Fimasartan reduces neointimal formation and inflammation after carotid arterial injury in apolipoprotein E knockout mice

Figures

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  Fig. 1. Regression of CA injury-induced neointimal hyperplasia in ApoE KO mice by Fimasartan administration. a Chemical structure of fimasartan (2-n-butyl-5-dimethylamino-thiocarbonyl-methyl-6-methyl-3-{[2-(1H-tetrazole-5-yl)biphenyl-4-yl]methyl}pyrimidin-4(3H)-one). b Schematic depiction of the study protocol. c Representative H&E-stained images of the CA cross sections of ApoE KO mice. Boxed regions were magnified in the under panels. Scale bar: 100 μm. d Quantitative analysis of the incidence of neointimal hyperplasia areas in CA after CA injury and/or fimasartan administration. Data shown represent mean ± SD on 40 sections (two fields per section, two sections per CA, n = 10 for each group). *p < 0.05. e Representative immunofluorescence images and quantitative analysis results showing CD68+ macrophage (red) expression in CA at 28 days after CA injury and/or fimasartan administration. White arrows indicate CD68+ macrophages in CA. Nuclei were stained with DAPI. Scale bar: 100 μm. *p < 0.05. All experimental data are from n = 10 of ApoE KO mice in each group

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  Fig. 2. Effects of Fimasartan on CD4+ T cells, Treg cells, and anti-inflammatory cytokines in the peripheral blood of ApoE KO mice. a Immunohistochemical images showing the expression of CD4 (brown) at 28 days after CA injury and/or fimasartan administration. Scale bar: 100 μm. b Representative pictures showing that CD4+ T cell subsets were gated using flow cytometry. The numbers indicate positive percentages of CD4+ T cells. c Results of statistical analyses of Treg cells in peripheral blood at 28 days after CA injury and/or fimasartan administration. d Plasma levels of Treg-related cytokines, including IL-10 and TGFβ were measured by ELISA. All experimental data are from n = 12 of ApoE KO mice in each group. The values are presented as the mean ± SD. *p < 0.05. NS, not significant

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  Fig. 3. Effects of Fimasartan on CD8+ T cells, SMCs, and cytokines/chemokine expression after CA injury. a Immunohistochemical images showing the expression of CD8+ T cells (brown) in CA at 28 days after CA injury and/or fimasartan administration. Scale bar: 100 μm. b Representative pictures showing that CD8+ T cell subsets were gated using Flow cytometry. Results of statistical analysis of CD8+ T cells in peripheral blood at 28 days after CA injury and/or fimasartan administration. c Representative immunohistochemical images of α-SMA+ expression in CA, showing reduced SMCs (brown) after fimasartan administration. Boxed regions were magnified in the under panels. Yellow arrows indicate α-SMA+ cells in CA. Scale bar: 100 μm. d Quantification of SMCs in CA at 28 days after CA injury and/or fimasartan administration. Data shown represent the mean ± SD on 48 sections (two fields per section, two sections per CA, n = 12 for each group). e Fluorescent images of CD8+ T cell lytic activity assay against SMCs. CFDA SE-labeled mSMCs were co-cultured for 4 h at a CD8+ T cell to mSMC ratio of 0:1, 1:1 or 3:1. f CD8+ T cell lytic activity assay against SMCs examined by flow cytometry. Quantification…

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  Fig. 4. Changes in Fimasartan on splenomegaly and immune cells in spleens after CA injury. a Representative H&E-stained images showing the size and distribution of splenic pulp at 28 days after CA injury and/or fimasartan administration. Scale bar: 100 μm. b-d Quantitative analyses of relative spleen weight, body weight, and ratio of spleen weight/body weight were performed at 28 days after CA injury and/or fimasartan administration. e Representative immunofluorescence images showing CD68+ macrophages (green) and CD4+ T cells (green) expression in the spleen at 28 days after CA injury and/or fimasartan administration. Boxed regions were magnified in the under panels. Nuclei were stained with DAPI. Scale bar: 100 μm. f-h Flow cytometry analyses of the number of CD4+, Treg, and CD8+ T cells from spleen tissues at 28 days after CA injury and/or fimasartan administration. All experimental data are from n = 9, 10, 12, and 12 of ApoE KO mice in each group. The values are presented as the mean ± SD. *p < 0.05. NS, not significant