Reprogramming through micrornas Stefanie Dimmeler
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1 Klinikum der Johann Wolfgang Goethe Universität Frankfurt am Main Reprogramming through micrornas Stefanie Dimmeler Conflict of interest: T2cure GmbH, Miragen
2 Non-coding DNA & RNA and micrornas Human Genome Project: Graig Venter, x10 9 bp Proteins % non-coding DNA Human 97 % Mammals % Fungi and Plants 7-75 % Human Genome: ca Genes (only 2 x more than worms or flies) Eurkaryotes % Junk DNA Source: Wikipedia
3 micrornas: Processing and functions Intronic mirnas microrna Exon Exon Exon Intergenic mirnas microrna Gene Gene sirna: One target mrna PrimarymiRNA Single mirna Cluster microrna: Nucleus Pre-miRNA Drosha up to hundreds of mrnas Dicer mirna Duplex RISC Complex Translational Repression mrna Degradation
4 MicroRNAs in the heart mir-92a (Van Rooij et al., Circ Res 2008)
5 micrornas and cell therapy Reprogramming: Induce pluripotency micrornas and stem cells Direct reprogramming: Induce differentiation Functional reprogramming: Improve cell functionality
6 micrornas: ESC and ips cells micrornas: induce pluripotency maintain pluripotency allow differentiation Somatic Cell mir-302 mir-367 Pluripotent Stem Cell Progenitor Cell Reprogramming Differentiation facilitate induce inhibit mir-21, mir-29a Let-7 family mir-25 cluster mir-106 cluster mir-34 mir-130, mir-301, mir-721 Maintenance of Pluripotency Cell cycle DNA methylation mir-290 family mir-302 cluster Oct4 Nanog Sox2 Klf4 Lin28 Down-regulation of pluripotency genes mir-134, mir-145, mir-470 mir-134. mir-296, mir-470 mir-134, mir-145, mir-200c mir-145, mir-200c Let-7 Heinrich & Dimmeler, Circ Res 2012
7 micrornas control self renewal, pluripotency and differentiation Differentiated cell mir-302 mir-367 Pluripotent Stem Cell ips Maintenance of Pluripotency Cell cycle DNA methylation mir-290 family mir-302 cluster (Anakye-Danso et al Cell Stem Cell 2011)
8 micrornas and cell therapy Reprogramming: Induce pluripotency micrornas and stem cells Direct reprogramming: Induce differentiation Functional reprogramming: Improve cell functionality
9 mirnas regulated during EC differentiation mirnas regulated during SMC differentiation Heinrich & Dimmeler, Circ Res 2012 mirnas regulated during CM differentiation mirs and cardiovascular lineage irnas: Induce differentiation Enhance maturation commitment Progenitor Cell mir-10a mir-145 mir-1 mir-499 Notch ligands HDAC4 Sox6 mir-133 Endothelial cells Smooth muscle Cardiac muscle Regulated mirnas mir-126 mir-17~92 mir-130 mir-210 Let-7 family mir-181 Function Vascular integrity & Lumenization mir-17 mir-20 mir-92 mir-17~92 inhibit sprouting mir-17/20: promote proliferation Pro-angiogenic Promotes deprogramming of lymphatic ECs towards vascular ECs Regulated mirnas mir-1 mir-10a mir-143 mir-145 mir-21 Function Required for SMC differentiation Required for SMC differentiation Induce contractile SMC phenotype Overexpression of mir-145 increases smooth muscle differentiation Endothelial-Mesenchymal transition Regulated mirnas mir-1 mir-133 mir-208 mir-499 Function Regulates proliferation and differentiation of progenitor cells Targets notch ligands and HDAC4 Blocks CM differentiation Regulates cardiomyocyte hypertrophy, fibrosis Increases cardiac differentiation in vitro
