R Ferrari, WJ Remme, M Simoons, M Bertrand, K FOX, On behal

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PERTINENT PERindopril Thrombosis, InflammatioN,
Endothelial dysfunction and neurohormonal
activation Trial
R Ferrari, WJ Remme, M Simoons, M Bertrand, K
FOX, On behalf of the EUROPA investigators.
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• 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)

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Primary endpoint
CV death, MI or cardiac arrest
EUROPA Study Investigators Lancet 2003362782-788
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• The background hypothesis for EUROPA trialwas a
  possible vascular and anti-atheroscleroticeffect
  of perindopril (8 mg/day)
• The PERindopril - Thrombosis, InflammatioN,Endoth
  elial dysfunction and Neurohormonalactivation
  Trial (PERTINENT) is a sub-study ofEUROPA
  designed to test this hypothesis

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Methodology
PERTINENT
Endothelial Function
1. Human Umbilical Vein Endothelial Cells
(HUVECs) wereisolated and incubated for 72 h
with serum from healthyage matched volunteers
(n45) or EUROPA patientsat baseline and after 1
year of treatment with eitherperindopril (n43)
or placebo (n44) 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
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Methodology
PERTINENT
Isolation of human endothelium
Incubated (72 h) with serum from
Healthy subjects
Europa Patients
ecNOSApoptosis
To mimic the effects of circulating blood on
endothelial function
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PERTINENT
Baseline characteristics
PERTINENT
EUROPA
placebo perindopril
placebo perindopril
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ecNOS expression
PERTINENT
Effects of HUVECs incubation with serum from
Controls
CAD PERTINENT patients
1 year
baseline
plt0.01
p ns
10
7.5
5
7.4
9.8
7.6
8.7
7.1
2.5
0
Controlsn 45
Placebon 44
Perindopril n 43
Placebon 44
Perindopriln 43
pcontrols vs baseline p ? perindopril vs ?
placebo
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ecNOS activity
PERTINENT
Effects of HUVECs incubation with serum from
Controls
CAD PERTINENT patients
baseline
1 year
p lt0.01
p lt 0.05
4
3
2
2.5
3.3
3.5
2.4
2.9
1
0
Controlsn 45
Placebon 44
Perindopril n 43
Placebon 44
Perindopril n 43
pcontrols vs baseline p ? perindopril vs ?
placebo
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BAX / Bcl2 Ratio(pro-) / (anti-) apoptosis
PERTINENT
Effects of HUVECs incubation with serum from
Controls
CAD PERTINENT patients
baseline
1 year
plt0.05
p lt 0.01
Bax /Bcl-2 ratio
0.7
0.8
0.4
0.9
Placebon 44
Placebon 44
Perindopril n 43
Perindopriln 43
Controlsn 45
pcontrols vs baseline p ? perindopril vs ?
placebo
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Apoptosis
PERTINENT
Effects of HUVECs incubation with serum from
Controls
CAD PERTINENT patients
pcontrols vs baseline p ? perindopril vs ?
placebo
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Methodology
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
after HPLC separation bradykinin (BK) by
radioimmunoassay after HPLC separation
tumor necrosis factor (TNF)-alpha by ELISA
as all these substances are known to modulate
ecNOS and the rate of endothelial apoptosis
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Angiotensin II
PERTINENT
pcontrols vs baseline p ? perindopril vs ?
placebo
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Bradykinin
PERTINENT
CAD PERTINENT patients
Controls
baseline
1 year
p lt0.05
plt0.01
20
15
Bradykinin (pg/mL)
10
14.8
12.4
18.3
12.3
17.7
5
0
Placebon 44
Perindopriln 43
Placebon 44
Perindopril n 43
Controlsn 45
pcontrols vs baseline p ? perindopril vs ?
placebo
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TNF- ?
PERTINENT
pcontrols vs baseline p ? perindopril vs ?
placebo
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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 (r0.43)
• bradykinin vs. ecNOS activity (r0.45)

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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
ecNOS EXPRESSION
ecNOS ACTIVITY
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Messages
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
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Methodology
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 (n591)or placebo (n566)
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von Willebrand factor
PERTINENT
P ? perindopril vs ? placebo
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PERTINENT
Significant Prognostic Role for vWf
Low (?142 / Unit)
High (gt142 / Unit)
outcome
plt0.01
0
2
3
4
1
Years
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Conclusions
PERTINENT
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
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PERTINENT
Conclusions
These data show that the vascular
andanti-atherosclerotic effects of perindopril
may beimportant at least in partexplaining the
results of EUROPA
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Acknowledgements
The PERTINENT patients and Investigators The
PERTINENT corelabs for the investigations Gussago
(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)