Renin Angiotensin Aldosterone system and ischemia-induced angiogenesis
Bernard I. Levy Cardiovascular Research Centre Lariboisière, Inserm U689
ACEI
Bradykinins
Angiotensinogen Renin
Angiotensin I
Products of dégradation
Ang Convert Enzyme
Angiotensin II
Ac-SDKP
Products of degradation
Angiotensin II AT
1
Vasoconstriction Aldosterone secretion Oxydative Stress Vasopressine secretion Stimulation of sympathetic system Inhibs renin secretion Reabsorbtion of Na and water Vascular proliferation Ventricular Hypertrophy Favors thrombosis Reduce thrombolysis
AT
2
Vasodilatation Antiproliferative Enhances bradykinines Stimulates NO
+ Angiotensin II In vitro
AT1
+
VEGF VEGF-R2 (Otani A, Circ Res, 1998) bFGF (Fischer TA, Am J Physiol, 1997)
+ MMP-9 (Rouet P, J Mol Cell Cardiol, 2000)
Angiotensin II
AT1-R
AT2-R
+ Endothelial and SM cells proliferation SMC proliferation Vascular density
(Nakajima M, PNAS USA, 1995) (Prescott MF, Am J Pathol, 1991) (Munzemaier DH, Hypertension, 1996) (Le Noble FA, Am J Physiol, 1993)
AT1
PKC PLD NADPH Oxidase ROS
Hypertrophy Inflammation Apoptosis
Adhesion Proliferation Matrix remodeling Dzau VJ. 2000
Angiotensin II
AT2-R
AT1-R
?
? VEGF eNOS MMPs
? in vivo angiogenesis
In Vivo matrigel Model Matrigel = basal membrane from Engelbreth-Holm-Swarm tumoral cells MATRIGEL
Groups: Matrigel control, Matrigel + Ang II (10-7 M) Matrigel + Ang II + Candesartan (10-6 M) Matrigel + Ang II + PD123 319 (10-6 M) Quantification of angiogenesis: immuno-histological analysis (scores) Measurement of DNA content (Cyquant) Molecular mechanisms: Western-Blot: VEGF, eNOS Zymography: MMP-2 and MMP-9 activity
Matrigel
Cont (X20)
Score 1: Score 2: Score 3:
Ang II (X20)
No or Few vascular cells Massive cellular infiltration Vascular structures containing RC
Score **
DNA concentration (ng DNA/ml) **
3
40
*** **
30
†
††
2
20 10
1 Cont
AII
AII + Cand
AII + PD
0
Cont
AII
AII + Cand
AII + PD
AII
AII αSMC Actin
Matrigel erythrocytes
(x 4)
(x 40) (x 40)
CD31 (Endo cells)
(x 20)
(x 40)
eNOS
VEGF Cont
400 300
AII
AII + Cand
AII + PD
Cont
AII
AII + Cand
AII + PD
%
%
500
**
**
***
400
**
300 200
†† 100 0
††
200 100
Cont AII
AII + AII + Cand PD
0
Cont
AII
AII + AII + Cand PD
VEGF localization
** Endothelial cells
VEGF
Interface
Matrigel
(Dapi)
Score
DNA concentration (ng DNA/ml)
**
40
3
**
30 2 † ††
†
††
20
† ††
10
1 Cont
AII
AII + AII + a-VEGF L-NAME
AII + eNOS -/-
0
Cont
AII
AII + AII + AII + a-VEGF L-NAME eNOS -/-
Angiotensin II and inflammation Cont
AII
AII + Cand
AII + PD Macrophage (MOMA-2)
COX-2
250
%
AII
200 **
* 150 † 100 0
Cont
AII
AII + Cand
AII + PD
Cont
Score
DNA concentration (ng DNA/ml)
**
40
3
**
30 2 †
†
††
20
†
10
1 Cont
AII
AII + a-VEGF
AII + Nimesulide
0
Cont
AII
AII + AII + a-VEGF Nimesulide
Angiotensin II
AT1-R
+ VEGF eNOS Inflammation
Tamarat et al, Lab Invest, 2002
Angiogenesis in Matrigel
Ischemia-induced angiogenesis Protocol Wistar rats (250-300g, n=7) Surgical ligature of femoral artery, 28 days Control Ang II (5 ng/kg/min) Ang II + Valsartan (20 mg/kg/day) Ang II + neutralizing antibody VEGF (2.5 µg ip twice/week) Quantification of angiogenesis Microangiography Capillary density in skeletal muscle (Anti-fibronectine) Skin blood flow (Laser Doppler) VEGF and NOSe expression Western-Blot
Non Ischemic
Ischemic
Ligature
X-ray source
transducer Digital X-ray
ligatures Barium sulfate
Image recording and analysis
Quantification of vessel density in the delimited zones
Capillaries
Non Ischemic
Ischemic
Angiographic score (Isch.-N.Isch.) 2
**
1.5 1
†
†
0.5 0
Cont
Ang II
Cont
Ang II
Ang II + Val
Ang II + a-VEGF
Capillary density (Isch./N.Isch.) 1.5
**
Cont
1
†
†
0.5
0
Ang II
Cont
Ang II
Ang II Ang II + + Val a-VEGF
II g An al t n l +V Co Va
I+ I g I An EGF I g a-V An
II g An al t n l +V Va Co
I+ I g I An EGF I g a-V An
eNOS (ischemic leg)
VEGF (ischemic leg) (% of control) 200
300 **
250 200
150
** ††
††
**
150
††
100
100 50
50 0
Cont
Ang II Ang II Ang II + + Val a-VEGF
0
Cont Ang II Ang II Ang II + + Val a-VEGF
Conclusion I Angiotensin II
AT1-R
+ VEGF
+
?
