Plasminogen activator inhibitor-1 and apolipoprotein E gene polymorphisms and diabetic angiopathy

Lise Tarnow1,, Coen D. A. Stehouwer2, Jef J. Emeis3, Odette Poirier4, François Cambien4, Birgitte V. Hansen1 and Hans-Henrik Parving1

1 Steno Diabetes Center, Gentofte, Denmark, 2 University Hospital and Institute for Cardiovascular Research Vrije Universiteit, Amsterdam, The Netherlands, 3 Gaubius Laboratory, TNO PG, Leiden, The Netherlands and 4 INSERM U525, Paris, France



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. A point mutation in the plasminogen activator inhibitor-1 (PAI-1) gene and a three-allelic variation in the apolipoprotein-E (ApoE) gene have been suggested as risk factors for the development of diabetic micro- and macrovascular complications.

Methods. We studied 198 type 1 diabetic patients with diabetic nephropathy [121 men, age (mean±SD) 41±10 years, diabetes duration 28±8 years] and 192 patients with persistent normoalbuminuria (118 men, age 43±10 years, diabetes duration 27±9 years).

Results. Male patients with nephropathy had elevated plasma PAI-1 levels [geometric mean (95% CI)], 70 (62–79) ng/ml, compared with normoalbuminuric men, 43 (38–47) ng/ml, P<0.001. Even though nephropathic patients with the 4G4G genotype tended to have higher plasma PAI-1 levels, P=0.06, no difference in allele frequency (4G/5G) was seen between patients with and without nephropathy: 0.538/0.462 vs 0.539/0.461, respectively. Nor did ApoE allele frequencies ({varepsilon}2/{varepsilon}3/{varepsilon}4) differ between nephropathic and normoalbuminuric patients: 0.099/0.749/0.152 vs 0.081/0.745/0.174, respectively. Genotype distributions were also similar, n.s. Coronary heart disease was more prevalent (36%) among nephropathic patients carrying the atherogenic {varepsilon}4-allele compared with 12% in patients with the {varepsilon}3,{varepsilon}3 genotype, P<0.001. No associations between diabetic retinopathy and PAI-1 or ApoE polymorphisms were observed, n.s.

Conclusions. The ApoE polymorphism may accelerate the development of coronary heart disease often seen in Caucasian patients with type 1 diabetes and diabetic nephropathy, a condition characterized by elevated plasma PAI-1 in men. Neither the PAI-1 nor the ApoE gene polymorphism contributes to the genetic susceptibility to diabetic nephropathy or retinopathy.

Keywords: ApoE polymorphism; coronary heart disease; diabetic complications; diabetic nephropathy; PAI-1 polymorphism; type 1 diabetes



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Several studies of familial clustering in diabetic nephropathy have suggested a genetic component in the pathogenesis of this devastating microvascular complication [1–3]. The increased cardiovascular mortality and morbidity observed, not only in diabetic patients with kidney disease [4], but also in their non-diabetic parents [5], have led investigators to assume a possible analogous process leading to atherosclerosis and glomerulosclerosis in diabetic patients, a hypothesis supported by experimental studies [6]. As a consequence, the candidate genes investigated in association studies have predominantly been cardiovascular risk factors, i.e. genes encoding components of the renin–angiotensin system, genes of the fibrinolytic cascade and genes regulating lipid metabolism.

Plasminogen activator inhibitor-1 (PAI-1) plays a critical inhibitory role in the regulation of intravascular fibrinolysis in addition to being involved in tissue repair and remodelling. Increased PAI-1 expression has been associated with matrix accumulation in glomerular disease [7] and with coronary heart disease (CHD) [8]. A deletion–insertion (4G/5G) polymorphism in the promoter region of the PAI-1 gene has been described which relates to plasma PAI-1 levels in some [9–12], but not all [13–15], studies. Reports from studies of the PAI-1 gene and its expression and diabetic micro- and macrovascular complications have been inconsistent [15–22]. Another atherogenic risk factor, the lipid profile, is in part determined by genetic variation in the apolipoproteins (Apo). Three common ApoE alleles, {varepsilon}2, {varepsilon}3 and {varepsilon}4 are inherited codominantly and encode three different isoforms E2, E3 and E4, that differ in their affinity to the low-density lipoprotein (LDL)-receptor. The {varepsilon}4-allele appears to be an important marker for the dyslipidaemia associated with CHD in non-diabetic [23,24] and type 2 diabetic populations [25].

