© 1999 American Heart Association, Inc.
Original Contribution |
Anti–Monocyte Chemoattractant Protein-1/Monocyte Chemotactic and Activating Factor Antibody Inhibits Neointimal Hyperplasia in Injured Rat Carotid Arteries
From the Department of Cardiovascular Medicine (Y.F., A.M., N.O., T.S., K.O., S.S.), Kyoto University Graduate School of Medicine, Kyoto, Japan, and the Department of Molecular Preventive Medicine (A.H., K.M.), School of Medicine, University of Tokyo, Tokyo, Japan.
Correspondence to Akira Matsumori, Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawaracho, Shogoin, Sakyo-ku, Kyoto 606-8397, Japan. E-mail amat{at}kuhp.kyoto-u.ac.jp
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Abstract—Monocyte chemoattractant protein-1 (MCP-1)/monocyte chemotactic and activating factor (MCAF) has been suggested to promote atherogenesis. The effects of in vivo neutralization of MCP-1 in a rat model were examined in an effort to clarify the role of MCP-1 in the development of neointimal hyperplasia. Competitive polymerase chain reaction analysis revealed maximum MCP-1 mRNA expression at 4 hours after carotid arterial injury. Increased immunoreactivities of MCP-1 were also detected at 2 and 8 hours after injury. Either anti–MCP-1 antibody or nonimmunized goat IgG (10 mg/kg) was then administered every 12 hours to rats that had undergone carotid arterial injury. Treatment with 3 consecutive doses of anti–MCP-1 antibody within 24 hours (experiment 1) and every 12 hours for 5 days (experiment 2) significantly inhibited neointimal hyperplasia at day 14, resulting in a 27.8% reduction of the mean intima/media ratio (P<0.05) in experiment 1 and a 43.6% reduction (P<0.01) in experiment 2. This effect was still apparent at day 56 (55.6% inhibition; P<0.05). The number of vascular smooth muscle cells in the neointima at day 4 was significantly reduced by anti–MCP-1 treatment, demonstrating the important role of MCP-1 in early neointimal lesion formation. However, recombinant MCP-1 did not stimulate chemotaxis of vascular smooth muscle cells in an in vitro migration assay. These results suggest that MCP-1 promotes neointimal hyperplasia in early neointimal lesion formation and that neutralization of MCP-1 before, and immediately after, arterial injury may be effective in preventing restenosis after angioplasty. Further studies are needed to clarify the mechanism underlying the promotion of neointimal hyperplasia by MCP-1.
Key Words: monocyte chemoattractant protein-1/monocyte chemotactic and activating factor • angioplasty • restenosis • macrophage • smooth muscle cell
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Late restenosis after balloon angioplasty remains the most limiting factor with regard to the long-term effectiveness of the procedure, and it is partially attributed to neointimal hyperplasia.1 2 Despite some success of various treatments3 4 5 in reducing neointimal hyperplasia in animal models, no sufficiently effective therapy has been confirmed in clinical trials. In the development of neointima, several cell types, such as monocytes/macrophages, vascular smooth muscle cells (VSMCs), and T lymphocytes, play diverse roles. Growth factors and cytokines mediate the interactions among these cells.6 7 One of these soluble factors, monocyte chemoattractant protein-1 (MCP-1)/monocyte chemotactic and activating factor (MCAF), has been reported to be expressed early in the injured arterial wall.8 MCP-1 is a potent chemotactic factor of monocytes9 10 and is produced by activated endothelial cells11 and VSMCs.12 It has also been detected in human atheromatous plaques using immunohistochemical staining and in situ hybridization.13 Monocytes/macrophages recruited into injured arterial walls become foam cells, the focal accumulation of which may play a pivotal role in atherogenesis,6 14 including in restenosis. A recent clinical study found that activation of blood monocytes before angioplasty promotes late lumen loss after percutaneous transluminal coronary angioplasty.15 Thus, MCP-1 may have a promoting role in neointimal hyperplasia. However, 2 opposite effects have been reported with respect to the mitogenic activity of MCP-1 on cultured VSMC proliferation.16 17 This suggests diverse effects of MCP-1 during the development of neointimal hyperplasia.
The purpose of this study was to clarify which action, facilitating or inhibitory, of MCP-1 is more prominent in vivo. First, the time course of the expression of MCP-1 in injured artery was studied by competitive reverse transcriptase–polymerase chain reaction (PCR) and tissue ELISA. On the basis of these results, the effects of anti–MCP-1 treatment in a rat carotid arterial injury model were investigated.
