Plasma Activity and Insertion/Deletion Polymorphism of Angiotensin I–Converting Enzyme
A Major Risk Factor and a Marker of Risk for Coronary Stent Restenosis
Background—Tissue proliferation is almost invariably observed in recurrent lesions within stents, and ACE, a factor of smooth muscle cell proliferation, may play an important role. Plasma ACE level is largely controlled by the insertion/deletion (I/D) polymorphism of the enzyme gene. The association among restenosis within coronary stents, plasma ACE level, and the I/D polymorphism is analyzed in the present prospective study.
Methods and Results—One hundred seventy-six consecutive patients with successful, high-pressure, elective stenting of de novo lesions in the native coronary vessels were considered. At follow-up angiography, recurrence was observed in 35 patients (19.9%). Baseline clinical and demographic variables, plasma glucose and serum fibrinogen levels, lipid profile, descriptive and quantitative angiographic data, and procedural variables were not significantly different in patients with and without restenosis; mean plasma ACE levels (±SEM) were 40.8±3.5 and 20.7±1.0 U/L, respectively (P<.0001). Diameter stenosis percentage and minimum luminal diameter at 6 months showed statistically significant correlation with plasma ACE level (r=.352 and −.387, respectively P<.001). Twenty-one of 62 patients (33.9%) with D/D genotype, 13 of 80 (16.3%) with I/D genotype, and 1 of 34 (2.9%) with I/I genotype showed recurrence; the restenosis rate for each genotype is consistent with a codominant expression of the allele D.
Conclusions—In a selected cohort of patients, both the D/D genotype of the ACE gene, and high plasma activity of the enzyme are significantly associated with in-stent restenosis. Continued study with clinically different subsets of patients and various stent designs is warranted.
Recurrence of lesions after CS occurs in 22% to 32% of patients.1 2 Compared with restenosis after balloon angioplasty, less is known about the mechanisms of restenosis after intracoronary stent placement; it is likely due to a predominant proliferative model of restenosis3 4 because stent diameter remains constant after placement and arterial remodeling cannot occur. In fact, analyses with ultrasounds have shown that restenoses after CS and after balloon PTCA differ5 in the amount of tissue proliferation, which is almost invariably observed within stents.6 7 ACE may play an important role in the proliferation of vascular smooth muscle cells through activation of angiotensin II (an inducer of cell proliferation) and inhibition of bradykinin (an inhibitor of growth).8 Plasma ACE activity is under genetic control: a functional mutation located within, or close to, the ACE locus, in almost complete linkage disequilibrium with the ACE I/D polymorphism, has been suggested to account for half of the ACE level variance.9 Therefore, both ACE level and I/D genotypes can be predictive risk markers for in-stent restenosis.
In this prospective angiographic study of restenosis after elective CS, plasma ACE level and the I/D polymorphism were assessed, along with factors currently implicated in restenosis after balloon angioplasty.
Between December 1993 and October 1996, 196 consecutive patients were enrolled. Each of them had a de novo lesion in a native coronary artery successfully treated with elective placement of one or more Palmaz-Schatz stents and they had received no medication with ACE inhibitors in the week before the procedure or during the follow-up period because even minimal doses of these drugs may interfere with basal plasma ACE level.10 Clinical and angiographic criteria for exclusion of patients were primary and rescue PTCA, PTCA within 2 days of acute myocardial infarction, insulin-dependent diabetes mellitus, a severe comorbid status, ostial lesions of the right coronary or left main stem artery, total coronary occlusions older than 2 weeks, lesions longer than 30 mm, and angiographic follow-up beyond 12 months. Twenty patients initially included in the study were excluded for the following reasons: (1) 11 patients received treatment with ACE inhibitors after the procedure, (2) 2 patients had subacute stent thrombosis, and (3) 7 patients (5 for personal reasons and 2 for medical reasons) did not undergo angiographic control. Therefore, we analyzed a total of 176 subjects with successful stent implantation, no treatment with ACE inhibitors, and angiographic follow-up at 6 months (or earlier when restenosis was suspected on clinical grounds).
