Pleiotropic effects of cytokines on left ventricular remodelling and cardiovascular stem cell therapy W. Wojakowski, MD, FESC Silesian School of Medicine, Katowice, Poland

Financial disclousure:  Supported by the grants from Polish Ministry of Science and Higher Education, Servier Research Grant and European Vascular Genomics Network (EVGN)  No conflict of interest

HEMATOPOIETIC AND INFLAMMATORY CYTOKINES  MOBILIZATION OF BONE MARROW-DERIVED  CARDIOGENIC DIFFERENTIATION OF BM- DERIVED CELLS,  DIRECT EFFECTS ON CARDIOMYOCYTES,  MARKERS OF MYOCARDIAL INJURY,  NON-SPECIFIC MARKERS OF TISSUE INJURY

* p< vs. CTRL ** p<0.003 vs. CTRL *** p=ns vs. CTRL * *** * ** * p<0.05 vs. baseline ** p< vs. baseline *** p=ns vs. baseline *** * ** Wojakowski, Circulation 2004;110:3213 Massa, Blood 2005,105:199 Mieno, Circulation 2006;114:186

Wojakowski, Circulation 2004;110:3213

CD34/CXCR4 + [cells/µL] p<0.03 p<0.01 p<0.03 SDF-1 [pg/mL]

Leone et al. Eur Heart J 2005; 26 Correlation between CD34+ cells and LV function at 1 year follow-up

Baseline CD34/CXCR4+ cells:  negatively correlated with LV remodeling: LVEDV (r=-0.31; p<0.03), LVESV (r=-0.34; p<0.04). Baseline CD34/CXCR4+ cells:  negatively correlated with the mean delayed enhancement after 1 year (r=-0.30; p<0.03)  number of segments displaying impaired stress first pass perfusion (r=-0.57; p<0.05) 1-YEAR FOLLOW-UP BY cMRI Wojakowski, AHA 2006

1-YEAR FOLLOW-UP (cMRI) CD34/CXCR4+; R=0.48, p=0.004 CD34/CXCR4+; R=0.51, p=0.002 ACUTE FOLLOW-UP Wojakowski, AHA 2006 Baseline and 12 month SDF-1 levels:  no correlations with LVEF, LV remodeling,  myocardial perfusion  no correlation with cardiopulmonary exercise test

SDF-1 IN PERIINFARCT MYOCYTES AND VESSELSREMOTE MYOCARDIUM Abbott, Circulation 2004,110:3300 Kucia, M. et al. Circ Res 2004;95:1191

Kucia, Circ Res 2004;95:1190 BMMNC chemoattracted to the infarcted myocardium in SDF-1-, HGF- and LIF-dependent manner

Kucia, Circ Res 2004;95:1191

CXCR4 C-Met LIF-R SDF-1 HGF LIF gradient Peripheral Blood Bone Marrow Skeletal Muscle Other Tissues Myocardium Brain Very Small TCSC Number Low Expression of Cardiac/Endothelial Markers Ratajczak, Biol Cell 2005, 97, Wojakowski, Heart 2007

CXCR4 c-Met LIF-R SDF-1 HGF LIF gradient Peripheral Blood Bone Marrow Skeletal Muscle Other Tissues Infarcted Myocardium Increased Number Increased Expression of Cardiac/Endothelial Markers Mobilization Homing/Engraftment Ratajczak, Biol Cell 2005, 97, Wojakowski, Heart 2007

BONE MARROW ASPIRATE ( mL) BMCells TREATMENT NON-SELECTED BM MNC CONTROL STEMI + LVEF≤40% (PRIMARY PCI <12 hrs.) randomization MRI, LV-ANGIO, ECHO, CONTRAST ECHO, CARDIOPULMONARY EXERCISE TEST CD34/CXCR4+ CELLS 6 MO. MRI, LV-ANGIO, ECHO, CONTRAST ECHO, CARDIOPULMONARY EXERCISE TEST IMMUNOMAGNETIC SELECTION 12 MO. ECHO, CLINICAL F-U REGENT TRIAL NCT

IT SEEMS THAT SDF-1 ATTRACTS LOTS OF INTERESTING THINGS… CXCR4

Medium aloneSDF-1 Chemotaxis to SDF-1 FACS SORTER CD45 - CD45 + Bone Marrow Mononuclear Cells CXCR4 + CD45 - CXCR4 + CD45 +

Which cells are attracted by SDF-1 ? Biol Cell 2005, 97,

FSC SSC Sca-1 PE-Cy5 Lineage PE CD45 APC-Cy7 Counts 50.5% 0.16% 0.009% 0.15% Sca-1 + lin - CD45 + Sca-1 + lin - CD45 -

Sca-1 + lin - CD45 + Sca-1 + lin - CD45 - Leukemia 2006,20: Very Small Embryonic Like (VSEL) Stem Cell Hematopoietic Stem Cell

