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Annual meeting of the Rembrandt Institute for Cardiovascular Science (RICS)

7th Rembrandt Symposium

4 November 2016

NH Conference Centre Leeuwenhorst, Noordwijkerhout

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Welcome to the 7th Rembrandt Symposium! In spring 2010, the boards of AMC and LUMC decided to combine their research efforts and resources in the Rembrandt Institute for Cardiovascular Science (RICS) in order to promote ground breaking cardiovascular research in the Netherlands. In the meantime, the LACDR, VUmc and Sanquin have also become part of the RICS. The name Rembrandt comes from the famous Dutch painter, who was born in Leiden, but spent much of his working life in both Leiden and Amsterdam and thus truly symbolizes our collaboration. The mission of the RICS is to carry out and to stimulate excellent fundamental and translational research within the field of (cardio-) vascular biology and disease (e.g. stem cells, vessel wall biology, immunometabolism, thrombosis, molecular cell biology, genetics, electrophysiology and developmental biology). Within this context, our main tasks are:

• To perform fundamental and translational research in the field of (cardio-) vascular biology and disease

• To obtain collaborative (consortium) grants to finance these research efforts • To function as interlocutor for external granting agencies such as the

Netherlands Heart Foundation (NHF), ZonMw or NWO • To translate fundamental research into clinically applications • To valorise research within the context of the Leiden Bio Science Park and

the Life Sciences Centre Amsterdam The Rembrandt Symposium is dedicated to promote the exchange of recent research findings in the form of oral and poster presentations. A highlight are the poster sessions with dedicated time to discuss projects and ideas with your fellow researchers and to set up collaborations. At the end of this meeting, the winners of the Rembrandt Research Grant 2016 will be announced and the best oral presentations and posters will be awarded. We look forward to an exciting scientific day! The organizing committee

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Content General information 5

Location of meeting rooms and floor plans 6

Organizers 7

Programme 8

Abstracts selected oral presentations 11 Poster presentations (alphabetical order) 19

Abstracts poster presentations 23 Contact details oral and poster presentations 102

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General information Meeting venue NH Conference Centre Leeuwenhorst Langelaan 3 2211 XT Noordwijkerhout +31 (0)252 378424 www.nh-hotels.nl How to get there By bus/train: From Leiden Central Station, board bus 20 (direction Noordwijk) and transfer to bus 90 in Noordwijk (direction Lisse). From Den Haag Central Station, take the train to Leiden and then the bus as described. Alternatively, take bus 90 (direction Lisse) from Den Haag Central Station. Get off the bus at “Langelaan". You will reach the meeting venue after a 5 minutes’ walk. By car: From Amsterdam/Utrecht Take the A4 Motorway towards Schiphol/Den Haag. At the A4/A44 interchange, keep right to join the A44 towards Leiden West/Sassenheim/Den Haag Centrum Leave the motorway at junction 3 for Noordwijkerhout/Sassenheim/Noorwijk (N208) At the end of the slip road turn right Stay in this road : (N443) for 5 km After 5 km follow the sign for “Congrescentrum” The entrace to the hotel is on your left. From Rotterdam/The Hague Take the A13 motorway towards Den Haag/Amsterdam At the A13/A4 interchange (Prins Clausplein), follow signs for Amsterdam A4 Leave the motorway at junction 8, for Leidschendam/Wassenaar (N14) Follow signs for Wassenaar until you reach the A44 motorway Join the motorway in the direction of Leiden/Amsterdam Leave the motorway at junction 8, for Leiden/Katwijk/Noordwijk. At the end of the slip road, turn left towards Katwijk/Noordwijk (N206) Stay on this road until you reach Noordwijkerhout. In Noordwijkerhout, follow the signs for “Congrescentrum” Take the exit for Noordwijkerhour/Sassenheim, then turn left. At the Roundabout continue straight ahead. The entrance to the hotel is on your left. Internet access Internet access is free. For access codes ask at the hotel reception. Contact Secretariat M.H.Sjardin-van_Leeuwen@lumc.nl Organizing committee P.H.A.Quax@lumc.nl

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Location of meeting rooms and floor plans

• The Regristration desk is located at the ‘Gaudi Lounge’.

• Oral presentations will take place at ‘Sorbonne 2’.

• Posters will be presented in the ‘Gaudi Lounge’ and ‘Sorbonne 8’.

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Organizers Organizing committee M.J.T.H. Goumans LUMC, Molecular Cell Biology P.H.A. Quax LUMC, Vascular Surgery M. Scharpfenecker LUMC, Pathology A.J. van Zonneveld LUMC, Internal Medicine Leiden Vascular Medicine (LVM) I.M. Bajema LUMC, Pathology M. van Eck LACDR, Biopharmaceutics M.J.T.H. Goumans LUMC, Molecular Cell Biology L.M. Havekes LUMC, Endocrinology M.V. Huisman LUMC, Thrombosis and Haemostasis J.W. Jukema LUMC, Cardiology C.L. Mummery LUMC, Anatomy P.H.A. Quax LUMC, Vascular Surgery P.H. Reitsma LUMC, Thrombosis and Haemostasis P.C.N. Rensen LUMC, Endocrinology M. Scharpfenecker LUMC, Pathology A.J. van Zonneveld LUMC, Internal Medicine Amsterdam Rembrandt Committee C.R. Bezzina AMC, Experimental Cardiology H.R. Büller AMC, Vascular Medicine V.M. Christoffels AMC, Anatomy, Embryology and Physiology M. Coppens AMC, Vascular Medicine M.J.A.P. Daemen AMC, Pathology M. Nieuwdorp AMC, Vascular Medicine Y.M. Pinto AMC, Experimental Cardiology C.A. Remme AMC, Experimental Cardiology E.S. Stroes AMC, Vascular Medicine C.J.M. de Vries AMC, Medical Biochemistry VU Medical Center H.J. Bogaard ICaR-VU/VUmc, Pulmonology E.C. Eringa ICaR-VU/VUmc, Physiology P.L. Hordijk VUmc, Physiology J. van der Velden ICaR-VU/VUmc, Physiology Sanquin J.D. van Buul Sanquin, Molecular Cell Biology Secretariat M. H. Sjardin-v. Leeuwen LUMC, Endocrinology

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Programme 09:00-09:30 Registration with coffee/tea 09:30-09:35 Opening Paul Quax (LUMC) 09:35-11:05 PLENARY SESSION 1 Chairs: Carol Ann Remme (AMC), Marion Scharpfenecker (LUMC) 09:35-10:05 Keynote lecture 1

Prof. dr. M. J. T. H. Goumans (Dept. of Molecular Cell Biology, LUMC) Balancing TGF-β signaling in PAH Marie-José Goumans received her PhD in 1999 from the Hubrecht Laboratory, where she investigated the role of TGFβ in cardiovascular development. She did her postdoctoral training at the Ludwig Institute for Cancer research in Uppsala, Sweden and the Netherlands Cancer Institute in Amsterdam, where she made important contributions to how TGFβ affects endothelial cell behaviour. In 2003, Marie-José Goumans was appointed assistant professor at the department of cardiology at the UMC Utrecht. In 2004, she was awarded a prestigious NWO VIDI grant to unravel the role of cardiac progenitor cells in heart regeneration. In 2008, she moved to the LUMC to continue her studies on cardiac progenitor cell biology, in particular the role of the TGFβ superfamily. In 2009, she became a member of the Young Academy of the Royal Dutch Academy of Science. Beginning 2012, she was appointed professor of molecular cardiovascular cell biology at the University of Leiden.

10:05-10:20 Roel Bijkerk (LUMC): MicroRNA-132 regulates renin synthesis via targeting Cox-2/Prostaglandin-E2 signaling: a novel posttranscriptional mechanism in blood pressure regulation

10:20-10:35 Rosa van den Berg (LUMC): Biological clock strongly regulates

brown adipose tissue activity: implications for postprandial triglyceride metabolism

10:35-10:50 Danielle Leuning (LUMC): Regeneration of kidney vasculature with

human kidney-derived endothelial cells in decellularized rat and human kidneys

10:50-11:05 Thijs Beldman (AMC): A novel hyaluronan-based nanostrategy for

imaging and therapy of atherosclerosis 11:05-12:15 Poster presentations (odd numbers) with coffee/tea 12:15-13:00 Lunch

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13:00-14:30 PLENARY SESSION 2 Chairs: Harm-Jan Bogaard (VUmc), Miranda van Eck (LACDR) 13:00-13:30 Keynote lecture 2

Dr. J.R. de Groot (Dept. of Cardiology, AMC) Autonomic modification in advanced atrial fibrillation Joris R. de Groot studied Medicine at the University of Amsterdam (MD 1997, cum laude), and defended his PhD thesis at that same institution in 2001. He was a Research Fellow with Michael R. Rosen MD (Columbia University, New York, NY) and with José Jalife (SUNY Upstate Medical University, Syracuse, NY). After his training as Cardiologist (2008), he subspecialized as Clinical Electrophysiologist (2010), both at the Academic Medical Center, Amsterdam. The main research topic of his group comprises mechanism and treatment of atrial fibrillation. Joris de Grootis a VIDI laureate and received a personal grant from the Dutch Heart Foundation.

13:30-13:45 Lejla Medzikovic (AMC): Orphan Nuclear Receptor Nur77 modulates

cardiomyocyte function via Neuropeptide Y 13:45-14:00 Tom Seijkens (AMC): TRAF-STOPs ameliorate atherosclerosis 14:00-14:15 Dorine Elffers (LUMC): In obese women, but not in obese men,

body fat distribution, in particular the presence of visceral fat, is associated with cardiometabolic risk factors

14:15-14:30 Emile Nyns (LUMC): Optogenetic termination of ventricular

arrhythmias in the whole heart: Towards biological cardiac rhythm management

14:30-15:45 Poster presentations (even numbers) with coffee/tea 15:45-17:05 PLENARY SESSION 3 – REMBRANDT PROJECT REPORTS Chairs: Marie-José Goumans (LUMC), Stephan Huveneers (AMC) 15:45-16:10 Final report Rembrandt project 2012: "Intestinal microbiota as

modifiable factor in the metabolic syndrome and associated cardiovascular disease; role of short and long chain fatty acids"

Awardees: Erik Stroes (AMC), Ko Willems van Dijk (LUMC) Kristien Bouter (AMC): The role of butyrate on insulin sensitivity in healthy and metabolic syndrome males Saeed Katiraei (LUMC): Intestinal microbiota modulate the inflammatory response of splenocytes

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16:10-16:35 Final report Rembrandt project 2012: “Brown adipose tissue meets the inmmune system:implications for the treatment of obesity and associated disorders” Awardees: Patrick Rensen (LUMC), Esther Lutgens (AMC), Menno de Winther (AMC) Susan van den Berg (AMC)/Andrea van Dam (LUMC): Brown adipose tissue meets the inmmune system

16:35-16:50 PLENARY SESSION 4 - AWARDS 16:35-16:45 Oral and poster presentation awards 2016 Marie-José Goumans (LUMC), Anton Jan van Zonneveld (LUMC) 16:45-16:55 Rembrandt project award 2016 Pieter Reitsma (LUMC) 16:55-17:00 Closing remarks Wouter Jukema (LUMC) 17:00-18:00 Drinks

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Abstracts selected oral presentations Plenary session 1: oral presentation 1 Vascular Biology MicroRNA-132 regulates renin synthesis via targeting Cox-2/Prostaglandin-E2 signaling: a novel posttranscriptional mechanism in blood pressure regulation. R. Bijkerk1, W. Stam1, S. van Gelderen1, T.J. Rabelink1, and A.J. van Zonneveld1 1Dept. of Internal Medicine (Nephrology) and the Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center (LUMC), Leiden. Hypertension is a main risk factor for cardiovascular disease. Renin synthesis and release is a rate-limiting step in the activation of the renin–angiotensin system (RAAS), a signaling pathway that is central to the regulation of body fluid and electrolyte homeostasis and blood pressure. In response to low blood pressure or salt, the cells of the macula densa (MD) cells generate prostaglandin-E2 (PGE2), in a cyclo-oxygenase-2 (Cox-2)-dependent manner, which subsequently stimulates the release of renin. Little is known about the underlying post-transcriptional mechanisms that control renin release. Using in situ hybridization, we identified miR-132 to be strongly expressed in the MD and found miR-132 to directly target Cox-2 in vitro. In vivo silencing of miR-132 resulted in increased production of PGE2 and renin by the kidney. Blocking PGE2 synthesis using the selective Cox-2 inhibitor Celecoxib abrogated the mir-132-antagonist induced increase in renin, supporting a regulatory role for miR-132 in the Cox-2/PGE2 dependent release of renin. To confirm this, we demonstrated that antagomir-132 de-represses Cox-2 in vitro in the MD cell-line MMDD-1. Moreover, under conditions of high salt, miR-132 expression by the MMDD-1 cells markedly increased with a concomitant decrease of Cox-2 expression, further validating an active role for miR-132 in RAAS regulation. Taken together, we demonstrated a crucial posttranscriptional regulatory role for miR-132 in the synthesis and release of renin through Cox-2 mediated PGE2 synthesis which is essential for regulating plasma salt concentration and blood pressure. Therapies aimed at inhibiting miR-132 deserve attention as potential treatments for hypertension.

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Plenary session 1: oral presentation 2 Metabolism Biological clock strongly regulates brown adipose tissue activity: implications for postprandial triglyceride metabolism Rosa van den Berg1,2, Sander Kooijman1,2, Lauren L. Tambyrajah1,2, Gustavo Abreu-Vieira1,2, Raymond Noordam4, Ashna Ramkisoensing3, Claudia P. Coomans3, Joke H. Meijer3, Diana van Heemst4, Nienke R. Biermasz1, Patrick C.N. Rensen12. 1Dept. of Medicine, Div. of Endocrinology, 2Einthoven Laboratory for Experimental Vascular Medicine, 3Dept. of Molecular Cell Biology, 4Dept. of Geriatrics and Gerontology, Leiden University Medical Center, Leiden, The Netherlands Aim: Disturbed circadian rhythms associate with hypertriglyceridemia, and disruption of the biological clock reduces the capacity of brown adipose tissue (BAT) to combust triglyceride (TG)-derived fatty acids (FA) into heat (PNAS USA 2015). Here, we hypothesized that a circadian rhythm in BAT activity determines circadian plasma lipid metabolism. Methods: Wild-type mice and dyslipidemic APOE*3-Leiden.CETP mice were exposed to short (8h), regular (12h), or long (16h) days during 5 weeks. The rhythm of BAT was determined by its capacity to take up plasma TG-derived FA at 6 time points during a 24h period. Postprandial lipid excursions were determined following an oral olive oil bolus. In 37 healthy volunteers, postprandial lipid excursions following three identical isocaloric meals were determined in 24h blood samples taken every 30 min. Results: In both wild-type and dyslipidemic mice, BAT FA uptake displayed a pronounced circadian rhythm as compared to other metabolically organs. The highest uptake was observed at wakening, which was independent of light exposure regimes and persistent at thermoneutrality (30°C). This rhythm coincided with circadian expression patterns of thermogenic and lipid genes in a cell-autonomous manner. Interestingly, circadian rhythmicity in BAT activity dictated circadian plasma lipid clearance by BAT as well as plasma lipid levels. Strikingly, in mice as well as humans postprandial lipid excursions were nearly absent at wakening and high before sleep, consistent with circadian BAT activity patterns. Conclusion: BAT displays a strong diurnal rhythm in TG-derived FA uptake, which strongly determines differences in postprandial TG metabolism throughout the daily light cycle. Since BAT activity is highest at wakening, accompanied by lowest postprandial lipid excursions, we anticipate that restriction of food intake to the early wakeful period improves metabolic health.

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Plenary session 1: oral presentation 3 Metabolism

Regeneration of kidney vasculature with human kidney-derived endothelial cells in decellularized rat and human kidneys. Daniëlle Leuning1, Annemarie de Graaf 1, Cathelijne W. van den Berg1, Ellen Lievers1, Loes Wiersma1, Hetty de Boer1, Cristina Avramut2, Bernard van den Berg1, Minoru Takasato3, Melissa Little4,5, Marten Engelse1 and Ton Rabelink1

1Nephrology, LUMC, Leiden, the Netherlands. 2Cell Biology, LUMC, Leiden, the Netherlands. 3RIKEN Center for Developmental Biology, Kobe, Japan.4 Murdoch Childrens Research Institute, Melbourne, Australia.5 Pediatrics, The University of Melbourne, Australia. Objective As there is a shortage of donor organs, there is an urgent need for new alternatives. One future alternative could be a bioengineered kidney by recellularization of a kidney scaffold with patient-derived cells. In order to achieve a functional bioengineered kidney, the vasculature should be intact. Here we study regeneration of kidney vasculature with human kidney-derived endothelial cells in both rat- and human kidney scaffolds. Methods Rat- and human kidneys (n=3) were decellularized with 1% SDS and 0.1% TritonX-100. Preservation of structure, integrity and glycosaminoglycan (GAG) landscape of the scaffolds was analyzed with immunofluorescence for extracellular matrix proteins, heparan sulfate proteoglycans and hyaluronan. Scaffolds were preloaded with resp. 0, 10 and 100 ng/ml vascular endothelial growth factor (VEGF). Rat whole organ and slices of human decellularized kidney were recellularized with human primary glomerular-derived microvascular endothelial cells (hgMVEC). Rat kidney scaffolds were recellularized via the renal artery and vein and cultured for 7 days in a custom-made organ chamber. Cell coverage was analyzed by confocal microscopy and quantified in ImageJ. Results Human and rat kidneys showed a preservation of structure, integrity and GAG landscape after decellularization. Site specific binding of VEGF and basic fibroblast growth factor (bFGF) was observed on these scaffolds. hgMVEC cell adherence of 20% coverage was observed in the absence of VEGF, while preloading the human scaffold slices with 100 ng/mL increased cell adherence to 55% coverage (P<0.001). Rat decellularized kidneys showed intact vascular integrity as shown by fluorescent bead perfusion. We show that recellularization of rat kidney scaffolds with hgMVEC via both the renal artery and vein gave the highest coverage of endothelial cells in both the glomeruli and the peritubular vessels without leakage towards the tubuli. Conclusions Here we show an extensive characterization of both rat and human kidney scaffolds for the GAG landscape and established an increase in human kidney-derived endothelial cell adherence after loading the scaffold with VEGF. Moreover, we show a novel recellularization method of rat kidney scaffolds in an organ chamber where long term organ-culture could be achieved. These results are a promising step towards a bioengineered kidney.

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Plenary session 1: oral presentation 4 Atherosclerosis Experimental, Imaging A novel hyaluronan-based nanostrategy for imaging and therapy of atherosclerosis Thijs Beldman1, Max Senders1, Amr Alaarg2, Carlos Perez‐Medina2, Jun Tang2, Francois Fay2, Fortune Cohen3, Elena Kartvelishvily3, Michal Neeman3, Esther Lutgens1, Willem J.M.Mulder1,2 and Ewelina Kluza1 1 Academic Medical Center, Amsterdam, The Netherlands; 2 Mount Sinai School of Medicine, New York, USA; 3 Weizmann Institute of Science, Rehovot, Israel Introduction: Hyaluronan is an important extracellular matrix component and immune regulator.1 In this study, we exploit hyaluronan suprachemistry to formulate highly biocompatible nanoparticles (HA-NPs), which we propose for targeting of atherosclerosis-associated inflammation. Methods: Targeting properties of HA-NPs were studied in bone marrow derived macrophages (BMDMs) stimulated towards a pro- and anti-inflammatory phenotype and, in vivo, in atherosclerotic ApoE-/- mice. HA-NPs were labeled with either a fluorescent dye or 89Zr and investigated by flow cytometry or nuclear techniques, respectively. Furthermore, 89Zr-HA-NPs were imaged in a rabbit atherosclerosis model using PET/MRI. Finally, we investigated the atheroprotective effects of HA-NPs, by treating mice for 12 weeks with weekly doses of HA-NPs (50mg HA/kg/week). Results: BMDMs with a pro-inflammatory phenotype displayed a threefold higher uptake of HA-NPs compared to naive and anti-inflammatory macrophages, which, however, was decreased by 30% in the presence of pro-atherogenic oxLDL. Consistently, a prolonged high fat diet resulted in a lower uptake efficacy of HA-NPs by aortic macrophages compared to the early disease stage (3060 ± 836 and 697 ± 142 a.u., respectively). Autoradiography showed that 89Zr-HA-NPs are localized at the typical sites of atherosclerotic plaque formation in mice. Quantification of the radioactive signal by gamma counting revealed a 30% higher percentage of injected dose of 89Zr‐HA-NPs in atherosclerotic compared to wild type mice. In vivo PET/MRI in rabbits showed the association of 89Zr‐HA-NPs with the abdominal aorta and six fold higher aortic SUV (standardized uptake value) compared to the skeletal muscle (p = 0.038). Lastly, we demonstrated that a weekly treatment regimen with HA-NPs significantly lowers the number of aortic immune cells by circa 40% compared to free HA- and PBS-treated mice. Conclusion: HA-NPs efficiently target the atherosclerotic plaques in both atherosclerotic mice and rabbits. Furthermore, weekly doses of HA-NPs induce atheroprotective effects by lowering the immune cell infiltration in atherosclerotic aorta. References: 1: Noble et al. Physiol Rev 2011

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Plenary session 2: oral presentation 1 Cardiac function Orphan Nuclear Receptor Nur77 modulates cardiomyocyte function via Neuropeptide Y. Lejla Medzikovic1, Cindy van Roomen1, Antonius Baartscheer2, Duco Koenis1, Carol Ann Remme2, Carlie JM de Vries1, E Karin Arkenbout3,4, Vivian de Waard1 1Dept. of Medical Biochemistry, 2Dept. of Experimental Cardiology, 3Dept. of Cardiology, Academic Medical Center Amsterdam, Amsterdam, The Netherlands 4Dept. of Cardiology, Tergooi Hospital, Blaricum, The Netherlands Cardiac function is modulated by the sympathetic nervous system via cathecholamines released from cardiac sympathetic nerve endings and adrenal chromaffin cells. Co-transmitters such as neuropeptide Y (NPY) are co-released with catecholamines and thus may also modulate cardiac function. NPY has previously been reported as an independent predictor of mortality in heart failure patients. In experimental models, NPY induces cardiac hypertrophy and elevates intracellular Ca2+ concentrations, L-type Ca2+ current density and Ca2+-dependent signalling. Furthermore, NPY potentiates the deleterious effects of sympathetic overstimulation on cardiomyocytes. However, not much is known yet about the upstream regulation of NPY in the context of cardiac function modulation. Interestingly, a robust upregulation of NPY gene expression has been reported in macrophages lacking orphan nuclear receptor Nur77. Previously, we have shown that Nur77 deficiency in mice leads to elevated intracellular Ca2+ concentrations in healthy cardiomyocytes and to adverse cardiac remodelling in response to isoproterenol. As these effects may be explained by enhanced NPY action in Nur77-deficient (Nur77-KO) mice, we hypothesized that Nur77 is a regulator of NPY. We now show that Nur77 down-regulates NPY expression in and secretion from sympathetic cells in vitro. Furthermore, NPY levels are significantly higher in adrenal glands, blood plasma and hearts from Nur77-KO mice compared to wildtype mice. Moreover, antagonism of NPY receptor 1 attenuates cardiomyocyte hypertrophy and elevated Ca2+ responses caused by Nur77 deficiency. Thus, Nur77, at least in part, may affect cardiomyocyte function by modulation of NPY. Furthermore, these results imply a novel role for Nur77 in cardiac disease involving elevated sympathetic activity.

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Plenary session 2: oral presentation 2 Atherosclerosis Experimental TRAF-STOPs ameliorate atherosclerosis Tom Seijkens (1), Claudia M. van Tiel (1), Pascal J. H. Kusters (1), Barbara Zarzycka (2), Oliver Soehnlein (3,4,11), Svenja Meiler (1,4), Marten Hoeksema (1), Marion J. Gijbels (1,5,6), Roy Schrijver (2), Louis Boon (8), Francois Fay (9), Gert Vriend (10), Boris Bleijlevens (1), Norbert Gerdes (4), Menno P.J. de Winther (1,4), Gerry A. Nicholaes (2), Willem J. Mulder (1,9), Christian Weber (4,5,11), Esther Lutgens (1,4,11) 1 Department of Medical Biochemistry, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands, 3 Department of Pathology, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands, 4 Institute for Cardiovascular Prevention (IPEK), Ludwig Maximilians University, Munich, Germany, 5 Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands, 6 Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands, 8 Bioceros BV, Utrecht, The Netherlands, 9 Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA, 10 Centre for Molecular and Biomolecular Informatics (CMBI), Radboud University Medical Center, Nijmegen, The Netherlands, 11 German Centre for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Munich, Germany. Inhibition of the costimulatory CD40-CD40L receptor/ligand dyad drastically reduces atherosclerosis. However, its long-term blockage can result in immune suppression. We recently identified small molecule inhibitors that block the interaction between CD40 and TNF Receptor Associated Factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity. We further characterized the TRAF-STOPs 6877002 and 6860766 and found that both reduced initial and established atherosclerosis in ApoE-/- mice and induced a stable plaque phenotype with increased collagen and VSMC content, decreased lipid core, and a decrease in macrophage number, indicative of plaque stabilization. There were no signs of immune suppression or toxicity. In vitro experiments showed that the TRAF-STOPs especially reduced inflammation in macrophages, but not in T- or B cells, endothelial cells or vascular smooth muscle cells. Additionally, the CD36-mediated uptake of ox-LDL by macrophages and foam cell formation was inhibited by TRAF-STOPs. Intravital microscopy demonstrated that the TRAF-STOPs also hampered monocyte recruitment to the plaque. Transcriptomics analysis and Ingenuity pathway analysis of TRAF-STOP-treated bone marrow-derived macrophages revealed that the top ranking canonical pathways for both TRAF-STOPs involved pro-inflammatory immune responses and cholesterol biosynthesis. 6877002 also affected cell cycle regulation. Surface plasmon resonance experiments and mutation studies demonstrated that 6877002 and 6860766 had a different interaction site within the TRAF6 C-domain, which explained the additional effect of 6877002. To target TRAF-STOPs specifically to macrophages, 6877002 was incorporated into rHDL nanoparticles. Flowcytometry and fluorescent microscopy demonstrated accumulation of rHDL-6877002 in plaque macrophages after a single dosis. Six weeks of rHDL-6877002 treatment hampered the atherosclerosis in ApoE-/- mice. TRAF-STOP 6877002 and 6860766 can overcome the current limitations of long-term CD40 and CD40L inhibition and nanoparticle-mediated delivery TRAF-STOP to plaque macrophages may become a future therapy for atherosclerosis.

