CX3CR1 Deficiency Confers Protection From Intimal Hyperplasia ...

2058 Arterioscler Thromb Vasc Biol. September 2006
Statistical Analysis
Numerical data presented in text and figure are expressed as
meanSEM. All sections were analyzed by 2 investigators, 1
blinded and 1 unblinded, with an inter-rater reliability of 0.95 to
0.99. Fisher’s exact test was utilized to determine incidence. Student
unpaired t test was used to compare average numbers of cells or
percentages between experimental groups. In all cases, P0.05 was
considered significant.
Results
Characterization of Intimal Hyperplasia
After Injury
Wire injury resulted in endothelial denudation as evidenced
by an absence of endothelial cells in femoral arteries from
WT mice 24 hours after injury. The contralateral, uninjured
control vessels retained their normal histology. Platelet adhesion
to the injured vessel wall at day 1 was followed by
monocyte recruitment to the intima by 5 days after injury
(Figure 1). At 14 days, the neointima was a mixture of
monocytes and VSMCs, and by 28 days, the neointima was
primarily composed of VSMC (Figure 1). Taken together,
intimal hyperplasia was readily apparent by 5 days and it was
continuously detected between 5 and 28 days after injury.
Monocyte recruitment to the intima occurred earlier than
VSMC accumulation, which was the primary cellular component
of neointima in the late stage (day 28). Eighty-six
percent of WT mice developed intimal hyperplasia with an
average Intima/Media (I/M) ratio of 0.8.
CX 3CL1 Expression Is Induced in Injured Arteries
To define the role of CX 3CR1 in the response to arterial
injury, we first examined the expression pattern of its ligand,
CX 3CL1, by immunohistochemistry. CX 3CL1 expression was
undetectable in non-injured, control arteries at any stage of
the injury response. In injured arteries, CX 3CL1 was not
detected at day 1. At day 5, CX 3CL1 was expressed in
endothelial cells and in a subset of the intimal SMC in WT
and CX 3CR1 / arteries (Figure 2). This pattern of expression
continued through the injury response. These data provide the
evidence that CX 3CL1 may be involved in the pathogenesis
of guide wire induced vascular injury.
CX 3CR1-Deficient Mice Are Protected From
Intimal Hyperplasia
To assess whether CX 3CR1 plays a role in mediating intimal
hyperplasia after arterial injury, we measured the incidence
and extent of neointima formation in CX 3CR1 / and WT
mice. The overall incidence of intimal hyperplasia at 5, 14,
and 28 days was decreased in CX 3CR1 / mice by 58% (8/21
Figure 1. Cellular composition of injured
arteries. Shown are immunohistochemical
analyses of WT arteries at 0, 1, 5, 14,
and 28 days after arterial injury. Platelets
(brown), monocytes (brown), and VSMCs
(red) were identified by anti-thrombocyte,
anti-F4/80, and anti--actin antibodies,
respectively.
KO versus 26/29 WT, P0.0017). Compared with the
average intimal area in WT mice, the average intimal area in
CX 3CR1 / mice was reduced by 84%, 56%, and 74% at 5,
14, and 28 days, respectively (Figure 3). In contrast, no
significant differences in luminal and medial areas were
detected between WT and CX 3CR1 / arteries (data not
shown). These data indicate that CX 3CR1 deficient mice are
protected from intimal hyperplasia after acute arterial injury,
and that the arterial injury model is appropriate to study the
functional roles of CX 3CR1.
Normal Platelet Function in CX 3CR1 / Mice
To define whether platelet CX 3CR1 is functionally relevant in
guide wire induced injury, we examined platelet accumulation
on the injured luminal surface by immunostaining. By
morphometric analysis, there was no difference in the area of
the platelet thrombus that accumulated along injured
CX 3CR1 / and WT arteries (Figure 4A and 4B). We also
examined platelet function under near-physiological conditions
in vitro using blood collected from CX 3CR1 / and WT
mice. As shown in Figure 4C and 4D, the absence of CX 3CR1
did not affect shear-induced platelet thrombus formation.
These data suggest that platelet CX 3CR1 may be not an
essential mediator of platelet adhesion or aggregation in
response to arterial injury or high shear.
Defective Monocyte Recruitment in
CX 3CR1 / Mice
CX 3CR1 is believed to be critically important for monocyte
recruitment to the inflamed or injured vessel. We tested this
hypothesis by examining monocyte infiltration into the vascular
wall by F4/80 antigen staining (Figure 5A). There was
a 100% and an 87% decrease in monocyte accumulation in
CX 3CR1 / mice at 5 and 14 days, respectively, compared
with WT mice (Figure 5B). Monocyte adhesion to CX3CL1
Figure 2. CX 3CL1 staining in injured vessels. Immunostaining of
CX 3CL1 and endothelial cells (vWF) in WT mice is shown in control
and injured arteries at 5 days after injury. Control arteries
have no CX3CL1 expression and injured arteries show expression
of CX3CL1 by vWF endothelial cells.
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