© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Probucol Enhances Selective Uptake of HDL-Associated Cholesteryl Esters In Vitro by a Scavenger Receptor B-I–Dependent Mechanism
From the College of Physicians and Surgeons of Columbia University, Department of Medicine, Division of Molecular Medicine, New York, NY. Dr Rinninger is presently at the Universität Hamburg, Krankenhaus Eppendorf, Medizinische Kernklinik und Poliklinik, Hamburg, Germany.
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Abstract |
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Abstract—Recently, the class B, type I scavenger receptor (SR-BI) has been shown to mediate the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CEs), ie, lipid uptake independent of HDL holoparticle uptake. In vivo, this selective uptake delivers CEs to the liver for excretion and to steroidogenic tissues for hormone synthesis. Probucol, a hydrophobic antioxidant drug, lowers plasma cholesterol in humans and rodents and may inhibit progression of atherosclerosis and postangioplasty restenosis. In this study, the effect of probucol on HDL selective CE uptake was investigated in mice and in cells expressing SR-BI. Probucol feeding lowered plasma HDL cholesterol and markedly increased selective CE uptake from HDL in the liver and adrenal glands. However, probucol did not alter SR-BI protein levels in membranes from these organs. When incubated with control Chinese hamster ovary (CHO) cells, HDL isolated from probucol-treated mice (P-HDL) and HDL from control mice (C-HDL) showed similar low selective uptake of CEs. However, when incubated with SR-BI–transfected CHO cells, P-HDL showed a 2-fold increase in selective uptake compared with C-HDL. In an adrenal cell line (Y1-BS1), which expresses SR-BI in an adrenocorticotropic hormone–inducible manner, P-HDL showed significantly greater selective CE uptake than did C-HDL, and the differential response was amplified by adrenocorticotropic hormone treatment. In contrast to P-HDL, incorporation of this compound into HDL in vitro did not result in stimulation of selective CE uptake by SR-BI–transfected CHO cells, even though a significant mass of probucol could be detected in the HDL preparation. The specific interaction of P-HDL with SR-BI in cell culture could be observed after only 24 hours of probucol feeding, when there were minimal changes in HDL size and composition. Thus, probucol or one of its metabolites increases selective CE uptake in vivo by modifying HDL in a way that causes enhanced interaction with SR-BI. The increased interaction of P-HDL with SR-BI in the liver and arterial wall may be partly responsible for the effects of probucol on atherosclerosis and restenosis.
Key Words: HDL • selective uptake • scavenger receptor BI • probucol
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Introduction |
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Plasma HDL plays a critical role in cholesterol metabolism in vivo. HDL removes cholesterol from cells in culture and from peripheral tissues.1 2 After cholesterol esterification, HDL-associated cholesteryl esters (CEs) can be transferred to other lipoprotein fractions by cholesteryl ester transfer protein (CETP) in some species3 or directly delivered to tissues.1 In rats, HDL-associated CEs can be taken up by the liver and steroidogenic tissues without parallel uptake of HDL apolipoproteins, and this metabolic pathway has been designated the selective CE uptake pathway.4 This selective uptake delivers CEs to steroidogenic tissues for hormone synthesis and to the liver, where HDL-derived cholesterol is either secreted into the bile, used for bile acid synthesis, or secreted in newly synthesized lipoproteins.5 The HDL-mediated transport of cholesterol from extrahepatic tissues to the liver, designated reverse cholesterol transport, is believed to play a critical role in whole-body cholesterol homeostasis.1
The class B, type I scavenger receptor (SR-BI) is a cell surface HDL receptor that mediates selective lipid uptake.6 In cultured cells, murine SR-BI, an 82-kDa glycoprotein that appears to be clustered in caveolae,7 binds HDL and mediates selective CE uptake from this lipoprotein fraction.6 In mice and rats, SR-BI is most abundantly expressed in the liver and steroidogenic tissues, which are the most active sites of HDL selective CE uptake.4 6 8 9 SR-BI expression in rodent steroidogenic tissues is coordinately regulated with steroid hormone synthesis by trophic hormones.10 11 Hepatic overexpression of murine SR-BI in mice substantially reduces plasma HDL and increases biliary cholesterol.12 Moreover, mice with induced mutations that attenuate expression of SR-BI show increased HDL levels and proportionately reduced tissue-selective uptake of HDL CEs.13 14 In summary, these studies show that SR-BI is a physiologically regulated HDL receptor that modifies HDL levels and mediates selective uptake of HDL lipids in vivo.
