This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsimikas, S. Articles by Witztum, J. L. Search for Related Content PubMed PubMed Citation Articles by Tsimikas, S. Articles by Witztum, J. L. Related Collections Lipids Animal models of human disease Pathophysiology Genetically altered mice Imaging Other diagnostic testing Lipid and lipoprotein metabolism Oxidant stress Mechanism of atherosclerosis/growth factors

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:95.)
© 2001 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Circulating Autoantibodies to Oxidized LDL Correlate With Arterial Accumulation and Depletion of Oxidized LDL in LDL Receptor–Deficient Mice

Sotirios Tsimikas; Wulf Palinski; Joseph L. Witztum

From the Divisions of Cardiovascular Diseases (S.T.) and Endocrinology and Metabolism (W.P., J.L.W.), Department of Medicine, University of California, San Diego.

Correspondence to Sotirios Tsimikas, MD, Department of Medicine, Division of Cardiology, University of California San Diego, 9500 Gilman Dr, BSB 1080, La Jolla, CA 92093-0682. E-mail stsimikas{at}ucsd.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—Autoantibodies to oxidized low density lipoprotein (OxLDL) are elevated in some human populations with increased risk of atherosclerosis. To determine whether autoantibody levels to epitopes of OxLDL reflect the extent of aortic atherosclerosis and the content of OxLDL, we measured IgG and IgM autoantibody titers to malondialdehyde (MDA)-LDL and copper-oxidized LDL (Cu-OxLDL) in 43 LDL receptor–deficient mice consuming atherogenic and regression diets. Antibody titers were correlated to percent atherosclerotic surface area, aortic weight, and aortic OxLDL content, measured as the in vivo uptake of ¹²⁵I-MDA2, a monoclonal antibody to MDA-LDL. All mice were fed an atherogenic diet for 6 months, and 1 group was euthanized. The other 3 groups were fed an atherogenic diet (fat/CHOL group), normal mouse chow (chow group), or mouse chow supplemented with vitamins E and C (chow+VIT group) for an additional 6 months. After dietary intervention, compared with their own baseline, autoantibody titers to MDA-LDL and Cu-OxLDL increased significantly in the fat/CHOL group, whereas they did not change or decreased significantly in the chow and chow+VIT groups. Aortic weight and surface area showed significant progression in the fat/CHOL group, mild progression in the chow group, and no progression in the chow+VIT group (P<0.001), whereas OxLDL content actually decreased in the latter 2 groups (P<0.001). Significant correlations were seen with MDA-LDL autoantibody titers and OxLDL content (IgM, R=0.64 and P=0.0009; IgG, R=0.52 and P=0.009), as well as with percent surface area and aortic weight. These data support the hypothesis that autoantibody titers to OxLDL reflect changes in OxLDL content in atherosclerotic lesions of LDL receptor–deficient mice. Whether autoantibody titers to OxLDL will provide similar valuable insights into the extent of human atherosclerosis, particularly anatomic measurements of plaque burden and OxLDL content, remains to be determined.

Key Words: regression • progression • atherosclerosis • lipoproteins • autoantibodies

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
It is generally accepted that oxidative modification of LDL is intimately involved in the initiation and progression of atherosclerotic lesions.¹ Human atherosclerotic lesions have been shown to contain abundant quantities of oxidized lipoproteins within foam cells and in the extracellular space.^{2 3 4 5 6} In patients with myocardial infarction, atherosclerotic lesions undergoing plaque rupture, erosion, and thrombosis often contain large lipid pools with significant amounts of extracellular oxidized lipids and oxidized LDL (OxLDL).^{7 8} Indeed, recent evidence suggests that plasma levels of malondialdehyde (MDA)-LDL, an epitope of OxLDL derived from lipid peroxidation, is elevated in patients with coronary artery disease and is a strong predictor of acute coronary syndromes.^{9 10}

The immune system plays a significant role in modulating the development of atherosclerosis.^{11 12} Our laboratory originally demonstrated that OxLDL is highly immunogenic and that autoantibodies to various epitopes of OxLDL are prevalent in humans and animals with atherosclerosis.^{2 13 14} Because LDL undergoes oxidative modification in vivo in atherosclerotic lesions, it is therefore likely that OxLDL within atherosclerotic tissues may induce the generation of circulating autoantibodies.^{2 3 14 15 16 17 18} This is also supported by the observation that a surprisingly high percentage of T lymphocytes isolated from human atherosclerotic lesions specifically recognizes OxLDL, suggesting that they are involved in immune activation and inflammation of atherosclerotic plaques.¹⁹ A corollary of these observations is that the autoantibody titers may reflect the atherogenic process. Indeed, previous studies have shown that autoantibody titers to OxLDL are elevated in humans and animals with atherosclerosis.^{11 20} However, the relationship between the extent of atherosclerosis and the humoral immune response is complex. Most of the human studies have been hampered by the lack of a precise cumulative assessment of atherosclerosis, and the autoantibody correlations have, in large part, been based on clinical surrogates. In addition, only very limited data from animal models are available on the relationship between autoantibody titers and the overall extent of atherosclerosis,²¹ and no published studies exist addressing the relationship between autoantibody titers and the progression or regression of atherosclerosis.

