- Email: [email protected]
Effect of verapamil, trandolapril and their combination on vascular function and structure in essential hypertensive patients
Atherosclerosis 205 (2009) 214–220
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Effect of verapamil, trandolapril and their combination on vascular function and structure in essential hypertensive patients D. Versari, A. Virdis, L. Ghiadoni, E. Daghini, E. Duranti, S. Masi, A. Magagna, S. Taddei ∗ Department of Internal Medicine, University of Pisa, Italy
a r t i c l e
i n f o
Article history: Received 16 July 2008 Received in revised form 21 October 2008 Accepted 25 November 2008 Available online 3 December 2008 Keywords: Endothelium Endothelial function Nitric oxide Verapamil Trandolapril
a b s t r a c t Aim: The aim of the present study is to evaluate the effect of treatment with verapamil, trandolapril and their combination on peripheral microcirculation vasoreactivity. Methods: Twenty hypertensive patients were randomized to receive oral trandolapril (4 mg oid; TRA) or verapamil (240 mg oid; VER) for 6 months and then the combination of the two drugs for additional 6 months. At baseline, 6 months and 12 months, peripheral microcirculation vasoreactivity was evaluated by forearm blood flow technique (venous plethysmography), as vasodilation to an endotheliumdependent (acetylcholine) and an endothelium-independent stimulus (sodium nitroprusside, SNP); minimal forearm vascular resistances (MFVR) were also evaluated. Results: Blood pressure decreased similarly and progressively in both groups throughout the study period. In VER, 6-month verapamil treatment significantly increased vasodilation to acetylcholine, but not to SNP. The superimposition of trandolapril increased the response to SNP, and less to acetylcholine. In TRA group, 6-month treatment with trandolapril improved the response to SNP, but not to acetylcholine. In this group, the superimposition of verapamil caused a significant improvement in the response to acetylcholine, but not to SNP. At the end of the study, MFVR were significantly reduced in both groups, but to a greater extent in TRA. Conclusion: The present study demonstrates that chronic treatment with verapamil ameliorates endothelial function in the forearm microcirculation of essential hypertensive patients, while trandolapril protects microcirculation from structural alterations. The combination of the two drugs is potentially a powerful tool to counteract hypertension-related microvascular dysfunction and damage. © 2009 Published by Elsevier Ireland Ltd.
1. Introduction Essential hypertension is characterized by impaired endothelium-dependent vasodilation [1–3] as a consequence of an increased vascular generation of reactive oxygen species (ROS), which in turn destroy nitric oxide (NO) [2,3]. It is widely accepted that NO protects the vessel wall against the onset of atherosclerosis and thrombosis, whereas a dysfunctioning endothelium secondary to NO deficiency is an early indicator of atherothrombotic damage and of cardiovascular events [4,5]. Beyond functional vascular abnormalities, hypertension also alters the physical properties of arterial blood vessels [6]. Indeed, microvascular remodeling, hypertrophy and rarefaction have been documented at very early stages of hypertensive disease [6]. These structural alterations are considered as the crucial pathogenic mechanism for the develop-
∗ Corresponding author at: Department of Internal Medicine, University of Pisa, Via Roma, 67, 56100 Pisa, Italy. Tel.: +39 050 992914; fax: +39 050 553407. E-mail address: [email protected] (S. Taddei). 0021-9150/$ – see front matter © 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.atherosclerosis.2008.11.023
ment and progression of the hypertensive process, including the end-organ damage and the occurrence of vascular events [6,7]. For these reasons it is conceivable that, in addition to lowering arterial pressure, the effect of reversing structural abnormalities or restoring NO availability have recently been emphasized as an additional target of antihypertensive therapy. Of importance, the mere normalization of blood pressure fails to restore microvascular function and structure, which is strictly dependent on specific properties of the antihypertensive agent employed [6,8]. Dihydropyridine calcium channel blockers (CCBs) have been demonstrated to be effective in improving endothelium-dependent vasodilation in several vascular districts including peripheral microcirculation [8], a beneficial effect exerted mainly through antioxidant properties leading to the restoration of NO availability [9–11]. However, at the present time, no data are available on the effects of chronic verapamil administration on endothelial function in essential hypertensive patients. Conversely, the role of ACE-inhibitors on endothelial function of essential hypertensive patients is not conclusive. Indeed, although ACE-inhibitors are the most effective antihypertensive drug class in improving endothelium-dependent vasodilation in peripheral [12,13] and coronary macrocirculation
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
[14], their effects on endothelial function in peripheral microcirculation are controversial [15–19]. However, ACE-inhibitors, through the blockade of the angiotensin II-dependent vascular inflammation and smooth muscle cell proliferation and migration, are able to improve vascular remodeling and fibrosis [6]. Considering the different peculiarities of these two drug classes towards the vessel wall, it is conceivable that the association of them might provide optimal protection for the microcirculation in hypertension. Therefore, in the present work we aimed to investigate the effect of the combination of the CCB verapamil and the ACE-inhibitor trandolapril as compared to either of the two drugs alone, on microvascular endothelial function and minimal forearm vascular resistances (MFVR), an integrated index of vascular structural changes, in essential hypertensive patients. 2. Methods 2.1. Patients The study population included 10 normotensive control subjects and 20 matched patients with essential hypertension. Subjects with smoking history (more than 5 cigarettes per day), severe hypercholesterolemia (total cholesterol greater than 6.2 mmol/L), diabetes mellitus, cardiac and/or cerebral ischemic vascular disease, impaired renal function, heart failure, conductance disturbances or other major diseases were excluded. Subjects were defined as normal according to the absence of family history of essential hypertension and to blood pressure (BP) values. Essential hypertensive patients were recruited among the newly diagnosed cases in our out-patient clinic and enrolled if they reported the presence of positive family history of essential hypertension, whenever supine BP (after 10 min of rest) measured with a mercury sphygmomanometer three times at 1-week intervals, was consistently greater than 140 mmHg/90 mmHg. Secondary forms of hypertension were excluded by routine diagnostic procedures. After baseline vascular reactivity evaluation, hypertensive patients were randomized to either oral verapamil 240 mg oid (VER, n = 10) or oral trandolapril 4 mg oid (TRA, n = 10; Fig. 1). After 6 months (step 1) all patients underwent a vascular reactivity evaluation. Then, patients from VER group received trandolapril 4 mg in addition to verapamil, and patients from TRA group received verapamil 240 mg in addition to trandolapril for 6 months. At the end of this second period (step 2), patients underwent a final vascular reactivity evaluation (Fig. 1). At baseline and at step 1 and step 2 anamnesis, physical examination as well as blood sampling were performed. The protocol was approved by the local Ethical Committee and all participants gave their written consent to the study.
215
2.2. Vascular reactivity procedures Vascular reactivity was assessed by the perfused forearm technique, as previously described [2]. Briefly, the brachial artery was cannulated for drug infusion at systemically ineffective rates and for intra-arterial BP and heart rate monitoring. Forearm blood flow (FBF) was measured in both forearms by strain-gauge venous plethysmography. Circulation to the hand was excluded 1 min before FBF measurement by inflating a pediatric cuff around the wrist at suprasystolic blood pressure. Sensitivity and reproducibility of the method in our laboratory have been elsewhere described [20]. Endothelium-dependent forearm vasodilation was estimated by a dose–response curve to intra-arterial acetylcholine (ACH, from 0.15 to 15 g/100 mL forearm tissue/min; 5 min each dose). Endothelium-independent vasodilation was assessed by a dose–response curve to intra-arterial sodium nitroprusside (SNP, from 1 to 4 g/100 mL forearm tissue/min, 5 min each dose). NO availability was assessed by repeating ACH in the presence of intra-arterial NG -monomethyl-l-arginine (L-NMMA, 100 g/100 mL forearm tissue/min), a specific NO-synthase (NOS) inhibitor. Since L-NMMA modifies blood flow, the effect of ACH in the presence of the NO-clamp system, which allows assessment of endothelial agonists in the presence of NOS blockade without changes in blood flow, was also evaluated, as already published [21]. Thus, after 10 min of L-NMMA infusion, SNP was co-infused (Normotensives: 0.4; hypertensives: 0.3 g/100 mL tissue/min for 5 min) to neutralize the L-NMMA-induced vasoconstriction and restore baseline FBF. SNP and L-NMMA were then continued throughout ACH infusion. A 30-min washout was allowed between each dose–response curve, whereas a 60-min period was allowed after L-NMMA. Minimal forearm vascular resistances (MFVR), an integrated index of microvascular structure, were evaluated by calculating the ratio between mean intra-arterial BP and maximal forearm vasodilation induced by 13 min of forearm ischemia (forearm cuff inflated to 300 mmHg) plus 1 min of hand exercise [22]. 2.3. Oxidative stress assessment In all study individuals, at baseline, step 1 and step 2, measurements of malondialdehyde (MDA) and lipoperoxides (LOOH) (colorimetric assay) [23,24] were performed, as index of oxidative stress. Moreover, antioxidant capacity was measured as plasma ferric reducing ability of plasma (FRAP, colorimetric assay) [25]. 2.4. Data analysis Data were analyzed in terms of changes in FBF. Differences between two means were compared by the paired Student’s t test.
Fig. 1. Study design. Patients were randomized to either verapamil (VER, n = 10) or trandolapril (TRA, n = 10) for 6 months, then trandolapril and verapamil were added, respectively for 6 additional months. At baseline, step 1 and step 2 patients were evaluated (physical evaluation, blood samples, vascular reactivity experiments).
