Journal of Atherosclerosis and Thrombosis Vol. 22, No. 2
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Original Article
The Cardio-Ankle Vascular Index and Ankle-Brachial Index in Young Russians Alexander Sorokin 1, Kazuhiko Kotani 2, Olga Bushueva 3, Nobuyuki Taniguchi 2 and Viktor Lazarenko 4 1
Department of Internal Medicine, Kursk State Medical University, Kursk, Russia Department of Clinical Laboratory Medicine, Jichi Medical University, Tochigi, Japan 3 Department of Biology, Medical Genetics and Ecology, Kursk State Medical University, Kursk, Russia 4 Department of Surgery, Kursk State Medical University, Kursk, Russia 2
Aim: A noninvasive approach to assess atherosclerosis in young people is of great concern. The cardio-ankle vascular index (CAVI) and ankle-brachial index (ABI) reflect the arterial conditions, although the CAVI has not fully been studied in Russian populations. This study aimed to determine the CAVI and ABI in young Russians, to compare these findings with those in their Japanese peers and to investigate the lifestyle correlates and genetic associations with the CAVI and ABI in the Russians. Methods: In addition to several atherosclerotic parameters and self-reported lifestyle factors, the CAVI and ABI levels were measured in 114 Russians (mean 21 years). Four gene polymorphisms, including cholesteryl ester transfer protein (CETP) Taq1B polymorphism, were typed in some of the subjects. Results: The Russians exhibited significantly higher CAVI levels compared to their Japanese counterparts (5.87 vs. 5.36; p ＜ 0.05), while the ABI levels were similar between the two populations. In the Russians, the ABI was significantly correlated with the mean blood pressure (r =−0.26) and heart rate (r =−0.43), while the CAVI did not show such correlations. No significant associations existed between lifestyle-related factors and the CAVI or ABI levels. A lower ABI level was found in carriers with the T-allele of CETP Taq1B in the Russians. Conclusions: The reference CAVI value can be specified for individual ethnic populations. Our findings suggest that Russians may develop atherosclerosis-related conditions at a younger age compared to Japanese subjects, although this must be verified in additional studies. The possible association between CETP polymorphisms and the ABI deserves further investigation. J Atheroscler Thromb, 2015; 22:211-218. Key words: Cardio-ankle vascular index, Arterial stiffness, Lifestyle, Gene polymorphism
Introduction Cardiovascular disease (CVD) remains the main cause of death and disability in Europe and in developed countries worldwide 1). According to a recent report, the death rate for both males and females younger than 65 living in the Russian Federation is Address for correspondence: Sorokin Alexander, Department of Internal Medicine, Kursk State Medical University, K. Marx St.3, 305041, Kursk, Russia E-mail: [email protected] Received: May 12, 2014 Accepted for publication: August 10, 2014
much higher than that in other European countries 2). However, a decrease in CVD-related mortality has been seen in modern Russia, where the primary health care system has functionally changed 3). Cost-effective and clinically relevant tools for the early detection and/or strategies to prevent CVD in younger people are needed 4). Atherogenesis is the basic cause underlying the development of CVD 5). Arterial stiffness and endothelial dysfunction are associated with the progression of atherosclerosis, including vascular plaque formation, along with arterial media remodeling 6, 7), and these are predictive of damage to target organs such as
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the heart, kidneys and brain 8). The cardio-ankle vascular index (CAVI) and ankle-brachial index (ABI) are clinically used as noninvasive methods to detect such arteriosclerotic pathologies 9-11). These indices have both shown clinical significance in CVD-related practice 9-11). When evaluating the arterial conditions, the CAVI has the advantage of being less dependent on the blood pressure (BP) measurement 12, 13). Despite the general acceptance of the CAVI and ABI for assessing the extent of arteriosclerosis, very few measurements of the CAVI are currently performed in the clinical setting in Russia. In contrast, CAVI assessment has been extensively incorporated into the routine clinical practice in several other countries, especially in Japan, where the CAVI was developed 10, 11). The vast majority of academic publications about the CAVI are thus from Asian countries, but these studies lack specific data on the ethnic variability of the CAVI values and the related factors 9). Ethnic characteristics can affect the arterial conditions, and likely influence the reference values of the CAVI for healthy people 4). In fact, the age-standardized death rate for CVD (per 100.000) is 366 in both Russian males and females, compared to 41 in Japan according to a recent database of mortality maintained by the World Health Organization (WHO) 14). Additionally, several CVD-related single nucleotide polymorphisms (SNPs) in gene may contribute to the arterial conditions. Here, we examined representative SNPs in angiotensin-converting enzyme (ACE), nitric oxide synthase 3 (NOS3), cholesteryl ester transfer protein (CETP) and lipoprotein lipase (LPL) for a detailed analysis. The current study aimed to determine the CAVI and ABI levels in young Russian subjects in comparison to their Japanese peers, and to investigate the lifestyle correlates and genetic associations of the CAVI and ABI levels in young Russians. Materials and Methods Subjects A total of 115 consecutive healthy volunteers (25 males, 90 females), aged 19-29 years, were enrolled in a study at Kursk State Medical University (KSMU) in Kursk, Russia. All recruited subjects were asymptomatic, and had no known history of cardiovascular, cerebrovascular, kidney or liver diseases. This study was approved by the Ethics Committee of KSMU, and each participant gave informed consent. In a comparative study of the CAVI and ABI levels, the data for Japanese peers from a previous study 4) were used in an age- and gender-matched fashion, with one Japanese subject for every two Russians. Because the number of
Russian males was odd, one Japanese subject was allocated to one Russian subject in one case. Both Russian and Japanese subjects were volunteers recruited from local medical students. Measurements of Atherosclerotic Parameters The body mass index (BMI) was calculated based on the weight and height in each subject wearing light clothing without shoes. The heart rate (HR), blood pressure (BP), CAVI and ABI were measured using the VaSera VS-1500N vascular screening system (Fukuda Denshi Co. Ltd, Tokyo, Japan) 11). Briefly, these indices were measured in the morning after 10 minutes of rest, with cuffs applied to the bilateral upper and lower extremities while the subject was in the supine position. The CAVI was estimated from the brachial and ankle pulse wave forms by using appropriate cuffs for each subject’s arm according to the manufacturer’s instructions. Electrocardiography, phonocardiography and BP measurements were performed simultaneously. The mean BP (MBP) was calculated based on the systolic BP (SBP) and diastolic BP (DBP) values. The stiffness parameter was calculated by the following equation: CAVI = In (Ps/Pd)× 2p/ΔP×PWV2, where Ps is the SBP, Pd is the DBP, p is the blood viscosity, ΔP is Ps-Pd and PWV is the pulse wave velocity from the aortic origin to the ankle via femoral artery. The ABI was measured based on the SBP for both the upper (brachial artery) and lower (tibial artery) BP, and was then was obtained by dividing the ankle SBP by the brachial SBP. The obtained data were automatically analyzed using the VaSera(R) Data Management software program (version: V1001, Fukuda Denshi Co. Ltd, Tokyo, Japan). Lifestyle Factors The subject’s lifestyle-related factors were obtained via a self-administered questionnaire. The study used a formulated questionnaire about the current status of smoking, alcohol intake, physical activity, sports experience, salt intake, meat intake, dairy intake, vegetable intake, carbohydrate intake, fat intake, meal frequency, water intake, personality type, mental conditions at home and mental conditions in the work place. SNPs Typing Approximately 8-10 mL of venous blood was collected into EDTA-coated tubes from each subject in this study (n = 90), and samples were stored at −20 ℃ until they were analyzed. Genomic DNA was isolated using a standard phenol/chloroform procedure. DNA samples were genotyped for the four selected SNPs:
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Table 1. A comparison of the demographics, CAVI and ABI between young Russian and Japanese subjects Parameter Subjects, n Gender, male/female, n Age, years Current smoking, n (%) Body mass index, kg/m2 Heart rate, bpm Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Mean blood pressure, mmHg CAVI ABI
Russian
Japanese
p
114 25/89 21.5±1.9 12 (11%) 21.9±3.5 70±12 124±11 75±7 92±8 5.87±0.80 1.10±0.10
57 13/25 21.3±1.6 2 (4%) 20.8±2.3 58±10 110±10 65±6 80±7 5.36±0.52 1.10±0.07
1.00 0.61 0.15 0.02＊ ＊ ＜ 0.01 ＊ ＜ 0.01 ＊ ＜ 0.01 ＊ ＜ 0.01 ＊a ＜ 0.01 0.67
−
CAVI: cardio-ankle vascular index, ABI: ankle-brachial index. The data are presented as the means±standard deviation or subject numbers (%). ＊p ＜ 0.05: t -test or Fisher’s exact test between the two groups. a p ＜ 0.01 in the age-, gender- and smoking-adjusted comparison; age-, gender-, smoking- and body mass index-adjusted comparison; age-, gender-, smoking-, body mass index- and mean blood pressure-adjusted comparison or age-, gender-, smoking-, body mass index-, mean blood pressure- and heart rate-adjusted comparison.
