Abstract
Objective
The influence of T-786C polymorphism in the promoter region of endothelial nitric oxide synthase (eNOS) on arteriosclerotic parameters by age and gender were examined.
Methods
Brachial-ankle pulse wave velocity (baPWV), heart-rate adjusted augmentation index (AIx@75), pulse pressure (PP) and albumin–creatinine ratio (ACR) were assessed as arteriosclerotic parameters in addition to non-high-density lipoprotein cholesterol (non-HDL-C) to HDL-C (non-HDL-C/HDL-C) ratio in 1499 participants. T-786C polymorphism (rs2070744) was screened using a TaqMan allelic discrimination assay. Analyses of covariance were carried.
Results
Women with the non-C allele showed significantly lower AIx@75 in participants aged <65 years and baPWV in participants aged ≥65 years than those with C allele. In contrast, men with the non-C allele showed significantly higher PP in participants aged <65 years, and higher ACR and non-HDL-C/HDL-C ratio in participants aged ≥65 years. In men on cholesterol-lowering medication, the non-C allele carriers showed significantly higher non-HDL-C compared to those in the C allele carriers.
Conclusions
eNOS T-786C polymorphism is significantly associated with arteriosclerotic parameters accompanied with age and gender differences, possibly involving antioxidative and/or endothelial signaling other than inflammatory signaling.
Keywords: eNOS, T-786C, Arteriosclerotic parameters
Introduction
Arterial stiffness is a hallmark of natural aging process, and contributes to progression of atherosclerosis, diabetes, chronic renal insufficiency, and further, cardiovascular diseases (CVD); which is the major death risks in the world [1]. Structural and cellular changes in vessel wall are considered to be major underlying mechanisms of stiffening [2]. Structural change is defined as compliance and distensibility and cellular change in vessel wall is mainly induced by alteration of endothelial function [3]. The predominant mediator of endothelial function is nitric oxide (NO). The endothelial nitric oxide synthase (eNOS) is expressed not only in the endothelium, but also in platelets, cardiomyocytes, kidney, red blood cells and elsewhere [4, 5]. The eNOS is considered to protect vessel wall from stiffening and contributes to maintain vascular homeostasis [6]. Among major polymorphisms in the eNOS gene, T-786C mutation in the promoter region is considered to have critical role on CVD [7]. It has been originally reported to be associated with coronary spasm [8]. Several meta-analysis reports suggest positive associations between this polymorphism and hypertension, ischemic stroke, diabetic nephropathy and coronary artery disease (CAD) accompanied with age and racial differences [9–12].
Non-invasive methods have been developed to evaluate the degree of arterial stiffness, including pulse wave velocity (PWV), augmentation index (AIx), and pulse pressure (PP). These indices are considered to reflect structural changes in vessel; however, it has been reported that AIx and brachial-ankle PWV (baPWV) reflect not only structural change in vessel but also endothelial dysfunction. Significant decrease of AIx@75 by NO stimulator β2-agonist salbutamol [13] and significant positive association between AIx and asymmetric dimethylarginine (ADMA), an endogenous NO inhibitor [14, 15], suggest that AIx may reflect NO bioavailability. Recent reports showed that significant association of arginine/ADMA level and baPWV [16] suggests baPWV also may reflect NO bioavailability. Furthermore, these indices are considered to be useful surrogate markers of atherosclerosis [17].
Besides these noninvasive indices, biochemical markers have also been reported to be useful to assess endothelial dysfunction. High-density lipoprotein cholesterol (HDL-C) induces NO by activating eNOS through Akt and mitogen-activated protein (MAP) kinases [18]. Thus, it is not unreasonable to consider non HDL-C, subtracting HDL-C from total cholesterol (T-chol) to HDL-C and triglyceride to HDL-C (TG/HDL-C) ratios can reflect endothelial dysfunction. Indeed, they have been reported as good markers for arterial stiffness [19] and coronary heart disease [20]. Furthermore, it has been reported urinary albumin to creatinine ratio (ACR) is significantly associated with arterial stiffness [21].
In the meantime, the influence of the eNOS T-786C polymorphism on arterial stiffness in general population has been rarely studied. We hypothesize that this polymorphism would contribute to prediction and prevention of arteriosclerotic diseases in general population depending on age and gender.
Here, we have investigated this polymorphism with markers related to arterial stiffness such as baPWV, AIx@75, PP and ACR together with non-HDL-C/HDL-C ratio in Japanese general population, to clarify the influence of eNOS genotype on arterial stiffness by age and gender.
Materials and methods
Study population
The participants were inhabitants living in a rural town of Wakayama Japan (9844 people ≥35 years old) who participated in annual health checkups conducted in July 2011 and July 2012. In 2011, among 1496 people who had health checkups, 987 people (474 male and 513 female) gave written informed consent for this study. In 2012, among 1801 people who had health checkups, 547 people (257 male and 290 female) who did not participate in this study in 2011 gave written informed consent for this study. In total, 1534 participants (731 male and 803 female) were recruited for this study. Among them, 1499 participants who fulfilled all variables were included in the present study. This study was approved by the ethical committee for analytical research on the human genome of Wakayama Medical University (Approval No. 92).
Questionnaire and measurements
A self-administered questionnaire including items on alcohol drinking consumption, cigarette smoking and waling status was used for data collection. Participants were also asked whether they took prescription medication for hypertension, diabetes, or higher cholesterol. Body height and weight were measured using a standard protocol to obtain body mass index (BMI). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured twice in seated position quietly using an automated sphygmomanometer (HEM-907; Omron, Kyoto, Japan) and mean value was calculated. A venous blood sample was taken for measuring TG, T-chol, and HDL-C. Of total 1499 participants, we excluded 91 participants who were not fasting for TG analysis (N = 1408). TG and T-chol were measured by enzymatic method (Determiner C TG and Determiner L TC II; Kyowa Medex, Tokyo, Japan). HDL-C and LDL-C were measured by direct method (Cholestest N HDL and Cholestest LDL; Sekisui Medical, Tokyo, Japan). Non HDL-C was calculated as subtracting HDL-C from T-chol. Urine sample was collected on site to measure urinary albumin (UA) and urinary creatinine. UA was measured by latex agglutination method (LX-Alb-II; Eiken Chemical, Tokyo, Japan). Urinary creatinine was measured using enzymatic method (Accuras Auto CRE; Shino-Test, Tokyo, Japan). Because the urine sample was spot urine, albumin creatinine ratio (ACR) was calculated using the formula, .