Probucol is a nonpolar antioxidant that substantially lowers plasma cholesterol.15 16 Besides LDL cholesterol, this drug reduces HDL cholesterol in humans and rodents.17 18 In parallel with a decrease in plasma HDL cholesterol, probucol increases the selective CE uptake from HDL by the liver in rats and to some extent in hepatic cells in culture.19 20 The antioxidant activity of probucol may prevent cellular lipid loading and the formation of foam cells in atherosclerotic lesions by limiting oxidative modification of LDL, thus minimizing macrophage uptake of cholesterol-rich lipoproteins.21 22 23 Interest in probucol was raised substantially by the recent demonstration that this compound significantly reduces the rate of coronary artery restenosis after coronary balloon angioplasty in humans.24
The hypothesis of the present study was that the increased selective uptake of HDL-associated CEs due to probucol treatment of mice would be mediated by an increase in SR-BI expression. When no change in SR-BI expression was found, we further explored the potential mechanism(s) by examining the interaction of HDL from probucol-treated animals (P-HDL) with cells expressing increased levels of SR-BI. These studies revealed that P-HDL enhances selective CE uptake by facilitating the interaction of HDL with SR-BI. These findings illuminate an important biological property of probucol that may account for some of its novel effects on restenosis and atherosclerosis.
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Methods |
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Animals
Adult female mice (C57BL/6, Taconic Farms, Germantown, NY) of 20 to 25 g body weight were used in all experiments. These animals were caged with alternating 12-hour light (7 AM to 7 PM) and 12-hour dark cycles. The mice had free access to food and water. In most cases, the animals were fed for 1 day or for 14 days a regular powdered mouse chow diet with no addition (control) or a chow diet containing 0.2% probucol (wt/wt, Sigma Chemical Co).18 All experiments with mice were approved by the Institutional Animal Care and Use Committee of Columbia University.
Lipoprotein Preparations
Mice were fasted for at least 4 hours before blood drawing.
Murine HDL was prepared in the density range d=1.063 to 1.21
g/mL from anticoagulated (EDTA) plasma of control mice by sequential
preparative ultracentrifugation according to standard
procedures.25 HDL was finally dialyzed against
phosphate-buffered saline (PBS) (pH 7.4) containing EDTA
(0.3 mmol/L) and NaN3 (0.02%). In some
cases, blood was obtained in parallel from control and probucol-treated
mice. Before blood harvest, the animals were fed for 24 hours a chow
diet supplemented or not with probucol (0.2% wt/wt).18
Compared with control mice, total plasma cholesterol was at
least 51% lower in mice fed for 24 hours a diet containing
probucol.
Preparation of Doubly Radiolabeled HDL
In brief, HDL protein was initially labeled by covalent
attachment of the intracellularly trapped
125I-N-methyl tyramine cellobiose
(125I-NMTC) ligand.26
Thereafter, 125I-NMTC-HDL was labeled with
[3H]cholesteryl oleyl ether
([3H]CEt, Amersham) to trace the CE
moiety.5 27 [3H]CEt was introduced
in a liposomal preparation and exchanged (6 hours, 37°C) into
125I-NMTC-HDL by using highly purified
recombinant human plasma CETP. The donor liposomes were separated from
labeled HDL by ultracentrifugation at
d=1.063 g/mL, and then HDL was reisolated by another spin at
d=1.21 g/mL to remove CETP. Thereafter the HDL preparation
was exhaustively dialyzed against PBS (pH 7.4) containing EDTA
(0.3 mmol/L) and sterile-filtered (0.45 µm). P-HDL was
radiolabeled exactly as described above for C-HDL, and this procedure
was done in strict parallel.
