© 1997 American Heart Association, Inc.
Articles |
Plasminogen Activator Inhibitor-1 (PAI-1) Antigen Plasma Levels in Subjects Attending a Metabolic Ward: Relation to Polymorphisms of PAI-1 and Angiontensin Converting Enzyme (ACE) Genes
From the Unita' di Trombosi e Aterosclerosi, IRCCS Casa Sollievo della Sofferenza, S. Giovanni Rotondo, Servizio di Epidemiologia, Istituto Tumori, Clinica Medica, Dipartimento di Medicina Clinica e Sperimentale, and Cattedra di Gerontologia e Geriatria, Università di Palermo, Italy.
Correspondence to Giovanni Di Minno, MD, Clinica Medica, Dipartimento di Medicina, Clinica e Sperimentale, Via S. Pansini, 5, 80131, Napoli, Italy.
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Abstract |
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Abstract Plasminogen activator inhibitor 1 (PAI-1) is a determinant of vascular events. Subjects in metabolic wards are at high risk for these events. The renin-angiotensin system modulates plasma PAI-1 levels. An insertion (4G)/deletion (5G) polymorphism is involved in the regulation of the circulating levels of PAI-1. We have evaluated the levels of plasma PAI-1 in 208 individuals from our metabolic ward and correlated these levels with the 4G/5G genotype as well as with a genotype (homozygosity for a deletion polymorphism, DD genotype) of the angiotensin-converting enzyme (ACE) gene. Homozygosity for the insertion genotype (5G/5G) was associated with PAI-1 levels lower than those associated with the deletion genotype (4G/4G) (26.2x/:1.6 versus 33.7x/:1.7 ng/mL, P=.036). Plasma PAI-1 levels appeared to depend on the genotype (P=.014) as much as on age (P=.044), t-PA (P=.0001), or triglyceride levels (P=.005). The association between triglycerides and PAI-1 was significant in subjects carrying the 4G/4G and the 4G/5G genotypes (P=.013 and .036, respectively) but not in those with the 5G/5G genotype. When stratified according to PAI-1 and ACE genotypes, individuals homozygous for both deletions (4G/4G-DD genotypes) exhibited higher plasma PAI-1 levels compared with those of individuals without such homozygosities. However, this difference did not reach statistical significance. We conclude that in a group of subjects from a metabolic ward, a 4G/5G polymorphism of the PAI-1 gene exerts effects on plasma PAI-1 antigen levels comparable to those of established determinants. The association between triglycerides and plasma PAI-1 levels is genotype dependent. A trend to a positive interaction between ACE DD and PAI-1 4G/4G in the regulation of circulating plasma PAI-1 levels is present in this setting.
Key Words: cross-sectional study • tissue plasminogen activator • tissue plasminogen activator • inhibitor 1 • genotype-environment interaction
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Introduction |
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Prospective studies have documented that an impaired fibrinolysis is a strong determinant of vascular events.1 2 3 4 An impaired fibrinolysis may accelerate the atherosclerotic process by allowing fibrin deposition and thrombosis within developing lesions5 6 and is commonly related to elevated levels of the principal inhibitor of the fibrinolytic system, plasminogen activator inhibitor-1 (PAI-1).7 A variety of factors have been shown to affect PAI-1 synthesis and secretion in vitro.5 6 7 8 However, modulation of its in vivo plasma concentrations is not fully understood. Recently, circulating plasma PAI-1 levels have been related to a common single base pair guanine insertion/deletion polymorphism (4G/5G).9 Among subjects from our metabolic ward, we have reported raised levels of PAI-1 that significantly correlated with circulating levels of tissue plasminogen activator (t-PA).10 t-PA is released from disturbed endothelial cells.11 12 By means of a newly developed polymerase chain reaction (PCR) protocol,13 we have evaluated the interrelationships between the 4G/5G genotype, t-PA, and PAI-1 antigen levels in this setting.
