ESC Congress 2012
Ang-2 induced pericyte dropout and increase in
vascular permeability is abolished by PDGF-B
transfection
Tilman Ziegler
Medizinische Klinik und Poliklinik I, Klinkum
Großhadern / LMU, München
Disclosure: none
van Meurs et al. Critical Care 2009
Generating a Mouse overexpressing
Ang-2 endotelialcell specific
tTA
VE-Cadherin promotor
Tet-Off tTA
- Dox
pTet
hAng2
Pan-endothelial
human Ang2
hAng2
pTet
tTA responsive promotor
Human angiopoietin 2
+Dox
pTet
hAng-2
Control
Ang-2 on
Experimental Protocol
Groups:
12 wk
18 wk
24 wk
Control
Ang-2 on
Ang-2 on + treatment
•
•
Echocardiography
non invasive blood pressure
measurement (NIBP)
•
•
Invasive haemodynamic Measurement
Histological Analysis
1 10
25 50ng
Norepinephrine stimulation
Pericyte loss and vessel rarification under
Ang-2 overexpression
PECAM-1
NG-2
Merge
PECAM-1
NG-2
Merge
Control
Control
NG-2
PECAM-1
NG-2
*
*
500
*
*
counts / hpf
EC-Ang2-on
EC-Ang2-on
400
300
200
EC-Ang2-off
EC-Ang2-off
100
0
Control
EC-Ang2-on EC-Ang2-off
Vessel permeability following Ang-2
overexpression
fluorescence intensity
[percentage of baseline]
Ang-2 on
Control
250
200
150
*
* * *
* * *
*
*
* *
100
50
Control
EC-Ang2-on
0
0
1
2
time [minutes]
3
Impairment of cardiovascular function
following Ang-2 overexpression
n.s.
*
100
80
60
40
120
100
80
20
0
*
*
*
weeks
24
*
*
Control
EC-Ang2-on
EC-Ang2-off
60
12
Rest 1
10
25
50
Norepinephrine [ng]
20
*
LVEDP [mmHg]
8
Cardiac output [ml/min]
MAP [mmHg]
140
LvDP [mmHg]
120
160
EC-Ang2-on
Control
6
4
2
Control
EC-Ang2-on
EC-Ang2-off
15
10
5
0
0
Rest
Control
EC-Ang2-on
1
10
25
Norepinephrine [ng]
50
Ang-2 induces leftventricular dilation
and myocardial fibrosis
0,60
Control
EC-Ang2-on
LvEDD [cm]
*
0,40
Control
0,20
EC-Ang2-on
EC-Ang2-off
0,00
EC-Ang2-on
12
EC-Ang2-off
Lens
Control
18
weeks
*
10
24
n.s.
40x
HW/BW [mg/g]
10x
8
6
4
2
0
Control
EC-Ang2-on EC-Ang2-off
Both Ang-1 and PDGF transfection reduce
microcirculatory changes in Ang-2 on mice
NG-2
Merge
EC-Ang2-on
PECAM
*
counts / hpf
EC-Ang2-on
+ Ang1
PECAM-1
400
*
NG-2
200
EC-Ang2-on
+ PDGF-B
0
EC-Ang2-on
250
*
150
* * *
* * *
*
*
* *
100
EC-Ang2-on
EC-Ang2-on + Ang-1
EC-Ang2-on + PDGF-B
50
0
0
1
2
time [minutes]
*
EC-Ang2-on +
PDGF-B
*
10
*
8
0,4
0,2
6
4
2
0
0,0
3
EC-Ang2-on +
Ang-1
HW/BW [mg/g]
200
*
0,6
LvEDD [cm]
fluorescence intensity
[percentage of baseline]
*
*
600
EC-Ang2-on
EC-Ang2-on +
Ang1
EC-Ang2-on +
PDGF-B
EC-Ang2-on
EC-Ang2-on + EC-Ang2-on +
Ang1
PDGF-B
increased vascular
permeability
