Vascular complications in patients with hyperhomocysteinemia

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1 Relation of a Common Methylenetetrahydrofolate Reductase Mutation and Plasma Homocysteine With Intimal Hyperplasia After Coronary Stenting Tai Kosokabe, MD; Kenji Okumura, MD; Takahito Sone, MD; Junichiro Kondo, MD; Hideyuki Tsuboi, MD; Hiroaki Mukawa, MD; Takahito Tomida, MD; Tomomichi Suzuki, MD; Hiroki Kamiya, MD; Hideo Matsui, MD; Tetsuo Hayakawa, MD Background Hyperhomocysteinemia has been identified as an independent risk factor for coronary artery disease. Recent studies have shown that a common mutation (nucleotide 677 C3 T) in the methylenetetrahydrofolate reductase (MTHFR) gene may contribute to mild hyperhomocysteinemia and, therefore, to the incidence of coronary artery disease. No information exists, however, regarding the association between the mutation of the MTHFR gene or plasma homocysteine levels and morphological analysis of coronary atherosclerosis using intravascular ultrasound. Methods and Results To examine the potential influence of and homocysteine on coronary arteries morphologically, we screened 62 patients with 65 lesions that were treated with 93 Palmaz-Schatz stents. The plasma homocysteine levels in the patients with the TT genotype were not significantly higher than those in the patients with non-tt (CC CT) genotypes ( versus mmol/l, P 0.16). Angiographic analysis showed that the percent diameter stenosis in the patients with the TT genotype was significantly greater than that in those with non-tt genotypes ( % versus %, P 0.015). Intravascular ultrasound analysis showed that the TT genotype was significantly associated with greater intimal hyperplasia area ( versus mm 2, P 0.001). In multiple stepwise regression analysis, the number of the T alleles was the only independent predictor of intimal hyperplasia after intervention (r , P 0.004). Conclusions The homozygous mutant genotype of the MTHFR gene may increase the risk of in-stent restenosis more than does the normal homozygous or heterozygous genotype. (Circulation. 2001;103: ) Key Words: angiography genes restenosis stents ultrasonics Vascular complications in patients with hyperhomocysteinemia are well known. To date, numerous studies have described the relationship between plasma homocysteine levels and vascular disease, including coronary atherosclerosis. Except for a few studies, 1,2 almost all published data have indicated that hyperhomocysteinemia is a significant independent risk factor for the development of coronary artery disease (CAD) and venous thrombosis. 3 6 Plasma homocysteine concentrations are influenced by many environmental and genetic factors. Plasma homocysteine levels also increase with age. 7 In general, men have higher plasma homocysteine levels than women. 7 Although the mechanism is unclear, renal function elevates plasma homocysteine levels. 8 Deficiencies in vitamins B 6 and B 12 and folate, which are essential coenzymes in homocysteine metabolism, elevate plasma homocysteine levels. 9,10 Several studies 11,12 have indicated that the disease itself may elevate plasma homocysteine concentrations. Egerton et al 11 reported that an analysis of samples obtained at the time of a myocardial infarction and up to 180 days later indicated an increase in homocysteine concentration by 27%. Lindgren et al 12 reported that samples collected within 2 days of a stroke and up to 645 days later exhibited a rise in homocysteine concentration by 27%. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, the methyl donor for the remethylation of homocysteine to methionine. Kang et al 13 reported that up to 5% of the general population has an inherited thermolabile form of MTHFR, one that is associated with reduced activity of the enzyme. Frosst et al 14 identified a mutation (nucleotide 677 C to T; ie, alanine to valine substitution in the enzyme) in the MTHFR gene that correlated with thermolability and reduced MTHFR activity. They concluded that individuals with homozygosity for the mutation have significantly elevated plasma homocysteine levels. These findings suggest that this homozygous mutant gene may be a risk factor for Received September 6, 2000; revision received January 24, 2001; accepted January 26, From Internal Medicine II, Nagoya University School of Medicine (T.K., K.O., T.T., T. Suzuki, H.K., H. Matsui, T.H.), Nagoya, Japan; and Department of Cardiology (T. Sone, J.K., H.T., H. Mukawa), Ogaki Municipal Hospital, Ogaki, Japan. Reprint requests to Kenji Okumura, MD, Internal Medicine II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya , Japan. kenji@med.nagoya-u.ac.jp 2001 American Heart Association, Inc. Circulation is available at

