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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:441-445.)
© 1995 American Heart Association, Inc.

Articles

Patients With Early-Onset Peripheral Vascular Disease Have Increased Levels of Autoantibodies Against Oxidized LDL

C. Bergmark; R. Wu; U. de Faire; A. K. Lefvert; J. Swedenborg

From the Departments of Surgery (C.B., J.S.) and Medicine (R.W., U. de F., A.K.L.), Karolinska Hospital, Stockholm, Sweden.

Correspondence to Jesper Swedenborg, MD, Department of Surgery, Karolinska Hospital, S-171 76 Stockholm, Sweden.

	Abstract

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Abstract Oxidative modification of LDL has been proposed as an early and crucial step in the development of atherosclerosis, and antibodies against such modified LDL are found in both healthy individuals and patients with atherosclerosis. In this study, 62 patients who were surgically treated for peripheral arterial occlusive disease below the age of 50 were investigated and compared with age- and sex-matched healthy individuals in a case-control study. Autoantibodies against oxidized LDL were measured with an enzyme-linked immunosorbent assay method. Risk factors such as smoking, hypertension, family history of premature cardiovascular events, and lipoprotein levels were also determined. The patients had significantly higher levels of autoantibodies against oxidized LDL; significantly higher levels of total cholesterol, LDL cholesterol, triglycerides, and apo A-I; and significantly lower levels of HDL cholesterol than did control subjects. In multivariate analyses autoantibodies against oxidized LDL discriminated better between patients and control subjects than did any of the different lipoprotein analyses. Among patients, the presence of hypertension and a family history of cardiovascular events were the only factors significantly associated with increased levels of autoantibodies against oxidized LDL.

Key Words: lipoproteins • oxidation • risk factors • premature atherosclerosis • antibodies

	Introduction

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Oxidation of LDL has been suggested to be an important event in the development of atherosclerosis. Macrophages, which constitute the foam cells of the atherosclerotic plaque, do not take up LDL unless it undergoes modification, which occurs in vivo by oxidation. Oxidized LDL, however, is readily taken up by the scavenger receptor on the macrophage.^{1 2} The lipid content of the plaque mainly consists of oxidized lipids.³ Furthermore, the plasma of patients with coronary heart disease or peripheral arterial disease has increased levels of lipid peroxides,⁴ and LDL from patients who have experienced a myocardial infarction before the age of 45 is more readily oxidized in vitro than is the LDL from corresponding control subjects.⁵ Additional support for the concept that oxidized LDL is important in the development of atherosclerosis is that treatment with antioxidants prevents development of intimal thickening in hypercholesterolemic animals.^{6 7}

Oxidation of LDL is believed to take place mainly in the intima of the arterial wall, because the plasma compartment has an effective antioxidant defense system.⁸ Thus, evaluation of LDL oxidation must be performed by indirect methods, mainly the susceptibility of LDL to oxidation in vitro.⁸ This method of assessing oxidation susceptibility measures the total antioxidant capacity of LDL. The level of autoantibodies against oxidized LDL^{9 10} has also been suggested to reflect the in vivo oxidation of LDL, since such modification of LDL results in the appearance of new epitopes that render LDL more antigenic.¹¹ In addition, the presence of autoantibodies against oxidized LDL reflects the capacity of the immune system to respond to modified LDL. The latter aspect is important, because it has been suggested that the immunologic response per se may be significant for the development of atherosclerosis.^{11 12 13 14 15 16 17}

In this study, we report on the autoantibody levels against oxidized LDL in patients who were treated surgically for peripheral arterial occlusive disease (PAOD) before the age of 50. If LDL oxidation is an early and necessary step in atherosclerotic disease, then it could be assumed that factors contributing to LDL oxidation are more easily expressed in younger rather than older victims of atherosclerosis. The aim of the study was to relate the autoantibody levels against oxidized LDL to other risk factors for PAOD, such as hereditary factors, lipoprotein levels, and smoking.

	Methods

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Patients
The patient group consisted of 62 individuals, 30 males and 32 females, with PAOD that required surgical reconstruction before the age of 50. The mean age was 42 years at onset of symptoms, 44 years at surgery, and 49 years at follow-up. The indication for surgery was lower-extremity ischemia in 58 patients. Two patients each had carotid stenosis and renal artery stenosis. Of the patients with lower-extremity ischemia, preoperative angiograms revealed that 28 had their occlusive disease confined to the suprainguinal area, and 17, to the infrainguinal vessels; in 13, both suprainguinal and infrainguinal arteries were involved (multilevel disease). At the time of operation, 16 patients had hypertension, 6 had diabetes, and 7 had signs of coronary heart disease, which was defined as angina pectoris, previous myocardial infarction, and/or electrocardiographic signs of a myocardial infarction.