ACE I/D (rs4646994), NOS3 -786T/C (rs2070744), CETP Taq1B (rs708272) and LPL HindIII (rs320). The I and D alleles of ACE were detected using an amplified fragment length polymorphism assay according to a previously described method 15). The PCR products were analyzed using electrophoresis on 2% agarose gels containing ethidium bromide, and were visualized under UV light on the GDS-8000 Computer Detection System (UVP Inc, CA, USA). For NOS3 -786T/C, CETP Taq1B and LPL HindIII, genomic DNA fragments were amplified by PCR in a 25 μL PCR reaction mixture containing 20-40 ng of DNA, deoxynucleotide triphosphates (0.2 mM of each), 10 pmol of each of the primers, 5 pmol of each of the probes, 1 x PCR-buffer (67 mM Tris-HCl pH 8.8, 16.6 mM (NH4)2SO4, 0.01% Tween-20) and 1.5 U of Taq DNA polymerase (SibEnzyme, Novosibirsk, Russia). The reactions contained 2.5 mM MgCl2 for NOS3 -786T/C, 2 mM MgCl2 for CETP Taq1B and 3.5 mM MgCl2 for LPL HindIII. The genotypes were detected by the CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad Laboratories, CA, USA). Statistical Analysis Differences between the groups were assessed by t -tests, Fisher’s exact tests or adjusted comparison tests (using general linear models). The correlations between parameters were calculated using a Pearson’s correlation test and a multiple regression analysis. Mutated alleles were determined according to the methodology of previous reports 16-20). Values of p ＜ 0.05 were considered to be statistically significant.
Results The clinical characteristics of the study subjects (Russian subjects: n = 114; one subject was excluded from the original cohort because of missing data) are summarized in Table 1. The mean age and gender proportions were similar between the Russian subjects and their Japanese peers (based on the age- and gender-matched cohorts). There was a non-significant difference in the prevalence of current smoking between the two populations. Significant differences were observed for the BMI (higher in Russians), HR (higher in Russians) and BP (higher in Russians). The ABI level was ＞ 0.9 in all subjects. The CAVI levels were significantly higher in the Russian subjects compared to their Japanese counterparts, while the ABI levels were similar between the two populations. The difference in the CAVI between the two populations remained independently significant, even after adjusting for age, gender and smoking (Adjusted model 1: p ＜ 0.01); age, gender, smoking and BMI (Adjusted model 2: p ＜ 0.01); age, gender, smoking, BMI and MBP (Adjusted model 3: p ＜ 0.01) or age, gender, smoking, BMI, MBP and HR (Adjusted model 4: p ＜ 0.01). The correlations of the CAVI and ABI with the atherosclerotic parameters in the Russian subjects are shown in Table 2. The CAVI was not significantly correlated with the MBP (r =−0.16). The CAVI showed an inverse correlation with the HR (r =−0.22), but after adjusting for age, gender, smoking, BMI and MBP, the correlation was decreased to a non-signifi-
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Table 2. The correlation coefficients for conventional factors and the CAVI or ABI in young Russian subjects Parameter Gender, male Age, years Current smoking, n (%) Body mass index, kg/m2 Heart rate, bpm Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Mean blood pressure, mmHg
CAVI
ABI
0.08 (0.42)
0.12 (0.22)
−0.11 (0.25)
−0.07 (0.45)
−0.15 (0.12)
−0.02 (0.80)
−0.10 (0.28)
0.11 (0.23)
−0.22 (0.02)
−0.43 (＜ 0.01)
−0.09 (0.35)
−0.35 (＜ 0.01)
−0.19 (0.04)
−0.15 (0.11)
−0.16 (0.09)
−0.26 (＜ 0.01)
＊a
＊d
＊b ＊c
＊e
CAVI: cardio-ankle vascular index, ABI: ankle-brachial index. Statistical data are given as correlation coefficients (p-values). ＊p ＜ 0.05: Pearson’s correlation test. aβ=−0.18 (p = 0.09) after adjusting for age, gender, smoking, body mass index and mean blood pressure. bβ=−0.37 (p ＜ 0.01) after adjusting for age, gender, smoking, body mass index and mean blood pressure. cβ=−0.45 (p ＜ 0.01) after adjusting for age, gender, smoking, body mass index and heart rate. dβ=−0.14 (p = 0.18) after adjusting for age, gender, smoking, body mass index and heart rate. eβ=−0.26 (p ＜ 0.01) after adjusting for age, gender, smoking, body mass index and heart rate.
cant level (β=−0.18: p = 0.09). The CAVI also showed an inverse correlation with the DBP (r =−0.19); however, after adjusting for age, gender, smoking, BMI and HR, the correlation became non-significant (β=−0.14: p = 0.18). The ABI was significantly and inversely correlated with the SBP (r =−0.35) and MBP (r =−0.26). The correlation remained significant, even after adjusting for age, gender, smoking, BMI and HR (SBP: β= −0.45: p ＜ 0.01, MBP: β=−0.26: p ＜ 0.01). In addition, the ABI was also significantly and inversely correlated with the HR (r =−0.43), and even after adjusting for age, gender, smoking, BMI and MBP, the correlation remained significant (β=−0.37: p ＜ 0.01). The associations between lifestyle factors and the CAVI or ABI levels in the Russian subjects are shown in Table 3. Overall, there were no significant differences between the lifestyle-related factors and CAVI levels. Similarly, no significant differences between the ABI were found for the lifestyle-related factors. The detection of the ACE, NOS3, LPL and CETP genotypes was completed in 85, 87, 79 and 83 subjects, respectively (the PCR products could not been obtained from the samples of some subjects due to material and technical reasons). The distributions for all four gene SNPs were in Hardy-Weinberg equilibrium. Non-significant associations were observed between these SNPs and the CAVI levels, as shown in Table 4. While non-significant associations were found between the SNPs of ACE, NOS3 or LPL and the ABI, a significant difference was found for the CETP Taq1B SNPs and ABI levels. The frequency distribution was 14 (16.9%) CC-homozygotes of CETP, 40 (48.2%) CT-heterozygotes and 29 (34.9%) TT-homozygotes. A significantly lower ABI level was observed
