© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Monocyte Chemoattractant Protein-1 Accelerates Atherosclerosis in Apolipoprotein E-Deficient Mice
From the Departments of Metabolic Disease (R.J.A., P.-A.K.B., S.L., W.W.), Molecular Sciences (P.M.M.), and Animal Health (E.N.), Central Research Division, Pfizer Inc, Groton, Conn; and Dana-Farber Cancer Institute (B.J.R.), Harvard Medical School, Boston, Mass.
Correspondence to Dr Robert J. Aiello, Pfizer Inc, Central Research, Eastern Point Road, Groton, CT 06340. E-mail robert_j_aiello{at}groton.pfizer.com
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Abstract—The pro-inflammatory chemokine, monocyte chemoattractant protein-1 (MCP-1), plays a fundamental role in monocyte recruitment and has been implicated as a contributing factor to atherosclerosis. The predominant cell types within the vessel wall—endothelial cells, smooth muscle cells, and macrophages—all contribute to overexpression of MCP-1 in atherosclerotic tissue. In this report we assess the role of MCP-1 expression by leukocytes on lesion progression in a murine model susceptible to atherosclerosis. Bone marrow cells from mice overexpressing a murine MCP-1 transgene on a background of apoE-deficiency or from control mice were transplanted into irradiated apoE-knockout mice. After repopulation of apoE-knockout mice with bone marrow containing the MCP-1 transgene, macrophages expressing the MCP-1 transgene were found in several tissues, including the aorta. Qualitative assessment of atherosclerosis in these mice revealed increased lipid staining, a 3-fold (P<0.001) increase in the amount of oxidized lipid, and increased immunostaining for macrophage cell surface markers with anti-F4/80 and anti-CD11b antibodies. There were no differences in plasma lipids, plasma lipoprotein profiles, or body weight between the 2 groups. These results provide the first direct evidence that MCP-1 expression by leukocytes, predominately macrophages, increases the progression of atherosclerosis by increasing both macrophage numbers and oxidized lipid accumulation.
Key Words: bone marrow • CD11b • F4/80 • oxidized lipid • chemokines
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Introduction |
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Monocyte/macrophage cells have an essential role in orchestrating the complex sequence of events involved in the initiation and progression of atherosclerosis.1 Cells of monocytic origin are present in the developing foam cell lesion, where they engulf lipid and form most of the volume of the fatty streak.2 3 This early stage of atherosclerosis is characterized by the focal attachment of monocytes to the endothelium and their subsequent transendothelial migration into the vessel wall.1 4 Once in the tissue, macrophages potentiate the inflammatory response by producing various inflammatory mediators, such as reactive oxygen species and growth factors, including basic fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and other proinflammatory cytokines and chemokines.2 5 Monocytes themselves promote additional monocyte adherence and emigration from the vascular space into sites of inflammation.6 7
The generation of a murine model in which the apoE gene has been disrupted has provided an important small animal model for the study of atherosclerosis.8 9 ApoE-deficient mice (apoE-KO) exhibit hypercholesterolemia and develop complex atheromatous lesions similar to those seen in humans.10 11 These mice develop a full range of lesions, from fatty streaks to raised fibrous plaques, making this model suitable for investigating the pathogenesis of atherosclerosis. To demonstrate the involvement of the macrophage in this process, Smith et al8 generated a double-mutant mouse by crossing the apoE-KO mouse to the osteopetrotic mouse (op/op), which lacks the expression of macrophage-colony stimulating factor (M-CSF) because of a structural gene mutation. The subsequent M-CSF deficiency in apoE-KO mice resulted in a macrophage deficiency and significant reduction in atherosclerotic lesion size, demonstrating that decreases in circulating monocytes or a reduced tissue macrophage concentration reduces atherosclerosis in this mouse model.8 9
In addition to absolute numbers of circulating monocytes, chemotaxis
undoubtedly plays an important role in the recruitment and migration of
such monocytes to sites of inflammation. Several molecules have been
described that have chemotactic activity for monocytes, including
N-formylmethionyl-leucyl-phenylalanine, complement fraction
C5a, leukotriene B4, tumor necrosis
factor-
, 12-hydroxyeicosatetraenoic
acid, and monocyte chemoattractant protein-1
(MCP-1).12 MCP-1, a monomeric polypeptide of
molecular weight 9000 to 15 000 Da, is the prototype of the C-C
chemokine ß subfamily and exhibits its most potent chemotactic
activity toward monocytes.13 In addition to promoting the
transmigration and emigration of circulating monocytes into tissues,
MCP-1 exerts various effects on monocytes, including the induction of
superoxide anions and expression of the various proinflammatory genes.
MCP-1 and its murine homolog JE, which was identified initially as a
platelet-derived growth factor–inducible gene,14 are
produced by various cell types within the arterial wall
including endothelial cells, smooth muscle cells, and
fibroblasts.15 Increased MCP-1 has been detected in
atherosclerotic lesions but not in normal arteries, suggesting its
potential role in the recruitment of monocytes and the progression of
atherosclerosis.16 Although MCP-1 has been
associated with atherosclerosis as well as several
other inflammatory diseases, a cause and effect relationship has been
difficult to prove.
