Atherosclerosis 154 (2001) 51 – 60
www.elsevier.com/locate/atherosclerosis

The LDL receptor is the major pathway for b-VLDL uptake bymouse peritoneal macrophages
Stéphane Perrey a, Shun Ishibashi a,*, Tetsuya Kitamine a, Jun-ichi Osuga a,
Hiroaki Yagyu a, Zhong Chen a, Futoshi Shionoiri a, Yoko Iizuka a, Naoya Yahagi a,
Yoshiaki Tamura a, Ken Ohashi a, Kenji Harada a, Takanari Gotoda a,
Nobuhiro Yamada bbaDepartment of Metabolic Diseases, Faculty of Medicine, Uni6ersity of Tokyo, 7 -3 -1 Hongo, Bunkyo-ku, Tokyo 113 -8655, Japan
Department of Endocrinology, Metabolism and Atherosclerosis, Institute of Clinical Medicine, Uni6ersity of Tsukuba, 1 -1 -1 Tennodai, Tsukuba,
Ibaraki 305 -8575, Japan

Received 9 August 1999; received in revised form 7 February 2000; accepted 2 March 2000

Abstract
In order to determine the contribution of the low density lipoprotein receptor (LDL-R) to the removal of apoB-containing
native lipoproteins by macrophages, we compared the uptake of b-VLDL in peritoneal macrophages (MPM) from wild type miceand mice lacking the LDL-R. The dB1.006 g/ml lipoproteins obtained from apoE deficient mice fed a high fat diet were poorly
degraded by macrophages and caused only a slight formation of CE in macrophages from both types of mice. On the other hand,
dB1.006 g/ml lipoproteins obtained from LDL-R deficient mice fed a high fat diet, b-VLDL with apoE, were avidly taken up
by and markedly stimulated CE formation in wild type macrophages, but not in macrophages lacking the LDL-R. The
degradation of 125I-labeled-apoE-containing b-VLDL by wild type MPM was poorly inhibited by unlabeled human LDL, andb-VLDL without apoE had no effects. In conclusion, we propose that the in vitro uptake of native apoE-enriched lipoproteins
by murine macrophages is primarily mediated by the LDL receptor and not by other apoE-recognizing receptor systems such as:
the LDL receptor related protein, the VLDL receptor or the triglyceride-rich lipoprotein receptor. © 2001 Elsevier Science Ireland
Ltd. All rights reserved.
Keywords: Foam cell formation; Knock-out mice; Receptor

1. Introduction
An early event in the development of an atherosclerotic lesion is the intimal accumulation of lipid-laden
foam cells which originate from either monocytemacrophages or vascular smooth muscle cells [1]. It isbelieved that foam cells are generated by cellular up-

take of lipoproteins through pathways independent of
the low density lipoprotein receptor (LDL-R), because
massive accumulation of foam cells is found even inatherosclerotic lesions of patients who completely lackthe LDL-R, i.e. homozygous familial hypercholesterolemia (see Refs. [2,-4] for review). Furthermore, the
LDL-R is barely detectable in lesions from patients

Abbre6iations: apo, apolipoprotein; BSA, Bovine Serum Albumin; CE, cholesterol ester; CHO, Chinese Hamster Ovary; DMEM, Dulbeco
modified Eagle’s medium; EDTA, Ethylenediaminetetra-acetic; FCS, fetal calf serum; FH, familial hypercholesterolemia; LDL, low density
lipoproteins; LDL-R( − /−), LDL-R deficient; LPDS, lipoprotein deficient serum; LPL, lipoprotein lipase; LRP, low density lipoprotein
receptor-related protein; MPM, mouse peritoneal macrophages; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline;
PMSF, phenylmethylsulphonylfluoride; SDS, Sodium Dodecyl Sulphate; TGRLP, triglyceride-rich lipoproteins; TLC, thin layer chromatography;
WHHL, Watanabe Heritable Hyperlipidemic; VLDL, very low density lipoprotein; b-VLDL, b-migrating very low density lipoprotein;b-VLDL-E( +), d less than 1.006 g/ml lipoproteins obtained from the LDL receptor knockout mice fed an atherogenic diets; b-VLDL-E(+ ), dless than 1.006 g/ml lipoproteins obtained from apolipoprotein E knockout mice fed an atherogenic diets.
* Corresponding author. Tel.: +81-3-3815-5411 (ext. 33113, 33129); fax: + 81-3-5802-2955.
E-mail address: ishibash-tky@umin.ac.jp (S. Ishibashi).
0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0021-9150(00)00457-3