The patients were compared, in a case-control study, to sex- and age-matched control subjects selected from the population register. Investigations included clinical examination with measurement of blood pressure, interviews concerning smoking habits, and family history of premature cardiovascular events, which were defined as myocardial infarction or PAOD requiring surgical correction before the age of 55 in blood siblings. Hypertension was defined as ongoing use of medication for high blood pressure and/or a resting arm blood pressure 170 mm Hg systolic and/or 95 mm Hg diastolic. Control subjects also completed a health declaration.

At surgery, all patients but two were smokers, with a mean consumption rate of 20 cigarettes/d for 20 years. At follow-up, 32 still smoked, but 30 had stopped. Among control subjects 19 (28%) were smokers.

At follow-up and after an overnight fast, venous blood samples were taken from patients and control subjects for measurement of autoantibodies against oxidized LDL and plasma concentrations of HDL cholesterol, LDL cholesterol, triglycerides, total cholesterol, apo A-I and B, and lipoprotein(a) [Lp(a)].

Isolation and Modification of LDL
LDL was isolated from human plasma collected in Na₂-EDTA (1 mg/mL). Plasma was recovered by means of low-speed centrifugation at 1400g for 20 minutes at 1°C and kept at this temperature throughout the separation. LDL was isolated from plasma in the density interval 1.025 to 1.050 kg/L by sequential preparative ultracentrifugation in a 50.3 Ti Beckman fixed-angle rotor (Beckman L8-80 ultracentrifuge) for 48 hours.¹⁸ The protein content of the LDL preparation was determined by the method of Lowry et al.¹⁹ Before oxidation LDL was dialyzed against PBS, pH 7.4, for 24 hours. LDL (200 µg/mL) was oxidized by exposure to 10 mmol/L CuSO₄ at 37°C overnight. The preparation was sterilized by filtration (0.22 µm), kept in RPMI-1640 medium (GIBCO Laboratories) containing 1% human type AB Rh-positive serum, and stored in the dark at 4°C.

Determination of Autoantibodies Against Native and Oxidized LDL
Antibodies of IgG, IgA, and IgM isotypes were determined by an enzyme-linked immunosorbent assay (ELISA). Ninety-six–well plates (Costar) were coated with 0.2 µg of either native or oxidized LDL per well in 100 µL coating buffer (carbonate-bicarbonate buffer, 50 mmol/L, pH 9.7) and incubated overnight at 4°C. The plates were washed three times with PBS containing 0.05% polyoxyethylenesorbitan monolaurate (Tween 20) and incubated overnight at 4°C with 100 µL serum diluted 1/10 in sample buffer (PBS, pH 7.4, containing 0.05% Tween 20). After they were washed three times, the plates were incubated at 37°C for 2 hours with alkaline phosphatase–conjugated goat anti-human immunoglobulin diluted 1/1000 (Sigma 18758). The plates were developed using the substrate disodium p-nitrophenyl phosphate, 1 mg/mL, in 1 mol/L diethanolamine buffer, pH 9.8, for 30 minutes at room temperature. The absorbance was read at 405 nm. The intra-assay coefficient of variation between triplicate tests was <9%, and the interassay coefficient was 12.5%. The results were obtained with native and oxidized LDL preparations that were less than 1 week old.

Serum Lipids and Lp(a)
Serum cholesterol and triglycerides were measured with an enzymatic colorimetric assay system (Boehringer-Mannheim automatic analyses for Hitachi system 717; Diagnostica). HDL cholesterol was measured after lipoproteins that contained apo B were precipitated with phosphotungstate–magnesium chloride. LDL cholesterol was calculated according to the formula of Friedewald et al. Lp(a), apo A, and apo B were measured with Tint-Elize (Biopool, Umeå, Sweden).

Statistical Methods
Continuous variables for patients and control subjects are expressed either as median values with ranges or as means±SEM. Wilcoxon's nonparametric test was used for calculating differences between means; for multivariate analyses, JMP software (SAS Institute) was used. Continuous variables that were not normally distributed were logarithmically transformed before inclusion in the statistical analysis. Residuals for the full model in multivariate analysis were tested for normal distribution. When discriminating factors were analyzed a multiple logistic regression model was used, and when factors associated with autoantibody levels were analyzed a multiple regression model was used. Both models were tested stepwise, forward and backward, and also tested for influence of outliers.

