From the Departments of Clinical Biochemistry, and
** Medicine, the Center for Research, Prevention, and
Treatment of Atherosclerosis, Hadassah University Hospital and Hebrew
University-Hadassah Medical School, Jerusalem 91120, Israel,
Departments of ¶ Pathology and Laboratory Medicine and
Pediatrics, University of Pennsylvania, Philadelphia 19104, Pennsylvania,
Tel-Aviv University, Sackler
School of Medicine, and Sheba Medical Center, Institute for Autoimmune
Diseases, Tel-Aviv 69978, Israel, and the
§§ Lipid Research Laboratory, Rambam Medical
Center and Rappaport Institute for Research in the Medical Sciences,
Technion Faculty of Medicine, Haifa 31096, Israel
Received for publication, August 30, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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Accumulation of low-density lipoprotein
(LDL)-derived cholesterol by macrophages in vessel walls is a
pathogenomic feature of atherosclerotic lesions. Platelets contribute
to lipid uptake by macrophages through mechanisms that are only
partially understood. We have previously shown that platelet factor 4 (PF4) inhibits the binding and degradation of LDL through its receptor,
a process that could promote the formation of oxidized LDL (ox-LDL). We have now characterized the effect of PF4 on the binding of ox-LDL to
vascular cells and macrophages and on the accumulation of cholesterol esters. PF4 bound to ox-LDL directly and also increased ox-LDL binding
to vascular cells and macrophages. PF4 did not stimulate ox-LDL binding
to cells that do not synthesize glycosaminoglycans or after enzymatic
cleavage of cell surface heparan and chondroitin sulfates. The effect
of PF4 on binding ox-LDL was dependent on specific lysine residues in
its C terminus. Addition of PF4 also caused an ~10-fold increase in
the amount of ox-LDL esterified by macrophages. Furthermore, PF4 and
ox-LDL co-localize in atherosclerotic lesion, especially in
macrophage-derived foam cells. These observations offer a potential
mechanism by which platelet activation at sites of vascular injury may
promote the accumulation of deleterious lipoproteins and offer a new
focus for pharmacological intervention in the development of atherosclerosis.
The development of atherosclerosis has been attributed to an
interplay among several factors, including endothelial cell denudation, adherence and activation of platelets, genetic and acquired
hyperlipidemia, oxidation of lipoproteins, infiltration of the vessel
wall with macrophages and their conversion to foam cells, and smooth
muscle cell proliferation and migration (1). Activated platelets may contribute to the progression of atherosclerosis through diverse pathways, including the release of potent smooth muscle cell mitogens such as platelet-derived growth factor, epidermal growth factor, and
transforming growth factor- One potential candidate that might be involved in the accumulation of
LDL is platelet factor 4 (PF4), a cationic protein that is released in
large amounts when platelets are activated and that is found in
atherosclerotic lesions (15, 16). Mature human PF4 is a 70-amino acid,
lysine-rich platelet-specific protein that belongs to the CXC (or PF4 secreted by platelets binds primarily to endothelial heparan
sulfate-rich proteoglycans, inhibiting the activity of anti-thrombin III (23), to the chemokine receptor Duffy on erythrocytes and endothelial cells (24, 25) and to coagulation proteins such as
thrombomodulin (26) and activated protein C (27, 28). PF4 also binds to
exposed subendothelium (29), where it may function as an inhibitor of
endothelial cell proliferation and angiogenesis (30). However, its
effects on vessel wall biology and the formation of atherosclerosis are
less clear. Although platelet activation promotes the accumulation of
LDL by macrophages, PF4 inhibits the uptake of LDL by fibroblasts (31).
In an attempt to elucidate the mechanism by which PF4 may facilitate
the incorporation of LDL into cells, we previously reported that PF4
diverts the uptake of LDL from the LDL receptor to the
relatively inefficient PG-dependent endocytic pathway,
likely increasing its residency time in tissue and its propensity to
undergo oxidation (47).
LDL that has not been internalized and degraded is subject to oxidation
in the vascular microenvironment (32). Unlike LDL, which is
internalized primarily through the LDL receptor, oxidized LDL is
internalized by the scavenger receptor and several other, more recently
described receptors (33-35). In the present study we extend these
studies by showing that, in contrast to LDL, PF4 stimulates the
binding of ox-LDL by vascular cells and promotes its esterification. We
propose a novel mechanism by which platelet activation may contribute
to the development of atherosclerosis through the release of PF4.
Recombinant PF4 and PF4 Variants--
A number of recombinant
PF4-like proteins that had been previously expressed and purified from
prokaryotic cells and characterized for their heparin affinity were
used. These include wild type (WT) PF4 and neutrophil-activating
protein-2 (NAP-2), a series of lysine to alanine substitutions in the
lysine-rich C terminus of PF4, and chimeric proteins between PF4 and
NAP-2 (36). PF4 was radiolabeled with 125I using
lactoperoxidase (Sigma) as described (37). Labeled protein was
separated from the free iodine using gel filtration (PD-10; Amersham Biosciences), and the protein concentration was
determined using Bio-Rad microprotein enzyme-linked immunosorbent assay.