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S. Perrey et al. / Atherosclerosis 154 (2001) 51–60

with the LDL-R [5,6]. In support of this notion, numerous receptors, which recognize and take up denatured
lipoproteins have been identified in macrophages. These
include scavenger receptor A [7,8], CD36 [9], Fc receptor [10], macrosialin [11] and others [12]. Furthermore,
modified lipoproteins have been demonstrated inatherosclerotic lesions [13,14].
In the development of atherosclerosis, lipoprotein
retention precedes lipoprotein denaturation [15]. Therefore, the mechanisms by which lipoproteins depositedin the extracellular milieu are eliminated before they are
denatured are no less important than are those of foamcell formation. Receptor systems, which mediate theuptake of native lipoproteins, account for the elimination by surrounding cells of native lipoproteins, which
are retended in the extracellular matrix. The LDL-R isexpressed in macrophages and mediates the uptake of
LDL, b-VLDL and apoE-rich b-migrating VLDL [16–19] The LRP is widely expressed in various tissues
including macrophages [20 – 22] and potentially mediates the uptake of lipoproteins that are rich in apoE or
LPL [23–25]. Finally, the VLDL receptor (LR8) also
binds to b-VLDL [26,27]. In addition to these receptors
belonging to the LDL receptor gene family, other receptors such as lipolysis-stimulated receptor (LSR) [28]and triglyceride-rich lipoprotein receptor (TGRLP receptor) [29,30] have been postulated to be involved in
the uptake of triglyceride-rich lipoproteins, although
their molecular identity is yet to be determined. Amongthese receptors for native lipoproteins, LRP [3,31] andthe VLDL receptor [32], but not the LDL-R [3,4], havebeen demonstrated to be present in atherosclerotic lesions, implicating their involvement in atherosclerosis.
b-VLDL is an atherogenic lipoprotein that is foundin the plasma of animals, which are fed a high cholesterol diet. In vitro, b-VLDL has been shown to cause
the conversion of macrophages into foam cells [16].
Murine models of both LDL-R [33] and apoE deficiency [34,35] have been created by targeted disruptionof these genes in embryonic stem cells, providing valuable tools for the detailed analysis of the pathways
involved in the uptake of native lipoproteins [36,37]. In
the current study, we made use of these animals to
re-examine the roles of the LDL-R and apoE in cellularcholesterol
homeostasis
inmouse
peritoneal
macrophages (MPM) from wild-type and LDL-R
knockout mice.

2. Materials and methods

2.1. cDNA
A plasmid containing mouse LDL-R cDNA [33] wasdigested with EcoRI, religated and used as a template
to synthesize an antisense riboprobe spanning fromnt233 to nt801 of the cDNA sequence. Polyclonal antibody against the human LDL-R was a generous giftfrom Drs Ho, Goldstein and Brown.

2.2. Animals
Generation of the mutated mice lacking the LDL-R[33] and apoE [34] was described in the indicatedreferences. These animals were allowed access to foodand water ad libitum. Two diets were used: (i) a normalchow (MF from Oriental Yeast, Tokyo); and (ii) an
atherogenic diet which contained 1.25% cholesterol
7.5% cocoa butter, 7.5% casein, and 0.5% cholic acid
(Oriental Yeast, Tokyo). The wild-type mice had agenetic background similar to that of the other two
mutant mice; hybrids of C57BL6 and 129Sv.

2.3. RNase protection assay
The 33P-labeled riboprobes for the LDL-R and bactin were synthesized from linearized plasmid templates by reverse transcriptase using MAXI scripts™
(Ambion, Austin, TX). The specific activity of theb-actin riboprobe was one tenth that of the LDL
receptor riboprobe. An RNase protection assay was
performed using RPAII™ (Ambion, Austin, TX) according to the manufacturer’s instruction.

