Valsartan Attenuates Atherosclerosis via Upregulating the Th2 Immune Response in Prolonged Angiotensin II-Treated ApoE(-/-) Mice

Abstract

Valsartan has a protective effect against hypertension and atherosclerosis in humans and experimental animal models. This study aimed to determine the effect of prolonged treatment with angiotensin II (Ang II) on atherosclerosis and the effect of valsartan on the activity of CD4(+) T lymphocyte subsets. The results showed that prolonged treatment (8 wks) with exogenous Ang II resulted in an increased atherosclerotic plaque size and a switch of stable-to-unstable plaque via modulating on CD4(+) T lymphocyte activity, including an increase in the T helper cell type 1 (Th1) and Th17 cells and a decrease in Th2 and regulatory T (Treg) cells. In contrast, valsartan treatment efficiently reversed the imbalance in CD4(+) T lymphocyte activity, ameliorated atherosclerosis and elicited a stable plaque phenotype in addition to controlling blood pressure. In addition, treatment with anti-interleukin (IL)-5 monoclonal antibodies weakened the antiatherosclerotic effects of valsartan without affecting blood pressure.

Figure 1

Blood pressure of Ang II infusion in ApoE^−/− mice. (A) Time course of systolic blood pressure (SBP) in the three groups. (B) Heart rate in the three groups. Data are expressed as the mean ± standard error of the mean (SEM); n = 7–9. *p < 0.05 versus group II, **p < 0.01 versus group II.

Figure 2

Valsartan ameliorates the development of atherosclerosis in Ang II–treated ApoE ^−/− mice (I). (A) Representative sections of aortic sinus stained with oil red O in the three groups. Black bar = 200 μm. (B) Representative photographs of oil red O staining in the surface lesion area of the entire aorta. (C) Quantitative analysis of the lesion size of aortic sinus staining in the three groups (*p < 0.05 versus group II). (D) Quantitative analysis of the percent lesion area of the entire vessel area (*p < 0.05 versus group II, **p < 0.01 versus group II). The values are presented as the mean ± SD; n = 5–7.

Figure 3

Valsartan ameliorates the development of atherosclerosis in Ang II–treated ApoE ^−/− mice (II). (A) Representative sections of aortic sinus stained with antibodies to α-SMA, MOMA-2, CD4 and collagen staining in ApoE ^−/− mice in the three groups. Black bar = 200 μm. (B–E) Quantitative analysis of α-SMA, MOMA-2, CD4 and collagen staining in the three groups (*p < 0.05 versus group II, **p < 0.01 versus group II). The values are presented as the mean ± SD; n = 7.

Figure 4

Valsartan reduces the expression of MMP-2 and MMP-9. (A) mRNA expression of MMP-2 and MMP-9 in the three groups. (B, C) Protein expression of MMP-2 and MMP-9. Treatment with Ang II induced mRNA and protein expression of MMP-2 and MMP-9, which were significantly increased compared with the control group in the aorta of the ApoE^−/−mice. Group I was set as the control group (*p < 0.05 versus group II, **p < 0.01 versus group II). The values are presented as the mean ± SD; n = 4–5.

Figure 5

Effect of valsartan administration on Th1, Th2 and Th17 cells and Tregs in the spleen of Ang II–treated ApoE ^−/− mice. (A) CD4⁺ T-cell subsets were gated (a). Representative pictures of Th1 cells (b), Th2 cells (c), Th17 cells (d) and Tregs (e) are shown. The numbers in the upper right quadrants indicate positive percentages of these cells. (B) Results of statistical analysis of Th1 cells (**p < 0.01 versus group II). (C) Results of statistical analysis of Th2 cells (*p < 0.05 versus group II; **p < 0.01 versus group II). (D) Results of statistical analysis of Th17 cells (**p < 0.01 versus group II). (E) Results of statistical analysis of Tregs (*p < 0.05 versus group II). The values are presented as the mean ± SD; n = 5–7.

Figure 6

The levels of serum cytokines among the three groups and the mRNA expression in the aorta. (A) The levels of serum Th1- and Th17-related cytokines, including IFN-γ, IL-18, IL-17 and IL-23, in the three groups. (B) The levels of serum Th2- and Treg-related cytokines, including IL-4, IL-5, IL-33, IL-10 and TGF-β1, in the three groups. (C) Expression of IFN-γ, TBX21, IL-18, RORγT, IL-17 and IL-23 mRNA in aorta in the three groups. (D) Expression of IL-4, GATA3, IL-5, IL-33, IL-10, TGF-β1 and FOXP3 mRNA in aorta in the three groups. The values are presented as the mean ± SD; n = 5–7. *p < 0.05 versus group II, **p < 0.01 versus group II, ^#p > 0.05 versus group II.

Figure 7

(A) The levels of serum IL-5 in the two groups (**p < 0.01 versus Ang II⁺Val⁺Rat IgG2a). (B) Blood pressure of two groups of ApoE^−/− mice. Time course of systolic blood pressure in the three groups is shown. Data are expressed as the mean ± SEM; n = 5–6. SBP, systolic blood pressure.

Figure 8

Anti-IL-5 mAb abrogates the effect of valsartan on atherosclerosis in Ang II–treated ApoE ^−/− mice. (A) Representative sections of the aortic sinus stained with oil red O, and antibodies specific to α-SMA, MOMA-2 and collagen in ApoE ^−/− mice. Black bar = 200 μm. (B–E) Quantitative analysis of the lesion size, α-SMA and MOMA-2 staining. The values are presented as the mean ± SD; n = 6. **p < 0.01, ^#p > 0.05.