Yonago Acta medica 2006;49:59–62

Effects of Glu298Asp Polymorphism of Endothelial NitricOxide Synthase (eNOS) Gene on eNOS mRNA and Protein
Expressions in Cultured Human Vascular Endothelial Cells
Takuji Kishimoto, Akihiko Kaetsu, Yoneatsu Osaki, Mikizoh Okamoto, MariyaNagai, Yoichi Kurosawa* and Soichi Yoshida†
Division of Environmental and Preventive Medicine and *Division of Health Administration andPromotion, Department of Social Medicine and †Division of Reproductive-Perinatal Medicineand Gynecologic Oncology, Department of Surgery, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503 JapanThe relationship between cardiovascular disease and Glu298Asp polymorphism of nitricoxide synthase (eNOS) gene in vascular endothelial cells is controversial due to conﬂicting results. To demonstrate the effects of Glu298Asp polymorphism of the eNOS geneon eNOS gene expression by excluding the effects of various confounding factors, eNOSmRNA and protein expressions were measured using cultured human umbilical vein
endothelial cells (HUVECs) according to genotypes of Glu298Asp polymorphism of theeNOS gene. eNOS mRNA expression was measured by real-time PCR/reverse trasnscription-PCR. eNOS protein expression was measured by an immunoassay method.
HUVECs with Glu298Asp tended to show higher eNOS mRNA and protein expressions
than those with Glu298Glu, but the difference was not signiﬁcant. These results suggest that the genotypic difference of Glu298Glu and Glu298Asp has no effect on eNOSmRNA and protein expressions.
Key words: gene expression; nitric oxide synthase; polymorphism; vascular endothelial cell

(7q35-36), spans 21 kb, and consists of 26 exons(Marsden et al., 1993). Among several reported
polymorphisms of the eNOS gene, the involvement of Glu298Asp polymorphism (glutamic acidto aspartic acid substitution at codon 298) in coronary artery disease and essential hypertension has
been discussed (Miyamoto et al., 1998; Kato et al.,
1999; Shoji et al., 2000; Benjaﬁeld et al., 2000;
Colombo et al., 2002). The conﬂicting results areconsidered to be caused by confounding factors
such as differences in the genetic backgrounds ofstudy populations and various environmental factors.
It is important to experimentally investigate
the functional effects of Glu298Asp polymor-

Nitric oxide produced from vascular endothelial
cells is synthesized from L-arginine by the functions of nitric oxide synthase (eNOS). Major
functions of vascular endothelial cell-derived NO
include relaxant and antiproliferative effects on
vascular smooth-muscle cells, antiaggregant effects on the platelets, and inhibitory effects on adhesion of leukocytes to vascular endothelial cells(Kader et al., 2000; Ozaki et al., 2002). In addition, the relationship between NO production in
vascular endothelial cells and various pathologic
conditions such as inﬂammatory disease, hypertension, and coronary artery disease has been reported.
The eNOS gene is located on chromosome 7Abbreviations: CTPP, confronting 2-pair primers for polymorphism; eNOS, nitric oxide synthase; HUVEC, human umbilical vein endothelial cell59

T. Kishimoto et al.

with confronting 2-pair primers for polymorphism(PCR-CTPP) method (Hamajima et al., 2000) wasperformed using 4 primers (primer 1: 5'-CATGAG GCT CAG CCC CAG AAC-3', primer 2:5'-AGT CAA TCC CTT TGG TGC TCAC-3',primer 3: 5'-GAA GGA AGA GTT CTG GGGGA-3', primer 4: 5'-GCT GCA GGC CCC AGATGA G-3'). In the primary cultures of 29 cases,polymorphisms were identified. As a result, 26
cases with the Glu298Glu genotype and 3 caseswith the Glu298Asp genotype were identiﬁed.

phism on eNOS gene expression by excludingthese confounding factors. Thus, we identified
Glu298Asp polymorphism using cultured humanvascular endothelial cells, and investigated thefunctional effects of Glu298Asp polymorphism
on eNOS mRNA and protein expressions.

Materials and Methods
EthicsThis study was reviewed and approved by theEthical Committee of the Faculty of Medicine,Tottori University. Biomaterials (human umbilical cords) were collected after obtaining informed
consent from the pregnant women.

