Elimination of Cholesterol Ester from Macrophage Foam Cells by Adenovirus-mediated Gene Transfer of Hormone-sensitive Lipase

Abstract

Cholesterol ester (CE)-laden foam cells are a hallmark of atherosclerosis. To determine whether stimulation of the hydrolysis of cytosolic CE can be used as a novel therapeutic modality of atherosclerosis, we overexpressed hormone-sensitive lipase (HSL) in THP-1 macrophage-like cells by adenovirus-mediated gene delivery, and we examined its effects on the cellular cholesterol trafficking. We show here that the overexpression of HSL robustly increased neutral CE hydrolase activity and completely eliminated CE in the cells that had been preloaded with CE by incubation with acetylated low density lipoprotein. In these cells, cholesterol efflux was stimulated in the absence or presence of high density lipoproteins, which might be at least partially explained by the increase in the expression of ABCA1. Importantly, these effects were achieved without the addition of acyl-CoA:cholesterol acyltransferase inhibitor, cAMP, or even high density lipoproteins. Furthermore, the uptake and degradation of acetylated low density lipoprotein was significantly reduced probably by decreased expression of scavenger receptor A and CD36. Notably, the cells with stimulated CE hydrolysis did not exhibit either buildup of free cholesterol or cytotoxicity. In conclusion, increased hydrolysis of CE by the overexpression of HSL leads to complete elimination of CE from THP-1 foam cells not only by increasing efflux but also by decreasing influx of cholesterol. Cholesterol ester (CE)1 -laden macrophage foam cells are a hallmark of fatty streak lesions in atherosclerotic plaques.Besides cleaning up extracellularly deposited atherogenic lipoproteins, macrophage foam cells secrete a wide variety of substances, which include inflammatory cytokines, chemokines, growth factors, and proteases (1). Furthermore, lipidrich lesions that are characterized by a plethora of macrophage foam cells are associated with massive infiltration with activated T lymphocytes and prone to rupture, thereby leading to thrombotic coronary occlusion (2). Thus, elimination of CE from foam cells is potentially a promising therapeutic strategy to stabilize rupture-prone atherosclerotic plaques.Foam cells are generated by the uptake of modified lipoproteins through scavenger receptors, such as scavenger receptor A (SR-A) and CD36 (3). Hydrolysis of the lipoprotein-associated CE by lysosomal acid lipase liberates free cholesterol (FC), which is subsequently re-esterified by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) to form CE for storage in the cytoplasmic lipid droplets (4, 5). The cytoplasmic CE is in turn hydrolyzed by neutral CE hydrolase (NCEH) to generate FC, which is transported to a compartment for re-esterification by ACAT1. The rest of the FC is released out of the cells primarily through ATP-binding cassette transporter A1 (ABCA1) (6, 7). Thus, the balance between synthesis and hydrolysis of CE conceivably governs the level of CE in macrophages.Inhibition of ACAT activity is shown to suppress CE accumulation with concomitant promotion of the net hydrolysis of ...

Identification of Neutral Cholesterol Ester Hydrolase, a Key Enzyme Removing Cholesterol from Macrophages

“…Second, HSL expression is regulated coordinately with nCEH activity in murine macrophages (13). Third, we (14) and others (15) demonstrated that overexpression of HSL is associated with increased hydrolysis of CE stores in THP-1 and RAW264.7 macrophages.…”

“…Tri[ 3 H]oleoylglycerol, cholesterol [1-14 C]oleate, and [1-14 C]oleic acid were purchased from GE Healthcare. LDL was isolated by ultracentrifugation and acetylated to prepare acetylated LDL (acLDL) as described previously (14).…”

“…The adenoviral vectors were linearized using restriction enzyme and transfected into HEK293 using Superfect reagent (Promega). Large scale production of high titer recombinant Ad-NCEH, Ad-HSL, or Ad-LacZ was performed as described elsewhere (14). The purified viruses were stored in 10% (v/v) glycerol/phosphate-buffered saline (PBS) at Ϫ80°C.…”

“…Probes for murine NCEH were originally prepared by reverse transcription-PCR from mouse peritoneal macrophage mRNA. Probes for HSL (exon 8 probe), TGH-1 were constructed from cDNA fragments amplified by reverse transcription-PCR using cDNA obtained from mouse adipose tissue as a template (14). After hybridization and washing, signals were visualized by exposure to a PhosphorImager Screen (FUJIFILM) and analyzed using BASTATION (FUJIFILM) software.…”

“…The supernatant was used as S-40 or S-100 cytosolic fraction, and the precipitates were resuspended and used as the microsomal fraction (14). Ten micrograms of proteins of various cellular fractions were separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane.…”