Cell Culture--
All cell cultures were maintained at 37 °C
under 5% CO2. Chinese hamster ovary (CHO) cells lacking
xylosyl transferase (XT Lipoprotein Preparation--
125I-LDL was prepared,
radiolabeled, and characterized as described (40, 41). Briefly, LDL was
isolated by ultrafiltration at d = 1.019-1.63 g/ml
from human plasma containing 1 mg/ml EDTA. The preparation was filtered
under sterile conditions, aliquoted into plastic tubes and kept under
N2 at 4 °C without exposure to light. LDL was oxidized
as described in Ref. 41. Briefly, LDL was dialyzed against
phosphate-buffered saline (PBS) at 4 °C to remove EDTA, then diluted
with PBS to 0.1 mg of protein per ml prior to oxidation. LDL was
incubated with 10 µM CuSO4 at 37 °C for
18 h. LDL oxidation was terminated by adding 25 µM butylated hydroxytoluene and 100 µM EDTA and by
refrigerating the LDL samples at 4 °C. The extent of LDL oxidation
was analyzed by the thiobarbituric acid-reactive substances assay,
which measures malondialdehyde equivalents (42), and by the lipid
peroxides assay that analyzes their capacity to convert iodide to
iodine (43). The amount of cholesteryl linoleate hydroperoxides was measured by high pressure liquid chromatography (44). Electrophoresis of the lipoproteins was performed on 1% agarose using a Hydragel-Lipo kit (Sebia). The results of the characterization of the ox-LDL are depicted in Table I. Acetylated LDL
(Ac-LDL) was prepared by addition of acetic anhydride to a stock
solution of LDL (3 mg of protein/ml) diluted 1:1 v:v with a solution of
saturated ammonium acetate at 4 °C by the method of Basu et
al. (45).
Binding of LDL and PF4 to Cells--
Cells were grown to
confluence in 48-well Falcon multiwell tissue culture plates (Becton
Dickinson, Lincoln Park, NJ) to a final density of ~5 × 104 cells/well. The cells were prechilled to 4 °C for 30 min and washed twice with cold binding buffer composed of PBS
containing 0.5% bovine serum albumin (BSA). The cells were then
incubated with 125I-LDL alone or in the presence of PF4
for 3 h at 4 °C. The incubation was terminated by washing
the cells four times with binding buffer to remove unbound ligand.
Radiolabeled ligand bound to the cell surface was released with 50 mM glycine-HCl, pH 2.8. The cells were then solubilized by
adding 0.1 N NaOH, and the residual cell-associated radioactivity was measured. Nonspecific binding was determined by
measuring cell-associated radioactivity in the presence of 20-fold
molar excess unlabeled LDL. Specific binding was defined as the
difference between total and nonspecific binding. The same cells and
conditions were used to measure the binding 125I-PF4 as
were used to determine the binding of 125I-LDL.
In other experiments, the formation of complexes between
125I-PF4 with LDL and with ox-LDL was examined.
125I-rPF4 (25 nM) was incubated with LDL,
ox-LDL, or BSA (50 nM) for 30 min at 37 °C in PBS. The
mixture was applied to a Bio-spin 30-gel filtration column (Bio-Rad),
and the radioactivity in the excluded volume was measured. To examine
the binding of 125I-ox-LDL/PF4 complexes to cells,
125I-ox-LDL was incubated with rPF4 or BSA for 30 min at
37 °C in PBS. The complexes were separated on Sephadex G-75. These
and all experiments described below were performed in triplicates and
were repeated a minimum of three times, unless otherwise noted. All
data are presented as mean ± S.D. of the three experiments.
Confocal Microscopy--
Cells grown on coverslips were
incubated in DMEM containing ox-LDL with or without PF4 and
supplemented with 10% fetal calf serum for 30 min at 37 °C. The
cells were washed three times with PBS, fixed for 10 min with 4%
formaldehyde in PBS, and permeabilized with 0.2% Triton x-100 in
PBS-BSA buffer for 3 min. The coverslips were overlaid for 20 min with
2% normal horse serum and then incubated for 40 min with anti-ox-LDL
antibodies diluted 1:500 with PBS. After four washes with PBS, the
cells were stained for 40 min with Alexa 488-labeled goat anti-rabbit
serum (Molecular Probes), washed four times with PBS and mounted in
80% glycerol and 20% PBS supplemented with 3% DABCO
(1,4-diazabicyclo-(2, 2)-octane) as anti-bleaching agent. No staining
was seen when either anti-defensin or the secondary antibody was omitted.
Confocal microscopy was performed using a Zeiss LSM 410 confocal laser
scanning system attached to the Zeiss axiovert 135 M
inverted microscope 40×/1.3 plain oil immersion lens. The system was
equipped with a 25-mW argon laser (488 nm excitation line with 515 nm
low pass barrier filter) for the excitation of Alexa 488 green
fluorescence. The differential interference contrast images according
to Nomarski were collected simultaneously using a transmitted light
detector. Autofluorescence of the specimen was set to background level.
Cholesterol Esterification--
J774 murine macrophages were
cultured in DMEM supplemented with 2 mM glutamine and 10%
fetal calf serum. The cells were washed, and the medium was replaced
with DMEM containing 200 nM native or ox-LDL in the
presence or absence of 100 nM PF4. 3H-oleic
acid (100 µM) was added in the form of
oleate-albumin complex (3:1 molar ratio) for 24 h at
37 °C, and the incorporation of 3H-oleic acid was
measured as a marker of cholesterol ester formation. To do so, the
medium was collected, the cells were washed four times with PBS, and 1 ml of absolute ethanol was then added for 2 h. The ethanol extract
was separated by thin layer chromatography on silica gels. The bands
were identified and isolated, and the radioactivity incorporated into
the cholesterol ester fraction was counted.
