Cardiovascular diseases are the main causal factor of mortality in the world (1). Arterial stiffening may be an independent risk factor for cardiovascular events and mortality (2). Pathogenesis of arterial stiffness is associated with a decline in endothelial function, resulting in hypertension, atherosclerosis, congestive heart failure, and stroke (2). Nitric oxide (NO), a potent vasodilator, is produced from l-arginine by endothelial NO synthase (eNOS) in endothelial cells (3). Impaired NO production in endothelial cells via downregulation of eNOS is related to a decline in NO bioavailability and results in endothelial dysfunction (4,5). Therefore, elevation of arterial NO production after eNOS activation is important to reduce arterial stiffness.
Many studies have demonstrated that habitual exercise is a promising first-line candidate for preventing and treating arterial stiffening (6,7). Arterial NO-derived vasodilation in response to aerobic exercise training (AT) participates in the mechanism underlying reduction in arterial stiffness (8). Indeed, in the aorta of young rats, the expression of eNOS protein and messenger RNA elevates after exercise training and concomitantly reduces arterial stiffness, which can be estimated by aortic pulse wave velocity (PWV) (9). Furthermore, a negative association between AT-induced decrease in arterial stiffness and elevation of circulating nitrite/nitrate (measured as the stable end-product of NO, i.e., NOx) level has been shown in human studies (8). Taken together, AT is an established countermeasure for arterial stiffness.
Several recent studies have investigated a difference of effect on arterial stiffness between continuous AT and other exercise training types such as resistance training (RT) and high-intensity interval training (HIIT) (10,11). No beneficial effects of high- and moderate-intensity RT on arterial stiffness and NO production were seen in these intervention studies (12). In contrast, HIIT may be an effective exercise program to reduce arterial stiffness rather than continuous AT in patients with cardiovascular diseases (13). Although the effect on arterial stiffness may be different in different types of exercise, the molecular mechanism underlying the difference in the training effect remains unclear. If different exercise programs induced difference change in arterial NO production, those findings may facilitate exercise-induced reduction of arterial stiffness.
The effect of HIIT on arterial stiffness is similar to that of continuous AT training or is greater (14,15). Even shorter HIIT may lead to an effective reduction in arterial stiffness. In various HIIT, shorter-lasting exhaustive HIIT (total duration 4 min) consisting of six to seven sets of 20-s exercises interspersed with a rest period of 10 s significantly increases aerobic capacity in 6 wk, and was shown to be comparable to that induced by conventional AT despite a decrease in total exercise volume (16). Therefore, HIIT may lower arterial stiffness by promoting NO production in central artery effectively even in a short time. However, an effect of shorter HIIT on arterial stiffness and its molecular mechanisms remains unclear.
Therefore, we hypothesized that different exercise programs induced different changes in arterial NO bioavailability and central arterial stiffness. To test these hypotheses, we examined and compared the resting levels of arterial eNOS signaling pathway and central arterial stiffness among aerobic, resistance, and high-intensity intermittent–trained Sprague–Dawley male rats. Furthermore, the effects of these training approaches on arterial stiffness and NO production were confirmed by a human study using healthy young men.
Experiment 1: Animal Study
Animals and protocol
The ethical approval for this study was obtained from the Committee on Animal Care at Ritsumeikan University. Forty 10-wk-old male Sprague–Dawley rats were obtained (CLEA Japan, Tokyo, Japan) and cared for according to the Guiding Principles for the Care and Use of Animals, based on the Declaration of Helsinki. The rats were housed individually in an animal facility under controlled conditions (12/12-h light/dark cycle). They were randomly divided into four groups: sedentary control (CON), AT, HIIT, and RT groups (n = 10 in each group). All groups were given access to water and fed normal chow (CE-2; CLEA Japan) ad libitum during the experimental period. Posttreatment experiments in trained rats were performed 72 h after the last exercise session to avoid acute effects of the exercise. After measuring body weight and aortic PWV, blood samples were obtained from the abdominal aorta under general anesthesia. After sacrifice, the abdominal aorta, epididymal fat, soleus, gastrocnemius, and plantaris muscles were quickly resected, rinsed in ice-cold saline, weighed, and frozen in liquid nitrogen.
