid X Receptor (FXR) is activated by bile acids and controls multiple metabolic processes, including bile acid, lipid, carbohydrate, amino acid and energy metabolism. Vitamin A is needed for proper metabolic and immune control and requires bile acids for efficient intestinal absorption and storage in the liver. Here, we analyzed whether FXR regulates vitamin A metabolism. Compared to control animals, FXR-null mice showed strongly reduced (>90%) hepatic levels of retinol and retinyl palmitate and a significant reduction in lecithin retinol acyltransferase (LRAT), the enzyme responsible for hepatic vitamin A storage. Hepatic reintroduction of FXR in FXR-null mice induced vitamin A storage in the liver. Hepatic vitamin A levels were normal in intestine-specific FXR-null mice. Obeticholic acid (OCA, 3 weeks) treatment rapidly reduced (>60%) hepatic retinyl palmitate levels in mice, concurrent with strongly increased retinol levels (>5-fold). Similar, but milder effects were observed in cholic acid (12 weeks)-treated mice. OCA did not change hepatic LRAT protein levels, but strongly reduced all enzymes involved in hepatic retinyl ester hydrolysis, involving mostly post-transcriptional mechanisms. In conclusion, vitamin A metabolism in the mouse liver heavily depends on the FXR and FXR-targeted therapies may be prone to cause vitamin A-related pathologies.


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Session 4 Non-canonical regulation of glycogenolysis and the Warburg phenotype by soluble adenylyl cyclase J.C. Chang1, S. Go1, E.H. Gilgioli2, H.L. Li3, H.L. Huang3, L.R. Levin4, J.C. Buck4, A.J. Verhoeven1, R.P.J. Oude Elferink1 1Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands. 2University of Maringá, Dept. of Biochemistry, Paraná, Brazil. 3National Taiwan University, Taipei, Taiwan. 4Dept. of Pharmacology, Weill Cornell Medical College, New york city, United States of America.

Background: In mammalian cells, Cyclic AMP is produced by two different types of adenylyl cyclases: the canonical transmembrane adenylyl cyclases (tmACs, ADCY1~ADCY9) and the evolutionarily more conserved soluble adenylyl cyclase (sAC, ADCY10). While the tmACs generate cAMP at plasma membrane as the second messenger for G protein-coupled receptors, basal sAC activity is maintained by physiologic concentrations of bicarbonate and can be further stimulated by free Ca2+. Due to its 10-fold lower affinity for ATP, sAC activity is also sensitive to changes in cellular ATP level. In this study we investigated the regulation of energy metabolism by sAC. Methods: Human hepatoma cell line HepG2, immortalized human cholangiocyte H69 (known to have high sAC activity), and primary mouse hepatocytes were used as models. The second-generation sAC inhibitor LRE-1 was used and its effects were confirmed with shRNA knockdown. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured by Seahorse XF96 extracellular flux analyzer. ATP production rates were derived from OCR and ECAR. Glycogen and medium metabolites were assayed enzymatically. Cytosolic NADH/NAD+ ratio was monitored by biosensor Peredox-mCherry. Results: Acute inhibition of sAC in the presence of glucose stably reprogrammed cellular metabolism to a Warburg-like phenotype that is characterized by increased glycolysis, decreased oxidative phosphorylation, and an elevated cytosolic NADH/NAD+ ratio. Importantly, the cytosolic adenylate energy charge and the estimated total cellular ATP production rate was not affected by sAC inhibition. While glucagon activates the tmACs-cAMP-PKA axis to induce glycogenolysis, we found that sAC-generated cAMP suppressed glycogenolysis in multiple cell lines tested as well as in primary mouse hepatocytes. Examination of the responsible cAMP effectors revealed that inhibition of Epac1, but not Epac2 or PKA, phenocopied the metabolic phenotype of a sAC-inhibited state. Conclusions: sAC is a metabolic switch that regulates energy homeostasis of cells by balancing the ATP production by glycogenolysis and glycolysis and ATP production by oxidative phosphorylation. In addition, we found that glycogen metabolism is regulated in opposite directions by cAMP depending on its location of synthesis and downstream effectors. While the canonical tmAC-cAMP-PKA axis promotes glycogenolysis, the novel sAC-cAMP-Epac1 axis suppresses glycogenolysis.


