Effect of Cyp27A1 gene dosage on atherosclerosis development in ApoE-knockout mice

Abstract

In humans, sterol 27-hydroxylase (CYP27A1) deficiency leads to cholesterol deposition in tendons and vasculature. Thus, in addition to its role in bile acid synthesis, where it converts cholesterol to 27-hydroxycholesterol (27-OHC), CYP27A1 may also be atheroprotective. Cyp27A1-deficient (Cyp27A1(-/-)) mice were crossed with apolipoprotein E (apoE)-deficient mice. Cyp27A1(+/+)/apoE(-/-) [ApoE-knockout (KO)], Cyp27A1(+/-)/apoE(-/-) heterozygous (het), and Cyp27A1(-/-)/apoE(-/-) [double-knockout (DKO)] mice were challenged with a Western diet (WD) for 3 and 6 mo. ApoE-KO mice fed a chow diet or a WD were used as the control. The severity of atherosclerosis in DKO mice was reduced 10-fold. Compared with the control, the DKO mice had no 27-OHC, total plasma cholesterol and low-density lipoprotein and very low density lipoprotein (LDL/VLDL) concentrations were reduced 2-fold, and HDL was elevated 2-fold. Expression of hepatic CYP7A1, CYP3A, and CYP8B1 were 5- to 10-fold higher. 3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) activity increased 4-fold. Fecal cholesterol was increased. In contrast, het mice fed a WD developed accelerated atherosclerosis and severe skin lesions, possibly because of reduced reverse cholesterol transport due to diminished 27-OHC production. CYP27A1 activity is involved in the control of cholesterol homeostasis and development of atherosclerosis with a distinct gene dose-dependent effect.

Keywords: 27-hydroxycholesterol; CYP3A11; LDL/VLDL fraction.

Figure 1.

Effect of CYP27A1 deficiency on the development of atherosclerosis: en face analysis. ApoE-KO, het, and DKO mice were fed for 3 or 6 mo with a CD or WD before they were euthanized, and aorta was analyzed en face. A, B) Quantification of atherosclerotic lesions after 3 or 6 mo of CD (A) or WD (B). Data are means ± sem (n=4–6). *P < 0.05, **P < 0.01, ***P < 0.001. C) Representative en face Sudan IV staining of sections of aortas of mice fed the WD for 3 mo. Scale bars = 1 mm. ND, not done.

Figure 2.

Effect of CYP27A1 deficiency on the development of atherosclerosis: analysis of aortic root sections. ApoE-KO, het, and DKO mice were fed for 3 or 6 mo with a CD or WD before they were euthanized. Atherosclerotic lesions in sections of aortic root were analyzed as described in Materials and Methods. A, B) Quantification of atherosclerotic lesions after 3 or 6 mo of CD (A) or WD (B).*P < 0.05, **P < 0.01, ***P < 0.001. Data are means ± sem (n=5–7). C) Representative photomicrographs of aortic root in paraffin-embedded sections stained with H&E in mice fed the WD for 3 mo. Scale bars = 200 μm. ND, not done.

Figure 3.

Effect of CYP27A1 deficiency on macrophage accumulation in atherosclerotic lesions. ApoE-KO, het, and DKO mice were fed for 3 to 6 mo with a CD or WD. Macrophages were stained with anti- MAC-1 antibody. A, B) Quantification of abundance of macrophages in atherosclerotic lesions after 3 or 6 mo of CD (A) or WD (B). *P < 0.05, **P < 0.01, ***P < 0.001. Data are means ± sem (n=4). C) Representative photomicrographs of aortic root in paraffin-embedded sections stained with anti-MAC-1 antibody in mice fed the WD for 3 mo. Arrows indicate the cholesterol cleft. Scale bars = 20 μm. ND, not done.

Figure 4.

