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J. Biol. Chem., Vol. 277, Issue 32, 29116-29124, August 9, 2002
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From the Shionogi Research Laboratories, Shionogi and Co., Ltd.,
Sagisu 5-12-4, Fukushima-ku, Osaka 553-0002, Japan
Received for publication, March 25, 2002, and in revised form, April 22, 2002
The deposition of cholesterol ester within foam
cells of the artery wall is fundamental to the pathogenesis of
atherosclerosis. Modifications of low density lipoprotein (LDL), such
as oxidation, are prerequisite events for the formation of foam cells.
We demonstrate here that group X secretory phospholipase
A2 (sPLA2-X) may be involved in this
process. sPLA2-X was found to induce potent hydrolysis of
phosphatidylcholine in LDL leading to the production of large amounts
of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which
contrasted with little, if any, lipolytic modification of LDL by the
classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative
charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in
oxidized LDL. The sPLA2-X-modified LDL was efficiently
incorporated into macrophages to induce the accumulation of cellular
cholesterol ester and the formation of non-membrane-bound lipid
droplets in the cytoplasm, whereas the extensive accumulation of
multilayered structures was found in the cytoplasm in oxidized
LDL-treated macrophages. Immunohistochemical analysis revealed marked
expression of sPLA2-X in foam cell lesions in the arterial
intima of high fat-fed apolipoprotein E-deficient mice. These findings
suggest that modification of LDL by sPLA2-X in the arterial
vessels is one of the mechanisms responsible for the generation of
atherogenic lipoprotein particles as well as the production of various
lipid mediators, including unsaturated fatty acids and
lyso-PC.
Initiation of atherosclerosis is characterized by the appearance
of fatty streaks underlying the endothelium of large arteries. Recruitment of macrophages and their subsequent uptake of low density
lipoprotein (LDL)1-derived
cholesterol are the major cellular events contributing to fatty streak
formation (1, 2). Oxidative modifications in the lipid and
apolipoprotein B (apoB) components of LDL are thought to drive the
formation of fatty streaks (2, 3), because oxidized LDL can be
incorporated into the macrophages via scavenger receptors leading to
the formation of foam cells that contain massive amounts of cholesterol
esters. In addition, there is substantial evidence that LDL oxidation
occurs in both animals and humans during the progression of
atherogenesis (4). However, prospective clinical trials with
antioxidants, such as vitamin E and beta carotene, in patients with
pre-existing atherosclerosis, have thus far been disappointing (5).
These findings suggest that other types of LDL modifications, such as
that resulting from lipolytic enzymes (6), also play pivotal roles in
the formation of foam cells.
Phospholipase A2 (PLA2) are a diverse family of
lipolytic enzymes that hydrolyze the sn-2 fatty acid ester
bond of glycerophospholipids to produce free fatty acids and
lysophospholipids (7, 8). Over the past two decades, a number of
PLA2s have been identified and classified into different
families based on their biochemical features and primary structures (9,
10). Among them, secretory PLA2 (sPLA2) have
several characteristics, including a low molecular mass (13-18 kDa)
and an absolute catalytic requirement for millimolar concentrations of
Ca2+ (10, 11). At present, nine different groups of
sPLA2s have been identified in humans (IB, IIA, IID, IIE,
IIF, III, V, X, and XII) (10, 12-14). Recent studies have shown that
group IIA sPLA2 (sPLA2-IIA) is expressed in the
atherosclerotic arterial intima and is associated with extracellular
matrix structures and lipid droplets (15-18). In addition,
sPLA2-IIA was shown to induce the lipolysis of LDL leading
to enhanced retention of LDL to human aortic proteoglycans (19, 20),
suggesting a potential role of sPLA2-IIA in the
accumulation of LDL in the proteoglycan matrix on the subendothelial
layer of the arterial intima. However, potent modifications of LDL
leading to increased uptake by macrophages were reported with the type
III bee venom sPLA2 but not with sPLA2-IIA (21). The differences in the potency of LDL modification might be due
to discrepancies in substrate specificity in the mixed micelle assay,
because sPLA2-IIA preferably hydrolyzes anionic phospholipids (22) such as phosphatidylglycerol and phosphatidylserine and has a very low enzymatic activity toward phosphatidylcholine (PC),
a major phospholipid component of LDL (23). Recently, we and other
groups (24) have shown that, among the endogenous sPLA2s in
mammals, group X sPLA2 (sPLA2-X) is one of the
enzymes with a potent hydrolyzing activity toward PC.
sPLA2-X has 16 cysteine residues located at positions
characteristic of the classic types of group IB sPLA2
(sPLA2-IB) and sPLA2-IIA and has an amino acid C-terminal extension that is typical of group II sPLA2
subtypes (25). We have shown that sPLA2-X can induce potent
release of arachidonic acid leading to cyclooxygenase
(COX)-dependent prostaglandin formation, as well as marked
production of lysophosphatidylcholine (lyso-PC) in various cell types,
including macrophages, spleen cells, and colon cancer cells (26-28).
During the process of these cell-based experiments, we found that
sPLA2-X elicits potent release of unsaturated fatty acids
from the culture medium containing fetal calf serum (FCS) in cell-free
systems. These observations prompted us to examine its potential role
in lipolysis of human serum lipoproteins.
In the present study, we first evaluated the potencies of three types
of human sPLA2s (sPLA2-IB, -IIA, and -X) with
respect to the release of fatty acids and the contents of PC and
lyso-PC in LDL. We then compared the characteristics of
sPLA2-X-modified LDL with oxidized LDL in terms of
phospholipid composition, negative charge, and apoB aggregation as well
as for the efficacy in uptake into macrophages. We found that
sPLA2-X induced potent lipolysis of LDL leading to the
formation of numerous lipid droplets in the macrophages. Finally, we
showed elevated expression of sPLA2-X in the foam cells in
the atherosclerotic arterial wall in high fat-fed mice deficient in
apolipoprotein E (apoE).
Materials--
Purified recombinant human sPLA2-IB,
sPLA2-X, and mouse sPLA2-X proteins were
prepared as described previously (24, 27). Recombinant human
sPLA2-IIA was a generous gift from Dr. Ruth Kramer (Eli
Lilly, Indianapolis, IN). Rabbit anti-human sPLA2-X Ab was
prepared as described previously (24), and anti-sPLA2-IB and anti-sPLA2-IIA Abs were purchased from Cayman
Chemicals. Bovine serum albumin (BSA), indomethacin (COX inhibitor),
and nordihydroguaiaretic acid (NDGA, lipoxygenase (LOX) inhibitor) were
obtained from Sigma Chemical Co. Indoxam (sPLA2 inhibitor)
was synthesized at Shionogi Research Laboratories (29).
Preparation of Human LDL and Modification with sPLA2s
and CuSO4 Oxidation--
Very low density lipoprotein
(VLDL, density less than 1.006 g/ml), LDL (d = 1.019-1.063 g/ml), and high density lipoprotein (HDL,
d = 1.085-1.210 g/ml) were isolated from plasma of
healthy and fasting donors by sequential ultracentrifugation, as
described previously (30). For modification of LDL with
sPLA2s, 1 mg/ml LDL was incubated with various
concentrations of sPLA2-IB, -IIA, or -X at 37 °C in
buffer composed of 1 mM CaCl2, 12.5 mM Tris-HCl (pH 8.0), 0.25 M NaCl, and 0.0125%
BSA. The reaction was stopped by addition of EDTA at a final
concentration of 5 mM. For oxidative modification, 1 mg/ml
LDL was incubated with 20 µM CuSO4 at
37 °C and then dialyzed against 150 mM NaCl containing
0.24 mM EDTA (pH 7.4). LDL prepared by incubation without
any modification was used as native LDL.
