Human Prostacyclin Synthase Gene and Hypertension
The Suita Study
Background—Prostacyclin (prostaglandin I2) is a strong vasodilator that inhibits the growth of vascular smooth muscle cells and is also the most potent endogenous inhibitor of platelet aggregation. Therefore, it has been considered to play an important roles in cardiovascular disease. On the basis of the hypothesis that variations of the prostacyclin synthase gene may also play an important role in human cardiovascular disease, we performed a screening for variations in the human prostacyclin synthase gene.
Methods and Results—We have detected a repeat polymorphism in the promoter region of the human prostacyclin synthase gene. The number of 9-bp (CCGCCAGCC) repeats in the promoter region, which encodes a tandem repeat of Sp1 transcriptional binding sites, varied between 3 and 7 in Japanese subjects. Luciferase reporter analysis indicated that the alleles of 3 and 4 repeats (R3 and R4, respectively) had less promoter activity in cultured human umbilical vein endothelial cells. We then investigated the possible association of this repeat polymorphism with blood pressure in a large population-based sample (the Suita Study), which consisted of 4971 Japanese participants. Multivariate models indicated that participants with the R3R3, R3R4, or R4R4 genotype (SS genotype, n=80) had significantly higher systolic pressure (P=0.0133) and pulse pressure (P=0.0005). The odds ratio of hypertension (140/90 mm Hg) for the SS genotype was 1.942 (95% confidence interval 3.20 to 1.19, P=0.0084).
Conclusions—Repeat polymorphism of the human prostacyclin synthase gene seems to be a risk factor for higher pulse pressure and is consequently a risk factor for systolic hypertension in the Japanese population.
Prostacyclin (prostaglandin I2) is a strong vasodilator that inhibits the growth of vascular smooth muscle cells and is also the most potent endogenous inhibitor of platelet aggregation.1 2 3 Therefore, it has been considered to play an important role in cardiovascular disease.4 On the basis of the hypothesis that variations of the prostacyclin synthase gene, which may modulate prostacyclin synthesis, may also play an important role in human cardiovascular disease, we performed a screening for variations in the promoter region and the entire coding region of the human prostacyclin synthase gene. We also investigated the physiological significance of the identified variation in vitro and then examined the association between the variation and hypertension by using a large population-based sample consisting of 4971 Japanese participants (the Suita Study).
The selection criteria and study design of the Suita Study have been described previously.5 The sample consisted of 14 400 men and women aged 30 to 79 years stratified by sex and age groups of 10 years selected randomly from the municipal population registry. The basic sampling of the population started in 1989 with a cohort study base, and 51.7% (n=7347) of the subjects had paid their initial visit to the National Cardiovascular Center by February 1997. The participants have visited the National Cardiovascular Center every 2 years for regular health checkups. DNA from leukocytes was collected from participants who visited the National Cardiovascular Center between May 1996 and February 1998. All of the participants were Japanese, and only those who gave their informed consent for genetic analysis were enrolled in the present study. Of the 5011 DNA samples collected during this period, 40 were not accessible for genotyping (samples were depleted in previous studies) and therefore excluded.
Genomic DNA from 12 subjects aged <40 years with myocardial infarction and 8 healthy subjects aged 60 to 70 years were used as templates in polymerase chain reaction (PCR). Sense and antisense PCR primers were prepared from the intron or 5′ promoter sequences (Table 1⇓). The PCR product was gel-purified and directly sequenced by use of automated protocols for the fluorescent detection of dideoxy termination products with a model ABI 310 genetic analyzer (PE Applied Biosystems).
The repeat polymorphism in the promoter region was determined by PCR using the sense (5′-CGGGGAACTTTACCTGGGAGTG-3′, from −70 to −49 according to the numbering scheme of Yokoyama et al6 ) and antisense (5′-CTCAGTAGCAGC- AGCAGCAGCAACAGTG-3′, from +59 to +35) primers with an annealing temperature of 68°C. The PCR product (≈120 bp in length, depending on the number of repeats) was resolved on 4% Metaphor agarose gel.