Assay of Cholesterol and Protein Content of Cells--
The total
and free cholesterol contents of J774 cells were determined using an
enzymatic kit (Roche Molecular Biochemicals). For these assays, J774
cells were harvested by scraping into distilled water and processed as
described previously (46). J774 protein content was determined by the
method of Lowry et al. using BSA as a standard.
Immunohistochemistry--
Human atherosclerotic arteries were
obtained anonymously from either surgically removed or autopsy
specimens from the Cooperative Human Tissue Network, Eastern division,
after approval from the University of Pennsylvania School of Medicine
Institutional Review Board. Formalin-fixed, paraffin-embedded 5-µm
sections were deparaffinized in xylene and rehydrated. Endogenous
peroxidase activity was quenched with 0.9% peroxide in methanol and
unreactive sites were blocked with 10% goat serum in 1× automation
buffer (Biomeda Corp.) for 20 min at 37 °C. Slides were then
incubated overnight at 4 °C with either rabbit anti-PF4 antibody (1 µg/ml polyclonal affinity-purified antibody from PeproTech), rabbit
anti-ox-LDL antibody (1:250 dilution of rabbit serum from CHEMICON
International), or normal rabbit serum as control. Sections were then
washed in 1× automation buffer and incubated for 30 min with
biotinylated goat-anti-rabbit IgG (Jackson Laboratories, West Grove,
PA) diluted 1:200 at 37 °C. Slides were washed and incubated with
streptavidin-horseradish peroxidase (Research Genetics) for 30 min at
37 °C and washed, and stable diaminobenzine chromogen
(Research Genetics) was applied for 5 min at 20 °C. Slides were
counterstained with dilute hematoxylin.
Effect of PF4 on the Binding and Internalization of ox-LDL--
We
have previously reported that PF4 diverts the uptake of LDL from the
LDL receptor to the relatively inefficient
PG-dependent endocytic pathway, likely increasing its
residency time in tissue and its propensity to undergo oxidation (47).
LDL that has not been internalized and degraded is subject to oxidation
in the vascular microenvironment (32). Unlike LDL, which is
internalized primarily through the LDL receptor, oxidized LDL is
internalized by the scavenger receptor and several other, more recently
described receptors (33-35). Therefore, based on its effects on LDL,
we examined the effect of PF4 on the uptake of ox-LDL by cells involved
in the pathogenesis of atherosclerosis.
In contrast to its inhibitory effect on the binding of LDL to cells
(47), PF4 promoted the binding of ox-LDL to cultured endothelial cells
and smooth muscle cells ~5-fold, and to J774 cells ~7-fold (Fig.
1A). Binding of ox-LDL to
HUVEC was increased by PF4 in a dose-dependent and
saturable manner (Fig. 1); half-maximal enhancement was observed at a
PF4 concentration of 1.63 µM. Lower concentrations of PF4
(50 nM) were sufficient to enhance the internalization of
ox-LDL as assessed by confocal microscopy (Fig. 1B).
These data are consistent with the known enhancement of
platelet activation on LDL metabolism and cholesterol ester
accumulation by macrophages (9-12).
Mechanism of the PF4 Stimulation of ox-LDL Binding--
The
finding that PF4 has opposing effects on the binding of LDL and ox-LDL
suggests that it may interact differently with the two forms of the
lipoprotein. To explore this possibility, we examined the binding of
125I-PF4 to LDL or ox-LDL directly using gel filtration.
Indeed, ox-LDL, but not WT LDL or BSA, bound radiolabeled PF4 (Fig.
2A). To examine the
consequences of this interaction, we next examined the binding of
ox-LDL/PF4 complexes to vascular cells and macrophages. Radiolabeled
ox-LDL was incubated with PF4, the complexes were isolated by gel
filtration, and binding to cells was measured. 125I-ox-LDL/PF4 complexes exhibited ~5-fold more binding
to endothelial cells than did 125I-ox-LDL alone (Fig.
2B). We observed a comparable increase in the binding of
PF4-ox-LDL complexes to HVSMC and J774 cells (data not shown).
These data suggest that PF4 and ox-LDL bind to cells as a complex.
Based on this finding, our next aim was to define the portion of the
complex (ox-LDL or PF4) that mediates cell binding. To address this
question, we examined the effect of ox-LDL on the binding of
125I-PF4 to cells. ox-LDL did not stimulate the binding of
PF4 (data not shown). The finding that PF4 stimulated the binding of
ox-LDL, whereas ox-LDL did not stimulate the binding of PF4, is
consistent with a model wherein PF4 mediates the binding of the complex
to the cells and binding of ox-LDL itself does not contribute to this process.
To understand the mechanism by which PF4-ox-LDL complexes bind to
cells, we took advantage of the fact that PF4 binds to heparin (48) and
to cell surface-associated proteoglycans (49) and that the binding to
proteoglycans is inhibited by heparin. Furthermore, heparin does not
bind ox-LDL (50). Based on this, we examined the effect of heparin on
the binding of ox-LDL/PF4 to cells. Heparin inhibited the binding of
ox-LDL/PF4 to HUVEC to essentially the same level as ox-LDL alone (Fig.
2B). Taken together, these data suggest that components on
the cell surface that share properties with heparin and that bind PF4
are engaged in the binding of the ox-LDL/PF4 complexes.
