Oxidized Low Density Lipoprotein Induces Apoptosis in
Cultured Human Umbilical Vein Endothelial Cells by Common and
Unique Mechanisms*
Mariko
Harada-Shiba
§,
Mikio
Kinoshita
§,
Hiroshi
Kamido¶, and
Kentaro
Shimokado
From the
National Cardiovascular Center Research
Institute, 7-1 Fujishirodai 5-chome, Suita, Osaka 565-8565 and the
¶ Department of Medicine, Kurume University School of Medicine,
Asahi-Machi, Kurume, Fukuoka 830-0011, Japan
 |
ABSTRACT |
Oxidized low density lipoprotein (oxLDL)
induces apoptosis in vascular cells. To elucidate the mechanisms
involved in this apoptosis, we studied the apoptosis-inducing activity
in lipid fractions of oxLDL and the roles of two common mechanisms,
ceramide generation and the activation of caspases, in apoptosis in
human umbilical vein endothelial cells treated with oxLDL. We also
studied the effects of antioxidants and cholesterol. oxLDL induced
endothelial apoptosis in a time- and dose-dependent
fashion. Apoptosis-inducing activity was recovered in the neutral lipid
fraction of oxLDL. Various oxysterols in this fraction induced
endothelial apoptosis. Neither the phospholipid fraction nor
its component lysophosphatidylcholine induced apoptosis. oxLDL induced
ceramide accumulation temporarily at 15 min in a
dose-dependent fashion. Two inhibitors of acid sphinogomyelinase inhibited both the increase in ceramide and the
apoptosis induced by oxLDL. Furthermore, a membrane-permeable ceramide
(C2-ceramide) induced endothelial apoptosis. These
findings demonstrated that ceramide generation by acid sphingomyelinase is indispensable for the endothelial apoptosis induced by oxLDL. Inhibitors of both caspase-1 and caspase-3 inhibited the
apoptosis, suggesting that oxLDL induced apoptosis by activating
these cysteine proteases. The antioxidants butylated hydroxytoluene and
superoxide dismutase but not catalase inhibited the apoptosis induced
by oxLDL or 25-hydroxycholesterol. This suggests not only that
superoxide plays an important role but also that a critical interaction
between oxLDL and the cell takes place on the outer surface of the
membrane, because superoxide dismutase is not membrane-permeable.
Exogenous cholesterol also inhibited the apoptosis. Our study
demonstrated that neutral lipids in oxLDL induce endothelial apoptosis
by activating membrane sphingomyelinase in a
superoxide-dependent manner, as well as by activating
caspases.
 |
INTRODUCTION |
Oxidized low density lipoprotein
(oxLDL)1 is a key substance
in atherogenesis (1, 2). oxLDL is generated by auto-oxidation in the
presence of transition metals (3, 4), by cell-mediated mechanisms
(5-7), and by enzyme-mediated mechanisms (8-11). It induces the early
changes of atherosclerosis: the expression of adhesion molecules on
endothelial cells (12), a decrease in production of endothelial
cell-derived relaxing factor (13) and prostacyclin (14), the
transformation of macrophages and smooth muscle cells to foam cells
(15), the production of various proinflammatory cytokines and growth
factors by almost all vascular cells (16, 17), the proliferation and
migration of vascular cells (18-20), the retardation of endothelial
regeneration (21), and changes in the balance between procoagulant and
anticoagulant activity on the vascular cell surface (22). These changes
consequently trigger a series of cellular responses in the arterial
wall that result in the formation of atheromatous lesions. oxLDL also
affects the later stage of atherosclerosis by its toxicity. oxLDL and its lipid components cause the release of lactic dehydrogenase from
cultured vascular smooth muscle cells, endothelial cells, and
fibroblasts (6) and decrease the number of these cells (6). This
cytotoxicity of oxLDL is one of the factors that make atheromatous
plaques unstable and prone to rupture (23).
Recently, the cytotoxicity of oxLDL has been partly attributed to
induction of apoptosis. oxLDL induces both the morphological changes
and DNA fragmentation characteristic of apoptosis in cultured smooth
muscle cells (24), macrophages (24, 25), endothelial cells (26, 27),
and lymphoid cells (28). The apoptosis of vascular cells plays a role
in both the progression and the regression of atherosclerotic lesions
(23, 29-31). However, it is not clear how oxLDL induces apoptosis in
endothelial cells and other vascular cells. Different agents, such as
tumor necrosis factor-
, ionizing radiation, UV radiation, hydrogen
peroxide (32), high glucose (33), and growth factor deprivation (34),
induce apoptosis in many cell types by both unique and common pathways
(35-37). For example, Fas and tumor necrosis factor receptor family
members transduce the signal of apoptosis through death
domain-containing molecules, such as FADD/MORT1 (37), whereas many
other apoptosis-inducing agents do not use molecules with a death
domain. On the other hand, almost all known apoptosis-inducing agents
share the activation of caspases (formerly called ICE family proteases)
(35-37). Typically, caspase-1 (ICE) activates caspase-3
(CPP32/apopain/YAMA), which then cleaves death substrates such as PARP
and lamins (37). Ceramide accumulation has been proposed to be a common
pathway of apoptosis; most apoptosis-inducing agents induce an
accumulation of intrinsic ceramide and a concomitant decrease in
sphingomyelin, a precursor of ceramide; C2-C8
ceramides, which are membrane-permeable synthetic analogues of
ceramide, activate caspases and induce apoptosis in many cells (35,
38-40). However, some of the increase in ceramide has been suggested
to be a result rather than a cause of cell death (40, 41).
