Abstract 402: HDL Mimetic Peptides ATI-5261 and CS-6253 Induce ABCA1 Oligomerization, Cellular Cholesterol Efflux and Generate Nascent HDL-Like Lipoproteins

Abstract

Novel approaches that target HDL biogenesis may increase nascent HDL-like particle formation that can be used for therapeutic purposes. Here, we evaluated two 26-amino acid apoA-I mimetic peptides designed from c-terminus of apoE, ATI-5261 and CS-6253 in their ability to promote cellular cholesterol efflux via the ABCA1 transporter. Using mifepristone-inducible ABCA1 BHK cells and cAMP-stimulated J774 macrophages, we examined ABCA1 oligomerization with dithiobis[succinimidyl propionate] (DSP) as a cross linker. Like native apoA-I, ATI-5261 and CS-6253 induced ABCA1 oligomerization at equimolar concentrations, and appear to interact with native oligomers of ABCA1, similar to apoA-I, as judged by crosslinking revealed by SDS-PAGE. By sucrose density gradient fractionation, we show that ABCA1 oligomerization occurs in non-raft domains. ATI-5261 and CS-6253 promoted cellular cholesterol efflux in a dose-dependent saturable manner. In BHK cells, the efficiency of cholesterol efflux (Km) on an equimolar basis was: apoA-I (0.16±0.02), ATI-5261 (0.45±0.08) and CS-6253 (0.68±0.15) and the efflux capacity (Vmax) (% in 24h) was: apoA-I (14.54±0.61), ATI-5261 (11.98±0.51) and CS-6253 (10.87±0.66), indicating a higher efflux efficiency for the apoA-I mimetics but with less capacity than native apoA-I. A similar trend was observed in J774 cells. Desorption of cellular cholesterol at 45 min occurred in both non-raft and raft-like membrane domains. We then examined by HPLC and 2-dimentional PAGGE the size and types of HDL-like particles formed. In 3[H]-cholesterol loaded BHK-ABCA1 cells, incubation with ATI-5261 and CS-6253 for 45 min and 12 hours yielded α-migrating particles with an apparent size ranging between 9 and 17 nm, as revealed by specific antibodies directed against ATI-5261 and CS-6253. Taken together, these observations suggest that ATI-5261 and CS-6253 induce cellular cholesterol efflux via ABCA1 oligomerization at molar concentrations similar to apoA-I and with higher efficiency. The particles generated by ATI-5261 and CS-6253 have α-migrating mobility with a size ranging between 9 and 17 nm and contain cholesterol, similar to nascent HDL particles.