EuCAR. Euregio Cardiovascular International Research Training Group GRK 1508 (EuCAR) Arterial Remodeling

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1 EuCAR Euregio Cardiovascular International Research Training Group GRK 1508 (EuCAR) Arterial Remodeling RWTH Aachen University Maastricht University Spokesman: Prof. Dr. med. Christian Weber Institute for Molecular Cardiovascular Research (IMCAR) Co-Spokesman: Prof. Mat Daemen, MD/PhD Cardiovascular Research Institute Maastricht (CARIM)

2 GRK 1508 Participating Applicants and Institutions (SC member steering committee) EuCAR RWTH Aachen University Univ.-Prof. Dr. Christian Weber Institute for Molecular Cardiovascular Research (IMCAR) Univ.-Prof. Dr. Jürgen Bernhagen (SC) Department of Biochemistry Univ.-Prof. Dr. Jürgen Floege (SC) Medical Clinic II (Nephrology) Univ.-Prof. Dr. Willi Jahnen-Dechent Institute for Biomedical Technology - Biomaterials Univ.-Prof. Dr. Malte Kelm Medical Clinic I (Cardiology/Angiology) Priv.-Doz. Dr. Andreas Ludwig IMCAR - Division of Cardiovascular Biochemistry Univ.-Prof. Dr. Thomas Schmitz-Rode Institute for Biomedical Technology Technology Univ.-Prof. Dr. Christian Trautwein Medical Clinic III (Gastroenterology) Univ.-Prof. Dr. Stefan Uhlig Institute for Pharmacology Univ.-Prof. Dr. Martin Zenke Institute for Biomedical Technology Cell Biology University of Maastricht Prof. Dr. Mat Daemen Cardiovascular Research Institute Maastricht Prof. Dr. Erik Biessen CARIM, Pathology Prof. Dr. Wim Buurman (SC) NUTRIM, Surgery Prof. Dr. Menno de Winther CARIM, Molecular Genetics Prof. Dr. Tilman Hackeng (SC) CARIM, Biochemistry Prof. Dr. Johan Heemskerk CARIM, Biochemistry Prof. Dr. Michael Jacobs Department of Vascular Surgery Prof. Dr. Marc Post CARIM, Physiology / Biophysics Prof. Dr. Cees Vermeer CARIM, Biochemistry Prof. Dr. Johannes Waltenberger Department of Cardiology

3 GRK 1508 From common scientific profiles towards an integrated model for a European University Hospital EuCAR Research & Teaching & Clinical Services Medizin & Technik EuCAR Medizinische Fakultät Kardiovaskuläre Forschung Klinische Neurowissenschaften In March 2007, the state minister for Innovation, Science, Research and Technology of North Rhine- Westphalia, released a statement concerning University Medicine and Medical Research. With regards to RWTH Aachen University, an integration of both clinical, research and teaching activities with University Hospital Maastricht in terms of an intense cooperation was viewed essential for future developments and structures aiming at a the first European University Hospital. Entzündung & Folgen

4 GRK 1508 The inflammatory pathogenesis of atherosclerosis EuCAR Endothelial dysfunction: monocyte adhesion Formation of fatty-streak lesions Fibroproliferative growth Unstable plaque: rupture & thrombosis R. Ross, P. Libby G. Hansson R. Virchow (Endarteritis chronica deformans): Atherosclerosis is caused by plasma components (including lipids) eliciting an inflammatory response in the arterial wall.

5 GRK 1508 Immunobalance of T cell regulation in inflammatory plaques EuCAR pro-inflammatory: Th1-cells IFN-γ IL-12 IL-18 IL-4 CD40/CD40L dendritic cells? MCP-1 RANTES MIF anti-inflammatory: regulatory T cells IL-10 TGF-β Th2-cells? IL-5 IL-4? Hansson, NEJM (2005) Tedgui & Mallat, Phys. Rev. (2006)

6 GRK 1508 Inspiration by closely related subdisciplines EuCAR Vascular Biology Immunology Technology - Mast cells - Costimulatory CD40L - Monocyte subsets - Complement Vascular Biology - Gene profiling - 2-photon microscopy - Sheddom proteomics - Tissue engineering Cardio-/Nephrology Immunology Arterial Remodeling Technology - EPCs, growth factors - IKK and signaling - Platelet microparticle - Arteriogenesis - Matrix calcification - Arterial grafts in uremia - Nitrite & cytoprotection Cardio- Nephrology

