CX3CR1 Deficiency Confers Protection From Intimal Hyperplasia ...

CX3CR1 

Deficiency Confers Protection From Intimal Hyperplasia After Arterial Injury 

Peng Liu, Sarita Patil, Mauricio Rojas, Alan M. Fong, Susan S. Smyth and Dhavalkumar D. 

Patel 

Arterioscler Thromb Vasc Biol. 2006;26:2056-2062; originally published online June 29, 2006; 

doi: 10.1161/01.ATV.0000234947.47788.8c 

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CX 3CR1 Deficiency Confers Protection From Intimal 

Hyperplasia After Arterial Injury 

Peng Liu, Sarita Patil, Mauricio Rojas, Alan M. Fong, Susan S. Smyth, Dhavalkumar D. Patel 

Objective—A functional polymorphism in the chemokine receptor CX 3CR1 is associated with protection from vascular 

diseases including coronary artery disease and internal carotid artery occlusive disease. We investigated the mechanisms 

by which CX 3CR1 may be involved by evaluating the inflammatory response to arterial injury in 

CX 3CR1-deficient animals. 

Methods and Results—Femoral arteries of CX 3CR1 / and wild-type (WT) mice were injured with an angioplasty guide 

wire. After 1, 5, 14, and 28 days, arteries were harvested and evaluated by histology, morphometry, and 

immunohistochemistry. Arterial injury upregulated the CX 3CR1 ligand CX 3CL1. In CX 3CR1 / compared with WT 

animals, the incidence of neointima formation was 58% lower (P0.0017), accompanied by no difference in the area 

of platelet accumulation at day 1 (P0.48) but a significant decrease in intimal monocyte infiltration at day 5 

(P0.006), vascular smooth muscle cell (VSMC) proliferation at days 5 and 14, and intimal area at day 28 (P0.009). 

Conclusions—In an endothelial denudation injury model, CX 3CR1 deficiency protects animals from developing intimal 

hyperplasia as a result of decreased monocyte trafficking to the lesion. CX 3CR1 deficiency decreases VSMC 

proliferation and intimal accumulation either directly or indirectly as a result of defective monocyte infiltration. 

(Arterioscler Thromb Vasc Biol. 2006;26:2056-2062.) 

Key Words: CX3CR1 monocytes smooth muscle cells intimal hyperplasia vascular biology 

Arterial injury elicits vessel wall inflammation by triggering 

platelet adhesion, leukocyte recruitment, and vascular 

smooth muscle cell (VSMC) proliferation and migration. 

1–3 These stereotypic cellular responses serve as the basis 

for many vascular diseases, including atherosclerosis, restenosis, 

and vasculitis. The molecular mechanisms by which 

these responses are regulated are of tremendous interest. 

Both animal models and human genetic association studies 

have implicated an important role for the chemokine receptor 

CX3CR1 and its ligand fractalkine (CX3CL1) in vascular 

disease. Mice that lack both CX3CR1 and apolipoprotein E 

(apoE) exhibit a reduction in atherosclerotic lesion formation 

in the aorta and aortic root compared with apoE-deficient 

mice. 4,5 CX3CL1-deficient mice have a reduction in atherosclerotic 

plaque burden in the innominate artery. 6 In humans, 

the V249I/T280M variant of CX3CR1 is associated with 

protection from coronary artery disease7–9 and internal carotid 

artery occlusive disease. 10 

While CX3CR1 and CX3CL1 are emerging as important 

mediators of vascular disease, the specific mechanisms regulating 

their involvement remain unclear. CX3CL1 is a 

transmembrane chemokine on activated endothelium that also 

exists in a soluble form. 11–13 Soluble CX3CL1 is a potent 

chemoattractant, and membrane-tethered CX3CL1 promotes 

cell adhesion by supporting the capture and firm adhesion of 

circulating CX 3CR1-expressing cells. 14,15 CX 3CR1 is expressed 

on monocytes, 16 VSMCs, 17,18 and platelets. 19 Thus, 

each of these cell types is a candidate to mediate the effects 

of CX 3CR1 on vascular inflammatory responses. 