216
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
Table 1 Clinical characteristics of patients throughout the study. Controls (n = 10)
Age (years) Sex (M/F) BMI (kg/m2 ) SBP (mmHg) DBP (mmHg) HR (bpm) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) Glycemia (mg/dL) Creatinine (mg/dL)
47.6 ± 7.5 6/4 26.1 ± 2.7 120.5 ± 4.6 79.5 ± 2.9 71.4 ± 3.4 198.8 ± 24.8 49.5 ± 8.2 129.6 ± 18.7 95.4 ± 27.8 82.5 ± 9.3 0.9 ± 0.2
VER group (n = 10)
TRA group (n = 10)
Baseline
Verapamil (6 mo)
Verapamil + trandolapril (12 mo)
Baseline
Trandolapril (6 mo)
Trandolapril + verapamil (12 mo)
45.9 ± 10.4 6/4 27.1 ± 2.6 146.2 ± 11.8 98.2 ± 2.7 72.5 ± 6.3 202.9 ± 25.2 47.1 ± 11.5 136.8 ± 21.5 96.9 ± 50.0 79.1 ± 10.9 0.9 ± 0.1
– – 27.0 ± 2.7 132.4 ± 7.9* 84.8 ± 5.6* 61.6 ± 5.0* 199.8 ± 35.0 46.1 ± 11.0 128.5 ± 19.0 117.8 ± 89.2 82.0 ± 9.2 0.9 ± 0.1
– – 27.0 ± 2.8 124.9 ± 5.9* , † 81.8 ± 2.3* , † 61.0 ± 3.8 208.8 ± 31.0 45.4 ± 13.2 133.9 ± 18.2 150.9 ± 94.7 83.4 ± 12.6 0.8 ± 0.1
46.6 ± 13.2 7/3 26.6 ± 4.4 150.0 ± 12.1 98.8 ± 1.8 69.8 ± 8.3 201.5 ± 34.5 47.8 ± 14.9 133.0 ± 36.0 100.6 ± 28.1 81.3 ± 10.3 0.9 ± 0.1
– – 26.3 ± 4.0 135.8 ± 6.6* 90.1 ± 3.5* 67.0 ± 8.8 203.6 ± 37.4 52.9 ± 14.15 122.2 ± 25.2 134.3 ± 71.8 84.9 ± 8.8 1.0 ± 0.2
– – 26.3 ± 4.0 128.2 ± 5.7* , † 82.0 ± 3.3* , † 62.6 ± 5.2† 207.5 ± 40.9 55.4 ± 17.2 125.6 ± 37.7 125.8 ± 69.7 80.4 ± 9.3 0.9 ± 0.1
BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; VER: verapamil; TRA: trandolapril. * p<0.05 vs. baseline. † p<0.05 vs. step 1.
Responses to ACH and SNP and change in clinical parameter were analyzed by ANOVA for repeated measures, followed by Bonferroni post hoc test. Multiple regression was applied, where specified. Since basal FBF proved to be different in the various experimental steps, data were also analyzed as percent increase or decrease from baseline. To distinguish the endothelium-dependent from the endothelium-independent component of the vasodilatory effects of acetylcholine, the ratio of the area under the dose–response curve to acetylcholine to the area under the dose–response curve to sodium nitroprusside were calculated. Results are expressed as mean ± SD. Differences were considered statistically significant at a value of P < 0.05. Computations for the statistical method were performed with NCSS 2004.
hypertensive patients, no significant difference was seen in lipid profile, blood glucose and creatinine between the VER and the TRA groups at baseline and throughout the treatment period (Table 1). 3.1. Effect of the study drugs on hemodynamics At baseline, BP and heart rate values were similar between VER and TRA groups (Table 1). A significant reduction in heart rate was observed at the first step visit in the VER group and at the step 2 visit in the TRA group, as result of the effect of verapamil therapy onset. BP values decreased similarly and progressively in both groups throughout the study period (Table 1). 3.2. Comparison of endothelium-dependent vasodilation in normotensive subjects and hypertensive patients at baseline
2.5. Drugs ACH (Farmigea S.p.A., Pisa, Italy), NG -monomethyl-l-arginine (Clinalfa AG, Läufelfingen, Switzerland) and SNP (Malesci, Milan, Italy) were obtained from commercially available sources and diluted freshly to the desired concentration by adding normal saline. SNP was dissolved in glucose solution and protected from light by aluminum foil.
In normotensive subjects, the FBF increase induced by ACH was significantly (P < 0.01) blunted by L-NMMA infusion (Table 2). On the contrary, in hypertensive patients, the vasodilation induced by ACH was significantly (P < 0.01) reduced as compared to normotensive subjects and resistant to L-NMMA (Table 2). In contrast, dose-dependent vasodilation to SNP was similar in normotensive subjects and hypertensive patients (Table 2).
3. Results 3.3. Effect of therapy on vascular functional responses Normotensive subjects and patients with essential hypertension showed similar basal systemic demographic, clinical and humoral characteristics, except for BP values (Table 1 and Table 2). Among
Six-month verapamil treatment (step 1) significantly (P < 0.0001) increased the vascular response to ACH (Table 2,
Table 2 Vascular responses in study populations. Absolute forearm blood flow (FBF) values and percent changes after challenges in normotensive subjects and essential hypertensive patients in the VER and TRA groups at the various study time points. Basal FBF
ACH FBF (%)
Basal FBF
L-NNMA + ACH FBF (%)
Basal FBF
SNP FBF (%)
Normal subjects VER baseline VER 6 months
2.4 ± 0.4 2.8 ± 0.2 2.9 ± 0.6
17.7 ± 4.6 (+556%) 12.6 ± 2.6 (+348%)† 16.1 ± 3.7 (+469%)‡
2.7 ± 0.4 2.8 ± 0.2 2.8 ± 0.6
9.6 ± 1.9 (+255%)* 12.2 ± 2.4 (+339%)† 9.3 ± 3.1 (+239%)* , ‡
2.8 ± 0.4 2.8 ± 0.2 2.8 ± 0.5
14.0 ± 2.3 (+396%) 12.0 ± 1.5 (+328%) 12.3 ± 2.5 (+337%)
VER 12 months TRA baseline TRA 6 months
2.8 ± 0.4 2.7 ± 0.5 2.7 ± 0.4
19.4 ± 3.7 (+601%)‡ , o 10.9 ± 2.5 (+313%)† 12.8 ± 2.6 (+370%)
2.8 ± 0.4 2.7 ± 0.4 2.8 ± 0.4
11.2 ± 2.8 (+298%)* , ‡ 10.8 ± 2.7 (+302%)† 12.6 ± 2.9 (+367%)†
2.7 ± 0.4 2.6 ± 0.4 2.7 ± 0.4
15.0 ± 2.2 (+444%)‡ , § 10.5 ± 1.9 (+295%) 13.3 ± 1.7 (+392%)‡
TRA 12 months
2.6 ± 0.4
2.7 ± 0.3
8.9 ± 0.6 (+237%)* , ‡
2.6 ± 0.4
12.7 ± 1.0 (+385%)‡
14.6 ± 1.1 (+461%)‡ , §
FBF is expressed as mL/min/100 g of tissue and responses to challenges are also presented as percent change from basal FBF, in brackets. * p < 0.05 vs. ACH FBF. † p < 0.05 vs. normal subjects. ‡ p < 0.05 vs. baseline. o p = 0.05 vs. 6 months.
§ p < 0.05 vs. 12 months (for exact p values, see text).
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
217
Fig. 2. Endothelium-dependent vasodilation to ACH (A, C) and endothelium-independent vasodilation to SNP (B, D) at baseline, step 1 and step 2 in VER group (A, B) and TRA group (C, D). *, P < 0.05 step 2 vs. baseline; †, P < 0.05 step 1 vs. baseline; ◦ , P < 0.05 step 1 vs. step 2.
Fig. 2A). At this step, L-NMMA significantly blunted the vasodilation to ACH (Table 2). The inhibitory effect of L-NMMA on vasodilation to ACH, while not detected at baseline, was present after verapamil administration and was similar to that seen in normotensive subjects (Fig. 3). On the contrary, the vasodilation to SNP was not affected by verapamil administration (Table 2, Fig. 2B). The superimposition of trandolapril (step 2) further increased the response to ACH (Table 2, Fig. 2A), without affecting the inhibitory effect of L-NMMA (Table 2, Fig. 3). Moreover, the combination of trandolapril significantly increased the vasodilation to SNP (Table 2, Fig. 2B). When the improving effect of trandolapril on the response to ACH was corrected for the response to SNP, no net effect on the response to muscarinic agent was observed (data not shown). In TRA group, at baseline, the vasodilation to ACH was similar to that in VER group and not modified by L-NMMA (Table 2). Six-month treatment with trandolapril (step 1), slightly but not significantly increased the vasodilating response to ACH (Table 2, Fig. 2C), and this partial improvement disappeared when a cor-
Fig. 3. Percent inhibition by L-NMMA on endothelium-dependent vasodilation to ACH in patients from VER and TRA group, throughout the study. *, P < 0.01 vs. baseline and TRA group at step 1.