In vivo14 and experimental15 studies suggest a role for angiotensin II in the regulation of plasma PAI-1 levels. Homozygosity for a deletion polymorphism of the ACE gene (DD genotype) is associated with high serum16 and cellular17 levels of ACE. The angiotensin-converting enzyme (ACE) is involved in the regulation of angiotensin II levels.14 15 16 17 In addition to raised levels of PAI-1, we have reported an elevated frequency of the DD genotype in these subjects.18 We have now evaluated potential interactions between these molecular variations of PAI-1 and ACE genes with respect to PAI-1 plasma levels in this setting.
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Methods |
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Subjects
After approval of the ethical committee, the studies were carried out according to the Principles of the Declaration of Helsinki; informed consent was obtained from all subjects. Demographic and clinical characteristics of the subjects and inclusion/exclusion criteria have been reported in detail elsewhere.19 Because of untypability, two subjects could not be evaluated. Thus the present study was carried out in 208 subjects (107 men, 101 women, mean age 63.6 years, range 31-86). They were chosen among patients in the metabolic ward of the outpatient clinic of our institution. All had been following an isocaloric Mediterranean diet for at least 6 months before being enrolled. The majority of subjects had been referred from the divisions of medicine or neurology. According to institutional practice, patients are referred to the metabolic ward because of a previous ischemic episode and/or because of one or more risk factors. In this respect, 61 had diabetes mellitus (most had type II); 114 had high blood pressure; 50 had LDL cholesterol levels >1.35 g/L; 51 were current smokers; 66 had a positive family history for ischemic events; and 42 had cardiovascular disease. Moreover, from 8 to 12 months before being enrolled in the study, 100 of the subjects had survived an ischemic stroke. This had been documented by nuclear magnetic resonance imaging and/or computerized tomography scan. None of the subjects had clinical evidence of cancer or acute or chronic inflammatory disease. We collected 18 mL of blood from each subject between 9 and 9:30 A.M. after 12 to 15 hours of overnight fasting. This was done without venous stasis, from the antecubital vein via a 19-gauge scalp vein needle into sterile tubes containing 2 mL of sterile 3.8% trisodium citrate and was immediately processed. A clinical summary was obtained from all subjects, with emphasis on personal and family history for vascular events and risk factors and for drugs.
Materials
Deoxynucleotide triphosphatase (dNTP), KCl,
MgCl2, gelatin, and mineral oil were from Perkin
Elmer-Cetus, Milano, Italy; proteinase K was from USB Corp; Lymphoprep
(d=1.077), from Nyegaard, Oslo, Norway; and HEPES, Tris-HCl,
EDTA, ethidium bromide, and SDS, were from Sigma Chemical Co.
Restriction enzyme Bsl I was from New England Biolabs Inc. The
concentrations of total cholesterol,
triglycerides, HDL cholesterol, and glucose
were detected enzymatically (Roche, Milan, Italy).20 The
Friedwald equation was used to calculate LDL
cholesterol.19 20 PAI-1 and t-PA antigens
(Imulyze(TM)) were assayed by ELISA methods using kits from
Biopool-Menarini, Florence, Italy. Reference-pooled normal plasma from
216 apparently healthy volunteers (29-70 years of age) who were
instructed to avoid any medication for at least 1 week was prepared and
stored under the same conditions applied to the study's subject
samples. Prior to pooling, PAI-1 ranged between 16.7 and 32.1
ng/mL, the geometric mean being 29.3 ng/mL. The intra-
and interassay coefficients of variation of PAI-1 and t-PA did not
exceeded 4.5%.
