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Ankle–brachial index in relation to the natriuretic peptide system polymorphisms and urinary sodium excretion in Chinese
Atherosclerosis 230 (2013) 86e91
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Ankleebrachial index in relation to the natriuretic peptide system polymorphisms and urinary sodium excretion in Chinese Bang-Chuan Hu a, b, Yan Li a, Ming Liu a, Chang-Sheng Sheng a, Ji-Guang Wang a, * a
Centre for Epidemiological Studies and Clinical Trials, The Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Ruijin 2nd Road 197, Shanghai 200025, China b Intensive Care Unit, Zhejiang Provincial People’s Hospital, 158 Shangtang Road, Hangzhou 310014, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 January 2013 Received in revised form 15 June 2013 Accepted 24 June 2013 Available online 10 July 2013
Objective: Recent studies have demonstrated that the natriuretic pepetides induce endothelial regeneration and angiogenesis after vascular injury through the autocrine or paracrine action, and might have an inhibitory effect on atherosclerosis. We therefore systematically investigated single nucleotide polymorphisms (SNPs) in the natriuretic peptide system in relation to ankleebrachial index (ABI) in a Chinese population. Methods: The study population was recruited from a mountainous area 500 km south of Shanghai from 2003 to 2009. Using the SNapShot method, we first genotyped 951 subjects enrolled in 2005 for 16 SNPs and then the remaining 1355 subjects as validation for 5 SNPs selected from the primary study. ABI and plasma proBNP were measured using the Omron VP-2000/1000 device and the Elecsys proBNP immunoassay, respectively. Results: Overall, the genetic associations were not significant (P 0.07). However, in the primary study, there was significant (Pint 0.045) interaction between 3 SNPs (rs6668352, rs198388, and rs198389) at the NPPAeNPPB locus and urinary sodium excretion in relation to ABI, and the rs6668352 polymorphism had the strongest association (Pint ¼ 0.018). In the primary combined with the validation study populations, the interaction between the rs6668352 polymorphism and urinary sodium excretion in relation to ABI remained statistically significant (Pint ¼ 0.0036). After adjustment for covariates, the rs6668352 A allele carriers, compared with GG homozygotes, had a higher ABI (mean standard error, 1.103 0.006 vs. 1.084 0.004, P ¼ 0.009) and lower risk of peripheral arterial disease (PAD, defined as an ABI < 0.90, odds ratio 0.37, 95% confidence interval: 0.14e0.98, P ¼ 0.04) in the subjects of high sodium intake. Conclusion: The minor alleles of 3 SNPs at the NPPAeNPPB locus are associated with a lower risk of PAD, especially in the subjects of high sodium intake. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Genetic polymorphism Nartiuretic peptide Urinary sodium excretion ABI Population
1. Introduction Natriuretic peptides consist of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) and elicit various biological effects by binding to two guanylyl cyclase-coupled receptors (natriuretic peptide receptors A [NPRA] and B [NPRB]) [1,2]. ANP and BNP preferentially bind to NPRA. CNP has a higher affinity with NPRB. Corin is a recently identified transmembrane serine protease, is highly expressed in cardiomyocytes, and cleaves inactive pro-ANP and pro-BNP into smaller biologically active molecules [3,4]. Natriuretic peptides play
* Corresponding author. Tel.: þ86 21 6437 0045x610911; fax: þ86 21 6466 2193. E-mail address: [email protected] (J.-G. Wang). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.06.020
an important role in the regulation of blood pressure through diuretic, natriuretic, and vasodilatory properties [5], and have beneficial roles in the cardiovascular system through the autocrine or paracrine action [1,2]. ANP at physiological concentrations induces proliferation and migration of endothelial cells and enhances endothelial regeneration in the vascular wall [6], where it also exerts anti-inflammatory properties [7]. Genetically increased BNP can promote vascular regeneration and accelerate the restoration of blood flow after the removal of a hindelimb artery in mice through the activation of the NPRA/cGMP/cGMP dependent protein kinase (cGKI) pathway [8]. CNP as an endothelium-derived relaxing peptide also accelerates re-endothelialization and suppresses neointimal hyperplasmia in vein grafting or balloon injuries in rabbits, which stimulate atherosclerotic lesions in humans [9,10]. These observations suggest that natriuretic peptides have the potential to promote vascular regeneration in peripheral arterial disease (PAD)
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and to inhibit the progression of atherosclerotic lesions. Indeed, in several population studies, circulating levels of natriuretic peptides have been demonstrated to be correlated positively with endothelial function [11,12] and inversely with early atherosclerosis [12]. The ankleebrachial index (ABI) is a widely used measure for detecting low extremity PAD. A decreased ABI indicates target organ damage [13e15] and predicts cardiovascular and all-cause mortality [14,16,17]. Although a number of atherosclerotic risk factors contribute to PAD and lower ABI, genetic factors account for approximately 21e48% of the variability in both ABI and the presence of PAD, defined according to ABI [18,19]. Genes that have potent vasodilatory and anti-atherosclerotic properties might be associated with ABI and the risk of PAD. Identification of these genes may provide useful clues in understanding the complex quantitative cardiovascular traits. Recent studies revealed significant associations of several polymorphisms at the NPPAeNPPB locus with circulating natriuretic peptides and incident hypertension in White populations [20e23]. The minor alleles of two tightly linked polymorphisms (T555I/Q568P) in the Corin gene were also associated with an increased risk of hypertension [24] and an enhanced cardiac hypertrophic response to pressure overload in African Americans [25]. However, to date, no previous study has ever investigated the relationship between genetic variants of the natriuretic peptide system and ABI. In addition, there is evidence that high sodium intake may affect the progression of atherosclerosis [26e28] and increase the level of plasma BNP. Sodium excretion is also a modulator of genetic associations with cardiovascular phenotypes [29]. In the present study, we therefore systematically investigated the association of 16 SNPs of 6 genes in the natriuretic peptide system (NPPA, NPPB, NPPC, NPRA, NPRC and Corin) with ABI and urinary sodium excretion, and explored the potential geneeenvironment interaction in relation to ABI and the risk of PAD in a Chinese population. 2. Methods 2.1. Study populations The study population was recruited in the framework of an ongoing longitudinal population-based genetic study in hypertension. From 2003 to 2009, we visited all homes in 18 villages randomly selected from JingNing County, a mountainous rural area approximately 500 km south of Shanghai. We invited all inhabitants at least 12 years of age to take part. Of the 3402 invited, 2392 (70.3%) participated. The study protocol was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. All participants gave informed written consent. Our primary genetic study included 981 subjects enrolled in 2005, for whom we genotyped 16 SNPs in the natriuretic peptide system. We excluded 30 subjects from the present analysis because of missing information on genotype (n ¼ 8) and phenotype (n ¼ 22). Thus, the total number of subjects in the primary study was 951. Our validation genetic study included the remaining 1411 subjects, for whom we genotyped 5 SNPs selected from the primary study. We excluded 56 subjects because of missing information on genotype (n ¼ 12) and phenotype (n ¼ 44). Thus, the total number of subjects included in the validation study was 1355. 2.2. Field work One experienced physician measured each participant’s brachial blood pressure five times consecutively by mercury
87
sphygmomanometry, after the subject had rested for at least 5 min in the sitting position. These five blood pressure readings were averaged for analysis. During the home visit, the same observer also administered a standardized questionnaire to collect information on medical history, smoking habits, alcohol consumption, and the use of medications. Hypertension was defined as a blood pressure of at least 140 mmHg systolic or 90 mmHg diastolic or as the use of antihypertensive drugs. Venous blood samples were taken after overnight fast for the measurement of plasma glucose concentration and for measurements of serum concentrations of total cholesterol and triglycerides. A 24-h urine sample was collected in a wide-neck plastic container for the measurement of urinary electrolytes. To ensure the complete collection of urine, urinary samples less than 600 ml were excluded. Thus, 847 and 918 urinary samples were available for measurement of urinary electrolytes in the primary and validation studies, respectively. 2.3. Genotyping The 16 SNPs in 6 genes (NPPA, NPPB, NPPC, NPRA, NPRC, and Corin) of the natriuretic peptide system were genotyped using the ABI PRISMÒ SNapShotÒ method (Applied Biosystem, CA, USA). In brief, the SNapShot reactions were carried out in a 10 ml final volume containing SNapShot Multiplex Ready Mix (5 ml), primer mix (0.02e0.6 mmol/l), and templates (4 ml) consisting of the multiplex PCR products, which had been purified with the QIAquick PCR Purification Kit (QIAGEN, GmbH, Germany). The cycling program included 25 cycles of 96 C for 10 s, 50 C for 5 s, and 60 C for 30 s. Extension products were purified by a 15-min incubation with1 U of shrimp alkaline phosphatase (Promega, Madison, WI, USA) at 37 C and a subsequent 15-min incubation at 80 C to inactivate the enzyme. The purified products (0.5 ml) were mixed with 9 ml of formamide and 0.5 ml of GeneScan-120 LIZ Size Standard (Applied Biosystems) and separated by capillary electrophoresis (ABI PRISM310 Genetic Analyzer; Applied Biosystems). The results were analyzed with GeneMapper 3.0 software (Applied Biosystems). All PCR and SNapShot primers are listed in supplementary Table 1. More than 98% of the total samples were successfully genotyped for all SNPs. To confirm the genotyping results, 30 samples were randomly selected and re-genotyped by direct sequencing using a BigDye terminator (Applied Biosystem). All assays were 100% concordant. 2.4. Measurements of ABI To ensure steady state, one trained physician performed all the ABI measurements under standardized conditions in a quiet examination room after the subjects had rested for 10 min in the supine position. The participants were asked to refrain from smoking, vigorous exercise, and drinking alcohol or caffeinecontaining beverages for at least 2 h before the examination. We used the Omron VP-2000/1000 device (Omron Healthcare, Kyoto, Japan) to obtain simultaneous oscillometric blood pressure readings at the right and left brachial and posterior tibial arteries. ABI was defined as a ratio of the ankle-to-brachial systolic blood pressure measurements and calculated for both sides. The lower of the 2 ABI values was determined (low ABI) and used in subsequent statistical analyses. In line with current recommendations, an ABI of 0.9 or less in either leg was defined as possible indicative of PAD. Those with an ABI >1.4 were excluded because it is indicative of incompressible, calcified arteries, and inclusion of these individuals could have led to misclassification of arterial extremity disease.
