Effects of chronic mild stress on the development of atherosclerosis and expression of toll-like receptor 4 signaling pathway in adolescent apolipoprotein E knockout mice

Abstract

Here, we investigated the effect of chronic mild stress (CMS) on the development of atherosclerosis as well as the expression of Toll-like receptors (TLRs) signaling pathway in adolescent apolipoprotein E knockout (apoE-/-) mice. Mice were subjected to daily CMS for 0, 4, and 12 weeks, respectively. To identify the expression of Toll-like receptor 4 signaling pathway in adolescent apolipoprotein E knockout mice subjected to CMS, we compared gene expression in aortas of stressed and unstressed mice using TLRs signaling pathway real-time PCR microarrays consisting of 87 genes. We found that atherosclerosis lesions both in aortic tress and sinuses of CMS mice were significantly increased linearly in response to duration of CMS exposure. Among 87 genes analyzed, 15 genes were upregulated in stressed mice, especially TLR4, myeloid differentiation factor 88 (MyD88), and IL-1beta, and 28 genes were downregulated compared with nonstressed mice. CMS mice demonstrated markedly increased aortic atherosclerosis that were associated with significant increases in levels of expression of TLR4, MyD88, nuclear factor kappaB (NF-kappaB), MCP-1, IL-1beta, TNF-alpha, and sICAM-1. Taken together, our results suggest an important role for TLR4 signaling pathway in atherosclerosis in a CMS mouse model.

Figure 1

1% sucrose solution preference in CMS and control groups (n = 20 per group) following different weeks of CMS. Sucrose intake was significantly reduced in the CMS group compared with the corresponding control group. There was a significant stress × week interaction on sucrose intake (n = 20, P < .001 by ANOVA). An ANOVA performed on sucrose intake yielded a main effect of group (P < .001). Values are means ± SEM. *P < .05, **P < .01, ***P < .001 versus respective CMS value.

Figure 2

Body weight (in grams) in CMS and control groups during the CMS period (n = 20 per group). All mice were weighted weekly from Week 0 (initial week) to Week 12 of the procedure every Monday following 12-hour period of food and water deprivation. Values are means ± SEM. *P < .05 versus the stressed group.

Figure 3

Serum corticosterone levels in CMS and control groups following at different weeks of CMS (n = 20 per group). Serum corticosterone concentrations were assayed using a radioimmunoassay (RIA). Corticosterone levels were higher in the CMS group versus the control group. Data are mean ± SEM. *P < .05, **P < .05, ***P < .001 versus respective control value.

Figure 4

CMS markedly increases the extent of aortic atherosclerosis development in apoE-/- mice. (A)–(C) Representative photographs of aortic sinuses from control apoE-/- (top) and CMS (bottom) apoE-/- mice. (a) Aortas of apoE-/- mice subjected to CMS or normal condition (control) for 0 week (A), 4 weeks (B), and 12 weeks (C) were isolated and stained for lipid deposition Soudan IV. Representative specimens from the two groups are shown. (b) Quantification of plaque areas in whole aortas in CMS and control apoE-/- mice stained for lipid deposition with Soudan IV. Each bar represents mean ± SEM (n = 10 per group). Total plaque area in CMS mice was significantly increased compared with corresponding control mice. **P < .01, ***P < .001 versus the control group.

Figure 5

CMS markedly increases the extent of atherosclerosis development in aortic sinus of apoE-/- mice. (A)–(F) Representative photographs of aortic sinuses from control apoE-/- (top) and CMS (bottom) apoE-/- mice. 6 μm frozen sections of apoE-/- mice subjected to CMS or normal condition (control) for 0 week (A), (D), 4 weeks (B), (E), 12 weeks (C), (F) were stained with H&E (A)–(C) and oil red O (D)–(F), respectively. (b), (c) Quantification of plaque areas in aortic sinuses in CMS and control apoE-/- mice stained for lipid deposition with oil red O. Each bar represents mean ± SEM (n = 10 per group). Total plaque area in CMS mice was significantly increased compared with corresponding control mice. **P < .01, ***P < .001 versus the control group. (Magnification × 100).

Figure 6

Validation of changes in TLR-4 (73 KD) and NF-κB p65 (80 kDa) protein expression by western blotting. Proteins extracted were prepared from arteries (n = 6) of mice subjected to CMS or normal conditions for 0, 4, and 12 weeks, respectively. Each lane shows representative western blots using anti-TLR4 or NF-κB p65 and anti-GAPDH bodies in (a1) and (a2), respectively. Each panel summarizes densitometric readings of band intensities normalized to GAPDH, which was measured by densitometry with Image J image analysis software. (b1), (b2) densitometric measurements TLR4 and NF-κB p65 from Western blots, respectively. Data are mean ± SEM. C: control group; S: chronic mild stress group (n = 6 per group). *P < .05, **P < .01 compared with the control. The data are representative of three experiments.

Figure 7

(a)–(c) Immunohistochemical evidence for TLR-4 expression within atherosclerotic plaque of aortic sinuses from control apoE-/- (top) and CMS (bottom) apoE-/- mice. 5 μm frozen sections of the apoE-deficient mouse aortic root were fixed with acetone for 5 minutes at room temperature and then immunostained with Rabbit antimouseTLR4 antibody (1 : 100). Rabbit IgG was used as a negative control. (a)–(c) represent TLR-4 expression within aortic sinus of apoE-/- mice exposed to normal condition (control) or CMS for 0 week, 4, and 12 weeks, respectively (brown staining); (d) represents rabbit IgG staining for negative control. In (a) there is minimal immunoreactivity to TLR-4 in nonatherosclerotic aortic sinus of apoE-/- mouse (magnification × 400).

Figure 8

MCP-1, sICAM-1, and TNF-a levels in the serum of apoE-/- mice were analyzed by ELISA. IL-1β (a), TNF-a (b), sICAM-1 (c), and MCP-1 (d) levels markedly increased in the serum of apoE mice following different weeks of CMS compared with the corresponding control group (n = 20 per group). These cytokines were measured by ELISA according to the manufacturer's instructions. Data are mean ± SEM. *P < .05, **P < .01, ***P < .01 versus respective control value.