Comparative quantiﬁcation of eNOS genemRNA expression
mRNA was extracted from HUVECs using ISOGEN (Nippon Gene, Toyama, Japan). eNOSmRNA expression was measured by a real-time
PCR/reverse transcription-PCR method. A Fast
Start DNA Master SYBER Green I Kit (RocheDiagnostics, Tokyo, Japan) was used as a reagent,and the primers used were as follows: 5'-CATGAG GCT CAG CCC CAG AAC-3' and 5'-AGTCAA TCC CTT TGG TGC TCAC-3'. A Takara
PCR Thermal Cycler (Takara Bio, Ootsu, Japan)
was used for cDNA synthesis under the following
reaction conditions: annealing at 30˚C for 10 min,reverse transcription at 42˚C for 15 min, and inactivation of reverse transcriptase at 99˚C for 5 min.
A LightCycler (Roche Diagnostics) was used forreal-time PCR. PCR was performed under thefollowing conditions: after denaturing at 95˚Cfor 10 min, 50 cycles of denaturing at 95˚C for 15s, annealing at 60˚C for 5 s, and extension at 72˚C for 10 s with a temperature transition rate of 20
˚C/s. Fluorescence was recorded following theextension reaction stages of each cycle. β-actin
gene mRNA expression was also measured forcomparative quantiﬁcation, and eNOS and β-actinmRNA expressions were quantiﬁed comparatively
by calculating the ratio between them. The primers used for β-actin gene were as follows: 5'-CGTGAC ATT AAG GAG AAG CTG TGC-3' and5'-GTC ATA CTC CTG CTT GOT GAT CCA-3'.

Cell culture
Human umbilical cords were aseptically obtainedimmediately after delivery, and human umbilicalvein endothelial cells (HUVECs) were collectedby enzyme treatment with 0.5 g/L collagenase
type IV (Wako Pure Chemical, Osaka, Japan).
The collected HUVECs were plated onto gelatincoated plastic culture dishes, and were staticallycultured in a CO 2 incubator (5% CO 2 in 95%
humidiﬁed air). An Endothelial Cell Growth Medium 2 Kit (Promo Cell, Heidelberg, Germany)
was used as the growth medium. The kit includes
the following: endothelial cell basal medium, 2%
fetal calf serum, 5.0 ng/mL epidermal growthfactor, 0.2 µg/mL hydrocortisone, 5.0 ng/mL vascular endothelial growth factor, 10 ng/mL basicﬁbroblast factor, 20 ng/mL R3 insulin-like growth
factor-1, 1 µg/mL ascorbic acid, 22.5 µg/mL
heparin, 50 ng/mL amphotericin B and 50 µg/mL
gentamicin.
Identiﬁcation of Glu298Asp polymorphism
DNA was extracted from umbilical cord tissue
using an automatic nucleic acid extraction instrument (MFX-2000, Toyobo, Osaka), and a PCR60

Glu298Asp of eNOS and mRNA/protein expression

Quantiﬁcation of eNOS protein expression
An immunoassay method by a Human eNOS
Immunoassay Kit (R&D Systems, Minneapolis,MN) was used to measure the eNOS protein expression. In brief, cells were trypsinized for collection, and were precipitated by centrifugation at300 × g for 5 min. Then cells were washed twicewith phosphate-buffered saline, and after the addition of cell lysis buffer, the cell solution wascentrifuged at 300 × g for 5 min, and the concentration of eNOS protein in the supernatant wasmeasured by a microplate reader.

Fig. 1. eNOS mRNA expression (A) and protein concentrations (B) according to genotypes of eNOS polymorphism Glu298Asp in exon 7. Results are expressed asmeans (SE; bars). Values for eNOS mRNA expression (A)are given as the ratio of the amounts of eNOS and β-actinmRNA expression measured by real time PCR/reverse
transcription-PCR. Values for eNOS protein (B) are measured by immunoassay method. Measurement was conducted on different dates using different HUVECs derived
from 5 and 3 individuals for Glu298Glu and Glu298Asp
genotypes, respectively.

Experiments according to Glu298Asp genotypes
HUVECs with the Glu298Glu or Glu298Asp
genotype were plated onto 2 gelatin-coated 60
mm plastic dishes, and were cultured in growth
medium to reach confluence. Subsequently, themedium was replaced with fresh growth medium,and 24 h later, mRNA and protein expressions
were measured as described above. Measurement
was conducted on different dates using different
HUVECs derived from 5 and 3 individuals for
Glu298Glu and Glu298Asp genotypes, respectively.

expression tended to be higher in HUVECs withthe Glu298Asp genotype than in those with
Glu298Glu (Fig. 1B), but the difference was notsigniﬁcant.

Statistical analysis

Discussion

SPSS software (version 11.0J) was used for statistical analysis. Student’s t-test was used as a statistical method for comparison. The signiﬁcancelevel was set to 5%.

