Macrophagic CD146 promotes foam cell formation and retention during atherosclerosis

Abstract

The persistence of cholesterol-engorged macrophages (foam cells) in the artery wall fuels the development of atherosclerosis. However, the mechanism that regulates the formation of macrophage foam cells and impedes their emigration out of inflamed plaques is still elusive. Here, we report that adhesion receptor CD146 controls the formation of macrophage foam cells and their retention within the plaque during atherosclerosis exacerbation. CD146 is expressed on the macrophages in human and mouse atheroma and can be upregulated by oxidized low-density lipoprotein (oxLDL). CD146 triggers macrophage activation by driving the internalization of scavenger receptor CD36 during lipid uptake. In response to oxLDL, macrophages show reduced migratory capacity toward chemokines CCL19 and CCL21; this capacity can be restored by blocking CD146. Genetic deletion of macrophagic CD146 or targeting of CD146 with an antibody result in much less complex plaques in high-fat diet-fed ApoE^-/- mice by causing lipid-loaded macrophages to leave plaques. Collectively, our findings identify CD146 as a novel retention signal that traps macrophages within the artery wall, and a promising therapeutic target in atherosclerosis treatment.

Figure 1

CD146 is upregulated in macrophage foam cells. (A) Human carotid artery (n = 3) atherosclerotic lesions staining for CD146 (red) and CD68 (green) and their co-localization (yellow merge; see arrows). (B) Atherosclerotic plaques from ApoE^−/− mouse (n = 5) that was fed a Western diet (WD) for 18 weeks staining for CD146 (red) and Mac-3 (green) and their co-localization (yellow merge; see arrows). The nuclei were stained with DAPI (blue). The dashed lines indicate the lesion borders. The scale bars in A and B are 50 μm. (C, D) Flow cytometric analysis (C) or western blot (D) of CD146 expression in CD11b⁺F4/80⁺ peritoneal macrophages isolated from wild-type C57BL/6J mice fed a normal diet (chow) or ApoE^−/− mice (n = 5) fed a normal diet or a WD. Bottom, quantification of the mean fluorescent intensity (MFI) of CD146 in each group (n = 5). CD146 expression in D (bottom) is presented relative to that of GAPDH (loading control). (E) Flow cytometric analysis of CD146 expression in F4/80⁺ peritoneal macrophages and bone marrow-derived macrophages (BMDMs) that were treated with or without oxLDL (50 μg/ml) for 24 h. Bottom panel: quantification of the MFI of CD146 in each group (n = 5). (F) Flow cytometric analysis of CD146 expression in F4/80⁺ BMDMs that were treated with LDL, acetylation LDL (AcLDL) or oxLDL (50 μg/ml) for 24 h. Bottom panel: quantification of the MFI of CD146 in each group (n = 5). (G, H) Real-time PCR analysis of mRNA level of Cd146 (G) or western blot analysis of CD146 protein level (H) in peritoneal macrophages and BMDMs that were treated with oxLDL (50 μg/ml) for 24 h in the presence or absence of NF-κB inhibitor BAY11-7082 (20 μM) (n = 3). (I) Cd146 promoter-luciferase reporter activity in HEK293 cells treated with oxLDL (50 μg/ml) in the presence or absence of the NF-κB inhibitor, presented relative to luciferase activity in unstimulated cells, set as 1. (J) Dual luciferase assay of putative NF-κB binding sites in the Cd146 promoter. The luciferase activity of these constructs was measured and normalized to that of the unstimulated wild-type construct (pGL3-WT) (n = 5). (K) ChIP assay of p65 binding to Cd146 promoter using an anti-p65 antibody or an isotypic control, followed by PCR amplification of the genomic DNA fragments covering the binding site -621 and -420. GAPDH was used as the internal control. (L) Quantification of the relative level of PCR product to that of input. Two-way ANOVA test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. The data represent three independent experiments.

Figure 2

CD146 is required for oxLDL-induced macrophage activation. (A-H) Western blot analysis of IκBα, phosphorylated (p-) and total NF-κB p65, Src and JNK in oxLDL-stimulated (50 μg/ml) BMDMs isolated from CD146^WT or CD146^M-KO mice (A-D) or BMDMs with or without pretreatment with anti-CD146 AA98 (50 μg/ml) (E-H). GAPDH was used as a loading control. The upper panel indicated the quantification of CD146 expression. (I, J) Quantitative real-time (RT) PCR analysis of mRNA levels of the inflammatory factors Mcp-1, Mmp-9, Tnf-α, Ifn-γ and Il1-β in BMDMs that were treated with oxLDL (50 μg/ml) for 24 h. (I) BMDMs were isolated from CD146^WT or CD146^M-KO mice and stimulated as indicated. (J) CD146^WT BDMDs were stimulated as indicated in the presence of control mIgG or anti-CD146 AA98 (50 μg/ml). Two-way ANOVA test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. The data represent three independent experiments.

