Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:689-697

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsimikas, S.
Right arrow Articles by Palinski, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsimikas, S.
Right arrow Articles by Palinski, W.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CHOLESTEROL
*MALONALDEHYDE
Related Collections
Right arrow Cardiovascular imaging agents/Techniques
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:689.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

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

Sotirios Tsimikas; Brian P. Shortal; Joseph L. Witztum; Wulf Palinski

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

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


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—To determine whether labeled antibodies against oxidized LDL (OxLDL) offer advantages for quantifying atherosclerosis, we compared in vivo aortic uptake of 125I-labeled MDA2, a monoclonal antibody against malondialdehyde-lysine epitopes), atherosclerotic surface area, and aortic weight in Watanabe heritable hyperlipidemic and New Zealand White rabbits and in low density lipoprotein receptor–deficient (LDLR-/-) and apolipoprotein E–deficient (apoE-/-) mice. Absolute and specific uptakes of 125I-MDA2 were significantly greater in plaque than in normal aortas. Uptake of 125I-MDA2 significantly correlated with aortic weight and percent atherosclerotic surface area in rabbits and mice. To assess whether 125I-MDA2 uptake reflects changes in lesion content of OxLDL, in a separate study, extensive atherosclerosis was induced in 4 groups of LDLR-/- mice by feeding them a high fat/cholesterol diet for 6 months. A baseline group was euthanized at this time. The remaining groups were fed "regression" diets (chow or chow+1% vitamin E+0.05% vitamin C) or the high fat/cholesterol diet for 6 more months. When atherosclerosis was measured as percent surface area or aortic weight, there was strong progression in the high fat/cholesterol group, moderate progression in the chow group, and no progression in the chow+vitamin E+vitamin C group compared with the baseline group. The 125I-MDA2 method also yielded a significant increase in atherosclerosis in the high fat/cholesterol group but significant decreases in the chow and chow+vitamin E+vitamin C groups. Immunocytochemistry showed fewer oxidation-specific epitopes in lesions from the chow and chow+vitamin E+vitamin C groups. Thus, the uptake of 125I-MDA2 correlates well with traditional measures of atherosclerosis but also reflects reduced plaque OxLDL content after hypocholesterolemic intervention.


Key Words: atherosclerosis • lipoproteins • oxidation • radioisotopes • imaging


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Quantification of atherosclerosis in animal models and human post mortem studies is usually performed by planimetry of lipid-stained aortas or by analysis of limited cross sections of the aortic root.1 2 3 4 5 6 7 8 Morphometry of cross sections can accurately quantify lesion area and volume but is too labor intensive to determine atherosclerosis in larger arterial segments or in the entire aorta. Planimetry does not measure lesion volume and, therefore, is insensitive to the progression of fatty streaks to advanced lesions. Without further histological and immunocytochemical analyses, neither of these methods is sensitive to qualitative changes in lesions. A method that preferentially quantifies lesions rich in oxidized LDL (OxLDL) would be highly desirable for studies in animal models and for the clinical environment because it is increasingly recognized that lipid-rich atheromas give rise to acute coronary syndromes and because multiple pathogenic effects have been attributed to lipid peroxidation.9 For example, OxLDL is cytotoxic, promotes vasoconstriction and platelet aggregation, and induces endothelial cells and macrophages to express adhesion molecules and cytokines that may affect cellular growth and extracellular matrix stability.9 10 11 12

The presence of OxLDL in atherosclerotic lesions of humans and animals is well documented.5 13 14 15 16 17 18 MDA2, a well-characterized monoclonal antibody (MAb) to malondialdehyde-lysine epitopes (present on OxLDL and other oxidatively modified proteins but not on normal LDL), has been extensively used for immunocytochemistry of human and rabbit atherosclerotic lesions.14 16 18 We have previously shown that 125I-labeled MDA2 (125I-MDA2) can be used to detect atherosclerotic lesions in rabbit aortas by autoradiography and that 99mTc-labeled MDA2 can be used to noninvasively image atherosclerotic lesions in live rabbits.19 However, OxLDL content of lesions varies, depending on their stage, and some of the labeled antibody detected in atherosclerotic lesions undoubtedly reflects increased permeability to macromolecules in general rather than specific binding to OxLDL. Therefore, a careful assessment of how much of the arterial MDA2 uptake represents specific binding to oxidation-specific epitopes and a comparison of MDA2 uptake with traditional measures of plaque burden are necessary.

In the present study, we determined specific uptake and evaluated to what degree quantification of atherosclerosis by 125I-MDA2 correlates with other measures of atherosclerosis, such as the percent surface area covered by lesions and the aortic weight. We also investigated whether this method is sensitive to changes in the amount of oxidation-specific epitopes within atherosclerotic lesions. To test this, we compared the in vivo aortic uptake of 125I-MDA2 with other parameters of atherosclerosis in rabbits and mice during progressive atherosclerosis and after hypocholesterolemic and antioxidant intervention.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Radiolabeled Antibodies
MDA2 is a murine MAb (IgG1) that recognizes MDA-lysine epitopes on OxLDL or other proteins modified by breakdown products of oxidized polyunsaturated fatty acids.20 Immunocytochemistry with MDA2 shows extensive presence of these epitopes in atherosclerotic lesions but not in normal arteries. An isotype-matched murine MAb to human albumin (Halb), which does not cross-react with rabbit or mouse albumin, was used as a control (Pierce). This antibody was given preference over a protein control or a nonspecific Fab antibody control, because it has the same molecular weight and size as MDA2 and can be expected to provide a good indication of nonspecific binding and binding to the Fc receptor on macrophages. Thus, by subtracting Halb uptake from MDA2 uptake, the specific uptake of MDA2 can be calculated. MDA2 and Halb were iodinated with 125I or 131I by using the lactoperoxidase/glucose oxidase method.20 More than 99% of radioactivity was trichloroacetic acid precipitable, and specific activity averaged 1.0 µCi/µg. The in vivo uptake of radiolabeled MAbs was determined by intravenously injecting {approx}100 µCi in rabbits and 10 µCi in mice 24 hours before euthanasia. We previously demonstrated by ELISA that radiolabeling does not significantly affect antigen binding of MDA2.19