In this study, we investigated the mechanisms involved in the
endothelial apoptosis induced by oxLDL. We found that ceramide generation, as well as caspase activation, is indispensable for the
endothelial apoptosis induced by oxLDL and that some unique mechanisms
are also involved in this process.
 |
Experimental Procedures |
Materials--
The inhibitor for caspase-3 (CPP32/apopain),
ac-DEVD-CHO, and the inhibitor for caspase-1
(interleukin-1
-converting enzyme), ac-YVAD-CHO, were purchased from
Peptide Institute, Inc. (Osaka, Japan). Cholesterol
(5-cholesten-3
-ol), 7
-hydroxycholesterol (5-cholesten-3
,7
-diol), 25-hydroxycholesterol
(5-cholesten-3
,25-diol), cholesterol-5
,6
-epoxide
(5
,6
-epoxycholestan-3
-ol), 3
,5
,6
-trihydroxycholestane (cholestane-3
,5
,6
-triol),
L-lysophosphatidyl-choline palmitoyl (lysoPC), butylated
hydroxytoluene (BHT), superoxide dismutase (SOD) (EC 1.15.1.1; bovine
erythrocytes), and catalase (EC 1.11.1.6; bovine liver) were purchased
from Sigma, and 7-keto-cholesterol (5-cholesten-3
-ol-7-one) was from
Makor Chemical Ltd. (Jerusalem, Israel). C2-ceramide
(N-acetylsphingosine) was from BIOMOL Research Laboratory
(Plymouth Meeting, PA).
Cells--
Human umbilical vein endothelial cells (HUVECs) (42)
were seeded at a density of 5 × 104/cm2
and cultured in Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), 20% (v/v) fetal calf serum
(FCS) (Life Technologies, Inc.), and 10 ng/ml recombinant human basic
fibroblast growth factor (bFGF) (Pepro Tech Inc., Rocky Hill, NJ) for
24 h before the medium was changed to DMEM supplemented with 0.1%
bovine serum albumin (BSA), 10 ng/ml bFGF, and various concentrations
of oxLDL. The number of cells in this confluent culture did not change
significantly in the serum-free medium for the next 48 h unless
oxLDL was added. The cells were used for the experiments between
passages 2 and 7.
Preparation of Modified LDL--
LDL (1.019 < d < 1.063) was prepared from normal human serum by sequential
ultracentrifugation as described previously (43). Fifteen ml of DMEM
was mixed with LDL (500 µg/ml) and Cu(II) sulfate (20 µM), and incubated with a confluent culture of HUVECs in
a 15-cm dish at 37 °C, 5% CO2, and 100% humidity for
48 h. The medium was centrifuged at 3500 rpm for 10 min,
sterilized with a 0.45 µm Millipore filter, and used as oxLDL. The
incubation of LDL in DMEM without either HUVECs or Cu(II) sulfate did
not cause significant oxidation of LDL. Acetylated LDL was prepared as
reported previously (44). All procedures were carried out under sterile conditions. The endotoxin was measured by a radioimmunoassay (Endospec SP test, Seikagaku-kogyo, Tokyo, Japan) and was under the detection limit in all native and oxLDL preparations. The oxidation of LDL was
evaluated by agarose gel electrophoresis (Universal gel, Ciba Corning
Diagnostic Corp., Alameda, CA) and by the measurement of thiobarbituric
acid reacting substance (6). oxLDL had a thiobarbituric acid reacting
substance value of 23.2 nmol of malondialdehyde/mg of protein (the
mean ± S.E. of four separate preparations), whereas native and
acetylated LDL each had no detectable thiobarbituric acid reacting
substance. The oxLDL and acetylated LDL had 2-3-fold higher
Rf values on agarose gel electrophoresis compared with the native LDL. Gas chromatography (45) revealed that a typical
preparation of ox LDL contained 4.0 µg of 7
-hydroxycholesterol/mg of LDL protein, 13.1 µg of epoxide/mg of LDL protein, 14.9 µg of
7-keto-cholesterol/mg of LDL protein, and 1.1 mg of unidentified oxycholesterol/mg of LDL protein.
Quantitative Analysis of Apoptotic Cells--
HUVECs were seeded
at 5 × 104 cells/well in gelatin-coated 8-well
chamber slides (Nunc, Inc., Naperville, IL) and cultured for 24 h
before the medium was changed to DMEM containing 0.1% BSA, 10 ng/ml
bFGF, and the indicated concentration of oxLDL. The cells were then
cultured in the presence of oxLDL for the indicated periods, followed
by fixation in 3% paraformaldehyde in phosphate-buffered saline for 20 min and staining with a solution of 4',6-diamidino-2-phenylindole (10 mM Tris-HCl pH 7.4, 10 mM EDTA, 100 mM NaCl, 500 ng/ml 4',6-diamidino-2-phenylindole) for 10 min at room temperature (46). The number of apoptotic cells was counted
in nine high power fields under a fluorescent microscope (approximately
1000-2000 cells/well). The percentage of apoptotic cells was
calculated as the number of apoptotic cells/number of total cells × 100%. Each experiment was conducted in triplicate and repeated at
least twice.
Fractionation of Lipids--
Total lipid was extracted from
oxLDL with chloroform/methanol and resolved by TLC (Silica gel 60, Merk, Darmstadt, Germany) using heptane/isopropyl ether/acetic acid
(60:40:4) as the developing solvent. Neutral lipids and phospholipids
were extracted from the TLC with chloroform/methanol (1:1).