218
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
Fig. 4. Bars show minimal forearm vascular resistances (MFVR) in hypertensive patients, at baseline and at the end of the study (step 2). Data shown as mean ± SEM. *, P < 0.01 vs. baseline counterpart; †, P < 0.05 vs. VER group at step 2.
rection for the response to SNP was applied. In contrast, vascular response to SNP was significantly increased by trandolapril administration (Table 2, Fig. 2D). Nevertheless, the lack of L-NMMA effect on vasodilation to ACH was confirmed (Table 2, Fig. 3). In this group, the superimposition of verapamil (step 2) caused a significantly further improvement in the response to ACH (Table 2, Fig. 2C) together with a restoration of an inhibitory effect of LNMMA on ACH (Table 2, Fig. 3). At step 2, the inhibition by L-NMMA on vasodilation to ACH was not different between TRA and VER groups (Table 2, Fig. 3). The presence of verapamil did not change the response to SNP as compared to step 1 (Table 2, Fig. 2D). 3.4. Effect of the therapy on vascular structural changes At baseline, MFVR resulted significantly (P < 0.01) higher in hypertensive patients (3.5 ± 0.6 SU) than in controls (1.8 ± 0.2 SU). No differences were observed between VER and TRA groups, at baseline (Fig. 4). In both groups, at step 2 the MFVR were reduced as compared to baseline (2.7 ± 0.3 and 2.1 ± 0.3, respectively), but the reductions were significantly greater in TRA than in VER group (Fig. 4). 3.5. Effect on oxidative stress At baseline, FRAP, MDA and LOOH plasma levels (Table 3) were similar between VER and TRA. Among patients from VER group, 6-month verapamil treatment significantly increased FRAP plasma levels (Table 3). Verapamil administration also slightly decreased MDA and LOOH levels, although the statistical significance was not achieved (Table 3). The superimposition of trandolapril further increased FRAP values, and significantly reduced MDA and LOOH (Table 3).
Fig. 5. Correlation between the percent change in FRAP and the percent change in the inhibitory effect of L-NMMA on ACH.
In TRA group, 6-month trandolapril administration failed to significantly change FRAP, but significantly decreased MDA and LOOH values (Table 2). The superimposition of verapamil determined a significant increment of FRAP, together with a further decrement of MDA and LOOH plasma levels (Table 2). In order to explain which clinical parameters might determine the change in NO availability, we evaluated the correlation between the percent changes in the inhibitory effect of L-NMMA on ACH and the clinical parameters modifications. We included the percent changes from baseline to step 1 and from step 1 to step 2 in both VER and TRA groups, separately. A significant correlation between the change in the inhibitory effect of L-NMMA and the change in FRAP (r = 0.59; p < 0.001) (Fig. 5) and DBP (r = −0.46; p < 0.05) were observed. When a multiple regression analysis was applied, including in the model the changes in oxidative stress, lipid profile, glycemia, blood pressure, creatinine and BMI, FRAP resulted the only variable which significantly explained the change in the inhibitory effect of L-NMMA (r2 = 0.21; p < 0.005). 4. Discussion The present study highlights the effectiveness of the combination of trandolapril and verapamil in ameliorating vascular structural and functional abnormalities associated with hypertension. In particular 6-month treatment with the diphenylalkylamine CCB verapamil is able to improve endothelium-dependent vasodilation in the forearm microcirculation of essential hypertensive patients, while the protective effect of ACE-inhibition on peripheral microcirculatory endothelial function was not significant. In contrast, trandolapril was able to reduce minimal vascular resistances and to increased vascular response to sodium nitroprusside, suggesting vascular structure improvement. These
Table 3 Systemic plasma markers of oxidative stress in patients throughout the study. ER group (n = 10)
FRAP (mmol/L) Maiondialdehyde (mol/L) Lipoperoxides (mol/L) * † ‡
§
TRA group (n = 10)
Baseline
Verapamil (6 mo)
Verapamil + trandolapril (12 mo)
Baseline
Trandolapril (6 mo)
298 ± 62 4.1 ± 2.2 5.7 ± 3.7
426 ± 69* 2.8 ± 1.1* 2.6 ± 1.4
545 ± 67† 2.7 ± 1.6 3.4 ± 2.7
329 ± 47 5.3 ± 1.8 6.4 ± 2.7
350 ± 54 3.3 ± 1.7* 3.2 ± 1.9*
P < 0.05 vs. baseline. P < 0.001 vs. baseline. P < 0.01 vs. baseline. P < 0.05 vs. trandolapril alone.
Trandolapril + verapamil (12 mo) 493 ± 87‡ , § 2.0 ± 0.9‡ 2.5 ± 1.9*
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
differential, and partly additive beneficial effects exerted by these two different compounds support a potential synergistic benefit deriving from the combination of ACE-inhibitors and CCB. Previous works clearly demonstrated the beneficial effect of dihydropyridines nifedipine [9], lacidipine [10] and lercanidipine [11] in improving endothelium-dependent vasodilation, virtually leading to a normalization of endothelial function in the peripheral microcirculation of essential hypertensive patients. Importantly, this effect was achieved through a restoration of NO availability by the oxidative stress scavenging action exerted by these drugs. In the present study we demonstrate for the first time that verapamil is also able to restore the inhibitory effect of L-NMMA on ACH, indicating that the restoration of NO availability by CCBs is not limited to a specific compound but is probably a class effect, thus extending this concept to non-dihydropyridines CCB. Our data are also in line with previous studies in different animal models of hypertension, where the effect of verapamil on vascular reactivity was evaluated. Indeed, in aortic rings from L-NAME-induced hypertensive rats, relaxation to ACH, but not that to SNP was increased by long-term verapamil treatment [26]. Similarly, verapamil was found to improve endothelium-dependent relaxation in aorta from stroke-prone spontaneously hypertensive rats [27]. In the present study we also observed that plasma FRAP levels were decreased by verapamil, a finding underscoring the antioxidant properties of this compound. The cross-talk between the antioxidant effects and the ability to restore the NO pathway by verapamil was evidenced in the present study by the relationship between the changes of FRAP plasma levels and the modifications in the inhibitory effect of L-NMMA on ACH response. Taken together, these findings suggest that the restored NO availability induced by verapamil could be related to its antioxidant activity and address FRAP as a possible indicator of NO availability. The ameliorated NO availability caused by verapamil was accompanied by a significant reduction in MDA, but not in LOOH levels. Indeed, experimental studies clearly showed that these plasma markers of lipid peroxidation might not reliably reflect the oxidative stress level in the artery wall [28,29]. The present results confirm this inconsistency and suggest FRAP as a better indicator of oxidative stress within the vascular wall. Concerning the effect of trandolapril on vascular reactivity, we observed that 6-month treatment with the ACE-inhibitor failed to significantly modify the inhibitory effect of L-NMMA on the muscarinic agonist, suggesting no effect of the drug on NO bioavailability. Despite the observed improvement in the vasodilation to ACH, this was in parallel to an amelioration in the response to the endothelium-independent stimulus SNP as well, and when this parameter was used as correction, no net effect of 6 month trandolapril treatment on the endothelium-dependent response was observed. These negative findings are consistent with previous reports, demonstrating no effect of different ACE-inhibitors on endothelium-dependent vasodilation to ACH or methacholine in the forearm microcirculation of essential hypertensive patients [16–18]. Additionally, in a recent paper [30] ramipril was found to ameliorate forearm post-ischemic hyperemia, a phenomenon largely independent from endothelial function and consistent with our finding of improved vasodilation to SNP. Indeed, the improvement in the vascular response to SNP by trandolapril indicates a direct effect of this compound on vascular smooth muscle cell vasoreactivity, and suggests a vascular structure amelioration. This possibility is supported by results from MFVR measurements. Indeed, at baseline, as expected, MFVR proved to be higher in hypertensive patients as compared to normotensive subjects. At the end of the combination treatment, a significant reduction of MFVR was observed in both study groups, but this result was significantly wider in TRA group which had been treated with trandolapril for
219
1 year. Taken in conjunction, these results demonstrate a regression, although not a normalization, of peripheral vascular structural alterations by trandolapril administration. These findings are in line with a large amount of data showing a high efficacy of ACEinhibitors in inducing a regression of vascular structural changes in the forearm microcirculation [6,17,31], as well as in subcutaneous small arteries [6,19,32,33]. Interestingly, trandolapril did not change FRAP but induced a significant reduction in plasma MDA and LOOH levels, suggesting that these latter markers of lipid oxidation might be associated with the presence of structural changes of the microcirculation. The combination of ACE-inhibitors and CCB has attracted increasing interest in recent years, since from a clinical point of view the two drugs show additive effects in term of blood pressure reduction and lower incidence of side effects [34]. Moreover, although, overall, no differences in terms of cardiovascular events reduction seems to be present among different antihypertensive drugs [35], recent data (ACCOMPLISH trial) suggest that a dihydropyridine CCB plus an ACE-inhibitor may significantly improve prognosis as compared to an ACE-inhibitor plus a diuretic combination, independently from blood pressure reduction [36]. The results of the present study support these evidence from a mechanistic point of view, since the combination of verapamil and trandolapril, as compared to single drugs, was associated with a better microvascular function and structure, as well as with a wider antioxidant capacity. In conclusions, the present study represents the first demonstration that chronic treatment with verapamil ameliorates endothelial function by restoring NO availability together with an antioxidant effect in the forearm microcirculation of essential hypertensive patients. In addition, our study also demonstrates a protective role of trandolapril toward the vascular wall, an effect mainly related to structural vascular changes. Finally, our results highlight a synergistic effect of verapamil and trandolapril in improving microvascular vasodilating function and systemic oxidative stress. The present findings can have important clinical implications. Indeed, if considering the antiatherogenic properties of NO and that a dysfunctioning endothelium promotes the atherosclerotic process, the beneficial effect of verapamil on NO availability would help to prevent the development of atherosclerosis. This hypothesis is supported by the results from the Verapamil in Hypertension and Atherosclerosis Study (VHAS), showing the effectiveness of verapamil in promoting regression of thicker carotid lesions in hypertensive patients [37]. Recently, Rizzoni et al [7] demonstrated that media to lumen ratio of subcutaneous small arteries, an index of structural alteration in the microcirculation, is a potent predictor of cardiovascular events in different groups of patients including hypertensive patients. Therefore, our results strongly support the potential beneficial effect of trandolapril mediated by the protection on microcirculatory structural improvement. However, in a recent clinical trial (INVEST), the verapamil–trandolapril-based strategy was as clinically effective as the atenolol-hydrochlorothiazide-based strategy in hypertensive patients with coronary artery disease [38]. This apparent discrepancy might be likely due to the fact that this trial was performed in high risk patients, characterized by an irreversible late stage atherosclerotic process. In contrast, an amelioration of vascular function, and of NO availability in particular, might play a more important role in the early phases of the disease. In this view, when considering the differential beneficial effect of verapamil and trandolapril toward vasculature in the present study, it might be speculated that verapamil–trandolapril is a desirable combination to maintain vascular wall health. Additionally, in patients with stable coronary artery disease of the INVEST, atenolol and verapamil were similarly effective in controlling blood pressure and preventing cardiovascular events but with verapamil having