The prevailing hypothesis is that the mechanism for 

CX 3CR1 in the development of vascular diseases such as 

atherosclerosis is the adherence of CX 3CR1-expressing 

monocytes to inflamed endothelium. 5,14 However, recent 

evidence that smooth muscle cells (SMCs) found in human 

atherosclerotic plaques also express CX 3CR1 suggests an 

alternative mechanism. 18 Stimulation of aortic smooth muscle 

cells with soluble CX 3CL1 leads to an increase in cell 

survival and proliferation. 20 Monocyte adhesion and arrest on 

neointimal SMC is dependent on CX 3CL1, 21 and VSMCs 

undergo chemotaxis toward CX 3CL1. 17 Thus, CX 3CL1 may 

act not only on monocytes, but also may function to stimulate 

SMC proliferation and recruitment into the neointima. 

In the present study, we employed an endoluminal arterial 

injury model to investigate the role of CX 3CR1 in the 

pathogenesis of vascular injury. This model elicits a stereotypic 

response that allows for assessment of the functional 

roles of platelets, monocytes, and VSMCs. Our data show 

enhanced expression of CX 3CL1 after injury and a role for 

Original received January 17, 2006; final version accepted June 19, 2006. 

From the Department of Medicine (P.L., M.R., A.M.F., S.S.S., D.D.P.), Thurston Arthritis Research Center (P.L., S.P., A.M.F., D.D.P.), and Carolina 

Cardiovascular Biology Center (M.R., S.S.S.), University of North Carolina at Chapel Hill, Chapel Hill, NC. 

Correspondence to Dhavalkumar D. Patel, Novartis Institutes for BioMedical Research, WSJ 386.11.25, CH-4002 Basel, Switzerland. E-mail 

dhavalkumar.patel@novartis.com 

© 2006 American Heart Association, Inc. 

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000234947.47788.8c 


http://atvb.ahajournals.org/ 2056 by guest on March 25, 2013

CX 3CR1 in the development of intimal hyperplasia. A deficiency 

in CX 3CR1 does not affect platelet function, but does 

significantly influence monocyte recruitment. CX 3CR1 deficiency 

also plays a role in regulating SMC activities. 

Materials and Methods 

Animal Care 

CX 3CR1 / (KO) mice (a gift from Dr Philip Murphy [NIH]) were 

backcrossed onto the C57BL/6J background for 12 generations, and 

age-matched CX 3CR1 / (WT) mice generated from littermates were 

used as controls. All mice were bred and maintained in the barrier 

facility at the University of North Carolina and fed Prolab RMH- 

3000 (PMI Nutrition International, Richmond, Ind), a normal rodent 

chow diet. All experimental protocols were in compliance with 

Institutional Animal Care and Use Committee (IACUC) guidelines 

and were approved by the IACUC at the University of North 

Carolina at Chapel Hill. 

Femoral Artery Injury 

Femoral arteries of 8- to 12-week-old male CX 3CR1 / and WT mice 

were injured by endoluminal passage of an angioplasty guide wire as 

previously described. 22 Arteries on one side were not injured and 

served as negative controls. Briefly, mice were anesthetized with 

inhaled isoflurane and femoral arteries were exposed by a longitudinal 

groin incision and viewed under a surgical microscope. The 

distal portion of the artery was encircled with an 8-0 nylon suture, a 

vascular clamp was placed proximally at the level of the inguinal 

ligament, and a 0.01-inch diameter guide wire (CrossIT-200XT; 

Guidant Corporation, Indianapolis, Ind) was introduced into the 

arterial lumen through an arteriotomy made in the distal perforating 

branch. After release of the clamp, the guidewire was advanced to 

the level of the aortic bifurcation and immediately pulled back 3 

times to denude the endothelium. After removal of the wire, the 

arteriotomy site was ligated and skin was closed. Animals were 

routinely monitored after surgery. 