2.4. Macrophage collection
One ml of 5% thioglycollate broth was injected into
the peritoneal cavities of mice aged between 2 and 6
months. After 4 days, the peritoneal cavities werelavaged with 10 ml of ice-cold saline. The cells were
washed three times with PBS and resuspended in
DMEM to give a concentration of 106 cells/ml. A totalof 1 ml/well and 10 ml/dish were used for plating in 12
well-plates and in 55 mm2 dishes (Corning), respectively. After incubation at 37°C for 2 h, the non-adherent cells were removed by washing three times with
pre-warmed PBS and the adherent cells were incubatedwith DMEM containing 5 mg/ml of LPDS for 36 h at
37°C in an atmosphere of 5% CO2 and 95% air unlessotherwise stated.

2.5. Immunoblot analysis
MPM from five mice were scraped in 1 ml of ice-cold
Buffer A containing 20 mM Tris–HCl, pH 8.0, 1 mM
CaCl2, 150 mM NaCl and a cocktail of protease inhibitors (1 mM PMSF, 1 mM o-phenanthroline, 1 mM
leupeptin, 5 mg/ml aprotinin and 1 mg/ml pepstatin) andhomogenized in a Potter–Elvehjem homogenizer. The
homogenate was centrifuged at 2000× g for 10 min andthe resulting supernatant was centrifuged at 100 000×g
for 60 min. Pellets were solubilized in an ice-cold buffer

S. Perrey et al. / Atherosclerosis 154 (2001) 51–60

B containing 250 mM Tris – maleate pH 6.0, 2 mM
CaCl2 and 1% (v/v) Triton X-100 supplemented withthe mixture of protease inhibitor. Resuspended pellets
were centrifuged at 100 000× g for 60 min. The supernatant, the Triton X-100 soluble fraction, was stored at− 70°C for up to 2 weeks. Thirty micrograms ofprotein was subjected to 7% SDS-polyacrylamide gelelectrophoresis. After transfer to a nitrocellulose membrane (Hybond ECL™, Amersham), the LDL receptor
was detected with the polyclonal antibody using the
ECL Western blotting system (Amersham) according to
the manufacturer’s procedure.

2.6. LipoproteinsMice were fed an atherogenic diet for 2 weeks, andblood was collected from the retro-orbital venous
plexus into tubes containing EDTA after an overnight
fast. The dB1.006 g/ml lipoproteins were isolated byultracentrifugation at a density of 1.006 g/ml for 16 h.
b-VLDL containing apoE (b-VLDL-E( + )) and bVLDL without apoE (b-VLDL-E( −)) were preparedfrom the LDL-R deficient mice and apoE knock-out
mice, respectively. Blood was drawn from healthy volunteers and LDL (d= 1.019 – 1.063 g/ml) and LPDS
(d \ 1.21 g/ml) were prepared by step-wise ultracentrifugation as described [38]. After re-centrifugation, thelipoprotein and LPDS solutions were dialyzed against 2
l of 2 mM sodium phosphate, pH 7.4, 150 mM NaCl,
0.01% EDTA and 0.01% NaN3. After dialysis against
saline, protein concentrations were determined using a
BCA™ protein assay reagent kit (Pierce, IL), Totalcholesterol, free cholesterol, triglycerides and phospholipids were determined using the determiner TC-555,
determiner L FC, determiner TG-S555 and determiner
L PL (Kyowa Medex, Tokyo, Japan), respectively.
Lipoproteins were radioiodinated by the iodine
monochloride method as described [39]. LDL wereacetylated as described [40]. These lipoproteins wereused within 1 week after preparation.

2.7. Uptake and degradation of the radioiodinatedlipoproteins
The cells were incubated with a medium containing
radioiodinated lipoprotein with or without unlabeled
lipoproteins and 5 mg/ml of BSA for 5 h. The medium
was removed and the radioiodinated lipoproteins degraded by the cells were measured according to Basu etal. [40].

