Thirty-one healthy middle-aged and older subjects (total, n = 31, 66.4 ± 1.3 years [range, 53-79 years]; male, n = 13, 69.8 ± 1.6 years [range, 61-79 years]; female, n = 18, 63.9 ± 1.7 years [range, 53-79 years]) volunteered to participate in this study. All volunteers provided written informed consent before participating in the study, which was approved by the Ethics Committee of Ritsumeikan University and was conducted in accordance with the Declaration of Helsinki. This study was registered in the University Hospital Medical Information Network Clinical Trials Registry (UMINCTR, UMIN000035520). All subjects were free from overt signs and symptoms of chronic disease and did not smoke. Subjects taking medications such as anti-hyperlipidemics, anti-hypertensives, or anti-hyperglycemics as well as those with a history of stroke, diabetes, hypertension, hyperlipidemia, cardiac disease, chronic renal failure, and mental disorders were excluded from the study. None of the subjects regularly performed resistance and aerobic exercise . The subjects in this study drank very little alcohol. Since estrogen hormones influence arterial function [27], we recruited postmenopausal women. Subjects were randomly divided into 2 groups: a training group (n = 16 [male = 6/female = 10], 64.8 ± 2.0 years) and a control group (n = 15 [male = 7/ female = 8], 68.1 ± 1.6 years).

For all subjects, peak oxygen uptake (V˙O2peak), body weight, body fat, height, resting systolic blood pressure (SBP), resting diastolic blood pressure (DBP), resting heart rate (HR), resting plasma ADMA concentration, resting plasma NOx concentration, as well as serum concentrations of total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were measured at the beginning and end of the experiment. Carotid β-stiffness was examined as an index of arterial stiffness. Before the subjects were tested, they sat quietly for 30 min. Resting brachial SBP, DBP, and HR were measured in duplicate in the supine position using a vascular testing device (OMRON COLIN Co., Tokyo, Japan). At the beginning and end of the study period, fasting blood samples were drawn following at least 48 h of rest after the last exercise training session. All subjects were instructed not to eat or drink fluids other than water for at least 12 h prior to blood sampling. In addition, we confirmed that participants did not consume any dietary sources of NOx over the 24 h prior to testing in either group since NOx can be affected by diet. Thus, both acute effects from the most recent bout of exercise and oral sources of NOx other than NO should be avoided. Serum and plasma samples were immediately centrifuged (1500 × g, 15 min, 4 °C). Blood samples were stored at -80 °C until use. Room temperature was maintained at 24 °C throughout the experiments.

Subjects attended the AT program, which consisted of cycling on a leg ergometer at 60%-70% V˙O2peak for 45 min/day, 3 days/week for 8 weeks. For the first two periods of the exercise program, subjects performed at 50% V˙O2peak for 40 min. The warm-up and cool-down periods consisted of 5 min of cycling at a 40% V˙O2peak. Exercise compliance was monitored carefully under direct supervision. Additionally, sedentary control subjects were encouraged to rest during the experimental period.

The V˙O2peak was measured using an incremental cycle exercise test on a cycle ergometer (MINATO, AE-310SRD, Osaka, Japan). The incremental cycle exercise began at a work rate of 60 W (30-90 W) for men and 30 W (0-60 W) for women. Power output was increased by 15 W/min until the subjects could not maintain a fixed pedaling frequency of 60 rpm. The subjects were encouraged to exercise at maximum intensity during the ergometer test. HR and rating of perceived exertion (RPE) were monitored minute by minute during the exercise. RPE was obtained using a modified Borg scale. V˙O2 was monitored during the last 30 s of each period of the increased work rate. The highest value of V˙O2 during the exercise test was designated as V˙O2peak if three out of four of the following criteria were met: (I) a plateau in V˙O2 with an increase in external work, (II) maximal respiratory exchange ratio ≥ 1.1, (III) maximal HR ≥ 90% of the age-predicted maximum (208 − 0.7 × age) [28], and (IV) RPE ≥ 18.

Carotid β-stiffness was evaluated as an indicator of arterial stiffness. A combination of ultrasound imaging of the pulsatile common carotid artery and simultaneous applanation of tonometrically obtained arterial pressure from the contralateral carotid artery allowed noninvasive determination of arterial compliance [8]. The carotid artery diameter was measured from images obtained using an ultrasound system equipped with a high-resolution linear array transducer [28]. A longitudinal image of the cephalic portion of the common carotid artery was acquired 1-2 cm proximal to the carotid bulb. All image analyses were performed by the same investigator.