Tissue Preparation, Histology, and Morphometry 

Mice were euthanized at days 1 (n20), 5 (n16), 14 (n8), and 28 

(n26) after arterial injury. Animals were perfused with phosphatebuffered 

saline for 5 minutes, followed by 4% paraformaldehyde for 

20 more minutes at 100 cm H 2O via cannulation of the left ventricle. 

The hind limbs were then harvested en bloc, fixed in 4% paraformaldehyde 

overnight, and decalcified in formic acid bone decalcifier 

(Immunocal; Decal Corporation, Tallman, NY) for 24 hours. Tissues 

containing the femoral artery were embedded in paraffin and cut into 

5-m sections for further analysis. 

Six to 10 sections per femoral artery at 100-m intervals were 

screened with H&E staining, and sections from the area with 

maximal injury response were further evaluated by staining with the 

Combined Masson’s elastin (CME) stain to visualize the arterial wall 

layers or processed for immunohistochemistry. The intima and 

media areas were measured by computerized morphometry (Image J, 

National Institutes of Health). Intimal hyperplasia was defined as the 

formation of a neointimal layer within the internal elastic lamina 

(IEL). Media area was calculated as the area encircled by the 

external elastic lamina (EEL) minus the area encircled by IEL. The 

intima-to-media (I/M) ratio was calculated as the intimal area 

divided by the media area. Arteries with a broken IEL or thrombosis 

by CME stain were excluded from the study. 

Immunohistochemistry 

For characterizing the molecular and cellular composition of arteries, 

immunohistochemical analysis was used to identify monocytes by 

mAb F4/80 (Serotec Inc, Raleigh, NC), VSMCs by alkaline phosphatase 

conjugated anti--actin (Sigma, St. Louis, Mo), platelets by 

anti-thrombocyte (Inter-Cell Technologies, Princeton, NJ), endothelial 

cells by anti-von Willebrand factor (vWF) (DakoCytomation Inc, 

Carpinteria, Calif), and CX 3CL1 by anti-fractalkine (R&D Systems, 

Liu et al CX 3CR1 Deficiency in Arterial Injury 2057 

Minneapolis, Minn). Recombinant mouse fractalkine/CX 3CL1 

(R&D Systems) was used to validate the specificity of CX 3CL1 

staining. Antigen retrieval was performed for F4/80 staining with 

trypsin digestion and for CX 3CL1 and vWF staining by steaming in 

a citrate buffer (0.01 mol/L, pH 6.0) for 40 minutes. Endogenous 

peroxide activity was quenched using 3% H 2O 2 in methanol for 10 

minutes at room temperature, and sections were blocked using 4% 

serum (goat, rabbit, or rat) for 10 to 60 minutes at room temperature. 

Primary antibodies and their respective controls were incubated 

overnight at 4°C. After washing, sections were incubated with a 

species-specific biotinylated secondary antibody (anti-mouse, antirabbit, 

anti-rat, and anti-goat, 1:200; Vector Laboratories, Burlingame, 

Calif) for 60 minutes at room temperature, followed by 

washes, and a subsequent 60-minute incubation at room temperature 

with streptavidin-horseradish peroxidase (Peroxidase Vectastain 

ABC Kit) to amplify antibody signal. After another wash, 3, 

3-diaminobenzidine (DAB) (Sigma, St. Louis, Mo) was used as a 

substrate for the peroxidase. Alkaline phosphatase staining by 

-actin antibody was visualized with the Vector Red Alkaline 

Phosphatase Substrate Kit (Vector Laboratories). 

VSMC Proliferation Analysis 

Four days after arterial injury, mice were injected intraperitoneally 

with three doses of bromodeoxyuridine (BrdU) (Roche Diagnostics, 

Basel, Switzerland) of 30 mg/kg at 8-hour intervals before euthanasia. 

