Download Arterioscler Thromb Vasc Biol. - Arteriosclerosis, Thrombosis, and

Humoral Immune Response Against Defined Oxidized
Low-Density Lipoprotein Antigens Reflects Structure and
Disease Activity of Carotid Plaques
Isabel Gonçalves, Marie-Louise M. Gronholdt, Ingrid Söderberg, Mikko P.S. Ares,
Borge G. Nordestgaard, Jacob F. Bentzon, Gunilla Nordin Fredrikson, Jan Nilsson
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Background—Immune responses against oxidized low-density lipoprotein (LDL) play an important role in atherosclerosis.
The aim of this study was to investigate if humoral immune response against specific oxidized LDL antigens, such as
aldehyde-modified peptide sequences of apolipoprotein B-100, reflects disease activity and structure of atherosclerotic
plaques.
Methods and Results—Plaques were obtained from 114 symptomatic subjects referred to carotid endarterectomy and
characterized immunohistochemically and histologically. Plasma levels of IgG and IgM against aldehyde-modified
apolipoprotein B-100 amino acid sequences 661 to 680, 3136 to 3155 (peptide 210), and 3661 to 3680 (peptide 240)
were determined by enzyme-linked immunosorbent assay. High levels of IgG against peptide 210 were associated with
increased plaque content of lipids (r⫽0.24, P⬍0.05) and hemorrhage (r⫽0.27, P⫽0.005), with decreased content of
fibrous tissue (r⫽⫺0.25, P⫽0.01), but also with lower total plaque volume (r⫽⫺0.21, P⬍0.05). In contrast, high levels
of IgM against peptide 240 were associated with plaques with more fibrous tissue (r⫽0.35, P⬍0.001), less lipids
(r⫽⫺0.34, P⬍0.001), and less macrophages (r⫽⫺0.24, P⬍0.05). IgM against peptide 210 were found to be associated
with plaque fibrous tissue (r⫽0.20, P⬍0.05), less lipids (r⫽⫺0.21, P⬍0.05), and less macrophages (r⫽⫺0.27,
P⫽0.01).
Conclusion—These findings support the notion that immune responses against oxidized LDL epitopes are involved in
atherosclerosis and that the level of circulating antibodies against these structures may reflect disease activity in the
arterial wall. (Arterioscler Thromb Vasc Biol. 2005;25:1250-1255.)
Key Words: antibodies 䡲 atherosclerosis 䡲 carotid plaque 䡲 carotid stenosis 䡲 modified low-density lipoprotein
I
nnate and adaptive immune responses against oxidatively
modified low-density lipoprotein (LDL) are believed to
play important roles in atherosclerosis.1,2 This escape from
self-tolerance is dependent on formation of oxidized phospholipids3,4 and aldehyde-modified breakdown fragments of
apolipoprotein B-100 (apoB-100).5 Antigen presenting cells
take up oxidized LDL through the scavenger receptor pathway and initiate an adaptive immune response by possibly
presenting lipid antigens on CD 1 receptors6 and peptide
antigens on human leukocyte antigen-DR (HLA-DR) receptors.7 The existence of a pre-existing, natural immune response against oxidized LDL phospholipids mediated by IgM
produced by B-1 cells has also been identified.4 The role of
adaptive immunity in atherosclerosis is complex and remains
to be fully understood. Mice lacking functional T and B cells,
such as SCID and RAG-1 mice, generally have less athero-
sclerosis.8,9 However, hyperactivation of oxidized LDL autoimmunity through immunization with LDL modified in vitro
as well as with aldehyde-modified apoB-100 peptide sequences results in a marked inhibition of atherosclerosis.10 –14
This atheroprotective effect of immunization has, in some
studies, been associated with expression of specific IgG.13,14
Treatment of apolipoprotein E⫺/⫺ mice with recombinant IgG
against aldehyde-modified apoB-100 peptide fragments inhibit the development of atherosclerosis.15
The emerging understanding of the importance of immune
responses against oxidized LDL in atherosclerosis has focused attention on the possibility that they could be used to
assess disease activity and risk for development of clinical
events in humans. Several studies have been performed to
investigate the association between autoantibodies against
oxidized LDL and the severity of atherosclerosis as assessed
Original received September 24, 2004; final version accepted March 30, 2005.