Measurement of arteriosclerotic parameters
The detail measurement of baPWV were described elsewhere [22]. Briefly, measurement was performed using a volume-plethysmographic apparatus (BP-203RPE II form 2, Omron Colin, Tokyo, Japan). The higher value of baPWV was used in the analyses. Measurements of baPWV with this apparatus have been reported to have good interobserver and intraobserver reproducibility [23]. AIx was assessed by computerized radial artery pulse wave analysis using an automated tonometric system (HEM-9000AI; Omron Colin). AIx was defined as the ratio of first systolic peaks to late systolic peaks. Since AIx was reported to be dependents on heart rate [24], AIx@75 as normalized for a heart rate of 75 bpm was used in the analyses. Because of technical difficulty, we were not able to perform AIx measurement to participants over 75 years (N = 1329). PP was calculated as subtracting DBP from SBP.
eNOS genotyping
Genomic DNA was extracted from whole blood using NucleoSpin Blood (Macherey–Nagel, Duren, Germany). Genotyping for the eNOS T-786C (rs2070744) was performed using TaqMan SNP Genotyping Assays (AssayID: C_15909863_10, Applied Biosystems, Carlsbad, CA) with real-time PCR on 7300 Real-time PCR System (Applied Biosystems). PCR cycle was carried out according to the manufacturer’s instructions. Results were analyzed by SDS 1.3 software for ABI 7300 (Applied Biosystems).
Statistical analysis
All statistical analyses were performed using STATA statistical software version 13 (STATA Corp., College Station, TX). Participants were categorized into two groups by their age; as older (≥65 years) and younger group (<65 years). When the distributions of biochemical values were skewed, data were transformed logarithmically before performing analyses. Differences in gender were tested using the Student’s t test. Proportions of current alcohol consumption, cigarette smoking, walking status and medication were tested using Chi-square test. Correlations among arteriosclerotic parameters were tested by using partial correlation analysis adjusted for age, BMI, gender (0: men, 1: women), current alcohol consumption (0: never or rarely, 1: habitually), cigarette smoking habit (0: ex/non-smoker, 1: current smoker), walking habit (0: regularly walking ≥1 h/day, 1: <1 h/day) and medication (0: no, 1: yes). Differences in the means for arteriosclerotic parameters according to the eNOS genotypes (non-C allele carriers vs C allele carriers) were statistically tested by analysis of covariance (ANCOVA) adjusted for age, BMI, gender, medication, current alcohol consumption, cigarette smoking and walking habits. P-values of <0.05 were considered statistically significant.
Results
The genotype frequency was within the Hardy–Weinberg equilibrium (total: χ2 = 0.081, P > 0.05; men: χ2 = 0.098, P > 0.05; women: χ2 = 0.007, P > 0.05).
The basic characteristics of participants according to age groups and gender were shown in Table 1. Overall, baPWV was significantly higher in men, whereas, AIx@75 and ACR were significantly higher in women. Non-HDL-C/HDL-C ratios were significantly higher in men. These tendencies were consistent in younger group. In older group, AIx@75 was significantly higher in women; however, no significant differences were seen in baPWV, ACR and non-HDL-C/HDL-C ratio. PP showed no significant gender difference.
Table 1.
Basic characteristics of participants
| Total | Men | Women | P | |
|---|---|---|---|---|
| N | 1499 | 714 | 785 | |
| Age (years) | 61.5 ± 11.7 | 61.2 ± 11.9 | 61.8 ± 11.4 | 0.295 |
| BMI (kg/m2) | 22.3 ± 3.1 | 22.7 ± 3.0 | 21.9 ± 3.2 | <0.001 |
| baPWV (m/s) | 16.0 ± 3.9 | 16.2 ± 3.7 | 15.8 ± 4.0 | 0.037 |
| AIx@75 (%)a) | 82.8 ± 11.5 | 78.8 ± 11.0 | 86.5 ± 10.7 | <0.001 |
| ACR (mg/g Cre) | 24.5 (14.3; 35.8) | 21.9 (13.4; 32.5) | 27.4 (15.6; 38.1) | 0.003 |
| SBP (mmHg) | 128.5 ± 18.4 | 130.4 ± 17.7 | 126.6 ± 19.0 | <0.001 |
| DBP (mmHg) | 74.1 ± 9.9 | 76.2 ± 9.5 | 72.2 ± 9.9 | <0.001 |
| PP (mmHg) | 54.4 ± 13.1 | 54.3 ± 13.0 | 54.5 ± 13.2 | 0.808 |
| T-chol (mg/dL) | 199.0 ± 32.8 | 192.6 ± 32.2 | 204.9 ± 32.3 | <0.001 |
| HDL-C (mg/dL) | 61.5 ± 15.6 | 57.2 ± 14.7 | 65.4 ± 15.4 | <0.001 |
| LDL-C (mg/dL) | 114.7 ± 28.3 | 111.2 ± 28.1 | 117.9 ± 28.2 | <0.001 |
| Non-HDL-C (mg/dL) | 137.5 ± 31.5 | 135.4 ± 30.7 | 139.5 ± 31.2 | 0.012 |
| Non-HDL-C/HDL-C | 2.40 ± 0.90 | 2.54 ± 0.94 | 2.28 ± 0.85 | <0.001 |
| <65 (years) | ||||
| N | 872 | 423 | 449 | |
| Age (years) | 53.7 ± 8.2 | 53.5 ± 8.4 | 54.1 ± 7.9 | 0.284 |
| BMI (kg/m2) | 22.3 ± 3.2 | 23.0 ± 3.1 | 21.6 ± 3.0 | <0.001 |
| baPWV (m/s) | 14.2 ± 2.7 | 14. ± 2.5 | 13.8 ± 2.8 | <0.001 |
| AIx@75 (%)a) | 81.2 ± 11.9 | 77.1 ± 11.5 | 85.2 ± 10.9 | <0.001 |
| ACR (mg/g Cre) | 23.2 (14.6; 32.8) | 20.5 (12.6; 29.9) | 25.5 (17.1; 34.2) | 0.001 |
