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Clinical Chemistry 49: 847-852, 2003; 10.1373/49.6.847
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(Clinical Chemistry. 2003;49:847-852.)
© 2003 American Association for Clinical Chemistry, Inc.

Genotype-specific Influence on Nitric Oxide Synthase Gene Expression, Protein Concentrations, and Enzyme Activity in Cultured Human Endothelial Cells

Junghan Song1, Yeomin Yoon2, Kyoung Un Park1, Junwan Park1, Young Joon Hong3, Seung Ho Hong4 and Jin Q. Kim1,a

1 Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea.

2 Department of Laboratory Medicine, Cheju National University College of Medicine, Jeju 690-716, Korea.

3 Department of Laboratory Medicine, Korea Cancer Center Hospital, Seoul 139-706, Korea.

4 Department of Science Education, Jeju National University of Education, Jeju 690-016, Korea.

aAddress correspondence to this author at: Department of Laboratory Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. Fax 82-2-745-6653; e-mail jqkim{at}plaza.snu.ac.kr.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The results of studies on the association of ecNOS polymorphisms and vascular diseases are inconsistent. To explore the nature of this interaction in the absence of confounding factors, such as smoking, we measured ecNOS mRNA, protein, and enzyme activity in cultured human umbilical vein endothelial cells (HUVECs) with and without ecNOS polymorphisms.

Methods: We identified a T-786->C polymorphism in the promoter region, the intron 4 variable number of tandem repeats (VNTR), the E298A polymorphism in exon 7, and the G10-T polymorphism in intron 23 of the ecNOS gene in the DNA from 43 human umbilical cords. We measured ecNOS and GAPDH mRNA from the cultured HUVECs by reverse transcription-PCR and ecNOS protein and enzyme activity by Western blotting (as ratio to positive control band) and by determining the conversion of [3H]arginine to [3H]citrulline, respectively.

Results: The T-786->C polymorphism showed the same allelic distribution as the intron 4 VNTR. Mean (SD) ecNOS protein from the cultured HUVECs was significantly lower in the 4a/4b genotype [0.84 (1.23); n = 9] of the intron 4 VNTR than in the 4b/4b genotype [2.14 (2.26); n = 34; P = 0.0300]. The enzyme activity was also significantly lower in the 4a/4b genotype [0.84 (0.21) pmol · min-1 · mg protein-1; n = 9] than in the 4b/4b genotype [1.07 (0.31) pmol · min-1 · mg protein-1; n = 34; P = 0.0197].

Conclusions: ecNOS gene expression, protein concentrations, and enzyme activity are genotype-dependent in HUVECs. The intron 4 VNTR has a consistent influence that may be mediated by the T-786->C polymorphism in the promoter region.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NO is synthesized from L-arginine by a family of enzymes, the NO synthases (NOS), 1 via the L-arginine/NO pathway (1). Synthesis of NO by the vascular endothelium is responsible for vasodilator tone, which is essential for the regulation of blood pressure (2). NO also contributes to the control of platelet aggregation and regulation of cardiac contractility (3). These actions are all mediated by the activation of soluble guanylate cyclase and the consequent increase in the concentration of cyclic GMP in target cells (4). Emerging evidence suggests that coronary artery disease (CAD) is related to defects in the generation or in the action of NO. On being released from cells, NO rapidly autooxidizes to yield NO2-, which interacts with hemoglobin to yield NO3- (5); moreover, these two species [NO2- plus NO3-, termed NOx] are relatively stable in blood, and the concentration of NOx in blood may be an indicator of the endogenous formation of NO (6).

There are at least three isoenzymes of NOS; inducible NOS, constitutive neuronal NOS, and constitutive endothelial NOS (ecNOS) (7), which constitute a "gene family" located on different chromosomes and expressed in different cell lines. The gene encoding ecNOS is located on chromosome 7q35–36 and is composed of 26 exons spanning 21 kb (8). Since the ecNOS gene was sequenced, several sequence variations have been identified. In its promoter region, three linked single-nucleotide polymorphisms (SNPs; -1468T->A, -922A->G, and -786T->C) have been detected (9)(10)(11), and within the gene, SNPs have been identified in intron 2 (IVS2 + 43G->A), intron 11 (IVS11 + 174A->G), intron 12 (IVS12 + 52G->T), intron 18 (IVS18 + 27A->C), intron 22 (IVS22 + 15A->G), and intron 23 (IVS23 + 11G->T) (11). Tandem repeats are located in introns 2 and 8 (32-bp repeats), intron 4 (27-bp repeat), and intron 13 [IVS13 + 81(CA)17–44] (8)(12). Polymorphisms in exons, including E298D in exon 7 and a silent mutation in exon 6, have also been found (11)(13).

