Atherosclerosis and Lipoproteins |
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 |
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Key Words: regression progression atherosclerosis lipoproteins autoantibodies
| Introduction |
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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 receptordeficient (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 |
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Determination of Antibody Binding to MDA-LDL
and Cu-OxLDL
The levels of IgM and IgG autoantibodies binding to
MDA-LDL (antiMDA-LDL) and Cu-OxLDL (antiCu-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 phosphataselabeled 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 suppliers
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 |
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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 antiMDA-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 antiMDA-LDL
and antiCu-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.
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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 antiMDA-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
antiCu-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 antiCu-OxLDL IgM and
125I-MDA2 uptake
(R=0.37,
P=0.09), but there was no
correlation with lesion area or weight.
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| Discussion |
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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 Edepleted 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 Edepleted. 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 |
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Received July 17, 2000; accepted October 6, 2000.
| References |
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