The relationship between cardiovascular disease and Glu298Asp polymorphism of nitric oxide synthase (eNOS) gene in vascular endothelial cells is controversial due to conflicting results. To demonstrate the effects of Glu298Asp polymorphism of the eNOS gene on eNOS gene expression by excluding the effects of various confounding factors, eNOS mRNA and protein expressions were measured using cultured human umbilical vein endothelial cells (HUVECs) according to genotypes of Glu298Asp polymorphism of the eNOS 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 eNOS mRNA and protein expressions

Kaetsu, Akihiko

Kishimoto, Takuji

Kurosawa, Yoichi

Nagai, Mariya

Okamoto, Mikizoh

Osaki, Yoneatsu

Yoshida, Soichi

Yonago Acta Medica

Tottori University Research Result Repository

Yonago Acta medica 2006;49:59–62 59Effects of Glu298Asp Polymorphism of Endothelial Nitric Oxide Synthase (eNOS) Gene on eNOS mRNA and Protein Expressions in Cultured Human Vascular Endothelial CellsTakuji Kishimoto, Akihiko Kaetsu, Yoneatsu Osaki, Mikizoh Okamoto, Mariya Nagai, Yoichi Kurosawa* and Soichi Yoshida†Division of Environmental and Preventive Medicine and *Division of Health Administration and Promotion, Department of Social Medicine and †Division of Reproductive-Perinatal Medicine and Gynecologic Oncology, Department of Surgery, School of Medicine, Tottori University Fac-ulty of Medicine, Yonago 683-8503 JapanThe relationship between cardiovascular disease and Glu298Asp polymorphism of nitric oxide synthase (eNOS) gene in vascular endothelial cells is controversial due to conﬂict-ing results.  To demonstrate the effects of Glu298Asp polymorphism of the eNOS gene on eNOS gene expression by excluding the effects of various confounding factors, eNOS mRNA and protein expressions were measured using cultured human umbilical vein endothelial cells (HUVECs) according to genotypes of Glu298Asp polymorphism of the eNOS gene.  eNOS mRNA expression was measured by real-time PCR/reverse trasn-scription-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 sug-gest that the genotypic difference of Glu298Glu and Glu298Asp has no effect on eNOS mRNA and protein expressions.Key words:  gene expression; nitric oxide synthase; polymorphism; vascular endothelial cellNitric oxide produced from vascular endothelial cells is synthesized from L-arginine by the func-tions of nitric oxide synthase (eNOS).  Major functions of vascular endothelial cell-derived NO include relaxant and antiproliferative effects on vascular smooth-muscle cells, antiaggregant ef-fects on the platelets, and inhibitory effects on ad-hesion of leukocytes to vascular endothelial cells (Kader et al., 2000; Ozaki et al., 2002).  In addi-tion, the relationship between NO production in vascular endothelial cells and various pathologic conditions such as inﬂammatory disease, hyper-tension, and coronary artery disease has been re-ported. The eNOS gene is located on chromosome 7 (7q35-36), spans 21 kb, and consists of 26 exons (Marsden et al., 1993).  Among several reported polymorphisms of the eNOS gene, the involve-ment of Glu298Asp polymorphism (glutamic acid to aspartic acid substitution at codon 298) in coro-nary 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 are considered to be caused by confounding factors such as differences in the genetic backgrounds of study populations and various environmental fac-tors. It is important to experimentally investigate the functional effects of Glu298Asp polymor-Abbreviations: CTPP, confronting 2-pair primers for polymorphism; eNOS, nitric oxide synthase; HUVEC, human um-bilical vein endothelial cellT. Kishimoto et al.60phism on eNOS gene expression by excluding these confounding factors.  Thus, we identified Glu298Asp polymorphism using cultured human vascular endothelial cells, and investigated the functional effects of Glu298Asp polymorphism on eNOS mRNA and protein expressions. Materials and MethodsEthicsThis study was reviewed and approved by the Ethical Committee of the Faculty of Medicine, Tottori University.  