Angioplasty and Stenting Technique
Treatment with 250 mg ticlopidine BID started 2 days before the procedure and continued for 2 months. Balloon dilatation was performed according to the conventional technique. Palmaz-Schatz stents were hand-crimped onto undersized compliant balloons and deployed at nominal pressures; all the prostheses were then expanded with a noncompliant balloon of the same diameter as the reference vessel segment, with inflations in the range of 14 to 20 atm for 60 to 90 sec.
“Multiple Palmaz-Schatz stenting” was used for long lesions or dissections (>15 mm), with angiographic overlapping of the stent edges. “Stenting” was “elective” in all cases: it did not obviate an acute or impending vessel occlusion after balloon dilatation. “Immediate angiographic success” was considered the deployment of the stent or stents in the target lesion, with a Thrombolysis in Myocardial Infarction grade 3 coronary flow11 and a residual stenosis of <20%.
Angiographic Assessment and QCA
The American College of Cardiology/American Heart Association classification as modified by Ellis et al12 was used to evaluate the morphology of coronary lesions. QCA was performed before balloon angioplasty, after high-pressure CS, and at follow-up coronary angiography using an online system (Philips DCI). Images of lesions were displayed in at least two orthogonal projections with the 13-cm image intensifier field after the administration of 0.5 mg intracoronary nitroglycerin. Measurements were made with the DCI Host Automated Coronary Analysis package (release 1.1.2).13 The D-Ref, MLD, and %DS were calculated as the mean of values obtained in two orthogonal views. Follow-up angiography used the same projections as the original procedure. “Acute gain” was defined as the increase in MLD achieved immediately after high-pressure CS. “Late loss” was defined as the decrease in MLD of the same segment observed on the follow-up angiogram. The “net gain” was the difference between the acute gain and the late loss. The definition of restenosis was %DS of ≥50% at the site of the lesion treated with the stent or stents observed in at least one of two orthogonal projections, one of which always including the “worst view” of the segment being analyzed (ie, in-stent restenosis). QCA of follow-up angiograms was performed by one of the cardiologists of our unit with experience in QCA who had no knowledge of other clinical, biochemical, and genetic data for the patient).
Conventional clinical and laboratory risk factors determined for all patients were age, sex, body mass index, family history of coronary artery disease (occurrence of unambiguous acute myocardial infarction, death from coronary artery disease, coronary artery bypass surgery, or PTCA), current smoking habits (>10 cigarettes/d), hypertension (diastolic blood pressure >90 mm Hg or systolic blood pressure >160 mm Hg), and non–insulin-dependent diabetes mellitus.
Blood samples collected in the morning of the procedure after 12-hour fasting were tested for levels of plasma glucose, total cholesterol, HDL cholesterol, and triglycerides with the use of enzymatic-colorimetric methods. Fibrinogen levels were measured with a fibrometer, and apolipoprotein B was measured with a nephelometric method. Plasma ACE activity was measured through quantitative kinetic determination at 340 nm with the use of FAPGG substrate (Sigma Diagnostics). Fasting blood samples were drawn from the femoral sheath immediately after the end of the CS procedure. In 40 consecutive patients, five blood samples were sequentially collected before PTCA, immediately after PTCA, and then 12, 24, and 48 hours later. The enzyme curves thus obtained were used to investigate possible deviations from basal level, as consequences of ACE release from injured vessel walls or ruptured plaques. ACE determinations were performed at the time of angiographic follow-up in 84 patients.