Pressure waveforms and amplitudes were obtained from the common carotid artery using a pencil-shaped probe with a high-fidelity strain gauge transducer (SPT-301; Millar Instruments, Houston, TX, USA) [8]. Because baseline blood pressure levels are subjected to hold-down forces, the pressure signal obtained via tonometry was calibrated by equating the carotid mean arterial blood pressure and DBP to brachial artery values [8,29]. The carotid β-stiffness index was calculated using the equation [ln(P1/P0)]/[(D1 − D0)/D0], where D1 and D0 are the maximum (systolic) and minimum (diastolic) diameters and P1 and P0 are the highest (systolic) and lowest (diastolic) blood pressures, respectively.

NOx levels in the plasma were measured using the Griess assay (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. All samples were assayed in duplicate. The optical density at 540 nm was measured using a microplate reader (xMark microplate spectrophotometer; Bio-Rad Laboratories, Hercules, CA, USA). The day-today coefficient of variation of plasma NOx levels was 1.0 ± 0.3%.

ADMA levels in the plasma were measured using an enzyme-linked immunosorbent assay (ELISA; Immundiagnostik AG, Bensheim, Germany) according to the manufacturer’s protocol. All samples were assayed in duplicate. The optical density at 450 nm was quantified using a microplate reader (xMark microplate spectrophotometer; Bio-Rad Laboratories). The day-to-day coefficient of variation of plasma ADMA levels was 0.6 ± 0.2%.

Fasting serum levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were determined using standard enzymatic techniques.

Values are expressed as mean ± standard errors (SE). The differences between the groups and the two time points were assessed by a two-way repeated-measure analysis of variance followed by Fisher’s post hoc test, which was applied when a measurement was significantly different. The relationships between AT-induced changes in plasma ADMA levels and plasma NOx levels or carotid β-stiffness as well as changes in the NOx/ADMA ratio and carotid β-stiffness were determined using the Pearson correlation coefficient. Sex differences in the changes in these parameters before and after the AT intervention were compared using a paired Student’s t-test. Statistical significance was set at P < 0.05. All statistical analyses were performed using StatView (5.0, SAS Institute, Tokyo, Japan).

Before AT, there were no significant differences in V˙O2peak between the training and control groups. However, there was a significant interaction between the group with intervention on V˙O2peak (P < 0.05, Table 1). Specifically, in the training group, V˙O2peak significantly increased after AT intervention (P < 0.05, Table 1). Although there was no significant difference in the carotid β-stiffness between the training and control groups before the AT, there was a significant interaction of the group with intervention on carotid β-stiffness (P < 0.01, Fig 1-A). Carotid β-stiffness significantly decreased after AT intervention (P < 0.01, Fig. 1-A). However, no significant differences between the groups or times were noted for height, body weight, BMI, HR, SBP, DBP, serum total cholesterol levels, HDL cholesterol levels, or triglyceride levels (Table 1).

Before AT, there were no significant differences in plasma ADMA or NOx levels and NOx/ADMA ratio between the training and control groups. There was significant interaction between plasma NOx levels in the intervention group (Fig. 1-B, P < 0.01). After AT, plasma NOx levels were significantly higher in the training group than in the control group (Fig. 1-B, P < 0.01). The interaction between groups and time points was significant for plasma ADMA levels (Fig. 1-C, P < 0.05). After AT, plasma ADMA concentrations were significantly lower in the training group than in the control group (Fig. 1-C, P < 0.01). Notably, there was a significant interaction between the intervention group and the NOx/ADMA ratio (Fig. 1-D, P < 0.01). After AT, the NOx/ADMA ratio was significantly higher in the training group than in the control group (Fig. 1-D, P < 0.01).

Changes in plasma ADMA levels were negatively correlated with changes in plasma NOx levels (Fig. 2-A, r = -0.414, P < 0.05). Moreover, there was a negative correlation between changes in plasma NOx levels and carotid β-stiffness (Fig. 2-B, r = -0.493, P < 0.01). Furthermore, changes in the NOx/ADMA ratio were negatively correlated with changes in carotid β-stiffness (Fig. 2-C, r = -0.514, P < 0.01).

There was no sex difference with regard to changes in carotid β-stiffness between male and female subjects (Fig. 3-A, male: -2.400 ± 0.411 vs. female: -2.390 ± 0.254). In addition, no sex differences were observed in the changes in plasma ADMA and NOx levels or in NOx/ADMA ratio between male and female subjects (Fig. 3-B, NOx; male: 24.412 ± 7.997 vs. female: 30.203 ± 5.810, Fig. 3-C, ADMA; male: -0.138 ± 0.024 vs. female: -0.142 ± 0.029, Fig. 3-D, NOx/ADMA ratio; male: 67.831 ± 16.707 vs. female: 87.964 ± 17.026).