From the Departments of Medicine (I.G., I. S., M.P.S.A., G.N.F., J.N.) and Cardiology (I.G.), Lund University, Wallenberg Laboratory, University
Hospital MAS, Malmö, Sweden; the Department of Vascular Surgery (M.L.G.), Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; the
Department of Clinical Biochemistry (B.N.), Herlev University Hospital, University of Copenhagen, Copenhagen, Denmark; and the Department of
Coronary Pathology Research (J.F.B.), Skejby University Hospital, Århus, Denmark.
Consulting Editor for this article was Peter Libby, MD, Brigham and Women’s Hospital, Boston, Mass.
Correspondence to Isabel Gonçalves, University of Lund, Wallenberg Laboratory, University Hospital MAS, SE-20502 Malmö, Sweden. E-mail
[email protected]
© 2005 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1250
DOI: 10.1161/01.ATV.0000166518.96137.38
Gonçalves et al
by coronary angiography16,17 and carotid intima-media thickness.18 –20 We have recently identified aldehyde-modified
peptide sequences in apoB-100 that are targeted by autoantibodies present in human plasma.18 In this study, we have
investigated the association between the plasma levels of 3 of
these autoantibodies specific for apoB-100 amino acids 661
to 680 (peptide 45), 3136 to 3155 (peptide 210), and 3661 to
3680 (peptide 240), respectively, by determining binding to
the corresponding aldehyde-modified synthetic polypeptides
in enzyme-linked immunosorbent assay (ELISA), and atherosclerotic carotid plaque structure as assessed by ultrasonography (grayscale median values), histology, and immunohistochemistry. These peptide sequences were selected because
their effect when used in active immunization of experimental animals is well-characterized and because autoantibodies
against these sequences are common in humans.
Materials and Methods
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Patients
This study included 114 patients (77 males, 37 females), aged
60.9⫾7.8 (mean⫾SD) years, referred to carotid endarterectomy at
Rigshospitalet, University of Copenhagen, Denmark. These patients
had previously experienced ipsilateral hemispheric neurological
symptoms (20 transient ischemic attack, 49 amaurosis fugax, and 45
stroke) in the last 93⫾59 days. Plaques were removed from patients
with internal carotid artery stenosis ⬎50%. The severity of carotid
stenosis was assessed by duplex imaging by the same observer using
internationally established criteria.21 Cardiovascular risk factors such
as hypertension (systolic blood pressure ⬎140 mm Hg), diabetes,
clinical history of coronary artery disease, claudication, tobacco use
(in the past or current), and lipid-lowering medication were recorded.
Laboratory analyses including total cholesterol, high-density lipoprotein cholesterol, LDL cholesterol, and triglycerides were performed in fasting blood samples. The study was approved by the
local research ethics committee. Patients with excessive alcohol
intake, liver disease, cancer, infectious diseases, systemic inflammatory disease, or cerebral hemorrhage were excluded. One or more
computer tomography scans were performed to exclude cerebral
hemorrhage as the cause for the neurological symptoms. Blood
sampling for determination of oxidized LDL autoantibodies and
fasting lipoproteins was performed the day before surgery.
Ultrasound Evaluation
Carotid high-resolution ultrasonography (Apogee Interspec 400
scanner; ATL Ultrasound Bothell, Wash; 5- to 10-MHz linear array
probe) of the plaques was blindly performed preoperatively by one
observer. Ultrasonographic data of 9 plaques were accidentally lost.