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Anti–MCP-1 Antibody (Ab) and Other Reagents
Anti–MCP-1 polyclonal Ab (anti-rat MCP-1 goat IgG) and control (nonimmunized) goat IgG were generously provided by Toray Basic Research Laboratory (Kanagawa, Japan). The neutralizing activity of this purified polyclonal anti-rat MCP-1 Ab was confirmed by monocyte chemotaxis assay.18 This Ab shows no cross-reactivity against recombinant rat RANTES and detected a single band in the same fraction as recombinant rat MCP-1 on Western blot analysis, when concanavalin A–stimulated spleen cell culture supernatant was fractionated by heparin-HPLC.18
Recombinant rat MCP-1 that contained <0.1 ng endotoxin/mg of rat MCP-1 protein (data from the manufacturer) was obtained from DIACLONE Research. Recombinant human platelet-derived growth factor (PDGF) was purchased from Gibco/BRL.
Animal Model Preparation
Male Sprague-Dawley rats 11 to 12 weeks old were obtained from
Shizuoka Agricultural Cooperation Association. The animals were housed
in plastic and stainless-steel cages, with controlled 12-hour
light/12-hour dark cycles and access to food and water as desired. They
were anesthetized with sodium pentobarbital (50 mg/kg IP). The
endothelium of the left common carotid artery was
denuded by 3 passages of an inflated 2F Fogarty embolectomy catheter
(Baxter Health Care) with a modification of the method of Clowes et
al.19
RNA Preparation and cDNA Synthesis
Carotid arteries were harvested at 1, 2, 4, 8, 24, 72, or 120
hours after balloon injury (n=3 for each time period). Noninjured left
common carotid arteries of Sprague-Dawley rats were used as normal
controls (n=3). Total RNA was prepared from the arteries by the
guanidinium thiocyanate/phenol/chloroform/isoamylalcohol isolation
method.20 One microgram of total RNA template was
subjected to first-strand cDNA synthesis with dNTP (Perkin-Elmer Corp)
and Moloney murine leukemia virus reverse transcriptase (Gibco/BRL)
under supplier-recommended conditions.
Competitive PCR
To estimate MCP-1 mRNA expression quantitatively, competitive
PCR analysis was performed as previously
described.21 Gene-specific oligonucleotide
primers and mimic PCR primers for the MCP-1 and GAPDH genes were
purchased from Oligos Etc, Inc. A sense primer (A) and an antisense
primer (B) for each were synthesized using the published cDNA sequences
for MCP-122 and GAPDH23 as follows.
Gene-Specific Primers
MCP-1 (A): 5'-CGGAATTCCGAACTCTCACTGAAGCCAGATCTCT-3'
MCP-1 (B): 5'-CCAAGCTTGGAGGTGAGTGGGGCATTAACTGCAT-3'
GAPDH (A): 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3'
GAPDH (B): 5'-CATGTAGGCCATGAGGTCCACCAC-3'
Mimic PCR Primers
- Mimic MCP-1 (A): 5'-AACTCTCACTGAAGCCAGATCTCTCGCAAGTGAAATCTCCTCCG-3'
- Mimic MCP-1 (B): 5'-AGGTGAGTGGGGCATTAACTGCATGGGACAAGATACTCATCTGC-3'
- Mimic GAPDH (A): 5'-TGAAGGTCGGTGTGAACGGATTTGGCCGCAAGTGAAATCTCCTCCG-3'
- Mimic GAPDH (B): 5'-CATGTAGGCCATGAGGTCCACCACTTGAGTCCATGGGGAGCTTT-3'
PCR-mimic cDNA (internal control) was created according to the
manufacturer's instructions (PCR MIMIC construction kit, Clontech).