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8 GRK 1508 DFG Program Project Grant FOR809 Chemokines and adhesion molecules in cardiovascular pathogenesis EuCAR TP1 JAB CD74 CXCR2 MIF EC SMC DC TP2 RANTES PF4 platelets CXCL16 CX3CL1 TP3 CXCR6 CX3CR1 CCL17 CCR4 T-cell monocyte LFA1 JAB metalloproteases TP5 TP6 EPC SPC JAM-A TP4 SDF-1 CXCR4 macrophage ery NO TP7 reperfusion/ infarct CCR1/2/5 JAM-A Molecular Genetics Transgenic Mice ZP Spokesman: Prof. Dr. Christian Weber Nat Med (2003/2003/2007); Nat Immunol (2002/2006); Nat Gen (2005); Nat Chem Biol (2005), Nat Neurosc (2007)

9 Aachen Maastricht EuCAR Applicants Professors Applicants Professors Lecture Series Master Classes Tenure-track Mittelbau Tenure-track Assistants MD/PhD program MD curriculum RWTH Aachen PhD program CARIM Maastricht Graduate Students PhD, MD and MD/PhD Graduate Events Method Modules Progress Reports Annual Workshop elearning Platform EVGN Summer School Intake Interview 100 days in 3 years / biweekly supervising group / bimonthly thesis committee PhD CDS / Soft Skills Annual CDS / Soft Skills Annual CDS / Soft Skills Method Modules Work- Method Modules Work- Method Modules Shop Shop 100 points points points Annual Work- Shop 2011

10 DFG FOR809 Technology: transgenic mice Cre-lox recombination: platelets: PF4-cre leukocytes: LysZ-cre SMCs: SM22-cre1Her arterial EC: Bmx-creERT2 (Cancer UK) microvasc. EC: VECad-creERT2 monocytes: c-fms-gfp dendritic cells: CCL17-eGFP CXCR4 flox CXCR2 flox JAM-A flox LacZ SM22-LacZ Tie2-LacZ ApoE-/- LDLR-/- CCR1-/- CCR2-/- CCR4-/- CCR5-/- CCR7-/- CXCR6 e/e CX3CR1 e/e MIF-/- CCL17 e/e Generation of transgenic mice: CX 3 CL1sol/nc PF4-/- PF4alt SDF-1 flox doxycyclin SM22α CMV t rtta CX3CL1 polya rtta polya Inducible expression of CX3CL1

11 Chemokine und Adhäsionsmoleküle bei Atherosklerose und Gefäßverletzung lumen monocyte selectins chemokines integrins injury platelets (neo-)intimal hyperplasia LDL RANTES NFκB KC TNF, IL-1 ROS macrophage modified LDL arterial wall MCP-1 scavenger receptor VCAM-1 ICAM-1 foam cell CMV TNF MCSF PDGF tissue factor matrix proteases fibrous plaque smooth muscle cell proliferation Weber et al., ATVB (2004); Zernecke & Weber, Basic Res. Cardiol. (2005)

12 Chemokine, ihre Funktionen und heptahelikale Rezeptoren > 50 chemotaktische Cytokine (Chemokine) lösliche 8-10kDa Polypeptide 20 heptahelikale Rezeptoren extrazellulär Chemokine bei Atherosklerose CC Chemokine Rezeptor MCP-1/CCL2 CCR2 RANTES/CCL5 CCR1/3/5 intrazellulär CXC Chemokine KC/CXCL1, IL-8 IP-10 SDF-1α/CXCL12 Rezeptor CXCR2 CXCR3 CXCR4 Migration, Adhäsion Differenzierung, Proliferation Angiogenese, Angiostase Entzündungsmediatoren Fraktalkin/CX3CL1 CX3CR Weber et al., ATVB (2004) Charo et al., Circ. Res. (2004)

13 Rolle von MCP-1 und dessen Rezeptor CCR2 bei der Bildung nativer atherosklerotischer Läsionen LDLR-/- CCR2+/+ MCP-1-/- CCR2-/- MCP-1+/+ apoe-/- Gu et al., Mol. Cell (1998) KM-Transplantation zeigt Rolle von CXCR2 bei Läsionsbildung und Makrophageninfiltration Boring et al., Nature (1998) Boisvert et al., J. Clin. Invest. (1998)

