Apolipoprotein E Receptor Binding Versus
Heparan Sulfate Proteoglycan Binding in Its Regulation of Smooth Muscle
Cell Migration and Proliferation*
Debi K.
Swertfeger
and
David Y.
Hui§
From the Department of Pathology and Laboratory Medicine,
University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267-0529
Received for publication, March 15, 2001, and in revised form, May 2, 2001
 |
ABSTRACT |
This study showed that synthetic peptides
containing either a single copy or tandem repeat of the receptor
binding domain sequence of apolipoprotein (apo) E, or a peptide
containing its C-terminal heparin binding domain, apoE-(211-243), were
all effective inhibitors of platelet-derived growth factor
(PDGF)-stimulated smooth muscle cell proliferation. In contrast, only
the peptide containing a tandem repeating unit of the receptor binding
domain sequence of apoE, apoE-(141-155)2, was
capable of inhibiting PDGF-directed smooth muscle cell migration.
Peptide containing only a single unit of this sequence,
apoE-(141-155), or the apoE-(211-243) peptide were ineffective in
inhibiting PDGF-directed smooth muscle cell migration. Additional
experiments showed that reductively methylated apoE, which is incapable
of receptor binding yet retains its heparin binding capability, was
equally effective as apoE in inhibiting PDGF-stimulated smooth muscle
cell proliferation. However, reductively methylated apoE was unable to
inhibit smooth muscle cell migration toward PDGF. Additionally, the
receptor binding domain-specific apoE antibody 1D7 also mitigated the
anti-migratory properties of apoE on smooth muscle cells. Finally,
pretreatment of cells with heparinase failed to abolish apoE inhibition
of smooth muscle cell migration. Taken together, these data documented
that apoE inhibition of PDGF-stimulated smooth muscle cell
proliferation is mediated by its binding to heparan sulfate
proteoglycans, while its inhibition of cell migration is mediated
through apoE binding to cell surface receptors.
 |
INTRODUCTION |
Research in the past two decades has clearly demonstrated that
apolipoprotein (apo)1 E
protects against vascular disease (1). Mice deficient in apoE emphasize
the importance of apoE in protection against atherosclerosis. The
apoE-deficient mice generated by gene targeting approach have plasma
cholesterol levels of 400-500 mg/dl and develop severe atherosclerosis
when fed a chow diet (2-5). In contrast, wild type mice have plasma
cholesterol levels of 60-85 mg/dl and do not develop atherosclerosis
under normal conditions. Atherosclerotic lesions in mice with only one
copy of the apoE gene (apoE+/
) were 10 times more severe than lesions
in wild type mice that were fed a high fat/high cholesterol diet,
despite relative similar cholesterol levels between the two groups of
animals (326 versus 238 mg/dl). In these studies, a direct
correlation between serum cholesterol levels and atherosclerotic lesion
size was not observed. Additionally, transgenic expression of apoE in
the arterial wall inhibited atheroma formation and severity without
affecting plasma cholesterol level and lipoprotein profile in
cholesterol-fed C57BL/6 mice (6). Taken together, the data showing an
inverse relationship between apoE level and atherosclerotic lesion
size, but a lack of correlation between total cholesterol and
atherosclerosis lesion size, have led to the hypothesis that apoE may
have direct impact on vascular occlusive diseases in a manner in
addition to, and independent of, its property as a
cholesterol-transporting apolipoprotein (7).
Recent data from our laboratory have indicated that apoE protection
against vascular occlusive disease may be directly related to its
modulation of vascular smooth muscle cell response to stimulation. We
have shown that apoE inhibits platelet-derived growth factor (PDGF)- or
oxidized LDL-induced smooth muscle cell migration and proliferation
(8). ApoE inhibition of smooth muscle cell proliferation was shown to
be due to cell cycle arrest resulting from the inhibition of growth
factor-induced cyclin D1 activation (8). The mechanism of this
inhibition appeared to be mediated through inducible
nitric-oxide synthase signaling pathways (9). In
contrast, apoE inhibition of growth factor-induced smooth muscle cell
migration was independent of inducible nitric-oxide synthase activation
(9). Thus, it is likely that apoE inhibition of smooth muscle cell
migration is mediated through mechanism(s) distinct from the pathways
involved with its anti-proliferative effects. The hypothesis that apoE inhibits growth factor-induced smooth muscle cell migration and proliferation via distinct mechanisms was further supported by data
showing that low doses (0.1-5 µg/ml) of apoE were capable of
inhibiting smooth muscle cell migration, while higher doses (25 and 50 µg/ml) were necessary for apoE inhibition of smooth muscle cell
proliferation (9).