Genotyping of the ACE Gene I/D Polymorphism
Genomic DNA was extracted from 200 μL of whole blood with a QUIAmp Blood Kit (QUIAGEN). The I/D polymorphism of the ACE gene was determined according to the method of Rigat et al,14 with slight modifications. The sequences of the sense and antisense primers were 5′-CTG GAG ACC ACT CCC ATC CTT TCT-3′ and 5′-GAT GTG GCC ATC ACA TTC GTC AGA T-3′, respectively. PCR was performed in a final volume of 50 μL that contained ≈500 ng genomic DNA, 12.5 pmol of each primer, 500 μM dNTP, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 5% DMSO, and 1 U AmpliTaq DNA Polymerase (Perkin-Elmer Cetus). Amplification was performed with a 9600 Perkin Elmer Thermal Cycler. Samples were denatured for 1 minute at 94°C and then cycled 30 times through the following steps: 45 seconds at 94°C, 1 minute at 62°C, and 1 minute at 72°C. PCR products were electrophoresed in 1.6% agarose gel and visualized directly with ethidium bromide staining. The insertion allele (I) was detected as a 490-bp band, and the deletion allele (D) was detected as a 190-bp band. DMSO was included in the PCR to prevent underestimation of heterozygotes and overestimation of D/D genotype15 ; moreover, each D/D type was subjected to a second, independent PCR amplification with a primer pair that recognizes an insertion-specific sequence (5′-TGG GAC CAC AGC GCC CGC CAC TAC-3′; 5′-TCG CCA GCC CTC CCA TGC CCA TAA-3′), with identical PCR conditions except for an annealing temperature of 67°C and the absence of 5% DMSO.16
Student’s t test, and one-way ANOVA with Bonferroni’s correction for pairwise comparisons were used to test differences between mean values of continuous variables. The χ2 statistic with Yates’ correction, or Fisher’s exact test when appropriate, was used to test associations of noncontinuous variables. Potential associations among clinical, angiographic, biochemical variables, I/D genotypes, and restenosis were first tested by univariate methods (Student’s or χ2 tests).
Acute and follow-up QCA parameters were correlated with plasma ACE levels and tested. QCA parameters were transformed to avoid possible skewedness in their distribution. Log-transformation has been used for all variables, but %DS, which is a percentage, was transformed in arcsin %DS, the angular transformation that makes constant the sampling error of a percentage regardless of its value. Kruskal-Wallis nonparametric one-way ANOVA was used to evaluate differences within ACE genotypes, and the Least-Significant Difference multiple-comparison test (at a significance level of .05) was used to detect statistically significant differences of continuous parameters between genotypes (SPSS statistical package, release 5.01 for Windows).
This survey was approved by the ethical review committee of the relevant hospital division, and all patients gave informed consent to their inclusion in the study.
Patient Baseline Values
The cohort of the study included 151 men and 25 women aged 34 to 80 years old (mean age, 60±9.1 years). The I/D polymorphism genotype proportions fit the Hardy-Weinberg equilibrium law with allele frequencies p (D)=.58 and q (I)=.42, which was very similar to those from surveys with larger sample sizes.17 The genotype distribution and the corresponding mean plasma ACE level are shown in Table 1⇓. No significant difference was found among plasma ACE level at 12, 24, and 48 hours after CS and in basal determinations in 40 consecutive patients receiving single stenting (21.5±9, 19.7±10, 20.3±11, and 19.9±9 U/L, respectively). Fig 1⇓ shows that in 84 patients at 6-month angiography, plasma ACE levels were highly and significantly correlated with basal levels (r=.872, P<.001). With the only exception of mean plasma ACE level, no significant basal clinical, angiographic, or biochemical difference was found among patients with D/D, I/D, and I/I genotypes.
Follow-up coronary angiography for the whole cohort was performed at a mean of 6.17±2.3 months after the procedure (range, 2.74 to 10.24 months). Angiographic restenosis (R group) was present in 35 patients (19.9%) and absent (NR group) in 141; the mean time of follow-up was not statistically different for each genotype: 6.2±1.8 for the D/D, 6.4±1.8 for the I/D, and 6.3±1.1 months for the I/I group. QCA analysis did not show differences in the D-Ref between R and NR patients. Differences in MLD, %DS, late loss, and net gain are shown in Table 2⇓. Cardiovascular medication used during the follow-up period did not differ in the two groups.
