Nitric Oxide 55-56 (2016) 62e69

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Nitric Oxide
journal homepage: www.elsevier.com/locate/yniox

Endothelial nitric oxide synthase tagSNPs influence the effects of

enalapril in essential hypertension
Gustavo H. Oliveira-Paula a, Riccardo Lacchini b, Marcelo R. Luizon a, Vanessa Fontana b,
Pamela S. Silva b, Celso Biagi c, Jose E. Tanus-Santos a, *
a
Department of Pharmacology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil
b
Department of Pharmacology, State University of Campinas, Campinas, SP, Brazil
c
Santa Casa of Araçatuba, Araçatuba, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The antihypertensive effects of angiotensin-converting enzyme inhibitors (ACEi) are associated with up-
Received 16 November 2015 regulation of endothelial nitric oxide synthase (NOS3) activity. This mechanism may explain how
Received in revised form polymorphisms in NOS3 gene affect the antihypertensive responses to ACEi. While clinically relevant
18 March 2016
NOS3 polymorphisms were previously shown to affect the antihypertensive responses to enalapril, no
Accepted 22 March 2016
study has tested the hypothesis that NOS3 tagSNPs influence the antihypertensive effects of this drug. We
examined whether the NOS3 tagSNPs rs3918226, rs3918188, and rs743506, and their haplotypes, affect
the antihypertensive responses to enalapril in 101 patients with essential hypertension. Subjects were
Keywords:
Enalapril
prospectively treated only with enalapril for 8 weeks. Genotypes were determined by Taqman® allele
Endothelial nitric oxide synthase discrimination assay and real-time polymerase chain reaction (PCR) and haplotype frequencies were
Haplotypes estimated. We compared the effects of NOS3 tagSNPs on changes in blood pressure after enalapril
Hypertension treatment. To confirm our findings, multiple linear regression analysis was performed adjusting for age,
NOS3 gender, ethnicity, and alcohol consumption. We found that hypertensive patients carrying the AA ge-
tagSNPs notype for the tagSNP rs3918188 showed lower decreases in blood pressure in response to enalapril.
Moreover, the TCA haplotype was associated with improved decreases in blood pressure in response to
enalapril compared with the CAG haplotype. Adjustment for covariates in multiple linear regression
analysis did not change these effects. In addition, when patients were stratified according to the dose of
enalapril used, we found that the carries of the T allele for the functional tagSNP rs3918226 showed more
intense decreases in blood pressure in response to enalapril 20 mg/day. Our findings suggest that NOS3
tagSNPs influence the effects of enalapril in essential hypertension.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction However, the antihypertensive effects of ACEi also involve up-

Hypertension is a multifactorial disorder causing more than
seven million deaths per year, and the most modifiable risk factor
for cardiovascular events, which can be markedly prevented by
antihypertensive drug therapy [1,2]. Angiotensin-converting
enzyme inhibitors (ACEi) are commonly prescribed antihyperten-
sive drugs and their mechanism of action include decreased
angiotensin II formation and increased bradykinin levels [3].
* Corresponding author. Department of Pharmacology, Ribeirao Preto Medical
School, University of Sao Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeirao Preto,
SP, Brazil.
E-mail addresses: tanus@fmrp.usp.br, tanussantos@yahoo.com (J.E. Tanus-
Santos).
regulation of endothelial nitric oxide synthase (NOS3) activity,
thus resulting in the vasodilation mediated by nitric oxide (NO)
[4e8].
NO plays a major role in vascular homeostasis by controlling the
vascular tone, cellular proliferation, leukocyte adhesion, and
platelet aggregation [9,10]. It is synthesized from L-arginine by at
least three isoforms of NO synthases including NOS3 [11]. Given the
essential role of NO in the control of blood pressure, it is now clear
that impaired NOS3 activity results in NO deficiency and hyper-
tension [12]. ACEi, in turn, stimulate endogenous NO generation
resulting from increased levels of bradykinin, which activates bra-
dykinin B2 receptor (BR2) on endothelial cells and activates NOS3
to produce NO [4]. Therefore, it is possible that polymorphisms in
NOS3 gene influence the antihypertensive effects of ACEi.