Measurement of Released Fatty Acids, PC, and Lyso-PC in
sPLA2-treated LDL--
Human LDL (1 mg/ml) was
preincubated for 10 min at 37 °C and stimulated with various
concentrations of sPLA2 enzymes in a final volume of 40 µl. The reaction was stopped by the addition of 160 µl of Dole's
reagent, and the released fatty acids were extracted, labeled with
9-anthryldiazomethane (Funakoshi, Japan), and analyzed by reverse-phase
high-performance liquid chromatography (HPLC) on a LiChroCART 125-4 Supersphere 100 RP-18 column (Merck), as described previously
(24, 31).
For measurement of the amounts of PC and lyso-PC in LDL, lipids were
extracted with organic solvent as described previously (26). The
extracted phospholipids were then separated by normal-phase HPLC on
Ultrasphere silica 4.6 × 250 mm (Beckman) with a guard column of
4.6 × 45 mm using a solvent of acetonitrile/methanol/sulfuric acid (100:7:0.05, v/v) with a flow rate of 1 ml/min at room
temperature. Fractions corresponding to authentic PC or
L- Analysis of Oxidation, Electrophoretic Mobility, and ApoB
Modification in LDL Modified with sPLA2s and
CuSO4--
Following modification with sPLA2s
and CuSO4 oxidation, lipid peroxidation was assessed by the
following procedures. The peroxides were quantified in terms of
thiobarbituric acid-reactive substances (TBARS) according to the method
of Nagano et al. (33). Conjugated dienes were determined by
monitoring the changes in absorbance of A234 at
a final concentration of 200 µg/ml LDL, as reported previously (34).
The electrophoretic mobility of LDL was analyzed by agarose gel
electrophoresis (Titan Gel Lipoproteins, Helena Laboratories, Japan),
as described previously (35). For analysis of apoB modification, LDL
was delipidated and analyzed by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) in 4% acrylamide, as described previously (36).
Measurement of the Amount of Cholesterol Ester in
Macrophages--
Mouse peritoneal macrophages were obtained from the
peritoneal cavity of male C57BL/6J mice (8 weeks) 5 days after
injection of 3% thioglycolate (Difco Laboratories). The cells were
washed, resuspended in serum-free medium, X-VIVO 15 (BioWhittaker), and plated in 24-well plates (Costar) (5 × 105
cells/well). Non-adherent cells were removed by washing and adherent macrophages were incubated with native LDL or modified LDL (200 µg/ml) for 48 h. The lipid extracts of macrophages were
prepared, evaporated, dissolved with isopropanol and the cholesterol
mass was quantified by enzyme fluorometry (37). The amount of
esterified cholesterol was calculated by subtracting the free
cholesterol from total cholesterol. The amounts of cellular proteins
were quantified with BCA Protein Assay reagent (Pierce) after
dissolving the cells in 0.2 N NaOH.
Oil Red O Staining of Macrophages Incubated with
sPLA2-treated and Oxidized LDL--
Mouse peritoneal
macrophages were prepared as described above and cultured in four-well
tissue culture chambers (Iwaki, Japan, 1.25 × 105
cells/well). The adherent macrophages were incubated with either native
LDL or modified LDL (200 µg/ml) for 48 h at 37 °C. After incubation, the cells were washed three times with phosphate-buffered saline (PBS) and fixed with 11% formaldehyde in PBS for 15 min. They
were then stained with oil red O for 30 min and counterstained with
Meyer's hematoxylin for 1 min. The stained cells were examined by
light microscopy. All procedures were performed at room temperature.
Electron Microscopic Analysis of Lipid Droplets in Macrophages
Incubated with sPLA2-X-treated, Acetylated, or Oxidized
LDL--
Macrophages were prepared as described above and cultured in
four-well glass slides (Lab-Tek II chamber slide, Nalge Nunc International Corp.). Acetylation of LDL was performed as described previously (38). After incubation with native LDL or modified LDL (200 µg/ml) for 24 or 48 h at 37 °C, the cells were fixed with
0.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.2. Samples were rinsed in 7% sucrose in 0.1 M phosphate buffer, post-fixed in 1% OsO4,
dehydrated, and then embedded in epoxy resin. Ultrathin sections were
cut, stained with uranyl acetate and lead citrate, and examined with a
Jeol 1200EX microscope.
Immunohistochemistry--
ApoE-deficient mice (8 weeks) were
obtained from The Jackson Laboratory, and age-matched C57BL/6J mice
were obtained from Clea Japan. They were fed a high fat and high
cholesterol diet (15.8% cocoa butter, 1.25% cholesterol, 0.5% sodium
cholate) for 9 weeks then flushed with PBS via the abdominal aorta
under pentobarbital anesthesia and perfused with 4% paraformaldehyde
in PBS. Segments of the proximal aorta and the portions of the heart
containing the aortic arches were swiftly removed and cut into small
pieces, which were then immersed in the same fresh fixative at 4 °C
overnight. Fixed samples were thoroughly rinsed with PBS and
subsequently dehydrated by passage through an alcohol series diluted
with double-distilled water followed by n-butyl alcohol. The
tissue preparations were then passed into paraffin at 56 °C.
Transversal tissue sections (6-µm thickness) were cut from embedded
paraffin blocks and mounted on slides freshly thin-coated with
3-aminoprophyltriethoxysilane. Immunohistochemistry was performed after
paraffin dewaxing. The tissue slides were incubated in methanol
containing 0.3% H2O2 for 30 min and then
treated with 5% normal rabbit serum for 20 min. The slides were
incubated with anti-sPLA2-X Ab (6 µg/ml), anti-sPLA2-IB Ab (5 µg/ml), or anti-sPLA2-IIA
Ab (7 µg/ml) in PBS containing 0.1% BSA for 1 h at room
temperature. After rinsing with PBS, they were incubated with
biotin-conjugated goat anti-rabbit IgG for 30 min followed by treatment
with horseradish peroxidase avidin-biotin complex reagent (Vector
Laboratories). After washing, the peroxidase activity was visualized by
10-min incubation in 50 mM Tris-HCl (pH 7.6) containing 200 µg/ml 3,3'-diaminobenzidine and 0.006% H2O2.
After counterstaining of the nuclei with 0.4% hematoxylin, the
preparations were mounted in Malinol resinous medium (Muto Pure
Chemicals, Japan). Positive signals were detected as dark brown
diaminobenzidine deposits. Neutralization of
sPLA2-X-specific signals was performed by incubating
anti-sPLA2-X Ab with purified mouse sPLA2-X
protein (60 µg/ml) for 2 h at room temperature followed by
addition to the slides. In separate experiments, the slides were
incubated with rat anti-mouse macrophage F4/80 Ab (Serotec, UK) and
then incubated with biotin-conjugated rabbit anti-rat IgG Ab followed
by treatment with peroxidase avidin-biotin complex reagent.
Potent Release of Unsaturated Fatty Acids from LDL by
sPLA2-X--
We first examined the potency of three types
of human sPLA2 for the release of fatty acids in human
plasma at a concentration of 50 nM and found that
sPLA2-X induced the most potent release of unsaturated
fatty acids (data not shown). We then prepared three types of
lipoprotein fraction (VLDL, HDL, and LDL) from freshly isolated human
plasma and then examined the potencies of sPLA2s for the
release of fatty acids. As shown in Fig.
1, sPLA2-X elicited marked
release of various types of unsaturated fatty acids from human LDL in
the following order: linoleic acid (C18:2) > arachidonic acid
(C20:4) > oleic acid (C18:1) Effect of sPLA2-X on the PC and Lyso-PC Contents in
LDL--
Because PC is a major component of phospholipids in LDL (23),
we next examined the PC contents in LDL after treatment with sPLA2s or CuSO4. As shown in Fig.