The Role of Cell Surface Proteoglycans in PF4-mediated Binding of
ox-LDL--
Given the above data, proteoglycans containing
glycosaminoglycans that share similarity with heparin and bind PF4 are
likely candidates to be the cell surface binding sites for ox-LDL/PF4 complexes. To examine this possibility, we compared the binding of the
ox-LDL/PF4 complexes to cells with altered proteoglycan cell surface
expression. In these experiments we used two independent approaches. In
the first approach, we used a XT Determinants in PF4 Required to Stimulate ox-LDL Binding--
As a
third, independent approach toward understanding the requirements for
PF4 to promote the binding of ox-LDL to cells, we studied the effect of
NAP-2, another member of the CXC chemokine family that shares extensive
sequence homology with PF4 and demonstrates significant affinity for
heparin (51, 52). NAP-2 did not affect the binding of ox-LDL (Fig.
4A).
Two chimeric constructs in which sequences from PF4 had been exchanged
with an analogous region of NAP-2 were then used to further define the
structural elements in PF4 involved ox-LDL metabolism. These constructs
were NPPP, in which the N-terminal eight amino acids before the first
of the four conserved cysteine residues in PF4 was replaced with the
homologous NAP-2 sequence, and NNNP, in which the complete PF4 sequence
from the N terminus to the fourth cysteine residue was replaced with
the homologous sequence from NAP-2. Previous studies showed that NPPP
binds heparin with the same affinity as PF4, while NNNP binds similarly
to both NAP-2 and the lysine to alanine PF4 C-terminal substitutions
discussed below (36). NPPP was as effective as PF4 in stimulating
ox-LDL binding by HUVEC, whereas NNNP was as ineffective as NAP-2 (Fig. 4A). These studies showed that the N terminus of PF4 is not
critically involved in modulating ox-LDL binding.
Consistent with the ability of heparin to block PF4/ox-LDL complexes
from binding to cell surfaces (Fig. 2B), the importance of
surface heparanoid molecules in complex binding (Fig. 3), and the fact
that NAP-2 binds heparin with somewhat lower affinity than does PF4
(37), we next examined the determinants in PF4 required to bind heparin
in greater detail and to consider their impact on ox-LDL binding. Each
PF4 monomer contains four lysine residues that have been implicated in
the binding of heparin and that are located in the C-terminal
The capacity of NAP-2 and PF4 variants to form complexes with ox-LDL
paralleled their capacity to stimulate binding of ox-LDL to cells.
Specifically, ox-LDL bound to the chimera NPPP but not to NAP-2 or NNNP
(Fig. 5). None of the WT or chimeric
chemokines bound to LDL (data not shown), suggesting that the capacity
of PF4 to bind to ox-LDL, but not LDL, underlies its divergent effect on the cellular binding of the two lipoproteins. The capacity of each
active PF4 variant and the NPPP chimera to stimulate ox-LDL binding was
negated in cells genetically lacking or enzymatically stripped of
proteoglycans (data not shown), suggesting that each act by the same
mechanism as PF4.
Effect of PF4 on ox-LDL Metabolism--
Even early atherosclerotic
lesions are characterized by macrophage-derived foam cells enriched in
cholesterol ester. Therefore, we next examined the effect of PF4 on the
esterification of cholesterol from ox-LDL. PF4 increases esterification
of ox-LDL cholesterol by J774 cells almost 10-fold (Fig.
6), while NAP-2 had no effect. Furthermore, Fig. 6 shows that PF4 stimulates the esterification of
Ac-LDL cholesterol but not that from native LDL. To study the role of
proteoglycans in the PF4-mediated stimulation of ox-LDL esterification,
we incubated the cells with heparanase and chondroitinase. Enzymatic
cleavage of proteoglycans decreased the capacity of J774 cells to
esterify cholesterol from the ox-LDL/PF4 complex by more than 70%
(data not shown). These studies show that PF4 binds ox-LDL in
vitro, and the resultant complex shows enhanced binding to
vascular cells and promote esterification of ox-LDL by J774 cells. To
confirm the effect of PF4 on ox-LDL metabolism after incubation of J774
cells with ox-LDL in the presence and absence of PF4, the cell content
of cholesterol and cholesterol ester was determined. The presence of
PF4 during the incubation increased the cell content of cholesterol
from 17 ± 3.3 nmol/mg cell protein at time zero to 104 ± 7.3 after 24 h. In the absence of PF4 the increase of cellular
cholesterol was only to 37 ± 4.5 nmol/mg cell protein. Similarly,
at the end of the incubation, in the presence of PF4, the cellular
content of cholesterol ester from ox-LDL was 9.8 ± 1.4 nmol/mg
cell protein compared to 1.3 ± 0.7 in its absence. Similar
results were obtained with Ac-LDL (data not shown).
PF4 and ox-LDL in Atherosclerotic Lesions--
Lastly, to evaluate
the relevance of these in vitro findings, we examined the
localization of PF4 and ox-LDL in human atherosclerotic lesions.
Immunohistochemical staining of serial sections of human arteries
involved by atherosclerosis demonstrated the presence of both PF4 and
ox-LDL in macrophages (Fig. 7). This was
true for early lesions (Fig. 7) as well as more advanced lesions (data not shown). That PF4 is present with ox-LDL in early atherosclerotic lesions supports our hypothesis that PF4 may contribute to early lesion
formation by increasing the vascular accumulation of ox-LDL.
