Figure 5.

SMCs within the fibrous cap preserve a strategic positioning of plaque MΦs and secure homeostatic MΦ functions

(A and B) Reanalyzed single-cell RNA-seq data from mouse aortic roots from atherosclerotic SMC^lin mice from Wirka et al., GEO: GSE131780. (A) UMAP based dimensionality reduction of analyzed cells (left), heatmap illustrating cytokine and chemokine expression of different SMC subsets (right). (B) Marker genes of SMC clusters illustrated in a heatmap, composed by ClustVis.

(C) Representative confocal image depicting the spatial distribution of the key cSMC marker PDGFRβ within an atherosclerotic valve in SMC^lin; Cx3cr1-MΦ^GFP-rep mice after 22–24 weeks of western diet, SMC^lin cells in red, MΦs in green, and PDGFRβ in white. Scale bars: 30 μm (left) and 15 μm (right images).

(D) Illustration of the experimental setup of the migration assay: macrophages undergo a migratory decision either moving toward the artificially composed SMC-rich fibrous cap below or residing at the artificially composed, necrotic cell rich, necrotic core. SMCs (representing the fibrous cap) are located in the lower chamber, whereas peritoneal macrophages have been attached on the transwell of the upper chamber. Necrotic Jurkat cells (representing the necrotic core) have been added to the upper chamber.

(E) Number of peritoneal MΦs from Lyz-MΦ^GFP-rep mice that transmigrated toward the lower chamber per field of view (FOV). Isotype or anti-CCL2 blocking antibody was simultaneously added to the lower chamber. MΦ numbers per FOV counted at 4 subsequent time points (n = 4 independent experiments).

(F) Distribution of macrophages as percentage of LGALS3⁺ area in 30 μm plaque surface area in percentage of total plaque LGALS3⁺ area at three subsequent locations (n = 10 each).

(G) Left: quantification of LGALS3⁺ surface macrophage content as relative LGALS3⁺ area in percentage of total plaque surface area (defined as the upper 30 μm of the plaque) from BCA sections at three consecutive locations (n = 10 each). Right: representative immunofluorescent images of BCA sections for ACTA2 (green), LGALS3 (red), and Hoechst (blue) with highlighted 30 μm plaque surface area from Ccl2^SMC+/+ and Ccl2^SMCΔ/Δ littermates after 14 weeks of western-diet feeding. Scale bars, 100 μm.

(H) Volcano plot depicting differentially regulated genes analyzed by RNA-seq of FACS-sort enriched peritoneal MΦs, coincubated either with live or dead Jurkat cell supernatant for 12 h.

(I) Quantification of peritoneal macrophages 12 h after addition of live or dead Jurkat cell supernatant (n = 6).

(J–L) Efferocytosis assay, analyzing the efferocytotic capacity of the MΦ population, isolated from Lyz-MΦ^GFP-rep mice. Apoptotic Jurkat cells were added for 1 h after 6 h incubation either with or without CCL2. (J) Quantification of MΦs with engulfed apoptotic cells upon presence or absence of CCL2 (n = 5 independent experiments). (K) Quantification of the total number of engulfed apoptotic cells upon CCL2 presence of absence. (L) Representative epifluorescence images of the efferocytosis assay with peritoneal macrophages (green) and apoptotic Jurkat cells (red), 1 h after Jurkat cell addition. Scale bars, 50 μm.

(M–O) Necrotic core analysis as total necrotic area in μm² (M) and in percentage of plaque area (N), assessed with Masson Trichrom’s staining of valve sections, from Ccl2^SMC+/+ (n = 9) and Ccl2^SMCΔ/Δ (n = 10) littermates after 14 weeks of western diet. (O) Left: representative images of necrotic core content analyzed by Masson Trichrom’s staining of valve sections from Ccl2^SMC+/+ and Ccl2^SMCΔ/Δ littermates after 14 weeks of western diet. ^∗ indicates necrotic areas. Scale bars, 100 μm. Right: representative images of immunofluorescence stainings of valve sections from Ccl2^SMC+/+ and Ccl2^SMCΔ/Δ littermates after 14 weeks of western diet for ACTA2 (green), LGALS3 (red), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) (yellow), and DAPI (blue). Scale bars, 100 μm.

(P and Q) Quantification of cell apoptosis as total amount of TUNEL⁺ LGALS3⁺ Hoechst⁺ MΦs in plaque (P) and as total amount of TUNEL⁺ Hoechst⁺ apoptotic cells (Q) in Ccl2^SMC+/+ (n = 9) and Ccl2^SMCΔ/Δ (n = 10) individual littermates in total after 14 weeks of western diet, only including plaques at the proximal and intermediate BCA, without distal BCA areas with its early lesions.

(R) Quantification of valve atherosclerotic plaques for (left) total and relative TUNEL⁺ cells. Data are shown as mean and SEM.

(I, J, K, M, N, and R) Student’s t test was used for normally distributed data and Wilcoxon matched-pairs signed rank test for not normally distributed data. (E, F, G, P, and Q) Repeated measure two-way ANOVA or mixed-effects model was used. ^∗p < 0.05; ^∗∗ p < 0.01; NS, not significant. Bar graphs show mean with SEM.