Combined treatment with 9-cis β-carotene and 22R-hydroxycholesterol augments cholesterol efflux in macrophages

High plasma levels of LDL-cholesterol play a causative role in atherogenesis. The first step in atherogenesis is characterized by oxidation of retained LDL particles in the arterial wall, and scavenger receptors-mediated entry of oxidized-LDL into macrophages, resulting in the accumulation of cholesterol within macrophages. Therefore, cholesterol efflux from macrophages to HDL particles is an essential process in inhibiting the formation of atherosclerotic plaques. Previous in-vitro experiments conducted in our laboratory, revealed that 9-cis β-carotene (9CBC) enhanced the efflux of radioactively labeled cholesterol from macrophages to HDL. However, the mechanism by which 9CBC enhances cholesterol efflux remained obscure. We found, in a controlled in-vitro assay, that treating macrophages both with the Dunaliella bardawil alga derived carotenoid 9CBC, and 22R-hydroxycholesterol (22-HC), two ligands of the nuclear receptors Retinoid X Receptor (RXR) and Liver X Receptor (LXR) respectively, resulted in a synergistically increase in fluorescently-labeled cholesterol efflux. Moreover, concurrent treatment of 9CBC with liarozole, which blocks the metabolism of Retinoic Acid (RA), increased cholesterol efflux from macrophages. This result strengthens our hypothesis that 9CBC activates the RXR nuclear receptor by its conversion to RA. Furthermore, cholesterol efflux was greater in macrophages isolated from alga Dunaliella-fed WT or apoE-/- mice compared to chow diet-fed mice. Our results suggest that simultaneous activation of nuclear receptors affects regulation of cellular cholesterol and may accelerate reverse cholesterol transport, by increasing cholesterol efflux from macrophages and thus, reducing the risk for atherogenesis.