Therefore, the aim of this study was to investigate the relationship between the 4G/5G polymorphism of the PAI-1 gene and the ApoE polymorphism on the one hand, and diabetic nephropathy on the other hand, in a group of type 1 diabetic patients. In addition, we examined the association between these polymorphisms and CHD in nephropathic patients with type 1 diabetes mellitus.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
During 1993, 198 type 1 diabetic patients with diabetic nephropathy, whose glomerular filtration rate had been measured during the same year, were recruited from the outpatient clinic at Steno Diabetes Center for a case–control study [26,27]. Diabetic nephropathy was diagnosed clinically based on the following criteria: persistent albuminuria >300 mg/24 h in at least two of three consecutive 24-h urine collections, presence of retinopathy, and no clinical or laboratory evidence of kidney or renal tract disease other than diabetic glomerulosclerosis [28]. A total of 192 patients with long-standing type 1 diabetes and persistent normoalbuminuria, matched for sex, age and duration of diabetes, served as controls. Five women with nephropathy and six with normoalbuminuria received oestrogen therapy. None of the patients was taking lipid-lowering drugs, whereas 13 and 3 patients had low-dose aspirin (75–150 mg daily) prescribed in the group with and without nephropathy, respectively. The study was approved by the local ethics committee, and all patients gave their informed consent.

All investigations were performed in the morning following an overnight fast—in an attempt to avoid diurnal variation. Venous blood was drawn with minimal stasis from an antecubital vein into EDTA tubes. Centrifugation was performed within 1 h and plasma was stored at -80°C.

Lymphocytes were isolated from peripheral blood and DNA was prepared using standard techniques. A polymerase chain reaction (PCR) was used to detect the two alleles of the 4G/5G polymorphism and the three alleles of the restriction fragment length ApoE polymorphism. DNA was amplified and genotyped as described in the CANVAS Internet Site (http:ifr69.vjf.inserm.fr/~canvas). Genotyping was performed in 197 of 198 patients with nephropathy and 191 (4G/5G) and 192 (ApoE) patients in the normoalbuminuric group. Subjects were classified into one of three PAI-1 genotype groups: 4G4G, 4G5G or 5G5G and one of six ApoE genotypes: {varepsilon}2/{varepsilon}2, {varepsilon}2/{varepsilon}3, {varepsilon}2/{varepsilon}4, {varepsilon}3/{varepsilon}3, {varepsilon}3/{varepsilon}4 or {varepsilon}4/{varepsilon}4. Patients with the {varepsilon}2/{varepsilon}4 genotype (n=10) were excluded from the analyses comparing ApoE allele carriers.

Plasma PAI-1 antigen was determined for all samples in a single assay run, using the Thrombonostika PAI-1 ELISA kit (Organon Teknika, Turnhout, Belgium). In parallel assays (n=11) using pooled normal plasma, the intra-assay coefficient of variation was 6.8%.

Patients were interviewed using the World Health Organization (WHO) cardiovascular questionnaire [29]. A 12-lead electrocardiogram (ECG) was recorded and subsequently coded independently by two trained observers, who were blinded to the clinical status of the patients, using Minnesota Rating Scale [30]. CHD was diagnosed if the ECG showed signs of probable myocardial infarction (Minnesota Rating Scale 1.1–2) or possible myocardial ischaemia (Minnesota Rating Scale 1.3, 4.1–4, 5.1–3 or 7.1), or if patients reported a history of either angina pectoris, defined in accordance to Rose [31], or of myocardial infarction according to WHO criteria [29].