To reveal the effects of Glu298Asp polymorphismof the eNOS gene on eNOS mRNA and protein
expressions, eNOS mRNA and protein expressions were measured using HUVECs with theGlu298Glu or Glu298Asp genotype. Both eNOSmRNA and protein expressions tended to behigher in HUVECs with Glu298Asp than in thosewith Glu298Glu, but the difference was not signiﬁcant.
Mainly in case-control studies, the relationship between the Glu298Asp polymorphism ofthe eNOS gene and cardiovascular disease iscontroversial due to conﬂicting results (MiyamotoResults
The effects of genotypes on eNOS mRNA andprotein expressions are shown in Fig. 1. eNOSmRNA expression tended to be higher in HUVECs with the Glu298Asp genotype than inthose with Glu298Glu (Fig. 1A), but the difference was not significant. Also, eNOS protein61T. Kishimoto et al.

et al., 1998; Kato et al., 1999; Shoji et al., 2000;
Benjaﬁeld et al., 2000; Colombo et al., 2002). We
conducted a cohort study on the relationship between the Glu298Asp polymorphism of the eNOS
gene and hypertension in Japanese, but found norelationship (Kishimoto et al., 2004).
Also, there are several studies on the effectsof the eNOS Glu298Asp genotype on the functions of the circulatory system. Schneider et al.
(2000) investigated the dilation reaction of theforearm artery by administering acetylcholine
to 80 patients, and reported no difference due tothe eNOS Glu298Asp polymorphism. Jesung etal. measured plasma nitric oxide levels derived
from vascular endothelial cells, and reported nodifference due to the eNOS Glu298Asp polymorphism. Our study supports these results, showing
no effects of the Glu298Asp polymorphism of theeNOS gene on the circulatory system.
This study was limited by the unavailability ofHUVECs with the homozygotic Asp298Asp genotype. Our cohort study included 85.3% Glu298Glu,14.0% Glu298Asp and 0.7% Asp298Asp (n = 2,042).
It is considered necessary to obtain HUVECs
with the homozygotic Asp298Asp genotype byincreasing the sample size in the future.
In conclusion, the results of this study suggest the possibility that the Glu298Glu andGlu298Asp polymorphisms of the eNOS gene
have no effect on eNOS mRNA and protein expressions in HUVECs, and also on the circulatory
system.

tion of a common variant of the endothelial nitricoxide synthase gene (Glu298 Asp polymorphism) tothe presence, extent, and severity of coronary arterydisease. Heart 2002;87:525–528.
3 Ha maji ma N, Sa ito T, Mast uo K , Koza k i K ,Takahashi T, Tajima K. Polymerase chain reaction
with confronting two-pair primers for polymorphism
genotyping. Jpn J Cancer Res 2000;91:865–868.
4 Jesung M, Suin Y, Eunkyung K, Chol S, Sangmee
AJ, Inho J. Lack of evidence for contribution ofGlu298Asp (G894T) polymorphism of endothelial
nitric oxide to plasma nitric oxide levels. Thromb
Res 2002;107:129–134.
5 Kader KN, Akella R, Ziats NP, Lakey LA, Harasaki
H, Ranieri JP, et al. eNOS-overexpressing endothelial cells inhibit platelet aggregation and smoothmuscle cell proliferation in vitro. Tissue Eng
2000;6:241–251.
6 Kato N, Sugiyama T, Morita H, Nabika T, Kurihara
H, Yamori Y, et al. Lack of evidence for association
between the endothelial nitric oxide synthase geneand hypertension. Hypertension 1999;33:933–936.
7 Kishimoto T, Misawa Y, Kaetu A, Nagai M, Osaki Y,Okamoto M, et al. eNOS Glu298Asp polymorphismand hypertension in a cohort study in Japanese. Prev
Med 2004;39:927–931.
8 Marsden PA, Heng HH, Scherer SW, Stewart RJ,Hall AV, Shi XM, et al. Structure and chromosome localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem
1993;268:17478–17488.
9 Miyamoto Y, Saito Y, Kajiyama N, Yoshimura M,
Shimasaki Y, Nakayama M, et al. Endothelial nitricoxide synthase gene is positively associated with essential hypertension. Hypertension 1998;32:3–8.
10 Ozaki M, Kawashima S, Yamashita T, Hirase T,Namiki M, Inoue N, et al. Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deﬁcient mice. J Clin
Invest 2002;110:331–340.
11 Schneider MP, Erdmann J, Delles C, Fleck E,
Regitz-Zagrosek V, Schmieder RE. Functional genetesting of the Glu298Asp polymorphism of the endothelial NO synthase. J Hypertens 2000;18:1767–
1773.
12 Shoji M, Tsutaya Shoji, Saito R, Takamatu H,
Yasujima M. Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan. Life Sci 2000;66:2557–2562.

Acknowledgments: This research was supported in partby a Grant-in-Aid from Japan Society for the Promotionof Science.
References
1 Benjafield AV, Morris BJ. Association analysesof endothelial nitric oxide synthase gene polymorphisms in essential hypertension. Am J Hypertens
2000;13:994–998.
2 Colombo MG, Andreassi MG, Paradossi U, Botto N,Manfredi S, Masetti S, et al. Evidence for associa-Received December 16, 2005; accepted January 17, 2006
Corresponding author: Takuji Kishimoto, MD62