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Introduction
Despite significant advance in medical treatments, atherosclerotic disorders, such as stroke and acute coronary syndrome, are still leading causes of mortality in developed countries. The pathophysiology of these catastrophic events involves impaired endothelial function and increase inflammation and coagulation activity.1)2) Endogenous nitric oxide (NO) production, which mediated endothelial dependent vasodilatation, is a key mediator of these changes in endothelial function, inflammation and coagulation.3-5)
Repeated application of whole-body periodic acceleration (WBPA) upregulates NO synthase in animals and improves brachial artery endothelial function in humans, as measured by brachial artery flow-mediated vasodilatation (FMD).6) Recent reports suggest that single application of WBPA can result in improved coronary microcirculation.7) However, the acute effect of single application of WBPA on brachial artery endothelial function in humans has not been fully characterized.
We have developed a novel semi-automatic vessel chasing system (UNEXEF18G; UNEX Corporation, Nagoya, Japan) identify time to peak vasodilatation, and determine the area under the vasodilatation curve.8)9) UNEXEF18G can easily identify the time to peak vasodilatation as well as the area under the vasodilatation curve. The area under the vasodilatation curve is a more accurate assessment of brachial artery endothelial function than FMD alone and may be a good quantitative reflection of NO production. In addition, although a novel UNEXEF18G has now been developed in Japan,9) the direct comparison of UNEXEF18G with a conventional method for FMD measures has not been conducted even if UNEXEF18G has already been utilized in a relatively large scale study.8)9)
Thus, the purpose of this study was to: 1) investigate the acute effect of single application of WBPA on brachial artery endothelial function in humans, and 2) to validate UNEXEF18G by comparing UNEXEF18G with conventional methods for measurement of brachial artery endothelial function. For this purpose, 1) we performed 45 min of WBPA in healthy volunteers, and 2) we compared the results of UNEXEF18G with that of conventional measures of brachial artery endothelial function at rest in same time in random conditions.
Methods
Study population
The study population consisted of 20 healthy volunteers (12 men, 8 women; age, 34 ± 12 years). Twelve of these subjects had at least one of Framingham's coronary risk factors, including cigarette use (n = 6), hyperlipidemia (n = 2), family history of coronary artery disease (n = 2), and obesity with borderline diabetes mellitus (n = 1). None of the subjects were receiving any medications for their conditions.
All subjects were enrolled in Iruma Heart Hospital and all studies were conducted also in Iruma Heart Hospital. However, all analyses were performed in National Defense Medical College.
Fourteen non-smoking subjects (7 men, 7 women) from the above 20 subjects were also used for the UNEXEF18G validation study.
Study protocol
This was a random crossover study protocol (Fig. 1 and 2). All studies were done from 14:00 to 17:00 in a temperature-controlled room (25℃) with the subject in a fasting, resting, and supine state. After at least 30 min of bed rest, subjects underwent 45-min of WBPA or 45 min of bed rest (control). WBPA was performed according to a protocol described previously;6) in brief, periodic acceleration was applied with the motion platform (Fig. 2) at a frequency of 2-3 Hz with approximately ± 0.25 g for 45 min. Before and after the intervention (WBPA or bed rest), blood pressure was measured by Korotkoff's method with the patient in the supine position. CM5 lead electrocardiogram was continuously monitored, and heart rate was determined at the time of blood pressure measurements. Following their assigned intervention (WBPA or bed rest), patients crossed-over to receive the other intervention, and experimental parameters were measured in the same fashion (Fig. 1 and 2).
For the UNEXEF18G validation study, UNEXEF18G FMD measurement and conventional FMD measurement were performed in a random order, only at rest. Since the simultaneous measures of FMD are difficult because once 5-minutes occlusion-induced ischemia precludes the continuous measurement of FMD, the effect of WBPA on FMD were evaluated by UNEXEF18G system only. The validation of measurement system between UNEXEF18G and conventional method was performed at baseline only with random conditions. And two measures of FMD by different method were conducted about 30 min interval that is empirically confirmed to be enough time periods to cancel the effect of 5 min ischemia on FMD values. In addition, FMD values are easily changed by the measurement time during a day and the meals taken before the test, suggested by published FMD measurement standard,1)2) so that the comparison of two measurement methods was conducted only one point at rest with random measurement times of the day and conditions.
