r ferrari, wj remme, m simoons, m bertrand, k fox, on behalf of the europa investigators. a sub...
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R Ferrari, WJ Remme, M Simoons, M Bertrand, K FOX, R Ferrari, WJ Remme, M Simoons, M Bertrand, K FOX,
On behalf of the EUROPA investigators.On behalf of the EUROPA investigators.
A sub study ofA sub study of
PERTINENTPERTINENTPERPERindopril indopril – – TThrombosishrombosis,, IInflammationflammatioNN,, EEndothelial dysfunction andndothelial dysfunction and
neurohormonal activation neurohormonal activation TTrialrial
EUROPA
Randomised 12,218 patients with stable coronaryartery disease (CAD) and a broad range of risk forcardiovascular complications
Showed the benefit of long-term (mean 4.2 years)
ACE-inhibition (perindopril 8 mg/day)
Primary endpoint
% CV death, MI or cardiac arrest% CV death, MI or cardiac arrest
Placebo annual event rate: 2.4%Placebo annual event rate: 2.4%
PerindoprilPerindopril
PlaceboPlacebo
p = 0.0003p = 0.0003RRR: 20%RRR: 20%
YearsYears00
22
44
66
88
1010
1212
1414
00 11 22 33 44 55
n = 12,218n = 12,218
EUROPA Study InvestigatorsEUROPA Study Investigators Lancet 2003;362:782-788 Lancet 2003;362:782-788
The background hypothesis for EUROPA trialwas a possible vascular and anti-atheroscleroticeffect of perindopril (8 mg/day)
The PERindopril - Thrombosis, InflammatioN,Endothelial dysfunction and Neurohormonalactivation Trial (PERTINENT) is a sub-study ofEUROPA designed to test this hypothesis
1. Human Umbilical Vein Endothelial Cells (HUVECs) wereisolated and incubated for 72 h with serum from healthyage matched volunteers (n=45) or EUROPA patientsat baseline and after 1 year of treatment with eitherperindopril (n=43) or placebo (n=44)
2. Measurements:
• protein expression and activity of endothelial nitric
oxide synthase (ecNOS)• ratio between 2 cytosolic proteins: Bcl2 (anti-apoptotic)
and Bax (pro-apoptotic)
• rate of HUVECs apoptosis
Endothelial Function
PERTINENT Methodology
PERTINENT
Healthy subjectsHealthy subjects
IncubatedIncubated (72 h)(72 h) with serum from with serum from
Europa PatientsEuropa Patients
ecNOSecNOSApoptosisApoptosis
To mimic the effects of circulating blood on endothelial functionTo mimic the effects of circulating blood on endothelial function
Isolation of human endotheliumIsolation of human endothelium
Methodology
Age (mean) 61 60 60 60
Male (%) 93 93 85 85
Previous MI (%) 77 65 65 65
Diabetes mellitus (%) 14 7 13 12
SBP (mmHg) 138 139 137 137
DBP (mmHg) 82 81 82 82
Lipid lowering therapy (%) 32 35 57 58
Blockers (%) 61 63 61 62
Calcium channel blockers (%) 39 44 31 32
Baseline characteristicsPERTINENT EUROPA
placebo perindopril placebo perindopril
PERTINENT
Effects of HUVECs incubation with serum from:Effects of HUVECs incubation with serum from:
0
10
2.5
7.5
ecN
OS
exp
ress
ion
(arb
itra
ry u
nit
s/m
g p
rote
in)
Controls
pp<0.01<0.01##
ControlsControlsn = 45n = 45
9.8
CAD PERTINENT patients1 year
p = nsp = ns‡‡
PlaceboPlacebon = 44n = 44
PerindoprilPerindopriln = 43n = 43
7.6 8.7
baseline
PlaceboPlacebon = 44n = 44
PerindoprilPerindopriln = 43n = 43
7.4 7.1
## p=controls vs baseline p=controls vs baseline‡‡ p= p= perindopril vs perindopril vs placebo placebo
ecNOS expression
5
PERTINENT
Controlsn = 45
3.5
p <0.01# p < 0.05 ‡
Placebon = 44
2.5
Perindopriln = 43
2.4
Placebon = 44
2.9
Perindopriln = 43
3.3
1 yearbaseline
# p=controls vs baseline‡ p= perindopril vs placebo
0
4
1
3
ecN
OS
act
ivit
y(p
mo
l/min
/mg
pro
tein
)
Effects of HUVECs incubation with serum from:Effects of HUVECs incubation with serum from:Controls CAD PERTINENT patients
2
PERTINENT ecNOS activity
p < 0.01 p < 0.01 ‡‡
CAD PERTINENT patients
baseline 1 year
0.7
PlaceboPlacebon = 44n = 44
0.9
PlaceboPlacebon = 44n = 44
0.8
PerindoprilPerindopriln = 43n = 43
0.4
PerindoprilPerindopriln = 43n = 43
0.3
Controls
ControlsControlsn = 45n = 45
p<0.05 p<0.05 ##
Bax
/Bcl
-2 r
atio
0
1
0.5
# p=controls vs baseline‡ p= perindopril vs placebo
BAX / Bcl2 Ratio(pro-) / (anti-) apoptosis
Effects of HUVECs incubation with serum fromEffects of HUVECs incubation with serum from
PERTINENT
1.3
Controlsn = 45
p<0.01 #
# p=controls vs baseline‡ p= perindopril vs placebo
p < 0.05 ‡
baseline 1 year
Placebon = 44
7.0
Perindopriln = 43