3A, PC contents were
time-dependently decreased after treatment with 50 nM sPLA2-X and oxidation. Over half of the PC
was diminished in LDL by sPLA2-X within 3 h, and PC
was completely degraded after 24-h treatment. Corresponding to the
reduction of PC contents, the amounts of lyso-PC in LDL was increased
up to 24 h after sPLA2-X treatment (Fig.
3B). Incubation with the sPLA2-specific
inhibitor indoxam (10 µM) or
anti-sPLA2-X Ab (100 µg/ml), both of which have been
shown to block the enzymatic activity of sPLA2-X (24),
resulted in significant suppression of sPLA2-X-induced
lipolysis of LDL (data not shown). In contrast, treatment with
sPLA2-IB or -IIA caused little change in either PC or
lyso-PC contents in LDL. Oxidation of LDL with CuSO4 also caused significant production of lyso-PC. However, the amount of
lyso-PC produced during 24-h oxidation was about 30% of that induced
by sPLA2-X, although PC was degraded similarly by both treatments. In addition, there was no significant release of long chain
unsaturated fatty acids examined during oxidation of LDL (data not
shown). Treatment of LDL with CuSO4 caused an increase in
TBARS (Table I) as well as the production
of conjugated dienes (data not shown), whereas treatment with three
types of sPLA2 did not alter these oxidative parameters.
Taken together, these findings demonstrate that sPLA2-X
induces PC hydrolysis in LDL leading to the production of large amounts
of lyso-PC and unsaturated fatty acids without any oxidative
modification.
Effects of sPLA2-X on Electrophoretic Mobility and ApoB
in LDL--
Modifications of apolipoproteins or surface lipids in LDL
were shown to affect the cellular uptake of LDL and hence the formation of foam cell macrophages (2). We then examined the effects of
sPLA2-X treatment on the electronic charge of LDL by
agarose gel electrophoresis. As shown in Fig.
4, oxidized LDL was characterized by
increased anodic migration compared with native LDL. Treatment with
sPLA2-X also caused enhanced mobility of LDL. Small but
significant migration was detected after 3 h of treatment, and
marked migration was observed after 24 h of treatment with
sPLA2-X. In contrast, the mobility of LDL after treatment
with sPLA2-IB or -IIA was not changed during 24-h
incubation. Addition of anti-sPLA2-X Ab (100 µg/ml)
resulted in complete blockade of sPLA2-X-induced
mobilization (data not shown), demonstrating that the increase of
negative charge in LDL is dependent on the enzymatic activity of
sPLA2-X.
Next, we examined the effects of sPLA2-X on the
modification of apoB by SDS-PAGE analysis. As shown in Fig.
5, excessive aggregation and proteolytic
fragmentation of apoB was detected in oxidized LDL even at 3 h of
incubation. In contrast, apoB in the sPLA2-X-treated LDL
was almost intact with slight aggregation at 24 h of treatment compared with native LDL. There were no changes in apoB of LDL treated
with sPLA2-IB and -IIA even at 500 nM (data not
shown). These findings demonstrate that sPLA2-X induced an
increase in the negative charge of LDL with little modification of
apoB.
Cellular Uptake of sPLA2-X-treated LDL by
Macrophages--
Next, we examined the potency of
sPLA2-X-treated LDL for uptake into macrophages. After
exposure of mouse peritoneal macrophages to modified LDL for 48 h,
the cellular levels of free and esterified cholesterol were measured.
As shown in Fig. 6, the esterified cholesterol mass was significantly increased in the macrophages after
incubation with sPLA2-X-treated LDL, and its level was
about 6-fold higher than that induced by native LDL but half that
evoked by oxidized LDL. The cellular lipid droplets were then stained with oil red O and analyzed by light microscopy. As shown in Fig. 7A, there was little staining
in the macrophages treated with native LDL. In contrast, the formation
of numerous intracellular lipid droplets was observed in macrophages
incubated with sPLA2-X-modified LDL (Fig. 7B),
and lipid droplets were obviously larger than those observed in
macrophages treated with oxidized LDL (Fig. 7C). Lipid droplets could not be detected after incubation with
sPLA2-IB- or sPLA2-IIA-treated LDL (Fig.
8, A and B). When
anti-sPLA2-X Ab (100 µg/ml) was added during the
modification of LDL with sPLA2-X followed by incubation
with macrophages, the formation of lipid droplets was completely
abolished (Fig. 8C). In contrast, lipid droplet formation
was not affected when anti-sPLA2-X Ab was added to block
the enzymatic activity of sPLA2-X only during incubation with macrophages (Fig. 8D). Furthermore, pretreatment of
macrophages with sPLA2-X alone did not cause lipid droplet
formation after incubation with native LDL, and the COX inhibitor
indomethacin and LOX inhibitor NDGA did not affect lipid droplet
formation in the macrophages treated with sPLA2-X-modified
LDL (data not shown). These findings suggest that
sPLA2-X-induced formation of intracellular lipid droplets
is completely dependent on the modification of LDL and is not related
to either the activation of scavenger functions of macrophages or the
action of eicosanoid metabolites.
Electron Microscopic Analysis of Lipid Droplets in Macrophages
Incubated with Modified LDL--
To clarify the morphological
differences in oil red O staining studies (Fig. 7), we further examined
the cellular lipid droplets by electron microscopy. In contrast to the
macrophages incubated with native LDL (Fig.
9A), numerous
non-membrane-bound lipid droplets with about 1-µm profile diameters
were detected in the cytoplasm of macrophages incubated with
sPLA2-X-treated or acetylated LDL (Fig. 9, B and
C). Especially, accumulation of cholesterol crystals was
detected in the lysosomes of the cells incubated with acetylated LDL
for 48 h (data not shown). In contrast, there were few
non-membrane-bound lipid droplets in the macrophages incubated with
oxidized LDL (Fig. 9D). Instead, extensive accumulation of
multilayered structures was found in their cytoplasm (Fig.
9E). Similar structures have been reported in the
lipid-laden cells of atherosclerotic lesions or in the oxidized
LDL-treated macrophages, and these have been designated as lysosomal
lipid bodies (39) or multilamellar lipoid structures containing large
amounts of cholesterol (40). However, these multilamellar structures
were not found in cells incubated with sPLA2-X-treated or
acetylated LDL, thus suggesting that sPLA2-X-modified LDL
undergo similar intracellular processing to acetylated LDL.
Expression of sPLA2-X in Atherosclerotic Lesions of
ApoE-deficient Mice--
To examine the localization of
sPLA2-X in atherosclerotic lesions, we used apoE-deficient
mice, a commonly used rodent model of atherogenesis (41). Although
apoE-deficient mice develop atherosclerotic lesions with normal diet,
an atherogenic diet dramatically accelerates the development of
atherosclerotic lesions (41). Therefore, we examined the expression
profiles of sPLA2 enzymes in apoE-knockout mice fed with a
high fat and high cholesterol diet for 9 weeks. In the vessels isolated
from age-matched normal C57BL/6J mice, there were no atherosclerotic
lesions and few, if any, positive signals for sPLA2-X
expression (Fig. 10A). In the sections prepared from apoE-deficient mice, positive signals were
detected in the multilayered foam cell lesions present in the arterial
intima as well as in smooth muscle cells in the medial layer of the
artery wall (Fig. 10B). These signals were specific for
sPLA2-X, because pretreatment of the Ab with a 10-fold
excess of mouse sPLA2-X protein abolished the signals (Fig.
10C), and no signals were detected in parallel control
samples with non-immune IgG (data not shown). To identify the
sPLA2-X-expressing cell types, the macrophages were stained
with rat anti-mouse macrophage F4/80 Ab. The sPLA2-X
signals in the arterial intima were found to coincide well with the
locations of the macrophages (data not shown), similar to the location
in mouse splenic macrophages (27). There were no detectable signals
with anti-sPLA2-IB Ab (Fig. 10D), although its
availability for immunohistochemical analysis had been confirmed in the
pancreas. Because apoE-deficient mice have a mixed genetic background
derived from the two inbred strains with naturally disrupted
sPLA2-IIA genes (42), there was no signal with
anti-sPLA2-IIA Ab (data not shown).