220
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
a higher tolerability and tending to reduce more effectively angina and stroke [39]. In the future, we will need clinical trials to investigate the impact of this drug combination on long-term prognosis in low-risk populations. Without such information, at the present time, whether the synergistic beneficial effect on vascular function and structure might lead to a better prognosis, remains an attractive hypothesis. Conflicts of interest No conflict of interest exists. References [1] Taddei S, Virdis A, Mattei P, et al. Effect of insulin on acetylcholine-induced vasodilation in normotensive subjects and patients with essential hypertension. Circulation 1995;92:2911–8. [2] Taddei S, Virdis A, Ghiadoni L, Magagna A, Salvetti A. Vitamin C improves endothelium-dependent vasodilation by restoring nitric oxide activity in essential hypertension. Circulation 1998;97:2222–9. [3] Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation 1993;87:1468–74. [4] Lerman A, Zeiher AM. Endothelial function: cardiac events. Circulation 2005;111:363–8. [5] Taddei S, Salvetti A. Endothelial dysfunction in essential hypertension: clinical implications. J Hypertens 2002;20:1671–4. [6] Schiffrin EL. Remodeling of resistance arteries in essential hypertension and effects of antihypertensive treatment. Am J Hypertens 2004;17:1192–200. [7] Rizzoni D, Porteri E, Boari GE, et al. Prognostic significance of small-artery structure in hypertension. Circulation 2003;108:2230–5. [8] Taddei S, Virdis A, Ghiadoni L, Sudano I, Salvetti A. Effects of antihypertensive drugs on endothelial dysfunction: clinical implications. Drugs 2002;62:265–84. [9] Taddei S, Virdis A, Ghiadoni L, et al. Restoration of nitric oxide availability after calcium antagonist treatment in essential hypertension. Hypertension 2001;37:943–8. [10] Taddei S, Virdis A, Ghiadoni L, et al. Lacidipine restores endothelium-dependent vasodilation in essential hypertensive patients. Hypertension 1997;30: 1606–12. [11] Taddei S, Virdis A, Ghiadoni L, et al. Calcium antagonist treatment by lercanidipine prevents hyperpolarization in essential hypertension. Hypertension 2003;41:950–5. [12] Ghiadoni L, Versari D, Magagna A, et al. Ramipril dose-dependently increases nitric oxide availability in the radial artery of essential hypertension patients. J Hypertens 2007;25:361–6. [13] Ghiadoni L, Magagna A, Versari D, et al. Different effect of antihypertensive drugs on conduit artery endothelial function. Hypertension 2003;41:1281–6. [14] Antony I, Lerebours G, Nitenberg A. Restoration of normal coronary vasomotricity after intravenous infusion of angiotensin converting-enzyme inhibitor (perindoprilat) in hypertensive patients. Arch Mal Coeur Vaiss 1995;88:1125–8. [15] Schiffrin EL, Deng LY. Comparison of effects of angiotensin I-converting enzyme inhibition and beta-blockade for 2 years on function of small arteries from hypertensive patients. Hypertension 1995;25:699–703. [16] Creager MA, Roddy MA. Effect of captopril and enalapril on endothelial function in hypertensive patients. Hypertension 1994;24:499–505. [17] Taddei S, Virdis A, Ghiadoni L, Mattei P, Salvetti A. Effects of angiotensin converting enzyme inhibition on endothelium-dependent vasodilatation in essential hypertensive patients. J Hypertens 1998;16:447–56. [18] Kiowski W, Linder L, Nuesch R, Martina B. Effects of cilazapril on vascular structure and function in essential hypertension. Hypertension 1996;27:371–6. [19] Rizzoni D, Muiesan ML, Porteri E, et al. Effects of long-term antihypertensive treatment with lisinopril on resistance arteries in hypertensive patients with left ventricular hypertrophy. J Hypertens 1997;15:197–204. [20] Pedrinelli R, Taddei S, Graziadei L, Salvetti A. Vascular responses to ouabain and norepinephrine in low and normal renin hypertension. Hypertension 1986;8:786–92.
[21] Virdis A, Pinto S, Versari D, et al. Effect of oral contraceptives on endothelial function in the peripheral microcirculation of healthy women. J Hypertens 2003;21:2275–80. [22] Pedrinelli R, Spessot M, Salvetti A. Reactive hyperemia during short-term blood flow and pressure changes in the hypertensive forearm. J Hypertens 1990;8:467–71. [23] Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol 1990;186:407–21. [24] Jiang ZY, Hunt JV, Wolff SP. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal Biochem 1992;202:384–9. [25] Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem 1996;239: 70–6. [26] Kung CF, Moreau P, Takase H, Luscher TF. L-NAME hypertension alters endothelial and smooth muscle function in rat aorta. Prevention by trandolapril and verapamil. Hypertension 1995;26:744–51. [27] Novosel D, Lang MG, Noll G, Luscher TF. Endothelial dysfunction in aorta of the spontaneously hypertensive, stroke-prone rat: effects of therapy with verapamil and trandolapril alone and in combination. J Cardiovasc Pharmacol 1994;24:979–85. [28] Versari D, Daghini E, Rodriguez-Porcel M, et al. Chronic antioxidant supplementation impairs coronary endothelial function and myocardial perfusion in normal pigs. Hypertension 2006;47:475–81. [29] Keaney Jr JF, Gaziano JM, Xu A, et al. Low-dose alpha-tocopherol improves and high-dose alpha-tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J Clin Invest 1994;93:844–51. [30] Brili S, Tousoulis D, Antoniades C, et al. Effects of ramipril on endothelial function and the expression of proinflammatory cytokines and adhesion molecules in young normotensive subjects with successfully repaired coarctation of aorta: a randomized cross-over study. J Am Coll Cardiol 2008;51: 742–9. [31] Virdis A, Ghiadoni L, Lucarini A, et al. Presence of cardiovascular structural changes in essential hypertensive patients with coronary microvascular disease and effects of long-term treatment. Am J Hypertens 1996;9:361–9. [32] Schiffrin EL, Deng LY, Larochelle P. Progressive improvement in the structure of resistance arteries of hypertensive patients after 2 years of treatment with an angiotensin I-converting enzyme inhibitor. Comparison with effects of a beta-blocker. Am J Hypertens 1995;8:229–36. [33] Rizzoni D, Porteri E, De Ciuceis C, et al. Effect of treatment with candesartan or enalapril on subcutaneous small artery structure in hypertensive patients with noninsulin-dependent diabetes mellitus. Hypertension 2005;45:659– 65. [34] Kirchengast M. Preclinical considerations and results with the combination of verapamil and trandolapril: blood pressure reduction and beyond. J Hypertens Suppl 1997;15:S27–33. [35] Turnbull F, Neal B, Algert C, et al. Effects of different blood pressure-lowering regimens on major cardiovascular events in individuals with and without diabetes mellitus: results of prospectively designed overviews of randomized trials. Arch Intern Med 2005;165:1410–9. [36] Jamerson KA. Avoiding cardiovascular events in combination therapy in patients living with systolic hypertension. In: Paper presented at: American College of Cardiology 57th Annual Scientific Session. 2008. [37] Zanchetti A, Rosei EA, Dal Palu C, et al. The Verapamil in Hypertension and Atherosclerosis Study (VHAS): results of long-term randomized treatment with either verapamil or chlorthalidone on carotid intima-media thickness. J Hypertens 1998;16:1667–76. [38] Pepine CJ, Handberg EM, Cooper-DeHoff RM, et al. A calcium antagonist vs a non-calcium antagonist hypertension treatment strategy for patients with coronary artery disease The International Verapamil-Trandolapril Study (INVEST): a randomized controlled trial. JAMA 2003;290:2805–16. [39] Bangalore S, Messerli FH, Cohen JD, et al. Verapamil-sustained release-based treatment strategy is equivalent to atenolol-based treatment strategy at reducing cardiovascular events in patients with prior myocardial infarction: an INternational VErapamil SR-Trandolapril (INVEST) substudy. Am Heart J 2008;156:241–7.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Effect of verapamil, trandolapril and their combination on vascular function and structure in essential hypertensive patients D. Versari, A. Virdis, L. Ghiadoni, E. Daghini, E. Duranti, S. Masi, A. Magagna, S. Taddei ∗ Department of Internal Medicine, University of Pisa, Italy
a r t i c l e
i n f o
Article history: Received 16 July 2008 Received in revised form 21 October 2008 Accepted 25 November 2008 Available online 3 December 2008 Keywords: Endothelium Endothelial function Nitric oxide Verapamil Trandolapril
a b s t r a c t Aim: The aim of the present study is to evaluate the effect of treatment with verapamil, trandolapril and their combination on peripheral microcirculation vasoreactivity. Methods: Twenty hypertensive patients were randomized to receive oral trandolapril (4 mg oid; TRA) or verapamil (240 mg oid; VER) for 6 months and then the combination of the two drugs for additional 6 months. At baseline, 6 months and 12 months, peripheral microcirculation vasoreactivity was evaluated by forearm blood flow technique (venous plethysmography), as vasodilation to an endotheliumdependent (acetylcholine) and an endothelium-independent stimulus (sodium nitroprusside, SNP); minimal forearm vascular resistances (MFVR) were also evaluated. Results: Blood pressure decreased similarly and progressively in both groups throughout the study period. In VER, 6-month verapamil treatment significantly increased vasodilation to acetylcholine, but not to SNP. The superimposition of trandolapril increased the response to SNP, and less to acetylcholine. In TRA group, 6-month treatment with trandolapril improved the response to SNP, but not to acetylcholine. In this group, the superimposition of verapamil caused a significant improvement in the response to acetylcholine, but not to SNP. At the end of the study, MFVR were significantly reduced in both groups, but to a greater extent in TRA. Conclusion: The present study demonstrates that chronic treatment with verapamil ameliorates endothelial function in the forearm microcirculation of essential hypertensive patients, while trandolapril protects microcirculation from structural alterations. The combination of the two drugs is potentially a powerful tool to counteract hypertension-related microvascular dysfunction and damage. © 2009 Published by Elsevier Ireland Ltd.