Several studies using MCP-1 transgenic mice have suggested that the ability of MCP-1 to elicit monocyte infiltration depends on MCP-1 being expressed at specific sites. In transgenic mice in which MCP-1 overexpression was driven by the mouse mammary tumor virus promoter (MMTV), which directs expression in a broad range of tissues, there were no significant increases in monocyte infiltration in a variety of tissues examined.17 However, MCP-1 transgenic mice generated using tissue-specific promoters, including the rat insulin II promoter,18 the myelin basic protein promoter,19 or the K14 keratin promoter,20 developed considerable increases in monocyte infiltration in tissues in which MCP-1 expression was detected. Therefore, we sought to develop a model that would selectively address the contribution of MCP-1 expression by macrophages in atherosclerosis.
Circulating monocytes and tissue macrophages within atherosclerotic lesions are originally derived from hematopoietic cells residing in the bone marrow. Thus, bone marrow transplantation provides a means to restrict the expression of a particular gene or transgene to bone marrow-derived cells.21 Boisvert et al22 demonstrated that macrophages transplanted from wild-type mouse bone marrow into apoE-KO mice expressed apoE in sufficient quantities to lower plasma lipid levels. Recently, LDL receptor-deficient mice that were irradiated and repopulated with bone marrow cells expressing the interleukin-8 receptor (CXCR-2) yielded smaller lesions than the unmanipulated mice.23 However, increased lesion size in the CXCR-2 mice with transplant was inferred by comparison with the CXCR-2 mice without transplant. Recently, MCP-1–deficient mice24 and MCP-1 receptor (CCR2)–deficient mice25 were shown to have decreased lesion formation in atherosclerotic mice. To determine whether the localized overexpression of MCP-1 by macrophages would result in subsequent amplification of atherosclerosis, irradiated apoE-deficient mice were repopulated with bone marrow cells from MCP-1 transgenic mice and a qualitative and quantitative assessment of atherosclerotic lesions was undertaken.
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Methods |
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Animals
Transgenic mice used in the present study were described previously.17 26 Homozygous C57bl/6 apoE-KO mice were established from progenitor stock with a mixed background (129ola x C57bl/6) as described previously.26 These apoE-KO homozygous mice were backcrossed for 8 generations to C57bl/6 mice and used for atherosclerotic studies. To obtain mice homozygous for both the MMTV-MCP-1 transgene and apoE deficiency, male apoE-KO mice were mated to heterozygous female MMTV/MCP-1(±) mice. The resulting MMTV-MCP-1(±)xapoE-KO progeny were identified by Southern blot analysis and inbred to produce test donor mice, MMTV MCP-1(+/+)xapoE-KO, or control donor mice, MMTV-MCP-1(-/-) x apoE-KO. Thus, the strain backgrounds were similar for both donor mice ([FVBX B6]xB6) F1. All mice were maintained on a 12-hour light/dark cycle and fed a chow diet.
Bone Marrow Transplantation
At 4 weeks of age, recipient apoE-KO mice were divided into 2
groups. Bone marrow was harvested from femurs and tibias of donor mice
as previously described.22 Bone marrow cells were washed
and resuspended in RPMI 1640 (Gibco Life Technologies) supplemented
with 2% FBS and heparin (5 U/mL). The cells were administered by tail
vein injection 4 hours after lethal irradiation (1000 rad from a cesium
gamma source). The recipients received 5x106
bone marrow cells (0.3 mL) from either test donor mice,
MMTV-MCP-1(+/+)xapoE-KO, or from control donor mice,
MMTV-MCP-1(-/-)xapoE-KO. These 2 groups of mice are referred to as
apoE-KO treated (EO-T) and apoE-KO control (EO-C) mice, respectively.
To ensure that the exposure dose was sufficient to ablate the bone
marrow, a third group of mice (n=6) was irradiated and not given a bone
marrow injection. These mice died within 3 days whereas all mice
receiving bone marrow survived and appeared in good health.
Tissue Preparation
At 20 weeks of age, mice were fasted for 4 hours,
anesthetized with ketamine and xylazine, and
sacrificed. Blood was collected in heparin, and bone marrow, liver,
lung, spleen, and aorta were removed and snap-frozen in liquid
N2. The heart was perfused in situ (80
mm Hg) with 10 to 15 mL of PBS, pH 7.4, followed by 4%
paraformaldehyde for 3 to 5 minutes. Perfusion-fixed
hearts were removed and fixed for an additional 1 to 2 hours in 4%
paraformaldehyde followed by infiltration with 30% gum
sucrose (1% gum arabic, 30% sucrose in PBS) for 24 hours at 4°C and
embedded in either OCT or paraffin.
