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Association of body flexibility and carotid atherosclerosis in Japanese middle-aged men: a cross-sectional study

Objective This study examined the associations of body flexibility with carotid arterial remodelling, including intima–media thickness (IMT) and plaque formation in middle-aged men. Methods The subjects of this cross-sectional study included 1354 Japanese men aged 35–59 years without histories of stroke or cardiac diseases. The arm extensibility test, which can estimate flexibility of the upper extremity (composed of shoulder external rotation and forearm supination), and the sit-and-reach test were performed. Common carotid IMT and plaque formation (≥1.1 mm) were estimated by ultrasound. Results The proportion of subjects who fully completed the arm extensibility test was 55.0%, and who had plaques in the common carotid artery was 37.8%. IMT was associated with poor arm extensibility (β=–0.073, 95% CI –0.02224 to –0.00041, P=0.004), while plaque formation was associated with poor sit-and-reach (OR 0.98579, 95% CI 0.97257 to 0.99919, P=0.038) after adjustment by all covariates. Conclusions This study demonstrated that poor upper extremity and trunk flexibility were associated with characteristics of early onset of atherosclerosis. Furthermore, these associations were independent of covariates such as age, blood pressure, blood lipids glucose levels and abdominal fat accumulation, handgrip strength and lifestyle, including sleeping, drinking, exercise and smoking habits. Poor flexibility may reflect subclinical atherosclerosis in middle-aged men.


Medical examinations 89
After an overnight fast of ≥11 h, subjects underwent measurement of anthropometry, systolic blood 90 pressure (SBP), diastolic blood pressure (DBP), blood chemistry analyses, ultrasound examination, and 91 computed tomography (CT). Pulse pressure was calculated reducing DBP from SBP. Height and body 92 mass were measured using an automated measuring instrument (BF-220; Tanita, Tokyo, Japan), from 93 which the body mass index was calculated. Percentage of body fat was determined by biochemical 94 impedance (BF-220; Tanita). Waist circumference was measured at the level of the umbilicus in a standing 95 position while breathing normally (at the end of expiration while breathing gently). Blood samples were 96 drawn from the antecubital vein from seated subjects. 97 98

Assessment of carotid IMT and plaques by ultrasound images 99
All estimations of carotid IMT and plaque were performed by well-trained clinical technologists who 100 were blinded to other clinical information. Common carotid IMT and plaque presence were estimated with 101 an Aplio 500 ultrasound device (TUS-A500; Toshiba Medical Systems Co., Otawara, Tochigi, Japan) by 102 manual method using a 7.5 MHz probe frequency. All subjects were examined in the supine position. A 103 long axial section of the carotid artery was probed to show the common carotid artery on both sides to 104 measure IMT and to detect plaques. IMT was measured in the far wall at ~20 mm from the carotid 105  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   7   bifurcation using recorded images of the carotid artery. If plaques were observed around the point, IMT  106 was determined at a point avoiding the plaques. Furthermore, if a defective image of the far wall was 107 observed, IMT was determined in the near wall. The mean IMT of each side was used for analysis. Plaques 108 were defined as elevated lesions with a maximum thickness of ≥1.1 mm, and having a point of inflection 109 on the surface of the intima-media complex as previously reported. 14 The plaque was measured on a short 110 axial section. If multiple plaques were observed, the maximum value was used for analysis.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   9 Upper extremity flexibility was determined by the arm extensibility test, which can estimate the ranges 144 of shoulder external rotation and forearm supination. Details of the arm extensibility test are shown in 145 Figure 2. In brief, the first trial estimated whether an individual was able to fully extend both elbows while 146 interlocking their hands in the right-arm-crossing over position. Next, a similar trial of the 147 left-arm-crossing over position was performed. If the individual fully extended both elbows in both trials, 148 the subject was judged as 'complete'. We confirmed the reproducibility of this test. Observed percent 149 agreements between observers 1 and 2 (n = 58) and between observers 1 and 3 (n = 76) were 98.3% and 150 97.4%, respectively, and κ coefficients between them were 0.96 and 0.94. 151 Trunk flexibility was assessed by a sit-and-reach test using a device (T.K.K. 5412; Takei Scientific 152 Instruments Co., Ltd., Niigata, Japan). Subjects sat on the floor with their hips, back, and occipital region 153 of the head touching a wall, and their legs held straight at 90° in front of the upper body. The zero point of 154 the device was set in this position. Subjects then slowly bent forward and as far as possible while pushing 155 the device with both hands, and the moving distance of the device was measured. Measurements were 156 performed twice, and the maximum value was used in final analyses. 157 Handgrip strength was measured using a handgrip dynamometer (T.K.K. 5401; Takei Scientific 158 Instruments). Measurements were performed in duplicate in each hand, and the mean maximum force of 159 each hand was used in final analyses.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y 10 Data are shown as mean ± standard deviation (SD). An unpaired t-test was used to compare mean 163 values between subjects who did not complete and those who completed in both arm positions in the arm 164 extensibility test, and between subjects who had plaques in the common carotid artery and those who did 165 not. Arm extensibility (incomplete or complete), presence of plaque (yes or no), and lifestyle differences 166 including ≥30 min of exercise (none, or more than once/week), alcohol consumption (none, or more than 167 sometimes), and cigarette smoking (never, former, or current) were compared using the χ 2 test. 168 Multiple linear regression models were performed to estimate the association of IMT with arm 169 extensibility or sit-and-reach. Three models were used to assess these associations; Model 1 was crude, 170 Model 2 was adjusted for age and height, and Model 3 was adjusted for Model 2 covariates plus SFA, VFA, 171 DBP, pulse pressure, TG, HDL-C, LDL-C, FBG, sleeping time, exercise habit, drinking habit, smoking 172 habit, handgrip strength, and sit-and-reach (arm extensibility analysis) or arm extensibility (sit-and-reach 173 analysis). 174 The relationships between the presence of plaque and arm extensibility or sit-and-reach were analyzed 175 using multiple logistic regression models. The same three models used for multiple linear regression 176 analyses for estimating IMT were used. 177 All statistical analyses were performed using statistical software (SPSS v23.0; IBM, Armonk, NY, 178 USA). Differences were considered statistically significant if P < 0.05.    Table 1. 183 The proportion of subjects who fully completed the arm extensibility test was 55.0%, and the proportion of 184 those who had a common carotid plaque was 37.8%. Mean IMT (−6.9%) and the proportion of subjects 185 with a plaque (−26.2%) were significantly lower, while the sit-and-reach test distance (+16.0%) was 186 significantly higher, in subjects who completed the arm extensibility test compared with subjects who did 187 not complete. Furthermore, the proportion of subjects who fully completed the arm extensibility test 188 (−20.3%) and the distance of the sit-and-reach test (−4.5%) were significantly lower, while the mean IMT 189 was significantly higher, in subjects who had a plaque compared with those without plaques. 190

