ORIGINAL RESEARCH ARTICLE

Am J Cardiovasc Drugs 2011; 11 (3): 189-198 1175-3277/11/0003-0189/$49.95/0

2011 Adis Data Information BV. All rights reserved.

ACE Inhibition Modulates Endothelial Apoptosis and Renewal via Endothelial Progenitor Cells in Patients with Acute Coronary Syndromes
` Elisa Cangiano,1 Jlenia Marchesini,1 Gianluca Campo,1 Gloria Francolini,2 Cinzia Fortini,3 Giacomo Carra,3 Matteo Miccoli,1 Claudio Ceconi,1 Luigi Tavazzi 3 and Roberto Ferrari 1,2
1 Department of Cardiology, University of Ferrara, Ferrara, Italy 2 Cardiovascular Research Centre, Salvatore Maugeri Foundation, IRCCS, Ferrara and Lumezzane, Italy 3 GVM Hospitals of Care and Research, Cotignola, Ravenna, Italy

Abstract

Background: The equilibrium between endothelial apoptosis and endothelial renewal is altered in acute coronary syndromes and may be related to differences in the beneficial effects of angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists (angiotensin receptor blockers). Methods: We evaluated the effect of treatment on endothelial function in post-myocardial infarction (MI) patients treated with perindopril (group 2, n = 16) or valsartan (group 3, n = 17) at baseline and after 7, 15, and 30 days and in normal controls (group 1, n = 20). Endothelial apoptosis was determined by cultivating serum samples in vitro with human umbilical vein endothelial cells (HUVECs), while endothelial renewal was assessed by mobilization of CD34+ bone marrow cells. Results: At baseline, post-MI patients had significantly elevated rates of apoptosis (16.6 5.0% and 16.5 8.4% in groups 2 and 3, respectively [both p = 0.01] vs 1.6 0.7% in group 1), which declined in group 2 (10.5 4.4% at 30 days, p = 0.04), but not in group 3. Similar results and trends were found for the Bax/Bcl-2 ratio. CD34+ mobilization was significantly increased in group 2 (3.0 1.0 at baseline to 6.2 1.6 at 15 days, p = 0.03), whereas in group 3 CD34+ mobilization did not change significantly. The findings in group 2 were accompanied by an increase in vascular endothelial growth factor at 15 days, and a reduction in tumor necrosis factor-a and its soluble receptors, versus no change in group 3. Similar findings were observed for angiotensin II and bradykinin. Conclusion: Our results indicate that perindopril, but not valsartan, reduces the proapoptotic effect of serum on the endothelium and increases endothelial renewal in patients with acute coronary syndromes.

Introduction Meta-analysis of multicenter trials of treatment with angiotensin-converting enzyme (ACE) inhibitors indicates a reduction in cardiovascular mortality and myocardial infarction (MI) in patients with coronary artery disease (CAD).[1,2] The same effects have not been confirmed for angiotensin II receptor antagonists (angiotensin receptor blockers [ARBs]).[2,3] The background hypothesis for the beneficial effects of ACE inhibitors is a specific endothelial and vascular protection, which leads to an antiatherosclerotic effect.[4-7] These effects, includ-

ing both reduced endothelial apoptosis and increased endothelial renewal mobilizing endothelial progenitor cells (EPCs) by bone marrow,[8,9] are additional to the antihypertensive effect of ACE inhibitors. It has been suggested that a mismatch between an abnormal rate of apoptosis and reduced endothelial renewal may be involved in the pathogenesis of a number of cardiovascular diseases, including atherosclerosis, restenosis, and heart failure (HF).[10,11] This implies that the equilibrium between these opposing effects may be altered in patients with MI and is relevant to the eventual beneficial effect of ACE inhibitors or ARBs.

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In the present study we evaluated the effect on endothelial function of acute and short-term (1 month) treatment with the ACE inhibitor perindopril or the ARB valsartan in patients with non-ST-segment elevation MI. A control group of 20 healthy subjects was also enrolled.