Isolation of DNA and Genotype Analysis
The polymerase chain reaction (PCR) technique used to detect the
I/D polymorphysm of the ACE gene has been previously
described.18 The primers and the experimental conditions
employed for these studies were the ones suggested by Rigat et
al.21 The amplification products were
electrophoretically resolved in a 2% agarose gel by a 40-mmol
Tris-acetate buffer, pH 7.7, containing 1 mmol of EDTA, stained
with 0.5 µg/mL of ethidium bromide and visualized by
ultraviolet light. To achieve a rapid evaluation of the common guanine
insertion/deletion polymorphism of the PAI-1 gene, a newly
developed protocol based on PCR technique and endonuclease digestion
was employed.13 A mutated oligonucleotide
was synthetized that inserted in the PCR product, a restriction
site for the Bsl I enzyme. Such a restriction site enables the
identification of the extra G base. In this respect, a 22-mer
(-697/-676) forward oligonucleotide with a
G>A-681 A substitution (5'-CACAGAGAGAGTCGGCCAGGT-3') and a
21-mer (-598/-619) reverse oligonucleotide
(5'-CCAACAGAGGACTCTTGGTCT-3') were synthetized (Espedite(TM), Millipore
Corp). PCR was carried out on 50-µL volume samples in a Perkin
Elmer-Cetus thermal cycler. Each sample contained 0.5 µg of genomic
DNA, 15 pmol of each primer, 100 µmol of dNTP, 10 mmol of
Tris HCl, pH 8.3, 50 mmol of KCl, 1.5 mmol of
MgCl2, 0.001% (w/v) gelatin, and 1 U of thermostable Taq
polymerase. The solution was overlaid with 50 µL of mineral oil. The
30 cycles consisted of steps at 95°C for 1 minute, at 60°C for 1
minute, and at 72°C for 2 minutes. Then, 20-µL volumes of the
amplification products (99 bp for the 5G and 98 bp for the 4G) were
digested for 150 minutes at 55°C with 5 U of the Bsl I restriction
enzyme and the fragments (one of 98 bp for the 4G allele and two of
77 and 22 bp, respectively for the 5G allele) were fractionated by
4% agarose gel electrophoresis (MethaPhor(TM), FMC
Bioproducts) in a 40-mmol Tris acetate buffer, pH 7.7 that
contained 1 mmol of EDTA and 0.5 µg/mL of ethidium
bromide and visualized under ultraviolet light.
Statistical Analysis
All analyses were performed according to the Statistical
Package for Social Sciences for personal computers, version 6.1,
following the recommended procedures.22 Plasma PAI-1 means
in different categories were evaluated by the Mann-Whitney U test.
Differences between PAI-1 genotypes related to categorical
variables were analyzed by chi-square statistics, those
related to continuous variables, by the univariate
ANOVA. If the test was statistically significant (P<.05),
differences between pairs of genotypes were evaluated by the
Scheffé test. Variables with high skewness were
logarithmically transformed before being evaluated in the ANOVA and in
the regression models. Comparisons of regression slopes were used to
evaluate the effect of the genotypes on the association between
triglycerides and plasma PAI-1. A multiple linear
regression analysis was carried out to evaluate factors that
affected plasma PAI-1 concentrations as well as the possibility of
interactions between the factors. In the model, different PAI-1 and ACE
genotypes were coded by dummy variables. Their values were
as follows: PAI-1 4G/4G was 1 for 4G/4G individuals and 0 for 4G/5G and
5G/5G individuals; PAI-1 5G/5G was 1 for 5G/5G individuals and 0 for
4G/5G and 4G/4G individuals; ACE DD was 1 for DD individuals and 0 for
ID and II individuals; ACE II was 1 for II individuals and 0 for DD and
ID individuals. Thus, as far as PAI-1 genotype was concerned,
0-0 identified 4G/5G individuals, 1-0 identified 4G/4G ones, and 0-1
identified 5G/5G individuals. Likewise, as far as ACE genotype
was concerned, 0-0 identified ID individuals, 1-0 identified DD ones,
and 0-1 identified II individuals. In each case, significance was
established as a probability of <.05.
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Results |
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Determinants of PAI-1 Antigen Plasma Levels
Consistent with data from the literature,6 8 9 10 11 12 plasma PAI-1 antigen levels were related to a series of variables such as LDL cholesterol >1.35 g/L, alcohol consumption, t-PA, triglycerides >1.80 g/L, a history of stroke, and to the PAI-1 4G/5G genotype (Table 1
). No
significant association was found with age >70 years, sex, a positive
family history for ischemic events, smoking habits,
hypertension, or diabetes mellitus.