2.8. Cholesterol ester formation assay
Cholesterol ester (CE) formation from [1-14C]oleate
was determined essentially as described [40]. Briefly, thecells were incubated in a medium containing lipo-

53

proteins, the [1-14C]oleate-albumin complex and 5 mg/
ml of BSA at 37°C for 24 h. Cells were washed twicewith PBS containing 2 mg/ml BSA and once with PBS.
CE was extracted with hexane-isopropanol (3:2) towhich [3H]cholesteryl-oleate and unlabeled oleic acid
were added as the internal standard and carrier, respectively. The organic phase was evaporated to dryness
under flowing N2. Cholesterol [1-14C]oleate was separated on silica coated TLC plates (c5583, Merck,
FRG) using a solvent system composed of heptane/
ethyl ether/acetic acid (90:30:1; v/v/v), their position
was identified using a BAS2000 phosphoimager (FujiFilm, Tokyo, Japan). The spots corresponding to CE
were scraped and their radioactivities were quantified
by liquid scintillation. Cellular protein was dissolved in
0.1 N NaOH and its concentration was determined by
the Lowry method [41].

2.9. Cellular cholesterol mass measurement
Lipids were extracted from cultured monolayers of
MPM by hexane-isopropanol. TC and FC were determined by fluorometric microassay according to amodified method of Heider and Boyett [42]. Cellular
protein was dissolved in 0.1 N NaOH and its concentration was determined by the Lowry method [41].

2.10. Statistics
A two-tailed Student’s t-test was used to assess thesignificance of difference between two groups.

3. Results

3.1. Lipoproteins
The results of the SDS/PAGE analysis of theapolipoprotein composition of the lipoproteins used forthe experiments are shown in Fig. 1(A). ApoB-100,
apoB-48 and apoE were the major apolipoprotein constituents of the b-VLDL-E( +) and the apoB-48 andapoB-100 concentrations were similar. In contrast, theb-VLDL-E(− ) contained apoB-48, apoA-IV andapoA-I, but lacked apoE. Upon agarose gel electrophoresis (Fig. 1(B)), b-VLDL-E(+) had b-mobility,
while b-VLDL-E( − ) migrated faster and had mobility
between the b and pre-b positions, suggesting thatb-VLDL-E(− ) was charged more negatively than bVLDL-E(+ ). The ratios of free cholesterol, cholesterolester and phospholipids to the proteins of b-VLDLE(+ ) were similar to those of b-VLDL-E(− ) (Table1). The ratio of triglycerides were higher in b-VLDLE(+) however this difference was not statistically significant (P=0.064).

S. Perrey et al. / Atherosclerosis 154 (2001) 51–60

54

3.3. Degradation of 125I-b-VLDL-E(+) and125
I-b-VLDL-E( − ) by MPM

3.2. Expression of the LDL-R
RNase protection assays were performed to confirm
the expression of the LDL-R mRNA (Fig. 2). After
treatment with LPDS for 36 h, a band corresponding
to the LDL-R was observed in the wild type
MPM (614 bp). The mRNA for the disrupted
LDL-R was detected in the LDL-R(−/ − ) MPM (401bp) and was markedly up-regulated (between 50- and100-fold) compared to the band for the wild-typemRNA.
Immunoblot analysis was performed to estimate the
LDL-R protein expression in the wild type MPM (Fig.
3). Without treatment with LPDS, the LDL receptor
protein was not detectable. After treatment with LPDS
for 36 h, LDL receptor expression was substantiallyinduced.

The cellular degradation of 125I-b-VLDL-E( +) andI-b-VLDL-E( − ) were compared in MPM obtained
from the LDL-R(− /−) and wild type mice (Fig. 4).
The MPM possessing functional LDL-R degraded substantial amounts of 125I-b-VLDL-E( + ). In contrast,
the degradation of 125I-b-VLDL-E( + ) in LDL-R( −/− ) MPM was less than 10% of that in wild type MPM.
In wild type MPM, 125I-b-VLDL-E( − ) were significantly less degraded than 125I-b-VLDL-E( + ) and thelevel was comparable to that of 125I-b-VLDL-E( +)degraded by LDL-R( − /− )MPM. There was no difference in the degradation of 125I-b-VLDL-E( −) between the two types of cells. These results indicate thatboth the LDL-R on the cells and apoE on the lipo125

