Transcript EUROPA
PERTINENT PERindopril – Thrombosis, InflammatioN, Endothelial dysfunction and neurohormonal activation Trial A sub study of R Ferrari, WJ Remme, M Simoons, M Bertrand, K FOX, On behalf of the EUROPA investigators. 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 14 Placebo 12 10 Perindopril 8 6 RRR: 20% p = 0.0003 4 2 0 0 1 2 3 Placebo annual event rate: 2.4% EUROPA Study Investigators Lancet 2003;362:782-788 4 5 Years n = 12,218 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 Methodology PERTINENT Endothelial Function 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 PERTINENT Methodology Isolation of human endothelium Incubated (72 h) with serum from Healthy subjects Europa Patients ecNOS Apoptosis To mimic the effects of circulating blood on endothelial function PERTINENT Baseline characteristics PERTINENT EUROPA placebo perindopril placebo perindopril 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 ecNOS expression PERTINENT Effects of HUVECs incubation with serum from: Controls CAD PERTINENT patients baseline 1 year p = ns‡ p<0.01# (arbitrary units/mg protein) ecNOS expression 10 7.5 5 9.8 7.1 7.6 8.7 2.5 0 # 7.4 Controls n = 45 p=controls vs baseline ‡ p= perindopril vs placebo Placebo Perindopril n = 44 n = 43 Placebo Perindopril n = 44 n = 43 ecNOS activity PERTINENT Effects of HUVECs incubation with serum from: Controls CAD PERTINENT patients baseline (pmol/min/mg protein) ecNOS activity 4 3 2 3.5 2.5 2.4 2.9 3.3 1 0 # p <0.01# 1 year p < 0.05 ‡ Controls n = 45 p=controls vs baseline ‡ p= perindopril vs placebo Placebo Perindopril n = 44 n = 43 Placebo Perindopril n = 44 n = 43 BAX / Bcl2 Ratio (pro-) / (anti-) apoptosis PERTINENT Effects of HUVECs incubation with serum from Controls CAD PERTINENT patients baseline p<0.05 # 1 year p < 0.01 ‡ Bax /Bcl-2 ratio 1 0.5 0.3 0.9 0.8 0.7 0.4 0 Controls n = 45 # p=controls vs baseline ‡ p= perindopril vs placebo Placebo Perindopril n = 44 n = 43 Placebo Perindopril n = 44 n = 43 Apoptosis PERTINENT Effects of HUVECs incubation with serum from Controls 10.0 p<0.01 # CAD PERTINENT patients baseline 1 year p < 0.05 ‡ (%) Apoptosis 7.5 5.0 7.8 2.5 6.8 7.0 4.7 1.3 0 # Controls n = 45 p=controls vs baseline ‡ p= perindopril vs placebo Placebo Perindopril n = 44 = 43 Placebo Perindopril n = 44 n = 43 Methodology 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 Angiotensin II Controls CAD PERTINENT patients baseline 25 1 year p <0.05 ‡ p<0.01 # 20 (pg/mL) Angiotensin II PERTINENT 15 10 5 10.8 15.8 17.1 14.4 12.5 0 Controls n = 45 # p=controls vs baseline ‡ p= perindopril vs placebo Placebo Perindopril n = 44 n = 43 Placebo Perindopril n = 44 n = 43 Bradykinin Controls PERTINENT CAD PERTINENT patients baseline 1 year p <0.05 ‡ p<0.01 # 15 (pg/mL) Bradykinin 20 10 18.3 12.4 14.8 12.3 17.7 5 0 # Controls n = 45 p=controls vs baseline ‡ p= perindopril vs placebo Placebo Perindopril n = 44 n = 43 Placebo Perindopril n = 44 n = 43 TNF- Controls 40 35 PERTINENT CAD PERTINENT patients baseline 1 year p <0.05 ‡ p<0.01 # TNF-a (pg/mL) 30 25 20 27.7 15 10 27.1 28.9 24.6 18.0 5 0 # Controls n = 45 p=controls vs baseline ‡ p= perindopril vs placebo Placebo Perindopril n = 44 n = 43 Placebo Perindopril n = 44 n = 43 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 ecNOS EXPRESSION Baseline ecNOS ACTIVITY ICATIBANT Without With Baseline 10.0 7.5 5.0 7.4 8.7 7.0 2.5 (pmol/min/mg protein) (arbitrary units/mg protein) 10.0 ICATIBANT Without With 7.5 5.0 2.5 2.5 0 n = 87 Perindopril Perindopril n = 43 n = 20 0 n = 87 3.3 2.1 Perindopril Perindopril n = 43 n = 20 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 Methodology 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) von Willebrand factor PERTINENT CAD PERTINENT patients baseline 1 year 200 p <0.05 # 180 160 vWf (%/Unit) 140 120 100 80 145 142 135 128 Placebo n =566 Perindopril n = 591 Placebo n = 566 Perindopril n = 591 60 40 20 0 # P = perindopril vs placebo PERTINENT Significant Prognostic Role for vWf 1.0 Low (142% / Unit) outcome 09 High (>142% / Unit) p<0.01 0.8 0.7 0 1 2 Years 3 4 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 Conclusions 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 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)