Measurement of Ceramide--
The cellular ceramide content was
measured by an Escherichia coli diacylgylcerol kinase assay
(47, 48). Briefly, total lipids were extracted from HUVECs (2 × 106 cells) by chloroform/methanol (1:1) and dried under
N2 gas. The lipid was solubilized by sonication for 2 min
into 20 µl of aqueous 7.5%
n-octyl-
-D-glucopyranoside (Dojindo), 5 mM cardiolipin (Sigma), and 1 mM
diethylenetriaminepentaacetic acid (Dojindo, Kumamoto, Japan). The
solubilized lipid was mixed with 20 µl of reaction buffer (250 mM Tris-HCl, 500 mM NaCl, 10 mM
EGTA, 25 mM MgCl2, pH 7.0), 10 µl of 1 mg/ml
E. coli diacylglycerol kinase (Calbiochem), 20 µl of 10 mM ATP (Sigma), and 5 µl of [-32P]ATP (111 TBq/mmol, DuPont). The reaction was allowed to proceed at room
temperature for 40 min and then was stopped by the addition of 1 ml
chloroform/methanol/1 N HCl (100:100:1, v/v), 340 µl of buffered saline solution, and 60 µl of 100 mM EDTA. The
lower organic phase was dried under N2. Labeled ceramide
was resolved by TLC using chloroform/acetone/methanol/acetic acid/water
(10:4:2:2:1, v/v) as the developing solvent. The autoradiogram was
analyzed by a Fuji Bas 2000 Bioimaging analyzer, and the ceramide
content was determined with a standard curve of 0-1.7 nmol of
ceramides (type III, Sigma). The ceramide content was standardized with total cellular phospholipid determined by a commercial kit (Wako Phospholipid Test, Wako Pure Chemicals, Osaka, Japan).
Statistical Analysis--
Statistical analysis was conducted
with Student's t test. Differences were considered
significant when probability values less than 5% were obtained.
 |
Results |
oxLDL Induces Apoptosis in HUVECs--
oxLDL induced the apoptosis
in a time- and dose-dependent manner (Fig.
1). oxLDL induced significant apoptosis
in HUVECs as early as at 12 h of incubation, and the number of
apoptotic cells increased up until 48 h of incubation (Figs. 1 and
2). Twelve to 18% of the HUVECs became
apoptotic after 48 h incubation with 25 µg of protein/ml of
oxLDL. This value is approximately the same as that obtained with the
deprivation of both serum and bFGF (Fig. 2A). At higher
concentrations of oxLDL, the percentage of apoptotic cells decreased
(Fig. 2B), as did the total cell number (data not shown).
After 72 h, the HUVECs became apoptotic regardless of the presence
or absence of oxLDL, probably due to serum starvation, and the effect
of oxLDL became less prominent (data not shown). Incubation with native
LDL or acetylated LDL did not induce apoptosis beyond the control level
(less than 5% of total cells) (data not shown). These findings
indicated that oxLDL induces apoptosis in cultured human endothelial
cells.

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Fig. 1.
Endothelial apoptosis induced by oxLDL.
HUVECs were cultured in 8-well Lab-Tek chamber slides at 5 × 104 cells/cm2 in DMEM containing 20% FCS and
10 ng/ml bFGF. After 24 h, the medium was changed to DMEM
containing 0.1% BSA and 10 ng/ml bFGF, cultured further for 48 h
in the presence or absence of oxLDL (25 µg of protein/ml), and then
stained with 4',6-diamidino-2-phenylindole as described under
"Experimental Procedures." A, cells cultured with oxLDL;
B, the same cells with a higher magnification; C,
cells without oxLDL. Bar indicates 10 µm.
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Fig. 2.
Time- and dose-dependent
induction of apoptosis in HUVECs by oxLDL. A, time
dependence. HUVECs were seeded as described in the legend to Fig. 1,
and cultured in the presence ( ) or absence ( ) of oxLDL (25 µg
of protein/ml) for the indicated periods. As a positive control ( ),
cells were cultured in the absence of bFGF. The number of apoptotic
cells was counted in nine high power fields under a fluorescent
microscope. Each point represents the mean ± S.E. of quintuple
samples for the negative control and oxLDL groups and of quadruplicate
samples for the positive control. B, dose dependence. HUVECs
were seeded and incubated with indicated concentrations of oxLDL for
48 h. As a positive control, cells were cultured in the absence of
bFGF. The values shown are the mean ± S.E. of quadruplicate
samples.
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Lipid Components in oxLDL Induce Apoptosis--
The cytotoxicity
of oxLDL has been attributed to its lipid components (49, 50). To
examine whether the apoptotic activity of oxLDL is also due to the
lipid fractions, oxLDL was extracted with organic solvent, and neutral
lipids and phospholipids were separated by TLC. All of the
apoptosis-inducing activity of oxLDL was recovered in the total lipid
fraction (Table I). More than 90% of the
activity was recovered in the neutral lipid fraction, and no
significant activity was recovered in the phospholipid fraction. Gas
chromatography revealed that the major components of the neutral lipid
fraction generated in oxLDL were a series of oxysterols, such as
7-ketocholesterol and 7-hydroxycholesterol (see preparation of modified
LDL under "Experimental Procedures"). We therefore investigated the
apoptosis-inducing activity of various oxysterols found in oxLDL. All
oxysterols examined induced apoptosis, to various degrees; significant
apoptosis was induced by 5 µg/ml 7-keto-cholesterol,
25-hydroxycholesterol, or triol and by 20 µg/ml 7-hydroxycholesterol
or epoxide (Table II). Oxysterol tended to induce apoptosis to a greater degree compared with oxLDL.
Cholesterol did not induce apoptosis at 20 µg/ml. LysoPC did not
induce significant endothelial apoptosis (Table II). These data suggest
that neutral lipids, such as oxysterols, are responsible for the
apoptosis-inducing activity of oxLDL.
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Table I
Apoptosis-inducing activity of various lipid fractions of oxLDL
oxLDL was fractionated as described under "Experimental
Procedures." Each fraction was tested at a concentration of lipid
equivalent to 25 µg of protein/ml of oxLDL. Values are the mean ± S.E. of six samples.
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Table II
Oxysterols induce apoptosis in HUVECs
The cells were seeded as described in the legend to Fig. 1. On day 2, the medium was changed to DMEM containing the indicated concentration
of oxysterol. After 48 h, apoptotic cells were counted. Each value
represents the mean ± S.E. of quadruplicate samples.
7 -OH-cholesterol, 7 -hydroxycholesterol; 7-keto-cholesterol,
5-cholesten-3 -ol-7-one; epoxide, cholesterol 5 , 6 -epoxide;
25-OH-cholesterol, 25-hydroxycholesterol; Triol,
cholestane-3 ,5 ,6 -triol. NS, not significant.