| SBP (mmHg) | 122.9 ± 17.0 | 126.0 ± 16.2 | 120.0 ± 17.1 | <0.001 |
| DBP (mmHg) | 73.1 ± 10.4 | 76.0 ± 10.0 | 70.4 ± 10.0 | <0.001 |
| PP (mmHg) | 49.8 ± 10.2 | 50.0 ± 9.9 | 49.5 ± 10.4 | 0.474 |
| T-chol (mg/dL) | 201.1 ± 32.7 | 197.0 ± 32.4 | 205.0 ± 32.5 | <0.001 |
| HDL-C (mg/dL) | 62.8 ± 16.0 | 58.0 ± 14.9 | 67.4 ± 15.6 | <0.001 |
| LDL-C (mg/dL) | 115.5 ± 28.1 | 113.5 ± 28.1 | 117.4 ± 28.1 | 0.044 |
| Non-HDL-C (mg/dL) | 138.2 ± 31.8 | 139.0 ± 33.0 | 137.5 ± 30.6 | 0.488 |
| Non-HDL-C/HDL-C | 2.37 ± 0.88 | 2.58 ± 0.98 | 2.16 ± 0.73 | <0.001 |
| ≥65 (years) | ||||
| N | 627 | 291 | 336 | |
| Age (years) | 72.3 ± 5.7 | 72.4 ± 5.8 | 72.2 ± 5.7 | 0.627 |
| BMI (kg/m2) | 22.3 ± 3.1 | 22.4 ± 2.8 | 22.3 ± 3.3 | 0.731 |
| baPWV (m/s) | 18.6 ± 3.9 | 18.7 ± 3.9 | 18.4 ± 3.9 | 0.450 |
| AIx@75 (%)a | 85.6 (10.1) | 82.1 ± 9.2 | 88.9 ± 9.8 | <0.001 |
| ACR (mg/g Cre) | 27.2 (13.8; 41.4) | 24.0 (14.9; 37.6) | 29.8 (13.4; 43.5) | 0.412 |
| SBP (mmHg) | 136.2 ± 17.6 | 136.9 ± 17.7 | 135.6 ± 17.6 | 0.354 |
| DBP (mmHg) | 75.4 ± 9.0 | 76.4 ± 9.6 | 74.5 ± 9.2 | 0.010 |
| PP (mmHg) | 60.8 ± 14.0 | 60.5 ± 14.3 | 61.1 ± 13.7 | 0.627 |
| T-chol (mg/dL) | 196.2 ± 32.9 | 186.3 ± 30.8 | 204.7 ± 32.1 | <0.001 |
| HDL-C (mg/dL) | 59.6 ± 14.9 | 56.1 ± 14.5 | 62.6 ± 14.7 | <0.001 |
| LDL-C (mg/dL) | 113.5 ± 28.5 | 107.8 ± 27.7 | 118.5 ± 28.3 | <0.001 |
| Non-HDL-C (mg/dL) | 136.6 ± 31.1 | 130.2 ± 29.0 | 142.1 ± 31.8 | <0.001 |
| Non-HDL-C/HDL-C | 2.46 ± 0.93 | 2.48 ± 0.87 | 2.44 ± 0.97 | 0.540 |
Data were shown in mean ± SD. Skewed values were shown in median (25; 75th quantile). P: men vs. women
aSubjects number for AIx@75 were total 1329 (men 642 and women 687), <65 years (men 423 and women 447) and ≥65 years (men 219 and women 240)
Table 2 showed the proportion of selected lifestyle factors and medication among participants. None of these proportions were statistically significant between eNOS genotypes irrespective of age and gender groups. The proportion of who were on medication was higher in older participants compared to those in younger participants, independent from eNOS genotype or gender (P < 0.005 for all comparisons).
Table 2.
Prevalence of selected life style factors in participants by eNOS T-786C genotypes
| Total | <65 (years) | ≥65 (years) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| TT | TC | CC | C carrier | TT | TC | CC | C carrier | TT | TC | CC | C carrier | ||
| Men | |||||||||||||
| N (%) | 714 (47.6) | 587 | 120 | 7 | 127 | 349 | 70 | 4 | 74 | 238 | 50 | 3 | 53 |
| Alcohol consumption (%)a | 507 (71.1) | 71.9 | 68.3 | 42.9 | 66.9 | 73.9 | 67.1 | 50.0 | 66.2 | 68.9 | 70.0 | 33.3 | 67.9 |
| Smoking habit (%)b | 183 (25.6) | 24.5 | 31.7 | 14.3 | 30.7 | 31.8 | 40.0 | 0.0 | 37.8 | 13.9 | 20.0 | 33.3 | 20.8 |
| Walking habit (%)c | 368 (51.5) | 51.8 | 51.7 | 28.6 | 50.4 | 55.0 | 57.1 | 0.0 | 54.1 | 47.1 | 44.0 | 66.7 | 45.3 |
| Medicationd | 278 (38.9) | 39.0 | 40.0 | 14.3 | 38.6 | 26.1 | 28.6 | 0.0 | 27.0 | 58.0 | 56.0 | 33.3 | 54.7 |
| Women | |||||||||||||
| N (%) | 785 (52.4) | 628 | 148 | 9 | 157 | 363 | 83 | 3 | 86 | 265 | 65 | 6 | 71 |
| Alcohol consumption (%)a | 257 (32.7) | 32.0 | 35.8 | 33.3 | 35.7 | 36.4 | 42.2 | 66.7 | 43.0 | 26.0 | 27.7 | 16.7 | 26.8 |
| Smoking habit (%)b | 21 (2.7) | 2.9 | 2.0 | 0.0 | 1.9 | 4.4 | 2.4 | 0.0 | 2.3 | 0.8 | 1.5 | 0.0 | 1.4 |
| Walking habit (%)c | 403 (51.3) | 52.6 | 47.3 | 33.3 | 46.5 | 53.7 | 47.0 | 33.3 | 46.5 | 50.9 | 47.7 | 33.3 | 46.5 |
| Medicationd | 311 (39.6) | 38.5 | 43.2 | 55.6 | 44.0 | 25.3 | 28.9 | 0.0 | 27.9 | 56.6 | 61.5 | 83.3 | 63.4 |
aPrevalence of whom drink alcohol beverage regularly
bPrevalence of current smoker
cPrevalence of whom walk regularly (≥1 h/day)
dPrevalence of participants on medication
Table 3 showed estimated mean (SE) of each variables according to their genotypes in ANCOVA analyses. In men, the non-C allele carriers showed significantly higher non-HDL-C/HDL-C ratio compared to those in the C allele carriers. The non-C allele carriers also showed trend of higher PP. In younger men, the non-C allele carriers showed significantly higher PP compared to those in the C allele carriers. In older men, the non-C allele carriers showed significantly higher ACR and non-HDL-C/HDL-C ratio compared to those in the C allele carriers. In contrast, in younger women, the non-C allele carriers showed significantly lower AIx@75 compared to those in the C allele carriers. In older women, the non-C allele carriers also showed significantly lower baPWV and trend of lower PP compared to those in the C allele carriers. Meanwhile, in older women, the non-C allele carriers showed trend of higher non-HDL-C/HDL-C ratio compared to those in the C allele carriers.