in carriers with the ‘T’ allele (mutated allele) of CETP than in those without. This difference remained significant (p = 0.04), even after adjusting for age, gender, smoking, BMI, HR and MBP. Discussion In the current study, we observed ethnic differences in the CAVI levels between the young Russian and Japanese subjects. Although the Russian subjects had higher HR and BP levels, these levels were independent of the differences in the CAVI among the two populations. While this is the first report using the CAVI for healthy younger Russian people, a previous study, which used the brachial-ankle pulse wave velocity (baPWV: an indicator of arterial stiffness), showed that healthy Russian individuals might have higher arterial stiffness values compared to their Japanese counterparts 21). In that report, the authors simply described the findings in Russian subjects under 39 years of age 21). Additionally, some differences are known to exist between the baPWV and the CAVI levels, because the CAVI has less dependence on the BP 11, 13). Thus, it is hard to directly compare those data 21) with our present data. Another ethnic comparison study of the CAVI and ABI levels was carried out between young Japanese and Mongolian subjects 4). That study demonstrated that the CAVI levels were low in Japanese people (the value was 5.4) and high in their Mongolian peers (6.5), while the ABI levels showed a non-significant difference between the two populations. In addition to that study 4), the current study between Japanese and Russian subjects further suggests the exis-
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Table 3. The associations of the lifestyle factors with the CAVI or ABI in young Russian subjects Factors CAVI Current smoking Alcohol intake ( ＞ light-to-moderate) Physical activity ( ＞ once/week) Sports experience ( ＞ periodic) Salt intake (high) Meat intake ( ＞ four times/week) Dairy intake ( ＞ four times/week) Vegetable intake ( ＞ four times/week) Carbohydrate intake ( ＞ four times/week) Fat intake ( ＞ four times/week) Meal frequency (three times/day) Meal regularity (regular everyday) Water intake ( ＜ one liter/day) Personality type (aggressive) Mental condition at home ( ＞ good) Mental condition at work ( ＞ good) ABI Current smoking Alcohol intake ( ＞ light-to-moderate) Physical activity ( ＞ once/week) Sports experience ( ＞ periodic) Salt intake (high) Meat intake ( ＞ four times/week) Dairy intake ( ＞ four times/week) Vegetable intake ( ＞ four times/week) Carbohydrate intake ( ＞ four times/week) Fat intake ( ＞ four times/week) Meal frequency (three times/day) Meal regularity (regular everyday) Water intake ( ＜ one liter/day) Personality type (aggressive) Mental condition at home ( ＞ good) Mental condition at work ( ＞ good)
Yes (number)
No (number)
p
5.53±0.77 (n = 12) 5.86±0.80 (n = 69) 5.63±0.97 (n = 15) 5.88±0.83 (n = 91) 5.42±0.87 (n = 4) 6.03±0.79 (n = 45) 5.90±0.89 (n = 27) 5.79±0.78 (n = 54) 6.01±0.82 (n = 37) 6.45±0.33 (n = 3) 5.87±0.91 (n = 57) 5.84±0.91 (n = 12) 5.70±0.69 (n = 19) 5.77±0.85 (n = 32) 5.87±0.80 (n = 109) 5.86±0.81 (n = 112)
5.91±0.80 (n = 102) 5.88±0.81 (n = 45) 5.90±0.77 (n = 99) 5.83±0.68 (n = 23) 5.88±0.80 (n = 110) 5.76±0.80 (n = 69) 5.86±0.78 (n = 60) 5.94±0.82 (n = 60) 5.80±0.79 (n = 77) 5.85±0.81 (n = 111) 5.87±0.69 (n = 57) 5.87±0.79 (n = 102) 5.90±0.82 (n = 95) 5.91±0.78 (n = 82) 5.76±0.98 (n = 5) 6.16±0.69 (n = 2)
0.12 0.87 0.23 0.78 0.26 0.07 0.84 0.34 0.19 0.20 0.98 0.91 0.32 0.42 0.75 0.61
1.09±0.12 (n = 12) 1.10±0.10 (n = 69) 1.12±0.12 (n = 15) 1.10±0.10 (n = 91) 1.08±0.07 (n = 4) 1.12±0.11 (n = 45) 1.09±0.10 (n = 27) 1.10±0.10 (n = 54) 1.09±0.10 (n = 37) 1.15±0.15 (n = 3) 1.09±0.10 (n = 57) 1.11±0.10 (n = 12) 1.08±0.10 (n = 19) 1.07±0.10 (n = 32) 1.10±0.10 (n = 109) 1.10±0.10 (n = 112)
1.10±0.10 (n = 102) 1.10±0.09 (n = 45) 1.10±0.09 (n = 99) 1.07±0.09 (n = 23) 1.10±0.10 (n = 110) 1.08±0.09 (n = 69) 1.10±0.10 (n = 87) 1.09±0.10 (n = 60) 1.10±0.10 (n = 77) 1.10±0.10 (n = 111) 1.11±0.09 (n = 57) 1.10±0.10 (n = 102) 1.10±0.10 (n = 95) 1.11±0.10 (n = 82) 1.04±0.09 (n = 5) 1.13±0.03 (n = 2)
0.80 0.90 0.36 0.14 0.69 0.10 0.53 0.66 0.46 0.35 0.21 0.65 0.39 0.10 0.22 0.63
CAVI: cardio-ankle vascular index, ABI: ankle-brachial index. The data are expressed as the means±standard deviation. ＊p ＜ 0.05: t -test between the two groups.
tence of differences in the reference values of the CAVI that are specific for each ethnic population. Additionally, considering the clinical significance of the CAVI for the development of CVD 10, 11), as well as the ethnic diversity in the prevalence and impact of CVD 4, 14), the possible ethnic differences in the CAVI values indicate the presence of possible populationrelated differences in the atherosclerotic burden among younger subjects. Namely, Russians may develop atherosclerosis-related conditions at a younger age than Japanese subjects, which is supported by the WHO data for CVD 14).
In the current study, the CAVI was not dependent on the BP, which is in agreement with the previous findings (again, this is a known characteristic of the CAVI) 11, 13). The ethnic differences observed in the occurrence of CVD may be, at least in part, attributable to lifestyle/environmental factors 22-24); however, the current study did not find any associations between the lifestyle-related factors examined and the CAVI levels. Although the questionnaire was not the same as the one that was applied previously 4), non-significant associations were also noted between lifestylerelated factors and the CAVI levels in younger Japa-
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Table 4. The association of gene polymorphisms with the CAVI or ABI levels in young Russian subjects Factors CAVI ACE (D allele) NOS3 (C allele) LPL (G allele) CETP (T allele) ABI ACE (D allele) NOS3 (C allele) LPL (G allele) CETP (T allele)
Present (number)
Absent (number)
p
5.84±0.83 (n = 61) 5.75±0.69 (n = 50) 5.80±0.97 (n = 32) 5.84±0.80 (n = 69)
5.78±0.76 (n = 24) 5.90±0.93 (n = 37) 5.84±0.73 (n = 47) 5.97±0.62 (n = 14)
0.77 0.41 0.83 0.57
1.09±0.09 (n = 61) 1.10±0.10 (n = 50) 1.12±0.11 (n = 31) 1.09±0.09 (n = 68)
1.12±0.12 (n = 23) 1.10±0.09 (n = 36) 1.10±0.09 (n = 47) 1.15±0.13 (n = 14)
0.30 0.98 0.40 a 0.02＊
ACE: angiotensin converting enzyme, NOS: endothelial nitric oxide synthase, LPL: lipoprotein lipase, CETP: cholesteryl ester transfer protein, CAVI: cardio-ankle vascular index, ABI: ankle-brachial index. The data are presented as the means±standard deviation. ＊p ＜ 0.05: t -test between the two groups. a p = 0.04 after adjusting for age, gender, smoking, body mass index, heart rate and mean blood pressure.