AT. Eight-week treadmill running protocol that has been previously shown to cause functional adaptation in the aorta was used (17). The rats were trained 5 d·wk−1 on a motor-driven treadmill for 15 min·d−1 at 15 m·min−1 for the first 2 d. Duration and intensity increased daily for 1 wk until the rats were running for 60 min at 30 m·min−1, 0% grade. Thereafter, the trained group continued running training for 7 wk at this workload level.
RT. RT was performed 3 d·wk−1 on alternate days for 8 wk using a ladder with a length of 1.1 m, a grid step of 2.0 cm, and an incline of 80° according to a previous study (18). The rats climbed the ladder for three sets of four repetitions each, and were allowed to rest between sets for 1 min.
HIIT. When animals were 12 wk old, the HIIT group repeated a 20-s swimming session (10–14 times), while bearing a weight equivalent to 16% of their body weight with a 10-s pause allowed between exercise sessions 4 d·wk−1 for 6 wk according to a previous study (19).
CON. CON rats were confined to their cages for 8 wk but were handled daily.
Aortic PWV and blood pressures
Under general anesthesia, aortic PWV and blood pressures were measured while maintaining the body temperature of rats at 37°C using an animal heat mat. A catheter (carotid artery; SP45 and femoral artery; SP31, Natume, Tokyo, Japan) with pressure transducer (DT4812; Nihon Kohden, Tokyo, Japan) was implanted at 2 points in the aortic arch via the left carotid artery and in the proximal abdominal aortic bifurcation via the left femoral artery (9). Pulse pressure waves obtained from the two pressure transducers were simultaneously captured in an amplifier and displayed on a data acquisition system (PEG-1000) at a sampling rate of 10,000 Hz. After the experiment was completed, the pulse wave propagation distance of the aorta was measured and then the straight distance between the tips of the two catheters was also measured. Propagation time from the aortic arch to abdominal aortic bifurcation is the difference in time between the beginning of the upstroke of each pulse wave form. Finally, aortic PWV was calculated by dividing the propagation distance by the propagation time. In addition, systolic and diastolic blood pressures were measured simultaneously with pulse pressure waves in the left carotid artery.
Western blot analysis was performed as previously described (20). Briefly, the aortic proteins (20 μg) were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were treated for 1 h with blocking buffer (3% skim milk in phosphate-buffered saline with 0.1% Tween 20 (PBS-T)) and then incubated for 12 h in blocking buffer at 4°C with antibodies (diluted 1/1000 in blocking buffer) against phosphor-eNOS (Ser1177; sc-12972; Santa Cruz Biotechnology), total eNOS (No. 610297; BD Biosciences), phosphor-Akt (Ser473; No. 9271; Cell Signaling Technology), and total Akt (No. 9272; Cell Signaling Technology). β-Actin antibody (No. 4967; Cell Signaling Technology) was used as a loading control. The membranes were washed three times with PBS-T and then incubated for 1 h at room temperature with horseradish peroxidase–conjugated secondary antibody and antirabbit (GE Healthcare Biosciences, Piscataway, NJ) and antimouse (GE Healthcare Biosciences) immunoglobulins diluted (1/3000) in blocking buffer. The membranes were then washed three times with PBS-T. Finally, the proteins were detected using the Enhanced Chemiluminescence Plus system (GE Healthcare Biosciences), visualized on an LAS4000 imager (GE Healthcare Biosciences), and quantified by densitometry using ImageQuant TL 7.0 software (GE Healthcare Biosciences).
The soleus muscles (50 mg) were homogenized with 10 vol of 250 mM sucrose, 1 mM Tris–HCl (pH 7.4), and 130 mM NaCl on ice using a Teflon homogenizer. The homogenate was centrifuged at 9000g for 20 min at 0°C, and the pellet was resuspended with homogenate buffer and centrifuged at 600g for 10 min at 0°C. The supernatant was centrifuged at 8000g for 15 min at 0°C, and the pellet was resuspended with 250 mM sucrose. To determine citrate synthase activity, 50 μL of each sample (mitochondrial-enriched fractions) was incubated for 2 min at 30°C in a 900-μL incubation mixture containing 100 mM Tris–HCl (pH 8.0), 1 mM 5,5′-dithiobis(2-nitrobenzoic acid), and 10 mM acetyl-CoA. The reaction was initiated by the addition of 50 μL of 10 mM oxaloacetate and then was determined spectrophotometrically at 412 nm for 3 min (21).