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Session 4 Differential toxicity of oleate and palmitate on liver sinusoidal endothelial and vascular endothelial cells in vitro Y. Geng, H. Blokzijl, M. Buist-Homan, H. Moshage Universitair Medisch Centrum Groningen, Groningen, The Netherlands. Introduction: Lipotoxicity, the toxicity of free fatty acids (FFAs) to various cell types, is one of the most important factors driving the progression of non-alcoholic fatty liver disease (NAFLD). Liver sinusoidal endothelial cells (LSECs) are known to play an important role in the initiation and progression of chronic liver diseases, including NAFLD. However, the effect of FFAs on LSECs has not been systematically investigated. Aim of this study was to investigate the effects of FFAs on primary rat LSECs in comparison to the effects on human umbilical vein endothelial cells (HUVECs), frequently used as a model for LSECs. Methods: HUVECs were purchased from Lonza. LSECs were isolated from rats. Cells were exposed to the free fatty acids palmitate (0.5mM) and/or oleate (0.25mM). Necrosis was determined by LDH release and apoptosis by caspase-3 activity assay or Western-blot for p-JNK, cleaved-caspase 3 and cleaved-PARP. Intracellular ATP level was determined by luciferase-based assay. Lipid droplets were visualized by Oil red O staining and intracellular triglyceride (TG) was measured. Expression of mRNAs (ICAM-1, VCAM-1, e-Selectin, eNOS) was measured by quantitative PCR. Results: Palmitate induced apoptosis in both HUVECs and LSECs. In HUVECs, oleate was not toxic and protected against palmitate-induced apoptosis and inhibited palmitate-induced gene expression of ICAM1, VCAM1, e-Selectin and eNOS. However, in freshly isolated LSECs, oleate induced necrosis, a sharp drop of intracellular ATP level and the dysfunction of mitochondrial respiration. Surprisingly, the combination of oleate and palmitate was not toxic and induced lipid droplet formation as well as intracellular TG level in LSECs. Discussion: Our results show that oleate acts differently in HUVECs and LSECs. Oleate is not toxic and protects against palmitate-induced lipotoxicity in HUVECs, whereas it causes acute necrosis in LSECs. Palmitate induces apoptosis in both HUVECs and LSECs, however, the combination of oleate and palmitate does not induce cell death. Of note, the non-toxic conditions are always accompanied by increased lipid droplet formation, suggesting that lipid droplets protect against fatty acid-induced toxicity.


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Session 4 A specific role for Retriever and Retromer in hepatocytes to control plasma lipid levels D.Y. Vos, M.E. Wijers, M. Smit, N. Huijkman, N.J. Kloosterhuis, K. Wolters, J.A. Kuivenhoven, B. van de Sluis University Medical Centre Groningen, Groningen, The Netherlands. Background: We recently found that the CCC and WASH complexes coordinate the endosomal transport of various lipoproteins receptors, such as LDLR, LRP1 and SR-BI. Hepatic loss of any of these two complexes results in impaired recycling of these receptors and, ultimately, in hypercholesterolemia in mice and humans. In vitro studies have implicated that both complexes are recruited to endosomes through a binding with the retromer subunit VPS35. A dominant-negative mutation in VPS35 has been correlated with Parkinson’s disease but to our knowledge, this mutation has not been associated with dyslipidemia. Interestingly, another multiprotein complex, called retriever, has also been linked to CCC and WASH in order to regulate endosomal receptor recycling. Altogether, retromer and retriever are strongly associated with the CCC-WASH axis but their contribution in lipid metabolism remains unclear. Methods: Using somatic CRISPR/Cas9 gene editing approach we generated liver-specific deficient mice for either VPS35 or the retriever component VPS26C. Proteomics and western blot analysis were used to study the protein expression of the different protein complexes, LDLR, LRP1 and SR-BI. Plasma triglyceride (TG) and cholesterol (TC) concentrations were measured and lipoprotein profiles were determined by FPLC. Results: Hepatic loss of VPS26C (retriever) did not affect plasma TG or TC levels but markedly increased hepatic LDLR levels. Although the formation of the CCC complex was not affected by the loss of VPS26C, a significant reduction in the retromer component VPS35 was seen. Acute hepatic VPS35 inactivation did not alter body weight, liver weight, or cause overt liver pathology, but it decreased plasma TG levels by ~50% compared to control mice. This decrease in plasma TG levels was accompanied by increased hepatic LDLR levels, suggesting that this increase in LDLR might enhance postprandial clearance of TG-rich particles. Conclusion: Although additional research is needed, our data suggest that the retriever and retromer have a specific role in the endocytic system to regulate the endosomal trafficking of lipoprotein receptors, and subsequently, to control plasma lipid levels. Furthermore, here, we clearly show that endosomal transport of receptors is tightly coordinated by different multiprotein complexes.


