Lipid Metabolism Related Receptor As A Target For Anti-atherosclerosis Drug Screening And Drug Action Mechanism

Atherosclerosis is a progressive disease that is characterized by the accumulation of lipid-rich plaques within the walls of arteries.Despite substantial therapeutic progress resulting from the widespread use of statins,which primarily lower plasma levels of low density lipoprotein(LDL) cholesterol,atherosclerosis is still one of the leading causes of mortality in industrialized and developing nations.In recent years,the reverse cholesterol transport(RCT) pathway and modified low density lipoprotein (mLDL) induced foam cell formation,two processes other than LDLR pathway were extensively studied and concerned as major targets for the development of novel therapies.Plasma concentrations of high density lipoprotein(HDL) cholesterol(HDL-C) are inversely proportional to the risk for atherosclerotic cardiovascular disease.One of the major atheroprotective actions of HDL particles involves the transport of excess cholesterol from peripheral tissues to the liver for excretion,a process known as reverse cholesterol transport.Scavenger receptor class B typeⅠ(SR-BI) and its human homologue CLA-1 are the high-affinity HDL receptor.CLA-I/SR-BI,which plays an important role in reverse cholesterol transport,has been suggested as a new preventative and/or therapeutic target for atherosclerosis.Using a previously established cell-based CLA-1 up-regulator screening assay,one of the positive strains,04-9179,presented potent activity in elevating CLA-1 transcriptional level.Three pure compounds,designated as 9179A,9179B and 9179C, were purified from the strain.The structures and bioactivities of these compounds were analyzed,and 9179A showed highest activity.9179A was demonstrated chemically and biologically identical to a known compound trichostatin A(TSA).The effects of 9179A on CLA-1/SR-BI expression both in HepG2 human hepatoma cells and RAW 264.7 murine macrophage cells were detected in vitro.The results showed that the mRNA and protein level of CLA-1/SR-BI were significantly up-regulated by 9179A both in HepG2 and RAW 264.7 cells.Corresponding to this,the uptake of fluorescent labeled-HDL(DiI-HDL) was increased by 9179A in dose-dependent manner in HepG2 and RAW 264.7 cells.The cholesterol efflux was also increased by 9179A in RAW 264.7 cells.Using a combination of reporter assays with various deletion in CLA-1 promoter and electrophoretic mobility shift assay,we demonstrated that -419～-232 bp fragment of the CLA-1 promoter mediated the effects of 9179A (i.e.,TSA).Combined treatment of CLAp-LUC HepG2 cells with 9179A and rosiglitazone(ROS) confirmed that they up-regulated the promoter activity through different cis-elements.Together,these studies identified a novel up-regulator of CLA-1/SR-BI both in HepG2 and RAW 264.7 cells,suggesting that TSA should be useful in further understanding of the transcriptional regulation of CLA-1/SR-BI,and might serve as a starting point for the development of novel antiatherosclerotic agents. Macrophage foam cell formation is a characteristic event that occurs in the early stage of atherosclerosis.A critical step in foam cell formation is recognition and internalization of oxidized low density lipoprotein(oxLDL) by scavenger receptors CD36 and SR-A.In ex vivo models it has been shown that 60～90%of macrophage foam cell formation may be CD36 dependent.In vivo studies of CD36-null mice showed a major negative impact of CD36 on lesion development.Extensive evidences point to a significant role of CD36 in atherosclerotic lesions and suggest that it could be a lead target for therapeutic treatment.In this study,we constructed recombinant plasmid pET-sCD36 using pET-30a(+),and expressed soluble CD36(sCD36) in E.coli BL21(DE3).The protein of sCD36 was expressed in form of inclusion body and refolded by dilution.The ligand binding activity of sCD36 was validated by ligand binding assay and Oil Red O assay.With oxLDL itself as a positive control,sCD36 was used to establish a specific ELISA-like assay in a 96-well microplate format to screen CD36 antagonists.The conditions for the high-throughput screening(HTS) assay were optimized.The evaluating parameter Z' value of 0.84 showed that this cell-free HTS assay was robust and reliable. Screening of 640 chemical compounds identified 27 positive compounds.Active compounds were further validated by Oil Red O assay and DiI-acLDL uptake assay. The active compounds originated from this HTS assay may be developed to drug candidates or lead compounds for new antiatherosclerotic agents.