Human Molecular Genetics, 2000, Vol. 9, No. 18 2629-2637
© 2000 Oxford University Press
Replication protein A1 reduces transcription of the endothelial nitric oxide synthase gene containing a –786T
C mutation associated with coronary spastic angina
Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyou-ku, Kyoto 606-8507, Japan, 1Department of Cardiovascular Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan, 2Department of Obstetrics and Gynecology, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan and 3National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-0873, Japan
Received 26 June 2000; Revised and Accepted 1 September 2000.
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ABSTRACT |
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We recently reported that a mutation (–786T
C) in the promoter region of the endothelial nitric oxide synthase (eNOS) gene reduced transcription of the gene and was strongly associated with coronary spastic angina and myocardial infarction. To elucidate the molecular mechanism for the reduced eNOS gene transcription, we have now purified a protein that specifically binds to the mutant allele in nuclear extracts from HeLa cells. The purified protein was identical to replication protein A1 (RPA1), known as a single-stranded DNA binding protein essential for DNA repair, replication and recombination. In human umbilical vein endothelial cells, inhibition of RPA1 expression using antisense oligonucleotide restored transcription driven by the mutated promoter sequence, whereas, conversely, overexpression of RPA1 further reduced it. RPA1 was similarly detected in placenta and eNOS mRNA levels in placentas carrying the –786TC mutation were significantly lower than in placentas without it. The functional importance of the diminished eNOS expression was revealed by the finding that serum nitrite/nitrate levels among individuals carrying the –786TC mutation were significantly lower than among those without the mutation. RPA1 thus apparently functions as a repressor protein in the –786TC mutation-related reduction of eNOS gene transcription associated with the development of coronary artery disease. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Coronary spasm plays a central role in the pathogenesis of coronary artery diseases such as coronary spastic angina (CSA), exertional angina pectoris and acute myocardial infartion (MI) (1–3). In patients with CSA, the basal tone of their coronary arteries is elevated (4–6), and basal, acetylcholine-stimulated and flow-dependent nitric oxide (NO) production are diminished in both the coronary and brachial arteries (5,7,8). The fact that several lines of evidence indicate the prevalence of CSA to be higher among the Japanese than among Caucasians (9,10) suggests that a genetic factor is involved in its pathogenesis. We therefore hypothesized that mutation of the endothelial nitric oxide synthase (eNOS) gene is involved in the pathogenesis of CSA and searched for polymorphisms of the eNOS gene in CSA patients.
We subsequently discovered three single nucleotide polymorphisms (SNPs) (–786TC, –922AG and –1468TA) in the 5'-flanking region of the eNOS gene (11) and a Glu298Asp missense variant in exon 7 (12–14). In addition, we were able to show that the SNPs are not in linkage disequilibrium with the Glu298Asp variant and that they are completely linked with each other and strongly associated with CSA (odds ratio: 5.65) and MI, especially when there is no organic stenosis (odds ratio: 11.19) (11,15). Among the SNPs, the –786TC mutation reduces transcription of the eNOS gene in human umbilical vein endothelial cells (HUVECs) by 
40% (11).
To clarify the molecular mechanism underlying the mutation-related reduction in eNOS gene transcription, nuclear extracts from HeLa cells were used as a starting material to purify a protein that specifically binds to the mutant allele of the eNOS gene. Here we demonstrate that the purified protein is identical to the replication protein A1 (RPA1), which represses transcription of the eNOS gene harboring the mutation. We also confirmed that similar levels of RPA1 are present in placentas, with and without the –786TC mutation, but that eNOS gene transcription was significantly lower in placentas with the mutation than in those without it. Thus, RPA1 appears to play a key role in the pathogenesis of the coronary artery diseases significantly associated with the –786TC eNOS gene mutation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Presence of a protein binding to the eNOSM1 element
We first determined whether any nuclear factors were bound to the mutant sequences. Nuclear extracts from HUVECs were incubated with double-stranded oligonucleotides made up of either wild-type [eNOSW1 (–786T), eNOSW2 (–922A) or eNOSW3 (–1468T)] or mutant [(eNOSM1 (–786C), eNOSM2 (–922G) or eNOSM3 (–1468A)] sequences that subsequently served as probes in a series of gel mobility shift assays (GMSAs). It turned out that incubation with only the eNOSM1 fragment resulted in a retarded complex, the formation of which was specifically antagonized by a molar excess of unlabeled competitor (Fig. 1a, lanes 3 and 4). No other probes, including the eNOSW1 fragment, formed specific complexes (Fig. 1a, lanes 1, 2 and 5–12), which is consistent with our recent finding that, among the three SNPs, only the –786T
C mutation is functional (11). The GMSAs (Fig. 1b) and luciferase reporter gene assays (Fig. 2b) yielded similar results in human aortic artery endothelial cells (HAECs) and human coronary artery endothelial cells (HCAECs), making it likely that a repressor protein commonly exists in human endothelial cells and binds to the eNOSM1 element with higher affinity than to the eNOSW1.