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Session 4 WASHC2 - a regulator of receptor recycling and plasma cholesterol W. van Zwol1, A. Rimbert2, J.C. Wolters1, N. Kloosterhuis1, N. Huijkman1, B. van de Sluis1, J.A. Kuivenhoven1 1UMCG/RUG, Groningen, The Netherlands. 2University of Nantes, Nantes, France. Background: The importance of the recycling of lipoprotein receptors in the liver to control plasma cholesterol levels in mice and human is becoming increasingly apparent. Our group uncovered that several multiprotein complexes of the endosomal sorting machinery in hepatocytes, are essential in the regulation of hepatic cholesterol metabolism and, consequently, in whole-body cholesterol homeostasis. We recently reported that the WASH complex, a protein complex consisting of 5 subunits, is involved in the hepatic recycling of SRB1, LDLR and LRP1 in mice. Interestingly, a common variant close to the WASHC2 gene locus in humans is associated with decreased WASHC2 expression (p=1.45x10-99), increased plasma high-density lipoprotein (HDL) cholesterol (p=1.72.10-26) but not low-density lipoprotein (LDL) cholesterol or triglycerides. In the current study, we set out to investigate the specific contribution of WASHC2 to lipid metabolism through investigating the effects of hepatic specific knock down of WASHC2 in mice. Methods: With use of the CRISPR-Cas9 system, adenoviral delivery of gRNAs in mice expressing Cas9 specifically in hepatocytes resulted in hepatic specific knock down of WASHC2 in female mice (n=6/7 per group) on chow diet. Cholesterol and triglyceride levels in plasma were analysed after 3 weeks. WASHC2 levels were determined with proteomics. Results: In line with the human data, the mice with 25-40% reduction of the protein showed an isolated increase of 28% in HDL cholesterol (p=0.007) but not LDL cholesterol or triglyceride levels in plasma. Conclusion: Downregulation of WASHC2 results in increased HDL levels in mice, similar to human findings. These results lead us to hypothesise that WASHC2 specifically regulate SRB1 recycling and suggest that different components of the WASH complex can ‘fine-tune’ receptor recycling in the liver.


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Session 4 Modelling the tumor micro-environment in Hepatocellular carcinoma with multicellular tumor spheroids: a tool to investigate (nano) therapeutics A. Ortiz Perez, J.B.G.M. Blonde, E.K. Geervliet, S. Le Gac, R. Bansal University of Twente, Enschede, The Netherlands. Background: Hepatocellular carcinoma (HCC) is a global health burden with alarming incidence. To date, there is no effective treatment for HCC, and the development of new therapies is hindered by the lack of understanding of this complex disease and the unsuitability of the current experimental models to recapitulate human HCC and enable drug screening. To have a physiologically relevant in vitro HCC model, it is imperative to mimic the tumor microenvironment (TME); involved in all stages of cancer (from initiation to progression) and a major contributor to chemoresistance. In this study, we aimed at developing multicellular HCC spheroids, containing key stromal cells mimicking the TME, for understanding the disease biology and evaluating targeted and combination therapies. Methods: Multicellular tumor spheroids (MCTS) are assembled using a dedicated microwell array. Pivotal elements of the tissue micro-environment such as extracellular matrix capsule, vascularization, and cell polarization are characterized using imaging (confocal/optical and scanning electronic microscopy), protein and gene expression techniques. To validate the developed MCTS platform, the effect of Doxil (liposomal doxorubicin) is evaluated alone and in combination with collagen degradation (nano) therapeutics (collagenase and collagenase-decorated polymersomes) and examined for viability and immunostainings. Results: Hepatic stellate cells, endothelial cells and macrophages were co-cultured with HepG2 cells (hepatocellular carcinoma cell line) in physiological and pathophysiological ratios to form viable spheroids of 400 μm in size using custom-designed round-bottom microwell array culture dishes. The MCTS displayed a dense capsule of extracellular matrix around the tumoral core - rich in collagen type I - and other hallmarks of HCC including hypoxia. Liposomal doxorubicin in combination with collagenase treatment is under current evaluation using this model; this combination is expected to increase the therapeutic efficacy of Doxil due to improved penetration and accessibility to the cancerous tissue owing to the disruption of the collagen-rich capsule responsible for chemo-resistance. Conclusion: We envision this platform as a tool to (a) recapitulate essential features of HCC and (b) investigate nanoparticle-based therapies in a highly relevant human-mimic HCC compared to other conventional models (2D cultures, and in vivo models); including penetration of the stroma, TME remodeling or cellular targeting.


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Session 5

SPX1 - a novel lipid gene affecting VLDL secretion in mice W. van Zwol1, A. Rimbert2, J.C. Wolters1, M. van der Toorn1, N. Kloosterhuis1, N. Huijkman1, B. van de Sluis1, J.A. Kuivenhoven1, 1UMCG/RUG, Groningen, The Netherlands. 2University of Nantes, Nantes, France. Background: We recently identified a novel lipid gene, SPX1, via contextual co-expression analysis to be closely associated with other genes that are validated targets for pharmaceutical intervention to treat hypercholesterolemia and hypertriglyceridemia (APOB, MTTP, ANGPTL3). SPX1 is almost exclusively expressed in the liver and small intestine and is predicted to encode for a small (107aa) transmembrane protein. In this study, we elucidated the role of SPX1 in lipid metabolism. Methods: Using somatic CRISPR/Cas9 gene editing technology, we targeted Spx1 in mouse livers (both male and female, n=10 per group) on chow diet. VLDL secretion rate is based on triglyceride levels in plasma after 0, 30, 60, 120, 180 and 240 min upon intraperitoneally injected poloxamer. Protein levels were measured with proteomics. Results: Liver-specific knock-down of SPX1 by 67% resulted in reduced cholesterol and triglycerides levels in plasma ( ̴50%), but increased hepatic lipid levels. Moreover, a reduction in the VLDL secretion rate by 45% was observed. Future perspective: We currently focus on the biological function via localization experiments and analysing proteins of different pathways involved in VLDL assembly and secretion. Future biological and safety studies will learn whether SPX1 can be regarded as new pharmaceutical target to reduce cholesterol and triglycerides in plasma.