Materials and Methods

Study Population

We enrolled 20 healthy subjects (normal controls, group 1) and 33 consecutive patients referred to the Coronary Care Unit of the University Hospital of Ferrara (groups 2 and 3) for nonST-segment elevation MI. Group 1 comprised sex- and agematched healthy subjects, none of whom had clinical signs of acute or chronic illness or were receiving any treatment. Inclusion criteria for groups 2 and 3 were typical chest pain lasting for 20 minutes in the last 12 hours, ST-segment depression 0.5 mm and elevation of myocardial necrosis markers (CK-MB and troponin I) above the normal range (5 and 0.15 ng/mL, respectively) in two or more separate blood samples. Exclusion criteria were symptoms lasting >12 hours before hospitalization, presence of any known neoplastic disease, diseases affecting the immune system, ongoing infectious disease, and prior use of ACE inhibitors and ARBs. Of note, patients showing symptoms and signs of heart failure were also excluded. All patients received medical therapy, coronary artery angiography, and, when indicated, percutaneous coronary angioplasty according to current guidelines.[12] At entry, before any intervention, they were randomly allocated to receive perindopril (target dose 10 mg/day, group 2) or valsartan (target dose 160 mg twice daily, group 3). After hospitalization, discharged patients underwent two follow-up visits (after 15 and 30 days, respectively). The study was approved by the local ethics committee, and all participants gave informed consent.
Blood Samples

were collected. One was stored for 1 hour, and centrifuged at 1700 g for 15 minutes at room temperature. The serum obtained was frozen at -80C and was used for subsequent cytokine determination and Western blot analysis. The other aliquot was placed in a prechilled plastic tube containing (final concentrations): 3.3 mM Na2EDTA.2H20 and, as protease inhibitors, 0.3 mM aprotinin, 4.2 mM leupeptin, 0.5 mg/mL perindoprilat, and 1 mM Ro 42-5292. These inhibitors block the degradation of bradykinin and the delivery of angiotensin II by endogenous enzymatic systems after sampling. The blood solution was gently mixed and immediately centrifuged at 4C for 15 minutes at 1700 g. The separated plasma was frozen at -80C until subsequent analysis of bradykinin and angiotensin II.
Human Umbilical Vein Endothelial Cell (HUVEC) Culture

HUVECs were isolated from umbilical cords according to the technique of Jaffe et al.[13] and resuspended in M199 RPMI 1640 1 : 1 containing 20% of pooled human serum and seeded on culture flasks without additional growth factors, as previously described.[4] HUVECs were von Willebrand factor-positive and showed typical cobblestone morphology by microscope observation. As a positive control, some of the cells were treated without serum for 48 hours. Others were incubated for 72 hours with 20% serum from normal controls or from patients at each of the study times (T0, T1, T2, and T3).
Quantitative and Qualitative Assessment of Apoptosis by Flow Cytometry and Fluorescence Microscopy

Blood samples were collected from fasting patients at four time points: at baseline (T0) on entry to the Coronary Care Unit before treatment, and after 7, 15, and 30 days (T1, T2, and T3, respectively). Blood samples were collected in the morning before administration of the daily dose of drug treatment. Blood samples were collected from fasting healthy volunteers at entry (T0) and after 30 days (T3). Blood was drawn from the antecubital vein using a 21-gauge needle and immediately utilized for CD34+ cell assay and for cultivation of human umbilical vein endothelial cells (HUVECs). Two further aliquots
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Apoptotic cells were detected by flow cytometry following a slightly modified method of Nicoletti et al.[14] In brief, endothelial cells were fixed in cold methanol and then incubated with 50 mg/mL propidium iodide (PI) and RNAsi, 0.1% Triton X-100 in sodium citrate 0.1%, pH 7.4. Analysis was performed by means of a Coulter Epics XL-MCL flow cytometer equipped with an argon laser at 488 nm wavelength. A minimum of 10 000 cells were analyzed from each sample. Apoptotic cells were detected on a PI histogram of cells as a hypodiploid peak. Dead cells and debris were excluded by setting an appropriate threshold trigger. Data analysis was performed with Multicycle for Windows Software, Phoenix Flow System, Inc. For qualitative assessment, cells were washed and spun on slides. PIpositive apoptotic cells showing typical nuclear fragmentation and chromatin condensation were viewed using a fluorescence microscope (Nikon Optiphot-2 equipped with mercury lamp excitation [HBO 100 W] and a 510- to 560-nm long-pass filter for PI fluorescence detection).
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Western Blot Analysis