14 Spezialisierte Funktion von KC/CXCR2 (Arrest) und MCP-1/CCR2 (Emigration) bei atherogener Monozytenrekrutierung (örtlich, zeitlich) Weber et al., Eur. J. Immunol. (1999); Huo et al., J. Clin. Invest. (2001) Veillard et al., Circulation (2005); Lutgens et al., Circulation (2005)

15 CX3CL1 und SDF-1 im Plaque potenzieren Plättchenaktivierung Thrombozytäre Chemokine CX3CL1 CX3CL1 SDF-1 SDF-1 CC Chemokine RANTES (CCL5) MCP-1 (CCL2) MIP-1α (CCL3) MCP-3 (CCL7) Mach et al., JCI (1999) Abi-Younes et al., Circ. Res. (2000) Schäfer et al., Blood (2004) CXC Chemokine und Vorstufen PF4 (CXCL4) GRO-α (CXCL1) ENA-78 (CXCL5) CTAP-III/β-TG (NAP-2/CXCL7) IL-8 (CXCL8) SDF-1 (CXCL12) Beteiligung von CX3CR/L1 an der Atherogenese Charo et al., JCI (2003); Combadiere et al. Circulation (2003) Teupser et al., PNAS (2004) Gear et al., Microcirculation (2003)

16 Plättchen und Chemokine: Komplizen in der Atherosklerose (TP2) Platelet-induced chemokine secretion in EC and SMC platelet monocyte Deposition of platelet chemokines on EC triggering monocyte arrest monocyte IL-1ß EC CD40L MCP-1 SMC platelet P-selectin EC RANTES PF4 Potentiation of platelet activation by vascular chemokines Presentation of vascular chemokines by adherent platelets monocyte progenitor cell MDC / TARC thrombin ADP MCP-1 SDF-1α SDF-1α fractalkine SMC collagen SMC ECM platelets Weber, Circ. Res. (2005)

17 Das pleiotrope Zytokin MIF in der Atherosklerose (TP1) Strukturhomologie von macrophage migration inhibitory factor (MIF) Monomer und IL-8 Dimer control anti-mif macrophages SMC MIF MIF collagen I IL8 IL8 > neue MIF Rezeptoren identifiziert Bernhagen et al., PNAS (1996) Bernhagen et al., Nat. Rev. Drug Dev. (2006) Schober et al., Circulation (2004)

18 Gendefekt von VCAM-1 nicht aber ICAM-1 reduziert Atherosklerose: Effekt junktionaler Adhäsionsmoleküle (JAM)? (TP6) Cybulsky et al., J. Clin. Invest. (2001) Ostermann et al., ATVB (2005) Imhof et al., Nat. Rev. Immunol. (2004) Chavakis et al., J. Exp. Med. (2003) Ostermann et al., Nat. Immunol. (2002)

19 Äquilibrium vaskulärer Umbauprozesse neovascularization arteriogenesis arteriogenesis SPC SPC EPC EPC Offene Fragen: SDF-1 KC/IL-8 PF4 MCP-1 - Hierarchie der Protagonisten? restenosis transplant SDF-1 PDGF SDF-1 SMC SMC SPC SPC repair matrix matrix EPC EPC KC/IL8 KC/IL-8 EPC EPC native native plaque plaque - Regulative Mechanismen? - Interaktionen von Chemokinen? MCP-1 MCP-1 RANTES VEGF VEGF - Therapeutische Strategien? unstable unstable EPC? SMC? DC? SDF-1 SDF-1? SMC MIF? stable Schober et al., Trends Cardiovasc. Med. (2006) Schober et al., Circulation (2003) Zernecke et al., Circ. Res. (2005)

20 DFG Forschergruppe FOR809 Chemokine und Adhäsionsmoleküle in der kardiovaskulären Pathogenese TP1 JAB CD74 CXCR2 MIF EC SMC DC TP2 RANTES PF4 Plättchen CXCL16 CX3CL1 TP3 CXCR6 CX3CR1 CCL17 CCR4 T-Zelle Monozyt LFA1 JAB Metalloproteasen TP5 TP6 EPC SPC JAM-A TP4 SDF-1 CXCR4 Makrophage Ery NO TP7 Reperfusion/ Infarkt CCR1/2/5 JAM-A Molekulargenetik TP8 Ziel: Modellübergreifendes Studium der pathophysiologischen Rolle dieser Moleküle im Kontinuum von initialer Atherosklerose, Gefäßumbau bis zum Myokardinfarkt