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Additional GMSAs were then performed to confirm the specific binding site of the repressor protein in the eNOSM1 fragment. Six fragments (µ1–µ6) containing mutated nucleotides in various regions of eNOSM1 were synthesized and utilized as probes (Fig. 1c). As shown in Figure 1d, incubation of labeled µ1 with nuclear extract from HUVECs resulted in a retarded complex similar to that seen in the GMSA probed with eNOSM1, whereas incubation of nuclear extract with µ2–µ6 yielded more weakly shifted bands. In particular, the µ4 probe, which did not contain the –786C mutation, yielded the weakest shift, comparable to the band seen in the GMSA probed with eNOSW1. From these results, we concluded that a specific nuclear protein binding site was present within the eNOSM1 sequence.
The eNOSM1 element negatively regulates eNOS and SV40 promoter activities
As we reported previously using the HUVEC culture system (11), not only mutant eNOS promoter (pPGV–eNOSmt) but also an eNOS promoter containing only a –786TC mutation (pPGV–eNOSmt1) reduced transcription activity in comparison with the wild-type eNOS promoter (pPGV–eNOSwt) in HUVECs, HAECs and HCAECs (Fig. 2a and b). To test the hypothesis that the eNOSM1 element negatively regulates promoter activity, we first examined transcription driven by a deleted promoter (pPGV–eNOSdel), in which an 11 bp fragment (–791 to –781) was deleted from the mutant eNOS gene promoter sequence of pPGV–eNOSmt (Fig. 2a). We found that the deletion restored the transcription efficiency of pPGV–eNOSdel nearly to the level of that of pPGV–eNOSwt (Fig. 2c). In a second experiment, ligation of three tandem repeats of the eNOSM1 element to an SV40 promoter, in either the forward or inverted orientation (Fig. 2d), reduced SV40 promoter activity by 50% in HUVECs (Fig. 2e).
Purification of a protein binding to the eNOSM1 sequence
A database search failed to detect any known consensus cis-repressor element within the eNOSM1 sequence. Therefore, to gain additional information about the molecular mechanism responsible for the negative regulation of the eNOS gene by the –786C mutation, we purified the protein(s) bound to the eNOSM1 sequence. Because large quantities of nuclear extract were required for the protein purification, we selected HeLa cells, which are often used for purification of transcription factors, as the source. We first confirmed the presence of the expected binding protein in HeLa cells (Fig. 3a) and that pPGV–eNOSmt1 drove significantly less transcriptional activity than pPGV–eNOSwt (Fig. 3b). On the basis of these findings, we decided to purify the repressor protein from the nuclear extract by monitoring binding activity in GMSAs.
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Ten 30 mg samples of nuclear extract were each fractionated (Fig. 3c), after which the fractions were assayed by GMSA using the eNOSM1 probe. The fraction containing the strongest binding activity (no. 21) was acquired from each sample and pooled, and a 50 kDa peptide was isolated as described in Materials and Methods (Fig. 3d). The isolated peptide was then digested with lysyl endopeptidase and trypsin and sequenced, and the peptides in the resultant digest were found to be identical to the partial amino acid sequences of RPA1 (Table 1). Immunoblot assays also readily detected the presence of RPA1 in HUVECs, HAECs and HCAECs (Fig. 3e).
