Characterization of fluorescent NBD‑cholesterol efflux in thp‑1‑derived macrophages

Song,Wei; Wang,Wei; Wang,Yu; Dou,Liyang; Chen,Lianfeng; Yan,Xiaowei

doi:10.3892/mmr.2015.4154

Characterization of fluorescent NBD‑cholesterol efflux in thp‑1‑derived macrophages

Authors:
- Wei Song
- Wei Wang
- Yu Wang
- Liyang Dou
- Lianfeng Chen
- Xiaowei Yan
View Affiliations

Affiliations: Department of Cardiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, P.R. China
Published online on: July 30, 2015 https://doi.org/10.3892/mmr.2015.4154
Pages: 5989-5996

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Macrophage cholesterol efflux is important in maintaining cellular lipid homeostasis and preventing the formation of lipid‑laden foam cells. Although radioactive [3H]‑cholesterol is widely used as a tracer in cholesterol efflux assays, the lengthy and labor‑intensive assay procedure, and the radioactivity disposal procedure limit the use of this assay for high‑throughput screening. In the present study, a novel procedure using fluorescent N‑(7‑nitrobenz‑2‑oxa‑1,3‑diazol‑4‑yl)amino)‑23,24‑bisnor‑5‑xholen‑3β‑ol (NBD)‑cholesterol was developed as a substitute for [3H]‑cholesterol for the measurement of cholesterol efflux in THP‑1‑derived macrophages. NBD‑cholesterol uptake and metabolism in the THP‑1 cells were characterized using fluorescent microscopy and spectrophotometry. NBD‑cholesterol distributed rapidly into the cell organelles, with the exception of the nucleus. The uptake of NBD‑cholesterol in the THP‑1 macrophages was concentration‑ and time‑dependent, and reached a plateau following 4 h incubation. The present study subsequently investigated whether NBD‑cholesterol efflux was correlated with [3H]‑cholesterol efflux in THP‑1 derived macrophages and in human peripheral blood mononuclear cells (PBMCs). The results demonstrated that the percentage of efflux of NBD‑cholesterol in the THP‑1 cells was significantly correlated with that of [3H]‑cholesterol, at various concentrations of HDL or apoA‑1 as lipid acceptors (R2=0.876 for HDL; R2=0.837 for apoA‑1; P<0.001). In the PBMCs, NBD‑cholesterol efflux also correlated significantly with [3H]‑cholesterol efflux (R2=0.887 for HDL; R2=0.872 for apoA‑1; P<0.001). Furthermore, NBD‑cholesterol efflux in the THP‑1 cells exhibited a similar trend to that obseved in the PBMCs. In conclusion, the results of the present study suggested that fluorescent NBD‑cholesterol can be used as a sensitive and specific probe in cholesterol efflux assays in THP‑1‑derived macrophages.

