The vascular endothelium is involved in the release of various vasodilators, including nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factor (EDHF), as well as vasoconstrictors.1,2 NO plays an important role in the regulation of vascular tone, inhibition of platelet aggregation, and suppression of smooth muscle cell proliferation.3,4 Endothelial dysfunction is an initial step in the development of atherosclerosis, resulting in cardiovascular complications.5 Peripheral arterial disease (PAD) is 1 of the major manifestations of atherosclerosis. PAD is associated with increased cardiovascular morbidity and mortality.6-9 It has been reported that patients with PAD have severe endothelial dysfunction.10-12 Recently, several investigators have shown that endothelial dysfunction in patients with PAD is reversible.13-15 It is expected that some interventions may improve endothelial function in PAD patients.
Serotonin (5-hydroxytryptamine, 5-HT), released from activated platelets, has various subtypes of receptors,16,17 mediates both vasoconstriction and vasodilation,18,19 and promotes platelet aggregation. Vasoconstricting effects of serotonin are mediated by 5-HT2A receptors on vascular smooth muscle cells and platelets.16,20-22 PAD patients have higher plasma 5-HT concentrations than those found in healthy subjects.23 Sarpogrelate hydrochloride, a selective 5-HT2A antagonist, has been widely used as an anti-platelet agent for the treatment of PAD.24,25 By the blockade of 5-HT2A receptors, sarpogrelate inhibits thrombus formation,26,27 suppresses platelet aggregation,28,29 and inhibits vascular smooth muscle cell proliferation.30 Several investigators have reported that sarpogrelate improves ischemic symptoms such as intermittent claudication, pain and cold sensation of the lower extremities, and objective indices such as ankle-brachial pressure index (ABPI).31-34 Although the precise mechanisms of these beneficial effects of sarpogrelate remain unknown, an anti-atherosclerotic effect of sarpogrelate has been reported.35 We hypothesized that sarpogrelate might improve endothelial function. There have been no studies aimed at determining whether endothelial function is improved after initiation of sarpogrelate treatment in patients with PAD.
In the present study, we evaluated the effects of the 5-HT2A antagonist sarpogrelate hydrochloride on vascular function before and after 12 and 24 weeks of treatment in patients with PAD.
Twenty-one Japanese subjects with PAD (10 men and 11 women aged from 67 to 92 years) were enrolled in this study. Subjects with ABPI less than 0.90 and with a history of PAD, including peripheral bypass surgery and radiological intervention, were defined as having PAD. All PAD patients were angiographically proven to have stenosis lesions in arteries of the lower extremities. All patients abstained from taking drugs for 24 hours before the study. The study protocol was approved by the Ethics Committees of Hiroshima University Graduate School of Biomedical Sciences. Written informed consent for participation was obtained from all subjects.
Measurement of FBF and LBF
The FBF and LBF were measured with a mercury-filled Silastic strain-gauge plethysmography (EC-5R, D. E. Hokanson, Inc., Issaquah, WA), as previously described.36,37 Briefly, a strain gauge was attached to upper part of the left arm and the diseased leg and then connected to a plethysmographic device and supported above the right atrium. A wrist cuff and an ankle cuff were inflated to 50 mm Hg above the systolic blood pressure to exclude hand and foot circulation from the measurement taken 1 min before measurement of FBF and LBF. Congesting cuffs of the upper arm and leg were inflated to 40 mm Hg for 7 seconds in each 15 second cycle to occlude venous outflow from the arm and leg by using a rapid cuff inflator (EC-20, D. E. Hokanson, Inc., Issaquah, WA). The blood flow output signals were transmitted to a recorder (U-228, Advance Co., Nagoya, Japan). The FBF and LBF were expressed as mL/min per 100 mL of forearm and leg tissue volume. The FBF and LBF were calculated by 2 independent observers blinded to the study protocol from the linear portions of the plethysmographic recordings. The intraobserver coefficients of variation were 3.0% and 2.5%.
All enrolled patients received antiplatelet agents (100 mg of aspirin once per day or 100 mg of ticlopidine 3 times per day) before the study. The patients were randomly divided into 2 groups. Patients in the sarpogrelate group (n = 10; 3 men and 7 women; mean age, 81.0 ± 2.2 years; body mass index, 18.9 ± 1.0 kg/m2) were treated with sarpogrelate hydrochloride (Mitsubishi Pharma Co., Osaka, Japan) at a dose of 100 mg 3 times per day for 24 weeks in addition to previous antiplatelet agents. Patients in the control group (n = 11; 7 men and 4 women; mean age, 75.8 ± 2.0 years; body mass index, 21.6 ± 1.1 kg/m2) were treated by continuing with previous antiplatelet agents for 24 weeks. There was no significant difference between aspirin and ticlopidine treatment in the 2 groups. All patients continued to receive conventional therapy throughout the study.