http://dx.doi.org/10.1016/j.niox.2016.03.006
1089-8603/© 2016 Elsevier Inc. All rights reserved.
 G.H. Oliveira-Paula et al. / Nitric Oxide 55-56 (2016) 62e69
We have previously examined the effects of three clinically
relevant NOS3 polymorphisms (rs2070744, 4b/4a VNTR and
rs1799983) on the antihypertensive responses to ACEi enalapril
[13]. However, it is possible that other polymorphisms covering a
broader range of genetic variability in NOS3 gene also affect the
responses to these drugs. In this regard, recent studies have focused
on tagSNPs, which represent the information of neighboring SNPs
in linkage disequilibrium in the NOS3 gene [14e17]. Interestingly,
despite a previous study reporting that the tagSNP rs3918226 does
not affect plasma nitrite levels [18], this tagSNP was shown to affect
NOS3 promoter activity [19], to modify the susceptibility to hy-
pertension [20,21], and to predict cardiovascular mortality and
morbidity [22]. However, no previous study examined whether
these tagSNPs in the NOS3 gene affect the antihypertensive re-
sponses to ACEi.
In this study, we examined whether the tagSNPs (rs3918226,
rs3918188, and rs743506) in the NOS3 gene affect the responses to
enalapril in patients with essential hypertension. In addition, we
have also examined whether the combined effect of haplotypes
formed by these tagSNPs affect the responses to enalapril.
2. Materials and methods
2.1. Study population
The study population included a total of 101 hypertensive pa-
tients with systolic blood pressure (SBP)
140 and  179 mmHg
and diastolic blood pressure (DBP)
90 and 109 mmHg, recruited
from the Cardiology Division of the Araçatuba Health Center (Ara-
çatuba, SP, Brazil). Office blood pressure measurement was ob-
tained from the average of three blood pressure readings on at least
two office visits with the individuals in the seated position, ac-
cording to the Seventh Report of the Joint National Committee on
Prevention, Detection, Evaluation and Treatment of High Blood
Pressure [2]. Subjects with evidence of severe or secondary hy-
pertension, diabetes, hepatic or renal dysfunction were excluded
from the present study. This study was approved by the Institu-
tional Review Board at the Ribeirao Preto Medical School, Univer-
sity of Sao Paulo, Brazil and each subject signed an informed
consent. The present study was performed in accordance with the
ethics standards of the Helsinki Declaration.
2.2. Blood pressure measurement
Office SBP and DBP were measured with a semi-automatic blood
pressure monitor (OMRON® - HEM-433 INT, Bannockburn, Illinois,
USA) before and after the use of enalapril. Mean blood pressure
(MBP) was calculated using the averages of three different systolic
and diastolic blood pressure measurements, with intervals of 1 min
between each other. MBP was calculated as MBP ¼ (SBP þ 2*DBP)/3.
The patients were never-treated (n ¼ 75) or underwent a wash-out
period for at least 2 weeks (n ¼ 26). This wash-out period, which
has been used in several studies [23e26], is sufficient to abolish the
effects of previous antihypertensive treatments on physiological
parameters [27].
2.3. Antihypertensive responses to enalapril
After physical examination, laboratorial analysis, and evaluation
of medical history, patients were treated for 8 weeks with enalapril
10 mg/day (n ¼ 48) or 20 mg/day (n ¼ 53), according to the judg-
ment of the physician, considering the number of risk factors and
the previous history of treatment of patients that underwent
washout. Values of SBP, MBP and DBP were used to calculate