The most commonly used measure of atherosclerosis in animal models is percent atherosclerotic surface area of the aorta. In a previous study in LDL receptor–deficient (LDLR^-/-) mice, we have shown that OxLDL content may be measured by intravenous injection of radiolabeled oxidation-specific antibodies (¹²⁵I-MDA2).²² In that study, we induced lesion progression and regression and measured 3 parameters of atherosclerotic burden, ie, percent atherosclerotic surface area, aortic weight, and aortic uptake of ¹²⁵I-MDA2. Focusing on the relationship between these parameters, we showed that in progressing atherosclerosis induced by a high-fat high-cholesterol diet, plaque uptake of ¹²⁵I-MDA2 was correlated exceedingly well with the percent atherosclerotic surface area and the aortic weight (which reflects plaque volume). After prolonged exposure of mice with preexisting lesions to regular mouse chow with or without antioxidants, the ¹²⁵I-MDA2 uptake indicated "regression," ie, reduced lesion content of OxLDL, which was confirmed by immunostaining. In contrast, the percent surface area and aortic weight showed slight or no progression of atherosclerosis rather than regression. The ¹²⁵I-MDA2 uptake technique therefore complements the traditional anatomic measurements of plaque size during lesion progression but is more sensitive to decreases in lesion content of OxLDL.

Using the sera and data on atherosclerosis from that study, we now address the hypothesis that autoantibodies to epitopes of OxLDL reflect the progression and regression of experimental atherosclerosis assessed by anatomic measurements of atherosclerosis and determination of OxLDL content.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animal Models and Dietary Intervention
The design of the intervention study has been described in detail previously²² and is summarized in Figure 1. In brief, 6-month-old male LDLR^-/- mice (n=43) were placed on an atherogenic diet containing 21% milk fat and 1.25% cholesterol for 6 months to induce atherosclerosis. The mice were then divided into 4 groups. Group I (n=13) was euthanized as the baseline control group. For an additional 6 months, groups II (chow group, n=10) and III (chow+VIT group, n=10) were placed on hypocholesterolemic "regression diets" consisting of regular chow and of regular chow supplemented with antioxidants (1.0% vitamin E added to the chow and 0.05% vitamin C added to the drinking water), respectively. Group IV (fat/CHOL group, n=10) was continued on the high-fat/cholesterol diet for 6 months. Atherosclerosis was measured in terms of percent Sudan-positive aortic surface area, aortic weight, and uptake of ¹²⁵I-MDA2, as previously described.²² Throughout the study, blood samples were obtained from the retro-orbital plexus and placed in EDTA tubes, and the plasma was isolated and stored at -70°C. These studies were approved by the Animal Subjects Committee of the University of California, San Diego.

View larger version (10K): [in this window] [in a new window]   Figure 1. Schematic representation of the protocol used in the mouse progression/regression study.

Determination of Antibody Binding to MDA-LDL and Cu-OxLDL
The levels of IgM and IgG autoantibodies binding to MDA-LDL (anti–MDA-LDL) and Cu-OxLDL (anti–Cu-OxLDL) were determined in murine sera by use of a chemiluminescence ELISA.²³ Autoantibody levels were determined in individual samples from all 4 groups of mice after the initial 6 months of the atherogenic diet and in the chow, chow+VIT, and fat/CHOL groups after 6 more months of dietary intervention. Antigens for the ELISA, MDA-LDL or Cu-OxLDL, were generated as described.²⁴ In this assay, 5 µg/mL of the antigen in 50 mmol/L Tris-buffered saline (TBS), pH 7.5, containing 0.27 mmol/L EDTA, 0.02% azide, and 20 µmol/L butylated hydroxytoluene (dilution buffer), was added to each well of a 96-well white round-bottomed MicroFluor microtitration plate (Dynatech Laboratories, Inc) and incubated overnight at 4°C. Plates were washed 4 times with washing buffer (TBS containing 0.27 mmol/L EDTA, 20 µmol/L butylated hydroxytoluene, 0.02% NaN₃, and 0.001% aprotinin) with the use of an automated plate washer. Murine sera were diluted 1:500 in dilution buffer containing 1% BSA, and 50 µL was added to each well and incubated for 1 hour at room temperature. After 4 washes, plates were incubated with 50 µL per well of an alkaline phosphatase–labeled goat anti-mouse IgG (-chain specific) or phosphatase-labeled goat anti-mouse IgM (µ-chain specific, both from Sigma) for 1 hour at room temperature. These antibodies were diluted in 1% BSA/TBS according to the supplier’s specifications. After the plates were washed, 25 µL of a 50% solution of Lumi-Phos 530 (Lumigen) was added to each well, and the plates were incubated for 1 to 2 hours at room temperature in the dark, and luminescence was determined. Antibody binding was measured as relative light units (RLU) in 100 milliseconds. Triplicate determinations were performed for each plasma sample. Measurement of antibody binding to a given antigen was performed in a single assay. A high and a low standard serum was included on each plate of a given assay to detect potential variations between microtitration plates. The intra-assay coefficients of variation for these assays were 6% to 10%.