nese and Mongolian subjects in a previous study. The influences of lifestyle-related factors on the arterial conditions, as expressed by the CAVI values, are apparently minimal in healthy younger populations, and may only be detected upon disease progression in older populations. The ABI is helpful for determining the arterial conditions linked to the lower limbs (i.e., the existence of peripheral arterial disease) 25), and is a predictor of CVD events 26). Even when the ABI level is ＞ 0.9 (as in our study subjects), the ABI measurement is useful for predicting the development of CVD 26). The clinical significance of the ABI generally differs from that of the CAVI. However, similar to the CAVI, we did not observe any associations between lifestyle-related factors and the ABI levels in the current study. The correlations between atherosclerotic parameters and the ABI showed different patterns of changes from those of the CAVI; namely, the ABI was inversely correlated with the HR and BP (the SBP in particular) in the Russian subjects, which is in agreement with the findings of the previous study in Mongolian and Japanese subjects 4). Four gene SNPs, which are considered to affect the development of CVD, were investigated in the Russian subjects in the present study. The ACE molecule is a key enzyme of the renin-angiotensin system, which regulates the systemic circulatory system 27). The SNPs leading to an insertion/deletion in intron 16 (ACE I/D) have been commonly studied, and have been shown to have a substantial effect on ACE gene expression 28). The NO molecule acts as a pro-inflammatory and pro-atherogenic factor, for instance, via the suppression of vascular contraction and attenua-
tion of oxidative stress and inflammation 7, 18, 29). The NOS3 gene is responsible for the endothelial NOS production 17), and NOS3 -786T＞ C has been reported to be associated with CVD 16). The LPL molecule is an enzyme that controls lipid metabolism by hydrolyzing triglyceride-rich particles via the generation of free fatty acids and glycerol 30), and LPL HindIII was previously reported to be associated with CVD 20). Although all of these SNPs are important risk factors for the development of CVD, the current study did not find any association between the CAVI/ABI and these gene SNPs. However, the ‘T’ allele carriers of CETP Taq1B (rs708272) had lower ABI levels, while there were no association between the CAVI and CETP SNPs in this population. The CETP molecule facilitates cholesteryl ester exchange between high-density lipoproteins and triglyceride-rich lipoproteins 30), and the CETP Taq1B SNPs, located in intron 1 of the CETP gene, have been extensively studied 31). Based on the previous reports showing that some CETP SNPs were associated with blood pressure and CVD (the relationship was noted to be present regardless of the CETP activity) 19, 32), our finding of an association between the CETP Taq1B SNPs and ABI is reasonable, although further biological studies should be conducted to confirm this association. There are some limitations associated with the current study. First, this study was conducted with a small sample and a cross-sectional design. Second, we did not obtain blood chemistry data. Unfortunately, the Japanese subjects were not examined with respect to their lifestyle-related factors using the same questionnaire used for the Russians in the current study, so
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no comparison of the lifestyle-related factors is possible. In addition, the small sample size, especially in the substudy of SNPs, might have resulted in reduced statistical power. Finally, although we think that Russians may develop atherosclerotic conditions at a younger age relative to Japanese subjects, this should be confirmed based on future atherosclerosis manifestations. These limitations should be addressed in future studies.In summary, the current study demonstrated higher CAVI levels in young Russian people compared to their Japanese peers. These data indicate that the reference values for the CAVI should be considered separately for different ethnic populations, and suggests that Russians may develop atherosclerosisrelated conditions at a younger age compared to Japanese people. The possible association between the CETP SNPs and the ABI deserves further study. More research using the CAVI and ABI is warranted in Russian and other populations. Acknowledgement I would like to express my sincere gratitude to Boris Vaisman, PhD, from the Lipoprotein Metabolism Section, Cardio-Pulmonary Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, United States, for his suggestions on the manuscript. Funding No specific funding was provided for the study. Conflict of Interest None declared. Abbreviations ABI – ankle-brachial index; ACE: angiotensinconverting enzyme; baPWV: brachial-ankle pulse wave velocity; CAVI – cardio-ankle vascular index; CETP: cholesteryl ester transfer protein; CVD: cardiovascular disease; HR – heart rate; LPL: lipoprotein lipase; NOS3: nitric oxide synthase 3; SNP – single nucleotide polymorphism. References 1) Nichols M, Townsend N, Scarborough P, Luengo-Fernandez R, Leal J, Gray A, Rayner M: Mortality and Morbidity. In: European Cardiovascular Disease Statistics 2012 Ed. ed Susanne Løgstrup and Sophie O’Kelly, pp 10-45,
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European Heart Network, Brussels, European Society of Cardiology, Sophia Antipolis, 2012 2) Nichols M, Townsend N, Scarborough P, Rayner M: Trends in age-specific coronary heart disease mortality in the European Union over three decades: 1980-2009. Eur Heart J, 2013; 34: 3017-3027 3) Boĭtsov SA, Kalinina AM, Ipatov PV: New clinical and organizational approaches to preventing cardiovascular diseases in the primary health care system. Ter Arkh, 2013; 85: 8-13 4) Uurtuya S, Taniguchi N, Kotani K, Yamada T, Kawano M, Khurelbaatar N, Itoh K, Lkhagvasuren T: Comparative study of the cardio-ankle vascular index and anklebrachial index between young Japanese and Mongolian subjects. Hypertens Res, 2009; 32: 140-144 5) Riccioni G, Sblendorio V: Atherosclerosis: from biology to pharmacological treatment. J Geriatr Cardiol, 2012; 9: 305-317 6) Mattace-Raso FU, van der Cammen TJ, Hofman A, van Popele NM, Bos ML, Schalekamp MA, Asmar R, Reneman RS, Hoeks AP, Breteler MM, Witteman JC: Arterial stiffness and risk of coronary heart disease and stroke: the Rotterdam Study. Circulation, 2006; 113: 657-663 7) van Varik BJ, Rennenberg RJ, Reutelingsperger CP, Kroon AA, de Leeuw PW, Schurgers LJ: Mechanisms of arterial remodeling: lessons from genetic diseases. Front Genet, 2012; 3: 290 8) Nürnberger J, Keflioglu-Scheiber A, Opazo Saez AM, Wenzel RR, Philipp T, Schäfers RF: Augmentation index is associated with cardiovascular risk. J Hypertens, 2002; 20: 2407-2414 9) Sun CK: Cardio-ankle vascular index (CAVI) as an indicator of arterial stiffness. Integr Blood Press Control, 2013; 6: 27-38 10) Kotani K, Remaley AT: Cardio-AnkleVascular Index (CAVI) and its Potential Clinical Implications for Cardiovascular Disease. Cardiol Pharmacol, 2013; 2: 108 11) Shirai K, Utino J, Otsuka K, Takata M: A novel blood pressure-independent arterial wall stiffness parameter; cardio-ankle vascular index (CAVI). J Atheroscler Thromb, 2006; 13: 101-107 12) Nakamura K, Tomaru T, Yamamura S, Miyashita Y, Shirai K, Noike H: Cardio-ankle vascular index is a candidate predictor of coronary atherosclerosis. Circ J, 2008; 72: 598-604 13) Ibata J, Sasaki H, Kakimoto T, Matsuno S, Nakatani M, Kobayashi M, Tatsumi K, Nakano Y, Wakasaki H, Furuta H, Nishi M, Nanjo K: Cardio-ankle vascular index measures arterial wall stiffness independent of blood pressure. Diabetes Res Clin Pract, 2008; 80: 265-270 14) Organization WH: WHO Mortality Database. http:// apps.who.int/healthinfo/statistics/mortality/whodpms/ (March 2012) 15) Rigat B, Hubert C, Corvol P, Soubrier F: PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res, 1992; 20: 1433 16) Piccoli JCE, Manfredini V, Hamester FI, Bandinelli JB, Turkienicz IM, Chies JA, Peres A, Bodanese LC, Bogo MR: Interaction between endothelial nitric oxide synthase