In some cases, probucol was incorporated into HDL in vitro after preparation. In brief, in one approach, probucol was introduced in an ethanolic solution. In this case, doubly radiolabeled HDL (1 mg) was incubated with (10 µmol/L) or without probucol in 26 mL of Dulbecco's modified Eagle's medium (DMEM) containing BSA (5 mg/mL), ethanol (1% vol/vol), and NaN3 (0.02%).20 28 After incubation (4 hours, 37°C), HDL was subsequently reisolated by ultracentrifugation and dialyzed (PBS). Alternatively, a lipid microemulsion with or without probucol and [3H]CEt was prepared as outlined by Tsujita and Yokoyama.29 This lipid emulsion was incubated (37°C) in PBS containing 125I-NMTC-HDL and CETP similarly as described above for double radiolabeling of HDL.5
Metabolism of Doubly Radiolabeled HDL in Mice
Experiments to investigate the plasma decay (fractional
catabolic rate, or FCR) of both HDL tracers and their tissue sites of
uptake were carried out as previously described.4 8 31 In
brief, mice were fasted for 12 hours before tracer injection and
throughout the 24-hour study period but had free access to water.
Doubly radiolabeled HDL was injected intravenously (iliac
vein) at 10 AM into the mice. Thereafter periodic blood
samples were drawn from a tail vein at 0.16, 0.5, 2.0, 5.0, 10.0, and
24.0 hours. Plasma samples were directly radioassayed for
125I and analyzed for
3H after lipid extraction.32
Twenty-four hours after tracer injection, the animals were anesthestized with isoflurane and exsanguinated. Then the vasculature was perfused with saline (50 mL per animal). Organs and gut contents were collected, weighed, homogenized, and radioassayed. The tissue content of 125I radioactivity was directly assayed and that of 3H was analyzed by liquid scintillation spectrometry after lipid extraction.4 32 As in previous studies, tracers in the gut contents were attributed to primary uptake by the liver.4 26
Calculations for the Experiments Performed With Mice
FCRs for both HDL-associated tracers
(125I-NMTC and [3H]CEt)
in mice were calculated by fitting the data from each animal separately
by two 2-pool models, 1 for each tracer. Each 2-pool model had a
circulating pool in equilibrium with a noncirculating
pool.33 In each case, it was confirmed that 2 pools fit
the data significantly better than did a 1-pool model; a third pool was
not required for any study.
Uptake of both HDL tracers by murine tissues was expressed in terms of organ FCRs.4 These organ FCRs were obtained by multiplying the plasma FCR of a given HDL tracer with the fraction of the tracer recovered in a specific tissue. The fraction of tracer uptake attributed to an organ was calculated as the radioactivity recovered in that organ divided by the total radioactivity recovered from all extravascular sources, including all tissues and gut contents.4 Thus, the organ FCRs represent the fraction of the plasma pool of the traced HDL component cleared per hour by a specific organ. 125I-NMTC, ie, the tracer of the protein moiety, represents HDL particle metabolism.5 Selective CE uptake was obtained as the rate of CE tracer uptake minus that due to HDL particle uptake (ie, 125I-NMTC).4 31
Detection of SR-BI Protein and mRNA
Anti–SR-BI antisera were prepared by immunization of rabbits
with a recombinant murine SR-BI fragment (amino acids 315 to 412) that
was prepared in a bacterial expression system and
purified.10 After murine tissue harvest, membranes were
prepared by ultracentrifugation from the liver or from
6 pooled adrenal glands.10 Tissues were
homogenized in PBS in the presence of protease
inhibitors (0.5 µg/mL leupeptin, 1 µg/mL aprotinin, 1
µg/mL pepstatin A, 0.2 mmol/L PMSF, and 1 mmol/L EDTA).
Immunoblotting was performed with liver and adrenal membrane preparations.10 Routinely, 50 µg of liver membrane protein or 20 µg of adrenal membrane protein was loaded per lane. Membranes were subjected to 7.5% SDS–polyacrylamide gel electrophoresis (PAGE) under reducing conditions. SR-BI protein immunoreactivity was identified at its authentic molecular size (82 kDa).6 SR-BI expression levels were determined by chemiluminescence detection (Amersham). SR-BI mRNA abundance in the adrenal glands and liver was determined by RNAse protection assay as previously described10 using ß-actin as an internal standard.