Promoter Activity Analysis
The sequence analysis revealed variations in the number of 9-bp repeats (3 to 7) in the promoter regions. The promoter regions (−604 to +23) of these 5 alleles were amplified by PCR with use of the sense primer (5′-GGTGGGAAAGCTCCTCCTGAC-3′) and the antisense primer (5′-CCGAGGAGCGCGGCCCAAGCCAACGC-3′). The underlined adenine nucleotide (instead of thymidine) was mismatched to abolish the intrinsic methionine initiation codon of the prostacyclin synthase gene. The PCR product was blunt-ended by Klenow and T4 polynucleotide kinase and subcloned into the SmaI site, one of multiple cloning sites, of pGV-B (Toyo Ink Co), a promoterless luciferase reporter vector. The promoter regions of the plasmids of these 5 alleles were confirmed by sequencing. For transfection experiments, plasmids were purified by a Wizard PureFection purification system to remove endotoxin (Promega).
Human umbilical vein endothelial cells were cultured as described previously.7 Transfection was performed with Lipofectamine-plus reagent according to the manufacturer’s recommendations (GIBCO-BRL). As an internal standard, pRL-cytomegalovirus (pRL-CMV, Toyo Ink Co), which expresses sea pansy luciferase under the control of CMV immediate-early enhancer promoter, was cotransfected. At 36 hours after the transfection, the medium was replaced with medium either with or without interleukin-6 (IL-6, 1 ng/mL; Sigma), and cells were harvested 40 hours after transfection. Luciferase and sea pansy luciferase activities were assayed with a Dual Sea Pansy Luminescence Kit (Toyo Ink Co).
All statistical analyses were performed using the JMP statistical software package (SAS Institute Inc, Cary, NC). Linear regression analyses were performed with and without other covariates (sex, age, body mass index [BMI], waist/hip ratio, consumption of alcohol, cigarette smoking, diabetes mellitus, presence of major cardiovascular complications including cerebrovascular accidents and ischemic heart diseases, and use of antihypertensive treatment). Prevalence of hypertension was evaluated by a multivariate logistic regression. Hypertension was defined as systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg or current use of antihypertensive medication. Differences in numerical data among the groups were analyzed by 1- or 2-way ANOVA. Differences in frequency among the groups were analyzed by contingency table analysis.
Screening for Variations in Human Prostacyclin Gene
We found a C to T substitution at nucleotide 591 (according to the numbering scheme in Reference 88 ) in exon 5, a G to A substitution at nucleotide 768 in exon 6, and an A to C substitution at nucleotide 1117 in exon 8. However, because none of these polymorphisms caused an amino acid substitution, they were not studied further in the present study. The sequence immediately upstream from the translational initiation site of the human prostacyclin synthase gene has very GC-rich and pyrimidine-rich regions. Sequence analysis in this region revealed that the number of repeats of the 9-bp sequence (CCGCCAGCC) varied from 3 to 7. The allele with 3 repeats (repeat 3 [R3]) contained 2 Sp1 sites, and that with 7 repeats (R7) contained 5 Sp1 sites (Figure 1⇓). The R6 and R7 alleles have 1 aberrant repeat: CCGTCAGCC instead of CCGCCAGCC (Figure 1⇓).
Functional Significance of Repeat Polymorphism
To investigate the functional significance of the variation in the number of 9-bp repeats, the promoter activity of these 5 alleles was assayed in human umbilical vein endothelial cells with the luciferase reporter vector. Two-way ANOVA (IL-6 and repeat polymorphism) indicated that IL-6 (P<0.0001) and the polymorphism (P<0.0001) significantly affected promoter activity. Moreover, the effects of IL-6 and the polymorphism showed a significant interaction (P<0.001) (Figure 2⇓). The promoter activity of the R3 allele was significantly lower than those of the alleles of R4, R5, R6, and R7 under no IL-6 stimulation (P<0.01, Scheffé F test; Figure 2⇓). Under IL-6 stimulation, the promoter activities of the alleles of R3 and R4 were significantly lower than those of the alleles of R5, R6, and R7 (P<0.01 and P<0.01, respectively, by Scheffé F test; Figure 2⇓).