Animal Models
Rabbits
Sixteen Watanabe heritable hyperlipidemic (WHHL) rabbits ({approx}1.5 years old) with extensive aortic atherosclerosis and 8 New Zealand White (NZW) rabbits (6 months old) free of atherosclerosis (both strains fed regular rabbit chow) were used to assess in vivo aortic uptake of MAbs. The average weight of the rabbits was {approx}3.0 kg, and total plasma cholesterol ranged from 600 to 1000 mg/dL in WHHL rabbits and from 60 to 110 mg/dL in NZW rabbits. Sodium iodide (1 mg/kg) was injected intravenously 1 hour before injection of MAbs to block thyroid uptake of 125I. To detect absolute aortic uptake, 125I-MDA2 was injected in 11 WHHL and 4 NZW rabbits, and 125I-Halb was injected in 5 WHHL and 4 NZW rabbits. The MAbs were injected in a volume of 250 to 500 µL through a marginal ear vein, and pharmacokinetics were assessed by blood sampling at 12, 24, 36, and 240 minutes and 24 hours. Initially, each rabbit was injected with only 1 antibody, so that autoradiographs could be obtained. However, the WHHL rabbits were well matched, and there was no statistically significant difference in percent atherosclerotic surface area (63% versus 55%, P=0.47) or the aortic weight (1060 versus 820 mg, P=0.24) between the rabbits receiving 125I-MDA2 and those receiving 125I-Halb. Specific uptake in each tissue was defined as absolute 125I-MDA2 uptake minus absolute 125I-Halb uptake. In additional experiments, 4 WHHL rabbits were coinjected with equal amounts (40 µg protein) of 125I-MDA2 and 131I-Halb to simultaneously assess aortic uptake of both MAbs within the same rabbits.

Mice
In the first study assessing in vivo aortic uptake of 125I-MDA2, 6- to 12-month-old LDL receptor–deficient (LDLR-/-) and apoE-deficient (apoE-/-) mice (n=38, mean weight 42.4±13.2 g) were fed a high fat/cholesterol diet containing 21% milk fat and 1.25% (LDLR-/-) or 0.15% (apoE-/-) cholesterol (TD 96121, Harlan Teklad) for 6 months. This raised their mean plasma cholesterol levels to 1000 to 2200 mg/dL and plasma triglyceride levels to 240 to 600 mg/dL. Absolute plaque uptake of 125I-MDA2 in mice was assessed by injection of 125I-MDA2 into 16 LDLR-/- and 8 apoE-/- mice and injection of 125I-Halb into 6 LDLR-/- and 7 apoE-/- mice. The radiolabeled MAbs (100 µL) were injected through the tail vein, and blood was collected to assess pharmacokinetics in EDTA-containing tubes from the retro-orbital plexus at 15, 30, 45, and 240 minutes and 24 hours after injection.

In a second study designed to assess whether in vivo aortic uptake of 125I-MDA2 reflects changes in arterial content of OxLDL and other oxidation-specific epitopes, 43 LDLR-/- mice were placed on the same atherogenic diet described above for 6 months, starting at 6 months of age. Mice were then randomly divided into 4 groups, and group I (n=13) was euthanized as the baseline group. For an additional 6 months, groups II (chow, n=10) and III (chow+vitamin E+vitamin C, n=10) were placed on low cholesterol "regression diets" consisting of either regular chow (Harlan Teklad Rodent Diet 8604) or regular chow supplemented with antioxidants (1.0% vitamin E added to the chow and 0.05% vitamin C added to the drinking water). Group IV (high fat/cholesterol, n=10) was continued on the high fat/cholesterol diet for the same time period. One mouse each from groups III and IV died before the end of the study, and 2 mice from group II were excluded from analysis because of poor tail vein injections.

Studies were approved by the Animal Subjects Committee of the University of California, San Diego.

Preparation of Aortas for Gamma Counting, Image Analysis, and Autoradiography
At the specified time points, rabbits and mice were euthanized by an overdose of pentobarbital, and the systemic circulation was perfused with isotonic PBS containing 2 mmol/L EDTA, pH 7.4, from a cannula inserted into the left ventricle, with blood exiting from an incision in the right atrium (rabbits) or vena cava (mice) until the effluent was clear. This was followed by in situ fixation at physiological pressure with formal sucrose (4% paraformaldehyde, 5% sucrose, 20 µmol/L butylated hydroxytoluene, and 1 mmol/L EDTA, pH 7.4) for 20 minutes. The aortas were then dissected, opened longitudinally, thoroughly cleansed of adventitial tissue, and stained with Sudan IV, as previously described.5 Sudan-positive adventitial tissue remaining after the initial dissection of the aorta was then removed, aortas were pinned out on a black wax pan, and the percentage of atherosclerotic surface area was determined by computer-assisted image analysis, as previously described in detail.7

Autoradiographs of the aortas were generated with Kodak Biomax high-speed film using an intensifying screen after 5 days (rabbits) or 14 days (mice) of exposure at 4°C. After autoradiography, the aortas of the rabbits and mice were rehydrated in PBS for 2 hours, blotted dry with tissue paper, weighed (wet weight), and counted for 125I in an LKB Wallac 1282 Compugamma Universal Gamma counter. Tissue uptake was expressed as the absolute aortic uptake (percent injected dose [% ID]) and as the percent injected dose per gram aortic tissue (% ID/g) after correction for radioisotope decay. In rabbits, the segments of the aortas containing plaques were separated from visually normal aortic segments to determine the differential uptake of 125I-MDA2 in plaque versus normal tissue. No attempt was made to separate the atherosclerotic intima from the underlying media in plaque tissue, because it is difficult to obtain a clean separation. Areas that could not be unequivocally classified as "normal" or "plaque" were not used. Plaque was defined as tissue containing Sudan-stained lesions, and normal was defined as tissue without Sudan-positive regions. No attempt was made to separate plaque and normal tissue in mouse aortas because of the small size of the aortas.

