Secretome of apoptotic cells causes cardioprotection and ...
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Wien, 11.01.2012 Secretome of apoptotic cells causes cardioprotection and inhibits ventricular remodeling after acute myocardial infarction Doctoral viva Dr.med.univ. Michael Lichtenauer Supervisor Univ.Doz. Dr.med.univ. Hendrik Jan Ankersmit Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Vienna – Austria Medical University Vienna, Department of Thoracic Surgery - Vienna – Austria
Transcript of Secretome of apoptotic cells causes cardioprotection and ...
Wien, 11.01.2012
Secretome of apoptotic cells causes cardioprotection and inhibits ventricular remodeling after
acute myocardial infarction
Doctoral viva
Dr.med.univ. Michael Lichtenauer
Supervisor Univ.Doz. Dr.med.univ. Hendrik Jan Ankersmit
Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Vienna – Austria
Medical University Vienna, Department of Thoracic Surgery - Vienna – Austria
Wien, 11.01.2012
Can Stem Cells Repair a Damaged Heart? . In Stem Cell Information. Bethesda, MD: National Institutes of
Health, U.S. Department of Health and Human Services <http://stemcells.nih.gov/info/scireport/chapter9>
Background
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The Dying Stem Cell Hypothesis by Anker et al.
up to 25% of all transplanted cells are in the state of apoptosis
apoptotic cells induce transient immunosuppression
Myocardial Infarction
Necrosis
Attraction of immune cells
Secretion of pro-inflammatory cytokines
Amplification of inflammation
IL-1 IL-6 TNF-α
J Am Coll Cardiol. 2005 Nov 15;46(10):1799-802. J Clin Invest. 2001 Oct;108(7):957-62.
Background
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Experimental Design
anesthetized and mechanically ventilated rat
intercostal thoracotomy
ligation of the coronary artery
Stem Cell
x Peripheral Blood
Mononuclear Cells
(PBMC)
Irradiation
&
Induction of
Apoptosis
Cell Culture
for 18-24h
Flow Cytometry Annexin-positivity >70%
Model of
Experimental
AMI
Intramyocardial
Injection Intravenous Injection
Controls ↓
Injection of
Non-irradiated
viable PBMC
Cell Culture
Medium
Sham
Operation
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Results
Histology and Immunohistology 3 days after induction of MI
Medium Control Viable PBMC IV Apoptotic PBMC IV Apoptotic PBMC IM
n=5-6 per group
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Results
Scar Dimension 6 Weeks after Induction of MI
IA-PBMC suspensions of irradiated apoptotic peripheral blood mononuclear cells
Intravenous
Administration
of IA-PBMC
Intramyocardial
Injection of
IA-PBMC
Intravenous Administration
of viable PBMC Control
medium IV
viable PBMC IV
apoptotic
PBMC IV
apoptotic
PBMC IM
0
10
20
30
40
50
60
**
*
**
Are
a o
f fi
bro
sis
aft
er
6 w
ee
ks
% o
f le
ft v
en
tric
le
n=10-12
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Results
Composition of Scar Tissue
IA-PBMC suspensions of irradiated apoptotic peripheral blood mononuclear cells
Intravenous
Administration
of viable PBMC Control
Intravenous
Administration
of IA-PBMC
Intramyocardial
Injection
of IA-PBMC
medium IV
viable PBMC IV
apoptotic
PBMC IV
apoptotic
PBMC IM
0
5
10
15
20
25 ***
***
***
*
Perc
en
tag
e o
f ela
sti
c f
ibre
s
in
fib
roti
c s
car
tissu
e
n=10-12
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Results
Evaluation of Cardiac Function
sham
medium IV
viable PBMC IV
apoptotic
PBMC IV
apoptotic
PBMC IM
6
8
10
12
14
*
****
**
LV
ED
D (
mm
)
sham
medium IV
viable PBMC IV
apoptotic
PBMC IV
apoptotic
PBMC IM
0
2
4
6
8
10
12
14
16
*
****
***
*
LV
ES
D (
mm
)
n=10-12
sham
medium IV
viable PBMC IV
apoptotic
PBMC IV
apoptotic
PBMC IM
0
10
20
30
40
50
60
70
80
90
100
**
**
*
Eje
cti
on
Fra
cti
on
%
sham
medium IV
viable PBMC IV
apoptotic
PBMC IV
apoptotic
PBMC IM
0
10
20
30
40
50
60
**
**
**
Sh
ort
en
ing
Fra
cti
on
%
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Conclusion
Administration of irradiated apoptotic PBMC after myocardial infarction induces …
Reduction of Pro-inflammatory Signals
Increased Homing of CD68+ and c-kit+ Cells
Up-regulation of Pro-angiogenic mediators
Better Recovery of Cardiac Function
Il-1β ↓ IL-6 ↓
Interleukin-8 ↑ MMPs ↑
Favorable Elastin/Collagen Ratio
Ejection Fraction ↑ Shortening Fraction ↑
Dilation ↓
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Experimental Set-up
Suspensions of apoptotic PBMC control
Eur J Clin Invest. 2009 Jun;39(6):445-56. Irradiated cultured apoptotic peripheral blood mononuclear cells regenerate infarcted myocardium.