Statistical Analysis
Statistical analyses were performed with ANOVA and paired t test for post hoc analysis when appropriate. Correlations between parameters of atherosclerosis and autoantibody titers were tested by linear regression analysis. Data shown are mean±SD, unless otherwise indicated.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Autoantibody Determinations
The mean autoantibody levels in the entire cohort of 43 mice after 6 months of the atherogenic diet were as follows: anti–MDA-LDL IgG, 2525±1228 RLU; anti–MDA-LDL IgM, 15182±6412 RLU; anti–Cu-OxLDL IgG, 1285±587 RLU; and anti–Cu-OxLDL IgM, 3562±1777 RLU. These levels are generally much higher than those found in chow-fed C57B6 mice.^{14 24} At the end of the initial 6 months on a high-fat diet, mice were divided into the 4 experimental groups. There were no significant differences in the autoantibody titers among the groups except for the anti–MDA-LDL IgM titers, which were higher in the mice assigned to the chow and chow+VIT groups (P=0.02, data not shown). Note that the assignment of animals to the groups was aimed at matching cholesterol levels (and thus, presumably, atherosclerosis) at baseline, whereas no matching for antibody levels was performed as part of the original design.²² The baseline control group was euthanized at this time, whereas the other 3 groups were continued under the study protocol (Figure 1) for another 6 months. At the end of the study, levels of anti–MDA-LDL and anti–Cu-OxLDL autoantibody titers were consistently higher (1.2- to 1.5-fold) in the fat/CHOL group compared with the other groups (Table).

View this table: [in this window] [in a new window]   Table 1. OxLDL Autoantibody Titers in LDLR-/- Mice at End of Study

Because this comparison uses mice at different time points (the baseline control group was 6 months younger than the other 3 groups at the terminal autoantibody measurement) and because starting levels of autoantibodies were not equal, for all subsequent analyses, we compared the changes in autoantibody levels for each individual animal within each group, ie, before and after the dietary intervention. Figure 2A summarizes the mean changes in OxLDL autoantibody levels in the 3 intervention groups. When changes were compared within each group, in the fat/CHOL group, there was a significant increase in all autoantibodies to OxLDL, with anti–MDA-LDL IgG showing the greatest increase (81%, P<0.0001 compared with the preintervention values in the same group of mice). In contrast, in the chow and chow+VIT groups, the IgG autoantibody titers for anti–MDA-LDL and anti–Cu-OxLDL did not change, whereas the IgM titers actually decreased significantly after the regression/antioxidant diet. The corresponding changes in the cumulative measures of atherosclerosis and plaque OxLDL content are shown in Figure 2B. Because only 1 measurement was possible in these parameters (at the time of euthanasia), the atherosclerosis indices were compared with those in the baseline control group. As expected, because of the continued progression of lesions in the fat/CHOL group, all 3 atherosclerosis indices increased significantly compared with those in the baseline control group (P<0.001). There was reduced progression of atherosclerosis measured by surface area (P<0.001) but not aortic weight in the chow group. No significant changes in surface area or aortic weight were seen in the chow+VIT group. In contrast, significant reductions were noted in the OxLDL content, as reflected by ¹²⁵I-MDA2 aortic uptake, in the chow and chow+VIT groups (P<0.001 for both). Therefore, MDA-LDL and Cu-OxLDL autoantibody levels qualitatively reflected the changes seen in the atherosclerosis indices and OxLDL content.

View larger version (28K): [in this window] [in a new window]   Figure 2. A, For each mouse, autoantibody titers were measured at baseline and after completion of the experimental protocol outline in the scheme. The bar graph represents the mean percent change in OxLDL autoantibody titers in the 3 groups of mice after dietary/antioxidant intervention or continued high-fat/cholesterol diet. The probability value represents paired t test comparing autoantibody levels at baseline and after dietary/antioxidant intervention. B, Corresponding changes in percent atherosclerotic surface area, aortic weight, and 125I-MDA2, a measure of plaque OxLDL content, are shown for the same groups. Because only 1 measurement of atherosclerosis for each mouse was possible in this analysis, the changes in atherosclerosis indices are compared with the baseline control group values. The probability value represents the differences compared with the baseline control group. *P<0.001.

Correlations Between Autoantibody Levels and Atherosclerosis Indices
Correlations were carried out between atherosclerosis indices and the percent change in OxLDL autoantibody levels. Figure 3 shows the correlations between MDA-LDL autoantibody titers and the percent surface area, aortic weight, and uptake of ¹²⁵I-MDA2. Highly significant correlations were seen with atherosclerosis parameters and anti–MDA-LDL autoantibodies, particularly with aortic uptake of ¹²⁵I-MDA2 (for IgM, R=0.64 and P=0 0.0009; for IgG, R=0.52 and P=0.009). The correlations with anti–Cu-OxLDL IgG were more modest (for ¹²⁵I-MDA2, R=0.42 and P=0.044; for aortic weight, R=0.45 and P=0.033; and for percent surface area, R=0.37 and P=0.075). A trend was noted for the correlation between anti–Cu-OxLDL IgM and ¹²⁵I-MDA2 uptake (R=0.37, P=0.09), but there was no correlation with lesion area or weight.

