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INTRODUCTION |
High density lipoprotein
(HDL)1 may exert its
anti-atherogenic effects by various mechanisms (1, 2). First, reverse
cholesterol transport, as originally proposed by Glomset (3), is a
widely accepted mechanism of anti-atherogenic action. In this concept, HDL accepts excessive cholesterol from extrahepatic cells for transport
to the liver parenchymal cells (3, 4). The direct uptake of native HDL
cholesteryl ester (HDL-CE) by liver parenchymal cells is fundamentally
different from that of the classical LDL receptor pathway in that
HDL-CEs are taken up selectively without simultaneous uptake of the
holoparticle (5, 6). This so-called selective uptake of HDL-CE in the
liver parenchymal cells is efficiently coupled to bile acid formation
and secretion (4). A second mechanism for the anti-atherogenic action
of HDL is related to its antioxidative properties (7). Several
mechanisms have been proposed by which HDL can prevent oxidative damage
of LDL. One of the possible mechanisms that have been suggested
involves the transfer of reactive oxidized lipids from LDL to HDL and
the subsequent efficient transport to the liver for biliary secretion
(2, 8, 9). The rapid removal of the oxidized lipids may prevent the
propagation of an oxidation cascade in LDL. Bowry et al.
(10) showed that HDL is the predominant carrier of cholesteryl ester hydroperoxides (CEOOH) in humans. The first step in LDL oxidation involves hydroperoxide formation, and it is suggested that HDL may
accept CEOOH from LDL and that this process is possibly mediated by
cholesteryl ester transfer protein (11). Moreover, a HDL-associated hydroperoxide-reducing activity was found that converted the reactive CEOOH to the less reactive cholesteryl ester hydroxides (CEOH) (9).
Previous studies (2) have shown that oxidized cholesterol esters are
taken up extremely efficiently as compared with native cholesterol
esters by the liver parenchymal cells and this uptake is coupled to an
increased rate of biliary secretion, indicating that the efficient
liver uptake indeed might lead to the irreversible removal from serum
of the reactive oxidized cholesterol esters in vivo. The
hypothesis was put forward that this antioxidant mechanism may use the
same selective uptake pathway as that used during normal reverse
cholesterol transport but with a much higher efficiency (2).
Acton et al. (12) recently provided the first evidence that
scavenger receptor class BI (SR-BI), a member of the CD 36 family (13),
binds HDL and can also mediate the selective uptake of HDL-CE. SR-BI
was found to bind a broad spectrum of ligands, including both modified
and native lipoproteins and anionic phospholipids (14). The binding of
HDL to SR-BI was shown to be mediated by the major apolipoproteins of
HDL, e.g. apoA-I, apoA-II, and apoC-III (15). We showed
recently that the selective uptake of HDL-CE by isolated rat liver
parenchymal cells can be inhibited completely by ligands specific for
SR-BI (16), indicating that the expression of SR-BI can be solely
responsible for the selective HDL-CE uptake in this cell type.
In vivo, SR-BI is expressed in the steroidogenic organs and
liver of rodents (12, 17, 18), which all display selective uptake of
HDL-CE. In the steroidogenic tissues, SR-BI expression is coordinately
regulated with the steroidogenesis by adrenocorticotropic hormone,
human chorionic gonadotropin, and estrogen (17, 19). Furthermore, SR-BI
expression in adrenals is up-regulated in apoA-I knockout mice, hepatic
lipase knockout mice, and lecithin:cholesterol acyltransferase knockout
mice (18, 20). Unlike the steroidogenic tissues, SR-BI expression in
the liver is down-regulated by estrogen treatment of rats (17). We
showed recently that the down-regulation of SR-BI expression in the
liver is limited to the parenchymal cells and is correlated with a
decrease in the selective HDL-CE uptake (21). Surprisingly, SR-BI
expression and the selective HDL-CE uptake is up-regulated in Kupffer
cells after 17-
-ethinyl estradiol (EE) treatment or a high
cholesterol diet, pointing to a different regulatory response in tissue
macrophages (Kupffer cells) as compared with parenchymal cells
(21).
Our present goal was to analyze whether SR-BI may mediate the increased
hepatic uptake of oxidized cholesterol esters in vitro and
in vivo. We investigated the potential role of scavenger
receptor BI in the efficient removal of HDL-CEOH by rat liver cells by performing in vitro competition studies with ligands
specific for scavenger receptor BI. In vivo, the effect of
the regulation of SR-BI in liver cells by EE treatment or a high
cholesterol diet on the selective uptake in vivo of oxidized
cholesterol esters was studied. Finally, the uptake of both native and
oxidized HDL cholesterol esters was determined in SR-BI-transfected CHO
cells. These experiments indicate that SR-BI can mediate the highly
efficient selective uptake of oxidized cholesterol esters from HDL.