Arterial blood pressure was measured in the supine position after 10 min rest using a Hawksley random zero sphygmomanometer and an appropriate cuff size. Diastolic blood pressure was recorded at the disappearance of Korotkoff sounds (phase V). Retinopathy was assessed by fundus photography after pupillary dilatation and graded: nil, simplex or proliferative diabetic retinopathy. Smokers were defined as persons smoking more than one cigarette/cigar/pipe a day, all others were classified as non-smokers. Urinary albumin concentration was measured by enzyme immunoassay [32] from 24-h urine collections. Haemoglobin (Hb) A1c was measured by HPLC (DIAMAT, Bio-Rad, CA, USA) (normal range 4.1–6.1%). Serum creatinine concentration was assessed using a kinetic Jaffé method. Serum total cholesterol and triglyceride concentrations were determined enzymatically from a venous blood sample, and ApoA-1 and B by endpoint turbidimetry. High-density lipoprotein (HDL)-cholesterol was determined after precipitation of ApoB containing lipoprotein with phosphotungstic acid. LDL-cholesterol was calculated using the Friedewald formula [33].

Statistical analysis
Normally distributed variables are given as means±SD. Urinary albumin excretion rate, serum creatinine, triglyceride and PAI-1 concentrations were log transformed before statistical analysis, because of their positively skewed distribution, and given as medians (range) or geometric means (95% CI). Comparisons between groups were performed using an unpaired Student's t-test or analysis of variance (ANOVA). Frequencies are given as percentage and 95% confidence interval. A chi-square test was used to compare genotype and allele distributions in cases and controls, and was also used for comparison between groups of non-continuous variables. Allele frequencies were estimated by the gene counting method, and Hardy-Weinberg equilibrium was checked by a chi-square test. A P-value (two-tailed) <0.05 was considered statistically significant. All calculations were made using commercially available programs (Statgraphics, STSC, Rockville, MD, USA).



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The group of patients with nephropathy and the normoalbuminuric group were well matched with regard to sex, age and duration of diabetes. Clinical data from these patients are shown in Table 1Go.


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Table 1. Clinical characteristics of 198 type 1 diabetic patients with diabetic nephropathy and 190 type 1 diabetic patients with persistent normoalbuminuria

 
Patients with nephropathy had elevated systolic and diastolic blood pressure and raised serum creatinine and HbA1c, in addition to increased serum cholesterol and triglycerides compared with patients with normoalbuminuria (P<0.001).

Nephropathic patients had a higher prevalence of proliferative retinopathy [137 (69%)] compared with the normoalbuminuric group [18 (10%)] (P<0.001).

Male patients with diabetic nephropathy had elevated plasma PAI-1 levels, 70 (62–79) ng/ml compared with normoalbuminuric men, 43 (38–47) ng/ml, P<0.001. PAI-1 levels were similarly elevated in nephropathic men with duration of persistent proteinuria above and below the median value of 8 years, 63 (52–76) vs 75 (64–87) ng/ml, n.s. Within the nephropathic group, no difference in plasma PAI-1 was seen when data were dichotomized according to median serum creatinine, nor did PAI-1 levels differ between sexes or between patients who were or were not receiving antihypertensive medication or aspirin at the time of examination (n.s.). In women no significant difference in plasma PAI-1 levels was observed between patients with and without nephropathy: 66 (57–76) vs 56 (50–64) ng/ml, respectively.

There was a borderline significant difference in plasma PAI-1 levels between PAI-1 genotypes in the group of patients with nephropathy (P=0.05), in which patients with the 4G4G genotype tended to have a higher concentration of PAI-1 in plasma 79 (65–96) ng/ml, than in patients carrying the 5G allele 65 (58–72) ng/ml, P=0.06. No relationship between PAI-1 genotypes and plasma PAI-1 levels was detected in the normoalbuminuric group. There was no difference in the regression slopes of PAI-1 levels on serum triglycerides between genotype groups (n.s.). Serum cholesterol, LDL-cholesterol and ApoB levels were higher in nephropathic patients with the {varepsilon}4-allele, whereas HDL-cholesterol and triglycerides did not differ significantly between carriers of ApoE {varepsilon}2-alleles, {varepsilon}3-alleles and {varepsilon}4-alleles (Table 2Go).