The molecular basis of the development of atherosclerosis is
complex, multifaceted, and incompletely understood. Platelets may
contribute to this process by stimulating lipid uptake by macrophages
(10-12). However, the mediators responsible for this activity and the
mechanism by which they act have not been defined and merit detailed
investigation in an era where chronic administration of inhibitors of
platelet activation has become possible.
One potential mediator of platelet involvement in atherosclerosis is
PF4, a chemokine that comprises 2-3% of the total protein releasate
on a molar basis (21). The biological role of PF4 is unclear, although
it clearly binds to heparin and heparanoid side-chains with high
affinity. The potential involvement of PF4 in LDL metabolism has been
controversial because of two apparently contradictory observations: PF4
inhibits the uptake of LDL by fibroblasts (47), while, on the other
hand, platelet activation stimulates macrophage uptake of
cholesterol (9-12). Our finding that PF4 inhibits the binding
and degradation of LDL by cells but stimulates the binding of ox-LDL
offers an explanation for these apparently divergent findings. The
effect of PF4 on both forms of the lipoprotein involves its capacity to
bind heparin-like molecules. Mutation of the amino acid residues
involved in heparin binding decreases its capacity to affect the
binding of both subtypes of LDL. Moreover, heparin inhibits the effect
of PF4 on both species of LDL.
How does the interaction of PF4 with heparin inhibit the binding of LDL
to cells while stimulating ox-LDL binding to the same cells? Our
studies provide insight into this aspect of the process by showing that
one striking difference is that PF4 binds to ox-LDL but not to LDL. The
stimulatory effect of PF4 depends on its ability to bind heparin and
ox-LDL simultaneously. Only WT PF4 and mutants that bind heparin and
form complexes with ox-LDL enhance lipoprotein binding to cells.
Similarly, chimeric PF4 constructs that bind heparin weakly and/or that
do not bind ox-LDL had no stimulatory effect. The mechanism we propose
to explain the effect of PF4 on ox-LDL binding to cells is consistent
with studies suggesting that several cell types express a low affinity,
high capacity pathway for the uptake of cationized lipoproteins
(45).
Taking into consideration our studies and those previously reported, we
propose that PF4 released from activated platelets in the vicinity of a
perturbed vascular wall stimulates atherosclerosis through the
following mechanism: PF4 blocks LDL uptake by the LDL receptor
expressed by all of the prevalent cell types in the vascular wall,
increasing its retention within the vascular space. Increased residence
time in the vasculature permits the lipoprotein to undergo oxidative
and non-oxidative modifications. PF4 binds to nascent and preformed
ox-LDL. PF4 bridges ox-LDL and cell surface proteoglycans promoting
vascular retention. PF4 also enhances the endocytosis and subsequent
esterification of the cell-bound ox-LDL in macrophages, accelerating
the formation of foam cells. Our data lead us to propose a multistep
process by which PF4 accelerates the rate of plasma-derived LDL
cholesterol accumulation in the vascular tissue.
PF4 has been imputed to participate in diverse other biologic functions
through its strong affinity for heparin-like molecules in addition to
those reported in this study. Some of these, such as its chemotactic
properties for inflammatory cells (6, 54) and its ability to induce
monocytes to differentiate into macrophages (53) may also contribute to
the development of atherosclerosis. The fact that PF4 and ox-LDL were
found to be co-localized in human atherosclerotic lesions supports the
conclusions of our in vitro studies. Nevertheless, in
vivo studies underway in our laboratory in which PF4
concentrations are enhanced or eliminated in transgenic animal models
are needed to determine the biological relevance of PF4 for the
development of atherosclerosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(2-6). In accord with these observations, interventions that reduce platelet number and/or inhibit
platelet activation retard the formation of atherosclerotic lesions in
several experimental systems (7, 8). Platelet activation also promotes
the accumulation of low-density lipoproteins (LDL)1 by macrophage foam
cells (for review see Refs. 9-13). The mechanism(s) by which platelets
contribute to the accumulation of LDL in the vessel wall are less clear
(14).
)
chemokine subfamily, in which the first two of the four conserved
cysteine residues are separated by one amino acid residue (17). Human
PF4 has been sequenced (18) and cloned (19), and its x-ray
crystallographic structure has been defined (20). PF4 is synthesized by
megakaryocytes and comprises 2-3% of the total protein in mature
platelets (21). When platelets are activated, PF4 is secreted in
concentrations approaching ~25 µg/ml (4 µM) in the
vicinity of the vessel wall (22). PF4 exists as a tetramer with the
three
-sheets of each subunit facing inwards and the N and C termini
lying on the surface of the molecule. The four lysine-rich C-terminal
-helices form a circumferential band around the tetramer, where they
and other cationic residues have been implicated in the binding of
heparin (22, 23).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), and therefore incapable of
initiating the synthesis of heparan sulfate or chondroitin sulfate
chains, were the kind gifts of Dr. J. Esko, University of California,
San Diego, CA (38). WT and mutant CHO cells were grown in Ham's F-12
media supplemented with fetal calf serum (10%), glutamine (200 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml). Cultured human umbilical vein endothelial cells (HUVEC) and
smooth muscle cells were cultured as previously described (39).
Ox-LDL characteristics
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PF4 stimulates the binding of ox-LDL
to HUVEC, HVSMC, and macrophages. A,
125I-oxidized LDL (200 nM) was incubated with
HUVEC, HVSMC, or J774 cells for 3 h at 4 °C in the absence
(empty bars) presence (filled bars) of 10 µM PF4, and the cell-associated radioactivity was
measured. The mean ± S.D. of three experiments is shown for this
and all the figures below. The inset shows the binding of
ox-LDL to HUVEC incubated with increasing concentrations of PF4.