Apolipoprotein E binds to all members of the LDL receptor gene family
as well as to heparan sulfate proteoglycans (HSPG) on the surface of
mammalian cells (10-12). Recent studies from several laboratories
showed that ligand binding to LDL receptor gene family members can
mediate signal transduction events (13-19). Likewise, ligand binding
to heparan sulfate proteoglycans may also induce signal transduction
events (20-22). Smooth muscle cells are known to express HSPG as well
as many receptors of the LDL receptor gene family, including the LDL
receptor, LRP, the VLDL receptor, and the apoE receptor-2 LR8 (10, 11,
23). The goal of the current study is to determine whether apoE
inhibition of smooth muscle cell migration and proliferation is
mediated through its interaction with cell surface receptors or with
HSPG.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Type I collagenase, elastase, and heparinase III
were obtained from Sigma. Dulbecco's modified Eagles' medium
(DMEM), fetal bovine serum, and PDGF-BB were purchased from Life
Technologies, Inc. [3H]Thymidine was obtained from
PerkinElmer Life Sciences, and Transwell polycarbonate membrane
filters were purchased from Corning Costar Corp. (Cambridge, MA).
Antibodies against smooth muscle-specific
-actin were obtained from Sigma.
Human ApoE and ApoE Peptides--
Human apoE was isolated from
fresh plasma from healthy volunteers by the method of Cardin et
al. (24). The purity of apoE was assessed by SDS-polyacrylamide
gel electrophoresis, and samples containing only a single band with
Mr = 34,000 were used. Purified apoE was
resuspended in phosphate-buffered saline and added directly to the
culture medium without reconstitution with lipids. In selected experiments, lysine residues in apoE were modified by reductive methylation according to the procedure of Weisgraber et al.
(25). The reductive methylated apoE was extensively dialyzed against phosphate-buffered saline and stored at 4 °C for no more than 1 week
prior to use.
Peptides containing residues 141-155, heretofore designated as
apoE-(141-155), or a tandem repeat of the same sequence,
apoE-(141-155)2, or residues 211-243, designated as
apoE-(211-243), were synthesized chemically by the Synpep Co.
(Hopkinton, MA). The sequence of each peptide was verified by mass
spectrometry. The lyophilized peptides were reconstituted in
phosphate-buffered saline, dialyzed extensively against the same
buffer, aliquoted in small quantities, and stored at
20 °C until use.
Isolation of Primary Mouse Smooth Muscle Cells--
Mouse aortic
smooth muscle cells were isolated using a modification of Mimura's
procedure (26). Briefly, thoracic aortas were dissected from eight mice
and the adventitial tissue trimmed away. The aortas were then incubated
in Hanks' solution containing 1 mg/ml collagenase and 3.3 units/ml
elastase for 30 min at 37 °C. Remaining adventitial tissue was
dissected away, and the remaining tissue was incubated in Hanks'
solution containing collagenase (1 mg/ml) and elastase (3.3 units/ml)
for 1 h at 37 °C. Cell clumps were dissociated by aspiration
through a 10-ml pipette. The cell suspension was centrifuged at
150 × g for 5 min at room temperature and resuspended
in 10 ml of DMEM containing 10% fetal bovine serum. The isolated cells
were characterized as smooth muscle cells based on positive
immunohistochemistry staining with anti-
-actin antibodies and by
morphological characteristics similar to that observed with human
smooth muscle cells. The primary smooth muscle cells were cultured in
DMEM containing 10% fetal bovine serum, 100 units/ml penicillin, and
0.1 mg/ml streptomycin. Cells between passage 1 and 5 were used for experiments.