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2.5. Measurement of plasma proBNP concentration In 1386 individuals of the JingNing study from 2003 to 2005, we collected venous blood samples in chilled ethylenediaminetetraacetic acid tubes. After centrifugation, the clarified plasma samples were frozen, stored at 80 C, and thawed just before assays. Plasma proBNP was measured using an automated, commercially available immunoassay (Elecsys proBNP, Roche Diagnostics, Indianapolis, Indiana, USA) according to the manufacturer’s instructions. The inter-assay coefficient of variation in our study was 1.2%. 2.6. Statistical methods We used SAS version 9.13 (SAS institute, Cary, NC, USA) for database management and statistical analyses. Comparisons of means and proportions relied on the Student t-test and Fisher’s exact test, respectively. Continuous measurements with a skewed distribution were normalized by logarithmic transformation and represented by geometric mean and 95% confidence interval (CI). HardyeWeinberg equilibrium was tested using the c2 test. We identified covariates of the phenotypes under study using stepwise multiple regressions with a P-value for independent variables to enter and stay in the model set at 0.10. We studied genetic associations using the analysis of covariance, while controlling for covariates. The value of P < 0.05 was considered to indicate statistical significance. 3. Results 3.1. Characteristics of the primary study population In the primary study, the 951 participants included 470 men (49.4%), 222 (23.3%) hypertensive patients, and 31 patients (3.3%) with PAD. Table 1 shows the characteristics of the study participants by sex. Men and women were similar in characteristics, except that men were slightly older (þ3.0 years, P ¼ 0.003) and had Table 1 Characteristics of the primary study population. Characteristic
Men (n ¼ 470)
Women (n ¼ 481)
P
Age (years) Body mass index (kg/m2) Current smoking, n (%) Alcohol intake 5 g/week, n (%) Prevalence of hypertension, n (%) Took antihypertensive drugs, n (%) Serum total cholesterol (mmol/l) Serum triglycerides (mmol/l) Fasting plasma glucose (mmol/l) Serum creatinine (mmol/l) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Pulse pressure (mmHg) ProBNP (pg/ml)
46.5 15.9 22.0 2.8 285 (60.6) 297 (63.2) 98 (20.9) 20 (4.2) 4.87 0.96 1.48 2.08 4.38 1.76 78.7 22.0 129.2 24.4 75.9 12.4 53.3 20.5 32.3 (30.2e34.6) 1.106 0.084 13 (2.8)
43.5 15.0 22.1 2.9 0 139 (28.9) 124 (25.8) 40 (8.3) 4.78 0.97 1.24 0.95 4.45 0.82 64.5 15.1 129.8 26.6 75.8 13.9 54.0 19.7 46.9 (45.1e49.9) 1.069 0.093 18 (3.7)
0.003 0.46 <0.001 <0.001 0.08 <0.001 0.13 0.02 0.40 <0.001 0.74 0.88 0.60 <0.001 <0.001 0.40
1.33 0.58 181.0 86.6 25.4 9.7
1.14 0.45 178.0 78.7 24.4 10.0
<0.001 0.54 0.14
Ankleebrachial index Prevalence of peripheral arterial disease, n (%) 24-h urinary measurements Volume (l) Sodium excretion (mmol/day) Potassium excretion (mmol/day)
Values are mean SD or number of subjects (percentage of the column total). P values are for gender difference. 24-h urinary measurements were available in 420 men and 427 women. Plasma proBNP concentration was measured in 676 men and 710 women recruited in the primary and validation studies.
higher serum concentrations of triglycerides (þ0.24 mmol/l, P ¼ 0.02) and creatinine (14.2 mmol/l, P < 0.001). Men, compared with women, had a significantly (P < 0.001) greater urinary volume (þ0.19 l) and ABI (þ0.039) and lower plasma proBNP level (14.6 pg/ml). However, they had similar (P 0.14) urinary sodium (179.2 82.9 mmol/day) and potassium excretions (24.8 9.9 mmol/day) and similar prevalence of PAD (2.8% vs. 3.7%, P ¼ 0.40). 3.2. ABI and SNPs in the natriuretic peptide system In the primary study, the genotype frequencies of the 16 SNPs did not deviate from the HardyeWeinberg equilibrium (P 0.08), and were similar in men and women (P 0.12) and in the presence (n ¼ 31) and absence of PAD (n ¼ 920, P 0.26). With adjustments for age, sex, body mass index, mean arterial pressure, current smoking, alcohol intake, serum creatinine, and the use of antihypertensive drugs, we did not find any significant association between ABI and 16 SNPs in the natriuretic peptide system (P 0.07, Table 2). However, with similar adjustments applied, there was significant interaction (Pint 0.045) between the 3 SNPs (rs6668352, rs198388, and rs198389) at the NPPAeNPPB locus and urinary sodium excretion in relation to ABI, and the rs6668352 polymorphism had the strongest association (Pint ¼ 0.018). In subjects above the median of urinary sodium excretion (182.1 mmol/day), the rs6668352 A allele carriers, compared with GG homozygotes, had an higher ABI (1.105 0.004 vs. 1.086 0.006, P ¼ 0.03). However, in subjects below the median of sodium excretion, there was no difference in ABI between A allele carriers and GG homozygotes (1.094 0.004 vs. 1.086 0.006; P ¼ 0.27). 3.3. The validation study We performed a validation study in the remaining 1355 individuals of the JingNing study. Of the 5 SNPs genotyped (rs198358, rs6668352, rs3811544, rs700923, and 9662664), none had significant association with ABI (P 0.09, Table 3) or with the prevalence of PAD (n ¼ 46 [3.4%], P 0.13). In the combined analysis of the primary and validation studies, no significant differences in genotype distributions of the 5 SNPs were found between the subjects with PAD (n ¼ 77) and those without (n ¼ 2229, P 0.19, Table 4). However, the interaction between the rs6668352 polymorphism and urinary sodium excretion in relation to ABI remained statistically significant (Pint ¼ 0.0036). In subjects above the median of sodium excretion (180.8 mmol/l), the A allele carriers, compared with GG homozygotes, had a higher ABI (1.103 0.006 vs. 1.084 0.004, P ¼ 0.009) and a lower risk of PAD (odds ratio [OR] 0.37, 95% CI: 0.14e0.98, P ¼ 0.04). However, in subjects below the median of sodium excretion, the A allele carriers and GG homozygotes had a similar ABI (1.092 0.003 vs. 1.084 0.005; P ¼ 0.18) and risk of PAD (OR 1.23, 95% CI: 0.51e3.01, P ¼ 0.64, Fig. 1). 3.4. Plasma proBNP level and ABI In 1386 participants, plasma proBNP level was positively correlated with ABI (r ¼ 0.11, P < 0.0001). In multivariate regression analyses adjusted for age, sex, body mass index, current smoking, alcohol intake, mean arterial pressure, serum creatinine, and the use of antihypertensive drugs, plasma proBNP was an independent determinant of ABI (regression coefficient SE: 0.0158 0.007, P ¼ 0.024). In further analyses stratified for the tertile distributions of plasma proBNP, both before and after adjustment for covariates, ABI was significantly (P 0.007) different across tertiles of proBNP.