2 Kosokabe et al MTHFR Gene and Homocysteine Effects on Restenosis 2049 CAD through mild hyperhomocysteinemia. Numerous studies have been conducted to examine the relationship between this mutation and the incidence of CAD. Although some studies reported that the mutation was associated with an increased risk of CAD, 15,16 others reported that the mutation were not associated with increased risk of CAD. 17,18 However, few morphological analyses of coronary atherosclerosis using intravascular ultrasound (IVUS) have been reported. The purpose of this study was to analyze morphologically whether the MTHFR 677 C to T mutation correlates with an increased risk of in-stent restenosis, as assessed by IVUS analysis. Methods Patient and Lesion Population From September 1997 to December 1998, 64 patients were enrolled in this study. The inclusion criterion for patients was successful stent implantation with a proximal reference diameter of at least 3.0 mm. We analyzed 65 lesions (left anterior descending, 35; left circumflex, 9; and right coronary artery, 21) in 62 patients (aged 64 9 years; 49 men). The exclusion criterion was a serum creatinine concentration 2.0 mg/dl. Written, informed consent was obtained from all study participants before intervention. All subjects received 200 mg of ticlopidine and 160 mg of aspirin per day after coronary intervention, and no significant difference existed between the groups in terms of frequency of use of other cardiac medication. Follow-up angiography at 6 months was part of the study protocol. During the follow-up period, 3 patients received follow-up angiography because of recurrent symptoms. These results were used as the 6-month results. IVUS analysis was available in 45 lesions (69%). At the time of stent implantation, 9 patients presented with acute myocardial infarction and 15 with unstable angina. Forty-five lesions were treated with a single Palmaz-Schatz stent, 12 were treated with 2 stents, and 8 were treated with 3 stents. Of all stents, 57 were 3.0 mm, 27 were 3.5 mm, and 9 were 4.0 mm. Palmaz-Schatz stents were implanted according to standard protocols. 19,20 IVUS-guided coronary angioplasty and stenting were performed in all patients. All stents were implanted using highpressure adjunct balloon angioplasty ( atm) to achieve the targeted stent expansion. The targeted stent expansion was a minimal area 80% of the average of the proximal and distal reference lumen areas by IVUS, as well as complete stent-vessel wall apposition. Angiographic Analysis Angiography was performed after the administration of 0.2 mg of intracoronary nitroglycerin. All films of cineangiograms were analyzed by an independent, experienced core angiographic laboratory without knowledge of the results of ultrasound analysis. Using an automated edge detection algorithm (QCA-CMS System, MEDIS Inc), the minimum lumen diameter, reference diameter, and percent diameter stenosis (%DS) were measured from multiple projections; the results in the worst view were recorded. User-defined reference segments were selected as the mean of 10-mm-long segments proximal and distal to the lesion. Angiographic restenosis was defined as a DS 50%. Late loss was calculated as postintervention minus follow-up minimum lumen diameter. The late loss index was calculated by dividing the late loss by the early gain. IVUS Image Acquisition and Analysis IVUS imaging was performed using a 30-MHz mechanical ultrasound transducer (Ultra Cross TM 3.2, Boston Scientific SCIMED). The transducer was withdrawn within the stationary imaging sheath at a speed of 0.5 mm/s using a motorized transducer pullback device after the administration of 0.2 mg of intracoronary nitroglycerin. Using computerized planimetry, quantitative IVUS analysis was performed by a single individual who was blinded to angiographic and genetic results. Validation of cross-sectional measurements by IVUS has been reported previously External elastic membrane, stent, and lumen areas were measured. In addition to the analysis of the narrowest cross-section, proximal and distal reference segments were analyzed. A reference segment was defined as the most normal looking cross-sections within a 10-mm segment proximal or distal to the stent that did not crossing any large side branches. Reference segment areas were calculated as the mean value of the proximal and distal reference areas. Late lumen loss was calculated as postintervention lumen area minus follow-up lumen area. Intimal hyperplasia (IH) area was calculated as stent minus lumen area at follow-up. Relative IH was calculated as IH area divided by follow-up stent area. Plaque area was calculated as external elastic membrane area minus lumen area, because ultrasound cannot accurately measure media thickness. 