Many studies have explored the association between ecNOS polymorphisms and vascular diseases (14). In a previous study, we reported that the G allele frequency of the G10-T polymorphism in intron 23 was significantly higher in CAD patients (15), although other polymorphisms showed no differences in terms of their allele frequencies between CAD patients and healthy controls. We found that in the controls, the E298D polymorphism of the ecNOS gene was associated with increased plasma NO, but controversy remains on the relationship between ecNOS gene polymorphisms and vascular diseases. Inconsistencies between the genotype-phenotype association studies may be attributable to the presence of several confounding factors, such as phenotypic differences between cases, differences in the genetic backgrounds of cases and controls (particularly in multiracial areas), and methodologic difficulties (e.g., artifacts and lack of specificity), or to an insufficient number of cases. To evaluate the causal relationships between the polymorphism and plasma NOx concentrations, more studies involving ecNOS gene expression, protein concentrations, and enzyme activity and their associations with genotypes may be mandatory.

A recent report discussed the relationship between the ecNOS polymorphism and gene expression and/or enzyme activity (16), but because the study was performed on postpartum placental tissues, the associations arrived at might have been modified by a history of maternal smoking and other environmental factors. We therefore analyzed ecNOS polymorphisms, mRNA and protein concentrations, and enzyme activity in cultured human umbilical vein endothelial cells (HUVECs) to elucidate more exactly base interactions in a model free from many of these confounding factors,


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
huvec culture
HUVECs were obtained from freshly discarded umbilical veins by mild collagenase treatment (collagenase type IV; 0.5 g/L). Detached endothelial cells were seeded into human fibronectin-coated plastic T-25 flasks and grown to confluence in complete culture medium [medium 199 containing 0.025 mmol/L HEPES buffer (pH 7.4), 0.002 mmol/L fresh L-glutamine, 100 kilounits/L penicillin, 100 mg/L streptomycin, 100 mL/L heat-inactivated fetal bovine serum, 90 mg/L heparin, and 50 mg/L endothelial cell growth factor]. All experiments were carried out with individual vessel-derived, third-passage, post-confluent (2–3 days after reaching their stable confluence density) cultured HUVECs. ecNOS is relatively unstable; we therefore harvested the cultured cells quickly and froze the harvested cells at -70 °C before homogenization for mRNA measurement and Western blot analysis.

dna isolation and identification of ECNOS polymorphisms
Total genomic DNA was isolated from pieces of fresh human umbilical cords (17). Analyses of the intron 4 variable number of tandem repeats (VNTR) polymorphism, the E298A polymorphism in exon 7, and the G10-T polymorphism in intron 23 of the ecNOS gene were performed as we have previously described (15). The T-786->C polymorphism was detected by PCR amplification as described by Hyndman et al. (18). PCR products were incubated overnight with MspI, after which the restriction products were separated by electrophoresis on 4% NuSieve agarose gels.

rna isolation
Total RNA was extracted from the confluent monolayer of individual genotyped endothelial cells after the third passage. Cultured cells were trypsinized and washed twice in phosphate-buffered saline (PBS; Dulbecco), and total RNA was extracted by a single-step method with a Qiagen RNeasy Total RNA extraction reagent set (Qiagen), according to the manufacturer’s instructions.

measurement of ECNOS MRNA
ecNOS mRNA concentrations were analyzed by reverse transcription-PCR (RT-PCR), as described previously, with minor modifications (19). Briefly, 1 µg of total RNA was reverse-transcribed for 1 h at 37 °C in a 20-µL volume with use of oligo(dT) primer. The resulting cDNA (2 µL) was amplified with ecNOS-specific primers in a 20-µL reaction volume. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutively expressed housekeeping gene, was used as an internal control and was coamplified with GAPDH-specific primers. PCR was performed for 30 cycles (1 cycle of 94 °C for 1 min, 53 °C for 90 s, and 72 °C for 90 s). Final extension was for 8 min at 72 °C. PCR products were analyzed on a 2% agarose-ethidium bromide gel. Gels were photographed, and the intensities of the ecNOS and GAPDH mRNA bands were quantified by densitometric scanning using a Image Analyzer and expressed as the ratio of the ecNOS band intensity to the GAPDH band intensity.

