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Reduction of atherosclerosis in low-density lipoprotein receptordeficient mice by passive administration of antiphospholipid antibody.

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Vol. 48, No. 10, October 2003, pp 2974–2978
DOI 10.1002/art.11255
© 2003, American College of Rheumatology
Reduction of Atherosclerosis in
Low-Density Lipoprotein Receptor–Deficient Mice by
Passive Administration of Antiphospholipid Antibody
Danielle Nicolo, Bruce I. Goldman, and Marc Monestier
Objective. Patients with antiphospholipid syndrome (APS) are at a high risk of developing atherosclerotic complications. Conversely, individuals with
primary atherosclerosis have an increased prevalence of
antiphospholipid antibodies (aPL) and antibodies to
oxidized low-density lipoproteins (ox-LDL). Several
studies suggest that these two antibody populations may
in fact overlap, although it is unclear how aPL contribute to pathogenesis. In this study, we characterized an
IgG monoclonal aPL and assessed its ability to modulate atherosclerosis in low-density lipoprotein receptor–
deficient (LDLRⴚ/ⴚ) mice.
Methods. The cardiolipin-reactive monoclonal
antibody FB1 was obtained from an (NZW ⴛ BXSB)F1
mouse, a strain with APS features that make it prone to
fatal myocardial infarctions. Using an enzyme-linked
immunosorbent assay, we investigated the binding of
this antibody to phospholipid and LDL antigens. We
also passively administered FB1 to atherosclerosisprone mice to determine its effect on atherogenesis.
Results. In contrast to earlier studies of aPL that
were specific for oxidized forms of LDL, FB1 crossreacted with both native LDL and ox-LDL. In vivo,
passive administration of FB1 significantly reduced
plaque formation in atherosclerosis-prone LDLRⴚ/ⴚ
Conclusion. These results indicate that some aPL
may play a protective role in atherogenesis and suggest
a novel approach to the prevention of atherosclerosis.
Autoantibodies that react with negatively
charged phospholipids are encountered in a variety of
systemic autoimmune diseases, including systemic lupus
erythematosus and antiphospholipid syndrome (APS).
Several studies indicate that some antiphospholipid antibodies (aPL) can react with oxidized low-density lipoproteins (ox-LDL) (1). Some authors have suggested
that similar antigenic determinants can be created by the
oxidation of phospholipids and LDLs (2,3). Because the
oxidation process can create a great diversity of
epitopes, it is likely that aPL possess different specificities. Antibodies to ox-LDL are seen in patients with
atherosclerosis, a condition associated with a progressive
oxidation of lipoprotein particles (4). It is unclear how
autoantibodies affect the progression of the disease.
Some investigators have suggested that antibodies to
ox-LDL may contribute to the pathogenesis of atherosclerosis (5). In contrast, recent studies suggest that
these antibodies may protect against the disease, possibly because they can scavenge the ox-LDL particles
To further explore the relationship between aPL
and atherosclerosis, we studied an IgG monoclonal
antibody (mAb) from an (NZW ⫻ BXSB)F1 mouse, a
cross that develops autoimmune and pathologic manifestations that are reminiscent of APS (8). This mAb was
originally selected for its ability to bind cardiolipin (CL),
but it also reacts with both native and oxidized forms of
LDL. Administration of this mAb to atherosclerosisprone mice paradoxically resulted in a decrease in
plaque formation. These data suggest that the manipulation of humoral immunity represents a therapeutic
approach to atherosclerosis.
Supported by grants from the NIH and the American Heart
Danielle Nicolo, PhD, Bruce I. Goldman, MD, Marc Monestier, MD, PhD: Temple University School of Medicine, Philadelphia,
Address correspondence and reprint requests to Marc Monestier, MD, PhD, Temple University School of Medicine, Department
of Immunology and Microbiology, 3400 North Broad Street, Philadelphia, PA 19140. E-mail:
Submitted for publication August 27, 2002; accepted in
revised form June 13, 2003.
FB1 antiphospholipid monoclonal antibody. FB1 is a
CL-reactive IgG2b mAb that was obtained from a 5-month-old
female (NZW ⫻ BXSB)F1 mouse (9). FB1 was purified from
supernatant by affinity chromatography on protein G–
Sepharose (Amersham Biosciences, Piscataway, NJ).