B, confocal microscopic studies of ox-LDL (50 nM) uptake by HUVEC in the absence (i) or in
presence of 50 nM (ii) or 100 nM PF4
(iii). In iv no ox-LDL was added. The uptake of
ox-LDL was done using a rabbit anti-ox-LDL primary antibody and a
fluorescein isothiocyanate-labeled anti-rabbit secondary antibodies.
Studies using an irrelevant primary antibody to defensin or leaving out
the secondary antibody were done and were similar to iv
(data not shown).
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Fig. 2.
PF4 directly binds ox-LDL, and the complex
binds to HUVEC. A, formation of complexes between PF4 and
ox-LDL or LDL was determined using 125I-recombinant PF4 (25 nM) incubated with ox-LDL, BSA, or LDL (50 nM
each) for 30 min at 37 °C in PBS. The mixture was applied to a
Bio-spin 30 gel filtration column, and the radioactivity in the
excluded volume was measured. B, complexes between PF4 (25 nM) and 125I ox-LDL (50 nM) or
125I ox-LDL alone were isolated by a gel filtration column
and incubated with HUVEC in the absence or presence of heparin (0.2 units/ml).
CHO cells that are
incapable of synthesizing heparan or chondroitin sulfates. These cells
bound ox-LDL/PF4 complexes poorly compared with WT CHO cells (Fig.
3). Second, simultaneous digestion of heparan and chondroitin sulfates from the cell surface neutralized the
stimulatory effect of PF4 on ox-LDL binding to WT CHO cells by more
than 80% (data not shown). Enzymatic cleavage of heparan and
chondroitin sulfates also greatly decreased the capacity of HUVEC
to bind ox-LDL/PF4 complexes (Fig. 3).
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Fig. 3.
The role of proteoglycans in the binding of
PF4/ox-LDL complexes to cells. Ox-LDL (200 nM) and PF4
(10 µM) were incubated with WT or XT /
CHO
cells expressing neither heparan sulfate nor chondroitin sulfate
proteoglycans. In another set of experiments, ox-LDL (200 nM) and PF4 (10 µM) were incubated with HUVEC
that had (HUVEC+PG) or had not (HUVEC) been
treated with heparan and chondroitin sulfatases.
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Fig. 4.
Role of the C terminus of PF4 in the binding
of ox-LDL. A, HUVEC were incubated with
125I-ox-LDL for 3 h at 4 °C in the presence of WT
PF4 (100% activity), NAP-2, NNNP, or NPPP (10 µM each),
and the cell-associated radioactivity was measured. B, HUVEC
were incubated as above in the presence of WT PF4 or PF4 variants with
single substitutions (Lys to Ala) at positions 62, 65, or 66 (10 µM each).
-helical domain (20). The results of a previous study (36) show that
mutation of any of the four amino acids residues from lysine to alanine
reduced heparin-binding affinity of the PF4 tetramer. In the current
study, we asked whether these lysine residues were also involved in the
capacity of PF4 to stimulate binding of ox-LDL to cells. To do so, we
compared the capacity of recombinant PF4 variants with individual Lys
to Ala substitutions at positions 62, 65, or 66 to inhibit the binding of 125I-LDL to the cells. (PF4K63A did not
express well, and no protein was available to test its function).
Whereas PF4K62A and PF4K65A were almost as
potent as PF4 both in terms of inhibiting LDL binding and promoting
ox-LDL binding to endothelial cells, PF4K66A was less
potent than PF4 but more potent than NAP-2 (Fig. 4B). These
data suggest that the heparin-binding lysine residues in the C-terminal
-domain of PF4 contribute to the stimulation of ox-LDL binding.
However, the data also indicate that although the binding to heparin is
important, it appears not to be the only feature as retention of
certain Lys residues are more critical than others with
PF4K65A retaining nearly normal activity despite having a
reduced heparin-binding capacity comparable to NAP-2.
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Fig. 5.
Binding of ox-LDL to PF4 and NAP2
chimeras. Formation of complexes between PF4 variants and
ox-LDL was determined as in Fig. 2A using PF4, NAP-2, the
chimeric PF4/NAP-2 molecules NNNP or NPPP (25 nM each).
Each recombinant chemokine was incubated with 50 nM
oxidized LDL (full bars) or BSA (empty
bars).
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Fig. 6.
Stimulation of cholesterol esterification in
macrophages by PF4. J774 cells were incubated with
3H-oleic acid (100 µM) and 200 nM
ox-LDL, acetylated LDL (Ac-LDL), or wild type LDL
(LDL) in the absence or presence of 10 µg/ml of PF4 or
NAP-2.
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[in a new window]
Fig. 7.
Immunohistochemical localization of
PF4 and ox-LDL in human atherosclerotic vessels. Shown are
sections of a carotid artery containing an early atherosclerotic
lesion. Sections were stained with anti-PF4 (A and
C), anti-ox-LDL (B and D) or preimmune
rabbit serum (insets in A and C). Higher
magnifications show that both PF4 (B) and ox-LDL
(D) in foam cells correspond to the boxes
outlined in A and B. Magnification, 50×
(A and B), 400× (C and
D).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* 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.
§ Both authors contributed equally to this work.