Twofold dilutions of each PCR mimic between 10-2
and 10-5 were added to the PCR amplification
reaction mixture containing 1 µL of sample cDNA and an aliquot of
[
32P]dCTP for each reaction. Both MCP-1 and
GAPDH cDNA were analyzed by 30 cycles of amplification in a
thermal cycler (Perkin-Elmer Corp). Each cycle consisted of
denaturation at 94°C for 45 seconds, annealing at 50°C for 45
seconds, and extension at 72°C for 90 seconds. A portion of each PCR
product was electrophoresed on a 4% polyacrylamide gel,
and the densitometric values of 32P-labeled
target and internal control were analyzed with a FUJIX
bioimaging analyzer (BAS 2000). The molar ratio between target
and internal control was calculated with the following formula:
target/internal
control=(VT/VC)x(CC/CT),
where VT and VC
represent the densitometric value of the PCR product from
target and internal control, respectively, and
CC and CT
represent the dCTP content in the PCR product from internal
control and target. The amount of target gene was determined as that of
the internal control at the point of an equal molar ratio between the
target and the internal control (Figure 1A
). The relative amount of MCP-1 cDNA
was corrected by the amount of GAPDH cDNA. The calculated values were
finally normalized for each by assigning a standard number of 1 to the
sample that demonstrated the highest normalized MCP-1 expression.
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Enzyme-Linked Immunosorbent Assay
Carotid arteries were harvested at 2, 8, 24, or 120 hours after
injury (n=3 for each time period). Right common carotid arteries were
used as controls. The total length of each common carotid artery was
homogenized in 1 mL of PBS containing 0.05%
NaN3 with an ultrasonic processor, ASTRASON
(Misonix Inc), and then centrifuged. MCP-1 in the
supernatant was quantified using an ELISA kit (Biosource
International). All measurements were performed in duplicate. The
values were corrected by protein concentrations measured by a
modification of Lowry's method.24
Experiment 1
Animals were randomly assigned to an anti–MCP-1 treatment group
or to a control group. Rats in the anti–MCP-1 treatment group (n=5)
received 10 mg/kg anti–MCP-1 Ab, via the tail vein, 30 minutes before
arterial injury and 12 and 24 hours after the first
injection. Control rats (n=5) were given 10 mg/kg nonimmunized goat
IgG.
Experiment 2
Rats in the anti–MCP-1 treatment group (n=6) and control
group (n=6) received 10 mg/kg anti–MCP-1 Ab or nonimmunized goat IgG,
respectively, via the tail vein, every 12 hours for 5 days starting at
30 minutes before injury. This second series of experiments was
performed to test the value of a longer treatment with anti–MCP-1
Ab.
Experiment 3
To determine the persistence of the inhibitory
effects of anti–MCP-1 Ab on neointimal hyperplasia, the
arteries were examined 56 days after carotid arterial
injury, when cell proliferation in the injured arterial
walls returns to the basal level.19 Rats in the
anti–MCP-1 treatment group (n=5) and the control group (n=5) received
5 doses of 10 mg/kg anti–MCP-1 Ab or nonimmunized goat IgG,
respectively, via the tail vein, every 12 hours starting at 30 minutes
before injury. This third series of experiments was performed to test
whether the inhibitory effect was limited to a delay of
lesion progression.
Light-Microscopic Examination and Morphometry of Neointima
Fourteen days after injury in experiments 1 and 2, and 56 days
after injury in experiment 3, the rats were anesthetized with
sodium pentobarbital (50 mg/kg, IP) and received 200 µL of 2% Evans
blue dye in PBS injected into the tail vein. They then received heparin
(100 U/rat) intravenously, and after perfusion with saline,
the left common carotid arteries were perfusion fixed with 10% neutral
buffered formalin as described previously.25 The carotid
arteries were removed and fixed further. Central portions of the
blue-stained areas were embedded in paraffin. Five cross sections of
each artery situated 2 mm and 1 mm proximal to the center, at
the center, and 1 mm and 2 mm distal to the center, were
stained with elastic van Gieson stain. Intimal and medial areas were
blindly measured with a computer-based image analyzing system (LUZEX3U,
Nikon). The mean intimal and medial areas of each artery were
determined from these 5 sections.
Immunohistochemical Staining
Immunohistochemical staining was performed to evaluate the
effects of anti–MCP-1 Ab on (1) accumulation of macrophages in
the neointima, (2) the number of VSMCs in the early
neointimal lesion, and (3) initial medial proliferation of
VSMCs. For these 3 experiments, rats in the anti–MCP-1 treatment group
and in the control group received 3 doses of 10 mg/kg anti–MCP-1 Ab
and nonimmunized goat IgG, respectively, as described in experiment 1.