Western blotting was performed by using mouse monoclonal anti-human Fas and Bax (R&D Systems Europe, Ltd, Abingdon, UK, 1 : 500 and 1 : 1000, respectively) and Bcl-2 antibodies (Serotec, Oxford, UK, 1 : 1000) as primary antibodies. To compare baseline with follow-up protein expression, results are expressed as a ratio to the internal control obtained by serum subtraction. Results are also reported as Bax/Bcl-2 ratio.[4,15]
Cytokine Determinations

Vascular endothelial growth factor (VEGF) was measured by R&D Systems Europe, Ltd (Abingdon, UK) solid phase immunoassay ELISA. The minimum detectable dose was <9.0 pg/mL, the intra- and interassay coefficients of variations were <6.7% and <8.8%, respectively. Antigenic tumor necrosis factor-a (TNFa) was determined according to the manufacturers instructions (Biosource TNFa EASIA kit, Biosource Europe, SA, Nivelles, Belgium) by a solid phase enzymeamplified sensitivity immunoassay in which the use of a blend of monoclonal antibodies allowed the measurement of the total circulating TNFa. The sensitivity of the assay was 3 pg/mL. Intra- and interassay coefficients of variation were 5.2% and 9.9%, respectively. TNF-soluble receptors TNFR1 and TNFR2 were assessed according to the manufacturers specifications by ELISA kit Quantikine, available from R&D Systems Europe, Ltd (Abingdon, UK). The minimum detectable dose was <1.2 pg/mL for soluble TNFR1 and <1.0 pg/mL for soluble TNFR2. Intra- and interassay coefficients of variation were 5% and 8.8% for soluble TNFR1 and 2.5% and 5.1% for soluble TNFR2, respectively.
Bradykinin and Angiotensin II Determination

established by running eight aliquots of a single plasma sample containing 20 pg/mL Ang-(18) octapeptide in the same radioimmunoassay. The coefficient of variation (12.5%) for interassay precision was determined by measuring the Ang-(18) octapeptide content of the same plasma on 15 different assays. For bradykinin, the coefficient of variation (9.0%) for intraassay precision was established by running eight aliquots of a single plasma sample containing 20 pg/mL BK-(19) in the same radioimmunoassay. The coefficient of variation (14.1%) for interassay precision was determined by measuring the BK(19) content of the same plasma on 15 different assays. Normal angiotensin II and bradykinin values in our laboratory are in the range of 417.5 pg/mL and 1125 pg/mL, respectively.
CD34+ Cell Quantification

Quantification of peripheral blood CD34+ cells was performed by using double-labeling, with fluorescein isothiocyanate (FITC)-anti-CD45 and phycoerythrin-(PE)-anti-CD34 monoclonal antibodies (Becton Dickinson, Franklin Lakes, NJ, USA) on a FACSCalibur flow cytometer (Becton Dickinson) according to standardized procedures.[18] At least 100 000 CD45+ events were acquired. Gating was based on the ISHAGE guidelines and used the CD34 and CD45 expression patterns as well as their morphologic qualities for detection.[18] Our previous studies showed that CD34+ cells strongly correlate with CD34+AC133+ VEGFR2+ cells.[19,20]
Statistical Analysis

The determination of angiotensin II and bradykinin peptides consisted of a three-step procedure. First, solid-phase extraction was used to recover angiotensin II and bradykinin from plasma according to the method of Hermann et al.[16] modified using Oasis/Waters cartridges. Second, the dry residue was dissolved in 0.3 M acetate buffer, filtered (0.45 mm), and 200 mL used for high-performance liquid chromatography (HPLC) separation according to the procedure of Campbell et al.,[17] modified as previously described.[4] Finally, evaporated specimens were used for peptide quantification by standard radioimmunoassay, using two kits from Peninsula Laboratories Inc. (Belmont, CA, USA). For angiotensin II, the coefficient of variation (7.1%) for intra-assay precision was
2011 Adis Data Information BV. All rights reserved.

