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. 2022 Nov 16;44(4):304–318. doi: 10.1093/eurheartj/ehac647

Figure 6.

Figure 6

Administrating a GRK2-specific inhibitor to ApoE−/− mice reduces atherosclerotic lesions and increases plaque stability. ApoE−/− mice received vehicle control or GRK2-specific inhibitor (GRK2-I, 10 mg/kg IV every 3 d) for 6 (A and B), 12 (C and D), or 18 weeks (E–I). (A) Mouse aortas were subjected to en-face co-immunostaining for VCLS721p and VE-cadherin with DAPI nuclear counterstains (n = 6). The inter-endothelial gaps between VE-cadherin junctions in the IA region were quantified. (B) Mice (n = 4) were subjected to in vivo endothelial permeability assay using FITC-conjugated microspheres. The intensities of leaked microspheres in the endothelium of the IA region were quantified. (C) Whole aortas were dissected and stained with Oil-red O to quantify lesion areas. (D) Serial longitudinal sections of aortas were stained with hematoxylin and eosin (upper panel). Macrophages infiltrated in lesions were determined by co-immunostaining for the macrophage marker CD68 and the SMC marker αSMA with DAPI nuclear counterstains (lower panel). The number of macrophages infiltrated in lesions was quantified (n = 8). (E–I) Quantifications of collagen content by PicroSirus red staining (E), fibrous cap thickness by Movat pentachrome staining (F), necrotic core areas (identified as the regions that lack DAPI-positive nuclei) by DAPI counterstaining (G), synthetic SMCs within the lesions by co-immunostaining for αSMA and vimentin (VIM) (H), and percentage of TUNEL-positive ECs on the lesions by co-immunostaining with TUNEL and vWF (I). The right panels in I are the magnified views of the indicated areas (dashed line box). The representative images of A, B, E, F, G, and H are shown in Supplementary material online, Figure S11A, B, D, E, and F, respectively. All analyses in E–I were performed on the sections of the innominate artery and the IA region of mice (n = 8 per group). Data are means ± SEM, and the images represent four to eight independent experiments with similar results. A two-tailed Student t-test was applied to A and C–I. One-way ANOVA with the Tukey multiple comparison test was applied to B.