Figure 4. Macrophage dynamics show Ly6c^hi monocyte recruitment is the key kinetic change impairing regression in Ccr2^–/– recipient mice.

Aortic arches from Apoe^–/– donors fed WD for 14 weeks were transplanted into recipients. (A) Schematic of timeline for EdU and bead injections into recipient mice to assess recruitment of Ly6C^hi and Ly6C^lo monocytes, respectively, into transplanted aortic arches under regression conditions. (B) Representative flow cytometry plots of CD45⁺CD115⁺ circulating monocytes showing Ly6C versus EdU, and (C) quantification of EdU incorporation in circulating Ly6C^hi versus Ly6C^lo populations showing that EdU is preferentially incorporated into Ly6C^hi monocytes in WT, Ccr2^–/–, and Ccr5^–/– mice (n = 4–8 per group). (D) Analysis of EdU⁺ cells/section of atherosclerotic plaques transplanted into WT, Ccr2^–/–, or Ccr5^–/– recipients showing significantly reduced recruitment into plaques of Ly6C^hiEdU⁺ cells into Ccr2^–/– compared with WT recipients. (E) Representative flow cytometry plots of CD45⁺CD115⁺ circulating monocytes showing Ly6C versus beads, and (F) quantification of bead incorporation in circulating Ly6C^hi versus Ly6C^lo monocyte populations showing that beads are preferentially incorporated into Ly6C^lo monocytes in WT, Ccr2^–/–, and Ccr5^–/– mice (n = 8–10 per group). (G) Analysis of bead⁺ cells/section of atherosclerotic plaques transplanted into WT, Ccr2^–/–, or Ccr5^–/– recipients showing significantly reduced recruitment of Ly6C^lo bead⁺ cells into Ccr5^–/– compared with WT mice. Quantification in plaque sections of (H) Ki67 (to assess proliferation) and (I) cleaved caspase 3 (to assess apoptosis) showed no significant differences in proliferation or apoptosis between transplant recipient groups. Quantifications in D, G, H, and I were done in aortic arch plaques from mice 5 days after transplantation (n = 8–9 per group). ^#P = 0.05, *P < 0.05 when compared with WT group using 1-way ANOVA with Dunnett’s multiple comparisons testing.