B.-C. Hu et al. / Atherosclerosis 230 (2013) 86e91 Table 2 Ankleebrachial index in relation to 16 SNPs in the natriuretic peptide system in the primary study. Gene and polymorphism Ankleebrachial index NPPAeNPPB locus rs198358
P
AA (n ¼ 697) GA (n ¼ 237) GG (n ¼ 17) 1.086 0.003 1.084 0.006 1.112 0.022 0.54
rs5063
GG (n ¼ 724) GA (n ¼ 211) AA (n ¼ 16) 1.086 0.003 1.083 0.006 1.118 0.023 0.34
rs198375
AA (n ¼ 622) GA (n ¼ 304) GG (n ¼ 25) 1.085 0.004 1.086 0.005 1.092 0.018 0.93
rs6668352
GG (n ¼ 674) GA (n ¼ 262) AA (n ¼ 15) 1.085 0.004 1.084 0.006 1.120 0.024 0.31
rs198388
CC (n ¼ 636) CT (n ¼ 291) TT (n ¼ 24) 1.085 0.004 1.086 0.005 1.093 0.019 0.92
rs198389
AA (n ¼ 647) GA (n ¼ 279) GG (n ¼ 25) 1.084 0.004 1.086 0.005 1.109 0.005 0.42
NPPC rs5262 rs3811544
NPRA rs28730726 rs9662664
NPRC rs2270915
GG (n ¼ 674) GA (n ¼ 251) AA (n ¼ 26) 1.086 0.004 1.086 0.006 1.085 0.018 0.99 CC (n ¼ 902) CT (n ¼ 49) 1.086 0.003 1.077 0.013
GG (n ¼ 803) GC (n ¼ 143) CC (n ¼ 5) 1.084 0.003 1.094 0.008 1.106 0.043 0.47 GG (n ¼ 422) GT (n ¼ 420) TT (n ¼ 109) 1.082 0.004 1.093 0.004 1.071 0.009 0.07
AA (n ¼ 693) AG (n ¼ 242) GG (n ¼ 16) 1.086 0.003 1.084 0.006 1.107 0.022 0.57 AA (n ¼ 586) AG (n ¼ 332) GG (n ¼ 33) 1.085 0.004 1.084 0.005 1.104 0.016 0.49
rs2292026
CC (n ¼ 605) CT (n ¼ 316) TT (n ¼ 30) 1.088 0.004 1.083 0.005 1.103 0.016 0.51
CC (n ¼ 929) CT (n ¼ 22) 1.085 0.003 1.102 0.020
Table 3 Ankleebrachial index in relation to 5 SNPs in the natriuretic peptide system in the validation study. Gene and polymorphism Ankleebrachial index NPPAeNPPB locus rs198358 rs6668352
NPPC rs3811544
NPRA rs9662664
NPRC rs700923
P
AA (n ¼ 927) GA (n ¼ 403) GG (n ¼ 25) 1.091 0.003 1.093 0.004 1.104 0.017 0.76 GG (n ¼ 864) GA (n ¼ 446) AA (n ¼ 45) 1.094 0.003 1.087 0.004 1.117 0.013 0.09
CC (n ¼ 1273) CT (n ¼ 82) 1.093 0.003 1.082 0.010
0.27
GG (n ¼ 587) GT (n ¼ 601) TT (n ¼ 167) 1.091 0.004 1.093 0.004 1.097 0.007 0.68
AA (n ¼ 849) AG (n ¼ 457) GG (n ¼ 49) 1.094 0.003 1.090 0.004 1.100 0.013 0.61
Values are mean SE. Ankleebrachial index was adjusted for sex, age, body mass index, mean arterial pressure, current smoking, alcohol intake, serum creatinine, and the use of antihypertensive drugs.
0.49
rs700923
Corin rs11934749
89
0.40
rs3749585
TT (n ¼ 329) TC (n ¼ 436) CC (n ¼ 186) 1.089 0.005 1.084 0.004 1.083 0.007 0.76
rs2289433
AA (n ¼ 470) GA (n ¼ 397) GG (n ¼ 84) 1.086 0.004 1.084 0.005 1.092 0.010 0.75
Values are mean SE. Ankleebrachial index was adjusted for sex, age, body mass index, mean arterial pressure, current smoking, alcohol intake, serum creatinine, and the use of antihypertensive drugs.
The unadjusted ABI was 1.084 (0.083), 1.094 (0.088), and 1.107 (0.09) in the top (<24.7 pg/ml, n ¼ 464), middle (24.7e55.3 pg/ml, n ¼ 468) and bottom tertiles (>55.3 pg/ml; n ¼ 454), respectively (Fig. 2).
4. Discussion The main finding was first to demonstrate the interaction between the 3 SNPs (rs198389, rs198388, rs6668352) at the NPPAe NPPB locus, which are in tight linkage disequilibrium, and urinary sodium excretion in relation to ABI. Our validation study was basically confirmatory. Few studies investigated the association between the abovementioned 3 SNPs and cardiovascular phenotypes. In a recent caseecontrol study including 697 patients of European descent undergoing primary coronary artery bypass graft, the minor alleles
of these 3 SNPs were associated with a lower risk of left ventricular dysfunction (P 0.03) [30]. In a larger French population cohort (n ¼ 3216), Meirhaeghe and colleagues [31] reported that the rs198389 CC subjects, compared with T allele carriers, had a lower risk of impaired fasting glucose (P ¼ 0.005) and type 2 diabetes (OR 0.69; 95% CI: 0.47e1.00, P ¼ 0.05). These genetic associations were replicated in 4 additional caseecontrol study populations for type 2 diabetes mellitus [31]. In addition, these investigators found that the rs198389 polymorphism might be a functional variant, with a higher transcriptional activity in the C allele carriers [31]. Our and previous studies consistently demonstrated that the minor alleles of these 3 SNPs were associated with an elevated circulating proBNP level in the general population [22]. Taken together, these findings suggested that the protective associations of the SNPs at the NPPAeNPPB locus with left ventricular dysfunction, diabetes mellitus or ABI might be attributable to increased circulating levels of BNP. However, we observed that, of the 3 SNPs, the rs6668352 polymorphism, not the rs198389 polymorphism, had the strongest
Table 4 Genotype frequencies according to the presence and absence of peripheral arterial disease (PAD) in the primary and validation populations. Polymorphism
Genotypes
P
rs198358 PAD () PAD (þ)
AA 1568 (70.3) 56 (72.7)
GA 620 (27.8) 20 (26.0)
GG 41 (1.9) 1 (1.3)
0.87
rs6668352 PAD () PAD (þ)
GG 1481 (66.4) 57 (74.0)
GA 688 (30.9) 20 (26.0)
AA 60 (2.7) 0 (0.0)
0.19
rs3811544 PAD () PAD (þ)
CC 2105 (94.4) 70 (90.9)
CT þ TT 124 (5.6) 7 (9.1)
rs9662664 PAD () PAD (þ)
GG 972 (43.6) 37 (48.0)
GT 991 (44.5) 30 (39.0)
TT 266 (11.9) 10 (13.0)
0.63
rs700923 PAD () PAD (þ)
AA 1386 (62.2) 49 (63.6)
AG 763 (34.2) 26 (33.8)
GG 80 (3.6) 2 (2.6)
0.88
0.20
Values in the parentheses are percentage of the column total. Fisher’s exact test was used to compare the subjects with PAD and those without.
90
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Odds of PAD 3.5
A allele carriers vs GG homozygotes (rs6668352)
3.0
Odds ratio
2.5 2.0 1.5 P=0.64
1.0 0.5
P=0.23 P=0.04
0.0 All subjects
UNa
UNa>P50
n=1765
883
882
Fig. 1. Odds ratio of peripheral arterial disease (PAD) associated with the rs6668352 A allele in the primary and validation study populations in all subjects and in subjects above or below the median (P50) of urinary sodium excretion (Una). The size of squares is proportional to the number of subjects (bottom). Vertical lines denote 95% confidence intervals. For each odds ratio, the P value is given.
interaction with sodium excretion on ABI. It is possible that the rs6668352 polymorphism located in the promoter region of NPPB gene may be involved in regulating NPPB gene expression. In addition to diuretic, natriuretic, and vasodilatory properties, there is accumulating evidence that natriuretic peptides induce endothelial regeneration and modulate angiogenesis through the autocrine or paracrine action after vascular injury, and thus may have an inhibitory effect of atherosclerosis. In human coronary arterial and umbilical vein endothelial cells, ANP at physiological concentrations induced endothelial regeneration [6]. In animal experiments, activation of the natriuretic peptides/cyclic GMP/cyclic GMP-dependent protein kinase (PKG) pathway accelerated
Fig. 2. Ankleebrachial index according to the tertile distributions of plasma proBNP concentration. Values are mean adjusted for age, sex, body mass index, mean arterial pressure, current smoking, alcohol intake, serum creatinine and the use of antihypertensive drugs. The P-value was derived from the analysis of covariance. Vertical lines denote standard error (SE). For each genotype, the number of subjects is given.
