Figure 5. Deletion of CD163 in mice reduces intraplaque neovascularization and plaque progression.

(A) Immunoblotting of mouse macrophages with quantitation of densitometry for HIF1α in macrophages isolated from WT or CD163^–/– mice stimulated with Hb (n = 4 per group). (B) Analysis of macrophage supernatant for VEGF-A by ELISA (n = 4 per group). (1: WT macrophages, 2: Hb-stimulated WT macrophages, 3: CD163^–/– macrophages, 4: Hb-stimulated CD163^–/– macrophages). (C) Representative H&E staining of BCA plaque with a of gross inset photograph of the aortic arch from 1-year-old ApoE^–/– and ApoE^–/– CD163^–/– mice. Scale bars: 100 μm. (D–G) Quantitative measurements of lesion size, percentage of stenosis, lesion pathological scores, and necrotic areas in the BCA plaque (n = 8–12 per group). (H) Representative immunofluorescence confocal microscopic images of BCA plaque stained with VE-cadherin (green), CD163 (red), and DAPI (blue). Scale bars: 50 μm and 10 μm (enlarged images of boxed areas on the left). (I) Microvessel density quantification calculated by the number of microvessels per plaque area identified by VE-cadherin immunofluorescence confocal microscopy (n = 6–7 per group). (J) Representative immunofluorescence confocal microscopic images of intraplaque FITC-dextran (green) as a marker for permeability. Scale bars: 100 μm. Total intraplaque FITC fluorescence was quantified from confocal images of BCA plaques perfused with FITC-dextran to determine permeability (n = 7 per group). Bars and plots indicate the mean ± SEM (A and B) or the mean ± SD (D–G, I, and J). (A and B) *P < 0.05 versus other groups, by 1-way ANOVA , and, if the 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. (D–G, I, and J) P < 0.01 and P < 0.05, by 2-sided Student’s t test.