The development of foam cells is a hallmark of both early and late
atherosclerotic lesions, and cholesterol accumulation in macrophages is
mediated primarily by uptake of modified forms of LDL (2). The present
study demonstrated that sPLA2-X induces lipolytic
modification of LDL leading to the enhanced accumulation of cholesterol
ester in macrophages. In addition, the elevated expression of
sPLA2-X was detected in atherogenic lesions in
apoE-deficient mice. These findings suggest that, in addition to
oxidative modifications, enzymatic modification by sPLA2-X
is one of the mechanisms for the generation of atherogenic LDL.
sPLA2-X-modified LDL shows features in common with oxidized
LDL in terms of the reduction of PC associated with increased lyso-PC
production as well as the increase in negative charge. However, marked
differences were observed in several aspects between both
modifications. The increase in lyso-PC production during
sPLA2-X treatment was accompanied with release of large
amounts of unsaturated fatty acids, which was in contrast with the lack
of release responses during LDL oxidation. Because oxidation causes
lipid peroxidation and oxidized fatty acids are efficiently cleaved by
lipoprotein-associated PLA2 (43), the polyunsaturated fatty
acids could not be released during this process. Higher levels of
lyso-PC production were detected in LDL with sPLA2-X
treatment than with oxidized LDL despite the similar PC degradation
(Fig. 3), which might also be due to differences in the
PLA2 subtypes involved. Because lyso-PC is believed to play
an important role in atherosclerosis and inflammatory diseases by
altering various cellular functions, such as the induction of various
chemokines and cell adhesion molecules (44, 45), sPLA2-X-modified LDL might evoke proatherogenic cellular
events similarly to oxidized LDL (3, 46).
Another difference between LDL modifications with sPLA2-X
and oxidation is the modification of apoB (Fig. 5). Oxidation with cupric ions caused exaggerated aggregation and fragmentation of apoB in
contrast to few, if any, changes seen following sPLA2-X treatment. It has been shown that LDL oxidation elicits the production of fatty acid hydroperoxides that can directly cause oxidation of apoB
associated with an increase in the negative charge of LDL as well as
the generation of structures recognized by the scavenger receptors
(47). The absence of chemical modification of apoB during
sPLA2-X treatment suggests the existence of different mechanisms for the increase in the negative charge of LDL. We found
that the majority of unsaturated fatty acids released during sPLA2-X treatment are nonspecifically bound to LDL.
Furthermore, the addition of 4% BSA resulted in the removal of fatty
acids from sPLA2-X-modified LDL leading to a decrease in
its negative charge. However, the accumulation of cholesteryl ester as
well as the formation of intracellular lipid droplets in macrophages (Figs. 7 and 8) were also observed in LDL treated with
sPLA2-X in the presence of 4%
BSA.2 These findings strongly
suggest that the negative charge of LDL due to the bound fatty acids
does not account for the increased uptake into the macrophages.
Although the precise mechanisms remain uncertain, potent lipolysis of
LDL might alter the conformation of apoB leading to the increased
uptake into macrophages, as reported for LDL treated with bee venom
PLA2 (48). Alternatively, the released unsaturated fatty
acids might be incorporated into the macrophages leading to the
production of bioactive lipid mediators. For example, linoleic acid
could be metabolized by 12/15-LOX leading to the production of
13-hydroxyoctadecadienoic acid that can act as an endogenous ligand for
peroxisome proliferator-activated receptor In the atherosclerotic lesions in vivo, three types of
intracellular lipid deposits have been reported in macrophage foam cells: non-membrane-bound lipid droplets, lysosomal lipid bodies and
cholesterol crystals (39, 51-54). In the present study, numerous non-membrane-bound lipid droplets and cholesterol crystals were detected in the cytoplasm of macrophages incubated with acetylated LDL
(Fig. 9), whereas numerous multilamellar structures were found in the
cytoplasm of the cells treated with oxidized LDL. This observation was
consistent with previous reports demonstrating that acetylated and
oxidized LDL are distributed in different intracellular compartments
after internalization into macrophages (55-57). The differences in
compartmentalization may be attributable to the inefficient degradation
of oxidized LDL in macrophage lysosomes in ways that lead to lysosomal
lipid accumulation (58) or the differences in uptake pathways between
acetylated and oxidized LDL (56). The formation of non-membrane-bound
lipid droplets by sPLA2-X-treated LDL (Fig. 9B)
suggests a degradation pathway similar to that of acetylated LDL. The
large lipid droplets having more than 0.4-µm profile diameters have
been observed in the fatty streak regions in human arteries (59). In
addition, the non-membrane-bound lipid droplets have been demonstrated
to be the main form of lipid accumulation at the early stage of the
lesions, whereas the lysosomal lipid accumulation found in oxidized LDL
treatment was more prominent in the advanced stage of atherosclerotic
lesions in animal models (51, 52). In this context, sPLA2-X
might be one of the endogenous molecules involved in the initiation of
atherogenesis, because acetylated LDL is an artificial product.
In the present study, we focused on the differences between
sPLA2-X-treated and oxidized LDL in various aspects.
However, these modifications can work synergistically in
vivo, because sPLA2-IIA has been shown to cause
oxidative modification of LDL by the cooperative action with 12/15-LOX
that is present in the atherogenic lesions (60-62). Because abrogation
of the 12/15-LOX gene caused marked reduction in lipid peroxidation and
atherogenesis in apoE-deficient mice (63, 64), the synergistic action
of oxidation and sPLA2 enzymes in LDL modification deserves
further examination. In apoE-deficient mice, the expression of
sPLA2-X was elevated in foam cells in the arterial intima
as well as in vascular smooth muscle cells (Fig. 10). Because
sPLA2-X is a secreted enzyme (24, 25), it could be released
from foam cells and smooth muscle cells to act in the circulation
and/or in the local area in atherogenic lesions. Although we analyzed
the expression profiles of sPLA2-X in apoE-deficient mice
after the extreme treatment by feeding a high fat and high cholesterol
diet, we also detected its expression in foam cell lesions of the
arterial intima in Watanabe heritable hyperlipidemic
rabbits.3 Further studies of
the expression of sPLA2-X in human atherosclerotic lesions
as well as its circulating levels in patients with atherosclerosis are
required to understand its pathological roles in humans.
Among the human sPLA2s examined, potent lipolysis of LDL
was observed only on treatment with sPLA2-X, which may have
been due to its higher hydrolyzing activity toward PC, the major
phospholipid species in LDL (23, 24). Weak fatty acid release was
detected during treatment of LDL with higher concentrations of
sPLA2-IB (Fig. 2), which was consistent with previous
reports describing the hydrolyzing potency of porcine
sPLA2-IB on LDL (65). However, sPLA2-IB is
mainly expressed in the pancreas, and its expression was not detected
in the atherosclerotic lesions of humans (15) or apoE-deficient mice
(Fig. 9), indicating that its role in atherogenesis is quite minor.
With regard to sPLA2-IIA, significant modification of LDL
was not detected even at 500 nM during 24-h incubation. However, Hakala et al. (19) have recently shown that
incubation of LDL with proteoglycan-bound sPLA2-IIA for a
longer time (48 h) results in weak lipolysis of LDL leading to particle
fusion and enhanced retention of LDL to human aortic proteoglycans. It has also been reported that the expression of sPLA2-IIA is
markedly elevated in human atherosclerotic arterial intima, where the
majority of sPLA2-IIA is localized along the extracellular
matrix, associated with collagen fibers and other structures (16, 18).
