Inhibition of RORα/γ suppresses atherosclerosis via inhibition of both cholesterol absorption and inflammation

Abstract

Objective: Cardiovascular diseases (CVDs) are the leading cause of mortality in Western countries. Atherosclerosis is a multi-step inflammatory disease characterized at early stages by accumulation of cholesterol in the arterial wall followed by recruitment of immune cells. We sought to determine if pharmacological suppression of RORα/γ activity is beneficial in treatment of atherosclerosis.

Methods: To identify the role of RORα and RORγ in atherosclerosis, we used the LDL-R(-/-) mouse model of atherosclerosis placed on a high cholesterol diet treated with SR1001, a RORα/γ inverse agonist, for four weeks.

Results: Our results demonstrate that treatment with the ROR inverse agonist substantially decreases plaque formation in vivo. The mechanism of the anti-atherogenic activity of the inhibition of RORα/γ activity appeared to be due to targeting two distinct pathways. SR1001 treatment reduced plasma low density lipoprotein (LDL) level without affecting high density lipoprotein (HDL) via increasing intestinal cholesterol excretion. Treatment with SR1001 also induced an anti-atherogenic immune profile that was characterized by a reduction in Th17 cells and an increase in Treg and Th2 cells. Our data suggest that RORα and RORγ play a critical role in atherosclerosis development by regulating at least two major pathways important in the pathology of this disease: cholesterol flux and inflammation.

Conclusion: Our data suggest that pharmacological targeting of RORα/γ may be an effective method for treatment of atherosclerosis offering a distinct mechanism of action relative to statins.

Keywords: Atherosclerosis; Cholesterol; Drug discovery; Heart disease; Nuclear receptor; ROR.

Figure 1

RORα deficient mice show an anti-inflammatory profile. (A) Weight and body composition of single housed 12 week-old males RORαWT (white bar, n = 6) or RORα Hypo (black bar, n = 6) littermate. (B) Blood cytokines profile form RORαWT (n = 6) or RORα Hypo (n = 6) male mice. (C) CD4 (upper) or CD8 (lower) expression in splenocytes freshly isolated from the spleen of 12 week-old male RORαWT (white, n = 5) or RORα Hypo (black, n = 5) mice. *p < 0.05, **p < 0.01.

Figure 2

SR1001 prevents atherosclerosis formation in LDL-R deficient mice. En face aortas of LDL-R^−/− mice fed 10 days (A) or a month (B) with a high cholesterol (HC) diet and treated for one moth with SR1001 (n = 9) or vehicle (n = 9). Lipid deposits were stained with Oil Red O and total plaque surface was calculated relative to the total surface of the aorta. (C) Arterial roots from LDL-R^−/− mice fed one month with HC diet and then treated with SR1001 (n = 5) or vehicle (n = 5) for a month. Sections were stained for lipid (Oil Red O) and cholesterol (Masson Trichrome) deposit or immune cell infiltration (CD4 or Moma). Quantification of positive staining was done using ImageJ. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

SR1001 decreases inflammatory profile in LDL-R^−/−mice. Splenocytes from LDL-R^−/− mice fed 10 days with a HC diet and treated for one moth with SR1001 (black bar, n = 6) or vehicle (white bar, n = 6) were analyzed by flow cytometry for CD4⁺ (left panel A), CD8⁺ (middle panel A) or Th17 (SSC-A/IL-17⁺) (right panel A). Splenocytes form LDL-R^−/− mice fed one month with a HC diet and treated for one moth with SR1001 (black bar n = 6) or vehicle white bar, (n = 6) were analyzed by flow cytometry for Th2 (CD4⁺ Gata3⁺ (B, upper right)), Treg (CD4⁺ FoxP3⁺ (B, upper left)) or Th17 (CD4⁺/IL-17⁺(B, lower left)) or Th1 (CD4⁺/IFNγ⁺ (B, lower right)). Plasma form LDL-R^−/− mice fed one month with a HC diet and treated for one month with SR1001 (black bar, n = 9) or vehicle (white bar, n = 9) was analyzed by Luminex for pro (IFNγ and IL-17) or anti-inflammatory cytokines (C). *p < 0.05, **p < 0.01.

Figure 4

SR1001 decreases plasma cholesterol levels and increases intestinal cholesterol excretion in mice. Plasma form LDL-R^−/− mice fed one month with a HC diet and treated for one month with SR1001 (black bar, n = 9) or vehicle (white bar, n = 9) was analyzed by chemistry analyzer for total (CHOL), LDL, HDL and triglycerides levels (A). Body weight (B) and fat mass (C) of single housed LDL-R^−/− mice fed one month with a HC diet over the month of SR1001 (black bar, n = 9) or vehicle (white bar, n = 9) treatment. (D) Liver and (E) intestinal gene expression of cholesterol absorption/excretion form LDL-R^−/− mice fed one month with a HC diet and treated for one month with SR1001 (black bar, n = 9) or vehicle (white bar, n = 9). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

SR1001 decreases plasma cholesterol levels and increases intestinal cholesterol excretion in WT mice. (A) Body weight, food, cholesterol intake and fecal cholesterol excretion of single housed WT mice fed with HC diet for 10 days and then treated with SR1001 (black bar, n = 10) or vehicle (white bar, n = 10) for 3 weeks. (B) Total plasma cholesterol level as quantify in SR1001 (black bar, n = 10) or vehicle (white bar, n = 10) treated animals. Intestinal gene (C) and protein (D) expression of cholesterol excretion. Relative level of ABCG5 protein compare to actin for SR1001 (black bar, n = 4) or vehicle (white bar, n = 4) animals. *p < 0.05.