+ eNOS
?
Neo-vascularisation
Experiments NOSe+/+ and NOSe -/- mice (n=5 per group) Ligature of the femoral artery, 28 days eNOS+/+ eNOS+/+ + Ang II (0.3 mg/kg/day) eNOS -/eNOS -/- + Ang II (0.3 mg/kg/day)
Quantification of the neo-vascularisation Microangiography Capillary density (Anti-fibronectine) Skin blood flow (Laser Doppler) VEGF and eNOS expression Western-Blot
Angiographic score (Isch./N.Isch.) 1.5 ** 1
*
0.5
0
eNOS+/+ + Ang II
*
eNOS+/+ eNOS+/+ eNOS -/- eNOS -/+ Ang II + Ang II
eNOS -/- + Ang II
VEGF
+
++
+
++
Angiotensin II
AT2-R
AT1-R
+
VEGF eNOS
Neo-vascularisation Tamarat et al, Hypertension, 2002
?
Myocardial infarction in AT1a KO mice - capillary and arteriole densities, - Granulocytes & Macrophages infiltration, - MMP-2 et MMP-9 - Akt & eNOS - TGF-β β, MCP-1, inflammatory cytokines
Toko H et al ATVB 2004;24:664-670
Angiotensin II
AT2-R
AT1-R
+
VEGF eNOS
Neo-vascularisation Tamarat et al, Hypertension, 2002
?
Protocol Male C57BL/6J and Agtr2 -/Y (AT2 KO), n=7 Ligature of the femoral artery, 28 days +/- Ang II (0.5 ng/kg/min) Quantification Microangiography Capillary density Skin blood flow Laser Doppler Molecular mechanisms VEGF, eNOS - Western-Blot Apoptosis: Tunel, Bcl-2, Bax
Angiographic score (Isch/N.Isch) *
1.5
*
*
1
0.5
0
Cont
AT2 KO
Cont
Cont AT2 + AII KO
AT2 KO + AII
Day 3
Cont
AT2KO
D 14
D 21
D 28
Isch
NIsch
AT2 and Apoptosis
Bcl-2/Bax
Cont + Cont AT2KO AT2KO Ang II + Ang II
(Ischemic leg) 5
Bcl-2
††† 4
†††
3
Bax Actine
2 1 0
Cont Cont AT2 AT2 KO + + AII KO AII
Apoptosis 0.2
Cont 100 µm
Cells/mm2 (Isch)
0.15 *
0.1
*
0.05
0
Cont AT2 KO
Cont AT2 + AII KO
AT2 KO + AII
Angiotensin II
AT1-R
AT2-R
+
+
VEGF Apoptosis eNOS Inflammation
+ Silvestre et al, Circ Res 2002
-
Neo-vascularisation
Inactive peptide Angiotensin I
Angiotensin Converting Enzyme
Angiotensin II
+
Bradykinin
+ AT2
AT1
ACE Inhibitor B2-R
Apoptosis
VEGF eNOS Cox-2 Inflammation
Tamarat R et al, Lab Invest 2002 Tamarat R et al, Hypertension 2002 Silvestre JS et al, Circ Res 2001 Silvestre JS et al, Circ Res 2002 Silvestre JS et al, JPET 2002
+ Angiogenesis
+ eNOS
+