Cell Culture
Control Chinese hamster ovary (CHO) cells or CHO cells stably
transfected with a murine SR-BI cDNA (overexpressing SR-BI CHO cells)
were obtained and maintained as previously described.34
Y1-BS1 cells5 11 35 were maintained in Ham's F-10 medium
(Gibco) supplemented with heat-inactivated horse serum
(12.5% vol/vol, Gibco), heat-inactivated FBS (2.5%
vol/vol, Gibco), L-glutamine (2 mmol/L), penicillin
(100 U/mL), and streptomycin (100 µg/mL). Human Hep 3B hepatoma cells
were cultured in DMEM supplemented with FBS (10% vol/vol) and
antibiotics.27
Uptake Assay for Doubly Radiolabeled HDL by Cells in
Culture
When the cells had reached near-confluence, the culture medium
was removed and cells were washed twice with PBS. Thereafter the
cells were preincubated (37°C) for 16 hours in the respective
serum-free medium (Ham's F-12, Ham's F-10, or DMEM) supplemented with
5 mg/mL BSA (Sigma) and antibiotics. In some experiments with Y1-BS1
cells, adrenocorticotropic hormone (ACTH, 100 nmol/L, Sigma) was
present in this incubation medium as well. After this 16-hour
preincubation period, the medium was removed and the cells were washed
twice with PBS. Uptake of HDL tracers was then initiated by incubation
(4 hours, 37°C) of the cells in the respective medium containing BSA
(5 mg/mL) and doubly radiolabeled HDL (10 µg HDL protein per
mL).27 Thereafter the medium was aspirated and cells were
washed twice with PBS. Cells were then released from the wells by
incubation (5 minutes, 37°C) in saline containing trypsin/EDTA
(0.05% wt/vol trypsin and 0.53 mmol/L EDTA, total volume
of 1 mL; Gibco). Trypsin activity was quenched by the addition of 1 mL
PBS containing 50 mg/mL BSA. The cell suspension was transferred to
tubes by rinsing the wells with PBS and pelleted by low-speed
centrifugation. After aspiration of the supernatant,
the cell pellets were washed with 5 mL PBS. Finally, the cell pellets
were dissolved in 0.1N NaOH solution, followed by sonication. Aliquots
of the cell suspensions were directly assayed for
125I in a gamma spectrometer.
[3H]CEt was extracted32 from the
cell suspensions, followed by liquid scintillation spectrometry, and
protein was analyzed as described.36
Calculations for Uptake of HDL-Associated Tracers by Cells in
Culture
In experiments with cultured cells, the cellular uptake of each
lipoprotein tracer is shown in terms of apparent HDL particle uptake
expressed in terms of HDL protein.5 This allows direct
comparison of uptake of both tracers on a common basis. HDL selective
CE uptake is calculated as the difference in
[3H]CEt uptake in excess of
125I-NMTC, assuming that
125I-NMTC uptake traces HDL particle
uptake.5 27
Nondenaturing Gradient PAGE
Mouse HDL and mouse plasma were analyzed by gradient
PAGE performed under nondenaturing conditions37 (Lipogel,
Zaxis). Lipoprotein size was estimated by comparison with standard
proteins (high-molecular-weight calibration kit, Pharmacia). Finally,
the gels were stained with the lipoprotein-specific stain Sudan black
B.
Other Techniques
Protein was determined according to the method described by
Lowry and coworkers.36 For analytical purposes, HDL was
isolated by ultracentrifugation.25 Total
cholesterol, unesterified cholesterol,
phospholipid, and triglycerides were measured using
commercial kits (Wako). Esterified cholesterol
represents the difference between total and unesterified
cholesterol. SDS-PAGE followed the procedure described by
Laemmli38 and coworkers. From HDL, after extraction with
chloroform/methanol, probucol was analyzed by
high-performance liquid chromatography.
Statistical Analysis
Values shown are mean±SEM. Statistical significance was
determined by two-tailed Student's t test for unpaired
data.
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Results |
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Mice were fed for 14 days a chow diet supplemented with probucol (0.2% wt/wt) or control chow.18 After this period, probucol decreased total plasma cholesterol by 83% and plasma HDL cholesterol by 82% compared with corresponding values in control mice (Table 1
).