Fig. 1. Biochemical characteristics of b-VLDL-E(+ ) and b-VLDL-E(−). b-VLDL-E(+ ) and b-VLDL-E(−) were isolated by ultracentrifugation
at a density of 1.006 g/ml from plasma obtained from LDL-R deficient and apoE deficient mice which had been fed an atherogenic diet for 2
weeks. Twenty-five micrograms of the lipoproteins were delipidated with ethanol/ether and subjected to 5 – 15% gradient polyacrylamide SDS gelelectrophoresis (A). Proteins were visualized with Coomassie Brilliant Blue R250. Rainbow marker (Amersham) was used to calibrate themolecular weight. Lane 1, b-VLDL-E(+ ); lane 2, b-VLDL-E(−). One microliter of the concentrated d B1.006 g/ml lipoproteins were subjected
to agarose gel electrophoresis (B). Lipids were visualized with Fat Red 7B. The migratory positions are indicated. Lane 3, b-VLDL-E(+ ); lane
4, b-VLDL-E(− ).
Table 1
Lipid composition of b-VLDL used in the cell studiesa
Lipoprotein

b-VLDL-E(+)
b-VLDL-E(−)

Lipid/protein ratio
TC

FC

CE

TG

PL

3.119 0.73
3.989 1.14

0.67 90.11
0.81 90.21

2.43 90.63
3.17 90.95

1.339 0.73
0.24 9 0.01

1.01 9 0.19
0.95 9 0.27

The dB1.006 g/ml lipoproteins were isolated by ultracentrifugation at a density of 1.006 g/ml for 16 h. b-VLDL (dB1.006 g/ml) containing
apoE [b-VLDL-E(+)] and b-VLDL without apoE [b-VLDL-E(−)] were prepared from five LDL-R deficient mice and five apoE knock-out mice,
respectively. The protein, total cholesterol (TC), free cholesterol (FC), triglyceride (TG) and phospholipid (PL) concentrations were determined
as described in Section 2. The cholesterol ester values were obtained by subtracting free from total cholesterol values. Results represent the meanin mg lipid/mg protein ratio 9S.D. of five separate samples.
aS. Perrey et al. / Atherosclerosis 154 (2001) 51–60

Fig. 2. RNase protection assay for the LDL receptor mRNA. MPM
were incubated with DMEM containing 5 mg/ml of LPDS. Ten
micrograms of total RNA was used for RNase protection assay formouse LDL receptor and b-actin. Seven percent polyacrylamide gel
was used for separation of the protected probes. The sizes of theprotected RNA are 614, 401 and 245 bp, for the wild-type, mutated
LDL receptor and b-actin, respectively.

55

5). The degradation of 125I-b-VLDL-E( + ) by wild type
MPM was almost completely inhibited by excess
amounts of unlabeled b-VLDL-E(+ ), while even 125fold excess of b-VLDL-E( − ) had no inhibitory effects
on its degradation (Fig. 5(A)). These results indicatethat the degradation process for b-VLDL-E( +) primarily depends upon the presence of apoE. As expected, 125-fold excess of human LDL reduced thedegradation by 50%. This confirms that human LDL
have lower affinity for receptors expressed by MPM
than b-VLDL-E(+ ) [17]. On the other hand, the degradation of 125I-b-VLDL-E( − ) by wild type MPM,
which was substantially lower than that of 125I-bVLDL-E(+ ), was displaced by both unlabeled bVLDL-E(− ) and b-VLDL-E(+ ) to a similar extent,
but not by LDL, suggesting that the degradation process for b-VLDL-E( − ) involves a molecule(s) common
to both b-VLDL-E(− ) and b-VLDL-E( +) (Fig. 5(B)).

Fig. 3. Western blot analysis for LDL receptor protein. MPM weretreated with DMEM containing either 10% (v/v) FCS (1) 5 mg/ml of
LPDS (2) or for 36 h. The membrane fractions were prepared and 30
mg were subjected to 7% polyacrylamide SDS gel electrophoresis.
Immunoblot analysis was performed as described in Section 2. Rainbow marker (Amersham) was used to calibrate molecular weight.

proteins are required for the specific uptake and degradation of dB1.006 lipoproteins. The amounts of 125ILDL degraded by wild type MPM was two-fold larger
than that of 125I-b-VLDL-E( −) by the same cells (datanot shown). Yet, the LDL-R(−/ − ) MPM did notdegrade any human 125I-LDL above background levels
and this degradation could not be displaced by excess
unlabeled LDL (data not shown).
Competition assays were performed to determine thespecificity of the degradation of the lipoproteins (Fig.