10 mir-1 and mir-499 control cardiac differentiation of stem cells (see also: Sluijter et al ATVB 2010 (mir-1, mir-499); Ivey et al, Cell Stem Cell 2008 (mir-1)) Wilson et al Circ Genetics 2010
11 In vivo cardiac reprogramming In vitro: Cell 2010 JMCC Ieda et al, Cell (3): In vivo: GATA4, MEF2C, TBX5 GATA4, HAND2, MEF2C, TBX5
12 Combination of: mir-1, mir-133, mir-208, mir JAK inhibitor Circ Res 2012
13 micrornas and cell therapy Reprogramming: Induce pluripotency micrornas and stem cells Direct reprogramming: Induce differentiation Functional reprogramming: Improve cell functionality
14 Impairment of EPC and BMC in patients with CAD Risk factors for CAD (Age) Diabetes Heart failure Vasa Circ Res 2001; Tepper Circ 2002; Loomans 2004;Seeger 2005; Kissel JACC 2006, Sorrention Circ 2007 EPC & BMC (cardiac stem cells) Number (EPC) Functions: Migration towards VEGF & SDF-1 Colony forming capacity Vascular network formation Incorporation & Homing Differentiation to cardiac myocytes
15 Genes: Cell enhancement strategies Akt & upstream kinases (ILK) Akt downstream kinases (GSK3ß) VEGF enos E-selectin Small molecules: Statins Targeting micrornas? p38-inhibitors enos enhancer EPAC Sphingosine-1-Phosphate HMGB1 soluble E-selectin PPARg agonists Hormones/Cytokines Growth hormone; IGF Chavakis et al. 2007; Carmona et al. 2009; Walter et al. 2007; Cho et al. 2005; Choi et al. 2004; Iwaguro et al. 2002; Kong et al. 2004; Dimmeler et al. 2001; Walter et al. 2002; Sasaki et al. 2006; Nishiwaki et al 2006; Thum et al. 2007
16 Identification of disease-associated mirnas mir Expression in BMC from healthy donors versus patients with cardiac diseases (N=6 per group) Xu et al, JACC 2012
17 Tzatsos, Cell Stem Cell 2008 Validation of dysregulated mirnas Healthy controls (HC) n=11 Ischemic Heart Failure (ICM) n= 11 Acute myocardial infarction (AMI) n= 8 Dilative Cardiomyopathy (DCM) n=7
18 Cardiac disease regulate expression of mirnas in BMC mir-34a in BMC * p<0.05 vs Healthy Aging mir ±5 % (p<0.05) 15 mir-34a Human hearts Healthy controls (HC) n=11 Ischemic Heart Failure (ICM) n= 11 Acute myocardial infarction (AMI) n= 8 Dilative Cardiomyopathy (DCM) n=7 Relative Expression 10 5 *** AGE
19 mir-34 regulates cell survival and proliferation mir-34: Pro-apoptotic Prevent reprogramming Yamakuchi et al. (2009) Cell Cycle 8,
20 Inhibition of mir-34a inhibits cell death and improves BMC functions mir-34a AntimiRs (LNA-34a): Reduce mir-34a expression Reduce cell death Improve migration
21 Co-miR mir-34a Proliferation Cell death Cyclins (CCND1) SIRT1 Cdk4 mir-34a Bcl2
22 Inhibition of mir-34a improves in vivo functions of BMC Acute myocardial infarction Pre-treatment of BMC with mir-34a AntimiRs (LNA-34a) Fractional shortening Ejection fraction nude mouse BMC + AntimiR-34a for 24 h wash 2X10 6 i.m. v 2 Weeks Recovery
23 Support: Deutsche Forschungsgemeinschaft (SFB553, TR-SFB23) Leducq Foundation: Transatlantik Network of Excellence Excellence Cluster ECCPS European Union: IP Heart Repair; ERC Advanced Grant
24 Klinikum der Johann Wolfgang Goethe Universität Frankfurt am Main Quan-Fu Xu Jessica Castillo Kazuma Iekushi Reinier Boon Andreas Zeiher F. Seeger B. Assmus
25
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