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To explore the mechanism of the probucol-induced decrease in HDL cholesterol, C-HDL isolated from control mice was doubly radiolabeled in the protein and CE moieties with 125I-NMTC and [3H]CEt, respectively.5 26 These HDL tracers are intracellularly trapped at their sites of tissue uptake.5 26 This labeled HDL was injected intravenously into both control and probucol-fed mice. Previous experiments suggested a rapid equilibration of control HDL with HDL in probucol-treated mice.19
In control mice, the plasma FCR for HDL-associated
[3H]CEt was higher compared with
125I-NMTC, ie, the protein tracer that
represents the metabolism of holo-HDL particles
(Figure 1 and Table 2). The difference between both FCRs
represents lipid catabolism independent of holoparticle
catabolism, ie, selective CE uptake from HDL by the whole
body.8 Probucol treatment of mice increased the plasma FCR
for HDL-associated [3H]CEt 
3-fold compared
with control mice (Figure 1 and Table 2). This drug also
enhanced the plasma FCR of the protein tracer
125I-NMTC by 2-fold (Table 2). The
fractional increase in FCR due to probucol was thus higher for
[3H]CEt than for
125I-NMTC. The difference in catabolic rates
between the 2 tracers ([3H]CEt minus
125I-NMTC), ie, whole-body selective lipid
uptake, increased 6-fold in probucol-treated mice (Table 2).
These results show that probucol increases the selective catabolism of
plasma HDL CEs in mice.
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Twenty-four hours after HDL injection, the tissue sites of tracer
uptake were analyzed.4 31 For the liver of control
mice, the FCR for HDL-associated [3H]CEt was
substantially higher than that of 125I-NMTC
(Figure 2, left). Previous studies
established that the organ FCR for 125I-NMTC
represents HDL holoparticle
metabolism.5 Therefore, the higher rate of
hepatic [3H]CEt clearance indicates that this
organ selectively takes up CEs from HDL in control
mice.4 8 Probucol treatment of mice increased the hepatic
FCR for HDL-associated [3H]CEt by 4-fold,
and this compound stimulated uptake of 125I-NMTC
by 2.5-fold (Figure 2, left). Again, the difference between
[3H]CEt and 125I-NMTC
represents selective CE uptake from HDL by the liver, and this
metabolic parameter increased 4-fold as a
result of probucol treatment.
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Uptake of HDL-associated tracers by mouse adrenal glands in vivo is
shown in Figure 2
(right). In control mice, the adrenal FCR for
HDL-associated [3H]CEt was 5-fold in excess
of that of 125I-NMTC, indicating selective CE
uptake from HDL (Figure 2, right). Probucol treatment of mice
increased the adrenal FCRs for [3H]CEt and
125I-NMTC by 9- and 5-fold, respectively. As a
result of these changes, selective CE uptake from HDL, ie, the
difference between [3H]CEt and
125I-NMTC, by adrenal glands increased 10-fold
in probucol-treated mice compared with control animals (Figure 2, right). Changes in uptake rates were minor in the kidneys,
lungs, heart, spleen, muscle, adipose tissue, and carcass (data not
shown). In summary, in mice, probucol induced a significant decrease in
plasma HDL cholesterol and markedly increased the selective
CE uptake from HDL in the liver and adrenal glands.
SR-BI plays an essential role in selective CE uptake from HDL. To see
whether the expression of this membrane protein was altered in
probucol-fed mice, Western blotting was performed on membranes prepared
from the liver and adrenal glands by using an SR-BI–specific antiserum
(Figure 3). There was no detectable
effect of probucol treatment on SR-BI expression levels. Similar data
were obtained when SR-BI mRNA was quantified in these organs by RNAse
protection analysis (data not shown).10 Thus, even
though the liver and adrenal glands demonstrated a substantial increase
in HDL selective CE uptake in response to probucol, this increment was
not the result of an increase in SR-BI expression.