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Table 2. Lipids and lipoproteins according to the ApoE polymorphism in type 1 diabetic patients with and without diabetic nephropathy

 
Table 3Go shows that no difference in PAI-1 genotype distribution was observed between type 1 diabetic patients with diabetic nephropathy and those with normoalbuminuria. Furthermore, no difference in ApoE genotype distribution, allele frequency or allele carrier status was observed between patients with and without nephropathy (Table 3Go). The distributions of PAI-1 and ApoE genotypes were similar in men and women, n.s.


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Table 3. Distribution of ApoE({varepsilon}2,{varepsilon}3,{varepsilon}4) and PAI-1(4G5G) genotypes and alleles in type 1 diabetic patients with nephropathy and with persistent normoalbuminuria

 
Overall the distribution of PAI-1 genotypes did not differ from Hardy-Weinberg equilibrium (n.s.), whereas in the normoalbuminuric group, the number of patients with the 4G5G genotype was smaller (n=80) than expected (n=95), P=0.03. The distribution of ApoE genotypes overall, as in cases and controls separately, did not differ from Hardy-Weinberg equilibrium (n.s.).

The prevalence of CHD was elevated in patients with nephropathy, 19 (16–22)% vs 8 (4–12)% in normoalbuminuric patients, P<0.001. Among patients with nephropathy, those suffering from CHD were older 45.1±9.7 vs 40.0±9.3 years (P<0.005) and had a longer duration of diabetes 29.3±8.9 vs 26.1±7.6 years (P=0.02). In addition, systolic blood pressure and serum creatinine were elevated: 160±23 vs 150±22 mmHg (P<0.01) and 127 (73–684) vs 96 (54–403) µmol/l (P<0.001), respectively. The majority [95 (88–100)%] of nephropathic patients with CHD received antihypertensive medication, compared with 72 (65–79)% of nephropathic patients without CHD, P<0.005. In the nephropathic group, serum cholesterol (P=0.02) and triglycerides (P=0.01) were raised in patients with CHD: 6.1±1.4 and 1.44 (0.74–5.07) vs 5.5±1.2 and 1.19 (0.31–9.87) mmol/l in patients without CHD. Sex distribution was similar and BMI, HbA1c, urinary albumin excretion rate, diastolic blood pressure and serum HDL-cholesterol did not differ between nephropathic patients with and without CHD.

No relationship between plasma PAI-1 and the presence/absence of CHD was seen in the group of patients with nephropathy (data not shown). Furthermore, the distribution of PAI-1 genotypes was similar in nephropathic patients with vs without CHD: 7, 24 and 7 vs 47, 80 and 32 patients had 4G4G, 4G5G and 5G5G genotypes respectively, n.s. In contrast, a significant association existed between the ApoE polymorphism and CHD (chi-square=17.96, 2 df: P<0.001) the frequency of the {varepsilon}4-allele being higher among type 1 diabetic patients with diabetic nephropathy and CHD, 0.303 vs 0.116 in nephropathic patients without CHD, P<0.001. Accordingly, 36% (17/47) of nephropathic patients carrying the {varepsilon}4-allele had CHD, whereas only 12% (13/112) of patients with the wild-type {varepsilon}3/{varepsilon}3 genotype showed signs of CHD, P<0.001.

Finally, no differences in PAI-1 levels or PAI-1 and ApoE genotype distributions were seen between patients with proliferative retinopathy, simplex or no diabetic retinopathy in either the nephropathic or the normoalbuminuric group (data not shown).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Our case–control study revealed no associations between the PAI-1 (4G/5G) or the ApoE polymorphism and diabetic nephropathy or retinopathy in Caucasian type 1 diabetic patients. The atherogenic {varepsilon}4-allele was seen more frequently in patients with CHD and diabetic nephropathy, a condition characterized by elevated plasma PAI-1 in men.

PAI-1 is a potent inhibitor of fibrinolysis. PAI-1 levels are increased in the type 2 diabetic state per se [34,35]. In uncomplicated type 1 diabetes plasma PAI-1 did not differ from non-diabetic controls [16,34], whereas two studies [16,17] including fewer than 25 micro- and/or macroalbuminuric patients with and without retinopathy found increased values in type 1 diabetic patients with an increased urinary albumin excretion rate. The present study of 198 patients with persistent macroalbuminuria and diabetic retinopathy confirms and extends the finding of elevated PAI-1 levels in type 1 diabetic men early in the course of diabetic nephropathy.