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EXPERIMENTAL PROCEDURES |
Materials--
[Cholesteryl-1,2,6,7-3H(N)]-linoleate
was purchased from NEN Life Science Products.
[1
,2
(n)-3H]Cholesteryl linoleate
([3H]CH18:2) and 125I (carrier free) in NaOH
were obtained from Amersham Pharmacia Biotech.
2,2'-Azobis-(2,4-dimethyl-valeronitrile) was purchased from Polyscience
(Warrington, Florida).
22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)(NBD)-23,24-bisnor-5-cholen-3
-yl linoleate and carboxyfluorescein diacetate was obtained from Molecular Probes (Eugene, OR). Egg yolk phosphatidylcholine was obtained from
Fluka (Buchs, Switzerland) the phospholipid, cholesterol oxidase-peroxidase aminophenazone, and glycerolphosphate
oxidase-peroxidase aminophenazone kits from Boehringer Mannheim.
Ethylmercurithiosalicylate (thimerosal), bovine serum albumin (BSA)
(fraction V), and collagenase type I and type IV were from Sigma.
Dulbecco's modified Eagle's medium (DMEM) was from Life Technologies,
Inc. All other chemicals were of analytical grade.
Label
Preparation--
[3H]Cholesteryl-linoleate-hydroperoxide
([3H] Ch18:2-OOH) was prepared by peroxidation of
(cholesteryl-1,2,6,7-3H(N))-linoleate with a
lipid-soluble peroxyl radical generator 2,2'-azobis-(2,4-dimethyl-valeronitrile). 200 µCi of
[3H]Ch18:2 in toluene (final volume, 1 ml) was
peroxidized with 2,2'-azobis-(2,4-dimethyl-valeronitrile) (0.4 M at 37 °C for 5 h). The incubation mixture was
dried under nitrogen and purified by reversed phase HPLC on an LC-18
column as described in Ref. 22. The fraction eluting between 8 and 9 min (corresponding to the retention time of an unlabeled Ch18:2-OOH
standard) was collected and dried in a SpeedVac (Savant, Bierbeek,
Belgium). The resulting [3H]Ch18:2-OOH was then
chemically reduced to the corresponding [3H]Ch18:2-OH by
NaBH4 (10 mg) reduction in methanol (1 ml) on ice (1 h) as
described by Van Kuijk et al. (23).
[3H]Ch18:2-OH was extracted into hexane and purified in a
second HPLC step (LC-18 column, mobile phase
acetonitrile/isopropanol/H2O, 22:27:1, v/v) (24).
Fluorescent NBD-cholesteryl linoleate was oxidized with 70%
tert-butyl OOH and 100 µM
Fe(II)SO4 over night at 45 °C. This method yielded a
100% oxidation of the fluorescent cholesterol ester as checked by TLC.
Isolation and Labeling of HDL--
Human HDL was isolated from
the blood of healthy volunteers by differential ultracentrifugation as
described by Redgrave et al. (25). HDL (1.063<
d < 1.21) was dialyzed against phosphate-buffered saline/1 mM EDTA and labeled with either
[3H]Ch18:2 or [3H]Ch18:2-OH by exchange
from donor particles as reported previously (1).
HDL was labeled by incubating HDL with donor particles (mass ratio of
HDL protein:particle phospholipid, 8:1) in the presence of human
lipoprotein-deficient serum as cholesteryl ester transfer protein
source (1:1 v/v) for 8 h at 37 °C in a shaking water bath under
argon as described (2, 16). Subsequently, the labeled HDL was dialyzed
against phosphate-buffered saline/1 mM EDTA and passed
through a heparin-Sepharose affinity column to remove apoE-containing particles (26). After the labeling procedure, the radiolabeled HDL was
checked for hydrolysis of the cholesteryl ester labels by a Bligh and
Dyer extraction (27) followed by thin layer chromatography. The effect
of the labeling procedure on HDL was analyzed by measurement of
phospholipid, cholesterol, cholesteryl ester, and triglyceride content
(with the phospholipid, cholesterol oxidase-peroxidase aminophenazone,
and glycerolphosphate oxidase-peroxidase aminophenazone kits,
respectively). The density, electrophoretic
-mobility, and particle
size (photon correlation spectroscopy, System 4700 C, Malvern
Instruments) were also analyzed. Labeled HDL was only used when there
was no change observed in the measured composition or physical
characteristics as compared with the original unlabeled HDL batch.
Additionally, to exclude the possibility that endogenous HDL lipids or
tracers were oxidized during the labeling procedure, HPLC analyses of
lipid extracts obtained from [3H]Ch18:2-labeled HDL were
performed. HDL lipids were extracted into methanol/hexane (1:5), and
the hexane phase, containing the neutral lipids, was dried and analyzed
by HPLC as described (2). HDL was iodinated by the ICl method of
McFarlane (28) as modified by Bilheimer et al. (29).