Enzyme-linked immunosorbent assay (ELISA). An
ELISA was used to assess the binding of FB1 to CL (Avanti
Polar Lipids, Alabaster, AL) or to LDL and its oxidized
derivatives. Briefly, polyvinyl chloride microtiter plates (Becton Dickinson, Franklin Lakes, NJ) were coated with 20 ␮g/ml
of native LDL, minimally modified LDL, or ox-LDL. When
native LDL was used as the antigen, EDTA (0.1 mg/ml) and
butylated hydroxytoluene (20 ␮M) (both from Sigma, St.
Louis, MO) were included throughout to prevent oxidation.
When CL was used as the antigen, the plates were coated with
20 ␮g/ml of CL in ethanol. After blocking, purified FB1
(diluted in phosphate buffered saline [PBS] containing 1%
bovine serum albumin [Sigma] and 0.05% Tween 20 [Sigma]),
was added to the wells at varying concentrations and incubated
for 2 hours at room temperature. After washing, binding was
detected with goat anti-mouse IgG–alkaline phosphatase
(Southern Biotechnology, Birmingham, AL) followed by color
development with the appropriate substrate.
Animal studies. LDL receptor–deficient (LDLR⫺/⫺)
mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facilities. All animal
research protocols were approved by the Temple University
Institutional Animal Care and Use Committee. In experiment
1, 16-week-old mice received intraperitoneal injections of
purified FB1 twice a week for 12 weeks (0.75 mg/injection),
whereas control animals received PBS. Mice were fed a
high-fat Western-type diet containing 20% fat with 1.25%
cholesterol and 0.5% cholic acid. In experiment 2, 10-week-old
mice received an identical regimen of FB1 for 12 weeks, but
control animals received equivalent injections of mAb 51A9,
an isotype-matched mAb to the influenza virus (a gift of Dr. T.
Moran, Mount Sinai School of Medicine, New York, NY).
These mice were also fed a Western-type diet. In both
experiments, total serum cholesterol levels were determined at
the beginning and end of the experiments.
Quantification of atherosclerotic lesions. At the end of
the experiments, each mouse was killed, and the aorta was
perfused with PBS via the left ventricle of the heart and
then removed. Dissections were performed in a blinded manner using a Leica MZ 12.5 dissecting microscope (Leica
Instruments, Allendale, NJ). The heart and ascending and
descending aorta were dissected to the iliac bifurcation. Adventitial and adipose tissues were removed, and the outer
curvature of the arch was cut longitudinally (10). Aortas
were briefly rinsed with 70% ethanol, stained with Sudan IV
for 5 minutes, and then destained for 5 minutes (10). Aortas
were pinned, with the intimal surface up, on a black wax
surface. Images were captured using a Leica DC 200 digital
camera, and the surface area of the red-stained lesions was
quantified with Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD). Results were expressed as the
percentage of intimal surface involved.
Immunohistochemistry. The aortic arch was
transected at the level of the aortic sinuses, snap-frozen in
embedding compound, and stored at ⫺80°C. Serial 6 ␮m–thick
transverse cryosections from the aortic arch through the aortic
root were taken at 300-␮m intervals. Sections were air-dried,
stained with oil red O, counterstained with hematoxylin, and
Figure 1. Cross-reaction of antiphospholipid monoclonal antibody
FB1 with native and oxidized low-density lipoproteins (LDLs).
Enzyme-linked immunosorbent assay was performed with 20 ␮g/ml of
antigen and varying concentrations of FB1, as described in Materials
and Methods. OD ⫽ optical density; MM-LDL ⫽ minimally modified
LDL; CL ⫽ cardiolipin; ox-LDL ⫽ oxidized LDL.
examined with a light microscope to assess the overall architecture and severity of atherosclerosis.
Binding of FB1 to LDLs at various stages of
oxidation. To determine the binding specificity of FB1,
we tested the mAb against LDL at various stages of
oxidation. The process of oxidation occurred continuously, affecting both the lipid and protein moieties of
LDL (7). FB1 reacted with all forms of LDL in a
dose-dependent manner (Figure 1). However, the level
of FB1 binding was inversely correlated with the oxidation of LDL, since FB1 reacted best with native LDL
and least with ox-LDL. For purposes of comparison,
Figure 1 also shows FB1 binding to CL.