Peritoneal Macrophages
A subset of mice (n=3) were injected
intraperitoneally with 1 mL of sterile 6% casein,
and peritoneal exudate cells were harvested after 4 days by washing the
peritoneal cavity with HBSS (Gibco Laboratories). The peritoneal cells
were washed 3 times in HBSS and lysed in TRIzol reagent (Gibco
Laboratories), and mRNA was prepared as described below.
Plasma Lipoproteins and Lipids
Separation of plasma lipoproteins using fast protein liquid
chromatography (FPLC) was performed as previously
described.27 Total plasma cholesterol and
triglycerides were measured using
colorimetric methods with commercially available
Cholesterol/HP (Boehringer-Mannheim) and
Triglyceride-G (Wako Chemicals) kits.
RNA Preparation and Analysis
Frozen tissues were pulverized in liquid
N2 with a mortar and pestle on dry ice, and total
RNA was prepared using TRIzol reagent (GIBCO Laboratories). RNA, 8 µg
for liver, lung, and spleen and 10 µg for aorta, was brought up to 20
µL in H2O and reverse transcribed to cDNA using
a random hexamer priming obtained in a cDNA synthesis kit provided by
Pharmacia, Biotech Inc. To detect MCP-1 the cDNA was then amplified by
PCR using the MCP-1 specific primer 1149 to 5'-GCTGGTGAATGAGTAGCAGC-3'
(nt 208 to 189) in combination with
[
-32P]dATP–labeled MCP-1 primer 1148 to
5'-GCCAACTCTCACTGAAGCC-3' (nt 47 to 66) for the detection of
endogenous MCP-1 cDNA and the transgenic RNA or with
[-32P]dATP primer 1344 to
5'-CGTCTCCGCTCGTCACTTATCC-3' derived from the transgene MMTV
long-terminal repeat (LTR) cap for the unique detection of the
transgenic RNA (see Figure 1
). PCR
reactions were carried out for 30 cycles in 50 µL with 5 µL of
cDNA, 100 µmol/L dNTPs in PCR buffer (100 mmol/L Tris-HCl,
15 mmol/L MgCl2, 500 mmol/L KCl, pH
8.3), 10 pmol of primers, and 2.5 U of native Taq polymerase
(Perkin Elmer). PCR amplification was performed as follows: denaturing
at 95°C for 2 minutes followed by 30 cycles of 57°C (30 seconds),
72°C (1 minute), and 95°C (1 minute). PCR amplification from 30
cycles results in a 161-bp fragment for the endogenous
MCP-1 and a 215-bp fragment for the MCP-1 transgene. The DNA
products from the PCR reactions were analyzed on a 6%
polyacrylamide gel in TBE buffer, using
-32P-labeled pBR 322 DNA Msp I
fragments (New England Biolab, Beverly, Mass) as a molecular
weight standard. The polyacrylamide gels were then dried on a
slab gel dryer supplied by Enprotech and exposed to X-OMAT x-ray film
(Eastman Kodak).
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Lesion Analysis
To determine cross-sectional lesion area, hearts were embedded
in OCT compound (Baxter) and sectioned at 10 µm using a cryostat
at -18°C, as previously described.27 Cryostat sections
were stained with oil red O (Polyscientific) and counterstained with
hematoxylin, Gill No. 3 (Sigma Chemical Company). Each section of the
aortic valve was evaluated for oil red O staining area by capturing
images directly from an RGB camera attached to an Olympus BX-50 light
microscope and displaying them on a Trinitron RGB monitor. Image
analysis was determined using Optimas 4.1 software (Image
Processing Solutions) as described previously.27 Results
are expressed as the average lesion size per section or as the percent
of the total cross-sectional vessel wall area (normal+diseased
area/section, excluding the lumen) stained with oil red O. For each
animal, the average of 12 to 16 sections was determined, and data are
expressed as lesion size or mean percent lesion area. The percent of
the proximal aortic surface covered by lesions was determined using an
en face preparation as previously
described.27 Briefly, aortas infiltrated with gum
sucrose, as described above, were cleaned of adventitia, and a
longitudinal cut was made from the arch down toward the femoral branch.
A second longitudinal cut was made between the coronary and
carotid arteries in the aortic arch, and the aorta was laid open on a
piece of polystyrene. Each aorta was evaluated for lesion area by
direct image capture from a CCD camera attached to a copy stand and
displayed on a Trinitron monitor. The lesion area was determined in
unstained tissue using Optimas 4.1 image analysis. Areas of
atherosclerotic plaques in aortas cleaned of adventitia appeared as
yellowish-white areas. This area was quantitated by manually setting
thresholds for shades of black (background), gray (normal tissue), and
white (lesion area).