Table 3.
Association of eNOS T-786C genotypes with arterial stiffness indices
| Total | <65 (years) | ≥65 (years) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Non-C carrier | C carrier | P | Non-C carrier | C carrier | P | Non-C carrier | C carrier | P | |
| Men (N) | 587 | 127 | 349 | 74 | 238 | 53 | |||
| baPWV (m/s)a | 15.3 (1.0) | 15.0 (1.0) | 0.251 | 14.8 (1.0) | 14.7 (1.0) | 0.578 | 15.3 (1.0) | 14.8 (1.0) | 0.261 |
| AIx@75 (%) | 77.9 (1.0) | 77.2 (1.3) | 0.489 | 80.8 (1.3) | 79.0 (1.7) | 0.177 | 76.4 (2.2) | 77.6 (2.5) | 0.427 |
| PP (mmHg) | 52.4 (1.0) | 50.5 (1.3) | 0.069 | 49.8 (1.2) | 47.3 (1.5) | 0.037 | 45.5 (2.5) | 44.0 (3.0) | 0.434 |
| ACR (mg/g Cre)a | 19.5 (1.1) | 16.7 (1.1) | 0.135 | 22.7 (1.1) | 22.0 (1.2) | 0.807 | 16.0 (1.2) | 11.5 (1.3) | 0.039 |
| Non-HDL-C/HDL-C | 2.87 (0.08) | 2.69 (0.10) | 0.029 | 2.95 (0.12) | 2.80 (0.15) | 0.189 | 2.86 (0.16) | 2.61 (0.19) | 0.048 |
| Women (N) | 628 | 157 | 363 | 86 | 265 | 71 | |||
| baPWV (m/s)a | 14.5 (1.0) | 14.8 (1.0) | 0.169 | 14.2 (1.0) | 14.1 (1.0) | 0.649 | 14.4 (1.0) | 15.1 (1.0) | 0.031 |
| AIx@75 (%) | 87.0 (0.8) | 88.5 (1.1) | 0.129 | 86.1 (1.1) | 88.6 (1.5) | 0.045 | 91.3 (2.0) | 91.3 (2.5) | 0.972 |
| PP (mmHg) | 52.1 (0.7) | 53.0 (1.1) | 0.342 | 50.9 (1.0) | 50.1 (1.4) | 0.483 | 47.8 (1.9) | 50.7 (2.4) | 0.090 |
| ACR (mg/g Cre)a | 22.9 (1.1) | 23.4 (1.1) | 0.788 | 24.2 (1.1) | 22.5 (1.1) | 0.499 | 22.3 (1.2) | 25.7 (1.2) | 0.299 |
| Non-HDL-C/HDL-C | 2.44 (0.05) | 2.38 (0.08) | 0.384 | 2.51 (0.07) | 2.59 (0.10) | 0.366 | 2.69 (0.14) | 2.47 (0.18) | 0.071 |
Estimated mean (SE) adjusted for age, BMI, medication, current alcohol consumption, smoking and walking habits
aSkewed values were analyzed with logarithmic transformation
Then, we examined the effects of medication on the associations of eNOS T-786C genotypes with HDL and non-HDL cholesterols and PP which showed inverse associations with T-786C genotypes (Table 4). Overall, in men without medication, the non-C allele carriers showed lower HDL-C, higher non-HDL-C compared to those in the C allele carriers independent of age, but differences were not statistically significant. In younger men, the non-C allele carriers showed trend of higher PP compared to those in the C allele carriers. In men on cholesterol-lowering medication, the non-C allele carriers showed significantly higher non-HDL-C compared to those in the C allele carriers. This tendency was seen in both younger and older group, but the differences were not statistically significant. In men on anti-hypertensive medication, the non-C allele carriers showed higher PP compared to those in the C allele carriers, but the differences were not statistically significant. In women without cholesterol-lowering medication, the non-C allele carriers showed lower HDL-C and lower non-HDL-C except older group, in which the non-C allele carriers showed higher non-HDL-C compared to those in the C allele carriers; however, none of these differences were statistically significant. In contrast, women with medication, the non-C allele carriers showed higher HDL-C and higher non-HDL-C, but the differences were not statistically significant.
Table 4.