Biomaterials (human umbili-cal cords) were collected after obtaining informed consent from the pregnant women.Cell cultureHuman umbilical cords were aseptically obtained immediately after delivery, and human umbilical vein endothelial cells (HUVECs) were collected by enzyme treatment with 0.5 g/L collagenase type IV (Wako Pure Chemical, Osaka, Japan). The collected HUVECs were plated onto gelatin-coated plastic culture dishes, and were statically cultured in a CO2 incubator (5% CO2 in 95% humidiﬁed air).  An Endothelial Cell Growth Me-dium 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 growth factor, 0.2 µg/mL hydrocortisone, 5.0 ng/mL vas-cular endothelial growth factor, 10 ng/mL basic ﬁbroblast 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 polymorphismDNA was extracted from umbilical cord tissue using an automatic nucleic acid extraction instru-ment (MFX-2000, Toyobo, Osaka), and a PCR with confronting 2-pair primers for polymorphism (PCR-CTPP) method (Hamajima et al., 2000) was performed using 4 primers (primer 1:  5'-CAT GAG 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 GGG GA-3', primer 4:  5'-GCT GCA GGC CCC AGA TGA G-3').  In the primary cultures of 29 cases, polymorphisms were identified.  As a result, 26 cases with the Glu298Glu genotype and 3 cases with the Glu298Asp genotype were identiﬁed.Comparative quantiﬁcation of eNOS gene mRNA expressionmRNA was extracted from HUVECs using ISO-GEN (Nippon Gene, Toyama, Japan).  eNOS mRNA expression was measured by a real-time PCR/reverse transcription-PCR method.  A Fast Start DNA Master SYBER Green I Kit (Roche Diagnostics, Tokyo, Japan) was used as a reagent, and the primers used were as follows:  5'-CAT GAG GCT CAG CCC CAG AAC-3' and 5'-AGT CAA 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 inac-tivation of reverse transcriptase at 99˚C for 5 min. A LightCycler (Roche Diagnostics) was used for real-time PCR.  PCR was performed under the following conditions:  after denaturing at 95˚C for 10 min, 50 cycles of denaturing at 95˚C for 15 s, 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 the extension reaction stages of each cycle.  β-actin gene mRNA expression was also measured for comparative quantiﬁcation, and eNOS and β-actin mRNA expressions were quantiﬁed comparatively by calculating the ratio between them.  The prim-ers used for β-actin gene were as follows:  5'-CGT GAC ATT AAG GAG AAG CTG TGC-3' and 5'-GTC ATA CTC CTG CTT GOT GAT CCA-3'.Glu298Asp of eNOS and mRNA/protein expression61Quantiﬁcation of eNOS protein expressionAn immunoassay method by a Human eNOS Immunoassay Kit (R&D Systems, Minneapolis, MN) was used to measure the eNOS protein ex-pression.  In brief, cells were trypsinized for col-lection, and were precipitated by centrifugation at 300 × g for 5 min.  Then cells were washed twice with phosphate-buffered saline, and after the ad-dition of cell lysis buffer, the cell solution was centrifuged at 300 × g for 5 min, and the concen-tration of eNOS protein in the supernatant was measured by a microplate  reader.Experiments according to Glu298Asp geno-typesHUVECs 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, the medium 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, respec-tively.Statistical analysisSPSS software (version 11.0J) was used for statis-tical analysis.  Student’s t-test was used as a sta-tistical method for comparison.  The signiﬁcance level was set to 5%.ResultsThe effects of genotypes on eNOS mRNA and protein expressions are shown in Fig. 1.  eNOS mRNA expression tended to be higher in HU-VECs with the Glu298Asp genotype than in those with Glu298Glu (Fig. 1A), but the differ-ence was not significant.  Also, eNOS protein expression tended to be higher in HUVECs with the Glu298Asp genotype than in those with Glu298Glu (Fig. 1B), but the difference was not signiﬁcant.DiscussionTo reveal the effects of Glu298Asp polymorphism of the eNOS gene on eNOS mRNA and protein expressions, eNOS mRNA and protein expres-sions were measured using HUVECs with the Glu298Glu or Glu298Asp genotype.  Both eNOS mRNA and protein expressions tended to be higher in HUVECs with Glu298Asp than in those with Glu298Glu, but the difference was not sig-niﬁcant. Mainly in case-control studies, the relation-ship between the Glu298Asp polymorphism of the eNOS gene and cardiovascular disease is controversial due to conﬂicting results (Miyamoto Fig. 1.  