Experiment 2: Human Study
In experiment 2, a human study was conducted to confirm the beneficial effects of HIIT and AT on arterial stiffness and NO production.
Twenty-one young male subjects 22.5 ± 1.3 yr of age volunteered to participate in this study. Each subject was randomly divided into one of the three groups: AT (n = 7), HIIT (n = 7), and CON (n = 7) groups. The subjects were sedentary or moderately active and did not participate in vigorous sport activities. The subjects in this study did not smoke and hardly drank alcohol (average alcohol intake, 38.0 g·wk−1) were free of chronic diseases, and did not use any medications. All subjects were given an oral and written briefing of the study and provided a written informed consent. The study was approved by the Ethics Committee of Ritsumeikan University and was conducted in accordance with the Declaration of Helsinki.
Before and after each intervention (AT for 8 wk, HIIT for 6 wk, and CON for 8 wk), body composition, maximal oxygen uptake (V˙O2max), carotid–femoral PWV (cfPWV; examined as an index of aortic arterial stiffness), blood pressure, heart rate, and plasma NOx concentration were measured. All subjects were instructed not to eat or drink fluids other than water at least 12 h before blood sampling. At the end of the study period, cfPWV as an index of arterial stiffness was measured, and fasting blood samples were drawn after at least 48 h of rest after the last exercise-training session to avoid the acute effects. In addition, we ensured that participants did not consume any dietary sources of NOx over 24 h before testing in both groups, because NOx can be affected by diet. Blood samples were immediately centrifuged at 1500g for 15 min at 4°C. Serum samples were stored at −80°C until measurement. During the experiment, room temperature was maintained at 24°C.
Exercise training interventions
AT. Subjects in the AT group performed exercise consisting of cycling on a cycle ergometer (828E; Monark, Stockholm, Sweden) for 45 min at 60%–70% V˙O2max intensity 3 d·wk−1 for 8 wk. Subjects performed warm-up and cool-down for 5 min with a relatively low intensity (40% V˙O2max) for each exercise session. Compliance of exercise was carefully monitored by direct supervision.
HIIT. Subjects in the HIIT group performed each exhaustive exercise session consisting of six to seven sets of 20-s cycling on a cycle ergometer (828E; Monark) at an intensity of approximately 170% of V˙O2max, with a 10-s rest between each bout 4 d·wk−1 for 6 wk according to a previous study (16). When subjects could bike at the prefixed intensity for more than eight sets, exercise intensity was increased so that the intensity of the exercise exhausted the subject within six to seven sets of 20-s exercise. Compliance was carefully monitored by direct supervision.
CON. Subjects in the control group were instructed not to change their level of physical activity. All subjects in the three groups were encouraged to maintain their food intake as usual during the experiment.
Measurement of V˙O2max
V˙O2max was determined during an incremental cycle exercise test on a cycle ergometer by monitoring breath-by-breath oxygen consumption and carbon dioxide production (AE-310SRD; Minato, Osaka, Japan). The subjects were instructed to maintain a pedaling rate at 60 rpm. Incremental cycle exercise began at a work rate of 60 W (30–90 W); the subject started with a workload of 15 W each minute until a fixed pedaling frequency of 60 rpm could not be maintained (7). During the ergometer test, the subjects were encouraged to exercise at a maximum intensity. Rating of perceived exertion (using the modified Borg scale) and heart rate were monitored each minute during the exercise. The highest 30-s averaged value of V˙O2 during the exercise test was designated as V˙O2max, if three of four following criteria were met: (I) plateau in V˙O2 with an increase in external work, (II) maximal heart rate of ≥90% of the age-predicted maximum (208 − 0.7 × age), (III) maximal respiratory exchange ratio of ≥1.1, and (IV) rating of perceived exertion of ≥18.