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4G/5G Genotype and PAI-1 Antigen Plasma Levels
Among the 208 individuals, 62 (29.8%) carried the 4G/4G
genotype, 44 (21.2%) the 5G/5G genotype, and 102
(49%) the 4G/5G genotype. The frequencies observed for the 4G
and 5G allele were 54.3% and 45.7%, respectively, and resembled
those reported in other Caucasian populations.9 These
frequencies were compared with those predicted from the Hardy-Weinberg
equilibrium and no significant differences were found (chi-square
test). No difference in the PAI-1 genotype was found with
respect to age being above or below 60 or 70 years, to diabetes
mellitus, to hypertension, to family history for ischemic
events, to LDL cholesterol plasma levels >1.35 g/L,
to smoking habit, to t-PA, to Lp(a), or to plasma
triglycerides (Table 2). In
contrast, the 5G/5G genotype was associated with significantly
higher levels of HDL cholesterol and significantly lower
numbers of males compared with the 4G/4G genotype. Furthermore,
the circulating levels of PAI-1 antigen were significantly lower in the
5G/5G genotype compared with the 4G/4G ones but not to the
4G/5G genotype.
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A stepwise analysis (Table 3)
revealed the independent nature of the PAI-1 4G/5G polymorphism
with respect to plasma PAI-1 antigen levels. This analysis also
showed that when dummy variables are employed, only the 4G/4G
variable (see Statistical Analysis Section for details) is
significantly associated to PAI-1 plasma concentrations
(P=.015), the 4G/4G individuals carrying significantly
higher PAI-1 plasma levels compared with 4G/5G and 5G/5G individuals.
Finally, a genotype-dependent association between
triglycerides and PAI-1 plasma levels was observed in the
4G/4G individuals (r2 0.32, P=.013)
and in the 4G/5G individuals (r2 0.21,
P=.036) but not in the 5G/5G setting
(r2 0.15, P=.38). Similar results for
the 4G/4G genotype were obtained when the 61 diabetic patients
were excluded from the analysis (not shown).
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Relation between ACE and PAI-1 Genotypes and PAI-1 Antigen
Plasma Levels
The extent to which an insertion/deletion polymorphism of the
ACE gene might affect the association between the 4G/5G
polymorphism and the circulating levels of PAI-1 was also
analyzed. Subjects carrying the ACE DD genotype and
homozygotes for the single base pair deletion of the PAI-1
genotype (4G/4G) had plasma PAI-1 levels of 36.0x/:1.8
ng/mL. This figure was lower in the ACE non-DD (DI+II)
individuals who were nonhomozygotes for the PAI-1 gene deletion
(4G/5G+5G/5G) (29.1x/:1.6 ng/ml). This difference was not
statistically significant (P=.09). Trends across the
genotypes are summarized in Table 4.