eNOS mRNA expression (A) and protein concen-trations (B) according to genotypes of eNOS polymor-phism Glu298Asp in exon 7.  Results are expressed as means (SE; bars).  Values for eNOS mRNA expression (A) are given as the ratio of the amounts of eNOS and β-actin mRNA expression measured by real time PCR/reverse transcription-PCR.  Values for eNOS protein (B) are mea-sured by immunoassay method.  Measurement was con-ducted on different dates using different HUVECs derived from 5 and 3 individuals for Glu298Glu and Glu298Asp genotypes, respectively.T. Kishimoto et al.62et 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 be-tween the Glu298Asp polymorphism of the eNOS gene and hypertension in Japanese, but found no relationship (Kishimoto et al., 2004). Also, there are several studies on the effects of the eNOS Glu298Asp genotype on the func-tions of the circulatory system.  Schneider et al. (2000) investigated the dilation reaction of the forearm artery by administering acetylcholine to 80 patients, and reported no difference due to the eNOS Glu298Asp polymorphism.  Jesung et al. measured plasma nitric oxide levels derived from vascular endothelial cells, and reported no difference due to the eNOS Glu298Asp polymor-phism.  Our study supports these results, showing no effects of the Glu298Asp polymorphism of the eNOS gene on the circulatory system. This study was limited by the unavailability of HUVECs with the homozygotic Asp298Asp geno-type.  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 by increasing the sample size in the future. In conclusion, the results of this study sug-gest the possibility that the Glu298Glu and Glu298Asp polymorphisms of the eNOS gene have no effect on eNOS mRNA and protein ex-pressions in HUVECs, and also on the circulatory system.Acknowledgments:  This research was supported in part by a Grant-in-Aid from Japan Society for the Promotion of Science.References 1 Benjafield AV, Morris BJ.  Association analyses of endothelial nitric oxide synthase gene polymor-phisms 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-tion of a common variant of the endothelial nitric oxide synthase gene (Glu298 Asp polymorphism) to the presence, extent, and severity of coronary artery disease.  Heart 2002;87:525–528. 3 Hamajima N, Sa ito T, Mastuo K, Kozak 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 of Glu298Asp (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 endo-thelial cells inhibit platelet aggregation and smooth muscle 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 gene and hypertension.  Hypertension 1999;33:933–936. 7 Kishimoto T, Misawa Y, Kaetu A, Nagai M, Osaki Y, Okamoto M, et al.  eNOS Glu298Asp polymorphism and 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 chromo-some localization of the human constitutive en-dothelial 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 nitric oxide synthase gene is positively associated with es-sential hypertension. Hypertension 1998;32:3–8.10 Ozaki M, Kawashima S, Yamashita T, Hirase T, Namiki M, Inoue N, et al.  Overexpression of endo-thelial nitric oxide synthase accelerates atheroscle-rotic 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 gene testing of the Glu298Asp polymorphism of the en-dothelial NO synthase.  J Hypertens 2000;18:1767–1773.12 Shoji M, Tsutaya Shoji, Saito R, Takamatu H, Yasujima M.  Positive association of endothelial ni-tric oxide synthase gene polymorphism with hyper-tension in northern Japan.  Life Sci 2000;66:2557–2562.Received December 16, 2005; accepted January 17, 2006Corresponding author:  Takuji Kishimoto, MD

Effects of Glu298Asp Polymorphism of Endothelial Nitric Oxide Synthase (eNOS) Gene on eNOS mRNA and Protein Expressions in Cultured Human Vascular Endothelial Cells

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Effects of Glu298Asp Polymorphism of Endothelial Nitric Oxide Synthase (eNOS) Gene on eNOS mRNA and Protein Expressions in Cultured Human Vascular Endothelial Cells

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