Fig. 4. Cellular degradation of 125I-b-VLDL-E(+) and 125I-b-VLDLE( − ) in MPM. MPM were prepared from mice as described in
Section 2. After incubation with DMEM containing 10% (v/v) FCS
for 36 h, the cells were incubated with DMEM containing 5 mg/ml of
LPDS for 36 h. A total of 2 mg/ml of 125I-b-VLDL-E(+ ) (open bar)and 125I-b-VLDL-E(−) (hatched bar) were incubated with and without 100 mg/ml of unlabeled lipoproteins for 5 h. Data are themean 9S.D. of four wells for the wild type MPM, and the mean of
two wells for the LDL-R(−/ −) MPM. The total degradation by thewild type macrophages was inhibited by 85% with an excess amounts
of unlabeled b-VLDL-E(+) (data not shown), indicating that a
specific endocytic pathway is involved in the uptake and degradationof the 125I-b-VLDL-E(+ ).

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S. Perrey et al. / Atherosclerosis 154 (2001) 51–60

3.4. Cholesterol ester formation in MPM stimulated bylipoproteins
CE formation stimulated by LDL, acetyl LDL, bVLDL-E(+ ) or b-VLDL-E( − ) was compared between wild type and LDL-R(− /−) MPM (Fig. 6).
b-VLDL-E(+ ) substantially stimulated CE formationin wild type MPM, but not in LDL-R(− /−) MPM. In
LDL-R(− /−) MPM, however, there was no difference in CE formation between b-VLDL-E(+ ) andb-VLDL-E(− ). Similarly, b-VLDL-E( − ) induced
comparable CE formation in both types ofmacrophages. Although b-VLDL, irrespectively of thepresence of apoE, caused CE accumulation to occur ina dose-dependent saturable manner in wild type
macrophages, its effects on CE accumulation in the
LDL-R(− /−) MPM was linear (data not shown).
Compared to b-VLDL-E( +), acetyl LDL and native
LDL induced smaller CE formation.

3.5. Cellular cholesterol mass in MPM stimulated bylipoproteinsFig. 5. Inhibition of the degradation of 125I-b-VLDL-E(+ ) (A) and125
I-b-VLDL-E(− ) (B) by unlabeled b-VLDL-E(+ ), b-VLDL-E(− )and LDL in MPM. A total of 2 mg/ml of 125I-b-VLDL-E(+ ) (A) and125
I-b-VLDL-E(− ) (B) were incubated without and with 2, 10, 50,
250 mg/ml of unlabeled b-VLDL-E(+ ) (open circle), b-VLDL-E(−)(closed circle) and LDL (square) for 5 h. Data are presented as themeans of duplicate wells. The amounts of 125I-b-VLDL-E(+ ) and125
I-b-VLDL-E(− ) degraded without excess unlabeled lipoproteins
were 351 and 64 ng/mg, respectively.

Wild type and LDL-R(− /−) macrophages wereincubated with either b-VLDL-E( + ) or b-VLDL-E(−) and CE mass was measured (Fig. 7). While b-VLDLE(+ ) remarkably increased the CE mass in wild type
MPM, it had only a small effect in LDL-R(−/−)
MPM. b-VLDL-E( −) induced poor accumulation of
CE mass irrespective of the expression of the LDL-R
by the macrophages. Free cholesterol mass was increased by the addition of b-VLDL independently of
the MPM phenotype or the origin of the lipoprotein.

4. Discussion

Fig. 6. Stimulation of cholesterol ester formation by b-VLDL-E(+),
b-VLDL-E(− ), LDL or acetyl LDL. MPM were harvested from wild
type and LDL-R deficient mice. After incubation with DMEM
containing 5 mg/ml LPDS for 36 h, the cells were incubated with 10
mg/ml of b-VLDL-E(+) (solid), b-VLDL-E(− ) (black hatched),
LDL (white hatched), acetyl LDL (white) and [1-14C]oleate-albumincomplex for 24 h. The values are presented as the means9 S.D. offour wells. *P B 0.001.