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gene polymorphisms (-786T ＞ C, 894G ＞ T and intron 4 a/b) and cardiovascular risk factors in acute coronary syndromes. Arch Med Res, 2012; 43: 205-211 17) Casas JP, Cavalleri GL, Bautista LE, Smeeth L, Humphries SE, Hingorani AD: Endothelial nitric oxide synthase gene polymorphisms and cardio-vascular disease: a huge review. Am J Epidemiol, 2006; 164: 921-935 18) Piccoli JCE, Manfredini V, Faoro D, Farias F, Bodanese L, Bogo M: Association between endothelial nitric oxide synthase gene polymorphism (-786T ＞ C) and interleukin-6 in acutecoronary syndrome. Hum Exp Toxicol, 2014; 33: 396-402 19) Lu Y, Tayebi N, Li H, Saha N, Yang H, Heng CK: Association of CETP Taq1B and -629C ＞ A polymorphisms with coronary artery disease and lipid levels in the multiethnic Singaporean population. Lipids Health Dis, 2013; 12: 85 20) Sagoo GS, Tatt I, Salanti G, Butterworth AS, Sarwar N, van Maarle M, Jukema JW, Wiman B, Kastelein JJ, Bennet AM, de Faire U, Danesh J, Higgins JP: Seven lipoprotein lipase gene polymorphisms, lipid fractions, and coronary disease: a HuGE association reviewand meta-analysis. Am J Epidemiol, 2008; 168: 1233-1246 21) Liu H, Yambe T, Zhang X, Saijo Y, Shiraishi Y, Sekine K, Maruyama M, Kovalev YA, Milyagina IA, Milyagin VA, Nitta S: Comparison of brachial-ankle pulse wave velocity in Japanese and Russians. Tohoku J Exp Med, 2005; 207: 263-270 22) Razvodovsky YE: Alcohol-attributable fraction of ischemic heart disease mortality in Russia. ISRN Cardiol, 2013; 2013: 1-4 23) Kesse-Guyot E, Vergnaud AC, Fezeu L, Zureik M, Blacher J, Péneau S, Hercberg S, Galan P, Czernichow S: Associations between dietary patterns and arterial stiffness, carotid artery intima-media thickness and atherosclerosis. Eur J Cardiovasc Prev Rehabil, 2010; 17: 718724 24) Gluckman PD, Hanson MA, Buklijas T, Low FM, Beedle AS: Epigenetic mechanisms that underpin metabolic and cardiovascular diseases. Nat Rev Endocrinol, 2009; 5: 401-408 25) Alzamora MT, Forés R, Pera G, Torán P, Heras A, Sorribes M, Baena-Diez JM, Urrea M, Alegre J, Viozquez M, Vela
C: Ankle-brachial index and the incidence of cardiovascular events in the Mediterranean low cardiovascular risk population ARTPER cohort. BMC Cardiovasc Disord, 2013; 13: 119 26) Fowkes F, Murray G, Butcher I, Folsom A, Hirsch AT, Couper DJ, Debacker G, Kornitzer M, Newman AB, Sutton-Tyrrell KC, Cushman M, Lee AJ, Price JF, D'Agostino RB Sr, Murabito JM, Norman P, Masaki KH, Bouter LM, Heine RJ, Stehouwer CD, McDermott MM, Stoffers HE, Knottnerus JA, Ogren M, Hedblad B,Koenig W, Meisinger C, Cauley JA, Franco O, Hunink MG, Hofman A, Witteman JC, Criqui MH, Langer RD, Hiatt WR, Hamman RF; Ankle Brachial Index Collaboration: Development and validation of an ankle brachial index risk model for the prediction of cardiovascular events. Eur J Prev Cardiol, 2013; 21: 310-320 27) Aroor AR, Demarco VG, Jia G, Sun Z, Nistala R, Meininger GA, Sowers JR: The Role of Tissue ReninAngiotensin-Aldosterone System in the Development of Endothelial Dysfunction and Arterial Stiffness. Front Endocrinol (Lausanne), 2013; 4: 161 28) Mizuiri S, Hemmi H, Kumanomidou H, Iwamoto M, Miyagi M, Sakai K, Aikawa A, Ohara T, Yamada K, Shimatake H, Hasegawa A: Angiotensin-converting enzyme (ACE) I/D genotype and renal ACE gene expression. Kidney Int, 2001; 60: 1124-1130 29) Dejam A, Hunter CJ, Schechter AN, Gladwin MT: Emerging role of nitrite in human biology. Blood Cells Mol Dis, 2004; 32: 423-429 30) Rosenson RS, Brewer HB Jr, Davidson WS, Fayad ZA, Fuster V, Goldstein J, Hellerstein M, Jiang XC, Phillips MC, Rader DJ, Remaley AT, Rothblat GH, Tall AR, Yvan-Charvet L: Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport. Circulation, 2012; 125: 1905-1919 31) Hassanzadeh T, Firoozrai M, Zonouz AE, Zavarehee A, Paoli M: Taq1B polymorphism of cholesteryl ester transfer protein (CETP) gene in primary combined hyperlipidaemia. Indian J Med Res, 2009; 129: 293-298 32) Tabas I: Macrophage death and defective inflammation resolution in atherosclerosis. Nat Rev Immunol, 2010; 10: 36-46
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Original Article
The Cardio-Ankle Vascular Index and Ankle-Brachial Index in Young Russians Alexander Sorokin 1, Kazuhiko Kotani 2, Olga Bushueva 3, Nobuyuki Taniguchi 2 and Viktor Lazarenko 4 1
Department of Internal Medicine, Kursk State Medical University, Kursk, Russia Department of Clinical Laboratory Medicine, Jichi Medical University, Tochigi, Japan 3 Department of Biology, Medical Genetics and Ecology, Kursk State Medical University, Kursk, Russia 4 Department of Surgery, Kursk State Medical University, Kursk, Russia 2
Aim: A noninvasive approach to assess atherosclerosis in young people is of great concern. The cardio-ankle vascular index (CAVI) and ankle-brachial index (ABI) reflect the arterial conditions, although the CAVI has not fully been studied in Russian populations. This study aimed to determine the CAVI and ABI in young Russians, to compare these findings with those in their Japanese peers and to investigate the lifestyle correlates and genetic associations with the CAVI and ABI in the Russians. Methods: In addition to several atherosclerotic parameters and self-reported lifestyle factors, the CAVI and ABI levels were measured in 114 Russians (mean 21 years). Four gene polymorphisms, including cholesteryl ester transfer protein (CETP) Taq1B polymorphism, were typed in some of the subjects. Results: The Russians exhibited significantly higher CAVI levels compared to their Japanese counterparts (5.87 vs. 5.36; p ＜ 0.05), while the ABI levels were similar between the two populations. In the Russians, the ABI was significantly correlated with the mean blood pressure (r =−0.26) and heart rate (r =−0.43), while the CAVI did not show such correlations. No significant associations existed between lifestyle-related factors and the CAVI or ABI levels. A lower ABI level was found in carriers with the T-allele of CETP Taq1B in the Russians. Conclusions: The reference CAVI value can be specified for individual ethnic populations. Our findings suggest that Russians may develop atherosclerosis-related conditions at a younger age compared to Japanese subjects, although this must be verified in additional studies. The possible association between CETP polymorphisms and the ABI deserves further investigation. J Atheroscler Thromb, 2015; 22:211-218. Key words: Cardio-ankle vascular index, Arterial stiffness, Lifestyle, Gene polymorphism
Introduction Cardiovascular disease (CVD) remains the main cause of death and disability in Europe and in developed countries worldwide 1). According to a recent report, the death rate for both males and females younger than 65 living in the Russian Federation is Address for correspondence: Sorokin Alexander, Department of Internal Medicine, Kursk State Medical University, K. Marx St.3, 305041, Kursk, Russia E-mail: [email protected] Received: May 12, 2014 Accepted for publication: August 10, 2014
much higher than that in other European countries 2). However, a decrease in CVD-related mortality has been seen in modern Russia, where the primary health care system has functionally changed 3). Cost-effective and clinically relevant tools for the early detection and/or strategies to prevent CVD in younger people are needed 4). Atherogenesis is the basic cause underlying the development of CVD 5). Arterial stiffness and endothelial dysfunction are associated with the progression of atherosclerosis, including vascular plaque formation, along with arterial media remodeling 6, 7), and these are predictive of damage to target organs such as