The best longitudinal B-mode image of the carotid plaque and the
corresponding color Doppler image showing the outlines of the
plaque were recorded on S-VHS, later digitized, and processed by
the software program Image-Pro Plus 1.2.01 for Windows (Media
Cybernetics, Silver Spring, Md). The carotid plaque was outlined
carefully, excluding acoustic shadowing when present. The median
of the grayscale of the outlined pixels (grayscale median) was
determined according to previously described and validated methodology.22–27 The degree of stenoses was determined by Doppler
criteria, separating into 0% to 15%, 16% to 49%, 50% to 79%, and
80% to 99% stenosis and occlusion.21
Peptide ELISA
Malondialdehyde (MDA)-modified peptides were prepared as described.28 MDA-modified peptide 45 (amino acids 661 to 680), 210
(amino acids 3136 to 3155), or 240 (amino acids 3661 to 3680)18
were used in the ELISAs. Peptide 45 contained 2 lysine residues and
the MDA content of the modified peptide was 0.047 mol/mol
peptide, peptide 210 contained 3 lysine and 1 histidine residues, and
Antibodies Against oxLDL and Plaque Structure
1251
the MDA content of the modified peptide was 0.080 mol/mol peptide
and peptide 240 contained 2 lysine residues, and the MDA content of
the modified peptide was 0.048 mol/mol peptide. The peptides were
diluted in phosphate-buffered saline, pH 7.4 (20 ␮g/mL), and used
for coating of microtiter plates (Nunc MaxiSorp; Nunc, Roskilde,
Denmark) in an overnight incubation (4°C). The coated plates were
washed with phosphate-buffered saline containing Tween-20,
blocked with Superblock in Tris-buffered saline (Pierce, Rockford,
Ill) for 10 minutes at room temperature, incubated with human serum
diluted 1:100 in TBS 0.1% Tween for 2 hours at room temperature,
and thereafter overnight at 4°C. The deposition of antibodies was
detected by adding biotinylated rabbit anti-human IgM or IgG
antibodies (Jackson ImmunoResearch, Westgrove, Pa) for 2 hours at
room temperature. After washing, bound antibodies were detected by
alkaline phosphatase-conjugated streptavidin (DakoCytomation,
Glostrup, Denmark) as described.18 The results are presented as
absorbance (abs) units with the background value (binding to
nonpeptide-coated plastic wells) subtracted. Specificity testing of the
ELISAs was performed using plasma pooled from healthy controls
(n⫽33, age 42.7⫾14.0 years). In this reference, plasma IgM levels
for peptides 45, 210, 240, and MDA–LDL were 1.05 abs units, 1.47
abs units, 1.08 abs units, and 1.67 abs units, respectively. The
interassay coefficients of variation for the IgM ELISAs were ⬍5%.
Pre-incubation of control plasma with 500 ␮g of peptide 210 for 1
hour at room temperature and overnight at 4°C competed 31% of
binding to peptide 45, 57% of binding to peptide 210, 53% of
binding to peptide 240, and 21% of binding to MDA–LDL. IgG
levels for peptides 45, 210, 240, and MDA–LDL were 0.44 abs units,
0.22 abs units, 0.05 abs units, and 0.26 abs units, respectively. The
interassay coefficients of variation for the IgG ELISAs were ⬍15%.
Pre-incubation of control plasma with 500 ␮g of peptide 210
competed 33% of binding to peptide 45, 70% of binding to peptide
210, 100% of binding to peptide 240, and 0% of binding to
MDA–LDL.
Histological Procedure
Carotid plaques were removed en bloc by carotid endarterectomy.
The specimen was then cut transversally in 3-mm-thick blocks (4 to
14 per patient), paraffin-embedded, and 4-␮m sections were cut from
the blocks. Sections were then stained with hematoxylin and eosin,
Van Gieson stain, and Verhoeff stain for fibrous tissue. Plaque
constituents (lipid-rich core, hemorrhage, and fibrous tissue) in all
sections were measured morphometrically using the semi-automatic
image analyzing software Leitz Texture Analyzing System (TAS,
Cambridge, UK). The microscopic image of the plaque section was
transferred through a video camera and digitized to a computer
screen. Plaque constituents were then outlined manually using a light
pen by a pathologist to determine the relative plaque area in each
section. The total plaque volume is calculated as the sum of the
plaque area in all sections times the plaque length of each section.29
The largest variation on reproducibility testing based on 10 randomly
chosen plaques for measurements of plaque constituents was found
for hemorrhage (0.5% ⫾ 0.3%; mean⫾SD). All analyses were
performed blindly.