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Session 5 Fibroblast growth factor 21 reduces hepatic steatosis and protects against atherosclerosis in hypercholesterolemic APOE*3-Leiden.CETP mice C. Liu1,2, Milena Schönke1,2, Enchen Zhou1,2, Zhuang Li1,2, Sander Kooijman1,2, Mikael Larsson3, Kristina Wallenius3, Xiao-Rong Peng3, Yanan Wang1,2,4, Patrick C.N. Rensen1,2,4 1Division of Endocrinology, Dept. of Medicine, Leiden University Medical Center, Leiden, The Netherlands. 2Eindhoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands. 3Cardiovascular, Renal and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden. 4Dept. of Endocrinology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an Jiaotong University, Xi’an, China.

Background & Aims: Nonalcoholic fatty liver disease (NAFLD) and atherosclerosis are intimately associated with increased cardiovascular mortality. Fibroblast growth factor 21 (FGF21), the key regulator of energy metabolism, is currently evaluated in humans for treatment of cardiometabolic diseases. However, its role in NAFLD and atherogenesis remains elusive. Therefore, the aim of this study is to investigate the therapeutic effects of FGF21 on NAFLD and atherosclerosis and the underlying mechanisms in hypercholesterolemic APOE*3-Leiden.CETP mice, a well-established translational model with a human-like lipoprotein metabolism. Methods: Female APOE*3-Leiden.CETP mice were fed a cholesterol-enriched Western-type diet for 3 weeks, and subcutaneously injected with either vehicle or FGF21 at a pharmacological dose (1 mg/kg body weight) 3 times per week for 16 weeks. At the end of the study, kinetic lipid clearance was studied and atherosclerotic lesion area as well as severity and composition were assessed in the aortic root. Hepatic lipid content was determined and liver histology was assessed. Results: FGF21 treatment reduced body fat mass as explained by highly increased energy expenditure. Mechanistically, FGF21 promoted brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning, thereby enhancing selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT and into beige adipocytes of WAT, consequently accelerating the hepatic clearance of the cholesterol-enriched remnants as evidenced from kinetic studies with radiolabeled lipoprotein-like particles. Accordingly, FGF21 reduced plasma total cholesterol and non-HDL cholesterol levels. Within the liver, FGF21 increased hepatic fatty acid oxidation and triglyceride secretion, and markedly reduced hepatic steatosis. Together, these actions of FGF21 largely decreased atherosclerotic lesion area and improved lesion severity. Moreover, FGF21 increased atherosclerotic plaque stability as indicated by reduced macrophage content without affecting collagen and smooth muscle cell content. Conclusions: FGF21 activates BAT and induces browning of WAT, thereby accelerating lipoprotein turnover, improving hypercholesterolemia and protecting against atherosclerosis development. Additionally, FGF21 increases hepatic fatty acid oxidation and VLDL-triglyceride secretion resulting in improvement of hepatic steatosis. We have thus provided mechanistic evidence to support the clinical use of FGF21 in the treatment of NAFLD and atherosclerotic cardiovascular diseases.


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Session 5 A role for GalNac-T2 dependent glycosylation in the regulation of insulin sensitivity

R.C. Verzijl, N. Loaiza Velasquez, F. Oldini, Y. Guan, S.A. Ljunggren, T.H. van Dijk, N.J. Kloosterhuis, J.C. Wolters, M. Smit, D. Struik, V.W. Bloks, H. Karlsson, A. Fernandez, N.L. Samara, L.A. Tabak, J.W. Jonker, J.A. Kuivenhoven UMCG, Groningen, The Netherlands.