View larger version (27K): [in this window] [in a new window]   Figure 3. Correlations between anti–MDA-LDL IgM (A through C) and IgG (D through F) autoantibody titers and percent atherosclerotic surface area, aortic weight (reflecting plaque volume), and in vivo aortic uptake of 125I-MDA2 (reflecting OxLDL content).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we demonstrate that autoantibody titers to OxLDL are significantly influenced by diet and antioxidant supplementation in atherosclerotic LDLR^-/- mice, and we provide evidence that they indicate lesion progression as well as depletion of OxLDL during dietary regression. Plasma autoantibody levels to OxLDL increased significantly in mice continually fed an atherogenic diet but did not change or actually decreased in mice that were switched to a regression diet consisting of normal mouse chow or chow supplemented with vitamins E and C. A good correlation with traditional measures of atherosclerosis, such as percent atherosclerotic surface area or aortic weight, was noted for anti–MDA-LDL IgG and IgM autoantibodies. An even stronger correlation was noted with the in vivo aortic uptake of ¹²⁵I-MDA2, which reflects the in vivo OxLDL content of lesions. These data provide evidence that in vivo ¹²⁵I-MDA2 aortic uptake accurately reflects the presence of MDA-LDL and malondialdehyde-lysine epitopes within the plaque in atherosclerosis progression and "regression", ie, depletion of MDA-LDL and malondialdehyde-lysine epitopes.

In humans, elevated titers of OxLDL autoantibodies have been documented in patients with myocardial infarction and angiographically documented coronary artery disease, peripheral vascular disease, and accelerated progression of carotid atherosclerosis.^{20 25 26 27 28} Elevated titers also seem to predict impaired endothelial function by demonstrating an inverse correlation with forearm blood flow in hypercholesterolemic smokers and with reduced coronary flow reserve assessed by positron emission tomographic scanning.^{29 30 31} In animal models, apoE-deficient and LDLR^-/- mice have been shown to have elevated titers of autoantibodies to multiple epitopes of OxLDL.^{13 14 21} Freigang et al³² have also shown that immunization of LDLR^-/- mice with homologous MDA-LDL results in markedly increased antibody titers to OxLDL, which were associated with a protective role against atherosclerosis. More recently, Cyrus et al³³ showed that disruption of the 12/15-lipoxygenase gene in apoE-deficient mice resulted in reduced aortic root lesions and markedly diminished autoantibody titers to MDA-LDL and Cu-OxLDL. These human and animal studies clearly demonstrate that the humoral responses to OxLDL reflect changes within atherosclerotic plaques.

The potential role of autoantibodies to OxLDL as indicators of the regression of atherosclerosis has not been previously studied. Our data demonstrate that autoantibody titers to epitopes of OxLDL are well correlated with changes in atherosclerotic lesions, particularly their content of OxLDL. We have previously shown that in vivo aortic uptake of ¹²⁵I-MDA2 is a sensitive marker of the progression and regression of atherosclerosis. The fact that the uptake of ¹²⁵I-MDA2 decreased as a result of regression diets clearly suggests that the lesion content of OxLDL decreases, and this was confirmed by immunohistochemistry.²² Although antigen formation may also occur elsewhere in lipid-rich tissues and may equally be affected by the regression diets, our results support the assumption that the decrease in antibody titers is at least in part the result of the reduced presence of OxLDL in the aorta. The analogy in the changes in autoantibody titers and atherosclerosis (Figure 2) and the correlations between the these parameters (Figure 3) also support this conclusion.

The autoantibody titers were qualitatively similar in the chow and chow+VIT groups, but the surface area and aortic weight were different, although only the surface area was statistically significant between the 2 groups. This apparent discrepancy likely reflects the fact that the autoantibody titers more accurately indicate the content of OxLDL within the lesions, which were similarly reduced in both groups to the same extent, rather than other lesion components that contribute to plaque mass.

The presumed etiology of the bulk of the "oxidation-specific" antigens responsible for the generation of these autoantibodies is thought to be the vessel wall. However, it is possible that the marked hypercholesterolemia could have induced increased oxidation-specific epitopes elsewhere as well.³⁴ In addition, it has been shown that oxidized cholesterol and oxidized lipids in the diet accelerate the development of atherosclerosis in mice and rabbits.^{35 36 37} In those studies, the dietary levels of oxidation byproducts were significantly increased by heating vitamin E–depleted corn oil or cholesterol to 100°C for several hours. In contrast, in the present study, all diets were refrigerated until time of use and were not vitamin E–depleted. Although we did not measure the levels of dietary peroxides in the present study, the plasma vitamin E levels (corrected to total plasma cholesterol) were higher in the fat/CHOL group than in the chow group, which may have provided some antioxidant protection.²²

It is not surprising that the autoantibody titers to MDA-LDL showed the best correlation with the in vivo aortic uptake of ¹²⁵I-MDA2, because of the identity of the antigens. However, the observation that antibodies to Cu-OxLDL are also correlated to some extent with ¹²⁵I-MDA2 aortic uptake emphasizes the fact that the uptake of ¹²⁵I-MDA2 provides a representative measure of OxLDL in the artery wall, not just a measure of its content in MDA-lysine epitopes. Indeed, although MDA-LDL is only 1 of many epitopes of OxLDL, it is clearly ubiquitous in atherosclerotic lesions.^{3 5 6 13 15 16 38 39} The correlations with lesion size were also much better for MDA-LDL than for Cu-OxLDL autoantibodies. Besides MDA-lysine epitopes, copper-induced oxidation of LDL also generates many other epitopes, such as oxidized phospholipids. Therefore, some differences between these autoantibody measurements would be expected. Additionally, the measured plasma autoantibody levels reflect an equilibrium between rates of formation and removal as well as immune complex formation (which we did not measure in the present study), which may more accurately reflect the development or regression of lesions.¹¹

Measurements of autoantibody titers to OxLDL have not yet been reported in human studies of dietary or pharmacological regression. Whether these measurements will be useful in the clinical arena remains to be seen. The cholesterol levels in the LDLR^-/- mice after a Western-type diet are very high and are not usually seen in human subjects. The dietary regimens also resulted in drastic reductions in total cholesterol levels. The lesions induced, however, do reflect human lesions in many respects; therefore, we believe that the relevance of this model in humans is valid in principle. It is likely, however, that changes in humans will be much more modest. Nevertheless, depletion in the content of OxLDL may occur in patients treated with hypolipidemic agents and antioxidants over longer periods of time; this depletion will, in turn, be reflected in reduced autoantibody titers.