Reduction of atherosclerosis in LDLRⴚ/ⴚ mice
by FB1. Because FB1 reacts with LDL in various forms,
we investigated whether FB1 could modulate disease in
atherosclerosis-prone mice. FB1 was administered to
groups of LDLR⫺/⫺ mice consuming a Western-type
diet as described above. In experiment 1, the passive
administration of FB1 reduced the extent of atherosclerosis from 28% lipid lesions in PBS-treated mice to 20%
in FB1-treated mice (P ⫽ 0.03), representing a 28%
reduction in atherosclerosis (Figure 2A). The total serum cholesterol levels were unaffected by FB1 treatment
(Figure 2B).
To exclude any nonspecific effect of FB1, we
repeated this experiment while administering an isotypematched irrelevant mAb to the control group. In experiment 2, treating mice with mAb FB1 again reduced the
Figure 2. Reduction of atherosclerosis in low-density lipoprotein receptor–deficient (LDLR⫺/⫺)
mice by antiphospholipid monoclonal antibody FB1. A, In experiment 1, 16-week-old LDLR⫺/⫺
mice were injected intraperitoneally for 12 weeks with FB1 at a dosage of 1.5 mg/week, while
control mice were injected with an equal volume of sterile phosphate buffered saline (PBS). Values
are the mean and SEM percentage of atherosclerosis in the aortas of both groups of mice that were
fed a Western-type diet beginning at the start of the injections. Lesions in the aortic root area were
quantitated morphometrically, as described in Materials and Methods. The difference in the
percentage of intimal involvement in lipid lesions between the PBS- and FB1-treated mice was
statistically significant (P ⫽ 0.03). B, Scatterplot showing total serum cholesterol levels in the same
mice as in A. Cholesterol levels were not significantly different between the 2 groups of mice. Each
symbol represents an individual mouse. C, In experiment 2, 10-week-old LDLR⫺/⫺ mice were
injected intraperitoneally for 12 weeks with FB1 at a dosage of 1.5 mg/week, while control mice
received injections of 51A9, an irrelevant isotype-matched control antibody. The difference in lipid
lesions between the 51A9- and FB1-treated mice was statistically significant (P ⫽ 0.007). Mice in
experiment 1, which compared FB1 and PBS treatments, were 1.5 months older and, consequently,
had a higher percentage of lipid lesions than the mice used in this experiment. Values are the mean
and SEM. D, Scatterplot of total serum cholesterol levels in the same mice as in C.
extent of atherosclerosis compared with mice treated
with the anti-influenza virus control mAb 51A9. Figure
2C shows that FB1 treatment reduced the lipid lesions
from 23% in control mAb–treated mice to 13% in
FB1-treated mice (P ⫽ 0.007), representing a 43%
reduction in atherosclerosis. Again, this reduction in
atherosclerosis in FB1-treated mice was independent of
alterations in total serum cholesterol levels (Figure 2D).
The results of both of these experiments indicated that
treatment with FB1 significantly reduces the extent of
aortic lesions in LDLR⫺/⫺ mice.
We compared the morphology and architecture
of the lesions between the 51A9- and FB1-treated mice
with the use of a light microscope to examine the oil
red O–stained cryosections. Figure 3 shows representative sections taken from the aortic sinuses of both groups
in experiment 2. The extent and severity of the lesions in
the FB1-treated mice were qualitatively reduced compared with those in the 51A9-treated mice (Figures 3A
and C). These histochemical staining results are consistent with those obtained using en face staining of the
aortas from these 2 groups of mice (Figure 2C). In
addition, we observed differences in the aortic medial
layer between the 2 groups of mice. The aortic medial
layer of the FB1-treated mice tended to be thicker than
that of the 51A9-treated mice (Figures 3B and D).
Moreover, lipid-containing cells were present in the
aortic medial layer of the FB1-treated mice, whereas
Figure 3. Effect of antiphospholipid monoclonal antibody (mAb)
FB1 treatment on atherosclerosis in LDLR⫺/⫺ mice. Transverse
cryosections were cut from aortic roots and stained with oil red O and
hematoxylin. A, Representative section of aortic root from a control
LDLR⫺/⫺ mouse receiving irrelevant isotype-matched control antibody. B, High-power photomicrograph of the area of lesion indicated
by the asterisk in A, showing intimal atherosclerotic plaque composed
of extracellular matrix with numerous lipid-containing cells (arrows)
and small lipid droplets, which occupy most of the luminal surface.
