Introduction

Endothelial dysfunction is known to have a pivotal role in the initiation/progression of atherosclerosis.1 In patients with hypertension, flow-mediated vasodilatation of the brachial artery (FMD), a marker of conduit arterial endothelial function, is known to be a predictor of future cardiovascular events.2, 3, 4 The reactive hyperemia index (RHI), a marker of resistance arterial endothelial function measured by peripheral arterial tonometry, is available in clinical settings. Although a significant correlation has been observed between the FMD and RHI in subjects with coronary artery disease,5, 6 a weak to almost no association between the two indicators has been found in the general population.7, 8 Thus, it is still unknown whether the FMD and RHI might reflect similar pathophysiological abnormalities associated with endothelial function, and whether the significance of the relationship between these two indicators may differ among different pathophysiological conditions such as cardiovascular disease, dyslipidemia, obesity, diabetes mellitus and hypertension.

Nitric oxide (NO), derived from the vascular endothelium, is one of the major factors contributing to vasodilatation caused by reactive hyperemia, and its effect is more prominent in the conduit arteries than in the peripheral resistance arterioles.9 However, Hijmering et al.10 demonstrated that autonomic nervous system activation was also associated with FMD. Several other studies have shown an association between autonomic nervous system activity not only with FMD, but also with RHI.10, 11, 12, 13 The 5-min forearm clamping procedure to induce reactive hyperemia is indispensable for both assessments, and it is possible that this procedure affects autonomic nervous system activity.14 Such activation may influence the tone of the peripheral resistance arterioles rather than that of the conduit arteries. Several studies have also demonstrated that mental stress may affect the peripheral hemodynamics in subjects with hypertension.15, 16

We hypothesized that FMD may not be closely associated with the RHI, and furthermore, that autonomic nervous system activation induced by 5-min forearm clamping may exert a greater influence on the measured value of the RHI by applanation tonometry than on the FMD in subjects with hypertension. The present study was conducted to clarify whether autonomic nervous activity induced by 5-min forearm clamping may have a greater influence on the measured RHI than on the FMD in subjects with hypertension.

Methods

Study subjects

The study subjects were hypertensive patients, well-controlled with treatment (blood pressure<150/95 mm Hg), who were in follow-up care at the outpatient department of the Department of Cardiology of Tokyo Medical University Hospital for at least 6 months. The exclusion criteria were: patients taking beta-blocker medication; patients with coronary artery disease including a history of percutaneous coronary intervention or coronary bypass surgery, severe valvular heart disease, arrhythmia requiring treatment (that is, atrial fibrillation, atrial flutter, permanent pacemaker implantation or frequent ventricular premature beats) or impaired left ventricular systolic function (left ventricular ejection fraction <40%); patients with malignancy; patients receiving treatment with steroids, nonsteroidal anti-inflammatory drugs or immunosuppressive drugs; patients with a serum creatinine level greater than 2.5 mg dl−1; patients with stroke, aortic disease (except peripheral arterial disease) or serious liver disease. Written informed consent was obtained from all the subjects before their participation in the study. The protocol of the study was approved by the Ethics Committee of Tokyo Medical University.

From April 2012 through February 2013, a total of 132 patients with hypertension were enrolled in the study, and their FMD, RHI and heart rate variability (HRV) were measured simultaneously. Of the 132 patients, 17 were excluded because they were being treated with beta-blockers that are well-known to affect autonomic nervous system activity.

Measurements

Flow-mediated vasodilatation of the brachial artery

The method of measurement of FMD is described elsewhere in detail.17 The subjects were instructed to fast overnight and to abstain from alcohol, smoking, caffeine and antioxidant vitamins for at least 12 h before the measurements. They were asked to rest in the sitting position in a quiet, dark, air-conditioned room (22 °C to 25 °C) for 5 min. Then, they were asked to rest again for at least 15 min in the supine position in the same room before the measurement of FMD. Blood pressure was then measured by the oscillometric method (UA 767; A&D, Saitama, Japan). We performed ultrasound measurements according to the guidelines for ultrasound assessment of FMD.18

Using high-resolution ultrasound with a 10-MHz linear array transducer, longitudinal images of the right brachial artery were recorded at baseline and then continuously from 30 s before to 2 min or more after cuff deflation following suprasystolic compression (50 mm Hg over the systolic blood pressure) of the right forearm for 5 min. The diastolic diameter of the brachial artery was determined semi-automatically using an instrument equipped with a software for monitoring the brachial artery diameter (Unex, Nagoya, Japan). In brief, continuous recordings of two-dimensional gray-scale images and A-mode waves of the brachial artery in the longitudinal plane were obtained with a novel stereotactic probe-holding device. A segment with clear anterior (intima–lumen) and posterior (lumen–intima) interfaces was manually determined. These border interfaces were identified automatically on the A-mode waves as a signal from the intima–media complex, and the diastolic diameter of the brachial artery beats was synchronized with the electrocardiographic R-waves and tracked automatically. The changes in the diastolic diameter were continuously recorded. Then, FMD was estimated as the percent change of the diameter of the brachial artery at maximal dilatation during reactive hyperemia compared with the baseline value. The reproducibility of the measurement of FMD at our institute is described elsewhere.18