Arteries were harvested on day 5 after injury, and proliferating 

cells were identified by immunostaining with an anti-BrdU antibody 

(Roche Diagnostics). For all times other than day 5 after injury, an 

anti-PCNA antibody (Santa Cruz Laboratories, Santa Cruz, Calif) 

was used to identify proliferating cells. Proliferating VSMCs were 

defined as -actin–positive cells that were also positive for BrdU or 

PCNA staining in series sections. The proliferation index was 

calculated as the percentage of BrdU-stained or PCNA-stained nuclei 

of the total number of nuclei in the indicated area. VSMC proliferation 

was also determined in vitro utilizing primary cultures of 

VSMC isolated from aortas of CX 3CR1 / and WT mice with a 

colorimetric assay based on the uptake of MTT by viable cells (Cell 

Proliferation Kit; Roche Applied Science, Indianapolis, Ind). 

Platelet Function 

Platelet function was tested in vitro with the Cone and Plate(let) 

Analyzer (CPA) system using a DiaMed Impact-R machine (DiaMed 

Israel Ltd) according to the manufacture’s instructions. Briefly, fresh 

samples of sodium citrated anticoagulated blood (130 L) from 

CX 3CR1 / and WT mice were placed in polystyrene wells and 

subjected to defined shear (1800/second) for 2 minutes. The samples 

were then thoroughly washed with phosphate-buffered saline, 

stained with May-Grünwald stain, and quantitated with an image 

analyzer. Platelet deposition on the polystyrene surface was evaluated 

by examining: (1) the percentage of total area covered with 

platelets designated as surface coverage; and (2) average size in m 2 

of surface-bound platelet thrombi. 

Monocyte Adhesion 

Monocyte adhesion was evaluated as previously described. 14 Briefly, 

spleens from CX 3CR1 / and WT mice were homogenized in 

RPMI-1640 with 10 mmol/L HEPES using a manual tissue homogenizer. 

Red blood cells were lysed with red blood cell lysis buffer 

(0.14 mol/L NH 4CI, 0.017 mol/L Tris-HCl pH7.5, adjust to pH7.2). 

Cells were washed with RPMI-1640 with 10% fetal bovine serum 

(FBS), passed through a 70-m nylon filter, and suspended in 

RPMI-1640 with 10% FBS. Adhesion of the splenocytes to fractalkine 

under physiological flow conditions was then tested by the 

parallel plate flow chamber assay with recombinant fractalkinesecreted 

alkaline phosphatase fusion proteins immobilized on a glass 

coverslip. After the run, adherent cells were stained with MOMAfluorescein 

isothiocyanate (FITC) antibody (Beckman Coulter, Inc, 

Fullerton, Calif) and the number of monocytes bound was quantified 

by counting MOMA cells. 



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. 



under physiological flow conditions was also substantially 

lower with CX 3CR1 / compared with WT cells (Figure 5C). 

Taken together, these results provide strong evidence that 

CX 3CR1 plays a critical role in monocyte recruitment to the 

inflamed vessel wall. 

Role of CX 3CR1 in VSMC Response to 

Vascular Injury 

To assess whether CX 3CR1 is important for the function of 

VSMC in response to arterial injury, we took 3 approaches. 

First, we evaluated the numbers of VSMC in the vessel wall 

by immunohistochemistry for -actin. Second, we evaluated 

VSMC proliferation by BrdU incorporation and by immunohistochemistry 

for PCNA expression in -actin positive cells. 

Third, we evaluated the in vitro proliferation of primary 

VSMC isolated from CX 3CR1 / and WT mouse aortas. 

In injured arteries, substantial VSMC proliferation was 

observed at 5 days and 14 days (Figure 6). At 5 days, most 

proliferating cells were localized in the media. The proliferation 

index in CX 3CR1 / mice was decreased by 45% 

compared with WT mice (8.52.7% versus 15.33.4%; 

P0.07). As the lesion progressed at 14 days, an 85% 

decrease in proliferating cells was detected in the intima of 


Figure 3. Injury-induced intimal hyperplasia 

in wild type (WT) and CX 3CR1 / 

(KO) mice. Unilateral wire injury was performed 

in femoral arteries of WT and 

CX 3CR1 / mice, with the contralateral 

uninjured side used as a negative control. 