Aortic media (M) shows essentially no lipid deposits. C, Representative section from aortic root of FB1-treated LDLR⫺/⫺ mouse. The
extent of luminal involvement by atherosclerosis is reduced in comparison with control mAb 51A9–treated mice, with reduction comparable with that seen in en face studies. D, High-power photomicrograph of the area of lesion indicated by the asterisk in C, showing
relatively reduced intimal plaque thickness and lipid-containing cells
(arrow) in the media. (Original magnification ⫻ 40 in A and C; ⫻ 400
in B and D.)
none were seen in the aortic medial layer of the 51A9treated mice (Figures 3B and D).
Various studies suggest that autoantibodies to
lipid antigens are present during both APS and atherosclerosis, and several mechanisms have been invoked to
explain the epitope specificities common to both aPL
and anti–ox-LDL antibodies. The molecular basis for
this cross-reactivity may be related to the presence of
phospholipids, such as CL, in the LDL particle (11).
Some studies have suggested that the recognition of
oxidation-specific epitopes is the explanation for this
cross-reactivity (2). Those investigators generated a
panel of mAb obtained from apolipoprotein E–deficient
(Apo E⫺/⫺) mice (3). The mAb display preferential
binding to oxidation-specific epitopes since they bind
ox-LDL, but not native LDL. Likewise, the mAb from
Apo E⫺/⫺ mice react with spontaneously oxidized CL,
but not with reduced CL. FB1, the mAb used in the
present study, displayed a different pattern of crossreactivity. FB1 reacted with both native LDL and oxLDL. In fact, FB1 binding to ox-LDL was decreased
relative to native LDL. Likewise, FB1 reacted with both
naturally oxidized and hydrogenated CL (data not
shown). The differences in binding specificities between
the mAb FB1 and Apo E⫺/⫺ may be related to their
different origins. Consistent with our current findings, a
recent study showed that human aPL react equally well
with reduced and oxidized forms of CL (12).
A remarkable property of FB1 is its ability to
decrease atherosclerosis in LDLR⫺/⫺ mice. This effect
may seem paradoxical since this mAb comes from
diseased animals. The role of antibodies during atherosclerosis remains unclear and has led to contradictory
suggestions. Autoantibodies could play a role in the
disease by forming immune complexes and aggravating
the inflammatory process. Alternatively, antibodies may
not participate in the pathogenesis and could merely be
an epiphenomenon resulting from the hyperlipidemia
during the disease. More recently, several authors have
suggested that some antibodies may in fact improve
atherosclerotic manifestations, although their mechanisms of action have not been elucidated. Some have
suggested that autoantibodies to ox-LDL may act as
scavengers and clear ox-LDL from the circulation or
prevent their uptake by macrophages (6,7). Because FB1
can bind to both native LDL and ox-LDL, it is likely that
FB1 has additional effects besides preventing ox-LDL
The finding of increased medial foam cells in the
aortas of FB1-treated mice was unexpected, and the
mechanism underlying this effect is not currently obvious. Potentially involved mechanisms could include altered permeability characteristics and/or ligand–
receptor interactions of FB1-modified lipoproteins, or
FB1-related changes in adhesion molecule expression in
the aortic vasa vasorum of treated animals. Elucidation
of the underlying mechanism will require identification
of the cell type(s) of the medial foam cells, which is
beyond the scope of current studies.
Studies in atherosclerosis-prone mice suggest
that humoral immunity can be beneficial, since immunization with ox-LDL can reduce the severity of disease
(13,14). However, it is likely that within the same
atherosclerotic individual, several antibody populations
coexist that can have beneficial or detrimental effects
upon the disease. Harnessing humoral immunity to
reduce plaque formation in human atherosclerosis will
depend upon identifying the fine specificities of protective antibodies.
We thank M. Fowler, Z. Khan, H. Bradford, and Drs.
A. Varachadhary, T. Moran, R. Colman, R. Pixley, D. Rader,
and R. Tangirala for assistance with various aspects of this
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