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Plenary session 2: oral presentation 3 Metabolism In obese women, but not in obese men, body fat distribution, in particular the presence of visceral fat, is associated with cardiometabolic risk factors Theodora W Elffers1,2, Renée de Mutsert1, Hildo J Lamb3, Arie C Maan2, Peter W Macfarlane4, Ko Willems van Dijk5, Frits R Rosendaal1,6, J Wouter Jukema2, Stella Trompet2,7 Objective Obesity and body fat distribution are associated with several cardiometabolic risk factors in the general population. It is unclear whether fat distribution remains important in the obese. We investigated the associations between different measures of body fat distribution and cardiometabolic risk factors in obese individuals. Methods In this cross-sectional analysis of baseline measurements of obese individuals included in the Netherlands Epidemiology of Obesity Study anthropometry was measured, in addition to abdominal subcutaneous adipose tissue (aSAT) and visceral adipose tissue (VAT) by MRI. We calculated odds ratios (OR) with 95% confidence intervals of the associations of waist circumference (WC), waist to hip ratio (WHR), aSAT, and VAT with high blood pressure, triglycerides, glucose concentrations and low HDL-cholesterol using logistic regression, analysis were adjusted for age, sex, ethnicity, education, smoking, alcohol consumption, and physical activity. Associations of WHR, WC and VAT were additionally adjusted for total body fat. Results After exclusion of non-obese participants (n=3,655) and participants with missing cardiometabolic risk factors (n=33), 2,983 participants (57% women) with a mean (SD) age of 56 (6) years and BMI of 34.0 kg/m2 (4.0) were analysed. In the multivariate model in obese women all measures except for aSAT (OR: 0.89, 95% CI: 0.65-1.21) were associated with the risk of having at least one cardiometabolic risk factor, of which VAT most strongly. Per SD VAT (64.0cm2) the OR was 5.77 (3.02-11.01) and one SD higher WC (10.9cm) with an OR of 1.29 (1.03-1.63). In the multivariate model in obese men, the measures of body fat distribution were not associated with having at least one cardiometabolic risk factor. (aSAT:0.73, 0.46-1.16; WC:1.15, 0.71-1.86; VAT:1.42, 0.84-2.41) Conclusions In obese women, but not in obese men, measures of body fat distribution, of which VAT most strongly, are associated with cardiometabolic risk factors. Future studies should aim at unravelling the underlying mechanisms of the detrimental metabolic effects of visceral fat in women. The NEO study is supported by the participating Departments, the Division and the Board of Directors of the Leiden University Medical Center, and by the Leiden University, Research Profile Area ‘Vascular and Regenerative Medicine’.

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Plenary session 2: oral presentation 4 Cardiac arrhythmia Optogenetic termination of ventricular arrhythmias in the whole heart: Towards biological cardiac rhythm management. Emile C.A. Nyns1, Annemarie Kip1, Cindy I. Bart1, Jaap J. Plomp2, Katja Zeppenfeld1, Martin J. Schalij1, Antoine A.F. de Vries1, Daniël A. Pijnappels1

1Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center 2Department of Neurology and Neurophysiology, Leiden University Medical Center Background Current treatments of ventricular arrhythmias rely on modulation of cardiac electrical function through drugs, ablation or electroshocks, which are all non-biological and rather unspecific, irreversible or traumatizing interventions. Optogenetics, however, is a novel, biological technique allowing electrical modulation in a specific, reversible and trauma-free manner using light-gated ion channels. The aim of our study was to investigate optogenetic termination of ventricular arrhythmias in the whole heart. Methods and results Systemic delivery of cardiotropic adeno-associated virus vectors, encoding the light-gated depolarizing ion channel red-activatable channelrhodopsin (ReaChR), resulted in global cardiomyocyte-restricted transgene expression in adult Wistar rat hearts allowing ReaChR mediated depolarization and pacing. Next, ventricular tachyarrhythmias (VTs) were induced in the optogenetically modified hearts by burst pacing in a Langendorff setup, followed by programmed, local epicardial illumination. A single 470-nm light pulse (1000 ms, 2.97 mW/mm2) terminated 96% of monomorphic and 53% of polymorphic VTs vs 0% without illumination, as assessed by electrocardiogram recordings. Optical mapping showed significant prolongation of voltage signals just before arrhythmia termination. Pharmacological action potential duration (APD) shortening almost fully inhibited light induced arrhythmia termination indicating an important role for APD in this process. Conclusion Brief local epicardial illumination of the optogenetically modified adult rat heart allows contact- and shock-free termination of ventricular arrhythmias in an effective and repetitive manner after optogenetic modification. These findings could lay the basis for the development of fundamentally new and biological options for cardiac arrhythmia management.

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Poster presentations (alphabetical order) 1. Suzanne A.B.M. Aarts (AMC) Cell type specific inhibition of co-stimulatory CD40

interactions in obesity 2. Jacob Amersfoort (LACDR) Lipocalin-2 contributes to experimental atherosclerosis in

a stage-dependent manner 3. Jeroen Baardman (AMC) ATP citrate lyase links cellular metabolism and the

epigenetic landscape to macrophage function 4. Jeroen Baardman (AMC) Mitochondrial dysfunction prevents repolarization of

inflammatory macrophages 5. Erik Bakker (AMC) Paravascular clearance pathways: a unique feature of the brain

vasculature 6. Siroon Bekkering (AMC) Innate immune cell activation in symptomatic and

asymptomatic atherosclerosis in humans in vivo 7. Siroon Bekkering (AMC) The cholesterol synthesis pathway is essential for trained

innate immunity 8. Mathijs C. Bodde (LUMC) Higher LDL-cholesterol levels are independently associated

with larger infarct size expressed by peak CK level in patients with ST-segment elevation myocardial infarction undergoing primary percutaneously coronary intervention.

9. Natalija Bogunovic (VUmc) Transdifferentiation of human dermal fibroblasts to smooth muscle like cells: a novel method to study the effect of MYH11 and ACTA2 variants in the aortic aneurysm wall

10. Ilse A.E. Bollen (Vumc) Commonalities and differences between dilated and peripartum cardiomyopathy; titin, fibrosis and myomesin

11. Ilze Bot (LACDR) Systemic mastocytosis associates with cardiovascular events despite lower plasma lipid levels

12. Jeroen T Buijs (LUMC) Direct anticoagulants do not inhibit growth and metastasis of orthotopically growing human MDA-MB-231 breast cancer cells in immune-deficient mice.

13. Andrea D. van Dam (LUMC) Impaired interferon signalling may underlie high prevalence diabetes and cardiovascular disease in South Asians

14. Calinda KE Dingenouts (LUMC) Inhibiting DPP4 in a mouse model of HHT-1 causes a shift in macrophage balance and decreases fibrosis after myocardial infarction

15. Charlotte E.A. Dronkers (LUMC) Association of disease prevalence and failure rate in diagnostic management studies in suspected pulmonary embolism: towards a new tailored standard for future studies

16. Janine van Duijn (LACDR) CD39-mediated exhaustion of CD8+ T cells in atherosclerotic lesions: role of the arterial microenvironment

17. Johanne (Rianne) H. Ellenbroek (LUMC) A-Kinase Anchoring Proteins in the Protection of Pancreatic Beta-cells from Cytokines

18. Iolanda Feola (LUMC) Optogenetic ablation of spiral wave arrhythmias by creating "light lesions".

19. Barend W. Florijn (LUMC) miRNA-224 and miRNA-452 display an oestrogen responsive decrease in male- to female transgenders and male endothelial cells

20. Barend W. Florijn (LUMC) Altered distribution of HDL, extracellular vesicle and Argonaute-2 associated circulating microRNAs in diabetic nephropathy and systemic microvascular damage

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21. Rick van der Geest (LACDR) Heat shock protein alpha B-crystallin increases atherosclerotic lesion development in LDLR KO mice

22. Niels Harlaar (LUMC) Utilizing excised human atrial and ventricular cardiac tissue for ex vivo studies into patient-specific disease and treatment

23. Merel L. Hartgers (AMC) Familial hypercholesterolemia: classification of mutation severity according to percentile LDL-cholesterol useful for predicting coronary artery disease risk.

24. Marco Heestermans (LUMC) Silencing of Anticoagulant Protein C Evokes Low Incident but Spontaneous Atherothrombosis in Apolipoprotein E Deficient Mice

25. Ntsiki Held (AMC) Fluxomic tools for characterization of metabolic reprogramming upon brown adipose tissue activation

26. Geerte Hoeke (LUMC) Cold exposure beneficially modulates HDL metabolism in mice and humans

27. Maurice J B van den Hoff (AMC) Deletion of Follistatin-like 1 from the endocardial lineage causes ongoing growth of the aortic leaflet of the mitral valve

28. Lisa Hoving (LUMC) Yeast-derived mannan oligosaccharide attenuates atherosclerosis development

29. Xu Hu (VUmc) Heat shock protein activators accelerate recovery from contractile dysfunction in experimental Atrial Fibrillation

30. Matthijs F Jansen (AMC) Development of anti-Galectin-2 antibodies to improve arteriogenesis.

31. Annika de Jong (LUMC) Improvement of a severe von Willebrand factor multimerization defect by efficient and allele-specific small interfering RNAs

32. Marjolein Caroline de Jongh (LUMC) Serial ECG Analysis after Myocardial Infarction:When Heart Failure Develops the ECG Becomes Increasingly Discordant.

33. Mohsin AF Khan (AMC) RBM20 regulates circular RNA production from the titin gene 34. Duco S Koenis (AMC) Genome-wide profiling reveals role of nuclear receptor Nur77 in

macrophage and skeletal muscle metabolism 35. H. Ibrahim Korkmaz (VUmc) Neutrophil Extracellular Traps: Contributors in Burn-

induced Microvascular Damage and Thrombosis? 36. Boudewijn PT Kruithof (LUMC) A new ex vivo model to study cardiac fibrosis 37. Eline N Kuipers (LUMC) Short term high fat feeding impacts on mitochondrial function

in brown adipose tissue 38. Pascal J.H. Kusters (AMC) Constitutive CD40-signaling in Dendritic Cells limits

atherosclerosis by provoking inflammatory bowel disease and ensuing cholesterol malabsorption

39. Zhuang Li (LUMC) Butyrate via the gut-brain circuit reduces appetite and activates brown adipose tissue

40. Jia Liu (LUMC) Protection from drug-induced arrhythmias by a novel allosteric modulator in a new validated rat ventricular cardiomyocyte model

41. Claire Mackaaij (AMC) Intracranial endothelial cells differ from their extracranial counterparts.

42. Andrea Mattiotti (AMC) Regulation of Follistatin-Like 1 Expression 43. Thijs E van Mens (AMC) Cochrane Diagnostic Test Accuracy Review: Imaging for the

exclusion of pulmonary embolism in pregnancy 44. Joya E. Nahon (LACDR) Glucose handling and white adipocyte phenotype are altered

in proteoglycan 4 deficient mice 45. Vera van de Pol (LUMC) Patients with Bicuspid Aortic Valve show increased aortic

inner media pathology at the high flow side

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46. Vincent Portero (AMC) Microtubule-associated protein RP/EB family member 1 modulates sodium channel trafficking and cardiac conduction

47. Marianne G Pouwer (LUMC) AT04A vaccine against PCSK-9 (Proprotein Convertase Subtilisin/Kexin Type 9) reduces total cholesterol, vascular inflammation and atherosclerosis in APOE*3Leiden.CETP mice

48. Maaike van Putten (LUMC) Effect of voluntary exercise on heart function in mice with low dystrophin levels

49. Timo Rademakers (SANQUIN) ADAM10-mediated cleavage of ICAM-1 is required for neutrophil release from the endothelial surface and enter the diapedesis step

50. Baoyan Ren (LACDR) Hematopoietic dual specificity phosphatase 4 deficiency promotes early atherosclerotic lesion development in LDL receptor knockout mice

51. Luca Sala (LUMC) A new hERG allosteric modulator rescues genetic and drug-induced Long-QT Syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells

52. Maaike Schilperoort (LUMC) G protein-coupled receptor 120 signaling activates brown adipocytes

53. Lilian Schimmel (SANQUIN) Endothelial DLC1 mediates the transition from rolling to adhesion stage of leukocytes prior to transendothelial migration

54. Johan G. Schnitzler (AMC) Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy.

55. Mark Schreuder (LUMC) Uncovering the unique functional APC-resistant modifications of P. Textilis FV

56. Tom T.P. Seijkens (AMC) Deficiency of the T cell regulator CBL-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death

57. Annelie Shami (AMC) GITR activation promotes regulatory CD4+ T-cell responses and stimulates leukocyte recruitment in atherosclerotic plaques

58. Annelie Shami (AMC) Macrophage CD40 signaling in atherosclerotic plaque development

59. Karin Hendrika Simons (LUMC) Interferon Regulatory Factors 3 and 7 regulate vein graft remodeling via TNF induced macrophage accumulation.

60. Ronald J van der Sluis (LACDR) LOWERING VLDL/LDL-C LEVELS IN APOE KNOCKOUT MICE DECREASES GLUCOCORTICOID PRODUCTION

61. Anke M. Smits (LUMC) Comparing fetal to adult epicardial cell activation to identify differences relevant for the epicardial post-injury response

62. Olga S.C. Snip (LACDR) Leukocyte ABCA1 Impedes Progression of Established Atherosclerotic Lesions after Dietary Cholesterol Lowering in LDLr-/- Mice

63. Katka Szilagyi (SANQUIN) SIRPalpha is a novel immune checkpoint on B1 cells regulating IgM production and atherosclerosis development

64. Alexander Teplenin (LUMC) Ectopic activity from localized oxidative stress zone violates traditional sink-source mismatch paradigm.

65. Claudia M van Tiel (AMC) CD40-Filamin A interactions are required for translocation of CD40 to lipid rafts in endothelial cells and for endothelial cell activation

66. Minh Khang Tran (AMC) Role of FHL2 in obesity and adipocyte differentiation 67. Laween Uthman (AMC) A cardiac mechanism of the kidney-targeted diabetes drug

EMPAgliflozin unveiled: EMPA decreased cytosolic sodium/calcium, increased mitochondrial calcium by inhibiting NHE-1 in cardiomyocytes and increased oxygen consumption and vasorelaxation in the isolated heart

68. Anna M.D. Végh (LUMC) Developing a gene transfer-platform for delivery of ion channel-based biological pacemakers

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69. Daniël Verhoef (LUMC) Side-by-side evaluation of clotting parameters in human, porcine, rabbit and rat plasma.

70. Simone Verweij (AMC) Remnant cholesterol as a direct mediator of arterial and cellular inflammation in humans

71. Dianne Vreeken (LUMC) The role of human genetic variations in neuronal guidance cues in premature atherosclerosis

72. Linde Woudstra (VUmc) Lymphocytic myocarditis occurs with myocardial infarction and coincides with increased inflammation, hemorrhage and instability in coronary artery atherosclerotic plaques.

73. Huayu Zhang (LUMC) The neuronal guidance cue semaphorin 3F is highly expressed by endothelial cells upon laminar flow and is important for endothelial barrier function

74. Kang He Zheng (AMC) Evaluation of ultrasmall superparamagnetic particles of iron-oxide (USPIO) enhanced MRI with ferumoxytol to quantify arterial wall inflammation.

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Abstracts poster presentations Abstract 1 Metabolism Cell type specific inhibition of co-stimulatory CD40 interactions in obesity Suzanne Aarts, Susan van den Berg, Annelie Shami, Myrthe den Toom, Milou Valk, Linda Beckers, Esther Lutgens. Academic Medical Center, Amsterdam, The Netherlands. The co-stimulatory molecule pair CD40-CD40L plays a central role in fine-tuning immunological reactions, including obesity-induced inflammation. Genetic ablation of CD40L results in amelioration of adipose tissue inflammation, but in contrast the CD40-/- mice displayed worsened insulin resistance and excessive inflammation of adipose tissue compared with wild-type mice. To investigate which cells are causing the phenotype observed in the CD40-/- mice, we have generated cell type specific CD40 knock out mice for macrophages (LysMcreCD40flfl), dendritic cells/adipose tissue macrophages (CD11CcreCD40flfl), adipocytes (AdipoQcreCD40flfl), and CD40L knock out mice for T cells (CD4creCD40Lflfl) which are fed a high fat diet for 18 weeks. So far, the LysMcreCD40flfl mice show that loss of macrophage CD40 has no effect on adipose tissue mass and body weight increase, insulin tolerance, and plasma triglyceride concentrations. The CD11CcreCD40flfl mice show that CD40 deficiency on DCs and adipose tissue macrophages doesn’t affect body weight, but decreases visceral adipose tissue weight, increases lipid content in the liver and slightly decreases leukocyte numbers and proinflammatory gene expression in the adipose tissue. To this point, the first experiments show that macrophage CD40 does have a minor effect in obesity-induced inflammation, but is probably not the key player. Future experiments with the AdipoQcreCD40flfl and the CD4creCD40Lflfl mice will have to provide clarity on the exact role of CD40-CD40L interactions in diet-induced obesity.

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Abstract 2 Atherosclerosis Experimental, Atherosclerosis Clinical Lipocalin-2 contributes to experimental atherosclerosis in a stage-dependent manner Jacob Amersfoort1, Frank H. Schaftenaar1, Hidde Douna1, Gijs H.M. van Puijvelde1, Margreet R. de Vries2, Paul H.A. Quax2,3, Johan Kuiper1, Ilze Bot1 1Division of Biopharmaceutics, LACDR, Leiden University, Leiden, The Netherlands, 2Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands, 3Einthoven Laboratory for Experimental Vascular Medicine Lipocalin-2 (Lcn2) is an antibacterial glycoprotein, which is secreted by activated neutrophils, monocytes and macrophages upon infection. It is associated with different parameters of coronary artery disease, including severity of disease, while its pathophysiological mechanisms remain relatively unexplored. We aimed to examine these in an Lcn2 knock-out (KO) model in experimental diet-induced atherosclerosis. Lcn2 expression is upregulated during Western-type diet in carotid arteries with collar-induced atherosclerosis from female LDLr deficient mice (LDLr KO) as compared to non-atherosclerotic control carotid arteries (t0: 123.8±12.14, t2: 14822±6936.4, t6: 1549±977.3, t10: 1541±924.5). Female LDLr (KO) or Lcn2 LDLr-dKO (dKO) mice were fed a Western-type diet for 6 or 12 weeks to examine early and advanced atherosclerosis, respectively. Early lesion development in the 3-valve area was increased in the dKO group (KO: 2,42±0,54*105 µm2 vs dKO: 2,90±0,55*105 µm2, p<0.05) while advanced lesion size was not significantly altered (KO: 8,25±1,67*105 µm2 vs dKO: 8,60±1,43*105 µm2, p=0.58). Interestingly, increased lesion stability as assessed by necrotic core size was observed in advanced lesions from dKO mice (KO: 31,2±6,62% vs dKO: 24,3±6,17%, p<0.01), while intraplaque MMP9 activity appeared decreased. Flow cytometry analysis suggests Lcn2 KO increases monocyte recruitment as circulating pro-inflammatory CD11b+Ly6G-Ly6Chi monocytes were increased during early (KO: 39,6±5,79% vs dKO: 50,6±7,33%, p<0.05) and advanced lesions (KO: 41,7±4,27% vs dKO: 48,4±5,45%, p<0.05). Also, monocyte levels were decreased in bone marrow (KO: 42,6±5,17% vs dKO: 30,0±2,17%, p<0.01). Interestingly, no effects were observed on CD11b+Ly6G+Ly6Cint neutrophil abundance or activation status in the circulation or bone marrow, while Lcn2 is a potent chemokine for neutrophils. In conclusion, in early-stage experimental atherosclerosis Lcn2 KO increases lesion development, presumably by increasing monocyte recruitment. In advanced stage atherosclerosis Lcn2 KO mainly affects plaque stability. These findings suggest Lcn2 has a stage-dependent contribution to experimental atherosclerosis.

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Abstract 3 Atherosclerosis Experimental, Metabolism ATP citrate lyase links cellular metabolism and the epigenetic landscape to macrophage function Jeroen Baardman1, Menno P.J. de Winther1 and Jan Van den Bossche1 1Experimental Vascular Biology, Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam Introduction: Macrophage activation and function is regulated by alterations in gene expression and epigenetic mechanisms now arise as key controllers of macrophage activity. Epigenetic enzymes require metabolites to support their chromatin-modifying activity. For example, histone acetyltransferases (HATs) utilizing acetyl-coenzyme A (Acetyl-CoA) as substrate for histone acetylation. Several enzymes are capable to generate acetyl-CoA in mammalian cells, including ATP citrate lyase (Acly), and possibly involved in macrophage activation. Modulating the epigenetic landscape in macrophages could be a therapeutic tool to dampen chronic inflammatory diseases such as atherosclerosis. Results: We found that transcript and protein levels of Acly are not affected after LPS activation of BMDMs and RAW 264.7 cells. However, LPS activation induced phosphorylation of Acly on serine residue 455, a mark of enhanced catalytic activity. In addition to the cytosol, Acly was found to be localized in the nucleus, possibly involved in nuclear acetyl-CoA synthesis for histone acetylation. Indeed, knockdown of Acly in RAW 264.7 cells using shRNAs resulted in a reduction of global histone 3 (H3) and H4 acetylation. In addition, knockdown of Acly diminished the expression of several LPS-induced genes related to atherosclerosis (e.g. Ccl2 ,Ccl5 and Cxcl10). Conclusion: LPS induces the activity of Acly, an enzyme that is localized in both cytosol and nucleus in macrophages and able to synthesizes acetyl-CoA. This metabolite have been found by others to be crucial in modulating the epigenetic landscape in a cell. Reduced global histone acetylation and reduced expression of genes associated with the pathogenesis of atherosclerosis are observed after knockdown of Acly, probably via aberrant acetyl-CoA levels in the cell.

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Abstract 4 Atherosclerosis Experimental, Metabolism Mitochondrial dysfunction prevents repolarization of inflammatory macrophages Jan Van den Bossche1,*‡, Jeroen Baardman1, Natasja A. Otto1,2,3, Saskia van der Velden1, Annette E. Neele1, Susan M. van den Berg1, Rosario Luque-Martin1, Hung-Jen Chen1, Marieke C.S. Boshuizen1, Mohamed Ahmed1, Marten A. Hoeksema1, Alex F. de Vos2,3, and Menno P.J. de Winther1 1Department of Medical Biochemistry, Experimental Vascular Biology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam, 1105AZ, the Netherlands; 2Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam, 1105AZ, the Netherlands; 3Center for Infection and Immunity, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam, 1105AZ, the Netherlands. * supported by a Junior Postdoc grant, provided by the Netherlands Heart Foundation (grant number 2013T003) and by a NWO Veni grant ‡ Correspondence : j.vandenbossche@amc.uva.nl Macrophages determine the outcome of atherosclerosis by propagating inflammation, foam cell formation and necrotic core development. Yet, the pathways that regulate their atherogenic functions remain ill-defined. Macrophages are innate immune cells that adopt diverse activation states in response to their microenvironment. Editing macrophage activation is of high interest to dampen inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages to anti-inflammatory (M2) macrophages. Here, we found that mouse and human M1 macrophages completely failed to convert into M2 cells upon IL-4 exposure in vitro and in vivo. In sharp contrast, M2 macrophages were more plastic and readily repolarized into an inflammatory M1 state. We identified M1-associated inhibition of mitochondrial oxidative phosphorylation as the factor responsible for preventing M1→M2 repolarization. Inhibiting nitric oxide production, a key effector molecule in M1 cells, dampened the decline in mitochondrial function and improved metabolic and phenotypic reprogramming to M2 macrophages. Thus, inflammatory macrophage activation blunts oxidative phosphorylation, thereby preventing repolarization. Therapeutically restoring mitochondrial function might be useful to improve the reprogramming of inflammatory macrophages into anti-inflammatory cells to control cardiovascular disease.

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Abstract 5 Imaging, Vascular Biology Paravascular clearance pathways: a unique feature of the brain vasculature Beatrice Bedussi1, Monique GJTB van Lier1, Judith de Vos1, Nicole N. van der Wel2, Henk van Veen2, Maria Siebes1, Ed VanBavel1 and Erik NTP Bakker1. 1Dept. of Biomedical Engineering and Physics, 2Electron Microscopy Centre Amsterdam, Dept. of Cell Biology and Histology, Academic Medical Center, Amsterdam, the Netherlands.

Cardiovascular diseases increase the risk for dementia, including Alzheimer’s disease. The mechanisms behind this association are not fully known. It has been speculated that impaired function of the recently discovered paravascular clearance pathways may play a role herein. These pathways may function as the brains’ lymphatic system in maintaining fluid balance and removal of waste products, including amyloid β. The aim of the current study was to visualize and characterize the paravascular pathways in rodent brain. Therefore, we injected FITC-dextran (500kD) in the cisterna magna of rats and mice and allowed these tracers to spread for 30 minutes. Horizontal and coronal brain sections were imaged using confocal microscopy, a 3D imaging cryomicrotome, and Correlative Light Electron Microscopy (CLEM) to reveal the pattern of distribution. After injection in the cisterna magna, tracers revealed an extensive, continuous CSF compartment that consisted of the ventricles, subarachnoid space and cisterns, and extended into the parenchyma along arteries (not veins). Paravascular penetration of tracer into the tissue could be observed down to the capillary level. In conclusion, cerebrospinal fluid penetrates the brain along paravascular spaces. Thus, the CSF compartment has an intricate spatial relationship with the vasculature, which may facilitate clearance of the brain from amyloid β and other waste products.

100 µm

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Abstract 6 Atherosclerosis Clinical, Metabolism, Autoimmunity and Inflammation Innate immune cell activation in symptomatic and asymptomatic atherosclerosis in humans in vivo. Siroon Bekkering3, Inge van den Munckhof1, Tim Nielen2, Joost Rutten1, Jacqueline de Graaf1, Leo AB Joosten1, Mihai G Netea1, Marc ER Gomes2, Niels P Riksen1

1 Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands 2 Department of Cardiology, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands 3 Department of Vascular Medicine, Academic Medical Centre, Amsterdam, The Netherlands Aim – We have recently reported that monocytes can adopt a long-term pro-inflammatory phenotype after brief exposure to atherogenic stimuli, such as oxidized LDL. This is mediated by epigenetic reprogramming and a metabolic switch towards increased aerobic glycolysis. We hypothesize that this mechanism (‘trained immunity’) contributes to atherogenesis. We now studied the inflammatory phenotype and epigenetic remodeling of monocytes from patients with atherosclerosis. Methods – Monocytes were isolated from 20 patients with severe symptomatic coronary atherosclerosis (total plaque score >4 on cardiac computed tomography angiography) and 17 patients with asymptomatic carotid atherosclerosis and matched controls. We measured cytokine production after 24 hours of ex vivo stimulation with LPS and analyzed histone modifications with Chromatin Immunoprecipitation. Results Monocytes from patients with symptomatic atherosclerosis have a higher production of pro-inflammatory cytokines upon LPS stimulation than healthy controls (TNFα 499 ± 102 versus 267 ± 45 pg/ml, p=0.01). This was associated with lower histone 3 lysine 4 trimethylation (H3K4me3) (19% vs 33%, p=0.002) and lower H3K27me3 (0.005% vs 0.8%, p<0.0001) on the tnfα promoter. Furthermore, relative mRNA expression of the glycolytic rate limiting enzymes Hexofructokinase 2 and Phospho-fructokinase was higher in patients (0.7±0.2 vs 0.3±0.06 resp. 1.7±0.2 vs 1.0±0.1, P=0.007 resp. 0.003). Interestingly, this pro-inflammatory phenotype was only present in patients with symptomatic atherosclerosis, and not in patients with asymptomatic carotid atherosclerosis. Conclusion Circulating monocytes of patients with symptomatic, but not asymptomatic, atherosclerosis have a pro-inflammatory phenotype and increased glycolysis, associated with epigenetic remodeling at the level of histone methylation.

