Characterization of fluorescent NBD-cholesterol efflux in THP-1-derived macrophages

Abstract

Abstract. Macrophage cholesterol efflux is important in maintaining cellular lipid homeostasis and preventing the formation of lipid-laden foam cells. Although radioactive [ 3 H]-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 [ .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. IntroductionCoronary artery disease (CAD) is the leading contributor to morbidity and mortality rates worldwide, and results in a mortality rate of >7,000,000 per year (1). Atherosclerotic plaques are key in the pathophysiology of CAD, in which macrophages initially become foam cells and initiate plaque formation. The imbalance of cholesterol uptake and efflux in macrophages may cause excessive accumulation of cholesterol and foam cell formation, which is a hallmark in the development of atherosclerosis (2). Cholesterol efflux from macrophages is a critical step in reverse cholesterol transport, which may prevent macrophages from becoming foam cells and thus reduce atherosclerosis (3,4). Free cholesterol is transported from macrophages in the arterial atherosclerotic plaque to an extracellular acceptors, including apoliporotein (apo) A-1 and high-density lipoprotein (HDL), and then back to the liver for bile acid synthesis and excretion (5). Previously, Khera et al reported a reverse correlation between cholesterol efflux capacity and atherosclerosis (6). To understand the underlying mechanism regulating cholesterol efflux, it is important to develop a reliable assay for measuring cholesterol efflux in vitro.Traditionally, radioisotope-labeled cholesterols have been used as probes in cholesterol efflux assays. However, the procedure is lengthy and labor-intensive, and the radioactivity disposal procedure can limit the use of this assay in high-throughput screening. By contrast, fluorescent

Oligomeric proanthocyanidins and epigallocatechin gallate aggravate autophagy of foam cells through the activation of Class III PI3K/Beclin1‐complex mediated cholesterol efflux

“…In vitro generation of PMA‐induced monocytes to macrophage differentiation showed well‐established molecular characteristics of mature macrophages such as decreased rate of proliferation, increased adherence, improved phagocytosis, and expression of scavenger receptors . Macrophages present in the atherosclerotic plaque give rise to a special type of cells which could ingest OxLDL and become overloaded with lipids called foam cells.…”

“…In vitro generation of PMA-induced monocytes to macrophage differentiation showed well-established molecular characteristics of mature macrophages such as decreased rate of proliferation, increased adherence, improved phagocytosis, and expression of scavenger receptors. 17 Macrophages present in the atherosclerotic plaque give rise to a special type of cells which could ingest OxLDL and become overloaded with lipids called foam cells. This study also showed the transdifferentiation of PMA derived macrophages into foam cells with the increased expression of upregulated scavenger receptors: SR-A1, SR-B1, and LOX-1 and with the reduced expression of transporter proteins ABCA1/ABCG1.…”

DBZ (Danshensu Bingpian Zhi), a Novel Natural Compound Derivative, Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice

“…Cholesterol efflux was measured using the methods modified from Song et al . In brief, THP‐1 macrophages were equilibrated with NBD cholesterol (5 μmol/L) for 4 hours.…”

Heat shock protein 27–derived atheroprotection involves reverse cholesterol transport that is dependent on GM‐CSF to maintain ABCA1 and ABCG1 expression in ApoE − / − mice.

“…As previously described, differentiated THP‐1 cells were equilibrated with NBD cholesterol {22‐[ N ‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐23,24‐bisnor‐5‐cholen‐3β‐Ol; Thermo Fisher Scientific} for 18 h, then washed with PBS and treated with rHSP‐27 overnight (26). Thereafter, cells were incubated with the extracellular cholesterol acceptors, ApoA1 (15 mg/ml; Cedarlane) or HDL (50 mg/ml HDL; Cedarlane) in serum‐free medium for 6 h. The fluorescence‐labeled cholesterol released from cells into the medium was measured with a microplate reader at excitation l = 485 nm and emission l = 535 nm.…”