Immunocytochemistry
To assess the lesion content of oxidation-specific epitopes in the 4 groups of LDLR-/- mice in the dietary intervention study, atherosclerotic segments of the aortas were embedded in paraffin. Serial 8-µm-thick sections were rehydrated and immunostained with MAL2, a guinea pig antiserum with the same specificity for MDA-lysine epitopes as MDA2,14 20 by use of an avidin–biotin–alkaline phosphatase system.5

Plasma Cholesterol and Vitamin E Levels
Plasma total cholesterol and triglycerides were measured by enzymatic assays on an automated Abbott VP Super System bichromatic analyzer. Vitamin E levels were measured by high-performance liquid chromatography as described.21

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


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Study I: Comparison of Different Parameters of Atherosclerosis
In Vivo Uptake in Aortic Plaque
Rabbits
Complete data for the absolute and specific uptake of 125I-MDA2 and 125I-Halb in all aortic segments of rabbits are provided in TableDown I (published online only, http://atvb.ahajournals.org/cgi/content/full/20/3/689/DC1). In the entire aorta of WHHL rabbits, the uptake of 125I-MDA2 was significantly higher (2.7-fold) than that of 125I-Halb (0.072±0.026% ID/g versus 0.027±0.007% ID/g, P<0.001). The absolute aortic uptake of either MAb in NZW rabbits was very low (0.004% ID/g and 0.001% ID/g for MDA2 and Halb, respectively). The uptake of 125I-MDA2 was approximately 17-fold higher in WHHL than NZW rabbits (0.072±0.026% ID/g versus 0.004±0.002% ID/g, P<0.001). Specific uptake of 125I-MDA2, defined as 125I-MDA2 uptake minus 125I-Halb uptake, was {approx}20-fold higher in the entire aorta of WHHL rabbits than NZW rabbits (0.045% ID/g versus 0.002% ID/g, P<0.001). Similar data were obtained in the aortic arch and thoracic and abdominal aortas. The above results were obtained from carefully matched rabbits (similar extent of atherosclerosis) injected with either 125I-MDA2 or 125I-Halb. In a separate experiment, similar results were obtained in 4 rabbits injected simultaneously with 125I-MDA2 and 131I-Halb. In these rabbits, the aortic uptake of 125I-MDA2 was 3.1-fold higher than that of 131I-Halb (0.043% ID/g versus 0.014% ID/g), comparable to the 2.7-fold increase noted above.


View this table:
[in this window]
[in a new window]
  		Table 1. Absolute and Specific Uptake of 125I-MDA2 in WHHL Rabbit Plaque and Normal Arterial Tissue

Table 1Up shows the differences in uptake of both MAbs between plaque and visually normal tissue of WHHL rabbits. NZW rabbits were not included because they did not have lesions. Plaque uptake of 125I-MDA2 was significantly higher than uptake of 125I-Halb in all segments of the aorta. In contrast, in normal segments, no significant differences were noted between 125I-MDA2 and 125I-Halb uptake, except in the thoracic aorta, where 125I-MDA2 uptake was greater. Specific 125I-MDA2 uptake was significantly higher in plaque than in normal tissue (P<0.001) in all aortic segments. Within the same WHHL rabbit aortas, the ratio of plaque to normal tissue uptake of 125I-MDA2 was 7.1, whereas for 125I-Halb, it was 2.9 (P<0.001).

As a whole, these data demonstrate that MDA2 accumulates to a significantly greater extent in plaque tissue than does a nonspecific antibody. The fact that the nonspecific MAb Halb has very low uptake in NZW aortas but increased uptake in WHHL aortas may reflect a generalized increase in permeability of atherosclerotic tissue to macromolecules22 23 24 25 26 as well as antibody binding to Fc receptors on macrophage/foam cells.27

Mice
Because mouse aortic lesions were so small that they could not reliably be separated into plaque and normal tissue, measurements of antibody uptake were made only in the entire aorta. 125I-MDA2 uptake in atherosclerotic mouse aortas was 1.6-fold greater than uptake of 125I-Halb (3.3% ID/g versus 2.2% ID/g). Interestingly, the uptake of 125I-MDA2 and 125I-Halb was significantly higher in mouse aortas than in rabbit aortas (42-fold for MDA2 and 75-fold for Halb, P<0.001 for both MAbs), suggesting that hypercholesterolemia-induced mouse lesions are even more permeable than rabbit lesions.

Correlation of In Vivo Uptake of Radiolabeled Antibodies With Percent Atherosclerotic Surface Area and Aortic Weight
To compare the different parameters of atherosclerosis, the extent of atherosclerosis measured as percent aortic surface area stained with Sudan IV was first correlated with the in vivo uptake of MAbs in rabbits. Figure 1ADown shows an excellent correlation between the percent surface area and the uptake of 125I-MDA2 (r=0.85, P<0.0001) and 125I-Halb (r=0.94, P<0.0001). However, the slope of the regression line of 125I-MDA2 was steeper than that of 125I-Halb, indicating that the specific uptake of 125I-MDA2 increased to a much greater degree with increasing atherosclerosis. A similar correlation was seen in mice injected with 125I-MDA2 (r=0.90, P<0.001) and 125I-Halb (r=0.87, P<0.001) (data not shown).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Correlation between the absolute aortic uptake of radiolabeled monoclonal antibodies (% ID) and the percent atherosclerotic surface area staining with Sudan IV (A) or the aortic weight (B) of all WHHL and NZW rabbits injected with 125I-MDA2 (n=15) and 125I-Halb (n=9).