MCP-1 In Situ Hybridization
Hearts were fixed in 4% paraformaldehyde and
embedded in paraffin, and the aortic valves were sectioned at 10
µm. To construct riboprobes, the 630-bp EcoRI fragment of
the mouse MCP-1 cDNA obtained from ATCC (ATCC 37590) was inserted into
pBSII SK+ to generate pMMCP-1. The antisense or
sense RNA probes were generated by T7 or T3 RNA polymerase with
SmaI- or EcoRV-linearized plasmid DNA,
respectively, using a DIG/Genius RNA labeling kit (Boehringer
Mannheim). After deparaffinization and hydration through a series of
graded ethanols, sections were postfixed with 4%
paraformaldehyde, and ribosomes were disrupted by
treatment with 0.2N HCl for 10 minutes. Sections were treated with
0.5% acetic anhydride to reduce nonspecific background, and
deproteinization of sections was performed with 20 µg/mL proteinase K
for 20 minutes at 55°C. Sections were dehydrated through a series of
graded alcohols and dried in chloroform. Dried sections were then
heated to 55°C for 30 minutes before adding 10 ng/mL of probe in a
hybridization buffer consisting of 2x SSC, 10% dextran sulfate,
0.01% sheared salmon sperm DNA, 0.02% SDS, and 50% formamide. After
a 5-minute incubation with the probe at 95°C, the sections were
incubated overnight at 62°C. After hybridization, the sections were
rinsed twice using 2x SSC at room temperature, followed by two
10-minute washes with 50% formamide in 1x SSC at 55°C. Nonspecific
binding was reduced by a 30-minute treatment with 20 µg/mL RNase A at
37°C. The slides were incubated with a horseradish
peroxidase-conjugated anti-digoxigenin antibody (Boehringer
Mannheim) diluted 1:1500 in 0.5% blocking reagent mix (NEN-Life
Science) and 1% sheep serum in 100 mmol/L Tris, 150 mmol/L
NaCl buffer. The signal was then amplified for 5 minutes using a TSA
kit (NEN-Life Science), visualized with 3,3'-diaminobenzidine (DAB,
Vector Laboratories) and counterstained with hematoxylin, Gill No.
3.
Immunohistochemistry
Paraffin-embedded sections of aortic valves (10 µm) were
immunostained for MCP-1 with a primary rabbit anti-human
polyclonal antibody (Genzyme Diagnostics) in a buffer
containing 0.1% saponin at 4°C for 12 hours. The secondary
biotinylated antibody, a goat anti-rabbit IgG provided by Jackson
Immunoresearch Laboratories, was applied at a dilution of 1:200 for 30
minutes, followed by incubation with horseradish peroxidase-conjugated
streptavidin (1:1500, Jackson Immunoresearch Laboratories). An
alternative primary rabbit IgG antibody (Jackson Immunoresearch
Laboratories) was used as a negative control. Serial cryostat sections
(10 µm) of the aortic valve were immunostained for
macrophages using rat monoclonal antibodies
(IgG2b)against F4/80 (a generous gift from Dr E.
Weringer, Pfizer Inc) and CD11b (Pharmingen). An alternative primary
rat IgG2b antibody (Jackson Immunoresearch
Laboratories) was used as a negative control. Endogenous
biotin and peroxidase activity were blocked by incubating each section
with an avidin/biotin solution (Vector Laboratories), and 0.3%
H2O2 in 1% bovine serum,
respectively. Sections were then incubated in 3% bovine nonfat milk
(Sigma) for 30 minutes at room temperature. Rat anti-mouse
F4/802b was applied to each section in a dilution
of 1:10, and then followed by a biotinylated mouse, anti-rat
IgG2b secondary antibody (1:400, Pharmingen) and
incubation with horseradish peroxidase-conjugated streptavidin
(1:1500). An alternative primary rat IgG2b
antibody (Pharmingen) was used as a negative control. Rat anti-mouse
CD11b2b (Pharmingen) was used at a dilution of
1:100, followed by the biotinylated mouse anti-rat
IgG2b secondary antibody (1:200, Pharmingen) and
incubation with horseradish peroxidase-conjugated streptavidin
(1:1500). An alternative primary rat IgG2b
antibody (Pharmingen) was used as a negative control. For
immunohistochemical staining of oxidized lipids, a mouse monoclonal
antibody against MDA2 (a gift from Dr J. Witztum, Scripps Institute, La
Jolla, Calif) was used. Either mouse anti-MDA2 or mouse IgG (Jackson
Immunoresearch Laboratories) was applied in a dilution of 1:50,
followed by a biotinylated horse anti-mouse secondary antibody and a
horseradish peroxidase complex (Vector ABC kit) according to
manufacturer's specifications (Vector Laboratories). Antibody binding
was visualized with DAB (Vector Laboratories), and all sections were
counterstained with hematoxylin, Gill No. 3. Results are expressed as
the percent of the total cross-sectional vessel wall area
(normal+diseased area/section, excluding the lumen) stained with DAB.
For each animal, the average of 2 to 3 sections was determined and data
are expressed as mean percent.
Statistical Analysis
ANOVA was used to test for statistically significant differences
between the groups with regard to treatment, serum lipids, and lesion
size. All data are expressed as means±SD
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Results |
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MCP-1 Expression in Recipient Mice
To determine the contribution of macrophage MCP-1 expression in the development of atherosclerosis, apoE-deficient mice were irradiated and repopulated with bone marrow obtained from mice that expressed or lacked an MCP-1 transgene. Successful repopulation of the bone marrow and subsequent differentiation of these cells was confirmed by the demonstration of transgenic MMTV-MCP-1 mRNA in several tissues of EO-T mice including bone marrow (Figure 2
, top), liver, lung,
spleen (Figure 2, middle), and aorta (Figure 2, bottom)
after transplantation.