Significant effort has gone into developing techniques that provide clinically useful information on atherosclerotic lesions in patients. Unfortunately, most of the current gold-standard techniques are invasive, and we lack noninvasive techniques to either image the vessel wall or to ascertain plaque composition. The present data suggest that measurement of autoantibody titers to OxLDL can provide at least a cumulative measure of plaque OxLDL content. Whether measurement of autoantibody titers to OxLDL will be useful in assessing the regression of human atherosclerotic lesions needs to be evaluated in studies using quantitative techniques (such as intracoronary ultrasound) that completely assess the atherosclerotic plaque burden.⁴⁰

	Acknowledgments

 
This investigation was supported by an National Heart, Lung, and Blood Institute (NHLBI) Mentored Clinical Scientist Development Award (HL-07444 to S.T.), by a New Investigator Award from the California Tobacco-Related Diseases Research Project (HT-0106 to S.T.), and by an NHLBI grant (HL-56989, La Jolla Specialized Center of Research in Molecular Medicine and Atherosclerosis to W.P. and J.L.W.). We would like to thank Jennifer Pattison, Florencia Casanada, Mercedes Silvestre, Elizabeth Miller, and Joseph Juliano for excellent technical assistance.

Received July 17, 2000; accepted October 6, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Tsimikas S, Witztum JL, The oxidative modification hypothesis of atherosclerosis. In: Keaney JF, ed. Oxidative Stress and Vascular Disease. Boston, Mass: Kluwer Academic Publishers; 2000:49–74.
2. Palinski W, Rosenfeld ME, Ylä-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A. 1989;86:1372–1376.[Abstract/Free Full Text]
3. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086–1095.
4. Carpenter KL, Taylor SE, van der Veen C, Williamson BK, Ballantine JA, Mitchinson MJ. Lipids and oxidised lipids in human atherosclerotic lesions at different stages of development. Biochim Biophys Acta. 1995;1256:141–150.[Medline] [Order article via Infotrieve]
5. Piotrowski JJ, Shah S, Alexander JJ. Mature human atherosclerotic plaque contains peroxidized phosphatidylcholine as a major lipid peroxide. Life Sci. 1996;58:735–740.[Medline] [Order article via Infotrieve]
6. Suarna C, Dean RT, May J, Stocker R. Human atherosclerotic plaque contains both oxidized lipids and relatively large amounts of alpha-tocopherol and ascorbate. Arterioscler Thromb Vasc Biol. 1995;15:1616–1624.[Abstract/Free Full Text]
7. Davies MJ, Richardson PD, Woolf N, Katz DR, Mann J. Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage, and smooth muscle cell content. Br Heart J. 1993;69:377–381.[Abstract/Free Full Text]
8. Felton CV, Crook D, Davies MJ, Oliver MF. Relation of plaque lipid composition and morphology to the stability of human aortic plaques. Arterioscler Thromb Vasc Biol. 1997;17:1337–1345.[Abstract/Free Full Text]
9. Holvoet P, Vanhaecke J, Janssens S, van de Werf F, Collen D. Oxidized LDL and malondialdehyde-modified LDL in patients with acute coronary syndromes and stable coronary artery disease. Circulation. 1998;98:1487–1494.[Abstract/Free Full Text]
10. Holvoet P, Collen D, Van de Werf F, Malondialdehyde-modified LDL as a marker of acute coronary syndromes. JAMA. 1999;281:1718–1721.[Abstract/Free Full Text]
11. Palinski W, Witztum JL. Immune responses to oxidative neoepitopes on LDL and phospholipids modulate the development of atherosclerosis. J Intern Med. 2000;247:371–380.[Medline] [Order article via Infotrieve]
12. Libby P, Hansson GK, Pober JS, Atherogenesis and inflammation. In: Chien KR, ed. Molecular Basis of Cardiovascular Disease. Philadelphia, Pa: Saunders; 1999:349–366.
13. Palinski W, Ord VA, Plump AS, Breslow JL, Steinberg D, Witztum JL. ApoE-deficient mice are a model of lipoprotein oxidation in atherogenesis: demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Arterioscler Thromb. 1994;14:605–616.[Abstract/Free Full Text]
14. Palinski W, Hörkko S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK, Witztum JL. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice: demonstration of epitopes of oxidized low density lipoprotein in human plasma. J Clin Invest. 1996;98:800–814.[Medline] [Order article via Infotrieve]
15. Haberland ME, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science. 1988;241:215–218.[Abstract/Free Full Text]
16. Rosenfeld ME, Palinski W, Ylä-Herttuala S, Butler S, Witztum JL. Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis. 1990;10:336–349.[Abstract/Free Full Text]
17. Boyd HC, Gown AM, Wolfbauer G, Chait A. Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe heritable hyperlipidemic rabbit. Am J Pathol. 1989;135:815–825.[Abstract]
18. Tsimikas S, Palinski W, Halpern SE, Yeung DW, Curtiss LK, Witztum JL. Radiolabeled MDA2, an oxidation-specific, monoclonal antibody, identifies native atherosclerotic lesions in vivo. J Nucl Cardiol. 1999;6:41–53.[Medline] [Order article via Infotrieve]
19. Stemme S, Faber B, Holm J, Wiklund O, Witztum JL, Hansson GK. T lymphocytes from human atherosclerotic plaques recognize oxidized low density lipoprotein. Proc Natl Acad Sci U S A. 1995;92:3893–3897.[Abstract/Free Full Text]
20. Ylä-Herttuala S. Is oxidized low-density lipoprotein present in vivo? Curr Opin Lipidol. 1998;9:337–344.[Medline] [Order article via Infotrieve]