1. Introduction Essential hypertension is characterized by impaired endothelium-dependent vasodilation [1–3] as a consequence of an increased vascular generation of reactive oxygen species (ROS), which in turn destroy nitric oxide (NO) [2,3]. It is widely accepted that NO protects the vessel wall against the onset of atherosclerosis and thrombosis, whereas a dysfunctioning endothelium secondary to NO deficiency is an early indicator of atherothrombotic damage and of cardiovascular events [4,5]. Beyond functional vascular abnormalities, hypertension also alters the physical properties of arterial blood vessels [6]. Indeed, microvascular remodeling, hypertrophy and rarefaction have been documented at very early stages of hypertensive disease [6]. These structural alterations are considered as the crucial pathogenic mechanism for the develop-
∗ Corresponding author at: Department of Internal Medicine, University of Pisa, Via Roma, 67, 56100 Pisa, Italy. Tel.: +39 050 992914; fax: +39 050 553407. E-mail address: [email protected] (S. Taddei). 0021-9150/$ – see front matter © 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.atherosclerosis.2008.11.023
ment and progression of the hypertensive process, including the end-organ damage and the occurrence of vascular events [6,7]. For these reasons it is conceivable that, in addition to lowering arterial pressure, the effect of reversing structural abnormalities or restoring NO availability have recently been emphasized as an additional target of antihypertensive therapy. Of importance, the mere normalization of blood pressure fails to restore microvascular function and structure, which is strictly dependent on specific properties of the antihypertensive agent employed [6,8]. Dihydropyridine calcium channel blockers (CCBs) have been demonstrated to be effective in improving endothelium-dependent vasodilation in several vascular districts including peripheral microcirculation [8], a beneficial effect exerted mainly through antioxidant properties leading to the restoration of NO availability [9–11]. However, at the present time, no data are available on the effects of chronic verapamil administration on endothelial function in essential hypertensive patients. Conversely, the role of ACE-inhibitors on endothelial function of essential hypertensive patients is not conclusive. Indeed, although ACE-inhibitors are the most effective antihypertensive drug class in improving endothelium-dependent vasodilation in peripheral [12,13] and coronary macrocirculation
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
[14], their effects on endothelial function in peripheral microcirculation are controversial [15–19]. However, ACE-inhibitors, through the blockade of the angiotensin II-dependent vascular inflammation and smooth muscle cell proliferation and migration, are able to improve vascular remodeling and fibrosis [6]. Considering the different peculiarities of these two drug classes towards the vessel wall, it is conceivable that the association of them might provide optimal protection for the microcirculation in hypertension. Therefore, in the present work we aimed to investigate the effect of the combination of the CCB verapamil and the ACE-inhibitor trandolapril as compared to either of the two drugs alone, on microvascular endothelial function and minimal forearm vascular resistances (MFVR), an integrated index of vascular structural changes, in essential hypertensive patients. 2. Methods 2.1. Patients The study population included 10 normotensive control subjects and 20 matched patients with essential hypertension. Subjects with smoking history (more than 5 cigarettes per day), severe hypercholesterolemia (total cholesterol greater than 6.2 mmol/L), diabetes mellitus, cardiac and/or cerebral ischemic vascular disease, impaired renal function, heart failure, conductance disturbances or other major diseases were excluded. Subjects were defined as normal according to the absence of family history of essential hypertension and to blood pressure (BP) values. Essential hypertensive patients were recruited among the newly diagnosed cases in our out-patient clinic and enrolled if they reported the presence of positive family history of essential hypertension, whenever supine BP (after 10 min of rest) measured with a mercury sphygmomanometer three times at 1-week intervals, was consistently greater than 140 mmHg/90 mmHg. Secondary forms of hypertension were excluded by routine diagnostic procedures. After baseline vascular reactivity evaluation, hypertensive patients were randomized to either oral verapamil 240 mg oid (VER, n = 10) or oral trandolapril 4 mg oid (TRA, n = 10; Fig. 1). After 6 months (step 1) all patients underwent a vascular reactivity evaluation. Then, patients from VER group received trandolapril 4 mg in addition to verapamil, and patients from TRA group received verapamil 240 mg in addition to trandolapril for 6 months. At the end of this second period (step 2), patients underwent a final vascular reactivity evaluation (Fig. 1). At baseline and at step 1 and step 2 anamnesis, physical examination as well as blood sampling were performed. The protocol was approved by the local Ethical Committee and all participants gave their written consent to the study.
215
2.2. Vascular reactivity procedures Vascular reactivity was assessed by the perfused forearm technique, as previously described [2]. Briefly, the brachial artery was cannulated for drug infusion at systemically ineffective rates and for intra-arterial BP and heart rate monitoring. Forearm blood flow (FBF) was measured in both forearms by strain-gauge venous plethysmography. Circulation to the hand was excluded 1 min before FBF measurement by inflating a pediatric cuff around the wrist at suprasystolic blood pressure. Sensitivity and reproducibility of the method in our laboratory have been elsewhere described [20]. Endothelium-dependent forearm vasodilation was estimated by a dose–response curve to intra-arterial acetylcholine (ACH, from 0.15 to 15 g/100 mL forearm tissue/min; 5 min each dose). Endothelium-independent vasodilation was assessed by a dose–response curve to intra-arterial sodium nitroprusside (SNP, from 1 to 4 g/100 mL forearm tissue/min, 5 min each dose). NO availability was assessed by repeating ACH in the presence of intra-arterial NG -monomethyl-l-arginine (L-NMMA, 100 g/100 mL forearm tissue/min), a specific NO-synthase (NOS) inhibitor. Since L-NMMA modifies blood flow, the effect of ACH in the presence of the NO-clamp system, which allows assessment of endothelial agonists in the presence of NOS blockade without changes in blood flow, was also evaluated, as already published [21]. Thus, after 10 min of L-NMMA infusion, SNP was co-infused (Normotensives: 0.4; hypertensives: 0.3 g/100 mL tissue/min for 5 min) to neutralize the L-NMMA-induced vasoconstriction and restore baseline FBF. SNP and L-NMMA were then continued throughout ACH infusion. A 30-min washout was allowed between each dose–response curve, whereas a 60-min period was allowed after L-NMMA. Minimal forearm vascular resistances (MFVR), an integrated index of microvascular structure, were evaluated by calculating the ratio between mean intra-arterial BP and maximal forearm vasodilation induced by 13 min of forearm ischemia (forearm cuff inflated to 300 mmHg) plus 1 min of hand exercise [22]. 2.3. Oxidative stress assessment In all study individuals, at baseline, step 1 and step 2, measurements of malondialdehyde (MDA) and lipoperoxides (LOOH) (colorimetric assay) [23,24] were performed, as index of oxidative stress. Moreover, antioxidant capacity was measured as plasma ferric reducing ability of plasma (FRAP, colorimetric assay) [25]. 2.4. Data analysis Data were analyzed in terms of changes in FBF. Differences between two means were compared by the paired Student’s t test.
Fig. 1. Study design. Patients were randomized to either verapamil (VER, n = 10) or trandolapril (TRA, n = 10) for 6 months, then trandolapril and verapamil were added, respectively for 6 additional months. At baseline, step 1 and step 2 patients were evaluated (physical evaluation, blood samples, vascular reactivity experiments).