western blot analysis
Confluent cultured cells were trypsinized, washed twice in PBS (Dulbecco), and homogenized in 0.25 mol/L sucrose containing complete-protease inhibitor. We used 30 µg of the protein from this homogenate and 5 µg of ecNOS-positive control to perform sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% gels and transferred the proteins to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech) with use of a Hoefer SemiDry Transfer Unit (Amersham Pharmacia Biotech). After each blot was blocked with 50 g/L nonfat dried milk, it was incubated in primary mouse monoclonal antibody specific for human ecNOS (1:2000 dilution). After washing, the blots were incubated with rabbit anti-mouse IgG1 secondary antibody (1:2000 dilution) and washed. Blots were developed using enhanced chemiluminescence (Amersham Pharmacia Biotech), according to the manufacturer’s instructions, exposed to x-ray film, developed in an automated radiograph developer, and evaluated by densitometry (Molecular Dynamics). The primary monoclonal antibody and positive control ecNOS were purchased from Transduction Laboratories; the antibody recognized 140-kDa human ecNOS, especially C-terminal amino acids 1025–1203. Standardized amounts of protein from the endothelial cell homogenate (30 µg) and ecNOS-positive control (5 µg) were used, and the ratio of the net intensity of the sample homogenate band to the positive control band was used to estimate ecNOS protein concentration.

ECNOS activity
Cultured endothelial cells were washed in ice-cold PBS, scraped, pelleted, and resuspended in homogenization buffer [25 mmol/L Tris-HCl (pH 7.4) containing 1 mmol/L EDTA and 1 mmol/L EGTA]. Cells were disrupted by repeated pipetting and centrifuged at 100 000g for 10 min. The pellets were resuspended in homogenization buffer, and ecNOS activity in the preparation was measured by the conversion of [3H]arginine to [3H]citrulline with use a Nitric Oxide Synthase Assay Kit (Calbiochem), according to the manufacturer’s instructions. The enzyme activity was expressed as picomoles of substrate liberated per mg of cell protein per minute.

statistical analysis
Statistical analyses were performed with the Statistical Analysis System package (SAS Institute Inc.), Ver. 8.0. All data are expressed as the mean (SD) of replicate experiments performed in each assay. Statistical differences were evaluated using the Student t-test. P <0.05 was taken to indicate a statistically significant difference.


   Results
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 Abstract
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 Materials and Methods
 Results
Discussion
 References
 
ECNOS polymorphisms in human umbilical cords
In the case of the intron 4 VNTR, 9 of 43 individuals had the 4a/4b genotype, and the others had the 4b/4b genotype. The T-786->C polymorphism showed the same allelic distribution as for the intron 4 VNTR. Because the concordance was 100%, the C substitution in the T-786->C polymorphism was always linked to the possession of the 4a allele of intron 4. For the E298A polymorphism, we identified 12 individuals with the GT genotype and 31 individuals with the GG genotype. The frequency of the T allele of the G10-T polymorphism in intron 23 is known to be extremely low in Koreans (15), and we did not identify any GT or TT individuals among the 43 participants.

MRNA quantification by rt-pcr
To quantify differences in ecNOS expression according to the genotypes of the intron 4 VNTR and the E298A polymorphism, ecNOS and GAPDH mRNA were reverse-transcribed and amplified in the same tube (multiplex RT-PCR). The ratios of the intensities of the ecNOS and GAPDH bands were determined according to the genotypes of ecNOS polymorphisms and are summarized in Fig. 1A . We observed no significant difference in ecNOS mRNA concentrations between the 4a/4b [2.08 (1.81); n = 9] and the 4b/4b [2.47 (2.33); n = 34] genotypes for the intron 4 VNTR or between the GT [2.29 (2.04); n = 12] and GG [2.58 (2.11); n = 31] genotypes of the E298A polymorphism in exon 7.



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  		Figure 1. ecNOS mRNA (A) and protein concentrations (B) and enzyme activities (C) according to genotypes of the ecNOS polymorphisms in the intron 4 VNTR and the E298A polymorphism in exon 7.