The section from the most stenotic area and the 2 adjacent ones
(upstream and downstream) were used for macrophage staining.
After rehydration and proteolytic pretreatment with trypsin, the 3
sections were immunostained by sequential incubation with blocking
rabbit serum (DakoCytomation), primary monoclonal antibody
against human macrophages (CD 68, 1:200; DakoCytomation),
secondary biotinylated rabbit antibody (1:300; DakoCytomation),
ABComplex-alkaline phosphatase (DakoCytomation), chromogen
substrate (Vector Laboratories, Burlingame, Calif), and counterstained with hematoxylin. The microscopic images of the immunostained sections were captured with a Sony 3-chip color video
camera (Tokyo, Japan) and analyzed blindly with an automated
image analysis equipment. The brightness and color tone were
adjusted and a 24-color palette containing 3 red colors specific for
the macrophage staining was applied in PaintShop Pro 5 (Jasc
Software, Minnetonka, Minn). The percentage of plaque having the
macrophage-specific colors was quantified in Sigma Scan Pro 3.0
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Arterioscler Thromb Vasc Biol.
June 2005
TABLE 1.
Clinical Characteristics of the Patients
Patients (n⫽114)
Age, y
60.9⫾7.8
Sex
37 F/77 M
Degree of stenosis, %
80.4⫾12.1
Hypertension, %
54 (47)
Diabetes, %
12 (11)
Coronary artery disease, %
24 (21)
Smoking (in the past or currently), %
67 (59)
Fasting lipoproteins
Total cholesterol, mmol/L
6.3⫾1.6
HDL cholesterol, mmol/L
1.4⫾0.5
LDL cholesterol, mmol/L
3.5⫾1.1
Triglycerides, mmol/L
1.9⫾1.1
Statin use, %
19 (17)
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Values are presented as mean⫾standard deviation.
F indicates female; HDL, high-density lipoprotein; LDL, low-density lipoprotein; M, male.
(Jandel Corporation, San Rafael, Calif). An average value of the
macrophage density in the 3 sections was used in statistical analysis.
Statistical Analysis
Values are presented as mean⫾SD. ␹2 analyses or Fisher exact test
analysis was performed to investigate associations with dichotomous
variables. Two-group comparisons were performed using the unpaired Student t or Mann–Whitney test, according to the distribution
of the samples. Spearman correlation and partial correlations controlling for age and gender were used. Linear regression models
considering the histological parameters as the dependent variables
were used. When the regression was multivariate, the backward
elimination was performed. Values of P⬍0.05 were considered to
indicate statistically significant findings. Statistical analysis was
performed using SPSS 12.0.1.
Results
Basic Characteristics and Autoantibodies
The study group consisted of 77 men and 37 women with
symptomatic carotid disease and internal carotid artery stenosis ⬎50% referred to carotid endarterectomy. The clinical
characteristics of the study group are described in Table 1.
The most avidly expressed autoantibodies were IgM against
peptide 210 (Figure 1). Significant correlations were found
between the different IgM autoantibodies as well as between
the different IgG. In contrast, there were no correlations
between the respective IgM and IgG autoantibodies for each
peptide (Table 2). IgG against peptide 210 was lower in men
versus women (0.32⫾0.46 versus 0.49⫾0.40 abs units;
P⬍0.02) and in patients with diabetes versus those without
(0.10⫾0.10 versus 0.41⫾0.49 abs units; P⬍0.05). Otherwise,
there were no statistically significant differences in the
antibody levels for patients with or without hypertension,
history of coronary artery disease, use of tobacco, or statin
medication. With the exception of a positive association
between IgG against peptide 45 and LDL cholesterol levels
(r⫽0.21, P⬍0.05), there were no associations between antibodies and plasma lipoproteins.