It has been shown that b-VLDL-E( + ) strongly stimulates CE accumulation in macrophages including
MPM [16]. By using blocking antibody against the
LDL-R, Koo et al. [17] and Ellsworth et al. [18] haveshown that the uptake of b-VLDL-E( + ) by MPM islargely mediated by the LDL-R. However, other receptors (i.e. LSR [28] and TGRLP receptor [29,30]) havebeen postulated to be involved in the uptake of triglyceride-rich lipoproteins, although their molecular identity is yet to be determined. The use of MPM obtained
from mice whose LDL-R is genetically ablated, is ideal
to address the question of whether the LDL-R pathway
is involved in the uptake of b-VLDL-E(+ ). Comparison of the degradation of 125I-b-VLDL-E( + ) between
wild type and LDL-R(− /− ) MPM clearly indicatesthat the LDL-R pathway accounts for as much as
80–90% of the degradation of b-VLDL-E( + ) (Fig. 4).
Previously, Whitman et al. [43] described that bVLDL prepared from apoE deficient mice stimulates

S. Perrey et al. / Atherosclerosis 154 (2001) 51–60

CE accumulation only slightly in J774A.1 cells, which
do not express endogenous apoE. This confirmed theresults of other studies using human lipoprotein inwhich apoE amount or ability to bind to receptors play
an important role in the CE accumulation in
macrophages [44–47]. In line with this, our current
results showed that the presence of apoE on lipoproteins is absolutely required for LDL-R-mediateddegradation of 125I-b-VLDL (Fig. 4). Furthermore, bVLDL-E(+), but not b-VLDL-E( − ), completely inhibited the degradation of 125I-b-VLDL-E( +), andhuman LDL had intermediate effects (Fig. 5). These
results implicate that the other receptor pathways,
which recognize apoE, such as the LRP and VLDL
receptors, play negligible roles in the uptake of apoErich lipoproteins by MPM.
Lipoprotein lipase is secreted by MPM [48] andpotentially may mediate the direct uptake of lipoproteins by bridging the lipoprotein to the receptor [49].
However our current results indicate that the contribution of the bridge function [50] of LPL is minimal interms of overall uptake of the apoE-rich lipoproteinscompared with the LDL-R-dependent pathway.
Similar studies addressing the role of the LDL-R
have been performed with cells from other species suchas humans and rabbits with natural mutations in the
LDL-R. Koo et al. [19] used monocyte-derived

57

macrophages from homozygous FH individuals andconcluded that the LDL-R accounts for the uptake ofb-VLDL-E(+) in these cells. Daugherty and Rateri[51] used alveolar macrophages from WHHL rabbits,
an animal model of FH, however the LDL-R in
WHHL rabbits retains binding affinity to b-VLDLE(+ ) [52] and this limits the interpretation of theresults.
Although the degradation of b-VLDL-E( − ) is lower
than that of b-VLDL-E( + ) in wild type MPM, themechanism involved remains an interesting question.
Zsigmond et al. [53] reported that serum VLDL in
apoE(− /−) mice fed a chow diet are resistant tolipolysis by LPL, and this may affect their uptake bymacrophages. However, the poor induction of cholesterol ester formation by b-VLDL-E(− ) compared tob-VLDL-E(+ ) may not be explained by increased
triglyceride content since in our experimental condition
where mice were fed a high fat diet, triglycerides were
not higher in b-VLDL-E(− ). Another potential explanation for the degradation of the b-VLDL-E(−) is thatradiolabeled apolipoprotein may be selectively taken up
and therefore the degradation data do not reflect lipoprotein particle endocytosis. However b-VLDL-E( −)at 50 mg/ml induced a significant increase in CE mass
independently of the LDL-R expression by the MPM(Fig. 7). Therefore, we assume that degradation reflects,

Fig. 7. Cholesterol ester mass measurement in MPM. MPM were harvested from wild type and LDL-R deficient mice. After incubation withDMEM containing 5 mg/ml LPDS for 36 h, the cells were incubated with 10 or 50 mg/ml of b-VLDL-E(+ ) (closed circle), or b-VLDL-E(− )(open circle) for 24 h. Lipid extraction and quantification were performed as described in Section 2. The values are presented as the means 9 S.D.
of three wells: (1) difference not significant compared to no b-VLDL addition; (2) P B0.05 compared to no b-VLDL addition; (3) P B 0.001
compared to no b-VLDL addition; and (4) difference not significant compared to wild type MPM.