Ultrasound FMD measurements in the brachial artery by UNEXEF18G
Except validation studies, all ultrasound studies were performed from 14:00 to 17:00 in a temperature-controlled room (25℃) with the subject in a fasting, resting, and supine state. Heavy meals, including a high-fat diet and caffeine-containing beverages, were prohibited beginning the night before the study. Patients were not allowed to have lunch on the day of ultrasound study. Blood pressure (BP) and heart rate were recorded from the left arm every 3 minutes with an automatic sphygmomanometer (Nihon Korin, BP-203, Tokyo, Japan) during the ultrasound procedure. Vasodilatation responses of the brachial artery were determined by the ultrasound technique using a semi-automatic device (UNEXEF18G; UNEX, Nagoya, Japan) (Fig. 3-5). Briefly, the diameter of the brachial artery was measured from B-mode ultrasound images using a 10-MHz linear array transducer. Then, a BP cuff was inflated to 50 mmHg above the systolic BP over the proximal portion of the right forearm for 5 min. The diastolic diameter of the brachial artery was determined semi-automatically using an instrument equipped with software for monitoring the brachial artery diameter. The changes in the diastolic diameter were continuously recorded. Then, FMD was determined as the maximum change in diameter after cuff release normalized to the baseline diameter (% of baseline diameter). The novel UNEXEF18G equipment consisted of three ultrasound probes with an appropriately flexible stabilizing arm that supports and fixes the ultrasound probes to the human arm. There were two H-shaped ultrasound probes and one longitudinal ultrasound probe (Fig. 4), which enabled the technician to easily track target brachial artery images during the FMD study. In addition, this machine has specially designed software that can measure FMD on-line, identify time to peak vasodilatation, and determine the area under the vasodilatation curve (Fig. 5). In details, as shown in right upper portion of Fig. 3, UNEXEF18G identifies both the intima media and the main body of brachial artery vessel walls of both near and far wall of the brachial artery as A-mode ultrasound signals. The signals of either intima media or the main body of brachial artery vessel walls can be re-selected, if necessary, by operators. These signals are automatically chasing and being recorded continuously to obtain the maximum vasodilatation of brachial artery after the reactive hyperemia. By using manual techniques, the interpreters or the operators can modify or re-selected the appropriate measuring points on either the intima media or the main signals of the brachial artery vessel walls on both near and far wall images. All measurements are automatically performed by sophisticated software installed in UNEXEF18G in order to indentify the changes in the vessel diameter, the values of FMD, the time to peak vasodilatation and the area under the vasodilatation curve.
Ultrasound FMD measurements in the brachial artery using the conventional method
All studies were performed according to a previously reported method.10)11) Briefly, all evaluations were conducted in a temperature-controlled room (25℃) with the subject in a supine position. The electrocardiogram (ECG) was monitored continuously. Blood pressure was recorded from the left arm every 3 min with an automatic sphygmomanometer. The subject's right arm (the dominant arm) was comfortably immobilized in the extended position to allow for consistent access to the brachial artery for imaging purposes. The brachial artery diameter and flow velocity were imaged using a 7.5 MHZ linear array transducer ultrasound system (Hewlett Packard, SONOS 1500, Andover, MA, USA). First, baseline two-dimensional images were obtained; then, pulsed-Doppler blood flow velocity was determined. Brachial arterial flow velocity was obtained using a Doppler signal at a 70-degree angle to the vessel, with the range gate (1.5 mm) in the center of the artery. After performing baseline measurements, a small-width blood pressure cuff was inflated on the most proximal portion of the forearm to occlusive pressure (systolic blood pressure + 50 mmHg) for 5 min in order to induce hyperemia. Next, the cuff was rapidly deflated. Immediately after deflation, pulsed-Doppler signals were recorded for 15 sec. Two-dimensional images of the brachial artery were obtained for 60 sec after cuff deflation. All images were recorded on super VHS videotape