Cholesterol Acceptors Regulate the Lipidome of Macrophage Foam Cells - PubMed (original) (raw)
Cholesterol Acceptors Regulate the Lipidome of Macrophage Foam Cells
Antoni Paul et al. Int J Mol Sci. 2019.
Abstract
Arterial foam cells are central players of atherogenesis. Cholesterol acceptors, apolipoprotein A-I (apoA-I) and high-density lipoprotein (HDL), take up cholesterol and phospholipids effluxed from foam cells into the circulation. Due to the high abundance of cholesterol in foam cells, most previous studies focused on apoA-I/HDL-mediated free cholesterol (FC) transport. However, recent lipidomics of human atherosclerotic plaques also identified that oxidized sterols (oxysterols) and non-sterol lipid species accumulate as atherogenesis progresses. While it is known that these lipids regulate expression of pro-inflammatory genes linked to plaque instability, how cholesterol acceptors impact the foam cell lipidome, particularly oxysterols and non-sterol lipids, remains unexplored. Using lipidomics analyses, we found cholesterol acceptors remodel foam cell lipidomes. Lipid subclass analyses revealed various oxysterols, sphingomyelins, and ceramides, species uniquely enriched in human plaques were significantly reduced by cholesterol acceptors, especially by apoA-I. These results indicate that the function of lipid-poor apoA-I is not limited to the efflux of cholesterol and phospholipids but suggest that apoA-I serves as a major regulator of the foam cell lipidome and might play an important role in reducing multiple lipid species involved in the pathogenesis of atherosclerosis.
Keywords: ABCA1; ABCG1; LDL; atherosclerosis; cholesterol; foam cells; lipidome; lipidomics; macrophage; mass-spectrometry; oxysterols; reverse cholesterol transport.
Conflict of interest statement
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Figures
Figure 1
Oxidized low-density lipoprotein (LDL) enriches oxysterols in macrophage foam cells. (A) Foam cell formation. Mouse peritoneal macrophages (MPMs) plated on a coverslip remained untreated (left) or were loaded with either acLDL (middle) or hi-oxLDL (right) for 24 h to generate low-density lipoprotein (LDs). LDs were visualized with Bodipy 483/503 staining (green) and nuclei were stained with DAPI (blue). (B) Total Bodipy fluorescence intensity per cell represents total lipid contents (left). Area of LD per cell was measured and presented in μm2/cell. n = 88 for acLDL and n = 55 for Hi-oxLDL-treated MPMs. (C) Sterol contents of foam cells treated with modified LDLs. MPMs were treated with acLDL for 24 h, and oxLDL or hi-oxLDL for 48 h. Detected sterols using mass-spectrometry (MS) was normalized by mg protein and shown as pmol of lipids over μg of protein. The data were presented as mean ± standard error of mean (SEM).
Figure 2
ApoA-I increases efflux of free cholesterol (FC) from hi-oxLDL-loaded foam cells. (A) MPMs were loaded with 3H-cholesterol-coupled acLDL or hi-oxLDL. Cholesterol efflux was initiated with 50 μg/mL of apoA-I. The cells without apoA-I loading were used as a control. The net percentage of cholesterol efflux at each time point compared to time 0 was presented as % efflux (n = 4 per each treatment). (B,C) Hi-oxLDL-loaded MPMs were loaded with 10 or 50 μg/mL of apoA-I or HDL. Immunoblotting with anti-ABCA1 and anti-tubulin were performed, and the relative level of ABCA1 was normalized with tubulin and quantified (n = 3) using Fiji software. The data for both cholesterol efflux and WB were presented as mean ± SEM. * p < 0.05, ** p < 0.005.
Figure 3
ApoA-I significantly reduces total sterols, cholesterol, and oxysterols in foam cells. (A) Experimental procedure. MPMs were loaded with hi-oxLDL (50 μg/mL) for 24 h. The media was refreshed without hi-oxLDL followed by incubation with or without apoA-I (10 μg/mL and 50 μg/mL) or HDL (50 μg/mL) for 24 h. Lipids were extracted, and sterol species were identified by MS. (B) The levels of total sterols, cholesterol, and oxysterols were presented as abundance obtained as pmol of lipids normalized with μg of protein. The data were presented as mean ± SEM (n = 4 per group). * p < 0.05, ** p < 0.005. *** p < 0.0005.
Figure 4
Targeted sterol lipidomics. (A) Relative levels of 12 sterols were presented based on their % of abundance to total sterols detected in untreated MPMs. (B) Increased levels of oxysterols upon hi-oxLDL loading were presented as abundance (C–M) Abundance of individual sterol species with apoA-I and HDL treatments. The abundance of lipids in (B–M) was calculated by pmol of lipid normalized to μg of protein. The data were presented as mean ± SEM (n = 4 per group). * p < 0.05, ** p < 0.05, *** p < 0.0005.
Figure 5
Untargeted global lipidomics. (A) Total lipids detected in each treatment group. (B) Heat map showing the relative abundance of five lipid categories as % of each category to total detected lipids. (C) Phospholipids. Total amount of phospholipids detected in each treatment group (top) and each class of phospholipids regulated by cholesterol acceptors (bottom). (D–G) The levels of sterol lipids, glycerolipids, sphingolipids, and non-esterified fatty acids (NEFA) in each group. The amount of lipid detected was presented as pmol lipid normalized to mg protein. TG; triglyceride, DG; diglyceride, MA; monoglyceride. The data were presented as mean ± SEM (n = 4 per group). * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005. Hi-oxLDL versus hi-oxLDL + A-I 50: ## p < 0.005, #### p < 0.00005. Hi-oxLDL versus hi-oxLDL+ HDL: ☨☨ p < 0.005, ☨☨☨☨ p < 0.0005.
Figure 5
Untargeted global lipidomics. (A) Total lipids detected in each treatment group. (B) Heat map showing the relative abundance of five lipid categories as % of each category to total detected lipids. (C) Phospholipids. Total amount of phospholipids detected in each treatment group (top) and each class of phospholipids regulated by cholesterol acceptors (bottom). (D–G) The levels of sterol lipids, glycerolipids, sphingolipids, and non-esterified fatty acids (NEFA) in each group. The amount of lipid detected was presented as pmol lipid normalized to mg protein. TG; triglyceride, DG; diglyceride, MA; monoglyceride. The data were presented as mean ± SEM (n = 4 per group). * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005. Hi-oxLDL versus hi-oxLDL + A-I 50: ## p < 0.005, #### p < 0.00005. Hi-oxLDL versus hi-oxLDL+ HDL: ☨☨ p < 0.005, ☨☨☨☨ p < 0.0005.
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