For the staining of macrophages, the carotid arteries were
removed 14 days after injury and embedded in OCT compound tissue medium
(Miles Inc). Short axial 4-µm cryostat sections were cut from
proximal, middle, and distal segments for each sample and fixed for 10
minutes in acetone at 4°C. To label macrophages, ED1 Ab (BMA
Biomedicals Ltd)26 was used as primary Ab. Labeling of
VSMCs or proliferating cells was performed using the 20 or the 5
formalin-fixed, deparaffinated sections of carotid arteries obtained at
day 4 or 48 hours, respectively. As primary Ab, mouse monoclonal Ab
against muscle actin (HHF35, ENZO Diagnostics Inc) or mouse
monoclonal Ab against proliferating cell nuclear antigen (PCNA; PC10,
YLEM Srl) was used. The sections were incubated with the
following: primary Ab at a dilution of 1:50 overnight at 4°C,
biotinylated secondary Ab (rabbit anti-mouse IgG; DAKO) at 1:500 for 30
minutes at room temperature, and Vector Elite ABC
biotin-avidin-peroxidase complex for 30 minutes. The sections were
developed with 3,3'-diaminobenzidine (Dojindo) solution and
counterstained with hematoxylin. For negative controls, normal mouse
IgG or normal rabbit IgG was used instead of primary or secondary Ab.
Cell number was counted for each sample at a magnification of
x400.
Cell Culture
Rat aortic VSMCs for migration assay were prepared by the
explant method.27 VSMCs migrated from dissected rat aorta
were grown in DMEM (Nissui) supplemented with 10% FCS, 100 µg
streptomycin/mL, and 100 units penicillin/mL (Gibco/BRL) in a
humidified atmosphere (5% CO2/95% air) at
37°C. Cells from the third through the seventh passages were used in
each assay.
VSMC Migration Assay
Migration of rat aortic VSMCs was assayed with a 48-well
modified Boyden-chamber apparatus (Neuro Probe
Inc).28 29 The wells were covered with a
polyvinylpyrrolidone-free filter with 8-µm pores (Nuclepore Corp),
coated with 2.7 µg/well Matrigel (Collaborative
Research),29 or 100 µg/mL type I collagen (Sigma).
Cultured VSMCs were trypsinized and suspended at a concentration of
5.0x105 cells/mL in serum-free DMEM with
streptomycin and penicillin. Fifty microliters of the cell suspension
was added to each upper chamber. DMEM containing recombinant rat MCP-1
at the final concentration of 20, 50, or 100 ng/mL was added to the
lower chamber in a volume of 50 µL. As negative and positive
controls, DMEM without MCP-1 and DMEM with recombinant human PDGF-BB
(Gibco/BRL), with a final concentration of 20 ng/mL were used,
respectively. To test whether MCP-1 can augment the chemotactic
activity of VSMCs when PDGF-BB was used as a chemoattractant, MCP-1 was
added to the upper chamber at a final concentration of 50 ng/mL in some
of the experiments. Six filters were used for each treatment. After
incubation in a humidified atmosphere (5%
CO2/95% air) at 37°C for 12 hours, the cells
on the upper membrane surface were removed, and those on the lower
surface were fixed in methanol and stained with Diff-Quik staining
solution (International Reagents Corp). The number of cells per four
200x high-power fields was counted under a microscope, and the mean
number of cells represented migration activity.
Statistical Analysis
Values for relative MCP-1 gene–product levels and MCP-1
immunoreactivity are expressed as mean±SEM. Values for intimal areas,
medial areas, intima/media ratio, numbers of cells, percentage
ED1-positive cells, percentage proliferating cells, and migration
activity are expressed as mean±SD. Values for intimal areas and
intima/media ratio in experiment 3 were compared using the Mann-Whitney
U test, since they were nonparametrically
distributed. Values for migration activity were compared using ANOVA.
Other values were compared using the 2-tailed unpaired Student
t test.
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Time Course of MCP-1 Gene Expression in Injured Rat Carotid Arteries
Coamplification of sample cDNA with serially diluted mimic cDNA showed a close correlation between the amount of mimic cDNA and target cDNA/mimic cDNA ratio for each reaction (Figure 1A
). Induction
of MCP-1 mRNA was demonstrable as early as 1 hour, and peak expression
occurred at 4 hours after arterial injury (Figure 1B). MCP-1 mRNA level decreased at 8 hours, although relatively
low but upregulated MCP-1 gene expression was still present 120
hours after injury.