Before use, LDL was dialyzed against phosphate-buffered saline with 10 µM EDTA. Acetylation of LDL was performed by acetic anhydride as described (30). LDL was oxidized by exposure to CuSO4 as described in detail earlier (31).
Phospholipid Liposome Preparation--
Unilamelar liposomes were
obtained by sonication of egg yolk
phosphatidylcholine/phosphatidylserine/cholesterol in a molar ratio of
1:1:1 as described previously (16).
Liver Association and Tissue Distribution--
Male Wistar WU
rats (200-250 g) were anesthetized by intraperitoneal injection of 20 mg of Nembutal. Body temperature was maintained with a heating lamp.
After opening of the abdomen, radiolabeled HDL was injected into the
inferior vena cava. At the indicated time point, blood sampling and
adrenal and liver excision were performed as described previously
(2).
Hepatic Cellular Distribution--
The hepatic cellular
distribution of HDL was studied by using a low temperature cell
isolation technique as described (32). By means of centrifugal
elutriation, the Kupffer cells were purified from the non-parenchymal
cell preparation (33). The purity of each cell fraction was checked by
light microscopy after staining for peroxidase activity. Cellular
cholesterol concentrations were measured with a commercial kit as
mentioned above after a Bligh and Dyer extraction (27) of the cellular lipids.
In Vitro Studies with Freshly Isolated Rat Liver
Cells--
Liver cells were isolated by perfusion of the livers of
male Wistar WU rats (200-250 g) with collagenase at 37 °C as
described (33). By means of centrifugal elutriation, the Kupffer cells were purified from the non-parenchymal cell preparation (33). The
viability (>95%) of the obtained parenchymal cells was checked by
trypan blue exclusion. The cells from the last centrifugation step were
resuspended in oxygenated DMEM supplemented with 2% BSA, pH 7.4. For
in vitro competition studies, the liver cells were incubated
with the indicated amount of radiolabeled HDL and competitors for 180 min in 1 ml of DMEM containing 2% BSA at 37 °C. The temperature
dependence studies were performed at the indicated temperatures for 15 min. All cell incubations were performed in a circulating laboratory
shaker (Adolf Kühner AG, Basel, Switzerland) at 150 rpm. Every
hour, the incubations were briefly oxygenated. The viability of the
cells remained higher than 88% during these long term incubations
(33). After incubation, the cells were centrifuged for 2 min at 50 × g for parenchymal cells and 500 × g for
Kupffer cells in an Eppendorf centrifuge and washed two times in 50 mM Tris-HCl, 0.15 M NaCl, 0.2% BSA, pH 7.4 at
4 °C. Subsequently, the cell pellet was washed in a similar medium
without BSA. The cells were lysed in 0.1 N NaOH, and the
protein content and radioactivity were determined.
SR-BI Assays in Transfected CHO Cells--
ldlA cells (clone 7),
an LDL receptor-deficient CHO line (provided by M. Krieger) were
cultured in Ham's F-12 medium containing 5% (v/v) fetal bovine serum,
2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml
streptomycin. Stable transfectants expressing mouse SR-BI were prepared
as described previously using the expression vector pCMV5 (37).
Expressing lines were isolated and maintained in medium containing 0.5 mg/ml G-418. Binding and uptake assays with transfected CHO cells were
carried out following the procedure of Acton et al. (12) as
described previously (37).
Confocal Microscopy--
Freshly isolated parenchymal cells were
incubated on glass coverslips in 6-well plates (Costar, Cambridge MA)
for 2 h at 4 °C in DMEM supplemented with 2% BSA and the
indicated amount of oxidized NBD cholesteryl linoleate-labeled HDL or
carboxyfluorescein diacetate. Cells on the coverslips were transferred
to a Zeiss IM-35 inverted microscope (Oberkochen, Germany) with a × 63, NA 1.4 planapochromatic objective that was equipped with a
Bio-Rad MRC600 confocal visualization system. The microscope was fitted with a incubation chamber to allow incubation of the cells at 37 °C.
Quantification of SR-BI Expression--
The expression levels of
SR-BI in membranes of isolated parenchymal and Kupffer cells were
measured on Western blot as described (21).
Protein Determination--
Protein was determined according to
Lowry et al. (34) with BSA as standard.