Association between body flexibility indicators 192
The association between sit-and-reach and arm extensibility tests was estimated using linear regression 193 models. Sit-and reach was inversely associated with arm extensibility in non-adjusted (β = -0.190, P < 194 0.001) and age-adjusted (β = -0.086, P = 0.001) models. 195

Association of IMT and body flexibility 197
The associations of IMT with the arm extensibility or sit-and-reach tests using multivariate linear 198 regression models are shown in Table 2. Overall arm extensibility was inversely associated with IMT,199 whereas the sit-and-reach was unrelated to IMT. 200

Association of plaque formation and body flexibility 202
The associations of presence of plaques with the arm extensibility or sit-and-reach tests using 203 multivariate logistic regression models are shown in Table 3. For arm extensibility, there was a significant 204 positive association observed in Model 1, which was no observed after adjusting for covariates. The In the present study, we examined the association of flexibility with subclinical atherosclerosis in 210 middle-aged men. Interestingly, our findings suggested that poor flexibility of the upper extremity was 211 positively associated with IMT, while poor trunk flexibility was associated with presence of plaques in the 212 common carotid artery. These associations remained significant even after adjusting for age, blood 213 pressure, blood lipids, abdominal fat accumulation, handgrip strength, and life styles. Because the arm 214 extensibility and sit-and-reach tests are simple, easy, safe, and inexpensive methods for evaluating physical 215 fitness, we propose that poor flexibility may be a useful marker for subclinical atherosclerosis. 216 Previous studies have reported an association of arterial stiffness with poor trunk flexibility in 217 middle-aged men (40−59 years), 10 11 which may support our findings as arterial stiffness is linked with 218 arterial remodeling. Increased carotid-femoral PWV is also associated with progression of IMT and plaque 219  16 Furthermore, the CAVI score is significantly correlated with carotid 220 IMT 12 and plaque formation. 17 Atherosclerosis is an inflammatory process related to thickening of the 221 arterial walls and plaque formation, which are, at least in part, accompanied by increased atheroma and 222 smooth muscle cells in the intima, increased collagen and decreased elastin in the media, and deposition of 223 calcium and advanced glycation end-products. 18 These characteristics can modify the mechanical 224 properties of arteries. 19 225 A potential explanation for the relationship between flexibility and arterial remodeling is that the 226 exercises that improve flexibility may also regulate remodeling. Indeed, stretching and yoga exercise 227 training can reduce arterial stiffness or enhance arterial compliance. 20-22 Four weeks of static stretching 228 training was reported to reduce the brachial-ankle PWV and CAVI score, and with improved sit-and-reach 229 test performance, in middle-aged men. 20 Furthermore, in older adults, stretching training for 13 weeks 230 increased carotid artery compliance, while strength training or combination of strength and aerobic 231 exercise training, did not. 21 Similarly, in older subjects, 12 weeks of yoga training significantly reduced 232 carotid-femoral PWV and brachial-ankle PWV while there was no effect of brisk walking training. 22 It is 233 likely that good flexibility is associated with low arterial stiffness and possible prevention of 234 atherosclerosis, at least in part, via habitual exercises that can modify flexibility. Although our study design 235 did not allow us to determine the exact cause, we found a potential role of exercises that improve 236 flexibility on arterial remodeling, which partially affected the associations observed in this study. 237 The current study showed that arm extensibility predicted IMT, but not the presence of plaques, while 238  Health-related physical fitness is composed of cardiorespiratory fitness, body composition, muscular 247 strength and endurance, and flexibility. 