Activation of aryl hydrocarbon receptor induces vascular inflammation and promotes atherosclerosis in apolipoprotein E-/- mice

Abstract

Objective: Exposure to dioxins has been shown to contribute to the development of inflammatory diseases, such as atherosclerosis. Macrophage-mediated inflammation is a critical event in the initiation of atherosclerosis. Previously, we showed that treatment of macrophages with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to aryl hydrocarbon receptor (AhR)-dependent activation of inflammatory mediators and the formation of cholesterol-laden foam cells. However, the mechanisms responsible for the formation of atherosclerotic lesions mediated through AhR have not been identified.

Methods and results: An in vitro macrophage and an apolipoprotein E (ApoE)-/- mouse model were used to determine whether chemokines and their receptors are responsible for the AhR-mediated atherogenesis. Exposure of ApoE-/- mice to TCDD caused a time-dependent progression of atherosclerosis, which was associated with induction of inflammatory genes, including interleukin-8, as well as F4/80 and matrix metalloproteinase-12. A high-fat diet enhanced the TCDD-mediated inflammatory response and aggravated the formation of complex atheromas. Treatment with a CXCR2 inhibitor and an AhR antagonist reduced the TCDD-induced progression of early atherosclerotic lesions in ApoE-/- mice.

Conclusion: The results suggest that CXCR2 mediates the atherogenic activity of environmental pollutants, such as dioxins, and contributes to the development of atherosclerosis through the induction of a vascular inflammatory response by activating the AhR-signaling pathway.

Figure 1

(A) Expression of foam cell marker genes in macrophages treated with TCDD or CSE in absence or presence of MNF. Significantly different from control (*) or from CSE and TCDD alone (**). (B–C) Expression of (B) VEGF and (C) CYP1A1 in macrophages treated with TCDD for 24h after transient transfection with siRNA against AhR or IL-8 for 48h. Significantly different from control (*) or from TCDD treated siControl (**). (D) Cholesterol accumulation in macrophages treated with TCDD or CSE in the absence or presence of MNF, INCB3344 or SB225002. Significantly different from control (*) or from corresponding CSE or TCDD-treated cells (**). *P< 0.05.

Figure 2

Development of atherosclerotic lesions in ApoE−/− mice fed on regular chow after treatment with TCDD. (A–B) C57BL/6 and (C–D) ApoE−/− mice received (A&C) corn oil (Ctrl) or (B&D) TCDD for 60 days. (E–F) ApoE−/− mice received (E) corn oil or (F) TCDD for 7 months. Shown are representative cross sections from serial sections of aorta arches visualized by H&E staining. In each group, eight mice were evaluated showing similar phenotypes (See also Fig. SII). Arrows indicate formation of atherosclerotic lesions. (G–H) Expression of foam cell marker MMP-12 in aorta of (G) control and (H) 40-day TCDD treated ApoE−/− mice. Arrows indicate staining of MMP-12.

Figure 3

Development of atherosclerotic lesions in ApoE−/− mice fed a high fat diet. (A–B) ApoE−/− mice received coin oil (Ctrl) or (C–D) a single injection of 15 µg/kg TCDD. Representative images of eight mice are shown of serial tissue sections of aorta arches prepared 60 days after treatment with TCDD and stained with H&E. All animals in each group showed a similar phenotype (see also Fig. SIII).

Figure 4

Inhibition of CXCR2 and AhR decreases plaque formation in ApoE−/− mice. Mice fed regular chow and received (A&E) corn oil (Ctrl) or (B&F) TCDD for 60 days. ApoE−/− mice were treated with (C) SB225002 in the absence or (D) presence of TCDD or treated with (G) CH223191 in the absence or (H) presence of TCDD. Serial cross sections of representative aorta arches from six mice were visualized by H&E staining. The animals in each group showed a similar phenotype (see also Fig. SIV). Arrows indicate formation of early atherosclerotic lesions.

Figure 5

TCDD increased the amount of cholesterol accumulated in the aorta. ApoE−/− mice were treated with TCDD for 60 days in the absence or presence of SB225002 or CH223191 as described in Fig. 4. The amount of cholesterol accumulated in the aorta was measured via GC/MS from five animals in each group. Significantly different from vehicle control (*) or from TCDD-treated mice (**) (P < 0.05).