These findings suggest that sPLA2-IIA is involved in the
accumulation of extracellular lipoproteins in the proteoglycan-rich
subendothelial layer of the arterial intima, whereas
sPLA2-X induces more powerful lipolysis of LDL linked to
foam cell formation within the arterial wall. Most recently, Gesquiere
et al. (66) have reported that sPLA2-V, another
type of sPLA2 with a potent hydrolyzing activity toward PC
(10), can also hydrolyze lipoprotein PC with about 30 times more
efficient than sPLA2-IIA. Our preliminary studies also
revealed the potent lipolytic modification of LDL and HDL,2
although further studies are required to characterize the potency of
sPLA2-V in lipoprotein modifications linked to the lipid
droplet formation in macrophages. However, sPLA2-V was
shown to release more linoleic acid (over 10-fold) than arachidonic
acid from lipoproteins (66), which was consistent with its fatty acid
specificity using synthetic substrate (67). In contrast,
sPLA2-X can induce potent release of arachidonic acid from
LDL with on the level of more than half of linoleic acid (Fig.
1), suggesting its relevance to the eicosanoid biosynthesis. Although
sPLA2-V is known to be secreted by macrophages (68), there
are few reports on its expression in the pathological states, including
atherosclerotic lesions. This is possibly due to the difficulty in
generating sPLA2-V-specific Ab for immunohistochemistry,
because many of Abs for sPLA2-IIA have been shown to
cross-react with sPLA2-V (67). Further analysis in terms of
the potency for lipoprotein modifications and the expression in
atherosclerotic lesions is required to understand the involvement of
sPLA2-V and other novel types of sPLA2s, such as sPLA2-III and XII, in the progression of atherosclerosis.
High plasma levels of small, dense LDL have been shown to be associated
with increased risk of cardiovascular disease (69). Such modified LDL
contains less phospholipids and unesterified cholesterol in the surface
monolayer than LDL present in normal subjects (70) and is associated
with increased susceptibility to oxidation (71) and enhanced uptake
into macrophages (72). Sartipy et al. (73) have recently
shown that type III sPLA2 from bee venom can cause similar
modifications of LDL, although the identities of the endogenous
sPLA2 enzymes involved remain uncertain. In the present
study, sPLA2-X showed powerful lipolytic activity on LDL to
induce modification of LDL leading to enhanced uptake into macrophages.
Although morphological changes of LDL during sPLA2-X
treatment have not yet been studied, the contribution of this
sPLA2 subtype to the formation of small, dense LDL deserves further examination. Potent release of unsaturated fatty acids was also
observed in HDL and VLDL treated with sPLA2-X. Our
preliminary experiments revealed that sPLA2-X can induce
modification of HDL similarly to LDL in terms of the reduction of PC
contents and increases in lyso-PC and negative charge.3
These findings suggest that sPLA2-X is a key molecule that
induces the alternation of native lipoproteins into the atherogenic
phenotype. Recent studies have shown that sPLA2-IIA
transgenic mice have lower plasma levels of HDL compared with wild-type
littermates (74), because overexpression of this sPLA2
increased the rate of catabolism and altered sites of tissue uptake of
HDL (75). The contribution of sPLA2-X to lipoprotein
modifications should also be clarified in future genetic studies,
including the generation of knockout mice. Because sPLA2-X
elicits potent lipolytic effects in human plasma as well as FCS
in vitro, previous reports concerning sPLA2-X-induced lipid mediator production in cultured cell
systems, such as transfection experiments (76, 77), should be
re-evaluated with respect to the contribution of lipolysis of serum lipoproteins.
In conclusion, we have demonstrated here that sPLA2-X
induces modification of LDL leading to foamy macrophage formation
without oxidation or proteolysis of LDL. In addition, a variety of
lipid mediators are produced during lipolysis of LDL by
sPLA2-X, which might also contribute to the progression of
atherosclerosis. Given the atherogenic features of oxidized LDL,
prospective clinical trials with antioxidants have been performed but
to date have been unsuccessful (5). Because sPLA2
inhibitors, but not COX or LOX inhibitors, suppressed the
sPLA2-X-induced lipolysis of LDL, the availability of
sPLA2 inhibitors as anti-atherogenic drugs should be
evaluated in future studies.
We thank Dr. Ruth Kramer for the generous
gift of recombinant human sPLA2-IIA. We are grateful
to Kazumi Nakano, Ayako Terawaki, Keiko Kawamoto, Dr. Yasunori
Yokota, and Dr. Yasuhide Morioka for excellent technical assistance. We
also thank Dr. Seijiro Hara for fruitful discussions.
*
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.
Published, JBC Papers in Press, May 20, 2002, DOI 10.1074/jbc.M202867200
2
S. Yamamoto, Y. Ishimoto, and K. Hanasaki,
unpublished data.
3
Y. Ishimoto, S. Yamamoto, and K. Hanasaki,
manuscript in preparation.
The abbreviations used are:
LDL, low density
lipoprotein;
apoB, apolipoprotein B;
apoE, apolipoprotein E;
PLA2, phospholipase A2;
sPLA2, secretory PLA2;
sPLA2-IIA, group IIA
sPLA2;
PC, phosphatidylcholine;
sPLA2-X, group
X sPLA2;
sPLA2-IB, group IB sPLA2;
COX, cyclooxygenase;
lyso-PC, lysophosphatidylcholine;
FCS, fetal calf
serum;
BSA, bovine serum albumin;
LOX, lipoxygenase;
NDGA, nordihydroguaiaretic acid;
VLDL, very low density lipoprotein;
HDL, high density lipoprotein;
HPLC, high performance liquid chromatography;
TBARS, thiobarbituric acid-reactive substances;
PBS, phosphate-buffered
saline;
Ab, antibody..
Potent Modification of Low Density Lipoprotein by
Group X Secretory Phospholipase A2 Is Linked to Macrophage
Foam Cell Formation*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-lyso-PC (Sigma Chemical Co.), detected at the
wavelength of 202 nm, were pooled and subjected to quantitative
phosphorus analysis (32).
RESULTS
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DISCUSSION
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docosahexaenoic acid
(C22:6), whereas sPLA2-IB and -IIA caused little release. In contrast, there was little, if any, release of saturated fatty acids, including myristic acid, palmitic acid, and stearic acid, from
LDL after sPLA2-X treatment. The profiles of free fatty
acids released by sPLA2-X were almost identical among LDL,
HDL, and VLDL. In addition, there were no significant changes in the
contents of sphingomyelin in LDL and HDL (data not shown). As shown in Fig. 2A, sPLA2-X
induced time-dependent release of arachidonic acid from
LDL. In contrast, no significant release was observed during treatment
with sPLA2-IB or -IIA for 4 h. Fig. 2B
shows the dose-dependent release of arachidonic acid by
three types of sPLA2 during 1-h incubation.
sPLA2-X induced significant release at 5 nM,
whereas sPLA2-IB evoked slight but significant release at
500 nM. There was little, if any, release with
sPLA2-IIA treatment even at 500 nM,
demonstrating that sPLA2-X elicits more potent release of
unsaturated fatty acids from human LDL as compared with
sPLA2-IB and -IIA.
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Fig. 1.
Profiles of fatty acids released from LDL by
the action of human sPLA2s. LDL (1 mg/ml) was
incubated with 50 nM purified human sPLA2s for
4 h at 37 °C, and the released fatty acids were quantified as
described under "Experimental Procedures." Each point
represents the mean ± S.D. of triplicate measurements. The data
are representative of three experiments.
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Fig. 2.
Time- and dose-dependent release
of arachidonic acid by sPLA2s in LDL. A,
time-dependent release of arachidonic acid by human
sPLA2s. LDL (1 mg/ml) was incubated with 50 nM
purified human sPLA2s for various times at 37 °C.