25 For muscular strength, we provide novel data that handgrip 248 strength is not associated with IMT or plaque formation. Nevertheless, these findings do not rule out an 249 association of muscular strength with atherosclerosis, as we did not estimate the strength of other muscle 250 groups. Indeed, leg extension strength is inversely associated with brachial-ankle PWV, 11 suggesting that 251 reduced lower extremity muscular strength may predict atherosclerosis. Cardiorespiratory fitness is also 252 inversely associated with carotid IMT in older adults (≥60 years). 26 Furthermore, good cardiorespiratory 253 fitness is associated with slower progression of carotid arterial remodeling in middle-aged men (42−60 254 years). 27 It remains unknown whether the association of carotid atherosclerosis with flexibility observed in 255 the present study is mediated by other physical fitness variables. However, it was reported the association 256 of sit-and-reach performance with brachial-ankle PWV is independent of cardiorespiratory fitness and leg 257  This study has four main limitations with respect to generalizability and interpretation of results. 260 Because of its cross-sectional design, we were unable to determine the exact causes of the various 261 relationships in our study. Future longitudinal studies are required to determine the causal relationship 262 between flexibility and atherosclerosis. Second, the characteristics of the subjects in this study limited the 263 generalizability of our results. All participants were middle-aged, male Japanese employees, and our 264 findings cannot be applied to subjects of other ages, genders, or ethnicities. Third, this study estimated only 265 two types of flexibility. The arm extensibility test can evaluate the range of shoulder external rotation and 266 forearm supination, while the sit-and-reach test can assess hamstring and lower back flexibility. However, 267 the association of flexibility of other joints or muscle groups with atherosclerosis remains unknown. Fourth, 268 this study did not measure any indicators of arterial stiffness such as PWV, CAVI, or β stiffness. Therefore, 269 associations among body flexibility, atherosclerosis, and arterial stiffness have been still unclear. Instead, 270 the associations between subclinical atherosclerosis indicators and body flexibility were adjusted by pulse 271 pressure, which may partially reflect stiffened aorta that tends to increase SBP and decrease DBP through a 272 variety of mechanisms. 28 29 30 273 In summary, this cross-sectional study investigated the association of flexibility with subclinical 274 atherosclerosis in Japanese middle-aged men. Poor arm extensibility was associated with increased 275 common carotid IMT, while poor sit-and-reach performance was associated with carotid plaque formation. 276

BMJ Open
Multiple linear regression models were performed to estimate the association of IMT with arm 174 extensibility or sit-and-reach. Three models were used to assess these associations; Model 1 was crude, 175 Model 2 was adjusted for age and height, and Model 3 was adjusted for Model 2 covariates plus SFA, VFA, 176 DBP, pulse pressure, TG, HDL-C, LDL-C, FBG, sleeping time, exercise habit, drinking habit, smoking 177 habit, handgrip strength, and sit-and-reach (arm extensibility analysis) or arm extensibility (sit-and-reach 178 analysis). 179 The relationships between the presence of plaque and arm extensibility or sit-and-reach were analyzed 180 using multiple logistic regression models. The same three models used for multiple linear regression 181

Anthropometry and medical and fitness examinations 187
Anthropometry data and medical and fitness examinations in the study subjects are shown in Table 1. 188 The proportion of subjects who fully completed the arm extensibility test was 55.0%, and the proportion of 189 those who had a common carotid plaque was 37.8%. Mean IMT (−6.9%) and the proportion of subjects 190 with a plaque (−26.2%) were significantly lower, while the sit-and-reach test distance (+16.0%) was 191 significantly higher, in subjects who completed the arm extensibility test compared with subjects who did 192 not complete. Furthermore, the proportion of subjects who fully completed the arm extensibility test 193 (−20.3%) and the distance of the sit-and-reach test (−4.5%) were significantly lower, while the mean IMT 194 was significantly higher, in subjects who had a plaque compared with those without plaques. 195

