Original Contributions |
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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Key Words: fibrinolytic activity plasminogen activator inhibitor 1 angiotensin-converting enzyme gene variant
| Introduction |
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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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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
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Materials
dNTP, KCl, MgCl2, gelatin, and mineral oil
were obtained from Perkin ElmerCetus; 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 ElmerCetus 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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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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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 polymorphismPAI-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 settings17 18 36 but not in others37,43; 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 |
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Received July 8, 1997; accepted November 12, 1997.
| References |
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