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

GOOD THING ABOUT BEING

INTERVENTIONAL CARDIOLOGIST IS TO GET A LOT OF

EXPERIENCE IN HEMATOLOGY

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