As shown in Figure 1BUp, there was also a strong linear correlation between the in vivo uptake of 125I-MDA2 and the aortic weight (P<0.0001) when data from WHHL and NZW rabbits were pooled. The slope of the regression line for 125I-MDA2 uptake was again much steeper than that for 125I-Halb in the rabbits. When the NZW rabbits were removed from the analysis, the correlation remained significant for 125I-MDA2–injected WHHL rabbits (r=0.95, P<0.001) but not for 125I-Halb–injected WHHL rabbits (r=0.83, P=0.08). Similar results were obtained for individual segments of the aorta (data not shown). As shown in Table 2Down, in rabbits, the aortic uptake of 125I-MDA2 correlated better with the aortic weight than did the aortic uptake of 125I-Halb. When the uptake was normalized for the aortic weight (ie, expressed as % ID/g), the correlation remained statistically significant for 125I-MDA2 but not for 125I-Halb. In mice, similar correlations were noted between aortic weight and aortic uptake of 125I-MDA2 (Figure 2ADown; r=0.82, P<0.0001) and 125I-Halb (Figure 2BDown; r=0.86, P<0.0005). In analogy to rabbits, the correlation between the percent injected dose per gram and the aortic weight was significant only for 125I-MDA2 (Table 2Down).


View this table:
[in this window]
[in a new window]
  		Table 2. Correlation Between Aortic Uptake of 125I-MDA2 and 125I-Halb in Entire Aorta and Aortic Weight of All Rabbits and Mice



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Correlation between the absolute aortic uptake of radiolabeled antibodies (% ID) and the aortic weight in all LDLR-/- and apoE-/- mice injected with 125I-MDA2 (n=24, A) and 125I-Halb (n=13, B).

Study II: Diet/Antioxidant Intervention Study
Lipid Parameters and Vitamin E Levels
The second study with LDLR-/- mice was designed to determine the relation of the uptake of 125I-MDA2, presumably reflecting the lesion content of oxidation-specific epitopes, with other measures of atherosclerosis. For this purpose, experimental conditions that were expected to result in either regression or progression of lesions were chosen (see Methods for details). Forty-three mice were given a high fat/cholesterol diet for 6 months, starting at 6 months of age. Mice were then divided into 4 groups. This initial dietary intervention raised the total plasma cholesterol levels from {approx}250 to {approx}1800 mg/dL. As shown in Figure 3Down, there were no significant differences in cholesterol or triglyceride levels among groups at the beginning of the study or after 6 months of intervention. Group I (baseline) was euthanized at the end of the initial 6 months of intervention, whereas the other 3 groups were continued for another 6 months on the diets described in Methods. At the end of the study (ie, after 12 months of intervention), group IV (high fat/cholesterol) had significantly higher cholesterol levels than did groups II (chow) and III (chow+vitamin E+vitamin C); respective cholesterol levels were 1526±858 mg/dL versus 334±56 and 406±149 mg/dL (P<0.001). The cholesterol levels of groups II and III were not significantly different from each other. Similar results were obtained for the triglyceride levels (FigureUp I, published online only, http://atvb.ahajournals.org/cgi/content/full/20/3/689/DC1).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Plasma cholesterol levels in the dietary intervention study. Four groups of LDLR-/- mice were fed a high fat/cholesterol diet for 6 months. Group I (baseline, {square}, n=13) was then euthanized. For the following 6 months, group II (chow, {triangleup}, n=8) received regular mouse chow, group III (chow/vit E,C, {circ}, n=9) received regular mouse chow supplemented with 1.0% vitamin E and 0.05% vitamin C, and group IV (high fat/cholesterol, {blacksquare}, n=9) continued to receive the high fat/cholesterol diet. The inset shows the plasma levels of vitamin E normalized to the total plasma cholesterol. Data are mean±SEM.

Vitamin E levels were measured in groups II to IV at 6, 8, and 12 months. At 6 months, the plasma vitamin E levels were 34.0±12.0, 44.1±10.2, and 39.6±18.9 µg/mL in the chow, high fat/cholesterol, and chow+vitamin E+vitamin C groups, respectively (P=0.35). At the end of the study, the respective levels were 5.0±2.5, 39.6±17.9, and 21.1±12.1 µg/mL (P<0.001). Because vitamin E is carried in plasma in lipoprotein particles, results were also expressed as the plasma level normalized to total cholesterol (micrograms plasma vitamin E per milligram total plasma cholesterol). At 6 months, the vitamin E levels were similar (inset in Figure 3Up). At the end of the study, there was a 2.4-fold increase in vitamin E levels in the chow+vitamin E+vitamin C group (P=0.009 compared with the level at 6 months), whereas the high fat/cholesterol group showed no significant change. In the chow group, a slight decrease was observed (P=0.03 compared with its baseline). At the end of the study, significant differences in vitamin E levels were noted between all 3 groups (P<0.001).