24 Measurement of Plasma Homocysteine Levels and Genetic Analysis Plasma homocysteine levels were measured 3 months after coronary intervention. Fasting venous blood was drawn because plasma homocysteine levels have been shown to be influenced by meals. 25 Plasma homocysteine levels were determined as total homocysteine by high-performance liquid chromatography with fluorescence detection, as previously described. 26 Genomic DNA was isolated from nucleated blood cells using a phenol chloroform method. Identification of the C to T transition at nucleotide 677 was determined using the method of Frosst et al. 14 Because the C to T mutation at nucleotide 677 produces the Hinf I digestion site, the polymerase chain reaction product (198 bp) derived from the mutant gene is digested into 175-bp and 23-bp fragments by Hinf I. Statistics Statistical analysis was performed using StatView 5.0 (SAS Institute). Continuous variables are presented as mean SD, and categoric variables are presented as frequencies. Because our subject group was small and no significant differences existed in any variables between the wild-type (CC) and heterozygous genotype (CT), we established 2 genotype groups: the mutant homozygote (TT) and the others (non-tt: CC CT). Continuous variables were compared using Student s t test. The categoric data were compared using 2 analysis. Pearson product-moment correlation coefficients (r) were computed to identify the variables that were significantly associated with follow-up %DS, as measured by angiography, and IH area, as measured by IVUS. Forward stepwise multiple regression analysis was performed to examine significant contributions of the variables to the prediction of follow-up %DS and IH area. P 0.05 was considered statistically significant. Results Plasma Homocysteine Concentration and MTHFR Genotypes The allele frequency of the T mutation in the 62 patients was The distribution of the 3 genotypes was as follows: homozygous normal (CC) genotype, 30.7%; heterozygous (CT) genotype, 41.9%; and homozygous mutant (TT) genotype, 27.4%. These genotypic distributions were similar to those described previously in patients with CAD. 27 Clinical characteristics of all patients with each genotype are shown in Table 1. The plasma homocysteine concentration in the patients with the TT genotype was higher than that in those with non-tt genotypes ( versus mmol/l, P 0.16), but the difference was not statistically significant. No significant difference existed between the patients with TT and non-tt genotypes for any variables examined, including age, sex, and creatinine, that influenced plasma homocysteine levels.

3 2050 Circulation April 24, 2001 TABLE 1. Patient Characteristics in Each MTHFR Genotype CC (n 19) CT (n 26) TT (n 17) non-tt (n 45) P * Sex, male/female 15/4 20/6 14/3 35/ Age, y Body mass index, kg/m Acute myocardial infarction, n Unstable angina, n Smoker/nonsmoker, n 4/15 9/17 6/11 13/ Blood pressure, mm Hg Systolic Diastolic Glucose, mg/dl Total cholesterol, mg/dl Triglyceride, mg/dl Creatinine, mg/dl Homocysteine, mol/l Data are presented as mean SD or No. of patients. Angiographic Results Representative angiograms and IVUS images are shown in the Figure. Lesion characteristics are shown in Table 2. No significant differences in lesion site, lesion type, lesion length, number of stents, stent size, and maximal inflation pressure existed between the patients with the TT and non-tt genotypes. In addition, no significant differences existed in reference diameters or minimum lumen diameters before intervention, after intervention, and at follow-up between the TT and non-tt groups (Table 3). There was no significant difference in %DS before intervention or after intervention; however, %DS in the patients with the TT genotype was Representative angiograms (A and B) and IVUS images (C and D) of patient with TT genotype and right coronary artery lesions. Follow-up DS was 55.4% by quantitative coronary angiographic analysis (B). IVUS images (D) revealed a large amount of intimal hyperplasia at follow-up (IH area, 9.6 mm 2 ; relative IH, 82.8%).