21. Palinski W, Tangirala RK, Miller E, Young SG, Witztum JL. Increased autoantibody titers against epitopes of oxidized LDL in LDL receptor–deficient mice with increased atherosclerosis. Arterioscler Thromb Vasc Biol. 1995;15:1569–1576.[Abstract/Free Full Text]
22. Tsimikas S, Shortal BP, Witztum JL, Palinski W. In vivo uptake of radiolabeled MDA2, an oxidation-specific monoclonal antibody, provides an accurate measure of atherosclerotic lesions rich in oxidized LDL and is highly sensitive to their regression. Arterioscler Thromb Vasc Biol. 2000;20:689–697.[Abstract/Free Full Text]
23. Hörkkö S, Miller E, Dudl E, Reaven P, Curtiss L, Zvailfler NJ, Terkeltaub R, Pierangeli SS, Branch DW, Palinski W, et al. Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids: recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low-density lipoprotein. J Clin Invest. 1996;98:815–825.[Medline] [Order article via Infotrieve]
24. Palinski W, Ylä-Herttuala S, Rosenfeld ME, Butler SW, Socher SA, Parthasarathy S, Curtiss LK, Witztum JL. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis. 1990;10:325–335.[Abstract/Free Full Text]
25. Salonen JT, Ylä-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssonen K, Palinski W, Witztum JL. Autoantibody against oxidised LDL and progression of carotid atherosclerosis. Lancet. 1992;339:883–887.[Medline] [Order article via Infotrieve]
26. Puurunen M, Manttari M, Manninen V, Tenkanen L, Alfthan G, Ehnholm C, Vaarala O, Aho K, Palosuo T. Antibody against oxidized low-density lipoprotein predicting myocardial infarction. Arch Intern Med. 1994;154:2605–2609.[Abstract]
27. Wu R, Nityanand S, Berglund L, Lithell H, Holm G, Lefvert AK. Antibodies against cardiolipin and oxidatively modified LDL in 50-year-old men predict myocardial infarction. Arterioscler Thromb Vasc Biol. 1997;17:3159–3163.[Abstract/Free Full Text]
28. Bergmark C, Wu R, de Faire U, Lefvert AK, Swedenborg J. Patients with early-onset peripheral vascular disease have increased levels of autoantibodies against oxidized LDL. Arterioscler Thromb Vasc Biol. 1995;15:441–445.[Abstract/Free Full Text]
29. Heitzer T, Luoma J, Kurz S, Munzel T, Just H, Olschewski M, Drexler H. Cigarette smoking potentiates endothelial dysfunction of forearm resistance vessels in patients with hypercholesterolemia: role of oxidized LDL. Circulation. 1996;93:1346–1353.[Abstract/Free Full Text]
30. Heitzer T, Ylä-Herttuala S, Wild E, Luoma J, Drexler H. Effect of vitamin E on endothelial vasodilator function in patients with hypercholesterolemia, chronic smoking or both. J Am Coll Cardiol. 1999;33:499–505.[Abstract/Free Full Text]
31. Lehtimaki T, Lehtinen S, Solakivi T, Nikkilä M, Jaakkola O, Jokela H, Ylä-Herttuala S, Luoma JS, Koivula T, Nikkari T. Autoantibodies against oxidized low density lipoprotein in patients with angiographically verified coronary artery disease. Arterioscler Thromb Vasc Biol. 1999;19:23–27.[Abstract/Free Full Text]
32. Freigang S, Hörkkö S, Miller E, Witztum JL, Palinski W. Immunization of LDL receptor–deficient mice with homologous malondialdehyde-modified and native LDL reduces progression of atherosclerosis by mechanisms other than the induction of high titers of antibodies to oxidative neoepitopes. Arterioscler Thromb Vasc Biol. 1998;18:1972–1982.[Abstract/Free Full Text]
33. Cyrus T, Witztum JL, Rader D, Tangirala RK, Fazio S, Linton MF, Funk CD. Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice. J Clin Invest. 1999;103:1597–1604.[Medline] [Order article via Infotrieve]
34. Liao F, Andalibi A, Qiao JH, Allayee H, Fogelman AM, Lusis AJ. Genetic evidence for a common pathway mediating oxidative stress, inflammatory gene induction, and aortic fatty streak formation in mice. J Clin Invest. 1994;94:877–884.
35. Staprans I, Rapp JH, Pan XM, Hardman DA, Feingold KR. Oxidized lipids in the diet accelerate the development of fatty streaks in cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol. 1996;16:533–538.[Abstract/Free Full Text]
36. Staprans I, Pan XM, Rapp JH, Feingold KR. Oxidized cholesterol in the diet accelerates the development of aortic atherosclerosis in cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol. 1998;18:977–983.[Abstract/Free Full Text]
37. Staprans I, Pan XM, Rapp JH, Grunfeld C, Feingold KR. Oxidized cholesterol in the diet accelerates the development of atherosclerosis in LDL receptor- and apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2000;20:708–714.[Abstract/Free Full Text]
38. Palinski W, Koschinsky T, Butler S, Miller E, Silvestre M, Vlassara H, Cerami A, Witztum JL. Immunological evidence for the presence of advanced glycosylation end products in atherosclerotic lesions of euglycemic rabbits. Arterioscler Thromb Vasc Biol. 1995;15:571–582.[Abstract/Free Full Text]
39. Harland WA, Gilbert JD, Brooks CJ. Lipids of human atheroma, 8: oxidised derivatives of cholesteryl linoleate. Biochim Biophys Acta. 1973;316:378–385.[Medline] [Order article via Infotrieve]
40. Kapadia SR, Nissen SE, Ziada KM, Guetta V, Crowe TD, Hobbs RE, Starling RC, Young JB, Tuzcu EM. Development of transplantation vasculopathy and progression of donor-transmitted atherosclerosis: comparison by serial intravascular ultrasound imaging. Circulation. 1998;98:2672–2678.[Abstract/Free Full Text]