Results are expressed as means (SD; bars). Values for ecNOS mRNA (A) are given as the ratio of the intensity of the ecNOS band to the GAPDH band; values for ecNOS protein (B) are given as the ratio of the of the sample homogenate band to the positive control band. P values for differences between the genotypes of each polymorphism were obtained by the Student t-test.

The semiquantitative RT-PCR method used in this study has some limitations in terms of sensitivity and reproducibility, which might explain the high SD values of each mRNA concentration. To minimize these limitations, other quantitative method may be needed.

protein quantification by western blot
We measured ecNOS protein concentrations, according to the genotypes of the intron 4 VNTR and the E298A polymorphism, by Western blotting; the results are summarized in Fig. 1BUp . The protein concentrations are presented as the ratio of the net intensity of the sample homogenate band to that of the positive control band. The amount of ecNOS protein encoded by the 4b/4b genotype of the intron 4 VNTR [2.14 (2.26); n = 34] was significantly higher than the amount of protein encoded by the 4a/4b genotype [0.84 (1.23); n = 9; P = 0.030, Student t-test]. The amounts of ecNOS protein encoded by the different genotypes for the E298A polymorphism, however, were not significantly different [GT, 1.58 (2.45); n = 12; GG, 1.98 (2.04); n = 31].

ECNOS enzyme activity
The ecNOS enzyme activities in cultured HUVECs, according to the genotypes of the intron 4 VNTR and E298A polymorphism, are summarized in Fig. 1CUp . The 4b/4b genotype of the intron 4 VNTR produced significantly higher ecNOS activity [1.07 (0.31) pmol · min-1 · mg protein-1; n = 34] than the 4a/4b genotype [0.84 (0.21) pmol · min-1 · mg protein-1; n = 9; P = 0.0197, Student t-test]. For E298A polymorphism, however, the ecNOS enzyme activity did not differ significantly between the GT [1.02 (0.29) pmol · min-1 · mg protein-1; n = 12] and GG genotypes [1.02 (0.31) pmol · min-1 · mg protein-1; n = 31].


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CAD is a multifactorial disease that may be dependent on race or ethnic group; for example, the prevalence of CAD differs widely among different populations, and ecNOS gene polymorphisms have been reported to vary among ethnic groups (20). Several polymorphisms of the ecNOS gene associated with CAD have been identified. Wang et al. (21) found an association between homozygosity for the 4a allele of the intron 4 VNTR in the ecNOS gene and an increased risk of CAD only in current and ex-smokers in the Australian population, and our previous results showed that the frequency of the G allele for ecNOS gene polymorphism G10-T in intron 23 was significantly higher in CAD patients (15). However, no significant differences were found between patients and controls in terms of the allelic frequencies of the intron 4 VNTR or E298D polymorphism. We also found that plasma NOx concentrations in CAD patients with hypertension are higher than in CAD patients without hypertension and in controls (15), which might be attributable to a compensatory phenomenon developed in hypertension. A relationship between increased plasma NOx and polymorphisms of the ecNOS gene was observed in some groups with the intron 4 VNTR (in CAD patients with hypertension) and with the E298D mutation (in controls). However, some of our results differ from those of other reports (21)(22)(23), and there have been previous discrepancies among case-control studies on the genotype-phenotype associations. To evaluate the exact causal relationships between polymorphisms and plasma NOx, more studies on gene expression, ecNOS enzyme activity, and the nature of their dependence on genotype is required.

Recently, Wang et al. (16) demonstrated genotype-dependent effects on ecNOS gene expression and enzyme activity at the tissue level. They reported that ecNOS protein was lower in postpartum placental tissues possessing the rare allele but that ecNOS enzyme activity was approximately sevenfold higher in those tissues. In general, ecNOS expression is affected by various stimuli that modify ecNOS regulation at the mRNA level by inducing changes in the transcription kinetics and stability of ecNOS mRNA. Numerous factors affect basal expression of the ecNOS gene. ecNOS expression is regulated by shear stress-responsive elements, lipopolysaccharide, cyclic strain, agents that inhibit protein kinase C, an enhanced proliferative state, hydrogen peroxide, estrogen, vascular endothelial growth factor, insulin, basic fibroblast growth factor, epidermal growth factor, transforming growth factor-ß, oxidized LDL lysophosphatidylcholine, tumor necrosis factor-{alpha}, erythropoietin, and hypoxia (24). Placental tissue directly derived from the human body could be influenced by the many factors that regulate ecNOS mRNA expression. Wang et al. (16) mentioned that cigarette smoking and a complicated pregnancy affect ecNOS mRNA and/or protein concentrations. Rossmanith et al. (19) also reported that ecNOS mRNA expression was affected by gestation, fetal retardation, or maternal diabetes. It may therefore be difficult to verify the precise polymorphism-related influence on ecNOS mRNA expression and enzyme activity when placental tissue is used because of the presence of many confounding factors. Although the cell culture methodology also has an impact on ecNOS function and regulation (25)(26), the use of cultured endothelial cells provides a better platform from which to demonstrate the genotype-dependent effects on ecNOS expression and protein concentration because it excludes the influence of environmental factors that may affect ecNOS expression.