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Abstract 7 Metabolism, Autoimmunity and Inflammation The cholesterol synthesis pathway is essential for trained innate immunity Siroon Bekkering1, Rob JW Arts2, Boris Novakovic3, Rob ter Horst2, Julia van Tuijl2, Henk Stunnenberg3, Leo AB Joosten2, Mihai G Netea2, Erik SG Stroes1, Niels P Riksen2 1 Department of Vascular Medicine, Academic Medical Centre, Amsterdam, The Netherlands 2 Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands 3 Department of Molecular Biology, Radboud University, Nijmegen, The Netherlands Aim –Monocytes can adopt a longterm proinflammatory and proatherogenic phenotype after brief exposure to atherogenic stimuli, such as Lipoprotein(a) and oxidized LDL (oxLDL). This is mediated by epigenetic reprogramming of histones, and is called trained innate immunity. Here we studied the role of the cholesterol synthesis pathway in trained immunity. Methods – Isolated human monocytes were exposed to 10 μg/ml oxLDL or β-glucan as positive control for 24 hours in the presence of RPMI, 10 μM fluvastatin, 200 μM 6-Fluoromevalonate, 5 μM Zaragozic Acid or mevalonate (figure). After 6 days, we measured foam cell formation, ex vivo cytokine production, and histone methylation with ChIP-PCR and ChIP-sequencing. HIDS patients lacking Mevalonate Kinase were also studied for their inflammatory profile and epigenetic landscape. An in vivo study in patients with elevated plasma cholesterol on the effect of statins on trained immunity is ongoing. Results - 24-hour exposure of monocytes to oxLDL, followed by 6 days of rest, increased LPS-induced TNFα production on day 7 (747 pg/ml (-oxLDL) vs 4163 pg/ml (+oxLDL), p=0.01). This was associated with increased glycolysis, increased foam cell formation and increased Histone 3 lysine 4 trimethylation (H3K4me3) on promoters of inflammatory genes. The increased pro-inflammatory phenotype was prevented by fluvastatin (TNFα production 4163 pg/ml (-Fluva) vs 2296 pg/ml (+Fluva), P=0.004), and restored by adding mevalonate (p=0.008). Furthermore, 6-Fluoromevalonate increased TNFα production (p=0.02), and Zaragozic Acid did not result in inhibition or increase. Mevalonate alone (p=0.003), was also able to potentiate LPS-induced TNF production 6 days after initial priming, similarly to oxLDL, and showed a trained immunity phenotype when RNA sequencing was performed on cells on day 1 and 6 as well as confirmed by ChIP sequencing. Conclusion - Trained immunity is critically dependent on the cholesterol synthesis pathway (see figure), but not on the synthesis of cholesterol itself.

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Abstract 8 Atherosclerosis Clinical Higher LDL-cholesterol levels are independently associated with larger infarct size expressed by peak CK level in patients with ST-segment elevation myocardial infarction undergoing primary percutaneously coronary intervention. M.C. Bodde, M.P.J. Hermans, R. Wolterbeek, A. van der Laarse, M.J. Schalij, J.W. Jukema Institution: Department of Cardiology. Leiden University Medical Centre (LUMC). 2300 RC Leiden. The Netherlands and the department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Centre (LUMC). 2300 RC Leiden. Introduction: It is hypothesized that high levels of LDL-cholesterol (LDL-c) in patients with ST-segment elevation myocardial infarction (STEMI) cause more reperfusion damage during primary percutaneous coronary intervention (pPCI) and therefore larger infarct size. Plasma peak creatine kinase (CK) has proven to be a good measure of infarct size and clinical outcome. Infarct size in patients with STEMI has been shown to be associated with mortality. The aim of this study was to evaluate whether baseline LDL-cholesterol levels were associated with infarct size expressed by peak CK in plasma of patients with STEMI after pPCI. Methods: All patients admitted between 2004-2014 with STEMI and undergoing pPCI were treated according to current guidelines. Patients with an out hospital cardiac arrest (OHCA), time of ischemia ≥ 10 h, no complete reperfusion after pPCI in the culprit vessel and unknown levels of peak CK during admission were, were excluded from this analysis. Baseline blood samples were defined as first blood samples acquired during admission. Peak CK level was defined as the maximum measured value during admission. Variables with p-value <0.10 in univariate analysis were included in a multivariate regression model. Results: A total of 2248 patients were included in this retrospective analysis (mean age 61.8 ± 12.2 years; 25.0% female). The mean LDL-c level was 3.6 ± 1.1 mmol/L with a median peak CK level of 1275 U/L (IQR 564-2590 U/L) for the entire study population. On multivariable analysis baseline LDL-c levels (β =0.041; [CI 0.019-0.062]; p-value<0.001) were independently associated with peak CK level in plasma. Furthermore, LAD (as culprit vessel), baseline TIMI flow of the culprit vessel, male gender, time of ischemia and prior aspirin use were associated with peak CK levels (p<0.05, for all). Conclusion: In patients with STEMI treated with pPCI, baseline LDL-c levels are independently associated with peak CK level. This conclusion is in line with the hypothesis that elevated LDL-c levels result in augmented reperfusion damage during pPCI.

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Table 1 Independent correlates for infarct size measured by peak CK (log transformed). Variable Multivariate analysis β (95% CI)

P-value

LDL-c (mmol/L) 0.041 (0.019-0.062)

<0.001

LAD culprit lesion Baseline TIMI flow 0-1 Gender (male)

0.152 (0.108-0.195) 0.431 (0.384-0.478) 0.103 (0.052-0.154)

<0.001 <0.001 <0.001

Time of Ischemia (onset symptoms to balloon time) Aspirin use before STEMI

0.206 (0.102-0.309) -0.073 (-0.146-0.000)

0.032 0.050

Current smoker 0.036 (-0.009-0.082) 0.119

Age Beta-blocker use before STEMI

-0.001 (-0.003-0.001) -0.012 (-0.075-0.051)

0.315 0.705

Statin use before STEMI -0.003 (-0.072-0.065)

0.923

TIMI (Thrombolysis in Myocardial Infarction flow), LAD (Left anterior descending), * Symptom onset to balloon time (in minutes) Β = standardized regression coefficient

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Abstract 9 Vascular Biology Transdifferentiation of human dermal fibroblasts to smooth muscle like cells: a novel method to study the effect of MYH11 and ACTA2 variants in the aortic aneurysm wall Natalija Bogunovic1,2,, R.J.P. Musters2, D. Micha3, G.Pals3, W. Wisselink1, J.D. Blankensteijn1, B. Zandieh-Doulabi4 , K.K. Yeung1,2, 1 Department of Surgery, VU University medical center, Amsterdam, The Netherlands 2 Department of Physiology, VU University medical center, The Netherlands 3 Department of Clinical Genetics, VU University medical center, The Netherlands 4 Department of Oral Cell Biology, ACTA University of Amsterdam and VU University Amsterdam, The Netherlands Introduction Research on the pathogenesis of aortic aneurysms has revealed mutations in genes encoding the smooth muscle cell (SMC) contractile proteins as key underlying causes. Mutations associated with familial aortic aneurysms have been found in MYH11 (myosin heavy chain 11), ACTA2 (smooth muscle actin alpha 2) and MYLK (myosin light chain kinase) genes, which encode integral proteins of the SMC contractile apparatus. Currently, SMC can only be obtained by an invasive aortic biopsy. Therefore, the aim of this study is to transdifferentiate skin fibroblasts into SMC-like cells to provide a less invasive diagnostic test to study SMC function and mutations. Methods Dermal fibroblasts from 7 healthy donors and 7 patients with MYH11 or ACTA2 variants were transdifferentiated into SMC-like cells within 2 weeks by using 5ng/mL TGFβ1 and a scaffold containing collagen and elastin (matriderm). As control, cells were cultured without TGFβ1. SMC-specific markers were analyzed via qPCR, western blot and immunofluorescence. To investigate and classify the pathogenicity of the variants, cDNA sequencing was performed. Transdifferentiated SMC were additionally compared to primary SMC. Results The induced SMC-like cells were comparable to primary human aortic SMC in the expression of SMC specific markers on mRNA and protein level: ACTA2 (αSMA), SMTN (smoothelin) and CNN1 (calponin). Importantly, in patients with MYH11 or ACTA2 variants the effect on splicing can be demonstrated on the mRNA level in the induced SMC, allowing prediction and classification into pathogenic or non- pathogenic variants. Moreover, the variants showed different contractile protein expression, if compared to transdifferentiated SMC from the healthy donors. Conclusions Direct conversion of human dermal fibroblasts into SMC-like cells is a highly efficient method to investigate the pathogenic effect of variants in genes encoding the proteins of the SMC contractile apparatus. Our findings suggest the possible role of these variants in disturbed SMC contractility and aortic aneurysm formation.

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Abstract 10 Heart failure Commonalities and differences between dilated and peripartum cardiomyopathy; titin, fibrosis and myomesin Ilse AE Bollen1, Floor Bouwman1, Romy Fessl1, Elisabeth Ehler2, Karin Y van Spaendonck-Zwarts3, Yigal M Pinto4, Diederik WD Kuster1, Jolanda van der Velden1 1Department of Physiology, VU University Medical Center, Amsterdam, the Netherlands, 2The Randall Centre of Cell and Molecular Biophysics King’s College London. 3Department of genetics, Academic Medical Center, Amsterdam, the Netherlands. 4Heart Failure Research Center, Academic Medical Center, Amsterdam, The Netherlands. Introduction. Dilated cardiomyopathy (DCM) and peripartum cardiomyopathy (PPCM) share aspects of clinical presentation such as ventricle dilation and reduced systolic function. However, the underlying cause and disease progression are different. We therefore hypothesized that the pathomechanisms of the two cardiomyopathies are different. We investigated titin isoform composition, fibrosis and induction of fetal myomesin expression in end-stage PPCM and DCM patients compared with non-failing donors. Methods. Left ventricular tissue of 6 PPCM, 11 DCM patients and 13 non-failing donors was used. Titin isoform composition was determined by agarose gel electrophoresis stained with SYPRO ruby protein stain. Fibrosis and fetal myomesin expression were determined by immunohistochemical staining. Results and conclusions. Expression of the compliant titin isoform (N2BA/N2B ratio) was higher in PPCM and DCM patients (0.82±0.15 and 0.88±0.11 respectively) compared with donors (0.57±0.06). The size of N2BA titin was also increased in both cardiomyopathy groups. Increased fibrosis was seen in both PPCM (4.5%) and DCM (5.5%) patients compared with donors (1.5%). This indicates that the titin isoform shift might compensate for the increased fibrosis. Induction of the fetal isoform of myomesin was observed only in DCM patients and not in PPCM patients and donors. This provides a first indication that remodelling might be different in the two cardiomyopathies.

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Abstract 11 Atherosclerosis Clinical Systemic mastocytosis associates with cardiovascular events despite lower plasma lipid levels 1Swasti Indhirajanti, 2Paul L.A. van Daele, 1Sven Bos, 1Monique T. Mulder, 3Ilze Bot, 1Jeanine E. Roeters van Lennep 1Department of Internal medicine, division Vascular Medicine, and 2Department of Internal medicine, division Immunology, Erasmus MC, Rotterdam, The Netherlands 3Division of Biopharmaceutics, Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands Background Systemic mastocytosis (SM) is a disease characterized by an accumulation of mast cells in one or more organs. Mast cells have previously been shown to contribute to atherosclerotic lesion progression and destabilization both in mouse models and in human pathology studies. However, the prevalence of cardiovascular diseases (CVD) in patients suffering from SM has up to now not been analyzed. In this study, we thus aimed to compare the incidence of CVD between patients with systemic mastocytosis (SM) and controls. Methods In 50 patients with SM and 50 age- and sex-matched controls, the history of CVD and the presence of cardiovascular risk factors were assessed. Carotid ultrasound was performed to determine the presence of plaque and to analyze the carotid intima-media thickness (C-IMT). Also, plasma lipid levels were analyzed. Results The incidence of CVD events was significantly higher in SM patients compared to controls (20% versus 6%, p=0.04). The number of carotid artery plaques was similar between patients with SM and controls, and also C-IMT did not differ between the groups (0.65±0.11mm compared to 0.64±0.13 mm, p=0.65). In multivariate analysis, CVD events were significantly associated with SM (OR 7.0 (95% CI 1.3-37.6), p=0.02) and hypertension (OR 9.5 (95% CI 1.9-48.7), p=0.01). The prevalence of diabetes, hypertension, obesity and smoking was similar between the two groups. Interestingly, total cholesterol and LDL-C levels were actually significantly lower in the SM patients compared to the control group. (TC: 5.1±1.1 versus 5.9±0.9 mmol/L, p<0.05; LDL-C: 2.9±0.8 versus 3.5±0.7 mmol/L, p<0.05). Conclusions Despite lower plasma total cholesterol and LDL-C, the prevalence of CVD is higher in patients with SM compared to healthy controls. Therefore cardiovascular screening in patients with SM deems warranted. These data also indicate that mast cells actively contribute to atherosclerotic plaque destabilization. Funding: I. Bot is supported by a Dr. Dekker Senior Postdoc grant from the Dutch Heart Foundation (2012T083).

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Abstract 12 Stem cells, Thrombosis and Hemostasis Direct anticoagulants do not inhibit growth and metastasis of orthotopically growing human MDA-MB-231 breast cancer cells in immunodeficient mice Buijs JT 1, Tieken C 1, van der Molen K 1, Crooijmans J 1, Ünlü B 1, Lagmani EH 1, Kroone C 1, Swier N 1, Gabri van der Pluijm 2, le Dévédec SE 3, Versteeg HH 1 1 Dept. of Internal Medicine/ Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Centre, Leiden, The Netherlands 2 Dept. of Urology, Leiden University Medical Centre, Leiden, The Netherlands 3 Div. of Toxicology Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands Cancer is associated with a hypercoagulable state and a 4-6-fold increased risk for venous thromboembolism (VTE). Therefore, cancer patients with VTE are treated with anticoagulant therapy. Preclinical studies have suggested that coagulant factors may also have direct pro-tumorigenic effects, however, a meta-analysis did not show beneficial effects of prophylactic treatment with Low Molecular Weight Heparin (LMWH) on survival in cancer patients. Recently, two novel direct anticoagulant (DOACs) therapies are available in the clinic for treatment of VTE, namely dabigatran and rivaroxaban, a direct thrombin and FXa inhibitor, respectively. However, the effects of DOACs on cancer progression remain to be investigated. In this study, we investigated whether DOACs could inhibit tumor growth and metastasis of orthotopically injected MDA-MB-231 breast cancer cells in immune deficient mice. Mice put on a chow diet with Dabigatran (10g/kg) or Rivaroxaban (0.4g/kg) showed a significant (P<0.001) increase in coagulation time as measured by PT assay (13.5±0.5 sec vs 18.5±0.4 sec for Veh. vs. rivaroxaban, p<0.001) and aPTT assay (28.1±0.1 sec vs 68.9±1.1 sec for Veh. vs. Dabigatran, p<0.001) when compared to vehicle mice (chow diet). However, dabigatran and rivaroxaban did not result in significant inhibition in tumor growth or metastasis formation in immunodeficient NOD-SCID gamma mice. Likewise, dabigatran and rivaroxaban were also not able to significantly inhibit tumor growth or metastasis formation (One-Way ANOVA using Bonferroni posttest) in a pilot study using a immunodeficient, but NK-cell proficient, NOD-SCID mice. Furthermore, a variety of in vitro assays showed that the coagulant factors thrombin (10-50nM) and FXa (2-20nM) were unable to potently induce migration, proliferation or stemness in triple negative breast cancer (TNBC) cell lines MDA-MB231 and HCC1806. Taken together, this study showed that the coagulation factors FXa and thrombin are not critically important in tumor progression of TNBC. However, a role for DOACs as prophylactic treatment in other types of cancer may still be an interesting strategy to pursue.

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Abstract 13 Autoimmunity and Inflammation Impaired interferon signalling may underlie high prevalence diabetes and cardiovascular disease in South Asians Andrea D. van Dam1, Mark J.W. Hanssen2, Robin van Eenige1, Edwin Quinten1, Hetty C. Sips1, Joris Hoeks2, Ingrid M. Jazet1, Wouter D. van Marken Lichtenbelt2, Mariëlle C. Haks1, Patrick C.N. Rensen1, Mariëtte R. Boon1,2 1LUMC, 2MUMC South Asians have a higher risk of developing type 2 diabetes (T2D) and cardiovascular disease (CVD) compared to white Caucasians. Despite the presence of predisposing classical risk factors such as central obesity, insulin resistance and dyslipidemia, the high prevalence of T2D and CVD in South Asians cannot be explained by these factors alone. Since inflammation plays an important role in the pathogenesis of both T2D and CVD, we aimed to assess whether inflammation is altered in South Asians compared to white Caucasians. We determined inflammatory gene expression by dual color reverse transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA) in subcutaneous adipose tissue and skeletal muscle biopsies in ten obese, pre-diabetic South Asians and ten BMI-and age-matched white Caucasians. Whole body energy expenditure and mitochondrial oxygen consumption in muscle biopsies were also assessed. In white adipose tissue, the expression of most macrophage markers and T cell subsets, pattern recognition receptors and inflammasome components were similar in South Asians and white Caucasians. Surprisingly, South Asians exhibited lower expression of the interferon signalling genes IFI44 (-40%, p<0.01), IFIT5 (-28%, p<0.01), OAS1 (-33%, p<0.01), IFIT3 (-23%, p<0.05), IFIT2 (-22%, p<0.05) IFI35 (-22%, p<0.05) and STAT1 (-13%, p<0.05). In muscle, although expression of inflammatory markers was generally low, expression of the interferon signaling genes IFIT3 (-45%, p<0.01) and IFI44 (-51%, p<0.01) were also markedly lower in South Asians compared to white Caucasians. Interestingly, IFIT2 (R2=0.28, p<0.05), IFIT3 (R2=0.50, p<0.001) and IFIT5 (R2=0.45, p<0.01) expression in WAT correlated positively with mitochondrial respiration in skeletal muscle. In conclusion, impaired interferon signalling might contribute to the disadvantageous metabolic phenotype of South Asians. Future research should assess the link between interferon signalling and mitochondrial function as a contributor to the increased risk of T2D and CVD in this population.

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Abstract 14 Vascular biology Inhibiting DPP4 in a mouse model of HHT-1 causes a shift in macrophage balance and decreases fibrosis after myocardial infarction C.K.E. Dingenouts1, W. Bakker1, K. Lodder1, C.C. Wiesmeijer1, A.T. Moerkamp1, J.A. Maring1, H.M. Arthur2, A.M. Smits1, M.J. Goumans1. 1 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands 2 Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, United Kingdom Background: Hereditary Hemorrhagic Telangiectasia type 1 (HHT-1) is a genetic dominant vascular disorder caused by haploinsufficiency of the TGFβ co-receptor Endoglin. The pathology of HHT-1 suggests that mainly vascular endothelial cells are involved. However, we have previously shown dysfunctional homing of HHT-1 mononuclear cells (MNC) towards the infarcted myocardium (MI). This is most likely due to enhanced expression of dipeptidyl peptidase-4 (DPP4). DPP4 inactivates SDF-1α, thereby inhibiting recruitment of CXCR4 expressing cells. Purpose: Our aim is to increase homing of the HHT-1 MNC and improve cardiac recovery and function in Eng+/- mice, as a model of HHT-1, following MI by inhibiting DPP4 activity. Methods: MI was induced in wildtype (WT) and Endoglin heterozygous (Eng+/-) mice by ligation of the left anterior descending (LAD) coronary artery, followed by 5 days of daily treatment with the DPP4 inhibitor Diprotin A (2.5mg/kg/day). The infarcted hearts were functionally, morphologically and immunohistologically assessed at day 4 and at day 14 post MI. Results: DPP4 inhibition restored the number of CXCR4+ MNC present in the infarcted hearts and significantly reduced infarct size, as measured by myocardial collagen deposition. Analysis of cardiac function using ultrasound demonstrated that DPP4 inhibitor treatment of WT mice improved ejection fraction, but slightly deteriorating heart function in Eng+/- animals. Furthermore at day 4 post-MI, during the peak of inflammatory cell influx, Eng+/- mice have significant lower numbers of regenerative M2 macrophages in the heart compared to WT mice, which was still observed at day 14 post-MI, together with an overall increase in macrophage presence. DPP4 inhibition raised the M2 levels in Eng+/- at day 4 and even at 14 days post MI the increased M2 levels had persisted. Conclusions: In conclusion, systemic DPP4 inhibition does restore impaired MNC homing in Eng+/- mice but does not improve cardiac ejection fraction. Interestingly, cardiac repair is enhanced as demonstrated by a decreased fibrotic response, resulting in a reduced infarct size. Furthermore Eng+/- mice have a defect in macrophage differentiation and function. DPP4 inhibition results in an increase in angiogenesis and rescues the amount of M2 macrophages to wildtype levels. This project is funded by the Netherlands Institute for Regenerative Medicine, project 20743, and by the Dutch Heart Foundation, project 30710.

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Abstract 15 Thrombosis and Hemostasis Association of disease prevalence and failure rate in diagnostic management studies in suspected pulmonary embolism: towards a new tailored standard for future studies Charlotte E.A. Dronkers1, Tom van der Hulle1, Yvonne M. Ende-Verhaar1, Suzanne C. Cannegieter2, Menno V. Huisman1, Frederikus A. Klok1

1Department of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands 2Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands Background : Traditionally, the accepted failure rate of pulmonary embolism (PE) diagnostic studies is ≤2.7%, based on a meta-analysis of pulmonary angiography studies performed in the 1990’s. However, the disease prevalence in PE studies has decreased considerably over the past decade and differs between geographical regions. Considering Bayes’ theorem, this should imply that the failure rate margin of contemporary diagnostic tests or algorithms should be lowered as well. Aims : We set out to evaluate the association of PE prevalence and diagnostic failure rate in prior studies in order to set a new standard for future studies. Methods : We selected all high-quality diagnostic studies in acute PE from 1990 on, that included consecutive patients who were followed for at least three months and were subjected to an appropriate diagnostic standard, i.e. (an algorithm consisting of) a validated clinical decision rule combined with a highly sensitive D-dimer test, multi-slice CT pulmonary-angiography, ventilation/perfusion scintigraphy in accordance to the PIOPED criteria, and/or conventional pulmonary angiography . Results : Fifty studies including 28,848 patients were included, with a mean baseline PE prevalence of 23.3% (95%CI 20.5-26.1, range 0.28-44.5%). The reference line calculated by linear regression analysis adjusted for study sample size of the graph plotting failure rate against PE prevalence corresponded to a mean sensitivity of 99.6% across the studies. The background 3-month incidence of PE (virtual population with 0% PE at baseline, cross point of the y-axis) was 0.69%. The formula of the upper 95% confidence interval of the reference line is ‘1.85+ 0.0041*prevalence’. Conclusion : We propose that future PE diagnostic studies should incorporate this formula as reference standard for the minimum diagnostic accuracy of new diagnostic tests or algorithms and thus customize their power analysis to the expected PE prevalence in the studied cohort.

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Abstract 16 Atherosclerosis Experimental CD39-mediated exhaustion of CD8+ T cells in atherosclerotic lesions: role of the arterial microenvironment J. van Duijn1, M. van Elsas1, N. Benne2, E. Kritikou1, T. van der Heijden1, F.H. Schaftenaar1, M.J. Kröner1, G.H.M. van Puijvelde1, I. Bot1, A.C. Foks1, W. Jiskoot2, B. Slütter1,2and J. Kuiper1

1 Division of Biopharmaceutics, LACDR, Leiden University, the Netherlands 2 Division of Drug Delivery Technology, LACDR, Leiden University, the Netherlands

Atherosclerosis is a complex disease, in which the immune system plays an important role in driving plaque formation. CD8+ T lymphocytes may play an important role in atherogenesis, as they represent 29% of all lymphocytes in early human lesions, increasing up to 50% in advanced plaques. Nonetheless, their contribution to plaque initiation and progression remains unclear. Here we show that CD8+ T-cells derived from enzymatically digested atherosclerotic lesions of ApoE-/- mice display a unique phenotype. Most notably, lesion derived CD8+ T cells produce less IFN-γ after PMA/Ionomycin stimulation compared to their counterparts in the spleen (11.9%±5.9 vs. 31.72%±0.7). Importantly, this effect does not appear to be antigen specific, as ovalbumin-recognizing CD8+ T cells derived from OT.1 donors show similar cytokine exhaustion within the atherosclerotic lesion environment. Furthermore, this loss of cytokine production is not due to classical exhaustion of the T cells, as we found no difference in the expression of exhaustion markers such as PD-1 and LAG-3. Interestingly, we observed an increased expression of the ectonucleotidase CD39 in lesion- derived compared to splenic CD8+ T cells (23.7%±5.2 vs. 2.9%±0.3), which has recently been implied to play a role in exhaustion. Co-expression with the ecto-5'-nucleotidase CD73 on plaque derived CD8+ T cells indicates an adenosine-mediated mechanism of immunosuppression by these cells. Together, these results suggest that the specific atherosclerotic environment in the vessel wall decreases CD8+ T cell functionality, which provides a new understanding of CD8+ T cells and their role in the pathogenesis of atherosclerosis.

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Abstract 17 Metabolism A-Kinase Anchoring Proteins in the Protection of Pancreatic Beta-cells from Cytokines Johanne H. Ellenbroek*1,2, Lorna M. Dickson2, Rob C. Hoeben1, Eelco J.P. de Koning1,3, Chris J. Rhodes2, Barton Wicksteed2,4

1Leiden University Medical Center, Leiden, The Netherlands. 2University of Chicago, Chicago, IL, USA. 3Hubrecht Institute, Utrecht, The Netherlands. 4Northwestern University Feinberg School of Medicine, Chicago, IL, USA. Diabetes mellitus results from loss of functional insulin-producing beta-cells in the pancreas. Therefore, therapies are needed that improve beta-cell survival. cAMP-protein kinase A (PKA) signaling improves beta-cell survival and function. Signaling downstream of PKA is coordinated by A-kinase anchoring proteins (AKAPs) that determine where and when PKA meets downstream effectors. For example, in cardiomyocytes, AKAPs are located at specific cellular sites to coordinate critical functions of the heart, including excitation-contraction coupling, oxygen homeostasis and transcriptional control. In beta-cells, the full complement of AKAPs is unknown. Our aim is to elucidate the role of PKA-AKAP signaling in beta-cell survival. MIN6 cells were infected with adenoviruses expressing a dominant negative PKA or an AKAP inhibitory sequence to reduce PKA activity or PKA signaling through AKAPs, respectively. Cells were exposed to cytokines for 4 hours followed by detection of caspase 3 activity. Loss of PKA signaling through expression of PKA-DN or AKAP-IS aggravated cytokine-induced apoptosis of MIN6 cells. To identify AKAPs, MIN6 cells were transfected with a FLAG-tagged PKA regulatory subunit (PKA-RIIa-FLAG) to allow pull-down of associated proteins that where analyzed by mass spectrometry. Analysis of associated proteins led to the identification of 3 AKAPs (AKAP1, AKAP10 and AKAP13) and an endogenous AKAP disruptor, that have not been associated with beta-cells before. The role of the identified AKAPs in beta-cell survival was assessed by lentivirus-mediated shRNA knockdown in MIN6 followed by exposure to cytokines. Loss of AKAP1 and AKAP13 aggravated cytokine-induced apoptosis of MIN6 cells. In contrast, loss of the AKAP disruptor led to an improvement of MIN6 cell survival after cytokine exposure. We show that PKA-AKAP signaling in MIN6 cells is involved in cell survival. AKAP1, AKAP10, AKAP13 and an endogenous AKAP disruptor were associated with beta-cells for the first time. Elucidating the role of PKA-AKAP signaling in beta-cell biology is central to understand the mechanisms by which cAMP-PKA signaling improves beta-cell survival and function.

