Abca1 As A Target To Build New Anti-atherosclerosis Drug Screening Model And Applied Research

Atherosclerosis is the principal pathogenesis for many critical cardiovascular diseases (e.g.,coronary heart disease,angina pectoris,myocardial infarction and stroke).The characteristic component of the atherosclerotic plaque is the macrophage derived foam cell.Intracellular free cholesterol can be toxic to the cell and therefore an efficient cholesterol efflux mechanism in macrophage is mandatory to prevent cholesterol accumulation.The ATP-binding cassette transporter A1(ABCA1),which belongs to a superfamily of ATP-binding cassette transporters(ABCs),mediates the rate-controlling step in the HDL particle formation and in the assembly of free cholesterol and phospholipids with lipid-poor apolipoprotein A-I(apoA-I).Mutations in ABCA1 can cause an autosomal recessive genetic disorder called Tangier disease,which is characterized by absent HDL and premature atherosclerosis.Furthermore,recent data demonstrated that hepatic ABCA1 contributed approximately 70%to the steady-state plasma HDL cholesterol pool,and overexpression of hepatic ABCAI raises HDL cholesterol levels considerately.Therefore,identification of novel up-regulators of ABCA1 would be beneficial for atherosclerosis prevention and/or therapy due to its pivotal role in cholesterol homeostasis and HDL metabolism.To search for active compounds that can increase ABCA1 transcription,a cell-based high-throughput screening(HTS) method for up-regulator of ABCA1 was developed in the present study.The assay uses the human hepatoma HepG2 cells stably transfected with pGL3-ABCA1,in which a firefly luciferase reporter gene was fused at the downstream of the 5'-flanking promoter region of the human ABCA1 gene.The 9-cis-retinoic acid(9CRA),a natural retinoid X receptor(RXR) ligand,which may up-regulate ABCA1 expression at the transcriptional level via the liver X receptor(LXR) /RXR pathway,was used as a positive control for optimization and evaluation of the HTS assay for detection in a multiwell format.The luciferase-based assay system described herein consistently displayed an excellent Z' value(＞0.5).This,and in conjunction with other parameters(S/N ratio and S/B ratio),exhibits the high reliability of detecting compounds with ABCA1 up-regulatory activities.Application of this assay, 2200 compounds and 4000 microbial fermentation extracts were screened,and 7 positive compounds and 3 positive strains were identified as hits.We studied the primary taxology of positive strain I05-1143 and I05-1743,strain I05-1143 was identified as Bacillus pumilus and I05-1743 was identified as Acremonium.sp.We also studied fermentation,isolation of bioactive components of both strains,I05-1743 A was isolated from fermentation broth of I05-1743 and I05-1143A was isolated from fermentation broth of I05-1143.By means of several spectral analyses,the structure of 1143A was identified as 4',7-dihydroxyisoflavone(daidzein).4 microbial products,pyrromycin(2006-F7),aclarubicin(2008-F4),daidzein (1143A) and pratensein(4776B),showed potently up-regulating ABCA1 reporter gene expression in HepG2 cells.The ABCA1 up-regulatory activity of the 4 positive compounds on the transcriptional and translational levels was confirmed by real-time quantitative RT-PCR and western blot.Moreover,all of these 4 positive compounds were also found to inhibit the formation of foam cells at low micromolar concentrations. This study identified for the first time that pyrromycin,aclarubicin,daidzein and pratensein have up-regulating activity on ABCA1,which bears important theoretical and practical significance for further investigation of the transcriptional regulation of the ABCA1 gene and the discovery of drug candidates or lead compounds of new antiatherosclerotic agents.