21 Modell der Diät-induzierten Atherosklerose (ApoE-/-, LDLR-/-): Exazerbation durch aktivierte Plättchen & RANTES Deposition (TP1-6) Thorako-abdominelle Aorta RANTES Aortenwurzel CCR1/5 RANTES Oligomer EC Proteoglycan von Hundelshausen et al., Circulation (2001) Schober et al., Circulation (2002); Huo et al., Nat. Med. (2003); Baltus et al., Blood (2003); Zernecke et al., Blood (2006)

22 Modelle arterieller Gefäßverletzung: Deletion von CCR2 inhibiert und VEGF exazerbiert neointimale Hyperplasie und Infiltration (TP3-5) endotheliale Drahtverletzung CCR2 +/+ apoe -/- CCR2 -/- apoe -/- periarterielle Manschette Ad.empty Ad.hVEGF-A CCR5 -/- apoe -/- van der Thüsen et al., Circulation (2001) Schober et al., Circ. Res. (2004) Zernecke et al., Blood (2006) Lucerna et al., Blood (2006)

23 Tabelle 1: Herstellung und Nutzung transgener Mäusestämme durch die Forschergruppe Genprodukt Stamm Genetische Veränderung ADAM10 C57BL/6N tissue specific inducible ADAM17 tissue specific inducible CCL17 (egfp) with GFP knock-in CCL17 (egfp) apoe double with GFP knock-in CXCL4/PF4 in progess CXCL4/PF4alt in progess variant knock-in CX3CL1 C57BL/6N tissue-specific inducible overexpression CX3CL1nc in progess non-clevable variant knock-in CXCL12 flox/flox in progess conditional CCR2 CCR5 CCR7 Quelle/Kooperation P. Saftig, CA-Uni Kiel S. Rose-John, CA-Uni Kiel I. Förster, HHU Düsseldorf C. Weber, RWTH Aachen C. Weber, RWTH Aachen C. Weber, RWTH Aachen D. Brotner, GlaxoSmithKline S. Jung, Weizmann, Rehovot C. Weber, RWTH Aachen W. Kuziel, Protein Design Labs W. Kuziel, Protein Design Labs M. Lipp, MDC Berlin Projekt P5, P6 P5, P6 P3 P3 P2 P2 P5 P5 P1, P4 P3 P3 P3 CCR1 apoe double C. Weber, RWTH Aachen P3, P7 CCR2 apoe double C. Weber, RWTH Aachen P3, P7 CCR5 apoe double C. Weber, RWTH Aachen P3, P7 CD44 R. Bucala, Yale University P1, P2 CD74 R. Bucala, Yale University P1, P4 CXCR2 Balb/c Jackson Labs P1, P4 CXCR2 MIF in progess double J. Bernhagen / G. Fingerle P1, P4 CXCR4 D. Littman, HHMI, New York P4, P7 CXCR4 flox/flox conditional Y. Nie, Columbia University P4 CXCR6 (egfp) with GFP knock-in D. Littman, HHMI, New York P5, P3 CX3CR1 GFP knock-in D. Littman, HHMI, New York P5 c-met flox/flox -tg conditional C. Birchmeier, MDC Berlin P3 DO Balb/c TCR transgen OVA MHC II restringiert M. Lutz, Uni Erlangen P3 enos J. Schrader, HH-U Düsseldorf P7 Gqα S. Offermanns, Uni Heidelberg P2 Gq12 S. Offermanns, Uni Heidelberg P2 Gq13 S. Offermanns, Uni Heidelberg P2 Id2-/- 129Sv Y. Yokota, FMU, Japan P3 JAB flox/flox conditional J. Bernhagen, RWTH Aachen P1 JAM-A E. Dejana, MNI Mailand P6 JAM-A ape Double C. Weber, RWTH Aachen P6 JAM-A flox/flox conditional E. Dejana, MNI Mailand, T Sato P6 MIF R. Kleemann, TNO Leiden P1 MIF flox/flox conditional G. Fingerle, Uni Köln P1 MIF LDLR double R. Kleemann, TNO Leiden P1 Myoglobin NMRI J. Schrader, HHU Düsseldorf P6 OT-I TCR transgen OVA MHC I restringiert M. Lutz, Uni Erlangen P3 OT-II TCR transgen OVA MHC II restringiert M. Lutz, Uni Erlangen P3 OT-I/Rag-/- TCR transgen; Rag C. Kurts, Uni Bonn P3 Bmx-creERT2 tissue specific inducible expression R. Adams, London University P4, P5 LysZ-cre tissue specific expression Jackson Labs P4, P1 PF4-cre tissue specific expression R. Skoda, Uni Basel P4, P5 SM22-cre1Her /129Sv tissue specific inducible expression M. Mericksay, INSERM Paris P4, P5 SMMHC-creERT2 tissue specific inducible expression S. Offermanns, Uni Heidelberg P4 VECad-creERT2 tissue specific inducible expression R. Adams, London University P4, P5 egfp transgene Jackson Labs P4, P3 LacZ transgene Jackson Labs P4, P6 SM22-LacZ tissue specific expression Jackson Labs P4 Tie2-LacZ tissue specific expression Jackson Labs P4, P6 apoe C. Weber, RWTH Aachen P1-P7 LDLR R. Kleemann, TNO Leiden P1, P4