Smooth Muscle Cell Migration Assay--
The migration of mouse
vascular smooth muscle cells toward a PDGF-BB gradient was examined
according to the procedure of Law et al. (27), as described
previously (8, 9). Briefly, smooth muscle cells were made quiescent by
incubation with DMEM and 0.4% fetal bovine serum for 48 h. Cells
(2 × 105/ml) were incubated in solution with or
without apoE for 30 min at 37 °C, and 0.1-ml aliquots were added to
the top chamber of tissue culture-treated Transwell polycarbonate
membrane with 8-µm pores in 24-well plates. The lower
Transwell compartment contained 0.6 ml of DMEM, 0.4% fetal bovine
serum, and 0.2% bovine serum albumin, with or without 10 ng/ml
PDGF-BB. After incubating for 4 h at 37 °C, the upper surface
of the filters was washed with phosphate-buffered saline. The cells
were then fixed with methanol for 10 min at 4 °C followed by
hematoxylin staining. The number of cells that migrated to the lower
surface of the filter was determined by counting the cells in six high
power fields (× 320). Data are presented as the percentage of cells
that migrated to the lower surface as compared with PDGF treatment
alone without apoE. All experiments were performed in triplicate and
were repeated at least three times.
Determination of Smooth Muscle Cell Proliferation--
Smooth
muscle cell proliferation was measured based on the incorporation of
[3H]thymidine into cellular DNA as described previously
(8, 9). Briefly, cells were plated into 96-well plates at a density of 2.5 × 103 cells/well and allowed to attach for
24 h at 37 °C. Quiescence was induced by incubating the cells
in DMEM containing 0.4% fetal bovine serum for 48 h at 37 °C,
after which time the experiment was initiated. Test reagents, such as
apoE or apoE peptides, were diluted in DMEM containing 0.4% fetal
bovine serum, 0.2% bovine serum albumin, and 10 ng/ml PDGF-BB before
adding to the cell culture. Cells incubated in medium without PDGF-BB
served as control to determine basal [3H]thymidine
incorporation into cellular DNA during the quiescence phase. Five hours
prior to the end of the experiment, 1 µCi of [3H]thymidine was added to the culture medium. Cells were
harvested onto filters using a cell harvester to determine
[3H]thymidine incorporation 24 h after the addition
of the test reagents. Radioactivity was quantified by liquid
scintillation counting. Data shown are from an individual
representative experiment. All experiments were performed with five
replicates per experiment, and each experiment was repeated at least
three times.
 |
RESULTS |
Initial experiments compared peptides containing either a single
receptor binding domain sequence of apoE or a tandem repeat of the same
sequence with a peptide containing the C-terminal heparin binding
domain sequence of apoE for their inhibition of PDGF-induced smooth
muscle cell migration and proliferation. While none of these peptides
were cytotoxic, as determined by lactate dehydrogenase release, the
incubation of smooth muscle cells with 3-12 µM
concentration of any of these apoE peptides resulted in the
suppression of PDGF-stimulated cell proliferation (Fig.
1). The apoE-(211-243) peptide was found
to be the most effective in inhibiting smooth muscle cell
proliferation, with complete abolishment of [3H]thymidine
incorporation into cellular DNA observed at 12 µM. The
apoE-(141-155) and apoE-(141-155)2 peptides were less
active than the apoE-(211-243) peptide, with ~50% inhibition
observed at 12 µM (Fig. 1).

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Fig. 1.
Effect of apoE peptides on PDGF-stimulated
proliferation of aortic smooth muscle cells. Serum-starved mouse
smooth muscle cells were incubated for 24 h in 96-well plates
(2.5 × 103 cells/well) in the presence of PDGF-BB and
apoE synthetic peptides: apoE-(141-155)2 (filled
circles), apoE-(141-155) (open circles), or
apoE-(211-243) (filled triangles). Cell proliferation was
determined by liquid scintillation counting of
[3H]thymidine incorporated into cellular DNA during the
last 5 h of incubation. Data are represented as percent of maximum
proliferation induced by PDGF-BB in the absence of apoE. * indicates
significant difference from results with other peptides at
p < 0.05. The data are the mean ± S.E. from five
different determinations in a single representative experiment. Similar
trends were observed in at least three different experiments.
|
|
The effect of the apoE peptides on smooth muscle cell migration toward
PDGF was dramatically different from that observed for their inhibition
of cell proliferation. The peptide with the tandem repeating sequence
of the receptor binding domain, apoE-(141-155)2, inhibited
PDGF-directed smooth muscle cell migration when used at a concentration
greater than 0.75 µM (Fig.