Values are expressed as means SD. Comparisons between two groups were performed with Students t-test or the MannWhitney test in the case of nonparametric variables. Continuous variables were tested for normal distribution with the Kolmogorov-Smirnov test. Fishers exact test was used for categoric variables. Comparisons among more than two groups were performed by two-tailed ANOVA and post hoc comparisons by Tukey honest significance difference test. Correlations between variables were tested by Pearson analysis. Probability was significant at a level of p < 0.05. Results Table I reports baseline characteristics and medical therapy of the enrolled population. Patients (n = 33) and normal controls (n = 20) were well matched for sex and age, but differed for cardiovascular risk factors, previous cardiovascular events, and parameters related to the infarct. The age of the patients in groups 2 and 3 was 66 13 years and 22 were male (67%), while
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Table I. Baseline characteristics of the study populationa Characteristic Normal controls group 1 (n = 20) Age (y) Male [n (%)] Cardiovascular risk factors [n (%)] current smoker diabetes hypertension dyslipidemia family history of CAD prior myocardial infarction prior percutaneous coronary intervention prior coronary artery bypass graft Clinical and laboratory data systolic BP (mmHg) diastolic BP (mmHg) left ventricular ejection fraction (%) C-reactive protein (ng/mL) white blood cells (u/mm ) CK-MB at peak (ng/mL) troponin I at peak (ng/mL) Coronary angiography data [n (%)] multivessel disease diseased vessel LAD LCx RCA multivessel PCI CABG Medical therapy before T0 [n (%)] statin ACE inhibitor angiotensin II receptor blocker b-adrenoceptor antagonist Medical therapy at discharge [n (%)] statin ACE inhibitor angiotensin II receptor blocker target dose reached b-adrenoceptor antagonist a Values are mean SD unless otherwise indicated. ACE = angiotensin-converting enzyme; CK-MB= creatine kinase MB fraction; MI = myocardial infarction; CABG = coronary artery bypass graft; CAD = coronary artery disease; LAD = left anterior descending; LCx = left circumflex artery; PCI = percutaneous coronary intervention; RCA = right coronary artery; T0 = baseline; / indicates not relevant; * p 0.05 vs group 1.
2011 Adis Data Information BV. All rights reserved. Am J Cardiovasc Drugs 2011; 11 (3)
3

Post-MI patients group 2 perindopril (n = 16) 66 12 11 (69) group 3 valsartan (n = 17) 66 14 11 (65)

65 14 13 (65)

1 (5%) 0 2 (10%) 1 (5%) 3 (15%) 0 0 0

8 (50)* 4 (25)* 14 (78)* 13 (75)

*

8 (47)* 5 (29)* 14 (82)* 12 (71)* 9 (53)* 2 (12)

8 (50)* 1 (6) 4 (25) 1 (6)

*

5 (29)* 0

145 8 92 6 65 7 0.1 0.4 7.3 2.6 / / 45 8

*

143 9 91 9 46 10* 2.2 0.6* 9.8 4.0 38 10 6.9 2.5

2.1 0.5* 10.1 3.9 35 8 7.3 2.1

8 (50)

8 (47)

/ / / / /

8 (50) 6 (37) 10 (62) 3 (19) 1 (6)

9 (53) 6 (35) 12 (70) 4 (23) 0 (0)

0 0 0 0

9 (56) 0 0 10 (63)

9 (53) 0 0 9 (53)

0 0 0

15 (94) 16 (100) 0 13 (81)

16 (94) 0 17 (100) 13 (76) 13 (76)

12 (75)

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group 1 comprised healthy subjects aged 65 14 years and 13 were male (65%). As expected, several cardiovascular risk factors were present in the treated groups 2 and 3, but were well balanced between them. Necrosis was confirmed in all patients by an increase in CK-MB and troponin I, which were both twice the upper normal limit during hospitalization. Coronary artery angiography was performed in all patients, and all patients subsequently underwent percutaneous coronary intervention, except for one patient with three-vessel disease (in group 3) who underwent coronary artery bypass grafting. Prior to admission, 18 patients were being treated with statins and 19 with b-blockers. After entry, all patients were treated with medical therapy according to current guidelines (table I). Particular care was paid to treat each group with the same type and dosage of statins. BP was reduced by treatment, with no difference between groups 2 and 3 in the values measured at T1, T2, and T3 (126 10/80 10 and 128 10/78 11 mmHg in groups 2 and 3, respectively, at T3).
Occurrence of Apoptosis at Entry