	Results

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Comparison Between Patients and Control Subjects
The level of autoantibodies against oxidized LDL was significantly higher in patients compared with that of control subjects (P<.0001). The actual levels were 0.36±0.007 absorbance units for patients versus 0.30±0.007 for control subjects (mean±SEM) (Fig 1). There was no corresponding significant difference between the level of autoantibodies against native LDL: 0.23±0.013 absorbance units for the control subjects and 0.25±0.014 for patients (P=.5). When individual differences between levels of autoantibodies against native and oxidized LDL were compared, there was also a significant difference between the two groups: 0.11±0.010 absorbance units for patients and 0.07±0.014 for control subjects (mean±SEM, P=.006).

View larger version (15K): [in this window] [in a new window]   Figure 1. Box plot of autoantibody levels against oxidized LDL in patients and control subjects, showing a significant difference (P<.0001, Wilcoxon) between the two groups. The box includes observations from the 25th to the 75th percentile; the horizontal line within the box represents the median value. Lines outside the box represent the 10th and 90th percentiles. The width of the box is proportional to the number of observations.

Significant differences between patients and control subjects for total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and apo A-I were also found, but there were no differences in the levels of Lp(a) and apo B (Table 1).

View this table: [in this window] [in a new window]   Table 1. Serum Lipid and Lipoprotein Levels in Patients and Control Subjects

A multiple logistic regression analysis was performed to determine which factors discriminated between patients and control subjects. In the full model all parameters in Table 1, in addition to the factors: levels of autoantibodies against oxidized LDL, hypertension, diabetes, and smoking habits, were included. In Table 2, parameters that significantly discriminate between patients and control subjects are shown.

View this table: [in this window] [in a new window]   Table 2. Multivariate Analysis of Parameters Discriminating Between Patients and Control Subjects

With this set of tests it was possible to correctly classify 80% of the patients and 85% of control subjects (prediction value).

Risk Factors in Patients
At follow-up 6 patients had diabetes mellitus, 28 had hypertension, and 13 had coronary heart disease. Twenty patients had a positive family history for premature vascular disease, and its influence was analyzed. Patients with a positive family history had significantly higher levels of autoantibodies against oxidized LDL (Fig 2).

View larger version (16K): [in this window] [in a new window]   Figure 2. Box plot of autoantibody levels against oxidized LDL in patients with and without a positive family history of premature vascular disease, showing a significant difference (P=.048, Wilcoxon) between the two groups. For additional explanation of box plot symbols, please refer to the legend to Fig 1.

The influence of hypertension and family history was analyzed in the patient group (those with lower-extremity ischemia). There was no difference in autoantibody level between patients with and those without hypertension, but in a multiple regression analysis of patients that examined the effect of different risk factors on levels of autoantibodies against oxidized LDL, both family history and hypertension were found to be the only significant factors. This finding can probably be explained by a nonadditive effect when hypertension and family history are combined. The factors included in this model were those in Table 1 in addition to age, sex, hypertension, family history, level of disease, diabetes, and smoking habits (Table 3). Furthermore, patients with both a positive family history and hypertension had higher levels of autoantibodies against oxidized LDL than did patients with only one or neither of these risk factors (Fig 3).

View this table: [in this window] [in a new window]   Table 3. Factors of Significance for Levels of Autoantibodies Against Oxidized LDL Among Patients in a Multiple Linear Regression Analysis

View larger version (20K): [in this window] [in a new window]   Figure 3. Box plot of autoantibody levels against oxidized LDL in patients with and without hypertension and a positive family history of cardiovascular disease. For additional explanation of box plot symbols, please refer to the legend to Fig 1.

It has been proposed that the levels of antibodies against oxidized LDL reflect the total surface area of diseased arteries.²⁰ To test this hypothesis, we compared patients with lower-extremity ischemia at different levels of disease. There was a tendency for higher autoantibody levels in patients with more extensive atherosclerotic lesions, but the differences between groups were not significant.

	Discussion

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Reports concerning the oxidative state of LDL in atherosclerotic vessel wall lesions are not uniformly conclusive. Thus, complete evidence for the biological importance of oxidative modification of LDL, based on its uptake by the scavenger receptor of macrophages, is still lacking.²¹ The present study is the first to report data on a population of patients with PAOD and to relate the levels of autoantibodies against oxidized LDL to other risk factors.