2). Thus, the effectiveness of the
apoE-(141-155)2 peptide in inhibiting smooth muscle cell migration was similar to that observed for intact apoE (Fig. 2 and Ref.
9). In contrast, the peptide containing only a single copy of the
receptor binding domain of apoE, apoE-(141-155), and the peptide
containing only the C-terminal heparin binding domain sequence,
apoE-(211-243), were ineffective in inhibiting PDGF-directed smooth
muscle cell migration even when higher concentrations were used (Fig.
3).

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Fig. 2.
Effect of a tandem repeat peptide of the apoE
receptor binding domain on PDGF-directed migration of smooth muscle
cells. Quiescent mouse smooth muscle cells were incubated with
either native apoE (open circles) or the apoE tandem peptide
apoE-(141-155)2 (filled circles) for 30 min at
37 °C prior to adding the cells to the top chamber of Transwell
membranes at a density of 2 × 104 cells/well in a
24-well plate. The lower chamber of the plate contained basal medium
(DMEM with 0.4% fetal bovine serum and 0.2% bovine serum albumin) and
PDGF-BB (10 ng/ml). Cells were incubated in the Transwells for 4 h
at 37 °C. The number of cells that migrated to the lower surface was
counted. Basal migration was determined in the absence of PDGF-BB. Data
are presented as a percent of the cells that migrated to the lower
surface relative to cells incubated with PDGF-BB only. Data are
expressed as the mean ± S.E. from three samples in a single
representative experiment. Each experiment was repeated at least three
times with similar results.
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Fig. 3.
The effect of apoE peptides on PDGF-directed
smooth muscle cell migration. Quiescent smooth muscle cells were
incubated with apoE-(141-155)2 (filled
circles), apoE-(141-155) (open circles), or
apoE-(211-243) (filled triangles) for 30 min prior to
plating cells (2 × 104 cells/well) on the Transwell
membrane. The lower chamber of the Transwell contained basal medium
(DMEM with 0.4% fetal bovine serum and 0.2% bovine serum albumin) and
PDGF-BB (10 ng/ml). After a 4-h incubation at 37 °C on the
Transwells, cells that migrated to the lower surface were counted.
Basal migration was determined in the absence of PDGF-BB. Data are
presented as the percent of cells that migrated to the bottom chamber
in the presence of apoE peptides relative to the number of cells that
migrated toward PDGF-BB in the absence of apoE peptides. Data represent
the mean ± S.E. from three replicates in a single experiment. The
experiment was repeated at least three times with similar
results.
|
|
Previous studies have shown that both the receptor binding domain and
the domain between residues 211 and 243 are capable of heparin binding
(28, 29). Moreover, the receptor binding properties of apoE require
multiple copies of the receptor binding domain (30, 31). Thus, the
differential effects of the apoE peptides on PDGF-stimulated smooth
muscle cell proliferation and migration suggest that apoE inhibition of
smooth muscle cell migration may be mediated by its interaction with
cell surface receptors, while its inhibition of cell proliferation
requires its interaction with HSPG. This hypothesis was examined by
taking advantage of previous observations that reductive methylation of
lysine residues in apoE abolished its ability to bind receptors (25,
32), without interfering with its heparin binding properties (25, 33).
In the current experiments, reductively methylated apoE was found to be
equally effective as native apoE in inhibiting PDGF-stimulated smooth
muscle cell proliferation, with complete inhibition observed at 1.5 µM (Table I). In contrast
to the results observed on apoE inhibition of cell proliferation,
reductive methylation of apoE abolished its ability to inhibit smooth
muscle cell migration toward PDGF (Fig.