A, CME stains of representative sections 

of control and injured femoral arteries 

at 28 days after injury. Intimal 

hyperplasia is defined as the formation 

of a neointimal layer within the internal 

elastic lamina (arrows). B, Average intimal 

areas of injured WT and KO arteries 

at 5, 14, and 28 days after injury. Results 

are reported as meanSEM. **P0.05. 

CX 3CR1 / mice compared with WT animals (3.12.0% 

versus 206.0%; P0.03). In vitro, while WT aortic VSMC 

proliferated in response to CX3CL1 (P0.005), CX 3CR1 / 

aortic VSMC did not (PNS). These results suggest that 

CX 3CR1 deficiency diminishes VSMC proliferation in the 

early development of intimal hyperplasia in response to 

arterial injury. 

Discussion 

Inflammation is a driving force behind vascular diseases such 

as atherosclerosis and restenosis. Arterial injury is the initial 

stage of the pathohistological changes of these diseases. We 

found that guidewire-induced endothelial denudation of 

mouse femoral arteries elicits an inflammatory response with 

upregulation of CX 3CL1 expression. Therefore, we investigated 

the mechanisms of action of CX 3CR1 in vascular 

inflammation by testing the effects of CX 3CR1 deficiency in 

this injury model, which stimulates acute inflammatory responses 

similar to restenosis. Because CX 3CR1 is expressed 

on platelets, monocytes and VSMC, we focused on these cell 

types. 

CX 3CR1 does not appear to play a critical role in platelet 

accumulation along the denuded endothelial surface. In our 


http://atvb.ahajournals.org/ by guest on March 25, 2013 

Figure 4. CX 3CR1 effect on platelet 

adhesion. One day after injury, control 

and injured femoral arteries were immunostained 

for platelets (A, B). A, Platelets 

(brown) adhering to the luminal surface 

in vessels of injured WT and injured 

CX 3CR1 / (KO), but not control WT 

mice. B, Area of adherent platelets measured 

by morphometric analysis in 

injured CX 3CR1 / (n7) and WT arteries 

(n10). Results are expressed as 

meanSEM. Shear-induced platelet 

adhesion represented by surface coverage 

(C) and aggregation represented by 

average size (D) were measured in blood 

from WT (n4) and CX 3CR1 / (n4) 

mice using the CPA technology. Data are 

expressed as meanSEM.


model, platelets and neutrophils cover the denuded luminal 

surface within 24 hours. 22–24 The area of platelet accumulation 

was unaffected by the absence of CX 3CR1. Likewise, no 

significant differences were observed in shear-induced platelet 

thrombus formation in blood from WT and CX 3CR1deficient 

mice. These results suggest that platelet CX 3CR1 

does not play a major role in the initial phases of platelet 

adhesion and aggregation stimulated by the vascular injury. 

Likewise, CX 3CL1 was not highly expressed within the first 

24 hours after injury. Whether CX 3CR1 contributes to other 

platelet responses cannot be determined from our study. 

Regenerated endothelial cells expressed high levels of 

CX 3CL1. Similar to previous reports, 22 we observed regeneration 

of endothelial cells within 5 days of arterial injury. 