Association of IMT and body flexibility 202
The associations of IMT with the arm extensibility or sit-and-reach tests using multivariate linear 203 regression models are shown in Table 2. Overall arm extensibility was inversely associated with IMT, 204 whereas the sit-and-reach was unrelated to IMT. 205 206

Association of plaque formation and body flexibility 207
The associations of presence of plaques with the arm extensibility or sit-and-reach tests using 208 multivariate logistic regression models are shown in Table 3. For arm extensibility, there was a significant 209 positive association observed in Model 1, which was no observed after adjusting for covariates. The In the present study, we examined the association of flexibility with subclinical atherosclerosis in 215 middle-aged men. Interestingly, our findings suggested that poor flexibility of the upper extremity was 216 positively associated with IMT, while poor trunk flexibility was associated with presence of plaques in the 217 common carotid artery. These associations remained significant even after adjusting for age, blood  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  Previous studies have reported an association of arterial stiffness with poor trunk flexibility in 222 middle-aged men (40−59 years), 10 11 which may support our findings as arterial stiffness is linked with 223 arterial remodeling. Increased carotid-femoral PWV is also associated with progression of IMT and plaque 224 formation in the carotid artery. 16 Furthermore, the CAVI score is significantly correlated with carotid 225 IMT 12 and plaque formation. 17 Atherosclerosis is an inflammatory process related to thickening of the 226 arterial walls and plaque formation, which are, at least in part, accompanied by increased atheroma and 227 smooth muscle cells in the intima, increased collagen and decreased elastin in the media, and deposition of 228 calcium and advanced glycation end-products. 18 These characteristics can modify the mechanical 229

properties of arteries. 19 230
A potential explanation for the relationship between flexibility and arterial remodeling is that the 231 exercises that improve flexibility may also regulate remodeling. Indeed, stretching and yoga exercise 232 training can reduce arterial stiffness or enhance arterial compliance. 20-22 Four weeks of static stretching 233 training was reported to reduce the brachial-ankle PWV and CAVI score, and with improved sit-and-reach 234 test performance, in middle-aged men. 20 Furthermore, in older adults, stretching training for 13 weeks 235 increased carotid artery compliance, while strength training or combination of strength and aerobic 236 exercise training, did not. 21 Similarly, in older subjects, 12 weeks of yoga training significantly reduced 237 carotid-femoral PWV and brachial-ankle PWV while there was no effect of brisk walking training. 22   likely that good flexibility is associated with low arterial stiffness and possible prevention of 239 atherosclerosis, at least in part, via habitual exercises that can modify flexibility. Although our study design 240 did not allow us to determine the exact cause, we found a potential role of exercises that improve 241 flexibility on arterial remodeling, which partially affected the associations observed in this study. 242 The current study showed that arm extensibility predicted IMT, but not the presence of plaques, while 243 the sit-and-reach test predicted the presence of plaques, but not IMT. The causes of these contrasting 244 findings are unclear, although differences in the biological characteristics of the IMT and plaques may be Health-related physical fitness is composed of cardiorespiratory fitness, body composition, muscular 252 strength and endurance, and flexibility. 25 For muscular strength, we provide novel data that handgrip 253 strength is not associated with IMT or plaque formation. Nevertheless, these findings do not rule out an 254 association of muscular strength with atherosclerosis, as we did not estimate the strength of other muscle 255 groups. Indeed, leg extension strength is inversely associated with brachial-ankle PWV, 11 suggesting that 256 reduced lower extremity muscular strength may predict atherosclerosis. Cardiorespiratory fitness is also 257   In summary, this cross-sectional study investigated the association of flexibility with subclinical 279 atherosclerosis in Japanese middle-aged men. Poor arm extensibility was associated with increased 280 common carotid IMT, while poor sit-and-reach performance was associated with carotid plaque formation.

Methods 72
Ethical approval of the study protocol 73 The study was conducted in accordance with the Declaration of Helsinki. The study protocol was 74 approved by the Ethics Committee of Toyota Memorial Hospital (Aichi, Japan). All individuals provided 75 written consent to participate in this study. 76 77

Anthropometry and medical and fitness examinations 181
Anthropometry data and medical and fitness examinations in the study subjects are shown in Table 1. A 182 total of 745 subjects (55.0%) fully completed the arm extensibility test, and 512 subjects (37.8%) had a 183 common carotid plaque. Mean IMT (−6.9%) and the proportion of subjects with a plaque (−26.2%) were 184 significantly lower, while the sit-and-reach test distance (+16.0%) was significantly higher, in subjects who 185 completed the arm extensibility test compared with subjects who did not complete. Furthermore, the 186 proportion of subjects who fully completed the arm extensibility test (−20.3%) and the distance of the 187 sit-and-reach test (−4.5%) were significantly lower, while the mean IMT was significantly higher, in 188 subjects who had a plaque compared with those without plaques. 189 190