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Abstract 18 Cardiac arrhythmia Optogenetic ablation of spiral wave arrhythmias by creating “light lesions”. Iolanda Feola, Linda Volkers, Rupamanjari Majumder, Antoine A. F. de Vries and Daniël A. Pijnappels. Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center. The Netherlands. Background Atrial fibrillation (AF) is the most common sustained arrhythmia and may have electrical spiral waves as drivers. Such drivers may be terminated by the introduction of lesions (lines of permanently damaged tissue) in the affected regions through catheter ablation. The success rate of this procedure may depend on the spatial precision and characteristics of the lesions with respect to the location of the spiral waves. In this study we investigated these spatial features of ablation by mimicking conventional lesions, through patterned illumination and optogenetics, in a 2D model of AF. Methods and Results Neonatal rat atrial cardiomyocyte monolayers were transduced with lentiviral vectors encoding the light-activated, depolarizing ion channel called Ca2+-translocating channelrhodopsin (CatCh). Patch clamp analyses showed that during illumination (470nm, 250-500ms) a stable voltage plateau (-10 mV) was generated, leaving the cells inexcitable and thereby acting as a conduction block. A patterned illumination device was used to create block in any desired configuration and location, while the effects were investigated by optical mapping. Single and sustained spiral waves rotating around a functional core were induced. The core was targeted by illuminating a circular shape with ∅ of 3-6-12cm (n=12) or a single straight line of 1mm width (n=20). The lines were connecting the core with i) both physical boundaries of the well, ii) only one boundary or iii) not having any connections. Circular “lesions” created a new pathway to which spiral waves anchored, maintaining the arrhythmia. In contrast, creation of linear “lesions” did result in spiral wave termination, however, only if the core was crossed and connected to at least one boundary. Here it was shown that spiral waves anchored and moved to the boundary where they collided, allowing normal activity to regain. Conclusions This study shows that an essential requirement for termination of spiral wave, by means of ablation, is the connection of the core to an inexcitable boundary. These insights might help to develop novel ablation strategies with higher success rate of arrhythmia termination.

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Abstract 19 Heart failure, Metabolism, Vascular Biology miRNA-224 and miRNA-452 display an oestrogen responsive decrease in male- to female Barend W. Florijn (1,2), Maartje Klaver (3), Jacques M.G.J. Duijs (1,2), Roel Bijkerk (1,2), Ton Rabelink (1,2), Martin den Heijer (3) and Anton Jan van Zonneveld (1,2) (1) Department of Internal Medicine (Nephrology) and (2) Einthoven Laboratory for Experimental Vascular Research, Leiden University Medical Center, Netherlands (3) Department of Internal Medicine, Division of Endocrinology VU University Medical Center Heart failure in women predominantly presents with preserved ejection fraction (HFpEF). In women, non-cardiac comorbidities, like diabetes mellitus (DM), activate a systemic inflammatory state inducing microvascular endothelial dysfunction and the development of HFpEF. Currently, no reliable biomarkers can detect this female microvascular disease or explain its pathophysiology. In search of early biomarkers for HFpEF, the Queen of Hearts consortium aims for the discovery of circulating sex-biased microRNAs (miRNAs) that associate with microvascular disease. In search for sex-biased miRNAs we identified oestrogen responsive miRNAs in male-to-female (MtoF) transgenders in whom oestrogen treatment is known to induce insulin resistance. Next-generation sequencing of plasma-miRNAs from, MtoF transgenders (n=20, mean age 36.8 ± 13.4) at baseline and after one year of oestrogen administration, identified 2541 different circulating miRNAs. Upon quantitative PCR (qPCR) validation, miRNA-224, miRNA-452 and miRNA-133b displayed an oestrogen-responsive decrease after one year of oestrogen treatment of 7.6 fold (p=0.002), 13.0 fold (p=0.04) and 4.8 fold (p= 0.03) respectively. Decreased miRNA-224 and miRNA-452 levels were confirmed in a second MtoF transgender cohort (n=39, mean age 33,8 ± 11,6 ) but not in female to male (FtoM) transgenders (n=41, mean age 26,9 ± 10,2). Interestingly, miRNA-224 (R= -0.4, p=0.04) but not miRNA-452 correlated with increased levels of insulin. In vitro, miRNA-224 (p= 0.09) and miRNA-452 (p= 0,04) displayed a similar decrease in male human umbilical vein endothelial cells (HUVECs) (n= 6) subjected to 100 nM oestrogen. Oestrogen treatment of male HUVECs further increased thioredoxin interacting protein (TXNIP) expression, a regulator of cellular redox signalling and established target of both miRNA-224 and miRNA-452. miRNA-224 and -452 will be included in a panel of sex-biased miRNA to identify their biomarker potential in diagnosing female-specific cardiovascular phenotypes such as HFpEF. Additionally, the identification of TXNIP as a potential miRNA-224/452 target may provide a novel explanation for the hyperinsulinemic side effects of oestrogen administration in MtoF transgenders. Dutch Heart Foundation: Women and cardiovascular disease

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Abstract 20 Metabolism Altered distribution of HDL, extracellular vesicle and Argonaute-2 associated circulating Barend W. Florijn (1,2), Jacques M.G.J. Duijs (1,2), Johannes H. Levels (3), Geesje M. Dallinga-Thie (3), Anita N. Boing (4), Rienk Nieuwland (4), Ton J. Rabelink (1,2), Marlies E.J. Reinders (1,2), Roel Bijkerk (1,2) and Anton Jan van Zonneveld (1,2) European Foundation for the Study of Diabetes (EFSD) / Boehringer Ingelheim grant We previously demonstrated an association between total plasma levels of specific microRNAs (miRNAs) and microvascular injury in patients with diabetic nephropathy (DN). These circulating miRNAs are carried by extracellular vesicles (EVs), RNA-binding protein Argonaute2 (Ago2) or high-density lipoprotein (HDL). We hypothesized that identification of the carrier specificity of the selected miRNAs can enhance the biomarker potential of these miRNAs. In addition, carrier-specific transfer of miRNAs to vascular cells may play a causal role vascular homeostasis. Here we assessed the plasma carrier distribution of miRNAs in DN (n=21), diabetes mellitus (DM; n=15; eGFR of ≥ 30 mL/min) patients and healthy controls (n=15). EVs, HDL and Ago2 were isolated using size exclusion chromatography, KBr density gradient ultracentrifugation and immunoprecipitation, respectively. MiRNA expression was determined using TaqMan® miRNA Arrays and correlated to markers of vascular injury, including angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), soluble thrombomodulin (sTM) and capillary density. Specific miRNA-carrier complexes were identified to be associated with DN and vascular injury. EV-miR-21 and Ago2-miR-660 levels displayed a significant increase in both DM and DN groups compared to healthy controls and correlated with capillary tortuosity and sTM, respectively. Furthermore, HDL-miR-132 levels decreased in DN and correlated with levels of Ang2, while both HDL-miR-152 and Ago-miR-152 levels displayed a significant increase in DN. Interestingly, only Ago-miR-152 levels correlated with levels of Ang2 and sTM. Our data suggest that miRNAs in specific carriers may contribute to vascular injury and could improve selectivity and sensitivity of biomarkers for microvascular injury in DN.

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Abstract 21 Atherosclerosis Experimental Heat shock protein alpha B-crystallin increases atherosclerotic lesion development in LDLR KO mice R. van der Geest1, J.J. Geerling1, L.R. de Leeuw1, M. Hoekstra1, R. Verbeek2, J.M. van Noort2, and M. Van Eck1

1Division of BioPharmaceutics, Cluster BioTherapeutics, Leiden Academic Centre for Drug Research, Leiden, The Netherlands 2Delta Crystallon, Leiden, The Netherlands Background and aim: Alpha B-crystallin (HSPB5) is a small heat shock protein that exerts anti-inflammatory effects in various animal models of inflammatory disorders, which most likely occur through the induction of TLR2-mediated responses in macrophages and microglia. These anti-inflammatory properties suggest that HSPB5 may also have a beneficial impact on the development of atherosclerosis. Here we evaluated the effect of HSPB5 on atherosclerotic lesion development in low-density lipoprotein receptor knockout (LDLR KO) mice. Methods and results: LDLR KO mice were fed a Western-type diet to induce the development of atherosclerosis and received intraperitoneal HSPB5 injections every other day for 6 weeks. HSPB5-treated mice showed a two-fold increase in the number of peritoneal leukocytes (p<0.001) compared to control mice. The peritoneal infiltrate of HSPB5-treated mice, in line with previous reports, showed a more anti-inflammatory status, as judged from a lower percentage of effector CD4 (-15%; p<0.05) and CD8 T cells (-28%; p<0.01) and a marked increase in the number of regulatory T cells (3-fold; p<0.001). In addition, gene expression analysis of peritoneal cells showed an increase in the expression of the M2 macrophage marker arginase 1 (2-fold; p<0.001) and a decrease in the expression of TNFα (-59%; p<0.01) upon HSPB5 treatment. Strikingly, despite the observed anti-inflammatory effects, HSPB5 treatment resulted in a significant increase in atherosclerotic lesion size (+34%; p<0.05). The macrophage and collagen content of the lesions was not affected by the HSPB5 treatment. Plasma cholesterol levels and systemic inflammatory status were similar in control and HSPB5-treated mice and thus did not account for the increased atherosclerosis susceptibility upon treatment with HSPB5. Conclusion: We show that treatment with HSPB5 increases the development of atherosclerosis in LDLR KO mice. Current efforts focus on identifying the mechanism that underlies the increased susceptibility for atherosclerosis in response to HSPB5 treatment. Funding: The Netherlands CardioVascular Research Initiative: ‘the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences’ for the GENIUS project ‘Generating the best evidence-based pharmaceutical targets for atherosclerosis’ (CVON2011). The Netherlands Organization for Scientific Research (VICI Grant 91813603 to M.V.E).

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Abstract 22 Cardiac arrhythmia Utilizing excised human atrial and ventricular cardiac tissue for ex vivo studies into patient-specific disease and treatment Niels Harlaar1,2, Thomas J. van Brakel2, Lisa E. Starken1, Pim R.R. van Gorp1, Masaya Watanabe1, Robert J.M. Klautz2, Martin J. Schalij1, Katja Zeppenfeld1, Antoine A. F. de Vries1, Daniël A. Pijnappels1. 1. Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center, the Netherlands. 2. Department of Cardiothoracic Surgery, Leiden University Medical Center, the Netherlands. Background Currently, most mechanistic and pioneering studies into cardiovascular disease and treatment are performed in animal models. Despite the various advantages of these models, their translational potential to human (electro)physiology has proven to be limited. We strived to develop a broadly applicable human cardiac tissue model allowing for structural and functional studies into heart disease, including experimental interventions, with a focus on cardiac arrhythmias. Methods and results Human atrial and ventricular myocardial biopsies, ranging from fetal and neonatal to healthy and diseased adult patients, were retrieved after obtaining written informed consent if applicable. Following rapid transfer, biopsies were either directly processed for optical voltage mapping, or alternatively, cultured in a customized chamber after embedding in low-melting agarose and slicing into 300 μm-thick slices using a vibratome. Optical voltage mapping was performed using a conventional setup and a dynamic pacing protocol to characterize baseline electrical impulse generation and propagation. Current efforts are focussed on enabling long-term culture of these cardiac slices with minimal functional and structural deterioration, allowing for pharmacological and viral vector-based genetic modification. Subsequent molecular and immunohistochemical analyses into fibrosis, sarcomeric- and gap junctional proteins and major ion channels were employed and could be correlated to the optical mapping data. In the future, the combination of such structural and functional data obtained ex vivo with high density epicardial mapping obtained perioperatively and a wide range of clinical parameters, would create an integrative platform which could benefit diagnosis, prognosis and treatment of patients with cardiovascular disease. Conclusion We demonstrate how human cardiac tissue, from both the atria and ventricles, can be functionally studied ex vivo and combined with structural and clinical data to establish an integrative platform, serving as an alternative to traditional animal-based research. Moreover, with further improvement of such human models, it seems plausible that future patient-specific ex vivo studies could improve clinical decision making.

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Abstract 23 Metabolism Familial hypercholesterolemia: classification of mutation severity according to percentile LDL-cholesterol useful for predicting coronary artery disease risk. Merel L. Hartgers, G. Kees Hovingh, John J.P. Kastelein, Roeland Huijgen Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, the Netherlands Introduction: The clinical variability in patients diagnosed with familial hypercholesterolemia (FH) is largely related to severity of the underlying mutation. International guidelines advise to group these mutations according to class 1 and non-class 1 mutations, but this classification does not incorporate available data on observed severity in terms of LDL-cholesterol (LDL-c) levels and coronary artery disease (CAD) risk. Aims We set out to establish CAD risk in patients with confirmed pathogenic LDLR mutations, with stratification according to percentile LDL-c (PLDL). Methods Individuals were eligible if they underwent family screening for FH in the Netherlands between 1994 and 2010. We calculated the mean pre-treatment age and gender corrected PLDL of each FH mutation and stratified these in 6 strata using the 75th , 88th, 92nd, 96,5th and 98th percentile as boundaries. We used Cox proportional hazard model to estimate the risk of major CAD (myocardial infarction, percutaneous coronary intervention, coronary artery bypass graft) for different FH mutation carriers versus unaffected relatives before 1990 (pre-statin). The reference population consisted of non-mutation carriers from the same families. Results A total of 34197 persons were tested for 456 different pathogenic LDLR mutations, identified in the index patient of their family. Of these, 12245 (36%) were found carriers. CAD risk inclined gradually from 2.2 for those with mutations associated with PLDL below the 75th percentile to 13 for those with mutations with a mean PLDL higher than then the 98th percentile. Conclusions Mutation severity classified based on PLDL allows more adequate risk stratification in FH than classification according to the dichotomy of class 1 versus non-class 1 mutations. The current proposed approach will likely be of clinical use in counseling patients on the expected risk based on their FH mutation as well as in identifying high risk patients for expensive treatments.

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Association between mutation category based on mean PLDL and CAD risk in the pre-statin era

Controls

(p50)

< p75

p75 - p

88

p88 - p

92

p92 - p

96,5

p96,5

- p98

> p98

0

3

6

9

12

1515202530

A cox proportional hazard model was used to compare CVD riskbetween carriers and non-carriers (control), with adjustment for age,

sex, hypertension, diabetes, body mass index, and exposure ever to smoking.The controls served as reference group.

CA

D r

isk

(HR

95%

CI)

NOT INCLUDED: By comparison, the 4197 patients carrying a class 1 mutation had a 7.2 (5.4-9.7) higher risk of CAD than their unaffected relatives. The patients carrying non-class 1 mutation had a variable risk of CADon average 4.1 (3.2 to 5.3) higher than unaffected relatives. Familial hypercholesterolemia is a common genetic disorder caused by more than 1200 unique mutations in the LDLR, apoB and PCSK9 genes

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Abstract 24 Atherosclerosis Experimental Silencing of Anticoagulant Protein C Evokes Low Incident but Spontaneous Atherothrombosis in Apolipoprotein E Deficient Mice Marco Heestermans1,2*, Amber B. Ouweneel3*, Robin A.F. Verwilligen3, Marion Gijbels4, Pieter H. Reitsma1,2, Miranda Van Eck3**, Bart J. M. van Vlijmen1,2** 1Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, 2Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, 3Division of Biopharmaceutics, Cluster BioTherapeutics, Leiden Academic Centre for Drug Research, Leiden University, Leiden, 4Department of Pathology, Amsterdam Medical Center, Amsterdam, The Netherlands. *,**with equal contribution Mouse atherosclerosis models do not spontaneously develop atherothrombotic complications. Here, we investigated whether silencing of anticoagulation may allow murine pre-existing atherosclerotic plaques to progress towards an atherothrombotic phenotype. Upon establishing experimental conditions (n=4 per group), it appeared that when targeting antithrombin, using small interfering (si) RNA (siSerpinc1) in atherosclerotic apolipoprotein E deficient (Apoe-/-) mice, animals rapidly developed spontaneous thrombosis, restricted to the venous vasculature. This thrombotic phenotype was similar as described previously for wild type mice injected with siRNAs targeting Serpinc1 (Safdar et al. Blood, 2013). In contrast, WTD-fed Apoe-/- mice injected with an siRNA solely targeting Proc (siProc) remained fully healthy. Interestingly, one animal displayed a highly unique, organized, and large fibrin- and leukocyte-rich thrombus superimposed on an advanced atherosclerotic plaque located in the aortic root area. Although again at low incidence, comparable thrombi at the same location were observed in Apoe-/- animals during a second independent large scale experiment (in 3 out of 25 siProc treated mice). siProc treated Apoe-/- mice with thrombi on top of the atherosclerotic plaques did not show other abnormalities and had comparably low plasma protein C levels as siProc treated Apoe-/- animals without thrombi. Also, the fibrinogen and thrombin-antithrombin concentrations, and blood platelets numbers were unchanged. Plaques in siProc mice with thrombi had a composition and size comparable to plaques in siProc mice without thrombi. Our findings indicate that siRNA-mediated silencing of Proc in Apoe-/- mice creates a condition that allows the formation of spontaneous atherothrombosis, albeit at low incidence. Our unique approach can be a valuable tool to identify factors that increase atherothrombotic complications.

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Abstract 25 Vascular Biology Fluxomic tools for characterization of metabolic reprogramming upon brown adipose tissue activation Ntsiki M. Held1, Eline N. Kuipers2, Michel van Weeghel1, Patrick C.N. Rensen2, Ronald J. Wanders1, Arthur Verhoeven1, Mariëtte R. Boon2 and Riekelt H. Houtkooper1 1 Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, The Netherlands 2 Division of Endocrinology, and Einthoven Laboratory of Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands Brown adipose tissue (BAT) contributes to whole body energy expenditure through energy dissipation as heat. Imaging techniques have showed high rates of glucose and fatty acid uptake in activated BAT, suggesting an overall metabolic overload. Substrate overloading requires and induces metabolic reprogramming. This metabolic effect in BAT has primarily been studied after prolonged cold exposure through gene expression arrays, which showed increased expression of glycolysis, β-oxidation, glycogen and fatty acid synthesis genes. However, how these distinct processes are regulated to occur simultaneously is poorly understood. Using fluxomic tools, we aim to estimate metabolic fluxes in the T37i brown adipocyte cell line during β3-adrenergic activation. With a set of specific substrate inhibitors and an extracellular flux analyzer we found that the main substrates of BAT cells are fatty acids, which were equally oxidized during activation as well as during rest. Activated BAT cells could increase oxidation of other substrates, such as glutamine. Also, glycolysis rates increased by 60% upon activation, but interestingly most glucose is not converted to pyruvate to become fully oxidized. Therefore, in current studies we seek to further elucidate the metabolic pathways through which glucose is utilized. With [U-13C6]-D-glucose labeling we perform stable isotope tracer-based metabolomics. This allows us to accurately calculate metabolic fluxes based on tracer incorporation in most central carbon metabolites. Gaining insight in BAT metabolism holds potential to accelerate the quest for pharmacological targets that collectively expand BAT volume as well as its function.

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Abstract 26 Metabolism Cold exposure beneficially modulates HDL metabolism in mice and humans Geerte Hoeke1, Kimberly J Nahon1, Alexander Bartelt2, Donna Dinnes3, Diana Drettwan4, Philipp Pagel4, Ingrid M Jazet1, Maaike Kockx3, Leonard Kritharides3, Wendy Jessup3, Joerg Heeren2, Jimmy FP Berbée1, Mariëtte R Boon1, Patrick CN Rensen1 1Dept. of Medicine, Div. of Endocrinology, LUMC, Leiden, the Netherlands 2University Medical Center Hamburg, Hamburg, Germany 3University of New South Wales, Sydney, Australia 4Numares, Regensburg, Germany Aim: Upon cold activation, brown adipose tissue (BAT) combusts intracellular triglycerides (TG) into heat. Our mouse studies showed that BAT consequently internalizes VLDL-TG-derived fatty acids to replenish intracellular TG stores, resulting in the formation of surface remnants (Hoeke, Circ Res 2016). We now assessed the effect of cold-induced BAT activation on HDL metabolism in mice and humans. Methods: BAT was activated in APOE*3-Leiden.CETP transgenic mice, a well-established model for human-like lipoprotein metabolism, by the beta-3-adrenergic receptor (ADRB3) agonist CL316243 (3x20 µg/week; s.c.) or exposure to cold (7 days; 7°C). BAT was activated in human lean adolescent males (n=12) by a personalized water cooling protocol (average 9.9°C) for 3 h. Results: BAT activation of APOE*3-Leiden.CETP mice by ADRB3 agonism increased plasma HDL-C (+30%) with a negative correlation between HDL-C and atherosclerosis lesion area (R2=0.333; p<0.01). Short term ADRB3 agonism or cold exposure increased fecal output of [3H]cholesterol derived from i.p. injected macrophages (+100%; p<0.05). Likewise, cold exposure (3h) of adolescent human males (n=12) increased HDL particle concentration (+9%; p<0.05), apoAI (+13%; p<0.05) and HDL-C (+9%; p<0.05), specific for small HDL, and decreased the average HDL size (-0.1 nm), which is consistent with LPL-mediated generation of VLDL surface remnants. This was accompanied by an increased ABCA1-dependent efflux of [3H]cholesterol from ABCA1-transfected CHO cells to HDL in vitro. Interesting, we observed that LysoPC is a biomarker for BAT activity in these human subjects (R2=0.837; p<0.0001), which is in full accordance with LCAT-dependent cholesterol esterification accompanied by transfer of fatty acids from PC to cholesterol resulting in generation of LysoPC. Conclusions: Activation of BAT in by ADRB3 agonism/cold exposure in mice and humans generates LPL-derived surface remnants that enhance cholesterol efflux into the circulation resulting in the formation of small HDL. This may be the biological mechanism explaining that LysoPC is a biomarker for BAT activity.

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Abstract 27 Cardiac function Deletion of Follistatin-like 1 from the endocardial lineage causes ongoing growth of the aortic leaflet of the mitral valve Stuti Prakash1, Luis J. J. Borreguero2, Marc Sylva1, Fereshte Rezai1, Quinn D Gunst1, de la Pompa JL3, Jan Ruijter1, Maurice J.B. van den Hoff1 Follistatin-like 1 (Fstl1) is a secreted protein that is expressed in the atrioventricular valves (AV) throughout embryonic development, neonatal stages and adulthood. In this study, we investigated the effect of ablation of endocardial Fstl1 to determine the functional role of Fstl1 in the development and maturation of mitral AV valves after birth. The conditional ablation of Fstl1 led to an upregulation of Bmp and Tgfβ signaling. This results in ongoing proliferation and growth of the valves leading to deformed non-functional mitral valves and hypertrophic dilated hearts. Echocardiographic analysis of these hearts suggests that loss of Fstl1 leads to myxomatous valves and results in LV diastolic dysfunction with mitral regurgitation. Subsequently, Fstl1-cKO mice develop heart failure with preserved ejection fraction (HFpEF) which ultimately leads to the neonatal death of the 50% of mice around three weeks after birth.

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Abstract 28 Atherosclerosis Experimental Yeast-derived mannan oligosaccharide attenuates atherosclerosis development Lisa Hoving1, Amanda Pronk1, Lianne van Wee-Pals2, Trea Streefland2, Marieke Heijink3, Sam van Tuin1, Martin Giera3, Vanessa van Harmelen1, and Ko Willems van Dijk1,2 1Dept. of Human Genetics and Einthoven Laboratory for Experimental Vascular Medicine, 2Dept. of Medicine, division Endocrinology, 3Center of Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Albinusdreef 2, 2300RC Leiden, The Netherlands. BACKGROUND Alterations in the composition of the gut microbiota have been associated with the development of metabolic disease such as atherosclerosis. We exploited the potential of indigestible carbohydrates to induce significant changes in the gut microbiota composition. Mannan oligosaccharides (MOS) derived from the outer cell wall of the yeast Saccharomyces cerevisiae have proven effective at improving growth performance and protecting against infection through pathogen binding, while also lowering plasma lipid levels and reducing inflammation. Atherosclerosis is mainly a lipid and inflammatory driven disease. Therefore, this study aimed to investigate the effect of MOS atherosclerosis development in APOE*3-Leiden.CETP (E3L.CETP) mice, a well-established model for human-like lipid metabolism and atherosclerosis. METHODS Female E3L.CETP mice were fed a Western-type diet with 0.1% cholesterol with or without 1% MOS for 14 weeks. The effects of dietary MOS supplementation on plasma lipids, atherosclerosis development, plasma bile acids, and short-chain fatty acid (SCFA) production. Gut microbial composition will be assessed using 16S rRNA gene sequencing. RESULTS Remarkably, MOS supplementation decreased plasma total cholesterol levels in E3L.CETP mice. Accordingly, atherosclerotic lesion area was significantly reduced in MOS supplemented mice. Fecal samples of MOS supplemented E3L.CETP mice showed an increased in the excretion of primary bile acids, while total secondary bile acids excretion remained unchanged. Cecal SCFA analysis revealed a significant increase in butyrate production in the MOS supplemented mice. MOS supplementation did not result in differences in body weight, body composition, food intake, liver lipids, peripheral blood monocyte count, and plasma IL-1Ra levels. Whether MOS supplementation affect gut microbiota composition is currently being investigated. CONCLUSION MOS supplementation attenuates atherosclerosis development by lowering plasma total cholesterol levels in E3L.CETP mice, presumably mediated via alterations in gut microbiota composition and their products.

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Abstract 29 Cardiac arrhythmia Heat shock protein activators accelerate recovery from contractile dysfunction in experimental Atrial Fibrillation Xu Hu, Denise Van Marion, Bianca J.J.M. Brundel. Department of Physiology, Institute for Cardiovascular Research, VU University of Medical Center, The Netherlands. Background: Atrial Fibrillation (AF) is the most common tachyarrhythmia in clinical practice, which associated with electrical, structural remodeling and contractile dysfunction. The impaired protein homeostasis occurs during the progression of AF, contributing to the irreversible structural damages. The molecular chaperones heat shock proteins (HSPs) family play a critical role in facilitating correct protein folding and degradation, to maintain the functional quality control system in cardiomyocytes. Our previous studies found that HSP boosters GGA and its derivatives protect against contractile dysfunction in atrial cardiomyocyte and drosophila model, suggesting that HSP activators are promising upstream therapy for AF. It is also interesting to exam whether those HSP inducers can reverse contractile dysfunction in the AF experimental model, and explore the underlying mechanisms. Methods: HL-1 atrial cardiomyocytes were tachypaced (TP) for 10hrs with 5HZ, which induce calcium transient (CaT) loss and contractile dysfunction, followed by treatment with HSP inducers Nyken 41 for 8hrs, 24hrs, named posttreatment. CaT and western blot were mainly used to indicate the changes in cells function and protein expression. CaT was exclusively measured in 24hrs. Results: TP induced significant CaT reduction in cardiomyocytes, which did not recover during a 24h-recovery period. Interestingly, cardiomyocytes that were post-treated with Nyken 41 after TP revealed a dramatic recovery from CaT loss. Furthermore, HSP25 was dramatically boosted during the 8hrs posttreatment. In addition, the microtubule components α-tubulin and acetylated α-tubulin were significantly recovered by posttreatment. Interestingly, the expression level of contractile proteins, represented by troponin I (TnI) and troponin T (TnT), was also enhanced after 8hrs posttreatment. Conclusions: Our results imply that the HSP activators improved recovery from TP-induced contractile dysfunction in cardiomyocytes, and HSP activity was stimulated, contractile protein and microtubule network were restored during the posttreatment, which indicates that enhancement of HSP level might modulate function of contractile proteins and microtubule components, to promote the recovery of contractile dysfunction of cardiomyocytes, HSP induction could be a target for reversing AF progression.