Figure 3

CD146 promotes foam cell formation. (A) Lipid uptake was measured by Oil red O staining of CD146^WT or CD146^M-KO BMDMs that were stimulated with oxLDL (50 μg/ml) for 24 h with or without pretreatment with anti-CD146 AA98 (50 μg/ml). The scale bar is 100 μm. The lower panel shows quantification of oil red O content by means of the Image-Pro Plus software. (B-D) BMDMs from CD146^WT or CD146^M-KO mice or BMDMs with or without pretreatment with AA98 (50 μg/ml) were incubated with Dil-oxLDL (50 μg/ml). Dil-oxLDL was detected by either confocal microscopy (B) or flow cytometry (C, D). Right panel: quantification of the MFI of Dil-oxLDL. (E) A biochemical lipid quantitative assay was used to measure total cellular lipids (with two-way ANOVA; ^**P < 0.01, ^***P < 0.001). The data represent three independent experiments.

Figure 4

CD146 interacts with CD36. (A) Co-IP assay of CD36 and CD146 in BMDMs. CD146 and CD36 from cell lysates were immunoprecipitated with anti-CD146 mAb and anti-CD36, respectively. (B) Direct interaction between CD36 and CD146. CD146-ECD was first incubated with CD36-ECD. CD146-ECD was pull down by anti-CD146 ME-9F1, and then precipitated by protein G beads. Bound proteins were subsequently analyzed by western blotting. (C) Co-IP assay showed that the CD36 and CD146 interaction was enhanced in the presence of oxLDL in BMDMs. Immunoblot analysis of CD146-precipitated proteins from BMDMs treated with oxLDL (50 μg/ml) for the indicated times. Right panel: quantification of CD36 level relative to CD146. (D) Co-IP assay showed that the CD36 and CD146 interaction was decreased by anti-CD146 antibody AA98. Right panel: quantification of CD36 level relative to CD146. (E) Direct interaction of CD36/CD146 or CD36/CD146^D4-5. CD36-ECD was first incubated with CD146-ECD and CD146^D4-5, respectively. CD36-ECD was pull down by anti-CD36 antibody, and then precipitated by protein G beads. (F) AA98 blocked CD36 and CD146 interaction. His-CD146-ECD or His-CD146^D4-5 was first incubated with BMDM cell lysates in the presence of mIgG or AA98 (50 μg/ml). CD36 proteins bound to His-CD146-ECD or His-CD146^D4-5 were detected by immunoblot with anti-CD36 antibody. ^*P < 0.05, ^**P < 0.01. The data represent three independent experiments.

Figure 5

CD146 facilitates CD36 internalization. Western blot (A, B) and FACS analysis (C, D) of membrane CD146 and CD36 in oxLDL-stimulated BMDMs isolated from CD146^WT or CD146^M-KO mice (A, C) or BMDMs with or without pretreatment with anti-CD146 AA98 (50 μg/ml) (B, D). Membrane fractions were immunoblotted with the indicated antibodies. SP (sodium pump) served as a loading control for membrane fractions. Right panel: quantification of CD36 or CD146 levels relative to SP (A, B). Bottom panel: quantification of the MFI of CD36 or CD146 (C, D). (E, F) Western blots of CD146 and CD36 in endosomal fractions of BMDMs isolated from CD146^WT or CD146^M-KO mice (E) or BMDMs with or without pretreatment with anti-CD146 AA98 (50 μg/ml) (F). Endosomal fractions were immunoblotted with the indicated antibodies. Right panel: quantification of CD36 or CD146. (G, H) BMDMs from CD146^WT or CD146^M-KO mice or BMDMs with or without pretreatment with AA98 (50 μg/ml) were labeled with a CD36-cross-linking antibody. Then, the cells were incubated at 37 °C to cross-link CD36. The cells were then washed with cold acid wash buffer to deplete surface CD36. Confocal microscopy was used to detect and quantify CD36 internalization. Bottom panel: quantification of the MFI of CD36. ^*P < 0.05, ^**P< 0.01, ^***P< 0.001. The data represent three independent experiments.

Figure 6

CD146 controls the expression of macrophage migratory factors in response to oxLDL. (A, B) Quantitative real-time (RT) PCR analysis of mRNA levels of the macrophage migratory factors Cd36, Netrin-1, Sema 3E and Ccr7 in BMDMs that were incubated with oxLDL (50 μg/ml) for 24 h. (A) BMDMs were isolated from WT, CD146^M-KO or CD36^KO mice and stimulated as indicated; (B) WT BDMDs were stimulated as indicated in the presence of control mIgG or anti-CD146 AA98 (50 μg/ml). (C, D) Western blot analysis of the macrophage migratory factors CD36, Netrin-1 and CCR7 in BMDMs that were treated as indicated. GAPDH was used as a loading control. (C) BMDMs were isolated from WT, CD146^M-KO or CD36^KO mice and stimulated as indicated; (D) WT BDMDs were stimulated as indicated in the presence of control mIgG or anti-CD146 AA98 (50 μg/ml). Right panel: quantification of protein expression level relative to GAPDH. ^*P < 0.05, ^**P < 0.01. The data represent three independent experiments.