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RESULTS |
Inhibition of Selective Uptake of HDL-CEOH by Rat Liver Parenchymal
Cells by Substrates of Scavenger Receptor BI--
We showed earlier
that the selective uptake of HDL-CE by parenchymal cells could almost
be completely inhibited by OxLDL and liposomes containing
phosphatidylserine, e.g. known substrates for SR-BI (16). As
for native cholesterol esters, the increased selective uptake of
HDL-CEOH by the liver is mediated by the parenchymal cells in
vivo (2). To answer the question of whether SR-BI is mediating
this increased selective uptake of HDL-CEOH, the inhibitory effects of
known SR-BI substrates on HDL-CEOH uptake by isolated parenchymal cells
were compared with the inhibitory effects on the selective uptake of
HDL-CE and HDL particle association as measured by iodinated HDL.
Freshly isolated rat liver parenchymal cells were incubated for 3 h at 37 °C with HDL, either iodinated or labeled with
[3H]CE or [3H]CEOH. At this time point, the
apparent association as calculated according to Pitmann (5) of
[3H]CE-labeled HDL (202 ± 14 ng of HDL/mg of cell
protein) exceeded 125I-HDL (36 ± 3 ng of HDL/mg of
cell protein) association by 5.6 times, whereas the association of
[3H]CEOH-HDL (966 ± 150 ng of HDL/mg of cell
protein) apparently exceeded 125I-HDL association by 27 times. The ability of (modified) lipoproteins to compete for
[125I]HDL and selective HDL-CE and HDL-CEOH uptake was
tested by co-incubation with either 100 µg/ml LDL, acetylated LDL, or
OxLDL and either neutral liposomes or liposomes containing
phosphatidylserine (Fig. 1). Addition of
either 100 µg/ml LDL or modified LDL only marginally (<10%)
decreased the cell association of [125I]HDL.
[3H]CE-HDL and [3H]CEOH-HDL association was
also not significantly affected by addition of LDL. However, addition
of acetylated LDL led to a 35 and 30% inhibition of
[3H]CE-HDL and [3H]CEOH-HDL association,
respectively, whereas OxLDL decreased their uptake by 75 ± 2.4 and 75 ± 5.4%, respectively. Liposomes consisting of
phosphatidylcholine, cholesterol, and the anionic phospholipid
phosphatidylserine inhibited both HDL-CE and HDL-CEOH uptake by
almost 50% (Fig. 1), whereas [125I]HDL association was
increased. Neutral liposomes, consisting only of phosphatidylcholine
and cholesterol, did not influence HDL-CE and HDL-CEOH uptake. The
possibility of exchange of [3H]CE and
[3H]CEOH to the other lipoproteins and liposomes was
tested by isolating the lipoproteins after incubation for 3 h at
37 °C. Both gradient density ultracentrifugation and agarose gel
electrophoresis were performed. Less than 5% of both
[3H]CE and [3H]CEOH was recovered in the
LDL, acetylated LDL, OxLDL, or liposomal fraction after incubation for
3 h at 37 °C, establishing that exchange to competitors could
not explain the achieved results. Therefore, both OxLDL and the
phosphatidylserine liposomes were effective inhibitors of HDL-CEOH
uptake by the liver parenchymal cells, and the inhibition of HDL-CEOH
uptake was comparable to the inhibition of HDL-CE uptake. The effective
inhibition of the selective uptake of oxidized cholesterol esters, like
that of native cholesterol esters from HDL, did not correlate with a
similar inhibition of the HDL particle association (16). Apparently, the total amount of HDL binding sites in liver exceeds the amount of
SR-BI-mediated binding sites, albeit these latter are efficiently coupled to selective uptake. This is in agreement with the limited amount of poly I-insensitive binding sites for OxLDL on parenchymal cells (31) as compared the total amount of HDL binding sites (less than
10%).

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Fig. 1.
Effect of native and modified lipoproteins
and neutral and phosphatidylserine liposomes on the parenchymal cell
association of 125I-HDL, [3H]CE-, or
[3H]CEOH-labeled HDL. Rat liver parenchymal cells
were incubated for 3 h at 37 °C with 10 µg/ml labeled HDL in
the absence or presence of 100 µg/ml unlabeled competitors in DMEM
with 2% BSA. The 100% value for association of [3H]CE-
or [3H]CEOH-labeled HDL was 202 ± 14 and 966 ± 150 ng of HDL/mg of cell protein, respectively, and for
125I-HDL association, it was 36 ± 3 ng of HDL/mg of
cell protein. The association is expressed as the percentage of the
radioactivity obtained in the absence of competitor. The results are
given as means ± S.E. (n = 3).
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The inhibitory effect of OxLDL on the the selective uptake of HDL-CEOH
was further analyzed with respect to efficiency of competition.