Enhanced MCP-1 Immunoreactivity in the Injured Arterial
Wall
Immunoreactivity of MCP-1 in the injured arteries increased as
early as 2 hours after injury (129.0±6.8 pg/mg protein; mean±SEM),
and the upregulation was still detectable at 8 (118.5±18.3) and 24
hours (82.5±14.6). At 120 hours, MCP-1 immunoreactivity had returned
to the level of noninjured arteries. In the noninjured
arterial tissues, no significant changes were observed
throughout the observation period (Figure 2).
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Effects of Anti–MCP-1 Treatment on Neointimal Hyperplasia
Significant neointimal hyperplasia was observed in
injured arteries 14 days after injury. Intravenous
administration of 3 doses of anti–MCP-1 Ab significantly inhibited
neointimal hyperplasia, compared with the control group
(Table 1, Figure 3A). The mean intima/media ratio
was reduced to 0.679±0.174 (mean±SD), in contrast to 0.941±0.062 in
the control group (P<0.05), representing a
27.8% inhibition in the anti–MCP-1 treatment group. Administration of
anti–MCP-1 Ab every 12 hours for 5 days (10 doses) resulted in 43.6%
inhibition (Table 1, Figures 3B and 4). The mean intima/media ratio was
0.465±0.163 in the anti–MCP-1–treated group versus 0.825±0.093 in
the control group (P<0.01). In experiment 3, this
inhibitory effect was still present 56 days after
injury, and the mean intima/media ratio was reduced by 55.6% in the
anti–MCP-1 treatment group (P<0.05; Table 1,
Figures 3C and 4).
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Population of Macrophages in the Neointima
The number of nuclei and ED1-positive cells in the
neointima of 3 sections was counted for each sample, and
the percentage of ED1-positive cells was calculated. The number of
ED1-positive cells in the neointima tended to be lower in
the anti–MCP-1 treatment group (80±26 in the anti–MCP-1 treatment
group versus 115±27 in the control group; P=0.054). The
number of nucleated cells also tended to be lower (1646±273 in the
anti–MCP-1 treatment group versus 2074±419 in the control group;
P=0.071). As a result, the percentages of ED1-positive cells
relative to the nucleated cells in the neointima were
comparable in both groups (4.81±1.03% in the anti–MCP-1 treatment
group versus 5.50±0.65% in the control group; P=0.228,
Table 2).
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Effect of Anti–MCP-1 Ab on the Number of VSMCs in Early
Neointimal Lesions
In this rat model, VSMCs appear in the intima 4 days after
injury.19 The number of intimal VSMCs was thus compared
between the anti–MCP-1 treatment group and the control group at day 4,
representing migration activity and intimal proliferation
of VSMCs in the early neointimal lesion. Table 3 shows that the number of VSMCs in the
intima was decreased by treatment with anti–MCP-1 Ab
(P<0.05).
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Effect of Anti–MCP-1 Ab on Initial Medial Proliferation After
Mechanical Injury
Forty-eight hours after injury, immunohistochemical staining with
anti-PCNA Ab showed no differences, between the anti–MCP-1 treatment
group and the control group, in the population of proliferating cells
in the media (Table 4). Percentage
PCNA-positive cells was 35.8±2.9% in the anti–MCP-1 treatment group,
and that in the control group was 34.4±7.2%.
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Effects of MCP-1 on VSMC Migration In Vitro
Recombinant rat MCP-1 failed to stimulate chemotactic activity of
VSMCs into coated Matrigel, even at high concentrations, which have
been observed to exert a significant effect in a monocyte chemotaxis
assay.18 MCP-1 also failed to increase PDGF-BB–stimulated
VSMC migration activity when added directly into the upper chamber
(Figure 5). Similar results were obtained
in the assays using type I collagen as a coating material (data not
shown).
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The present study shows that anti–MCP-1 treatment before and soon after arterial injury reduced neointimal hyperplasia, an effect that persisted during the chronic stage. Rats that had undergone carotid arterial injury were treated with anti–MCP-1 Ab for different lengths of time. Treatment for 5 days (10 doses) seemed more effective in limiting the neointimal lesions than treatment limited to 3 doses, although there was not a notable difference in the inhibitory effects between the 2 treatment durations. This suggests that anti–MCP-1 treatment exerts beneficial effects mostly by neutralizing MCP-1 produced soon after arterial injury. We demonstrated in experiment 3 that the effects of early anti–MCP-1 treatment did not just delay the development of neointimal lesions, but also influenced the severity of neointimal hyperplasia in the chronic stage. As a possible mechanism of the inhibitory effect of anti–MCP-1 treatment, it is noteworthy that the number of VSMCs in the early neointimal lesions at day 4 decreased significantly. In addition, the accumulation of macrophages tended to decrease with anti–MCP-1 treatment. These observations suggest an important role of MCP-1 in the development of accelerated atherosclerosis.
