Author + information
- Received September 25, 2000
- Revision received January 23, 2001
- Accepted February 6, 2001
- Published online June 1, 2001.
- Teruo Inoue, MD, FACCa,* (, )
- Toshihiko Uchida, MDa,
- Hirotoshi Kamishirado, MDa,
- Kan Takayanagi, MD, FACCa and
- Shigenori Morooka, MDa
- ↵*Reprint requests and correspondence: Dr. Teruo Inoue, Department of Cardiology, Koshigaya Hospital, Dokkyo University School of Medicine, 2-1-50 Minamikoshigaya, Koshigaya City, Saitama 343-8555, Japan
This study aimed to elucidate whether an antibody against oxidized low density lipoprotein (anti-Ox-LDL) could predict short-term coronary artery atherosclerotic lesion progression.
It is still controversial whether higher levels of the anti-Ox-LDL titer are associated with atherosclerotic coronary artery disease.
In 52 patients undergoing coronary angioplasty and six-month follow-up angiography, we performed quantitative coronary angiographic analysis of a lesion on a branch away from the intervention site vessel and assessed lesion progression or regression using the Progression-Regression score calculated as the baseline minimal lumen diameter minus the follow-up minimal lumen diameter. The serum anti-Ox-LDL titer was measured using an enzyme-linked immunosorbent assay method just before the initial angiography in all patients.
The anti-Ox-LDL titer was 16.6 ± 1.5 AcU/ml in the progression group (Progression-Regression score >0.15 mm; n = 20), which was significantly higher (p < 0.001) than the value of 9.5 ± 1.2 in the regression group (≤−0.15 mm; n = 14) and also higher (p < 0.01) than the value of 11.4 ± 1.3 in the no-change group (−0.15 to 0.15 mm; n = 18). The Progression-Regression score was correlated with the antibody titer in all patients (r = 0.56, p < 0.001). Multiple regression analysis showed that the Progression-Regression score was independently correlated with the antibody titer (r = 0.44, p < 0.01) as well as lipoprotein (a) (r = 0.33, p < 0.05).
Anti-Ox-LDL may be an independent predictor of coronary atherosclerotic lesion progression in the short term.
Oxidized low density lipoprotein (LDL) is believed to play a key role in the development of atherosclerosis (1). Oxidative modification of LDL induces immunogenic epitopes in the LDL molecule (2), and the presence of antibodies against oxidized LDL (anti-Ox-LDL) has been demonstrated in human sera (3,4). Several studies indicated that higher levels of the anti-Ox-LDL titer were associated with the presence of atherosclerotic disease (5–8). In other recent studies, however, no such relationships between atherosclerotic disease and the antibody titer have been found (9). On the other hand, the anti-Ox-LDL titer has also been shown to correlate with the rate of atherosclerosis progression, rather than the presence of atherosclerosis itself (7). In this study, to clarify these controversial results we angiographically assessed lesion progression, or lesion regression of the coronary artery, and investigated whether short-term coronary artery lesion progression could be predicted by the anti-Ox-LDL titer.
From patients with atherosclerotic coronary artery disease undergoing elective coronary angioplasty for a single lesion and six-month follow-up coronary angiography, we selected for this study 52 consecutive patients (41 men and 11 women; age 63 ± 1 year), who had at least one lesion (25% to 75% diameter stenosis from a visual impression) on the proximal portion of other major branches besides the angioplasty site vessel. All patients had received the standard medications for angina, including nitrates, beta-blockers, calcium blockers and aspirin, which were continued during the intervention procedure and not changed until the follow-up angiography. Ticlopidine was given if coronary stents were implanted in the intervention site. Patients who received other cardioactive drugs including angiotensin-converting enzyme inhibitors, patients who had diabetes mellitus requiring oral hypoglycemic agents or insulin injection, or patients who had other cardiac or noncardiac complications that could have affected our analysis were also excluded. Each patient gave written informed consent to participate in this study, which was approved by the Dokkyo University Institutional Review Board and conformed to the principles outlined in the Declaration of Helsinki.
We selected the most proximal lesion from the lesions in the nonintervention vessels as the target lesion for assessment of lesion progression or regression during the six-month natural course. If the lesions existed in both of two nonintervention vessels, a lesion in the larger diameter vessel was nominated as the target lesion. On baseline coronary angiography just before coronary angioplasty, all lesions were visually assessed. In addition, the Gensini score, which indicates the severity of the total lesions in the entire coronary artery system, taking into consideration both stenosis severity and lesion distribution, was calculated as described previously (10). Next, quantitative coronary angiographic analysis of the target lesion was performed twice (a baseline coronary angiography and follow-up angiography) for assessment of lesion progression or regression. Quantitative coronary angiographic analysis was performed on the same projection and the minimal lumen diameter was measured. The progression or regression of the lesion was evaluated using the Progression-Regression score, calculated as the baseline minimal lumen diameter minus the follow-up minimal lumen diameter. The analyses were performed by one investigator who was unaware of the identity of the patient and the working hypothesis. Intraobserver variability for the measurement of the coronary artery diameter showed high reproducibility (r = 0.98, SEE = 3.6%, p < 0.001).