changes in blood pressure in response to enalapril. After blood
63
pressure measurements, venous blood samples were collected and
genomic DNA was extracted by a salting-out method and stored
at 20  C until analyzed.
2.4. Genotyping
Three NOS3 tagSNPs, rs3918226, rs3918188 and rs743506 were
selected from the Genome Variation Server of Seattle SNPs (http://
gvs.gs.washington.edu/GVS/) using the parameters of r2
0.8 as
threshold for clusters of linkage disequilibrium (LD) among poly-
morphisms, and minor allele frequency (MAF)
10% [18]. Geno-
types were determined by pre-designed Taqman Allele
discrimination assays (rs3918188: C_29193459-10; rs3918226:
primer forward: 50 -AGCGTGCGTCACTGAATGA-30 , reverse 50 -
ACACCCCCATGACTCAAGTG-30 and probes 50 -CAGGAAGCT[G/A]
CCTTC-30 ; rs743506: primer forward: 50 -GCTGTCCCCTCCCTCTG-30 ,
reverse 50 -GTGCCAGCTCCAGAGCAA-30 and probes 50 -CTCTCC[A/G]
AGGCTCC-30 ). TaqMan polymerase chain reaction (PCR) was per-
formed in a total volume of 10 ml (5 ng of template DNA, 1x Taqman
Genotyping Master Mix and 1x Taqman Allele Discrimination
Assay) placed in 96-well PCR plates. Thermal cycling was per-
formed in standard conditions and fluorescence was recorded by
the StepOne Plus Real Time PCR equipment (Applied Biosystems,
Foster City, CA, USA). Genotyping quality was checked using in each
experiment both previously determined positive controls (repre-
sentative of all genotypes) and negative controls (samples con-
taining no template). Moreover, we have also repeated 10% of the
whole sample randomly and have obtained 100% of consistency.
Results were analyzed with manufacturer's software.
2.5. Haplotype inference
Haplotypes frequencies were inferred using the Bayesian
statistical-based program PHASE (version 2.1; http://www.stat.
washington.edu/stephens/software.html) [28]. To further confirm
these haplotype estimates, we have also used the Haplo.stats
software (version 1.4.4; http://cran.r-project.org/web/packages/
haplo.stats/index.html). The possible haplotypes including the 3
NOS3 tagSNPs (rs3918188, rs3918226 and rs743506) were: CCA,
CCG, TCA, TCG, AAA, CAG, TAA, TAG. LD plots for the tagSNPs in the
present study along with the clinically relevant rs2070744 and
rs1799983 SNPs previously studied [29] were characterized by
calculating D0 and LOD (log of the likelihood odds ratio, a measure
of confidence in the value of D0 ) among the SNPs using the Hap-
loview software (version 4.2; http://www.broad.mit.edu/mpg/
haploview/).
2.6. Statistical analysis
Clinical and laboratory characteristics of the studied groups are
expressed as means ± SD, whereas the changes in blood pressure
after enalapril treatment are expressed as means ± SEM. Clinical
and laboratorial characteristics of patients were compared by un-
paired t-test (parametric data), by Mann Whitney test (non-para-
metric data), by chi-square test or Fisher's test (categorical
variables) when appropriate. Deviation from the Hardy-Weinberg
equilibrium was evaluated by chi squared test. The effects of
NOS3 genetic markers on changes in SBP, MBP and DBP were tested
by ANOVA (rs3918188, rs743506 and haplotypes) or unpaired t-test
(rs3918226). Moreover, the effects of NOS3 genotypes and haplo-
types on changes in SBP, MBP and DBP according to ethnicity were
evaluated by two-way ANOVA. To confirm our findings, we per-
formed a multiple linear regression analysis to assess the effects of
the NOS3 tagSNPs genotypes on responses to enalapril (changes in
SBP, MBP and DBP after 8 weeks of treatment). In addition, the
64 G.H. Oliveira-Paula et al. / Nitric Oxide 55-56 (2016) 62e69