Patients with and without restenosis showed no significant difference in age, sex, clinical, angiographic, and procedural parameters; QCA data before angioplasty and after CS were also similar in R and NR patients (Table 3⇓). Mean plasma ACE level was 40.8±3.5 U/L in the R group versus 20.7±1.0 in the NR group (P<.0001); their distribution is shown in Fig 2⇓. Correlation between ACE plasma level and %DS (after angular transformation) gives a value of r=.352, which is statistically different from zero (Table 4⇓). Similar values were obtained when ACE level is correlated with MLD (r=−.387) and late loss at angiographic follow-up (r=0.280). Fig 3A⇓ through 3C shows how basal plasma ACE level correlates with 6-month %DS for each genotype. The relationships among follow-up QCA results, plasma ACE level, and I/D genotypes are shown in Table 4⇓. Twenty-one of 62 patients (33.9%) with D/D genotype, 13 of 80 patients (16.3%) with I/D genotype, and 1 of 34 patients (2.9%) with I/I genotype showed restenosis. The D/D genotype was significantly more prevalent in the R group (21 of 35 [60%] versus 41 of 141 [29%], P=.026). An optimal cutoff value for plasma ACE level was calculated by maximizing the specificity for predicting restenosis: the resulting value of 34 U/L predicts occurrence and no occurrence of restenosis in 65% and 92% of the cases. Restenosis occurred in 11 of 139 patients (7.9%) with “low” (<34 U/L) ACE level and 24 patients of 37 patients (64.9%) with “high” (≥34 U/L) ACE levels (P<.00001), with the angiographic follow-up mean time between the two groups not statistically different (6.42±1.55 versus 6.10±1.37 months, respectively; P=NS). In 62 patients with D/D genotype, high and low plasma ACE levels were observed in 29 (46.8%) and 33 (53.2%) cases, respectively; restenosis occurred in 18 (62%) of the former and in 3 (9.1%) of the latter (P=.0001).
Stepwise logistic multiple regression analysis identified plasma ACE level and I/D polymorphism as the only significant predictors of restenosis. The relative risk of angiographic restenosis was 8.2 in the group of patients with high plasma ACE level (95% CI, 4.43 to 15.15). The relative risks for restenosis among patients carrying the I/I, I/D, and D/D genotype were 0.12, 0.71, and 2.75, respectively (95% CI, 0.02 to 0.86, 0.38 to 1.32, and 1.51 to 5.03). The I/D polymorphism, however, when considered together with the plasma ACE level, was no longer a significant predictive risk factor because of the correlation between the two variables.
Many variables have been associated with restenosis in balloon angioplasty, and a number of these are suspected to contribute to restenosis after stent implantation. The role of plasma level of ACE and ACE genotypes in the susceptibility to restenosis after elective coronary stent placement have been analyzed in 176 consecutive patients: our follow-up angiographic results showed a 19.9% global restenosis rate.
The association between restenosis after PTCA and the D/D genotype of the ACE gene was first observed by Ohishi et al.18 Three studies, however, failed to confirm these findings19 20 21 ; similar negative conclusions were reached by van Bockxmeer et al,22 who found a strong interaction between ACE and ε4 apoE genotypes with restenosis, an observation that supports a multifactorial basis for the etiology of restenosis. A report recently published in Circulation shows that the D allele of the ACE gene is associated with an increased restenosis rate after CS.23
To our knowledge, there was no other research aimed at evaluating the association between the I/D genotype and the plasma ACE level in patients treated with CS. To avoid the confounding effects of other variables, we used very strict criteria for patient selection: (1) only de novo lesions in native vessels and one type of prosthesis with high-pressure deployment were considered, and (2) subjects using ACE inhibitors throughout the follow-up period were excluded for two reasons: first, the possible effect of the drug on patients with high basal level of ACE, even though an inhibitory effect on endothelial vascular proliferation seems unlikely at conventional oral doses,24 and second, our intention to estimate the physiological basal levels of ACE for each genotype and, therefore, the fraction of the ACE level variance explained by the I/D polymorphism.