Measurement of FBF and LBF were performed at the beginning of the study (0 week) and after 12 and 24 weeks. This measurement began at 8:30 am. Subjects fasted the previous night for at least 12 hours. They were kept in the supine position in a quiet, dark, air-conditioned room (constant temperature 22°C to 25°C) throughout the study. Basal blood flow was measured after 30 minutes in the supine position, and then the effects of RH and sublingual NTG on FBF and LBF were measured. To obtain RH, FBF was occluded by inflating the cuff on the left upper arm to a pressure of 280 mm Hg for 5 min, and LBF was occluded by inflating of the cuff on the more diseased femoral region at a pressure of 280 mm Hg for 5 min. After the release of ischemic cuff occlusion, blood flow was measured for 3 min. NTG was sublingually administered at the dose of 0.3 mg by 1 tablet (Nihonkayaku Co. Tokyo, Japan). After the administration of NTG, blood flow was measured for 5 min. These studies were carried out in a randomized fashion. Each study proceeded after blood flow had returned to baseline. In the preliminary study, after the release of cuff occlusion or the sublingual NTG, blood flow returned to baseline within 10 min. Thus, the end of the response to RH or sublingual NTG was followed by a 15 min recovery period.
Baseline fasting serum concentrations of total cholesterol, high density lipoprotein (HDL) cholesterol, triglyceride, glucose, as well as plasma rennin activity, plasma norepinephrine, plasma angiotensin II, were obtained after a 30 min rest period before the study. Samples of venous blood were placed in polystyrene tubes containing sodium EDTA (1 mg/mL). The EDTA-containing tubes were immediately chilled in an ice bath. The plasma was separated by centrifugation at 3100 rpm at 4°C for 10 min. Serum was separated at 1000 rpm at room temperature for 10 min. Samples were stored at -80°C until assayed. Routine chemical methods were used to determine the serum concentration of total cholesterol, HDL cholesterol, triglyceride, and glucose. Plasma rennin activity and the concentration of angiotensin II were determined by radioimmunoassay (anti-angiotensin II antibody kit, SRL Co, Atsugi, Japan). The plasma concentration of norepinephrine was measured by high-performance liquid chromatography (catecholamine-plasma assay kit, SRL Co, Atsugi, Japan).38
Results are presented as the mean ± standard error. The Mann-Whitney U test was used to evaluate the differences in variables at the baseline between the sarpogrelate treatment group and the control group. Comparisons of time-course curves of the FBF and the LBF during RH were analyzed by 2 way analysis of variance (ANOVA) for repeated measures. Comparisons of clinical variables before and after treatment were performed with adjusted means by ANOVA using baseline data as covariates. A P level less than 0.05 was accepted as statistically significant. Data were processed using the software package Statview IV (SAS Institute, Cary, NC), or Super ANOVA (Abacus Concepts, Berkeley, CA).
Baseline clinical characteristics of the 10 PAD patients treated with sarpogrelate and the 11 PAD patients as control are shown in Table 1. Baseline values for all parameters were similar in the 2 groups.
Effects of Sarpogrelate on Clinical Characteristics
The effects of sarpogrelate on baseline variables are summarized in Table 1. In the sarpogrelate group, ABPI after 12 and 24 weeks tended to be higher than that before treatment, but the differences were not statistically significant. Other parameters, such as systemic hemodynamics, basal FBF and LBF, lipid profiles, and glucose metabolism, also remained unchanged at 12 and 24 weeks in the 2 groups.
Effects of Sarpogrelate on Vascular Function
Both FBF and FBF responses to RH and LBF response to RH were similar in the sarpogrelate group and the control group at baseline (Figure 1). Twelve weeks of oral sarpogrelate administration significantly increased the maximal FBF and LBF responses to RH from 13.2 ± 1.7 to 18.1 ± 2.2 mL/min per 100 mL tissue (P < 0.01) and from 8.2 ± 0.9 to 14.2 ± 2.1 mL/min per 100 mL tissue (P < 0.05), respectively, in the sarpogrelate group (Figure 1 A). Sarpogrelate-induced augmentation of blood flow response to RH was maintained at 24 weeks (Figure 1A). No change was observed in the control group at each follow-up time point (Figure 1B). Changes in FBF and LBF after sublingual administration of NTG were similar in the sarpogrelate group (Figure 2A) and control group (Figure 2B) during follow-up periods.
In the present study, we demonstrated (1) that the administration of sarpogrelate for 12 weeks increased both FBF and FBF responses to RH and LBF response to RH in PAD patients and (2) that sarpogrelate-induced augmentation of blood flow response to RH was maintained at 24 weeks.
After 12 weeks of oral sarpogrelate administration, FBF response to RH and LBF response to RH, an index of endothelium-dependent vasodilation, showed significant increases in the sarpogrelate-treated group, whereas no change was observed in the control group. However, FBF response to NTG and LBF response to NTG, an index of endothelium-independent vasodilation, were similar in the 2 groups at all time points. Our findings suggest that sarpogrelate improves endothelial function in patients with PAD. To our knowledge, there has been no study aimed at determining whether vascular function is improved after initiation of sarpogrelate treatment in patients with PAD. This is the first report on the effect of sarpogrelate on vascular function in PAD patients.