Increasing concentrations of OxLDL were added to freshly isolated
parenchymal cells in the presence of [3H]CEOH-labeled
HDL. Already at 20 µg/ml OxLDL, the uptake of HDL-CEOH was inhibited
by more than 70% (Fig. 2). Poly I, an
established inhibitor of scavenger receptor class A, did not lower the
cell association of [3H]CEOH-HDL. The simultaneous
presence of increasing concentrations of OxLDL and 100 µg/ml poly I
did not influence the inhibitory action of OxLDL, indicating that the
effect of OxLDL was not due to interaction with a poly I-sensitive
site.

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Fig. 2.
Effect of increasing concentrations of OxLDL
and poly I on the parenchymal cell association of
[3H]CEOH-labeled HDL. Rat liver parenchymal cells
were incubated for 3 h at 37 °C with 10 µg/ml labeled HDL in
the presence of the indicated amounts of OxLDL (  ) or poly I
(  ). When OxLDL was added together with poly I (  ), a
fixed 100 µg/ml of poly I was applied. The association is expressed
as the percentage of the radioactivity obtained in the absence of
competitor. The results are given as means ± S.E.
(n = 3).
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The selective uptake of HDL-CEOH was also analyzed by confocal laser
scanning microscopy using HDL labeled with a fluorescent analog of
CEOH. The uptake of fluorescent NBD-CEOH was followed by up to 3 h
of incubation with parenchymal cells. During the incubation period,
some parenchymal cell pairs, which were not separated during the
isolation procedure, regained their cellular polarity and formed active
bile canaliculi. These so-called parenchymal cell couplets (35) allow
to follow bile-directed transport of fluorescent cholesterol esters. At
3 h of incubation at 37 °C, most of the label inside the cell
was concentrated in the bile canalicular vacuole of the parenchymal
cell couplets, indicating a very efficient biliary secretion of
oxidized cholesterol esters (Fig. 3). A
very punctuate labeling in the cells was also observed. Addition of 100 µg/ml OxLDL abolished uptake of fluorescent CEOH almost
completely.

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Fig. 3.
Visualization of the interaction of oxidized
NBD cholesteryl linoleate-labeled HDL with liver parenchymal cell
couplets and the effect of OxLDL. Freshly isolated parenchymal
cells were preincubated on glass coverslips for 2 h at 4 °C in
DMEM supplemented with 2% BSA and 50 µg/ml HDL labeled with oxidized
NBD cholesteryl linoleate in the presence (B) or absence
(A) of 100 µg/ml OxLDL. Cells were analyzed with a
confocal microscope fitted with a incubation chamber to allow
incubation of the cells at 37 °C. The fluorescence of oxidized NBD
cholesteryl linoleate is the result of a 3-h incubation of the cells at
37 °C. As a control, 5 µg/ml carboxyfluorescein diacetate, a
substrate for an ATP-dependent organic anion transporter of
the bile canalicular membrane, was used and incubated for 3 h at
37 °C with the cells (C).
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As a control, carboxyfluorescein diacetate was used, which is a
substrate for an ATP-dependent organic anion transporter of the bile canalicular membrane and which is absent in TR
rats (42, 43). A concentration of fluorescence in the bile canalicular
vacuole similar to that of the oxidized cholesterol esters was noticed.
Uptake of HDL-CEOH by the Liver and Adrenals in Vivo--
The main
uptake site of [3H]CEOH-labeled HDL in vivo is
the liver (2). The liver uptake of [3H]CEOH-HDL 30 min
after injection was 27.0 ± 1.9 injected dose% (Fig.
4) and was 2.3-fold higher than the liver
uptake of native HDL-CE (n = 4). When the adrenal
uptake of [3H]CEOH-HDL was compared with the uptake of
[3H]CE-HDL, a similar 2.6-fold difference in uptake was
seen. Adrenal uptake at 30 min after injection was 2.0 ± 0.7 injected dose% and 5.1 ± 0.6% for [3H]CE-HDL and
[3H]CEOH-HDL, respectively (n = 4). No
other organs have a preferential uptake of HDL-CEOH as compared with
native HDL-CE (2).

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Fig. 4.
Tissue distribution of
[3H]CEOH-HDL and [3H]CE-HDL at 30 min after
injection in the rat. 300-400 µg (500000 dpm) of
[3H]CEOH-HDL or [3H]CE-HDL was injected
into the inferior vena cava of anaesthetized rats. 30 min after
injection, the amount of radioactivity was determined after combustion
in a Hewlett-Packard sample oxidizer 306 and counting for
radioactivity. The total recovery of label was 94 and 86% of the
injected dose for [3H]CEOH and [3H]CE-HDL,
respectively. Values are means ± S.E. of four experiments. **
indicates extremely significant difference (p <0.005). *
indicates significant difference (p < 0.05) (unpaired
Student's t test).