HUVEC Apoptosis and CD34+ Cells Mobilization during Follow-Up

The results for the HUVECs treated with serum subtraction (positive control), serum from normal controls (group 1), or serum from patients before randomization to perindopril (group 2) or valsartan (group 3) are shown in figure 1. The positive control showed an average of 24.4 2.0% apoptotic nuclei. As expected, incubation with serum from post-MI patients resulted in a significant increase in the rate of HUVEC apoptosis, from 1.6 0.7% in group 1 to 16.6 5.0% in group 2 (p = 0.01) and 16.5 8.4% in group 3 (p = 0.01).
* 24 * Apoptotic nuclei (%) 18

Figure 2a shows the variations of apoptosis with time in the three study groups. Incubation of HUVECs with serum from normal controls after 30 days (T3) did not change the percentage of apoptotic nuclei, which remained consistently low. Figure 2a also shows that treatment with perindopril caused a progressive decline in the rate of apoptosis (from 16.6 5.0% at T0 to 10.5 4.4% at T3, p = 0.04). The effect of perindopril on the rate of apoptosis is reflected by the relative reduction in the Bax/Bcl-2 ratio (table II). By contrast, treatment with valsartan did not modify Bax or Bcl-2 concentrations, or the rate of apoptosis, which remained elevated for the entire follow-up. Figure 2b shows results related to CD34+ cell mobilization measured in the same blood samples that were utilized for HUVEC incubation. In normal controls, the value of circulating CD34+ was 2.2 0.8 U/mL and remained constant throughout the observational period (non-significant at T3, p = 0.8). In both treated groups, there was already an increase in CD34+ stem cells at 7 days (T1). However, significant mobilization of CD34+ cells was only observed in perindopril-treated patients (from 3.0 1.0 at baseline to 6.2 1.6 at 15 days, p = 0.03), despite similar BP reduction, and similar dosage and type of statin. In perindopril, the peak of mobilization was observed at 15 days (T2).
Cytokine Values

12

0 Positive controls Group 1 Normal controls Group 2 Post-MI Group 3 Post-MI

Fig. 1. Percentage of apoptotic nuclei in human umbilical vein endothelial cells (HUVECs) incubated with serum subtraction (positive control), serum from normal controls (group 1), and serum from post-myocardial infarction (MI) patients at baseline prior to treatment (groups 2 and 3). * p < 0.01 vs group 1.
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Figure 3 summarizes the cytokine values at baseline and during follow-up. At baseline, all the tested cytokines were higher in patients (groups 2 and 3) than in normal controls (group 1), which remained constant throughout the observational period. At baseline, there were no significant differences between the two treated groups. In group 2 (treated with perindopril), there was a progressive, significant increase in VEGF, reaching a peak at 15 days (T2). In the same group, there was a constant progressive reduction from T0 to T3 in the values of TNFa and its soluble receptors TNFR1 and TNFR2. In group 3, values of all tested cytokines remained unchanged with respect to baseline (T0). The level of VEGF at 15 days (T2) was significantly higher in group 2 than in group 3 (923 314 vs 342 100 pg/mL, p = 0.03). Equally, the values of TNFa and its soluble receptors TNFR1 and TNFR2 were significantly lower in group 2 than in group 3 (18.2 5.8 vs 33 20 pg/mL, p = 0.02; 1167 292 vs 1861 925 pg/mL, p = 0.05; 1839 495 vs 2535 760 pg/mL, p = 0.04; respectively). The same findings were observed for the angiotensin II and bradykinin values (table II). Only in group 2 did we observe a significant modulation of
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Group 1 Group 2 Group 3 a 25

