Antiatherosclerotic effects of a novel synthetic tissue-selective steroidal liver X receptor agonist in low-density lipoprotein receptor-deficient mice

Abstract

Liver X receptor (LXR) agonists have the potential to treat atherosclerosis based on their ability to enhance reverse cholesterol transport. However, their side effects, such as induction of liver lipogenesis and triglyceridemia, may limit their pharmaceutical development. In contrast to the nonsteroidal LXR agonist N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317), 3alpha, 6alpha, 24-trihydroxy-24, 24-di(trifluoromethyl)-5beta-cholane (ATI-829), a novel potent synthetic steroidal LXR agonist, was a poor inducer of sterol regulatory element-binding protein 1c expression in hepatoma HepG2 cells, whereas both compounds increased ABCA1 expression in macrophage THP-1 cells. In male low-density lipoprotein receptor-deficient mice, ATI-829 selectively activated LXR target gene expression in mouse intestines and macrophages but not in the liver. A significant increase in liver triglyceride and plasma triglyceriderich small very low-density lipoprotein (VLDL) was observed in T0901317 but not ATI-829-treated mice. Compared with vehicle-treated mice, atherosclerosis development was significantly inhibited in the innominate artery after treatment with either compound. However, in the aortic root, inhibition of atherosclerosis was only observed in the right (right coronary artery-associated sinus) but not the left coronary-related sinus (left coronary artery-associated sinus; LC) of mice treated with either compound. Lesions in the innominate artery were less complex after treatment with either compound and contained mostly macrophage foam cells. In contrast, LC lesions were more complex and had a large collagen-positive fibrous cap and less macrophage foam cell area after treatment with either compound. The T0901317-induced hypertriglyceridemia was accompanied by an increase in small triglyceride-rich VLDL that may influence LXR agonist-mediated antiatherosclerotic effects at certain vascular sites. ATI-829, by selectively activating LXR in certain tissues without inducing hypertriglyceridemia, is a good candidate for drug development.

Fig. 1

Chemical structures of T0901317 and ATI-829 (A), nuclear receptor-specific transactivation by ATI-829 (B) and effect of T0901317, ATI-829, 22(R)-HC, 24(S)-HC, and 24(S), 25-EC on luciferase reporter gene expression in HEK293 cells expressing LXRα (C) or LXRβ (D) and co-transfected with a DR-4 luciferase reporter. The ability of ATI-829 to act as an agonist for other nuclear receptors was examined in HEK293 cells co-transfected with various nuclear receptors and their cognate luciferase reporter genes. Firefly luciferase expression was normalized using a co-transfected sea pansy luciferase expression vector and normalized firefly luciferase expression is presented relative to luciferase expression in transfections without added agonist. n = 9 in T0901317 and ATI-829 experiments and n = 6 in 22(R)-HC, 24(S)-HC, and 24(S), 25-EC experiments. Effect of T0901317 and ATI-829 on the induction of SREBP-1c mRNA expression in HepG2 cells (E) (n = 8) and ABCA1 expression in THP-1 cells (F) (n = 6). Specific mRNA levels were measured by quantitative RT-PCR, normalized with 36B4, and presented relative to vehicle control.

Fig. 2

Pharmacokinetics of LXR agonists. C57 BL/6 mice were orally administered with either 10 mg/kg ATI-829 (n=5), or 2 mg/kg (n=6) and 10 mg/kg T0901317 (n=3), and bled at different time points (from 10 minutes to 24 hours). Liquid chromatography with electrospray positive tandem mass spectrometry detection (LC/MS/MS) was used to determine the concentration of compounds in serum after extraction.

Fig. 3

Effect of LXR agonists on gene expression in mouse intestine (A), peritoneal macrophages (B), and liver (C) and mouse hepatic triglycerides (D). Male LDLR−/− mice on a Western diet were gavaged daily for 2 weeks with vehicle (control, n=8), 10 mg/kg ATI-829 (n=8), or 2 mg/kg T0901317 (n=7). Specific mRNA levels were measured by quantitative RT-PCR, normalized with 36B4, and presented relative to controls. Significant differences between control and treatment groups are indicated as * P<0.05.

Fig. 4

Effect of LXR agonist on lipoprotein profiles (A and B) and plasma lipid levels (C and D) in male LDLR−/− mice after 2 weeks of treatment with vehicle (control, n=8), 10 mg/kg/day of ATI-829 (n=8), or 2 mg/kg/day (n=7) of T0901317. Lipoprotein profiles were determined by FPLC. Significant differences between vehicle and treatment groups are indicated as * P<0.05.

Fig. 5

Effect of LXR agonist on plasma VLDL size. Male LDLR−/− mice were fed a Western diet and gavaged with 2 mg/kg/day of T0901317 for 1 week (A), 2 weeks (B), 2 months (C), or with 10 mg/kg/day of ATI-829 for 2 weeks (D), 2 months (E), or with vehicle for 2 months (F). FPLC fractions 7 to 14 were pooled for EM analysis. Size bar is 100 nm. The average size (G) and size distributions (H) of VLDL particles from mice treated with vehicle (n=3), 10 mg/kg/day of ATI-829 (n=3), or 2mg/kg/day of T0901317 (n=3) were determined after 2 months of treatment. Significant differences between vehicle and treatment groups are indicated as * P<0.05.

Fig. 6

Effect of LXR agonist on plasma lipid levels (A and B) and hepatic triglyceride levels (C) in male LDLR−/− mice after 12 weeks of treatment with vehicle (control, n=12), 10 mg/kg/day of ATI-829 (n=14), or 2 mg/kg/day (n=11) of T0901317. Significant differences between vehicle and treatment groups are indicated as * P<0.05.

Fig 7

Effect of LXR agonists on atherosclerosis lesion area in the innominate artery (A), aortic root (B) and individual sinuses of the aortic root (C-F). Male LDLR−/− mice on a Western diet were gavaged daily for 12 weeks with vehicle (control, n=12), 5 (n=12) or 10 mg/kg/day (n=14) of ATI-829, or 2 mg/kg/day (n=11) of T0901317. Atherosclerotic lesions were measured as described in Methods. LC and RC are the left and right coronary associated sinuses, respectively and NC is the sinus not associated with a coronary artery. Significant differences between vehicle and compound treated groups are indicated as * P<0.05.

Fig. 8

Analysis of atherosclerotic lesion composition. Macrophage foam cells in lesions of the innominate artery (A-D) and aortic root (E-H) from mice treated with vehicle (A, E), 5 (B, F) or 10 (C, G) mg/kg/day of ATI-829, or 2 mg/kg/day of T0901317 (D, H) were immunostained with MOMA-2 antibody to detect macrophages. LC lesions from mice treated with vehicle (I), 5 (J) or 10 (K) mg/kg/day of ATI-829, or 2 mg/kg/day of T0901317 (L) were stained for collagen using trichrome stain (Masson). Percentage of macrophage foam cell area in the innominate and sinus plaques (M) and the average thickness of fibrous cap in LC lesions (N) were also quantitated. Significant differences between vehicle and compound treated groups are indicated as * P<0.05. Arrows indicate the fibrous cap. Original magnification: 100 × (A-D), 40 × (E-H) and 200 × (IL).

Fig. 9

Expression of ABCA1 protein within aortic sinus plaques from male LDLR−/− mice. Aortic sinus sections were analyzed for ABCA1 and MOMA-2 protein expression by immunostaining. Original magnification: 200 ×.