Nitrovasodilators have been widely used for more than a century in the treatment of acute and chronic coronary artery disease (CAD). However, fundamental understanding of their beneficial effects is more recent and is still being actively studied. Liberation of nitric oxide by endothelial cells mediates vasodilation, and decreases platelet aggregation (1,2). In CAD, NO production is deficient (3), and endothelial function can be impaired at early stages of coronary atherosclerosis (4). Exogenous NO treatment is known to induce arterial vasodilation, especially in atherosclerosis (5). However, whether exogenous NO may affect dynamic regulation of endothelial function through modulation of endothelium-dependent vasodilation has not been determined.
Endothelial function plays an important role in vascular homeostasis, including regulation of vascular tone, regional blood flow, and inhibition of thrombosis. Endothelial function is impaired in most vessels in atherosclerosis, even before the onset of vascular macroscopic lesions (6,7). Because shear stress is believed to be the main physiologic stimulus responsible for basal and stimulated NO formation, flow-mediated vasodilation (FMD) appears to be a good parameter to evaluate endothelial function (8). Many physiologic or pathologic states are responsible for an endothelial dysfunction such as hypercholesterolemia (9), hypertension, heart failure, and diabetes (10-12). One of the most important cause of endothelial dysfunction is oxidative stress, which leads to inactivation of NO and subsequent formation of hydroxyl radicals (13). Indeed, antioxidants, such as vitamins C and E, reverse endothelial dysfunction in smoking, heart failure, and atherosclerosis (14).
Molsidomine is widely used in the symptomatic treatment of CAD and heart failure. It belongs to sydnonimine family and is an exogenous NO donor with specific pharmacologic characteristics. First, NO release by SIN-1, the active metabolite of molsidomine, is nonenzymatic, without the need of any cofactors (15). This feature is thought to explain, at least in part, the absence of tolerance after prolonged administration. Second, NO release is accompanied by superoxide anion release, which could lead to oxidative side reactions and interact with endothelial function (15,16). Thus we evaluated in this study the effect of molsidomine on endothelial function.
Twenty patients with CAD were included in this study. Inclusion criteria for patients were stable angina pectoris and at least one stenosis on a major coronary vessel (>50%). Exclusion criteria for patients were less than 3-months-old myocardial infarction, heart failure, creatinine clearance <30 ml/min, diabetes, ventricular arrhythmia, and any treatment that could not be discontinued ≥48 h before the beginning of the protocol. For ethical reasons, treatments with β-blockers were not interrupted, and aspirin was interrupted only 48 h before the study (all patients received β-blockers and aspirin). However, patients treated with >300 mg of aspirin a day were not included, because of the known effects of high doses of intravenous aspirin on endothelial function (17). Current smokers (two patients) refrained from smoking 2 days before the beginning of the protocol and until the study was completed. Patients were divided in two groups by a double-blinded randomization, receiving either molsidomine (the molsidomine group) or placebo (the placebo group). All participants gave written informed consent, and the ethics committee of the institution approved the protocol.
Measurement of flow-mediated dilation
A good correlation has been established between endothelial function in coronary arteries and peripheral arteries (18). The brachial artery is readily accessible to vascular ultrasound for stable measurements, and hyperemia reaction can be easily obtained through a short ischemia of the hand. For these reasons, this study was designed to explore the effects of molsidomine on endothelial function in the brachial artery, by using a high-resolution ultrasound Wall Track system (Pie Medical, Maastricht, Neederlands) with a 7.5-Hz linear probe to measure systolic and diastolic internal diameters of distal brachial artery. This echo-tracking system, analyzing the radiofrequency signal, has a precision for diastolic diameter measurements of 30 μm. FMD was measured as a percentage of increase of diastolic diameter of the brachial artery before and after an 8-min ischemia of the homolateral hand performed with an inflatable wrist cuff (hyperemia test). When the wrist cuff is deflated, blood flow and shear stress increase transiently, inducing NO release by the endothelium and FMD. Maximal diameter (DM) was defined as the greatest diastolic diameter after deflation of the cuff, measurements being made at deflation and every 30 s thereafter for 5 min. Measurement at deflation corresponds to the minimal diameter (DB for basal diameter). FMD is expressed as 100 × (DM − DB)/DB. This noninvasive method gives good reproducibility and precision, as it has been previously described (19). We found similar results with an intraobserver intersession coefficient of variability of FMD measured on brachial artery of 6.7%.
The study protocol is shown in Fig. 1. Treatment or placebo was given to patients in a double-blind randomized protocol. After a bed-rest period of ≥30 min, a first FMD measurement was made, just before the first molsidomine intake (H0). Then subjects took orally 4 mg molsidomine or placebo 3 times a day (10 a.m., 4 p.m., 10 p.m.) on day 1 and day 2 and a single dose at 10 a.m. on day 3. Measurements, including diastolic arterial diameters, were performed in the morning on day 1 and 3, before (corresponding to H0 on day one and H48 on day 3), and 2 h after (corresponding to H2 on day 1 and H50 on day 3) molsidomine intake. The 2-h delay between administration of treatment and FMD measurement was chosen in accordance with pharmacologic properties of molsidomine. Blood pressure was measured continuously during each FMD measurement.