Peripheral arterial tonometry

The methodology for measurement of the RHI is described elsewhere.3, 19 During the measurement of FMD, before the forearm cuff occlusion, a peripheral arterial tonometry device (Endo-PAT2000; Itamar Medical, Caesarea, Israel) was placed on the index finger of each hand. Then, the pulse amplitude detected by this device was electronically recorded throughout the FMD measurement. The extent of reactive hyperemia was calculated as the ratio of the average pulse amplitude of the device signal over a 1-min time interval starting 1.5 min after cuff deflation to the average pulse amplitude of the device signal over a 2.5-min time period before cuff inflation. The RHI value was calculated as the ratio of the reactive hyperemia between the two hands.

Heart rate variability

After a 10-minute rest in the supine position in preparation for the measurement of FMD, recording of the electrocardiographic R-R intervals was begun. The recording was not controlled for the respiratory phase. The parameters of HRV (HRVs) were determined from these sample recordings, and the power spectra of the R-R interval were calculated using the maximum entropy method using MemCalc software (Cross-well, Yokohama, Japan).20, 21 Then, the R-R interval, high-frequency power (HF) component, low-frequency power (LF) component and the ratio of the LF to HF (LF/HF ratio) were calculated. These parameters were calculated in three phases (that is, the HRVs calculated from the total of 15-min electrocardiographic recording during the measurement of FMD (HRVs total), from the 5-min electrocardiographic recording from the start of the measurement of FMD to just before forearm cuff occlusion (HRVs measured before clamping), and from the 10-min electrocardiographic recording from the start of cuff occlusion to the end of measurement of the FMD (HRVs after the clamping)). The changes of the HRVs induced by 5-min forearm clamping were calculated as follows: ((HRVs after clamping−HRVs before clamping)/HRVs before clamping) × 100 (%).

Laboratory measurements

Serum levels of high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides and creatinine, and blood glucose were measured enzymatically in fasting blood samples collected from the subjects.

Statistics

All the data are expressed as the mean±s.d. The relationships among the variables were assessed by univariate linear regression and multivariate linear regression analyses. The differences in the values of the variables measured before and after the 5-min forearm clamping procedure were assessed by the paired t-test. The differences between the groups were assessed by the Mann-Whitney U-test or χ2 test. In all the analyses, P-values less than 0.05 were considered to indicate a statistically significant difference. All the analyses were conducted using IBM SPSS (Statistical Package for the Social Sciences) software (19.0J for Windows, IBM/SPSS, Chicago, IL, USA).

Results

Table 1 shows the clinical characteristics of the study subjects. Univariate linear regression analysis did not reveal any significant relationship between FMD and the RHI (nonstandardized coefficient=0.027, 95% confidence interval=−0.015 to 0.069, standardized coefficient (β)=−0.121, P=0.200).

Table 1 Clinical characteristics of the study population
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In the overall study population, the 5-min forearm clamping procedure did not affect either the HRVs or the heart rate (from before the clamping procedure to after the clamping procedure: LF=from 334±625 to 403±1221 ms2, P=0.455; HF=from 196±266 to 229±473 ms2, P=0.223; LF/HF=from 2.4±1.7 to 2.3±1.7, P=0.626; heart rate=from 61±8 to 61±8 beats per minute, P=0.423). As shown in the horizontal axis of Figure 1, this procedure was associated with an increase of the LF/HF ratio in 61 of the 115 subjects (53%).

Figure 1
figure 1

Scatter-plots showing the changes in the ratio between the low-frequency power component and high-frequency power component induced by 5-min forearm clamping and the reactive hyperemia index measured by peripheral arterial tonometry. deltaLF/HF, change of the ratio between the low-frequency power component and high-frequency power component induced by 5-min forearm clamping; RHI, reactive hyperemia index measured by peripheral arterial tonometry.

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As shown in Figure 1, univariate linear regression analysis revealed a significant relationship between the change of the LF/HF ratio associated with 5-min forearm clamping and the RHI (nonstandardized coefficient=−0.002, 95% confidence interval=−0.004 to 0.000, β=−0.222, P=0.017), but not FMD (nonstandardized coefficient=0.002, 95% confidence interval=−0.006 to 0.010, β=0.053, P=0.574). Multivariate linear regression analysis with the adjustments revealed a significant relationship between the change of the LF/HF ratio associated with 5-min forearm clamping and the RHI (Table 2). However, no significant associations were observed between any of the HRV parameters (other than the change of LF/HF) and either the FMD or RHI.

Table 2 Results of multivariate linear regression analysis to assess the significance of the relationship of the change of LF/HF with the reactive hyperemia index
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The patients were classified into tertile ranges of the change of the LF/HF ratio associated with 5-min forearm clamping: (T1, 203–29%; T2, 28 to −18%; T3, −18 to −81%). Then, they were divided into two groups (that is, T1 vs. T2–3). Although clinical characteristics and the FMD were similar between both groups, the RHI was significantly lower in the T1 group than in the T2–3 group (Table 3).