Figure 1. IgG (gray box plot) and IgM (white box plot) autoantibodies against MDA-modified apoB-100 peptide sequences
(P45, P210, and P240). Box plots showing the median, 25th to
75th percentiles, and minimum–maximum observed absorbance
units in ELISA for each autoantibody.
Histology and Autoantibodies
Controlling for age and gender, significant correlations were
found between high plasma levels of IgM against peptide 210
and an increased plaque content of fibrous tissue (r⫽0.20,
P⬍0.05; Figure 2A). In contrast, high levels of IgG against
the same peptide sequence were associated with a decreased
content of fibrous tissue (r⫽⫺0.25, P⫽0.01; Figure 3A).
Moreover, high levels of IgM against peptide 210 were
associated with a decreased plaque content of lipids
(r⫽⫺0.21, P⬍0.05; Figure 2C) and macrophages (r⫽⫺0.27,
P⫽0.01; Figure 2E), whereas high IgG levels correlated with
an increased plaque content of lipids (r⫽0.24, P⬍0.05;
Figure 3B). There were also significant correlations between
IgG against peptide 210 and the severity of plaque hemorrhage (r⫽0.27, P⫽0.005; Figure 3C), as well as with a lower
total plaque volume (r⫽⫺0.21, P⬍0.05; Figure 3D).
Also, IgM against peptide 240 were found to be associated
with plaques containing more fibrous tissue (r⫽0.35,
P⬍0.001; Figure 2B), less lipids (r⫽⫺0.34, P⬍0.001; Figure
2D), and less macrophages (r⫽⫺0.24, P⬍0.05; Figure 2F).
There were no significant relations between IgM against
peptide 45 and plaque structure. Moreover, there were no
TABLE 2. Spearman Correlation Matrix for the Antibodies
Against MDA-Modified apoB-100 Peptide Sequences (P45,
P210, and P240)
P45
IgG
P45 IgG
P45
IgM
P210
IgM
P240
IgG
P240
IgM
1
P45 IgM
0.10
1
P210 IgG
0.21*
⫺0.05
P210 IgM
P210
IgG
⫺0.02
P240 IgG
0.26†
P240 IgM
0.11
0.37†
1
⫺0.05
⫺0.06
0.42†
0.19*
⫺0.21*
Results are expressed as r values.
*P⬍0.05; †P⬍0.001.
1
0.03
1
0.59†
⫺0.02
1
Gonçalves et al
Antibodies Against oxLDL and Plaque Structure
1253
together explaining 14% of the variation (P⬍0.005). Finally,
independent associations with plaque hemorrhage were found
for IgG against peptide 210 (P⬍0.005), age (P⬍0.05),
smoking (P⬍0.05), claudication (P⬍0.005), family history
for coronary heart disease (P⬍0.05), and degree of stenosis
(P⬍0.05) together explaining 27% of the variation
(P⬍0.001).
Discussion
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Figure 2. Correlations between IgM antibodies and carotid
plaque histological characteristics, controlling for age and gender. Correlations between peptide 210 (P210) IgM (logarithmically transformed) and the plaque area stained for fibrous tissue
(A), lipids (C), and macrophages (E). Correlations between peptide
240 (P240) IgM (logarithmically transformed) and the plaque area
stained for fibrous tissue (B), lipids (D), and macrophages (F).
significant correlations between IgG against peptides 45 and
240 and plaque structure. However, a significant association
was observed between IgG against peptide 240 and the
plaque grayscale median value at the pre-operative ultrasound
investigation (r⫽0.23, P⬍0.05).