In the pathogenesis of atherosclerosis, various vascular and inflammatory cells, including monocytes/macrophages and VSMCs, play important interacting roles.6 7 19 Soluble factors and adhesion molecules regulate the pathophysiological activities of these cells.30 31 32 33 34 35 As MCP-1 is one of the soluble factors that participate in atherogenesis, the pathophysiological roles of MCP-1 have been studied in cultured cells. Expression of MCP-1 is regulated by various stimuli such as growth factors36 37 and those from adhesion molecules.30
In this study, MCP-1 mRNA and protein were rapidly induced by mechanical arterial injury, with a time course comparable to that in previous experiments performed in a rabbit model.8 Because MCP-1 is a potent chemoattractant of monocytes,9 10 this induction of MCP-1 may contribute to early inflammatory responses in the injured arterial walls and local accumulation of monocytes/macrophages into the neointima. Recently, Boring et al38 reported that additional depletion of CCR2 gene, a receptor for MCP-1, to apolipoprotein E (ApoE) gene markedly attenuated atherosclerotic lesions in ApoE-deficient mice by inhibiting macrophage accumulation; these results strongly support the important role of MCP-1 in atherosclerotic lesion formation, particularly macrophage-rich lesions. In fact, in our experiments, the number of macrophages accumulated in the neointima tended to be decreased by anti–MCP-1 Ab treatment, although the population of macrophages is smaller in the rat than in other models of arteriosclerosis.39 40 This may partially explain the inhibitory effects of anti–MCP-1 Ab on neointimal hyperplasia in the rat arterial injury model.
In addition to its well-known chemotactic activity on monocytes/macrophages, other properties of MCP-1 have been described that may stimulate atherosclerotic-lesion formation. Some investigators have reported that MCP-1 stimulates chemotaxis of T lymphocytes41 42 and VSMCs.43 44 Ikeda et al45 reported that MCP-1 increased the expression of intercellular adhesion molecule-1 on rat aortic VSMCs. Another study has shown that MCP-1 has direct mitogenic effects on the proliferative response of cultured rat VSMCs,17 although controversy remains.16 Thus, several reports have shown biological effects of MCP-1 on VSMCs, despite the absence of a clear demonstration of receptors for MCP-1. Recent observations suggest the existence of chemokine receptors for MCP-1 on the surface of VSMCs. Schecter et al46 reported that human VSMCs may express a distinct receptor for MCP-1 from CCR2, which is expressed on monocytes and T lymphocytes, by binding assay and reverse transcriptase–PCR, and another group demonstrated the expression of CCR2 mRNA in unstimulated cultured human VSMCs.47 There is a discrepancy between these 2 reports with respect to the type of VSMC receptor. Nevertheless, they suggest that MCP-1 may modulate the function of VSMCs and that neutralization of MCP-1 may inhibit neointimal lesion formation not only by reducing the accumulation of monocytes/macrophages, but also by blocking the biological effects of MCP-1 on VSMCs. Since the intimal population of macrophages is much greater in humans than in this rat model, the clinical effects of anti–MCP-1 treatment cannot be immediately predicted. However, together with the reduction of macrophage-rich atherosclerotic lesions by CCR2 gene depletion in a recent study,38 the inhibitory effects of anti–MCP-1 treatment in this rat model suggest that such treatment may reduce the formation of neointimal lesions after balloon angioplasty, lesions that contain more VSMCs and fewer macrophages than primary atherosclerotic plaques.48
Previous studies have suggested that migration and intimal proliferation are more critical steps in the development of intimal hyperplasia than first-round medial VSMC proliferation in this model. Neutralization of basic fibroblast growth factor with anti–basic fibroblast growth factor Ab markedly inhibited initial medial VSMC proliferation but not the associated intimal lesion,31 whereas anti-PDGF treatment inhibited neointimal hyperplasia at day 8 after injury, with no difference observed in the proportion of proliferating VSMCs found in the media and intima between anti-PDGF–treatment and control groups.3 To investigate the role of MCP-1 in early neointimal lesion formation, we tested the effects of anti–MCP-1 Ab on the number of VSMCs in the neointima on day 