4).
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Table I
Effect of reductive methylation on apoE inhibition of smooth muscle
cell proliferation
Serum-starved mouse smooth muscle cells were incubated for 24 h in
96-well plates (2.5 × 103 cells/well) in the presence or
absence of 10 ng/ml PDGF-BB, 1.5 µM native apoE, or
reductively methylated apoE (CH3-apoE). [3H]Thymidine
(1 µCi) was added to each well 5 h prior to the end of the
incubation period. Cell proliferation was determined as the amount of
[3H]thymidine incorporated into cellular DNA. Maximum
stimulation was determined by subtracting the basal amount of
[3H]thymidine incorporated into cellular DNA in the absence
of growth factor stimulation from that observed with PDGF stimulation
in the absence of apoE. The data represent mean ± S.E. from
triplicate determinations in two separate experiments.
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Fig. 4.
Effects of reductive methylation on the
ability of apoE to inhibit PDGF-BB-directed smooth muscle cell
migration. Quiescent smooth muscle cells were incubated with apoE
(closed bars) or reductively methylated apoE (open
bars) for 30 min at 37 °C before adding to the top chamber of
Transwell membranes in 24-well dishes at a density of 2 × 104 cells/well. Cells that migrated toward the lower
chamber of the Transwells, which contained basal medium with PDGF-BB
(10 ng/ml), were determined after a 4-h incubation period. Basal
migration was determined in the absence of PDGF-BB. Maximum stimulation
was determined by incubating cells in the absence of apoE
(hatched bar). Data represent the mean ± S.E. of
triplicate samples from a representative experiment. The experiment was
repeated at least three times with similar results.
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|
The importance of the receptor binding domain in mediating the
anti-migratory properties of apoE was further tested by determining the
effect of the receptor binding domain-specific apoE antibody 1D7 (34,
35) on PDGF-induced smooth muscle cell migration. Results showed that
the 1D7 antibody alleviated the apoE inhibition of smooth muscle cell
migration toward PDGF (Fig. 5).
Importantly, 1D7 at similar concentrations did not alleviate apoE
inhibition of smooth muscle cell proliferation (data not shown).

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Fig. 5.
Antibody inhibition of apoE effects on
PDGF-directed smooth muscle cell migration. Serum-starved mouse
smooth muscle cells were preincubated in the presence or absence of
apoE and its receptor binding domain-specific antibody 1D7 for 30 min
at 37 °C prior to addition to the top chamber of Transwell membranes
in 24-well plates at a density of 2 × 104 cells/well.
Cells were allowed to migrate toward the PDGF-BB (10 ng/ml) in the
lower chamber for 4 h at 37 °C. Cells that migrated to the
lower surface of the membrane were counted and expressed as the percent
of cells that migrated toward PDGF in the lower chamber in the absence
of apoE pretreatment. Data represent the mean ± S.E. of
triplicate determinations in two separate experiments.
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Additional experiments were performed to further test the hypothesis
that apoE inhibition of smooth muscle cell migration is independent of
HSPG binding. In these experiments, smooth muscle cells were
preincubated with 1 unit/ml heparinase in the presence or
absence of apoE prior to the determination of PDGF-directed smooth
muscle cell migration. Heparinase pretreatment inhibited PDGF-stimulated smooth muscle cell proliferation (data not shown). Thus, this approach cannot be used to assess HSPG binding on cell proliferation. However, treating cells with 1 unit/ml heparinase had no effect on PDGF-directed smooth muscle cell migration (Fig. 6). Importantly, preincubation of cells
with heparinase also had no effect on apoE inhibition of PDGF-directed
smooth muscle cell migration (Fig. 6). These results document that apoE
inhibition of smooth muscle cell migration is independent of apoE
binding to HSPG.

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Fig. 6.