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To show that mature macrophages in the EO-T mice were
expressing the MCP-1 transgene, mRNA was isolated from
macrophages elicited from the peritoneal cavity 16 weeks after
transplantation. As shown in Figure 3, the mRNA corresponding to both the MMTV-MCP-1 transgene and total MCP-1
mRNA were overexpressed in the EO-T mice as compared with the EO-C
mice. As the DNA primers used to detect the endogenous
MCP-1 also amplify the MCP-1 transgene (Figure 1
), the absolute
levels of endogenous as compared with MMTV-MCP-1 mRNA
within the various tissues could not be assessed separately. For a more
quantitative assessment, sections of the aortic valve were
analyzed for MCP-1 mRNA expression using in situ hybridization.
The expression of MCP-1 mRNA and MCP-1 protein in the EO-T mice was
markedly increased compared with the EO-C mice (Figure 4) as evidenced by the differential brown
staining with the DAB reagent with the antisense probe, whereas no
staining was observed with the sense probe (data not shown). The MCP-1
staining appeared to be localized in cells that had the morphology, as
seen under light microscopy, of tissue macrophages and foam
cells. By quantifying the amount of positive staining using image
analysis, the level of MCP-1 expression (endogenous
MCP-1 mRNA+MMTV-MCP-1 mRNA) was approximately 4- to 5-fold higher in
the EO-T mice compared with EO-C (17.1±2% versus 3.3±1%,
respectively; P<0.001, n=6). This increased expression
resulted in a significant increase in MCP-1 protein as determined by
immunostaining for MCP-1
(Table).
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Lesion Area
ApoE-deficient mice spontaneously develop lesions in the aortic
valve and throughout the arterial tree.26 As
shown in Figure 5, lesions were observed
throughout the aorta in both EO-T and EO-C mice with lesions being more
extensive in the EO-T mice. The increased expression of MCP-1 by
leukocytes resulted in a significant increase in the percent of
atherosclerotic lesion area (10.4±0.3% versus 3.7±0.1%,
P<0.05, n=6) between the EO-T versus EO-C mice,
respectively (Table). When atherosclerotic lesions within serial
sections of the aortic valves of EO-T mice were stained for
intracellular and extracellular lipid with oil red O (Figure 6, top) and quantitative data were
expressed as a percentage of the total valve area (Figure 7), there was also significantly
(P<0.05) greater percent lesion area compared with EO-C
mice (Table). Corresponding lesion area was increased
approximately 50% in the EO-T mice compared with the EO-C mice
(106 890±13 571x103 versus
69 253±20 711x103
µm2, respectively).
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Oxidized Lipids
The increase in lesion area in the EO-T mice was paralleled by
an even greater increase in the amount of oxidized lipid within the
lesion (Table). Figure 6, bottom, shows aortic valves from
recipient mice stained for lipid with oil red O and
immunostained for oxidized LDL with a monoclonal antibody
directed against malondialdehyde (MDA) lysine-conjugated lipid. By
quantifying the amount of lesion area that stained positive for
oxidized lipid (eg, stained with DAB), we demonstrated a 3-fold greater
amount of lipid oxidation in the EO-T recipients compared with EO-C
recipients (Table). Although in vitro MCP-1 expression is increased by
oxidized LDL, the data reported herein demonstrate that MCP-1
expression increases the retention or oxidation of LDL in the vessel
wall.
Macrophage Immunostaining
In both groups of recipient mice, lesions at various stages of
development could be observed in a single animal. These vascular
lesions had a variety of histological appearances
ranging from multilayered foam cell deposits to advanced plaques. In
the less progressed lesions, macrophage/foam cell deposits were
sometimes subendothelial, consisting solely of small
groups of lipid-filled cells. In other more progressed lesions,
multilayered foam cell deposits were evident. Occasionally, smooth
muscle cells were also evident in the intima, and some of these lesions
displayed a fibrous cap formation. Although lesions comprised a mixture
of cells, immunohistochemical analysis showed a significant
increase in the number of cells immunostaining positive
for a monocyte/macrophage-specific surface marker,
F4/80 (3.8±0.4 versus 1.3±0.2 per lesion area, P<0.01) in
the EO-T compared with EO-C recipients.
In addition to increased F4/80 staining (Table), lesions from MCP-1
recipient mice also stained more intensely for the macrophage
marker CD11b, the alpha subunit of the ß2 integrin heterodimer,
CD11b/CD18 (Mac-1, CR3 complement receptor type 3). However, unlike
F4/80, which showed uniformed staining of macrophages (Figure 8), the CD11b staining appeared to be
concentrated in specific areas (Figure 9). As shown in Figure 9, most of
the positive CD11b staining occurred in areas around the necrotic core
in cells with the morphologic appearance of macrophages.