Association Study Between Repeat Polymorphism and Hypertension in the General Population
Next, we investigated the possible association of the repeat polymorphism of the human prostacyclin synthase gene with blood pressure in a population-based sample (the Suita Study) that consisted of 4971 participants. Table 2⇓ shows blood pressure levels, including systolic, diastolic, and pulse blood pressure, according to the genotypes of the repeat polymorphism. Significant differences in systolic, diastolic, and pulse blood pressure were observed among participants with different genotypes (Table 2⇓). The allele frequencies of R3, R4, R5, R6, and R7 were 1.0%, 10.3%, 3.7%, 83.8%, and 1.3%, respectively. The genotype frequencies were not significantly deviated from the expected values based on the allele frequencies. We separated R3 and R4 alleles (short allele, low responder to IL-6) from R5, R6, and R7 alleles (long allele, high responder to IL-6) into 2 groups based on a luciferase reporter analysis. The R3/L genotype was separated from the other groups on the basis of blood pressure levels.
Systolic and pulse blood pressure were significantly different among participants with different genotypes (SS, R3/L, and R4/L+LL) even after adjusting for other risk factors for hypertension, including sex, age, BMI, waist/hip ratio, alcohol consumption, presence of diabetes mellitus, presence of cardiovascular complications including cerebrovascular accidents and ischemic heart diseases, cigarette smoking, and treatment with antihypertensive drugs (Table 3⇓). Even in a subgroup analysis in participants without any drug treatment that might affect blood pressure (excluding participants with antihypertensive drug treatment and participants with cardiovascular complications including cerebrovascular accidents and/or ischemic heart diseases), systolic and pulse blood pressures were significantly different among participants with different genotypes (Table 3⇓).
The association of the SS genotype (R3/L+R4/L+LL as a reference group) with higher pulse blood pressure was observed in subgroup analyses including female, young (≤64 years), and old (≥65 years) subgroups (Table 3⇑) but not in male participants. In male participants, the repeat polymorphism tended to affect only systolic blood pressure(Table 3⇑). However, in male participants aged ≥65 years (n=1008; S/S=18, R3/L=17, and R4/L+L/L=973), the repeat polymorphism affected adjusted systolic pressure (P1=0.0714, P2=0.0306, and P3=0.322) and adjusted pulse pressure (P1=0.0544, P2=0.0488, and P3=0.7695) (see Table 3⇑ for definitions of P1 to P3).
The participants with the SS genotype were more likely to have antihypertensive treatment (20 [25%] of 80) than those with the other genotypes (707 [14.4%] of 4891, P=0.0081 by Pearson correlation). The prevalence of hypertension according to the repeat polymorphism was evaluated in a logistic regression analysis. The prevalence of hypertension in participants with the SS genotype was 44 (55.0%) of 80, which was significantly higher (P=0.001 by Pearson correlation) than that in participants with the other genotypes (1792 [36.6%] of 4891). After adjusting for other covariates (sex, age, BMI, waist/hip ratio, alcohol consumption, presence of diabetes mellitus, presence of cardiovascular complications including cerebrovascular accidents and ischemic heart diseases, and cigarette smoking), the odds ratio of hypertension for the SS group (R3/L+R4/L+LL as a reference group) was 1.942 (95% confidential interval 3.20 to 1.19, P=0.0084).
Intriguingly, the participants with the SS genotype were more likely to have major cardiovascular complications, including cerebrovascular accidents or ischemic heart diseases (8 [10.0%] of 80) than those with the other genotypes (203 [4.1%] of 4891, P=0.010 by Pearson correlation). The frequency of the SS genotype (R3R3+R3R4+R4R4) in our study population was 1.6% (80 of 4971), whereas that in the hypertensive population was 2.3%. Remarkably, the hypertensive participants with the SS genotype had a higher frequency (18.2%, P=0.0005 by Pearson correlation) of major cardiovascular complications (3 with angina pectoris, 4 with myocardial infarction, and 1 with cerebral infarction; 8 of 44 participants) than the hypertensive participants with the other genotypes (126 [7.03%] of 1792 hypertensive participants).