¶¶ To whom correspondence should be addressed: Children's Hospital of Philadelphia, 1 Civic Center, ARC, Rm. 317, Philadelphia, PA 19104. Tel.: 215-590-3574; Fax: 215-590-4834; E-mail: Poncz@email.chop.edu.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M208894200
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ABBREVIATIONS |
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The abbreviations used are: LDL, low-density lipoproteins; PF4, platelet factor 4; WT, wild type; NAP, neutrophil-activating protein; CHO, Chinese hamster ovary; XT, xylosyl transferase; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; HUVEC, human umbilical vein endothelial cells; ox-LDL, oxidized LDL; ac-LDL, acetylated LDL; BSA, bovine serum albumin; PG, proteoglycan; HVSMC, human vascular smooth muscle cell.
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REFERENCES |
---|
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---|
1. |
Williams, K. J.,
and Tabas, I.
(1995)
Arterios. Thromb. Vasc. Biol.
15,
551-558 |
2. | Woolf, N., and Carstairs, K. C. (1967) Am. J. Path. 51, 373-386[Medline] [Order article via Infotrieve] |
3. | Ross, R. (1993) Nature 362, 801-809[CrossRef][Medline] [Order article via Infotrieve] |
4. | Davies, M. J. (1996) Thromb. Res. 82, 1-32[CrossRef][Medline] [Order article via Infotrieve] |
5. | Hawiger, J. (1995) Thromb. Haemost. 74, 369-372[Medline] [Order article via Infotrieve] |
6. | Holvoet, P., and Collen, D. (1997) Curr. Opin. Lipid. 8, 320-328[Medline] [Order article via Infotrieve] |
7. | Harker, L., Ross, R., Slichter, S., and Scott, C. (1976) J. Clin. Invest. 58, 731-741[Medline] [Order article via Infotrieve] |
8. | Moore, S. R., Friedman, R. J., Singal, D. P., Gauldie, J., and Blajchman, M. (1976) Thromb. Diath. Haemorrhagica 35, 70-81 |
9. | Sevitt, S. (1986) Atherosclerosis 61, 107-115[Medline] [Order article via Infotrieve] |
10. | Kruth, H. S. (1985) Science 227, 1243-1245[Medline] [Order article via Infotrieve] |
11. | Curtiss, L. K., Black, A. S., Takagi, Y., and Plow, E. F. (1987) J. Clin. Invest. 80, 367-373[Medline] [Order article via Infotrieve] |
12. | Aviram, M. (1995) Thromb. Haemost. 74, 560-564[Medline] [Order article via Infotrieve] |
13. | Hussein, O., Brook, G. J., and Aviram, M. (1993) Isr. J. Med. Sci. 29, 453-459[Medline] [Order article via Infotrieve] |
14. | Osterud, B. (1997) Thromb. Res. 85, 1-22[CrossRef][Medline] [Order article via Infotrieve] |
15. | Chesterman, C. N., and Berndt, M. C. (1986) Clin. Hematol. 15, 323-353 |
16. | O'Brien, J. R., Etherington, M. D., and Pashley, M. (1984) Thromb. Haemost. 51, 354-357[Medline] [Order article via Infotrieve] |
17. |
Rollins, J. R.
(1997)
Blood
90,
909 |
18. | Deuel, T. F., Keim, P. S., Farmer, M., and Heinrikson, R. L. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2256-2258[Abstract] |
19. | Poncz, M., Surrey, S., LaRocco, P., Weiss, M. J., Rappoport, E. F., Conway, T. M., and Schwartz, E. (1987) Blood 69, 219-223[Abstract] |
20. | Zhang, X., Chen, L., Bancroft, D. P., Lai, C. K., and Maione, T. E. (1994) Biochemistry 33, 8361-8366[Medline] [Order article via Infotrieve] |
21. | Niewiarowski, S. (1993) in Hemostasis and Thrombosis (Bloom, A. L. , and Thomas, D. P., eds) , pp. 167-177, Churchill Livingstone, London |
22. | Stuckey, J., St., Charles, R., and Edwards, B. F. (1992) Proteins 14, 277-287[Medline] [Order article via Infotrieve] |
23. | Stern, D., Nawroth, P., Marcum, J., Handley, D., Kisiel, W., and Rosenberg, R. (1985) J. Clin. Invest. 75, 272-279[Medline] [Order article via Infotrieve] |
24. | Hadley, T. J., Lu, Z., Wasniowska, K., Martin, A. W., Peiper, S. C., Hesselgesser, J., and Horuk, R. (1994) J. Clin. Invest. 94, 985-991[Medline] [Order article via Infotrieve] |
25. | Peiper, S. C., Wang, Z. X., Neote, K., Martin, A. W., Showell, H. J., Conklyn, M. J., Ogborne, K., Hadley, T. J., Lu, Z. H., and Hesselgesser, J. (1995) J. Exp. Med. 181, 1311-1317[Abstract] |
26. |
Lecomte-Raclet, L.,
Alemany, M.,
Sequiera-Le Grand, A.,
Amiral, J.,
Quentin, G.,
Vissac, A. M.,
Caen, J. P.,
and Han, Z. C.
(1998)
Blood
91,
2772-2780 |
27. |
Slungaard, A.,
and Key, N. S.
(1994)
J. Biol. Chem.
269,
25549-25556 |
28. |
Dudek, A. Z.,
Pennell, C. A.,
Decker, T. D.,
Young, T. A.,
Key, N. S.,
and Slungaard, S.