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oxLDL Induces Apoptosis by Generating Ceramide--
Although
ceramide has been implicated in the apoptosis induced by a variety of
agents, its precise role is still controversial (35, 40). Therefore, we
studied whether ceramide was involved in oxLDL-induced apoptosis.
First, we examined whether oxLDL affected the cellular ceramide
content. Ceramides were detected as two major spots on TLC, as reported
previously (Fig. 3A) (48).
oxLDL (0-50 µg/ml) increased the cellular ceramide transiently at 15 min in a dose-dependent fashion (Fig. 3). After 24 h
of oxLDL treatment, the cellular ceramide increased again to a similar extent (data not shown). An oxysterol, 7-keto-cholesterol (5 µg/ml), also stimulated ceramide generation at 15 min (3.2-fold increase; average of two independent experiments). Second, we studied whether the
inhibition of ceramide generation prevented endothelial apoptosis. Two
different inhibitors of sphingomyelinase, desipramine and chlorpromazine (51), inhibited the oxLDL-induced apoptosis completely (Fig. 4A). At the same
concentration, these inhibitors inhibited the ceramide generation
induced by oxLDL (Fig. 4B). Third, we studied whether
exogenous ceramide induced apoptosis in our experimental conditions. In
accordance with a previous report with exogenous C6-ceramide (52), C2-ceramide, a
membrane-permeable form of ceramide, induced endothelial apoptosis
(Table III). These findings demonstrated
that the increase in cellular ceramide is indispensable for the
induction of endothelial apoptosis by oxLDL.

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Fig. 3.
oxLDL increases the cellular ceramide
content. A and B, time course. HUVECs were seeded
at 5 × 104 cm2 in DMEM supplemented with
20% FCS and 10 ng/ml bFGF and cultured for 24 h. The medium was
changed to DMEM supplemented with 0.1% BSA, 10 ng/ml bFGF, and oxLDL
(25 µg of protein/ml). At the indicated times, the total cellular
lipid was extracted, and the ceramide content was measured as described
under "Experimental Procedures" (A). The standard
(std) was 1.7 nmol of ceramide. Quantitative analysis was
conducted with BAS200 image analyzer (B). Each data
point indicates the mean ± S.E. of values obtained from
three independent experiments conducted in duplicate. Ceramide content
of time 0 was 2.9 ± 0.7 mol/104 mol of phospholipid
(the mean ± S.E. of six samples). C, dose dependence.
HUVECs were cultured in the presence of various concentrations of oxLDL
for 15 min. Ceramide content of the sample containing no oxLDL was
2.1 ± 0.6 mol/104 mol of phospholipid (the mean ± S.E. of six samples).
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Fig. 4.
Effect of sphingomyelinase inhibitors on
ceramide generation and apoptosis. A sphingomyelinase inhibitor
was added to the culture 22 h after seeding. After 2 h, the
medium was changed to DMEM supplemented with 0.1% BSA, 10 ng/ml bFGF,
oxLDL (25 µg of protein/ml), and the inhibitor. The percentage of
apoptotic cells (A) was determined after 48 h, and
ceramide content (B) was determined at 15 min. Each
column represents the mean ± S.E. of three independent
experiments conducted in duplicate.
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Table III
Effects of C2-ceramide on endothelial apoptosis
HUVECs were seeded as described in the legend to Fig. 1 and cultured in
the presence of C2-ceramide for 48 h. Values are the
mean ± S.E. of three independent experiments conducted in
duplicate.
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Other Mechanisms Involved in oxLDL-induced
Apoptosis--
To elucidate other mechanisms involved in
this endothelial apoptosis, we studied the effects of inhibitors of the
enzymes involved in apoptosis, antioxidants, and cholesterol. Although most apoptosis-inducing agents activate caspases (35-37), there are
some exceptions (53). Therefore, we tried to confirm whether oxLDL
induces apoptosis by activating caspases. The inhibitor of caspase-1
(ac-YVAD-CHO) and the inhibitor of caspase-3 (ac-DEVD-CHO) each
inhibited apoptosis (Fig. 5), confirming
that oxLDL induces apoptosis by activating caspases, as do most other
apoptosis-inducing factors. Because oxLDL could be further oxidized in
the presence of HUVECs and the radicals generated during this process
could induce apoptosis, we studied the effects of antioxidants on
endothelial apoptosis. Among the antioxidants, SOD and BHT inhibited
the apoptosis but catalase had no effect on oxLDL-induced apoptosis
(Fig. 6), suggesting that superoxide, but
not hydrogen peroxide, is responsible for the ability of oxLDL to
induce of apoptosis. Similarly, both SOD and BHT inhibited the
apoptosis-inducing activity of 25-hydroxycholesterol (data
not shown). A recent report that caspase-3 cleaves sterol regulatory
element binding proteins and may up-regulate cholesterol synthesis (54)
indicates a close relationship between apoptosis and cholesterol
metabolism, prompting us to study the effect of cholesterol on
endothelial apoptosis. Exogenous cholesterol potently inhibited the
apoptosis induced by both oxLDL and oxysterols (Fig. 7).

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Fig. 5.
Effects of caspase inhibitors on
oxLDL-induced apoptosis. HUVECs were seeded as described in
the legend to Fig. 1. The cells were cultured in DMEM supplemented with
0.1% BSA, 10 ng/ml bFGF, and oxLDL (25 µg of protein/ml) in the
presence or absence of an inhibitor for 48 h. The values shown are
the mean ± S.E. of quadruplicate samples.
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Fig. 6.
Effects of antioxidants on oxLDL-induced
apoptosis. HUVECs were seeded as described in the legend to Fig.
1. The cells were cultured in DMEM supplemented with 0.1% BSA, 10 ng/ml bFGF, and oxLDL (25 µg of protein/ml) in the presence or
absence of antioxidants for 48 h. For antioxidants, 50 µM of BHT, 100 µg/ml SOD, and 100 µg/ml catalase were
used. The vehicle of these antioxidants (ethanol and phosphate-buffered
saline) alone did not affect the apoptosis. BHT was dissolved in
ethanol, and SOD and catalase were dissolved in phosphate-buffered
saline. The values shown are the mean ± S.E. of quadruplicate
samples.
