R Ferrari, WJ Remme, M Simoons, M Bertrand, K FOX, On behalf of the EUROPA investigators. A sub...

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



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


0.9


0.8


0.4


0.3

Controls


p<0.05 p<0.05 ##

Bax

/Bcl

-2 r

atio

0

1

0.5


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 < 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 ‡‡


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 ##


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 ‡


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 #


Bradykinin

5

15

PERTINENT

0

5

10

15

20

25

30

35

40

TN

F-a

(pg

/mL

)


18.0

p<0.01 p<0.01 ##

Controls

baseline 1 year

p <0.05 p <0.05 ‡‡





27.127.7 28.9 24.6



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


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)

R Ferrari, WJ Remme, M Simoons, M Bertrand, K FOX, On behalf of the EUROPA investigators. A sub...

Documents

Transcript of R Ferrari, WJ Remme, M Simoons, M Bertrand, K FOX, On behalf of the EUROPA investigators. A sub...