20 Apoptotic nuclei (%)

15

10

0 T0 b 8 7 6 CD34+ cells (u/mL) 5 4 3 2 1 0 T0 T3 T0 T1 T2 T3 T3 T0 T1 T2 T3

Fig. 2. Variations in apoptosis (a) and CD34+ mobilization (b) in normal controls (group 1) at baseline (T0) and after 30 days (T3), and in post-myocardial infarction patients at baseline (T0) and after 7, 15, and 30 days (T1, T2, and T3, respectively) of treatment with perindopril (group 2) or valsartan (group 3). * p < 0.05 vs baseline (T0).

values induced by administration of perindopril (angiotensin II: from 20.3 5.5 at baseline to 16.2 5.1 pg/mL at T2, p = 0.04; bradykinin from 10.7 2.5 at baseline to 7.3 1.4 pg/mL at T3, p = 0.05, respectively).
Correlations

In our experiments, VEGF values were strongly correlated with CD34+ cells (r = 0.82, p < 0.01) and TNFa with the rate of apoptosis (r = 0.65, p < 0.01). No other correlation could be found.

Discussion Our results suggest that ACE inhibition with perindopril exerts a direct positive effect on the vascular endothelium. By
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using a previously validated methodology,[4,10,15] we were able to demonstrate that serum from acute coronary syndrome (ACS) patients displays a proapoptotic effect on HUVECs. This effect is counteracted by ACE inhibition (perindopril), but not by angiotensin II receptor blockade (valsartan). Moreover, in comparison with angiotensin II receptor blockade, ACE inhibition further increases the necrosis-induced CD34+ mobilization. This is in line with results with ACE inhibitor versus ARB in experimental models of ischemia, in which perindopril alone or in combination with the diuretic indapamide, but not ARB, increases the number of circulating EPCs and improves post-ischemic neovascularization and capillary density.[21,22] CD34+ cell mobilization has been associated with both better prognosis and reduced left ventricular remodeling in post-MI patients.[23,24] It follows that by reducing the proapoptotic
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charge of the serum from ACS patients and by enhancing the mobilization of the EPCs from the bone marrow, ACE inhibition is likely to minimize the mismatch between life and death of the endothelium, and this may contribute to beneficial vascular effects of ACE inhibitor therapy in CAD patients.[5,6] Equally, although this was not within the scope of the present study, we can hypothesize that this could explain the anti-

Table II. Plasma concentrations of Bcl-2 associated protein (Bax) and B-cell lymphoma/leukemia 2 (Bcl-2), angiotensin II, and bradykinin in normal controls (group 1) at baseline (T0) and after 30 days (T3), and in postmyocardial infarction (MI) patients at baseline (T0) and after 7, 15, and 30 days (T1, T2, and T3, respectively) of treatment with perindopril (group 2) and valsartan (group 3)a Variable Normal controls group 1 (n = 20) Post-MI patients group 2 perindopril (n = 16) group 3 valsartan (n = 17)

Bax (%) T0 T1 T2 T3 Bcl-2 (%) T0 T1 T2 T3 0.7 0.4 0.6 0.4 11.5 2.2 12.8 2.4 14.2 2.1 13.4 2.1
*

0.5 0.3

26.4 7.6 22.9 5.7 20.0 4.9

29.3 6.8 29.2 6.8 29.4 6.4

*

0.5 0.3

19.7 4.0

29.9 6.4

12.8 2.4 13.1 2.5 13.3 2.4 14 2.4

Bax/Bcl-2 ratio T0 T1 T2 T3 0.8 0.5 0.6 0.4 2.4 0.7 1.9 5.9 1.4 0.4 1.5 0.3* 2.4 0.7 2.3 0.7 2.3 0.6 2.2 0.5

Angiotensin II (pg/mL) T0 T1 T2 T3 6.1 0.6 6.0 0.5 20.3 5.5 18.8 5.7 16.6 5.2 16.7 5.7
*

20.4 6.7 20.7 6.3 20.8 6.7 20.0 6.6

Bradykinin (pg/mL) T0 T1 T2 T3
*

2.8 0.7

10.7 2.5 9.9 1.7 7.9 1.9

10.1 2.1 10.0 2.3 10.0 2.2 10.1 2.1

2.6 0.8

7.3 1.4

a Values are mean SD. p < 0.05 vs baseline (T0).