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Fig. 7.
Effect of cholesterol on apoptosis induced by
oxLDL or oxysterols. HUVECs were seeded as described in the legend
to Fig. 1. After 24 h, the medium was changed to DMEM supplemented
with 0.1% BSA, 10 ng/ml bFGF, and oxLDL (25 µg of protein/ml) or
oxysterols. 25-OH Chol, 25-hydroxycholesterol (5 µg/ml),
Triol, cholestane-3,5,6-triol (5 µg/ml). Cells were
cultured in the presence or absence of 50 µg/ml cholesterol for
48 h. Each column represents the mean ± S.E. of
quadruplicate values.
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DISCUSSION |
In this study, we observed that oxLDL induces apoptosis in
cultured human umbilical vein endothelial cells by both common and
unique mechanisms. The common mechanisms include the accumulation of
intrinsic ceramide and the activation of caspases. A unique mechanism
underlying oxLDL-induced apoptosis is the involvement of superoxide. We
also found that cholesterol inhibits oxLDL-induced apoptosis, although
its precise mode of action is unknown.
Previous reports indicated that oxysterols and oxLDL could induce
apoptosis in endothelial cells (26, 27, 55). We confirmed these
observations and showed that oxysterols (and potentially some other
lipids) in the neutral lipid fraction of oxLDL account for the
apoptosis-inducing activity of oxLDL. Most apoptosis-inducing activity was recovered in the neutral lipid fraction that contains a
series of oxysterols, as detected by gas chromatography. Oxysterols potently induced the apoptosis at concentrations comparable to those in
oxLDL (Table II). Although lysoPC in oxLDL has some important biological effects (12), neither the phospholipid fraction nor isolated
lysoPC induced endothelial apoptosis in the present study.
One of the mechanisms involved in this apoptosis is the accumulation of
intrinsic ceramide. This conclusion was drawn from three sets of
observations. First, oxLDL increased the cellular ceramide content, as
reported with other apoptosis-inducing agents such as tumor necrosis
factor-
, anti-FAS antibody, and ionizing radiation. This ceramide
generation did not need sphingomyelin in oxLDL as a substrate of
sphingomyelinase (56) because an oxysterol also increased the cellular
ceramide content (see "Results"). Second, two different nonspecific
inhibitors of acid sphingomyelinase completely inhibited both ceramide
generation and the apoptosis. These inhibitors do not inhibit other
lysosomal enzymes or neutral sphingomyelinase (51). Third, a
membrane-permeable homologue of ceramide, C2-ceramide,
induced apoptosis in HUVECs at concentrations comparable to those of
intrinsic ceramide. A similar membrane-permeable ceramide, C6-ceramide,
was reported to induce apoptosis in HUVECs (53). Although accumulating
data suggest that the increase in ceramide is a common mechanism among
various apoptosis-inducing agents (35-37), there are some concerns
about the role of ceramide in apoptosis. For example, although
Fas-induced apoptosis is independent of transcription or translation,
C2 ceramide-induced apoptosis is inhibited by blocking the
transcription of AT-1, suggesting that FAS, although it increases
ceramide generation, induces apoptosis by a mechanism unrelated to
this increase in ceramide. The inhibition of caspase-1 blocked both the
ceramide generation and apoptosis induced by REAPER, suggesting that
ceramide generation is a result rather than a cause of the activation
of caspase-1, a key enzyme of apoptosis (41). Our finding that cellular
ceramide increased temporarily at 15 min and again at 24 h after
stimulation with oxLDL suggests that cellular ceramide increases as
both a cause and a result of apoptosis. The increase of ceramide at 15 min is clearly a cause of apoptosis, because the inhibitor of
sphingomyelinase blocked both ceramide generation at 15 min and
apoptosis at 6 h later. The increase of ceramide at 15 min could
not be a result of apoptosis, because no apoptosis was detected at that
point. The significance of the increase in ceramide at early time
points agrees well with a recent report concerning the temporal profile of apoptosis in a cell-free system, in which ceramide caused
fragmentation of nuclei only when it was added in the first 90 min of
the time course (57). Interleukin-1
induces E-selectin in HUVECs
through a transient ceramide generation similar to that observed in the present study (48). Contrary to the ceramide increase at 15 min, the
ceramide increase after 24 h could be a result of apoptosis because significant apoptosis had occurred by this time.
Another controversy regarding the ceramide pathway of apoptosis centers
on the enzymes involved in the ceramide generation. Tumor necrosis
factor-
and Fas ligand activate acid sphingomyelinase in many cell
types (58-61), whereas ionizing radiation induces apoptosis in bovine
aortic endothelial cells by activating neutral sphingomyelinase (62).
Ceramide synthase accounts for ceramide generation in
daunorubicin-induced apoptosis (63). We employed two sphingomyelinase
inhibitors that inhibit acid sphingomyelinase (64, 65) and demonstrated
that acid sphingomyelinase is responsible for the ceramide generation
in oxLDL-induced apoptosis. Acid sphingomyelinase was recently found to
be localized not only in the lysosome but also in association with the
caveola, a membrane domain that can undergo an internalization cycle
(66). Most membrane sphingomyelin and ceramide are also localized in
the caveola, and interleukin-1 can activate acid sphingomyelinase and
increase ceramide in the caveola (66). As discussed later, our data
suggest that a critical interaction between oxLDL and endothelial cells
takes place on the outer surface of the cell. Taken together, the
present and above-mentioned findings suggest that acid sphingomyelinase
in the caveola plays a role in the ceramide generation and apoptosis induced by oxLDL.