significant effects of NOS3 tagSNPs haplotypes on responses to
enalapril, as shown by ANOVA, were confirmed by multiple linear
regression analysis. The regression models were adjusted for age,
gender, ethnicity and alcohol consumption. The analysis was car-
ried out using JMP® software (SAS Institute, Cary, NC). A value of
P < 0.05 was considered significant.
Genotype data for Africans and Europeans was obtained from
the GVS SeattleSNPs database and were also evaluated using Hap-
loview software.
3. Results
The clinical and laboratorial characteristics of the 101 hyper-
tensive patients enrolled in this study are shown in Table 1. Sig-
nificant decreases in office SBP and DBP were observed after
treatment with enalapril (P < 0.001). We found no differences in
gender, ethnicity, age, body mass index, alcohol consumption or in
any laboratorial parameters between the group of patients treated
with enalapril 10 mg/day and the group of patients treated with
enalapril 20 mg/day (Table 1; P > 0.05).
The distribution of genotypes for the three studied tagSNPs
showed no deviation from Hardy-Weinberg equilibrium (all
P > 0.05). The effects of rs3918226, rs3918188, and rs743506 ge-
notypes and haplotypes on changes in blood pressure after treat-
ment with enalapril are shown in Fig. 1. Although the rs3918226
and rs743506 genotypes did not affect blood pressure responses to
enalapril (Fig. 1A and C; P > 0.05), we found that subjects carrying
the AA genotype for the tagSNP rs3918188 showed lower decreases
in SBP after treatment with enalapril than individuals carrying the
homozygous ancestral genotype CC (Fig. 1B; P < 0.05). In addition,
subjects carrying the TCA haplotype showed more intense de-
creases in SBP after treatment with enalapril compared with car-
riers of the CAG haplotype (Fig. 1D; P < 0.05). Adjustment for age,
gender, ethnicity and alcohol consumption in multiple linear
regression analysis did not change these findings (Table 2). We also
performed allele analysis and the A allele for the rs3918188 poly-
morphism was associated with lower decreases in SBP (P < 0.05;
data not shown), whereas the other alleles for the rs3918226 and
rs743506 polymorphisms were not associated with significant
differences in responses to enalapril (P > 0.05; data not shown). In
addition, although there are interethnic differences in the distri-
bution of NOS3 variants [30e32], the effects of NOS3 tagSNPs ge-
notypes and haplotypes on changes in SBP, MBP and DBP in
response to enalapril were not different between whites and non-
whites (P > 0.05; data not shown).
We also evaluated the effects of NOS3 tagSNPs on changes in
blood pressure after enalapril according to enalapril doses. The
effects of rs3918226, rs3918188, and rs743506 genotypes on
changes in blood pressure after treatment with enalapril 10 mg/day
or 20 mg/day are shown in Fig. 1A-C and Fig. 1D-F, respectively.
Although the rs3918226 genotypes did not affect blood pressure
responses to enalapril 10 mg/day (Fig. 1A; P > 0.05), subjects car-
rying the T allele (CT þ TT genotypes) showed higher decreases in
SBP after treatment with enalapril 20 mg/day than individuals
carrying the homozygous ancestral genotype CC (Fig. 1D; P < 0.05).
These findings did not change in multiple linear regression analysis
(Table 3). When stratified by doses, in unadjusted analyses, the
genotypes for the rs3918188 polymorphism did not significantly
affect the changes in blood pressure of hypertensive patients in
response to enalapril treatment (Fig. 1B and E; P > 0.05), despite the
fact that carriers of the CC and CA genotypes marginally showed
more intense decreases in SBP after treatment with enalapril
20 mg/day (Fig. 1E; P ¼ 0.05). However, after adjusting for cova-
riates, the A allele was associated with lower decreases in SBP in
response to enalapril 10 mg/day (Table 3; P < 0.05) and, in line with
this finding, the C allele was associated with higher decreases in
SBP in response to enalapril 20 mg/day (Table 3; P < 0.05).
The effects of NOS3 tagSNPs haplotypes on changes in blood
pressure after enalapril treatment were also stratified according to
enalapril doses. NOS3 tagSNPs haplotypes produced no effects on
changes in blood pressure in response to enalapril 10 mg/day
(Fig. 2A; P > 0.05). However, subjects carrying the TCA haplotype
showed more intense decreases in SBP after treatment with ena-
lapril 20 mg/day compared with carriers of the CAG haplotype
(Fig. 2B; P < 0.05). Consistently, after adjusting for selected vari-
ables, the TCA haplotype was associated with more intense de-
creases in SBP in response to enalapril 20 mg/day (Table 2;
P < 0.05), while the CAG haplotype was associated with lower

