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<0.0001 vs. CTRL** p<0.003 vs. CTRL*** p=ns vs. CTRL
*
***
*
**
* p<0.05 vs. baseline** p<0.0001 vs. baseline*** p=ns vs. baseline ***
*
**
Wojakowski, Circulation 2004;110:3213Massa, Blood 2005,105:199Mieno, Circulation 2006;114:186
Wojakowski, Circulation 2004;110:3213
control
PBMNC 24h after infarct
PBMNC 48h after infarct
PBMNC 7 days after infarct
0
10
20
30
40
Nkx
2.5/
Csx
GA
TA
-4
ME
F2
C
Myf
5
Myo
D
Myo
gen
in
VE
-cad
her
in
Vo
n W
ille
nb
ran
d
cardiac markers muscle markers endothelial markers
fold
of
dif
fere
nce
CD34/CXCR4+ [cells/µL]CD34/CXCR4+ [cells/µL]
p<0.03
p<0.01p<0.03
SDF-1 [pg/mL]SDF-1 [pg/mL]
Leone et al. Eur Heart J 2005; 26
Correlation between CD34+ cellCorrelation between CD34+ cellss and LV and LV function function at 1 year follow-upat 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 cMRI1-YEAR FOLLOW-UP BY cMRI1-YEAR FOLLOW-UP BY cMRI1-YEAR FOLLOW-UP BY cMRI
Wojakowski, AHA 2006
1-YEAR FOLLOW-UP (cMRI)
CD34/CXCR4+; R=0.48, p=0.004 CD34/CXCR4+; R=0.48, p=0.004 CD34/CXCR4+; R=0.51, p=0.002 CD34/CXCR4+; R=0.51, p=0.002
ACUTE ACUTE FOLLOW-UPFOLLOW-UP
Wojakowski, AHA 2006
Baseline and 12 month SDF-1 levels:
no correlations with LVEF, LV remodeling,
myocardial perfusionno correlation with cardiopulmonary exercise test
SDF-1 IN PERIINFARCT MYOCYTES AND VESSELS REMOTE MYOCARDIUM
Abbott, Circulation 2004,110:3300Kucia, 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-1HGFLIFgradient
Peripheral Blood Bone MarrowSkeletal Muscle Other Tissues
MyocardiumBrain
Very Small TCSC NumberLow Expression ofCardiac/Endothelial Markers
Ratajczak, Biol Cell 2005, 97, 113-146. Wojakowski, Heart 2007
CXCR4
c-Met
LIF-R
SDF-1HGFLIFgradient
Peripheral Blood Bone MarrowSkeletal MuscleOther Tissues
InfarctedMyocardium
Increased NumberIncreased Expression ofCardiac/Endothelial Markers
MobilizationHoming/Engraftment
Ratajczak, Biol Cell 2005, 97, 113-146. Wojakowski, Heart 2007
BONE MARROW ASPIRATE (100-120 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 TRIALREGENT TRIALNCT 00316381NCT 00316381
IT SEEMS THAT SDF-1 ATTRACTS
LOTS OF INTERESTING
THINGS…
CXCR4
Medium alone SDF-1
Chemotaxis to SDF-1
FACS SORTERFACS SORTER
CD45- CD45+
Bone Marrow Mononuclear Cells
CXCR4+CD45- CXCR4+CD45+
0
80
160
240
320
400
Myf5 MyoD Myogenin
muscle markers
fold
of d
iffer
ence BM MNC control
Input CD45 positive
Input CD45 negative
SDF-1 CD45 positive
SDF-1 CD45 negative0
60
120
180
GFAP Nestin
neural markers
fold
of d
iffer
ence
BM MNC control
Input CD45 positive
Input CD45 negative
SDF-1 CD45 positive
SDF-1 CD45 negative
0
100
200
300
400
Fetoprotein CK19
liver markers
fold
of d
iffer
ence BM MNC control
Input CD45 positive
Input CD45 negative
SDF-1 CD45 positive
SDF-1 CD45 negative
0
60
120
180
240
Nkx2.5/Csx GATA-4 MEF2C VE-cadherin
Cardiac markers endothelialmarkers
fold
of d
iffer
ence
BM MNC control
Input CD45 positive
Input CD45 negative
SDF-1 CD45 positive
SDF-1 CD45 negative
Which cells are attracted by SDF-1 ?
Biol Cell 2005, 97, 113-146
FSC
SS
C
Sca-1 PE-Cy5
Lin
ea
ge
PE
CD45 APC-Cy7
Co
un
ts
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 2006,20:857-869
Very Small Embryonic Like (VSEL) Stem Cell
Hematopoietic Stem Cell
RQ-PCR analysis in Sca-1+lin-CD45- cells
TCSC markers Sca-1+lin-CD45+ Sca-1+lin-CD45-
cardiacNsx2.5/Csx 2.05 ± 0.12 98.63 ± 7.93*
GATA-4 2.74 ± 0.41 268.63 ± 31.42*
endothelium VE-cadherin 1.05 ± 0.38 142.36 ± 12.49*
Leukemia 2006,20:857-869
0
40
80
120
160
200
Oct-4 Nanog Rex-1 Dppa3 Rif1
PSC markers
mR
NA
(fo
ld d
iffe
ren
ce)
unpurified BMMNC
Sca-1+lin-CD45+
Sca-1+lin-CD45-
Leukemia 2006,20:857-869
Oct4
SSEA-1
Nanog
AP staining on ES-D3 cell-derived embryonic bodies
AP staining on VSEL derived embryonic like bodies
Leukemia 2006, 20, 18- 28
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 VSELMobilization of HSC and VSEL i into peripheral blood after AMInto peripheral blood after AMI
0
1
2
3
4
5
No
of
VS
EL
s /
ul o
f b
loo
dControl Sham
control24h 48h 7 days
0
2
4
6
8
10
12
14
16
No
of H
SC
s /
ul o
f bl
ood
A B
Control Shamcontrol
24h 48h 7 days
* #
* #
* #
* #
* vs. control # vs. sham controlP < 0.05
Courtesy of MZ Ratajczak
CONTROL CONTROL
0,08
0,05
0,12
Wojakowski, Circulation 2004;110:3213
STEMI 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-CSFG-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 thickeningGCSF 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;26:2238-2243
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-CSFG-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
0
0.5
1
1.5
2
2.5
3
corn oil CCL4 72h
liver
fold
of
dif
fere
nc
e
0
10
20
30
40
nonirradiated
24h afterirradiation
48h afterirradiation
nonirradiated
24h afterirradiation
48h afterirradiation
BM heart
fold
of
dif
fere
nce
0
50
100
150
200
250
corn oil CCL4 72h
kidney
fold
of
dif
fere
nce
A
B C
Leukemia 2004, 18: 29-40
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. RatajczakMagdalena KuciaJanina RatajczakEwa Zuba-SurmaRyan Reca
KATOWICEMichał Tendera Paweł BuszmanMarek KrólKasia MaślankiewiczAndrzej PyrlikRadek ParmaRafał WyderkaJoanna CiosekSebastian DworowyIwona Mróz
SZCZECINBogdan Machaliński, Maciek Halasa
KRAKÓWMarcin MajkaAnia Zebzda
BRISTOLPaolo MadedduNicole KraenkelConstanza Emanueli
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