Together these data are consistent with a proatherogenic role
of MCP-1 in enhancing the migration of monocytes into the vessel
wall.
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Plasma Lipids
Plasma cholesterol and triglyceride levels
did not differ between the EO-T and EO-C recipient mice (Table).
Similarly, the distribution of plasma lipoprotein
cholesterol, analyzed by FPLC, was also not
different between the EO-T and EO-C mice (Figure 10). Thus, the increased lesion area
and amount of oxidized lipids could not be attributed to any changes in
plasma lipid levels.
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Discussion |
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The objective of our study was to determine whether the overexpression of MCP-1 by macrophages would accelerate the progression and development of atherosclerosis using a well-established animal model system, the apoE-KO mouse.10 26 Our results show that expression of MCP-1 by leukocytes increases atherosclerotic lesion size and macrophages in apoE-KO mice. The overexpression of MCP-1 by leukocytes was accomplished by transplantation of bone marrow cells from donor mice expressing a murine MCP-1 transgene into apoE-KO recipient mice. This study is the first to clearly demonstrate that the expression of MCP-1 potentiates the inflammatory response and increases atherosclerotic lesion formation. Moreover, the increased expression of the integrin CD11b suggests that MCP-1 may play not only a pivotal role as a chemoattractant for monocytes, but may promote monocyte adhesion as well as contribute to the oxidative modification of lipids in vessels. Thus, factors that restrict the expression of MCP-1 may have therapeutic applications to the treatment of atherosclerosis.
Studies using mice that overexpress MCP-1 have shown variable results with regard to monocyte infiltration. In one model, Fuentes et al19 expressed MCP-1 in the brain under control of the myelin basic protein promoter. They observed an F4/80-positive mononuclear cell infiltrate in perivascular and meningeal regions coincident with transgene expression. In another model, MCP-1 was expressed in pancreatic islets under control of the rat insulin promoter.18 This work also found that the MCP-1–induced leukocyte infiltration was composed almost entirely of F4/80-positive mononuclear cells with the morphological appearance of blood monocytes rather than activated macrophages. In a third study, MCP-1 transgenic mice were generated using an MCP-1 transgene under control of the mouse mammary tumor virus long-terminal repeat (MMTV-LTR) promoter.17 In this model, despite high levels of MCP-1 expression, there was no increase in monocyte infiltrate in any of the expressing organs. The high circulating levels of MCP-1 may have led to desensitization of its receptor on monocytes, rendering them incapable of responding to locally produced MCP-1.17 To circumvent such nonphysiological plasma MCP-1 concentrations, we limited the expression of MCP-1 to cells derived from bone marrow in the apoE-KO mice. Because macrophages are derived from hematopoietic stem cells, transplanting bone marrow from these animals into apoE-KO mice provided a means to selectively test the effects of macrophage expression on atherosclerotic lesion progression. Previous studies using such a strategy demonstrated that apoE expression by macrophages lowers plasma lipid levels and decreases lesions in the apoE-deficient mouse.22
In our transplanted animal study, the ability of monocytes to migrate out of the bone marrow and infiltrate various tissues was evident by the detection of the MCP-1 transgene in several organs, including the aorta. Moreover, macrophages elicited from the peritoneal cavity expressed the MCP-1 transgene. Consistent with the role of MCP-1, the increase in lipid-laden lesions in EO-T mice showed much greater staining for both MCP-1 protein and F4/80 as well as a second macrophage marker, CD11b. However, the effect on lesions observed in this study could be a result of increased expression in the vessel wall or a consequence of elevated MCP-1 expression in other tissues. For example, the increased levels of oxidized LDL found in the EO-T animals may have resulted from increased TH2 cell production of interleukin-4,28 enhancing the expression of 15-lipoxgenase activity.29
Recent cell culture studies have shown that the expression of monocyte
chemokines MCP-1, macrophage inflammatory protein , and
RANTES increased the expression of the chain of 2 members of
the ß2 family of integrins, CD11a and CD11b. CD11b, one of 2 subunits
of the CD11b/CD18 (Mac-1, CR3 receptor) ß2 integrin, is highly
regulated and is expressed maximally in terminally differentiated
myeloid cells, including granulocytes, monocytes/macrophages,
and polymorphonuclear leukocytes, but is not expressed in lymphoid
cells.30 Because atherosclerotic lesions contain few
neutrophils,31 macrophages are the most likely
source for the CD11b in atherosclerotic lesions. It is interesting to
note that unlike the uniform staining observed with the F4/80 antibody
(Figure 8), only a small percentage of cells stained positive
for CD11b (Figure 9). Macrophages that showed the most
positive staining for CD11b appeared in necrotic cores of the lesions.
Currently, we do not know whether the increases in CD11b staining
resulted from an increased number of macrophages or from
increased CD11b expression by individual macrophages.
