Fas/Fas ligand-mediated death pathway is involved
in oxLDL-induced apoptosis in vascular smooth muscle
cells
Tzong-Shyuan
Lee and
Lee-Young
Chau
Division of Cardiovascular Research, Institute of Biomedical
Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China
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ABSTRACT |
Oxidized
low-density lipoprotein (oxLDL) is a potent inducer of
apoptosis for vascular cells. In the present study, we
demonstrate that the expression of death mediators, including p53, Fas,
and Fas ligand (FasL) was substantially upregulated by oxLDL in
cultured vascular smooth muscle cells (SMCs). The induction of these
death mediators was time dependent and was accompanied by an increase in apoptotic death of SMCs following oxLDL treatment. Two
oxysterols, 7
-hydroxycholesterol and 25-hydroxycholesterol, were
also effective to induce the expression of death mediators and
apoptosis.
-Tocopherol and deferoxamine significantly
attenuated the induction of death mediators and cell death induced by
oxLDL and oxysterols, suggesting that reactive oxygen species are
involved in triggering the apoptotic event. Incubation of cells
with FasL-neutralizing antibody inhibited the oxLDL-induced cell death
up to 50%. Furthermore, caspase 8 and caspase 3 activities were
induced time dependently in SMCs following oxLDL treatment.
Collectively, these data suggest that the Fas/FasL death pathway is
activated and responsible for, at least in part, the apoptotic
death in vascular SMCs upon exposure to oxLDL.
atherosclerosis; oxysterols; p53; caspase; oxidized low-density
lipoprotein
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INTRODUCTION |
APOPTOSIS IS A
PROMINENT FEATURE in atherosclerotic lesions of humans (8,
15, 16, 18, 24) and experimental animals (7, 27).
It occurs in macrophages, T lymphocytes, and smooth muscle cells (SMCs)
of the lesions and is implicated in the development and progression of
the disease. Increasing evidence has suggested that the apoptotic
death of SMCs in fibrous caps and media of the advanced lesions is one
of the crucial determinants leading to plaque rupture, which is the
cause of most of the severe clinical complications of atherosclerosis
(4, 30). It appears that the characterization of mediators
and molecular mechanisms underlying the apoptotic death of vascular
cells is fundamentally important for the better understanding of the
pathogenesis of vascular diseases. Recently, numerous studies showed
that oxidized low-density lipoprotein (oxLDL), which is an atherogenic
substance commonly present in atherosclerotic lesions, induces
apoptosis in cultured endothelial cells (10, 12, 19,
39), macrophages (20, 26, 35, 36), lymphoid cells
(13), and SMCs (25, 34). Nevertheless, oxLDL-induced apoptosis in different cell types is not
necessarily mediated by the same mechanism. For example, it has been
shown that oxLDL induces endothelial death through activating the
ceramide pathway (19) and increasing sensitivity to the
Fas-mediated pathway (39) by downregulating Fas-associated
death domain-like interleukin-1
-converting enzyme-inhibitory
protein, a cellular caspase inhibitor (38). A
study on macrophages has revealed that apoptosis induced by
oxLDL is associated with the induction of tumor suppressor p53 and
manganese superoxide dismutase (26). However, there is
little information regarding the underlying mechanism responsible for
oxLDL-induced apoptotic death in SMCs.
It has been shown that the initiation of apoptosis in various
cell systems frequently associates with the induction of some death-regulating mediators (33). Recent studies on human
atherosclerotic lesions and aneurysms revealed that the
death-regulating proteins, including p53 and Fas antigen, are detected
in apoptotic SMCs of the diseased vessels (5, 22, 23,
32). The localization of oxLDL immunoreactivity in apoptotic
SMCs was also observed in human carotid plaques (25). It
is of great importance to know whether p53-dependent and/or
Fas-mediated death pathways are involved in the apoptotic death of
SMCs induced by oxLDL in vivo. In an attempt to clarify the issue, in
the present study, we assess the expression profiles of these
death-associated mediators in vascular SMCs upon exposure to oxLDL in
vitro. The results clearly show that oxLDL upregulated the expression
of p53, Fas, and Fas ligand (FasL) in SMCs. The induction of these
death mediators was concurrent with apoptotic death of cells
following oxLDL treatment. 7
-Hydroxycholesterol and
25-hydroxycholesterol, the oxidative products present in oxLDL
(9), were also effective to induce the expression of
death-regulating proteins and cell death. Since the interaction between
Fas and FasL leads to the initiation of the death signaling, the
biological relevance of a Fas/FasL-mediated pathway in the
apoptotic event induced by oxLDL was further elucidated. The data
show that the oxLDL-induced cell death was markedly attenuated by the
treatment with neutralizing antibody to FasL. On the other hand, the
activity of caspase 8, which is a specific downstream target of a
Fas/FasL death-signaling pathway (2), was significantly induced by oxLDL treatment. Together, these observations strongly suggest that the Fas/FasL-mediated death pathway is activated and
contributes, at least in part, to the apoptosis of vascular SMCs induced by oxLDL.