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To further explore the mechanism of the probucol-induced increase in
HDL selective CE uptake, HDL was prepared from mice fed probucol for 24
hours (ie, P-HDL) or from control mice (ie, C-HDL) and doubly labeled
with 125I-NMTC and
[3H]CEt as described above. In a typical
preparation, doubly labeled P-HDL contained 14.8 µmol/L probucol
per mg HDL protein (determined by high-performance liquid
chromatography), whereas in C-HDL no probucol could be
detected. Doubly labeled P-HDL and C-HDL were incubated with control
CHO cells or CHO cells stably transfected with an SR-BI cDNA (Figure 4).34 Uptake of both tracers
by cells was determined, and the data were expressed in terms of
apparent HDL particle uptake.5 27 Previous studies
established that 125I-NMTC traces HDL
holoparticle metabolism, and the difference in apparent HDL
particle uptake between [3H]CEt and
125I-NMTC represents selective CE
uptake.5 For labeled C-HDL, in both lines of CHO cells,
apparent HDL particle uptake according to
[3H]CEt was in excess of that due to
125I-NMTC. Thus, both types of CHO cells
selectively took up HDL-associated CEs ([3H]CEt
minus 125I-NMTC). As expected, in CHO cells with
high SR-BI expression, the rate of apparent selective CE uptake from
HDL was increased 5-fold compared with control cells with low SR-BI
expression (Figure 4).
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CHO cells with low SR-BI expression were incubated in medium containing
labeled P-HDL (Figure 4). In this case, apparent HDL particle
uptake (125I-NMTC) and apparent HDL selective CE
uptake ([3H]CEt minus
125I-NMTC) were only marginally different between
P-HDL and C-HDL. However, for cells with high SR-BI expression, there
was an 9-fold increase in [3H]CEt uptake
from P-HDL. Compared with C-HDL, this represented a
2.1-fold increment in apparent HDL selective CE uptake. In contrast,
P-HDL and C-HDL yielded a similar rate for particle uptake
(125I-NMTC) in cells with high SR-BI expression
(Figure 4). This experiment indicates that P-HDL demonstrates
enhanced selective CE uptake as a result of its interaction with
SR-BI.
To further evaluate the possibility that SR-BI and P-HDL
synergistically increase the rate of HDL selective CE uptake by cells,
selective uptake from C-HDL and P-HDL was evaluated in murine Y1-BS1
adrenocortical tumor cells.5 11 35 These cells have a
relatively high basal expression of SR-BI and show induction of SR-BI
protein and mRNA after treatment with ACTH11 34 ; parallel
effects were also observed for selective CE uptake.5 In
these cells, P-HDL displayed somewhat higher (1.3-fold) selective
uptake than did C-HDL under basal conditions (Figure 5). However, after ACTH treatment, the
increment in selective uptake was more pronounced for P-HDL than for
C-HDL. Again, there was no difference in P-HDL and C-HDL particle
uptake, as assessed by the protein label
(125I-NMTC). In parallel experiments, ACTH
treatment resulted in a 2-fold induction of SR-BI protein levels as
determined by Western blots (data not shown) and as reported
previously.10 11 Also, this experiment suggests that P-HDL
interacts with SR-BI to enhance selective CE uptake from HDL.
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In the experiments described above, probucol was introduced
biologically into HDL. However, this drug can be incorporated in
lipoproteins in vitro.20 28 29 This was done by exchange
from a liposomal donor particle with CETP (see Methods). In a typical
preparation, the final doubly labeled P-HDL contained 76.2 µmol
probucol per mg HDL protein, whereas no probucol could be detected in
C-HDL. Control CHO cells or CHO cells overexpressing SR-BI were
incubated (37°C, 4 hours) with C-HDL (10 µg protein per mL) or
P-HDL (10 µg HDL protein per mL) under experimental conditions (data
not shown) similar to those in Figure 4.29 However,
in this case, there was no enhancement of selective CE uptake for P-HDL
compared with C-HDL in either cell type. An identical result was
obtained when probucol was incorporated into HDL by ethanol injection
(data not shown).20 In this case, in a typical
preparation, the final doubly labeled P-HDL preparation contained
240 µmol probucol per mg HDL protein, whereas no probucol could
be detected in C-HDL. These results indicate that in vivo, probucol
modifies HDL to enhance its interaction with SR-BI, but not in vitro.
To better understand this observation, P-HDL was analyzed and
compared with C-HDL.
