Exosomes secreted by Human Cardiac Progenitors contain mirna with cardioprotective and proangiogenic activities
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1 Exosomes secreted by Human Cardiac Progenitors contain mirna with cardioprotective and proangiogenic activities Elisabetta Cervio, PhD Molecular Cardiology Laboratory Cardiocentro Ticino, Lugano, CH International Society for Cellular Therapy 23 th -26 th April 2014 Paris, France
2 Mechanism of Stem Cell function after homing into damaged heart Stem Cells homing Endothelial and smooth muscle differentiation Cardiomyocytes differentiation Angiogenesis Cardiomyocytes apoptosis Activation of CSCs Cardiomyocite proliferation Adverse remodeling PARACRINE EFFECTS
3 Background & Aims It has been demonstrated that Exosomes secreted by MSC reduce myocardial Ischemia/reperfusion injury Cardiac Progenitor Cells (CPC)-exosomes stimulate migration of endothelial cells We investigated the role of Exosomes in paracrine secretion of human CPC
4 Exosomes Production and release of SF Exosomes SF & mirna Modified from Gyorgy et al, Cell Mol Life Sci 2011; 68:
5 Methods: isolation and characterization of human CPC FACS Analysis Biopsy Atrial Tissue Processing Explants CPC
6 Methods: isolation and characterization of CPC-derived Exosomes 48h Conditioned Medium Centrifuge x3000g Filtration 0,2 µm Exosomes pellet Dinamic light scattering: NanoSight FACS Analysis Western Blot CD81 Particle ranging from: 40nm up to 250nm Modal particle size: 70.8nm Concentration: particles/ml CD63
7 Methods: functional characterization of human CPC-derived exosomes Cell viability HL-1 Neonatal CMC Dead cells Live cells Cell culture Conditioned Medium (CM) 12h 48h 48h Apoptosis Live cells ExoQuick 48h 48h Caspase 3/7 +ve cells Exo DCM + Exo Exo-depleted CM (DCM)
8 Exosomes effects on cell viability & apoptosis 0.90 Cell viability Apoptosis Dead/Total Cells 0.45 * CM DCM DCM+ Exo GM-FBS BM CM DCM DCM+Exo Apoptotic/Total Cells 0.45 ** * 0.0 GM-FBS BM CM DCM DCM+Exo CM DCM DCM+ Exo *p<0.05 and **p<0.01 vs BM # p<0.05 vs DCM Submitted CVR
9 CPC-CM promotes angiogenesis HUVEC media Basal medium w/o FBS CM DCM DCM+ Exo Submitted CVR
10 CPC-exosomes promote angiogenesis and inhibit CMC apoptosis in vitro *p<0.05 vs BM & # p<0.05 vs Exo-F * p<0.05, **p<0.01 & *** p<0.001 vs Exo-F Total tube length 6000 * * Apoptotic/Tot cells EXO-F *** * EXO-CPC ** 0 BM EM-FBS Exo-CPC Exo-F g 30 g 300 g Submitted CVR
11 CPC-exosomes preserve cardiac function after MI Echocardiografic measurements (1Wk) PBS Low-Dose Changes in LVEF (follow-up vs. baseline) 10 * ΔLVEF% (EF week -EF baseline ) *p<0.05 vs PBS -30 PBS Sham Exo-CPC LD Exp-CPC HD Exo-F High-Dose Poster Session I April 24 th from 5:00PM to 6:30PM Poster 125 Barile et al. Submitted CVR
12 mirna content in CPC-exosomes vs fibroblasts-exosomes Relative mirna expression levels by Real-Time PCR Relative quantity (2- Ct) Angiogenesis Cytoprotection 0 mir132 mir133a mir146a-3p mir181a mir210 mir323-5p Submitted CVR
13 Gain & Loss-of-Function: cytoprotection CTRL mir210 Cel-miR-39 CTRL mir210 Cel-miR-39 Ephrin A3 PTP1b β-actin % Apoptotic cells σ σ * * FLASH β-actin 0 σ p<0.05 vs CTRL; *p<0.05 vs cel-mir-39
14 Gain of Function: angiogenesis Matrigel Assay FBS No FBS mir132 8 # Total tube length (mm) ** mir132 CTRL Cel-miR-39 p120rasgap β-actin 0 FBS w/o FBS mir132 **p<0.005 vs w/o FBS & # p<0.01 vs mirna132
15 Gain of function: cytoprotective effects of mirna on CPC 18h hypoxia/12h reoxigenation 18 CTRL Cel-miR-39 ** ** % Apoptotic cells 9 * * mirna132 mirna181 0 **p<0.001 vs CTRL; *p<0.05 vs cel-mir-39
16 Conclusions CPC-derived exosomes are rich in mirna and exert proangiogenic and cardioprotective effects in vitro CPC-derived exosomes preserve cardiac function after myocardial infarction in vivo Compared with Exo-F, Exo-CPC are markedly enriched for cardioprotective and pro-angiogenic mirna In gain- and loss-of-function experiments, forced overexpression of specific mirna leads to cardioprotection against hypoxia/reoxygenation injury Exosome as a cell-free approach may circumvent many of the limitations of cell therapy
17 Aknowledgments Molecular Cardiology Laboratory Victor Babes National Institute of Pathology, Bucharest, Romania Mihaela GHERGHICEANU Laurențiu M. POPESCU Scuola Superiore Sant Anna, Pisa, Italy Vincenzo LIONETTI ICGEB, Trieste, Italy Chiara COLLESI Mauro GIACCA University of Milano-Bicocca, Milano, Italy Prof. Dir. Tiziano Moccetti Claudia ALTOMARE Marcella ROCCHETTI Antonio ZAZA
18 THANK YOU for your attention
19
20 In-Vivo Study Fase I LAD & Exosomes injection Echo Echo & Sacrifice Pool of exoxomes from 4 patients Intramuscular Injection Ligation d0 d2 d7 60 min Injection 10 Rats/dose, total 2 doses + Saline + NHDF-derived exosomes Apoptosis evaluation (blind analysis)
21 Exosomes mirna profile change after hypoxia: hypoxamirs 10 Relative quantity (2- Ct) 5 * 0 mirna132 mirna133a mirna146a-3p mirna181a mirna210-3p mirna323-5p *p<0.05 Normoxia
22 Ultrastructural evidence of exosomes in CPC Submitted CVR
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