Different methods of LDL oxidation result in the appearance of different epitopes.²² The present study used oxidation by copper ions, which induces aldehyde epitopes and results in LDL oxidation products similar to those mediated by endothelial cells.²³ Salonen et al²⁴ have reported relations between ultrasound evaluations of carotid artery lesions in a population of men drawn from a population register and measurements of metabolic risk factors for PAOD and antibody titers against malondialdehyde-modified LDL. They found that individuals with the largest increases in intima-media thickness of the carotid artery over a 2-year observation period had the highest antibody titers against oxidized LDL. Their findings are in general agreement with ours.

The patients in the present study had elevated levels of total and LDL cholesterol and decreased levels of HDL cholesterol compared with those of control subjects. Levels of autoantibodies against oxidized LDL, however, discriminated better between patients and control subjects than any of the conventional lipoprotein measurements. These antibody levels were much lower when the results were expressed as the difference between oxidized and native LDL instead of the total antibody levels against oxidized LDL. Virella et al²⁵ and Schumacher et al²⁶ were unable to demonstrate differences in antibody levels against oxidized LDL when they compared patients with coronary disease and healthy control subjects. Neither were increased antibody levels found in a population of patients surviving a myocardial infarction before the age of 45 (A. Hamsten, personal communication, 1994). Patients with peripheral atherosclerotic disease have more widespread atherosclerotic lesions than do those with a myocardial infarction. This may provide one explanation for the difference in results, if one assumes that antibody levels against oxidized LDL are positively related to the extent of atherosclerosis, as suggested previously.²⁰ Alternatively, patients with more extensive disease have a larger mass of ischemic tissue capable of producing free radicals and thus an increased potential for lipid peroxidation.

Increases in diene production upon oxidation in vitro, as a measure of susceptibility to oxidation, have been demonstrated in LDL from young male survivors of myocardial infarction.⁵ Such an increased tendency for LDL oxidation in vitro was, however, not found in a patient group with claudication.²⁷ The tendency for LDL oxidation in vitro, however, is distinctly different from measurements of antibody titers against oxidized LDL, because the latter also evaluates the capacity of the immune system to react to modified LDL.

The finding of an association between high levels of autoantibodies against oxidized LDL and a family history of premature vascular disease among patients indicates that genetically controlled mechanisms are operating in the atherosclerotic process. The capacity to form autoantibodies is influenced by several lines of genetic control.¹¹ The present cross-sectional study cannot establish any causal relationships between formation of autoantibodies and development of atherosclerosis. Nor has an association between family history and levels of endothelial cell antibodies or cardiolipin antibodies been demonstrated in this patient group (S. Nitayand et al, unpublished observations, 1994). It has been reported that antibodies against oxidized LDL and cardiolipin partly recognize the same epitopes,²⁸ but no such correlation between the different autoantibody species could be demonstrated in the present group of patients.

An association between hypertension and oxidized LDL has been suggested by Martin-Nizard et al,²⁹ who demonstrated that human macrophages secrete endothelin after stimulation by oxidized LDL. Oxidized LDL may also impair endothelium-dependent arterial relaxation.³⁰ Both of these mechanisms provide a link between oxidation of LDL and hypertension. Moreover, the lysolecithin that is formed during oxidation of LDL has been proposed to contribute to hypertension.³¹ Recently Maggi et al³² reported elevated levels of antibodies against oxidized LDL in a population of middle-aged hypertensive patients. Their finding agrees with the present study, which has demonstrated an association between hypertension and levels of autoantibodies against oxidized LDL.

Smoking makes plasma LDL more susceptible to oxidation,³³ partly by decreasing the content of vitamin E, which prevents LDL oxidation.³⁴ In the present study the absence of an influence of current smoking on levels of autoantibodies against oxidized LDL may be explained either by the fact that all patients except two were heavy smokers at the time of surgery or that the autoantibody levels reflected the state of the arterial wall rather than of the plasma. Only a minor fraction of plasma LDL is considered to undergo oxidation, because plasma normally contains high levels of antioxidants.⁸ Smoking, as expressed in pack-years, could possibly cause LDL oxidation, but this was not shown in this study when tested for in the multiple regression model, possibly because of the small variation in this parameter in a patient population that was too small.

In conclusion, young patients who are surgically treated for PAOD have increased levels of autoantibodies against oxidized LDL. We found no association between these levels and classic risk factors, such as age, male sex, and plasma lipoproteins, but there was an association between levels of autoantibodies and the presence of hypertension and a family history of premature cardiovascular disease.

	Acknowledgments

 
This study was supported by the Swedish Heart-Lung Foundation, the King Gustav V Research Foundation, the Knut and Alice Wallenberg Foundation, the Nanna Schwartz Foundation, and the Petrus and Augusta Hedlund Foundation.

Received September 27, 1994; accepted January 20, 1995.

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