Another common mechanism involved in this apoptosis is the activation
of caspases (35-37). Typically, apoptotic agents activate caspase-1
and caspase-3 sequentially. However, there are many alternative
caspases to cleave death substrates (37), or even a pathway independent
of classical interleukin 1
-converting enzyme (53). oxLDL has been
found to activate caspase-3 (CPP32) and induce apoptosis in HUVECs
(27). Our findings with specific inhibitors confirm that both caspase-1
and caspase-3 are involved in the endothelial apoptosis induced by
oxLDL.
We identified a unique mechanism underlying the oxLDL-induced
endothelial apoptosis: the involvement of superoxide. Our finding that
SOD but not catalase inhibits oxLDL-induced apoptosis indicates that
superoxide play an important role in the apoptosis. Oxygen radical has
been proposed as a target of bcl-2, but now its role is questioned
based on observations that apoptosis is induced even under an anaerobic
condition (67, 68). An important difference between previous reports
and ours is that superoxide plays a role outside the cell in our
experimental system, whereas intracellular radical-associated
mechanisms have been studied as intracellular mechanisms of
apoptosis. Because SOD is not membrane-permeable, the site of
action of this enzyme would expected to be outside the plasma membrane.
Therefore, although oxLDL and oxysterols can get into the cell,
critical interactions between these lipids and the cell are thought to
take place on the outer surface of the cell. It is also noteworthy that
LDL modified with reactive oxygen species is not sufficient and needs
the further involvement of reactive oxygen species to induce
endothelial apoptosis. This is in contrast with a previous report that
toxicity of oxLDL is not blocked by antioxidants once LDL is oxidized
(3). Our findings suggest that oxysterols (and potentially other lipid
components) in oxLDL play a role in the propagation of the chain
reaction initiated by superoxide to induce apoptosis.
An interesting finding of our study is that exogenous cholesterol
inhibited the apoptosis induced by oxLDL or oxysterols. Although
previous investigators have pointed out a potential relationship between cholesterol metabolism and apoptosis (54), direct evidence of
an antiapoptotic effect of cholesterol has not been reported. Based on
an observation that caspase-3 was found to cleave sterol regulatory
element binding proteins and to potentially up-regulate cholesterol
metabolism (54), it was suggested that cholesterol is needed to
maintain membrane integrity during apoptosis. If so, cholesterol could
inhibit apoptosis by reducing the intrinsic cholesterol supply that is
needed for apoptosis. However, this is not likely, because
hydroxycholesterols are more potent negative regulators for HMG-CoA
reductase, whereas hydroxycholesterols induce rather than inhibit
apoptosis. Our findings that oxLDL activates caspase-3 and that
cholesterol inhibits apoptosis suggest that caspase-3 up-regulates
cholesterol synthesis as a negative feedback mechanism to prevent
apoptosis, whereas it cleaves the death substrate to induce apoptosis.
Although the precise mode of action of cholesterol remains to be
elucidated, cholesterol can inhibit sphingomyelin degradation and
therefore can potentially inhibit the ceramide pathway of
apoptosis.
In summary, we reported mechanisms involved in the oxLDL-induced
apoptosis in vascular endothelial cells. Our data suggest that neutral
lipids, such as oxysterols, in oxLDL activate acid sphingomyelinase on
the outer membrane of the cell in a superoxide-dependent manner to generate ceramide, which eventually activates caspases.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Atsushi Masamune for helpful
advice on ceramide measurement, Dr. Shigeko Takaichi for electron
microscopy, and Dr. Hisayuki Matsuo for encouragement during this
project.
 |
FOOTNOTES |
*
This study was supported by grants from the Science and
Technology Agency, from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare.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.
§
The first two authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
81-6-833-5012, ext. 2590; Fax: 81-6-872-7485; E-mail:
kshimoka{at}res.ncvc.go.jp.
1
The abbreviations used are: oxLDL, oxidized low
density lipoprotein; BHT, butylated hydroxytoluene; SOD,
superoxide dismutase; HUVEC, human umbilical vein endothelial
cell; DMEM, Dulbecco's modified Eagle's medium; lysoPC,
lysophosphatidylcholine; bFGF, basic fibroblast growth factor;
BSA, bovine serum albumin.
 |
REFERENCES |
-
Steinberg, D.,
Parthasarathy, S.,
Carew, T. E.,
Khoo, J. C.,
and Witztum, J. L.
(1989)
New Engl. J. Med.
320,
915-924[Medline]
[Order article via Infotrieve]
-
Ross, R.
(1993)
Nature
362,
801-809[CrossRef][Medline]
[Order article via Infotrieve]
-
Morel, D. W.,
Hessler, J. R.,
and Chisolm, G. M.
(1983)
J. Lipid Res.
24,
1070-1076[Abstract]
-
Lamb, D. J.,
Michinson, M. J.,
and Leake, D. S.
(1995)
FEBS Lett.
374,
12-16[CrossRef][Medline]
[Order article via Infotrieve]
-
Steinbrecher, U. P.,
Parthasarathy, S.,
Leake, D. S.,
Witztum, J. L.,
and Steinberg, D
(1984)
Proc. Natl. Acad. Sci. U. S. A.
81,
3883-3887[Abstract]
-
Morel, D. W.,
DiCorleto, P. E.,
and Chisolm, G. M.
(1984)
Arteriosclerosis
4,
357-364[Abstract]
-
Hiramatsu, K.,
Rosen, H.,
Heinecke, J. W.,
Wolfbauer, G.,
and Chait, A.
(1987)
Arteriosclerosis
7,
55-60[Abstract]
-
Ehrenwald, E.,
Chisolm, G. M.,
and Fox, P. L.
(1994)
J. Clin. Invest.
93,
1493-1501[Medline]
[Order article via Infotrieve]
-
Parthasarathy, S.,
Steinbrecher, U. P.,
Barnett, J.,
Witztum, J. L.,
and Steinberg, D.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
3000-3004[Abstract]
-
Daugherty, A.,
Dunn, J. L.,
Rateri, D. L.,
and Heinecke, J. W.