Effects of cholesterol-lowering or anti-hypertensive medications on the association between eNOS T-786C genotypes and cholesterols or PP
| Total | <65 (years) | ≥65 (years) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Non-C carrier | C carrier | P | Non-C carrier | C carrier | P | Non-C carrier | C carrier | P | |
| Men | |||||||||
| Without medication | |||||||||
| (N) | 521 | 110 | 323 | 67 | 198 | 43 | |||
| HDL-C (mmHg) | 52.2 (1.3) | 54.1 (1.7) | 0.173 | 52.5 (1.8) | 53.3 (2.3) | 0.660 | 54.1 (2.8) | 57.9 (3.3) | 0.095 |
| Non-HDL-C (mg/dL) | 144.3 (2.8) | 140.5 (3.8) | 0.235 | 148.3 (4.0) | 143.3 (5.3) | 0.243 | 148.9 (6.1) | 146.1 (7.0) | 0.563 |
| (N) | 392 | 86 | 273 | 57 | 119 | 29 | |||
| PP (mmHg) | 51.0 (1.0) | 49.5 (1.4) | 0.210 | 48.7 (1.2) | 46.3 (1.5) | 0.055 | 44.7 (3.1) | 44.7 (3.7) | 0.987 |
| On medication | |||||||||
| (N) | 66 | 17 | 26 | 7 | 40 | 10 | |||
| HDL-C (mmHg) | 57.5 (3.5) | 54.7 (4.9) | 0.515 | 60.5 (5.7) | 48.4 (6.7) | 0.089 | 58.5 (7.1) | 61.0 (10.3) | 0.699 |
| Non-HDL-C (mg/dL) | 125.3 (6.6) | 104.4 (9.3) | 0.012 | 130.4 (13.0) | 105.7 (15.1) | 0.120 | 114.8 (12.3) | 96.3 (17.8) | 0.098 |
| (N) | 195 | 41 | 76 | 17 | 119 | 24 | |||
| PP (mmHg) | 58.2 (2.1) | 55.2 (2.6) | 0.161 | 59.0 (2.8) | 55.5 (3.3) | 0.216 | 51.2 (4.1) | 47.7 (4.8) | 0.257 |
| Women | |||||||||
| Without medication | |||||||||
| (N) | 510 | 117 | 320 | 74 | 141 | 32 | |||
| HDL-C (mmHg) | 61.3 (1.0) | 62.3 (1.6) | 0.505 | 63.3 (1.5) | 63.9 (2.3) | 0.749 | 60.5 (2.5) | 62.4 (3.2) | 0.438 |
| Non-HDL-C (mg/dL) | 144.2 (1.9) | 145.6 (3.3) | 0.653 | 155.0 (2.7) | 160.7 (4.0) | 0.105 | 161.2 (5.2) | 152.9 (6.6) | 0.103 |
| (N) | 412 | 94 | 293 | 70 | 119 | 24 | |||
| PP (mmHg) | 52.3 (0.7) | 52.6 (1.1) | 0.784 | 51.2 (1.0) | 50.5 (1.4) | 0.607 | 47.7 (2.1) | 49.8 (2.9) | 0.353 |
| On medication | |||||||||
| (N) | 118 | 40 | 43 | 12 | 75 | 28 | |||
| HDL-C (mmHg) | 69.0 (2.1) | 65.6 (2.7) | 0.170 | 69.6 (3.1) | 68.1 (4.6) | 0.730 | 68.0 (3.4) | 63.3 (4.3) | 0.132 |
| Non-HDL-C (mg/dL) | 135.9 (4.2) | 128.8 (5.5) | 0.163 | 145.2 (7.7) | 134.3 (11.3) | 0.309 | 130.5 (5.8) | 125.7 (7.3) | 0.378 |
| (N) | 194 | 55 | 70 | 16 | 124 | 39 | |||
| PP (mmHg) | 58.2 (1.5) | 60.5 (2.1) | 0.235 | 58.6 (2.1) | 58.5 (3.6) | 0.984 | 56.9 (2.9) | 60.2 (3.3) | 0.181 |
Estimated mean (SE) adjusted for age, BMI, current alcohol consumption, smoking and walking habit
Discussion
The novel finding in the present study is that T-786C polymorphism in the promoter region in eNOS gene was significantly associated with arterial stiffness accompanied with age and gender differences. C allele is significantly associated with higher AIx@75 in women aged <65 years and with higher baPWV in women aged ≥65 years. In contrast, C allele is significantly and inversely associated with PP in men aged <65 years and with ACR and non-HDL-C/HDL-C ratio in men aged ≥65 years.
The present study subjects also had measurement of ankle-brachial index (ABI) and included 18 participants (14 male and 4 female) whose ABI values were <0.9. We re-examined associations of the eNOS polymorphism and baPWV excluding those with ABI <0.9, and we obtained similar results; C-allele was associated (P = 0.031) with higher baPWV in women aged ≥65 years (data not shown).
To our knowledge, there has been very limited number of studies reported the association of T-786C polymorphism with arteriosclerotic parameters. The study conducted with CAD patients failed to find the association of this polymorphism with AIx in the univariate analysis [25]. Another study showed significant positive association of T-786C polymorphism with albuminuria in participants containing diabetes patients (83 %) [26], which is inconsistent with our observation in the men aged ≥65 years in the present study. These previous studies were conducted with patients, thus, those results might not consistent with our findings in the present study, because the degree of arteriosclerotic parameters and endothelial function could be altered in patients with these diseases. Furthermore, these previous studies analyzed men and women together, and gender differences were not considered. Since NO production is affected by sex hormone, gender and age differences cannot be negligible. In fact, estrogen increases eNOS activity via phosphatidylinositol 3-kinase/Akt (PI3k/Akt) pathway or by reducing oxidative stress [27]. The expression of estrogen receptor associated with eNOS protein expression is greater in premenopausal women compared to those in postmenopausal women [28]. Although underlying mechanism is not as clear as estrogen, recent study revealed that androgens increase eNOS expression as well [29]. As those sex hormones decrease by aging, it is very important to consider age and gender differences when investigate the influence of eNOS polymorphisms. The details about menstrual status of the participants in the present study are unavailable; nevertheless, we assume the participants aged ≥65 years were less influenced by benefit of sex hormones.
The earliest study has shown reduction of promoter activity by 52 ± 11 % because of this mutation in HUVEC [8]. In consistent to in vitro study, the eNOS mRNA level in peripheral blood mononuclear cells shows a tendency towards a decrease with the number of C allele [30]. If this is the case for all, C allele should show positive association with arteriosclerotic parameters. It is actually true on AIx@75, baPWV, and PP in women in the present study. The age differences on the association of C allele with these indices can be explained partially by estrogen status. In addition, it has been reported that AIx is more sensitive in younger individuals [31].