The endothelium before injury did not express CX 3CL1, but 

the regenerated endothelium expressed high levels of 

CX 3CL1. In contrast to WT animals that had robust monocyte 

accumulation, CX 3CR1 / mice failed to recruit monocytes to 

the intima despite expression of CX 3CL1. Interestingly, 

monocytes accumulated in the adventitia of WT and 

CX 3CR1 / animals after injury, suggesting that monocyte 

recruitment to the adventitia may occur through mechanisms 

distinct from those required for intimal recruitment. A primary 

difference between these two sites is the presence of 

arterial shear forces, and monocyte capture and transmigration 

along the artery may require CX 3CR1-driven adhesion 

and/or migration to resist the shear. In contrast, recruitment of 

Figure 5. CX 3CR1 effect on monocyte 

adhesion. Monocyte recruitment to 

injured vessels in vivo was tested by 

immunohistochemical staining of F4/80 

antigen (A, B) and adhesion to CX3CL1 

in vitro was tested by the parallel plate 

flow chamber adhesion assay using 

splenocytes from WT and CX 3CR1 / 

mice (C). A, Monocyte staining (brown) in 

injured WT and CX 3CR1 / arteries at 5 

days after surgery. B, Average number of 

monocytes in the intima of WT and 

CX 3CR1 / (KO) arteries 5 days after 

injury (n16). Results are expressed as 

the meanSEM. **P0.05. C, Average 

number of Moma2-FITC monocytes 

bound to CX 3CL1. Results are expressed 

as the meanSEM. **P0.05. 

tissue macrophages or transvenous migration of monocyte to 

the adventitia may occur by CX 3CR1-independent mechanisms. 

While we cannot exclude the possibility that adventitial 

macrophages traffic and migrate toward soluble CX 3CL1 

through the vessel wall to the luminal surface, the data 

support a primary role for CX 3CR1 in the rapid capture and 

firm adhesion of monocytes under flow conditions. 14 Clinical 

cohorts have shown that 2 single nucleotide polymorphisms 

of CX 3CR1, V249I and T280M, are associated with reduced 

prevalence of atherosclerosis and coronary artery disease. 8,9 

In addition, the M280 allele is associated with a reduced risk 

of internal carotid artery (ICA) occlusive disease. 10 Our 

laboratory has shown that the protein encoded by the M280 

allele has impaired adhesive capacity, 7 suggesting that cell 

adhesion is an important mechanism by which CX 3CR1 

exerts its effect on recruiting cells during vascular 

inflammation. 

In addition to CX 3CR1, CCR2 appears to mediate the 

response to arterial injury as well. In the same mouse model 

of wire-induced femoral artery injury, CCR2 / mice had a 

phenotype comparable to CX 3CR1 / mice in terms of a 

reduced intimal hyperplasia and intima/media ratio 4 weeks 

after injury. 23 In the CCR2 study, no macrophage infiltration 

was seen by MOMA-2 or CD68 staining in either WT or 

CCR2 / animals and the authors concluded that CCR2 did 

not affect macrophage accumulation within the arterial wall 

after injury. Although both CX 3CR1 and CCR2 mediate 


http://atvb.ahajournals.org/ by guest on March 25, 2013 

Figure 6. VSMC proliferation in response to vascular 

injury. Shown is the proliferation of VSMCs in 

the intima and media of injured WT and CX3CR1 / 

arteries at 5 and 14 days after injury. Proliferating 

VSMCs were defined as those -actin positive 

cells that were BrdU (d5) or PCNA (d14) positive. 

Results are represented as meanSEM. *P0.07, 

**P0.03.

monocyte chemotaxis and trafficking to sites of inflammation, 

CX 3CR1 mediates direct monocyte binding to its 

membrane-bound ligand, CX 3CL1, without the help of other 

adhesion molecules, such as integrins. 15 Thus, monocyte 

trafficking to damaged tissue during arterial injury may favor 

the CX 3CR1–CX 3CL1 system. Recently, Schober et al reported 

an alternative mechanism for CCR2 in acute vascular 

injury. 25 In their study, CCL2 was found to be co-localized 

with platelets on the denuded carotid artery surface of 

apoE / mice fed with a high-fat diet within 24 hours after 

wire injury. Even though platelets do not contain CCL2, 26 

low-affinity CCR2-dependent binding of CCL2 to human 

platelets is detected in vitro, 27 suggesting that adherent 

platelets with immobilized CCL2 mediate monocyte recruitment 

after mechanical injury. Monocyte adhesion to the 

luminal surface is not detected within 24 hours under our 

experimental conditions or in similar wire-induced arterial 

injury models under either normolipidemic 23 or hyperlipidemic 

25,28 conditions. Nevertheless, platelet deposition may 

play a vital role in the pathogenesis of restenosis, 29 and CCR2 

may be involved in that process. 