View Figures

View References

1	Yusuf S, Reddy S, Ounpuu S and Anand S: Global burden of cardiovascular diseases: Part I: General considerations, the epidemiologic transition, risk factors and impact of urbanization. Circulation. 104:2746–2753. 2001. View Article : Google Scholar : PubMed/NCBI
2	Ross R: Atherosclerosis-an inflammatory disease. N Engl J Med. 340:115–126. 1999. View Article : Google Scholar : PubMed/NCBI
3	Fielding CJ and Fielding PE: Cellular cholesterol efflux. Biochim Biophys Acta. 1533:175–189. 2001. View Article : Google Scholar : PubMed/NCBI
4	Tall AR, Costet P and Wang N: Regulation and mechanisms of macrophage cholesterol efflux. J Clin Invest. 110:899–904. 2002. View Article : Google Scholar : PubMed/NCBI
5	Cuchel M and Rader DJ: Macrophage reverse cholesterol transport: Key to the regression of atherosclerosis? Circulation. 113:2548–2555. 2006. View Article : Google Scholar : PubMed/NCBI
6	Khera AV, Cuchel M, de la Llera-Moya M, Rodrigues A, Burke MF, Jafri K, French BC, Phillips JA, Mucksavage ML, Wilensky RL, et al: Cholesterol efflux capacity, high-density lipoprotein function and atherosclerosis. N Engl J Med. 364:127–135. 2011. View Article : Google Scholar : PubMed/NCBI
7	Zhang J, Cai S, Peterson BR, Kris-Etherton PM and Heuvel JP: Development of a cell-based, high-throughput screening assay for cholesterol efflux using a fluorescent mimic of cholesterol. Assay Drug Dev Technol. 9:136–146. 2011. View Article : Google Scholar :
8	Wustner D: Fluorescent sterols as tools in membrane biophysics and cell biology. Chem Phys Lipids. 146:1–25. 2007. View Article : Google Scholar : PubMed/NCBI
9	Ramirez DM, Ogilvie WW and Johnston LJ: NBD-cholesterol probes to track cholesterol distribution in model membranes. Biochim Biophys Acta. 1798:558–568. 2010. View Article : Google Scholar
10	Storey SM, Atshaves BP, McIntosh AL, Landrock KK, Martin GG, Huang H, Ross Payne H, Johnson JD, Macfarlane RD, Kier AB and Schroeder F: Effect of sterol carrier protein-2 gene ablation on HDL-mediated cholesterol efflux from cultured primary mouse hepatocytes. Am J Physiol Gastrointest Liver Physiol. 299:G244–G254. 2010. View Article : Google Scholar : PubMed/NCBI
11	Sankaranarayanan S, Kellner-Weibel G, de la Llera-Moya M, Phillips MC, Asztalos BF, Bittman R and Rothblat GH: A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol. J Lipid Res. 52:2332–2340. 2011. View Article : Google Scholar : PubMed/NCBI
12	Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T and Tada K: Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer. 26:171–176. 1980. View Article : Google Scholar : PubMed/NCBI
13	Tsuchiya S, Kobayashi Y, Goto Y, Okumura H, Nakae S, Konno T and Tada K: Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester. Cancer Res. 42:1530–1536. 1982.PubMed/NCBI
14	Liu XH, Xiao J, Mo ZC, Yin K, Jiang J, Cui LB, Tan CZ, Tang YL, Liao DF and Tang CK: Contribution of D4-F to ABCA1 expression and cholesterol efflux in THP-1 macrophage-derived foam cells. J Cardiovasc Pharmacol. 56:309–319. 2010. View Article : Google Scholar : PubMed/NCBI
15	Larrede S, Quinn CM, Jessup W, Frisdal E, Olivier M, Hsieh V, Kim MJ, Van Eck M, Couvert P, Carrie A, et al: Stimulation of cholesterol efflux by LXR agonists in cholesterol-loaded human macrophages is ABCA1-dependent but ABCG1-independent. Arterioscler Thromb Vasc Biol. 29:1930–1936. 2009. View Article : Google Scholar : PubMed/NCBI
16	Patel S, Drew BG, Nakhla S, Duffy SJ, Murphy AJ, Barter PJ, Rye KA, Chin-Dusting J, Hoang A, Sviridov D, et al: Reconstituted high-density lipoprotein increases plasma high-density lipoprotein anti-inflammatory properties and cholesterol efflux capacity in patients with type 2 diabetes. J Am Coll Cardiol. 53:962–971. 2009. View Article : Google Scholar : PubMed/NCBI
17	de Almeida MC, Silva AC, Barral A and Barral Netto M: A simple method for human peripheral blood monocyte isolation. Mem Inst Oswaldo Cruz. 95:221–223. 2000. View Article : Google Scholar : PubMed/NCBI