Not like the influence of T-786C mutation on eNOS mRNA expression, the influence of this mutation on NO production in human is rather complicated. Since NO is rapidly oxidized to nitrite and nitrate (NOx) in vivo, measurement of NOx rather than NO itself has been used to assess the endogenous NO production [32]. Study conducted with healthy male population showed no association between T-786C polymorphism and circulating NOx concentration [33]. In contrast, plasma NOx concentration was significantly higher in CC homozygous in control group but not in CAD patients [34]. The reason of this discrepancy is unclear; however, there are differences between these two studies. Earlier study was assessed among younger men (mean age TT: 31.1 ± 9.1, TC: 32.3 ± 9.8, CC: 32.2 ± 9.9 years); whereas later study assessed among older subjects including women (men 71.6 %, mean age 51.8 ± 8.9 years). Thus, age and gender differences on NO production might cause this discrepancy.
To clarify the unexpected inverse association of eNOS T-786C polymorphisms with arteriosclerotic parameters in men, we first simply hypothesized which might be caused by the effects of cholesterol-lowering or anti-hypertensive medications. It has been reported that statins, cholesterol-lowering drugs, improve endothelial function and inhibit oxidative stress by increasing eNOS expression and enhance NO production through PI3k/Akt signaling [35]. Several studies have shown that effects of statins on reduction of oxidative stress, inflammation and metabolic lipid parameters were greater in CC homozygous than those in TT homozygous by modulating NO bioavailability [36–38]. This might support our findings in which men with C allele on cholesterol-lowering medication showed significantly lower non-HDL-C compared to those in TT homozygous. With respect to PP, men without anti-hypertensive medication with C allele showed trend being associated with lower PP compared to those with the non-C allele carriers. Although the difference was not statistically significant, this tendency was also seen in men on anti-hypertensive medication. Thus, the effects of medications do not explain fully our finding in inverse association in men.
There are several limitations in the present study. First, since this is the cross-sectional design, causal relationship between eNOS T-786C polymorphism and arteriosclerotic parameters remains unclear. If C allele respond to stress more favorably compared to those in TT homozygous as described above, life style factors which affect NO production such as exercise might have influence on NO bioavailability in C allele. Accumulation of these effects by aging might alter arteriosclerotic parameters. Further follow-up studies will help examine our hypothesis and clarify the causal relationship. Second, there are other genetic variations on eNOS gene, such as a SNP in exon 7 (Glu298Asp, rs1799983) and a variable number of tandem repeats (VNTR) in intron 4. It has been reported that specific haplotype C-Glu-4b (“C” of T-786C, “Glu” of Glu298Asp and “4b” of VNTR) is significantly associated with lower circulating NOx concentrations [39]. Recently, it has been reported that carriers of C-Glu showed increases in NO2 concentration after training independent of VNTR [40]. Thus, haplotype analyses are warranted to clarify the influence of eNOS on arteriosclerotic parameters. Third, because we have made comparisons for several arteriosclerotic parameters between non-C and C allele carriers by age and gender, we need to keep in mind possibilities of the effects of multiple comparisons. It is of importance to examine reproducibility of the current results in future studies with larger number of the study subjects.
Because the eNOS T-786C polymorphism has been associated with hypertension, ischemic stroke, diabetic nephropathy and coronary artery disease (CAD) in recent meta-analyses [9–12, 41], it is of great importance to investigate relationships between the polymorphism and arteriosclerotic parameters such as PWV, AIx, and albuminuria. It is also important to investigate relationships between this polymorphism and relatively late-stage arteriosclerotic parameters such as ABI and carotid intima-media thickness (CIMT). Those investigations would provide collective idea for the effects of this polymorphism on stage-specific arteriosclerotic parameter changes. Furthermore, prospective studies to investigate the effects of this polymorphism and other factors on arteriosclerotic parameters with large number of study subjects are of great importance. We plan to do such studies in near future.
In conclusion, ENOS T-786C polymorphisms are associated with arteriosclerotic parameters accompanied with age and gender differences. In which, C allele showed positive association with arteriosclerotic parameters in women; whereas it showed inverse association with arteriosclerotic parameters in men. It suggests possible involvement of hormones and/or endothelial signaling other than inflammatory signaling.
Acknowledgments
This work was supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (25460811 and 25350894). We are grateful to Dr. Mitsuru Shiba, Dr. Yuji Uematsu and Ms. Momoko Buyo of Wakayama Medical University for their supports in the examination of participants. We are also grateful to Ms. Mami Murao and Mr. Yushiro Kagami for their participation in the genotyping. We also wish to thank Ms. Toshiko Hanamitsu, a public health nurse, and other members of the Public office on-site in for their assistances in locating and scheduling participants for examinations. We also thank Ms. Yuko Kuwabara for her assistance with data reduction and administration.
Compliance with ethical standards
Conflict of interest
No conflicts of interest, financial or otherwise, are declared by the authors.
