Figure 6. CD163⁺ macrophages increase intraplaque endothelial VCAM and leukocyte recruitment via VEGF.

Activation of VCAM and NF-κB in HAECs (A–J) and association of high CD163⁺ macrophages and inflammation in human atherosclerotic lesions (K–N). In A–C, HAECs were incubated with culture media supernatants collected from human macrophages differentiated in HH [M(Hb) Sup] or control media [M(con) Sup] overnight. The expression of VCAM, p-p65 (p–NF-κB), and total NF-κB was measured by immunoblotting. LPS (100 ng/ml) was used as a positive control (n = 4 per group). (D) Immunostaining for VE-cadherin (green) and VCAM (red) in BCA plaques from 1-year-old ApoE^–/– and ApoE^–/– CD163^–/– mice. Scale bars: 100 μm. (E) The colocalization of VE-cadherin and VCAM in confocal microscopic images was analyzed for correlation coefficiency (n = 6–7 per group). (F) Total macrophage content in BCA plaques as described in H was analyzed using macrophage marker Mac3 immunohistochemical staining (n = 5 per group). (G–I) Cells were transfected with VEGFR2 siRNA for 48 hours before incubation with culture media supernatants from macrophages differentiated with HH or control media. The expression of VCAM, ICAM, p-p65 (p–NF-κB), and total NF-κB was measured by immunoblotting (n = 4 per group). Scr siRNA, scrambled siRNA. (J) Monocyte infiltration into BCA plaques from 6- to 8-month-old mice after a 4-week treatment with a control or VEGF-blocking antibody. Monocyte tracing was performed by injecting Au-nanoparticle–labeled monocytes, and labeled monocytes were counted on histological images (n = 6 per group). (K) Representative images of H&E and CD163 immunohistochemical staining showing high CD163 and low CD163 areas. Scale bars: 500 μm and 100 μm. (L) Areas of high and low expression of CD163⁺ macrophages in human atherosclerotic plaques were scored for inflammation (n = 7–8 per group). (M) CD163⁺ macrophages and CD3⁺ T cells were detected surrounding microvessels in high CD163 areas (white dashed lines show microvessels). Scale bars: 20 μm. (N) Quantitation of CD3⁺ T cells in high and low CD163⁺ macrophages surrounding intraplaque microvessels (n = 8 per group). Data represent the mean ± SEM (B, C, and H–J), the mean ± SD (E, F, J, and N), or the median for the box and whisker plot (L). (H–J) *P < 0.05, by 1-way ANOVA, and, if the variance ratio test (F test) was significant, a more detailed post-hoc analysis of differences between groups was performed using a Tukey-Kramer honest significant difference test. (B and C) *P < 0.05, by 2-sided Student’s t test. (E, F, and N) P < 0.01 and P 0.05, by 2-sided Student’s t test. (L) P < 0.01, by Mann-Whitney-Wilcoxon test.