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Intrahepatic Cellular Uptake in Vivo of HDL-CEOH: Effect of
17
-Ethinyl Estradiol Treatment or a High Cholesterol Diet on SR-BI
Expression and HDL-CEOH Uptake--
Treatment of rats with EE for 5 consecutive days or a high cholesterol diet for 2 weeks lowered
expression of SR-BI in the liver parenchymal cells, whereas the
expression of SR-BI in the Kupffer cells was increased as previously
reported (Table I) (21). Furthermore, it
was found that the induced changes in hepatic SR-BI expression were
well correlated to changes in the selective uptake of HDL-CE (21). In
order to test whether the changes in SR-BI expression induced by either
EE treatment or a high cholesterol diet also affected the increased
selective uptake of HDL CEOH, the liver uptake of
[3H]CEOH-labeled HDL was determined, as well as the
association of iodinated HDL in order to analyze total particle
association. To identify the changes in the cellular uptake sites for
[3H]CEOH-HDL, parenchymal cells and the liver tissue
macrophages (Kupffer cells) were isolated (Fig.
5). Treatment of rats with EE for 5 days
resulted in a significant 50% decrease in [3H]CEOH-HDL
uptake by the liver, whereas uptake of 125I-labeled HDL was
not significantly changed (data not shown). Thus, the selective uptake
of HDL-CEOH was greatly inhibited by treatment of rats with EE, in
accordance with the supposed role of SR-BI as the mediator of selective
HDL-CEOH uptake. This decrease in selective uptake of HDL-CEOH by the
liver can be explained by a 64% decrease in [3H]CEOH-HDL
uptake by the parenchymal cells after EE treatment. In contrast, the
Kupffer cells showed a significant 6.6-fold increase (p < 0.05) in uptake of [3H]CEOH-HDL.
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Table I
SR-BI expression levels on parenchymal and Kupffer cell membranes
Cell membranes from parenchymal (PC) and Kupffer (KC) cells were
isolated from control rats, rats treated with EE (5 mg/kg) for 5 days,
or rats that had been fed a high-cholesterol diet for 2 weeks.
Solubilized membrane proteins were subjected to SDS-polyacrylamide gel
electrophoresis and blotted on to nitrocellulose membranes. SR-BI was
visualized by immunolabeling followed by enhanced chemiluminescence
detection and quantitated (21). Values are means ± S.E. of three
experiments.
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Fig. 5.
In vivo distribution of
[3H]CEOH-HDL between parenchymal and Kupffer cells at 10 min after injection in EE-treated rats, rats fed with a high
cholesterol diet, or control rats. Shown are the results for
control rats (open bars) and rats treated with EE (5 mg/kg)
for 5 days (hatched bars) or put on a high cholesterol diet
for 2 weeks (black bars). At 10 min after injection of
[3H]CEOH-HDL, the liver was perfused, and parenchymal
cells (PC) and Kupffer cells (KC) were isolated
at 4 °C. Values, expressed as the percentage of the injected
dose × 103/mg of cell protein, are means ± S.E.
of five experiments. ** indicates very significant difference
(p < 0.01). * indicates significant difference
(p < 0.05) (unpaired Student's t
test).
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Rats were also fed a high cholesterol containing diet for 2 weeks. This
diet increased the plasma cholesterol levels 20-fold as compared with
the control animals, as described previously (21), whereas total
cholesterol concentration in the liver increased more than 10-fold. The
total cellular cholesterol concentration in parenchymal cells increased
from the control value of 11 ± 0.9 ng/mg of cell protein up to
136 ± 18 ng/mg of cell protein (n = 3) (mean ± S.E.), whereas the cholesterol content in the Kupffer cells increased
from 6.8 ± 0.2 ng/mg of cell protein in the control animals up to
155 ± 66 ng/mg of cell protein after the 2-week diet
(n = 3). This diet resulted in a very significant (p < 0.01) 80% decrease in selective uptake of
[3H]CEOH-HDL by the liver (Fig. 5). The 2-week high
cholesterol diet inhibited only the parenchymal cell uptake of
[3H]CEOH-HDL (81%), whereas Kupffer cell uptake of
[3H]CEOH-HDL was almost 5-fold increased. The decrease in
HDL-CEOH uptake by the parenchymal cells and increase in HDL-CEOH
uptake by the Kupffer cells after either EE treatment or high
cholesterol diet is similar, as was found with native HDL-CE, and
correlates with the induced changes in hepatic SR-BI expression
(21).
The effects of EE treatment or a high cholesterol diet on the selective
uptake of HDL-CEOH was also studied in vitro. Hepatic parenchymal cells were isolated from both EE-treated rats and rats that
had been fed a high cholesterol diet for 2 weeks. The concentration
dependence of the cell association of [3H]CEOH-HDL was
studied (Fig. 6). Data are expressed in
terms of apparent particle uptake as originally devised by Pittman
et al. (5). Parenchymal cells isolated from both EE-treated
rats and from rats fed on a high cholesterol diet showed a significant decrease in [3H]CEOH-HDL association (p < 0.05, two-way analysis of variance) in vitro (Fig. 6).