Measurement of Atherosclerosis by 3 Parameters
Figure 4Down compares atherosclerosis in the 4 groups of mice, measured as uptake of 125I-MDA2, percent surface area, and aortic wet weight. When measured as the percent surface area (Figure 4ADown), compared with the baseline group (ie, the extent of atherosclerosis after 6 months on the atherogenic diet), extensive progression was noted in the high fat/cholesterol group (126%, P<0.001), moderate progression in the chow group (68%, P=0.009), but no significant progression in the chow+vitamin E+vitamin C group (12%, P=0.51). The aortic weights (Figure 4BDown) closely paralleled the progression of atherosclerosis as measured by surface area in the high fat/cholesterol group (149%, P=0.0005), chow group (49%, P=0.10), and chow+vitamin E+vitamin C group (4%, P=0.79). In contrast, when atherosclerosis was determined as uptake of 125I-MDA2 (Figure 4CDown), there was a significant increase in the high fat/cholesterol group (164%, P<0.001) but a significant decrease in the chow group (-32%, P<0.001) and the chow+vitamin E+vitamin C group (-45%, P<0.001). The extent of lesions measured by all 3 parameters tended to be smaller in the chow+vitamin E+vitamin C group than in the chow-only group, but differences did not reach statistical significance. This is consistent with a recent report by Praticò et al28 that provided evidence for lesion regression in apoE-/- mice treated with vitamin E.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Results of the dietary intervention study in LDLR-/- mice. Atherosclerosis was assessed as percent atherosclerotic surface area (A), aortic weight (B), and uptake of 125I-MDA2 (C). When measured as the percent surface area, compared with the baseline group (n=13), there was extensive progression in the high fat/cholesterol group (high fat/chol, n=9), moderate progression in the chow group (n=8), and no progression in the chow+vitamin E+vitamin C group (chow+vit E,C, n=9). Results using aortic weight were qualitatively similar. In contrast, uptake of 125I-MDA2 showed increased uptake in the high fat/chol group but decreased uptake in the chow and chow+vit E,C groups (*P<0.001 vs baseline). Data are mean±SEM.

Correlations were then repeated in these groups of mice between the percent surface area, aortic weight, and aortic uptake of 125I-MDA2. The correlations between aortic weight and the aortic uptake of 125I-MDA2 (% ID) remained significant for the baseline group (r=0.83, P<0.001) and the high fat/cholesterol group (r=0.85, P<0.001) but not for the chow (r=0.70, P<0.08) or chow+vitamin E+vitamin C group (r=0.14, P=0.71); ie, the lesion content of oxidation-specific epitopes in the latter 2 groups decreased disproportionately to other plaque components. Similarly, there were significant correlations between the percent surface area and the uptake of 125I-MDA2 (% ID) in the baseline group (r=0.81, P<0.001) and the high fat/cholesterol group (r=0.84, P<0.001) but not in the chow group (r=0.09, P<0.83) or chow+vitamin E+vitamin C group (r=0.24, P=0.18). These results confirm that all 3 methods detect progression of atherosclerosis (eg, baseline versus high fat/cholesterol groups). However, only the aortic uptake of 125I-MDA2 showed marked decreases as a result of the chow and chow+vitamin E+vitamin C diets, suggesting that this method is more sensitive to changes in lesion content of OxLDL (oxidation-specific epitopes) induced by low cholesterol diets or antioxidant intervention.

Examples of Sudan-stained aortas and the corresponding autoradiographs from the baseline and chow groups are provided in Figure 5Down. The autoradiograph from the baseline group (Figure 5ADown) clearly shows that 125I-MDA2 accumulates almost exclusively within atherosclerotic lesions and accurately reflects all lesions. The same was true for the high fat/cholesterol group (not shown). This is in agreement with our previous observation that autoradiographs of WHHL rabbit aortas accurately reflect Sudan-positive lesions.19 In contrast, in the chow (Figure 5BDown) and chow+vitamin E+vitamin C groups, some areas containing large Sudan-positive lesions did not show MDA2 uptake by autoradiography (arrow in Figure 5BDown), implying that these lesions contained fewer oxidation-specific epitopes. Note that to demonstrate differences in oxidation-specific epitopes detected by autoradiography, we deliberately selected arteries with a similar extent of atherosclerosis for Figure 5Down. Differences in the autoradiography might otherwise also reflect the fact that lesions of different stages contain different amounts of OxLDL.



View larger version (52K):
[in this window]
[in a new window]
  		Figure 5. En face preparations of the arch and thoracic part of Sudan-stained aortas (left) and corresponding autoradiographs (right) from LDLR-/- mice injected with 10 µCi 125I-MDA2. Panel A shows an aorta from group I (baseline) with relatively little atherosclerosis. Panel B shows a representative aorta from group II, in which a decrease in oxidation-specific epitopes is likely to have occurred as a result of chow feeding. Note that a large lesion in the arch of this Sudan-stained aorta does not yield a corresponding image in the autoradiograph (arrow).

Immunocytochemistry
Immunostaining with the guinea pig antiserum against MDA-lysine, MAL2, was performed under identical conditions in aortic sections from groups II to IV (Figure 6Down). Representative examples shown in panels A and B demonstrate extensive immunostaining in lesions from a mouse on the high fat/cholesterol diet. In contrast, in lesions from the chow and chow+vitamin E+vitamin C groups, there was markedly less staining for oxidation-specific epitopes (panels C and D, respectively). Control immunocytochemistry without the primary antibody yielded no staining. Thus, although the 2-dimensional (percent surface area) and 3-dimensional (aortic weight) assessment of atherosclerosis showed progression of atherosclerosis, the uptake of 125I-MDA2 and immunocytochemistry clearly indicated diminished OxLDL in lesions of the same stage.