SMC accumulate in the intima in both atherosclerosis and 

restenosis. 30,31 This process is also seen in animals following 

guidewire-induced arterial injury. CX 3CR1 is expressed in 

human SMC cultured from coronary arteries and in atherosclerotic 

lesions, 17,18 suggesting that CX 3CR1 may play a role 

in regulating SMC function. In our study, intimal hyperplasia 

was significantly decreased in CX 3CR1 / arteries after 

injury. Concurrently, there were decreased numbers of 

VSMC in the neointima. VSMC proliferation in response to 

injury was also impaired in CX 3CR1 / mice as measured by 

both BrdU incorporation and PCNA immunohistochemistry. 

Whether the VSMC effects are primary or secondary cannot 

be addressed by this study. However, several data point to a 

primary effect of CX 3CR1 on VSMC function in this model. 

In a recent report of interleukin (IL)-15’s effect on CX 3CR1/ 

CX 3CL1 expression and the response to arterial injury, 32 

IL-15 attenuated SMC proliferation and CX 3CR1/CX 3CL1 

mRNA expression on serum stimulation. Using a periadventitial 

injury model, the authors further demonstrated that 

IL-15 upregulation after injury reduced intimal thickening, 

and blockade of IL-15 increased CX 3CR1/CX 3CL1 expression. 

These results suggested that IL-15 up-regulation decreases 

neointimal formation in response to arterial injury via 

suppressing CX 3CR1 signaling in SMC. CX 3CR1 is a typical 

G protein-coupled receptor, and the binding of CX 3CL1 to 

CX 3CR1 initiates multiple signal transduction pathways that 

are associated with cell proliferation and survival, such as 

extracellular signal regulated kinase (ERK)/P38 MAPK pathways 

33 and the PI3K/Akt pathway. 34 In addition, tumor 

necrosis factor- stimulates CX 3CL1 expression in human 

aortic SMC, 35 and the induction of CX 3CR1 facilitates SMC 

proliferation via NF-B in aortic SMCs. 20 Additionally, 

CX 3CR1/CX 3CL1 may directly regulate SMC migration in 

response to vascular inflammation. Cultured human coronary 

artery SMC have been shown to express CX 3CR1 and 

undergo chemotaxis toward CX 3CL1. 17 US28, a viral receptor 

for CX 3CL1 can mediate SMC migration in vitro. 36 Although 

direct evidence for a role of CX 3CR1-mediated SMC migra- 


tion in arterial injury needs to be further demonstrated in 

vivo, our findings of substantially reduced intimal hyperplasia 

and SMC proliferation in CX 3CR1 / mice after mouse 

femoral artery denudation strongly suggest that, in addition to 

mediating monocyte infiltration at early stages of the injury 

response, CX 3CR1 may also play an important role in 

vascular remodeling in the late stage of injury by regulating 

SMC functions. 

In summary, we demonstrate that CX 3CR1 plays a critical 

role in the regulation of cellular responses to acute vascular 

injury. CX 3CL1 expression is upregulated on endothelial and 

smooth muscle cells after injury. Mice lacking CX 3CR1 have 

lower incidence and degree of intimal hyperplasia after 

injury. The role of CX 3CR1 in regulating vascular inflammation 

involves monocyte accumulation in the vessel wall and 

VSMC proliferation and migration. Combined with data that 

CX 3CR1 polymorphisms associated with defective cell adhesion 

protect humans from atherosclerotic diseases, our findings 

suggest that CX 3CR1 could be an effective drug target 

for both acute and chronic vascular injuries, such as restenosis 

and atherosclerosis. 

Acknowledgments 

The authors sincerely thank Gail Grossman and Kirk McNaughton 

for their technical assistance in tissue processing and staining, Rishi 

Rampersad for animal husbandry and Drs Teresa Tarrant and Maya 

Jerath for proofreading the manuscript. 

Sources of Funding 

This study was supported by National Institute of Health R01s 

CA098110 (D.D.P.) and HL074219 (S.S.S.). 

None. 

Disclosures 

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CX3CR1 Deficiency Confers Protection From Intimal Hyperplasia ...

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