Figure 7

CD146 facilitates the retention of macrophage foam cells. (A, B) Migration of BMDMs isolated from WT, CD146^M-KO or CD36^KO mice toward CCL19 (A) or CCL21 (500 ng/ml) (B) during oxLDL (50 μg/ml) stimulation was measured in a Transwell Boyden chamber (n = 5). The number of migrated cells was counted. (C, D) Migration assay of BMDMs toward CCL19 (C) or CCL21 (500 ng/ml) (D) during oxLDL (50 μg/ml) stimulation with or without anti-CD146 AA98 (50 μg/ml) using a Transwell Boyden chamber (n = 5) (two-way ANOVA test). (E) Representative photomicrograph of a lesional section from ApoE^−/− mouse at baseline where YG-bead⁺ monocyte-derived cells were observed. Red: Mac-3⁺ cells; blue: DAPI-stained nuclei; green: YG-beads (internal elastic lamina is also green due to autofluorescence). Arrows indicate cells containing fluorescent beads in the lesion section. lu, lumen of aorta. The scale bar is 100 μm. (F, G) In vivo analysis of the recruitment of bead-labeled macrophages (in green) to and retention in atherosclerotic plaques of CD146^WT→ApoE^−/− and CD146^M-KO→ApoE^−/− mice (F), or AA98- or mIgG-treated ApoE^−/− mice (G) using a monocyte tracking system. The results are presented as beads per area at 10 days (baseline, five mice per group) and 14 days (eight mice per group) after beads injection. At least 20 sections per mouse were analyzed. Two-way ANOVA test. ^*P < 0.05, ^**P< 0.01, ^***P< 0.001. The data represent three independent experiments.

Figure 8

Targeted deletion of CD146 in macrophages leads to lower atherosclerosis burden and less complex plaques. (A) Representative images of atherosclerosis in the aorta en face of CD146^WT→ApoE^−/− and CD146^M-KO→ApoE^−/− mice. The aorta was stained with oil red O. (B) Representative images of oil red O staining of lesions isolated from CD146^WT→ApoE^−/− and CD146^M-KO→ApoE^−/− mice. (C) H&E staining of atherosclerotic lesions isolated from CD146^WT→ApoE^−/− and CD146^M-KO→ApoE^−/− mice (n = 8). The dashed lines indicate the lesion borders. The scale bar is 100 μm. (D) Lesion area of atherosclerotic plaques of the aortic roots of CD146^WT→ApoE^−/− and CD146^M-KO→ApoE^−/− mice, presented for each genotype across the 400 μm of the aortic root (n = 8). (E) Quantification of lesion area of aortic plaques isolated from each genotype (n = 8). (F) The distribution of early, moderate and advanced plaques based on histological staging of the atherosclerotic lesions (n = 8). (G) Quantification of the number of Mac-3⁺ macrophages in the aortic plaques (n = 8, at least 10 sections per mouse). (H) Quantification of necrotic core areas of aortic plaques (n = 8, at least 20 sections per mouse). (I) Masson Trichrome (collagen) staining of aortic plaques (left). Right panel: quantification of staining (n = 8, at least 10 sections per mouse). The scale bar is 100 μm. One-way ANOVA test, *P < 0.05, ^**P < 0.01.

Figure 9

CD146 as a target for atherosclerosis therapy. (A-G) Preventive targeting of CD146 with antibody reduces atherosclerosis. ApoE^−/− mice were preventively injected with mIgG or AA98 when they began a Western diet (eight mice per group). All the quantifications were performed using at least 15 sections per mouse. (A) Intervention dosing regimen of CD146-targeted therapy using anti-CD146 AA98 or control IgG in high fat diet (HFD)-induced atherosclerosis. (B) Lesion area of plaques of the aortic roots of each group of mice, presented for each group across the 400 μm of the aortic root (n = 8). (C) Quantification of lesion area of aortic plaques isolated from ApoE^−/− mice. (D) The distribution of early, moderate and advanced plaques based on histological staging of the atherosclerotic lesions. (E) Quantification of immunostained aortic plaques for macrophages using the macrophage marker Mac-3. (F) Quantification of necrotic core areas of aortic plaques. (G) Quantification of Masson's trichrome (collagen) staining of aortic plaques. (H-N) Therapeutic targeting of CD146 with antibody reduces atherosclerosis (five mice per group). All the quantifications were performed using at least 15 sections per mouse. (H) Intervention dosing regimen of CD146-targeted therapy using anti-CD146 AA98 or control IgG in HFD-induced atherosclerosis. ApoE^−/− mice were fed a HFD for 12 weeks before antibody treatment for additional 6 weeks, the time at which the mice were sacrificed. (I) Lesion area of atherosclerotic plaques of the aortic roots of each group of mice, presented for each group across the 400 μm of the aortic root (n = 5). (J) Quantification of lesion area of aortic plaques isolated from ApoE^−/− mice. (K) The distribution of early, moderate and advanced plaques based on histological staging of the atherosclerotic lesions. (L) Quantification of immunostained aortic plaques for macrophages using the macrophage marker Mac-3. (M) Quantification of necrotic core areas of aortic plaques. (N) Quantification of Masson Trichrome (collagen) staining of aortic plaques. The scale bar is 100 μm. One-way ANOVA test, ^*P< 0.05, ^**P< 0.01, ^***P< 0.001.