A linear regression analysis was performed, including IgG
and IgM autoantibodies, age, gender, plasma lipoproteins,
smoking, hypertension, diabetes, claudication, and family
history of coronary heart disease. Significant independent
associations with total plaque volume were found for gender
(P⬍0.001), total cholesterol (P⫽0.001), high-density lipoprotein (P⫽0.005), triglycerides (P⬍0.05), and family
history for coronary heart disease (P⬍0.05), explaining 40%
of the variation, whereas IgG against peptide 210 did not
remain significantly associated after adjustment for the factors mentioned. Plasma levels of IgM against peptide 240,
IgG against peptide 210, and family history for coronary heart
disease together explained 17% of the variation in plaque
fibrous tissue content (P⫽0.001), but only IgM against
peptide 240 remained independently associated after adjustment for all other factors (P⬍0.02). IgG against 210 and IgM
against 240 together explained 13% of the variation of plaque
lipids (P⬍0.005), but only IgM against peptide 240 showed
independent significant association (P⫽0.01). Both LDL
(P⬍0.01) and IgM against peptide 210 (P⬍0.02) showed
independent association with plaque macrophage content,
The present findings suggest the interesting possibility that
monitoring humoral immune responses against oxidized
LDL-specific antigens could be used to determine the structure and disease activity of atherosclerotic lesions. High
levels of IgG against the aldehyde-modified apoB-100 peptide 210 was associated with small, lipid-rich, fibrous-poor
plaques frequently containing signs of hemorrhage. These
features are generally considered characteristic for vulnerable, rupture-prone plaques.30,31 In contrast, high levels of IgM
against aldehyde-modified peptides 210 and 240 were associated with fibrous, lipid-poor plaques containing less macrophages. These characteristics are considered to be typical
for stable plaques with little risk for development of clinical
events.30,31 Because evidence is accumulating of an important
role for immune responses against oxidized LDL in the
disease process, it seems reasonable that the activity of
human immune responses against oxidized LDL could reflect
the disease process within atherosclerotic plaques.
There were significant associations between the different
IgM, as well as between the different IgG. To a certain extent,
this was explained by cross-reactivity of the same antibodies
with different peptides most likely caused by recognition of
MDA adducts. Binding competition studies revealed that this
was particularly true for antibodies binding to peptides 210
and 240. However, the cross-reactivity of these antibodies
with peptide 45 was much less prominent despite a similar
degree of MDA modification, suggesting that the antibody
Figure 3. Correlations between IgG antibodies and carotid
plaque histological characteristics, controlling for age and gender. Correlations between peptide 210 (P210) IgG (logarithmically transformed) and the plaque area stained for fibrous tissue
(A), lipids (B), hemorrhage (C), and total plaque volume (D).
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Arterioscler Thromb Vasc Biol.
June 2005
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binding also depended on the peptide sequence. In contrast,
there was no association between IgG and IgM against the
same peptide. Moreover, immune responses against different
sites in apoB-100 were not consistent in their association with
plaque structure. The oxidative modification of LDL is a
complex process and its in vivo kinetics remains largely
unknown. However, it is known that LDL with minor
modifications is present in the circulation, whereas more
severely oxidized LDL are found inside atherosclerotic
plaques.32
High levels of IgG against peptide 210 were not only
associated with more lipid-rich lesions but also associated
with a smaller plaque size. The latter observation is in
agreement with several studies demonstrating an inverse
association between oxidized LDL IgG and carotid intimamedia thickness.19,20 However, in prospective studies, high
titers of IgG against oxidized LDL have also been associated
with an increased risk for development of cardiovascular
events.33,34 This is in accordance with the present observation
that IgG levels may reflect the presence of vulnerable
plaques.
