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1 DECLARATION OF CONFLICT OF INTEREST No disclosures

2 micrornas: Role in progression of atherosclerosis Christian Weber Institute for Cardiovascular Prevention (IPEK) Ludwig-Maximilians-University Munich & Munich/Maastricht Centre for Atherosclerosis Research (MCAR)

3 The inflammatory pathogenesis of atherosclerosis Endothelial dysfunction: monocyte adhesion Formation of fatty-streak lesions Fibroproliferative growth Unstable plaque: rupture & thrombosis R. Ross, NEJM (1999) 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.

4 Biogenesis and function of micrornas Urbich et al., Cardiovasc Res (2008)

5 mirs in arterial inflammation and remodeling Weber et al., Curr Drug Ther (2010)

6 CXCL12 expression in atherosclerosis, correlation of plasma levels with stable disease, and genome-wide association studies Signal-intensity plots of associations of SNPs with coronary atherosclerosis 9p21 10q11 (CXCL12) Abi-Younes et al. Circ Res (2000); Damas et al., Circulation (2002) Samani et al., N Engl J Med (2007)

7 Interference with CXCR4 unveils a protective role in atherosclerosis Zernecke et al., Circ. Res. (2008)

8 Chemokines in the homeostasis of vascular progenitor cells CD133 + CD34 + VEGFR2 + CD14 + CXCR2 + CD14 lo Tie-2 + EPCs/EOCs mobilization hypoxia / VEGF / CXCL12 erythropoietin / G(M)-CSF CXCL1 / CXCL7 CXCR2 / CXCR4 endothelial regeneration SPCs CD105+ a-sma, SM-MHC, SM22 PDGFR-b c-kit +/- sca-1 + lin - PDGFR-b CXCL12 / CXCR4 induction by apoptosis protection plaque growth plaque instability SMCs neovascularisation neointima formation plaque stability Weber et al., Nat Rev Immunol (2008)

9 Uptake of endothelial apoptotic bodies (AB) induces CXCL12 - mediated by transfer of RNA Zernecke et al., Sci Signal (2009)

10 Transfer of functional RNA from apoptotic bodies to recipient cells Horizontal transfer of mrna and microrna as a mechanism of genetic exchange Valadi et al., Nat Cell Biol (2007) Zernecke et al., Sci Signal (2009)

11 mir-126 is enriched in AB and can be confered to endothelial cells: induction of CXCL12 Absolute expression levels in AB (left) and after incubation of HUVECs (right) CXCL12 expression in HUVECs Zernecke et al., Science Sign. (2009); Fish et al., Science Sign. (2009)

12 Regulation of angiogenic signaling by mir-126 Wang et al., Developmental Cell (2008); Fish et al., Developmental Cell (2008)

13 B mir-126 induces CXCL12 expression by targeting RGS16, unleashing an autoregulatory feedback loop via CXCR4 H. sapiens RGS16 hsa-mir-126/mmu-mir-126 M. musculus Rgs16 5 -GCCAGUGUUUUUUGUGG -UAUGA-3 : : : : : : 3 -GCGUAAUA AUGAGUGCC -AUGCU-5 : : : : : 5 - AUCAGUUCUCUUUGUGGGUAUGA-3 / Zernecke et al., Sci Signal (2009)

14 Endothelial AB increase in hyperlipidemic mice and induce luminal CXCL12 expression and Sca-1 + cell recruitment to lesions Zernecke et al., Science Sign. (2009)

15 Transfer of mir-126 by endothelial AB induces CXCL12 expression and provides CXCR4- dependent protection against atherosclerosis CD31 APC CD31 PE-Cy pro-mir-126 pro-mir-126 U6 U6 A human plaque AB ultrasound-guided incubation of cartoid arteries annexin-v FITC 10 5 B mouse blood AB Zernecke et al., Science Sign. (2009)

16 Apoptotic bodies deliver mir-126 to elicit CXCL12-mediated vasoprotection lin - sca-1 + progenitor cell recruitment EC apoptosis release of AB VCAM-1 CXCL12 DNA fragmentation AB transfer of mir-126 SPRED1 RGS16 CXCL12 CXCR4 function P-ERK CXCL12 protection from atherosclerosis plaque stability - Mechanisms of AB enrichment? - Free mir-126 and uptake of mimic? - mir-126 -/- treatment

17 Circulating micrornas in patients with coronary artery disease: reduction in mir-126 Fichtlscherer et al., Circ Res (2010)

18 Plasma microrna profiling reveals loss of endothelial mir-126 confined to circulating plasma particles/vesicles in type 2 diabetes Zampetakiet al., Circ Res (2010)

19 Impaired Dicer expression in endothelial cells exacerbates neointima formation after arterial injury Nazari-Jahantigh et al., submitted

20 mir-126 deficiency promotes neointima formation by impairing endothelial recovery: relevance of mir-126-5p and DLK1 Nazari-Jahantigh et al., submitted

21 mir profiling in complex murine plaques versus healthy artery Nazari-Jahantigh et al., submitted

22 U6 snrna mir-105 mir-217 mir-196a mir-10b mir-15b mir-205 mir-98 mir-152 mir-30e-5p mir-26b mir-125a fold change (AU relative to U6) -log 10 (p-value) mir profiling in human atherosclerotic plaques versus healthy artery healthy vessel plaque logfc mir-520b mir-105 mir-520e mir-10b mir-218 mir-17-3p ambi-mir-7039 mir-186 mir-489 mir-143-mm2 mir-524-5p healthy vessel log2fc plaque mir-220 mir-147 mir-422a mir-15b mir-185 mir-181a mir mir-152 mir-422b 0 mir-30e-5p mir-26b mir-125a Bidzhekov et al., submitted

23 Acknowledgements IPEK Munich, IMCAR Aachen Rory Koenen Philipp v. Hundelshausen Alma Zernecke Mihail Hristov Kiril Bidzhekov Andreas Schober Oliver Söhnlein Remco Megens Esther Lutgens See you at abcam The Leukocyte in Cardiovascular Disease Conference Munich, October 2012 University of Texas, Dallas Eric Olson CARIM, Maastricht University Tilman Hackeng / Kevin Mayo EuCAR GRK1508 Arterial Remodeling FOR809 TRR57 GRK1035 SFB542 MSE06 GIF

24 SDF-1/CXCR4 recruit bone-marrow cells giving rise to neointimal SMC wire-injury of carotid arteries LacZ a-sma LacZ or CXCR4-/- bone marrow irradiation c-kit - /sca-1 + /lin - / PDGFR-b + SPC SM22-LacZ SM22 + /a-sma + / SMMHC + SMC Zernecke et al., Circ. Res. (2005); Massberg et al., J. Exp. Med. (2006)

25 Transfer of human/plaque endothelial AB CXCR4-dependently reduces atherosclerotic lesion formation, macrophage infiltration and apoptosis CD31 APC pro-mir-126 -mir-126 U6 A human plaque AB annexin-v FITC 10 5 B Zernecke et al., Sci Signal (2009) mouse blood AB

26 Local transfer of mir-126 limits plaque formation, macrophage content and apoptosis, while increasing CXCL12, SMC and collagen content collar and pluronic gel placement in cartoid arteries collagen content Zernecke et al., Sci Signal (2009)

27 Local exposure to mir-126-carrying but not mir-126-deficient mouse AB limits plaque formation, repressing RGS16 and inducing CXCL12 ultrasound-guided incubation of cartoid arteries Zernecke et al., Sci Signal (2009)

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