Finally, there is a growing acceptance of a role for
oxidatively modified lipoproteins in atherogenesis.32 33
All major vascular cell types are capable of oxidizing lipoproteins,
and several lines of evidence support the occurrence of oxidized
lipoproteins in atherosclerotic lesions in man, as well as the apoE-KO
mouse.34 Oxidized lipoproteins may modulate the expression
of genes involved in atherogenesis, eg, mediation of MCP-1 expression
by NF-
B.35 However, the specific factors determining
lipoprotein oxidation are relatively unknown. In this study, we showed
that lesions in the EO-T mice had a significant increase in staining
for a lipid oxidation-specific epitope, MDA.36 MDA is a
highly reactive dialdehyde generated during arachidonic
acid catabolism and is also known to result from lipid peroxidation
that occurs during phagocytosis by monocytes.37 Whether
the increase in MDA staining resulted from increased macrophage
numbers in the lesion, or from MCP-1 expression that subsequently
potentiated lipid oxidation, is currently being investigated.
As progenitors of tissue macrophages, monocytes play an important role in atherosclerosis, serving as scavengers, secretory cells, and regulators of lymphocyte function.1 The monocytes that migrate into the subendothelium via cell junctions respond to chemoattractants in the intima or media of the vessel wall.12 Although MCP-1 has been shown to be expressed in macrophage-rich areas of both human and animal atherosclerotic lesions,16 38 39 the data in this study suggest that MCP-1 expression by macrophages originating from plasma can further promote the infiltration of monocytes. In addition to acting as a chemoattractant, MCP-1 may further potentiate the inflammatory response by promoting lipid oxidation and integrin expression.
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Acknowledgments |
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The authors thank Maruja Lira for assistance in assay development, Donald Wilder for his assistance with FPLC separation, Dr Mark Kovacs, Frank Benso, and Paul McGill for help with the animal breeding and husbandry, Carol Petras for technical assistance, and Drs Omar Francone, Donald Mann, and Patricia Uelmen-Huey for their help and insightful comments during the preparation of this manuscript.
Received June 11, 1998; accepted November 25, 1998.
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B. Coles, A. Bloodsworth, S. R. Clark, M. J. Lewis, A. R. Cross, B. A. Freeman, and V. B. O'Donnell Nitrolinoleate Inhibits Superoxide Generation, Degranulation, and Integrin Expression by Human Neutrophils: Novel Antiinflammatory Properties of Nitric Oxide-Derived Reactive Species in Vascular Cells Circ. Res., September 6, 2002; 91(5): 375 - 381. [Abstract] [Full Text] [PDF]
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C. Viedt, J. Vogel, T. Athanasiou, W. Shen, S. R. Orth, W. Kubler, and J. Kreuzer Monocyte Chemoattractant Protein-1 Induces Proliferation and Interleukin-6 Production in Human Smooth Muscle Cells by Differential Activation of Nuclear Factor-{kappa}B and Activator Protein-1 Arterioscler. Thromb. Vasc. Biol., June 1, 2002; 22(6): 914 - 920. [Abstract] [Full Text] [PDF]
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 N. Tuaillon, D. F. Shen, R. B. Berger, B. Lu, B. J. Rollins, and C.-C. Chan MCP-1 Expression in Endotoxin-Induced Uveitis Invest. Ophthalmol. Vis. Sci., May 1, 2002; 43(5): 1493 - 1498. [Abstract] [Full Text] [PDF]
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 A. R. Silva, E. F. de Assis, L. F. C. Caiado, G. K. Marathe, M. T. Bozza, T. M. McIntyre, G. A. Zimmerman, S. M. Prescott, P. T. Bozza, and H. C. Castro-Faria-Neto Monocyte Chemoattractant Protein-1 and 5-Lipoxygenase Products Recruit Leukocytes in Response to Platelet-Activating Factor-Like Lipids in Oxidized Low-Density Lipoprotein J. Immunol., April 15, 2002; 168(8): 4112 - 4120. [Abstract] [Full Text] [PDF]
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M. Roque, W. J.H. Kim, M. Gazdoin, A. Malik, E. D. Reis, J. T. Fallon, J. J. Badimon, I. F. Charo, and M. B. Taubman CCR2 Deficiency Decreases Intimal Hyperplasia After Arterial Injury Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 554 - 559. [Abstract] [Full Text] [PDF]
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M. E. Rosenfeld Leukocyte Recruitment Into Developing Atherosclerotic Lesions: The Complex Interaction Between Multiple Molecules Keeps Getting More Complex Arterioscler. Thromb. Vasc. Biol., March 1, 2002; 22(3): 361 - 363. [Full Text] [PDF]