Heparinase treatment of smooth muscle cells
on apoE inhibition of PDGF-BB-directed migration. Serum-starved
mouse smooth muscle cells were preincubated in the presence or absence
of heparinase III (1 unit/ml) and apoE (1.5 µM) for 30 min at 37 °C prior to addition to the top chamber of Transwell
membranes in 24-well plates at a density of 2 × 104
cells/well. Cells were allowed to migrate toward the PDGF-BB (10 ng/ml)
in the lower chamber for 4 h at 37 °C. Cells that migrated to
the lower surface of the membrane were counted and expressed as the
percent of cells that migrated toward PDGF in the lower chamber in the
absence of apoE or heparinase pretreatment. Heparinase treatment of the
cells did not alter the ability of the cells to migrate toward the
PDGF-BB. Data represent the mean ± S.E. of triplicate
determinations in a single representative experiment. The experiment
was repeated at least three times with similar results.
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 |
DISCUSSION |
Previous studies have documented that residues 141-155 encompass
the receptor binding domain of apoE (34). This domain and the one
encompassing residues 211-243 have also been identified as heparin
interaction sites on apoE (28, 29). The difference between receptor and
heparin binding properties of apoE is that multiple copies of the
receptor binding domain are necessary for receptor interaction (30, 31,
36), while a single copy of either domain is sufficient to confer
heparin interaction (28, 29). Results of the current study showed that
peptides containing at least one copy of either the receptor binding
domain or the distal heparin binding site of apoE were capable of
inhibiting PDGF-stimulated smooth muscle cell proliferation. These
results indicated that apoE inhibition of smooth muscle cell
proliferation is mediated via its interaction with HSPG on the cell
surface. Interestingly, none of these peptides were as effective as
native apoE in inhibiting smooth muscle cell proliferation. This
observation suggested that optimal inhibition of smooth muscle cell
proliferation may involve multiple domain interactions between apoE and
HSPG.
The interactive role of apoE and HSPG in the modulation of cell
functions has been proposed previously. Obunike et al. (37) showed that endogenously expressed apoE enhances cellular proteoglycan synthesis. Exogenously added apoE also yielded a 2-fold induction of
proteoglycan synthesis and secretion by smooth muscle cells (38, 39).
The latter study further demonstrated a correlation between apoE
inhibition of cell proliferation and its induction of HSPG
synthesis. Anti-perlecan antibody was also shown to completely abrogate the anti-proliferative effect of apoE (38). Based on these
observations, these investigators proposed that apoE, like other
anti-proliferative agents such as transforming growth factor-
(40)
and heparin (41), inhibits smooth muscle cell proliferation by
promoting the synthesis and secretion of perlecan (38). However, the
mechanism by which apoE stimulates perlecan synthesis and the cell
surface molecules responsible for these apoE effects have not been
determined. The current study showed that apoE inhibits smooth muscle
cell proliferation via binding to cell surface HSPG. Thus, it is likely
that apoE-HSPG interaction induces intracellular signals required for
activation of perlecan synthesis and its subsequent inhibition of cell
proliferation. The observation that reductively methylated apoE, which
does not bind to receptors but remains active in HSPG binding, was
capable of inhibiting smooth muscle cell proliferation is supportive of
this conclusion.
In contrast to their effects on cell proliferation, peptides containing
either a single copy of the receptor binding domain or the distal
heparin binding domain of apoE did not inhibit PDGF-directed smooth
muscle cell migration. Only the peptide containing a tandem repeating
sequence of the receptor binding domain of apoE displayed anti-migratory effects. In fact, the apoE-(141-155)2
peptide was equally as active as native apoE in inhibiting
PDGF-directed smooth muscle cell migration. Moreover, our results also
showed that reductive methylation of apoE, which abolished its
receptor, but not its heparin, binding activities (25, 33), also
abolished the anti-migratory, but not the anti-proliferative, effects
of apoE. These results, taken together with observations showing the
receptor domain-specific apoE antibody abolished the anti-migratory properties of apoE, indicated that apoE inhibition of smooth muscle migration is mediated via its binding to cell surface receptors and is
independent of HSPG interaction.