4 Kosokabe et al MTHFR Gene and Homocysteine Effects on Restenosis 2051 TABLE 2. Lesion Characteristics Among Each MTHFR Genotype CC (n 20) CT (n 28) TT (n 17) non-tt (n 48) P * Intervals, days Lesion site, n RCA LAD LCX Lesion type (AHA/ACC), n A B B C Ostial/nonostial, n 3/17 1/27 1/16 4/ No. of stents Stent size, mm Lesion length, mm Inflation pressure, atm Data are presented as mean SD or No. of patients. RCA indicates right coronary artery; LAD, left anterior descending artery; and LCX, left circumflex artery. significantly greater than that in those with the non-tt genotypes ( % versus %, P 0.015) at follow-up. The late loss was mm in the TT group and mm in the non-tt group (P NS). The late loss index and restenosis rate were higher in the patients with the TT genotype than in those with the non-tt genotypes (59.6% versus 46.7% and 29.4% versus 12.5%, respectively), but the differences were not statistically significant. TABLE 3. Angiographic Characteristics in Each MTHFR Genotype CC (n 20) Serial IVUS Results IVUS variables were available in 45 lesions after intervention and at follow-up. Table 4 summarizes IVUS results in s. The subjects with the TT genotype had greater external elastic membrane and plaque areas in the reference segment after intervention (both P 0.016) but not at follow-up. At the narrowest cross-section, lumen area decreased from to mm 2 in the TT CT (n 28) TT (n 17) non-tt (n 48) P * Before intervention Reference diameter, mm MLD, mm DS, % After intervention Reference diameter, mm MLD, mm DS, % Follow-up Reference diameter, mm MLD, mm DS, % Late loss, mm Late loss index, % Restenosis rate, % Data are presented as mean SD. MLD indicates minimum lumen diameter.

5 2052 Circulation April 24, 2001 TABLE 4. IVUS Variables Among Each MTHFR Genotype CC (n 13) CT (n 21) TT (n 11) non-tt (n 34) P After intervention Reference EEM area, mm Lumen area, mm Plaque area, mm Narrowest site Lumen area, mm Follow-up Reference EEM area, mm Lumen area, mm Plaque area, mm Narrowest site Stent area, mm Lumen area, mm Late lumen loss, mm IH area, mm Relative IH, % Data are presented as mean SD. EEM indicates external elastic membrane. group and from to mm 2 in the non-tt group, indicating no significant difference between the 2 groups regarding the lumen area at follow-up. However, the late lumen loss in those with the TT genotype was significantly greater than that in those with non-tt genotypes. ( versus mm 2, P 0.002). IH area in those with TT genotypes was also significantly greater than that in those with non-tt genotypes ( versus mm 2, P 0.001). Univariate and Stepwise Multivariate Analysis of Determinant of Increased Follow-Up %DS and IH Area Univariate analysis using the Pearson correlation coefficients was performed to determine which variables were associated with follow-up %DS and IH area (Table 5). The number of T alleles was significantly associated with IH area by IVUS analysis and had a tendency to increase follow-up %DS (r 0.24, P 0.053), although plasma homocysteine levels were not associated with follow-up %DS or IH area. In terms of stepwise multiple regression analysis, the number of T alleles (r , P 0.004) was the only predictor of IH area (data not shown). Discussion Although stent implantation is expected to decrease restenosis after angioplasty and a high primary success rate has been obtained, the late restenosis rate still limits the long-term benefit of this procedure. 28 A number of studies have attempted to determine the clinical, angiographic, and genetic features that predict restenosis. Many variables have been suggested as candidates associated with restenosis, but only a few genetic predispositions have been reported previously. 29,30 In this study, a harmful effect of homozygosity for the mutation of the MTHFR gene was revealed after stent implantation, as determined using both quantitative coronary angiography and IVUS imaging. Interestingly, the number of T alleles was the only independent predictor for IH area by IVUS analysis after coronary stenting. Quantitative assessment with IVUS may be superior to angiography for the evaluation of coronary artery stenosis because it provides more detailed information from tomographic views of coronary arteries than does angiography. Numerous studies have reported that homozygosity for the mutation was associated with mild hyperhomocysteinemia In this study, however, we failed to find a significant correlation between TABLE 5. Univariate Analysis of Predictors for Follow-Up %DS and IH Area Variables Follow-up %DS IH area r P r P Homocysteine Number of T alleles Lesion length Number of stents Inflation pressure Post-reference plaque area Post-reference lumen area Post-lumen area at narrowest site