B, dose-dependent release of arachidonic acid by
human sPLA2s. LDL (1 mg/ml) was incubated with various
concentrations of purified human sPLA2s for 1 h at
37 °C. The released arachidonic acid was quantified as described
under "Experimental Procedures." The results are shown after
subtracting the values obtained with incubation in the absence of
sPLA2s at each time point. Each point represents
the mean ± S.D. of triplicate measurements. The data are
representative of three experiments.
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Fig. 3.
Effects of human sPLA2s and
Cu2+-oxidation on PC and lyso-PC contents in LDL.
A, effects on PC contents in LDL. LDL (1 mg/ml) was
incubated with 50 nM purified human sPLA2s or
20 µM CuSO4 for various times at 37 °C,
and the amounts of PC were quantified as described under
"Experimental Procedures." The results are expressed as the
percentage of the amounts of PC present in the LDL before the
incubation (1400 nmol/mg of protein). B, effects on lyso-PC
contents. After incubation, the amounts of lyso-PC were quantified as
described under "Experimental Procedures." Each point
represents the mean ± S.D. of triplicate measurements. The data
are representative of three experiments.
Amounts of TBARS in LDL treated with human sPLA2s or
CuSO4
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Fig. 4.
Agarose gel electrophoresis of LDL treated
with human sPLA2s or CuSO4. LDL (1 mg/ml)
was incubated with 50 nM purified human sPLA2s
(IB, IIA, or X) or 20 µM
CuSO4 (Cu2+) for the indicated times,
and the electrophoretic mobility was analyzed by agarose gel
electrophoresis (25 min at 90 V) followed by staining with fat red
7B.
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Fig. 5.
SDS-PAGE analysis of apoB in LDL treated with
human sPLA2s or CuSO4. LDL (1 mg/ml) was
incubated with vehicle (None), 50 nM human
sPLA2-X (X), or 20 µM
CuSO4 (Cu2+) for the indicated times.
After delipidation, LDL (5 µg) was analyzed by SDS-PAGE (4%
polyacrylamide gel) followed by staining with Coomassie Brilliant Blue.
Molecular mass markers (kDa) are indicated in the left
lane.
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Fig. 6.
Measurement of cholesterol ester mass in
macrophages. Mouse peritoneal macrophages were incubated with
native LDL, sPLA2-X-modified LDL (X-LDL), or
oxidized LDL (ox LDL) of 200 µg/ml for 48 h, and then
the amount of cellular cholesterol was quantified by enzyme
fluorometry. The amount of esterified cholesterol was calculated by
subtracting the free cholesterol from total cholesterol. Each
point represents the mean ± S.D. of triplicate
measurements. The data are representative of three experiments.
Statistical significance was determined by Student's t test
(*, p < 0.01 compared with macrophages treated with
native LDL).
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Fig. 7.
Oil red O staining of macrophages incubated
with modified LDL. sPLA2-X-treated LDL and
oxidized LDL were prepared by incubating LDL with 50 nM
sPLA2-X or 20 µM CuSO4 for
24 h at 37 °C, respectively. Mouse peritoneal macrophages were
then incubated with 200 µg/ml native LDL (A),
sPLA2-X-treated LDL (B), or oxidized LDL
(C) for 48 h. After incubation, macrophages were fixed
and stained with oil red O. The bar indicates 20 µm.
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Fig. 8.
Oil red O staining of macrophages incubated
with sPLA2s-treated LDL. LDL (1 mg/ml) was incubated
with 50 nM human sPLA2-IB (A) or
sPLA2-IIA (B) for 24 h at 37 °C. Mouse
peritoneal macrophages were then incubated with 200 µg/ml
sPLA2-treated LDL for 48 h. In separate experiments,
LDL (1 mg/ml) was incubated with 50 nM human
sPLA2-X in the presence of anti-sPLA2-X Ab
(C) or control rabbit IgG (D) for 24 h at
37 °C. Macrophages were then incubated with 200 µg/ml
sPLA2-X-treated LDL for 48 h in the presence of
control rabbit IgG (C) or anti-sPLA2-X Ab
(D). The final concentration of Ab used in both reactions
was 500 µg/ml. After incubation, the cells were fixed and stained
with oil red O and hematoxylin. The bar indicates 50 µm.
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Fig. 9.
Electron micrographs of macrophages incubated
with sPLA2-X-treated, acetylated, or oxidized LDL.
sPLA2-X-treated LDL and oxidized LDL were prepared by
incubating LDL with 50 nM sPLA2-X or 20 µM CuSO4 for 24 h at 37 °C,
respectively. Mouse peritoneal macrophages in four-well glass slides
were then incubated with 200 µg/ml native LDL (A),
sPLA2-X-treated LDL (B), acetylated LDL
(C), or oxidized LDL (D) for 48 h. After
fixation of the cells, Ultrathin sections of the cells were prepared,
stained with uranyl acetate and lead citrate, and examined with an
electron microscope. Numerous non-membrane-bound lipid droplets were
detected in the cytoplasm of macrophages incubated with
sPLA2-X-treated LDL and acetylated LDL (indicated by
L in B and C). In contrast, numerous
multilamellar structures were found in the cytoplasm of cells treated
with oxidized LDL (indicated by the arrow in D),
one of which was shown by further amplification (E).
Bars indicate 2 µm (A-D) and 0.2 µm (E).
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Fig. 10.
Immunostaining of atherogenic lesions of
apoE-deficient mice with anti-sPLA2-X Ab. Segments of
the proximal aorta and the portions of the heart containing the aortic
arches were removed from age-matched C57BL/6J (A) and
high-fat fed apoE-deficient mice (B-D). The
tissue preparations were incubated with anti-sPLA2-X Ab (6 µg/ml) in the absence (A, B) or presence
(C) of purified mouse sPLA2-X (60 µg/ml) or
with anti-sPLA2-IB Ab (D) and then analyzed as
described under "Experimental Procedures." The positive signals
were detected as dark brown diaminobenzidine deposits, and
the nuclei were counterstained with hematoxylin. The bar
indicates 50 µm.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
involved in foam cell
formation (49). Because fatty acids and oxidized LDL were reported to
induce the expression of adipocyte fatty acid-binding protein, aP2, one
of the key molecules involved in foam cell formation (50),
sPLA2-X might also regulate aP2 expression in macrophages
via modification of LDL. Further studies are required to understand the
cellular mechanisms underlying the potent uptake of
sPLA2-X-modified LDL.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 81-6-6455-2104;
Fax: 81-6-6458-0987; E-mail: kohji.hanasaki@shionogi.co.jp.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Ross, R.
(1993)
Nature
362,
801-809[CrossRef][Medline]
[Order article via Infotrieve]
2.
Glass, C. K.,
and Witztum, J. L.
(2001)
Cell
104,
503-516[CrossRef][Medline]
[Order article via Infotrieve]
3.
Navab, M.,
Berliner, J. A.,
Watson, A. D.,
Hama, S. Y.,
Territo, M. C.,
Lusis, A. J.,
Shih, D. M.,
Van Lenten, B. J.,
Frank, J. S.,
Demer, L. L.,
Edwards, P. A.,
and Fogelman, A. M.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
831-842 4.
Napoli, C.,
D'Armiento, F. P.,
Mancini, F. P.,
Postiglione, A.,
Witztum, J. L.,
Palumbo, G.,
and Palinski, W.
(1997)
J. Clin. Invest.
100,
2680-2690[Medline]
[Order article via Infotrieve]
5.
Yusuf, S.,
Dagenais, G.,
Pogue, J.,
Bosch, J.,
and Sleight, P.
(2000)
N. Engl. J. Med.
342,
154-160 6.
Hakala, J. K.,
Oorni, K.,
Ala-Korpela, M.,
and Kovanen, P. T.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
1276-1283 7.
Vadas, P.,
and Pruzanski, W.
(1986)
Lab. Invest.
55,
391-404[Medline]
[Order article via Infotrieve]
8.