remodeling effect of ACE inhibitors.[7,25-27] The proapoptotic substances released in serum during ACS are incompletely characterized. More than 30 inducers of apoptosis have been found, including neurohormones, cytokines, and newly discovered genes, thus making it difficult to identify the underlying triggers.[28] On the basis of previous experience,[10] we hypothesized that TNFa plays a crucial role in the biochemistry of apoptotic signaling. This cytokine induces its cytotoxic effect by binding to specific receptors present in almost all cells.[29] TNFR1 has a cytoplasmic signaling motive called the death domain, which induces caspase activation and apoptosis.[30] We found a 2-fold increase in soluble TNFR1, suggesting that circulating TNFa has interacted with this receptor and may have promoted apoptosis, as implied by the correlation found herein. Other agents linked to the renin angiotensin system may also play a major causal role in the death of the endothelium. For instance, angiotensin II, acting through the angiotensin II receptors and Bcl-2 family members, promotes apoptosis, while bradykinin and the Bax family members exert clear antiapoptotic effects.[31] Interestingly, all these proapoptotic substances were significantly increased in our ACS patients versus normal controls, while the antiapoptotic factors were reduced. It is likely that these changes in serum composition played, at least in part, a role in increasing HUVEC apoptosis. This suggestion is supported by our previous data showing that direct ex vivo administration of TNFa and bradykinin to the HUVECs at concentrations in the range of the changes observed in our patients exerted opposing actions on the rate of apoptosis, i.e. enhancement by TNFa and reduction by bradykinin.[4] The observation of an antiapoptotic effect of perindopril is not a new finding. PERTINENT (PERindopril Thrombosis, InflammatioN, Endothelial dysfunction and Neurohormonal activation Trial), a substudy of EUROPA (EUropean trial on Reduction Of cardiac events with Perindopril in stable coronary Artery disease), showed that treatment with perindopril 8 mg/day for 1 year in patients with chronic stable CAD brought about a significant reduction in serum levels of von Willebrand factor, and counteracted the downregulation of endothelial nitric oxide synthase (eNOS) protein expression and the proapoptotic activity of the serum.[4] The results described here constitute the first demonstration of the effect of perindopril on endothelial function during ACSs. The antiapoptotic effect of ACE inhibition with perindopril is confirmed by the increase of the plasma levels of Bcl-2 (antiapoptotic) and the reduction of Bax (proapoptotic). As expected, perindopril also increased the plasma concentration of bradykinin, and reduced that of angiotensin II and TNFa and its receptors, suggesting that perindopril reduces the
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Group 1 Group 2 Group 3 a 1250 * 55 50 1000 VEGF (pg/mL) 45 40 TNF (pg/mL) 750 35 30 25 20 15 250 10 5 0 T0 c 3000 2500 TNFR1 (pg/mL) TNFR2 (pg/mL) 2000 1500 1000 500 0 T0 T3 T0 T1 T2 T3 * 3500 3000 2500 2000 1500 1000 500 0 T0 T3 T0 T1 T2 T3 * T3 T0 T1 T2 T3 d 0 T0 T3 T0 T1 T2 T3 * b

500

Fig. 3. Variations in (a) vascular endothelial growth factor (VEGF), (b) tumor necrosis factor-a (TNFa), and (c and d) its soluble receptors TNFR1 and TNFR2 in normal controls (group 1) at baseline (T0) and after 30 days (T3), and in post-myocardial infarction patients at T0 and after 7, 15, and 30 days (T1, T2, and T3, respectively) of treatment with perindopril (group 2) or valsartan (group 3). * p < 0.05 vs T0.