These in vitro data thus illustrate that the changes in
in vivo uptake are also reflected in vitro with
the isolated parenchymal cells.

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Fig. 6.
Concentration dependence of
[3H]CEOH-HDL association to rat liver parenchymal cells
isolated from control rats, EE-treated rats, or cholesterol-fed
rats. Rat liver parenchymal cells were isolated from control rats,
rats treated with EE (5 mg/kg) for 5 days, or rats on a high
cholesterol diet for 2 weeks. Rat liver parenchymal cells were
incubated for 3 h at 37 °C with the indicated amount of labeled
HDL in DMEM with 2% BSA (w/v). Data are expressed in terms of apparent
particle uptake. The values are corrected for nonspecific cell
association in the presence of a 20-fold excess of HDL
(n = 2 separate cell isolations).
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SR-BI Transfection Studies--
To assess the ability of SR-BI to
mediate the uptake of oxidized cholesterol esters from HDL, stable CHO
cell transfectants expressing mouse SR-BI were used for cell
association studies. SR-BI-specific values were determined as the
difference between the values for the SR-BI-transfected cells and the
control ldlA cells. CHO cells expressing SR-BI showed a clear selective
uptake of both native and oxidized cholesterol esters (Fig.
7) wherein the apparent association of
[3H]CE-HDL and [3H]CEOH-HDL exceeded the
125I-HDL association 7.8 and 20 times, respectively (30-min
incubation). The rate of uptake of [3H]CEOH-HDL by
transfected cells was markedly greater (3.4 ± 0.6 (n = 3) at 30 min of incubation) than that of native
HDL cholesterol esters. The amount of cell-associated
[125I] HDL was also increased in SR-BI-transfected cells
as compared with mock-transfected cells, but it did not differ
significantly between CEOH-HDL and CE-HDL (Fig. 7, C and
D).

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Fig. 7.
Time dependence of the selective uptake
of [3H]CE and [3H]CEOH-HDL by
SR-BI-transfected CHO cells. Cells were incubated at 37 °C with
labeled HDL (10 µg of protein/ml) for the indicated times, and
cell-associated label was quantified as described under "Experimental
Procedures." A and B, cells were incubated with
[3H]CE-HDL (A) or [3H]CEOH-HDL
(B). C and D, cells were incubated
with 125I-labeled CE HDL (C) or
125I-labeled CEOH-HDL (D). Values represent the
mean of duplicate determinations. Similar results were obtained in two
additional experiments.
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DISCUSSION |
The generally accepted anti-atherogenic effect of HDL can be based
upon several mechanisms. The protective role of HDL is classically
associated with reverse cholesterol transport, as first proposed by
Glomset (3). In addition to this generally accepted concept, HDL may
also protect against oxidation of LDL (7), thereby preventing the
formation of an atherogenic form of LDL. The mechanism by which HDL
exerts its antioxidant action is still speculative. It was suggested
that the degree of protection during LDL oxidation was directly related
to the paraoxonase activity of HDL (36). In addition, Kunitake et
al. (38) showed that HDL-associated cearuloplasmin or transferrin
could scavenge metal ions and thus prevent catalysis of lipid
peroxidation in LDL. However, in the same studies, ultracentrifugally
prepared HDL, which looses most of its associated enzymes, still
retained antioxidant activity. In addition, reconstituted HDL, not
containing any undefined proteins, decreased the capacity of OxLDL to
form foam cells (39). Parthasarathy et al. (40) suggested
that HDL may act as a reservoir for lipid peroxides. HDL is suggested
to break the chain of lipid peroxide propagation by taking over LDL
lipid peroxides. Consistent with this theory, it was found that HDL is
the major carrier of lipid hydroperoxides in human blood. More than
85% of all detectable oxidized lipids in human plasma are associated
with HDL, whereas LDL is relatively peroxide-free (10). HDL-associated
lipid hydroperoxides are readily reduced by an intrinsic peroxidase
activity leading to less reactive hydroxides (9), an activity that may
be exerted by apoA-I and apoA-II (41).
The biological fate of HDL-associated oxidized cholesterol esters was
until recently unexplored. Sattler and Stocker (8) showed that oxidized
cholesterol esters in HDL are taken up to a greater extent by HepG2
cells in vitro than native cholesterol esters. Recently, we
showed that oxidized cholesterol esters show an increased serum decay
as compared with native cholesterol esters, whereas the liver uptake
exceeded particle uptake, thus indicating selective delivery (2). The
selective uptake of HDL-CEOH by the liver is 8.2-fold higher than
native cholesterol ester uptake at 2 min after injection, whereas after
30 min, the uptake is still 2.3-fold higher. This process is, within
the liver, selectively exerted by the parenchymal cells, and it is
coupled to efficient biliary excretion. However, the mechanism of the
highly efficient uptake of HDL-CEOH was not clear.