for later analysis. The brachial artery blood flow at rest and during reactive hyperemia was determined by previously described methods.11) The flow volume was calculated by multiplying the velocity-time integral of the Doppler flow signal (corrected for the angle and heart rate) and the vessel cross-sectional area (πr2), using public domain software (Hewlett Packard, SONOS 1500). The relative increase in blood flow at reactive hyperemia was calculated as the maximal flow recorded in the first 15 sec after cuff deflation divided by the flow at baseline scan. By playing back the recorded information on a videocassette recorder, a 10-20 mm segment of the brachial artery could be identified for analysis using the anatomic landmarks in each subject. To reproducibly select the images at the same point in the cardiac cycle, images at peak diastole (maximum dilation, close to the R wave on the ECG) were identified, and the diameter of the brachial artery was digitized. A quantitative coronary angiography analysis computer (Kontron Elektronik, Cardio 500, Boston, MA, USA) containing a digitizing board was used for these measurements. For each condition (baseline, reactive hyperemia at 60 sec after cuff deflation), three separate images from three different cardiac cycles were digitized. The average segment diameter of these three images was determined. All of these measurements were performed in a blinded manner. To conduct a blind measurement of brachial artery endothelial function, the technician performing the study was not informed of the study protocol. In addition, study subjects were instructed not to inform the technician of the study protocol. The percent diameter changes from baseline in response to hyperemia were calculated.
Statistical analysis
Data are expressed as the mean ± SD. The paired Student's t-test was used to compare data before and after each treatment. To examine the repeatability of FMD measurements by the UNEXEF18G or the conventional method, we used the method proposed by Bland and Altman,12) which analyses the plot of the differences in the parameter values between the two FMD measurements against their means. A coefficient of repeatability was then computed as twice the standard deviation of the differences.13) Differences or statistical values were considered significant at p < 0.05. Even if the sample size was small, the histogram of each sample were not skewed (data not shown) so that we presumed each samples in this study are drawn from normally distributed data. Parametric statistical methods were subsequently utilized.
Results
The effects of either 45-min of WBPA or 45-min of bed rest on hemodynamics and FMD parameters are summarized in Table 1. There was a significant decrease in heart rate following 45-min of WBPA when compared with that following 45-min of control bed rest. Three parameters of FMD significantly changed in response to 45-min of WBPA but not to 45-min of bed rest. Specifically, 45-min of WBPA resulted in a significant increase in FMD, a significant decrease in time to peak vasodilatation and a significant increase in the area under the vasodilatation curve (Table 1).
As shown in Fig. 6 and 7, 17 of 20 subjects experienced an increase in FMD and a decrease in the time to peak vasodilatation in response to WBPA. The three other subjects experienced a decrease in FMD and an increase in time to peak vasodilatation. Changes in the area under the vasodilatation curve and the shortening of the time to peak vasodilatation have similar clinical significance to increased FMD values (Table 1). Two of these subjects did not have any risk factors for atherosclerosis, and the remaining patient was a smoker. However, the decrement in FMD and the increment in the time to peak vasodilatation in response to WBPA were relatively small. No unique clinical characteristics were identified among these subjects.
The repeatability of the UNEXEF18G measurements, as assessed by Bland and Altman's method as well as by calculation of the correlation coefficient, deemed to be adequate, indicating that UNEXEF18G could replace the conventional method for measuring FMD (Fig. 8). UNEXEF18G was easy to use. Parameters other than FMD, such as the time to peak and the area under the vasodilatation curve, were adequately measured by the UNEXEF18G. In addition, online detection of the diameter changes after reactive hyperemia enabled easy and precise detection of the peak brachial vasodilatation easily (Fig. 3-5).