(1994)
J. Clin. Invest.
94,
437-444[Medline]
[Order article via Infotrieve]
-
Yla-Herttuala, S.,
Luoma, J.,
Viita, H.,
Hiltunen, T.,
Sisto, T.,
and Nikkari, T.
(1995)
J. Clin. Invest.
95,
2692-2698[Medline]
[Order article via Infotrieve]
-
Kume, N.,
Cybulsky, M. I.,
and Gimbrone, M. A., Jr.
(1992)
J. Clin. Invest.
90,
1138-1144[Medline]
[Order article via Infotrieve]
-
Mangin, E. L.,
Kugiyama, K.,
Nguy, J. H.,
Kerns, S. A.,
and Henry, P. D.
(1993)
Circ. Res.
72,
161-166[Abstract]
-
Thorin, E.,
Hamilton, C. A.,
Dominiczak, M. H.,
and Reid, J. L.
(1994)
Arterioscler. Thromb.
14,
453-459[Abstract]
-
Henriksen, T.,
Mahoney, E. M.,
and Steinberg, D.
(1981)
Proc. Natl. Acad. Sci. U. S. A.
78,
6499-6503[Abstract]
-
Kume, N.,
and Gimbrone, M. A., Jr.
(1994)
J. Clin. Invest.
93,
907-911[Medline]
[Order article via Infotrieve]
-
Nakano, T.,
Raines, E. W.,
Abraham, J. A.,
Klagsbrun, M.,
and Ross, R.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
1069-1073[Abstract]
-
Quinn, M. T.,
Parthasarathy, S.,
and Steinberg, D.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
2805-2809[Abstract]
-
Auge, N.,
Andrieu, N.,
Negre-Salvayre, A.,
Thiers, J.-C.,
Levade, T.,
and Salvayre, R.
(1996)
J. Biol. Chem.
271,
19251-19255[Abstract/Free Full Text]
-
Yui, S.,
Sasaki, T.,
Miyazaki, A.,
Horiuchi, S.,
and Yamazaki, M.
(1993)
Arterioscler. Thromb.
13,
331-337[Abstract]
-
Murugesan, G.,
and Fox, P. L.
(1996)
J. Clin. Invest.
97,
2736-2744[Abstract/Free Full Text]
-
Ishii, H.,
Kizaki, K.,
Horie, S.,
and Kazama, M.
(1996)
J. Biol. Chem.
271,
8458-8465[Abstract/Free Full Text]
-
Bjorkerud, S.,
and Bjorkerud, B.
(1996)
Am. J. Pathol.
149,
367-380[Abstract]
-
Bjorkerud, B.,
and Bjorkerud, S.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
416-424[Abstract/Free Full Text]
-
Reid, V. C.,
Mitchinson, M. J.,
and Skepper, J. N.
(1993)
J. Pathol.
171,
321-328[Medline]
[Order article via Infotrieve]
-
Escargueil-Blanc, I.,
Meilhac, O.,
Pieraggi, M.-T.,
Arnal, J.-F.,
Salvayre, R.,
and Negre-Salvayre, A.
(1997)
Arterioscler. Thromb. Vasc. Biol.
17,
331-339[Abstract/Free Full Text]
-
Dimmeler, S.,
Haendeler, J.,
Galle, J.,
and Zeiheer, A. M.
(1997)
Circulation
95,
1760-1763[Abstract/Free Full Text]
-
Escargueil-Blanc, I.,
Salvayre, R.,
and Negre-Salvayre, A.
(1994)
FASEB J.
8,
1075-1080[Abstract/Free Full Text]
-
Isner, J. M.,
Kearney, M.,
Bortman, S.,
and Passeri, J.
(1995)
Circulation
91,
2703-2711[Abstract/Free Full Text]
-
Han, D. K. M.,
Haudenschild, C. C.,
Hong, M. K.,
Tinkle, B. T.,
Leon, M. B.,
and Liau, G.
(1995)
Am. J. Pathol.
147,
267-277[Abstract]
-
Bochaton-Piallat, M.-L.,
Gabbiani, F.,
Redard, M.,
Desmouliere, A.,
and Gabbiani, G.
(1995)
Am. J. Pathol.
146,
1059-1064[Abstract]
-
de Bono, D. P.,
and Yang, W. D.
(1995)
Atherosclerosis
114,
235-245[CrossRef][Medline]
[Order article via Infotrieve]
-
Baumgartner-Parzer, S. M.,
Wagner, L.,
Pettermann, M.,
Grillari, J.,
Gessl, A.,
and Waldhausl, W.
(1995)
Diabetes
44,
1323-1327[Abstract]
-
Araki, S.,
Shimada, Y.,
Kaji, K.,
and Hayashi, H.
(1990)
Biochem. Biophys. Res. Commun.
168,
1194-1200[Medline]
[Order article via Infotrieve]
-
Hale, A. J.,
Smith, C. A.,
Sutherland, L. C.,
Stoneman, V. E. A.,
Longthorne, V. L.,
Culhane, A. C.,
and Williams, G. T.
(1996)
Eur. J. Biochem.
236,
1-25[Abstract]
-
Fraser, A.,
and Evan, G.
(1996)
Cell
85,
781-784[Medline]
[Order article via Infotrieve]
-
Nagata, S.
(1997)
Cell
88,
355-365[Medline]
[Order article via Infotrieve]
-
Obeid, L. M.,
Linardic, C. M.,
Karolak, L. A.,
and Hannun, Y. A.
(1993)
Science
259,
1769-1771[Medline]
[Order article via Infotrieve]
-
Jarvis, W. D.,
Kolesnick, R. N.,
Fornari, F. A.,
Traylor, R. S.,
Gewirtz, D. A.,
and Grant, S.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
73-77[Abstract]
-
Hannun, Y. A.
(1996)
Science
274,
1855-1859[Abstract/Free Full Text]
-
Pronk, G. J.,
Ramer, K.,
Amiri, P.,
and Williams, L. T.