Measurement of arterial stiffness, blood pressure, and heart rate
After the subject lay quietly in a supine position for 15 min, the cfPWV, blood pressure, and heart rate were simultaneously measured with a vascular testing device (form PWV/ABI; Omron Colin, Kyoto, Japan). The cfPWV was measured using applanation tonometry with an array of 15 transducers (form PWV/ABI; Omron Colin) as previously described (22). The PWV was calculated from the time delay between the carotid artery and the femoral artery blood pressure waveform and the distance between the two points, which was measured using a nonelastic tape measure. In this study, the coefficient of variation for interobserver reproducibility of cfPWV was 4.7%. The mean value of the systolic and diastolic blood pressures in the right and left arms was obtained for analysis.
Plasma NOx concentration in animal and human studies
Plasma NOx concentration was measured by the Griess assay (R&D Systems, Minneapolis, MN). Optical density at 540 nm was qualified using a microplate reader (xMark microplate spectrophotometer; Bio-Rad Laboratories). Samples were converted into concentration by a linear fit of the log–log plot of the standard curve in each assay. The day-to-day coefficients of variation of plasma NOx concentrations were 5.3% (animal samples) and 5.7% (human samples).
All values are expressed as the mean ± SE. In experiment 1, statistical analysis was performed using ANOVA or ANCOVA adjusted for diastolic blood pressure, followed by a Fisher post hoc test that was applied when a measurement was significantly different. Relationships between aortic PWV and arterial eNOS phosphorylation level and between arterial eNOS and Akt phosphorylation levels were determined using Pearson correlation coefficients. In experiment 2, comparisons of the amount of change between before and after intervention were conducted using ANOVA or ANCOVA adjusted for systolic, diastolic, and mean blood pressures and heart rate, followed by a Fisher post hoc test that was applied when a measurement was significantly different. P values of <0.05 were accepted as significant. All statistical analyses were performed using StatView (5.0; SAS Institute, Tokyo, Japan) software.
Experiment 1: animal study
Body weight was significantly decreased in the AT group compared with the CON and RT groups (Table 1). Moreover, weight of the epidydimal fat was significantly lower in the AT group than in the other three groups (Table 1). Soleus and plantaris muscle mass in the AT and RT groups was increased compared with the CON group, and gastrocnemius muscle mass increased only in the RT group (Table 1). Enzyme activity of citrate synthase in the soleus muscle in the AT and HIIT groups was increased compared with the CON group (Table 1). No significant difference was observed in the resting heart rate, systolic blood pressure, and diastolic blood pressure among the four groups (Table 1).
Aortic PWV was significantly reduced in the AT and HIIT groups compared with the CON and RT groups (Fig. 1A). Whereas there was no difference between the RT and CON groups (Fig. 1A). HIIT-induced reduction of aortic PWV was equal to that caused by AT (Fig. 1A). Furthermore, even after the adjustment for diastolic blood pressure, significant differences in PWV among the four groups were observed to be the same.
Compared with the CON group, the AT and HIIT groups showed significantly higher circulating NOx levels (Fig. 1B), whereas there was no difference between the RT and CON groups (Fig. 1B). Increased circulating NOx levels were the same in the HIIT and AT groups (Fig. 1B). In addition, arterial eNOS and Akt phosphorylation were significantly elevated in both the AT and HIIT groups compared with the CON and RT groups, whereas there was no difference between the RT and CON groups (Figs. 2A, B). HIIT-induced increase in eNOS and Akt phosphorylation in the aorta was similar to that caused by AT (Figs. 2A, B).
Arterial eNOS phosphorylation was positively correlated with arterial Akt phosphorylation in all groups (y = 0.32x + 0.40, r = 0.36, P < 0.05; Fig. 3). A significant negative correlation between aortic PWV and arterial eNOS phosphorylation was observed in all groups (y = −0.09x + 0.58, r = −0.38, P < 0.05; Fig. 3).
Experiment 2: human study
There was no significant difference in height, body weight, percent body fat, plasma NOx level, blood pressure, heart rate, cfPWV, and V˙O2max among the CON, AT, and HIIT groups before intervention. Furthermore, the amount of change in body weight, percent body fat, and heart rate before and after intervention significantly decreased in the AT group compared with the CON group (Table 2). The amount of change in V˙O2max before and after intervention significantly increased in the AT and HIIT groups as compared with the CON group (Table 2). The amount of change in cfPWV was significantly reduced in both AT and HIIT groups compared with the CON group, and HIIT-induced reduction of cfPWV was equal to that caused by AT (Fig. 4). Furthermore, even after the adjustment for systolic, diastolic, and mean blood pressures and heart rate, significant differences in PWV among the three groups were observed to be the same. Moreover, the amount of change in plasma NOx level was significantly elevated in both AT and HIIT groups compared with the CON group, and HIIT-induced elevation of plasma NOx level was equal to that caused by AT (Fig. 4).
