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

Abstract

Background: The beneficial effects of angiotensin II type 1 receptor blockers (ARBs) on atherosclerosis have been demonstrated in numerous studies. We investigated the effects of fimasartan on reducing neointimal formation and systemic inflammation after carotid artery (CA) injury in Apolipoprotein E knockout (ApoE KO) mice.

Methods: ApoE KO mice were randomly allocated to Group I (without CA injury), Group II (without CA injury + Fimasartan), Group III (CA injury), and Group IV (CA injury + Fimasartan). Fimasartan was orally administered everyday starting 3 days before iatrogenic left CA injury.

Results: At 28 days, neointimal hyperplasia and the inflammatory cytokines including TNFα, IL-6, ICAM, and MMP-9 in the peripheral blood were significantly reduced in Groups II and IV compared to Groups I and III, respectively. All fimasartan-administered groups revealed significant increases of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells with increased plasma levels of IL-10 and TGFβ. In addition, increased CD8⁺ T cells by fimasartan were correlated with reduced smooth muscle cell (SMC) proliferation in the neointima in Groups II and IV. Furthermore, the populations of Treg and CD8⁺ T cells in total splenocytes were increased in Groups II and IV compared to Groups I and III, respectively. The enlargement of spleens due to CA injury in the Group III was attenuated by fimasartan, as shown in the Group IV. These data indicate that fimasartan significantly reduced SMC proliferation in neointima and increased Treg cells in ApoE KO CA injury mice.

Conclusions: This study suggests fimasartan could be an efficient strategy for reduction of atherosclerotic progression, with a decrease in immune response and systemic inflammation.

Keywords: Angiotensin receptor blockers; Atherosclerosis; Fimasartan; Neointimal hyperplasia; Regulatory T cell.

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

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

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 of PI-positive and CFDA SE-labeled mSMCs. g The plasma levels of inflammatory cytokines/chemokines (TNFα, IL-6, ICAM, and MMP-9). All experimental data are from n = 12 of ApoE KO mice in each group. Values are presented as the mean ± SD. *p < 0.05. NS, not significant

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