vascular regeneration and blood flow recovery in murine legs, for which ischemia was induced by femoral arterial ligation as a PAD model [8]. In a study in mice, endogenous ANP and BNP played a role in reparative vascular remodeling in ischemic tissues [32]. In human subjects, circulating N-terminal pro-ANP [11] or pro-CNP concentration [12] was positively associated with brachial artery endothelial function [11,12] and negatively with early atherosclerosis [12]. In a therapeutic study, infusion of recombinant ANP, carperitide, accelerated blood flow recovery with increasing capillary densities in ischemic legs of both non-diabetic and streptozotocin-induced diabetic mice and improved ABI, intermittent claudication, rest pain, and leg ulcers in patients with PAD (n ¼ 13) [33]. The exact mechanism underlying the interaction between the 3 SNPs (rs6668352, rs198388, and rs198389) at the NPPAeNPPB locus and sodium excretion in relation to ABI remains to be elucidated. It is generally accepted that 24-h urinary sodium excretion may reflect an individual’s habitual sodium intake. High sodium intake in experimental studies could affect the progression of atherosclerosis [26e28], and by reducing nitric oxide production and enhancing NADPH oxidase activity, impair endothelial dysfunction [26,34]. In the apolipoprotein E-deficient mice, high sodium intake alone increased vascular superoxide formation and promoted atherogenesis [27], and high-sodium intake combined with elevated circulating angiotensin II accelerated progression of atherosclerosis [28]. The finding in our study subjects of high sodium intake suggests that the beneficial effects of natriuretic peptides on the cardiovascular system can be revealed in high-risk subjects but concealed in low-risk subjects. Other mechanisms may also play a part. Previous studies showed that high sodium intake increased plasma BNP levels in healthy subjects [35] and in patients with hypertension [36] or compensated heart failure [37]. Genetic associations therefore might have been exaggerated by high sodium intake. Our study should be interpreted within the context of its strengths and limitations. In an extensively phenotyped general population sample, we may systemically test interaction between genetic variants and various environmental factors. In addition, we studied several well-characterized genetic polymorphisms in the natriuretic peptide system. However, our study was cross-sectional, and hence no causal conclusion could be drawn. Second, our validation population was not an independent population. Our current findings therefore need to be replicated in other populations. Third, our study had a relatively small sample size, and included few subjects with PAD. The possibility of a chance finding therefore cannot be entirely ruled out. However, according to retrospective power calculations, our study had 83%, 81% and 77% of power to detect significant interaction between urinary sodium excretion and the 3 SNPs (rs6668352, rs198388, and rs198389) in relation to ABI in the primary population, and had 75% of power to detect significant association between the rs6668352 polymorphism and the risk of PAD in the entire population. In conclusion, the protective effects of the minor alleles of the genetic polymorphisms at the NPPAeNPPB locus on PAD were apparent in subjects of high sodium intake. If confirmed in prospective studies and in other populations, our findings may have important implications in genetic risk stratification and in understanding the role of the NPPAeNPPB genes and the interaction with lifestyle factors in the progression of atherosclerosis.
Conflict of interest None of the authors have a conflict of interest with regard to the data presented in this paper.
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Acknowledgments The authors gratefully acknowledge the voluntary participation of all study subjects and the support of the local public health authorities of JingNing County (ZheJiang Province). The authors also appreciate the expert assistance of Shou-Yu Mao, Gu-Liang Wang, Jie Wang, Jun-Mei Xu, Li Zheng, and Wei-Zhong Zhang (The Shanghai Institute of Hypertension, Shanghai, China). The study was financially supported by grants from the National Natural Science Foundation of China (grants 30871360, 30871081, 81170245 and 81270373), the Ministry of Science and Technology (grant 2013CB530701 and a special grant for the ChinaeEuropean Union Collaborations [1012]) and the Ministry of Education, Beijing, China (NCET-09-0544), the Shanghai Commissions of Science and Technology (grant 11QH1402000) and Education (the “Dawn” project 08SG20), the Shanghai Bureau of Health (XBR2011004), and Shanghai Jiaotong University School of Medicine (a grant of Distinguished Young Investigators to Yan Li). Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. atherosclerosis.2013.06.020. References [1] Potter LR, Abbey-Hosch S, Dickey DM. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev 2006;27:47e72. [2] Suga S, Nakao K, Hosoda K, Mukoyama M, Ogawa Y, Shirakami G, et al. Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology 1992;130: 229e39. [3] Yan W, Wu F, Morser J, Wu Q. Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme. Proc Natl Acad Sci U S A 2000;97:8525e9. [4] Wu F, Yan W, Pan J, Morser J, Wu Q. Processing of pro-atrial natriuretic peptide by corin in cardiac myocytes. J Biol Chem 2002;277:16900e5. [5] Rubattu S, Sciarretta S, Valenti V, Stanzione R, Volpe M. Natriuretic peptides: an update on bioactivity, potential therapeutic use, and implication in cardiovascular diseases. Am J Hypertens 2008;21:733e41. [6] Kook H, Itoh H, Choi BS, Sawada N, Doi K, Hwang TJ, et al. Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro. Am J Physiol Heart Circ Physiol 2003;284:H1388e97. [7] Moro C, Klimcakova E, Lolmède K, Berlan M, Lafontan M, Stich V, et al. Atrial natriuretic peptide inhibits the production of adipokines and cytokines linked to inflammation and insulin resistance in human subcutaneous adipose tissue. Diabetologia 2007;50:1038e47. [8] Yamahara K, Itoh H, Chun TH, Ogawa Y, Yamashita J, Sawada N, et al. Significance and therapeutic potential of the natriuretic peptides/cGMP/cGMPdependent protein kinase pathway in vascular regeneration. Proc Natl Acad Sci U S A 2003;100:3404e9. [9] Doi K, Ikeda T, Itoh H, Ueyama K, Hosoda K, Ogawa Y, et al. C-type natriuretic peptide induces redifferentiation of vascular smooth muscle cells with accelerated reendothelialization. Arterioscler Thromb Vasc Biol 2001;21:930e6. [10] Ohno N, Itoh H, Ikeda T, Ueyama K, Yamahara K, Doi K, et al. Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide. Circulation 2002;105:1623e6. [11] Kathiresan S, Gona P, Larson MG, Vita JA, Mitchell GF, Tofler GH, et al. Crosssectional relations of multiple biomarkers from distinct biological pathways to brachial artery endothelial function. Circulation 2006;113:938e45. [12] Vlachopoulos C, Ioakeimidis N, Terentes-Printzios D, Aznaouridis K, Baou K, Bratsas A, et al. Amino-terminal pro-C-type natriuretic peptide is associated with arterial stiffness, endothelial function and early atherosclerosis. Atherosclerosis 2010;211:649e55. [13] Lamina C, Meisinger C, Heid IM, Löwel H, Rantner B, Koenig W, et al., Kora Study Group. Association of ankleebrachial index and plaques in the carotid and femoral arteries with cardiovascular events and total mortality in a population-based study with 13 years of follow-up. Eur Heart J 2006;27: 2580e7.