Table 1
Clinical and laboratorial characteristics of hypertensive patients treated with enalapril 10 mg/day or 20 mg/day.

All Enalapril 10 mg Enalapril 20 mg

N 101 48 53
Male/Female 69/32 34/14 35/18
Ethnicity (non-Whites) 40 22 18
Age (years) 47 ± 12 46 ± 13 48 ± 12
BMI (Kg/m2) 29.4 ± 5.5 29.0 ± 4.3 29.7 ± 5.5
Alcohol consumption (current drinkers) 4 2 2
Total cholesterol (mg/dL) 202 ± 43 195 ± 43 208 ± 38
LDL cholesterol (mg/dL) 128 ± 38 123 ± 43 134 ± 35
HDL cholesterol (mg/dL) 46 ± 15 47 ± 15 46 ± 15
Triglycerides (mg/dL) 147 ± 73 143 ± 73 151 ± 69
Glucose (mg/dL) 95 ± 33 90 ± 33 100 ± 25
Creatinine (mg/dL) 0.91 ± 0.15 0.93 ± 0.15 0.90 ± 0.15
Potassium (mEq/L) 4.3 ± 0.4 4.2 ± 0.3 4.3 ± 0.4
SBP (mmHg)
Baseline 149 ± 10 148 ± 9 149 ± 9
After enalapril treatment 129 ± 9* 129 ± 9* 129 ± 9*
DBP (mmHg)
Baseline 94 ± 9 93 ± 8 95 ± 10
After enalapril treatment 80 ± 8* 81 ± 8* 80 ± 7*
HR (beats/min)
Baseline 79 ± 12 78 ± 12 79 ± 13
After enalapril treatment 76 ± 11 77 ± 10 75 ± 11

BMI: body mass index; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; SBP: systolic blood pressure; DBP: diastolic blood pressure; HR: heart rate.
Values are the mean ± S.D. *P < 0.001 vs baseline.
 G.H. Oliveira-Paula et al. / Nitric Oxide 55-56 (2016) 62e69 65

Fig. 1. Effects of NOS3 tagSNPs genotypes and haplotypes on changes in blood pressure of hypertensive patients in response to enalapril treatment. (A) Effects of the CC (n ¼ 87) and
CT þ TT (n ¼ 11) genotypes for the tagSNP rs3918226 on changes in systolic, mean, and diastolic blood pressure in response to enalapril. (B) Effects of the CC (n ¼ 41), CA (n ¼ 49),
and AA (n ¼ 11) genotypes for the tagSNP rs3918188 on changes in systolic, mean, and diastolic blood pressure in response to enalapril. (C) Effects of the AA (n ¼ 39), AG (n ¼ 42),
and GG (n ¼ 20) genotypes for the tagSNP rs743506 on changes in systolic, mean, and diastolic blood pressure in response to enalapril. (D) Effects of the CCA (n ¼ 83), CCG (n ¼ 34),
TCA (n ¼ 3), TCG (n ¼ 11), CAA (n ¼ 34), and CAG (n ¼ 37) haplotypes on systolic, mean, and diastolic blood pressure after treatment with enalapril. Data are shown as means ± SEM.
*P < 0.05 versus CC genotype for the tagSNP rs3918188. #P < 0.05 TCA versus CAG haplotype.

Table 2
Effects of NOS3 tagSNPs on changes in blood pressure of hypertensive patients in response to enalapril. Multiple linear regression analysis adjusted for age, gender, ethnicity,
and alcohol consumption.

Constant Change in SBP (mmHg) Change in MBP (mmHg) Change in DBP (mmHg)

R2 b SE P value R2 b SE P value R2 b SE P value

rs3918226 (T allele) 0.088 1.341 1.560 0.392 0.125 0.553 1.219 0.651 0.075 0.391 1.459 0.789
rs3918188 (A allele) 0.129 þ2.353 1.030 0.025a 0.139 þ1.049 0.817 0.202 0.106 þ1.768 0.969 0.071
rs743506 (G allele) 0.086 þ0.765 1.102 0.489 0.125 þ0.372 0.860 0.665 0.078 þ0.643 1.027 0.533
TCA haplotype 0.106 6.617 2.912 0.024a 0.133 3.375 2.288 0.141 0.092 5.342 2.725 0.051
CAG haplotype 0.104 þ2.063 0.942 0.029a 0.129 þ0.806 0.741 0.278 0.076 þ0.494 0.889 0.578
a
Statistically significant.

Table 3
Effects of NOS3 tagSNPs on changes in blood pressure of hypertensive patients in response to enalapril, stratified by dose. Multiple linear regression analysis adjusted for age,
gender, ethnicity, and alcohol consumption.