Elevated PAI-1 levels have been associated with myocardial infarction in non-diabetic [36] and type 2 diabetic [20,21] subjects. The relatively small number of nephropathic cases with CHD, diagnosed using questionnaires and Minnesota-coded ECGs, might help to explain the lack of association between this macrovascular complication and plasma PAI-1 concentration seen in the present study. Suppression of plasma PAI-1 due to ACE inhibition has been suggested in clinical studies [37,38], and because most of the nephropathic patients with CHD were treated with ACE inhibitors, such an effect would confound the association between PAI-1 and CHD.

Some [9,11], but not all [13], studies of non-diabetic populations have found an association between the 4G4G genotype of a polymorphism in the promoter region of the PAI-1 gene and elevated plasma PAI-1 activity. In two studies of type 2 diabetic Pima Indians no relationship between promoter genotype and plasma PAI-1 activity was found [14,15]. In the present study of type 1 diabetic patients with diabetic nephropathy, a weak association existed between plasma PAI-1 concentrations and promoter genotype, with the highest values being found in patients with the 4G4G genotype. In the normoalbuminuric group, with lower PAI-1 concentrations, no differences between genotypes were detected. Assuming a gene–environment interaction between PAI-1 genotype and plasma glucose, as suggested by Mansfield et al. [39], the poorer metabolic control in patients with nephropathy in the present study might help to explain the observed differences between groups.

The lack of an association between the PAI-1 (4G/5G) polymorphism and diabetic nephropathy in our Caucasian type 1 diabetic patients is in accordance with data from a cross-sectional study of type 2 diabetic Pima Indians [15]. Furthermore, our cohorts were sufficiently large to yield 98% power to detect a 20% deviation in 4G4G genotype frequency, with P<0.05. Our negative finding is thus not explained by insufficient statistical power.

Plasma PAI-1 levels were not elevated in patients with retinopathy in either the present or a previous study [15]. Whereas simplex retinopathy was more predominant in carriers of the 4G allele in a study of Pima Indians [15], no association between PAI-1 gene polymorphisms and retinopathy was found in Caucasian patients (this study; Ref. 19). Discrepancies between studies can be ascribed to different phenotypes, i.e. type of diabetes, severity of retinopathy and/or ethnical differences, and finally the possibility of chance findings—positive or negative. Future investigation of this topic in studies designed to investigate the genetics of diabetic retinopathy will be required.

With respect to macrovascular disease, the prevalence of the 4G allele was significantly higher in a group of 100 non-diabetic patients with myocardial infarction than in age-matched control subjects [10]. However, this relationship could not be confirmed in a subsequent larger study (470 cases) of a non-diabetic population [11]. No association between CHD and 4G/5G polymorphism was detected in nephropathic type 1 diabetic patients in the present study, but the number of patients studied was small (n=38). In a previous study [22] of a similar number of type 2 diabetic patients with a clinical history of CHD, an increased frequency of the 4G4G genotype was found among cases, 53% vs 30% in controls, P<0.05.

Analogous pathophysiological mechanisms have been suggested in glomerulosclerosis and arteriosclerosis, both conditions associated with dyslipidaemia [6]. Lipid levels are, in part, determined by genetic variation in apolipoproteins. The {varepsilon}4-allele of a common polymorphism in the ApoE gene appears to be an important marker for the dyslipidaemia associated with CHD in non-diabetic [23,24] and type 2 diabetic populations [25]. As a natural consequence of the increased receptor binding and increased metabolism of triglyceride-rich lipoproteins associated with the {varepsilon}4-allele, we found increased LDL-cholesterol and ApoB levels in nephropathic patients carrying the atherogenic {varepsilon}4-allele in the present study. A previous study [40] of type 1 diabetic patients found no associations between lipid levels and the ApoE genotypes, but data on LDL-cholesterol and apolipoproteins were not presented. As in our study, no difference in genotype distribution between patients with normo- and macroalbuminuria was found in that study [40]. These results are in contrast to a recent study from the UK, in which the authors reported an excess of the {varepsilon}2-allele in type 1 diabetic patients with nephropathy compared with a group of type 1 diabetic patients with normo-or microalbuminuria [41]. By including patients with microalbuminuria among the controls, this design harbours a substantial risk for misclassification of patients, who will subsequently progress to overt diabetic nephropathy, despite long-standing diabetes [42].

