Abstract 473: Development of High-throughput ABCA1, ABCG1 and SR-BI Mediated HDL Efflux Assays
Studies have shown an inverse association between HDL-efflux capacity and cardiovascular disease (CVD). The efflux of cholesterol from cells to HDL can occur by several mechanisms, including unmediated aqueous diffusion and ABCA1, ABCG1 and SR-BI specifically mediated process. We developed a high throughput assay to measure pathway specific HDL efflux capacity using BHK cells expressing inducible ABCA1, ABCG1, or SR-BI in 96-well plate format. We found that ABCA1-mediated cholesterol efflux in BHK cells correlated significantly with that of J774 macrophages when apoA-I, HDL or 2% apoB-depleted serum were used as acceptors (R2=0.98 to 0.99, p<0.0001). Compared with J774 macrophages, ABCA1-mediated efflux using BHK cells is more sensitive, consistent and has better reproducibility (Intro-assay CV=3.0%, R2=0.99; Inter assay CV=3.2%, R2=0.98). The consistence and reproducibility for ABCG1 and SR-BI mediated cholesterol efflux were also excellent as judged by intro-assay and inter-assay CV and R2. When pathway specific assay was employed in a high-throughput format to measure HDL efflux capacity among subjects with or without CVD, we found that ABCA1-mediated cholesterol efflux was significantly lower in CVD subjects when compared with that of control (p<0.01), while ABCG1 or SR-BI mediated cholesterol efflux was not significantly different among the two groups. ABCG1-mediated cholesterol efflux was significantly associated with HDL-C (R2=0.65, p<0.01), but ABCA1 or SR-BI mediated cholesterol efflux was not correlated with HDL-C. Those studies demonstrate that BHK cells with inducible ABCA1, ABCG1, or SR-BI expression provide a sensitive, reproducible high throughput efflux assay that can be used to screen large number of serums in mechanistic studies or drug discovery program.
Author Disclosures: C. Storey: None. C. Tang: None.
This research has received full or partial funding support from the American Heart Association.
- © 2015 by American Heart Association, Inc.