RQ-PCR analysis in Sca-1 + lin - CD45 - cells TCSC markersSca-1 + lin - CD45 + Sca-1 + lin - CD45 - cardiac Nsx2.5/Csx 2.05 ± ± 7.93 * GATA ± ± * endothelium VE-cadherin 1.05 ± ± * Leukemia 2006,20:

unpurified BMMNC Sca-1 + lin - CD45 + Sca-1 + lin - CD45 - Leukemia 2006,20: Oct4 SSEA-1 Nanog AP staining on ES-D3 cell-derived embryonic bodies AP staining on VSEL derived embryonic like bodies

Leukemia 2006, 20,  myosin heavy chain troponin I Kucia, Circ Res 2004;95:1190

Genes down-regulated in VSELs vs. differentiation series:  ION CHANNELS (K+, Ca2+), CONNEXINS  LIFR, c-kit, c-met  CELL MOTILITY,  FIBRONECTIN RECEPTOR COMPLEX,  CARDIAC AND VESSEL DEVELOPMENT Genes with higher expressionin VSELs vs. differentiation series:  CXCR4,  TGFβ SIGNALLING Transcriptional profile of VSEL-derived cardiomyocytes Wojakowski, ACC 2007

Mobilization of HSC and VSEL into peripheral blood after AMI Control Sham control 24h 48h7 days AB Control Sham control 24h 48h7 days * # * # * # * # * vs. control # vs. sham control P < 0.05 Courtesy of MZ Ratajczak

CONTROL 0,08 0,05 0,12 Wojakowski, Circulation 2004;110:3213 STEMI 0,21 0,06 0,32 Massa, Blood 2005,105:199

Fazel, J. Clin. Invest. 2006;116:1865

 SDF-1 / CXCR4  HGF / c-met  SCF / c-kit  LIF / LIFR  VEGF / VEGFR  G-CSF / G-CSFR  GM-CSF / GM-CSFR  M-CSF / M-CSFR  flt3 – FL  Epo - EpoR

G-CSF induced mobilization G-CSF G-CSF and HSC:  proliferation,  differentiation,  survival  G-CSF and non-HSC: cardiomyocytes,  endothelial cells,  neuronal cells

STEMMI Ripa et al.: Circulation 2006,113 Regional systolic wall thickening GCSF vs. placebo, p=NS MRI GCSF vs. placebo, p=NS REVIVAL-2 Zohlnhofer et al.: JAMA 2006,295,1003

Britten, Circulation 2003;108:2212 Migratory capacity of transplanted progenitor cells and infarct remodeling

Honold, ATVB 2006;26: Migratory activity of nonmobilized and G-CSF-mobilized EPC

CELL MOBILIZATION FOR IMPROVEMENT OF LV FUNCTION  IMPROVED CONTRACTILITY IN AREAS REMOTE FROM THE INFARCT AREA  MYOCARDIAL REGENERATION AND PROLIFERATION CONFINED TO INFARCTED AREA  RESPONSIVENESS OF BONE MARROW CELLS TO CHEMOATTRACTANTS DECLINES IN AGE-DEPENDENT FASHION  STANDAD MOBILIZATION AGENTS (G-CSF) IMPAIR THE RESPONSIVENES OF PROGENITOR CELLS TO PIVOTAL CHEMOATTRACTANTS

Dawn, B. et al. Circ Res 2006;98:1098 Combination of cytokines in murine model of reperfused MI G-CSF G-CSF + FL (flt3 ligand) G-CSF+SCF

Dawn, B. et al. Circ Res 2006;98:1098 lin-/Sca-1+/c-kit+ cells from untreated bone marrow and peripheral blood

 improved mobilization  qualitative differences in function of mobilized cells (adhesion molecules)  higher rate of cardiomyocyte proliferation  better LV lunction, reduced remodeling COMBINATION OF CYTOKINES

UPREGULATION OF SDF-1 A BC Leukemia 2004, 18: SDF-1 D Aicher, Circulation 2006;114:2823

 MOBILIZATION OF BM-DERIVED PROGENITORS - INCREASED EXTRAVASATION, HOMING - INCREASED EXPRSSION OF ADHESION MOLECULES  TRANSDIFFERENTIATION INTO CARDIOMYOCYTES(?)  MIGRATION AND PROLIFERATION OF RESIDENT CARDIAC STEM CELLS  PARACRINE EFFECTS OF THE RECRUITED CELLS - ANTIAPOPTOTIC EFFECT - FAVOURABLE CHANGES IN ECM - INCREASED ANGIOGENESIS HEMATOPOIETIC CYTOKINES

LOUISVILLE Mariusz Z. Ratajczak Magdalena Kucia Janina Ratajczak Ewa Zuba-Surma Ryan Reca KATOWICE Michał Tendera Paweł Buszman Marek Król Kasia Maślankiewicz Andrzej Pyrlik Radek Parma Rafał Wyderka Joanna Ciosek Sebastian Dworowy Iwona Mróz SZCZECIN Bogdan Machaliński, Maciek Halasa KRAKÓW Marcin Majka Ania Zebzda BRISTOL Paolo Madeddu Nicole Kraenkel Constanza Emanueli

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