References
- 1.Cecelja M, Chowienczyk P. Role of arterial stiffness in cardiovascular disease. JRSM Cardiovasc Dis. 2012;1(4):11. doi: 10.1258/cvd.2012.012016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Shirwany NA, Zou MH. Arterial stiffness: a brief review. Acta Pharmacol Sin. 2010;31:1267–1276. doi: 10.1038/aps.2010.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Vanhoutte PM, Shimokawa H, Tang EH, Feletou M. Endothelial dysfunction and vascular disease. Acta Physiol (Oxf) 2009;196:193–222. doi: 10.1111/j.1748-1716.2009.01964.x. [DOI] [PubMed] [Google Scholar]
- 4.Cortese-Krott MM, Kelm M. Endothelial nitric oxide synthase in red blood cells: key to a new erythrocrine function? Redox Biol. 2014;2:251–258. doi: 10.1016/j.redox.2013.12.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Lowenstein CJ, Michel T. What’s in a name? eNOS and anaphylactic shock. J Clin Invest. 2006;116:2075–2078. doi: 10.1172/JCI29406. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Peng X, Haldar S, Deshpande S, Irani K, Kass DA. Wall stiffness suppresses Akt/eNOS and cytoprotection in pulse-perfused endothelium. Hypertension. 2003;41:378–381. doi: 10.1161/01.HYP.0000049624.99844.3D. [DOI] [PubMed] [Google Scholar]
- 7.Rossi GP, Maiolino G, Zanchetta M, Sticchi D, Pedon L, Cesari M, et al. The T(-786)C endothelial nitric oxide synthase genotype predicts cardiovascular mortality in high-risk patients. J Am Coll Cardiol. 2006;48:1166–1174. doi: 10.1016/j.jacc.2006.05.046. [DOI] [PubMed] [Google Scholar]
- 8.Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, et al. T-786→C mutation in the 5′-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999;99:2864–2870. doi: 10.1161/01.CIR.99.22.2864. [DOI] [PubMed] [Google Scholar]
- 9.Li J, Wu X, Li X, Feng G, He L, Shi Y. The endothelial nitric oxide synthase gene is associated with coronary artery disease: a meta-analysis. Cardiology. 2010;116:271–278. doi: 10.1159/000316063. [DOI] [PubMed] [Google Scholar]
- 10.Niu W, Qi Y. An updated meta-analysis of endothelial nitric oxide synthase gene: three well-characterized polymorphisms with hypertension. PLoS One. 2011;6:e24266. doi: 10.1371/journal.pone.0024266. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Wang M, Jiang X, Wu W, Zhang D. Endothelial NO synthase gene polymorphisms and risk of ischemic stroke in Asian population: a meta-analysis. PLoS One. 2013;8:e60472. doi: 10.1371/journal.pone.0060472. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Zeng Z, Li L, Zhang Z, Li Y, Wei Z, Huang K, et al. A meta-analysis of three polymorphisms in the endothelial nitric oxide synthase gene (NOS3) and their effect on the risk of diabetic nephropathy. Hum Genet. 2010;127:373–381. doi: 10.1007/s00439-009-0783-x. [DOI] [PubMed] [Google Scholar]
- 13.Kals J, Kampus P, Kals M, Pulges A, Teesalu R, Zilmer M. Effects of stimulation of nitric oxide synthesis on large artery stiffness in patients with peripheral arterial disease. Atherosclerosis. 2006;185:368–374. doi: 10.1016/j.atherosclerosis.2005.06.014. [DOI] [PubMed] [Google Scholar]
- 14.Cakar M, Bulucu F, Karaman M, Ay SA, Kurt O, Balta S, et al. Asymmetric dimethylarginine and augmentation index in newly diagnosed patients with hypertension. Angiology. 2015;66:43–48. doi: 10.1177/0003319713513145. [DOI] [PubMed] [Google Scholar]
- 15.Weber T, Maas R, Auer J, Lamm G, Lassnig E, Rammer M, et al. Arterial wave reflections and determinants of endothelial function a hypothesis based on peripheral mode of action. Am J Hypertens. 2007;20:256–262. doi: 10.1016/j.amjhyper.2006.09.009. [DOI] [PubMed] [Google Scholar]
- 16.Masaki N, Hakuno D, Toya T, Shiraishi Y, Kujiraoka T, Namba T, et al. Association between brachial-ankle pulse wave velocity and the ratio of l-arginine to asymmetric dimethylarginine in patients undergoing coronary angiography. J Cardiol. 2015;65:311–7. doi: 10.1016/j.jjcc.2014.06.005. [DOI] [PubMed] [Google Scholar]
- 17.Rosenbaum D, Giral P, Chapman J, Rached FH, Kahn JF, Bruckert E, et al. Radial augmentation index is a surrogate marker of atherosclerotic burden in a primary prevention cohort. Atherosclerosis. 2013;231:436–441. doi: 10.1016/j.atherosclerosis.2013.10.004. [DOI] [PubMed] [Google Scholar]
- 18.Mineo C, Yuhanna IS, Quon MJ, Shaul PW. High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J Biol Chem. 2003;278:9142–9149. doi: 10.1074/jbc.M211394200. [DOI] [PubMed] [Google Scholar]
- 19.Zhao W, Gong W, Wu N, Li Y, Ye K, Lu B, et al. Association of lipid profiles and the ratios with arterial stiffness in middle-aged and elderly Chinese. Lipids Health Dis. 2014;13:37. doi: 10.1186/1476-511X-13-37. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Eliasson B, Cederholm J, Eeg-Olofsson K, Svensson AM, Zethelius B, Gudbjornsdottir S, et al. Clinical usefulness of different lipid measures for prediction of coronary heart disease in type 2 diabetes: a report from the Swedish National Diabetes Register. Diabetes Care. 2011;34:2095–2100. doi: 10.2337/dc11-0209. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Liu CS, Pi-Sunyer FX, Li CI, Davidson LE, Li TC, Chen W, et al. Albuminuria is strongly associated with arterial stiffness, especially in diabetic or hypertensive subjects–a population-based study (Taichung Community Health Study, TCHS) Atherosclerosis. 2010;211:315–321. doi: 10.1016/j.atherosclerosis.2010.02.015. [DOI] [PubMed] [Google Scholar]
- 22.Miyai N, Utsumi M, Gowa Y, Igarashi Y, Miyashita K, Takeda S, et al. Age-specific nomogram of brachial-ankle pulse wave velocity in Japanese adolescents. Clin Exp Hypertens. 2013;35:95–101. doi: 10.3109/10641963.2012.690473. [DOI] [PubMed] [Google Scholar]