proapoptotic charge of the serum by modifying the serum concentrations of these agents. This is supported by the relative lack of effects of angiotensin II receptor blockade with valsartan on endothelial apoptosis, since this agent does not modify the serum concentrations of bradykinin, TNFa, angiotensin II, or Bcl-2/Bax ratio. In addition, valsartan blocks the angiotensin I receptor without interfering with the synthesis of either bradykinin or angiotensin II. Indeed, by selectively blocking angiotensin I receptors, valsartan causes a receptorial shift, meaning that angiotensin II can only act at the angiotensin II receptor, which promotes apoptosis. The trigger for bone marrow mobilization of progenitor cells in ACSs is not well known. Surprisingly, the extension of necrosis does not seem to be involved.[31] On the other hand, inflammatory cytokines, particularly VEGF, appear to be linked to the mobilization of stem cells from the bone marrow,[31] and may contribute to the reduction in progenitor cell recruitment at the site of ischemia in these subjects. Recently, it has been suggested
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that bradykinin is important for homing of EPCs as well as endothelial repair, which, despite the sensitivity to bradykinin (kinin B2 receptor signaling), is reduced in progenitor cells of patients with cardiovascular disease.[32] The effect of perindopril on CD34+ cell mobilization is difficult to explain. In a small-scale clinical trial, treatment with ramipril improved both number and functional activity of EPCs in patients with stable CAD.[33] In our patients with ACS, perindopril reduces the levels of TNFa and its receptors, and TNFa has a bone marrow-suppressing activity.[19] It is, however, unlikely that the effects of TNFa on bone marrow would be evident over the short term as in our study. Perindopril also modulated the ACS-associated activation of VEGF, which is a known predictor of CD34+ mobilization. It is likely that perindopril, by increasing bradykinin, favors the activation of specific cytokines such as VEGF, which are in turn responsible for the mobilization of CD34+. Valsartan, on the other hand, is not expected to improve bradykinin concentration, which reAm J Cardiovasc Drugs 2011; 11 (3)

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mained low at 15 and 30 days. As a consequence, VEGF also remained low. In addition, bone marrow-derived CD34+ EPCs have been shown to express the angiotensin II type I receptor.[34] Imanishi et al.[35] showed that angiotensin II exerts detrimental effects on proliferation of CD34+ cells, but the same group suggested that angiotensin II stimulates VEGFR-2 mRNA resulting in VEGF-induced proliferation of EPCs.[36] There are therefore contradictions in the role of angiotensin II on CD34+ cells. We were unable to find any correlations between angiotensin II and CD34+ cells.
Study Limitations

organization or entity in conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

References
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This study has several limitations that should be considered when interpreting the data. Firstly, we used in vitro HUVECs, which are neonatal differentiated endothelial cells, and are only partially representative of the in vivo adult endothelium. Secondly, with the impossibility of measuring endothelial apoptosis in vivo, the effects of treatment with perindopril or valsartan on endothelial apoptosis have been measured ex vivo. Thirdly, a more complete evaluation of endothelial dysfunction may also include measurement of oxidative stress status, but this was not done in this study. Fourthly, we measured CD34+ cells, which, even though they were well related to CD34+AC133+ VEGFR2+ in previous studies,[20] are a heterogeneous cell population. Finally, our sample size was small and the study was single blinded. Future studies, probably also with a different design (e.g. crossover study), are clearly needed to confirm our findings and explain the possible clinical implications. Conclusion Our results provide evidence of several changes in serum from patients with acute coronary syndromes, which promote apoptosis of the endothelium and mobilization of CD34+ cells. Perindopril reduces the proapoptotic effect of the serum and increases the mobilization of bone marrow cells. Valsartan does not share the same properties as perindopril. Acknowledgments
The authors thank Drs Monia Monti, Stefania Gambetti, and Laura Bistrot (Medical Trials Analysis, Ferrara, Italy) for their assistance in the data collecting and laboratory tests. The study was supported by a Regional-University project of Emilia Romagna Area IB Innovative Research grant. Conflicts of interest: Roberto Ferrari and Claudio Ceconi have received fees from Servier for speaking and Roberto Ferrari for serving on the Executive Committee of the EUROPA and PREAMI trials. None of the authors have other relevant affiliations or financial involvement with any
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Correspondence: Prof. Roberto Ferrari, Department of Cardiology, University of Ferrara, Corso Giovecca 203, 44100, Ferrara, Italy. E-mail: fri@unife.it

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Am J Cardiovasc Drugs 2011; 11 (3)