Abstract 191: Microparticles Released From Various Cell lines are ABCA1 Dependent

Abstract

The physiological functions of microparticles (MPs) released from cells remain poorly defined. Here, we study MPs released from various cell lines; baby hamster kidney (BHK) cells, human THP-1 and hepG2 cells also expressing ABCA1. To assess the role of MPs in cholesterol homeostasis we incubated cells with apoA-I or CS-6253 (CS) a novel apoE derived mimetic peptide and potential cholesterol efflux and HDL modulator. When incubated with BHK cells expressing ABCA1 under mifepristone and Mock cells in time dependent manner (0,5 to 24h) we found that MPs are ABCA1 dependent. Accordingly the dependence of MPs release on ABCA1 activity is confirmed in human THP-1 cells and hepG2 cells after blocking ABCA1 with probucol (10 μM, 24h). Given the rate-limiting step of ABCA1 in cholesterol homeostasis, we hypothesized that MPs may have a role in cholesterol transport from cells. To address this point we isolated MPs by FPLC from media collected from 3 H-cholesterol labelled cells that were exposed or not to (1 μM) apoA-I, or CS in time dependent manner. We demonstrate that MPs accounted for ~ 1/3 of the total cholesterol released to the medium. In addition, we found that cholesterol carried by MPs is redistributed among resident lipoproteins under conditions when 3 H-MPs were incubated in plasma (1h, 37°C), as shown by FPLC profiles. In addition, the cholesterol regulatory role in cellular release of MPs was tested after depletion of plasma membrane cholesterol by methyl-β-cyclodextrin (CDX, 10 mM, for 30 min). There was a significant decrease in cholesterol counts from MPs released in CDX treated macrophages THP-1 cells when compared to cells not treated relative to each condition: cells alone (16%, p<0.05), cells incubated with; apoA-I (48%, p<0.01), or CS (66%, p<0.0001), indicating that MPs require membrane cholesterol. MPs isolated from cells do not contain apoA-I or CS in contrast to HDL particles as confirmed by Western blotting analysis. MPs characterization by nanoparticle tracking analysis demonstrated vesicles that display (67.76±4 to 920±26nm) average size. We conclude that MPs generation was markedly decreased by blocking ABCA1 suggesting that ABCA1 is critical determinant for MPs formation, and potentially cell communication.