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[14] Newman AB, Shemanski L, Manolio TA, Cushman M, Mittelmark M, Polak JF, et al., The Cardiovascular Health Study Group. Ankleearm index as a predictor of cardiovascular disease and mortality in the Cardiovascular Health Study. Arterioscler Thromb Vasc Biol 1999;19:538e45. [15] Busch MA, Lutz K, Röhl JE, Neuner B, Masuhr F. Low ankleebrachial index predicts cardiovascular risk after acute ischemic stroke or transient ischemic attack. Stroke 2009;40:3700e5. [16] Lee AJ, Price JF, Russell MJ, Smith FB, van Wijk MC, Fowkes FG. Improved prediction of fatal myocardial infarction using the ankle brachial index in addition to conventional risk factors: the Edinburgh Artery Study. Circulation 2004;110:3075e80. [17] Diehm C, Darius H, Pittrow D, Schwertfeger M, Tepohl G, Haberl RL, et al. Prognostic value of a low post-exercise ankle brachial index as assessed by primary care physicians. Atherosclerosis 2011;214:364e72. [18] Carmelli D, Fabsitz RR, Swan GE, Reed T, Miller B, Wolf PA. Contribution of genetic and environmental influences to ankleebrachial blood pressure index in the NHLBI Twin Study. Am J Epidemiol 2000;151:452e8. [19] Murabito JM, Guo CY, Fox CS, D’Agostino RB. Heritability of the ankleebrachial index: the Framingham Offspring study. Am J Epidemiol 2006;164:963e8. [20] Newton-Cheh C, Larson MG, Vasan RS, Levy D, Bloch KD, Surti A, et al. Association of common variants in NPPA and NPPB with circulating natriuretic peptides and blood pressure. Nat Genet 2009;41:348e53. [21] Maimaitiming S, Roussel R, Hadjadj S, Fumeron F, Aubert R, Emery N, et al. Association of common variants in NPPA and NPPB with blood pressure does not translate into kidney damage in a general population study. J Hypertens 2010;28:1230e3. [22] Takeishi Y, Toriyama S, Takabatake N, Shibata Y, Konta T, Emi M, et al. Linkage disequilibrium analyses of natriuretic peptide precursor B locus reveal risk haplotype conferring high plasma BNP levels. 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High sodium intake increases vascular superoxide formation and promotes atherosclerosis in apolipoprotein E-deficient mice. Blood Press 2005;14:373e82. [28] Johansson ME, Bernberg E, Andersson IJ, Bie P, Skøtt O, Gan LM, et al. High-salt diet combined with elevated angiotensin II accelerates atherosclerosis in apolipoprotein E-deficient mice. J Hypertens 2009;27:41e7. [29] Kuznetsova T, Staessen JA, Brand E, Cwynar M, Stolarz K, Thijs L, et al. Sodium excretion as modulator of genetic associations with cardiovascular phenotypes in the European Project on genes in hypertension. J Hypertens 2006;24: 235e42. [30] Fox AA, Collard CD, Shernan SK, Seidman CE, Seidman JG, Liu KY, et al. Natriuretic peptide system gene variants are associated with ventricular dysfunction after coronary artery bypass grafting. Anesthesiology 2009;110: 738e47. [31] Meirhaeghe A, Sandhu MS, McCarthy MI, de Groote P, Cottel D, Arveiler D, et al. 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Ankleebrachial index in relation to the natriuretic peptide system polymorphisms and urinary sodium excretion in Chinese Bang-Chuan Hu a, b, Yan Li a, Ming Liu a, Chang-Sheng Sheng a, Ji-Guang Wang a, * a
Centre for Epidemiological Studies and Clinical Trials, The Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Ruijin 2nd Road 197, Shanghai 200025, China b Intensive Care Unit, Zhejiang Provincial People’s Hospital, 158 Shangtang Road, Hangzhou 310014, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 January 2013 Received in revised form 15 June 2013 Accepted 24 June 2013 Available online 10 July 2013
Objective: Recent studies have demonstrated that the natriuretic pepetides induce endothelial regeneration and angiogenesis after vascular injury through the autocrine or paracrine action, and might have an inhibitory effect on atherosclerosis. We therefore systematically investigated single nucleotide polymorphisms (SNPs) in the natriuretic peptide system in relation to ankleebrachial index (ABI) in a Chinese population. Methods: The study population was recruited from a mountainous area 500 km south of Shanghai from 2003 to 2009. Using the SNapShot method, we first genotyped 951 subjects enrolled in 2005 for 16 SNPs and then the remaining 1355 subjects as validation for 5 SNPs selected from the primary study. ABI and plasma proBNP were measured using the Omron VP-2000/1000 device and the Elecsys proBNP immunoassay, respectively. Results: Overall, the genetic associations were not significant (P 0.07). However, in the primary study, there was significant (Pint 0.045) interaction between 3 SNPs (rs6668352, rs198388, and rs198389) at the NPPAeNPPB locus and urinary sodium excretion in relation to ABI, and the rs6668352 polymorphism had the strongest association (Pint ¼ 0.018). In the primary combined with the validation study populations, the interaction between the rs6668352 polymorphism and urinary sodium excretion in relation to ABI remained statistically significant (Pint ¼ 0.0036). After adjustment for covariates, the rs6668352 A allele carriers, compared with GG homozygotes, had a higher ABI (mean standard error, 1.103 0.006 vs. 1.084 0.004, P ¼ 0.009) and lower risk of peripheral arterial disease (PAD, defined as an ABI < 0.90, odds ratio 0.37, 95% confidence interval: 0.14e0.98, P ¼ 0.04) in the subjects of high sodium intake. Conclusion: The minor alleles of 3 SNPs at the NPPAeNPPB locus are associated with a lower risk of PAD, especially in the subjects of high sodium intake. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Genetic polymorphism Nartiuretic peptide Urinary sodium excretion ABI Population
1. Introduction Natriuretic peptides consist of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) and elicit various biological effects by binding to two guanylyl cyclase-coupled receptors (natriuretic peptide receptors A [NPRA] and B [NPRB]) [1,2]. ANP and BNP preferentially bind to NPRA. CNP has a higher affinity with NPRB. Corin is a recently identified transmembrane serine protease, is highly expressed in cardiomyocytes, and cleaves inactive pro-ANP and pro-BNP into smaller biologically active molecules [3,4]. Natriuretic peptides play
* Corresponding author. Tel.: þ86 21 6437 0045x610911; fax: þ86 21 6466 2193. E-mail address: [email protected] (J.-G. Wang). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.06.020
an important role in the regulation of blood pressure through diuretic, natriuretic, and vasodilatory properties [5], and have beneficial roles in the cardiovascular system through the autocrine or paracrine action [1,2]. ANP at physiological concentrations induces proliferation and migration of endothelial cells and enhances endothelial regeneration in the vascular wall [6], where it also exerts anti-inflammatory properties [7]. Genetically increased BNP can promote vascular regeneration and accelerate the restoration of blood flow after the removal of a hindelimb artery in mice through the activation of the NPRA/cGMP/cGMP dependent protein kinase (cGKI) pathway [8]. CNP as an endothelium-derived relaxing peptide also accelerates re-endothelialization and suppresses neointimal hyperplasmia in vein grafting or balloon injuries in rabbits, which stimulate atherosclerotic lesions in humans [9,10]. These observations suggest that natriuretic peptides have the potential to promote vascular regeneration in peripheral arterial disease (PAD)
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and to inhibit the progression of atherosclerotic lesions. Indeed, in several population studies, circulating levels of natriuretic peptides have been demonstrated to be correlated positively with endothelial function [11,12] and inversely with early atherosclerosis [12]. The ankleebrachial index (ABI) is a widely used measure for detecting low extremity PAD. A decreased ABI indicates target organ damage [13e15] and predicts cardiovascular and all-cause mortality [14,16,17]. Although a number of atherosclerotic risk factors contribute to PAD and lower ABI, genetic factors account for approximately 21e48% of the variability in both ABI and the presence of PAD, defined according to ABI [18,19]. Genes that have potent vasodilatory and anti-atherosclerotic properties might be associated with ABI and the risk of PAD. Identification of these genes may provide useful clues in understanding the complex quantitative cardiovascular traits. Recent studies revealed significant associations of several polymorphisms at the NPPAeNPPB locus with circulating natriuretic peptides and incident hypertension in White populations [20e23]. The minor alleles of two tightly linked polymorphisms (T555I/Q568P) in the Corin gene were also associated with an increased risk of hypertension [24] and an enhanced cardiac hypertrophic response to pressure overload in African Americans [25]. However, to date, no previous study has ever investigated the relationship between genetic variants of the natriuretic peptide system and ABI. In addition, there is evidence that high sodium intake may affect the progression of atherosclerosis [26e28] and increase the level of plasma BNP. Sodium excretion is also a modulator of genetic associations with cardiovascular phenotypes [29]. In the present study, we therefore systematically investigated the association of 16 SNPs of 6 genes in the natriuretic peptide system (NPPA, NPPB, NPPC, NPRA, NPRC and Corin) with ABI and urinary sodium excretion, and explored the potential geneeenvironment interaction in relation to ABI and the risk of PAD in a Chinese population. 2. Methods 2.1. Study populations The study population was recruited in the framework of an ongoing longitudinal population-based genetic study in hypertension. From 2003 to 2009, we visited all homes in 18 villages randomly selected from JingNing County, a mountainous rural area approximately 500 km south of Shanghai. We invited all inhabitants at least 12 years of age to take part. Of the 3402 invited, 2392 (70.3%) participated. The study protocol was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. All participants gave informed written consent. Our primary genetic study included 981 subjects enrolled in 2005, for whom we genotyped 16 SNPs in the natriuretic peptide system. We excluded 30 subjects from the present analysis because of missing information on genotype (n ¼ 8) and phenotype (n ¼ 22). Thus, the total number of subjects in the primary study was 951. Our validation genetic study included the remaining 1411 subjects, for whom we genotyped 5 SNPs selected from the primary study. We excluded 56 subjects because of missing information on genotype (n ¼ 12) and phenotype (n ¼ 44). Thus, the total number of subjects included in the validation study was 1355. 2.2. Field work One experienced physician measured each participant’s brachial blood pressure five times consecutively by mercury