(1997)
J. Biol. Chem.
272,
31785-31792 |
29. | Goldberg, I. D., Stemerman, M. B., and Handin, R. I. (1980) Science 209, 611-612[Medline] [Order article via Infotrieve] |
30. | Maione, T. E., Gray, G. S., Petro, J., Hunt, A. J., Donner, A. L., Bauer, S. I., Carson, H. F., and Sharpe, R. J. (1990) Science 247, 77-79[Medline] [Order article via Infotrieve] |
31. | Brown, M. S., Deul, T. F., Basu, S. K., and Goldstein, J. L. (1978) J. Supramol. Struc. 8, 223-234[Medline] [Order article via Infotrieve] |
32. | Witztum, J. L., and Steinberg, D. (1991) J. Clin. Invest. 88, 1785-1792[Medline] [Order article via Infotrieve] |
33. |
Hajjar, D. P.,
and Haberland, M. E.
(1997)
J. Biol. Chem.
272,
22975-22978 |
34. |
Steinberg, D.
(1997)
J. Biol. Chem.
272,
20963-20966 |
35. |
Ji, Z.-S.,
Dichek, H. L.,
Miranda, R. D.,
and Mahley, R. W.
(1997)
J. Biol. Chem.
272,
31285-31292 |
36. |
Ziporen, L., Li, Z. Q.,
Park, K. S.,
Sabnekar, P.,
Liu, W. Y.,
Arepally, G.,
Shoenfeld, Y.,
Kieber-Emmons, T.,
Cines, D. B.,
and Poncz, M.
(1998)
Blood
92,
3250-3259 |
37. |
Higazi, A. A.-R.,
Ganz, T.,
Kariko, K.,
and Cines, D. B.
(1996)
J. Biol. Chem.
271,
17650-17655 |
38. | Esko, J. D. (1991) Cur. Opin. Cell Biol. 3, 805-816[Medline] [Order article via Infotrieve] |
39. |
Grobmeyer, S. R.,
Kuo, A.,
Orishimo, M.,
Okada, S. S.,
Cines, D. B.,
and Barnathan, E. S.
(1993)
J. Biol. Chem.
268,
13291-13300 |
40. | Esterbauer, H., Striegl, G., Puhl, H., and Rotheneder, M. (1989) Free Rad. Res. Commun. 6, 67-75[Medline] [Order article via Infotrieve] |
41. |
Aviram, M.,
Dornfeld, L.,
Rosenblat, M.,
Volkova, N.,
Kaplan, M.,
Coleman, R.,
Hayek, T.,
Presser, D.,
and Fuhrman, B.
(2000)
Am. J. Clin. Nutr.
71,
1062-1076 |
42. | Buege, J., and Aust, S. (1978) Methods Enzymol. 52, 301-310 |
43. | El-Saadani, M., Esterbauer, H., El-, Sayed, M., Goher, M., Nassar, A., and Jurgens, G. (1989) J. Lipid Res. 30, 627-630[Abstract] |
44. |
Aviram, M.,
Rosenblat, M.,
Bisgaier, C.,
Newton, R.,
Primo-Parmo, S.,
and La Du, B.
(1998)
J. Clin. Invest.
101,
1581-1590 |
45. | Basu, S. K., Goldstein, J. L., Anderson, R. G., and Brown, M. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3178-3182[Abstract] |
46. | Kruth, H. S., Skarlatos, S., Gaynor, P., and G., W. (1994) J. Biol. Chem. 269, 12420-12423 |
47. |
Sachais, B. S.,
Kuo, A.,
Nassar, T.,
Morgan, J.,
Kariko, K.,
Williams, K. J.,
Feldman, M.,
Aviram, M.,
Shah, N.,
Jarett, L.,
Poncz, M.,
Cines, D. B.,
Higazi, A. A.-R.,
Sachais, B. S.,
Kuo, A.,
Nassar, T.,
Morgan, J.,
Kariko, K.,
Williams, K. J.,
Feldman, M.,
Aviram, M.,
Shah, N.,
Jarett, L.,
Poncz, M.,
Cines, D. B.,
and Higazi, A. A.-R.
(2002)
Blood
99,
3613-3622 |
48. | Nath, N., Lowery, C. T., and Niewiarowski, S. (1975) Blood 45, 537-550[Abstract] |
49. | Rucinski, B., Niewiarowski, S., Strzyzewski, M., Holt, J. C., and Mayo, K. H. (1990) Thromb. Haemost. 63, 493-498[Medline] [Order article via Infotrieve] |
50. |
Keidar, S.,
Kaplan, M.,
and Aviram, M.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
97-105 |
51. | Walz, A., and Baggiolini, M. (1990) J. Exp. Med. 171, 449-454[Abstract] |
52. |
Yan, Z.,
Zhang, J.,
Holt, J. C.,
Stewart, G. J.,
Niewiarowski, S.,
and Poncz, M.
(1994)
Blood
84,
2329-2339 |
53. |
Scheuerer, B.,
Ernst, M.,
Durrbaum-Landmann, I.,
Fleischer, J.,
Grage-Griebenow, E.,
Brandt, E.,
Flad, H. D.,
and Petersen, F.
(2000)
Blood
95,
1158-1166 |
54. |
Gawaz, M.,
Neumann, F. J.,
Dickfeld, T.,
Koch, W.,
Laugwitz, K. L.,
Adelsberger, H.,
Langenbrink, K.,
Page, S.,
Neumeier, D.,
Schomig, A.,
and Brand, K.
(1998)
Circulation
98,
1164-1171 |