View larger version (97K):
[in this window]
[in a new window]
  		Figure 6. Immunocytochemistry of representative atherosclerotic lesions in LDLR-/- mice from groups II to IV. Both early (A) and intermediate (B) lesions from the high fat/cholesterol group showed extensive staining with MAL2. In similar lesions from the chow (C) and chow+vitamin E+vitamin C groups (D), there was markedly less staining for these model epitopes of OxLDL. Bar=50 µm.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
It is now generally accepted that the composition of atherosclerotic lesions, in particular their content of lipids, OxLDL, foam cells, and smooth muscle cells, determines their propensity to cause clinical events. In addition to contributing to the progression of atherosclerosis, OxLDL may profoundly influence the mechanical stability of atherosclerotic plaques, because foam cells offer little mechanical stability and because activated macrophages may secrete factors that weaken the plaque, such as metalloproteinases.10 Human pathology studies have shown that atheromas containing a large necrotic core, thin fibrous cap, and large numbers of macrophage/foam cells in the shoulder area are more predisposed to plaque rupture and thrombosis.12 29 30 31 32 These lesions, which frequently appear as mild or moderate coronary stenoses in angiographic studies, are characterized by large lipid pools exceeding 40% of plaque areas.10 33 34 35 Diagnostic methods that provide a measure of the overall extent of atherosclerotic lesions with an emphasis on their lipid content would therefore be desirable. In the absence of a method that specifically detects lipids, oxidation-specific epitopes may be useful for determination of the overall degree of atherosclerosis, because lipid peroxidation occurs in atherosclerotic lesions of all stages, including the lipid pools of atheromas.13 14 15 16 17 18 36 37 38 39 40 Furthermore, the lipid core of atheromas can be assumed to contain extensively oxidized lipids that are accumulated within foam cells and set free when these cells undergo necrosis or apoptosis (reviewed in Reference 4141 ).

We previously described a novel technique that determines the extent of atherosclerosis by measuring the plaque uptake of injected radiolabeled MDA2, an oxidation-specific antibody, and showed that it is suitable for noninvasive in vivo diagnostic imaging. We have now quantitatively assessed the in vivo uptake of 125I-MDA2 in rabbit and murine models of atherosclerosis and compared it with 2 conventional parameters that provide a cumulative measure of atherosclerosis.

The present study confirms that the plaque uptake of MDA2 accurately reflects the overall extent of lesions in hypercholesterolemic animals with progressing atherosclerosis. This may be explained by the abundance of the epitopes recognized by MDA2 in atherosclerotic lesions of animals and humans. As previously shown by immunocytochemistry, these epitopes occur in a wide range of lesions, both extracellularly (eg, in the necrotic core) and intracellularly (in foam cells).14 16 18 39 40 In addition, MDA2 binds not only to MDA-lysine epitopes on OxLDL but also to other oxidized proteins and phospholipids.14 20 42 In the present study, aortic uptake data confirmed that 125I-MDA2 specifically accumulates in plaque tissue, with only minimal uptake in nonatherosclerotic segments of the aorta. Furthermore, the uptake of 125I-MDA2 showed highly significant correlations with both the aortic weight and the percent of Sudan-positive (ie, lipid-containing) surface area. Thus, the accumulation of radiolabeled MDA2 in lesions appears to be a good measure not only of oxidation-specific epitopes but also of the overall extent of atherosclerotic lesions induced by hypercholesterolemia.

The dietary intervention study in LDLR-/- mice suggests that the uptake of labeled MDA2 may also be more suitable than traditional measures to rapidly detect lesion regression induced by hypocholesterolemic intervention. We believe this conclusion to be correct, as long as the term "regression" is not used in the narrow sense of "substantially reduced physical dimensions." Once advanced atheromas have developed, they are unlikely to regress to a degree that would markedly reduce the surface area of lesions. In contrast, the uptake of 125I-MDA2 clearly detects a decrease in the presence of oxidation-specific epitopes. Further evidence that there was a significant difference in the lesion content of oxidation-specific epitopes was provided by the titers of circulating IgG and IgM autoantibodies binding to MDA-LDL. IgG and IgM titers increased by 81% (P<0.0001) and 25% (P=0.02), respectively, in the high fat/cholesterol group compared with the baseline group, whereas the IgG titer remained unchanged, and the IgM titer decreased by 28% and 27% (P<0.05) in the chow and chow+vitamin C+vitamin E groups (S.T., J.L.W., W.P., unpublished observations). The decrease in MDA2 uptake may be indicative of decreased lipid oxidation, a slower rate of atherogenesis, or even a decreased risk of clinical complications, as discussed below. Because preparation of the tissues for Sudan staining involved fixation and alcohol extraction, it was not possible to directly measure lipid content in these aortas. However, our results are consistent with a decreased lipid accumulation in the aortas of mice on regression diets, similar to that recently reported by Aikawa et al.43

When evaluating the potential of the MDA2 method for post mortem determination of atherosclerosis in animal models and, potentially, the application of labeled MDA2 for noninvasive imaging in humans,19 it has to be appreciated that the uptake into the arterial wall does not exclusively represent "specific" binding to oxidation-specific epitopes. As we have shown, MDA2 and nonspecific antibodies have similar uptake values in normal tissue (Table 1Up). Both increase linearly with increasing atherosclerosis, but the uptake of MDA2 is much greater (Figures 1Up and 2Up). The fact that uptake of both antibodies increases is consistent with similar observations in other studies and probably reflects increased permeability of atherosclerotic lesions to all macromolecules.22 23 24 25 26 For example, the transvascular leakage of albumin was significantly increased in patients with severe clinical atherosclerosis.23 The vascular permeability was also directly related to cholesterol levels in a rabbit model of diet-induced hypercholesterolemia.24 Similarly, the arterial influx of lipoproteins in cholesterol-fed rabbits has been reported to be inversely proportional to lipoprotein size.26

An additional mechanism of retention of whole antibodies in the lesion may be through binding to Fc receptors on foam cells and macrophages.19 27 However, plaque tissue clearly shows a markedly faster increase in specific uptake of labeled MDA2 compared with nonspecific MAb, consistent with its specific binding to oxidative neoepitopes. The binding to Fc receptors could be overcome in future studies by using antibody Fab fragments. Nevertheless, the overall binding of MDA2 due to both epitope-specific binding and the Fc-mediated mechanism led to a 20-fold increase in binding of MDA2 in lesions versus normal aortic tissue.