Effect of CYP27A1 expression and diet on plasma lipids and CYP27A1 activity. ApoE-KO, het, and DKO mice were fed for 3 mo with a CD or WD. Cholesterol (A), LDL/VLDL (B), HDL (C), 27-OHC (D), CYP27A1 activity measured as 27-OHC/cholesterol ratio (E), and TGs (F) were quantified in plasma of mice after overnight food withdrawal. Data are means ± sem (n=5–10). ND, not detectable (<1 ng/ml). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Effect of CYP27A1 expression on fecal lipid content. Daily excretion of cholesterol, TGs, and bile acids was measured in feces collected from ApoE-KO, het, and DKO mice placed for 24 h in metabolic cages after 3 mo of CD (A) or WD (B). Results are presented as means ± sem (n=5). *P < 0.01, **P < 0.001, ***P < 0.0001.

Figure 6.

Genotype- and diet-induced effects on expression of genes involved in lipid metabolism in liver and intestine. A–D) Using β-actin as internal standard, expression of cytochromes (A), nuclear receptors (B), regulators of cholesterol and TG biosynthesis (C), and reverse cholesterol transport (D) was assessed in livers of ApoE-KO, het, and DKO mice fed for 3 mo with CD or WD. E, F) Similarly, gene expression of enzymes involved in bile acid and lipid metabolism in the jejunum (E) and ileum (F) was investigated. Results are presented as means ± sem (n=5–14). *P < 0.01, **P < 0.001, ***P < 0.0001 vs. ApoE-KO; ^§P < 0.01, ^§§P < 0.001, ^§§§P < 0.0001 vs. CD.

Figure 7.

Effect of diet and CYP27A1 genotype on CYP7A1 and HMGR activities in the liver. Conversion of cholesterol to 7α-cholesterol (A) and HMGCo-A to mevalonic acid (B) was measured in liver microsomes of ApoE-KO, het, and DKO mice fed for 3 mo with CD or WD as described in Materials and Methods. Results are presented as means ± sd of triplicate determinations (n=3 animals/condition). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8.

Possible atheroprotective mechanisms in DKO mice fed a WD. Atherosclerosis is mainly driven by high circulating cholesterol concentrations, low HDL, and high LDL. In plasma of DKO mice fed the WD, cholesterol values were low, HDL increased, and LDL decreased when compared to levels in ApoE-KO mice. In the absence of 27-OHC in DKO mice, the negative feedback regulation of the rate-limiting enzyme of cholesterol biosynthesis HMGCoAR is suppressed, and cholesterol production is increased, but because of CYP7A1 and CYP8B1 up-regulation, cholesterol is metabolized. The low level of LDL can be attributed to the increase of SREBP2 in the liver, which increases LDLR expression and LDL uptake. 27-OHC is also a ligand for LXRα, which, on activation, stimulates ABCA1, ABCG5, ABCG8, SR-B1, CYP7A1, and SREBP1c. The LXRα activation induces TG biosynthesis and CYP7A1 activity, which increases bile acid biosynthesis. Bile acids are natural ligands for FXR, a mediator of SHP, which interferes with the activity of LXRα to induce SREBP1c and CYP7A1. FXR reduces bile acid toxicity in the liver by increasing CYP3A11. In DKO mice, SHP is down-regulated so that the inhibitory effect on SREBP1c and CYP7A1 is suppressed, and TGs and bile acid synthesis are increased. In the intestine, FXR promotes the release of FGF15 from the intestine to the liver. On binding to the hepatic receptor FGF4R, FGF15 inhibits CYP7A1 expression. In DKO mice fed the WD, FXR is down-regulated so that the release of FGF15 to the circulation followed by activation of FGF4R is reduced. As a consequence, CYP7A1 transcription is enhanced in the liver. The decreased expression of FXR leads also to a reduction in SHP inhibition of bile acid absorption, bile acid reabsorption is increased, and excretion is reduced. The reduced intestinal absorption attributed to reduced expression of ABCA1, ABCG5, and ABCG8 leads to an increase in cholesterol excretion in feces. Genes and molecules overexpressed are in green; those down-regulated are in red.