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MATERIALS AND METHODS |
Cell culture.
Rat aortic SMCs were isolated from thoracic aortas of Sprague-Dawley
rats using the explant technique (14). Briefly, after removal of endothelium and adventitia, the aortic explants were cultured in DMEM that contained 10% fetal calf serum. After 2 wk,
cells that migrated out of the explants were removed by trypsinization and subcultured successively. The identity and purity of the SMCs were
verified by immunostaining using antibody against smooth muscle
-actin. Cultured rat SMCs from 9 to 15 passages were used for
experiments. Human aortic SMCs (passage 4) were purchased from Clonetics and cultured in formulated SmGM medium that contained 5% serum (Clonetics). Human SMCs from 5 to 7 passages were used for
experiments. Cells at 50-60% confluency were changed to
serum-free medium for 24 h and then subjected to treatment with
various agents for indicated times. Human LDL and oxLDL were prepared
as described previously (41). The oxLDL contained
~30-60 nmol of thiobarbituric acid-reactive substances (TBARS)
as malondialdehyde equivalents per milligram of LDL proteins.
Western blots.
Cells were rinsed with ice-cold phosphate-buffered saline (PBS) twice
and lysed in 65 mM Tris · HCl, pH 6.8, that contained 2% SDS,
2% 2-mercaptoethanol, and 5% glycerol, followed by boiling for 10 min. After sonication for 5 × 15 s, using a microprobe sonicator at an output of five and a pulse cycle of 50%, 50 µg of
protein lysates were electrophoresed on a 10% SDS-polyacrylamide gel
and then transblotted onto an Immobilon-P membrane (Millipore). The
membranes were blocked in PBS that contained 0.1% Tween 20 and 1%
skim milk at room temperature for 30 min. For p53 protein detection,
blots were incubated with sheep anti-p53 polyclonal antibody (1:3,000
dilution; Oncogene) in the blocking buffer for 1 h at room
temperature. After three washes, blots were incubated with
biotinylated rabbit anti-sheep IgG (1:3,000; Oncogene) in the
same buffer for 1 h. Blots were then washed and incubated with
streptavidin conjugated with peroxidase (1:4,000 dilution) for an
additional 1 h. For detection of Fas or FasL, blots were incubated
with rabbit anti-Fas polyclonal antibody (1:2,000 dilution; Santa Cruz)
or rabbit anti-FasL polyclonal antibody (1:2,000 dilution; Santa Cruz)
at room temperature for 1 h, followed by incubation with
peroxidase-conjugated goat anti-rabbit IgG (1:3,000 dilution) for
another 1 h. Antigens were detected by the enhanced
chemiluminescence system (Pierce).
In situ detection of apoptotic cells.
Cells grown on cover slides were fixed with 4% paraformaldehyde in PBS
for 15 min at room temperature, followed by 2× 5-min washes with PBS.
The DNA fragmentation was determined by the TdT-mediated dUTP nick end
labeling (TUNEL) method (Boehringer Mannheim). Briefly, after
incubation with proteinase K (20 µg/ml) for 20 min, samples were
incubated with digoxigenin (DIG)-dUTP and terminal deoxynucleotidyl transferase, which catalyzes the addition of deoxyribonucleotide to
3'-OH ends of DNA fragments. The incorporation of DIG-dUTP into DNA was
determined by incubating the slides with peroxidase-conjugated antibody
against DIG at 37°C for 30 min. The positive stain was then
visualized by incubation with 0.01% H2O2-0.1%
3,3'-diaminobenzidinine for 2-5 min at room temperature. The
slides were counterstained with hematoxylin. For each slide, at least
800 cells were counted in random fields. Cells with clear nuclear
labeling were defined as apoptotic cells. To assess the effect of
anti-FasL antibody on oxLDL-induced apoptosis, cells were
treated with oxLDL in the presence of indicated amounts of rabbit
anti-rat FasL polyclonal IgG (C-178; Santa Cruz) or control rabbit IgG.
After a 12-h incubation, the extent of apoptotic death was
determined as described above.
TUNEL staining on aortic tissues.
Rat thoracic aorta was denuded, cut into 5-mm-long segments, and placed
in DMEM for 24 h at 37°C in culture. LDL or oxLDL at indicated
concentrations were then added into the medium, and incubation
continued for another 24 h. The aortic segments were fixed with
4% paraformaldehyde and paraffin embedded. Tissues were serially
sectioned at 5 µm and subjected to TUNEL staining as described above.
RT-PCR.
