PAI-1 Plasma Levels in a General Population Without Clinical Evidence of Atherosclerosis

Relation to Environmental and Genetic Determinants

From Unita' di Trombosi e Aterosclerosi, IRCCS Casa Sollievo della Sofferenza (M.M., G.C., M.d'A., D.C., N.G., G.V., G.M., E.G.), S Giovanni Rotondo; and Istituto di Gerontologia e Geriatria, Università di Palermo (G. Di M.), Italy.

Correspondence to Maurizio Margaglione, MD, Unità di Aterosclerosi e Trombosi, IRCCS Casa Sollievo della Sofferenza, viale Cappuccini, San Giovanni Rotondo (FG) 71013, Italy.

	Abstract

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Abstract—Plasminogen activator inhibitor-1 (PAI-1) plasma levels have been consistently related to a polymorphism (4G/5G) of the PAI-1 gene. The renin-angiotensin pathway plays a role in the regulation of PAI-1 plasma levels. An insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been related to plasma and cellular ACE levels. In 1032 employees (446 men and 586 women; 22 to 66 years old) of a hospital in southern Italy, we investigated the association between PAI-1 4G/5G and the ACE I/D gene variants and plasma PAI-1 antigen levels. None of the individuals enrolled had clinical evidence of atherosclerosis. In univariate analysis, PAI-1 levels were significantly higher in men (P<.001), alcohol drinkers (P<.001), smokers (P=.009), and homozygotes for the PAI-1 gene deletion allele (4G/4G) (P=.012). Multivariate analysis documented the independent effect on PAI-1 plasma levels of body mass index (P<.001), triglycerides (P<.001), sex (P<.001), PAI-1 4G/5G polymorphism (P=.019), smoking habit (P=.041), and ACE I/D genotype (P=.042). Thus, in addition to the markers of insulin resistance and smoking habit, gene variants of PAI-1 and ACE account for a significant portion of the between-individual variability of circulating PAI-1 antigen concentrations in a general population without clinical evidence of atherosclerosis.

Key Words: fibrinolytic activity • plasminogen activator inhibitor 1 • angiotensin-converting enzyme • gene variant

	Introduction

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In Western countries, the ischemic complications of atherosclerosis, acute myocardial infarction and ischemic stroke, are the commonest causes of morbidity and mortality.¹ Overweight, diabetes mellitus, arterial hypertension, and cigarette smoking are commonly associated with a high risk for such complications.² However, the development of myocardial ischemia can only be accounted for in part by such risk factors. In the WHO/MONICA Core Study, as much as 20% of the difference in mortality for coronary artery disease among populations was correlated with the coexistence of hypertension, dyslipidemia, and smoking.³ Moreover, differences in lifestyle, such as diet composition, smoking habits, stress, and obesity, may reflect only partially changes in the incidence rate.

It also clear that an increased risk for arterial thrombosis is associated with high plasma levels of coagulation and fibrinolytic factors.^{4 5 6} Raised plasma levels of the principal inhibitor of fibrinolysis, PAI-1, have been documented in subjects who subsequently develop myocardial infarction.^{7 8} Prospective studies have documented that impaired fibrinolysis is a strong determinant of vascular ischemic events.^{9 10 11 12} Impaired fibrinolysis may also accelerate the atherosclerotic process by allowing fibrin deposition and thrombosis within developing lesions.^{13 14}

In vitro, a variety of factors have been shown to affect PAI-1 synthesis and secretion.^{13 14 15} In vivo, plasma PAI-1 levels have been related to a common, single-base-pair guanine insertion/deletion polymorphism (4G/5G) within the promoter region of the PAI-1 gene,¹⁶ with homozygotes for the deleted allele (4G/4G) carrying the highest plasma levels of this inhibitor.^{17 18}