Constant Change in SBP (mmHg) Change in MBP (mmHg) Change in DBP (mmHg)

R2 b SE P value R2 b SE P value R2 b SE P value

Enalapril 10 mg
rs3918226 (T allele) 0.115 þ2.882 2.722 0.296 0.098 þ1.004 1.943 0.607 0.088 0.235 2.035 0.909
rs3918188 (A allele) 0.192 þ3.635 1.605 0.029a 0.145 þ1.849 1.168 0.121 0.172 þ2.445 1.197 0.048a
rs743506 (G allele) 0.093 0.610 1.833 0.740 0.097 þ0.579 1.294 0.667 0.096 þ0.833 1.348 0.540
Enalapril 20 mg
rs3918226 (T allele) 0.242 4.268 1.843 0.025a 0.275 2.213 1.590 0.170 0.217 1.416 2.050 0.493
rs3918188 (C allele) 0.311 6.351 1.958 0.002a 0.297 3.228 1.746 0.070 0.244 3.272 2.245 0.152
rs743506 (G allele) 0.203 þ2.367 1.407 0.099 0.246 þ0.318 1.208 0.793 0.211 þ0.595 1.532 0.700
TCA haplotype 0.206 6.429 2.661 0.018a 0.258 3.023 2.272 0.186 0.231 4.880 2.867 0.092
CAG haplotype 0.201 þ2.917 1.282 0.025a 0.257 þ1.418 1.092 0.197 0.213 þ1.024 1.394 0.465
a
Statistically significant.
66 G.H. Oliveira-Paula et al. / Nitric Oxide 55-56 (2016) 62e69

Fig. 2. Effects of NOS3 tagSNPs on changes in blood pressure of hypertensive patients in response to enalapril treatment, stratified by dose. (A) Effects of the CC (n ¼ 42) and CT þ TT
(n ¼ 6) genotypes for the tagSNP rs3918226 on changes in systolic, mean, and diastolic blood pressure in response to enalapril 10 mg/day. (B) Effects of the CC (n ¼ 21), CA (n ¼ 22),
and AA (n ¼ 5) genotypes for the tagSNP rs3918188 on changes in systolic, mean, and diastolic blood pressure in response to enalapril 10 mg/day. (C) Effects of the AA (n ¼ 16), AG
(n ¼ 23), and GG (n ¼ 9) genotypes for the tagSNP rs743506 on changes in systolic, mean, and diastolic blood pressure in response to enalapril 10 mg/day. (D) Effects of the CC
(n ¼ 45) and CT þ TT (n ¼ 8) genotypes for the tagSNP rs3918226 on changes in systolic, mean, and diastolic blood pressure in response to enalapril 20 mg/day. (E) Effects of the CC
(n ¼ 20), CA (n ¼ 27), and AA (n ¼ 6) genotypes for the tagSNP rs3918188 on changes in systolic, mean, and diastolic blood pressure in response to enalapril 20 mg/day. (F) Effects of
the AA (n ¼ 23), AG (n ¼ 19), and GG (n ¼ 11) genotypes for the tagSNP rs743506 on changes in systolic, mean, and diastolic blood pressure in response to enalapril 20 mg/day. Data
are shown as means ± SEM. *P < 0.05 versus CC genotype for the tagSNP rs3918226.