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To confirm that the expected protein was indeed RPA1, we performed the following experiments. Figure 3f shows that the purified protein was predominantly bound to the eNOSM1 probe, shifting its migration, whereas migration of the eNOSW1 probe was unaffected. Addition of anti-RPA1 antibody then supershifted the eNOSM1/protein complex. The recombinant glutathione S-transferase (GST)–RPA1 fusion protein was also selectively bound to the eNOSM1 probe and formation of the eNOSM1/GST–RPA1 complex was inhibited by anti-GST antibody. It thus appears that the protein bound to the eNOSM1 element was RPA1.
In vitro effects of RPA1 on eNOS promoter activity
We next analyzed the effect of RPA1 on eNOS gene promoter activity. When RPA1 expression was inhibited by an antisense oligonucleotide designed to target the translation initiation site of the primary transcript of RPA1, pPGV–eNOSmt1-driven transcription was restored to a level similar to that seen with pPGV–eNOSwt (Fig. 4a). Conversely, when COS-1 cells were co-transfected with FLAG-ligated RPA1 and pPGV–eNOSmt1, eNOS gene promoter activity was significantly lower than that in cells transfected with pPGV–eNOSmt1 alone (Fig. 4b).
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RPA1 expression and eNOS transcription
To investigate in vivo expression of RPA1 in human tissue, we measured levels of RPA1 protein in placentas genotyped as wild-type homozygous or heterozygous for the –786T
C mutation, where RPA1 was readily detected in placentas with similar levels in those with or without the mutation (Fig. 5a). We then used RNase protection assays to examine eNOS mRNA levels in the placentas and found that levels of eNOS mRNA in placentas carrying the mutation were significantly lower than in those without it (Fig. 5b and c).
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Serum nitrite/nitrate
The functional effect of the –786T
C mutation was investigated by assessing NO synthesis among individuals genotyped as wild-type homozygous or heterozygous for the –786TC mutation. When serum nitrite/nitrate levels in the two groups were compared, we found that serum nitrite/nitrate levels were significantly lower in the mutant group than in the wild-type group (43 ± 16 versus 57 ± 25 mmol/l, P = 0.0159) (Fig. 5d). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We previously reported that the –786T
C mutation in the 5'-flanking region of the eNOS gene is positively associated with CSA and MI and reduces eNOS gene expression assessed by in vitro luciferase reporter analysis (11,12). The present study demonstrates that the eNOSM1 sequence containing the mutation acts as a negative regulatory element on both the eNOS gene and SV40 promoters. To clarify the mechanism by which activity of the eNOS promoter containing the –786TC mutation is repressed, we purified the protein that specifically binds to the mutant allele of the eNOS gene and identified it to be identical to RPA1. We then showed that RPA1 functionally represses transcription of the eNOS carrying the –786TC mutation, that RPA1 protein is present not only in endothelial cells but also in placenta, which is rich in vasculature, and that the level of eNOS mRNA in placentas with the –786TC mutation is significantly lower than in placentas without the mutation. Furthermore, we confirmed that serum nitrite/nitrate, reflecting NO synthesis, was lower in individuals carrying the –786TC mutation than in those without the mutation. RPA1 is the 70 kDa subunit of RPA, a heterotrimeric (70, 32 and 14 kDa), single-stranded DNA binding protein (16) that participates in DNA replication, repair and recombination (17). Although Wold and Kelly (17) noted the presence of a possible fourth RPA subunit of 53 kDa, analysis of partial proteolysis led them to conclude that the 53 kDa polypeptide was a proteolytic fragment of RPA1. We similarly found that nuclear extracts from HeLa cells, HUVECs, HAECs and HCAECs contained both 50 and 70 kDa forms of RPA1 (Fig. 3e) and, given the aforementioned evidence, believe our purified 50 kDa protein is a proteolytically cleaved fragment of RPA1.
