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Comparative Study
. 2008 Apr 1;117(13):1642-8.
doi: 10.1161/CIRCULATIONAHA.107.743872. Epub 2007 Dec 28.

Fractalkine deficiency markedly reduces macrophage accumulation and atherosclerotic lesion formation in CCR2-/- mice: evidence for independent chemokine functions in atherogenesis

Noah Saederup  1 , Liana Chan, Sergio A Lira, Israel F Charo
Affiliations
Comparative Study

Fractalkine deficiency markedly reduces macrophage accumulation and atherosclerotic lesion formation in CCR2-/- mice: evidence for independent chemokine functions in atherogenesis

Noah Saederup et al. Circulation. .

Abstract

Background: Monocyte-derived foam cells are the hallmark of early atherosclerosis, and recent evidence indicates that chemokines play important roles in directing monocyte migration from the blood to the vessel wall. Genetic deletions of monocyte chemoattractant protein-1 (MCP-1, CCL2), fractalkine (CX3CL1), or their cognate receptors, CCR2 and CX3CR1, markedly reduce atherosclerotic lesion size in murine models of atherosclerosis. The aim of this study was to determine whether these 2 chemokines act independently or redundantly in promoting atherogenesis.

Methods and results: We crossed CX3CL1(-/-)ApoE(-/-) and CCR2(-/-)ApoE(-/-) mice to create CX3CL1(-/-)CCR2(-/-)ApoE(-/-) triple knockouts and performed a 4-arm atherosclerosis study. Here, we report that deletion of CX3CL1 in CCR2(-/-) mice dramatically reduced macrophage accumulation in the artery wall and the subsequent development of atherosclerosis. Deletion of CX3CL1 did not reduce the number of circulating monocytes in either "wild-type" ApoE(-/-) mice or CCR2(-/-)ApoE(-/-) mice, which suggests a role for CX3CL1 in the direct recruitment and/or capture of CCR2-deficient monocytes.

Conclusions: These data provide the first in vivo evidence for independent roles for CCR2 and CX3CL1 in macrophage accumulation and atherosclerotic lesion formation and suggest that successful therapeutic strategies may need to target multiple chemokines or chemokine receptors.

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Figures

Figure 1
Figure 1
Size fractionation of plasma lipoproteins. Plasma samples of WT, CX3CL1−/−,CCR2−/−, and CX3CL1−/−CCR2−/− mice, fed the Western diet for 8 weeks, with similar total cholesterol levels were pooled and fractionated by fast protein liquid chromatography (n=3 to 4 mice of each geno-type). All mice were on the ApoE−/− genetic background.
Figure 2
Figure 2
Atherosclerotic lesion areas (en face) in the aorta in WT, CX3CL1−/−, CCR2−/−, and CX3CL1−/−CCR2−/− mice fed the Western diet for 8 weeks. All mice were on the ApoE−/− genetic background. Each symbol depicts the percentage of the total aorta that stained for lipid with Sudan IV in a single mouse. Males (closed circles) and females (open circles) were combined for the analysis. “WT” denotes the ApoE−/− background, without the chemokine receptor deletions. Lesions in CCR2−/− mice were significantly smaller than in CX3CL1−/− mice (2.3% vs 2.9%). Lesion area in CX3CL1−/−CCR2−/− double-knockout mice was significantly less than in CCR2−/− mice (1.2% vs 2.3%). Compared with WT mice, lesion area in CX3CL1−/− CCR2−/− mice was reduced by >65%. Horizontal bars represent mean values for the group (*P≤0.05 and **P≤0.005).
Figure 3
Figure 3
En face photographs of Sudan IV–stained aortas from WT, CX3CL1−/−, CCR2−/−, and CX3CL1−/−CCR2−/− mice, all on the ApoE−/− background, fed the Western diet for 8 weeks.
Figure 4
Figure 4
Analysis of atherosclerosis in the aortic root in CX3CL1−/−, CCR2−/−, and CX3CL1−/−CCR2−/− mice fed the Western diet for 8 weeks. All mice were on the ApoE−/− genetic background. The degree of atherosclerosis was determined by staining serial (8 μm) cross sections through the aortic root with oil red O. Lesion size was quantified by digital morphometry, as described in Methods. A, Each symbol represents the mean lesion size in a single mouse, and the horizontal bar represents the mean of the group. Lesion areas in CCR2−/− mice were significantly less than in CX3CL1−/− mice in both males and females, and lesions in CX3CL1−/−CCR2−/− double-knockout mice were significantly smaller than in CCR2−/− mice in both males and females. Lesion areas in female CX3CL1−/−CCR2−/− mice were less than 25% of the areas in WT mice. (*P≤0.05 and **P≤0.005). B, Photomicrographs of oil red O–stained aortas from WT, CX3CL1−/−, CCR2−/−, and CX3CL1−/−CCR2−/− mice, all on the ApoE−/− background, fed the Western diet for 8 weeks.
Figure 5
Figure 5
Macrophage infiltration of the aortic root in WT, CX3CL1−/−, CCR2−/−, and CX3CL1−/−CCR2−/− female mice, all on the ApoE−/− background, fed the Western diet for 8 weeks. Sections from the aortic root were stained for macrophages with MOMA-2, and the stained area was quantified by digital morphometry, as described in Methods. A, CX3CL1−/−CCR2−/− mice had significantly less MOMA-2 staining than CCR2−/− mice (*P≤0.05 and **P≤0.005). Each symbol represents a single mouse, and the horizontal bar represents the mean value for each group. B, Representative aortic root sections stained with MOMA-2. Sections are adjacent to the ones shown in Figure 3B.
Figure 6
Figure 6
Peripheral blood leukocytes (PBLs) in WT, CX3CL1−/−, CCR2−/−, and CX3CL1−/−CCR2−/− mice on the ApoE−/− background fed the Western diet for 8 weeks. A, Total monocytes in each of the 4 genotypes. There was no decrease in the percentage of monocytes in CX3CL1−/− mice vs WT mice. Monocytes were significantly lower in CCR2−/− and CX3CL1−/− CCR2−/− mice than in WT mice, but there was no difference between CCR2−/− and CX3CL1−/−CCR2−/− mice. B, Quantification of Gr1hi and Gr1lo populations of monocytes. There was no difference in the percentage of Gr1hi monocytes in CX3CL1−/− mice vs WT mice. The percentage of Gr1hi monocytes was very significantly reduced in CCR2−/− and CX3CL1−/−CCR2−/− mice compared with WT mice, but there was no difference between CCR2−/− and CX3CL1−/−CCR2−/− mice. Similar results were seen in the Gr1lo population. (*P≤0.05 and **P≤0.005).
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