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the heart, kidneys and brain 8). The cardio-ankle vascular index (CAVI) and ankle-brachial index (ABI) are clinically used as noninvasive methods to detect such arteriosclerotic pathologies 9-11). These indices have both shown clinical significance in CVD-related practice 9-11). When evaluating the arterial conditions, the CAVI has the advantage of being less dependent on the blood pressure (BP) measurement 12, 13). Despite the general acceptance of the CAVI and ABI for assessing the extent of arteriosclerosis, very few measurements of the CAVI are currently performed in the clinical setting in Russia. In contrast, CAVI assessment has been extensively incorporated into the routine clinical practice in several other countries, especially in Japan, where the CAVI was developed 10, 11). The vast majority of academic publications about the CAVI are thus from Asian countries, but these studies lack specific data on the ethnic variability of the CAVI values and the related factors 9). Ethnic characteristics can affect the arterial conditions, and likely influence the reference values of the CAVI for healthy people 4). In fact, the age-standardized death rate for CVD (per 100.000) is 366 in both Russian males and females, compared to 41 in Japan according to a recent database of mortality maintained by the World Health Organization (WHO) 14). Additionally, several CVD-related single nucleotide polymorphisms (SNPs) in gene may contribute to the arterial conditions. Here, we examined representative SNPs in angiotensin-converting enzyme (ACE), nitric oxide synthase 3 (NOS3), cholesteryl ester transfer protein (CETP) and lipoprotein lipase (LPL) for a detailed analysis. The current study aimed to determine the CAVI and ABI levels in young Russian subjects in comparison to their Japanese peers, and to investigate the lifestyle correlates and genetic associations of the CAVI and ABI levels in young Russians. Materials and Methods Subjects A total of 115 consecutive healthy volunteers (25 males, 90 females), aged 19-29 years, were enrolled in a study at Kursk State Medical University (KSMU) in Kursk, Russia. All recruited subjects were asymptomatic, and had no known history of cardiovascular, cerebrovascular, kidney or liver diseases. This study was approved by the Ethics Committee of KSMU, and each participant gave informed consent. In a comparative study of the CAVI and ABI levels, the data for Japanese peers from a previous study 4) were used in an age- and gender-matched fashion, with one Japanese subject for every two Russians. Because the number of
Russian males was odd, one Japanese subject was allocated to one Russian subject in one case. Both Russian and Japanese subjects were volunteers recruited from local medical students. Measurements of Atherosclerotic Parameters The body mass index (BMI) was calculated based on the weight and height in each subject wearing light clothing without shoes. The heart rate (HR), blood pressure (BP), CAVI and ABI were measured using the VaSera VS-1500N vascular screening system (Fukuda Denshi Co. Ltd, Tokyo, Japan) 11). Briefly, these indices were measured in the morning after 10 minutes of rest, with cuffs applied to the bilateral upper and lower extremities while the subject was in the supine position. The CAVI was estimated from the brachial and ankle pulse wave forms by using appropriate cuffs for each subject’s arm according to the manufacturer’s instructions. Electrocardiography, phonocardiography and BP measurements were performed simultaneously. The mean BP (MBP) was calculated based on the systolic BP (SBP) and diastolic BP (DBP) values. The stiffness parameter was calculated by the following equation: CAVI = In (Ps/Pd)× 2p/ΔP×PWV2, where Ps is the SBP, Pd is the DBP, p is the blood viscosity, ΔP is Ps-Pd and PWV is the pulse wave velocity from the aortic origin to the ankle via femoral artery. The ABI was measured based on the SBP for both the upper (brachial artery) and lower (tibial artery) BP, and was then was obtained by dividing the ankle SBP by the brachial SBP. The obtained data were automatically analyzed using the VaSera(R) Data Management software program (version: V1001, Fukuda Denshi Co. Ltd, Tokyo, Japan). Lifestyle Factors The subject’s lifestyle-related factors were obtained via a self-administered questionnaire. The study used a formulated questionnaire about the current status of smoking, alcohol intake, physical activity, sports experience, salt intake, meat intake, dairy intake, vegetable intake, carbohydrate intake, fat intake, meal frequency, water intake, personality type, mental conditions at home and mental conditions in the work place. SNPs Typing Approximately 8-10 mL of venous blood was collected into EDTA-coated tubes from each subject in this study (n = 90), and samples were stored at −20 ℃ until they were analyzed. Genomic DNA was isolated using a standard phenol/chloroform procedure. DNA samples were genotyped for the four selected SNPs:
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Table 1. A comparison of the demographics, CAVI and ABI between young Russian and Japanese subjects Parameter Subjects, n Gender, male/female, n Age, years Current smoking, n (%) Body mass index, kg/m2 Heart rate, bpm Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Mean blood pressure, mmHg CAVI ABI
Russian
Japanese
p
114 25/89 21.5±1.9 12 (11%) 21.9±3.5 70±12 124±11 75±7 92±8 5.87±0.80 1.10±0.10
57 13/25 21.3±1.6 2 (4%) 20.8±2.3 58±10 110±10 65±6 80±7 5.36±0.52 1.10±0.07
1.00 0.61 0.15 0.02＊ ＊ ＜ 0.01 ＊ ＜ 0.01 ＊ ＜ 0.01 ＊ ＜ 0.01 ＊a ＜ 0.01 0.67
−
CAVI: cardio-ankle vascular index, ABI: ankle-brachial index. The data are presented as the means±standard deviation or subject numbers (%). ＊p ＜ 0.05: t -test or Fisher’s exact test between the two groups. a p ＜ 0.01 in the age-, gender- and smoking-adjusted comparison; age-, gender-, smoking- and body mass index-adjusted comparison; age-, gender-, smoking-, body mass index- and mean blood pressure-adjusted comparison or age-, gender-, smoking-, body mass index-, mean blood pressure- and heart rate-adjusted comparison.
ACE I/D (rs4646994), NOS3 -786T/C (rs2070744), CETP Taq1B (rs708272) and LPL HindIII (rs320). The I and D alleles of ACE were detected using an amplified fragment length polymorphism assay according to a previously described method 15). The PCR products were analyzed using electrophoresis on 2% agarose gels containing ethidium bromide, and were visualized under UV light on the GDS-8000 Computer Detection System (UVP Inc, CA, USA). For NOS3 -786T/C, CETP Taq1B and LPL HindIII, genomic DNA fragments were amplified by PCR in a 25 μL PCR reaction mixture containing 20-40 ng of DNA, deoxynucleotide triphosphates (0.2 mM of each), 10 pmol of each of the primers, 5 pmol of each of the probes, 1 x PCR-buffer (67 mM Tris-HCl pH 8.8, 16.6 mM (NH4)2SO4, 0.01% Tween-20) and 1.5 U of Taq DNA polymerase (SibEnzyme, Novosibirsk, Russia). The reactions contained 2.5 mM MgCl2 for NOS3 -786T/C, 2 mM MgCl2 for CETP Taq1B and 3.5 mM MgCl2 for LPL HindIII. The genotypes were detected by the CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad Laboratories, CA, USA). Statistical Analysis Differences between the groups were assessed by t -tests, Fisher’s exact tests or adjusted comparison tests (using general linear models). The correlations between parameters were calculated using a Pearson’s correlation test and a multiple regression analysis. Mutated alleles were determined according to the methodology of previous reports 16-20). Values of p ＜ 0.05 were considered to be statistically significant.
