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
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
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
Exosomes Production and release of SF Exosomes SF & mirna Modified from Gyorgy et al, Cell Mol Life Sci 2011; 68:2667-2688
Methods: isolation and characterization of human CPC FACS Analysis Biopsy Atrial Tissue Processing Explants CPC
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: 9.8 10 12 particles/ml CD63
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)
Exosomes effects on cell viability & apoptosis 0.90 Cell viability Apoptosis Dead/Total Cells 0.45 * CM DCM DCM+ Exo 0.90 0.0 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-2013-1305
CPC-CM promotes angiogenesis HUVEC media Basal medium w/o FBS CM DCM DCM+ Exo Submitted CVR-2013-1305
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 12000 Total tube length 6000 * * Apoptotic/Tot cells 1.0 0.8 0.6 0.4 0.2 EXO-F *** * EXO-CPC ** 0 BM EM-FBS Exo-CPC Exo-F 0 0.3 g 30 g 300 g Submitted CVR-2013-1305
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 ) 0-10 -20 *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-2013-1305
mirna content in CPC-exosomes vs fibroblasts-exosomes Relative mirna expression levels by Real-Time PCR Relative quantity (2- Ct) 100 10 Angiogenesis Cytoprotection 0 mir132 mir133a mir146a-3p mir181a mir210 mir323-5p Submitted CVR-2013-1305
Gain & Loss-of-Function: cytoprotection CTRL mir210 Cel-miR-39 CTRL mir210 Cel-miR-39 Ephrin A3 PTP1b 25 20 β-actin % Apoptotic cells 15 10 5 σ σ * * FLASH β-actin 0 σ p<0.05 vs CTRL; *p<0.05 vs cel-mir-39
Gain of Function: angiogenesis Matrigel Assay FBS No FBS mir132 8 # Total tube length (mm) 6 4 2 ** 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
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
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
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
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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)
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
Ultrastructural evidence of exosomes in CPC Submitted CVR-2013-1305