Assessment of coronary risk factors
Answers to questions on history of hypertension, diabetes and smoking habits were carefully noted for each patient. Blood pressure measured in the supine position in the morning on the day of coronary angiography was evaluated. Mean blood pressure was calculated as diastolic pressure plus one-third pulse pressure. For the obesity index, the body mass index was calculated as [weight/(height)2kg/m2]. To evaluate smoking habits, Brinkmann’s index was calculated as (cigarette number × smoking year).
Lipid profile and glucose metabolism
Fasting venous blood was collected from each patient early in the morning on the day of coronary angioplasty and was used for routine lipid profiling and glucose metabolism analysis. The residual serum was frozen at −80°C until measurement of anti-Ox-LDL. Serum total cholesterol and triglyceride levels were determined by automated enzymatic assays (11,12). Low density lipoprotein cholesterol was assayed from enzymatic measurements and high density lipoprotein cholesterol was determined using a precipitation method. Apolipoprotein (apo) A-I, apo B and apo E were quantified with a turbidimetric immunoassay. The assay method of lipoprotein (a) (Lp [a]) was a latex agglutination immunoassay. The assay of remnant-like lipoprotein particle (RLP) cholesterol was performed according to published methods (13,14)using an RLP cholesterol assay kit. Plasma glucose was determined by the glucose oxidase method and plasma insulin level with a radioimmunoassay.
Assay of anti-Ox-LDL
The quantification of anti-Ox-LDL was performed using an enzyme-linked immunosorbent assay (ELISA) kit (Ox-LDL IgG ELISA Test; Biodesign International Inc., Saco, Maine), as reported elsewhere (5–8). Briefly, serum was diluted 1:21 with a sample diluent. Microtiter ELISA plates were precoated with malonic dialdehyde (MDA)-induced Ox-LDL. One hundred milliliters of the diluted specimen was added to each well and incubated at 37°C for 30 min. After the wells were washed five times, enzyme-labeled antihuman IgG was added, followed by incubation at 37°C for 30 min. The wells were again washed five times. Then, a substrate containing p-nitrophenyl phosphate was added, with incubation at 37°C for 30 min. The reaction was stopped by 1.5 mol/L sodium hydroxide. The absorbance was read at a wavelength of 405 nm. In our laboratory, anti-Ox-LDL titer was 9.6 ± 0.8 AcU/ml in 25 age-matched control subjects who underwent diagnostic coronary angiography because they complained of chest pain but had no detectable coronary artery disease.
Data are expressed as mean ± SE. Comparisons among the three groups were performed with one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test for multiple comparisons for continuous variables, or Kruskal-Wallis ANOVA on ranks followed by the Dann test for categorical variables. Correlation of the Gensini score or the Progression-Regression score with anti-Ox-LDL was assessed by a simple linear regression. Multiple regression analysis was performed for variables of the lipid profiles and anti-Ox-LDL predicting the Progression-Regression score; p < 0.05 was considered to be significant.
Lesion progression or regression during six-month follow-up
Out of 52 study patients, the Progression-Regression score was >0.15 mm in 20 patients (progression group), ≥0.15 mm and >−0.15 mm in 18 patients (no-change group) and ≥−0.15 mm in 14 patients (regression group). Patient backgrounds and medications, including lipid-lowering statins, were identical among the progression group, the no-change group and the regression group (Table 1). Table 2shows a comparison of coronary risk factors among the three groups. Significant differences were observed in the fasting plasma insulin level and Lp(a), but other coronary risk factors were identical in the three groups.
Anti-Ox-LDL and lesion progression
The anti-Ox-LDL titer was 16.6 ± 1.5 AcU/ml in the progression group, which was significantly higher (p < 0.001) than the value of 9.5 ± 1.2 in the regression group and also higher (p < 0.01) than the value of 11.4 ± 1.3 in the no-change group. There was no significant difference in the anti-Ox-LDL titer between the regression group and the no-change group (Fig. 1). The Gensini score on baseline coronary angiography was 53 ± 4 in all patients. The anti-Ox-LDL titer did not correlate with the Gensini score, but correlated with the Progression-Regression score (r = 0.56, p < 0.001) (Fig. 2). Multiple regression analysis showed that the Progression-Regression score was independently correlated with the anti-Ox-LDL titer (r = 0.53, p < 0.001) as well as Lp(a) (r = 0.38, p < 0.05). The most powerful predictor of Progression-Regression score was the anti-Ox-LDL titer (Table 3).
In the present study, we evaluated lesion progression or regression during a relatively short term of six months using a marker of Progression-Regression score assessed by quantitative coronary angiographic analysis. The major findings of our study are that the anti-Ox-LDL titer was higher in the progression group than in the regression group and the no-change group, and that the antibody titer correlated with the Progression-Regression score. These results indicate that the anti-Ox-LDL titer can predict lesion progression or regression over a six-month period.
