Atherosclerosis 152 2000 347 – 357
Effects of probucol on cholesterol metabolism in mouse peritoneal macrophages: inhibition of HDL-mediated cholesterol efflux
Toru Takemura , Masakazu Sakai, Hirofumi Matsuda, Takeshi Matsumura, Takeshi Biwa, Yoshichika Anami, Takeshi Nishikawa, Takayuki Sasahara,
Motoaki Shichiri
Department of Metabolic Medicine, Kumamoto Uni6ersity School of Medicine, Honjo
1
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1
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1
, Kumamoto
860
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8556
, Japan Received 20 March 1999; received in revised form 16 November 1999; accepted 13 December 1999
Abstract
Macrophage-derived foam cells are known to play an essential role in the development and progression of atherosclerotic lesions. Probucol prevents oxidative modification of low-density lipoprotein LDL and lowers plasma contents of LDL and
high-density lipoprotein HDL. A recent report using apoE − − mice demonstrated that probucol treatment enhanced atherosclerosis in apoE − − mice more rapidly than that in untreated apoE − − mice, and a reduction in plasma cholesterol
by probucol was not the cause of enhancement of atherosclerotic lesions in probucol-treated apoE − − mice. Moreover, probucol was reported to inhibit apoA-I mediated cholesterol efflux from mouse macrophages. These reports suggested that
probucol might directly affect cholesterol metabolism in mouse macrophages. Thus, we investigated the effects of probucol on cholesterol metabolism in mouse resident peritoneal macrophages. Probucol did not affect degradation of acetylated LDL
Ac-LDL, degradation of LDL and endogenous cholesterol synthesis in mouse macrophages. However, it significantly inhibited HDL-mediated cholesterol efflux. Moreover, probucol partially 30 inhibited the binding of HDL to mouse macrophages, and
significantly activated acyl-coenzyme A:cholesterol acyltransferase ACAT. Our results suggested that probucol inhibited HDL-mediated cholesterol efflux by inhibiting the binding of HDL to mouse macrophages and reducing HDL-accessible free
cholesterol content by ACAT activation, thereby worsening atherosclerotic lesions in apoE − − mice. However, it remains unclear whether probucol inhibits HDL-mediated cholesterol efflux from human macrophages. © 2000 Elsevier Science Ireland
Ltd. All rights reserved.
Keywords
:
Probucol; Mouse peritoneal macrophage; Acetylated low-density lipoprotein; High-density lipoprotein; Acyl-coenzyme A: cholesterol acyltransferase
www.elsevier.comlocateatherosclerosis
1. Introduction
The presence of a massive cluster of foam cells is a characteristic feature of atherosclerotic lesions [1]. Re-
cent immunohistochemical and pathological studies have revealed that foam cells appear in the early stages
of atherosclerotic lesions and are mainly blood mono- cyte-derived macrophages [2,3]. These cells play an
important role in the development and progression of atherosclerosis via production of various bioactive
molecules, such as growth factors and cytokines [1]. Chemically modified low-density lipoproteins modified
LDLs, such as acetylated LDL Ac-LDL and oxidized LDL Ox-LDL, are recognized by so-called scavenger
receptors [4], and undergo receptor mediated endocyto- sis, followed by intracellular delivery to lysosomes [5].
In these structures, cholesteryl esters in modified LDLs are hydrolyzed to free cholesterol by acid cholesterol
esterase ACEH in lysosome [6]. Free cholesterol thus formed is then transported across lysosomal membrane
into cytoplasm [7] where it is delivered to the endoplas- mic reticulum. Free cholesterol is re-esterified to
cholesteryl esters by acyl-coenzyme A:cholesterol acyl- transferase
ACAT, leading
to accumulation
of cholesteryl esters in cytoplasm. Cytoplasmic cholesteryl
esters undergo a continual shuttle between hydrolysis by neutral cholesteryl esterase NCEH and re-esterifi-
Corresponding author. Tel.: + 81-96-3735169; fax: + 81-96- 3668397.
0021-915000 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 9 9 0 0 5 0 0 - 6
cation by ACAT. This cholesteryl ester turnover was discovered by Brown et al. [8] and was termed
‘cholesteryl ester cycle’, which closely related to choles- terol efflux by high-density lipoprotein HDL [9,10].
Probucol [bis3, 5-d-tert-butyl-4-hydroxyphenylthio- propane] is a lipophilic agent with anti-oxidative prop-
erties. It is carried by lipoproteins, mainly in LDL, and is known to prevent oxidative modification of LDL by
its radical-scavenging phenol structure [11]. Oxidative modification of LDL is an early pathogenic event in the
development of atherosclerotic lesions.
Moreover, probucol is also known to lower plasma LDL levels by
a mechanism other than LDL receptor-mediated path- way [12], because probucol lowers LDL cholesterol in
homozygous LDL receptor-deficient patients. There- fore, probucol is expected to inhibit or reduce the
development of atherosclerosis. In fact, subsequent studies showed that probucol reduced atherosclerotic
lesions in Watanabe heritable hyperlipidemic WHHL rabbits
[13,14]. Furthermore,
probucol treatment
caused regression of subcutaneous and tendinous xan- thomas in hypercholesterolemic patients [15]. However,
a large-scale European clinical trial probucol quantita- tive regression Swedish trial, PQRST demonstrated
that probucol treatment did not produce a significant effect on atherosclerotic lesions of human femoral
artery, although it reduced cholesterol levels [16]. More- over, a recent report using apoE − − mice demon-
strated
that probucol
treatment enhanced
the development of atherosclerotic lesions more rapidly
than untreated apoE − − mice. In addition, probucol treatment also accelerated lesion development in apoE
+ − mice fed an atherogenic diet, indicating that the
adverse effect is not dependent on the complete absence of apoE. Interestingly, the plasma lipoprotein profile in
mice lacking both apoE and apoA-I was very similar to probucol-treated apoE − − mice, but did not acceler-
ate atherosclerotic lesions [17]. These reports suggested that probucol might directly affect the development of
atherosclerotic lesions, thereby worsening atherosclero- sis in apoE − − mice. In this regard, Tsujita and
Yokoyama [18] recently reported that probucol selec- tively inhibited apoA-I-mediated cellular lipid efflux
from cultured mouse macrophage-derived foam cells. Thus, it is possible that probucol may also affect
cholesterol metabolism in mouse macrophages.
In the present study, we investigated the effects of probucol on cholesterol metabolism in mouse peri-
toneal resident macrophages, and demonstrated that probucol inhibited HDL-mediated cholesterol efflux
from macrophages, which might be due to the inhibi- tion of binding of HDL to cells and reduction of
HDL-accessible free cholesterol content via activation of ACAT activity. This may be one of the underlying
mechanisms of the harmful effects of probucol on the development of atherosclerosis in apoE − − mice.
2. Materials and methods