In vivo¹⁹ and experimental²⁰ studies also suggest a role for angiotensin II in the regulation of plasma PAI-1 levels. The hexapeptide angiotensin IV is the form of angiotensin that stimulates endothelial expression of PAI-1 via a specific endothelial receptor.²¹ In rats, ACE inhibitors lower PAI-1 expression induced by balloon injury.²² ACE is a key enzyme in the renin-angiotensin system. Homozygosity for a deletion polymorphism of the ACE gene (DD genotype) is associated with the highest serum²³ and cellular²⁴ levels of ACE. In a setting from a metabolic ward, we reported a trend for a positive interaction between ACE DD and PAI-1 4G/4G genotypes in the regulation of circulating PAI-1 levels.²⁵ In this study, we evaluated the effect of these molecular variations on PAI-1 plasma levels in a large cohort of southern Italian workers without any ischemic complication of atherosclerosis.

	Methods

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Subjects
After approval by the local Ethics Committee, the study was carried out according to the Principles of the Declaration of Helsinki; informed consent was obtained from all employees of the Casa Sollievo della Sofferenza Hospital, S Giovanni Rotondo, southern Italy. All subjects who reported a history of clinical atherosclerosis were excluded from the study. Thus, from January 1995 to October 1996, 1032 employees (22 to 66 years old) were enrolled. All were white, none were the offspring of consanguineous marriage, and all of their parents and grandparents had been born in the same region. The male-female ratio of the sample was .76 (men=446; 43.2%), (women=586; 56.8%).

Blood was collected by venipuncture between 9 and 11 AM after a 12- to 15-hour fast and abstinence from alcohol. Platelet-free plasma obtained by centrifugation (2000g for 10 minutes at room temperature) was immediately divided into aliquots of 500 µL in plastic tubes (Nunc) and frozen at -70°C until assayed within 12 months of collection. A detailed clinical summary with emphasis on personal and family history for angina pectoris, myocardial infarction, ischemic stroke, and peripheral arterial disease was obtained from all subjects by a specially trained staff employing a previously validated questionnaire²⁶ prepared according to World Health Organization criteria for cardiovascular disease. In addition to questions about symptoms of ischemic heart disease, peripheral vascular disease, and previous vascular surgery, information concerning stroke history and the risk factors diabetes mellitus, arterial hypertension, drug use, alcohol consumption, and smoking habits was obtained. Hypertension was defined as a systolic blood pressure >160 mm Hg and/or a diastolic blood pressure >95 mm Hg taken while the subject was seated on at least three different occasions. Subjects with either a positive history for diabetes mellitus or a fasting blood glucose level >7.8 mmol/L were considered diabetic. Alcohol habit was defined as abstainers, past consumers, and current consumers. Subjects with a smoking habit were divided into smokers, including subjects who had ceased smoking in the last 10 years, and nonsmokers. Demographic characteristics of the study sample analyzed as a whole and stratified according to sex are shown in Table 1.

Materials
dNTP, KCl, MgCl₂, gelatin, and mineral oil were obtained from Perkin Elmer–Cetus; proteinase K was from USB Corp; and HEPES, Tris-HCl, EDTA, ethidium bromide, and SDS were from Sigma Chemical Co. The restriction enzyme BslI was from New England Biolabs Inc. The concentrations of total cholesterol and triglycerides were detected enzymatically with commercially available reagents (Roche). PAI-1 antigen (Imulyze) was assayed by ELISA using kits from Biopool-Menarini. Reference pooled normal plasma from 216 apparently healthy male and female volunteers (29 to 70 years old) who had been instructed to avoid any medication for at least 1 week before blood collection was prepared and stored under the same conditions as the study subjects' samples. Prior to pooling of reference plasma, PAI-1 was measured individually and ranged between 16.7 and 32.1 ng/mL, the geometric mean being 29.3 ng/mL. The intra-assay and interassay coefficients of variation of PAI-1 antigen did not exceed 4.5%.