Immunologic response to oxidized LDL
Autoimmune factors have been shown to play a dominant role in the progression of atherosclerotic plaque (15). Autoantigens that have been suggested to play a role in the autoimmune process include oxidized LDL (16,17). Specific immunologic epitopes expressed on oxidized LDL were found in atherosclerotic lesions both in animals with experimental atherosclerosis and in humans (3,18). T-cell-mediated immune responses occur in such lesions. Thus, oxidized LDL is an important local antigen in atherosclerosis and such local T-cell responses may induce B-cell activation, with concomitant systemic antibody production (19). Therefore, anti-Ox-LDL can be considered a marker of LDL oxidation at the tissue level on the vascular wall.
Anti-Ox-LDL as a marker of atherosclerotic coronary artery disease
In previous reports, anti-Ox-LDL antibodies have been shown to correlate with atherosclerotic coronary (5,6), carotid (7)and peripheral artery disease (8). In several other studies, however, no such relationships have been found between atherosclerotic disease and the antibody titer (9). This discrepancy may be because the importance of anti-Ox-LDL responses varies depending on the stage of atherosclerosis. On the other hand, Salonen et al. (7)demonstrated, in a prospective ultrasound observation of carotid atherosclerosis, that the anti-Ox-LDL titer was correlated with the rate of lesion progression, but not with baseline intima-media thickness. This fact suggests that the anti-Ox-LDL titer may be a clinically useful marker for the rate of lesion formation or disease activity, rather than a marker for atherosclerosis severity. In the present study, lesion progression or regression was evaluated via a six-month follow-up. We speculate from our own study that anti-Ox-LDL may be associated with relatively short-term lesion progression.
We previously demonstrated that the anti-Ox-LDL titer level was elevated in severe coronary artery disease but not in mild coronary artery disease, using 126 consecutive patients suspected of coronary artery disease who underwent diagnostic coronary angiography. We also showed that the titer was higher in a subgroup of unstable angina than in other groups. We concluded in these previous studies that the anti-Ox-LDL titer level may be predictive of severe coronary artery disease or of plaque instability (20). In the present study, however, the anti-Ox-LDL titer did not correlate with the baseline severity of coronary artery disease assessed by the Gensini score. This was probably because the patient population in the present study largely included patients with relatively mild coronary artery disease. More interestingly, in multiple regression analysis in the present study, the anti-Ox-LDL titer independently correlated with the Progression-Regression score but the serum LDL level did not. This result indicates that the anti-Ox-LDL titer can predict lesion progression independently of the serum LDL level itself. In our results, Lp(a) was also higher in the progression group than in the other group. In addition, multiple regression analysis showed that Lp(a) correlated with the Progression-Regression score. Lipoprotein(a), which consists of LDL-like particles binding with apo B and apo (a), is thought to inhibit the activity of the fibrinolytic system and to be an independent coronary risk factor (21). It has been reported that Lp(a) could predict the severity and extension of coronary atherosclerosis (22). Our results suggest that Lp(a) can also predict short-term lesion progression. However, the predictive ability of anti-Ox-LDL for lesion progression was higher than that of Lp(a).
MDA-induced oxidation of LDL
In the present study, we used a commercial kit for anti-Ox-LDL measurement. In this assay, we measured the antibody against an epitope of MDA-induced Ox-LDL. Recently, MDA-modified LDL has been noticed because of its roles in the atherosclerotic process, and its plasma level is considered to be associated with plaque inflammation or plaque instability (23). In addition, the antibody against MDA-induced-Ox-LDL was not only elevated in patients with acute myocardial infarction (AMI) (24), but also could predict the occurrence of AMI (5,25). These results suggest that MDA-induced LDL oxidation may play an important role in the development of plaque instability. The present study indicates that MDA-induced LDL oxidation may also affect the short-term progression of coronary atherosclerosis and also suggests the importance of MDA-induced LDL oxidation.
We evaluated lesion progression or regression only by changes in the minimal lumen diameter, using quantitative coronary angiographic analysis. Measurement of the plaque area or plaque volume by intravascular ultrasound would have provided more detailed information about lesion progression or regression. Another limitation is the possibility that medications continued during the follow-up term, such as nitrates, beta-blockers, calcium blockers, aspirin, ticlopidine or lipid-lowering statins, affected lesion progression or regression. In our study population, however, these medications were identical in all three groups: the progression, no-change and regression groups.
The anti-Ox-LDL titer correlated with short-term lesion progression or regression, rather than the baseline severity of coronary artery disease. This antibody titer may be an independent predictor of short-term lesion progression.
We gratefully acknowledge the technical support services of TFB Incorporation. We thank Mr. Hideyuki Maeda of TFB Inc. and Mrs. Kumiko Inoue of Dokkyo University for their help in ELISA assay of the anti-Ox-LDL.
☆ This study was supported in part by a grant from the Vehicle Racing Commemorative Foundation, Japan.
- acute myocardial infarction
- analysis of variance
- antibody against oxidized low density lipoprotein
- enzyme-linked immunosorbent assay
- low density lipoprotein
- malonic dialdehyde
- remnant-like lipoprotein particle
- Received September 25, 2000.
- Revision received January 23, 2001.
- Accepted February 6, 2001.
- American College of Cardiology
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