The chemical composition of doubly radiolabeled C-HDL or P-HDL (in vivo feeding, 24 hours) was analyzed. Compared with C-HDL, triglycerides were increased by 43% in P-HDL (data not shown). However, protein, esterified and unesterified cholesterol, and phospholipid differed only marginally between both HDL preparations. The possibility was also considered that probucol modifies the apolipoprotein composition of HDL. Therefore, C-HDL and P-HDL were analyzed by SDS-PAGE under reducing conditions. Probucol had no effect on HDL apolipoprotein composition compared with C-HDL (data not shown). Moreover, native PAGE showed no change in HDL size in mice treated for 24 hours with probucol compared with C-HDL (data not shown). In contrast to these findings, HDL from mice fed probucol for 2 weeks showed a decrease in overall particle size as assessed by native PAGE (data not shown), consistent with previous reports.39
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Discussion |
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The recent discovery that SR-BI mediates selective uptake of HDL CEs by murine tissues suggested that this molecule might be responsible for the increased selective uptake previously described in probucol-treated animals18 19 and confirmed herein. Surprisingly, there was no effect of this drug on SR-BI expression level. However, P-HDL displayed increased selective CE uptake when incubated with cells in an SR-BI–dependent fashion. This effect could not be reproduced by direct addition of probucol to HDL in vitro, even though these HDL preparations contained substantially more probucol than did P-HDL. Thus, it appears that an in vivo modification of HDL substantially improves the SR-BI–mediated selective uptake process. Such an enhancement of the interaction between plasma lipoproteins and SR-BI may in part be responsible for the antiatherogenic properties of probucol observed in many animal models.22 40 41
Recent evidence obtained in cell culture and in genetically manipulated mice indicates that SR-BI is able to mediate the selective uptake of HDL CEs and is likely to be the major molecule responsible for this process in the liver and adrenal gland.6 10 11 12 14 Mice with attenuated expression of SR-BI show a proportional reduction of HDL selective uptake in the liver,14 and an SR-BI antibody largely blocks selective uptake in cultured adrenal cells.42 These results suggest that the effects of probucol on selective uptake are likely to involve SR-BI. This protein is upregulated in the adrenal gland and other steroidogenic tissues in response to hormonal stimulation and alterations in cellular cholesterol stores.9 10 11 Thus, we had anticipated that SR-BI might be upregulated by probucol treatment. Unexpectedly, there was no evidence for regulation of SR-BI in the liver or adrenal gland, even though selective CE uptake by these organs was substantially increased. These observations suggested that mechanisms independent of the regulation of SR-BI expression must contribute to the probucol-induced increase in HDL selective CE uptake.
A highly plausible explanation was provided by cell culture experiments that showed an enhancement of selective uptake for P-HDL compared with C-HDL. This enhancement was observed only in cells with relatively high levels of SR-BI expression, ie, SR-BI–transfected CHO cells and Y1-BS1 adrenocortical tumor cells.11 34 In the latter cells, the enhancement of selective uptake from P-HDL was increased by ACTH treatment, in parallel with increased SR-BI expression levels.11 34 By contrast, in cells with very low levels of SR-BI expression (control CHO cells), P-HDL and C-HDL showed similar low levels of selective uptake. These findings suggest a specific enhancement of SR-BI–mediated selective uptake by P-HDL. The mechanism of such an effect is uncertain, but it is interesting to note that P-HDL and C-HDL demonstrated similar uptake of the nondegradable protein label 125I-NMTC in SR-BI–transfected CHO cells and in ACTH-treated Y1-BS1 cells, suggesting that probucol does not increase binding or uptake of HDL protein.
Interestingly, only P-HDL showed enhanced interaction with SR-BI–expressing cells; this effect was not seen when probucol was added to HDL in vitro, even though substantial amounts of probucol could be detected in the final, labeled HDL preparations.20 29 This suggested either that HDL was modified in vivo as an indirect result of probucol administration or that P-HDL carries an active metabolite formed in vivo. Although prolonged probucol administration results in changes in HDL size and composition,18 39 murine P-HDL isolated after only a 24-hour treatment was identical in size and apolipoprotein composition to C-HDL and showed only a minor enrichment in triglycerides. Nonetheless, P-HDL displayed an enhancement of selective uptake in SR-BI–expressing cells. Although triglycerides have a fluidizing effect on lipoprotein core lipids,43 it seems unlikely that these minor compositional changes would be responsible for the enhanced interaction with SR-BI. It is known that the bulk of probucol transported in the blood is found in lipoproteins, along with several probucol metabolites.44 Some hydrophobic probucol metabolites have been shown to act as potent fluidizing agents when mixed with CEs.16 Thus, our results could indicate that a probucol metabolite carried in P-HDL may be responsible for enhanced interaction with cellular SR-BI, leading to increased selective uptake of HDL CEs.