18	Xu Y, Wang W, Zhang L, Qi LP, Li LY, Chen LF, Fang Q, Dang AM and Yan XW: A polymorphism in the ABCG1 promoter is functionally associated with coronary artery disease in a Chinese Han population. Atherosclerosis. 219:648–654. 2011. View Article : Google Scholar : PubMed/NCBI
19	Fan J, Rone MB and Papadopoulos V: Translocator protein 2 is involved in cholesterol redistribution during erythropoiesis. J Biol Chem. 284:30484–30497. 2009. View Article : Google Scholar : PubMed/NCBI
20	Sasaki H and White SH: A novel fluorescent probe that senses the physical state of lipid bilayers. Biophys J. 96:4631–4641. 2009. View Article : Google Scholar : PubMed/NCBI
21	Frolov A, Petrescu A, Atshaves BP, So PT, Gratton E, Serrero G and Schroeder F: High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. A single- and multiphoton fluorescence approach. J Biol Chem. 275:12769–12780. 2000. View Article : Google Scholar : PubMed/NCBI
22	Sengupta B, Narasimhulu CA and Parthasarathy S: Novel technique for generating macrophage foam cells for in vitro reverse cholesterol transport studies. J Lipid Res. 54:3358–3372. 2013. View Article : Google Scholar : PubMed/NCBI
23	Ohashi R, Mu H, Wang X, Yao Q and Chen C: Reverse cholesterol transport and cholesterol efflux in atherosclerosis. QJM. 98:845–856. 2005. View Article : Google Scholar : PubMed/NCBI
24	Zhang J, Cai S, Peterson BR, Kris-Etherton PM and Heuvel JP: Development of a cell-based, high-throughput screening assay for cholesterol efflux using a fluorescent mimic of cholesterol. Assay Drug Dev Technol. 9:136–146. 2011. View Article : Google Scholar :
25	Ohashi M, Murata M and Ohnishi S: A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein. Eur J Cell Biol. 59:116–126. 1992.PubMed/NCBI
26	Adams MR, Konaniah E, Cash JG and Hui DY: Use of NBD-cholesterol to identify a minor but NPC1L1-independent cholesterol absorption pathway in mouse intestine. Am J Physiol Gastrointest Liver Physiol. 300:G164–G169. 2011. View Article : Google Scholar :
27	Takahashi M, Murate M, Fukuda M, Sato SB, Ohta A and Kobayashi T: Cholesterol controls lipid endocytosis through Rab11. Mol Biol Cell. 18:2667–2677. 2007. View Article : Google Scholar : PubMed/NCBI
28	Portioli Silva EP, Peres CM, Roberto Mendonca J and Curi R: NBD-cholesterol incorporation by rat macrophages and lymphocytes: A process dependent on the activation state of the cells. Cell Biochem Funct. 22:23–28. 2004. View Article : Google Scholar
29	Nagy L, Tontonoz P, Alvarez JG, Chen H and Evans RM: Oxidized LDL regulates macrophage gene expression through ligand activation of PPARgamma. Cell. 93:229–240. 1998. View Article : Google Scholar : PubMed/NCBI
30	Boullier A, Bird DA, Chang MK, Dennis EA, Friedman P, Gillotre-Taylor K, Hörkkö S, Palinski W, Quehenberger O, Shaw P, et al: Scavenger receptors, oxidized LDL and atherosclerosis. Ann N Y Acad Sci. 947:214–222; discussion 222–223. 2001. View Article : Google Scholar
31	Yvan-Charvet L, Ranalletta M, Wang N, Han S, Terasaka N, Li R, Welch C and Tall AR: Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice. J Clin Invest. 117:3900–3908. 2007.PubMed/NCBI
32	Adorni MP, Zimetti F, Billheimer JT, Wang N, Rader DJ, Phillips MC and Rothblat GH: The roles of different pathways in the release of cholesterol from macrophages. J Lipid Res. 48:2453–2462. 2007. View Article : Google Scholar : PubMed/NCBI
33	Wang X, Collins HL, Ranalletta M, Fuki IV, Billheimer JT, Rothblat GH, Tall AR and Rader DJ: Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo. J Clin Invest. 117:2216–2224. 2007. View Article : Google Scholar : PubMed/NCBI
34	Wang N, Lan D, Chen W, Matsuura F and Tall AR: ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci USA. 101:9774–9779. 2004. View Article : Google Scholar : PubMed/NCBI
35	Tall AR, Yvan-Charvet L, Terasaka N, Pagler T and Wang N: HDL, ABC transporters and cholesterol efflux: Implications for the treatment of atherosclerosis. Cell Metab. 7:365–375. 2008. View Article : Google Scholar : PubMed/NCBI
36	Yvan-Charvet L, Wang N and Tall AR: Role Of HDL, ABCA1 and ABCG1 transporters in cholesterol efflux and immune responses. Arterioscler Thromb Vasc Biol. 30:139–143. 2010. View Article : Google Scholar :