87
sphygmomanometry, after the subject had rested for at least 5 min in the sitting position. These five blood pressure readings were averaged for analysis. During the home visit, the same observer also administered a standardized questionnaire to collect information on medical history, smoking habits, alcohol consumption, and the use of medications. Hypertension was defined as a blood pressure of at least 140 mmHg systolic or 90 mmHg diastolic or as the use of antihypertensive drugs. Venous blood samples were taken after overnight fast for the measurement of plasma glucose concentration and for measurements of serum concentrations of total cholesterol and triglycerides. A 24-h urine sample was collected in a wide-neck plastic container for the measurement of urinary electrolytes. To ensure the complete collection of urine, urinary samples less than 600 ml were excluded. Thus, 847 and 918 urinary samples were available for measurement of urinary electrolytes in the primary and validation studies, respectively. 2.3. Genotyping The 16 SNPs in 6 genes (NPPA, NPPB, NPPC, NPRA, NPRC, and Corin) of the natriuretic peptide system were genotyped using the ABI PRISMÒ SNapShotÒ method (Applied Biosystem, CA, USA). In brief, the SNapShot reactions were carried out in a 10 ml final volume containing SNapShot Multiplex Ready Mix (5 ml), primer mix (0.02e0.6 mmol/l), and templates (4 ml) consisting of the multiplex PCR products, which had been purified with the QIAquick PCR Purification Kit (QIAGEN, GmbH, Germany). The cycling program included 25 cycles of 96 C for 10 s, 50 C for 5 s, and 60 C for 30 s. Extension products were purified by a 15-min incubation with1 U of shrimp alkaline phosphatase (Promega, Madison, WI, USA) at 37 C and a subsequent 15-min incubation at 80 C to inactivate the enzyme. The purified products (0.5 ml) were mixed with 9 ml of formamide and 0.5 ml of GeneScan-120 LIZ Size Standard (Applied Biosystems) and separated by capillary electrophoresis (ABI PRISM310 Genetic Analyzer; Applied Biosystems). The results were analyzed with GeneMapper 3.0 software (Applied Biosystems). All PCR and SNapShot primers are listed in supplementary Table 1. More than 98% of the total samples were successfully genotyped for all SNPs. To confirm the genotyping results, 30 samples were randomly selected and re-genotyped by direct sequencing using a BigDye terminator (Applied Biosystem). All assays were 100% concordant. 2.4. Measurements of ABI To ensure steady state, one trained physician performed all the ABI measurements under standardized conditions in a quiet examination room after the subjects had rested for 10 min in the supine position. The participants were asked to refrain from smoking, vigorous exercise, and drinking alcohol or caffeinecontaining beverages for at least 2 h before the examination. We used the Omron VP-2000/1000 device (Omron Healthcare, Kyoto, Japan) to obtain simultaneous oscillometric blood pressure readings at the right and left brachial and posterior tibial arteries. ABI was defined as a ratio of the ankle-to-brachial systolic blood pressure measurements and calculated for both sides. The lower of the 2 ABI values was determined (low ABI) and used in subsequent statistical analyses. In line with current recommendations, an ABI of 0.9 or less in either leg was defined as possible indicative of PAD. Those with an ABI >1.4 were excluded because it is indicative of incompressible, calcified arteries, and inclusion of these individuals could have led to misclassification of arterial extremity disease.
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2.5. Measurement of plasma proBNP concentration In 1386 individuals of the JingNing study from 2003 to 2005, we collected venous blood samples in chilled ethylenediaminetetraacetic acid tubes. After centrifugation, the clarified plasma samples were frozen, stored at 80 C, and thawed just before assays. Plasma proBNP was measured using an automated, commercially available immunoassay (Elecsys proBNP, Roche Diagnostics, Indianapolis, Indiana, USA) according to the manufacturer’s instructions. The inter-assay coefficient of variation in our study was 1.2%. 2.6. Statistical methods We used SAS version 9.13 (SAS institute, Cary, NC, USA) for database management and statistical analyses. Comparisons of means and proportions relied on the Student t-test and Fisher’s exact test, respectively. Continuous measurements with a skewed distribution were normalized by logarithmic transformation and represented by geometric mean and 95% confidence interval (CI). HardyeWeinberg equilibrium was tested using the c2 test. We identified covariates of the phenotypes under study using stepwise multiple regressions with a P-value for independent variables to enter and stay in the model set at 0.10. We studied genetic associations using the analysis of covariance, while controlling for covariates. The value of P < 0.05 was considered to indicate statistical significance. 3. Results 3.1. Characteristics of the primary study population In the primary study, the 951 participants included 470 men (49.4%), 222 (23.3%) hypertensive patients, and 31 patients (3.3%) with PAD. Table 1 shows the characteristics of the study participants by sex. Men and women were similar in characteristics, except that men were slightly older (þ3.0 years, P ¼ 0.003) and had Table 1 Characteristics of the primary study population. Characteristic
Men (n ¼ 470)
Women (n ¼ 481)
P
Age (years) Body mass index (kg/m2) Current smoking, n (%) Alcohol intake 5 g/week, n (%) Prevalence of hypertension, n (%) Took antihypertensive drugs, n (%) Serum total cholesterol (mmol/l) Serum triglycerides (mmol/l) Fasting plasma glucose (mmol/l) Serum creatinine (mmol/l) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Pulse pressure (mmHg) ProBNP (pg/ml)
46.5 15.9 22.0 2.8 285 (60.6) 297 (63.2) 98 (20.9) 20 (4.2) 4.87 0.96 1.48 2.08 4.38 1.76 78.7 22.0 129.2 24.4 75.9 12.4 53.3 20.5 32.3 (30.2e34.6) 1.106 0.084 13 (2.8)
43.5 15.0 22.1 2.9 0 139 (28.9) 124 (25.8) 40 (8.3) 4.78 0.97 1.24 0.95 4.45 0.82 64.5 15.1 129.8 26.6 75.8 13.9 54.0 19.7 46.9 (45.1e49.9) 1.069 0.093 18 (3.7)
0.003 0.46 <0.001 <0.001 0.08 <0.001 0.13 0.02 0.40 <0.001 0.74 0.88 0.60 <0.001 <0.001 0.40
1.33 0.58 181.0 86.6 25.4 9.7
1.14 0.45 178.0 78.7 24.4 10.0
<0.001 0.54 0.14
Ankleebrachial index Prevalence of peripheral arterial disease, n (%) 24-h urinary measurements Volume (l) Sodium excretion (mmol/day) Potassium excretion (mmol/day)
Values are mean SD or number of subjects (percentage of the column total). P values are for gender difference. 24-h urinary measurements were available in 420 men and 427 women. Plasma proBNP concentration was measured in 676 men and 710 women recruited in the primary and validation studies.
higher serum concentrations of triglycerides (þ0.24 mmol/l, P ¼ 0.02) and creatinine (14.2 mmol/l, P < 0.001). Men, compared with women, had a significantly (P < 0.001) greater urinary volume (þ0.19 l) and ABI (þ0.039) and lower plasma proBNP level (14.6 pg/ml). However, they had similar (P 0.14) urinary sodium (179.2 82.9 mmol/day) and potassium excretions (24.8 9.9 mmol/day) and similar prevalence of PAD (2.8% vs. 3.7%, P ¼ 0.40). 3.2. ABI and SNPs in the natriuretic peptide system In the primary study, the genotype frequencies of the 16 SNPs did not deviate from the HardyeWeinberg equilibrium (P 0.08), and were similar in men and women (P 0.12) and in the presence (n ¼ 31) and absence of PAD (n ¼ 920, P 0.26). With adjustments for age, sex, body mass index, mean arterial pressure, current smoking, alcohol intake, serum creatinine, and the use of antihypertensive drugs, we did not find any significant association between ABI and 16 SNPs in the natriuretic peptide system (P 0.07, Table 2). However, with similar adjustments applied, there was significant interaction (Pint 0.045) between the 3 SNPs (rs6668352, rs198388, and rs198389) at the NPPAeNPPB locus and urinary sodium excretion in relation to ABI, and the rs6668352 polymorphism had the strongest association (Pint ¼ 0.018). In subjects above the median of urinary sodium excretion (182.1 mmol/day), the rs6668352 A allele carriers, compared with GG homozygotes, had an higher ABI (1.105 0.004 vs. 1.086 0.006, P ¼ 0.03). However, in subjects below the median of sodium excretion, there was no difference in ABI between A allele carriers and GG homozygotes (1.094 0.004 vs. 1.086 0.006; P ¼ 0.27). 3.3. The validation study We performed a validation study in the remaining 1355 individuals of the JingNing study. Of the 5 SNPs genotyped (rs198358, rs6668352, rs3811544, rs700923, and 9662664), none had significant association with ABI (P 0.09, Table 3) or with the prevalence of PAD (n ¼ 46 [3.4%], P 0.13). In the combined analysis of the primary and validation studies, no significant differences in genotype distributions of the 5 SNPs were found between the subjects with PAD (n ¼ 77) and those without (n ¼ 2229, P 0.19, Table 4). However, the interaction between the rs6668352 polymorphism and urinary sodium excretion in relation to ABI remained statistically significant (Pint ¼ 0.0036). In subjects above the median of sodium excretion (180.8 mmol/l), the A allele carriers, compared with GG homozygotes, had a higher ABI (1.103 0.006 vs. 1.084 0.004, P ¼ 0.009) and a lower risk of PAD (odds ratio [OR] 0.37, 95% CI: 0.14e0.98, P ¼ 0.04). However, in subjects below the median of sodium excretion, the A allele carriers and GG homozygotes had a similar ABI (1.092 0.003 vs. 1.084 0.005; P ¼ 0.18) and risk of PAD (OR 1.23, 95% CI: 0.51e3.01, P ¼ 0.64, Fig. 1). 3.4. Plasma proBNP level and ABI In 1386 participants, plasma proBNP level was positively correlated with ABI (r ¼ 0.11, P < 0.0001). In multivariate regression analyses adjusted for age, sex, body mass index, current smoking, alcohol intake, mean arterial pressure, serum creatinine, and the use of antihypertensive drugs, plasma proBNP was an independent determinant of ABI (regression coefficient SE: 0.0158 0.007, P ¼ 0.024). In further analyses stratified for the tertile distributions of plasma proBNP, both before and after adjustment for covariates, ABI was significantly (P 0.007) different across tertiles of proBNP.
