Figure 4.

SMCs exert chemotactic cues on plaque MΦs

(A–F) In vivo imaging of an atherosclerotic lesions within the carotid artery in atherosclerotic MC^RFP-rep; Lyz-MΦ^GFP-rep mice after 14 weeks western diet by multi-photon microscopy. (A) Time-series with a focus on the shoulder region of the plaque, arrows depicting locally confined but dynamic protrusions formed by Lyz⁺ MΦs (green) toward SMCs (red). Images from Video S3. Scale bars, 20 μm. (B) In vivo imaging of Lyz⁺ MΦ-SMC contacts during Lyz⁺ MΦ migration within the intima. Rendered illustration of Lyz⁺ MΦs (green) migrating along SMCs (red), including exemplary migration tracks of 2 cells. Scale bars, 20 μm.

(C) Analysis of the duration of the interactions between SMCs (red) and Lyz⁺ MΦs (green). (D) Analysis of the displacement length during interaction and during free migration. (E) Velocity profile of cells 1 and 2 (labeled in the migration tracks above under B) over time: boxes indicate interactions; horizontal lines indicate mean velocity of the time period included. (F) Left: meandering index (track straightness) of Lyz⁺ MΦs during interaction with SMCs and during free migration without interaction. Right: displacement rate of Lyz⁺ MΦs during interaction with SMCs and during free migration without interaction with SMCs. (C–F) n = 46–52 cell tracks covering free migration (−) or subsequent SMC interaction (+) or vice versa from n = 3 mice, Mann-Whitney test used to compare groups, ^∗∗∗p < 0.001, ^∗∗p < 0.001.

(G) In vivo imaging of static SMC-Cx3cr1⁺ MΦ contacts in a MC^RPF-rep; Cx3cr1-MΦ^GFP-rep mouse, arrow depicting a SMC embedded in two Cx3cr1⁺ MΦs. Scale bars, 20 μm.

(H) Ex vivo confocal imaging of cross-sections of atherosclerotic valves in MC^RFP-rep; Cx3cr1-MΦ^GFP-rep mice after 12 weeks western diet, MΦs in green, SMCs in red, nuclei in blue, arrowheads depicting SMC-MΦ contacts. Scale bars, 40 μm.

(I and J) Ex vivo confocal imaging of SMC^lin; Cx3cr1-MΦ^GFP-rep mice after 22–24 weeks of western diet, (I) atherosclerotic valve cross-sections, MΦs in green, SMC and SMC-progeny in red, nuclei in blue, arrowheads depicting SMC-MΦ contacts, dashed line outlining SMC enveloping MΦ. Scale bars, 10 μm. (J) En face confocal z stacks of the atherosclerotic intima in SMC-tdT^lin; Cx3cr1-MΦ^GFP-rep mouse, Cx3cr1⁺ MΦs in green, SMC^lin cells in red, blue arrowheads pointing toward SMC-MΦ contacts observable in the cross- and longitudinal-sections of the z stack, most frequent within the plaque surface. Scale bars, 50 μm (left), 5 μm (right) (K), left: representative immunohistochemical images of human aortic plaques stained for CD68 and α-SMA, scale bars, 300 μm, right: immunofluorescence staining for CD68 (red) and α-SMA (green) and with Hoechst (blue), dashed lines represent macrophage and SMCs, scale bars, 20 μm on the left and 10 μm on the right immunofluorescence image.

(L) Pearson correlation of the relative CD68⁺ area in fibrous cap environment (defined as the plaque area within the top 30% plaque surface) with, left: the relative necrotic core size, middle: the α-SMA content and right: the plaque vulnerability index (further elaborated in methods). Intermediate (n = 5) and advanced (n = 8) human plaques, graded accordingly by the pathology department, were included. Pearson r and two-tailed p value are included for every Pearson correlation.

(M) Summary illustration of the natural MΦ distribution within atherosclerotic lesions. MΦs mainly localize at areas of the plaque surface adjacent to SMCs in murine and in human atherosclerotic lesions. Bar graphs show mean with SEM.