The mechanism by which apoE interaction with receptors
results in inhibition of cell migration remains unknown at this time. However, there is increasing evidence to suggest that apoE interaction with LDL receptor gene family member proteins may lead to signal transduction events that can modulate cell functions. For example, apoE
binding to apoE receptor-2 (LR8) on platelets has been shown to induce
nitric oxide synthesis and inhibit agonist-induced platelet aggregation
(42, 43). Interestingly, LR8 contains a Src homology 3 (SH3) consensus
sequence, as well as domains that can serve as targets for cGMP and
cAMP-dependent protein kinases. Thus, it is possible that
apoE inhibits platelet aggregation and induction of nitric oxide
synthesis through LR8-mediated signal transduction events. This
receptor is also present in the brain and has been shown to be
important for reelin signaling and activation of the c-Jun N-terminal
kinase-signaling pathway (14, 15, 44). Likewise, both the VLDL
receptor and LRP have been shown to interact with cytosolic adaptor and
scaffold proteins and modulate the transmission of extracellular
signals to activate intracellular tyrosine kinases (16). Since LRP,
VLDL receptor, and LR8 are all present on the surface of smooth muscle
cells, it is possible that apoE inhibition of smooth muscle cell
migration is mediated through signaling events subsequent to apoE
binding to one or more of these receptors. The identification of the
receptor responsible for mediating apoE inhibition of smooth muscle
cell migration will contribute to our understanding in this regard.
Regardless of the exact mechanisms involved, our data
documented that the anti-proliferative and anti-migratory effects of apoE are distinct, requiring different concentrations of apoE (9), and
are mediated through its interaction with HSPG and cell surface
receptors, respectively. A concentration-dependent effect of
apoE on cell functions has been reported previously in studies
examining its role on regulation of androgen synthesis by the ovary.
Dyer and Curtiss (45) showed that low concentrations of apoE promoted
androgen synthesis, while high concentrations of apoE were inhibitory.
Subsequent studies revealed that the tandem repeat peptide
apoE-(141-155)2 mimicked the effect of intact apoE, but
the single-copy peptide encompassing apoE residues 129-162 did not
stimulate androgen production but rather inhibited androgen synthesis
at high concentrations (46). These authors suggested that apoE
stimulation of androgen production may be mediated through apoE binding
to members of the LDL receptor gene family (46) and that inhibition of
androgen synthesis at high apoE concentrations is mediated by a
mechanism independent of any LDL receptor-related proteins. Although
the exact mechanism by which apoE modulates androgen production in
ovaries has not been identified, it is possible that this cell
regulatory function of apoE is similar to that observed here,
i.e. through processes mediated by distinct cell signaling
mechanisms as a consequence of apoE interaction with receptors and HSPG
(8, 9).
In summary, this study adds to the expanding literature documenting a
direct role of apoE interaction with receptors and HSPG in signal
transduction and modulation of cell functions. The current study also
documented that apoE interaction with receptors and HSPG modulates
distinct cell functions, i.e. inhibition of cell migration
and proliferation, possibly through triggering of different signal
transduction pathways. Thus, apoE may protect against vascular occlusive diseases by multiple mechanisms, including reverse
cholesterol transport and inhibition of smooth muscle cell migration
and proliferation. We have previously shown that the apoE level in
circulation can directly influence neointimal hyperplasia in response
to vascular injury, a process that is independent of lipid transport
(47). Accordingly, designing therapeutic agents that can increase the apoE level in circulation may be beneficial in combating vascular disease via multiple mechanisms.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant HL61332.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Received Postdoctoral Fellowship 9920615V from the Southern and
Ohio Valley Consortium of the American Heart Association.
§
To whom correspondence should be addressed: Dept. of Pathology and
Laboratory Medicine, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0529. Tel.: 513-558-9152; Fax:
513-558-2141; E-mail: Huidy@email.uc.edu.
Published, JBC Papers in Press, May 11, 2001, DOI 10.1074/jbc.M102357200
 |
ABBREVIATIONS |
The abbreviations used are:
apo, apolipoprotein,
PDGF, platelet-derived growth factor;
HSPG, heparan sulfate
proteoglycans;
DMEM, Dulbecco's modified Eagles' medium;
LDL, low
density lipoprotein;
VLDL, very low density lipoprotein;
LRP, LDL
receptor-related protein.
 |
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