Arita, H.,
Nakano, T.,
and Hanasaki, K.
(1989)
Prog. Lipid Res.
28,
273-301[CrossRef][Medline]
[Order article via Infotrieve]
9.
Balsinde, J.,
Balboa, M. A.,
Insel, P. A.,
and Dennis, E. A.
(1999)
Annu. Rev. Pharmacol. Toxicol.
39,
175-189[CrossRef][Medline]
[Order article via Infotrieve]
10.
Six, D. A.,
and Dennis, E. A.
(2000)
Biochim. Biophys. Acta
1488,
1-19[Medline]
[Order article via Infotrieve]
11.
Lambeau, G.,
and Lazdunski, M.
(1999)
Trends Pharmacol. Sci.
20,
162-170[CrossRef][Medline]
[Order article via Infotrieve]
12.
Ishizaki, J.,
Suzuki, N.,
Higashino, K.,
Yokota, Y.,
Ono, T.,
Kawamoto, K.,
Fujii, N.,
Arita, H.,
and Hanasaki, K.
(1999)
J. Biol. Chem.
274,
24973-24979 13.
Suzuki, N.,
Ishizaki, J.,
Yokota, Y.,
Higashino, K.,
Ono, T.,
Ikeda, M.,
Fujii, N.,
Kawamoto, K.,
and Hanasaki, K.
(2000)
J. Biol. Chem.
275,
5785-5793 14.
Gelb, M. H.,
Valentin, E.,
Ghomashchi, F.,
Lazdunski, M.,
and Lambeau, G.
(2000)
J. Biol. Chem.
275,
39823-39826 15.
Elinder, L. S.,
Dumitrescu, A.,
Larsson, P.,
Hedin, U.,
Frostegard, J.,
and Claesson, H. E.
(1997)
Arterioscler. Thromb. Vasc. Biol.
17,
2257-2263 16.
Romano, M.,
Romano, E.,
Bjorkerud, S.,
and Hurt-Camejo, E.
(1998)
Arterioscler. Thromb. Vasc. Biol.
18,
519-525 17.
Schiering, A.,
Menschikowski, M.,
Mueller, E.,
and Jaross, W.
(1999)
Atherosclerosis
144,
73-78[CrossRef][Medline]
[Order article via Infotrieve]
18.
Sartipy, P.,
Johansen, B.,
Gasvik, K.,
and Hurt-Camejo, E.
(2000)
Circ. Res.
86,
707-714 19.
Hakala, J. K.,
Oorni, K.,
Pentikainen, M. O.,
Hurt-Camejo, E.,
and Kovanen, P. T.
(2001)
Arterioscler. Thromb. Vasc. Biol.
21,
1053-1058 20.
Hurt-Camejo, E.,
Camejo, G.,
Peilot, H.,
Oorni, K.,
and Kovanen, P.
(2001)
Circ. Res.
89,
298-304 21.
Aviram, M.,
and Maor, I.
(1992)
Biochem. Biophys. Res. Commun.
185,
465-472[CrossRef][Medline]
[Order article via Infotrieve]
22.
Ono, T.,
Tojo, H.,
Kuramitsu, S.,
Kagamiyama, H.,
and Okamoto, M.
(1988)
J. Biol. Chem.
263,
5732-5738 23.
Hevonoja, T.,
Pentikainen, M. O.,
Hyvonen, M. T.,
Kovanen, P. T.,
and Ala-Korpela, M.
(2000)
Biochim. Biophys. Acta.
1488,
189-210[Medline]
[Order article via Infotrieve]
24.
Hanasaki, K.,
Ono, T.,
Saiga, A.,
Morioka, Y.,
Ikeda, M.,
Kawamoto, K.,
Higashino, K.,
Nakano, K.,
Yamada, K.,
Ishizaki, J.,
and Arita, H.
(1999)
J. Biol. Chem.
274,
34203-34211 25.
Cupillard, L.,
Koumanov, K.,
Mattei, M. G.,
Lazdunski, M.,
and Lambeau, G.
(1997)
J. Biol. Chem.
272,
15745-15752 26.
Saiga, A.,
Morioka, Y.,
Ono, T.,
Nakano, K.,
Ishimoto, Y.,
Arita, H.,
and Hanasaki, K.
(2001)
Biochim. Biophys. Acta
1530,
67-76[Medline]
[Order article via Infotrieve]
27.
Morioka, Y.,
Saiga, A.,
Yokota, Y.,
Suzuki, N.,
Ikeda, M.,
Ono, T.,
Nakano, K.,
Fujii, N.,
Ishizaki, J.,
Arita, H.,
and Hanasaki, K.
(2000)
Arch. Biochem. Biophys.
381,
31-42[CrossRef][Medline]
[Order article via Infotrieve]
28.
Morioka, Y.,
Ikeda, M.,
Saiga, A.,
Fujii, N.,
Ishimoto, Y.,
Arita, H.,
and Hanasaki, K.
(2000)
FEBS Lett.
487,
262-266[CrossRef][Medline]
[Order article via Infotrieve]
29.
Hagishita, S.,
Yamada, M.,
Shirahase, K.,
Okada, T.,
Murakami, Y.,
Ito, Y.,
Matsuura, T.,
Wada, M.,
Kato, T.,
Ueno, M.,
Chikazawa, Y.,
Yamada, K.,
Ono, T.,
Teshirogi, I.,
and Ohtani, M.
(1996)
J. Med. Chem.
39,
3636-3658[CrossRef][Medline]
[Order article via Infotrieve]
30.
Hara, S.,
Shike, T.,
Takasu, N.,
and Mizui, T.
(1997)
Arterioscler. Thromb. Vasc. Biol.
17,
1258-1266 31.
Tojo, H.,
Ono, T.,
and Okamoto, M.
(1993)
J. Lipid Res.
34,
837-844[Abstract]
32.
Chalvardjian, A.,
and Rudnicki, E.
(1970)
Anal. Biochem.
36,
225-226[CrossRef][Medline]
[Order article via Infotrieve]
33.
Nagano, Y.,
Arai, H.,
and Kita, T.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
6457-6461 34.
Esterbauer, H.,
Striegl, G.,
Puhl, H.,
and Rotheneder, M.
(1989)
Free Radic. Res. Commun.
6,
67-75[Medline]
[Order article via Infotrieve]
35.
Noble, R. P.
(1968)
J. Lipid Res.
9,
693-700[Abstract]
36.
Tong, H.,
Knapp, H. R.,
and VanRollins, M.
(1998)
J. Lipid Res.
39,
1696-1704 37.
Gamble, W.,
Vaughan, M.,
Kruth, H. S.,
and Avigan, J.
(1978)
J. Lipid Res.
19,
1068-1070[Abstract]
38.
Zhang, H.,
Yang, Y.,
and Steinbrecher, U. P.
(1993)
J. Biol. Chem.
268,
5535-5542 39.
Lupu, F.,
Danaricu, I.,
and Simionescu, N.
(1987)
Atherosclerosis
67,
127-142[CrossRef][Medline]
[Order article via Infotrieve]
40.
Klinkner, A. M.,
Waites, C. R.,
Kerns, W. D.,
and Bugelski, P. J.
(1995)
J. Histochem. Cytochem.
43,
1071-1078[Abstract]
41.
Zhang, S. H.,
Reddick, R. L.,
Burkey, B.,
and Maeda, N.
(1994)
J. Clin. Invest.
94,
937-945[Medline]
[Order article via Infotrieve]
42.
MacPhee, M.,
Chepenik, K. P.,
Liddell, R. A.,
Nelson, K. K.,
Siracusa, L. D.,
and Buchberg, A. M.
(1995)
Cell
81,
957-966[CrossRef][Medline]
[Order article via Infotrieve]
43.