Allele and genotype frequencies in our cohort are identical to those found in previous studies on European populations,17 and mean plasma ACE levels for each of the three genotypes are in agreement with that reported in other studies.9 25
Baseline clinical and angiographic variables were not significantly different in our R and NR groups. Unstable angina,26 lipid levels,27 and elevated plasma fibrinogen28 have been proposed as risk factors for restenosis after balloon PTCA; their roles, however, in the recurrence of lesions after elective, high-pressure CS are unclear. Diabetes was identified as one predictor of recurrence after balloon PTCA,29 and there is emerging evidence that it may also predict restenosis after CS30 ; many of the mechanisms promoting restenosis in diabetics are related to higher glucose or insulin levels or both; glycemic control may generally reverse the process and reduce the restenosis rate in diabetic patients.31 In our study, non–insulin-dependent diabetes mellitus seems not to be associated with restenosis, but the exclusion of patients with insulin-dependent diabetes mellitus did not allow us to draw similar conclusions for insulin-dependent diabetes mellitus.
Although the role of other patient-related or lesion-related factors cannot be excluded, our study shows that both the I/D polymorphism of the ACE gene and high plasma ACE level are correlated with late luminal narrowing after CS.
Restenosis after balloon PTCA is a complex and partially understood phenomenon: early events after balloon injury include elastic recoil, platelet deposition, and thrombus formation, followed by subsequent smooth muscle cell proliferation and matrix formation.3 Restenosis occurs less frequently after CS than after balloon PTCA,1 2 and recent observations with intravascular ultrasound have partially explained this difference. After metallic scaffolding of the vessel wall, late recoil of Palmaz-Schatz stents rarely occurs.7 Stents inhibit negative arterial remodeling (a decrease in arterial or external elastic membrane cross-sectional area), with neointimal hyperplasia being the predominant responsible for in-stent restenosis. A different mechanism of restenosis is observed in nonstented lesions, in which 73% of late lumen loss is due to arterial remodeling and 27% is due to actual tissue growth.5 Two major mechanisms leading to restenosis after CS or PTCA appear not to be equally balanced, and in-stent proliferation seems to be the predominant one in restenosis after stent implantation. ACE may play a key role by inducing in-stent cell growth secondary to the production of angiotensin II and inhibition of bradykinin.24 32
If ACE activity is involved in the proliferative response causing recurrence of lesions after CS, plasma ACE level itself may be a more direct marker of this process than the ACE genotype. In fact, although plasma and cellular ACE level appear to be tightly controlled by a genetic polymorphism, the ACE I/D polymorphism is a genetic marker probably in strong linkage disequilibrium with a functional mutation (ACE S/s) located within or near the ACE gene.9 In previous studies, the S/s functional polymorphism and the I/D polymorphism accounted for 44% to 47% and 28%, respectively, of the interindividual variance of plasma ACE level9 33 ; the latter figure is identical to that calculated in our study. Mean plasma ACE level in our D/D patients was more than twice as high as that in the I/I patients and intermediate to that in the I/D patients. High (as defined above) concentrations of plasma ACE were found in 46.7% of D/D patients, 10% of I/D patients, and none of I/I patients. Different rates of restenosis were observed among the D/D, I/D, and I/I genotypes (33.9%, 16.3%, and 2.9%), this is consistent with recently published data.23 When restenosis rates are calculated only in patients with high plasma ACE levels, we found rates of 62%, 75%, and 0%, respectively. This suggests that plasma ACE level determination may be more predictive than ACE I/D genotyping for risk of restenosis after CS and possibly for other cardiovascular disorders as well.16 17 34 35 Plasma ACE level and %DS at follow-up are correlated, depending, as expected, on the ACE genotypes (Fig 3A⇑ through 3C).
