J Lipid Res. 2015 Sep;56(9):1720-6.
ABCA1 contributes to macrophage deposition of extracellular cholesterol.
Jin X1, Freeman SR1, Vaisman B2, Liu Y1, Chang J1, Varsano N3, Addadi L3, Remaley A2, Kruth HS1
1Section of Experimental Atherosclerosis National Institutes of Health, Bethesda, MD 20892.
2Lipoprotein Metabolism Section, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
3Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel.
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. Macrophage 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.
Atherosclerotic plaques develop as a result of an imbalance between cholesterol accumulation and cholesterol removal. The macrophage plays a central role in both of these processes; taking up and storing cholesterol as well as processing this cholesterol for removal from the vessel. It is believed that when a macrophage’s mechanisms for cholesterol removal become overwhelmed, atherosclerotic plaques progress due to excess cholesterol accumulation. How macrophages eliminate excess cholesterol has been of great interest, and is important for understanding the cholesterol accumulation process in developing atherosclerotic plaques. Atherosclerotic plaques contain cholesterol in both intracellular and extracellular forms. However, the origin and fate of extracellular cholesterol in plaques is far less understood compared with intracellular cholesterol. ABCA1 and ABCG1 are transporters that function in reverse cholesterol transport: the process by which excess cholesterol is transported from peripheral tissues such as atherosclerotic plaques to the liver for elimination or reutilization. Our research focus has been to investigate macrophage deposition of extracellular cholesterol, and to determine whether ABCA1 and ABCG1 transporters function in this cholesterol deposition.
We previously described a novel cholesterol efflux pathway in which macrophages deposit unesterified cholesterol into the extracellular matrix (1-3). A monoclonal antibody, 58B1, has been used in our studies to detect unique cholesterol microdomains consisting of an ordered array of cholesterol molecules (4). (The antibody does not label cholesteryl ester). Depending on the macrophage type and culture conditions, macrophages enriched with cholesterol show development of spherical cholesterol microdomains either associated with the plasma membrane or deposited into the extracellular matrix. Cholesterol microdomain deposition occurs through a two-step process in which macrophages first accumulate unesterified cholesterol in the form of plasma membrane microdomains and then these plasma membrane microdomains are subsequently deposited into the extracellular matrix (Figure 1)(3).
Figure 1. Macrophage deposition of extracellular cholesterol. When enriched with cholesterol, human monocyte-derived macrophages deposit cholesterol in the form of spherical particles into the extracellular space (monoclonal antibody 58B1 green fluorescence indicated by arrows). Nuclei are labeled blue. (adapted from (4)).
Enriching macrophage with cholesterol in the presence of either probucol (an inhibitor of ABCA1) or TO901317 (a stimulator of ABCA1 and ABCG1) decreases and increases, respectively, macrophage deposition of extracellular cholesterol. Studies with ABCG1- or ABCA1-deficient mice demonstrate that both these ABC transporter proteins mediate macrophage deposition of extracellular cholesterol (1, 2).
The extracellular cholesterol deposited by macrophages can be mobilized by cholesterol acceptors ApoA-I, HDL, and cyclodextrin, thus linking this cholesterol with the reverse cholesterol transport pathway. However, we have found that 58B1-labeled extracellular cholesterol accumulates within atherosclerotic plaques indicating that reverse cholesterol transport of this deposited extracellular cholesterol is not adequate (3).
Our discovery that macrophages export cholesterol into the extracellular matrix mediated by ABCA1 and ABCG1 represents another means by which macrophages maintain cholesterol homeostasis (5). Macrophages not only clear their excess cholesterol by cholesterol esterification and storage within lipid droplets, but also by depositing this excess cholesterol within the extracellular matrix (Figure 2). Extracellular cholesterol deposited by macrophages is a novel cholesterol pool that functions in reverse cholesterol transport. Future research is needed to assess whether buildup of this extracellular cholesterol promotes the development of atherosclerotic plaques.
Figure 2. Function of cholesterol microdomains in reverse cholesterol transport. When macrophages are enriched with cholesterol, ABCA1 and ABCG1 can mediate deposition of excess cellular cholesterol into the extracellular space, where it can be mobilized by HDL. (adapted from (4)).
- Freeman SR, Jin X, Anzinger JJ, Xu Q, Purushothaman S, Fessler MB, Addadi L, Kruth HS, 2014 ABCG1-mediated generation of extracellular cholesterol microdomains. Journal of Lipid Research 55:115-127
- Jin X, Freeman SR, Vaisman B, Liu Y, Chang J, Varsano N, Addadi L, Remaley A, Kruth HS, 2015 ABCA1 contributes to macrophage deposition of extracellular cholesterol. Journal of Lipid Research 56:1720-1726
- Ong DS, Anzinger JJ, Leyva FJ, Rubin N, Addadi L, Kruth HS, 2010 Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport. Journal of Lipid Research 51:2303-2313
- Addadi L, Geva M, Kruth HS, 2003 Structural information about organized cholesterol domains from specific antibody recognition. Biochim Biophys Acta 1610:208-216
- Jin X and Kruth HS, in press, Macrophage deposition of extracellular lipid particles with microdomains of ordered cholesterol arrays, in Handbook of Cholesterol: Biology, function and role in health and diseases, R. Watson and F. DeMeester, Editors, Wageningen Academics
Acknowledgements: This work was supported by the Intramural Research Program, National Heart, Lung, and Blood Institute, National Institutes of Health, and by the Binational Science Foundation (Grant 2013045).
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