Synthetic LXR agonist inhibits the development of atherosclerosis in New Zealand White rabbits.

The nuclear receptors Liver X receptors, LXRα and LXRß, regulate cholesterol and triglyceride metabolism. We and others have previously reported that synthetic LXR agonists reduced atherosclerosis in models of mouse with no detectable plasma cholesteryl ester transfer protein (CETP) activity, which plays an important role in reverse cholesterol transport. In the present study, we investigated the effect of LXR activation in rabbits to elucidate the influence of CETP activity. First, we cloned rabbit LXRs cDNA. The data indicated that rabbit LXRα was mostly highly expressed in the liver, whereas LXRß expression was ubiquitous. Next, we investigated the effect of LXR agonist on lipid levels. Treatment with LXR agonist T0901317 increased plasma CETP activity and consequently elevated LDL, but no change in HDL. High cholesterol (HC) diet-feeding, which is thought to provide oxysterols as the natural agonists, could also increase expression of CETP and other LXR target genes. Finally, we tested T0901317 in the atherosclerosis intervention study. Chronic administration of T0901317 significantly reduced atherosclerosis in HC diet-fed rabbits despite less favorable lipid profiles, i.e. increases of plasma triglycerides and no change of HDL. T0901317 induced ATP-binding cassette transporters ABCA1 and ABCG1 and suppressed inflammatory genes expression in the aorta, suggesting that direct actions of LXR agonist on vascular gene expression are likely to contribute to the antiatherogenic effect. The present work strongly supports the idea that LXR agonists could be beneficial as therapeutic agents for treatment of atherosclerosis.

LXRalpha regulates human CETP expression in vitro and in transgenic mice.

Liver X receptors (LXRs), LXRalpha and LXRbeta, are members of the nuclear receptor superfamily and regulate the expression of genes involved in the regulation of cholesterol and fatty acid metabolism. Human plasma, unlike mouse plasma, contains cholesteryl ester transfer protein (CETP), which plays an important role in reverse cholesterol transport (RCT). LXRs induce CETP transcription via a direct repeat 4 element in the CETP promoter. However, the specific roles of the individual LXR subtypes in CETP expression and their consequences on plasma lipoprotein metabolism are still unclear. Here we showed that synthetic LXR agonist enhanced plasma CETP activity and resulted in non-high density lipoprotein (non-HDL) increase and HDL decrease in cynomolgus monkeys and human CETP transgenic mice. To address the relative importance of the two LXR subtypes, we investigated the effect of the suppression of both LXR subtypes on CETP expression in HepG2 cells. CETP expression induced by the LXR agonist was significantly reduced by LXRalpha knock-down, but not by LXRbeta. Consistent with these data, CETP promoter activity was enhanced by LXRalpha activation, whereas LXRbeta activation had only a minor effect. Furthermore, we investigated the effect of genetic deficiency of both LXR subtypes in human CETP transgenic mice. LXRalpha deficiency abolished the augmentation of plasma CETP activity and hepatic CETP expression induced by the synthetic LXR agonist, consequently increasing HDL and decreasing non-HDL, whereas LXRbeta deficiency did not affect CETP activation. These findings indicate that LXRalpha has an essential role in the regulation of CETP expression and maintaining RCT.

Liver X receptor agonists inhibit tissue factor expression in macrophages.

Exposure of blood to tissue factor (TF) rapidly initiates the coagulation serine protease cascades. TF is expressed by macrophages and other types of cell within atherosclerotic lesions and plays an important role in thrombus formation after plaque rupture. Macrophage TF expression is induced by pro-inflammatory stimuli including lipopolysaccharide (LPS), interleukin-1beta and tumor necrosis factor-alpha. Here we demonstrate that activation of liver X receptors (LXRs) LXRalpha and LXRbeta suppresses TF expression. Treatment of mouse peritoneal macrophages with synthetic LXR agonist T0901317 or GW3965 reduced TF expression induced by pro-inflammatory stimuli. LXR agonists also suppressed TF expression and its activity in human monocytes. Human and mouse TF promoters contain binding sites for the transcription factors AP-1, NFkappaB, Egr-1 and Sp1, but no LXR-binding sites could be found. Cotransfection assays with LXR and TF promoter constructs in RAW 264.7 cells revealed that LXR agonists suppressed LPS-induced TF promoter activity. Analysis of TF promoter also showed that inhibition of TF promoter activity by LXR was at least in part through inhibition of the NFkappaB signaling pathway. In addition, in vivo, LXR agonists reduced TF expression within aortic lesions in an atherosclerosis mouse model as well as in kidney and lung in mice stimulated with LPS. These findings indicate that activation of LXR results in reduction of TF expression, which may influence atherothrombosis in patients with vascular disease.

Overexpression of Bop3 confers resistance to methylmercury in Saccharomyces cerevisiae through interaction with other proteins such as Fkh1, Rts1, and Msn2.

We found that overexpression of Bop3, a protein of unknown function, confers resistance to methylmercury in Saccharomyces cerevisiae. Bmh2, Fkh1, and Rts1 are proteins that have been previously shown to bind Bop3 by the two-hybrid method. Overexpression of Bmh2 and the homologous protein Bmh1 confers resistance to methylmercury in yeast, but overexpression of either Fkh1 or Rts1 has a minimal effect. However, the increased level of resistance to methylmercury produced by overexpression of Bop3 was smaller in Fhk1-deleted yeast as compared with that of the wild-type strain. In contrast, the degree of resistance was significantly elevated in Rts1-deleted yeast. Msn2 and Msn4 were previously reported as proteins that bind to Bmh1 and Bmh2. Overexpression of Msn2 conferred a much greater sensitivity to methylmercury in yeast, while deletion of the corresponding gene lowered the degree of resistance to methylmercury induced by overexpression of Bop3. These results suggest that multiple proteins are involved in minimizing the toxicity of methylmercury induced by overexpression of Bop3.

T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice.

Liver X receptors (LXR alpha and LXR beta) are nuclear receptors, which are important regulators of cholesterol and lipid metabolism. LXRs control genes involved in cholesterol efflux in macrophages, bile acid synthesis in liver and intestinal cholesterol absorption. LXRs also regulate genes participating in lipogenesis. To determine whether the activation of LXR promotes or inhibits development of atherosclerosis, T-0901317, a synthetic LXR ligand, was administered to low density lipoprotein receptor (LDLR)(-/-) mice. T-0901317 significantly reduced the atherosclerotic lesions in LDLR(-/-) mice without affecting plasma total cholesterol levels. This anti-atherogenic effect correlated with the plasma concentration of T-0901317, but not with high density lipoprotein cholesterol, which was increased by T-0901317. In addition, we observed that T-0901317 increased expression of ATP binding cassette A1 in the lesions in LDLR(-/-) mice as well as in mouse peritoneal macrophages. T-0901317 also significantly induced cholesterol efflux activity in peritoneal macrophages. These results suggest that LXR ligands may be useful therapeutic agents for the treatment of atherosclerosis.