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S. Perrey et al. / Atherosclerosis 154 (2001) 51–60

at least partially, lipoprotein uptake. Since both bVLDL-E(+ ) and b-VLDL-E( −), but not LDL, displaced the degradation of 125I-b-VLDL-E( −) in asimilar manner, molecules common to these lipoproteins mediate its degradation; apoB-48, apoA-I andapoA-IV are likely candidates (Fig. 1).
Further studies are needed to know whether the
receptor(s) responsible for the uptake of b-VLDL-E( −) are identical to certain known receptors such as the
TGRLP receptors [29]. Recently, Hendriks et al. [54]have reported that VLDL from apoE-deficient mice
were taken up by J774 macrophages, leading to anincrease in CE mass, and they speculated that thetriglyceride-rich lipoprotein (TGRLP) receptor proposed by Gianturco et al. accounted for the uptake.
However we cannot rule out the possibility that slightoxidation or aggregation of the b-VLDL-E( − ) lipoproteins might occur during preparation and storageand may in part be responsible for the LDL-R independent uptake of the lipoproteins.
In agreement with the results of the lipoprotein
degradation, the stimulation of CE formation as well as
the accumulation of CE by b-VLDL-E( + ) was similar
to that of b-VLDL-E( −) in the LDL-R( −/ − ) MPM(Figs. 6 and 7). Because apoE is secreted by the LDLR(− /− ) MPM [36], these results indicate that thesecreted apoE did not further enrich the b-VLDL-E( +) leading to uptake by the LRP that is expressed bythese cells (data not shown). Furthermore, the CEformation and accumulation stimulated by b-VLDLE( − ) in wild type MPM were similar to those in
LDL-R(− /−) MPM (Figs. 6 and 7). This indicatesthat macrophage-secreted apoE does not interact with
b-VLDL-E(−) nor does it direct b-VLDL-E( −) to the
LDL-R pathway as postulated in the secretion-recapture hypothesis [49,55,56]. In contrast, Kowal et al. [23]reported that b-VLDL enriched with apoE stimulates
CE formation in ldlA7 cells, CHO cells lacking the
LDL-R. Potential explanations to reconcile the discrepancy between Kowal et al.’s and our experiments
are as follows: (1) the amounts of apoE secreted by
MPM may not be sufficient to convert b-VLDL-E( −)to be active in the stimulation of CE formation; and (2)MPM may express a lower level of LRP than CHO
cells.
In conclusion, we demonstrate that the LDL-R is theprimary mechanism for b-VLDL uptake in mouse peritoneal macrophages and the presence of apoE at thesurface of the b-VLDL is critical for the uptake process. Furthermore the slight uptake of b-VLDL by
LDL-R(− /−) MPM indicates that other potential
secondary mechanisms such as the LRP, b-VLDL receptor or the TGLRP receptor which may or may not
require apoE for b-VLDL uptake, play only a minorrole.

Since lipoprotein retention has been described as animportant event in the initiation of the development of
atherosclerotic lesions, the LDL-R may be an important factor to remove native lipoproteins inside thearterial wall prior to denaturation. The cellular cholesterol-related regulation of the LDL-R may suggest abeneficial role since the down-regulation of its expression would allow cells to avoid becoming overwhelmed
beyond their capacity of accommodating increasedcholesterol. On the other hand, with LDL-R down-regulation native lipoprotein would become trapped by the
extracellular matrix and be more susceptible to oxidative modification, resulting in cellular uptake by scavenger receptors, which are not subject to regulation by
intracellular cholesterol.

Acknowledgements
This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science
and Culture, the Promotion of Fundamental Studies in
Health Science of The Organization for Pharmaceutical
Safety and Research (OPSR) and Health Sciences Research Grants (Research on Human Genome and Gene
Therapy) from the Ministry of Health and Welfare. We
thank Kimiko Saito, Emi Herai and Mihoko Kusubae
for technical assistance and secretarial work, Masako
Shimada and Hitoshi Shimano for helpful discussion
and Alyssa M. Hasty for critical reading of themanuscript.

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