There is no report concerning expression of RPA1 in human coronary arteries. In this study, we showed that HCAECs contain RPA1 (Fig. 3e) and that promoter activity of the mutant eNOS gene was decreased in comparison with that of the wild-type eNOS gene in HCAECs (Fig. 2b). We further confirmed expression of RPA1 in human placenta, which is rich in vascular tissues, and raised the possibility that RPA1 functions as a transcriptional repressor on the eNOS gene carrying the mutation in vivo. However, it would be important to show the significance of RPA1 in coronary arteries.
RPA1 homologs have been identified in organisms ranging from single-celled eukaryotes to higher mammals. In yeast, for example, RPA1 is the same as binding URS1 factor (BUF), a repressor protein that binds to the URS1 sequence of the yeast arginase (CAR1) gene, thereby repressing its expression (18,19). In mammals, however, it remains unclear whether RPA1 acts as a repressor protein or not. The human eNOSM1 element (CTGGCCGGCTG) contains a 5 nucleotide core sequence (CGGCT) that is highly homologous to the inverted URS1 sequence (GGCGGCTA), whereas the eNOSW1 element (CTGGCTGGCTG) contains a substitution (CT) in the URS1 core sequence, which would explain why RPA1 has greater affinity for the former.
RPA1 has multiple functional domains, including a DNA polymerase a stimulation domain, a single-stranded DNA binding domain and a conserved zinc-finger domain of the 4-cystein type (20). Further studies will be necessary to determine which of these domains participate in the binding of RPA1 to double-stranded DNA and in its function as a repressor protein.
Several lines of evidence indicate that mice heterozygous for a disrupted eNOS gene exhibit an altered cardiovascular phenotype, for example elevation of systemic and pulmonary blood pressure in the absence of environmental modification (21–23). In the present study, the –786TC mutation was found to induce a modest (40%) but significant decline in eNOS gene promoter activity in cultured human endothelial cells and to be related to reduced placental eNOS mRNA and serum nitrite/nitrate levels. Thus, eNOS gene transcription and synthesis of NO are apparently both diminished in individuals carrying the –786TC mutation in the 5'-flanking region of the eNOS gene. However, measurement of the serum nitrite/nitrate is altered by sampling conditions. We took special care to minimize this. All patients in our study took similar hospital meals and were clinically free from infection and trauma at the time of experiment.
The polymorphism 4b/4a variable number of tandem repeats in intron 4 (4b/4a VNTRs) in the eNOS gene has been reported to be significantly associated with smoking-dependent coronary disease (24). In that regard, we recently showed that the –786TC mutation is in almost complete linkage disequilibrium with 4b/4a VNTRs (25), whereas others have shown that patients with the 4a polymorphism (corresponding to –786C) exhibit lower NO production than those with the 4b polymorphism (corresponding to –786T) (26). Taken together, these results strongly suggest that the –786TC mutation is the functional locus of the 4b/4a VNTRs marker.
In summary, we have demonstrated that RPA1 represses expression of the eNOS gene containing the –786TC mutation, which confers a predisposition towards coronary artery disease to the carriers. Furthermore, the present study provides a new insight into understanding endothelial dysfunction in general with reduced-endothelium-derived NO and pathogenesis of atherosclerosis and various vascular diseases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmid construction
Luciferase reporter genes containing DNA fragments of the 5'-flanking region of the eNOS gene (nucleotide positions –1600 to +26), without and with the aforementioned three point mutations and with only a –786C mutation, were constructed and designated pPGV–eNOSwt, pPGV–eNOSmt and pPGV–eNOSmt1, respectively, as in our previous report (11). In addition, pPGV–eNOSdel was constructed by deleting 11 bp (from –791 to –781) from pPGV–eNOSmt. Two reporter gene constructs, respectively designated pSV40eNOSM1–Luc and pSV40eNOSM1rev–Luc, were created by cloning three tandem repeats of the eNOSM1 element in a forward or inverted orientation into pSV40–Luc, which contains the SV40 minimal promoter vector. The nucleotide sequence of each constructed plasmid was confirmed by sequencing both strands.
Cell culture and luciferase reporter assays
HUVECs, HAECs and HCAECs were purchased from Sanko-Junyaku (Tokyo, Japan) and cultured in medium supplemented with 2% fetal bovine serum. In addition, HeLa and COS-1 cells were cultured in medium supplemented with 10% fetal bovine serum. HUVECs, HAECs and HCAECs were used for up to three passages.