Results The clinical characteristics of the study subjects (Russian subjects: n = 114; one subject was excluded from the original cohort because of missing data) are summarized in Table 1. The mean age and gender proportions were similar between the Russian subjects and their Japanese peers (based on the age- and gender-matched cohorts). There was a non-significant difference in the prevalence of current smoking between the two populations. Significant differences were observed for the BMI (higher in Russians), HR (higher in Russians) and BP (higher in Russians). The ABI level was ＞ 0.9 in all subjects. The CAVI levels were significantly higher in the Russian subjects compared to their Japanese counterparts, while the ABI levels were similar between the two populations. The difference in the CAVI between the two populations remained independently significant, even after adjusting for age, gender and smoking (Adjusted model 1: p ＜ 0.01); age, gender, smoking and BMI (Adjusted model 2: p ＜ 0.01); age, gender, smoking, BMI and MBP (Adjusted model 3: p ＜ 0.01) or age, gender, smoking, BMI, MBP and HR (Adjusted model 4: p ＜ 0.01). The correlations of the CAVI and ABI with the atherosclerotic parameters in the Russian subjects are shown in Table 2. The CAVI was not significantly correlated with the MBP (r =−0.16). The CAVI showed an inverse correlation with the HR (r =−0.22), but after adjusting for age, gender, smoking, BMI and MBP, the correlation was decreased to a non-signifi-
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Table 2. The correlation coefficients for conventional factors and the CAVI or ABI in young Russian subjects Parameter Gender, male Age, years Current smoking, n (%) Body mass index, kg/m2 Heart rate, bpm Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Mean blood pressure, mmHg
CAVI
ABI
0.08 (0.42)
0.12 (0.22)
−0.11 (0.25)
−0.07 (0.45)
−0.15 (0.12)
−0.02 (0.80)
−0.10 (0.28)
0.11 (0.23)
−0.22 (0.02)
−0.43 (＜ 0.01)
−0.09 (0.35)
−0.35 (＜ 0.01)
−0.19 (0.04)
−0.15 (0.11)
−0.16 (0.09)
−0.26 (＜ 0.01)
＊a
＊d
＊b ＊c
＊e
CAVI: cardio-ankle vascular index, ABI: ankle-brachial index. Statistical data are given as correlation coefficients (p-values). ＊p ＜ 0.05: Pearson’s correlation test. aβ=−0.18 (p = 0.09) after adjusting for age, gender, smoking, body mass index and mean blood pressure. bβ=−0.37 (p ＜ 0.01) after adjusting for age, gender, smoking, body mass index and mean blood pressure. cβ=−0.45 (p ＜ 0.01) after adjusting for age, gender, smoking, body mass index and heart rate. dβ=−0.14 (p = 0.18) after adjusting for age, gender, smoking, body mass index and heart rate. eβ=−0.26 (p ＜ 0.01) after adjusting for age, gender, smoking, body mass index and heart rate.
cant level (β=−0.18: p = 0.09). The CAVI also showed an inverse correlation with the DBP (r =−0.19); however, after adjusting for age, gender, smoking, BMI and HR, the correlation became non-significant (β=−0.14: p = 0.18). The ABI was significantly and inversely correlated with the SBP (r =−0.35) and MBP (r =−0.26). The correlation remained significant, even after adjusting for age, gender, smoking, BMI and HR (SBP: β= −0.45: p ＜ 0.01, MBP: β=−0.26: p ＜ 0.01). In addition, the ABI was also significantly and inversely correlated with the HR (r =−0.43), and even after adjusting for age, gender, smoking, BMI and MBP, the correlation remained significant (β=−0.37: p ＜ 0.01). The associations between lifestyle factors and the CAVI or ABI levels in the Russian subjects are shown in Table 3. Overall, there were no significant differences between the lifestyle-related factors and CAVI levels. Similarly, no significant differences between the ABI were found for the lifestyle-related factors. The detection of the ACE, NOS3, LPL and CETP genotypes was completed in 85, 87, 79 and 83 subjects, respectively (the PCR products could not been obtained from the samples of some subjects due to material and technical reasons). The distributions for all four gene SNPs were in Hardy-Weinberg equilibrium. Non-significant associations were observed between these SNPs and the CAVI levels, as shown in Table 4. While non-significant associations were found between the SNPs of ACE, NOS3 or LPL and the ABI, a significant difference was found for the CETP Taq1B SNPs and ABI levels. The frequency distribution was 14 (16.9%) CC-homozygotes of CETP, 40 (48.2%) CT-heterozygotes and 29 (34.9%) TT-homozygotes. A significantly lower ABI level was observed
in carriers with the ‘T’ allele (mutated allele) of CETP than in those without. This difference remained significant (p = 0.04), even after adjusting for age, gender, smoking, BMI, HR and MBP. Discussion In the current study, we observed ethnic differences in the CAVI levels between the young Russian and Japanese subjects. Although the Russian subjects had higher HR and BP levels, these levels were independent of the differences in the CAVI among the two populations. While this is the first report using the CAVI for healthy younger Russian people, a previous study, which used the brachial-ankle pulse wave velocity (baPWV: an indicator of arterial stiffness), showed that healthy Russian individuals might have higher arterial stiffness values compared to their Japanese counterparts 21). In that report, the authors simply described the findings in Russian subjects under 39 years of age 21). Additionally, some differences are known to exist between the baPWV and the CAVI levels, because the CAVI has less dependence on the BP 11, 13). Thus, it is hard to directly compare those data 21) with our present data. Another ethnic comparison study of the CAVI and ABI levels was carried out between young Japanese and Mongolian subjects 4). That study demonstrated that the CAVI levels were low in Japanese people (the value was 5.4) and high in their Mongolian peers (6.5), while the ABI levels showed a non-significant difference between the two populations. In addition to that study 4), the current study between Japanese and Russian subjects further suggests the exis-
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Table 3. The associations of the lifestyle factors with the CAVI or ABI in young Russian subjects Factors CAVI Current smoking Alcohol intake ( ＞ light-to-moderate) Physical activity ( ＞ once/week) Sports experience ( ＞ periodic) Salt intake (high) Meat intake ( ＞ four times/week) Dairy intake ( ＞ four times/week) Vegetable intake ( ＞ four times/week) Carbohydrate intake ( ＞ four times/week) Fat intake ( ＞ four times/week) Meal frequency (three times/day) Meal regularity (regular everyday) Water intake ( ＜ one liter/day) Personality type (aggressive) Mental condition at home ( ＞ good) Mental condition at work ( ＞ good) ABI Current smoking Alcohol intake ( ＞ light-to-moderate) Physical activity ( ＞ once/week) Sports experience ( ＞ periodic) Salt intake (high) Meat intake ( ＞ four times/week) Dairy intake ( ＞ four times/week) Vegetable intake ( ＞ four times/week) Carbohydrate intake ( ＞ four times/week) Fat intake ( ＞ four times/week) Meal frequency (three times/day) Meal regularity (regular everyday) Water intake ( ＜ one liter/day) Personality type (aggressive) Mental condition at home ( ＞ good) Mental condition at work ( ＞ good)
Yes (number)
No (number)
p
5.53±0.77 (n = 12) 5.86±0.80 (n = 69) 5.63±0.97 (n = 15) 5.88±0.83 (n = 91) 5.42±0.87 (n = 4) 6.03±0.79 (n = 45) 5.90±0.89 (n = 27) 5.79±0.78 (n = 54) 6.01±0.82 (n = 37) 6.45±0.33 (n = 3) 5.87±0.91 (n = 57) 5.84±0.91 (n = 12) 5.70±0.69 (n = 19) 5.77±0.85 (n = 32) 5.87±0.80 (n = 109) 5.86±0.81 (n = 112)
5.91±0.80 (n = 102) 5.88±0.81 (n = 45) 5.90±0.77 (n = 99) 5.83±0.68 (n = 23) 5.88±0.80 (n = 110) 5.76±0.80 (n = 69) 5.86±0.78 (n = 60) 5.94±0.82 (n = 60) 5.80±0.79 (n = 77) 5.85±0.81 (n = 111) 5.87±0.69 (n = 57) 5.87±0.79 (n = 102) 5.90±0.82 (n = 95) 5.91±0.78 (n = 82) 5.76±0.98 (n = 5) 6.16±0.69 (n = 2)
0.12 0.87 0.23 0.78 0.26 0.07 0.84 0.34 0.19 0.20 0.98 0.91 0.32 0.42 0.75 0.61
1.09±0.12 (n = 12) 1.10±0.10 (n = 69) 1.12±0.12 (n = 15) 1.10±0.10 (n = 91) 1.08±0.07 (n = 4) 1.12±0.11 (n = 45) 1.09±0.10 (n = 27) 1.10±0.10 (n = 54) 1.09±0.10 (n = 37) 1.15±0.15 (n = 3) 1.09±0.10 (n = 57) 1.11±0.10 (n = 12) 1.08±0.10 (n = 19) 1.07±0.10 (n = 32) 1.10±0.10 (n = 109) 1.10±0.10 (n = 112)
1.10±0.10 (n = 102) 1.10±0.09 (n = 45) 1.10±0.09 (n = 99) 1.07±0.09 (n = 23) 1.10±0.10 (n = 110) 1.08±0.09 (n = 69) 1.10±0.10 (n = 87) 1.09±0.10 (n = 60) 1.10±0.10 (n = 77) 1.10±0.10 (n = 111) 1.11±0.09 (n = 57) 1.10±0.10 (n = 102) 1.10±0.10 (n = 95) 1.11±0.10 (n = 82) 1.04±0.09 (n = 5) 1.13±0.03 (n = 2)
0.80 0.90 0.36 0.14 0.69 0.10 0.53 0.66 0.46 0.35 0.21 0.65 0.39 0.10 0.22 0.63
CAVI: cardio-ankle vascular index, ABI: ankle-brachial index. The data are expressed as the means±standard deviation. ＊p ＜ 0.05: t -test between the two groups.