In accordance with a previous study in type 2 diabetes [25], our study of type 1 diabetic patients demonstrated an increased prevalence of CHD in nephropathic patients carrying the {varepsilon}4-allele.

Population stratification, selection, methodological problems and chance might result in a deviation from the Hardy-Weinberg equilibrium. In this study, patients for the cardiovascular low-risk normoalbuminuric control group were recruited from the same homogenous Danish diabetic population as cases. Furthermore, applied methods to determine genotypes are validated, and only one other gene polymorphism investigated [26] has been in Hardy-Weinberg disequilibrium in this normoalbuminuric group. Therefore, we consider the observed deviation in PAI-1 genotype in controls to be a chance finding.

In conclusion, the ApoE polymorphism may accelerate the development of CHD often seen in Caucasian patients with type 1 diabetes and diabetic nephropathy, a condition characterized by elevated plasma PAI-1. Neither the PAI-1 nor the ApoE gene polymorphism contributes to the genetic susceptibility to diabetic nephropathy or retinopathy.



   Acknowledgments
 
We acknowledge the excellent technical assistance of Ms C. M. van den Hoogen and the assistance of Ms B. R. Jensen and Ms U. M. Smidt in conducting this study, and Ms C. Souriam for DNA extraction. J. J. Emeis is a recipient of grant 28-2623 from the Praeventiefonds.