Discussion
The present study demonstrated that single application of WBPA improved brachial artery endothelial function in humans and that the UNEXEF18G, a novel apparatus for measurement of FMD, was valid for clinical use in healthy subjects when compared with a conventional method. WBPA resulted in a significant increase in FMD, a significant decrease in time to peak brachial artery vasodilatation, and a significant increase in area under the vasodilatation curve. The area under the vasodilatation curve and the time to peak brachial artery vasodilatation have recently been proposed as new indices for the assessment of brachial artery endothelial function.14-16) The clinical application of FMD as a marker of endothelial function was described more than two decades ago,17) and clinical guidelines for the use of FMD have been published.18) However, the wide intra- and inter-variability of FMD measurement between difference institutions and investigators has limited its utility in the clinical assessment of endothelial function.18) Recently, a novel apparatus (UNEXEF18G) has been developed for measurement of brachial FMD and has been used widely in Japan for the clinical assessment of endothelial function.8)9) The present study directly compared the UNEXEF18G to conventional methods for the measurement of brachial FMD. Our group has been involved in brachial FMD studies since the early phase, and one of our investigators (A.U.) was involved in development of FMD measurement methods19) and underwent training in the US for the measurement of FMD more than 20 years ago.20) Thus, our conventional technique for measuring FMD is considered to be the standard. However, the present study showed that the UNEXEF18G produced adequate measurement of endothelial function in humans, as demonstrated by calculation of the correlation coefficient and by Bland and Altman plotting.
The present study demonstrated that single application of WBPA resulted in acute improvement in brachial endothelial function in normal healthy volunteers with Framingham risk factors for atherosclerotic disease. This observation is consistent with data from previous reports, in which repeated application of WBPA produced beneficial effects in normal subjects and cardiovascular patients.6) However, those previous reports did not clearly characterize the acute effect of WBPA on FMD. In one report, the acute effect of WBPA resulted in improvements in coronary flow, as assessed by cardiac ultrasound,7) which is consistent with observations from the present study. Further, the present study showed that single application of WBPA resulted in an acute decrease in the time to peak vasodilatation, which may reflect improved endothelial function. A previous study reported that decreased time to peak vasodilatation in the brachial artery could be used as a marker of brachial endothelial function.16) In terms of the effect of WBPA on area under vasodilatation curve, father study is needed to clarify the clinical significance of this finding. Fortunately, the UNEXEF18G apparatus can automatically calculate the area under the FMD, and UNEXEF18G is likely to be employed in more than 400 institutions in Japan by the end of 2012 (data from UNEX, Nagoya, Japan).
The observation that WBPA may improve endothelial function suggests that in may improve cardiovascular outcome by increasing production of NO. Thus, both single and repeated application of WBPA may have clinical utility in patients with cardiovascular disease. The present study used the forearm occlusion method (rather than upper arm) for measurements of FMD. Thus, the FMD value likely reflects the production of NO in the brachial artery.18) NO is a direct vasodilator and has anti-atherosclerotic,21-24) anti-inflammatory and anti-tumorigenic actions.25) In addition, FMD or shear stress resulted in production of NO by the brachial arterial endothelium as well as increases prostacyclin and tissue plasminogen activator, which also affect vasomotor tone, fibrinolysis and coagulation.3-5)26) All of these effects are closely related to the pathogenesis of atherosclerosis.
In addition, WBPA significantly decreased resting values of heart rate as shown in Table 1. As we have presented,27) WBPA increases high frequency power of heart rate variability. We presume WBPA can possibly increase cardiac vagal autonomic activity and subsequently decreased heart rate following 45-min of WBPA compared with control arm.
This study has several limitations. First, the number of the subjects was small, and the study was conducted and analyzed in only two medical centers, including one academic medical school hospital. Although the control and crossover design of this study enhances the validity of our results despite the small subject numbers, this study should be duplicated with a multicenter randomized clinical trial design. Second, we assessed the acute effect of WBPA only in normal healthy subjects, even if most of the subjects had Framingham risk factors for atherosclerotic disease. Thus, future study should determine the acute effects of WBPA in patients with established cardiovascular disease.
In conclusion, these data suggest that single application of WBPA results in acute improvement in brachial artery endothelial function in humans and that the novel UNEXEF18G apparatus is valid for clinical measurement of FMD.
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