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R. J. Aiello, P.-A. Bourassa, S. Lindsey, W. Weng, A. Freeman, and H. J. Showell Leukotriene B4 Receptor Antagonism Reduces Monocytic Foam Cells in Mice Arterioscler. Thromb. Vasc. Biol., March 1, 2002; 22(3): 443 - 449. [Abstract] [Full Text] [PDF]
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 A. Goyal, Y. Wang, M. M. Graham, A. I. Doseff, N. Y. Bhatt, and C. B. Marsh Monocyte Survival Factors Induce Akt Activation and Suppress Caspase-3 Am. J. Respir. Cell Mol. Biol., February 1, 2002; 26(2): 224 - 230. [Abstract] [Full Text] [PDF]
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M. Namiki, S. Kawashima, T. Yamashita, M. Ozaki, T. Hirase, T. Ishida, N. Inoue, K.-i. Hirata, A. Matsukawa, R. Morishita, et al. Local Overexpression of Monocyte Chemoattractant Protein-1 at Vessel Wall Induces Infiltration of Macrophages and Formation of Atherosclerotic Lesion: Synergism With Hypercholesterolemia Arterioscler. Thromb. Vasc. Biol., January 1, 2002; 22(1): 115 - 120. [Abstract] [Full Text] [PDF]
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E. Lutgens and M. J.A.P. Daemen Transforming Growth Factor-{beta}: A Local or Systemic Mediator of Plaque Stability? Circ. Res., November 9, 2001; 89(10): 853 - 855. [Full Text] [PDF]
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K. A. Carnevale and M. K. Cathcart Calcium-Independent Phospholipase A2 Is Required for Human Monocyte Chemotaxis to Monocyte Chemoattractant Protein 1 J. Immunol., September 15, 2001; 167(6): 3414 - 3421. [Abstract] [Full Text] [PDF]
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K. Aragane, K. Fujinami, K. Kojima, and J. Kusunoki ACAT inhibitor F-1394 prevents intimal hyperplasia induced by balloon injury in rabbits J. Lipid Res., April 1, 2001; 42(4): 480 - 488. [Abstract] [Full Text]
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A. M. Schmidt and D. M. Stern Chemokines on the Rise : MCP-1 and Restenosis Arterioscler. Thromb. Vasc. Biol., March 1, 2001; 21(3): 297 - 299. [Full Text] [PDF]
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F. Cipollone, M. Marini, M. Fazia, B. Pini, A. Iezzi, M. Reale, L. Paloscia, G. Materazzo, E. D'Annunzio, P. Conti, et al. Elevated Circulating Levels of Monocyte Chemoattractant Protein-1 in Patients With Restenosis After Coronary Angioplasty Arterioscler. Thromb. Vasc. Biol., March 1, 2001; 21(3): 327 - 334. [Abstract] [Full Text] [PDF]
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J. W. Knowles and N. Maeda Genetic Modifiers of Atherosclerosis in Mice Arterioscler. Thromb. Vasc. Biol., November 1, 2000; 20(11): 2336 - 2345. [Abstract] [Full Text] [PDF]
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F. E. Thorngate, L. L. Rudel, R. L. Walzem, and D. L. Williams Low Levels of Extrahepatic Nonmacrophage ApoE Inhibit Atherosclerosis Without Correcting Hypercholesterolemia in ApoE-Deficient Mice Arterioscler. Thromb. Vasc. Biol., August 1, 2000; 20(8): 1939 - 1945. [Abstract] [Full Text] [PDF]
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C.-J. Kim, J. C. Khoo, K. Gillotte-Taylor, A. Li, W. Palinski, C. K. Glass, and D. Steinberg Polymerase Chain Reaction-Based Method for Quantifying Recruitment of Monocytes to Mouse Atherosclerotic Lesions In Vivo : Enhancement by Tumor Necrosis Factor-{alpha} and Interleukin-1{beta} Arterioscler. Thromb. Vasc. Biol., August 1, 2000; 20(8): 1976 - 1982. [Abstract] [Full Text] [PDF]
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 W. B Strawn, R. H Dean, and C. M Ferrario Novel mechanisms linking angiotensin II and early atherogenesis Journal of Renin-Angiotensin-Aldosterone System, March 1, 2000; 1(1): 11 - 17. [PDF]
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S. Reddy, S. Hama, V. Grijalva, K. Hassan, R. Mottahedeh, G. Hough, D. J. Wadleigh, M. Navab, and A. M. Fogelman Mitogen-activated Protein Kinase Phosphatase 1 Activity Is Necessary for Oxidized Phospholipids to Induce Monocyte Chemotactic Activity in Human Aortic Endothelial Cells J. Biol. Chem., May 11, 2001; 276(20): 17030 - 17035. [Abstract] [Full Text] [PDF]
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M. Roque, W. J.H. Kim, M. Gazdoin, A. Malik, E. D. Reis, J. T. Fallon, J. J. Badimon, I. F. Charo, and M. B. Taubman CCR2 Deficiency Decreases Intimal Hyperplasia After Arterial Injury Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 554 - 559. [Abstract] [Full Text] [PDF]
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M. K. Froberg, A. Adams, N. Seacotte, J. Parker-Thornburg, and P. Kolattukudy Cytomegalovirus Infection Accelerates Inflammation in Vascular Tissue Overexpressing Monocyte Chemoattractant Protein-1 Circ. Res., December 7, 2001; 89(12): 1224 - 1230. [Abstract] [Full Text] [PDF]
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