Different cellular traffic of LDL-cholesterol and acetylated LDL-cholesterol leads to distinct reverse cholesterol transport pathways

Abstract

Endocytosis of LDL and modified LDL represents regulated and unregulated cholesterol delivery to macrophages. To elucidate the mechanisms of cellular cholesterol transport and egress under both conditions, various primary macrophages were labeled and loaded with cholesterol or cholesteryl ester from LDL or acetylated low density lipoprotein (AcLDL), and the cellular cholesterol traffic pathways were examined. Confocal microscopy using fluorescently labeled 3,3 ¶-dioctyldecyloxacarbocyanine perchlorate-labeled LDL and 1,1 ¶-dioctyldecyl-3,3,3 ¶,3 ¶-tetramethylindodicarbocyanine perchlorate-labeled AcLDL demonstrated their discrete traffic pathways and accumulation in distinct endosomes. ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I) was much greater for AcLDLloaded macrophages compared with LDL. Treatment with the liver X receptor ligand 22-OH increased efflux to apoA-I in AcLDL-loaded but not LDL-loaded cells. In contrast, at a level equivalent to AcLDL, LDL-derived cholesterol was preferentially effluxed to HDL, in keeping with increased ABCG1. In vivo studies of reverse cholesterol transport (RCT) from cholesterol-labeled macrophages injected intraperitoneally demonstrated that LDL-derived cholesterol was more efficiently transported to the liver and secreted into bile than AcLDL-derived cholesterol. This indicates a greater efficiency of HDL than lipid-poor apoA-I in interstitial fluid in controlling in vivo RCT.These assays, taken together, emphasize the importance of mediators of diffusional cholesterol efflux in RCT.

Differential Regulation of ATP Binding Cassette Protein A1 Expression and ApoA-I Lipidation by Niemann-Pick Type C1 in Murine Hepatocytes and Macrophages

“…3C) labeled with LDL. Cholesterol efflux to m␤-CD at 37°C, which measures labeled cholesterol associated with both plasma membrane and recycling compartments (26,27), was also reduced by about 40% in LDLlabeled Npc1-null hepatocytes and macrophages. Similar results were obtained with hepatocytes and macrophages metabolically labeled with [ 3 H]mevalonate (Fig.…”

“…3, B and D). Cholesterol efflux to m␤-CD at 4°C, which measures labeled cholesterol associated with the plasma membrane (27), was significantly and similarly reduced by more than 30% in Npc1-null hepatocytes (Fig. 3A) and macrophages ( Fig.…”

“…Macrophages were generated by incubating bone marrow cells (10 6 cells/ml) with DMEM of 10% FBS complemented with 15% L929 conditioned medium for 7 days (27).…”

“…Efflux to apoA-I (10 g in 2 mg/ml bovine serum albumin medium) was monitored for 3-5 h (29). Efflux to m␤-CD (10 mM in 2 mg/ml bovine serum albumin DMEM) was carried out for 15 min at 37 or 4°C (27).…”

Importance of macrophage cholesterol content on the flux of cholesterol mass

“…Cholesterol mass and isotope fl ux studies were conducted on J774 (murine cell line) cells and mouse peritoneal macrophage (MPM) cells that contained both normal and elevated levels of cholesterol. The level of cell cholesterol infl uences fl ux in a number of ways, including modulating the expression level of cell proteins mediating effl ux such as ABCA1, ABCG1, and SR-BI, and proteins, such as LDL and scavenger receptors, which participate in lipoprotein uptake ( 7,10 ) or infl ux. The fractional effl ux values were determined using cells prelabeled with [ 3 H]cholesterol and net fl ux was measured directly by quantitating changes in cell cholesterol mass upon incubation with either human sera or apolipoprotein (apo)-B-depleted sera.…”