In the present study, we investigated ecNOS gene expression, protein concentrations, and enzyme activity in cultured HUVECs. Both the ecNOS protein concentration and enzyme activity in cultured HUVECs were significantly lower in the rare allele (the 4a allele) of the intron 4 VNTR than the common allele (the 4b allele), and the ecNOS mRNA also tended to be lower in the rare allele, but this was not statistically significant. Wang et al. (16) reported that ecNOS mRNA and protein concentrations were significantly lower in the rare allele of the intron 4 VNTR, which is concordant with our data. In contrast, however, they found that ecNOS enzyme activity was higher in the rare allele, which contradicts our findings. They did not explain the exact mechanisms for the association between the low ecNOS protein concentrations and high enzyme activities, but hypothesized that the changes in ecNOS protein could indirectly influence the bioavailability of cofactors or the effective formation of ecNOS dimers and, thus, ecNOS enzyme activity. The only obvious difference between the two studies is that Wang et al. (16) used postpartum placental tissues and we used cultured HUVECs, and it is possible that placental tissue derived directly from the human body could have been influenced by several environmental factors that regulate ecNOS enzyme activity after translation, such as smoking, gestation, fetal retardation, or maternal diabetes. In our experiment, these confounding factors were minimized by the use of cultured endothelial cells. We therefore suggest that the ecNOS genetic polymorphism has a consistent influence on gene expression, protein concentration, and enzyme activity.

Recently, a SNP affecting transcription of the ecNOS gene was described, namely, the T-786->C polymorphism in the ecNOS promoter (9). In persons with the C allele, promoter activity is less than one-half that in those with the T allele. Asakimori et al. (27) and Ordonez et al. (28) reported that the T-786->C polymorphism showed strong linkage disequilibrium with the intron 4 VNTR. In the present study, the T-786->C polymorphisms in 43 individuals showed the same allelic distribution as that for the intron 4 VNTR, which means that the C substitution in the T-786->C polymorphism is probably always linked to the presence of the rare 4a allele of the intron 4 VNTR. The effect of the intron 4 VNTR on ecNOS mRNA expression, protein concentration, and enzyme activity is therefore mediated by the transcriptional efficiency of the T-786->C polymorphism, which is closely linked with the intron 4 VNTR in the Korean population.

In our previous report, individuals with the E298D mutation in exon 7 had significantly higher plasma NOx concentrations than those without this mutation (15). We therefore hypothesized that the E298D polymorphism might be a regulatory polymorphism of ecNOS gene expression. However, we could not establish a relationship between the E298A polymorphism and ecNOS mRNA or ecNOS protein concentration and enzyme activity. Many reports have indicated that the association of the E298D polymorphism with NOx concentrations and/or vascular disease is controversial (14); thus, more studies are needed on the relationship between the E298A polymorphism and ecNOS mRNA expression.

In conclusion, this study demonstrates the genotype-dependent regulation of ecNOS gene expression and its effect on protein concentrations and enzyme activity in cultured HUVECs. The intron 4 VNTR was found to have a consistent influence on ecNOS mRNA expression, protein concentration, and enzyme activity, which may be mediated by the T-786->C polymorphism in the promoter region of the ecNOS gene.


   Footnotes
 
1 Nonstandard abbreviations: NOS, nitric oxide synthase; CAD, coronary artery disease; NOx, NO2- plus NO3-; ecNOS, constitutive endothelial NOS; SNP, single-nucleotide polymorphism; HUVEC, human umbilical vein endothelial cell; VNTR, variable number of tandem repeats; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-PCR; and GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


   References
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 Abstract
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 Discussion
 References
 

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Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
Right arrow Endocrinology and Metabolism

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