Transient transfection was performed using TransIT-LT2 (Pan Vera, Madison, WI) according to the manufacturer’s instructions. Each promoter/luciferase reporter plasmid was co-transfected with 
-actin-driven ß-galactosidase reporter plasmid. Luciferase activity and ß-galactosidase activity was measured as we previously reported (11). All data were normalized as relative light units/ß-galactosidase activity.
GMSAs
Nuclear extracts from each cell type were prepared according to the protocol of Dignam et al. (27). The final protein concentration was 5 mg/ml. The double-stranded oligonucleotides used were:
eNOSW1 (–796 to –776), 5'-AAGCTCTTCCCTGGCTGGCTGACCCTGCCTC-3';
eNOSM1 (–796 to –776), 5'-AAGCTCTTCCCTGGCCGCTGACCCTGCCTC-3';
eNOSW2 (–932 to –912), 5'-CCAGCCCCTCAGATGACACAGAACTACAAAC-3';
eNOSM2 (–932 to –912), 5'-CCAGCCCCTCAGATGGCACAGAACTACAAAC-3';
eNOSW3 (–1478 to –1458), 5'-GAAGCCAGACTTGGGTTCTGTTGTCTCCTCC-3';
eNOSM3 (–1478 to –1458), 5'-GAAGCCAGACTTGGGATCTGTTGTCTCCTCC-3'.
Five micrograms of nuclear extract were incubated at 20°C for 15 min with 1 mg of poly(dI·dC) (Amersham Pharmacia Biotech, Uppsala, Sweden) plus the end-labeled oligonucleotide (0.1 pmol, 15 000 c.p.m.) in the presence or absence of unlabeled oligonucleotides. The binding reaction was carried out in solution containing 20 mM Tris–HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 10% glycerol and 1 mM dithiothreitol (DTT). The reaction mixtures (final volume: 20 ml) were then directly loaded onto 5% non-denaturing polyacrylamide gels (29:1, acrylamide:bisacrylamide) containing 2.5% glycerol in 1x lowionic strength buffer (7 mM Tris–HCl pH 7.5, 3 mM sodium acetate and 1 mM EDTA) that had been pre-electrophoresed for 20 min. After electrophoresis (120 V for 2.5 h at 4°C), the gels were dried and autoradiographed with an intensifying screen. Additional GMSAs were performed using eNOSW1, eNOSM1 and the six mutated eNOSM1 probes µ1–µ6 (Fig. 1c).
Purification of DNA binding protein
A total of 300 mg of HeLa cell nuclear extract was used as a starting material and divided into 10 aliquots of 30 mg of protein. Each sample was loaded onto a Hitrap Q Column (Amersham Pharmacia Biotech). The column was washed with buffer A (10 mM Tris–HCl pH7.5, 10% glycerol, 50 mM NaCl, 1 mM APMSF and 1 mM DTT) and then eluted using a 0.05–1.0 M NaCl linear gradient in buffer A. The resultant fractions were assayed by GMSA using eNOSM1 and eNOSW1 probes. Active fractions were pooled, diluted and exposed to eNOSM1-bound magnet beads. After washing off unbound proteins with buffer A, bound proteins were eluted with 1.0 M NaCl in buffer A. The eluate was then re-exposed to the magnet beads, washed and eluted. Proteins in the final eluate were separated by SDS–PAGE and then blotted on a polyvinylidene difluoride (PVDF) membrane (ProBlot; ABI, Foster City, CA). The main protein band (50 kDa) was cut from the membrane and digested with lysyl endopeptidase and trypsin. The digests were then separated by reversed-phase high performance liquid chromatography and sequenced using a protein sequencer (494 model; ABI).
Purification of recombinant RPA1
Recombinant RPA1 was purified in a GST-fused form. Full-length human RPA1 cDNA, a gift from T. Kelly (Johns Hopkins University, Baltimore, MD), was ligated to a GST expression vector, pGEX-4T-3 (Amersham Pharmacia Biotech), after which GST–RPA1 was purified according to the manufacturer’s instructions.