Detection of Biochemical and Genetic Variables
PAI-1 antigen plasma levels, total cholesterol, and triglycerides were determined as described elsewhere.²⁷ Blood samples were collected and DNA extracted according to standard protocols.²⁶ The PAI-1 4G/5G polymorphism was evaluated as previously reported.²⁸ In brief, a mutated oligonucleotide was synthesized that inserts a site for the BslI enzyme within the amplification product. Polymerase chain reaction (PCR) was carried out in 50-µL samples in a Perkin Elmer–Cetus thermal cycler. Each sample contained 0.5 µg of genomic DNA, 15 pmol of each primer, 100 mmol/L of dNTP, 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl₂, and 1 U thermostable Taq polymerase. The 30 cycles consisted of steps at 95°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes. Then 20-µL volumes of the amplification products were digested for 2.5 hours at 55°C with 5 U of the BslI restriction enzyme. The fragments were fractionated by 4% agarose gel electrophoresis and visualized under UV light. The PCR technique, primers, and experimental conditions employed for the ACE genotyping were the ones suggested by Rigat et al²⁹ with some modifications.³⁰ The amplification products were resolved in 2% agarose gels with a 40 mmol/L Tris-acetate buffer, pH 7.7, containing 1 mmol/L EDTA, stained with 0.5 µg/mL of ethidium bromide, and visualized by UV light.

Statistical Analysis
All analyses were performed according to the Systat 5.2.1 statistical package.³¹ BMI, PAI-1 antigen, cholesterol, and triglyceride levels were logarithmically transformed to allow the use of parametric tests. Differences in baseline characteristics between sexes were evaluated by Student's t test and ² test for continuous and discrete variables, respectively. The allele frequencies were estimated by gene counting and genotypes were scored. The numbers observed for each PAI-1 and ACE genotype were compared with those predicted in a population by Hardy-Weinberg equilibrium using a ² test. Plasma PAI-1 means in different categories were evaluated by Student's ttest. Differences between PAI-1 4G/5G genotypes and different categorical variables were analyzed by the ² test; univariate ANOVA was employed for continuous variables. Differences between different genotypes were evaluated by Scheffe's test. The regression slope, its calculated SE, and CI for the relationships between triglycerides, BMI, and total cholesterol (on the x axis) and PAI-1 antigen (on the y axis) were calculated for each genotype, and the slopes were compared according to Armitage and Berry.³² Multiple linear regression analysis with stepwise selection of the variables, in which "P to enter" and "tolerance" values were set at .05 and .01, respectively, evaluated those factors related to plasma PAI-1 concentrations as well as the possibility of interactions between triglycerides, total cholesterol, BMI, and PAI-1 gene polymorphism. For all data, significance was established at P<.05.

	Results

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Genotype frequencies of the PAI-1 gene 4G/5G polymorphism in the whole sample were .26 (4G/4G n=264; 95% CI=0.22 to 0.29), .48 (4G/5G n=502; 95% CI=0.45 to 0.52), and .26 (5G/5G n=266; 95% CI=0.23 to 0.29). Those of the ACE gene I/D polymorphism were .13 (II, n=132; 95% CI=0.10 to 0.16), .44 (ID, n=451; 95% CI=0.40 to 0.47), and .43 (DD, n=449; 95% CI=0.40 to 0.47). These frequencies and the calculated allele frequencies (PAI-1 4G, .50 [95% CI=0.48 to 0.52]; 5G, .50 [95% CI=0.48 to 0.52]; ACE D, .65 [95% CI=0.64 to 0.66]; and ACE I, .35 [95% CI=0.34 to 0.36]) were similar to those observed in samples from the same region^{25 30} and in other white populations^{16 17 18 33} and did not differ from those predicted by Hardy-Weinberg equilibrium. When stratified according to some variables (Table 2), mean plasma levels of PAI-1 antigen were higher in men, hypertensives, alcohol drinkers, cigarette smokers, and PAI-1 4G/4G homozygotes. Pearson's correlation coefficients showed a close correlation between PAI-1 plasma levels and age (r=.174, P<.001), BMI (r=.401, P<.001), cholesterol (r=.177, P<.001), and triglycerides (r=.344, P<.001). When the whole sample was analyzed according to PAI-1 4G/5G polymorphism, there was no difference with respect to age, hypertension, sex, diabetes mellitus, alcohol consumption, cigarette smoking, total cholesterol, triglycerides, BMI, and ACE I/D polymorphism (P always >.05). In contrast, mean plasma PAI-1 levels varied significantly among different PAI-1 genotypes (ANOVA test: F=5.207, P=.006), and they were higher in 4G/4G subjects (14.09±2.01 ng/mL) than in 4G/5G and 5G/5G individuals (12.56±1.90 ng/mL, P=.070 and 11.78±1.85 ng/mL, P=.007, respectively, by Scheffé's test). A stepwise model was finally used to determine the effects of some personal factors, lifestyle, and lipids on PAI-1 plasma levels (dependent variable). In the model, 4G/4G individuals were compared with non-4G/4G ones (Table 3). Triglycerides, BMI, sex, PAI-1 4G/5G, smoking habit, and ACE I/D gene polymorphisms independently and significantly predicted plasma PAI-1 antigen levels. Adjusted geometric mean PAI-1 antigen levels were 14.62 ng/mL in PAI-1 4G/4G homozygotes and 12.85 ng/mL in subjects carrying the other PAI-1 genotypes. In this analysis, PAI-1 antigen levels were still significantly higher in men than in women (14.86 versus 12.65 ng/mL) and in smokers (14.32 ng/mL) compared with subjects who had never smoked (12.65 ng/mL). Likewise, ACE DD carriers exhibited higher mean levels of PAI-1 antigen than did non-DD individuals (14.19 versus 13.24 ng/mL). This model accounted for 23.4% of the variation in PAI-1 antigen levels between individuals.