This article has been cited by other articles:

				P. Sjogren, G. N. Fredrikson, A. Samnegard, C.-G. Ericsson, J. Ohrvik, R. M. Fisher, J. Nilsson, and A. Hamsten High plasma concentrations of autoantibodies against native peptide 210 of apoB-100 are related to less coronary atherosclerosis and lower risk of myocardial infarction Eur. Heart J., September 2, 2008; 29(18): 2218 - 2226. [Abstract] [Full Text] [PDF]

				E. Merki, M. J. Graham, A. E. Mullick, E. R. Miller, R. M. Crooke, R. E. Pitas, J. L. Witztum, and S. Tsimikas Antisense Oligonucleotide Directed to Human Apolipoprotein B-100 Reduces Lipoprotein(a) Levels and Oxidized Phospholipids on Human Apolipoprotein B-100 Particles in Lipoprotein(a) Transgenic Mice Circulation, August 12, 2008; 118(7): 743 - 753. [Abstract] [Full Text] [PDF]

				H. F. Langer, R. Haubner, B. J. Pichler, and M. Gawaz Radionuclide imaging a molecular key to the atherosclerotic plaque. J. Am. Coll. Cardiol., July 1, 2008; 52(1): 1 - 12. [Abstract] [Full Text] [PDF]

				K. C. Briley-Saebo, P. X. Shaw, W. J.M. Mulder, S.-H. Choi, E. Vucic, J. G. S. Aguinaldo, J. L. Witztum, V. Fuster, S. Tsimikas, and Z. A. Fayad Targeted Molecular Probes for Imaging Atherosclerotic Lesions With Magnetic Resonance Using Antibodies That Recognize Oxidation-Specific Epitopes Circulation, June 24, 2008; 117(25): 3206 - 3215. [Abstract] [Full Text] [PDF]

				B. Ky, A. Burke, S. Tsimikas, M. L. Wolfe, M. G. Tadesse, P. O. Szapary, J. L. Witztum, G. A. FitzGerald, and D. J. Rader The Influence of Pravastatin and Atorvastatin on Markers of Oxidative Stress in Hypercholesterolemic Humans J. Am. Coll. Cardiol., April 29, 2008; 51(17): 1653 - 1662. [Abstract] [Full Text] [PDF]

				M. Torzewski, V. Ochsenhirt, A. L. Kleschyov, M. Oelze, A. Daiber, H. Li, H. Rossmann, S. Tsimikas, K. Reifenberg, F. Cheng, et al. Deficiency of Glutathione Peroxidase-1 Accelerates the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., April 1, 2007; 27(4): 850 - 857. [Abstract] [Full Text] [PDF]

				S. Tsimikas, E. S. Brilakis, R. J. Lennon, E. R. Miller, J. L. Witztum, J. P. McConnell, K. S. Kornman, and P. B. Berger Relationship of IgG and IgM autoantibodies to oxidized low density lipoprotein with coronary artery disease and cardiovascular events J. Lipid Res., February 1, 2007; 48(2): 425 - 433. [Abstract] [Full Text] [PDF]

				V. K. Bhatia, S. Yun, V. Leung, D. C. Grimsditch, G. M. Benson, M. B. Botto, J. J. Boyle, and D. O. Haskard Complement C1q Reduces Early Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice Am. J. Pathol., January 1, 2007; 170(1): 416 - 426. [Abstract] [Full Text] [PDF]

				B. Mackness, R. Quarck, W. Verreth, M. Mackness, and P. Holvoet Human Paraoxonase-1 Overexpression Inhibits Atherosclerosis in a Mouse Model of Metabolic Syndrome Arterioscler. Thromb. Vasc. Biol., July 1, 2006; 26(7): 1545 - 1550. [Abstract] [Full Text] [PDF]