Background and Aim: Previously, a role for o-linked glycosylation mediated by GalNac-T2 has been established in HDL metabolism. Indeed, loss of GALNT2 has been shown to decrease HDL-cholesterol in humans, mice, rats and non-human primates. Recent human studies, however, have also suggested a role for GALNT2 in type 2 diabetes and obesity. In line with this we also observed increased body weight, liver weight and fasting blood glucose levels in Galnt2-/- mice compared to wild-type littermates in response to a 12 week high fat diet (HFD, 60% fat). In the current study we aimed to investigate the mechanism by which GalNac-T2 affects the development of metabolic disease. Methods: To investigate metabolic changes independent of differences in body weight, Galnt2-/- mice and wild-type littermates were fed a low fat diet (LFD, 11% fat) as control diet, followed by a only short term HFD challenge for 6 weeks. Analyses include indirect calorimetry, glucose homeostasis and o-glycosylation of (potential) target proteins. Results: As described previously, Galnt2-/- mice displayed decreased plasma levels of cholesterol. In response to the LFD control diet, Galnt2-/- mice displayed a decreased respiratory exchange ratio (RER), indicating that they were more dependent on lipid oxidation as compared to wild-type littermates. This difference, however, disappeared upon HFD feeding, where both groups of mice completely relied on lipid oxidation. Interestingly, Galnt2-/- mice also displayed decreased glycosylation of the insulin receptor (INSR) in liver, muscle and adipose tissue. In line with this observation Galnt2-/- mice were more insulin resistant and exhibited higher fasting blood glucose levels than controls. Increased insulin resistance was also supported by increased plasma non-esterified free fatty acid levels and decreased gonadal white adipose tissue weight, suggesting a reduced insulin-mediated suppression of adipose lipolysis. Finally, increased ectopic lipid accumulation was found as reflected by increased hepatic steatosis, liver mass and liver-to-BW ratio. Conclusion: Together, our findings suggest a novel role for GalNac-T2 in glucose and energy homeostasis through glycosylation of the INSR and ultimately affecting insulin sensitivity.


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Session 5 The role of sAC-Sec61β in Polycystin-1 biogenesis S. Go, J.C. Chang, A.J. Verhoeven, S.R. Piersma, R.R. de Goeij-de Haas, T.V. Phang, C.R. Jimenez, R.P.J. Oude Elferink Amsterdam UMC, Amsterdam, The Netherlands. Background: Intracellular cAMP signaling generated by the transmembrane adenylyl cyclases (tmACs) and the soluble adenylyl cyclase (sAC) are important for cholangiocyte homeostasis. cAMP signaling is affected in polycystic liver disease caused by mutations in several genes including SEC61β. Whereas the role of tmACs in cholangiocytes has received much attention, the role of sAC is much less well defined. Here we describe a novel sAC-Sec61β-Polycystin-1 axis that may be important for cholangiocyte homeostasis. Methods: The immortalized human cholangiocyte cell line H69 was used as a model because of its high expression of sAC. Pulse chase experiments with L-azidohomoalanine (a methionine analog) were used to determine the half-life of sAC protein. Using the sAC inhibitor LRE-1 and phosphoproteomics, sAC-specific phosphorylation of proteins was investigated. Lentiviral mediated knockdown of sAC and Sec61β was used to investigate the changes in sAC, Sec61β and Polycystin-1 protein levels. Results: Pulse chase labeling shows that sAC has a very high turnover (t1/2 ± 30 min). We have shown previously that sAC is a membrane-tethered and N-linked glycosylated protein, the latter which requires import into the ER. Indeed, we could confirm this by the observation that knockdown of SEC61β reduces sAC levels. Subsequent experiments have now shown that 1) inhibition of sAC induces strong Sec61β dephosphorylation at serine 17 and 2) inhibition of sAC reduces the protein levels of sAC. Therefore, we hypothesized that sAC regulates the entry of a select group of proteins (including itself) into the ER by regulating Sec61β phosphorylation. To test this, we looked at the effect of sAC inhibition and knockdown on Polycystin-1 protein levels, which also requires Sec61β. Indeed, we could show that knockdown of Sec61β decreased Polycystin-1, but more importantly, inhibition or knockdown of sAC also reduced Polycystin-1 levels. Conclusion: In H69 cholangiocytes, the sAC-Sec61β-Polycystin-1 axis regulates Polycystin-1 levels and therefore may play a role in proper cholangiocyte functioning. Future experiments are required to determine the function of sAC-specific phosphorylation of Sec61β at serine 17 and the implications of it in bile ductular fluid secretion.