The regression slopes of PAI-1 antigen levels with respect to triglycerides among the different PAI-1 4G/5G genotypes were as follows: 4G/4G=0.438, 95% CI=0.302 to 0.573; 4G/5G=0.438, 95% CI=0.336 to 0.539; and 5G/5G=0.363, 95% CI=0.229 to 0.496. No difference was found (F=0.444; P=NS). The same was true when the effect of BMI (4G/4G=1.891, 95% CI=1.415 to 2.026; 4G/5G=2.000, 95% CI=1.637 to 2.363; and 5G/5G=1.354, 95% CI=0.852 to 1.856) or total cholesterol (4G/4G=0.513, 95% CI=0.113 to 0.913; 4G/5G=0.404, 95% CI=0.147 to 0.661; and 5G/5G=0.923, 95% CI=0.534 to 1.322) were analyzed (F=2.179, P=NS; and F=2.433, P=NS, respectively).

To assess the interaction between triglycerides, BMI, total cholesterol, and PAI-1 4G/5G gene polymorphism, regression models with interaction terms were set up. Triglycerides, BMI, sex, PAI-1 (4G/4G versus non-4G/4G), and ACE terms (DD versus non-DD) still independently and significantly predicted PAI-1 antigen levels (P always >.05). Interaction terms of PAI-1 polymorphism (4G/4G versus non-4G/4G, and 5G/5G versus non-5G/5G) with triglycerides, BMI, and total cholesterol did not significantly enter the model (P always >.05).

	Discussion

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The 4G/5G polymorphism is related to differential binding of nuclear proteins that affect the rate of transcription of this fibrinolytic inhibitor.³⁴ In humans, such genotype/phenotype correlations have been documented in patients with diabetes mellitus or in survivors of juvenile myocardial infarction.^{16 17 18 34} In subjects from a metabolic ward, we have confirmed that the 4G/5G polymorphism is a consistent predictor of PAI-1 plasma levels.²⁵ The PAI-1 4G/5G polymorphism has been suggested to exert a role in myocardial ischemia. The frequency of the deletion allele was significantly higher in patients with a previous myocardial infarction than in control subjects.^{34 35 36} However, these findings have been challenged.^{37 38}