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Discussion |
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PAI-1 plasma levels are increased in several prothrombotic states.12 23 24 25 In vivo, raised plasma levels of PAI-1 have been documented in ischemic heart disease26 and in clinical conditions marked by abnormally high circulating levels of insulin or triglycerides.23 24 27 The PAI-1 gene locus has been localized to q21.3-q22 of chromosome 7.28 Recently, plasma PAI-1 levels have been related to a common single base pair guanine insertion/deletion polymorphism 675 base pair upstream from the start of the transcription of the PAI-1 gene locus.9 Expression studies have documented that in response to interleukin-1, the deletion variant (4G) produces six times more mRNA than the insertion allele (5G).9 This is related to a differential binding of nuclear proteins at the site of the 4G/5G polymorphism that affects the rate of transcription of this fibrinolytic inhibitor.29 When evaluated in humans, such genotype/phenotype correlation has been documented in patients with diabetes mellitus or in survivors of juvenile myocardial infarction.29 30 31 In subjects from our metabolic ward, we have reported raised levels of PAI-1 that correlated with the circulating levels of t-PA.10 Diabetes mellitus or a history of cardiovascular disease did not significantly account for PAI-1 levels in this setting. In this high-risk population, we find that the PAI-1 4G/5G polymorphism is a consistent predictor of PAI-1 plasma levels (Tables 2
and 3). In this setting, the 4G/4G individuals carry
significantly higher PAI-1 plasma levels compared with 4G/5G and 5G/5G
individuals, and this association is independent of the effect of known
determinants of PAI-1 plasma levels. It is now clear that the levels of t-PA antigen rise with the increase in PAI-1 inhibition, so that high levels of either factor reflect reduced fibrinolysis. This concept is based on a series of information: (1) concentrations of t-PA antigen above normal ranges are present in subjects with high plasma PAI-1 levels;32 (2) increases in t-PA antigen reflect the inhibitory effect of PAI-1 on t-PA activity;33 (3) there is a negative correlation between t-PA antigen and activity in plasma samples.34 In our setting, t-PA was the strongest determinant of plasma PAI-1 antigen. t-PA is released from perturbed endothelial cells6 and raised levels of it have been reported as a marker of preclinical atherosclerosis in apparently healthy individuals.3 4 Risk factors are known to interact cumulatively to determine vascular injury.19 20 Most of our subjects had more than one risk factor. Clinical conditions such as myocardial infarction or diabetes mellitus are associated with atherosclerotic vascular injury.
A significant univariate relationship between triglycerides and coronary heart disease has been reported.35 In a prospective 12-year study of the incidence of coronary heart disease triglycerides were a major predictor of early onset vascular disease.36 Raised PAI-1 plasma levels are thought to be the link between triglycerides and vascular risk.23 In subsets of non-insulin-dependent diabetic subjects, the relation between plasma PAI-1 and triglycerides was genotype dependent.30 Because of the concomitant vascular disease, most of our diabetic patients were receiving insulin treatment. Insulin,37 like metformin,38 lowers plasma PAI-1 levels. Thus a separate analysis on the influence of the 4G/5G genotype and triglycerides on PAI-1 plasma levels, could not be carried out in the diabetic individuals in our study. However, a 4G-dependent effect of triglycerides on plasma PAI-1 antigen was found in this setting when the sample was analyzed as a whole, as well as when the diabetic subjects were excluded from the analysis.
In its homozygous state, a deletion polymorphism in the ACE gene is associated with high circulating tissue and cellular levels of ACE enzyme.16 17 39 In vivo infusion of angiotensin II results in a substantial increase in the circulating levels of PAI-1.14 Recent data40 have extended this finding to show that hexapeptide angiotensin IV is the form of angiotensin that stimulates endothelial expression of PAI-1 via the stimulation of a specific endothelial receptor. The present data show a trend to an interaction in the regulation of plasma PAI-1 levels between molecular variations of ACE and PAI-1 genes. The frequency of the DD genotype in our population (46.2%) is somehow higher than that reported by others in settings of patients with coronary artery disease or in survivors of myocardial infarction.41 42 43 This may have facilitated the possibility of detecting the trend in spite of our small sample size. However, larger studies are needed to establish a relationship between plasma PAI-1 levels and different PAI and ACE genotypes.
In agreement with the present data, in patients with non-insulin-dependent diabetes mellitus, as well as in survivors of juvenile myocardial infarction, individuals homozygous for the 5G allele exhibit a weak or negative relationship between plasma triglycerides and PAI-1 activity.30 31 An important direction to be explored is the possibility that the 4G/5G genotype of the PAI-1 gene may be useful in identifying those at the highest risk for ischemic events among subjects with insulin resistance.
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Portions of this report have been presented at the American Heart Association Annual Meeting, Anaheim, CA, November 13-16, 1995, and published in abstract form (Circulation. 1995;92:623).
Received June 27, 1996; accepted April 30, 1997.
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