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Abstract 30 Vascular biology Development of anti-Galectin-2 antibodies to improve arteriogenesis. Authors: Matthijs F. Jansen1,2, Maurits R. Hollander3, Anja M. van der Laan4, Cansu Yildirim2, Ruud Fontijn2, Josefien M. Baggen2, Tineke van der Pouw-Kraan2, Anton Horrevoets2, Niels van Royen3

Affiliations: 1. Department of Medical Biochemistry, Academic Medical Center. 2. Department of Molecular Cell Biology and Immunology, VU University Medical Centre. 3. Department of Cardiology, VU University Medical Centre. 4. Department of Cardiology, Academic Medical Centre, University of Amsterdam. Background In previous studies we showed the correlation between impaired arteriogenesis and an increased mRNA expression of galectin-2 in monocytic cell in a total of 50 patients with a chronic total coronary occlusion. In this study we aim to confirm the function of Galectin-2 in arteriogenesis and subsequently inhibit Galectin-2 to improve arteriogenesis. To inhibit the function of Galectin-2, we choose to develop camelid derived single-chain antibody (VHH), which are more robust than normal antibodies. Methods A murine hind-limb model was performed with 32 C57BL/6 mice. The mice were divided in two groups, where one was administered galectin-2 and the other PBS. To assess perfusion restoration, laser-dopler perfusion imaging (LDPI) was performed throughout the experiment. Histological analysis was performed on the abductor muscle to asses arteriogenesis. To create the VHH’s a displaying phage library for both human and mice galectin-2 was used. By utilizing ELISA, the binding of the VHH’s to galectin-2 was determined. Using FACS analysis the inhibition of galectin-2 binding to CD14 was measured in both human and murine monocytes. Results Systemic treatment with galectin-2 impairs the perfusion restoration in mice at 7 days following left femoral artery coagulation (54.5% in the galectin-2-treated mice vs. 74.6% in the control mice, P< 0.01). The VHH’s inhibited the binding of galectin-2 to monocytes by 0% to ca. 75%. The 5 most promising VHH’s were chosen and are now under further development. Conclusion/Discussion ; Administration of galectin-2 markedly decreases arteriogenesis. The developed VHH’s show significant potential as a therapeutic method to inhibit galectin-2 and possibly improve arteriogenesis. We hope to soon start on a murine hind-limb model to test their potential in arteriogenesis.

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Abstract 31 Thrombosis and Hemostasis Improvement of a severe von Willebrand factor multimerization defect by efficient and allele-specific small interfering RNAs A. de Jong, R. Dirven, J. Oud, D. Tio, J. Eikenboom Einthoven Laboratory for Experimental Vascular Medicine, Department of Thrombosis and Hemostasis, Leiden University Medical Center Von Willebrand disease (VWD), the most common inherited bleeding disorder, is often caused by dominant-negative mutations in von Willebrand factor (VWF). We hypothesize that inhibition of mutant VWF, while maintaining normal VWF, will correct the VWD phenotype. We have designed small interfering RNAs (siRNAs) that specifically inhibit the mutant VWF allele and thereby reduce the dominant-negative effect of the mutation. The mutant and normal VWF allele will be distinguished by targeting heterozygous single nucleotide polymorphisms (SNPs) in VWF. Three SNPs in VWF with a high minor allele frequency have been selected and siRNAs were designed for both alleles of the three SNPs. Co-transfection of the siRNA and the two VWF constructs containing the two respective VWF alleles in HEK293 cells was used to select the most potent siRNAs and define the optimal siRNA concentration. The effect of allele silencing of the most potent siRNAs has been tested on co-transfections of the siRNA with a normal VWF construct and a VWF construct containing the VWF p.Cys2773Ser mutation, a dominant-negative mutation that causes a severe VWF dimerization and multimerization defect. Improvement of the multimerization pattern was evaluated by VWF multimer analysis. Co-transfections of mutant VWF p.Cys2773Ser and normal VWF constructs in HEK293 cells led to an equal expression of both constructs, which resulted in a severely affected multimer pattern. The addition of allele-specific siRNAs that target the VWF allele containing the VWF p.Cys2773Ser mutation, improved the multimerization pattern significantly for all tested siRNAs. For the most optimal siRNAs, even an almost complete restoration of the multimer pattern was observed. This study shows the proof of principle for allele-specific inhibition of VWF by siRNAs. The observation of the restoration of the VWF multimer pattern holds promise for the use of allele-specific siRNAs as a therapy for VWD patients with dominant-negative VWF mutations.

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Abstract 32 Cardiac function Serial ECG Analysis after Myocardial Infarction: When Heart Failure Develops the ECG Becomes Increasingly Discordant Marjolein C de Jongh, Arie C Maan, Enno T van der Velde, Cees A Swenne Leiden University Medical Center (LUMC), Leiden, The Netherlands

1. Purpose Heart failure (HF) is a major public health issue. Myocardial infarction (MI) is a risk factor for HF; more than 50% of patients who present with HF have a history of MI[1]. The current study validates the spatial angle (SA) between the QRS- and T-axes as an ECG variable that can be used to detect emerging HF in patients after MI. The more the intricate mechanism of the electrical activation of the heart is disturbed, the larger the spatial angle (SA) and the more discordant the ECG. We hypothesized that the ECG of patients who develop HF after MI becomes more discordant.

2. Methods We searched the electronic patient files for patients who initially presented with acute MI, recovered and became stable, and presented later with symptoms of HF. We computed SA in ECGs at baseline (after recovery, 6 months after MI) and when they presented with HF. All ECGs were analyzed by our research ECG analysis program LEADS [2].

3. Results Our study group consisted of 20 patients (15/5 male/female), mean±SD age was 64±10 years and time between two successive ECGs 3.8±2.4. SA increased by 22° from 97±36° to 119±34° (P=0.003); SA decreased only in 4 patients.

4. Conclusions We conclude that SA is potentially a suitable ECG monitoring variable for early detection of HF after MI. The clinical usefulness of frequent ECG checks (e.g., with 6-mo or 1-yr intervals) of post-MI patients should be prospectively studied. References 1. Mosterd and Hoes, Heart 2007;93:1137-46 2. Draisma et al, Comput Cardiol 2005;32:515-8

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Abstract 33 Cardiac function, Heart failure RBM20 regulates circular RNA production from the titin gene Mohsin A.F. Khan*, Simona Aufiero,* Yolan J. Reckman,* Maarten M.G. van den Hoogenhof, Ingeborg van der Made, Abdelaziz Beqqali, Yigal M. Pinto and Esther E. Creemers Dept. of Exp. Cardiology and Dept. of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, Amsterdam, The Netherlands. RNA Binding Motif protein 20 (RBM20) is essential for normal splicing of many cardiac genes, and loss of RBM20 causes dilated cardiomyopathy. Given its role in splicing, we hypothesized an important role for RBM20 in forming circular RNAs (circRNAs), a novel class of non-coding RNA molecules. In this study, we performed circRNA profiling on ribosomal-depleted RNA from human hearts and identified the expression of thousands of circRNAs, with some of them regulated in disease. Interestingly, we identified 80 circRNAs to be expressed from the titin gene, a gene which is known to undergo highly complex alternative splicing. We show that some of these circRNAs are dynamically regulated in dilated cardiomyopathy, but not in hypertrophic cardiomyopathy. In addition, in a cardiac sample from an RBM20 mutation carrier, titin circRNA production was severely altered. Interestingly, the loss of RBM20 caused only a specific subset of titin circRNAs to be lost. These circRNAs originated from the RBM20-regulated I-band region of the titin transcript. In addition, we generated Rbm20 null mice and performed RNA-sequencing on their hearts. We detected hundreds of circRNAs conserved in the human and mouse heart. Similar to the patient carrying an Rbm20 mutation, Rbm20 null mice completely lack circRNAs from titin’s I-band, thereby demonstrating a novel role for RBM20 in regulating the production of a subset of cardiac circRNAs.

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Abstract 34 Atherosclerosis Experimental, Metabolism, Autoimmunity and Inflammation Genome-wide profiling reveals role of nuclear receptor Nur77 in macrophage and skeletal muscle metabolism Duco S. Koenis1#, Inkie J.A. Evers - van Gogh2#, Pieter van Loenen1, Nicole Hamers2, Yongsoo Kim3, Marten A. Hoeksema1, Menno P.J. de Winther1, Lodewyk F.A. Wessels4, Wilbert Zwart3, Eric Kalkhoven2§, Carlie J.M. de Vries1§ 1Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 2Molecular Cancer Research and Center for Molecular Medicine, University Medical Centre Utrecht, Utrecht, The Netherlands. 3Division of Molecular Pathology, 4Division of Urology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. #§ These authors contributed equally to this work. The orphan nuclear receptor Nur77 (also known as NR4A1) regulates a large variety of cellular processes, including apoptosis, skeletal muscle metabolism and macrophage inflammatory signaling, but the molecular mechanisms underlying its cell type-specific actions remain largely undefined. We assessed the importance of cellular context in Nur77-mediated gene regulation by comparing genome-wide DNA binding (ChIP-seq) and gene expression profiles (RNA-seq) in C2C12 myotubes and RAW264.7 macrophages. These experiments revealed strong cell-type specific DNA binding of Nur77, with less than 16% of Nur77 binding sites being shared between the two cell types. De novo motif analyses revealed that Nur77 binding sites in each of the two cell lines were enriched for both the classical Nur77 response motif (NBRE; 5’-AAAGGTCA-3’) as well as for binding motifs of cell-type specific transcription factors, such as MyoD in myotubes and PU.1 in macrophages. Motif analysis of binding sites that were shared between the two cell lines, as well as promoter analysis of genes that were differentially regulated in both cell lines, showed enrichment of the binding motif for the transcription factor Yin-Yang 1 (YY1). Both YY1 and Nur77 have previously been shown to positively regulate glycolysis and oxidative phosphorylation in skeletal muscle. We show that Nur77 and YY1 interact with each other and that Nur77 regulates both macrophage and skeletal muscle glycolysis and oxidative phosphorylation. Regulation of macrophage metabolism, which has recently been shown to play an important role in macrophage-driven inflammation, may be an underlying mechanism by which Nur77 exerts its atheroprotective function in vivo.

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Abstract 35 Thrombosis and Hemostasis Neutrophil Extracellular Traps: Contributors in Burn-induced Microvascular Damage and Thrombosis? H. Ibrahim Korkmaz, Paul A.J. Krijnen, Magda M.W. Ulrich, Sanne Vogels, Tim de Wit, Paul P.M. van Zuijlen, Hans W.M. Niessen Introduction: Burn wounds cause extensive scarring due to the tissue loss and subsequently aberrant healing. This tissue loss is partly related to secondary expansion of necrosis into vital dermis neighbouring the initial burn injury, leading to increased burn depth and area. An important factor in this phenomenon is the severe loss of perfusion of the burn wound, probably caused by microvascular damage induced by the intense local inflammatory responses as well as burn-induced hypercoagulation. Neutrophils are central initiators and propagators of burn-induced microvascular damage and hypercoagulation and thereby facilitate secondary necrosis, wound expansion and delayed healing. We hypothesize that the formation of Neutrophilic Extracellular Traps (NETs) play an important role in this. The purpose of this study was to investigate post-burn intravascular thrombosis, NETs formation and the coagulant state in the microvasculature of burns in animal models and patients. Methods: We used two in vivo burn wound models: rats and pigs. In rats the entire wound was excised at day 14 post-burn and in pigs burn wound biopsies were collected at different time points up to 60 days post-burn. Eschar from the burn wound was obtained from burn wound patients at different time points after wounding. The number of intravascular thrombi, the presence of intravascular NETs and the number of TF positive blood vessels in the burn wound was determined. Results: In burned rats, a significant increase in intravascular thrombi and TF expression was observed 14 days post-burn and 70% of the thrombi were positive for NETs. In pigs, a significant increase in intravascular thrombi and TF expression was found over time up to 60 days post-burn, that in majority coincided with NETs. Also in eschar of burn wound patients, a significant increase in intravascular thrombi was noted, already 0.5 days post-burn and remained elevated up to … days. Conclusions: This study shows the presence of NETosis in the microcirculatory thrombosis in the burn wound and a switch in the microcirculatory endothelium towards a pro-coagulant phenotype.

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Abstract 36 Heart failure A new ex vivo model to study cardiac fibrosis Boudewijn P.T. Kruithof1, Marjan van de Merbel1,2, Marianna Kruithof-de Julio2,3, Marie-José Goumans1 1Department of Molecular Cell Biology and 2department of Urology, Leiden University Medical Center, Leiden, The Netherlands. 3Department of Urology, University of Bern, Bern, Switzerland. Background/Introduction: Fibrosis is a characteristic of several cardiac diseases that lead to heart failure. No effective treatment exists, therefore the urgent need is present to obtain new insights in this disease in order to develop better therapeutics that will aid in its prognosis and treatment. In vivo mouse models (aortic banding, coronary artery ligation) have offered great insights in the definition and characterization of this disease. However, controlled manipulation of single or specific combination of potential effectors cannot be achieved. Purpose: The goal of our study is to develop an ex vivo culture system in which cardiac fibrosis can be induced and regulated in the adult mouse heart. Methods: Mouse hearts are isolated and cultured in the Miniature Tissue Culture System. In this ex vivo flow system a closed circulatory system is created in which the heart is perfused with medium for up to 2 weeks. After culture, the hearts are processed for histopathological analysis. Myofibroblast accumulation and collagen production are measured as indicatives of the fibrotic load. Results: Cardiac fibrosis can be induced within a week throughout the ventricular myocardium as shown by the high number of collagen-expressing myofibroblasts (alpha smooth muscle actin-positive cells). After 2 weeks, the extracellular matrix accumulation is evident by the high collagen deposition in the ventricular myocardium. The fibrosis induced in the ex vivo cultured heart is age- and flowspeed-dependent. By selective inhibition of the receptor of the profibrotic growth factor TGFβ pathway, a strong reduction of the fibrotic load was observed. Conclusions: The ex vivo flow system allows induction of cardiac fibrosis ex vivo in intact adult mouse hearts. In this powerful tool mechanical or biochemical stimuli can be altered, individually or in combination, in order to model the different stages of cardiac fibrosis. Moreover, the use of genetically modified mice hearts will give the opportunity to understand the cellular and molecular mechanisms underlying fibroblast expansion and function, revealing new therapeutic targets, paving the road towards the treatment of cardiac fibrosis. The Netherlands Heart Foundation, The Netherlands, the Netherlands Institute for Regenerative Medicine and Smartcare, part of the research program of the BioMedical Materials Institute, co-funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation

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Abstract 37 Metabolism Short term high fat feeding impacts on mitochondrial function in brown adipose tissue Eline N Kuipers1,2, Ntsiki M Held3, Andrea D van Dam1,2, Geerte Hoeke1,2, Riekelt HL Houtkooper3, Patrick CN Rensen1,2, Mariëtte R Boon1,2

1Department of Medicine, Division of Endocrinology, LUMC, Leiden, the Netherlands, 2Einthoven Laboratory for Experimental Vascular Medicine, LUMC, Leiden, the Netherlands, 3Laboratory Genetic Metabolic Diseases, AMC, Amsterdam, the Netherlands. Background and aim: Mitochondria in brown adipose tissue (BAT) burn fatty acids to produce heat, thereby contributing to energy expenditure. In diet-induced obese (DIO) mice, less and dysfunctional mitochondria are present in BAT accompanied by reduced cold tolerance. The aim of the current project is to unravel the rate and mechanism by which high fat diet (HFD) induces mitochondrial dysfunction in BAT in the course of DIO development, with the eventual goal to discover novel therapeutic avenues in obesity. Methods and results: 12-week-old C57Bl/6J mice were fed a HFD (45% of calories derived from fat) for 0, 1, 3 or 7 days (n = 10 per group). The HFD increased body fat mass at 3 and 7 days. Of note, 1 day of HFD already increased BAT weight and lipid droplet content. This was accompanied by reduced uptake of [3H]oleate derived from glycerol tri[3H]oleate-labeled lipoprotein-like particles by BAT, suggesting that reduced mitochondrial activity rather than enhanced fatty acid uptake underlies the increased lipid content in BAT. Accordingly, HFD decreased mRNA expression of the mitochondrial biogenesis master regulator Pgc1α. In addition, HFD increased mRNA expression of mitochondrial dynamics markers Opa1, Mfn2 and Fis1 in BAT as well as Opa1 and p-Drp (S637) protein content in BAT. Conclusion: Short term HFD rapidly reduced BAT function as reflected by reduced uptake of fatty acids by BAT and rapidly increased BAT weight, accompanied by a marked reduction in Pgc1α and a gene and protein expression profile consistent with an increase in mitochondrial fusion.

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Abstract 38 Atherosclerosis Experimental, Autoimmunity and Inflammation Constitutive CD40-signaling in Dendritic Cells limits atherosclerosis by provoking inflammatory bowel disease and ensuing cholesterol malabsorption Pascal Kusters1*, Esther Smeets1*, Bart Legein2, Tom Seijkens1, Holger Winkels3, Marion Gijbels1,2, Christina Bürger3, Christian Barthels4, Erik Biessen2,5, Christian Weber3, Thomas Brocker4, Norbert Gerdes3 & Esther Lutgens1,3. 1 Department of Medical Biochemistry, Experimental Vascular Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 2 Department of Pathology, Experimental Vascular Pathology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands. 3 Institute for Cardiovascular Prevention (IPEK), Ludwig Maximilians University, Munich, Germany 4 Institute for Immunology, Ludwig Maximilians University, Munich, Germany 5 Institute for Molecular Cardiovascular Research (IMCAR), Klinikum RWTH Aachen, Aachen, Germany The co-stimulatory molecule CD40 is a major driver of atherosclerosis. It is expressed on a wide variety of cell types including mature dendritic cells (DCs) and required for optimal T cell activation and expansion. It remains undetermined if and how CD40 on DCs impacts the pathogenesis of atherosclerosis. Here we examined the effects of constitutively active CD40 in DCs on atherosclerosis, using low-density lipoprotein-deficient (Ldlr-/-) bone-marrow chimeras that transgenically express an engineered latent membrane protein 1 (LMP)/CD40 fusion protein conferring constitutive CD40 signaling under control of the DC-specific CD11c promoter (DC-CD40ca). As expected, DC-CD40ca/Ldlr-/- chimeras showed increased antigen presenting capacity on DCs and increased T cell numbers. However, they developed extensive neutrophilia compared to wt/ldlr-/- chimeras. Despite overt T cell expansion and neutrophilia we observed a reduction in cDC frequency and a dramatic (~80%) reduction in atherosclerosis. Further analyses revealed that cholesterol and triglyceride levels decreased by 37% and 60%, respectively, in DC-CD40ca/Ldlr-/- chimeras. Moreover, DC-CD40ca/Ldlr-/- chimeras developed inflammatory bowel disease characterized by massive transmural influx of leukocytes and lymphocytes, resulting in villous degeneration and lipid malabsorption. Constitutive activation of CD40 in DCs results in inflammation of the gastrointestinal tract, thereby impairing lipid uptake, which consequently results in attenuated atherosclerosis.

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Abstract 39 Metabolism Butyrate via the gut-brain circuit reduces appetite and activates brown adipose tissue Zhuang Li1,2, Sander Kooijman1,2, ChunXia Yi3, Chih kit Chuang1, Jimmy F.P. Berbée1,2, Ko Willems van Dijk2,4, Albert K. Groen5,6, Patrick C.N. Rensen1,2, Yanan Wang1,2,6 1Dept. Medicine, Div. Endocrinology, LUMC, Leiden; 2Einthoven Laboratory for Experimental Vascular Medicine, LUMC, Leiden; 3Dept. Endocrinology and Metabolism, AMC, Amsterdam; 4Dept. Human Genetics, LUMC,Leiden; 5Amsterdam Diabetes Center, Dept. Vascular Medicine, AMC, Amsterdam; 6Dept. Pediatrics, UMCG, Groningen. Background: Accumulating evidence suggests that butyrate has promising potential applications in combating obesity and metabolic syndrome, but the underlying mechanism(s) responsible for the beneficial effects of butyrate has not been fully delineated. The aim of this study was to verify the metabolic effects of butyrate in mice and to investigate underlying mechanisms. Methods and Results: Oral administration of butyrate to C57Bl/6J mice rapidly activated anorexigenic POMC neurons and suppressed orexigenic NPY neurons within the hypothalamus, and reduced food intake within 24h. In contrast, direct infusion of butyrate into the circulation did not affect food intake, suggesting butyrate induces satiety probably via acting on the gut-brain circuit thereby activating hypothalamic satiety signalling. Next, APOE*3-Leiden.CETP mice were fed a high-fat diet (HFD) alone or supplemented with sodium butyrate for 10 weeks. A third group of mice received the same amount of HFD as that of the butyrate group (pair fed group). Consistent with the acute effects, dietary butyrate caused a persisting reduction in food intake. Butyrate prevented diet-induced obesity and hepatic steatosis, mainly attributed to the reduced food intake. Additionally, butyrate appeared to increase the sympathetic outflow towards BAT as evident by increased protein expression of tyrosine hydroxylase, a marker of sympathetic nerve activity. As a result, butyrate increased the thermogenic capacity of brown adipose tissue, as it decreased the intracellular lipid vacuole size, increased the UCP-1 content within BAT, and increased the triglyceride-derived fatty acid uptake by BAT. To investigate the role of gut-brain neuronal circuit, APOE*3-Leiden.CETP mice received either vagotomy or sham surgery. After a recovery period of one week, both vagotomised and control mice were fed a HFD with or without sodium butyrate for 7 weeks. Vagotomy completely abolished the effect of butyrate on food intake and impaired butyrate-induced BAT activation. Conclusion: Butyrate acts on the vagal nerve between gut and the hypothalamus to improve energy metabolism, via reducing energy intake and enhancing fat energy expenditure by activating BAT, thereby preventing diet-induced metabolic syndrome.

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Abstract 40 Cardiac arrhythmia Protection from drug-induced arrhythmias by a novel allosteric modulator in a new validated rat ventricular cardiomyocyte model Jia Liu1, Zhiyi Yu2, Jacobus van Veldhoven2, Adriaan P. IJzerman2, Daniël A. Pijnappels1, Laura H. Heitman2, Antoine A. F. de Vries1 1Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center, The Netherlands 2Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, The Netherlands Purpose : A significant number of high-potential drugs has been withdrawn from market because of drug-induced arrhythmia, and thus potential lethality, via unintended blockade of IKr (Kv11.1; hERG). This study investigated the ability of an allosteric Kv11.1 modulator to prevent such pro-arrhythmia. Methods ; A newly synthesized compound (LUF7244) and two existing compounds (VU0405601 and ML-T531) were compared for their allosteric modulatory effects on Kv11.1 by [3H]dofetilide binding and displacement assays with cell membranes of HEK293 cells transfected with a human Kv11.1 expression plasmid. Dofetilide, astemizole and sertindole were used as typical Kv11.1 blockers. Next, the anti-arrhythmic potential of LUF7244 was studied by optical voltage mapping in a novel in vitro model of Kv11.1-blockade-induced arrhythmias using neonatal rat ventricular cardiomyocyte (nrvCMC) monolayers. Results : Of the three compounds tested, LUF7244 (10 µM) most strongly reduced Kv11.1’s affinity for each of the blockers. Treatment of nrvCMC monolayers with Kv11.1 blockers (100 nM astemizole or 1 μM sertindole) caused: 1) prolongation of action potential duration (APD), 2) an increase in APD40 dispersion, 3) followed by early afterdepolarizations (EADs) upon 1-Hz electrical point stimulation, finally resulting in: 4) unstable, self-terminating tachyarrhythmias. Importantly, pretreatment of the nrvCMCs with LUF7244 effectively prevented these proarrhythmic effects. Control cultures treated with LUF7244 alone displayed electrophysiological properties indistinguishable from untreated nrvCMC cultures. Moreover, prolonged exposure of the nrvCMCs to Kv11.1 blocker plus allosteric modulator did not have noticeable effects on their viability, excitability and contractility. Conclusion : This is the first study to proof that allosteric modulation of IKr efficiently suppresses drug-induced ventricular arrhythmias in vitro by preventing potentially arrhythmogenic changes in APD properties, raising the possibility to resume the clinical use of unintended Kv11.1 blockers via pharmacological combination therapy. Comparing fetal to adult epicardial cell activation to identify differences relevant for the epicardial post-injury response

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Abstract 41 Vascular biology Intracranial endothelial cells differ from their extracranial counterparts. Olga C.G. Stam1, Claire Mackaaij1, Per W.B. Larsen1,2, Onno J. de Boer1, Tsveta S. Malinova3, Stephan Huveneers3, Mat J.A.P. Daemen1 Affiliations 1Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands 2Electron Microscopy Center Amsterdam, Academic Medical Center, Amsterdam, The Netherlands 3Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands Objective As intracranial atherosclerosis develops later in life than extracranial atherosclerosis, we hypothesized that intracranial endothelial cells (EC) have protective mechanisms against atherogenic stimuli and that cell-cell junctions are involved. Therefore, we examined the number of cell-cell junctions and their protein expression levels in human EC’s of intra- and extracranial arteries. Methods Transmission Electron Microscopy (TEM) was performed on 5 unpaired human common carotid artery (CCA) and basilar artery (BA) samples obtained from autopsy. Photographs of cross-sections were taken at 19.000x magnification. Of each EC the number of cell-cell junctions was counted and the surface area was calculated. Immunofluorescence and Confocal Microscopy (ICM) were used to examine sixteen paired, formalin fixed paraffin embedded human CCA and BA samples obtained from autopsy. CD31, an EC marker was combined with one cell junction marker (Claudin-5, Occludin, Zona Occludens-1 or VE-cadherin). Image analysis software was used to quantify the mean fluorescence intensity per area of the cell junction markers. Results TEM: The CCA EC’s had significantly larger surface areas than the BA EC’s, median level respectively 13 and 4 µm2 (Mann-Whitney U test, p=0.000). No difference was found in the number of cell-cell junctions per EC. BA EC’s showed a significantly higher number of cell-cell junctions per surface area than CCA EC’s (median levels respectively 7 and 2 junctions/ 10 µm2, Mann-Whitney U test, p=0.000). ICM: All cell junction markers showed a higher fluorescence intensity in the EC’s of the BA compared to the CCA (Wilcoxon signed rank test, see figure). Interestingly, the cell-cell junction markers involving tight junctions (Claudin-5, Occludin and ZO-1) showed a relative larger difference in fluorescence intensity in comparison to the VE-cadherin, indicating tighter junctions in the BA EC’s. Conclusion The EC’s of human BA contain more and tighter cell-cell junctions compared to CCA EC’s. This may explain the lower susceptibility of intracranial arteries to atherosclerosis.

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Abstract 42 Cardiac function Regulation of Follistatin-Like 1 Expression Andrea Mattiotti, Marc Sylva, Quinn D Gunst, Phil Barnett, Maurice JB van dβen Hoff Department of Anatomy, Embryology & Physiology, AMC Follistatin-like 1 (Fstl1) is a glycoprotein belonging to the Secreted Protein Acidic Rich in Cysteins family and initially identified as TGFβ stimulated clone-36. From early embryo, Fstl1 is broadly expressed and becomes restricted to the mesenchymal component of most tissues with subsequent development. Fstl1 is upregulated during myocardial infarction and transiently throughout the infarcted area. The level of Fstl1 is correlated with survival after infarction and can be used as a prognostic marker. To gain insight into the transcriptional regulation of Fstl1 expression, we set out to identify regulatory elements in- and surrounding the gene. Based on Chip-seq data anaylsis twenty candidate genomic regions were selected, cloned into a luciferase reporter vector and tested in vitro, eight of which revealed enhancing capacity To identify TGF-β responsive enhancers, the cells were stimulated with TGFβ2 or co-transfected with Alk5 receptor, Smad3 and Smad4. One of the eight candidate enhancers also displayed a TGFβ-response Analysis of the lacZ pattern of this element in an F0 screen at E12.5 revealed expression in outflow tract and atrioventricular canal cushions in 4 out of 10 embryos. Taken together we identified eight enhancers of which one is active in the non-myocardial component of the heart and is TGFβ-responsive.

