© 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
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
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Abstract |
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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
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Introduction |
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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.
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Methods |
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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
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 (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 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.
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Results |
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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 Table
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 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.
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Table 1
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 1A 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).
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As shown in Figure 1B, 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 2, 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 2A; r=0.82,
P<0.0001) and 125I-Halb (Figure 2B; 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 2).
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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 250 to 1800 mg/dL. As shown
in Figure 3, 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 (Figure
I, published online only,
http://atvb.ahajournals.org/cgi/content/full/20/3/689/DC1).
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, n=13) was then euthanized. For the following 6 months,
group II (chow, 
, n=8) received regular mouse chow, group III
(chow/vit E,C, 
, n=9) received regular mouse chow supplemented with
1.0% vitamin E and 0.05% vitamin C, and group IV (high
fat/cholesterol, 
, 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 3). 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 4 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 4A), 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 4B) 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 4C), 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.
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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 5. The autoradiograph from the baseline
group (Figure 5A) 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 5B) and
chow+vitamin E+vitamin C groups, some areas containing large
Sudan-positive lesions did not show MDA2 uptake by
autoradiography (arrow in Figure 5B), 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 5. Differences in the autoradiography might
otherwise also reflect the fact that lesions of different stages
contain different amounts of OxLDL.
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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 6). 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.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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 1). Both increase linearly with increasing
atherosclerosis, but the uptake of MDA2 is much greater
(Figures 1 and 2). 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.
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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.
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