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Session 5 Elevated cAMP protects against diclofenac-induced toxicity in primary rat hepatocytes: a protective effect mediated by EPACs. F.A. Aguilar, A.J. Moshage The University of Groningen, Groningen, The Netherlands. Background: Diclofenac (DF) is a nonsteroidal anti-inflammatory drug that is widely used to treat mild to moderate pain. Although there is no consensus of a toxic dose in the liver, chronic or excessive consumption can lead to drug-induced liver injury (DILI), typically with a delayed onset of 1 to 3 months. The mechanism of DF-induced liver injury is partially elucidated and involves mitochondrial injury in hepatocytes. It is known that cyclic adenosine monophosphate (cAMP) may protect hepatocytes against hepatocyte injury, e.g., induced by hydrophobic bile acids. However, the mechanism of cAMP-mediated protection is unknown and may involve the classical downstream cAMP target protein kinase A (PKA) or the alternative pathways EPAC1 and/or EPAC2 (Exchange Protein Directly Activated by cAMP/cAMP-regulated guanine nucleotide exchange factors). At present, it is unknown whether cAMP protects against DF-induced hepatocyte injury. Aim: To investigate whether cAMP protects against DF-induced injury in primary rat hepatocytes. Methods: Primary rat hepatocytes were exposed to DF. Apoptosis was measured by caspase-3 activity assay and necrosis by sytox green staining. Mitochondrial membrane polarization (MMP) was measured by JC-10 staining. The cAMP-elevating agent forskolin, a direct adenylyl cyclase activator and IBMX, a pan-phosphodiesterase inhibitor, were used to elevate cAMP and the effect of the PKA inhibitor RP-8CPT-cAMPS and the EPAC1 and EPAC2 inhibitors CE3F4 and ESI-O5 on cell death and MMP were investigated. Results: DF at 400µM induced apoptotic cell death peaking at 12 hr, but not necrotic cell death. DF also induced loss of mitochondrial membrane potential within 2 hr after DF exposure. Both forskolin and IBMX prevented DF-induced caspase-3 activation and apoptosis. PKA inhibition did not abolish the protective effect of forskolin and IBMX. In contrast, both EPAC inhibitors abolished the protective effect of forskolin and IBMX. Forskolin and IBMX also preserved the MMP, and both EPAC inhibitors abolished this effect. Conclusion: Elevated cAMP levels protect hepatocytes against diclofenac-induced apoptotic cell death. This protective effect is not mediated via the classical PKA pathway but via the recently described EPAC pathway. Our results suggest the cAMP/EPAC pathway as a novel therapeutic target for the treatment of DILI.


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Session 6 GPR146 as a novel regulator of HDL-c in mice and human

N. Loaiza Velasquez1, N. Dalila2, L. Blauw3, A. Rimbert4, M. Schilperoort3, S. Kooijman3, P. Rensen3, M. Smit1, N. Kloosterhuis1, J.F. de Boer1, T. van Dijk1, M. Koehorst1, F. Kuipers1, B. van der Sluis1, R. Frikke-Schmidt2, A. Tybjærg-Hansen2, J.A. Kuivenhoven1 1Department of Pediatrics, University Medical Center Groningen, Groningen, The Netherlands. 2Section Molecular Genetics, Department of Clinical Biochemistry, Rigshospital, Copenhagen, Denmark. 3Eindhoven Laboratory, Leiden University Medical Center, Leiden, The Netherlands. 4L'institut du thorax, INSERM, CNRS, Univ Nantes, Nantes, France. Background: Variation at the GPR146 gene locus is associated with total cholesterol (TC), HDL-c and LDL-c in humans, and a recent study indicates that genetic ablation of Gpr146 in mice reduces LDL-c and atherosclerosis development. To date, there are no studies into the relation between GPR146 and atherosclerosis in humans. In addition, current data indicate that GPR146 may not play identical roles in humans and mice. Methods: We used human data from public data sources, LifeLines, the Copenhagen City Heart Study and the Copenhagen General Population to study the association of GPR146 gene variation with plasma lipid traits, plasma metabolomics and the risk of ischemic heart disease (IHD). In addition, we have studied Gpr146-/- mice to find an explanation for species-specific effects. Results: Rs1997243 is associated with increased GPR146 expression and increased TC, HDL-c, ApoAI and LDL-c but not apoB. In a meta-analysis of 667796 individuals, these changes in lipid parameters do not alter the risk of IHD. Plasma NMR analysis shows that this SNP is, remarkably, exclusively associated with large size HDL particles and ApoAI, while no differences are found for ApoB, VLDL or triglycerides. Compared to WT controls, Gpr146-/- mice (fed chow) show decreased levels of TC, LDL-c, HDL-c but also Tg without changes in cholesterol uptake by the liver. Gpr146-/- mice also show increased cholesterol synthesis and exhibit reduced neutral sterol excretion. Conclusion: A common genetic variant in GPR146 is mainly associated with increased TC and HDL-c without affecting risk of IHD. NMR analyses show that rs199743 is associated with an isolated effect on HDL metabolism. In mice on the other hand Gpr146 ablation also causes a reduction in plasma triglycerides. Ongoing studies into putative loss of function GPR146 variants that we identified in humans will hopefully shed led on the question whether GPR146 antagonism may render beneficial effects.