It appears likely that the markedly increased (5-fold in the liver and 10-fold in the adrenal gland) selective uptake of HDL CEs accounts for a major part of the HDL cholesterol–lowering effect of probucol. The cell culture results suggest that these in vivo effects may in part reflect the stimulation of selective uptake by SR-BI in the liver. However, the magnitude of the effect in vivo was larger than that seen in cell culture (2-fold). This could be, because the turnover studies were conducted after 2 weeks of probucol treatment, whereas P-HDL for cell culture experiments had to be used 1 day after initiating probucol treatment. Alternatively, there could be additional mechanisms operating in vivo.
Although the current studies deal with HDL, probucol also lowers LDL cholesterol and stimulates catabolism of LDL CEs by the liver.45 46 SR-BI is known to bind LDL as well as HDL.47 Recent studies in SR-BI–transgenic mice show a marked reduction in LDL cholesterol and apo B levels compared with those in controls.48 A mechanism similar to that observed here involving interaction of LDL and the hepatic SR-BI could be responsible for the LDL cholesterol–lowering effects of probucol, which can occur in the absence of LDL receptors.45 46
In many animal models, probucol shows marked antiatherogenic effects.22 40 41 Probucol also causes regression of xanthomas and xanthelasmas in familial hypercholesterolemic and non–familial hypercholesterolemic subjects.49 However, the effects of probucol on atherosclerosis are complex. In the human Probucol Quantitative Regression Swedish Trial (PQRST), probucol did not influence femoral artery atherosclerosis as assessed by angiography.50 Paradoxically, probucol actually increased the extent and developmental stage of atherosclerosis in apo E–knockout mice by an apo A-I–independent mechanism.51 In primates, rabbits, and mice, probucol appears to have distinctive effects on the cellularity of lesions by decreasing the population of macrophage foam cells while increasing the prominence of smooth muscle cells and extracellular matrix deposition.52 53 In numerous studies of rabbits, probucol reduced the extent of atherosclerosis and appeared to be superior to other antioxidants such as vitamin E or probucol analogues.22 40 54 55 This result suggested either that the effects of antioxidants on atherosclerosis need to surpass a threshold level and that probucol is more effective than other antioxidants or that there are distinct biological effects of probucol.44 55 The present study describes a novel biological effect of probucol and thus supports the latter explanation. Enhanced reverse cholesterol transport by the SR-BI–dependent mechanism or as a result of increased levels of plasma CETP56 may explain the more potent effects of probucol on atherosclerosis. SR-BI increases the transport of HDL cholesterol across the liver into bile.12 Moreover, SR-BI appears to be expressed in foam cells in atheromatous lesions,34 and SR-BI facilitates the HDL-mediated efflux of cholesterol from cells.34 HDL from probucol-treated humans shows enhanced ability to promote removal of cholesterol from macrophage foam cells.39 Thus, the enhanced interaction of P-HDL with SR-BI may favor cholesterol removal from macrophage foam cells, as well as increased cholesterol transport across the liver. The enhanced interaction between P-HDL and SR-BI may result in improved reverse cholesterol transport and may underlie the antiatherogenic properties of probucol.
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Acknowledgments |
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Can Bruce, Columbia University, donated purified CETP and assisted in the production of graphics. N-Methyl tyramine cellobiose for lipoprotein labeling was a gift of Ray C. Pittman, Datil, NM. Anatol Contush and Willi Weber, University of Hamburg, Germany, analyzed probucol by HPLC. Support by these scientists is gratefully acknowledged. Stimulating discussions with Yu Sun and Yong Ji, Columbia University, are acknowledged as well.
Received June 8, 1998; accepted October 21, 1998.
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