24 Zentrale Einheit: Transgene Mäuse (TP4) Cre-lox recombination: platelets: PF4-cre leukocytes: LysZ-cre SMCs: SM22-cre1Her arterial EC: Bmx-creERT2 microvasc. EC: VECad-creERT2 monocytes: c-fms-gfp dendritic cells: CCL17-eGFP CXCR4 flox CXCR2 flox JAM-A flox LacZ SM22-LacZ Tie2-LacZ ApoE-/- LDLR-/- CCR1-/- CCR2-/- CCR4-/- CCR5-/- CCR7-/- CXCR6 e/e CX3CR1 e/e MIF-/- CCL17 e/e Generierung eigener Mauslinien: CX 3 CL1sol/nc PF4-/- PF4alt SDF-1 flox SM22α doxycyclin t CMV rtta CX3CL1 polya rtta polya Induzierte Expression von CX3CL1

25 Modell des Myokardumbaus bei Ischämie/Reperfusion bzw. Infarkt: Effekte von Chemokinen auf Angiogenese/Entzündung? (TP7) MCP-1 CCR2 Schaper et al., Circ. Res. (1997;2004) Weber et al., ATVB (1999) Dewald et al., Circ. Res. (2005) Kaikita et al. Am. J. Pathol. (2004) B C A Infarct area (% LV) z * CCR1 +/+ CCR1 -/- Merx et al., unpublished (2006)

26 Zentrale Einheit: Genpolymorphismen im Chemokinsystem (TP8) Gen Maus Mensch Chemokine CCL2 CCL5 CCL17 CXCL12 + +?? G-2518A G-403A C-3079T 3 A/G CXCL16 CX3CL1 MIF A181V C-2979T -794 CATT Rezeptoren CCR2 CCR3 CCR5 +? + V61I T51C 32,G303A CXCR6? 3K CX3CR + T280M Proteasen MMP3 + C94T MMP9 MMP1 +? C-1562T A10920G prospektives Kollektiv mit invasiver, koronarer Phänotypisierung (IVUS) und in vitro Korrelation

27 Plattformtechnologien: Konfokale 2-Photonmikroskopie (TP3) (in Kooperation mit Dr. M. van Zandvoort, Biophysik, CARIM) pp FRET/TIRF Mikroskopie (TP6) Intravitalmikroskopie (TP3)

28 Technologie: Biophysik (über TP2) Genom > Proteom > Chemokin-Interaktom Gewebs-spezifische Chemokinkomplexe integrieren synergistische & antagonistische Signale für Leukozyten (Beispiel: RANTES/PF4 Heteromer) Biophysikalische Methoden (Kooperation mit K. Mayo, K. Hoffmann, A. Kungl): - Biacore Plasmonresonanz - Fluoreszenz-Anisotropie - Kristallographie - Strukturmodelle - NMR Spektroskopie RANTES/PF4 Heteromer RANTES Dimer TOCSY Weber & Koenen, Trends Immunol. (2006) Hundelshausen et al., Blood (2005)