Sepsis
systemic
hypotension
high cardiac
output
Role of Ang-2 in septic patients
Kümpers et al., Crit Care 2009
Sepsis-induced hyperpermeability is
reduced by Ang-2 antibody treatment
**
IL-6 [pg/ml]
1,E+05
160
1,E+04
1,E+03
1,E+02
1,E+01
120
100
80
60
40
Control Ab
20
Ang2 Ab
0
1,E+00
Sham
PECAM-1
* *
* * * *
*
*
* * *
140
fluorescence intensity
[percentage of baseline]
1,E+06
Control Ab
NG-2
Ang2 Ab
0
1
2
time [minutes]
3
Merge
Ang2 Ab
*
400
300
counts / hpf
Control Ab
*
200
100
0
Control Ab
PECAM-1
Ang2 Ab
NG-2
Sepsis-induced hypotension and mortality
is reduced by Ang-2 antibody treatment
120
100%
*
80
survival
MAP [mmHg]
100
*
60
40
50%
Ang2 Ab
20
Control Ab
0%
0
12
Control Ab
hrs
24
Ang2 Ab
0
6
12
24
36 hrs
Sepsis-induced hyperpermeability is
reduced by PDGF-B treatment
fluorescence intensity
[percentage of baseline]
**
1,E+04
1,E+03
1,E+02
1,E+01
1,E+00
Sham
rAAV.PDGF-B
rAAV.LacZ
PECAM-1
NG-2
200
* *
* *
* * *
150
100
50
rAAV.LacZ
rAAV.PDGF-B
0
rAAV.LacZ rAAV.PDGF
rAAV.PDGF-B
0
1
2
time [minutes]
3
Merge
counts / hpf
1,E+05
IL-6 (pg/ml)
250
**
1,E+06
200
180
160
140
120
100
80
60
40
20
0
*
rAAV.LacZ
PECAM-1
rAAV.PDGF-B
NG2
Sepsis-induced hypotension and mortality
is reduced by PDGF-B treatment
100
100%
*
survival
MAP [mmHg]
80
60
40
50%
20
0%
0
12h
rAAV.LacZ
24h
rAAV.PDGF-B
rAAV.PDGF
0h
6h
rAAV . LacZ
12h
24h
36h
rAAV. PDGF-B
Inflammation
Ang-1
Ang-2
Y
EC
Y
100
100
Y
Y
EC
Y
Thank you for your attention!
Med. Klinik I,
Klinikum Großhadern der
LMU
Walter Brendel Centre, LMU
Stefan Dietzel
Ulrich Pohl
Vanessa Pfetsch
Karolina Weinmann
Jan Horstkotte
Rabea Hinkel
Philipp Lange
Franziska Globisch
Teresa Trenkwalder
Georg Stachel
Seungmin Lee
Elena Gottlieb
Qin Di
Cuong Kieu
Elisabeth Raatz
MPI for Biochemistry,
Martinsried
Reinhard Fässler
Ina Rohwedder
Theo.-Kocher
-Institut Bern
Urban Deutsch
Claudia Schwab
IPEK, LMU
Christian Kupatt
Christian Weber
Cardiac overexpression reduces
capillary density without functional
impairment
Control
CM Ang-2 on
350
PECAM-1
NG2
9
8
$
250
HW/BW ratio (mg/g)
10x
Counts per Field
300
$
200
150
$
$
100
50
7
#
6
5
4
3
0,40
0,20
Control
2
CM Ang-2 on
1
0
40x
0,60
*
LvEDD (cm)
Lens
400
Control
Ang2 on
0
CM-Ang2
on
0,00
Control
CM Ang-2 on
Ang-2 on
12
18
24
weeks
Control
140
LvDP (mmHg)
120
MAP (mmHg)
20
160
CM Ang-2 on
90
60
120
100
Control
80
30
LvEDP (mmHg)
150
12
24
weeks
Co ntro l
CM A ng-2 o n
10
5
CM Ang-2 on
60
0
15
0
Baseline
1
10
25
50
Norepinephrine [ng]
B aseline
1
10
20
50
Norepinephrine [ng]