216
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
Table 1 Clinical characteristics of patients throughout the study. Controls (n = 10)
Age (years) Sex (M/F) BMI (kg/m2 ) SBP (mmHg) DBP (mmHg) HR (bpm) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) Glycemia (mg/dL) Creatinine (mg/dL)
47.6 ± 7.5 6/4 26.1 ± 2.7 120.5 ± 4.6 79.5 ± 2.9 71.4 ± 3.4 198.8 ± 24.8 49.5 ± 8.2 129.6 ± 18.7 95.4 ± 27.8 82.5 ± 9.3 0.9 ± 0.2
VER group (n = 10)
TRA group (n = 10)
Baseline
Verapamil (6 mo)
Verapamil + trandolapril (12 mo)
Baseline
Trandolapril (6 mo)
Trandolapril + verapamil (12 mo)
45.9 ± 10.4 6/4 27.1 ± 2.6 146.2 ± 11.8 98.2 ± 2.7 72.5 ± 6.3 202.9 ± 25.2 47.1 ± 11.5 136.8 ± 21.5 96.9 ± 50.0 79.1 ± 10.9 0.9 ± 0.1
– – 27.0 ± 2.7 132.4 ± 7.9* 84.8 ± 5.6* 61.6 ± 5.0* 199.8 ± 35.0 46.1 ± 11.0 128.5 ± 19.0 117.8 ± 89.2 82.0 ± 9.2 0.9 ± 0.1
– – 27.0 ± 2.8 124.9 ± 5.9* , † 81.8 ± 2.3* , † 61.0 ± 3.8 208.8 ± 31.0 45.4 ± 13.2 133.9 ± 18.2 150.9 ± 94.7 83.4 ± 12.6 0.8 ± 0.1
46.6 ± 13.2 7/3 26.6 ± 4.4 150.0 ± 12.1 98.8 ± 1.8 69.8 ± 8.3 201.5 ± 34.5 47.8 ± 14.9 133.0 ± 36.0 100.6 ± 28.1 81.3 ± 10.3 0.9 ± 0.1
– – 26.3 ± 4.0 135.8 ± 6.6* 90.1 ± 3.5* 67.0 ± 8.8 203.6 ± 37.4 52.9 ± 14.15 122.2 ± 25.2 134.3 ± 71.8 84.9 ± 8.8 1.0 ± 0.2
– – 26.3 ± 4.0 128.2 ± 5.7* , † 82.0 ± 3.3* , † 62.6 ± 5.2† 207.5 ± 40.9 55.4 ± 17.2 125.6 ± 37.7 125.8 ± 69.7 80.4 ± 9.3 0.9 ± 0.1
BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; VER: verapamil; TRA: trandolapril. * p<0.05 vs. baseline. † p<0.05 vs. step 1.
Responses to ACH and SNP and change in clinical parameter were analyzed by ANOVA for repeated measures, followed by Bonferroni post hoc test. Multiple regression was applied, where specified. Since basal FBF proved to be different in the various experimental steps, data were also analyzed as percent increase or decrease from baseline. To distinguish the endothelium-dependent from the endothelium-independent component of the vasodilatory effects of acetylcholine, the ratio of the area under the dose–response curve to acetylcholine to the area under the dose–response curve to sodium nitroprusside were calculated. Results are expressed as mean ± SD. Differences were considered statistically significant at a value of P < 0.05. Computations for the statistical method were performed with NCSS 2004.
hypertensive patients, no significant difference was seen in lipid profile, blood glucose and creatinine between the VER and the TRA groups at baseline and throughout the treatment period (Table 1). 3.1. Effect of the study drugs on hemodynamics At baseline, BP and heart rate values were similar between VER and TRA groups (Table 1). A significant reduction in heart rate was observed at the first step visit in the VER group and at the step 2 visit in the TRA group, as result of the effect of verapamil therapy onset. BP values decreased similarly and progressively in both groups throughout the study period (Table 1). 3.2. Comparison of endothelium-dependent vasodilation in normotensive subjects and hypertensive patients at baseline
2.5. Drugs ACH (Farmigea S.p.A., Pisa, Italy), NG -monomethyl-l-arginine (Clinalfa AG, Läufelfingen, Switzerland) and SNP (Malesci, Milan, Italy) were obtained from commercially available sources and diluted freshly to the desired concentration by adding normal saline. SNP was dissolved in glucose solution and protected from light by aluminum foil.
In normotensive subjects, the FBF increase induced by ACH was significantly (P < 0.01) blunted by L-NMMA infusion (Table 2). On the contrary, in hypertensive patients, the vasodilation induced by ACH was significantly (P < 0.01) reduced as compared to normotensive subjects and resistant to L-NMMA (Table 2). In contrast, dose-dependent vasodilation to SNP was similar in normotensive subjects and hypertensive patients (Table 2).
3. Results 3.3. Effect of therapy on vascular functional responses Normotensive subjects and patients with essential hypertension showed similar basal systemic demographic, clinical and humoral characteristics, except for BP values (Table 1 and Table 2). Among
Six-month verapamil treatment (step 1) significantly (P < 0.0001) increased the vascular response to ACH (Table 2,
Table 2 Vascular responses in study populations. Absolute forearm blood flow (FBF) values and percent changes after challenges in normotensive subjects and essential hypertensive patients in the VER and TRA groups at the various study time points. Basal FBF
ACH FBF (%)
Basal FBF
L-NNMA + ACH FBF (%)
Basal FBF
SNP FBF (%)
Normal subjects VER baseline VER 6 months
2.4 ± 0.4 2.8 ± 0.2 2.9 ± 0.6
17.7 ± 4.6 (+556%) 12.6 ± 2.6 (+348%)† 16.1 ± 3.7 (+469%)‡
2.7 ± 0.4 2.8 ± 0.2 2.8 ± 0.6
9.6 ± 1.9 (+255%)* 12.2 ± 2.4 (+339%)† 9.3 ± 3.1 (+239%)* , ‡
2.8 ± 0.4 2.8 ± 0.2 2.8 ± 0.5
14.0 ± 2.3 (+396%) 12.0 ± 1.5 (+328%) 12.3 ± 2.5 (+337%)
VER 12 months TRA baseline TRA 6 months
2.8 ± 0.4 2.7 ± 0.5 2.7 ± 0.4
19.4 ± 3.7 (+601%)‡ , o 10.9 ± 2.5 (+313%)† 12.8 ± 2.6 (+370%)
2.8 ± 0.4 2.7 ± 0.4 2.8 ± 0.4
11.2 ± 2.8 (+298%)* , ‡ 10.8 ± 2.7 (+302%)† 12.6 ± 2.9 (+367%)†
2.7 ± 0.4 2.6 ± 0.4 2.7 ± 0.4
15.0 ± 2.2 (+444%)‡ , § 10.5 ± 1.9 (+295%) 13.3 ± 1.7 (+392%)‡
TRA 12 months
2.6 ± 0.4
2.7 ± 0.3
8.9 ± 0.6 (+237%)* , ‡
2.6 ± 0.4
12.7 ± 1.0 (+385%)‡
14.6 ± 1.1 (+461%)‡ , §
FBF is expressed as mL/min/100 g of tissue and responses to challenges are also presented as percent change from basal FBF, in brackets. * p < 0.05 vs. ACH FBF. † p < 0.05 vs. normal subjects. ‡ p < 0.05 vs. baseline. o p = 0.05 vs. 6 months.
§ p < 0.05 vs. 12 months (for exact p values, see text).
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
217
Fig. 2. Endothelium-dependent vasodilation to ACH (A, C) and endothelium-independent vasodilation to SNP (B, D) at baseline, step 1 and step 2 in VER group (A, B) and TRA group (C, D). *, P < 0.05 step 2 vs. baseline; †, P < 0.05 step 1 vs. baseline; ◦ , P < 0.05 step 1 vs. step 2.
Fig. 2A). At this step, L-NMMA significantly blunted the vasodilation to ACH (Table 2). The inhibitory effect of L-NMMA on vasodilation to ACH, while not detected at baseline, was present after verapamil administration and was similar to that seen in normotensive subjects (Fig. 3). On the contrary, the vasodilation to SNP was not affected by verapamil administration (Table 2, Fig. 2B). The superimposition of trandolapril (step 2) further increased the response to ACH (Table 2, Fig. 2A), without affecting the inhibitory effect of L-NMMA (Table 2, Fig. 3). Moreover, the combination of trandolapril significantly increased the vasodilation to SNP (Table 2, Fig. 2B). When the improving effect of trandolapril on the response to ACH was corrected for the response to SNP, no net effect on the response to muscarinic agent was observed (data not shown). In TRA group, at baseline, the vasodilation to ACH was similar to that in VER group and not modified by L-NMMA (Table 2). Six-month treatment with trandolapril (step 1), slightly but not significantly increased the vasodilating response to ACH (Table 2, Fig. 2C), and this partial improvement disappeared when a cor-
Fig. 3. Percent inhibition by L-NMMA on endothelium-dependent vasodilation to ACH in patients from VER and TRA group, throughout the study. *, P < 0.01 vs. baseline and TRA group at step 1.