In this study, we revealed the difference in effects of arterial eNOS signaling pathway as well as central arterial stiffness after different exercise programs, including aerobic, resistance, and HIIT training, in the animal study. HIIT effect on aortic PWV, as an index of central arterial stiffness, was the same as the decrease in central arterial stiffness caused by AT. However, it did not decrease after RT. In addition, in the HIIT group, phosphorylation of eNOS and Akt and plasma NOx level increased. The increase was similar to that caused by AT, and a negative correlation between aortic PWV and eNOS phosphorylation or plasma NOx level was observed. However, arterial tissue in the RT group did not show changed phosphorylation of eNOS and Akt and plasma NOx level. Furthermore, we confirmed the effect of HIIT and AT interventions on central arterial stiffness and plasma NOx level in the human study, and showed a reduced cfPWV, as an index of central arterial stiffness, and elevated plasma NOx level after HIIT and AT. In addition, there was a negative correlation between cfPWV and NOx level. Thus, based on both animal and human studies, HIIT may reduce central arterial stiffness via an increase in aortic NO bioavailability despite it being done in a short time and short term and has effect of the same degree in reducing arterial stiffness as does AT.
HIIT used in this study is the shortest total 4- to 7-min exercise program (20-s HIT + 10-s rest) among the reported studies, and the exercise time is between 2 min 30 s and 4 min 40 s. Therefore, HIIT is a brief supramaximal intermittent exercise training. In this study, short-time and short-term HIIT for 6 wk reduced central arterial stiffness (cfPWV) and led to increased aortic production of NO via eNOS signaling pathway. In this human study, HIIT reduced cfPWV by 12.8%, which was comparable to a decrease of 14.7% caused by AT in the untrained healthy young men. Cocks et al. (23) have reported that cfPWV decreased by 7% in 6-wk HIIT program with a total exercise time of 15.5–25.5 min (30-s exercise using Wingate test + 4.5-min 30 W, 4–6 sets) in untrained healthy young men. In addition, in healthy older adults, 9-wk HIIT with a total exercise time of 30 min (6-min ventilation threshold level exercise + 1-min maximal exercise intensities) reduced cfPWV by 5.9% (24). In hypertensive patients, 16-wk HIIT with total exercise time of 40 min (2-min 50% HR reserve + 1-min 80% HR reserve intensities, where HR refers to hear rate) reduced cfPWV by 5.7% (13). Therefore, HIIT (brief supramaximal intermittent exercise training) used in this study may reduce central arterial stiffness more than other HIIT despite it being done in a short time. Therefore, the reduction of central arterial stiffness by HIIT could be sufficiently obtained even with short-time HIIT, if the exercise intensity of HIIT is higher.
A molecular mechanism of HIIT-induced reduction of central arterial stiffness remains unclear. This study revealed activation of Akt-eNOS signaling pathway in arterial tissue with elevation of NO release caused by HIIT and the negative correlation between eNOS phosphorylation and aortic PWV in the animal study. Moreover, HIIT-induced activation of eNOS signaling pathway showed the same effect to that of AT. It is well known that an underlying molecular mechanism for decreased arterial stiffness by AT involves an increase in NO production via activation of eNOS signaling pathway in vascular endothelial cells (25). eNOS in vascular endothelial cells is activated by an increase in shear stress via exercise-induced elevation of blood flow (26). Previous studies reported that AMP-activated protein kinase (AMPK) and peroxisome proliferative–activated receptor gamma coactivator-1α are related to eNOS activation in response to shear stress in vascular endothelial cells (27,28). In addition, AMPK and peroxisome proliferative–activated receptor gamma coactivator-1α are activated in an exercise intensity–dependent manner (27,29). Activation of AMPK was elevated in the aortas of mice that underwent regular high physical activity as compared with mice that underwent low physical activity resulting in elevated arterial AMPK phosphorylation (27). As compared with AT, HIIT has high exercise intensity and high cardiac output during exercise (30) leading to AMPK activation with high shear stress. Therefore, short-time HIIT may activate eNOS to the same degree as AT, if the exercise intensity increases shear stress even during short-time HIIT. In fact, HIIT program with a total exercise time of 15.5–25.5 min (30-s exercise using Wingate test + 4.5-min 30 W, 4–6 sets) increased microvascular eNOS expression in untrained healthy young men (23). Furthermore, in meta-analysis, HIIT elevated the value of flow-mediated dilatation, as an index of endothelial function (31).