6 Kosokabe et al MTHFR Gene and Homocysteine Effects on Restenosis 2053 plasma homocysteine levels and s. One reason for this may be the small population of patients enrolled. Another reason for the lack of relationship may be that plasma homocysteine levels are also determined by factors other than MTHFR, as mentioned above. Postprandial homocysteine levels may be higher in individuals with the homozygous mutant genotype than in those with the normal genotype. Therefore, despite a lack of a sufficient relationship between the to fasting homocysteine levels, hyperhomocysteinemia must be due, in part, to the mutation of the MTHFR gene. The hypothesis that the elevation of plasma homocysteine levels is a risk factor for CAD is of considerable interest. However, few morphological studies have focused on the relationship between hyperhomocysteinemia and coronary atherosclerosis. In our study, the plaque area in reference segments was correlated with plasma homocysteine levels after intervention (n 58, r 0.38, P 0.004; data not shown). In addition, we observed greater plaque areas in reference segments in the patients with mutant homozygosity for MTHFR than in those with the normal homozygous and heterozygous genotypes combined. These findings are consistent with the previous reports describing an association between hyperhomocysteinemia and increased incidence of CAD, 3 5 although a significant relationship between follow-up %DS to plasma homocysteine levels was not found in this study. The mechanisms by which hyperhomocysteinemia promotes the development of atherosclerosis are not fully understood. It has been shown that a short-term increase in homocysteine concentration induced by an oral methionine load leads to endothelial dysfunction, probably resulting from oxidative effects, including the generation of superoxide anion radicals and hydrogen peroxide. 31 The resultant endothelial dysfunction, such as an impairment of the release and/or effects of nitric oxide, may then contribute to the progression of atherosclerosis. Other putative mechanisms include smooth muscle proliferation, extracellular matrix modification, lipoprotein oxidation, cytotoxicity, and effects on platelets and coagulation. 32 Recent studies using IVUS have suggested that in-stent restenosis and late lumen loss were the results of IH caused by smooth muscle cell migration and matrix formation because the Palmaz-Schatz stent prevents remodeling processes such as elastic vessel recoil. 22 In this study, the stent areas of the patients with mutant homozygosity for the MTHFR gene were greater to some extent than those of the other patients. Although only a weak correlation existed between IH area and stent area, IH thickness at follow-up was independent of stent size, as assessed by IVUS analysis. 33 Therefore, stent area does not seem to be a confounding factor. Relative IH area, which may account for differences in IH between the 2 groups, was significantly higher in the mutant homozygote group than in the other groups. In stepwise multivariate regression, the only independent contributor predicting IH area was the number of T alleles, not plasma homocysteine levels. These findings may suggest that the effect of the increasing IH of the mutant homozygosity for the MTHFR gene was due to unknown factors, such as the greater plaque burden of this genotype before intervention, rather than the effect of hyperhomocysteinemia itself. However, our results suggest homocysteine-lowering therapy, such as folate supplementation, 34 in the patients with mutant homozygosity for MTHFR gene may prevent the development of restenosis after stent implantation. Study Limitations The number of patients in the present study was fairly small, and larger studies will be needed to confirm our findings. Only Palmaz-Schatz stents were included; the frequency and magnitude of these findings in regard to other stents are unknown. The narrowest site evaluated by angiography was different from that evaluated by IVUS. Although IVUSguided coronary angioplasty was performed in all subjects, we did not perform quantitative evaluation of IVUS before intervention. Therefore, the plaque burden at the narrowest site before intervention was undetermined. During this period, we measured plasma homocysteine concentrations only once at 3 months after intervention. Because homocysteine concentrations fluctuate according to the state of the disease, more frequent measurements of plasma homocysteine concentrations may provide more detailed information. Conclusions The present study was the first morphological analysis of the association between the and in-stent restenosis using IVUS. The mutant homozygote of the MTHFR gene might increase the risk of in-stent restenosis more than does the normal homozygous or heterozygous genotype. 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