In this report, we tested the relationship between PAI-1 gene variants and plasma levels of this inhibitor in a cohort of subjects without clinical evidence of atherosclerosis. PAI-1 4G/4G genotype carriers had the highest plasma values of PAI-1 antigen (Table 2). The univariate analysis also shows that mean plasma levels of PAI-1 antigen differed in subjects with or without arterial hypertension, with age >35 years, with total cholesterol >5.18 mmol/L, with triglycerides >2.05 mmol/L, with a BMI >24 kgm², with alcohol consumption, and with cigarette smoking. In addition, men showed significantly higher mean PAI-1 antigen values than did women. However, BMI, alcohol consumption, cigarette smoking, and blood lipids differed between sexes, men being older. Different lifestyles between sexes and/or sex-related variables may exert a significant effect on the regulation of PAI-1 levels.^{39 40} Accordingly, when information from univariate analysis was tested in a multivariate general linear model, indices of insulin resistance (eg, BMI and triglycerides), were the strongest predictors of PAI-1 antigen levels, accounting for as much as 20% of the between-individual variability. These data are in agreement with those from previous reports.^{17 18 37 41} An additional (1%) contribution to PAI-1 variability is accounted for by gene variants of PAI-1 and ACE gene loci. The extent of the effect of the PAI-1 4G/5G polymorphism on gene expression evaluated in this sample is consistent with that calculated in the ECTIM Study.³⁷ In other studies, the univariate genotype-phenotype association was disputed by multivariate models.¹⁷ However, the present report and the ECTIM Study included the largest numbers of individuals analyzed, with >1000 subjects enrolled in each study.

The present data show that the contribution of the PAI-1 4G/5G polymorphism to gene expression is small compared with major determinants. The ACE I/D polymorphism plays a far less important role in such regulation, reaching significance at rather borderline values in the multivariate analysis. However, it is clear that, in addition to sex and features of insulin resistance, gene variants within PAI-1 and ACE gene loci significantly and independently regulate plasma concentration of PAI-1. To the best of our knowledge, this is the first report documenting such an association. We also found an independent association between ACE I/D polymorphism and PAI-1 plasma levels. Such a relationship is at variance with previous findings.⁴² Differences in the genetic background of the populations analyzed, in the criteria used to select the groups, in the coexistence of major risk factors for ischemia, and in the statistical power of the studies may well account for these discrepancies. The subjects analyzed in this report were all white. The two populations differed in age and the presence of cardiovascular ischemic disease. Moreover, our estimate is based on the analysis of 1032 individuals, whereas in the earlier report, so far as the ACE I/D polymorphism–PAI-1 plasma level relationship is concerned, a subset of only 97 subjects was evaluated.

Significant interactions between triglycerides, BMI, and PAI-1 4G/5G polymorphism and their effects on plasma PAI-1 concentrations have been shown in some studies,^{17 18 36} in which univariate (differences in regression slope among genotypes) and multivariate (interaction terms) analyses were employed. In our sample, regression slopes of triglycerides, BMI, and total cholesterol on PAI-1 antigen according to PAI-1 4G/5G genotype did not differ significantly. A similar lack of interaction was found in the ECTIM Study.³⁷ These findings were further confirmed by a multiple regression analysis with stepwise selection of the variables. In this model, the relationships between BMI, triglycerides, sex, PAI-1 4G/5G, ACE I/D polymorphism, and PAI-1 antigen were not affected by the insertion of interaction terms. This result was not consistent with previous findings¹⁸ and may simply reflect the play of chance. Pearson' s correlation shows a multicollinearity among these variables (P always >.001), although other possibilities should also be taken into consideration. Unknown gene variants and/or loci loosely linked to the PAI-1 4G/5G polymorphism may be involved. In this respect, interaction between triglycerides and 4G/4G genotype was present in some settings^{17 18 36} but not in others^37,43; a relationship between triglycerides and not 4G/4G genotypes was also found⁴¹; and data concerning the interaction between BMI and PAI-1 4G/5G polymorphism are inconclusive as well.^{36 37} In spite of these uncertainties, our data demonstrate that gene variants within PAI-1 and ACE gene loci are independently involved in the regulation of the plasma concentration of PAI-1.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme BMI = body mass index CI = confidence interval PAI-1 = plasminogen activator inhibitor-1

Received July 8, 1997; accepted November 12, 1997.

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