The liver and steroid-forming tissues are mainly responsible for the
clearance of native HDL cholesteryl esters from the blood circulation
(1, 5, 6), and SR-BI is now held responsible for mediating the
selective uptake of native HDL-CE by these organs (12, 17, 18). The
presence of hydroxyl groups in oxidized cholesterol esters yields a
better solubility in water, enabling the cholesteryl hydroxides to
transfer more efficiently through an aqueous phase between HDL and
cellular membranes. This might explain the increased cellular uptake of
HDL-CEOH. However, based upon our previous in vivo
experiments (2), it was concluded that this increased solubility did
not lead to an increased uptake by all cell types but, within the
liver, apparently only by the parenchymal cells. In addition, the
adrenals, which are known to have a high expression of SR-BI, show a
similar increased uptake of HDL-CEOH as compared with native HDL-CE.
Apparently, a cell-specific process is mediating uptake of oxidized
cholesterol esters in vivo, similar to the uptake of native
cholesterol esters. We now suggest that indeed the increased selective
uptake of oxidized cholesterol esters as compared with native
cholesterol esters is mediated by SR-BI. We obtained five points of
evidence for this involvement. First, the increased selective uptake of
HDL-CEOH by isolated parenchymal cells can be blocked by oxidized LDL, acetylated LDL, and phosphatidylserine liposomes to an extent similar
to native cholesterol ester uptake. Second, the effect of oxidized LDL
is not influenced by the simultaneous presence of poly I, indicating a
poly I-insensitive site for OxLDL interaction. Third, in
vivo the increased uptake of oxidized versus native cholesterol esters in untreated rats is only exerted by the parenchymal cells of the liver and by the adrenals, the cellular sites where SR-BI
is expressed. Fourth, within the liver, the uptake of oxidized cholesterol esters and native cholesterol esters are regulated to a
similar extent by estradiol treatment and by a high cholesterol diet,
whereby the in vivo uptake by parenchymal cells is decreased and the uptake by Kupffer cells is increased. These latter changes parallel the expression of SR-BI as analyzed on Western blots. Fifth,
the SR-BI-transfected CHO cells show a very efficient selective uptake
of oxidized cholesterol esters, exceeding particle association by 20 times at 30 min of incubation, while oxidized cholesterol ester uptake
at this time point is 3.4 times higher than for native cholesterol esters.
Recently, Thuren et al. (44) observed that an antibody
against SR-BI does block the selective uptake of native cholesterol esters by isolated parenchymal cells by up to 75%, confirming our
initial findings that substrates for SR-BI inhibit the selective uptake
of HDL cholesterol esters (16) in these cell types. Furthermore, the
similarity in uptake behavior observed in this study between native and
oxidized cholesterol esters by rat parenchymal cells, together with the
antibody data, are in accordance with our suggestion for SR-BI
involvement for both native and oxidized cholesterol esters from
HDL.
Oxidized lipids might be recognized by types of scavenger receptors
other than SR-BI, and we have shown earlier that oxidized LDL is
rapidly removed from the blood circulation by Kupffer cells (31). In
untreated rats, we did not observe any selective uptake of CE-OH by the
Kupffer cells; thus, the OxLDL-specific recognition site of Kupffer
cells (probably macrosialin (45)) does not interact with the oxidized
cholesterol esters from HDL. Down-regulation of SR-BI by estradiol or a
high cholesterol diet leads to a 64 or 81% inhibition, respectively,
of the oxidized cholesterol ester uptake by parenchymal cells. It thus
appears that SR-BI is a major determinant for the selective uptake of
oxidized cholesterol esters, leaving only a low percentage of
contribution for other receptors.
The mechanism for the more effective cellular uptake of oxidized
cholesterol esters as compared with native cholesterol esters from HDL
by SR-BI is presently unclear. The higher solubility of oxidized
cholesterol esters as compared with native cholesterol esters might
either facilitate the interaction of CEOH from HDL with the active site
of SR-BI or facilitate its further transport into the cell. Whatever
its mechanism, it might be concluded that the association of enzymatic
activities with HDL together with the efficient clearance route of
HDL-associated oxidized cholesterol esters as exerted by SR-BI on liver
parenchymal cells may work synergistically to detoxify lipid
hydroperoxides and thereby protect LDL from oxidation in
vivo. This scavenging function of SR-BI for oxidized lipids is in
line with the expected function of a member of the scavenger receptor family.