B.-C. Hu et al. / Atherosclerosis 230 (2013) 86e91 Table 2 Ankleebrachial index in relation to 16 SNPs in the natriuretic peptide system in the primary study. Gene and polymorphism Ankleebrachial index NPPAeNPPB locus rs198358
P
AA (n ¼ 697) GA (n ¼ 237) GG (n ¼ 17) 1.086 0.003 1.084 0.006 1.112 0.022 0.54
rs5063
GG (n ¼ 724) GA (n ¼ 211) AA (n ¼ 16) 1.086 0.003 1.083 0.006 1.118 0.023 0.34
rs198375
AA (n ¼ 622) GA (n ¼ 304) GG (n ¼ 25) 1.085 0.004 1.086 0.005 1.092 0.018 0.93
rs6668352
GG (n ¼ 674) GA (n ¼ 262) AA (n ¼ 15) 1.085 0.004 1.084 0.006 1.120 0.024 0.31
rs198388
CC (n ¼ 636) CT (n ¼ 291) TT (n ¼ 24) 1.085 0.004 1.086 0.005 1.093 0.019 0.92
rs198389
AA (n ¼ 647) GA (n ¼ 279) GG (n ¼ 25) 1.084 0.004 1.086 0.005 1.109 0.005 0.42
NPPC rs5262 rs3811544
NPRA rs28730726 rs9662664
NPRC rs2270915
GG (n ¼ 674) GA (n ¼ 251) AA (n ¼ 26) 1.086 0.004 1.086 0.006 1.085 0.018 0.99 CC (n ¼ 902) CT (n ¼ 49) 1.086 0.003 1.077 0.013
GG (n ¼ 803) GC (n ¼ 143) CC (n ¼ 5) 1.084 0.003 1.094 0.008 1.106 0.043 0.47 GG (n ¼ 422) GT (n ¼ 420) TT (n ¼ 109) 1.082 0.004 1.093 0.004 1.071 0.009 0.07
AA (n ¼ 693) AG (n ¼ 242) GG (n ¼ 16) 1.086 0.003 1.084 0.006 1.107 0.022 0.57 AA (n ¼ 586) AG (n ¼ 332) GG (n ¼ 33) 1.085 0.004 1.084 0.005 1.104 0.016 0.49
rs2292026
CC (n ¼ 605) CT (n ¼ 316) TT (n ¼ 30) 1.088 0.004 1.083 0.005 1.103 0.016 0.51
CC (n ¼ 929) CT (n ¼ 22) 1.085 0.003 1.102 0.020
Table 3 Ankleebrachial index in relation to 5 SNPs in the natriuretic peptide system in the validation study. Gene and polymorphism Ankleebrachial index NPPAeNPPB locus rs198358 rs6668352
NPPC rs3811544
NPRA rs9662664
NPRC rs700923
P
AA (n ¼ 927) GA (n ¼ 403) GG (n ¼ 25) 1.091 0.003 1.093 0.004 1.104 0.017 0.76 GG (n ¼ 864) GA (n ¼ 446) AA (n ¼ 45) 1.094 0.003 1.087 0.004 1.117 0.013 0.09
CC (n ¼ 1273) CT (n ¼ 82) 1.093 0.003 1.082 0.010
0.27
GG (n ¼ 587) GT (n ¼ 601) TT (n ¼ 167) 1.091 0.004 1.093 0.004 1.097 0.007 0.68
AA (n ¼ 849) AG (n ¼ 457) GG (n ¼ 49) 1.094 0.003 1.090 0.004 1.100 0.013 0.61
Values are mean SE. Ankleebrachial index was adjusted for sex, age, body mass index, mean arterial pressure, current smoking, alcohol intake, serum creatinine, and the use of antihypertensive drugs.
0.49
rs700923
Corin rs11934749
89
0.40
rs3749585
TT (n ¼ 329) TC (n ¼ 436) CC (n ¼ 186) 1.089 0.005 1.084 0.004 1.083 0.007 0.76
rs2289433
AA (n ¼ 470) GA (n ¼ 397) GG (n ¼ 84) 1.086 0.004 1.084 0.005 1.092 0.010 0.75
Values are mean SE. Ankleebrachial index was adjusted for sex, age, body mass index, mean arterial pressure, current smoking, alcohol intake, serum creatinine, and the use of antihypertensive drugs.
The unadjusted ABI was 1.084 (0.083), 1.094 (0.088), and 1.107 (0.09) in the top (<24.7 pg/ml, n ¼ 464), middle (24.7e55.3 pg/ml, n ¼ 468) and bottom tertiles (>55.3 pg/ml; n ¼ 454), respectively (Fig. 2).
4. Discussion The main finding was first to demonstrate the interaction between the 3 SNPs (rs198389, rs198388, rs6668352) at the NPPAe NPPB locus, which are in tight linkage disequilibrium, and urinary sodium excretion in relation to ABI. Our validation study was basically confirmatory. Few studies investigated the association between the abovementioned 3 SNPs and cardiovascular phenotypes. In a recent caseecontrol study including 697 patients of European descent undergoing primary coronary artery bypass graft, the minor alleles
of these 3 SNPs were associated with a lower risk of left ventricular dysfunction (P 0.03) [30]. In a larger French population cohort (n ¼ 3216), Meirhaeghe and colleagues [31] reported that the rs198389 CC subjects, compared with T allele carriers, had a lower risk of impaired fasting glucose (P ¼ 0.005) and type 2 diabetes (OR 0.69; 95% CI: 0.47e1.00, P ¼ 0.05). These genetic associations were replicated in 4 additional caseecontrol study populations for type 2 diabetes mellitus [31]. In addition, these investigators found that the rs198389 polymorphism might be a functional variant, with a higher transcriptional activity in the C allele carriers [31]. Our and previous studies consistently demonstrated that the minor alleles of these 3 SNPs were associated with an elevated circulating proBNP level in the general population [22]. Taken together, these findings suggested that the protective associations of the SNPs at the NPPAeNPPB locus with left ventricular dysfunction, diabetes mellitus or ABI might be attributable to increased circulating levels of BNP. However, we observed that, of the 3 SNPs, the rs6668352 polymorphism, not the rs198389 polymorphism, had the strongest
Table 4 Genotype frequencies according to the presence and absence of peripheral arterial disease (PAD) in the primary and validation populations. Polymorphism
Genotypes
P
rs198358 PAD () PAD (þ)
AA 1568 (70.3) 56 (72.7)
GA 620 (27.8) 20 (26.0)
GG 41 (1.9) 1 (1.3)
0.87
rs6668352 PAD () PAD (þ)
GG 1481 (66.4) 57 (74.0)
GA 688 (30.9) 20 (26.0)
AA 60 (2.7) 0 (0.0)
0.19
rs3811544 PAD () PAD (þ)
CC 2105 (94.4) 70 (90.9)
CT þ TT 124 (5.6) 7 (9.1)
rs9662664 PAD () PAD (þ)
GG 972 (43.6) 37 (48.0)
GT 991 (44.5) 30 (39.0)
TT 266 (11.9) 10 (13.0)
0.63
rs700923 PAD () PAD (þ)
AA 1386 (62.2) 49 (63.6)
AG 763 (34.2) 26 (33.8)
GG 80 (3.6) 2 (2.6)
0.88
0.20
Values in the parentheses are percentage of the column total. Fisher’s exact test was used to compare the subjects with PAD and those without.
