Plasminogen Activator Inhibitor-1 and Angiotensin I Converting Enzyme Gene Polymorphism in Patients With Hypertension

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1 AJH 1998;11: Plasminogen Activator Inhibitor-1 and Angiotensin I Converting Enzyme Gene Polymorphism in Patients With Hypertension Jing-Ren Jeng, Horng-Jyh Harn, Kuo-Cheu Yueh, Chii-Yuan Jeng, and Shyh-Ming Shieh Deletion polymorphism of angiotensin I- converting enzyme (ACE) gene has been reported to be an independent risk factor for myocardial infarction. Plasminogen activator inhibitor-1 (PAI- 1) was proposed to be a link between the reninangiotensin system and thrombotic risk. This study was undertaken to investigate the possible association between the insertion/deletion (I/D) polymorphism of the ACE gene and plasma PAI-1 levels in 160 patients with mild-to-moderate hypertension. The I/D genotypes were determined by polymerase chain reaction with oligonucleotide primers flanking the polymorphic region in intron 16 of the ACE gene. Baseline levels of PAI-1 antigen and activity and tissue plasminogen activator (t-pa) antigen were determined in fasting morning plasma samples. It was found that patients with homozygote deletion (DD, n 37) ACE genotype did not have significantly higher plasma levels of PAI-1 antigen ( ng/ml v ng/ml or ng/ml, P.42), Evidence is now accumulating that the insertion/deletion (I/D) polymorphism of the angiotensin I converting enzyme (ACE) gene is associated with an increased risk of myocardial infarction (MI) 1,2 and cardiac death 3,4 even in PAI-1 activity ( IU/mL v IU/ ml or IU/mL, P.60), or t-pa antigen ( ng/ml v ng/ml or ng/ml, P.40) as compared to those with heterozygote (DI, n 67) or homozygote insertion (II, n 56) genotypes. On multiple regression analysis, the ACE genotypes did not appear to be significant predictors for plasma PAI-1 levels and t-pa antigen after adjustment with age, sex, body mass index, plasma triglyceride, cholesterol, and glucose. In conclusion, the results indicated that the I/D polymorphism of the ACE gene was not related to plasma PAI-1 levels in a Chinese population with hypertension. The ACE genotypes may not have a role in influencing the fibrinolysis in hypertension. Am J Hypertens 1998;11: American Journal of Hypertension, Ltd. KEY WORDS: Plasminogen activator inhibitor-1, angiotensin I converting enzyme, gene polymorphism, hypertension. otherwise low risk subjects, but it does not appear to be a risk factor for atherosclerosis 2,5 or hypertension. 6,7 The pathophysiologic mechanisms linking the I/D polymorphism of the ACE gene and the risk of MI remain hypothetical. One plausible explanation is that Received August 21, Accepted September 29, From the Divisions of Cardiology (J-RJ), Endocrinology and Metabolism (C-YJ), Department of Pathology (H-JH, K-CY), and Clinical Research Center (J-RJ, C-YJ), Tri-Service General Hospital, National Defense Medical Center, and Chung-Shan hospital (S-MS), Taipei, Taiwan, Republic of China. This study was supported by grants from the National Science Council (NSC B , NSC B ), Taipei, Taiwan, Republic of China. Address correspondence and reprint requests to Jing-Ren Jeng, MD, Division of Cardiology, Tri-Service General Hospital, No. 8, Sec. 3, Ting-Chou Rd., Taipei 100, Taiwan by the American Journal of Hypertension, Ltd /98/$19.00 Published by Elsevier Science, Inc. PII S (97)

2 236 JENG ET AL AJH FEBRUARY 1998 VOL. 11, NO. 2 elevated ACE activity associated with homozygote deletion (DD) ACE genotype 8,9 may have primary impact on the transition of atherosclerosis to MI through enhancements of angiotensin II production and bradykinin degradation in the local circulation. Angiotensin II 10 and bradykinin 11 have been shown to be potent stimulators of plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-pa) secretion, respectively, in vivo. Hence, patients with elevated ACE activity may have increased angiotensin II and decreased bradykinin concentrations resulted in enhanced PAI-1 and reduced t-pa productions. The combined effect could be expected to induce an unfavorable shift in fibrinolytic balance and increase the incidence of intravascular thrombosis. The hypothesis that the I/D polymorphism of the ACE gene may relate to the risk of MI through an effect on PAI-1 was examined recently in diabetic and nondiabetic subjects, 12 but no difference in plasma PAI-1 activity was found among the three ACE genotypes. Impaired fibrinolysis with elevated PAI-1 levels and depressed t-pa activity is also very common in patients with hypertension 13,14 and ACE inhibitor therapy 15 has been shown to associate with improved fibrinolysis. It seemed important to determine whether there was an association between the I/D polymorphism of the ACE gene and impaired fibrinolysis in patients with hypertension; however, this possible association has not yet been examined in the literature. Therefore, the present study was initiated to test the null hypothesis of no association between the ACE gene polymorphism and fibrinolysis, focusing on comparison of plasma PAI-1 levels and t-pa antigen