   Notes
 
Correspondence and offprint requests to: Lise Tarnow, MD, Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Seaquist ER, Goetz FC, Rich S, Barbosa J. Familial clustering of diabetic kidney disease: evidence of genetic susceptibility to diabetic nephropathy. N Engl J Med1989; 320: 1161–1165[Abstract]
  2. Borch-Johnsen K, Nørgaard K, Hommel E, et al. Is diabetic nephropathy an inherited complication? Kidney Int1992; 41: 719–722[ISI][Medline]
  3. Quinn M, Angelico MC, Warram JH, Krolewski AS. Familial factors determine the development of diabetic nephropathy in patients with IDDM. Diabetologia1996; 39: 940–945[ISI][Medline]
  4. Borch-Johnsen K, Kreiner S. Proteinuria: value as predictor of cardiovascular mortality in insulin dependent diabetes mellitus. Br Med J1987; 294: 1651–1654[ISI][Medline]
  5. Earle K, Walker J, Hill C, Viberti GC. Familial clustering of cardiovascular disease in patients with insulin-dependent diabetes and nephropathy. N Engl J Med1992; 326: 673–677[Abstract]
  6. Keane W, Kasiske BL, O'Donnell MP. Lipids and progressive glomerulosclerosis. Am J Nephrol1988; 8: 261–271[ISI][Medline]
  7. Nakamura T, Tanaka N, Higuma N, Kazama T, Kobayashi I, Yokota S. The localization of plasminogen activator inhibitor-1 in glomerular subepithelial deposits in membranous nephropathy. J Am Soc Nephrol1996; 7: 2434–2444[Abstract]
  8. Hamsten A, Eriksson P. Fibrinolysis and atherosclerosis. Bailliére's Clin Haematol1995; 8: 345–363[Medline]
  9. Dawson S, Hamsten A, Wiman B, Henney A, Humphries S. Genetic variation at the plasminogen activator inhibitor-1 locus is associated with altered levels of plasma plasminogen activator inhibitor-1 activity. Arterioscler Thromb1991; 11: 183–190[Abstract]
  10. Eriksson P, Kallin B, van't Hooft FM, Båvenholm P, Hamsten A. Allele-specific increase in basal transcription of the plasminogen-activator inhibitor 1 gene is associated with myocardial infarction. Proc Natl Acad Sci1995; 92: 1851–1855[Abstract]
  11. Ye S, Green FR, Scarabin PY, et al. The 4G/5G genetic polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene is associated with differences in plasma PAI-1 activity but not with risk of myocardial infarction in the ECTIM study. Thromb Haemostas1995; 74: 837–841[ISI][Medline]
  12. Panahloo A, Mohamed-Ali V, Lane A, Green F, Humphries SE, Yudkin JS. Determinants of plasminogen activator inhibitor 1 activity in treated NIDDM and its relation to a polymorphism in the plasminogen activator inhibitor 1 gene. Diabetes1995; 44: 37–42[Abstract]
  13. Henry M, Chomiki N, Scarabin PY, et al. Five frequent polymorphisms of the PAI-1 gene. Lack of association between genotypes, PAI activity, and triglyceride levels in a healthy population. Arterioscler Thromb Vasc Biol1997; 17: 851–858[Abstract/Free Full Text]
  14. McCormack LJ, Nagi DK, Stickland MH, et al. Promoter (4G/5G) plasminogen activator inhibitor-1 genotype in Pima Indians: relationship to plasminogen activator inhibitor-1 levels and features of the insulin resistance syndrome. Diabetologia1996; 39: 1512–1518[ISI][Medline]
  15. Nagi DK, McCormack LJ, Mohamed-Ali V, Yudkin JS, Knowler WC, Grant PJ. Diabetic retinopathy, promoter (4G/5G) polymorphism of PAI-1 gene, and PAI-1 activity in Pima Indians with type 2 diabetes). Diabetes Care1997; 20: 1304–1309[Abstract]
  16. Jensen T, Feldt-Rasmussen B, Bjerre-Knudsen J, Deckert T. Features of endothelial dysfunction in early diabetic nephropathy. Lancet1989; i: 461–463
  17. Gruden G, Cavallo-Perin P, Bazzan M, Stalla S, Vuolo A, Pagano G. PAI-1 and factor VII activity are higher in IDDM patients with microalbuminuria. Diabetes1994; 43: 426–429[Abstract]
  18. Kario K, Matsuo T, Kobyashi H, Matsuo M, Sakata T, Miyata T. Activation of tissue factor-induced coagulation and endothelial cell dysfunction in non-insulin-dependent diabetic patients with microalbuminuria. Arterioscler Thromb Vasc Biol1995; 15: 1114–1120[Abstract/Free Full Text]
  19. Mansfield MW, Stickland MH, Carter AM, Grant PJ. Polymorphisms of the plasminogen activator inhibitor-1 gene in type 1 and type 2 diabetes, and in patients with diabetic retinopathy. Thromb Haemostas1994; 71: 731–736[ISI][Medline]
  20. Gray RP, Yudkin JS, Patterson DL. Plasminogen activator inhibitor: a risk factor for myocardial infarction in diabetic patients. Br Heart J1993; 69: 228–232[Abstract]