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Session 6 The coumarin-derivative esculetin inactivates hepatic stellate cells by inducing senescence M.E.N.G.F. Zhang, T. Damba, M. Buist-Homan, K.N. Faber, A.J. Moshage University Medical Center Groningen, Groningen, The Netherlands. Background: Activation of hepatic stellate cells (HSC) leads to initiation and progression of liver fibrosis. Therefore, there is an ongoing search for bioactive compounds that are able to inhibit or reverse stellate cell activation. Furthermore, it has been shown that induction of stellate cell senescence is inversely correlated with hepatic stellate cell proliferation and activation. Therefore, induction of hepatic stellate cell senescence may be a strategy to treat liver fibrosis. The natural compound esculetin, a coumarin-derivative, has been shown to attenuate hepatic steatosis and inflammation but its effect on liver fibrosis remains unclear. The aim of the present study was to investigate the effect of esculetin on HSC activation and senescence in vitro. Methods: Primary HSCs were isolated from male Wistar rats and used for experiments. Real-time cell analyzer(xCELLigence) was used to measure proliferation of HSCs. Gene expression of the senescence-associated genes Cdkn1a (p21), P53 and activation-associated genes Pparγ, Acta2 and Col1a1 were measured by RT-qPCR and normalized to house-keeping gene 36b4. Senescence associated β-Galactosidase (SA-β-Gal) staining was used to investigate senescent cell ratio of HSCs. Esculetin was used at a concentration of 50 μmol/L in all experiments. Results: Esculetin reduced the proliferation of HSCs and reduced mRNA expression of fibrogenic genes Acta2 and Col1a1 by more than 50%, both in activated and in early activating (quiescent) HSCs. Esculetin induced a 4-fold increase of mRNA level of senescence marker Cdkn1a (p21) and an increased ratio of SA-β-Gal positive cells, again both in early activating and activated HSCs. Activated HSCs pre-treated with esculetin for 48h followed by washout for 48h still exhibited less proliferation, more than 50% reduction of mRNA level of Acta2 and Col1a1, 2-fold increase of mRNA expression of Cdkn1a and P53 and increased ratio of β-Galactosidase positive cells compared to solvent treated HSCs. Conclusions: Esculetin inhibits proliferation and expression of the activation markers Acta2 and Col1a1 in primary HSCs in vitro possibly via inducing senescence. Induced senescence by natural compounds may be a promising therapeutic intervention for the treatment of liver fibrosis.


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Session 6 Pharmacological targeting of CCL2-CCR2 axis using a novel CCR2 antagonist peptide as a prospective treatment for liver fibrosis H.M. Nijland1, A. Vrynas1, M. Grewer2, C. Holtke2, R. Bansal1 1University of Twente, Enschede, The Netherlands. 2University Hospital Münster, Münster, Germany. Background: Hepatic fibrosis is a growing health problem affecting millions of people worldwide. It has been shown that hepatic inflammatory macrophages are capable of activating hepatic stellate cells (HSCs), known as the main effector cells in hepatic fibrosis. Upon activation, HSCs undergo functional and morphological changes resulting in proliferative myofibroblasts that produce excessive ECM, thereby contributing to liver fibrogenesis. The chemokine (C-C motif) ligand 2-chemokine (C-C motif) receptor 2 (CCL2-CCR2) axis has been shown to mediate monocyte recruitment upon liver injury, leading to shaping of the inflammatory response in the injured liver. Implication of CCL2-CCR2 has been shown using CCR2 knockout mice and CCR2/CCR5 cenicriviroc that efficiently blocks CCL2-mediated monocyte recruitment and has an anti-fibrotic effect in the fibrosis mouse models. Peptides represent a unique class of pharmaceutical compounds, molecularly poised between small molecules and proteins. Therefore, in this study, we have designed and investigated the potential of a newly designed CCR2 antagonizing peptide for the therapeutic targeting of inflammatory macrophages as a prospective treatment for liver fibrosis. Methods: Expression of CCL2 and CCR2 was examined in RAW and bone-marrow derived macrophages following polarization using M1 stimulus (LPS/IFN-gamma) and M2 stimulus (IL-4/IL-13). A novel CCR2 peptide was designed using ligand-receptor docking and computational structural modelling. Different concentrations of the CCR2 antagonizing peptide was examined in vitro in murine RAW and bone-marrow derived macrophages for cell viability and efficacy using inflammatory gene expression analysis. Results: Upregulation of CCL2 and CCR2 gene expression was observed in vitro and in vivo in fibrosis mouse models. Furthermore, we observed increased expression of CCL2 and CCR2 expression in human disease livers from different etiologies suggesting the importance of CCL2-CCR2 axis in liver diseases. Increased CCR2 was observed in RAW macrophages and bone-marrow derived macrophages stimulated with M1 stimulus (LPS/IFN-gamma) as compared to unstimulated and IL-4/IL-13 stimulated M2 macrophages. Interestingly, treatment with CCR2 antagonizing peptide inhibited the activation of M1 macrophages as assessed by reduced expression of inflammatory parameters. Notably, CCR2 antagonizing peptide didn’t induce any effect on cell viability. Conclusion: CCR2-antagonizing peptide represents an interesting therapeutic approach for the treatment of liver fibrosis.


