Sterol 27-hydroxylase gene dosage and the antiatherosclerotic effect of Rifampicin in mice

Abstract

Sterol 27-hydroxylase (CYP27A1) catalyzes the hydroxylation of cholesterol to 27-hydroxycholesterol (27-OHC) and regulates cholesterol homeostasis. In Cyp27a1/ Apolipoprotein E (ApoE) double knockout (KO) mice fed with Western diet (WD), the atherosclerotic phenotype found in ApoE KO mice was reversed. As protective mechanism, up-regulation of Cyp3a11 and Cyp7a1 was proposed. Cyp27a1 heterozygote/ApoE KO (het) mice, with reduced Cyp27a1 expression and normal levels of Cyp7a1 and Cyp3a11, developed more severe lesions than ApoE KO mice. To analyze the contribution of Cyp3a11 to the protection of atherosclerosis development, Cyp3a11 was induced by Rifampicin (RIF) in ApoE KO and het mice. Males were fed with WD and treated daily with RIF (10 mg/kg ip) or vehicle for 4 weeks. Atherosclerosis was quantified in the aortic valve. Plasma lipids and 27-hydroxycholesterol (27-OHC), expression of cytochromes P450 and genes involved in cholesterol transport and bile acids (BAs) signaling in liver and intestine, and intestinal cholesterol absorption were analyzed. RIF increased expression of hepatic but not intestinal Cyp3a11 4-fold in both genotypes. In ApoE KO mice treated with RIF, we found a 2-fold decrease in plasma cholesterol, and a 2-fold increase in high-density lipoprotein/low-density lipoprotein ratio and CY27A1 activity. Intestinal cholesterol absorption remained unchanged and atherosclerotic lesions decreased approximately 3-fold. In het mice, RIF had no effect on plasma lipids composition, CYP27A1 activity, and atherosclerotic plaque development, despite a reduction in cholesterol absorption. In conclusion, the antiatherogenic effect of Cyp3a11 induction by RIF was also dependent on Cyp27a1 expression.

Keywords: 27-hydroxycholesterol; ApoE knock out mice; Western diet.

Figure 1. Effect of RIF on liver morphology

Representative photomicrographs of H&E liver sections of ApoE KO and het mice fed with WD and treated with 10 mg/kg RIF or vehicle i.p. daily for 4 weeks; scale bars: 20 μm.

Figure 2. Effect of RIF on development of atherosclerosis

Representative photomicrographs of aortic valve in paraffin-embedded sections stained with H&E in ApoE KO and het mice fed with WD and treated with 10 mg/kg RIF or vehicle i.p. daily for 4 weeks; scale bars: 200 μm. (A) Quantification of atherosclerotic lesions in [mm²] (B) and as relative amount of atherosclerotic plaque when compared with vehicle (C). Arrows indicate atherosclerotic plaques. Means with whiskers (minimum and maximum) are presented; *P<0.05, **P<0.01.

Figure 3. Effect of RIF on gene expression in the liver

The expression of genes encoding for cytochromes P450 (A), BAs signaling (B), and cholesterol efflux (C) was quantified in ApoE KO and het mice fed with WD and treated with 10 mg/kg RIF or vehicle i.p. daily for 4 weeks, using β-actin as housekeeping gene. Results are presented as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001, vs ApoE KO, ^#P<0.05, ^###P<0.001 vs vehicle.

Figure 4. Effect of RIF on cholesterol absorption

Cholesterol absorption was measured in ApoE KO and het mice fed with WD and following treatment with 10 mg/kg RIF or vehicle i.p. daily, using dual absorption method. Results are presented as mean ± SEM; *P<0.05, **P<0.01.

Figure 5. Effect of RIF on cholesterol homeostasis in the jejunum

The expression of genes encoding for cytochromes P450 (A), BAs signaling (B), and cholesterol efflux (C) was quantified in ApoE KO and het mice fed with WD and treated with 10 mg/kg RIF or vehicle i.p. daily for 4 weeks using β-actin as housekeeping gene. Results are presented as mean ± SEM; *P<0.05, vs ApoE KO, ^#P<0.05, ^##P<0.01, ^###P<0.001 vs vehicle.

Figure 6. Cyp27a1 expression and the antiatherosclerotic effect of RIF in mice

The effect of Cyp3a11 induction by RIF on atherosclerosis development was analyzed in ApoE KO mice with normal Cyp27a1 expression and 27-OHC plasma concentration, and in het mice with reduced Cyp27a1 expression and 27-OHC plasma concentration. RIF induced hepatic Cyp3a11 mRNA 3-fold and had no effect on intestinal Cyp3a11 expression in both genotypes. In ApoE KO mice, RIF increased hepatic Cyp27a1 mRNA, leading to an increase in CYP27A1 activity measured by the 27-OHC/Cholesterol ratio in plasma. RIF also decreased TC and LDL-C and increased HDL-C. In het mice, RIF reduced Cyp27a1 mRNA levels in the intestine and had no effect on plasma lipids, despite reduced cholesterol absorption. The different effect of RIF on atherosclerosis development in ApoE KO and het mice underline the importance of Cyp27a1 expression in the protection of atherosclerosis.