In the present study, we identified a repeat polymorphism in the promoter region of the human prostacyclin synthase gene that is associated with promoter activity. We then conducted an association study between this polymorphism and blood pressure in a population-based sample (the Suita Study) consisting of 4971 participants. Our results indicate that the SS (R3R3+R3R4+R4R4) genotype, which is associated with lower transcriptional activity, is associated with higher pulse pressure in the overall population, especially among females, and with a higher systolic blood pressure in relatively older participants.
Because this repeat polymorphism affects systolic and pulse blood pressure, but not diastolic blood pressure, it may mainly affect the stiffness of conduit vessels. The stiffness of conduit vessels seems to be determined by various factors, especially the content of the extracellular matrix, including collagen and elastin.8 9 Prostacyclin synthase is abundantly expressed in vascular endothelial and smooth muscle cells,10 11 12 and prostacyclin has been shown to inhibit collagen expression.13 14 Therefore, it is plausible that the accumulation of collagen may be enhanced in subjects with the SS genotype because of the lower induction of prostacyclin synthase by various physiological stimuli, including mechanical and inflammatory stress. In the present study, this repeat polymorphism had a greater effect on blood pressure in females than in males. Although the mechanism of this difference by sex is currently unclear, it may be related to the observation that pulse pressure increases more rapidly with age in women than in men.15
Epidemiological studies have indicated that pulse pressure is an independent risk factor for cardiovascular complications, especially myocardial infarction.16 17 Because prostacyclin is also the most potent endogenous inhibitor of platelet aggregation, individuals with the SS genotype may be susceptible to thrombotic diseases. Indeed, in the present study, hypertensive participants with the SS genotype were more likely to have cardiovascular complications. Although the frequency of the SS genotype is not high in the general population (1.6%), the present study indicates that hypertensive individuals with the SS genotype may be considered to be at high risk, and the identification of this repeat polymorphism may have great clinical implications in the treatment of hypertension.
Decreased prostacyclin biosynthesis has not been reported in human essential hypertension, although it has been reported to precede the clinical manifestation of pregnancy-induced hypertension.18 Because prostacyclin synthase is upregulated by various cytokines, prostacyclin biosynthesis has been reported to be increased in the presence of atherosclerosis and platelet activation.19 Therefore, it is likely that a subset of subjects with a less active promoter of the prostacyclin synthase gene might be difficult to identify under established arteriosclerosis.
Mice lacking the prostacyclin receptor have been reported to be susceptible to thrombosis but not to hypertension.20 However, this observation in the knockout mouse is not necessarily inconsistent with our observation. The effects of the repeat polymorphism were more evident in female participants. Moreover, the repeat polymorphism mainly affected pulse pressure, and its effects on systolic blood pressure were evident only in participants aged ≥65 years.
Recently, genome-wide linkage analyses using highly discordant siblings have revealed several genomic regions that contain genes that affect systolic blood pressure variation.21 One of the suggestive loci in this report, 3 cM proximal to D20S478, is in the vicinity of the prostacyclin synthase gene.
Limitations of the Present Study
The present study is an association study; therefore, we cannot exclude the possibility that the repeat polymorphism is simply a marker in proximity to other unidentified functional variations of the prostacyclin synthase gene or other genes very close to the prostacyclin synthase gene that affect blood pressure.
Moreover, because hypertension is a complex disorder that is affected by various genetic and environmental factors, the degree of the contribution of any gene to the genesis of hypertension may vary according to the study population. Therefore, additional studies with larger populations of patients and with populations of other ethnic backgrounds and/or cardiovascular risk factor profiles will be necessary to determine the significance of this repeat polymorphism in human essential hypertension.
- Received May 11, 1999.
- Revision received July 6, 1999.
- Accepted July 22, 1999.
- Copyright © 1999 by American Heart Association
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