In conclusion, quantification of atherosclerosis in animal models by labeled oxidation-specific antibodies provides valuable information on lipid peroxidation as well as an accurate overall measure of atherosclerosis. In addition to complementing established ex vivo parameters measuring atherosclerosis in experimental models of the disease, diagnostic methods using antibodies to oxidation-specific epitopes may be of clinical use in humans. For example, in studies of the regression of coronary artery disease, lipid-lowering agents induced "plaque stabilization," as demonstrated by markedly improved clinical outcomes despite minimal changes in arterial luminal dimensions.44 45 46 47 This is thought to be due, in part, to removal of cholesterol, cholesterol esters, and, presumably, OxLDL from atherosclerotic lesions. The method described in the present study may provide a means to test these hypotheses. We have already shown that 99mTc-labeled MDA2 noninvasively detects lipid-rich atherosclerotic lesions.19 With improvement of the imaging methods, MDA2 and/or other labeled oxidation-specific antibodies may allow us to noninvasively detect, quantify, and monitor the course of atherosclerotic lesions in animals and humans.


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

Received July 13, 1999; accepted September 28, 1999.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Carew TE, Schwenke DC, Steinberg D. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci U S A. 1987;84:7725–7729.[Abstract/Free Full Text]
  2. Armstrong ML, Warner ED, Connor WE. Regression of coronary atheromatosis in rhesus monkeys. Circ Res. 1970;27:59–67.[Abstract/Free Full Text]
  3. Insull WJ. Stain for differentiation and quantitation of gross lesions of atherosclerosis. Arch Pathol. 1967;83:474–478.[Medline] [Order article via Infotrieve]
  4. Nunnari JJ, Zand T, Joris I, Majno G. Quantitation of oil red O staining of the aorta in hypercholesterolemic rats. Exp Mol Pathol. 1989;51:1–8.[Medline] [Order article via Infotrieve]
  5. 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]
  6. Wissler RW. New insights into the pathogenesis of atherosclerosis as revealed by PDAY: pathobiological determinants of atherosclerosis in youth. Atherosclerosis. 1994;108(suppl):S3–S20.
  7. Tangirala RK, Rubin EM, Palinski W. Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice. J Lipid Res. 1995;36:2320–2328.[Abstract]
  8. Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987;68:231–240.[Medline] [Order article via Infotrieve]
  9. Steinberg D, Witztum JL. Lipoproteins, lipoprotein oxidation, and atherogenesis. In Chien KR (ed): Molecular Basis of Cardiovascular Disease. Philadelphia, Pa: WB Saunders Co; 1999:458–475.
  10. Lee RT, Libby P. The unstable atheroma. Circulation. 1997;17:1859–1867.
  11. Superko HR, Krauss RM. Coronary artery disease regression: convincing evidence for the benefit of aggressive lipoprotein management. Circulation. 1994;90:1056–1069.[Abstract/Free Full Text]
  12. Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation. 1995;92:657–671.[Free Full Text]
  13. 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]
  14. 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]
  15. 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]
  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. Jürgens G, Chen Q, Esterbauer H, Mair S, Ledinski G, Dinges HP. Immunostaining of human autopsy aortas with antibodies to modified apolipoprotein B and apoprotein(a). Arterioscler Thromb. 1993;13:1689–1699.[Abstract/Free Full Text]
  18. Ricote M, Huang J, Fajas L, Li A, Welch J, Najib J, Witztum JL, Auwerx J, Palinski W, Glass CK. Expression of the peroxisome proliferator-activated receptor gamma (PPAR{gamma}) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein. Proc Natl Acad Sci U S A. 1998;95:7614–7619.[Abstract/Free Full Text]
  19. 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]
  20. 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]
  21. Reaven PD, Grasse BJ, Tribble DL. Effects of linoleate-enriched and oleate-enriched diets in combination with {alpha}-tocopherol on the susceptibility of LDL and LDL subfractions to oxidative modification in humans. Arterioscler Thromb Vasc Biol. 1994;14:557–566.[Abstract/Free Full Text]
  22. Adams CW, Virag S, Morgan RS, Orton CC. Dissociation of [3H]cholesterol and 125I-labelled plasma protein influx in normal and atheromatous rabbit aorta: a quantitative histochemical study. J Atheroscler Res. 1968;8:679–696.[Medline] [Order article via Infotrieve]
  23. Jensen JS. Renal and systemic transvascular albumin leakage in severe atherosclerosis. Arterioscler Thromb Vasc Biol. 1995;15:1324–1329.[Abstract/Free Full Text]
  24. Wu CC, Chang SW, Chen MS, Lee YT. Early change of vascular permeability in hypercholesterolemic rabbits. Arterioscler Thromb Vasc Biol. 1995;15:529–533.[Abstract/Free Full Text]
  25. Smith EB, Staples EM. Distribution of plasma proteins across the human aortic wall: barrier functions of endothelium and internal elastic lamina. Atherosclerosis. 1980;37:579–590.[Medline] [Order article via Infotrieve]
  26. Stender S, Zilversmit DB. Transfer of plasma lipoprotein components and of plasma proteins into aortas of cholesterol-fed rabbits: molecular size as a determinant of plasma lipoprotein influx. Arteriosclerosis. 1981;1:38–49.[Abstract/Free Full Text]
  27. Fischman AJ, Rubin RH, Khaw BA, Kramer PB, Wilkinson R, Ahmad M, Needelman M, Locke E, Nossiff ND, Strauss HW. Radionuclide imaging of experimental atherosclerosis with nonspecific polyclonal immunoglobulin G. J Nucl Med. 1989;30:1095–1100.[Abstract/Free Full Text]
  28. Praticò D, Tangirala RK, Rader DJ, Rokach J, FitzGerald GA. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice. Nat Med. 1998;4:1189–1192.[Medline] [Order article via Infotrieve]
  29. 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]
  30. Davies MJ, Thomas A. Thrombosis and acute coronary-artery lesions in sudden cardiac ischemic death. N Engl J Med. 1984;310:1137–1140.[Abstract]
  31. Lendon CL, Davies MJ, Born GV, Richardson PD. Atherosclerotic plaque caps are locally weakened when macrophage density is increased. Atherosclerosis. 1991;87:87–90.[Medline] [Order article via Infotrieve]
  32. van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation. 1994;89:36–44.[Abstract/Free Full Text]
  33. 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]
  34. Fishbein MC, Siegel RJ. How big are coronary atherosclerotic plaques that rupture? Circulation. 1996;94:2662–2666.[Free Full Text]
  35. Little WC, Constantinescu M, Applegate RJ, Kutcher MA, Burrows MT, Kahl FR, Santamore WP. Can coronary angiography predict the site of a subsequent myocardial infarction in patients with mild-to-moderate coronary artery disease? Circulation. 1988;78:1157–1166.[Abstract/Free Full Text]
  36. 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.
  37. Palinski W, Hörkkö 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]
  38. 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]
  39. Napoli C, D’Armiento FP, Mancini FP, Postiglione A, Witztum JL, Palumbo G, Palinski W. Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia: intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions. J Clin Invest. 1997;100:2680–2690.[Medline] [Order article via Infotrieve]
  40. Napoli C, Glass CK, Witztum JL, Deutsch R, D’Armiento FP, Palinski W. Influence of maternal hypercholesterolaemia during pregnancy on progression of early atherosclerotic lesions in childhood: Fate of Early Lesions in Children (FELIC) study. Lancet.. 1999;354:1234–1241.[Medline] [Order article via Infotrieve]
  41. Kockx MM. Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects. Arterioscler Thromb Vasc Biol. 1998;18:1519–1522.[Abstract/Free Full Text]
  42. Hörkkö S, Miller E, Dudl E, Reaven P, Curtiss L, Zvailfler NJ, Terkeltaub R, Pierangeli SS, Branch DW, Palinski W, Witztum JL. 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]
  43. Aikawa M, Rabkin E, Okada Y, Voglic SJ, Clinton SK, Brinckerhoff CE, Sukhova GK, Libby P. Lipid lowering by diet reduces matrix metalloproteinase activity and increases collagen content of rabbit atheroma: a potential mechanism of lesion stabilization. Circulation. 1998;97:2433–2444.[Abstract/Free Full Text]
  44. Watts GF, Lewis B, Brunt JN, Lewis ES, Coltart DJ, Smith LD, Mann JI, Swan AV. Effects on coronary artery disease of lipid-lowering diet, or diet plus cholestyramine, in the St Thomas’ Atherosclerosis Regression Study (STARS). Lancet. 1992;339:563–569.[Medline] [Order article via Infotrieve]
  45. Buchwald H, Varco RL, Matts JP, Long JM, Fitch LL, Campbell GS, Pearce MB, Yellin AE, Edmiston WA, Smink RDJ. Effect of partial ileal bypass surgery on mortality and morbidity from coronary heart disease in patients with hypercholesterolemia: report of the Program on the Surgical Control of the Hyperlipidemias (POSCH). N Engl J Med.. 1990;323:946–955.[Abstract]
  46. Brown G, Albers JJ, Fisher LD, Schaefer SM, Lin JT, Kaplan C, Zhao XQ, Bisson BD, Fitzpatrick VF, Dodge HT. Regression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B. N Engl J Med. 1990;323:1289–1298.[Abstract]
  47. Blankenhorn DH, Nessim SA, Johnson RL, Sanmarco ME, Azen SP, Cashin-Hemphill L. Beneficial effects of combined colestipol-niacin therapy on coronary atherosclerosis and coronary venous bypass grafts. JAMA. 1987;257:3233–3240.[Abstract]