In this animal study, RT, which induced muscle hypertrophy, did not change central arterial stiffness and had no effect of eNOS signaling pathway in arterial tissue. In meta-analysis, high-intensity RT, which leads to muscle hypertrophy and increase in muscle strength, had no beneficial effect on arterial stiffness (12). In addition, plasma NOx level and eNOS phosphorylation in arterial tissue have not been changed by RT (32,33). The same results were obtained in this study, and it was considered that high-intensity RT does not affect central arterial stiffness because RT has no effect on activation of eNOS signaling pathway in endothelial cells. A previous study showed that high-intensity RT increased plasma levels of endothelin-1, which plays a role of vasoconstrictor and is produced by vascular endothelial cells (34). Because endothelin-1 declines in eNOS activity by interaction with NOS, even if RT stimulates eNOS signaling pathway, it may be suppressing eNOS activation. However, the effect on arterial stiffness differed with different exercise intensity of RT (12). Further study is needed to examine the molecular mechanism of effect of RT on arterial stiffness.
This animal study showed that AT and HIIT induced reduction of arterial stiffness via upregulation of arterial eNOS signaling pathway, but this effect was not achieved by RT. Furthermore, the beneficial effect of AT and HIIT on arterial stiffness was confirmed in human study. In the human study, shorter HIIT consisting of six to seven sets of 20-s exercises interspersed with a rest period of 10 s is an exercise program with a total duration of only 4 min. Our previous studies demonstrated that HIIT induced reduction of colon cancer risk (35) and an increase in muscle glucose uptake and lactic metabolism (36). Therefore, short-time HIIT may be an effective exercise, because its effects were comparable to those induced by conventional AT despite a decrease in total exercise volume (16). Although the effect of HIIT in this study was observed in healthy young men, the effect of HIIT on other populations remains unclear. Further studies are needed to examine HIIT effects in older adults and various patients.
Circulating NOx levels can be affected by dietary nitrates such as those found in green vegetables and processed meats (37). In this study, we instructed the subjects not to ingest any dietary sources of NOx over 24 h before testing, according to our previous studies (38), and checked their compliance to this instruction. However, we did not assess their diet record information. Therefore, a further study needs to record their dietary intakes in detail for 2 d.
The present study showed that AT and HIIT in healthy young adults and rats reduced the arterial stiffness with an increase in aortic NO bioavailability via upregulation of the arterial eNOS signaling pathway. These findings reveal a preventive mechanism against cardiovascular diseases caused by AT and HIIT. Cohort studies showed that the risk of cardiovascular diseases at a young age is related to future increment in carotid intima–media thickness (39,40). Therefore, the regular exercise–induced reduction in arterial stiffness at a young age may be important to decrease future cardiovascular disease events.
In conclusion, this study revealed the difference in effects of different exercise programs on arterial eNOS signaling pathway as well as central arterial stiffness in both animal and human studies. In the animal study, the effect of HIIT was the same as that of AT, where central arterial stiffness was decreased and eNOS signaling pathway was activated. However, RT did not induce such changes. Furthermore, we confirmed the beneficial effects of HIIT and AT interventions on central arterial stiffness and plasma NOx level in the human study. Thus, HIIT may reduce central arterial stiffness via an increase in aortic NO bioavailability despite it being done in a short time and short term. HIIT produces the same extent of reduction in arterial stiffness as AT.
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI: No. 17H02182 and No. 16K13059 for M. Iemitsu).
The authors have no conflicts of interest to report. The results of the present investigation do not constitute endorsement by the American College of Sports Medicine. The results of the study are presented clearly, honestly, and without fabrication or inappropriate data manipulation.
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