Centrifugation
Lyophilization
Production of APOSEC
(Cell culture supernatants of apoptotic PBMC)
Venous Blood Withdrawal
Ficoll Cell Separation
Irradiation
Centrifugation
Incubation for 24h
Dialysis
Lyophilization
Lyophilized Cell Culture Supernatant
- Aposec -
PBMC
PBMC peripheral blood mononuclear cells
Cell Pellet (is discarded)
Supernatant
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APOSEC AMI – Small Animal Model
Medium IV
APOSEC IV
Medium IV
APOSEC IV
0
10
20
300,017
Are
a o
f n
ec
ros
is a
fte
r 3
da
ys
% o
f le
ft v
en
tric
le
Results after 72h
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n=5-6 per group
APOSEC AMI – Small Animal Model
APOSEC IV Medium IV
Medium IV
APOSEC IV
0
10
20
30
40
0,0006
Are
a o
f fi
bro
sis
aft
er
6 w
ee
ks
% o
f le
ft v
en
tric
le
Sham
Medium IV
APOSEC IV
20
40
60
80
1000,59
0,0180,048
Eje
cti
on
Fra
cti
on
%
Results after 6 Weeks
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n=10 per group
APOSEC AMI – Large Animal Model
APOSEC Reperfused AMI
Large Animal Model
Results after 24 Hours
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n=5
APOSEC Reperfused AMI
Large Animal Model
Results after 30 Days
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n=7-9 per group
APOSEC Reperfused AMI
Large Animal Model
Results MRI Analysis
Membran Array – Angiogenic Factors
pos Activin A ADAMTS-1 Angiogenin
Angiopoietin-1
Angiopoietin-2
Plasminogen
Amphiregulin
Artemin
pos
TF CXCL16 CD26 EGF EG-VEGF CD105 Endostatin
Endothelin-1
FGF acidic
FGF basic
FGF-4 FGF-7
GDNF GM-CSF HB-EGF HGF IGFBP-1 IGFBP-2 IGFBP-3 IL-1β IL-8 TGF-β1 Leptin MCP-1
MMP-9
NRG1-β1
Pentraxin 3
PD-ECGF
PDGF-AA
PDGF-AB/BB
Persephin CXCL4
CXCL4 Prolactin
MIP-1a MMP-8
Serpin B5
PAI-1 Serpin F1
TIMP-1
TIMP-4
TSP-1 TSP-2 uPA Vasohibin
VEGF VEGF-
C
pos neg
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Analysis of Protein Content
of
APOSEC (Cell culture supernatants of apoptotic PBMC)
APOSEC Mechanism of Action
Cell Culture of human Cardiomyocytes
Cell Starvation Assay
n=3
APOSEC Mechanism of Action
Cell Culture of human Cardiomyocytes – Factor Inhibtion Assay
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APOSEC Mechanism of Action
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Special thanks
Medical University Vienna
Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration
Hendrik Jan Ankersmit Konrad Hoetzenecker Stefan Hacker Andreas Mangold Stefanie Nickl Tina Niederpold Matthias Zimmermann Andreas Mitterbauer Gregor Werba Moritz Rauch
Ludwig Boltzmann Cluster for Cardiovascular Research
Bruno Karl Podesser Wolfgang Dietl Andrea Baumgartner Matthias Hasun Christoph Inci
Department of Dermatology Michael Mildner
Department of Cardiology
Mariann Gyoengyoesi