Table 3 Clinical characteristics and endothelial functions between the groups divided by tertile ranges of the change of the ratio between the low-frequency power component and high-frequency power component induced by 5-min forearm clamping
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Discussion

To the best of our knowledge, the present study is the first to examine whether the change in autonomic nervous activity associated with the 5-min forearm clamping procedure utilized to induce reactive hyperemia might influence FMD or the RHI.

The findings of the present study were consistent with the results of the Framingham study in which only a weak association was found between the PAT and FMD.5 This outcome suggests that the pathophysiological abnormalities related to FMD may differ from those associated with the RHI. The reduced NO release in response to stimuli has a central role in the pathophysiology of endothelial dysfunction in the conduit arteries reflected by FMD,22 whereas metabolic and other factors such as endothelium-derived hyperpolarizing factors are thought be important in the pathophysiology of endothelial dysfunction in the peripheral resistance arteries that are reflected by the RHI.4, 23 The Framingham study suggested that conventional risk factors for cardiovascular disease had different associations with the FMD and RHI.5 However, thus far, no study has examined the difference in the association of autonomic nervous system activity with the FMD and PAT.

Mental stress has been reported to both increase24 and decrease the HRV.25 Thus, the effect of mental stress on autonomic nervous system activity might differ depending on the method used to induce mental stress. The 5-min forearm clamping procedure induces slight mental stress in participants; however, no study thus far has examined the effect of the 5-min forearm clamping procedure on autonomic nervous system activity. In the present study, although the 5-min forearm clamping procedure did not induce a change in the HRV in the entire study population, half of the patients showed a change in the HRV with the clamping procedure. Thus, although mental stress is thought to exert a more pronounced effect on autonomic nervous activity in subjects with hypertension than in normotensive subjects,26 in the present study, the change in autonomic nervous activity induced by the 5-min clamping procedure differed even among individuals with hypertension.

Sympathetic nervous system activity has a pivotal role in the pathophysiological abnormalities underlying the development of hypertension,27 and sympathetic activation attenuates the skin blood flow by causing microvascular vasoconstriction.13 Vasodilatation and vasoconstriction coordinate the control of blood flow in the peripheral resistance arteriole network,28 and reactive hyperemia causes vasodilatation in the resistant arterioles independent of sympathetic nervous activation.29 Several studies have demonstrated an effect of autonomic nervous activity on the endothelial function.10, 11, 12, 13 Ghiadoni et al.30 reported that mental stress attenuates FMD. Basically, RHI is programmed such that the values are normalized by simultaneous measurement in the contralateral arm.14 Nevertheless, Chen et al.12 reported that mental stress also attenuates the RHI. In the present study, the change in the LF/HF ratio, a marker of the sympathetic nervous tone, induced by 5-min forearm clamping was negatively associated with the RHI, but not with the FMD. Therefore, in the present study, sympathetic nervous system activation induced by 5-min forearm clamping appeared to affect the RHI, but not the FMD, presumably via a more pronounced effect on the peripheral resistance arterioles than on the conduit arteries.

Watanabe et al.31 recorded the HRV during the FMD measurements and reported that the LF/HF ratio calculated from the total recording of the R-R intervals during the FMD measurement was negatively correlated with the FMD in patients with coronary artery disease, although the authors did not evaluate the change of the HRV associated with the 5-min forearm clamping procedure. In the present study, unlike the change of the HRV induced by the 5-min forearm clamping procedure, the HRV recorded completely during simultaneous measurement of the FMD and RHI were related to neither FMD nor the RHI. Progressive impairment of the HRV and FMD has been reported to occur together with the progression of atherosclerosis.1, 4, 32 The subjects in the present study were patients with hypertension, whereas Watanabe’s study subjects had coronary artery disease. Therefore, some of the study subjects of Watanabe might have had advanced atherosclerosis. The HRV calculated from the total recording of the R-R intervals during the measurement of FMD might therefore have been related to FMD in the Watanabe study.

The present study has the following limitations: (1) Some prospective studies have demonstrated that the RHI may be a predictor of future cardiovascular events. A recent meta-analysis reported that acute mental stress adversely affected future cardiovascular risk.33 We proposed to conduct a study to clarify whether the change in autonomic nervous activity by 5-min forearm clamping might be one of the mechanisms that could explain the predictive values of RHI; (2) The findings of the present study, in which autonomic nervous activation induced by 5-min forearm clamping affected the RHI, need to be confirmed in a variety of conditions such as diabetes mellitus and coronary heart disease, as well as in healthy subjects.

Conclusions

Autonomic nervous system activation, especially sympathetic activation, induced by 5-min forearm clamping utilized to induce reactive hyperemia may significantly affect the RHI rather than FMD in subjects with hypertension.