29 Zentrale Einheit und Infrastruktur: IZKF IZKF Nachwuchsgruppe (Leiter: PD Dr. A. Ludwig) IZKF Nachwuchswissenschaftler im PhD Programm Hristov et al., ATVB (2003) Blood (2004), Atherosclerosis (2006) Liehn et al., ATVB (2004) Merx, Liehn et al., Circulation (2004;2005) DNA Chip Facility BD FACSAria Core Facility Adoptiver Transfer von DC und vaskulären Vorläuferpopulationen Live 2-Photonmikroskop (CARIM)

30 Nachwuchsausbildung (Sprecher Vorsitzender) Dauer 3 Jahre >2 Publikationen als Erstautor Abschluss als Dr. med.nat. Naturwissenschaftl. Doktorandenausbildung - aktuell 9 DFG-geförderte Doktoranden - Stellvertretender Sprecher Mitglied der Fachgruppe Biologie der Naturwissenschaftl. Fakultät der RWTH - Integration in DFG Graduiertenschule im Rahmen der Exzellenzinitiative CARIM PhD Programm (Sprecher Associate) Introductory courses Physiology of the heart and circulation Biophysics of the heart and circulation Advanced microscopy and vital imaging Molecular biology and genetics Clinical aspects of cardiovascular and peripheral vascular diseases Introduction into cardiovascular pharmacology PhD-training courses Cardiac Function and Adaptation Vascular Biology Thrombosis and Haemostasis EVGN summer school Ziel: Internationales Euregio-Graduiertenkolleg Vascular Biology RWTH - CARIM

31 Externe Kooperationen Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht Mat Daemen, Esther Lutgens (Pathology) Arjan Griffioen (Pathology) Tilman Hackeng (Biochemistry) Marc van Zandvoort (Biophysics) Johannes Waltenberger (Cardiology) Wim Buurman (NUTRIM) University of Leiden University of Virginia Yale University Mario Negri Institute, Milano University of Geneva University of London University of Düsseldorf University of Munich University of Frankfurt University of Cologne University of Kiel/Lübeck RWTH, Institut für Textiltechnik (ITA) Institut für Molekulare Biotechnologie Medizinische Klinik II und III Institut für Biochemie Erik Biessen, Robert Kleemann Klaus Ley Rick Bucala Elisabetta Dejana Francois Mach Ralf Adams, Toby Lawrence Jürgen Schrader, Irmgard Förster Axel Gödecke Peter Nelson Judith Händeler Günther Fingerle Stefan Rose-John, Paul Saftig, Michael Krawczak, Heribert Schunkert Thomas Gries Kurt Hoffmann Peter Mertens, Jürgen Floege Frank Tacke Gerd Müller-Newen Bernhard Lüscher

32 Eckpunkte: Publikationen, Kooperationen, Demographie, Strukturen ca. 300 themenbezogene Originalarbeiten (letzte 5 Jahre) - davon 25 Publikationen Projekt- und Abteilungs-übergreifend 3x Nat. Genetics 3x Nat. Immunol. 2x J. Clin. Invest. 4x Proc. Natl. Acad. Sci. USA 7x Circ. Res. 1x Nat. Rev. Drug Discov. Kooperationen: Fakultätsstrukturen: TP8 TP7 TP6 TP1 TP4 TP3 TP2 TP5 Institut für Kardiovaskuläre Molekularbiologie (IMCAR, W3-Professur) 2x Nat. Med. 2x JAMA 8x Circulation 11x Blood 10x J. Am. Coll. Cardiol. 1x Nat. Chem. Biol. IZKF Nachwuchsgruppe (geplante W2 Professur Kardiovaskuläre Biochemie) Berufung W3 Professur für Innere Medizin - Kardiologie (Prof. M. Kelm) W1 Professur für Kardiovaskuläre Akutmedizin (Medizinische Klinik I) Demographie: C4 / W3 / C3 apl / C2 / PD C1 / IIa / Ib Core Facilities für Cell Sorter, DNA Chip, Mikroskopie, Maus-Echokardiographie 5x 5x 5x