A statistically significant, albeit not high, correlation between restenosis and plasma ACE level is also expected (Table 4⇑). The latter is the phenotypic expression of a major gene that is clearly codominant because of an additive effect. Data in Table 1⇑ show the codominant effect of the D allele on the phenotype (ACE level). Even if restenosis rates differ among I/D genotypes, when D/D and I/I are contrasted with I/D genotypes the codominant expression of the D allele as risk factor for restenosis is suggested, but it does not reach statistical significance.
As stated in a recent meta-analysis published in Circulation,36 the identification of the genetic effect of the D allele in cardiovascular disorders remains an important unresolved issue; our results are consistent with this statement and point out that (1) restenosis is the effect of a complex mechanism in which neointimal proliferation is a predominant factor; (2) the ACE-related mechanism plays an important, but not exclusive, role in in-stent restenosis, which could be even more significant than in myocardial infarction; (3) subjects without the D allele seem to be protected from restenosis, but I/D subjects behave more as D/D than it is expected on the basis of their heterozygous genotype (codominance effect); (4) the I/D polymorphism is probably a marker for a functional variant that controls ACE level and, presumably, restenosis—its linkage disequilibrium with the letter one is unknown; and (5) plasma ACE level may be a more informative marker for this process.
Our results are not necessarily in conflict with those from the MERCATOR37 and MARCATOR38 trials. These studies have shown that oral therapy with ACE inhibitors does not reduce the incidence of restenosis after PTCA. Vessel wall remodeling seems to be the major factor of recurrence after balloon dilatation, and the effects of these drugs on vascular wall remodeling are still poorly known. The use of ACE inhibitors to antagonize tissue proliferation might be beneficial in some patients who are predisposed to proliferation after balloon PTCA. If a high plasma ACE level is taken to mark this predisposition, benefit from these drugs is likely to be observed in ≈20% of European patients (ie, nearly one half of the patients carrying the D/D genotype). Last, but not least, drug dosages being used in these trials, albeit effective in reducing blood pressure, may not be high enough to affect neointima formation. In fact, animal studies show24 that neointimal proliferation may be affected by ACE inhibitors only if treatment is started before balloon injury and at much higher doses than required for the inhibition of circulating ACE.
We agree with the conclusions of a recent editorial by Singer et al39 that further studies are needed to shed more light on the significance of positive associations between ACE D allele and disease and to search for alternative genetic models of restenosis, such as the mostly proliferative model described for CS.40 Although our findings await confirmation in studies with larger sample sizes, clinically different subsamples of patients, various types of coronary stents, and perhaps different implantation techniques, we suggest that elective CS in addition to PTCA can dramatically reduce restenosis rates to <8% in patients with a low proliferative risk, as identified by a low plasma ACE level or lack of the D allele. On the contrary, it can be speculated that high-pressure CS after PTCA may favor the recurrence of lesions in patients with high proliferative risk: for these cases, plain balloon PTCA or other forms of coronary revascularization may prove more beneficial.
Selected Abbreviations and Acronyms
|%DS||=||percent diameter stenosis|
|MLD||=||minimum luminal diameter|
|PCR||=||polymerase chain reaction|
|PTCA||=||percutaneous transluminal coronary angioplasty|
|QCA||=||quantitative coronary analysis|
This work was supported in part by a grant from the Istituto Superiore della Sanita’, Roma, Italy, and grant MURST 60% given by the University of Torino, Italy. We wish to thank Dr Ian Penn (Vancouver General Hospital) for his valuable comments and Maria Stefania Dutto, RN, and Marilena Tomatis, RN, for their help in data collection.
Reprint requests to Flavio Ribichini, MD, Laboratorio di Emodinamica, Ospedale Santa Croce, Via M Coppino, 26, 12100 Cuneo, Italy.
Preliminary data from this study were presented in part at the 68th Scientific Sessions of the American Heart Association, November 1995, Anaheim, Calif, and the 45th Scientific Sessions of the American College of Cardiology, March 1996, Orlando, Fla.
- Received July 16, 1997.
- Revision received September 9, 1997.
- Accepted September 25, 1997.
- Copyright © 1998 by American Heart Association
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