  21. Gray RP, Patterson LH, Yudkin JS. Plasminogen activator inhibitor activity in diabetic and nondiabetic survivors of myocardial infarction. Arterioscler Thromb1993; 13: 415–420[Abstract]
  22. Mansfield MW, Stickland MH, Grant PJ. Plasminogen activator inhibitor-1 (PAI-1) promoter polymorphism and coronary artery disease in non-insulin-dependent diabetes. Thromb Haemostas1995; 74: 1032–1034[ISI][Medline]
  23. Luc G, Bard JM, Arveiler D, et al. Impact of apolipoprotein E polymorphism on lipoproteins and risk of myocardial infarction. The ECTIM study. Arterioscler Thromb1994; 14: 1412–1419[Abstract]
  24. Wilson PWF, Myers RH, Larson MG, Ordovas JM, Wolf PA, Schaefer EJ. Apolipoprotein E alleles, dyslipidemia, and coronary heart disease. The Framingham Offspring Study. JAMA1994; 272: 1666–1671[Abstract]
  25. Laakso M, Kesäniemi A, Kervinen K, Jauhiainen M, Pyörälä K. Relation of coronary heart disease and apolipoprotein E phenotype in patients with non-insulin dependent diabetes. Br Med J1991; 303: 1159–1162[ISI][Medline]
  26. Tarnow L, Cambien F, Rossing P, et al. Lack of relationship between an insertion/deletion polymorphism in the angiotensin-1-converting enzyme gene and diabetic nephropathy and proliferative retinopathy in IDDM patients. Diabetes1995; 44: 489–494[Abstract]
  27. Tarnow L, Cambien F, Rossing P, et al. Insertion/deletion polymorphism in the angiotensin-1-converting enzyme gene is associated with coronary heart disease in IDDM patients with diabetic nephropathy. Diabetologia1995; 38: 798–803[ISI][Medline]
  28. Parving H-H, Andersen AR, Smidt UM, Svendsen PA. Early aggressive antihypertensive treatment reduces rate of decline in kidney function in diabetic nephropathy. Lancet1983; i: 1175–1179
  29. Rose GA, Blackburn H. Cardiovascular survey methods. WHO, Geneva, 1968; 1–188
  30. Blackburn H, Keys A, Simonson E, Rautaharju P, Punsar S. The electrocardiogram in population studies: a classification system. Circulation1960; 21: 1160–1175[ISI]
  31. Rose GA. The diagnosis of ischaemic heart pain and intermittent claudication in field surveys. Bull WHO1962; 27: 645–658[ISI][Medline]
  32. Feldt-Rasmussen B, Dinesen B, Deckert M. Enzyme immunoassay: an improved determination of urinary albumin in diabetics with incipient nephropathy. Scand J Clin Lab Invest1985; 45: 539–544[ISI][Medline]
  33. Friedewald WT, Levy RI, Frederickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifugation. Clin Chem1972; 18: 499–502[Abstract/Free Full Text]
  34. Auwerx J, Bouillon R, Collen D, Geboers J. Tissue-type plasminogen activator antigen and plasminogen activator inhibitor in diabetes mellitus. Arteriosclerosis1998; 8: 68–72[Abstract]
  35. Juhan-Vague I, Roul C, Alessi MC, Ardissone JP, Heim M, Vague P. Increased plasminogen activator inhibitor activity in non insulin dependent diabetic patients—relationship with plasma insulin. Thromb Haemostas1989; 61: 370–373[ISI][Medline]
  36. Hamsten A, de Faire U, Walldius G, et al. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet1987; 2: 3–9[ISI][Medline]
  37. Vaughan DE, Rouleau JL, Ridker PM, Arnold JM, Menapace FJ, Pfeffer MA. Effects of ramipril on plasma fibrinolytic balance in patients with acute anterior myocardial infarction. HEART Study Investigators. Circulation1997; 96: 442–447[Abstract/Free Full Text]
  38. Wright RA, Flapan AD, Alberti KG, Ludlam CA, Fox KA. Effects of captopril therapy on endogenous fibrinolysis in men with recent, uncomplicated myocardial infarction. J Am Coll Cardiol1994; 24: 67–73[ISI][Medline]
  39. Mansfield MW, Stickland MH, Grant PJ. Environmental and genetic factors in relation to elevated circulating levels of plasminogen activator inhibitor-1 in Caucasian patients with non-insulin-dependent diabetes mellitus. Thromb Haemostas1995; 74: 842–847[ISI][Medline]
  40. Onuma T, Laffel L, Angelico MC, Krolewski AS. Apolipoprotein E genotypes and risk of diabetic nephropathy. J Am Soc Nephrol1996; 7: 1075–1078[Abstract]
  41. Chowdhury TA, Dyer PH, Kumar S, et al. Association of apolipoprotein E2 allele with diabetic nephropathy in Caucasian subjects with IDDM. Diabetes1998; 47: 278–280[ISI][Medline]
  42. Forsblom CM, Groop P-H, Ekstrand A, Groop L. Predictive value of microalbuminuria in patients with insulin-dependent diabetes of long duration. Br Med J1992; 305: 1051–1053[ISI][Medline]
Received for publication: 13. 4.99
Revision received 13. 1.00.