among three genotypes of hypertensive patients and multiple regression analysis of fibrinolytic variables controlling potential confounders. SUBJECTS AND METHODS This study was performed in 160 Chinese patients with mild-to-moderate hypertension diagnosed as diastolic blood pressure (DBP) of 95 to 110 mm Hg on at least three separate occasions. They volunteered to participate in this study after a protocol approved by the National Defense Medical Center Human Subjects Committee. None of these subjects had a history or clinical evidence of coronary heart disease. There was no apparent cause of secondary hypertension on clinical grounds in all patients. Those with known diabetes or fasting plasma glucose concentration 140 mg/dl were excluded from this study. Genomic DNA was extracted from 200 L of buffy coat by using an IsoQuick nucleic acid extraction kit (MicroProbe, Bothell, WA). The I/D polymorphism of ACE gene was determined according to the method of Tiret et al. 8 The sequences of sense oligonucleotide primer was 5 -CTGGAGACCACTCCCATCCTT- TCT-3 and the antisense primer 5 -GATGTGGCCAT- CACATTCGTCAGAT-3. These two primers designed to flank the polymorphic region of the ACE gene allowed detection of a 490-bp fragment as the insertion allele and a 190-bp fragment as the deletion allele. The I/D portion in intron 16 of the ACE gene was amplified by polymerase chain reaction (PCR) and detected by electrophoresis in agarose gel and staining with ethidium bromide. 7,16 To reduce interference by the diurnal variation 17 of PAI-1 and t-pa, blood was drawn at about 9 am after a 15-min rest and an overnight fast and was collected with buffered 0.13 mol/l sodium citrate. Antihypertensive drugs were discontinued for 1 week if the patients had been regularly treated. Plasma was separated within 1 hour by centrifugation for 20 min at 3000 g and stored at 70 C until assay. Plasma PAI- 1 18,19 and t-pa 20 antigens were measured quantitatively with an enzyme-linked immunosorbent assay. Plasma PAI-1 activity 21 was determined with a twostage, indirect enzymatic assay based on the addition of excess t-pa (40 IU/mL) to the samples and measurement of the residual t-pa activity. One unit of PAI-1 activity was defined as the amount of PAI-1 that inhibits 1 IU of international t-pa standard. The reagent kits for assay of t-pa and PAI-1 antigens and activities were purchased from Biopool AB, Umeå, Sweden. All data are expressed as mean SD. The statistic significances of differences on mean levels of variables among three genotypes were examined by ANOVA. Multiple regression was used to evaluate the associations of fibrinolytic variables (PAI-1 and t-pa) with ACE genotypes (DD v II and DD v DI) controlling for age, sex, body mass index (BMI), fasting plasma glucose, total cholesterol, and triglyceride as potential confounders. The criteria for statistic significance of quantitative traits among genotype classes is taken as P.05. RESULTS Table 1 shows clinical characteristics and fibrinolytic data of 160 hypertensive patients with different ACE genotypes. There were no significant differences on age, gender, BMI, history of hypertension, percentage of antihypertensive treatment, systolic and diastolic blood pressures on visit, fasting plasma glucose, total cholesterol, and triglyceride among the three ACE genotype groups. In addition, it can be seen that the three ACE genotype groups had similar average plasma levels for PAI-1 antigen, PAI-1 activity, or t-pa antigen. The differences were not significant among the three genotype classes as tested with ANOVA. Thus, ACE/DD genotype was not associated with significantly higher plasma PAI-1 levels and t-pa an-

3 AJH FEBRUARY 1998 VOL. 11, NO. 2 PAI-1 AND ACE GENOTYPE IN HYPERTENSION 237 TABLE 1. CLINICAL CHARACTERISTICS AND FIBRINOLYTIC DATA OF PATIENTS WITH DIFFERENT ACE GENOTYPES Variables II (n 56) DI (n 67) DD (n 37) F P Age (yr) Gender (M/F) 28/28 40/27 23/ Body mass index (kg/m 2 ) Hypertension history (yr) Antihypertensive therapy (%) Systolic BP (mm Hg) Diastolic BP (mm Hg) Smoking habit (%) Fasting plasma glucose (mg/dl) Total cholesterol (mg/dl) Triglyceride (mg/dl) PAI-1 activity (IU/mL) PAI-1 antigen (ng/ml) tpa antigen (ng/ml) Data are expressed as means SD. F ratios and P values were obtained from one-way analysis of variance. tigen, and may not be a genetic determinant of impaired fibrinolysis in Chinese patients with hypertension. Table 2 showed multiple regression analysis of fibrinolytic variables in patients with hypertension. It can be noted that the significant predictors for plasma PAI-1 activity were triglyceride, BMI, fasting plasma glucose, and gender. The plasma PAI-1 and t-pa antigen were significantly correlated with triglyceride. However, ACE genotypes (DD v II and DD v DI) were not significant predictors for plasma levels of PAI-1 activity, PAI-1 antigen, or t-pa antigen after adjustment for age, gender, BMI, fasting plasma glucose, total cholesterol, and triglyceride on multiple regression analysis. It may indicate no significant association between ACE genotypes and plasma levels of PAI-1 