218
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
Fig. 4. Bars show minimal forearm vascular resistances (MFVR) in hypertensive patients, at baseline and at the end of the study (step 2). Data shown as mean ± SEM. *, P < 0.01 vs. baseline counterpart; †, P < 0.05 vs. VER group at step 2.
rection for the response to SNP was applied. In contrast, vascular response to SNP was significantly increased by trandolapril administration (Table 2, Fig. 2D). Nevertheless, the lack of L-NMMA effect on vasodilation to ACH was confirmed (Table 2, Fig. 3). In this group, the superimposition of verapamil (step 2) caused a significantly further improvement in the response to ACH (Table 2, Fig. 2C) together with a restoration of an inhibitory effect of LNMMA on ACH (Table 2, Fig. 3). At step 2, the inhibition by L-NMMA on vasodilation to ACH was not different between TRA and VER groups (Table 2, Fig. 3). The presence of verapamil did not change the response to SNP as compared to step 1 (Table 2, Fig. 2D). 3.4. Effect of the therapy on vascular structural changes At baseline, MFVR resulted significantly (P < 0.01) higher in hypertensive patients (3.5 ± 0.6 SU) than in controls (1.8 ± 0.2 SU). No differences were observed between VER and TRA groups, at baseline (Fig. 4). In both groups, at step 2 the MFVR were reduced as compared to baseline (2.7 ± 0.3 and 2.1 ± 0.3, respectively), but the reductions were significantly greater in TRA than in VER group (Fig. 4). 3.5. Effect on oxidative stress At baseline, FRAP, MDA and LOOH plasma levels (Table 3) were similar between VER and TRA. Among patients from VER group, 6-month verapamil treatment significantly increased FRAP plasma levels (Table 3). Verapamil administration also slightly decreased MDA and LOOH levels, although the statistical significance was not achieved (Table 3). The superimposition of trandolapril further increased FRAP values, and significantly reduced MDA and LOOH (Table 3).
Fig. 5. Correlation between the percent change in FRAP and the percent change in the inhibitory effect of L-NMMA on ACH.
In TRA group, 6-month trandolapril administration failed to significantly change FRAP, but significantly decreased MDA and LOOH values (Table 2). The superimposition of verapamil determined a significant increment of FRAP, together with a further decrement of MDA and LOOH plasma levels (Table 2). In order to explain which clinical parameters might determine the change in NO availability, we evaluated the correlation between the percent changes in the inhibitory effect of L-NMMA on ACH and the clinical parameters modifications. We included the percent changes from baseline to step 1 and from step 1 to step 2 in both VER and TRA groups, separately. A significant correlation between the change in the inhibitory effect of L-NMMA and the change in FRAP (r = 0.59; p < 0.001) (Fig. 5) and DBP (r = −0.46; p < 0.05) were observed. When a multiple regression analysis was applied, including in the model the changes in oxidative stress, lipid profile, glycemia, blood pressure, creatinine and BMI, FRAP resulted the only variable which significantly explained the change in the inhibitory effect of L-NMMA (r2 = 0.21; p < 0.005). 4. Discussion The present study highlights the effectiveness of the combination of trandolapril and verapamil in ameliorating vascular structural and functional abnormalities associated with hypertension. In particular 6-month treatment with the diphenylalkylamine CCB verapamil is able to improve endothelium-dependent vasodilation in the forearm microcirculation of essential hypertensive patients, while the protective effect of ACE-inhibition on peripheral microcirculatory endothelial function was not significant. In contrast, trandolapril was able to reduce minimal vascular resistances and to increased vascular response to sodium nitroprusside, suggesting vascular structure improvement. These
Table 3 Systemic plasma markers of oxidative stress in patients throughout the study. ER group (n = 10)
FRAP (mmol/L) Maiondialdehyde (mol/L) Lipoperoxides (mol/L) * † ‡
§
TRA group (n = 10)
Baseline
Verapamil (6 mo)
Verapamil + trandolapril (12 mo)
Baseline
Trandolapril (6 mo)
298 ± 62 4.1 ± 2.2 5.7 ± 3.7
426 ± 69* 2.8 ± 1.1* 2.6 ± 1.4
545 ± 67† 2.7 ± 1.6 3.4 ± 2.7
329 ± 47 5.3 ± 1.8 6.4 ± 2.7
350 ± 54 3.3 ± 1.7* 3.2 ± 1.9*
P < 0.05 vs. baseline. P < 0.001 vs. baseline. P < 0.01 vs. baseline. P < 0.05 vs. trandolapril alone.
Trandolapril + verapamil (12 mo) 493 ± 87‡ , § 2.0 ± 0.9‡ 2.5 ± 1.9*
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
differential, and partly additive beneficial effects exerted by these two different compounds support a potential synergistic benefit deriving from the combination of ACE-inhibitors and CCB. Previous works clearly demonstrated the beneficial effect of dihydropyridines nifedipine [9], lacidipine [10] and lercanidipine [11] in improving endothelium-dependent vasodilation, virtually leading to a normalization of endothelial function in the peripheral microcirculation of essential hypertensive patients. Importantly, this effect was achieved through a restoration of NO availability by the oxidative stress scavenging action exerted by these drugs. In the present study we demonstrate for the first time that verapamil is also able to restore the inhibitory effect of L-NMMA on ACH, indicating that the restoration of NO availability by CCBs is not limited to a specific compound but is probably a class effect, thus extending this concept to non-dihydropyridines CCB. Our data are also in line with previous studies in different animal models of hypertension, where the effect of verapamil on vascular reactivity was evaluated. Indeed, in aortic rings from L-NAME-induced hypertensive rats, relaxation to ACH, but not that to SNP was increased by long-term verapamil treatment [26]. Similarly, verapamil was found to improve endothelium-dependent relaxation in aorta from stroke-prone spontaneously hypertensive rats [27]. In the present study we also observed that plasma FRAP levels were decreased by verapamil, a finding underscoring the antioxidant properties of this compound. The cross-talk between the antioxidant effects and the ability to restore the NO pathway by verapamil was evidenced in the present study by the relationship between the changes of FRAP plasma levels and the modifications in the inhibitory effect of L-NMMA on ACH response. Taken together, these findings suggest that the restored NO availability induced by verapamil could be related to its antioxidant activity and address FRAP as a possible indicator of NO availability. The ameliorated NO availability caused by verapamil was accompanied by a significant reduction in MDA, but not in LOOH levels. Indeed, experimental studies clearly showed that these plasma markers of lipid peroxidation might not reliably reflect the oxidative stress level in the artery wall [28,29]. The present results confirm this inconsistency and suggest FRAP as a better indicator of oxidative stress within the vascular wall. Concerning the effect of trandolapril on vascular reactivity, we observed that 6-month treatment with the ACE-inhibitor failed to significantly modify the inhibitory effect of L-NMMA on the muscarinic agonist, suggesting no effect of the drug on NO bioavailability. Despite the observed improvement in the vasodilation to ACH, this was in parallel to an amelioration in the response to the endothelium-independent stimulus SNP as well, and when this parameter was used as correction, no net effect of 6 month trandolapril treatment on the endothelium-dependent response was observed. These negative findings are consistent with previous reports, demonstrating no effect of different ACE-inhibitors on endothelium-dependent vasodilation to ACH or methacholine in the forearm microcirculation of essential hypertensive patients [16–18]. Additionally, in a recent paper [30] ramipril was found to ameliorate forearm post-ischemic hyperemia, a phenomenon largely independent from endothelial function and consistent with our finding of improved vasodilation to SNP. Indeed, the improvement in the vascular response to SNP by trandolapril indicates a direct effect of this compound on vascular smooth muscle cell vasoreactivity, and suggests a vascular structure amelioration. This possibility is supported by results from MFVR measurements. Indeed, at baseline, as expected, MFVR proved to be higher in hypertensive patients as compared to normotensive subjects. At the end of the combination treatment, a significant reduction of MFVR was observed in both study groups, but this result was significantly wider in TRA group which had been treated with trandolapril for
219
1 year. Taken in conjunction, these results demonstrate a regression, although not a normalization, of peripheral vascular structural alterations by trandolapril administration. These findings are in line with a large amount of data showing a high efficacy of ACEinhibitors in inducing a regression of vascular structural changes in the forearm microcirculation [6,17,31], as well as in subcutaneous small arteries [6,19,32,33]. Interestingly, trandolapril did not change FRAP but induced a significant reduction in plasma MDA and LOOH levels, suggesting that these latter markers of lipid oxidation might be associated with the presence of structural changes of the microcirculation. The combination of ACE-inhibitors and CCB has attracted increasing interest in recent years, since from a clinical point of view the two drugs show additive effects in term of blood pressure reduction and lower incidence of side effects [34]. Moreover, although, overall, no differences in terms of cardiovascular events reduction seems to be present among different antihypertensive drugs [35], recent data (ACCOMPLISH trial) suggest that a dihydropyridine CCB plus an ACE-inhibitor may significantly improve prognosis as compared to an ACE-inhibitor plus a diuretic combination, independently from blood pressure reduction [36]. The results of the present study support these evidence from a mechanistic point of view, since the combination of verapamil and trandolapril, as compared to single drugs, was associated with a better microvascular function and structure, as well as with a wider antioxidant capacity. In conclusions, the present study represents the first demonstration that chronic treatment with verapamil