4, which reflects the migration of medial VSMCs into the neointima and the early intimal proliferation of migrated VSMCs. We also examined the effects of anti–MCP-1 Ab on the number of proliferating medial VSMCs at 48 hours, which reflects first-round proliferative response in the media. The results showed a significant difference in the number of intimal VSMCs on day 4 between anti–MCP-1 treatment group and control group, but no difference in the number of proliferating VSMCs at 48 hour, suggesting that MCP-1 stimulates migration or intimal proliferation of VSMCs in vivo. The effects of recombinant MCP-1 on migration of cultured VSMCs were then examined. In the migration assay, MCP-1 did not directly act as a chemoattractant for VSMCs, nor did it stimulate migration activity of VSMCs into Matrigel or collagen I. Our results differ from those of previous reports with regard to the effects of MCP-1 on VSMC migration.43 44 Differences in the species of experimental cells used, or in the preparation of the cells, may explain this discrepancy. It is also possible that the effects of MCP-1 on VSMC migration in vivo are different from those in a cell culture. Nevertheless, the results of the migration assay suggest other effects of MCP-1 on VSMCs causing an early decrease in the number of VSMCs in the intima. Since there was no difference in the number of proliferating medial VSMCs between the anti–MCP-1 treatment group and the control group, we considered the possibility that anti–MCP-1 treatment did not affect first-round medial VSMC proliferation. However, the possibility that anti–MCP-1 Ab inhibited second-round proliferation of VSMCs in the intima cannot be excluded, since the proliferative activity of VSMCs in the injured arterial walls is biphasic,49 and >70% of intimal VSMCs are proliferating at day 4.19 Thus, a decrease in the number of VSMCs in the neointima by anti–MCP-1 treatment at day 4 could result from inhibition of second-wave VSMC proliferation in the intima. Studies using cultured VSMCs revealed variable proliferative responses of VSMCs to MCP-1 depending on the status of the cells.16 17 It is, therefore, possible that differences exist in between the proliferative responses of intimal VSMCs versus medial VSMCs. Another possible biological effect of MCP-1 on VSMCs, which may enhance the second wave of proliferative response, consists of stimulation and prolongation of procoagulant activity. MCP-1 produced by VSMCs and macrophages may induce procoagulant activity and contribute to mural thrombus formation via tissue-factor induction in atherosclerotic lesions.46 Thrombi that are formed after arterial injury contain chemoattractants and mitogens for VSMCs and seem to function as a matrix for the migration and proliferation of VSMCs.50 Therefore, anti–MCP-1 treatment may attenuate neointimal hyperplasia by reducing procoagulant activity in the injured arterial walls.
In conclusion, this study demonstrated that the expression of MCP-1 was induced early in a rat model of carotid arterial injury. Anti–MCP-1 treatment resulted in a significant attenuation of neointimal hyperplasia in studies of 2 different treatment periods, and the inhibitory effect persisted long after injury. As a possible mechanism of this inhibitory effect, neutralization of MCP-1 may affect not only accumulation of macrophages but also an early increase of VSMCs in the intima. These results suggest that MCP-1 acts as an early promoting factor in neointimal hyperplasia after mechanical injury and that anti–MCP-1 treatment before and soon after angioplasty may inhibit postprocedural intimal hyperplasia. Further investigations are needed to clarify the diverse biological effects of MCP-1 on both leukocytes and vascular cells in neointimal lesion formation and to measure the efficacy of anti–MCP-1 treatment in the prevention of restenosis after balloon angioplasty.
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Acknowledgments |
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This work was supported by a research grant from the Ministry of Health and Welfare of Japan and a grant-in-aid for general scientific research from the Ministry of Education, Science, Sports and Culture of Japan. We would like to thank Masanobu Naruto and Jun Utsumi (Toray Basic Research Laboratory) for their generous gifts of anti–MCP-1 polyclonal Ab and nonimmunized goat IgG.
Received October 7, 1998; accepted November 18, 1998.