MacPhee, C. H.,
Moores, K. E.,
Boyd, H. F.,
Dhanak, D.,
Ife, R. J.,
Leach, C. A.,
Leake, D. S.,
Milliner, K. J.,
Patterson, R. A.,
Suckling, K. E.,
Tew, D. G.,
and Hickey, D. M.
(1999)
Biochem. J.
338,
479-487[CrossRef][Medline]
[Order article via Infotrieve]
44.
Kume, N.,
and Gimbrone, M. A., Jr.
(1994)
J. Clin. Invest.
93,
907-911[Medline]
[Order article via Infotrieve]
45.
Quinn, M. T.,
Parthasarathy, S.,
and Steinberg, D.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
2805-2809 46.
Steinberg, D.,
Parthasarathy, S.,
Carew, T. E.,
Khoo, J. C.,
and Witztum, J. L.
(1989)
N. Engl. J. Med.
320,
915-924[Medline]
[Order article via Infotrieve]
47.
Steinbrecher, U. P.,
Lougheed, M.,
Kwan, W. C.,
and Dirks, M.
(1989)
J. Biol. Chem.
264,
15216-15223 48.
Kleinman, Y.,
Krul, E. S,
Burnes, M.,
Aronson, W.,
Pfleger, B.,
and Schonfeld, G.
(1988)
J. Lipid Res.
29,
729-743[Abstract]
49.
Huang, J. T.,
Welch, J. S.,
Ricote, M.,
Binder, C. J.,
Willson, T. M.,
Kelly, C.,
Witztum, J. L.,
Funk, C. D.,
Conrad, D.,
and Glass, C. K.
(1999)
Nature
400,
378-382[CrossRef][Medline]
[Order article via Infotrieve]
50.
Makowski, L.,
Boord, J. B.,
Maeda, K.,
Babaev, V. R.,
Uysal, K. T.,
Morgan, M. A.,
Parker, R. A.,
Suttles, J.,
Fazio, S.,
Hotamisligil, G. S.,
and Linton, M. F.
(2001)
Nat. Med.
7,
699-705[CrossRef][Medline]
[Order article via Infotrieve]
51.
Jerome, W. G.,
and Lewis, J. C.
(1985)
Am. J. Pathol.
119,
210-222[Abstract]
52.
Lewis, J. C.,
Taylor, R. G.,
and Ohta, K.
(1988)
Exp. Mol. Pathol.
48,
103-115[CrossRef][Medline]
[Order article via Infotrieve]
53.
Shio, H.,
Haley, N. J.,
and Fowler, S.
(1979)
Lab. Invest.
41,
160-167[Medline]
[Order article via Infotrieve]
54.
Bocan, T. M. A.,
Schifani, T. A.,
and Guyton, J. R.
(1986)
Am. J. Pathol.
123,
413-424[Abstract]
55.
Jerome, W. G.,
Cash, C.,
Webber, R.,
Horton, R.,
and Yancey, P. G.
(1998)
J. Lipid Res.
39,
1362-1371 56.
Lougheed, M.,
Moore, E. D.,
Scriven, D. R.,
and Steinbrecher, U. P.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
1881-1890 57.
Itabe, H.,
Suzuki, K.,
Tsukamoto, Y.,
Komatsu, R.,
Ueda, M.,
Mori, M.,
Higashi, Y.,
and Takano, T.
(2000)
Biochim. Biophys. Acta
1487,
233-245[Medline]
[Order article via Infotrieve]
58.
Lougheed, M.,
Zhang, H. F.,
and Steinbrecher, U. P.
(1991)
J. Biol. Chem.
266,
14519-14525 59.
Guyton, J. R.,
and Klemp, K. F.
(1994)
Arterioscler. Thromb.
14,
1305-1314 60.
Sparrow, C. P.,
Parthasarathy, S.,
and Steinberg, D.
(1988)
J. Lipid Res.
29,
745-753[Abstract]
61.
Neuzil, J.,
Upston, J. M.,
Witting, P. K.,
Scott, K. F.,
and Stocker, R.
(1998)
Biochemistry
37,
9203-9210[CrossRef][Medline]
[Order article via Infotrieve]
62.
Yla-Herttuala, S.,
Rosenfeld, M. E.,
Parthasarathy, S.,
Glass, C. K.,
Sigal, E.,
Witztum, J. L.,
and Steinberg, D.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
6959-6963 63.
Cyrus, T.,
Witztum, J. L.,
Rader, D. J.,
Tangirala, R.,
Fazio, S.,
Linton, M. F.,
and Funk, C. D.
(1999)
J. Clin. Invest.
103,
1597-1604[Medline]
[Order article via Infotrieve]
64.
Cyrus, T.,
Pratico, D.,
Zhao, L.,
Witztum, J. L.,
Rader, D. J.,
Rokach, J.,
FitzGerald, G. A.,
and Funk, C. D.
(2001)
Circulation
103,
2277-2282 65.
Menschikowski, M.,
Lattke, P.,
Bergmann, S.,
and Jaross, W.
(1995)
Anal. Cell Pathol.
9,
113-121[Medline]
[Order article via Infotrieve]
66.
Gesquiere, L.,
Cho, W.,
and Subbaiah, P. V.
(2002)
Biochemistry
41,
4911-4920[CrossRef][Medline]
[Order article via Infotrieve]
67.
Chen, Y. J.,
and Dennis, E. A.
(1998)
Biochim. Biophys. Acta
1394,
57-64[Medline]
[Order article via Infotrieve]
68.
Balboa, M. A.,
Balsinde, J.,
Winstead, M. V.,
Tischfield, J. A.,
and Dennis, E. A.
(1996)
J. Biol. Chem.
271,
32381-32384 69.
Lamarche, B.,
Tchernof, A.,
Moorjani, S.,
Cantin, B.,
Dagenais, G. R.,
Lupien, P. J.,
and Despres, J. P.
(1997)
Circulation
95,
69-75 70.
McNamara, J. R.,
Small, D. M., Li, Z.,
and Schaefer, E. J.
(1996)
J. Lipid Res.
37,
1924-1935[Abstract]
71.
Tribble, D. L.,
Krauss, R. M.,
Lansberg, M. G.,
Thiel, P. M.,
and van den Berg, J. J.
(1995)
J. Lipid Res.
36,
662-671[Abstract]
72.
Hurt-Camejo, E.,
Camejo, G.,
Rosengren, B.,
Lopez, F.,
Wiklund, O.,
and Bondjers, G.
(1990)
J. Lipid Res.
31,
1387-1398[Abstract]
73.
Sartipy, P.,
Camejo, G.,
Svensson, L.,
and Hurt-Camejo, E.
(1999)
J. Biol. Chem.
274,
25913-25920 74.
de Beer, F. C.,
de Beer, M. C.,
van der Westhuyzen, D. R.,
Castellani, L. W.,
Lusis, A. J.,
Swanson, M. E.,
and Grass, D. S.
(1997)
J. Lipid Res.
38,
2232-2239[Abstract]
75.
Tietge, U. J.,
Maugeais, C.,
Cain, W.,
Grass, D.,
Glick, J. M.,
de Beer, F. C.,
and Rader, D. J.
(2000)
J. Biol. Chem.
275,
10077-10084 76.
Bezzine, S.,
Koduri, R. S.,
Valentin, E.,
Murakami, M.,
Kudo, I.,
Ghomashchi, F.,
Sadilek, M.,
Lambeau, G.,
and Gelb, M. H.
(2000)
J. Biol. Chem.
275,
3179-3191 77.
Murakami, M.,
Koduri, R. S.,
Enomoto, A.,
Shimbara, S.,
Seki, M.,
Yoshihara, K.,
Singer, A.,
Valentin, E.,
Ghomashchi, F.,
Lambeau, G.,
Gelb, M. H.,
and Kudo, I.
(2001)
J. Biol. Chem.
276,
10083-10096
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