Decreased atherosclerosis in CX3CR1-/- mice reveals a role for fractalkine in atherogenesis

Abstract

The hallmark of early atherosclerosis is the accumulation of lipid-laden macrophages in the subendothelial space. Circulating monocytes are the precursors of these "foam cells," and recent evidence suggests that chemokines play important roles in directing monocyte migration from the blood to the vessel wall. Fractalkine (FK) is a structurally unusual chemokine that can act either as a soluble chemotactic factor or as a transmembrane-anchored adhesion receptor for circulating leukocytes. A polymorphism in the FK receptor, CX(3)CR1, has been linked to a decrease in the incidence of coronary artery disease. To determine whether FK is critically involved in atherogenesis, we deleted the gene for CX(3)CR1 and crossed these mice into the apoE(-/-) background. Here we report that FK is robustly expressed in lesional smooth muscle cells, but not macrophages, in apoE(-/-) mice on a high-fat diet. CX(3)CR1(-/-) mice have a significant reduction in macrophage recruitment to the vessel wall and decreased atherosclerotic lesion formation. Taken together, these data provide strong evidence that FK plays a key role in atherogenesis.

Figure 1

Immunolocalization of FK in atherosclerotic lesions. Mice lacking apoE (apoE^–/– mice) were maintained on the Western diet for 10 weeks. Serial sections were cut at the level of the aortic valve leaflets and incubated with antibodies. (a) Staining of FK (visualized as red). (b) Staining of macrophages (visualized as green). (c) Merged image of a and b. (d) Nuclei were stained with DAPI (blue) and the image was captured with a multiband (DAPI/phycoerythrin/FITC) filter. Original magnification: ×200.

Figure 2

Determination of FK mRNA in primary vascular tissues. Mice lacking apoE were maintained on the Western diet for 15 weeks. Thoracic and abdominal aortas were opened longitudinally, and the luminal endothelial cells (n = 11 mice), aortic media (n = 8 mice), and the aortic root (n = 14 mice) were isolated as described in Methods. Real-time PCR was performed to quantify FK mRNA expression, and the results are expressed as the mRNA FK/β-GUS mRNA. Each data point represents the mean of duplicate measurements.

Figure 3

Determination of FK mRNA in SMCs and macrophages. Mice lacking apoE were maintained on the normal chow diet, and primary SMCs and bone marrow–derived macrophage cultures were established as described in Methods. (a) SMCs were incubated for 24 hours with IFN-γ (20 ng/ml), TNF-α (20ng/ml), or both cytokines, and FK mRNA levels were determined by quantitative real-time PCR, as described in the legend for Figure 2. (b) Bone marrow–derived macrophages (note y-axis scale change). SMC cultures were established from two different mice and were used before passage three. Bone marrow–derived macrophages were from three different donors and were used on day 8. Each data point represents the mean of duplicate measurements.

Figure 4

Immunostaining of atherosclerotic lesions in apoE^–/– mice. Mice were fed the Western-type high-fat diet for 10 weeks, and serial sections were cut through the proximal aorta and stained for FK (a), CD11b (b), or with Movat stain to visualize elastin (c). Original magnification: ×200.

Figure 5

Lipoprotein analysis. Mice were maintained on a Western-type high-fat diet for 10 weeks. Plasma was pooled, the lipoproteins were fractionated by FPLC, and the cholesterol content of each fraction was determined as described in Methods. Plasma from six CX₃CR1^–/– and six CX₃CR1^+/+ mice was pooled.

Figure 6

Atherosclerotic lesion areas in CX₃CR1^+/+, apoE^–/– and CX₃CR1^–/–, apoE^–/– mice fed the Western diet for 5, 10, or 15 weeks. Each symbol depicts the percentage of the aorta that stained for lipid with Sudan IV in a single mouse. (a) Total aorta. Lesions in the CX₃CR1^–/– mice were significantly smaller than in CX₃CR1^+/+ mice at each timepoint (43%, 49%, and 36% reduced at 5, 10, and 15 weeks, respectively). (b) Aortic arch. Lesions were smaller in the CX₃CR1^–/– mice at each of the timepoints (29%, 40%, and 11% reduced at 5, 10, and 15 weeks, respectively). (c) Thoracic and abdominal aorta. Lesions were smaller in the CX₃CR1^–/– mice at each of the timepoints (56%, 60%, and 72% reduced at 5, 10, and 15 weeks, respectively). The horizontal bars represent the mean values for the group. *P ≤ 0.02, **P ≤ 0.002 vs. wild type.

Figure 7

Quantitation of atherosclerosis in the aortic root area. CX₃CR1^+/+, apoE^–/– and CX₃CR1^–/–, apoE^–/– mice were fed the Western diet for 10 weeks, and the degree of atherosclerosis was determined by staining serial 10-μm cross sections through the aortic root with oil red O. Lesion area was quantified by digital morphometry. Each symbol represents the mean total lesion area in a single mouse, and the horizontal bar represents the mean of the group. The CX₃CR1^–/– mice (n = 8) had significantly smaller total lesion areas (32% smaller, *P = 0.007) than the CX₃CR1^+/+ mice (n = 8).

Figure 8

Macrophage infiltration of the aortic sinus in CX₃CR1^+/+, apoE ^–/– and CX₃CR1^–/–, apoE^–/– mice fed the Western diet for 10 weeks. Sections from the aortic sinus were stained for macrophages with MOMA-2 and quantified by digital morphometry. CX₃CR1^+/+ mice had significantly more MOMA-2 staining than the CX₃CR1^–/– mice (40% more, *P ≤ 0.001). Each symbol represents a single mouse, and the horizontal bar represents the mean value for each group.