Fig. 3. Effects of NOS3 tagSNPs haplotypes on changes in blood pressure of hypertensive patients in response to enalapril treatment, stratified by dose. (A) Effects of the CCA
(n ¼ 43), CCG (n ¼ 15), TCG (n ¼ 6), CAA (n ¼ 12), and CAG (n ¼ 20) haplotypes on systolic, mean, and diastolic blood pressure after treatment with enalapril 10 mg/day. (B) Effects of
the CCA (n ¼ 40), CCG (n ¼ 19), TCA (n ¼ 3), TCG (n ¼ 5), CAA (n ¼ 22), and CAG (n ¼ 17) haplotypes on systolic, mean and diastolic blood pressure after treatment with enalapril
20 mg/day. Data are shown as means ± SEM. *P < 0.05 TCA versus CAG haplotype.
 G.H. Oliveira-Paula et al. / Nitric Oxide 55-56 (2016) 62e69
decreases in SBP after treatment with enalapril 20 mg/day (Table 2;
P < 0.05) (see Fig. 3).
In order to assess the LD among the tagSNPs studied here and
the clinically relevant rs2070744 and rs1799983 SNPs of NOS3 gene
previously studied [29], we have compared the D0 and LOD values
between the tagSNPs studied with Europeans and Africans from the
SeattleSNPs database (Supplementary Fig. S1). Higher LD values
were observed in the enalapril study groups compared to Euro-
peans and Africans. Notably, higher LD values were observed be-
tween the tagSNP rs3918188 and the SNP rs1799983 for the
enalapril group 10 mg/day (D0 ¼ 1, LOD ¼ 2.08) and 20 mg/day
(D0 ¼ 0.85, LOD ¼ 1.44). In addition, a high LD values was observed
between the tagSNP rs3918188 and the promoter SNP rs2070744
for the enalapril group 20 mg/day (D0 ¼ 1, LOD ¼ 2.65).
4. Discussion
This is the first study to evaluate whether the NOS3 tagSNPs
rs3918226, rs3918188 and rs743506 and haplotypes may influence
the antihypertensive responses to enalapril in essential hyperten-
sion. The main findings of this study were that: (i) the A allele for
the rs3918188 tagSNP was associated with worse responses to
enalapril; (ii) while the TCA haplotype was associated with better
responses, the CAG haplotype was associated with worse responses
to enalapril; (iii) the T allele (CT þ TT genotypes) for the tagSNP
rs3918226 was associated with better responses to enalapril 20 mg/
day.
Our findings are consistent with the known influence of ACEi on
NOS3 regulation. Indeed, the antihypertensive effects of ACEi are
associated, at least in part, with prevention of bradykinin degra-
dation [3]. In endothelial cells, bradykinin induces activation of
bradykinin type 2 receptors (BR2), leading to calcium-calmodulin-
dependent activation of NOS3, as well as Akt activation and phos-
phorylation of Ser1177, thus resulting in increased NO production
[33]. In this regard, it was shown that approximately 70% of the
plasma nitrite is derived from the activity of NOS3 [34]. However,
enalapril had no effects on circulating nitrite levels [35], probably
due to the fact that it may increase NO production at tissue level,
which are not precisely reflected by the circulating nitrite levels
[35]. Conversely, NOS3 polymorphisms have been shown to affect
this marker of endogenous NO production [14,17,36e38]. Indeed,
blood nitrite levels were shown to be affected by the tagSNP
rs3918226 [14] and by NOS3 haplotypes formed by the clinically
relevant genetic polymorphisms rs2070744, 4b/4a VNTR and
rs1799983 [36e38]. In addition, we have recently shown that one
haplotype formed by NOS3 tagSNPs was associated with low
circulating blood nitrite concentrations [17].
NOS3 polymorphisms have been associated with hypertension
and in this respect the genotype frequencies for rs3918226,
rs3918188 and rs743506 polymorphisms found in hypertensive
patients in the present study were similar to those reported in
apparently healthy subjects from the same population [17,18].
However, this study was not designed to address this issue, and
there is evidence suggesting that the T allele for rs3918226 poly-
morphism is associated with hypertension in Europeans [21,39].
Given the interethnic differences in the distribution of NOS3 poly-
morphisms [30e32], further studies comparing the hypertensives
and normotensive from different populations are required to clarify
this issue.
We have found that the A allele for the NOS3 tagSNP rs3918188
was associated with worse antihypertensive responses to enalapril.
To our knowledge, there is no functional data showing the effects of
the tagSNP rs3918188 on NOS3 expression or activity. However, we
performed LD analysis in an attempt to correlate this tagSNP with
the clinically relevant rs2070744 and rs1799983 SNPs previously
67
studied [29]. Higher LD values were observed between rs3918188
and rs1799983 in both groups of patients treated with enalapril,
and between rs3918188 and rs2070744 in the enalapril 20 mg/day
group. Therefore, the molecular mechanisms explaining how ge-
notypes for the rs3918188 tagSNP or haplotypes may affect NOS3
require further studies. Moreover, lower LD values were observed
between the NOS3 tagSNPs in Europeans and Africans, mainly in
this latter population. LD patterns reflect a particular population
history, and the recent admixture occurred in the Brazilians may
explain the observed different LD patterns. Noteworthy, there are
major populational differences in the distribution of NOS3 poly-