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Abstract 43 Imaging, Thrombosis and Hemostasis Cochrane Diagnostic Test Accuracy Review: Imaging for the exclusion of pulmonary embolism in pregnancy Thijs E van Mens1, Luuk JJ Scheres1, Paulien G de Jong1, Mariska MG Leeflang2, Mathilde Nijkeuter1,3, Saskia Middeldorp1 1Department of Vascular Medicine, Academic Medical Center, Amsterdam, Netherlands 2Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands 3Department of Internal Medicine, University Medical Centre Utrecht, Utrecht, Netherlands Background: Pulmonary embolism (PE) is a leading cause of pregnancy related death. An accurate diagnosis is crucial to prevent untreated PE but also unnecessary anticoagulant treatment. Applied imaging techniques likely perform differently in pregnancy. Objective: To determine the diagnostic accuracy of computed tomography pulmonary angiography (CTPA), lung scintigraphy and magnetic resonance angiography for the diagnosis of PE during pregnancy. Methods: For this Cochrane Diagnostic Test Accuracy Review, we searched MEDLINE and EMBASE until July 2015. Other sources for the search were citation indexes, reference lists and experts in the field. Two authors performed screening, data extraction and quality assessment. We included consecutive series of pregnant patients suspected of PE who underwent one of the index tests and either clinical follow-up or pulmonary angiography as reference test. In the primary analysis inconclusive index test results were regarded as negative and treatment for PE after an inconclusive index test was regarded as a positive reference test. Results: We included eleven studies, with a total of 695 CTPA and 665 lung scintigraphy results. Lung scintigraphy was applied with different techniques. There were no magnetic resonance studies matching our inclusion criteria. Overall the risk of bias and concerns regarding applicability were high in all studies when judged in light of the review research question, as was heterogeneity in study methods. We did not undertake meta-analysis. All studies used clinical follow-up as a reference standard, only few in a manner that enabled identification of false positives. Sensitivity and negative predictive value (NPV) were therefore the only valid test accuracy measures. The median NPV for CTPA was 100% (range 96% - 100%). Median sensitivity was 83% (range 0% - 100%).The median NPV for lung scintigraphy was 100% (range 99% - 100%). Median sensitivity was 100% (range 0% - 100%).The median frequency of inconclusive results was 5.9% (range 0.9% - 36%) for CTPA; and 4.0% (range 0% to 23%) for lung scintigraphy. The overall median prevalence of PE was 3.3% (range 0.0% to 8.7%). Conclusions:CTPA and lung scintigraphy seem well able to exclude PE during pregnancy. The quality of the evidence mandates cautious adoption of this conclusion. Important limitations included poor reference standards, necessary assumptions in the analysis regarding inconclusive test results and the inability of the studies to identify false positives. It is as yet unclear which test has the highest accuracy.

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Abstract 44 Metabolism Glucose handling and white adipocyte phenotype are altered in proteoglycan 4 deficient mice J.E. Nahon1, M. Hoekstra1, V. Van Harmelen2, J.B. Van Klinken2, K. Willems van Dijk2, M.L. Warman3, M. Van Eck1

1Division of Biopharmaceutics, Cluster BioTherapeutics, LACDR, Gorlaeus Laboratories, Leiden University, The Netherlands 2Department of Human Genetics, LUMC 3Department of Orthopaedics, Boston Children's Hospital, United States of America Background and aim: ChIP sequencing data and in vitro stimulation assays identified proteoglycan 4 (PRG4) as a novel peroxisome proliferator-activated receptor gamma (PPARy)-responsive gene. PPARy is a master regulator of lipid metabolism and is associated with adipogenesis and obesity. Therefore, we aimed to investigate the role of PRG4 in diet-induced obesity in mice. Methods and results: PRG4 is mainly expressed in metabolically active tissues in wild-type (WT) C57Bl/6 mice. Moreover, mice lacking PRG4 display improved glucose handling, providing further support for a potential metabolic function. To induce obesity, male PRG4 knockout (KO) mice and WT littermates were fed a high fat diet (HFD) for 12 weeks. We found no significant difference in body weight gain in PRG4 deficient mice compared to WT controls (p>0.05). However, PRG4 deficient adipocytes from the gonadal fat pad showed a significant increase in cell diameter (+11%, p=0.04) compared to WT which translated in a trend towards an increase in adipocyte volume (+29%, p=0.09). Interestingly, PRG4 deficient adipocytes showed significantly reduced lipolysis (2way ANOVA genotype p=0.03). Basal lipolysis did not differ significantly, however the maximum lipolytic capacity was significantly reduced in PRG4 deficient adipocytes explaining the effect on lipolysis (-28%, p=0.04). A change in adipocyte function could lead to disturbances in glucose metabolism. Therefore, this change in adipocyte function after HFD could potentially explain why the improved glucose handling by PRG4 KO mice under normolipidemic conditions was lost upon HFD feeding. Importantly, in a human cohort of obese women with Type 2 Diabetes (T2D) compared to obese normal glucose tolerant controls PRG4 expression was significantly increased (+11%, p=0.03) thus also underlining a potential role of the proteoglycan in human glucose handling. Conclusions: We show for the first time that PRG4 deficiency affects glucose handling and adipocyte phenotype in mice. Our human data highlights the potential of a role for proteoglycan 4 in human glucose metabolism. Follistatin-like 1 (Fstl1) is a secreted protein that is expressed in the atrioventricular valves (AV) throughout embryonic development, neonatal stages and adulthood. In this study, we investigated the effect of ablation of endocardial Fstl1 to determine the functional role of Fstl1 in the development and maturation of mitral AV valves after birth. The conditional ablation of Fstl1 led to an upregulation of Bmp and Tgfβ signaling. This results in ongoing proliferation and growth of the valves leading to deformed non-functional mitral valves and hypertrophic dilated hearts. Echocardiographic analysis of these hearts suggests that loss of Fstl1 leads to myxomatous valves and results in LV diastolic dysfunction with mitral regurgitation. Subsequently, Fstl1-cKO mice develop heart failure with preserved ejection fraction (HFpEF) which ultimately leads to the neonatal death of the 50% of mice around three weeks after birth.

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Abstract 45 Vascular Biology Patients with Bicuspid Aortic Valve show increased aortic inner media pathology at the high flow side Vera van de Pol1, Joshua Peterson2, Nimrat Grewal3, Evaldas Girdauskas4, Adriana Gittenberger-de Groot2, Marco DeRuiter2 Marie-José Goumans1 1 Molecular cell biology, Leiden University Medical Centre, Leiden, The Netherlands 2 Anatomy and Embryology, Leiden University Medical Centre, Leiden, The Netherlands 3 Cardiothorasic surgery, Leiden University Medical Centre, Leiden, The Netherlands 4 Department of Cardiac Surgery, Central Hospital Bad Berka, Bad Berka, Germany Background: Bicuspid aortic valve (BAV) is a congenital heart defect in which the aortic valve has two leaflets instead of three. BAV is associated with an increased occurrence of aortic dilation and dissection. Understanding the causes of aortic dilation in BAV is important for risk prediction and potential intervention. Although the pathogenesis of aortic dilation can be related to intrinsic structural differences in the aortic wall, BAV related hemodynamic flow abnormalities in the proximal aorta might be partially responsible. Material and methods: To investigate the effect of high flow on the vessel wall, aortic samples were collected from dilated BAV aortas at the high flow (jet) side as well as the opposite side (non-jet side). Immunohistochemical stainings for von Willebrand factor (VWF) and immunofluorescent analysis of α-smooth muscle actin (αSMA) and PECAM-1 were performed. To quantify the intimal and inner media pathology, every 1000 μm the thickness of the intima (endothelial cells (ECs) until Lamina Elastica Interna (LEI)) and the thickness of the inner medial organization (LEI until well-structured layers of αSMA and elastic lamellae) were measured. To determine endothelial activation, the distance of the secreted VWF into the aortic wall was measured. From these measurements, surface area ratios between the high flow jet and non-jet side were calculated. Results and discussion: The surface area of poorly organized inner media was significantly larger on the jet side compared to the non-jet side (N=4, mean ratio 1 : 0,501 +/- 0,335, P=0,0384). Intimal thickness and the area of VWF secretion did not show a correlation with the flow, indicating that the endothelial cells are not more activated by the high flow. One limitation of the study is the relatively low numbers of samples analyzed so far. Conclusion: High flow affects the organization of the inner media in BAV patients with a dilated aorta.

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Abstract 46 Cardiac arrhythmia, Cardiac function Microtubule-associated protein RP/EB family member 1 modulates sodium channel trafficking and cardiac conduction Portero V1, Veerman C1, Podliesna S1, Verkerk AO2, Marchal GA1, Klerk M1, Lodder EM1, Mengarelli I1, Bezzina CR1, Remme CA1 Departments of 1Clinical and Experimental Cardiology and 2Anatomy, Embryology and Physiology, Academic Medical Center, Amsterdam, The Netherlands Introduction: Microtubule-associated protein RP/EB family member 1 (EB1) encoded by the gene MAPRE1 is part of a protein network which binds microtubules at their (+)-end extremities underneath the cell membrane. EB1 has been shown to regulate trafficking of connexin43 (Cx43), and is located in adult cardiomyocytes at the intercalated discs. Furthermore, EB1 is removed from intercalated discs in cardiac hypertrophy, heart failure and in the setting of Cx43 mutations. Recent studies have also demonstrated that EB1 is implicated in the subcellular localisation of sodium channels in neurons. We here investigated the effects of EB1 on cardiac sodium channel function and its modulatory effect on cardiac conduction. Methods and Results : eQTL experiments performed on an F2 population of mice of two separate inbred strains carrying a sodium channel mutation (Scn5a1798insD+/-) showed a strong negative correlation between the expression of the MAPRE1 gene and QRS duration on the surface ECG, suggesting a functional impact for EB1 on ventricular conduction. Co-immuno precipitation experiments confirmed the physical interaction between EB1 and the major cardiac sodium channel Nav1.5. Overexpression of MAPRE1/EB1 in HEK293 cells together with SCN5A/Nav1.5 led to an increase in sodium current density without affecting kinetic properties, indicating an increased membrane trafficking of the Nav1.5 protein. We produced lentiviruses in order to knock-down (KD) and overexpress EB1 aiming to characterize its modulatory effect on ion currents and action potential (AP) parameters in hiPSC-CM using the dynamic clamp technique. The EB1 overexpression leads to a significant increase of the AP upstroke velocity as well as an increased sodium current density in hiPSC-CM. Surprisingly, the KD of EB1 was not changing significantly any of the AP parameters. Conclusion : In this study we demonstrate a functional role for EB1 in cardiac conduction and we highlight its direct regulation of the cardiac sodium channel Nav1.5. Our results shows that EB1 increases the sodium channel trafficking in case of overexpression. The absence of effect of the KD in hiPSC-CM suggests a compensatory mechanisms in case of a lack of EB1. In order to prove this concept we are now testing the electrophysiological effects of the depolymerisation of the microtubules in hiPSC-CM.

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Abstract 47 Atherosclerosis Experimental AT04A vaccine against PCSK-9 (Proprotein Convertase Subtilisin/Kexin Type 9) reduces total cholesterol, vascular inflammation and atherosclerosis in APOE*3Leiden.CETP mice Marianne Pouwer1,2*, Christine Landlinger3*, Claudia Juno3, José W.A. van der Hoorn1, Elsbet Pieterman1, J. Wouter Jukema2, Marzieh Edjlalipour3, Guenther Staffler3, Gergana Galabova3*, Hans M.G. Princen1* * These authors contributed equally to this work 1 The Netherlands Organization of Applied Scientific Research (TNO) - Metabolic Health Research, Gaubius Laboratory, Leiden, The Netherlands 2 Department of Cardiology, LUMC, Leiden, The Netherlands 3 AFFiRiS AG, Karl-Farkas-Gasse 22, Vienna 1030, Austria Objective: Proprotein convertase subtilisin kexin type 9 (PCSK9) has emerged as a promising therapeutic target for the treatment of hypercholesterolemia and atherosclerosis. PCSK9 binds to the LDL-receptor (LDLR) and enhances its degradation, which leads to the reduced clearance of low density lipoprotein cholesterol (LDLc) and a higher risk of atherosclerosis. In this study AT04A anti-PCSK9 vaccine was evaluated for its therapeutic potential in ameliorating or even preventing cardiac heart disease (CHD) in the atherogenic APOE*3Leiden.CETP mouse model. Approach and Results: Control and AT04A vaccine-treated mice were fed Western-type diet for 18 weeks. Antibody titers, plasma lipids, and inflammatory markers were monitored throughout. The progression of atherosclerosis was evaluated by histological analysis of serial cross-sections from the aortic sinus. AT04A vaccine induced high and persistent antibody levels against PCSK9 causing a significant reduction in plasma total cholesterol (-53%, p<0.001), triglycerides (-46%, p<0.001), and non-HDLc compared to controls. Plasma inflammatory markers such as serum amyloid A, VEGF-A, SCF, MIP-1 a s w e ll a s

MDC were significantly diminished in AT04A-treated mice, as well as ICAM-1 expression (-38%, p=0.018) and monocyte adherence to the activated endothelium (-38%, p=0.014) . As a consequence, treatment with AT04A vaccine resulted in more lesion-free aortic segments (+119%, p=0.026), a decrease in atherosclerotic lesion area (-64%, p=0.004) and reduced necrotic core content (-77%, p<0.001). Conclusions: AT04A vaccine induces an effective immune response against PCSK9 in APOE*3Leiden.CETP mice leading to a significant reduction of plasma lipids and inflammatory biomarkers, and vascular inflammation and atherosclerotic lesions in the aorta.

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Abstract 48 Vascular Biology Effect of voluntary exercise on heart function in mice with low dystrophin levels Maaike van Putten, Bauke Kogelman, Christa Tanganyika-de Winter, Ralf Werring, Margriet Hulsker, Louise van der Weerd, Annemieke Aartsma-Rus Duchenne muscular dystrophy patients lack dystrophin due to mutations in the X-chromosomal DMD gene. Patients develop severe heart failure, often leading to death. Several therapeutic approaches aiming at dystrophin restoration are in clinical trials, resulting in dystrophin restoration in skeletal muscle, while the heart appears more difficult to target. We here investigated the effects of voluntary exercise on heart function and pathology in mice expressing low dystrophin levels (1-20%) in skeletal muscle and heart due to skewed X-inactivation (mdx-XistΔhs).

We subjected 15.5 months old mdx-XistΔhs mice, mdx mice and two wild type mouse strains (C57BL/10ScSnJ and XistΔhs) to voluntary wheel running (total of 28 nights) or no exercise for a duration of 2 months. Ejection fraction, cardiac output, stroke volume and end-diastolic volume of the left and right ventricle were assessed utilizing a 7.0 Tesla Bruker MRI scanner at the age of 18 months. Additionally, fibrosis was visualized upon intravenous administration of the contrast solution Dotarem.

Voluntary running did improve skeletal muscle function in all strains. Cardiac hypertrophy was observed in all mdx mice, regardless of the voluntary exercise, while this was prevented in a dystrophin level dependent manner in the mdx-XistΔhs mice. Voluntary exercise did improve cardiac output of both ventricles, not only in wildtype mice, but also in both mdx and mdx-XistΔhs mice. Collagen infiltration in the heart was also partly prevented in the mdx-XistΔhs mice, independent whether mdx-XistΔhs mice were subjected to voluntary running or not. Voluntary exercise did not have an effect on expression of genes involved in heart function, immunological and fibrotic processes.

These data indicate that low intensity exercise has a positive effect on skeletal muscle and heart function pathology in mdx mice and highlight the protective effects of low dystrophin levels in the heart.

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Abstract 49 Vascular biology ADAM10-mediated cleavage of ICAM-1 is required for neutrophil release from the endothelial surface and enter the diapedesis step Timo Rademakers1, Sanne Brouns1, Annemarieke van Stalborch1, Marjo Donners2,*, Jaap D. van Buul1,*. 1Molecular Cell Biology Lab, Dept. Plasma proteins, Sanquin Research and Landsteiner Laboratory at the University of Amsterdam, Amsterdam, the Netherlands. 2Dept. Pathology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands. *authors contributed equally To efficiently cross the endothelial barrier during inflammation, neutrophils first firmly adhere to the endothelial surface. The endothelial adhesion molecule ICAM-1 plays a crucial role in this step. To continue to the final diapedesis step, neutrophils need to be released from ICAM-1. Integrins LFA1/Mac1 deactivation is one step that leads to this. Additionally, direct cleavage of ICAM-1 from the endothelium may be a second option. We found that A Disintegrin And Metalloprotease 10 (ADAM10) cleaves the extracellular domain of ICAM-1 from the endothelial surface. Silencing or blocking endothelial ADAM10 activity increased the surface expression of ICAM-1, thereby promoting the number of neutrophils that adhered and increasing the migration distance and velocity of the neutrophils on the surface of the endothelium. Surprisingly, despite increased number of adherent neutrophils, silencing of endothelial ADAM10 levels impaired the efficiency of neutrophils crossing the endothelium under flow conditions. We hypothesized that neutrophils were not able to dissociate from ICAM-1 without the help of ADAM10. Indeed, when measuring the kinetics, neutrophils took almost twice as much time to finish the diapedesis step. To further underscore our hypothesis, we found neutrophils that had crossed the endothelium and were positive for the extracellular domain of ICAM-1, whereas neutrophils that crossed a bare filter did not show this increase. Additionally, no parts of extracellular VE-cadherin, also sensitive for ADAM10-mediated shedding, were detected on the surface of transmigrated neutrophils. Based on these findings, we conclude that endothelial ADAM10 plays an active part in the process of neutrophil transendothelial migration by regulating the release of neutrophil binding to ICAM-1 and thereby allowing the neutrophil to enter the final diapedesis step.

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Abstract 50 Atherosclerosis Experimental Hematopoietic dual specificity phosphatase 4 deficiency promotes early atherosclerotic lesion development in LDL receptor knockout mice Baoyan Ren1*, Peter Van Santbrink1, Robin Plevin2, Miranda Van Eck1 1 Leiden Academic Centre for Drug Research, Cluster BioTherapeutics, Division of Biopharmaceutics, Leiden, The Netherlands 2 Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Scotland, United Kingdom Dual specificity phosphatase 4 (DUSP4) negatively regulates the activity of mitogen-activated protein (MAP) kinases, such as ERK1/2, JNK and p38 by dephosphorylating their tyrosine and/or serine/threonine residues. While several studies have established the role of MAP kinases in atherosclerosis, the exact role of DUSP4 in the pathogenesis of the disease is still largely unknown. In vitro, DUSP4 deficiency enhanced macrophage foam cell formation via upregulation of the scavenger receptors SR-A (2.6-fold; P<0.01) and CD36 (2.2-fold; P<0.001). To investigate the effect of hematopoietic DUSP4 deficiency on atherosclerotic lesion development in vivo, male LDLr KO mice were transplanted (T) with either DUSP4 knockout (KO) bone marrow (BM) or wildtype (WT) BM. After 8 weeks of recovery, all chimeric mice were fed a Western-type diet for 9 weeks to induce atherosclerosis. BM ablation of DUSP4 in LDLr KO mice led to 1.3-fold larger atherosclerotic lesions in the aortic root (162×103±13×103 µm2 for WT BMT vs. 213×103±19×103 µm2 for DUSP4 KO BMT; p<0.05) and a 30% lower lesional collagen content (38.2±3.5 % for WT BMT vs. 26.8±2.1 % for DUSP4 KO BMT; p<0.05). Despite the observed increase in atherosclerosis susceptibility, DUSP4 KO BMT mice displayed a striking 6.9-fold increase in IL10 cytokine levels in plasma (DUSP4 KO BMT vs. WT BMT: 817±106 vs. 118±35.7 pg/mL; p<0.0001) along with a 70% decrease in IL12-p40 levels (DUSP4 KO BMT vs. WT BMT: 8.5±1.6 vs. 30.3±5.3 pg/mL; p<0.001), indicating enhanced ERK signaling. In conclusion, our results provide new insights in the role of DUSP4 in macrophage foam cell formation and atherosclerotic lesion development in LDLr KO mice.

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Abstract 51 Cardiac arrhythmia, Stem cells A new hERG allosteric modulator rescues genetic and drug-induced Long-QT Syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells Luca Sala1, Zhiyi Yu2, Dorien Ward-van Oostwaard1, Jacobus P.D. van Veldhoven2, Alessandra Moretti3, Karl-Ludwig Laugwitz3, Christine L. Mummery1,†, Adriaan P. IJzerman2,†, Milena Bellin1,†,* 1 Department of Anatomy and Embryology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden., The Netherlands 2 Leiden Academic Centre for Drug Research, Leiden University, Gorlaeus laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands 3 I. Department of Medicine (Cardiology), Klinikum rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, Munich 81675, Germany †These authors contributed equally to this work ABSTRACT: Long QT syndrome (LQTS) is an arrhythmogenic disorder characterised by prolongation of the QT interval in the electrocardiogram, which can lead to sudden cardiac death if not properly managed. Pharmacological treatments are far from optimal for congenital forms of LQTS, while the acquired form, often triggered by drugs that (sometimes inadvertently) target the cardiac hERG channel, is still a challenge in drug development because of cardiotoxicity. Current experimental models in vitro fall short in predicting proarrhythmic properties of new drugs in humans. Here we leveraged a series of isogenically-matched, diseased and genetically engineered, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients to test a novel hERG allosteric modulator for treating congenital LQTS, drug-induced LQTS or a combination of the two. By slowing IKr deactivation and positively shifting IKr inactivation, the small molecule LUF7346 effectively rescued all of these conditions, demonstrating in a human system that allosteric modulation of hERG may be useful as an approach to treat inherited and drug-induced LQTS. Furthermore, our study provides experimental support of the value of isogenic pairs of patient hiPSC-CMs as platforms for testing drug sensitivities and performing safety pharmacology.

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Abstract 52 Metabolism G PROTEIN-COUPLED RECEPTOR 120 SIGNALING ACTIVATES BROWN ADIPOCYTES Maaike Schilperoort1,2, Andrea van Dam1, Geerte Hoeke1, Sander Kooijman1, Mark Christian2, Patrick CN Rensen1.

1 Dept. Medicine, Div. Endocrinology, Leiden University Medical Center, Leiden, the Netherlands. 2 Dept. Metabolic and Vascular Health, the University of Warwick, Coventry, the United Kingdom. Aim: Brown adipose tissue (BAT) has recently been shown to contribute to total energy expenditure in human adults, which makes it an attractive target to combat obesity and related disorders. Recent studies have shown that BAT activation by cold exposure strongly increases expression of the G protein-coupled receptor 120 (GPR120). Interestingly, both GPR120-deficient mice and humans carrying a mutation associated with decreased GPR120 signaling are predisposed to obesity. Collectively, these data suggest a role of GPR120 in BAT activation. Therefore, the aim of this study was to investigate whether GPR120 signaling could have beneficial metabolic effects by increasing the activity of brown adipocytes. Methods & Results: Classical activation of murine brown adipocytes by a β3-adrenergic receptor agonist increased Gpr120 gene expression. Also, induction of browning of subcutaneous and mesenteric white adipocytes by rosiglitazone induced Gpr120 expression, underlining the importance of GPR120 for brown adipocyte function. In addition, the GPR120 agonist TUG-891 strongly increased the oxygen consumption rate of brown adipocytes, as demonstrated by using the Seahorse XF24 Analyzer. To elucidate the pathway by which GPR120 agonism activates brown adipocytes, we assessed the effects of TUG-891 on downstream targets of Gαq signaling. Indeed, TUG-891 stimulated intracellular calcium release as well as phosphorylation of both ERK and AKT. Pre-treatment with the calcium chelator BAPTA-AM completely abrogated the GPR120-dependent increase in oxygen consumption, while inhibition of the ERK or AKT pathways had no effect. A pilot study in which C57Bl/6J mice were injected intraperitoneally with TUG-891 demonstrated that TUG891 increased fat oxidation in vivo, which is fully consistent with BAT activation. Conclusion: GPR120 signaling stimulates the metabolic activity of brown adipocytes. Mechanistically, this effect is dependent on an increase in intracellular calcium levels as a downstream target of Gαq signaling. These data suggest that activation of GPR120 on brown adipocytes is a promising novel therapeutic strategy to increase energy expenditure and combat the obesity epidemic.

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Abstract 53 Vascular biologie Endothelial DLC1 mediates the transition from rolling to adhesion stage of leukocytes prior to transendothelial migration Lilian Schimmel1, Kalim Nawaz1, Anne-Marieke van Stalborch1, Vivian de Waard2, Stephan Huveneers2, Jaap D. van Buul1

1. Department of Plasma Proteins, Molecular Cell Biology lab, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066CX, the Netherlands. 2. Department of Medical Biochemistry, Academic Medical Center Amsterdam, 1105AZ, the Netherlands During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium. Prior to the actual passage through the endothelial cell layer, they are subjected to the well-defined stages of rolling and adhesion. Where the necessary adhesion molecules as E-selectin and P-selectin for the rolling, and ICAM-1 and VCAM-1 for the adhesion are already known, it is less clear what mediates the transition from one stage to the other. Here we present data that describe the role of the endothelial GTPase activating protein (GAP) DLC1 in regulating the transition from leukocyte rolling to firm adhesion. Our data show that silencing of the RhoGAP DLC1 in endothelial cells increases the rolling time of neutrophils under physiological flow conditions. The increase in rolling time, and as a consequence the decrease in the number of firmly adhesive neutrophils results in a decrease of transmigrating neutrophils. Also, endothelial DLC1 knockdown results in neutrophils with reduced spreading and a more round phenotype. Mechanistically, we found that DLC1 knockdown lowers the activity of the small GTPases RhoA, RhoC and Rac1 in endothelial cells. We are currently working on how these endothelial GTPases control the transition from rolling leukocytes to firm adhesion in order to allow leukocytes to transmigrate.

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Abstract 54 Atherosclerosis Clinical, Atherosclerosis Experimental, Imaging, Autoimmunity and Inflammation Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy. Johan G. Schnitzler1, Feiko Tiessens1, Sophie J. Bernelot Moens1, Geesje M. Dallinga-Thie1,2, Erik S.G. Stroes1 and Jeffrey Kroon1 Affiliations: 1Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 2Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Background The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recently, peripheral lipid uptake was shown to negatively alter the monocyte phenotype towards an inflammatory state which coincided with an increased lipid droplet (LD) content. Determination of lipid content of circulating monocytes is however not very well established, since other lipid dyes are either non-specific, time-consuming or costly. Based on Nile Red (NR) LD-imaging using confocal microscopy, we developed a Matlab-based Nile Red Quantifier (NRQ); a novel quantification method for quick and non-arbitrary assessment of lipid droplet content in circulating monocytes. Results Freshly isolated circulating monocytes were used for the NR-staining procedure. In monocytes stained with NR, we clearly distinguish, based on 3D-imaging, phospho- and exclusively intracellular neutral lipids. Extraction of LDs and subsequent LD-composition analysis using high performance liquid chromatography (HPLC) showed that LDs comprises of amongst others, cholesterol esters and trigylcerides. Next, we developed and validated NRQ, a semi-automated quantification program enabling us to detect alterations in monocyte lipid accumulation after exposure to freshly derived low-density lipoproteins (LDL) in a time- and dose dependent fashion. This increased lipid accumulation furthermore induced upregulation of pro-inflammatory β2-integrin CD11c/CD18, ligand for vascular adhesion molecule-1 (VCAM-1) in THP-1 cells and thereby validating this staining and quantification method in more detail. Conclusion Thus, NR-staining is a suitable procedure to detect small differences in lipid droplet content in circulating monocytes using NRQ which, can be of value since an increasing body of evidence supports that peripheral lipid uptake is accompanied with a proinflammatory monocyte phenotype. Therefore, NR in combination with NRQ, enables researchers with a universal and quick tool to assess LD content in circulating monocytes and could potentially be used as a novel biomarker for cardiovascular disease.