PAD is caused by atherosclerosis. Endothelial dysfunction is an initial step in the development of atherosclerosis.5 Several lines of evidence have shown that PAD is associated with endothelial dysfunction.10-12 Recently, we have also proved by using strain-gauge plethysmography that endothelial function in PAD patients was impaired in the leg resistance artery.39 Bode-Boger et al13 reported that intravenous infusion of L-arginine, a substrate of eNOS, improves NO-dependent vasodilation in PAD patients with critical limb ischemia (Fontaine III and Fontaine IV). Other investigators reported enhancement of NO production after arterial reconstruction14 and exercise rehabilitation.15 These findings suggest that endothelial dysfunction in PAD patients is reversible and that some treatment may improve endothelial function in PAD patients.
Several mechanisms by which sarpogrelate improves endothelial function in patients with PAD have been postulated. 5-HT is stored mainly in platelets, which adhere to subendothelial tissues and release 5-HT when the vascular endothelium is injured as a result of arteriosclerosis and other factors. Indeed, an increase in plasma 5-HT concentration has been observed in PAD patients.23 5-HT has various subtypes of receptors.16,17 5-HT2A receptors on vascular smooth muscle cells and platelets mediates 5-HT-induced vasoconstriction and platelet aggregation, respectively.16,20-22 Vanhoutte40 reported that 5-HT might enhance NO production via activation of 5-HT1B receptor on the endothelium. Experimental studies suggest that the endothelium-dependent vasodilatory effect of 5-HT is mediated by 5-HT1B and/or 5-HT2B receptors in pig and dog isolated coronary artery41,42 and pig isolated pulmonary artery.44 Taken together, these results suggest that the selective blockade of 5-HT2A receptors on vascular smooth muscle cells by sarpogrelate may inhibit 5-HT-induced vasoconstriction; alternatively, 5-HT may stimulate 5-HT1B and/or 5-HT2B receptors on the endothelium, resulting in enhanced NO production in the endothelium. In addition, the increase in blood flow due to vasodilation may augment shear stress, which enhances NO production in the endothelium. Sarpogrelate may contribute to the augmentation of the beneficial effects of 5-HT. The second possible mechanism is that sarpogrelate may inhibit the adhesion and proliferation of macrophage and suppress neutrophil function. Several investigators have shown that sarpogrelate decreases superoxide anion production from macrophages and neutrophils.34,44 Hence, sarpogrelate may inhibit the NO scavenging by inhibition of superoxide anion production. The mechanism by which sarpogrelate improves endothelial function in patients with PAD is complex and multifactorial; it may be through an increase in NO bioavailability, subsequent to an increase in its production, and/or a decrease in its inactivation.
Naftidrofuryl, 5-HT2 receptor antagonist, acts as an anti-vasoconstrictor and inhibits platelet and erythrocyte aggregations, thrombus formation, and vascular smooth muscle cell proliferation.45 Indeed, naftidrofuryl has been used for intermittent claudication for many years in Europe and improves pain-free walking distance and health-related quality of life.46-48 It is expected that naftidrofuryl will also have beneficial effects on vascular function in patients with PAD.
A definitive method to assess vascular function may involve direct invasive infusion of vasoactive agents into the forearm and leg arteries. We have previously reported that a non-invasive method for measuring RH is useful for assessment of resistance artery endothelial function.36,49 RH in peripheral arteries is mainly mediated by the release of NO.50 The use of NO synthase inhibitors such as NG monomethyl L-arginine and L-nitroarginine methylester would have allowed us to draw more specific conclusions concerning the role of the basal and stimulated release of NO in the forearm and leg circulation. However, because intra-arterial infusion of NO synthase inhibitors may reduce vascular blood flow and increase vascular resistance of diseased vessels, these agents may lead to adverse effects in PAD patients. Therefore, we did not use an NO synthase inhibitor because of ethical concerns.
RH can be mediated by endothelium-dependent and -independent factors, including NO, EDHF, prostacyclin, and adenosine, and may be multifactorial. Results of studies using inhibitors of these factors should enable more specific conclusions concerning the mechanism of impaired vascular response to RH in PAD patients to be drawn.
Some investigators, including us, have shown that angiotensin-converting enzyme inhibitors,51 angiotensin type II receptor blockers,52 and statins53 improve endothelial function. Although the patients in this study abstained from taking any drugs for 24 hours before the study, we cannot rule out the possibility that these agents influenced the results.
This study gives the first arguments that that sarpogrelate might affect vascular function, but large studies with large number of patients in a double-blind, randomized, placebo-controlled conditions should confirm the present results that sarpogrelate improves vascular function in PAD patients.
In conclusion, long-term oral administration of sarpogrelate increased FBF response to RH and LBF responses to RH in patients with PAD, most likely through an increase in NO bioavailability.
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