ABCA1 contributes to macrophage deposition of extracellular cholesterol.

We previously reported that cholesterol-enriched macrophages excrete cholesterol into the extracellular matrix. A monoclonal antibody that detects cholesterol microdomains labels the deposited extracellular particles. Macro-phage deposition of extracellular cholesterol depends, in part, on ABCG1, and this cholesterol can be mobilized by HDL components of the reverse cholesterol transport process. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. ABCA1 functioned in extracellular cholesterol deposition. The liver X receptor agonist, TO901317 (TO9), an ABCA1-inducing factor, restored cholesterol deposition that was absent in cholesterol-enriched ABCG1(-/-) mouse macrophages. In addition, the ABCA1 inhibitor, probucol, blocked the increment in cholesterol deposited by TO9-treated wild-type macrophages, and completely inhibited deposition from TO9-treated ABCG1(-/-) macrophages. Lastly, ABCA1(-/-) macrophages deposited much less extracellular cholesterol than wild-type macrophages. These findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular matrix.

ABCG1-mediated generation of extracellular cholesterol microdomains.

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 µg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space.

Surface characterization of nanoparticles using near-field light scattering.

The effect of nanoparticle surface coating characteristics on colloidal stability in solution is a critical parameter in understanding the potential applications of nanoparticles, especially in biomedicine. Here we explored the modification of the surface of poly(ethylene glycol)-coated superparamagnetic iron oxide nanoparticles (PEG-SPIOs) with the synthetic pseudotannin polygallol via interpolymer complexation (IPC). Changes in particle size and zeta potential were indirectly assessed via differences between PEG-SPIOs and IPC-SPIOs in particle velocity and scattering intensity using near-field light scattering. The local scattering intensity is correlated with the distance between the particle and waveguide, which is affected by the size of the particle (coating thickness) as well as the interactions between the particle and waveguide (related to the zeta potential of the coating). Therefore, we report here the use of near-field light scattering using nanophotonic force microscopy (using a NanoTweezerTM instrument, Halo Labs) to determine the changes that occurred in hydrated particle characteristics, which is accompanied by an analytical model. Furthermore, we found that altering the salt concentration of the suspension solution affected the velocity of particles due to the change of dielectric constant and viscosity of the solution. These findings suggest that this technique is suitable for studying particle surface changes and perhaps can be used to dynamically study reaction kinetics at the particle surface.