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Abstract 55 Coagulation Uncovering the unique functional APC-resistant modifications of P. Textilis FV Schreuder M, Verhoef D, Cheung KL, Reitsma PH, Bos MHA Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands Blood coagulation factor Va (FVa) is proteolytically inactivated by activated protein C (APC) to downregulate the procoagulant response, a key reaction of hemostasis. APC cleaves FVa at several positions throughout the A2-domain, with Arg306 and Arg506 as major cleavage sites. Interestingly, we previously reported the functional resistance of Australian snake P.textilis factor V (ptFV) to human APC. Sequence analysis revealed the absence of the 306 cleavage site and a non-conserved surrounding region. Here we aimed to assess the role of the structural element in ptFV that replaces the cleavage site homologous to the human APC cleavage site Arg306. To do so, the non-conserved ptFV region (GNPDTLT) was exchanged for the human Arg306 region (PKKTRNL), thereby generating ptFV-h306. Upon introduction of the human 306 cleavage site we observed APC cleavage of ptFV-h306 at position 306. While full proteolysis of human FV (500nM) was achieved following treatment with 10nM of APC, a 75-fold higher APC concentration (750nM) was required to obtain fully proteolyzed ptFV-h306, similar to ptFV. Functional analysis in a human plasma system revealed that while FVa cofactor activity was fully absent in APC-cleaved human FV, APC cleaved ptFV-h306 retained approximately 5% of FVa cofactor activity. Fully proteolyzed ptFV, on the other hand, maintained a residual FVa cofactor activity of approximately 20%. These findings indicate that, conversely to human FV, APC-dependent cleavage of ptFV at Arg306 does not fully abrogate the FVa cofactor function, but reduces its activity by approximately 4-fold. This may suggest that even following APC cleavage at the positions homologous to human 306 and 506, the functional integrity of the ptFV A2-domain is stabilized such that it is able to form productive interactions. As such, ptFV provides a biological model to further study structure-function requirements that may contribute to an enhanced procoagulant FV activity.

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Abstract 56 Atherosclerosis Experimental Deficiency of the T cell regulator CBL-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death Tom Seijkens1, Svenja Meiler1,2, Esther Smeets1, Holger Winkels2, Quinte Braster1,2, Myrthe den Toom1, Marc Tjwa3, Johannes Levels4, Marion Gijbels1, Jan Albert Kuivenhoven5, Ljubica Perisic Matic6, Gabrielle Paulsson-Berne7, Ulf Hedin6, Göran K Hansson7, Christian Weber2, Norbert Gerdes2, Menno de Winther1,2, and Esther Lutgens1,2 1 Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 2 Institute for Cardiovascular Prevention (IPEK), Ludwig Maximilian’s University, Munich, Germany, 3 Laboratory of Vascular Hematology/Angiogenesis, Institute for Transfusion Medicine, Goethe University Frankfurt, Germany, 4 Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 5 Department of Pediatrics, Section Molecular Genetics, University of Groningen, University Medical Center Groningen, The Netherlands, 6 Department of Molecular Medicine and Surgery, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden, 7 Department of Medicine and Center for Molecular Medicine, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden The E3-ligase CBL-B (Casitas B-cell Lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis. We investigated the effect of CBL-B deficiency in hyperlipidemic Apoe-/- mice in atherosclerosis. At the age of 20 weeks, chow diet-fed Cbl-b-/-Apoe-/- mice showed a 1.8-fold increase in plaque area in the aortic arch, due to greater macrophage infiltration. Cbl-b-/-Apoe-/- macrophages displayed strong recruitment towards MCP1 and showed an increase in oxidized (ox)LDL uptake. In the aortic root of the same Cbl-b-/-Apoe-/- mice, where more advanced plaques were present than in the aortic arch, plaque area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages, had larger necrotic cores, and harboured more CD8+ T cells. The CD8+ T cells of Cbl-b-/-Apoe-/- mice were less susceptible to apoptosis and less resistant to Treg suppression. The increase in CD8+ T cells in the plaque effected greater macrophage apoptosis, resulting in enhanced necrotic core formation. In human atherosclerotic plaques, CBL-B gene expression was downregulated, and positively correlated with FoxP3 expression, indicating an atheroprotective effect. CBL-B is an important regulator of innate and adaptive immune reactions in atherosclerosis, by mediating macrophage recruitment and activation, CD8+ T cell activation, and CD8+ T cell-induced macrophage death in atherosclerotic plaques.

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Abstract 57 Vascular Biology GITR activation promotes regulatory CD4+ T-cell responses and stimulates leukocyte recruitment in atherosclerotic plaques Annelie Shami, Svenja Meiler, Norbert Gerdes, Esther Lutgens Experimental Vascular Biology division Dept. of Medical Biochemistry Academic Medical Center, University of Amsterdam Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) – a costimulatory molecule – is expressed on CD4(+) effector memory T cells and regulatory T cells as well as antigen-presenting cells and mast cells; while its ligand (GITRL) is mainly found on antigen-presenting cells and endothelial cells. However, the definitive role of GITR in atherosclerosis is not fully understood. Low-density lipoprotein receptor-deficient mice (Ldlr-/-) with B-cell–restricted overexpression of GITRL (Gitrltg) fed a high-cholesterol diet showed a profound increase in both CD4(+) effector memory T cells and regulatory T cells in secondary lymphoid organs in comparison to wild-type controls. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased GITRL and interleukin-2 transcript levels. Atherosclerotic lesions of Ldlr-/-Gitrltg mice contained more total CD3+ T cells as well as Foxp3+ regulatory T cells overall, leading to significantly less severe atherosclerosis. Conversely, atherosclerosis was found to be less severe in mice doubly deficient in apolipoprotein E and GITR (ApoE-/- GITR-/-). Atherosclerotic lesions in these mice were found to contain less macrophages and CD3-positive T-cells. Two-photon excitation microscopy revealed less wild type leukocyte adhesion on GITR-deficient endothelium, with a further reduction in adhesion by GITR-deficient leukocytes to both wild type and GITR-deficient endothelia. Finally, expression of GITR expression in human plaque tissue was significantly increased in ruptured plaques. These data indicate that continuous GITR stimulation through B cell GITRL acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4(+) T cells, while GITR activation on endothelial cells promotes atherogenesis by stimulating leukocyte recruitment into the plaque.

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Abstract 58 Vascular Biology Macrophage CD40 signaling in atherosclerotic plaque development Annelie Shami, Claudia van Tiel, Suzanne Aarts, Esther Lutgens Experimental Vascular Biology division Dept. of Medical Biochemistry Academic Medical Center, University of Amsterdam Atherosclerosis progression and the propensity to develop clinical symptoms such as myocardial infarction and stroke are largely determined by the immune system. Co-stimulatory molecules direct immune cell phenotypes and we have shown in previous studies that inhibition of CD40L-CD40 is highly effective in reducing atherosclerosis. Though CD40-CD40L interactions are predominantly known to play a role in the adaptive immune system, we found that while significantly reducing Ly6Chigh monocytes and macrophage infiltration in the treated mice the effects of blocking of CD40/TRAF6 signaling in adaptive immune cells were minimal. This suggests that a driving force in atherosclerosis development is in fact CD40 signaling in an innate rather than adaptive immune cell – the macrophage. The NLR family, pyrin domain containing 3 (NLRP3) inflammasome – an interleukin (IL)-1β and IL-18 processing complex – is activated in inflammatory conditions, and a role for the NLRP3 inflammasome has been shown in atherosclerotic plaque progression. In a microarray performed on murine bone marrow-derived macrophages (BMDMs) treated with a CD40-activating antibody the inflammasome-related genes IL-1β, IL-18 and NLRP3 was up-regulated – in foam cells as well as in non-lipid loaded macrophages. In addition, an increased expression of the MMPs 2, 9, 13 and 14 was found indicating that CD40 signaling is also involved in facilitating an inflammatory response – as well as possibly destabilizing the plaque as a whole – by increasing ECM remodeling and/or breakdown in the plaques. Conditional CD40flfl mice backcrossed with the lysM-Cre-ApolipoproteinE-deficient (ApoE-/-) mouse indeed showed a tendency for reduced size of initial plaques – similar analysis of advanced plaques is underway along with investigation of plaque composition of initial and advanced plaques from CD40flfl lysM-Cre-ApoE-/- compared to CD40flfl ApoE-/ mice. In line with the micro-array, preliminary quantitative polymerase chain reaction (qPCR) results confirm a decreased expression of MMP-9 and -13, as well as the tissue inhibitor of metalloproteinase-2 (TIMP2), in CD40flfl lysM-Cre-ApoE-/- BMDMs stimulated with lipopolysaccharide and interferon-γ or cholesterol crystals. MMP-2, -9 and -14 expression, along will the inflammasome components NLRP3 and ASC, will be similarly explored. Cytokine release (with a special focus on IL-1β and IL-18) will be quantified through Luminex technology, while MMP activity will be further explored in atherosclerotic plaque sections and in vitro through in situ and gel zymography. These results suggest a direct role for CD40 signaling in promoting inflammasome activation and atherosclerotic plaque remodeling, representing one possible route through which CD40 drives atherosclerosis, and providing further understanding of how inflammation is mediated in an atherosclerotic plaque environment.

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Abstract 59 Vascular Biology Interferon Regulatory Factors 3 and 7 regulate vein graft remodeling via tumor necrosis factor induced macrophage accumulation. K.H. Simons1,2,M.R. de Vries1,2, H.A.B. Peters1,2, J.F. Hamming1, J.W.Jukema2,3, P.H.A. Quax1,2 1Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands 2Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands, 3Department of Cardiology, Leiden University Medical Center, Leiden Introduction Vein grafts are used to bypass atherosclerotic lesions; however, patency rates of these grafts are poor. Inflammation driven by toll like receptors (TLRs) is a major cause of vein graft failure. Interferon regulatory factors (IRF) 3 and 7 are transcription factors downstream TLR signaling, regulating type I interferons (IFN) and type I IFN responsive genes (IFNRS). Recent data indicate that type I IFN are necessary for sustaining tumor necrosis factor (TNF)-driven inflammation. Here we hypothesize that IRF3 and IRF7 play a causal role in vein graft remodeling via modulation of type I IFNs and subsequent modulation of TNF mediated macrophage accumulation. Methods and Results Male Irf3-/-, Irf7-/- and C57Bl/6 mice underwent vein graft surgery, by interpositioning of a donor vena cava in the art. carotis of a receiver mouse. In control vein grafts both IRF3 and IRF7 are expressed in inflammatory as well as vascular cells as shown by IHC. In Irf3-/-

and Irf7-/- mice, compared to control, an increased vein graft thickening is observed 28d after surgery (n=9/group, Irf3-/- 39%, Irf7-/- 68%). At 28d both Irf3-/- and Irf7-/- mice showed a significant higher influx of macrophages in the vein graft compared to controls. At 28d IFNa4 and IFNb1 mRNA levels were detectable, but not differentially regulated. However, RNA levels of typical IFNRS such as Mx1, Ifit1-3 and Oas2 were down regulated in the knockout vein grafts compared to controls. Stimulation of both Irf3-/- and Irf7-/- bone marrow derived macrophages with the TLR ligand LPS, resulted in a significant increase in TNF production, compared to control. The early response of IRF3 and IRF7 was studied in vein grafts of Irf3-/- and Irf7-/- mice, harvested 7d after surgery. However, apart from an increased fibrin content in Irf3-/- mice (p=0.04) no differences in vein graft thickening or macrophage content of the vessel wall of Irf3-/- and Irf7-/- mice were seen. Conclusions The increased vessel wall thickening 28d after surgery observed in Irf3-/- and especially Irf7-

/- vein grafts demonstrates that IRFs regulate vein graft remodeling, by late pro-inflammatory responses as reflected by altered TNF expression in Irf3-/- and Irf7-/-

macrophages and altered accumulation of macrophages in the vein graft wall.

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Abstract 60 Metabolism Lowering vldl/ldl-c levels in apoe knockout mice decreases glucocorticoid production Ronald J. van der Sluis, Marie A.C. Depuydt, Menno Hoekstra, Miranda Van Eck Division of Biopharmaceutics, cluster BioTherapeutics, Leiden Academic Centre for Drug Research, The Netherlands VICI Grant awarded to M. Van Eck Hyperlipidemic apolipoprotein E knockout (APOE KO) mice display elevated glucocorticoid stress hormone levels when compared to normolipidemic wildtype (WT) mice. Cholesterol serves as the essential building block in adrenal steroidogenesis. Therefore, we hypothesize that APOE KO mice exhibit increased cholesterol substrate availability for the production of glucocorticoids. To verify the impact of the hyperlipidemia on adrenal steroidogenesis, we lowered the cholesterol substrate availability in APOE knockout mice. Hereto, lethally irradiated APOE KO mice were transplanted with APOE positive wildtype bone marrow (N=10) or, as a control, APOE KO bone marrow (N=6) and subsequently fed a regular chow diet for 8 weeks. Mice were fasted overnight to induce physiological stress before sacrifice. Reconstruction of APOE through bone marrow transplantation (BMT) lowered total plasma cholesterol (TC) levels 6-fold (p<0.001). Lipoprotein distribution analysis revealed a 60-fold (p<0.001) decrease in VLDL/LDL-cholesterol levels (0.011 μg/μl for WT BMT vs 0.67 μg/μl for APOE KO BMT). HDL-cholesterol levels were not changed (0.07 μg/μl for WT BMT vs 0.06 μg/μl for APOE KO BMT). No significant change was found in the internal adrenal cholesterol ester or free cholesterol levels. Expression analysis of genes involved in adrenal cholesterol uptake and de novo synthesis (LDLR, HMGCR) revealed no significant differences. Steroidogenesis related genes (CYP11A1, 3bHSD, CYP21A1 and CYP11B1) were also not affected. However, gene expression of rate limiting steps SR-BI (cholesterol uptake) and StAR (steroidogenesis) display a minor but significant decrease (fold change WT BMT; SR-BI -0.7 p<0.01, STAR -0.8 p<0.05). A prominent finding is the 2.8-fold decrease (p<0.05) in glucocorticoid levels under stressed conditions in the mice reconstituted with APOE positive bone marrow. In support of an associated decrease in anti-inflammatory glucocorticoid signaling, white blood cell counts were 2.1-fold higher in WT BMT mice (p<0.01). In conclusion, plasma cholesterol levels lowering by reconstitution of APOE reduces glucocorticoid output in APOE KO mice. Our studies highlight an important role for VLDL/LDL as source for adrenal steroidogenesis under stressed conditions. Objective : Obesity and body fat distribution are associated with several cardiometabolic risk factors in the general population. It is unclear whether fat distribution remains important in the obese. We investigated the associations between different measures of body fat distribution and cardiometabolic risk factors in obese individuals. Methods : In this cross-sectional analysis of baseline measurements of obese individuals included in the Netherlands Epidemiology of Obesity Study anthropometry was measured, in addition to abdominal subcutaneous adipose tissue (aSAT) and visceral adipose tissue (VAT) by MRI. We calculated odds ratios (OR) with 95% confidence intervals of the

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associations of waist circumference (WC), waist to hip ratio (WHR), aSAT, and VAT with high blood pressure, triglycerides, glucose concentrations and low HDL-cholesterol using logistic regression, analysis were adjusted for age, sex, ethnicity, education, smoking, alcohol consumption, and physical activity. Associations of WHR, WC and VAT were additionally adjusted for total body fat. Results : After exclusion of non-obese participants (n=3,655) and participants with missing cardiometabolic risk factors (n=33), 2,983 participants (57% women) with a mean (SD) age of 56 (6) years and BMI of 34.0 kg/m2 (4.0) were analysed. In the multivariate model in obese women all measures except for aSAT (OR: 0.89, 95% CI: 0.65-1.21) were associated with the risk of having at least one cardiometabolic risk factor, of which VAT most strongly. Per SD VAT (64.0cm2) the OR was 5.77 (3.02-11.01) and one SD higher WC (10.9cm) with an OR of 1.29 (1.03-1.63). In the multivariate model in obese men, the measures of body fat distribution were not associated with having at least one cardiometabolic risk factor. (aSAT:0.73, 0.46-1.16; WC:1.15, 0.71-1.86; VAT:1.42, 0.84-2.41) Conclusions : In obese women, but not in obese men, measures of body fat distribution, of which VAT most strongly, are associated with cardiometabolic risk factors. Future studies should aim at unravelling the underlying mechanisms of the detrimental metabolic effects of visceral fat in women.

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Abstract 61 Cardiac function, Stem cells Comparing fetal to adult epicardial cell activation to identify differences relevant for the epicardial Anke M Smits, Asja T Moerkamp, Anna MD Végh, Esther Dronkers, Kirsten Lodder, Tessa van Herwaarden, Marie-José Goumans Department of Molecular Cell Biology, Leiden University Medical Center The epicardium actively contributes to the formation and differentiation of many cellular components of the myocardium during cardiac development. In the adult, the epicardium is a quiescent layer enveloping the heart. However, upon injury the epicardium is reactivated, resulting in partial recapitulation of the developmental response including epicardial to mesenchymal transition (EMT), re-expression of Wilms’ Tumor-1 (WT1), proliferation, and migration of epicardial-derived cells (EPDCs). Given their function during embryonic development, EPDCs represent an appealing source for endogenous cardiovascular repair. Unfortunately, adult epicardial cells appear to be less efficient in their contribution to cardiac homeostasis and repair than their embryonic counterparts. Therefore, we Aim to identify the differences in fetal and adult epicardial activation and EMT, to ultimately optimize the adult post-injury response. Methods: Human fetal and adult EPDCs are isolated from cardiac specimens obtained after informed consent. EPDCs are cultured in an epithelial-like morphology in the presence of Alk5 kinase inhibitor (A5ki). EMT was induced by adding TGFb. Immunofluorescent staining, qPCR, RT2-PCR arrays for human EMT genes and angiogenic cytokine arrays were performed. Adult EPDCs pre- and post-EMT were transplanted into NOD-SCID mice after inducing myocardial infarction. Results: Both fetal and adult EPDCs can be expanded for several passages. TGFb stimulation results in EMT, leading to morphological changes accompanied by downregulation of WT1 and E-cadherin, and upregulation of mesenchymal genes a-SMA, and F-actin. Removal of the Alk5ki in fetal EPDCs instantly causes spontaneous EMT, indicating a different state of EMT, which may reflect their activity during development. PCR arrays and subsequent cluster analyses corroborate this. The importance of EMT for efficient contribution of EPDCs to cardiac repair was shown by a higher ejection fraction 6 weeks post-MI after transplanting activated epicardial cells compared to pre-EMT EPDCs. This may be explained by the finding that adult EPDCs produce higher levels of pro-angiogenic cytokines after undergoing EMT. Conclusion: The activation state of EPDCs is important for their post-injury potential. Studying fetal activation will provide more insight into this process. This project is funded by a NWO VENI (016.146.079, AMS) and a LUMC Research Fellowship (AMS), and a Rembrandt grant (MJG)

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Abstract 62 Atherosclerosis Experimental Leukocyte ABCA1 Impedes Progression of Established Atherosclerotic Lesions after Dietary Cholesterol Lowering in LDLr-/- Mice O.S.C Snip, J.J. Geerling, M. Van Eck Division of Biopharmaceutics, Cluster BioTherapeutics, LACDR, Leiden University, the Netherlands Background The ATP-binding cassette transporter subfamily A member 1 (ABCA1) facilitates the efflux of cholesterol and phospholipids to lipid-free apolipoprotein A1 and small dense high-density lipoproteins. Various studies have shown that leukocyte ABCA1 is an anti-atherogenic factor. Dietary cholesterol lowering stabilizes atherosclerotic lesions, however the role of ABCA1 in this process remains to be elucidated. Therefore, in this study we aim to investigate the effect of leukocyte ABCA1 on diet-induced atherosclerotic lesions after withdrawal of the atherogenic diet. Methods In order to specifically study the effect of leukocyte ABCA1, bone marrow cells from donor mice that were either knock-out or wild-type for ABCA1 were transplanted into atherosclerosis-susceptible low-density lipoprotein receptor knock-out (LDLr-/-) mice. One day before the transplantation, the recipient LDLr-/- mice were exposed to myeloablative total body irradiation to induce bone marrow aplasia. After 8 weeks of recovery, both groups of chimeric mice were fed a Western-type diet (0.25% cholesterol, 15% cacao butter) for 6 weeks to induce atherosclerotic lesion development. After this period, half of each group was sacrificed to determine baseline (wild-type→LDLr-/- n=14; ABCA1-/-→LDLr-/- n=12) atherosclerosis development. The remainder of the chimeric mice was switched to a chow diet (regular rodent diet, low fat and no cholesterol) for 3 weeks in order to lower plasma cholesterol levels (wild-type→LDLr-/- n=14; ABCA1-/-→LDLr-/- n=14). Results Withdrawal of the atherogenic diet normalized plasma cholesterol levels and decreased systemic white blood cell levels 2.2-fold (p<0.0001) in both groups. As a result, lesion development was stabilized in wild-type→LDLr-/- mice after dietary cholesterol lowering. On the contrary, ABCA1-/-→LDLr-/- mice displayed a 1.5-fold increase (p<0.01) in atherosclerotic lesion size. Conclusion Leukocyte ABCA1 impedes progression of established atherosclerotic lesions after dietary cholesterol lowering in LDLr-/- mice.

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Figure 1. Effect of cholesterol lowering on diet-induced atherosclerotic lesions in wild-type→LDLr-

/- and ABCA1-/-→LDLr-/- mice. Quantification of Oil Red O stained atherosclerotic lesions. Each dot represents the mean lesion size in a single mouse. Statistically significant difference as compared to baseline **p<0.01 and NS = non-significant. Abbreviations: WTD = Western type diet; wk = weeks.

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Abstract 63 Atherosclerosis Experimental SIRPalpha is a novel immune checkpoint on B1 cells regulating IgM production and atherosclerosis development Katka Szilagyi1, Saravanan Y. Pillai2, Hanke Matlung1, Marion J.J. Gijbels3,4, Mark Hoogenboezem5, Judy Geissler1, Chantal Pottgens4, Patrick J. van Gorp4, Christoph J. Binder6, Taco W. Kuijpers, 1,7, Georg Kraal8, Rudi W. Hendriks2, Menno P.J. de Winther3, Timo K. van den Berg1

1Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands 2Department of Pulmonary Medicine, Erasmus MC, Rotterdam, the Netherlands 3Department of Medical Biochemistry, Experimental Vascular Biology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands 4Department of Pathology and Department of Molecular Genetics, CARIM, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands 5Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands 6Department of Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria 7Department of Pediatric Hematology, Immunology and Infectious Disease, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands 8Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, the Netherlands Background: Signal regulatory protein alpha (SIRPα) is an inhibitory receptor expressed mainly on myeloid and neuronal cells where it binds the broadly expressed CD47 ligand. In cancer, interaction of CD47 on a cancer cell and SIRPα on a myeloid cell, is considered to be an important immune checkpoint, negatively controlling eradication of cancer cells. Inhibition of this interaction either by blocking CD47 or SIRPα has shown a great therapeutic potential. However, importance of this pathway is less clear in other diseases. Atherosclerosis is a chronic inflammatory disease, in which the immune system plays a crucial role. One of the atheroprotective mechanisms of the immune system is production of so-called natural antibodies by B1 cells. However, little is known how B1 cell-mediated natural antibody production can be modulated. We identified SIRPα as a novel immune checkpoint on B1 cells negatively regulating production of natural antibodies. Methods: Genetically modified mice lacking cytoplasmic tail of SIRPα (SIRPα∆CYT) and wild type (WT) controls were used for in vitro and in vivo experiments. Results: We show for the first time a selective expression of SIRPα on B1 cells in the peritoneal cavity where it negatively regulates production of natural antibodies in the steady state and after immunization. Genetic interference with SIRPα signaling in a mouse model of atherosclerosis leads to a substantially less severe disease accompanied by increased production of natural antibodies. In vitro stimulation of B1 cells isolated from WT or SIRPα∆CYT mice causes increased formation of large aggregates in mice lacking SIRPα signaling, a process that appears to be dependent on functional integrin CD11b, which is known to mediate their migration capacity. These findings suggest that SIRPα functions as an immune checkpoint in atherosclerosis by controlling the production of natural antibodies by B1 cells.

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Abstract 64 Cardiac arrhythmia Ectopic activity from localized oxidative stress zone violates traditional sink-source mismatch paradigm. Alexander Teplenin1, Antoine A.F. de Vries1, Alexander Panfilov2, Daniël A. Pijnappels1

1. Laboratory of experimental cardiology, Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands. 2. Department of Physics and Astronomy, Ghent University, Ghent, Belgium. Background Ectopic arrhythmia is a common type of arrhythmia and often predestinates occurrence of reentrant activity. Such ectopic beats are often caused by triggered activity. Hypothetically, this triggered activity can arise from areas of local tissue damage. In previous studies we found that local optogenetic induction of oxidative stress (OS) can be used to produce triggered activity. There local OS caused occurrence of zones of ultra-long action potentials (AP) (1.5-20s long) and associated ectopic beats. The aim of this study is to investigate the relationship between the shape of OS induced ultra-long AP zone and initiation points of ectopic beats. We addressed phenomenon both experimentally and computationally. Methods and results We performed optical mapping on monolayers of neonatal rat ventricular myocytes. The optogenetic tool called miniSOG (mini singlet oxygen generator) was expressed in these monolayers by means of lentiviral delivery. Local oxidative stress production and subsequent appearance of ultra-long AP zone was achieved by 470 nm 0.3 mW/mm2 light projection using a patterned illuminator for 3-6 minutes. In line with previous results, ectopic beats were initiated from ultra-long AP/healthy tissue interface. Surprisingly, we found that emission points of ectopic beats coincide with areas of highest curvature on ultra-long AP pattern. Since ultra-long APs could be interpreted as quasi-stable depolarized state of the cell, this result contradicts to the conventional sink-source mismatch theory. However, we reproduced effects in silico using generic FitzHugh-Nagumo and Aliev-Panfilov models. In addition, from simulations we found that ectopic beats can originate purely due to electrotonic coupling between ultra-long AP zone and healthy tissue without triggered automaticity on single cell level, with early or delayed afterdepolarizations. Conclusion We found a condition of sink-source mismatch violation for ectopic beats induced by optogenetic production of localized oxidative stress in cardiac tissue. We showed the existence of such effect both in vitro and in silico. Due to the generic nature of the computational model, we expect this effect to be found also in other pathological conditions of cardiac tissue.

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Abstract 65 Vascular biology CD40-Filamin A interactions are required for translocation of CD40 to lipid rafts in endothelial cells and for endothelial cell activation Claudia M. van Tiel1, Patrick Burger1, Pieter B. van Loenen1, Carlie J.M. de Vries1, Noam Zelcer1, Peter L. Hordijk2, Jaap D. van Buul3, Esther Lutgens1

1Department of Medical Biochemistry, subdivision Experimental Vascular Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 2Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands

3Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands Background and Aim: CD40 is a member of the co-stimulatory tumor necrosis factor receptor superfamily that is constitutively expressed not only on professional antigen presenting cells, but also on endothelial cells. Activation of CD40 signaling plays a role in chronic diseases such as rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. Therefore, in search for new therapeutic targets, we aimed to identify new CD40-binding partners. Methods and Results: We created a cDNA library of murine aortas containing atherosclerotic plaques at various stages and performed a yeast-two-hybrid with the C-terminal domain of CD40 as bait. We identified filamin A as a novel CD40 binding partner in atherosclerosis. Confocal microscopy in endothelial cells showed that, upon activation of CD40, filamin A was recruited to CD40 at distinct sites in the plasma membrane. Knock-down of filamin in endothelial cells inhibits the translocation of CD40 to lipid rafts upon activation of CD40 signaling. This inhibition of CD40 translocation resulted in repression of CD40-mediated Akt signaling, inhibition of VCAM-1 and CCL-2 expression and in reduced adhesion of monocytes to the endothelium. Conclusions: CD40-filamin A interactions in endothelial cells are involved in translocation of CD40 to lipid rafts and CD40-mediated activation of the Akt pathway. The reduced upregulation of VCAM-1 and CCL-2 and of monocyte adhesion to endothelial cells makes this an interesting target for novel therapies where reduced leukocyte recruitment is favorable. Funding: The Netherlands Organization for Scientific Research, a Vici grant to E.L.