				M. Mayr, S. Kiechl, S. Tsimikas, E. Miller, J. Sheldon, J. Willeit, J. L. Witztum, and Q. Xu Oxidized Low-Density Lipoprotein Autoantibodies, Chronic Infections, and Carotid Atherosclerosis in a Population-Based Study J. Am. Coll. Cardiol., June 20, 2006; 47(12): 2436 - 2443. [Abstract] [Full Text] [PDF]

				S. Tsimikas, J. T. Willerson, and P. M. Ridker C-reactive protein and other emerging blood biomarkers to optimize risk stratification of vulnerable patients. J. Am. Coll. Cardiol., April 18, 2006; 47(8 Suppl): C19 - C31. [Abstract] [Full Text] [PDF]

				H. Arakawa, J.-Y. Qian, D. Baatar, K. Karasawa, Y. Asada, Y. Sasaguri, E. R. Miller, J. L. Witztum, and H. Ueno Local Expression of Platelet-Activating Factor-Acetylhydrolase Reduces Accumulation of Oxidized Lipoproteins and Inhibits Inflammation, Shear Stress-Induced Thrombosis, and Neointima Formation in Balloon-Injured Carotid Arteries in Nonhyperlipidemic Rabbits Circulation, June 21, 2005; 111(24): 3302 - 3309. [Abstract] [Full Text] [PDF]

				J. Nilsson, G. K. Hansson, and P. K. Shah Immunomodulation of Atherosclerosis: Implications for Vaccine Development Arterioscler. Thromb. Vasc. Biol., January 1, 2005; 25(1): 18 - 28. [Abstract] [Full Text] [PDF]

				M. Schneider, B. Verges, A. Klein, E. R. Miller, V. Deckert, C. Desrumaux, D. Masson, P. Gambert, J.-M. Brun, J. Fruchart-Najib, et al. Alterations in Plasma Vitamin E Distribution in Type 2 Diabetic Patients With Elevated Plasma Phospholipid Transfer Protein Activity Diabetes, October 1, 2004; 53(10): 2633 - 2639. [Abstract] [Full Text] [PDF]

				R. P. Choudhury, J. X. Rong, E. Trogan, V. I. Elmalem, H. M. Dansky, J. L. Breslow, J. L. Witztum, J. T. Fallon, and E. A. Fisher High-Density Lipoproteins Retard the Progression of Atherosclerosis and Favorably Remodel Lesions Without Suppressing Indices of Inflammation or Oxidation Arterioscler. Thromb. Vasc. Biol., October 1, 2004; 24(10): 1904 - 1909. [Abstract] [Full Text] [PDF]

				N. Traverso, S. Menini, E. P. Maineri, S. Patriarca, P. Odetti, D. Cottalasso, U. M. Marinari, and M. A. Pronzato Malondialdehyde, a Lipoperoxidation-Derived Aldehyde, Can Bring About Secondary Oxidative Damage To Proteins J. Gerontol. A Biol. Sci. Med. Sci., September 1, 2004; 59(9): B890 - B895. [Abstract] [Full Text] [PDF]

				S. Tsimikas, H. K. Lau, K.-R. Han, B. Shortal, E. R. Miller, A. Segev, L. K. Curtiss, J. L. Witztum, and B. H. Strauss Percutaneous Coronary Intervention Results in Acute Increases in Oxidized Phospholipids and Lipoprotein(a): Short-Term and Long-Term Immunologic Responses to Oxidized Low-Density Lipoprotein Circulation, June 29, 2004; 109(25): 3164 - 3170. [Abstract] [Full Text] [PDF]

				D. P. Faxon, V. Fuster, P. Libby, J. A. Beckman, W. R. Hiatt, R. W. Thompson, J. N. Topper, B. H. Annex, J. H. Rundback, R. P. Fabunmi, et al. Atherosclerotic Vascular Disease Conference: Writing Group III: Pathophysiology Circulation, June 1, 2004; 109(21): 2617 - 2625. [Full Text] [PDF]

				M. M. Engler, M. B. Engler, M. J. Malloy, E. Y. Chiu, M. C. Schloetter, S. M. Paul, M. Stuehlinger, K. Y. Lin, J. P. Cooke, J. D. Morrow, et al. Antioxidant Vitamins C and E Improve Endothelial Function in Children With Hyperlipidemia: Endothelial Assessment of Risk from Lipids in Youth (EARLY) Trial Circulation, September 2, 2003; 108(9): 1059 - 1063. [Abstract] [Full Text] [PDF]

				X. P. Yang, D. Yan, C. Qiao, R. J. Liu, J.-G. Chen, J. Li, M. Schneider, L. Lagrost, X. Xiao, and X.-C. Jiang Increased Atherosclerotic Lesions in ApoE Mice With Plasma Phospholipid Transfer Protein Overexpression Arterioscler. Thromb. Vasc. Biol., September 1, 2003; 23(9): 1601 - 1607. [Abstract] [Full Text] [PDF]

A. Mertens, P. Verhamme, J. K. Bielicki, M. C. Phillips, R. Quarck, W. Verreth, D. Stengel, E. Ninio, M. Navab, B. Mackness, et al. Increased Low-Density Lipoprotein Oxidation and Impaired High-Density Lipoprotein Antioxidant Defense Are Associated With Increased Macrophage Homing and Atherosclerosis in Dyslipidemic Obese Mice: LCAT Gene Transfer Decreases Atherosclerosis Circulation, April 1, 2003; 107(12): 1640 - 1646. [Abstract]