Abstract 650: Characterization of Microparticles Formed by ABCA1
Microparticles (MPS) are lipoprotein-sized structures created by the ABCA1 transporter. Their biological roles in health and in disease remain unknown. Here, we study MPS released from baby hamster kidney (BHK) cells stably expressing ABCA1 and human THP-1 cells also expressing ABCA1.
Media cell culture was first collected from BHK-ABCA1 expressing cells after (45min, 2, 4, 8, and 24h) incubation. After centrifugation (4000xg for 15min and 10000xg for 30min) to remove cell debris, the supernatant was passed through a 10kDa cutoff filter and subsequently subjected to analytical FPLC. FPLC analysis shows creation of a single peak in the presence of ABCA1 but not in mock-transfected BHK cells. In a time-course study, the estimated hydrodynamic diameter remained stable (≥20nm).
After 8h incubation of BHK cell with apoA-I or an apo-E mimetic peptide, CS-6253 (1μM), ABCA1 mediated formation of MPS of similar size with a significant increase in 3[H]-FC content than those generated by ABCA1 alone, (373±23 % cpm, P<0.05) and (277±60 % cpm, P<0.05) respectively. This was associated with highly lipidated nHDL-CS-6253 compararely to nHDL-apoA-I (4535±72 % cpm, P<0.001 versus 1059±14 % cpm) respectively. This data suggests that MPS formations are an integral component of cellular cholesterol efflux. Also, MPS do not contain CS-6253 when ABCA1 cells were incubated with the peptide as confirmed by western blotting similar to MPS generated from ABCA1 cells generated by apoA-I incubation. Cholesterol is effluxed more to nHDL-CS-6253 (14±7.68, % cpm, P<0.01 than to MPS 1±0.10, % cpm, 24h; P<0.01) similar to apoA-I. Depletion of membrane cholesterol by methyl-β-cyclodextrin treatment impaired HDL genesis and decrease MPS release. Detection of flotellin-2 protein enriched in MPS in total cell lysate indicated that these MPS may be related to exosomes. MPS generation was also characterized in THP-1 cells for further molecular characterization. We conclude that MPS are formed by ABCA1 in diverse cell types and the cholesterol content is dependent on activation by apo A-I or mimetic peptides. The physiological role of MPS remains to be understood. These particles may be transporters of lipids and nucleic acids.
Author Disclosures: A. Hafiane: None. J. Bielicki: None. J. Johansson: None. J. Genest: None.
- © 2015 by American Heart Association, Inc.