Statistical analysis was performed by an independent statistical institute (Versus, Levallois, France). Data are expressed as mean ± SEM. Means are compared by unpaired Student's t test or Scheffé's test. Analysis of variance (ANOVA) was used to compare repeated measures. Statistical significance was accepted at a level α = 0.05.
Study population characteristics
Study population characteristics are provided in Table 1. All subjects included were men. Baseline characteristics did not significantly differ between the placebo and molsidomine groups. Twelve patients (seven in the molsidomine group, five in the placebo group) had a hypercholesterolemia history and long-term treatment with statins, and cholesterol levels were normalized in all patients at the time of inclusion. Two patients in the molsidomine group did not complete the study because of adverse reactions (headaches occurring after molsidomine intake). In these patients, H0 and H2 determinations were performed and used for statistical analysis. Two patients were excluded because of the unreliability of their FMD measurement (patients unable to keep a steady position during measurement).
Effect of molsidomine
Treatment with molsidomine improved FMD. As shown in Fig. 2, ANOVA of repeated measures of FMD in the placebo and molsidomine groups showed a significant difference between the two groups (by ANOVA, p = 0.03). FMD was increased by 60% at H2 in the molsidomine group as compared with the placebo group (respectively 4.58 ± 0.7% vs. 2.88 ± 0.6%; p < 0.05 by t test). At H50, molsidomine-treated patients also showed the same trend toward improvement of their endothelial function (a 47% increase). Of note, patients in both groups exhibited a nonsignificant difference in FMD at H0 (3.7 ± 0.8% in molsidomine-receiving patients and 2.7 ± 0.6% in the placebo group; p = 0.4). No significant difference in FMD was observed before the first (H0) and the last (H48) molsidomine intake.
We confirmed that exogenous NO donor molsidomine increased arterial diameter in patients with CAD. At H50, we observed a 7% increase in diastolic diameter (+0.5 ± 0.25 mm in the molsidomine group, −0.06 ± 0.1 mm in the placebo group; p = 0.03; not shown).
These results show that molsidomine improves endothelial dysfunction in patients with CAD, in addition to its vasodilator effect.
To evaluate the molsidomine effect on FMD, any treatment other than β-blockers was discontinued 2 days before the beginning of the study, to avoid any interference with other treatment effects on arterial diameter and FMD. For example, members of the statin family and angiotensin-converting enzyme (ACE) inhibitors are known to improve endothelial function (20). In our study, discontinuation of these two treatments may have impaired endothelial function and thus played against reversal by molsidomine. Of note, both groups were homogeneous for treatment with statins (five patients in the placebo group, seven in the molsidomine group), and only one patient had long-term treatment with ACE inhibitors. Current smokers also refrained from smoking. Only two patients were current smokers (one in each group), and smoking discontinuation may have improved endothelial function because smoking is known to impair endothelial function (21).
We measured FMD by using a high-resolution A mode ultrasound determination of brachial artery diameters. Although coronary arteries are the main concern in CAD, the brachial artery is interesting for several reasons. This artery is easily explored with ultrasound, whereas invasive procedures must be used to explore coronary arteries. Moreover, atherosclerosis is general disease, and it has been shown that coronary and brachial artery endothelial function were closely related (18). Study of FMD in coronary arteries also uses drugs that increase blood flow, whereas only mechanical ischemia of the hand is required to increase brachial artery flow. We observed a significant improvement of FMD in the molsidomine-treated group as compared with the placebo group, as assessed by ANOVA analysis. As shown in Fig. 2, 60% at H2 and 47% at H50 increased FMD. The difference in FMD that already exists between the two groups at H0 may interfere with the interpretation of the results. However, this difference is not statistically significant, and most important, the difference between the two curves (Fig. 2) is statistically significant by using repeated measures ANOVA.
When differences between the two groups are analyzed at each time (Scheffé's test), FMD is significantly different between the placebo and the treated groups at H2 but not at H50 (p < 0.05 and p = 0.08, respectively). Thus the improved endothelial function at early time points could remain stable in time, but could also be a transient effect. If molsidomine facilitate endogenous NO bioavailability at the onset of treatment, long-term effects may be weakened by a decreased release and efficacy of endogenous NO after prolonged exposure (22). Further studies are needed to evaluate the effect of molsidomine at later time points.
Molsidomine is a direct NO donor widely used in the treatment of CAD. Specific characteristics of molsidomine are a nonenzymatic release of NO, and the production of superoxide anion (15). NO donors induce arterial vasodilation, especially in atherosclerosis (5). Possible explanations for increased efficiency in atherosclerosis include that endogenous NO decreased production and increased degradation. The first hypothesis is supported by the decreased expression of endothelial NO synthase in atherosclerosis (3), and by the reversibility (although inconstant) of endothelial dysfunction in hypercholesterolemia after L-arginine administration (23). In accordance with the second hypothesis, oxidant stress is known to induce degradation of NO (13), and antioxidant treatment restores endothelial function in many pathologic states (14).
The mechanism involved in molsidomine improvement of FMD remains to be explained. However, this study shows that, at least at the beginning of the treatment, SIN-1 has a positive effect on endothelial function. Restoration of endothelial function may have long-term beneficial effects on thrombosis, progression of atherosclerosis, and more generally cell-to-cell interactions with the endothelium in patients with CAD.
Acknowledgment: This work was supported by a grant from les Laboratoires Hoechst Houdé, France.
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