Immunoblotting RPA1
Human placentas were collected at vaginal delivery from healthy pregnant women who did not experience pregnancy-induced hypertension. Each placenta was homogenized and lysed in a homogenizing buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 mg/ml leupeptin and 1 mM PMSF). Samples containing equivalent amounts of total protein were loaded into each lane and separated by 10% SDS–PAGE under reducing conditions. The resolved proteins were then transferred to a PVDF membrane and probed with a murine monoclonal anti-human RPA1 antibody (Ab1; Oncogene Research Product, Cambridge, MA). Immunoreactive bands were visualized with horseradish peroxidase-conjugated anti-mouse IgG using an ECL detection kit (Nycomed Amersham, Little Chalfont, UK) and quantified by densitometry.
Preparation of total RNA and RNase protection assays
Total RNA was prepared from human placentas using TRIzol reagent (Life Technologies, Gaithersburg, MD), after which RNase protection assays were used to analyze the expression of human eNOS mRNA. The 500 bp EcoRI–BamHI fragment of human eNOS cDNA and the 138 bp fragment of human ß-actin cDNA were subcloned, linearized and transcribed in vitro using T7 RNA polymerase (Promega, Madison, WI). RNase protection assays were performed with a HybSpeed PRAII kit (Ambion, Austin, TX) according to the manufacturer’s instructions.
Measurement of serum nitrite/nitrate concentration
The patients providing blood samples for analysis of nitrite/nitrate measurement were consecutively hospitalized at the Department of Cardiovascular Medicine, Kumamoto University School of Medicine, from November 1998 to March 1999. Twenty-five subjects heterozygous for the –786TC mutation (13 men and 12 women, mean age 63 years) and 61 age-, sex- and smoking-matched controls lacking the mutation (32 men and 29 women, mean age 63 years) were selected. Patients with active infection or serious inflammatory condition were excluded. All medications taken by study participants were discontinued at least 48 h before blood sampling. Venous blood samples were obtained in the morning while subjects were in a fasting state.
All participants in the study were admitted to the hospital for at least 1 week and ate similar hospital meals, assuring that there was no difference in diet between the two groups. Moreover, all subjects in this study were either non-smokers or refrained from smoking for at least 1 week so that this factor could be discounted. In considering the clinical characteristics of the study subjects, hypertension was operationally defined as blood pressures of >140/95 mmHg, whereas diabetes mellitus was defined as fasting blood glucose levels of >140 mg/dl or >200 mg/dl in an oral glucose tolerance test. There was no difference in these risk factors between the two groups.
Serum nitrite/nitrate concentrations were measured using a flow injection autoanalyzer (TCI-NOX1000; Tokyo Kasei Kogyo, Tokyo, Japan) based on the Griess reaction (28).
Identification of the genotype of the eNOS gene
Genomic DNA was extracted from whole blood or placenta and genotyped for the –786TC mutation as previously reported (11).
Statistical analysis
Promoter activities were assessed as a function of normalized luciferase activity in pairs of experiments and compared with two-tailed unpaired t-tests. Continuous variables, such as serum nitrite/nitrate concentration or eNOS mRNA levels, were also compared using two-tailed unpaired t-tests. Values of P < 0.05 were considered significant.
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ACKNOWLEDGEMENTS |
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We thank S. Narumiya and K. Tashiro for their valuable advice, T. Okumura for secretarial work and R. Ueno for genome purification. We thank T. Kelly of Johns Hopkins University (Baltimore, MD) for providing us with a full-length human RPA1 construct. This work was supported in part by research grants from the Japanese Ministry of Education, Science and Culture, the Japanese Ministry of Health and Welfare, the Japanese Society for the Promotion of Science ‘Research for the Future’ program (JSPS-RFTF96I00204 and JSPS-RFTF98L00801), the Smoking Research Foundation, the Research Foundation for Community Medicine ‘Research Meeting on Hypertension and Arteriosclerosis’ and the Takeda Science Foundation.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 75 751 3180; Fax: +81 75 751 4351; Email: yssaito@kuhp.kyoto-u.ac.jp
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