ameliorates endothelial function by restoring NO availability together with an antioxidant effect in the forearm microcirculation of essential hypertensive patients. In addition, our study also demonstrates a protective role of trandolapril toward the vascular wall, an effect mainly related to structural vascular changes. Finally, our results highlight a synergistic effect of verapamil and trandolapril in improving microvascular vasodilating function and systemic oxidative stress. The present findings can have important clinical implications. Indeed, if considering the antiatherogenic properties of NO and that a dysfunctioning endothelium promotes the atherosclerotic process, the beneficial effect of verapamil on NO availability would help to prevent the development of atherosclerosis. This hypothesis is supported by the results from the Verapamil in Hypertension and Atherosclerosis Study (VHAS), showing the effectiveness of verapamil in promoting regression of thicker carotid lesions in hypertensive patients [37]. Recently, Rizzoni et al [7] demonstrated that media to lumen ratio of subcutaneous small arteries, an index of structural alteration in the microcirculation, is a potent predictor of cardiovascular events in different groups of patients including hypertensive patients. Therefore, our results strongly support the potential beneficial effect of trandolapril mediated by the protection on microcirculatory structural improvement. However, in a recent clinical trial (INVEST), the verapamil–trandolapril-based strategy was as clinically effective as the atenolol-hydrochlorothiazide-based strategy in hypertensive patients with coronary artery disease [38]. This apparent discrepancy might be likely due to the fact that this trial was performed in high risk patients, characterized by an irreversible late stage atherosclerotic process. In contrast, an amelioration of vascular function, and of NO availability in particular, might play a more important role in the early phases of the disease. In this view, when considering the differential beneficial effect of verapamil and trandolapril toward vasculature in the present study, it might be speculated that verapamil–trandolapril is a desirable combination to maintain vascular wall health. Additionally, in patients with stable coronary artery disease of the INVEST, atenolol and verapamil were similarly effective in controlling blood pressure and preventing cardiovascular events but with verapamil having
220
D. Versari et al. / Atherosclerosis 205 (2009) 214–220
a higher tolerability and tending to reduce more effectively angina and stroke [39]. In the future, we will need clinical trials to investigate the impact of this drug combination on long-term prognosis in low-risk populations. Without such information, at the present time, whether the synergistic beneficial effect on vascular function and structure might lead to a better prognosis, remains an attractive hypothesis. Conflicts of interest No conflict of interest exists. References [1] Taddei S, Virdis A, Mattei P, et al. Effect of insulin on acetylcholine-induced vasodilation in normotensive subjects and patients with essential hypertension. Circulation 1995;92:2911–8. [2] Taddei S, Virdis A, Ghiadoni L, Magagna A, Salvetti A. Vitamin C improves endothelium-dependent vasodilation by restoring nitric oxide activity in essential hypertension. Circulation 1998;97:2222–9. [3] Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation 1993;87:1468–74. [4] Lerman A, Zeiher AM. Endothelial function: cardiac events. Circulation 2005;111:363–8. [5] Taddei S, Salvetti A. Endothelial dysfunction in essential hypertension: clinical implications. J Hypertens 2002;20:1671–4. [6] Schiffrin EL. Remodeling of resistance arteries in essential hypertension and effects of antihypertensive treatment. Am J Hypertens 2004;17:1192–200. [7] Rizzoni D, Porteri E, Boari GE, et al. Prognostic significance of small-artery structure in hypertension. Circulation 2003;108:2230–5. [8] Taddei S, Virdis A, Ghiadoni L, Sudano I, Salvetti A. Effects of antihypertensive drugs on endothelial dysfunction: clinical implications. Drugs 2002;62:265–84. [9] Taddei S, Virdis A, Ghiadoni L, et al. Restoration of nitric oxide availability after calcium antagonist treatment in essential hypertension. Hypertension 2001;37:943–8. [10] Taddei S, Virdis A, Ghiadoni L, et al. Lacidipine restores endothelium-dependent vasodilation in essential hypertensive patients. Hypertension 1997;30: 1606–12. [11] Taddei S, Virdis A, Ghiadoni L, et al. Calcium antagonist treatment by lercanidipine prevents hyperpolarization in essential hypertension. Hypertension 2003;41:950–5. [12] Ghiadoni L, Versari D, Magagna A, et al. Ramipril dose-dependently increases nitric oxide availability in the radial artery of essential hypertension patients. J Hypertens 2007;25:361–6. [13] Ghiadoni L, Magagna A, Versari D, et al. Different effect of antihypertensive drugs on conduit artery endothelial function. Hypertension 2003;41:1281–6. [14] Antony I, Lerebours G, Nitenberg A. Restoration of normal coronary vasomotricity after intravenous infusion of angiotensin converting-enzyme inhibitor (perindoprilat) in hypertensive patients. Arch Mal Coeur Vaiss 1995;88:1125–8. [15] Schiffrin EL, Deng LY. Comparison of effects of angiotensin I-converting enzyme inhibition and beta-blockade for 2 years on function of small arteries from hypertensive patients. Hypertension 1995;25:699–703. [16] Creager MA, Roddy MA. Effect of captopril and enalapril on endothelial function in hypertensive patients. Hypertension 1994;24:499–505. [17] Taddei S, Virdis A, Ghiadoni L, Mattei P, Salvetti A. Effects of angiotensin converting enzyme inhibition on endothelium-dependent vasodilatation in essential hypertensive patients. J Hypertens 1998;16:447–56. [18] Kiowski W, Linder L, Nuesch R, Martina B. Effects of cilazapril on vascular structure and function in essential hypertension. Hypertension 1996;27:371–6. [19] Rizzoni D, Muiesan ML, Porteri E, et al. Effects of long-term antihypertensive treatment with lisinopril on resistance arteries in hypertensive patients with left ventricular hypertrophy. J Hypertens 1997;15:197–204. [20] Pedrinelli R, Taddei S, Graziadei L, Salvetti A. Vascular responses to ouabain and norepinephrine in low and normal renin hypertension. Hypertension 1986;8:786–92.
[21] Virdis A, Pinto S, Versari D, et al. Effect of oral contraceptives on endothelial function in the peripheral microcirculation of healthy women. J Hypertens 2003;21:2275–80. [22] Pedrinelli R, Spessot M, Salvetti A. Reactive hyperemia during short-term blood flow and pressure changes in the hypertensive forearm. J Hypertens 1990;8:467–71. [23] Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol 1990;186:407–21. [24] Jiang ZY, Hunt JV, Wolff SP. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal Biochem 1992;202:384–9. [25] Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem 1996;239: 70–6. [26] Kung CF, Moreau P, Takase H, Luscher TF. L-NAME hypertension alters endothelial and smooth muscle function in rat aorta. Prevention by trandolapril and verapamil. Hypertension 1995;26:744–51. [27] Novosel D, Lang MG, Noll G, Luscher TF. Endothelial dysfunction in aorta of the spontaneously hypertensive, stroke-prone rat: effects of therapy with verapamil and trandolapril alone and in combination. J Cardiovasc Pharmacol 1994;24:979–85. [28] Versari D, Daghini E, Rodriguez-Porcel M, et al. Chronic antioxidant supplementation impairs coronary endothelial function and myocardial perfusion in normal pigs. Hypertension 2006;47:475–81. [29] Keaney Jr JF, Gaziano JM, Xu A, et al. Low-dose alpha-tocopherol improves and high-dose alpha-tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J Clin Invest 1994;93:844–51. [30] Brili S, Tousoulis D, Antoniades C, et al. Effects of ramipril on endothelial function and the expression of proinflammatory cytokines and adhesion molecules in young normotensive subjects with successfully repaired coarctation of aorta: a randomized cross-over study. J Am Coll Cardiol 2008;51: 742–9. [31] Virdis A, Ghiadoni L, Lucarini A, et al. Presence of cardiovascular structural changes in essential hypertensive patients with coronary microvascular disease and effects of long-term treatment. Am J Hypertens 1996;9:361–9. [32] Schiffrin EL, Deng LY, Larochelle P. Progressive improvement in the structure of resistance arteries of hypertensive patients after 2 years of treatment with an angiotensin I-converting enzyme inhibitor. Comparison with effects of a beta-blocker. Am J Hypertens 1995;8:229–36. [33] Rizzoni D, Porteri E, De Ciuceis C, et al. Effect of treatment with candesartan or enalapril on subcutaneous small artery structure in hypertensive patients with noninsulin-dependent diabetes mellitus. Hypertension 2005;45:659– 65. [34] Kirchengast M. Preclinical considerations and results with the combination of verapamil and trandolapril: blood pressure reduction and beyond. J Hypertens Suppl 1997;15:S27–33. [35] Turnbull F, Neal B, Algert C, et al. Effects of different blood pressure-lowering regimens on major cardiovascular events in individuals with and without diabetes mellitus: results of prospectively designed overviews of randomized trials. Arch Intern Med 2005;165:1410–9. [36] Jamerson KA. Avoiding cardiovascular events in combination therapy in patients living with systolic hypertension. In: Paper presented at: American College of Cardiology 57th Annual Scientific Session. 2008. [37] Zanchetti A, Rosei EA, Dal Palu C, et al. The Verapamil in Hypertension and Atherosclerosis Study (VHAS): results of long-term randomized treatment with either verapamil or chlorthalidone on carotid intima-media thickness. J Hypertens 1998;16:1667–76. [38] Pepine CJ, Handberg EM, Cooper-DeHoff RM, et al. A calcium antagonist vs a non-calcium antagonist hypertension treatment strategy for patients with coronary artery disease The International Verapamil-Trandolapril Study (INVEST): a randomized controlled trial. JAMA 2003;290:2805–16. [39] Bangalore S, Messerli FH, Cohen JD, et al. Verapamil-sustained release-based treatment strategy is equivalent to atenolol-based treatment strategy at reducing cardiovascular events in patients with prior myocardial infarction: an INternational VErapamil SR-Trandolapril (INVEST) substudy. Am Heart J 2008;156:241–7.