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Session 6 Δ24-Dehydrocholesterol reductase (DHCR24): a novel target for the treatment of NASH E. Zhou1, H. Nakashima1, Z. Li1, E. Steenvoorden3, C. Müller4, F. Bracher4, P.C.N. Rensen1,2, M. Giera3, Y. Wang1,2 1Dept. of Medicine, Div. of Endocrinology, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands. 2Dept. of Endocrinology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an Jiaotong University, Xi’an, China. 3Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands. 4Dept. of Pharmacy, Center for Drug Research, Ludwig Maximilians University, Munich, Germany. Aim: 24-Dehydrocholesterol reductase (DHCR24) is the terminal enzyme in cholesterol biosynthesis converting the ultimate intermediate desmosterol into cholesterol. Desmosterol is an endogenous liver X receptor ligand with anti-inflammatory properties and reduces cholesterol and fatty acid biosynthesis. We aimed to investigate the therapeutic effects of DHCR24 inhibition by using the novel selective DHCR24 inhibitor SH42 on hepatic steatosis and inflammation, the two most important hallmarks of nonalcoholic steatohepatitis (NASH). Methods: Male APOE*3-Leiden.CETP mice, a well-established translational model for lipoprotein metabolism that develops diet-induced human-like NASH characteristics, were fed a high fat and high cholesterol diet with or without simultaneous SH42 treatment (3 x 0.5mg/week, i.p.). After 8 weeks, liver steatosis and inflammation were assessed. Lipidomics and lipid mediator analyses were carried out on plasma and liver. Results: DHCR24 inhibition via SH42 markedly increased plasma desmosterol levels (+5,600%), without influencing food intake nor affecting body weight and body composition. SH42 decreased plasma cholesteryl ester (-24%) and fatty acids (-19%) levels whilst not affecting diacylglycerol (DAG) and triacylglycerol (TAG) levels. Notably, SH42 largely increased plasma 19,20-epoxydocosapentaenoic acid (19,20-EpDPA) levels (+210%), a well described and potent anti-inflammatory/pro-resolving lipid mediator. In the liver, SH42 reduced DAG (-21%), TAG (-38%) and cholesteryl esters (-26%). Strikingly, liver histological assessment showed that SH42 abolished diet-induced hepatic steatosis, inflammation, ballooning and crown-like structure formation. Conclusion: Inhibition of DHCR24 by SH42 abolishes hepatic steatosis, inflammation and damage as induced by a diet containing high fat and high cholesterol. DHCR24 inhibition is a potential novel therapeutic strategy for the treatment of NASH by killing two birds with one stone, i.e. hepatic steatosis and inflammation.


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List of participants Name Institute

F.A. Aguilar Mora UMCG K. Alagere Krishnamurthy UMCG J.C. Arroyave UMCG R. Bansal University of Twente R.C. Bartfai Radboud University J.F. de Boer UMCG R.S. Booijink University of Twente M.C. Bouwmeester Utrecht University M. Buist UMCG J.C. Chang Amsterdam UMC, loc. AMC R.E. Eilers UMCG K.N. Faber UMCG E.K. Geervliet University of Twente Y. Geng UMCG S. Go Amsterdam UMC V.E. Gómez Mellado Amsterdam UMC, loc. AMC K.F.J. van de Graaf Amsterdam UMC, loc. AMC M.W. Haaker Universiteit Utrecht J. Heegsma UMCG A.H. Heida UMCG


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T. Houben Universiteit Maastricht M.V. Hovingh UMCG S.C.D. van IJzendoorn UMCG R. de Jager UMCG J.W. Jonker UMCG C.P. Kohlberger UMCG D. Kong UMCG F. Kuipers UMCG J.A. Kuivenhoven UMCG R.F. Kunst Tytgat Institute for Liver and Intestinal Research L. van der Laan Erasmus MC V. Lehmann UMCU Q. Li UMCG R. Li UMCG C. Liu LUMC N. Loaiza Velasquez UMCG L.M. Matakovic The University Medical Center Groningen, UMCG H. Moshage UMCG N.L. Mulder UMCG S.C. Niesten Erasmus MC H.M. Nijland University of Twente R.P.J. Oude Elferink Amsterdam UMC, loc. AMC A. Palmiotti UMCG


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C.C. Paulusma Amsterdam UMC, loc. AMC L.C. Penning Universiteit Utrecht B.P. Porteiro Tytgat Institute for Liver and Intestinal Research T. Qin UMCG F.J.M. Roos Erasmus MC M.G.S. Rutten UMCG A. Saeed UMCG B. van de Sluis UMCG R. Sverdlov MUMC E. Verkade UMCG R.C. Verzijl UMCG D.Y. Vos UMCG H.D. de Vries UMCG Y. Wang The First Affiliated Hospital of Xi'an Jiaotong University H. Wu Radboud University Z. Wu UMCG J. Yang UMCG M. Zhang UMCG E. Zhou LUMC T. van Zutphen UMCG W. van Zwol UMCG/RUG


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Notes


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The DLR aims to initiate and catalyze interactions between liver researchers, in particular to integrate the new researchers in the established networks of our field. Sponsoring by the NASL/NVH allows us to make the DLR accessible for all of you. Koos Kranenborg from BIOKÉ sacrificed his free Thursday evening to operate as our enthusiast DJ. We want to express our gratitude to these sponsors.

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