- 23.Yamashina A, Tomiyama H, Takeda K, Tsuda H, Arai T, Hirose K, et al. Validity, reproducibility, and clinical significance of noninvasive brachial-ankle pulse wave velocity measurement. Hypertens Res. 2002;25:359–364. doi: 10.1291/hypres.25.359. [DOI] [PubMed] [Google Scholar]
- 24.Wilkinson IB, MacCallum H, Flint L, Cockcroft JR, Newby DE, Webb DJ. The influence of heart rate on augmentation index and central arterial pressure in humans. J Physiol. 2000;525(Pt 1):263–270. doi: 10.1111/j.1469-7793.2000.t01-1-00263.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Lekakis JP, Ikonomidis I, Tsibida M, Protogerou A, Papada A, Papapanagiotou A, et al. Genetic variations of the endothelial nitric oxide synthase gene are related to increased levels of C-reactive protein and macrophage-colony stimulating-factor in patients with coronary artery disease. Thromb Haemost. 2006;96:520–528. [PubMed] [Google Scholar]
- 26.Liu Y, Burdon KP, Langefeld CD, Beck SR, Wagenknecht LE, Rich SS, et al. T-786C polymorphism of the endothelial nitric oxide synthase gene is associated with albuminuria in the diabetes heart study. J Am Soc Nephrol. 2005;16:1085–1090. doi: 10.1681/ASN.2004100817. [DOI] [PubMed] [Google Scholar]
- 27.Duckles SP, Miller VM. Hormonal modulation of endothelial NO production. Pflug Arch. 2010;459:841–851. doi: 10.1007/s00424-010-0797-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Gavin KM, Seals DR, Silver AE, Moreau KL. Vascular endothelial estrogen receptor alpha is modulated by estrogen status and related to endothelial function and endothelial nitric oxide synthase in healthy women. J Clin Endocrinol Metab. 2009;94:3513–3520. doi: 10.1210/jc.2009-0278. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Goglia L, Tosi V, Sanchez AM, Flamini MI, Fu XD, Zullino S, et al. Endothelial regulation of eNOS, PAI-1 and t-PA by testosterone and dihydrotestosterone in vitro and in vivo. Mol Hum Reprod. 2010;16:761–769. doi: 10.1093/molehr/gaq049. [DOI] [PubMed] [Google Scholar]
- 30.Venturelli E, Galimberti D, Lovati C, Fenoglio C, Scalabrini D, Mariani C, et al. The T-786C NOS3 polymorphism in Alzheimer’s disease: association and influence on gene expression. Neurosci Lett. 2005;382:300–303. doi: 10.1016/j.neulet.2005.03.032. [DOI] [PubMed] [Google Scholar]
- 31.McEniery CM, Yasmin Hall IR, Qasem A, Wilkinson IB, Cockcroft JR, et al. Normal vascular aging: differential effects on wave reflection and aortic pulse wave velocity: the Anglo-Cardiff Collaborative Trial (ACCT) J Am Coll Cardiol. 2005;46:1753–1760. doi: 10.1016/j.jacc.2005.07.037. [DOI] [PubMed] [Google Scholar]
- 32.Baylis C, Vallance P. Measurement of nitrite and nitrate levels in plasma and urine—what does this measure tell us about the activity of the endogenous nitric oxide system? Curr Opin Nephrol Hypertens. 1998;7:59–62. doi: 10.1097/00041552-199801000-00010. [DOI] [PubMed] [Google Scholar]
- 33.Nagassaki S, Metzger IF, Souza-Costa DC, Marroni AS, Uzuelli JA, Tanus-Santos JE. eNOS genotype is without effect on circulating nitrite/nitrate level in healthy male population. Thromb Res. 2005;115:375–379. doi: 10.1016/j.thromres.2004.09.003. [DOI] [PubMed] [Google Scholar]
- 34.Salimi S, Naghavi A, Firoozrai M, Zand H, Tavilani H, Nakhaee A, et al. Association of plasma nitric oxide concentration and endothelial nitric oxide synthase T-786C gene polymorphism in coronary artery disease. Pathophysiology. 2012;19:157–162. doi: 10.1016/j.pathophys.2012.04.003. [DOI] [PubMed] [Google Scholar]
- 35.Förstermann U, Munzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 2006;113:1708–1714. doi: 10.1161/CIRCULATIONAHA.105.602532. [DOI] [PubMed] [Google Scholar]
- 36.Nagassaki S, Sertorio JT, Metzger IF, Bem AF, Rocha JB, Tanus-Santos JE. eNOS gene T-786C polymorphism modulates atorvastatin-induced increase in blood nitrite. Free Radic Biol Med. 2006;41:1044–1049. doi: 10.1016/j.freeradbiomed.2006.04.026. [DOI] [PubMed] [Google Scholar]
- 37.Souza-Costa DC, Sandrim VC, Lopes LF, Gerlach RF, Rego EM, Tanus-Santos JE. Anti-inflammatory effects of atorvastatin: modulation by the T-786C polymorphism in the endothelial nitric oxide synthase gene. Atherosclerosis. 2007;193:438–444. doi: 10.1016/j.atherosclerosis.2006.07.020. [DOI] [PubMed] [Google Scholar]
- 38.Zago VH, Santos JE, Danelon MR, Silva RM, Panzoldo NB, Parra ES, et al. Effects of atorvastatin and T-786C polymorphism of eNOS gene on plasma metabolic lipid parameters. Arq Bras Cardiol. 2013;100:14–20. doi: 10.1590/S0066-782X2012005000120. [DOI] [PubMed] [Google Scholar]
- 39.Metzger IF, Souza-Costa DC, Marroni AS, Nagassaki S, Desta Z, Flockhart DA, et al. Endothelial nitric oxide synthase gene haplotypes associated with circulating concentrations of nitric oxide products in healthy men. Pharmacogenet Genom. 2005;15:565–570. doi: 10.1097/01.fpc.0000167328.85163.44. [DOI] [PubMed] [Google Scholar]
- 40.da Silva RF, Sertorio JT, Lacchini R, Trape AA, Tanus-Santos JE, Rush JW, et al. Influence of training status and eNOS haplotypes on plasma nitrite concentrations in normotensive older adults: a hypothesis-generating study. Aging Clin Exp Res. 2014;26:591–598. doi: 10.1007/s40520-014-0218-y. [DOI] [PubMed] [Google Scholar]
- 41.Liu D, Jiang Z, Dai L, Zhang X, Yan C, Han Y. Association between the—786T> C 1polymorphism in the promoter region of endothelial nitric oxide synthase (eNOS) and risk of coronary artery disease: a systematic review and meta-analysis. Gene. 2014;545:175–183. doi: 10.1016/j.gene.2013.09.099. [DOI] [PubMed] [Google Scholar]