The functional role of oxidized LDL autoantibodies remains to be fully understood. The atheroprotective effect of
immunization with oxidized LDL antigens has generally been
associated with expression of specific IgG.13 Direct evidence
for a protective effect of IgG has also been obtained from
studies in mice treated with recombinant human IgG specific
for the MDA-modified peptide 45 sequence.15 Because these
studies favor an atheroprotective role of oxidized LDL IgG,
the present observation of an association of these IgG with
more unstable plaques appears paradoxical. One possibility is
that this reflects a fundamental difference in the immune
response to oxidized LDL that is activated endogenously as
part of the atherosclerotic disease process and that activated
in response to immunization. Apolipoprotein E⫺/⫺ mice lacking functional CD4⫹ T cells have less atherosclerosis,35
suggesting that the net effect of adaptive immunity is
proatherogenic. Assuming that similar mechanisms are involved also in the human disease process, IgG levels against
oxidized LDL antigens may act as markers of this adaptive
immune response. Experimental studies evaluating the effect
of active immunization have consistently used adjuvants
favoring Th2 type immune responses characterized by induction of antiinflammatory cytokines and increased IgG secretion.10 –13 Accordingly, IgG may in this situation serve as
marker for a shift from a pro-inflammatory Th1 response
toward an antiinflammatory Th2 response and reach sufficiently high levels to have protective effects in itself.
IgM against oxidized LDL phospholipids inhibit the scavenger receptor-mediated uptake of oxidized LDL and apoptotic cells in macrophages.3 Immunization of apolipoprotein
E⫺/⫺ mice with Streptococcus pneumoniae has been shown to
result in increased expression of oxidized LDL-specific IgM,
inhibition of atherosclerosis, and reduced levels of oxidized
LDL in plasma.36 The latter observation suggests the possibility that these IgM may help to clear oxidized LDL from the
circulation. This notion has also been supported by clinical
studies demonstrating inverse associations between IgM
against MDA-modified apoB-100 peptides and plasma-
oxidized LDL.18 However, recent findings of an unaltered
clearance of oxidized LDL in immunodeficient mice argue
against this effect of oxidized LDL IgM.37 The possibility that
antibody opsonization of oxidized LDL in plaques may
influence its removal from the extracellular space by mediating uptake via Fc or complement receptors and that IgG and
IgM may differ in this respect should be considered.38 There
is also a possibility that formation of oxidized LDL immune
complexes in plaques may lead to complement activation and
tissue damage. It remains to be clarified if the association
between IgG against peptide 210 and plaque hemorrhage
reflects activation of such processes.
The present observations need to be interpreted with due
caution because they are based on a relatively small number
of samples and represent associations present at a single time
point. It would be of considerable interest to study the
association of these immune responses with plaque structure
over an extended time period using ultrasound, both extravascular and intravascular, or magnetic resonance imaging.
Moreover, antibody levels were only compared with plaque
tissue from a single arterial segment. It is uncertain how
representative these plaques are of lesions in other arteries. It
should also be kept in mind that the present findings only
demonstrate the existence of an association between antibodies against oxidized LDL antigens and plaque structure but do
not clarify whether these antibodies have a direct effect on
plaques or if they only serve as secondary markers.
In summary, these studies add further support to the notion
that immune responses against epitopes in oxidized LDL are
involved in atherosclerosis and suggest that the level of
circulating antibodies against these structures may reflect
disease activity in the artery wall.
Acknowledgments
This study was supported by grants from the Swedish Research
Council (grant number 8311), the Swedish Heart and Lung Foundation, the Swedish Medical Society, Ernhold Lundström Foundation,
Crafoord Foundation, Malmö University Hospital funds, The Royal
Physiographic Society, and Lars Hierta Foundation. We thank Britt
M. Wiebe and Henning Laursen, Department of Neuropathology,
Rigshospitalet, Copenhagen, Denmark, for help on the histopathologic analysis of plaque composition, and Hanne Damm, Department
of Clinical Biochemistry, for handling blood samples.
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Downloaded from http://atvb.ahajournals.org/ by guest on May 11, 2017
Humoral Immune Response Against Defined Oxidized Low-Density Lipoprotein Antigens
Reflects Structure and Disease Activity of Carotid Plaques
Isabel Gonçalves, Marie-Louise M. Gronholdt, Ingrid Söderberg, Mikko P.S. Ares, Borge G.
Nordestgaard, Jacob F. Bentzon, Gunilla Nordin Fredrikson and Jan Nilsson
Arterioscler Thromb Vasc Biol. 2005;25:1250-1255; originally published online April 14, 2005;
doi: 10.1161/01.ATV.0000166518.96137.38
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