activity, PAI-1 antigen, or t-pa antigen in patients with hypertension. DISCUSSION This study was initiated to determine whether the fibrinolytic variables were present to greater or lesser levels in hypertensive patients with the ACE/DD genotype. Our data showed that hypertensive patients with the DD genotype had insignificantly different average plasma levels in PAI-1 antigen and activity and t-pa antigen as compared with those with the DI or II genotype. This result indicated no apparent association between impaired fibrinolysis and the I/D polymorphism of the ACE gene in patients with hypertension. In addition, it was shown that ACE genotypes were not significant predictors for fibrinolytic variables after adjustment for potential confounders. The hypothesis we wanted to test was whether the increased MI risk of the ACE/DD genotype might be mediated through elevated circulating PAI-1 levels. TABLE 2. MULTIPLE REGRESSION ANALYSIS OF FIBRINOLYTIC DATA FROM 160 PATIENTS WITH HYPERTENSION Variables PAI-1 activity (IU/mL) PAI-1 antigen (ng/ml) t-pa antigen (ng/ml) ACE genotypes DD v DI (.43) (.49) (.32) DD v II (.91) (.36) (.71) Age (.68) (.71) (.92) Gender (.03) (.24) (.02) Body mass index (.01) (.06) (.22) Fasting plasma glucose (.02) (.23) (.28) Total cholesterol (.69) (.88) (.19) Triglyceride (.001) (.0001) (.02) R Data are expressed as B SE (P values), which were obtained from multiple regression analsysis.

4 238 JENG ET AL AJH FEBRUARY 1998 VOL. 11, NO. 2 Our results presented exclude that possibility and support the null hypothesis that the I/D polymorphism of the ACE gene had no significant effect on fibrinolytic variables in systemic circulation. It may also suggest that the ACE/DD genotype and elevated plasma PAI-1 levels could be two independent risk factors of MI in Chinese patients with hypertension. The evidence 11,22 24 that angiotensin II is a potent stimulator of PAI-1 production supports the existence of an important relationship between the renin-angiotensin system (RAS) and the fibrinolytic system. Recently, Lachurié etal 25 have shown no detectable influence of the ACE I/D polymorphism on the conversion of intravenously infused angiotensin I to angiotensin II and on subsequent angiotensin II-mediated biologic effects. It suggested that ACE appears to have no limiting influence on the systemic generation of angiotensin II. This may provide a reasonable explanation for our observed absence of influence of ACE genotypes on plasma PAI-1 levels. However, our data do not exclude the possibility that the I/D polymorphism of the ACE gene may indeed regulate tissue or platelet PAI-1 concentrations in spite of no effect on the plasma levels. More recently, Hamdan et al 24 also demonstrated that the RAS regulates neointimal PAI-1 expression and ACE inhibitors can reduce PAI-1 levels in the vessel wall in vivo. Therefore, it remains possible that the ACE/DD genotype may relate to risk of MI through enhanced effects on vascular PAI-1 expression. Elevated plasma levels of PAI-1 have been observed in survivors of acute MI 26 and in patients who develop restenosis after angioplasty, 27 and has been proposed as a risk factor for MI. 28 Furthermore, subjects with obesity, 29 hypertriglyceridemia, 30 hypertension, 14 or diabetes mellitus 31 also have been found to have increased plasma PAI-1 levels. At present, however, genetic regulators and environmental determinants of PAI-1 expression in these patients are not entirely clear. Epidemiologic studies have shown a strong and positive correlation of plasma PAI-1 activity with BMI, serum triglyceride, and insulin levels. Recently, a 4G/4G polymorphism in the promoter of the PAI-1 gene was found to be associated with higher PAI-1 activity in patients with MI 33,34 or diabetes mellitus 35 and may determine the effect of triglyceride on PAI-1 activity. Our data showed that the I/D polymorphism of the ACE gene was not a significant determinant of plasma PAI-1 levels in patients with hypertension. Nevertheless, in view of the lack association of ACE gene polymorphism with plasma PAI-1, the ACE/DD genotype may still exert a synergic effect on the risk of MI in the presence of elevated plasma PAI-1 levels. In conclusion, the present study showed that hypertensive patients with the ACE/DD genotype were not associated with significantly higher plasma PAI-1 levels, and may not affect the risk of MI through the mechanism of reduced fibrinolysis and enhanced thrombogeneity. It also suggested that the ACE/DD genotype and elevated plasma PAI-1 levels could be two independent risk factors of MI in hypertension. The I/D polymorphism of the ACE gene may play an unimportant role in influencing the fibrinolysis in Chinese patients with hypertension. ACKNOWLEDGMENTS We thank Professor H.-C. Kwaan, professor of medicine in Northwest University, for his suggestion and instruction about the fibrinolytic measurement and S.-C. Lee for her technical assistance. REFERENCES 1. Cambien F, Poirier O, Lecerf L, et al: Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. 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