(1996)
Science
271,
808-810[Abstract]
-
Jaffe, E. A.,
Nachman, R. L.,
Becker, C. G.,
and Minick, C. R.
(1973)
J. Clin. Invest.
52,
2745-2756[Medline]
[Order article via Infotrieve]
-
Havel, R.,
Eder, H.,
and Braigon, J.
(1955)
J. Clin. Invest.
39,
1345-1363
-
Brown, M.,
Goldstein, J.,
Krieger, M.,
Ho, Y.,
and Anderson, R.
(1979)
J. Cell Biol.
82,
597-613[Abstract]
-
Kamido, H.,
Kuksis, A.,
Marai, L.,
and Myher, J. J.
(1993)
Lipids
28,
331-336[Medline]
[Order article via Infotrieve]
-
Shimokado, K.,
Umezawa, K.,
and Ogata, J.
(1995)
Exp. Cell Res.
220,
266-273[CrossRef][Medline]
[Order article via Infotrieve]
-
Van Veldhoben, P. P.,
Bishop, W., R.,
Yurivich, D., A.,
and Bell, R., M.
(1995)
Biochem. Mol. Biol. Int.
36,
21-30[Medline]
[Order article via Infotrieve]
-
Masamune, A.,
Igarashi, Y.,
and Hakomori, S.
(1996)
J. Biol. Chem.
271,
9368-9375[Abstract/Free Full Text]
-
Chisolm, G. M.,
Ma, G.,
Irwin, K. C.,
Martin, L. L.,
Gunderson, K. G.,
Linberg, L. F.,
Morel, D. W.,
and DiCorleto, P. E.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11452-11456[Abstract/Free Full Text]
-
Sevanian, A.,
Hodis, H. N.,
Hwang, J.,
McLeod, L. L.,
and Peterson, H.
(1995)
J. Lipid Res.
36,
1971-1986[Abstract]
-
Masson, M.,
Spezzatti, B.,
Chapman, J.,
Battisti, C.,
and Baumann, N.
(1992)
J. Neurosci. Res.
31,
84-88[Medline]
[Order article via Infotrieve]
-
Slowik, M. R.,
De Luca, L. G.,
Min, W.,
and Pober, J. S.
(1996)
Circ. Res.
79,
736-747[Abstract/Free Full Text]
-
Woodle, E.,
Smith, D.,
Bluestone, J.,
Kirkman, W.,
Green, D.,
and Skowronski, E.
(1997)
J. Immunol.
158,
2156-2164[Abstract]
-
Wang, X.,
Zelenski, N. G.,
Yang, J.,
Sakai, J.,
Brown, M. S.,
and Goldstein, J. L.
(1996)
EMBO J.
15,
1012-1020[Abstract]
-
Lizard, G.,
Deckert, V.,
Dubrez, L.,
Moisant, M.,
Gambert, P.,
and Lagrost, L.
(1996)
Am. J. Pathol.
148,
1625-1638[Abstract]
-
Kinscherf, R.,
Claus, R.,
Deigner, H., P.,
Nauen, O.,
Gehrke, C.,
Hermetter, A.,
Russwurm, S.,
Daniel, V.,
Hack, V.,
and Metz, J.
(1997)
FEBS Lett.
405,
55-59[CrossRef][Medline]
[Order article via Infotrieve]
-
Farschon, D. M.,
Couture, C.,
Mustelin, T.,
and Newmeyer, D. D.
(1997)
J. Cell Biol.
137,
1117-1125[Abstract/Free Full Text]
-
Cifone, M. G.,
DeMaria, R.,
Roncaioli, P.,
Rippo, M. R.,
Azuma, M.,
Lanier, L. L.,
Santoni, A.,
and Testi, R.
(1993)
J. Exp. Med.
177,
1547-1552
-
Cifone, M. G.,
Roncaioli, P.,
De Maria, R.,
Camarda, G.,
Santoni, A.,
Ruberti, G.,
and Testi, R.
(1995)
EMBO J.
14,
5859-5868[Abstract]
-
Higuchi, M.,
Singh, S.,
Jaffrezou, J.-P.,
and Aggarwal, B. B.
(1996)
J. Immunol.
156,
297-304[Abstract]
-
Santana, P.,
Pena, L. A.,
Haimovitz-Friedman, A.,
Martin, S.,
Green, D.,
McLoughlin, M.,
Cordon-Cardo, C.,
Schuchman, E. H.,
Fuks, Z.,
and Kolesnick, R.
(1996)
Cell
86,
189-199[Medline]
[Order article via Infotrieve]
-
Haimovitz-Friedman, A.,
Kan, C.-C.,
Ehleiter, D.,
Persaud, R. S.,
McLoughlin, M.,
Fuks, Z.,
and Kolesnick, R. N.
(1994)
J. Exp. Med.
180,
525-535[Abstract]
-
Bose, R.,
Verheij, M.,
Haimovitz-Friedman, A.,
Scotto, K.,
Fuks, Z.,
and Kolesnick, R.
(1995)
Cell
82,
405-414[Medline]
[Order article via Infotrieve]
-
Albouz, S.,
Le Saux, F.,
Wenger, D.,
Hauw, J. J.,
and Baumann, N.
(1986)
Life Sci.
38,
357-363[CrossRef][Medline]
[Order article via Infotrieve]
-
Lister, M. D.,
Crawford-Redick, C. L.,
and Loomis, C. R.
(1993)
Biochim. Biophys. Acta
1165,
314-320[Medline]
[Order article via Infotrieve]
-
Liu, P.,
and Anderson, R. G. W.
(1995)
J. Biol. Chem.
270,
27179-27185[Abstract/Free Full Text]
-
Jacobson, M. D.,
and Raff, M. C.
(1995)
Nature
374,
814-816[CrossRef][Medline]
[Order article via Infotrieve]
-
Muschel, R.,
Bernhard, E.,
Garza, L.,
McKenna, W.,
and Koch, C.
(1995)
Cancer Res.
55,
995-998[Abstract]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.