R Ferrari, WJ Remme, M Simoons, M Bertrand, K FOX, On behalf of the EUROPA investigators. A sub study of PERTINENT PER indopril – T hrombosis, I nflammatio N, E ndothelial dysfunction and neurohormonal activation T rial

EUROPA Randomised 12,218 patients with stable coronary artery disease (CAD) and a broad range of risk for cardiovascular 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 Placebo annual event rate: 2.4% Perindopril Placebo p = RRR: 20% Years n = 12,218 EUROPA Study Investigators Lancet 2003;362:

The background hypothesis for EUROPA trial was a possible vascular and anti-atherosclerotic effect of perindopril (8 mg/day) The PERindopril - Thrombosis, InflammatioN, Endothelial dysfunction and Neurohormonal activation Trial (PERTINENT) is a sub-study of EUROPA designed to test this hypothesis

1. Human Umbilical Vein Endothelial Cells (HUVECs) were isolated and incubated for 72 h with serum from healthy age matched volunteers (n=45) or EUROPA patients at baseline and after 1 year of treatment with either perindopril (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 subjects Incubated (72 h) with serum from Europa Patients ecNOS Apoptosis To mimic the effects of circulating blood on endothelial function Isolation of human endothelium Methodology

Age (mean)6160 Male (%)93 85 Previous MI (%)7765 Diabetes mellitus (%) SBP (mmHg) DBP (mmHg) Lipid lowering therapy (%)  Blockers (%) Calcium channel blockers (%) Baseline characteristics PERTINENTEUROPA placebo perindopril PERTINENT

Effects of HUVECs incubation with serum from: ecNOS expression (arbitrary units/mg protein) Controls p<0.01 # Controls n = CAD PERTINENT patients 1 year p = ns ‡ Placebo n = 44 Perindopril n = baseline Placebo n = 44 Perindopril n = # p=controls vs baseline ‡ p=  perindopril vs  placebo ecNOS expression 5 PERTINENT

Controls n = p <0.01 # p < 0.05 ‡ Placebo n = Perindopril n = Placebo n = Perindopril n = year baseline # p=controls vs baseline ‡ p=  perindopril vs  placebo ecNOS activity (pmol/min/mg protein) Effects of HUVECs incubation with serum from: ControlsCAD PERTINENT patients 2 PERTINENT ecNOS activity

p < 0.01 ‡ CAD PERTINENT patients baseline1 year 0.7 Placebo n = Placebo n = Perindopril n = Perindopril n = Controls Controls n = 45 p<0.05 # Bax /Bcl-2 ratio # p=controls vs baseline ‡ p=  perindopril vs  placebo BAX / Bcl2 Ratio (pro-) / (anti-) apoptosis Effects of HUVECs incubation with serum from PERTINENT

1.3 Controls n = 45 p<0.01 # # p=controls vs baseline ‡ p=  perindopril vs  placebo p < 0.05 ‡ baseline 1 year Placebo n = Perindopril n = Perindopril = Placebo n = Apoptosis (%) CAD PERTINENT patients Controls Effects of HUVECs incubation with serum from Apoptosis PERTINENT

To draw further insights on the mechanisms of action of perindopril we have also measured in the plasma from the 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 Methodology PERTINENT

p <0.05 ‡ CAD PERTINENT patients baseline1 year Perindopril n = Placebo n = Placebo n = Perindopril n = Controls Controls n = p<0.01 # # p=controls vs baseline ‡ p=  perindopril vs  placebo Angiotensin II (pg/mL) Angiotensin II PERTINENT

Bradykinin (pg/mL) p <0.05 ‡ CAD PERTINENT patients baseline1 year Placebo n = 44 Perindopril n = Placebo n = 44 Perindopril n = Controls Controls n = p<0.01 # # p=controls vs baseline ‡ p=  perindopril vs  placebo Bradykinin 5 15 PERTINENT

TNF-a (pg/mL) Controls n = p<0.01 # Controls baseline1 year p <0.05 ‡ Placebo n = 44 Perindopril n = CAD PERTINENT patients # p=controls vs baseline ‡ p=  perindopril vs  placebo TNF-  PERTINENT

Correlations There was no correlation of any parameter with SBP, 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 EUROPA patients receiving perindopril with or without ICATIBANT in the incubation medium n = Baseline ICATIBANT Without Perindopril n = Perindopril n = With ecNOS EXPRESSIONecNOS ACTIVITY (arbitrary units/mg protein) (pmol/min/mg protein) n = 87 Baseline 2.5 ICATIBANT 2.1 Perindopril n = 20 With Perindopril n = 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 on endothelial function we have measured plasma levels of von Willebrand factor (vWf), a marker of endothelial cell damage, both at baseline and after 1 year of treatment with either perindopril (n=591) or placebo (n=566) Methodology PERTINENT

von Willebrand factor vWf (%/Unit) CAD PERTINENT patients baseline Placebo n = Perindopril n = year p <0.05 # Perindopril n = 591 Placebo n = # P =  perindopril vs  placebo PERTINENT

Years Significant Prognostic Role for vWf outcome Low (  142% / Unit) High (>142% / Unit) p<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 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)