morphisms and haplotypes [30e32], and LD among NOS3 poly-
morphisms should be explored in other populations to interpret
the association signals including the tagSNP rs3918188.
In addition to NOS3 tagSNPs genotypes, we analyzed NOS3
tagSNPs haplotypes, which may provide improved genetic infor-
mation than the analysis of genetic markers one by one [40].
Indeed, NOS3 tagSNPs haplotypes affect NO bioavailability [17] and
were associated with pathological conditions such as childhood
obesity [14] and migraine with aura [41]. In the present study,
better responses to enalapril were associated with the TCA haplo-
type. Conversely, the CAG haplotype was associated with worse
responses to enalapril. Interestingly, the TCA haplotype includes
the T and C alleles for the tagSNPs rs3918226 and rs3918188,
respectively, which are involved with better responses to enalapril,
while the CAG haplotype contains the alleles for the mentioned
tagSNPs involved with worse responses to this drug. Thus, although
there are no functional studies addressing the functionality of these
haplotypes, we could speculate that the TCA and CAG haplotypes
may affect NOS3 expression or activity in opposite directions,
consequently leading to opposite responses to enalapril. However,
the molecular basis underlying the effects of the haplotypes re-
mains to be evaluated.
In addition to the analysis including 101 hypertensive patients,
we also evaluated the effects of NOS3 tagSNPs on the responses to
enalapril according to the dose of enalapril used. Interestingly,
although the rs3918226 genotypes did not affect blood pressure
responses to enalapril in the analysis including the total number of
patients, we found that individuals carrying the CT þ TT genotypes
(containing the T allele) showed better responses to enalapril
20 mg/day. Indeed, the effects of some polymorphisms on the re-
sponses to antihypertensive drugs are more pronounced at higher
doses [42,43]. The NOS3 tagSNP rs3918226 has been regarded as a
highly relevant polymorphism for the cardiovascular system
[14,19e22,44]. Notably, two genome-wide association studies
found the TT genotype in association with increased hypertension
risk [20,21]. Consistently, a recent study showed that this genotype
predicts the cardiovascular mortality and morbidity in white Eu-
ropeans [22]. In addition, the T allele for the tagSNP rs3918226 was
associated with parameters reflecting central arterial stiffness and
wave reflection [44]. The results found in the present study agree
with functional studies focusing on the rs3918226 polymorphism.
Indeed, while we found no effects of this polymorphism on plasma
nitrite levels [18], a recent study showed that this polymorphism
affects NOS3 expression [19]. The authors used a luciferase reporter
assay in HeLa and HEK293T cells transfected with NOS3 promoter
carrying either the T or the C allele at the position 665 in the NOS3
gene to show that the C to T conversion reduces promoter activity
by 20e40% [19]. The rs3918226 polymorphism is located next to a
potential transcription factor binding site for the Ets (E-twenty six)
family domain [21], which is essential for the activation of the NOS3
promoter [45]. Therefore, it is possible that these transcription
factors bind to NOS3 promoter with lower affinity in the presence of
the T allele, resulting in less NOS3 transcription compared with the
C allele. Taking together, these findings suggest that reduced NOS3
68 G.H. Oliveira-Paula et al. / Nitric Oxide 55-56 (2016) 62e69
expression could increase the risk for hypertension and cardio-
vascular diseases in carriers of the T allele. On the other hand,
carriers of the T allele could show enhanced response to drugs that
reduce blood pressure and reverse the cardiovascular damage by
increasing NOS3 expression, such as ACEi. However, this suggestion
remains to be tested in further studies.
As a limitation of the present study, we should mention the
relatively low number of subjects included. However, it is necessary
emphasize that we included only hypertensive patients under no
current antihypertensive treatment. Another point that has not
been approached in this study includes other polymorphisms in
genes related to NO pathway that could affect the antihypertensive
responses to enalapril, such as NOS2 [46] and PRKG [47]. Therefore,
these issues remain to be assessed.
In conclusion, our findings suggest that NOS3 tagSNPs influence
the effects of enalapril in essential hypertension. The A allele for the
tagSNP rs3918188 and the CAG haplotype are associated with
worse antihypertensive responses to enalapril. Conversely, the TCA
haplotype is associated with better responses to enalapril. Addi-
tionally, the T allele for the tagSNP rs3918226 is associated with
better responses to enalapril 20 mg/day.
Conflict of interest
All authors declare no conflicts of interest.
Acknowledgements
This study was supported by Conselho Nacional de Desenvol-
vimento Científico e Tecnologico and Fundaçao de Amparo a Pes-
quisa do Estado de Sao Paulo (FAPESP).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.niox.2016.03.006.
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