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References |
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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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C. Horvath, F. G.P. Welt, M. Nedelman, P. Rao, and C. Rogers Targeting CCR2 or CD18 Inhibits Experimental In-Stent Restenosis in Primates: Inhibitory Potential Depends on Type of Injury and Leukocytes Targeted Circ. Res., March 8, 2002; 90(4): 488 - 494. [Abstract] [Full Text] [PDF]
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C. Hay, C. Micko, M. F. Prescott, G. Liau, K. Robinson, and H. De Leon Differential Cell Cycle Progression Patterns of Infiltrating Leukocytes and Resident Cells After Balloon Injury of the Rat Carotid Artery Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1948 - 1954. [Abstract] [Full Text] [PDF]
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E.-i. Okamoto, T. Couse, H. De Leon, J. Vinten-Johansen, R. B. Goodman, N. A. Scott, and J. N. Wilcox Perivascular Inflammation After Balloon Angioplasty of Porcine Coronary Arteries Circulation, October 30, 2001; 104(18): 2228 - 2235. [Abstract] [Full Text] [PDF]
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U. Ikeda, K. Shimada, F. Cipollone, and A. Mezzetti Elevated Circulating Levels of Monocyte Chemoattractant Protein-1 in Patients With Restenosis After Coronary Angioplasty Arterioscler Thromb Vasc Biol, June 1, 2001; 21(6): 1090 - 1091. [Full Text] [PDF]
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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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R. Shibata, H. Kai, Y. Seki, S. Kato, M. Morimatsu, K. Kaibuchi, and T. Imaizumi Role of Rho-Associated Kinase in Neointima Formation After Vascular Injury Circulation, January 16, 2001; 103(2): 284 - 289. [Abstract] [Full Text] [PDF]
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N. Ohashi, A. Matsumori, Y. Furukawa, K. Ono, M. Okada, A. Iwasaki, T. Miyamoto, A. Nakano, and S. Sasayama Role of p38 Mitogen-Activated Protein Kinase in Neointimal Hyperplasia After Vascular Injury Arterioscler Thromb Vasc Biol, December 1, 2000; 20(12): 2521 - 2526. [Abstract] [Full Text] [PDF]
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K. Miyata, H. Shimokawa, T. Kandabashi, T. Higo, K. Morishige, Y. Eto, K. Egashira, K. Kaibuchi, and A. Takeshita Rho-Kinase Is Involved in Macrophage-Mediated Formation of Coronary Vascular Lesions in Pigs In Vivo Arterioscler Thromb Vasc Biol, November 1, 2000; 20(11): 2351 - 2358. [Abstract] [Full Text] [PDF]
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S.-i. Hayashi, N. Watanabe, K. Nakazawa, J. Suzuki, K. Tsushima, T. Tamatani, S. Sakamoto, and M. Isobe Roles of P-Selectin in Inflammation, Neointimal Formation, and Vascular Remodeling in Balloon-Injured Rat Carotid Arteries Circulation, October 3, 2000; 102(14): 1710 - 1717. [Abstract] [Full Text] [PDF]
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J. M. Waugh, J. Li-Hawkins, E. Yuksel, M. D. Kuo, P. N. Cifra, P. R. Hilfiker, R. Geske, M. Chawla, J. Thomas, S. M. Shenaq, et al. Thrombomodulin Overexpression to Limit Neointima Formation Circulation, July 18, 2000; 102(3): 332 - 337. [Abstract] [Full Text] [PDF]
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 M. MAYR, C. LI, Y. ZOU, U. HUEMER, Y. HU, and Q. XU Biomechanical stress-induced apoptosis in vein grafts involves p38 mitogen-activated protein kinases FASEB J, February 1, 2000; 14(2): 261 - 270. [Abstract] [Full Text]
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K. Kobuke, Y. Furukawa, M. Sugai, K. Tanigaki, N. Ohashi, A. Matsumori, S. Sasayama, T. Honjo, and K. Tashiro ESDN, a Novel Neuropilin-like Membrane Protein Cloned from Vascular Cells with the Longest Secretory Signal Sequence among Eukaryotes, Is Up-regulated after Vascular Injury J. Biol. Chem., August 31, 2001; 276(36): 34105 - 34114. [Abstract] [Full Text] [PDF]
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 A. Turler, N. T. Schwarz, E. Turler, J. C. Kalff, and A. J. Bauer MCP-1 causes leukocyte recruitment and subsequently endotoxemic ileus in rat Am J Physiol Gastrointest Liver Physiol, January 1, 2002; 282(1): G145 - G155. [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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C. Horvath, F. G.P. Welt, M. Nedelman, P. Rao, and C. Rogers Targeting CCR2 or CD18 Inhibits Experimental In-Stent Restenosis in Primates: Inhibitory Potential Depends on Type of Injury and Leukocytes Targeted Circ. Res., March 8, 2002; 90(4): 488 - 494. [Abstract] [Full Text] [PDF]
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