This article has been cited by other articles:


Home page
 J Am Coll CardiolHome page
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]

Home page
 J Am Coll CardiolHome page
S. H. Choi, A. Chae, E. Miller, M. Messig, F. Ntanios, A. N. DeMaria, S. E. Nissen, J. L. Witztum, and S. Tsimikas
Relationship Between Biomarkers of Oxidized Low-Density Lipoprotein, Statin Therapy, Quantitative Coronary Angiography, and Atheroma Volume Observations From the REVERSAL (Reversal of Atherosclerosis with Aggressive Lipid Lowering) Study.
J. Am. Coll. Cardiol., July 1, 2008; 52(1): 24 - 32.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
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]

Home page
 Eur Heart JHome page
R. Cangemi, L. Loffredo, R. Carnevale, L. Perri, M. P. Patrizi, V. Sanguigni, P. Pignatelli, and F. Violi
Early decrease of oxidative stress by atorvastatin in hypercholesterolaemic patients: effect on circulating vitamin E
Eur. Heart J., January 1, 2008; 29(1): 54 - 62.
[Abstract] [Full Text] [PDF]

Home page
 JNMHome page
M. P. S. Dunphy, H. Schoder, and H. W. Strauss
Radionuclide Techniques for Identifying Vulnerable Plaque
J. Nucl. Med., November 1, 2007; 48(11): 1753 - 1755.
[Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
F. Martino, P. Pignatelli, E. Martino, F. Morrone, R. Carnevale, S. Di Santo, B. Buchetti, L. Loffredo, and F. Violi
Early Increase of Oxidative Stress and Soluble CD40L in Children With Hypercholesterolemia
J. Am. Coll. Cardiol., May 15, 2007; 49(19): 1974 - 1981.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
K. Hartvigsen, C. J. Binder, L. F. Hansen, A. Rafia, J. Juliano, S. Horkko, D. Steinberg, W. Palinski, J. L. Witztum, and A. C. Li
A Diet-Induced Hypercholesterolemic Murine Model to Study Atherogenesis Without Obesity and Metabolic Syndrome
Arterioscler. Thromb. Vasc. Biol., April 1, 2007; 27(4): 878 - 885.
[Abstract] [Full Text] [PDF]