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Abstract 66 Metabolism Role of FHL2 in obesity and adipocyte differentiation. Tran M.K.1, Tol M.J.1, Kalkhoven E2, C.J.M. de Vries1

1. Department of Medical Biochemistry, Academic Medical Center (AMC), Amsterdam, University of Amsterdam 2. Department of Metabolic & Endocrine Diseases, University Medical Center (UMC), Utrecht Four and a Half LIM-domain protein 2(FHL2) is a protein consisting of LIM-domains that facilitate protein-protein interactions to modulate protein functions. FHL2 is known to be expressed in various cell types and effects multiple physiological processes. In diet-induced obesity there is a strong positive correlation between FHL2 expression in adipose tissue and weight gain. In both epididymal fat and brown adipose tissue of obese C57Bl6 mice, we observed increased FHL2 mRNA expression. To assess whether FHL2 also plays a role in adipocyte differentiation, we analyzed FHL2 mRNA expression during mouse 3T3-L1 pre-adipocyte differentiation. Here, we observed a strong decline in FHL2 mRNA expression as differentiation progresses, whereas adipocyte marker PPARϒ increases as expected. We hypothesized that FHL2 modulates PPARϒ activity, so we analyzed the influence of FHL2 on PPARϒ promoter activity. With a PPAR Response Element (PPRE)-reporter showing decreased activity when FHL2 was co-transfected. Applying several FHL2 mutants, demonstrated that the LIM3 domain was solely responsible for the inhibition of PPARϒ transcriptional activity. Co-immunoprecipitation experiments will reveal whether FHL2 and PPARϒ interact directly. To unravel the role of FHL2 in obesity, we will expose FHL2-deficient mice to a high-fat diet and monitor weight gain and insulin-sensitivity in comparison to wild-type mice.

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Abstract 67 Cardiac function, Heart failure A cardiac mechanism of the kidney-targeted diabetes drug EMPAgliflozin unveiled: EMPA decreased cytosolic sodium/calcium, increased mitochondrial calcium by inhibiting NHE-1 in cardiomyocytes and increased oxygen consumption and vasorelaxation in the isolated heart Laween Uthman, A Baartscheer, CA Schumacher, M Jancev, B Bleijlevens, JR de Groot, AHG Driessen, NC Weber, MW Hollmann, RCI Wüst, JWT Fiolet, GJM Stienen, R Coronel, CJ Zuurbier Dept of Clinical and Experimental Cardiology, Cardiothoracic Surgery, Medical Biochemistry and Anesthesiology, L.E.I.C.A., AMC; Dep of Physiology, Institute for Cardiovascular Research, VU University Medical Centre, Dept of Physics and Astronomy, Faculty of Science, VU University, Amsterdam, The Netherlands; University of Bordeaux, L'Institut du Rythmologie et Modélisation Cardiaque (LIRYC), Bordeaux, France The kidney-targeted Empagliflozin (EMPA) causes a large reduction of cardiovascular death in T2D patients (Zinman B et al, NEJM 2015). The underlying mechanism is unknown, and cannot be ascribed to a reduction in general risk factors (glycemic status, body weight, blood pressure). Here, we examined whether EMPA 1) has direct cardiac effects by monitoring cytoplasmatic sodium/calcium concentration ([Na+]c, [Ca2+]c) and mitochondrial calcium ([Ca2+]m) in cardiomyocytes, 2) modifies sodium-hydrogen exchanger 1 (NHE-1) activity, and 3) changes cardiac oxygen consumption and vascular function in the intact heart. EMPA caused a reduction of [Na+]c and [Ca2+]c in rabbit, human and diabetic mouse cardiomyocytes, and increased [Ca2+]m in rat cardiomyocytes. When increasing extracellular glucose from 5.5 to 11 mM, thereby elevating [Na+]c and [Ca2+]c, EMPA still caused lowering of both [Na+]c and [Ca2+]c and elevated [Ca2+]m. Surprisingly, EMPA directly inhibited the NHE-flux upon acidic loading of the cell, similar to NHE-1 inhibitor cariporide. Besides, EMPA was unable to further reduce [Na+]c after pre-incubation with cariporide. Furthermore, molecular modelling supported the possibility of two binding regions for EMPA within the cardiac NHE-1. EMPA effects were independent of glucose, negating a role for SGLTs in the inhibitory effect of EMPA. Finally, EMPA administration increased oxygen consumption and induced vasodilation in Langendorff-perfused mouse hearts, yet preserved proper cardiac mechanic function and energetics (PCr/ATP). This data suggests that EMPA affects cardiac metabolism, through increased mitochondrial calcium and/or coronary vasculature dilatation. In conclusion, EMPA shows direct cardiac effects by lowering cytoplasmatic sodium/calcium, enhancing mitochondrial calcium, impairing NHE-1 activity, and activating cardiac oxygen consumption. Because myocardial sodium/calcium accumulation and mitochondrial inhibition are drivers of long term myocardial dysfunction in conditions of type 2 diabetes and heart failure, these direct cardiac actions of EMPA may contribute to its reported beneficial cardiovascular effects.

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Abstract 68 Cardiac arrhythmia Developing a gene transfer-platform for delivery of ion channel-based biological pacemakers A.M.D. Végh1, L. Cocera Ortega2, K. Lodder1, H.L. Tan2, A.M. Smits1, G.J.J. Boink2, M.J.T.H. Goumans1 1 Molecular Cell Biology, Leiden University Medical Center, Leiden, Netherlands 2 Clinical & Experimental Cardiology, Academic Medical Center Amsterdam, Netherlands a.m.d.vegh@lumc.nl Abstract Bradycardias are slow heart beats of below 50 beats per minute (bpm), giving symptoms of dizziness and fainting. This can be caused by a blockade in the conduction system, for instance when the atrioventricular node fires with a delay. Bradycardias are a major health concern, requiring ~200.000 electronic pacemaker implantations yearly in the USA. One of the great limitations of electronic pacemakers is chronotropic incompetence. Chronotropic incompetence is the lack of response to adrenergic stimulation, which leads to exercise intolerance and is a major cause of morbidity. To bypass hardware related problems, cell-based biological pacemakers are being explored. However for translation towards the clinic, there is a need for effective and safe long-term delivery of pacemaker currents. In order to deliver long term ion channel overexpression to the heart, adeno-associated viruses (AAVs) or lentiviruses (LVs) can be used. For safety reasons, LVs require delivery through ex vivo modification of cells. CMPCs provide an attractive platform, as they are cardiac-derived and safely persist in the heart at least three months after transplantation. Moreover, overexpression of the HCN4 pacemaker channel was sufficient to deliver a pacemaker current after coupling of the CMPCs with cardiomyocytes in vitro. Our aim is to establish long term delivery of ion channels by AAVs or LVs in vivo. First, sustained short-term cardiac expression of the injected virus in the heart was determined. Mice were injected with AAV-GFP directly or with CMPCs transduced or electroporated with LV-GFP. After one (CMPCs), four or eight weeks (AAVs), mice were sacrificed and hearts were retrieved. We are currently in the process of analyzing the expression of GFP in these tissues. Nevertheless, first results show AAV-GFP around the injection sites above and below the left anterior descending artery. In addition, human CMPCs were distinguished from mouse cardiac cells using a human-specific mitochondrial staining and a staining for human collagen deposits. The amount of GFP protein was determined by Western Blot. This work was funded by the Rembrandt Institute for Cardiovascular Disease.

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Abstract 69 Thrombosis and Hemostasis Side-by-side evaluation of clotting parameters in human, porcine, rabbit and rat plasma. Daniël Verhoef, Annabelle Tjalma, Pieter H. Reitsma, Mettine H.A. Bos

A wide variety of existing animal models allow for the preclinical assessment of anticoagulant drugs, procoagulant agents and hemostatic bypassing agents. To evaluate the interspecies differences in clotting parameters, we have performed a side-by-side comparison of PT, APTT, anti-FXa activity and CAT parameters in plasma of rat, rabbit, porcine, and human origin.

Following a 1:6 plasma dilution, PT and APTT analyses resulted in clotting times of rat plasma that were most similar to those of human plasma. Interestingly, while the PT clotting times were shorter in rabbit and porcine plasma, the APTT of rabbit plasma was extensively prolonged and notably short in porcine plasma. To produce detectable and robust thrombin generation (TG) curves after triggering with low tissue factor (TF) concentrations, all plasmas were diluted (1:3) in buffered saline. Initiation of TG with either a low (2pM), medium (6pM), or high (20pM) concentration of TF resulted in a dose-dependent increase in thrombin peak height and endogenous thrombin potential (ETP). Side-by-side comparisons of these parameters plus time to peak (TTP) showed that rabbit plasma behaved most similar to human plasma at 6pM of TF. On the other hand, both porcine and rat plasmas generated shorter TTPs and lower ETPs compared to human plasma. Strikingly, while addition of a pharmacological concentration of the DOAC apixaban (2μM) completely abolished TG in human and rabbit plasma, TG was present in porcine plasma and maintained in rat plasma. Finally, analysis of plasma anti-FXa activity revealed that rat (23.2mU/s) and rabbit (21.1mU/s) plasma inhibited FXa at significantly higher rates compared to human (9.5mU/s) and porcine (8.2mU/s) plasma.

Overall, our dataset underscores the wide variations between species in response to various pro- and anticoagulant stimuli, which directly impacts the use and interpretation of animal models in research and preclinical assessment.

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Abstract 70 Atherosclerosis Clinical Remnant cholesterol as a direct mediator of arterial and cellular inflammation in humans S.L. Verweij1*, S.J. Bernelot Moens1*, J.G. Schnitzler2, L.C.A. Stiekema1, M. Bos1, A. Langsted3, Carlijn Kuijk4, Carlijn Voermans4, Hein J. Verberne5, Borge Nordestgaard3, Erik S.G. Stroes1, Jeffrey Kroon1,2 Affiliations: 1Department of Vascular Medicine, AMC, Amsterdam, The Netherlands; 2Department of Experimental vascular Medicine, AMC, Amsterdam, The Netherlands; 3 The Copenhagen General Population Study and Department of Clinical Biochemistry, Herlev and Gentofte Hospital, Copenhagen University Hospital, Denmark; 4Department of Hematopoiesis, Sanquin Research and Lansteiner Laboratory, Universtity of Amsterdam, The Netherlands, 5 Department of Nuclear Medicine, AMC, Amsterdam, The Netherlands Introduction: Mendelian randomization studies validated a causal role for remnant cholesterol in cardiovascular disease. Remnant particles can enter the arterial wall, potentially propagating arterial inflammation. Also, they are proposed to accumulate in circulating immune cells and their progenitors, pushing them towards a pro-inflammatory phenotype. We evaluated the impact of elevated levels of remnant cholesterol on inflammatory activity of the arterial wall, phenotype of circulating monocytes, and bone marrow activity in patients with familial dysbetalipoproteinemia (FD). Methods and Results: We included 17 FD patients (age 60±8, 70% male) with elevated levels of remnant cholesterol (3.3[95%CI:2.1-5.7]mmol/L) and 17 controls (age 61±8, 60% male, remnant cholesterol 0.29[0.27-0.4]mmol/L). Arterial inflammation, measured with 18F-FDG PET/CT, was significantly increased in FD patients (Aortic target-to-background ratio: 2.83±0.42 vs 2.33±0.22 in controls, p=0.007). Phenotypic assessment of monocytes revealed a pro-adhesive transformation of monocytes isolated from FD patients compared with controls, with higher expression of surface integrins (CD11b, CD11c, CD18). Also, the percentage of lipid positive monocytes was significantly increased (FD patients 92[86-95%, controls 76[66-81]%, p=0.0008) with a higher number of lipid droplets per monocyte. Furthermore, FD patients exhibited a monocytosis and leukocytosis as well, with a concomitant 1.2-fold increase of 18F-FDG uptake in the bone marrow (representing progenitor cell activity). 18F-FDG uptake in the bone marrow correlated to circulating leukocyte levels (r=0.569, p=0.017). Finally, we found a strong correlation between remnant cholesterol levels and leukocyte counts in the Copenhagen General Population Study (n=103.953, p for trend=5*10-276) which validate the relationship between remnant cholesterol levels and hematopoietic activity. Conclusion: FD patients have increased arterial inflammation and are characterized by a pro-adhesive phenotype of circulating monocytes, which accumulated larger amount of lipids. Moreover, our observational study as well as validation in a large cohort support a link between remnant cholesterol and bone marrow activity. These findings indicate an important inflammatory component to the atherogenicity of remnant cholesterol, contributing to its relationship with increased CVD risk.

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Abstract 71 Atherosclerosis Experimental The role of human genetic variations in neuronal guidance cues in premature atherosclerosis D. Vreeken1, C.S. Bruikman2, J.C. van Capelleveen2, G.M. Dallinga-Thie2, G.K. van Hovingh2, A.J. van Zonneveld1 and J.M. van Gils1. 1 Department of Internal Medicine, Einthoven Laboratory of Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands, 2 Department of Vascular Medicine, Academic Medical Center (AMC), Amsterdam, the Netherlands. Background/aim: The initiation and progression of atherosclerosis involves a complex interplay between genetic and environmental factors. Neuronal guidance cues (NGCs) are differentially expressed during development where they direct the formation of neuronal networks. In mice, studies revealed a role for NGCs in atherogenesis. However, little is known on the role of NGCs in cardiovascular disease in human. Recently, we identified a number of genetic variations in NGCs in families of patients with premature atherosclerosis (PAS). Our project aims to employ these unique cohorts to explore and validate the causality of the observed genetic variations in NGCs in human atherosclerosis. Methods and results: First, we identified a selection of NGCs with potential loss-of-function mutations that were conserved in specific PAS patient families and were found to be expressed in human endothelial cells (expression array data). A particularly promising NGC mutation in this respect was found in ephrin B2 (EFNB2). EFNB2 is a membrane bound ligand that by either forward (through the receptor) or reverse signalling (via the ligand itself) might affect vasculogenesis, angiogenesis, vascular stability or monocyte migration, all processes important for atherosclerosis. The patient-associated mutation in EFNB2 leads to the loss of a phosphorylation site that may influence EFNB2 reverse signalling. Our current studies focus on the exploration of the role of EFNB2 in endothelial cells and how this may be affected by the mutation observed in the PAS-patients. Patient derived blood outgrowth EC were isolated and will be used to study the impact of the EFNB2 mutation on migration, wound healing, vascular stability and proliferation. Conclusions: We identified a mutation in EFNB2 that is associated with PAS. Studies are in progress to assess the importance of this mutation in the pathogenesis of premature atherosclerosis. Funded by a Rembrandt Research Grand to JG and KH in 2015.

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Abstract 72 Cardiac function Lymphocytic myocarditis occurs with myocardial infarction and coincides with increased inflammation, hemorrhage and instability in coronary artery atherosclerotic plaques Linde Woudstra, Msc1,2; P. Stefan Biesbroek, MD2,3,4; Reindert W. Emmens, Msc1,2; Stephane Heymans, MD, PhD5; Lynda J. Juffermans, PhD2,3; Albert C. van Rossum, MD, PhD2,3; Hans W. M. Niessen, MD, PhD1,2,6; Paul A.J. Krijnen, PhD1,2

1 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. 2 ICaR-VU, Institute for Cardiovascular Research, VU University Medical Center, The Netherlands. 3 Department of Cardiology, VU University Medical Center, The Netherlands. 4 ICIN, Inter-university Cardiology Institute of the Netherlands, Utrecht, The Netherlands. 5 Center for Heart Failure Research, Cardiovascular Research Institute Maastricht (CARIM), University Hospital

Maastricht, Maastricht, the Netherlands. 6 Department of Cardiothoracic Surgery, VU University Medical Center, The Netherlands. Objective: Although lymphocytic myocarditis (LM) clinically can mimic myocardial infarction (MI), they are regarded as distinct clinical entities. However, we observed a high prevalence (32%) of recent MI in patients diagnosed post-mortem with LM. This suggests that LM or its underlying causes may facilitate the development of MI. To investigate if LM changes coronary atherosclerotic plaque, we analyzed in autopsied hearts the inflammatory infiltrate and stability in coronary atherosclerotic lesions in patients with LM and/or MI. Methods: The three main coronary arteries were isolated at autopsy of patients with LM, with MI of 3-6 hours old, with LM and MI of 3-6 hours old (LM+MI) and controls. In tissue sections of atherosclerotic plaque-containing coronary segments inflammatory infiltration, plaque stability, intraplaque hemorrhage and thrombi were determined via (immuno)histological criteria. Results: In tissue sections of those coronary segments the inflammatory infiltrate was found to be significantly increased in patients with LM, LM+MI and MI compared with controls. This inflammatory infiltrate consisted predominantly of macrophages and neutrophils in patients with only LM or MI, of lymphocytes in LM+MI and MI patients and of mast cells in LM+MI patients. Moreover, in LM+MI and MI patients this coincided with an increase of unstable plaques and thrombi. Finally, LM and especially MI and LM+MI patients showed significantly increased intraplaque hemorrhage. Conclusions: This study demonstrates prevalent co-occurrence of LM with a very recent MI at autopsy. Moreover, LM was associated with remodeling and inflammation of atherosclerotic plaques indicative of plaque destabilization pointing to coronary spasm, suggesting that preexistent LM, or its causes, may facilitate the development of MI. Higher LDL-cholesterol levels are independently associated with larger infarct size expressed by peak CK level in patients with ST-segment elevation myocardial infarction undergoing primary percutaneously coronary intervention.

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Abstract 73 Vascular Biology The neuronal guidance cue semaphorin 3F is highly expressed by endothelial cells upon laminar flow and is important for endothelial barrier function H. Zhang, R. G. de Bruin, Dianne Vreeken, W. M. P. J. Sol, E. P. van der Veer, B. M. van den Berg, T. J. Rabelink, A. J. van Zonneveld and J. M. van Gils Department of Internal Medicine, Einthoven Laboratory of Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands Emerging data suggest that neuronal guidance cues, typically expressed during development, have roles outside the nervous system in vascular biology. Aim: In this study, we aim to elucidate the regulation of endothelial expressed neuronal guidance cue semaphorin 3F (SEMA3F) and its potential function on endothelial barrier function. Methods and Results: Of all semaphorin 3 family members, SEMA3F is abundantly expressed by endothelial cells (ECs). Since hemodynamic conditions are fundamental determinants of vascular homeostasis, we investigated the effect of flow conditions on endothelial SEMA3F expression. HUVECs were exposed to laminar flow or oscillatory flow for 1 or 7 days. SEMA3F expression was upregulated under laminar flow, but not under oscillatory flow. Moreover, overexpressing the shear-induced transcription factor KLF2 increased SEMA3F expression, suggesting upregulation of SEMA3F is KLF2-dependent. In addition, inflammatory stimuli, namely TNFα and IL1β, reduced expression of SEMA3F in HUVECs. In order to know the potential function of SEMA3F in ECs, 2 shRNAs targeted were applied to HUVECs via a lentiviral vector. Both the 2 shRNAs induced knockdown (KD) of SEMA3F expression. Using ECIS system, we determined barrier function of ECs after SEMA3F KD and found that SEMA3F KD leads to a faster adhering and spreading phase and worse barrier function in the stable phase. Conclusion: These data provide novel insights into the endothelial expression of SEMA3F under different hemodynamic conditions. SEMA3F plays a potentially important role in endothelial barrier function. Funded by the Dutch Heart Foundation, project 2013T127.

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Abstract 74 Atherosclerosis Clinical, Imaging Evaluation of ultrasmall superparamagnetic particles of iron-oxide (USPIO) enhanced MRI with ferumoxytol to quantify arterial wall inflammation. Kang He Zheng1; Loek P. Smits1; Feiko Tiessens1,2,3; Erik S. Stroes1; Aart J. Nederveen2; Bram F. Coolen2

1. Department of Vascular Medicine, Academic Medical Center, Amsterdam, the Netherlands 2. Department of Radiology, Academic Medical Center, Amsterdam, the Netherlands 3. The Netherlands MIRA institute for Biomedical Engineering and Technical Medicien, University of Twente, the

Netherlands Background: Inflammation in atherosclerotic plaques is an import determinant of plaque vulnerability, and can be detected non-invasively using ultrasmall superparamagnetic particle of iron-oxide (USPIO) enhanced MRI. Objectives: The aim of the current study was to establish and validate a protocol for quantitative USPIO-MRI of carotid artery plaques using ferumoxytol (a second generation USPIO), and to study the relation between USPIO and 18F-fluorodeoxyglucose (18F-FDG) uptake in atherosclerotic plaques. Methods: In 9 patients with a carotid artery stenosis >30% and 4 healthy controls a quantitative R2* MRI scan of both carotid arteries was performed before and 72 hours after USPIO administration (4 mg/kg ferumoxytol). R2* measurements of plaque and non-plaque regions of the carotid artery were by fitting the signal intensities over the different echo-times. USPIO uptake was quantified by the difference in R2* (ΔR2*) between baseline and post-USPIO scans. Subsequently, an 18F-FDG PET/CT scan was performed of both carotid arteries. MR and PET/CT images were co-registered, and 18F-FDG uptake was quantified in all slices containing atherosclerotic plaque. Results: Infusion of Ferumoxytol resulted in an R2* increase after 72 hours in atherosclerotic plaques (ΔR2* 24.6±19.8 s-1; p=0.0003), but not in the carotid artery from healthy controls (ΔR2* 2.6± 5.6 s-1, p=0.23). USPIO uptake in patients was higher in regions with atherosclerotic plaque compared to non-plaque regions (ΔR2* of 24.6±19.8 vs. 7,5± 9.3 s-1, p=0.004). No correlation was found between USPIO uptake and 18F-FDG uptake in atherosclerotic plaques (R2 0.03, p=0.55). Conclusion: Ferumoxytol is selectively taken up by atherosclerotic plaques, and can be used for carotid USPIO-MRI. As USPIO and 18F-FDG uptake in atherosclerotic plaque do not correlate in this cohort, these agents likely visualize different pathophysiological aspects of plaque inflammation

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Keywords: USPIO; MRI; atherosclerosis; inflammation; PET/CT

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Contact details oral and poster presentations Poster Name email 1 Suzanne A.B.M. Aarts s.a.aarts@amc.nl 2 Jacob Amersfoort j.amersfoort@lacdr.leidenuniv.nl 3 +4 Jeroen Baardman j.baardman@amc.uva.nl 5 Erik Bakker n.t.bakker@amc.uva.nl 6 + 7 Siroon Bekkering s.bekkering@amc.uva.nl Oral Thijs J Beldman t.j.beldman@amc.uva.nl Oral Rosa van den Berg R.van_den_Berg.ENDO@lumc.nl Oral Roel Bijkerk r.bijkerk@lumc.nl 8 Mathijs C. Bodde m.c.bodde@lumc.nl 9 Natalija Bogunovic n.bogunovic@vumc.nl 10 Ilse A.E. Bollen a.bollen@vumc.nl 11 Ilze Bot i.bot@lacdr.leidenuniv.nl 12 Jeroen T Buijs j.t.buijs@lumc.nl 13 Andrea D. van Dam a.d.van_dam@lumc.nl 14 Calinda KE Dingenouts c.k.e.dingenouts@lumc.nl 15 Charlotte E.A. Dronkers c.e.a.dronkers@lumc.nl 16 Janine van Duijn j.van.duijn@lacdr.leidenuniv.nl Oral T.(Dorine) W. Elffers t.w.elffers@lumc.nl 17 Johanne (Rianne) H. Ellenbroek jhellenbroek@lumc.nl 18 Iolanda Feola I.Feola@lumc.nl 19 + 20 Barend W. Florijn b.w.florijn@lumc.nl 21 Rick van der Geest r.van.der.geest@lacdr.leidenuniv.nl 22 Niels Harlaar n.harlaar@lumc.nl 23 Merel L. Hartgers m.l.hartgers@amc.uva.nl 24 Marco Heestermans m.heestermans@lumc.nl 25 Ntsiki Held n.m.held@amc.uva.nl 26 Geerte Hoeke g.hoeke@lumc.nl 27 Maurice J B van den Hoff m.j.vandenhoff@amc.uva.nl 28 Lisa Hoving l.r.hoving@lumc.nl 29 Xu Hu x.hu1@vumc.nl 30 Matthijs F Jansen mf.jansen@vumc.nl 31 Annika de Jong a.de_jong.stol@lumc.nl 32 Marjolein Caroline de Jongh M.C.de_Jongh@lumc.nl 33 Mohsin AF Khan m.a.khan@amc.uva.nl 34 Duco S Koenis d.s.koenis@amc.nl 35 H. Ibrahim Korkmaz h.korkmaz@vumc.nl 36 Boudewijn PT Kruithof b.p.t.kruithof@lumc.nl 37 Eline N Kuipers e.n.kuipers@lumc.nl 38 Pascal J.H. Kusters p.j.kusters@amc.uva.nl Oral Danielle Leuning d.g.leuning@lumc.nl 39 Zhuang Li Z.Li@lumc.nl 40 Jia Liu j.l.liu@lumc.nl

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41 Claire Mackaaij c.mackaaij@amc.uva.nl 42 Andrea Mattiotti a.mattiotti@amc.nl Oral Lejla Medzikovic l.medzikovic@amc.uva.nl 43 Thijs E van Mens t.e.vanmens@amc.nl 44 Joya, E. Nahon j.e.nahon@lacdr.leidnuniv.nl Oral Emile C.A. Nyns e.c.a.nyns@lumc.nl 45 Vera van de Pol v.van_de_pol@lumc.nl 46 Vincent Portero v.m.portero@amc.uva.nl 47 Marianne G Pouwer marianne.pouwer@tno.nl 48 Maaike van Putten maaike.vanputten@lumc.nl 49 Timo Rademakers t.rademakers@sanquin.nl 50 Baoyan Ren b.ren@lacdr.leidenuniv.nl 51 Luca Sala l.sala@lumc.nl 52 Maaike Schilperoort m.schilperoort@lumc.nl 53 Lilian Schimmel l.schimmel@sanquin.nl 54 Johan G. Schnitzler j.g.schnitzler@amc.uva.nl 55 Mark Schreuder M.Schreuder@lumc.nl 56 + oral Tom T.P. Seijkens t.t.seijkens@amc.uva.nl 57 + 58 Annelie Shami a.g.shami@amc.uva.nl 59 Karin Hendrika Simons k.h.simons@lumc.nl 60 Ronald J van der Sluis r.vandersluis@lacdr.leidenuniv.nl 61 Anke M. Smits a.m.smits@lumc.nl 62 Olga S.C. Snip o.s.c.snip@lacdr.leidenuniv.nl 63 Katka Szilagyi k.szilagyi@sanquin.nl 64 Alexander Teplenin A.Teplenin@lumc.nl 65 Claudia M van Tiel c.m.vantiel@amc.uva.nl 66 Minh Khang Tran m.k.tran@amc.nl 67 Laween Uthman l.uthman@amc.uva.nl 68 Anna M.D. Végh a.m.d.vegh@lumc.nl 69 Daniël Verhoef d.verhoef.stol@lumc.nl 70 Simone Verweij s.l.verweij@amc.uva.nl 71 Dianne Vreeken d.vreeken@lumc.nl 72 Linde Woudstra l.woudstra@vumc.nl 73 Huayu Zhang h.zhang@lumc.nl 74 Kang He Zheng k.h.zheng@amc.nl