4.7
Perindopril = 43
6.8
Placebon = 44
7.8
Ap
op
tosi
s(%
)
0
2.5
5.0
10.0
7.5
CAD PERTINENT patientsControlsEffects of HUVECs incubation with serum fromEffects of HUVECs incubation with serum from
Apoptosis PERTINENT
To draw further insights on the mechanisms of action ofperindopril we have also measured in the plasma fromthe same population:
• angiotensin II (Ang II) by radioimmunoassay afterHPLC separation
• bradykinin (BK) by radioimmunoassay after HPLCseparation
• tumor necrosis factor (TNF)-alpha by ELISA
as all these substances are known to modulate ecNOS and the rate of endothelial apoptosis
Methodology PERTINENT
p <0.05 p <0.05 ‡‡
CAD PERTINENT patients
baseline 1 year
Perindopriln = 43
17.1
Placebon = 44
15.8
Placebon = 44
14.4
Perindopriln = 43
12.5
Controls
Controlsn = 45
10.8
p<0.01 p<0.01 ##
# p=controls vs baseline‡ p= perindopril vs placebo
0
5
10
15
20
25
An
gio
ten
sin
II
(pg
/mL
)Angiotensin II PERTINENT
0
10
20
Bra
dyk
inin
(p
g/m
L)
p <0.05 ‡
CAD PERTINENT patients
baseline 1 year
Placebon = 44
Perindopriln = 43
14.812.4
Placebon = 44
Perindopriln = 43
12.3 17.7
Controls
Controlsn = 45
18.3
p<0.01 #
# p=controls vs baseline‡ p= perindopril vs placebo
Bradykinin
5
15
PERTINENT
0
5
10
15
20
25
30
35
40
TN
F-a
(pg
/mL
)
ControlsControlsn = 45n = 45
18.0
p<0.01 p<0.01 ##
Controls
baseline 1 year
p <0.05 p <0.05 ‡‡
PlaceboPlacebon = 44n = 44
PlaceboPlacebon = 44n = 44
PerindoprilPerindopriln = 43n = 43
PerindoprilPerindopriln = 43n = 43
27.127.7 28.9 24.6
CAD PERTINENT patients
# p=controls vs baseline‡ p= perindopril vs placebo
TNF- PERTINENT
Correlations
There was no correlation of any parameter withSBP, DBP nor with any concomitant medications
The only significant correlations observed are:
bradykinin vs. ecNOS expression (r=0.43)
bradykinin vs. ecNOS activity (r=0.45)
PERTINENT
ecNOS activity and expression in HUVECs incubated for 72 h with serum of EUROPApatients receiving perindopril with or without ICATIBANT in the incubation medium
n = 87
7.4
BaselineICATIBANT
Without
Perindopriln = 43
8.7
Perindopriln = 20
7.0
With
ecNOS EXPRESSION ecNOS ACTIVITY
(arb
itra
ry u
nits
/mg
pro
tein
)
0
2.5
5.0
10.0
7.5
0
2.5
5.0
10.0
7.5
(pm
ol/m
in/m
g p
rote
in)
n = 87
Baseline
2.5
ICATIBANT
2.1
Perindopriln = 20
With
Perindopriln = 43
Without
3.3
PERTINENT
Treatment with perindopril for 1 year results in:
Restoration of Angiotensin II/Bradykinin balance
Improvement of ecNOS Activity
Reduction of TNF activation
Reduction of the rate of endothelium apoptosis
Messages PERTINENT
To further investigate the role of perindopril onendothelial function we have measured plasmalevels of von Willebrand factor (vWf), a marker ofendothelial cell damage, both at baseline and after1 year of treatment with either perindopril (n=591)
or placebo (n=566)
Methodology PERTINENT
von Willebrand factor v
Wf
(%/U
nit)
0
20
40
60
80
100
120
140
160
180
200
CAD PERTINENT patients
baseline
Placebon =566
145
Perindopriln = 591
142
1 year
p <0.05 #
Perindopriln = 591
Placebon = 566
135 128
# P = perindopril vs placebo
PERTINENT
Years
Significant Prognostic Role for vWf
outc
ome
outc
ome
0.7
0.8
09
1.0
00 22 33 4411
Low (Low (142% / Unit)142% / Unit)
High (>142% / Unit)High (>142% / Unit)
p<0.01p<0.01
PERTINENT
Conclusions
In CAD patients, treatment with perindopril:
1) increases bradykinin which in turn up-regulates ecNOS activity
2) reduces angiotensin II and TNF levels
3) reduces rate of apoptosis
4) reduces von Willebrand factor levels which are predictive for outcomes
This results in improvement of endothelial dysfunction
PERTINENT
These data show that the vascular and
anti-atherosclerotic effects of perindopril may be
important at least in part
explaining the results of EUROPA
PERTINENT Conclusions
Acknowledgements
The PERTINENT patients and Investigators
The PERTINENT corelabs for the investigationsGussago (Italy) and Birmingham (UK)
The PERTINENT Steering Committee:F Arbustini (Italy), A Blann (UK), D Cokkinos (Greece), C Kluft ( The Netherlands), MPM de Maat (The Netherlands),J Tavazzi (Italy)
The PERTINENT Statistical Committee:A de Carli (Italy), G Parinello (Italy)
The EUROPA Executive Committee:KM FOX (UK), M Bertrand (France), WJ Remme (The Netherlands), ML Simoons (The Netherlands)