tence of differences in the reference values of the CAVI that are specific for each ethnic population. Additionally, considering the clinical significance of the CAVI for the development of CVD 10, 11), as well as the ethnic diversity in the prevalence and impact of CVD 4, 14), the possible ethnic differences in the CAVI values indicate the presence of possible populationrelated differences in the atherosclerotic burden among younger subjects. Namely, Russians may develop atherosclerosis-related conditions at a younger age than Japanese subjects, which is supported by the WHO data for CVD 14).
In the current study, the CAVI was not dependent on the BP, which is in agreement with the previous findings (again, this is a known characteristic of the CAVI) 11, 13). The ethnic differences observed in the occurrence of CVD may be, at least in part, attributable to lifestyle/environmental factors 22-24); however, the current study did not find any associations between the lifestyle-related factors examined and the CAVI levels. Although the questionnaire was not the same as the one that was applied previously 4), non-significant associations were also noted between lifestylerelated factors and the CAVI levels in younger Japa-
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Table 4. The association of gene polymorphisms with the CAVI or ABI levels in young Russian subjects Factors CAVI ACE (D allele) NOS3 (C allele) LPL (G allele) CETP (T allele) ABI ACE (D allele) NOS3 (C allele) LPL (G allele) CETP (T allele)
Present (number)
Absent (number)
p
5.84±0.83 (n = 61) 5.75±0.69 (n = 50) 5.80±0.97 (n = 32) 5.84±0.80 (n = 69)
5.78±0.76 (n = 24) 5.90±0.93 (n = 37) 5.84±0.73 (n = 47) 5.97±0.62 (n = 14)
0.77 0.41 0.83 0.57
1.09±0.09 (n = 61) 1.10±0.10 (n = 50) 1.12±0.11 (n = 31) 1.09±0.09 (n = 68)
1.12±0.12 (n = 23) 1.10±0.09 (n = 36) 1.10±0.09 (n = 47) 1.15±0.13 (n = 14)
0.30 0.98 0.40 a 0.02＊
ACE: angiotensin converting enzyme, NOS: endothelial nitric oxide synthase, LPL: lipoprotein lipase, CETP: cholesteryl ester transfer protein, CAVI: cardio-ankle vascular index, ABI: ankle-brachial index. The data are presented as the means±standard deviation. ＊p ＜ 0.05: t -test between the two groups. a p = 0.04 after adjusting for age, gender, smoking, body mass index, heart rate and mean blood pressure.
nese and Mongolian subjects in a previous study. The influences of lifestyle-related factors on the arterial conditions, as expressed by the CAVI values, are apparently minimal in healthy younger populations, and may only be detected upon disease progression in older populations. The ABI is helpful for determining the arterial conditions linked to the lower limbs (i.e., the existence of peripheral arterial disease) 25), and is a predictor of CVD events 26). Even when the ABI level is ＞ 0.9 (as in our study subjects), the ABI measurement is useful for predicting the development of CVD 26). The clinical significance of the ABI generally differs from that of the CAVI. However, similar to the CAVI, we did not observe any associations between lifestyle-related factors and the ABI levels in the current study. The correlations between atherosclerotic parameters and the ABI showed different patterns of changes from those of the CAVI; namely, the ABI was inversely correlated with the HR and BP (the SBP in particular) in the Russian subjects, which is in agreement with the findings of the previous study in Mongolian and Japanese subjects 4). Four gene SNPs, which are considered to affect the development of CVD, were investigated in the Russian subjects in the present study. The ACE molecule is a key enzyme of the renin-angiotensin system, which regulates the systemic circulatory system 27). The SNPs leading to an insertion/deletion in intron 16 (ACE I/D) have been commonly studied, and have been shown to have a substantial effect on ACE gene expression 28). The NO molecule acts as a pro-inflammatory and pro-atherogenic factor, for instance, via the suppression of vascular contraction and attenua-
tion of oxidative stress and inflammation 7, 18, 29). The NOS3 gene is responsible for the endothelial NOS production 17), and NOS3 -786T＞ C has been reported to be associated with CVD 16). The LPL molecule is an enzyme that controls lipid metabolism by hydrolyzing triglyceride-rich particles via the generation of free fatty acids and glycerol 30), and LPL HindIII was previously reported to be associated with CVD 20). Although all of these SNPs are important risk factors for the development of CVD, the current study did not find any association between the CAVI/ABI and these gene SNPs. However, the ‘T’ allele carriers of CETP Taq1B (rs708272) had lower ABI levels, while there were no association between the CAVI and CETP SNPs in this population. The CETP molecule facilitates cholesteryl ester exchange between high-density lipoproteins and triglyceride-rich lipoproteins 30), and the CETP Taq1B SNPs, located in intron 1 of the CETP gene, have been extensively studied 31). Based on the previous reports showing that some CETP SNPs were associated with blood pressure and CVD (the relationship was noted to be present regardless of the CETP activity) 19, 32), our finding of an association between the CETP Taq1B SNPs and ABI is reasonable, although further biological studies should be conducted to confirm this association. There are some limitations associated with the current study. First, this study was conducted with a small sample and a cross-sectional design. Second, we did not obtain blood chemistry data. Unfortunately, the Japanese subjects were not examined with respect to their lifestyle-related factors using the same questionnaire used for the Russians in the current study, so
CAVI and ABI in Young Russians
no comparison of the lifestyle-related factors is possible. In addition, the small sample size, especially in the substudy of SNPs, might have resulted in reduced statistical power. Finally, although we think that Russians may develop atherosclerotic conditions at a younger age relative to Japanese subjects, this should be confirmed based on future atherosclerosis manifestations. These limitations should be addressed in future studies.In summary, the current study demonstrated higher CAVI levels in young Russian people compared to their Japanese peers. These data indicate that the reference values for the CAVI should be considered separately for different ethnic populations, and suggests that Russians may develop atherosclerosisrelated conditions at a younger age compared to Japanese people. The possible association between the CETP SNPs and the ABI deserves further study. More research using the CAVI and ABI is warranted in Russian and other populations. Acknowledgement I would like to express my sincere gratitude to Boris Vaisman, PhD, from the Lipoprotein Metabolism Section, Cardio-Pulmonary Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, United States, for his suggestions on the manuscript. Funding No specific funding was provided for the study. Conflict of Interest None declared. Abbreviations ABI – ankle-brachial index; ACE: angiotensinconverting enzyme; baPWV: brachial-ankle pulse wave velocity; CAVI – cardio-ankle vascular index; CETP: cholesteryl ester transfer protein; CVD: cardiovascular disease; HR – heart rate; LPL: lipoprotein lipase; NOS3: nitric oxide synthase 3; SNP – single nucleotide polymorphism. References 1) Nichols M, Townsend N, Scarborough P, Luengo-Fernandez R, Leal J, Gray A, Rayner M: Mortality and Morbidity. In: European Cardiovascular Disease Statistics 2012 Ed. ed Susanne Løgstrup and Sophie O’Kelly, pp 10-45,
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