90
B.-C. Hu et al. / Atherosclerosis 230 (2013) 86e91
Odds of PAD 3.5
A allele carriers vs GG homozygotes (rs6668352)
3.0
Odds ratio
2.5 2.0 1.5 P=0.64
1.0 0.5
P=0.23 P=0.04
0.0 All subjects
UNa
UNa>P50
n=1765
883
882
Fig. 1. Odds ratio of peripheral arterial disease (PAD) associated with the rs6668352 A allele in the primary and validation study populations in all subjects and in subjects above or below the median (P50) of urinary sodium excretion (Una). The size of squares is proportional to the number of subjects (bottom). Vertical lines denote 95% confidence intervals. For each odds ratio, the P value is given.
interaction with sodium excretion on ABI. It is possible that the rs6668352 polymorphism located in the promoter region of NPPB gene may be involved in regulating NPPB gene expression. In addition to diuretic, natriuretic, and vasodilatory properties, there is accumulating evidence that natriuretic peptides induce endothelial regeneration and modulate angiogenesis through the autocrine or paracrine action after vascular injury, and thus may have an inhibitory effect of atherosclerosis. In human coronary arterial and umbilical vein endothelial cells, ANP at physiological concentrations induced endothelial regeneration [6]. In animal experiments, activation of the natriuretic peptides/cyclic GMP/cyclic GMP-dependent protein kinase (PKG) pathway accelerated
Fig. 2. Ankleebrachial index according to the tertile distributions of plasma proBNP concentration. Values are mean adjusted for age, sex, body mass index, mean arterial pressure, current smoking, alcohol intake, serum creatinine and the use of antihypertensive drugs. The P-value was derived from the analysis of covariance. Vertical lines denote standard error (SE). For each genotype, the number of subjects is given.
vascular regeneration and blood flow recovery in murine legs, for which ischemia was induced by femoral arterial ligation as a PAD model [8]. In a study in mice, endogenous ANP and BNP played a role in reparative vascular remodeling in ischemic tissues [32]. In human subjects, circulating N-terminal pro-ANP [11] or pro-CNP concentration [12] was positively associated with brachial artery endothelial function [11,12] and negatively with early atherosclerosis [12]. In a therapeutic study, infusion of recombinant ANP, carperitide, accelerated blood flow recovery with increasing capillary densities in ischemic legs of both non-diabetic and streptozotocin-induced diabetic mice and improved ABI, intermittent claudication, rest pain, and leg ulcers in patients with PAD (n ¼ 13) [33]. The exact mechanism underlying the interaction between the 3 SNPs (rs6668352, rs198388, and rs198389) at the NPPAeNPPB locus and sodium excretion in relation to ABI remains to be elucidated. It is generally accepted that 24-h urinary sodium excretion may reflect an individual’s habitual sodium intake. High sodium intake in experimental studies could affect the progression of atherosclerosis [26e28], and by reducing nitric oxide production and enhancing NADPH oxidase activity, impair endothelial dysfunction [26,34]. In the apolipoprotein E-deficient mice, high sodium intake alone increased vascular superoxide formation and promoted atherogenesis [27], and high-sodium intake combined with elevated circulating angiotensin II accelerated progression of atherosclerosis [28]. The finding in our study subjects of high sodium intake suggests that the beneficial effects of natriuretic peptides on the cardiovascular system can be revealed in high-risk subjects but concealed in low-risk subjects. Other mechanisms may also play a part. Previous studies showed that high sodium intake increased plasma BNP levels in healthy subjects [35] and in patients with hypertension [36] or compensated heart failure [37]. Genetic associations therefore might have been exaggerated by high sodium intake. Our study should be interpreted within the context of its strengths and limitations. In an extensively phenotyped general population sample, we may systemically test interaction between genetic variants and various environmental factors. In addition, we studied several well-characterized genetic polymorphisms in the natriuretic peptide system. However, our study was cross-sectional, and hence no causal conclusion could be drawn. Second, our validation population was not an independent population. Our current findings therefore need to be replicated in other populations. Third, our study had a relatively small sample size, and included few subjects with PAD. The possibility of a chance finding therefore cannot be entirely ruled out. However, according to retrospective power calculations, our study had 83%, 81% and 77% of power to detect significant interaction between urinary sodium excretion and the 3 SNPs (rs6668352, rs198388, and rs198389) in relation to ABI in the primary population, and had 75% of power to detect significant association between the rs6668352 polymorphism and the risk of PAD in the entire population. In conclusion, the protective effects of the minor alleles of the genetic polymorphisms at the NPPAeNPPB locus on PAD were apparent in subjects of high sodium intake. If confirmed in prospective studies and in other populations, our findings may have important implications in genetic risk stratification and in understanding the role of the NPPAeNPPB genes and the interaction with lifestyle factors in the progression of atherosclerosis.
Conflict of interest None of the authors have a conflict of interest with regard to the data presented in this paper.
B.-C. Hu et al. / Atherosclerosis 230 (2013) 86e91
Acknowledgments The authors gratefully acknowledge the voluntary participation of all study subjects and the support of the local public health authorities of JingNing County (ZheJiang Province). The authors also appreciate the expert assistance of Shou-Yu Mao, Gu-Liang Wang, Jie Wang, Jun-Mei Xu, Li Zheng, and Wei-Zhong Zhang (The Shanghai Institute of Hypertension, Shanghai, China). The study was financially supported by grants from the National Natural Science Foundation of China (grants 30871360, 30871081, 81170245 and 81270373), the Ministry of Science and Technology (grant 2013CB530701 and a special grant for the ChinaeEuropean Union Collaborations [1012]) and the Ministry of Education, Beijing, China (NCET-09-0544), the Shanghai Commissions of Science and Technology (grant 11QH1402000) and Education (the “Dawn” project 08SG20), the Shanghai Bureau of Health (XBR2011004), and Shanghai Jiaotong University School of Medicine (a grant of Distinguished Young Investigators to Yan Li). Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. atherosclerosis.2013.06.020. References [1] Potter LR, Abbey-Hosch S, Dickey DM. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev 2006;27:47e72. [2] Suga S, Nakao K, Hosoda K, Mukoyama M, Ogawa Y, Shirakami G, et al. Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology 1992;130: 229e39. [3] Yan W, Wu F, Morser J, Wu Q. Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme. Proc Natl Acad Sci U S A 2000;97:8525e9. [4] Wu F, Yan W, Pan J, Morser J, Wu Q. Processing of pro-atrial natriuretic peptide by corin in cardiac myocytes. J Biol Chem 2002;277:16900e5. [5] Rubattu S, Sciarretta S, Valenti V, Stanzione R, Volpe M. Natriuretic peptides: an update on bioactivity, potential therapeutic use, and implication in cardiovascular diseases. Am J Hypertens 2008;21:733e41. [6] Kook H, Itoh H, Choi BS, Sawada N, Doi K, Hwang TJ, et al. Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro. Am J Physiol Heart Circ Physiol 2003;284:H1388e97. [7] Moro C, Klimcakova E, Lolmède K, Berlan M, Lafontan M, Stich V, et al. Atrial natriuretic peptide inhibits the production of adipokines and cytokines linked to inflammation and insulin resistance in human subcutaneous adipose tissue. Diabetologia 2007;50:1038e47. [8] Yamahara K, Itoh H, Chun TH, Ogawa Y, Yamashita J, Sawada N, et al. Significance and therapeutic potential of the natriuretic peptides/cGMP/cGMPdependent protein kinase pathway in vascular regeneration. Proc Natl Acad Sci U S A 2003;100:3404e9. [9] Doi K, Ikeda T, Itoh H, Ueyama K, Hosoda K, Ogawa Y, et al. C-type natriuretic peptide induces redifferentiation of vascular smooth muscle cells with accelerated reendothelialization. Arterioscler Thromb Vasc Biol 2001;21:930e6. [10] Ohno N, Itoh H, Ikeda T, Ueyama K, Yamahara K, Doi K, et al. Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide. Circulation 2002;105:1623e6. [11] Kathiresan S, Gona P, Larson MG, Vita JA, Mitchell GF, Tofler GH, et al. Crosssectional relations of multiple biomarkers from distinct biological pathways to brachial artery endothelial function. Circulation 2006;113:938e45. [12] Vlachopoulos C, Ioakeimidis N, Terentes-Printzios D, Aznaouridis K, Baou K, Bratsas A, et al. Amino-terminal pro-C-type natriuretic peptide is associated with arterial stiffness, endothelial function and early atherosclerosis. Atherosclerosis 2010;211:649e55. [13] Lamina C, Meisinger C, Heid IM, Löwel H, Rantner B, Koenig W, et al., Kora Study Group. Association of ankleebrachial index and plaques in the carotid and femoral arteries with cardiovascular events and total mortality in a population-based study with 13 years of follow-up. Eur Heart J 2006;27: 2580e7.
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