Total RNA was extracted from cells using TRIzol reagent (GIBCO BRL)
according to the manufacturer's instructions. The integrity of the RNA
was monitored by ethidium bromide staining of 28S and 18S ribosomal
RNAs analyzed by electrophoresis on 1% agarose gel. The cDNA was
synthesized from 1 µg of total RNA by RT using 0.2 µg of random
hexamers (Promega) and 200 units of Moloney murine leukemia virus RT in
the presence of 0.4 mM of each deoxynucleotide triphosphate, 10 mM
dithiothreitol (DTT), and 10 units of RNasin in a final volume of 20 µl. After a 1-h incubation at 37°C, the reaction was terminated by
heating at 95°C for 5 min, followed by dilution with
H2O2 to 250 µl. One- and
three-microliter aliquots were then used for the PCRs of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and FasL,
respectively. The primers used for analysis of GAPDH were
5'-TCCCTCAAGATTGTCAGCAA-3' (sense) and
5'-AGATCCACAACGGATACATT-3'(antisense), and for FasL,
5'-GGAATGGGAAGACACATATGGAACTGC-3' (sense) and
5'-CATATCTGGCCAGTAGTGCAGTAATTC-3' (antisense). All PCR amplifications
were performed in 25 µl of reaction mixture that contained 0.5 mM
deoxynucleotide triphosphates and 1 unit of Taq DNA
polymerase. The reaction proceeded for 30 cycles with denaturation at
94°C for 1 min, annealing at 50°C (GAPDH) or 65°C (FasL) for 1 min, and extension at 72°C for 30 s. The PCR products were
visualized by electrophoresis on 1.5% agarose gel that contained
ethidium bromide. The PCR products for GAPDH and FasL were 309 and 238 bp in length, respectively.
Caspase activity assay.
Cells were harvested by centrifugation at 100 g for 10 min and washed with ice-cold hypotonic buffer that contained 20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM
DTT, and 0.1 mM phenylmethylsulfonyl fluoride. Cell pellet was
resuspended in the same buffer and incubated on ice for 20 min. After
sonication, cell extract was clarified by centrifugation at 12,000 g for 30 min at 4°C. The supernatant was stored at
20°C until used for assay. Cell lysates (500 µg) were diluted in
caspase buffer that contained 50 mM HEPES (pH 7.2), 100 mM NaCl, 1 mM
EDTA, 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 5 mM DTT,
and 10% sucrose.
N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (caspase
3 substrate) or
N-acetyl-Ile-Glu-Thr-Asp-p-nitroanilide (caspase
8 substrate) was then added to a final concentration of 20 µM. For
inhibition assay, cell lysates were preincubated with 10 µM of
benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val(OMe)-Asp(OMe)-fluoromethyl ketone (caspase 3 inhibitor) or
benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone
(caspase 8 inhibitor) at 37°C for 30 min before the addition of
caspase substrate. The reaction was carried out at 37°C for 2 h.
The release of p-nitroanilide was monitored colorimetrically at a wavelength of 405 nm.
Statistical analysis.
Results were expressed as means ± SD. Data were analyzed by
Student's t-test. P < 0.05 was considered
statistically significant.
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RESULTS |
Induction of apoptosis and expression of death-regulating
proteins in SMCs by oxLDL.
Incubation of cultured human or rat vascular SMCs with copper-oxidized
LDL for 12 h resulted in significant cell death. The DNA
fragmentation induced by oxLDL was clearly revealed by TUNEL assay,
which gave dark brown stains on the nuclei of apoptotic cells (Fig.
1). The oxLDL-induced
apoptosis was also observed in aortic explants placed in
culture. As shown in Fig. 2, when rat-denuded aortic segments were incubated with oxLDL in culture for
24 h, the TUNEL staining again detected the DNA fragmentation occurring in the nuclei of medial SMCs. These results imply that oxLDL
is a potent inducer of apoptosis for vascular SMCs in vivo. To
examine whether the oxLDL-induced cell death was associated with the
alteration in expression of the death-regulating proteins, the protein
levels of p53, Fas, and FasL in vascular SMCs following oxLDL treatment
were assessed by Western blot analysis. As shown in Fig.
3, incubation of human or rat vascular
SMCs with oxLDL for 12 h led to a significant increase in the
expression of these death mediators. It was noted that LDL also
upregulated the expression of these proteins, albeit to a much lesser
extent. Since it has been shown that FasL is rarely detected in SMCs
(15, 37), the induction of FasL expression by oxLDL was
further confirmed by semiquantitative RT-PCR. As illustrated in Fig.
4, treatment of rat vascular SMCs with
oxLDL, but not LDL, for 6 or 12 h resulted in upregulation of FasL
mRNA expression. Time-course experiments in rat vascular SMCs further
demonstrated that the protein induction was evident at 3 h after
oxLDL treatment, which was the earliest time point for detecting
significant cell death by TUNEL assay (Fig.
5). The increase in expression of these
death-associated proteins was in parallel with the increment in the
percentage of apoptosis over 12 h of incubation with
oxLDL. At 18 h incubation, >85% of cells were stained positive
with TUNEL assay, although the level of p53, but not Fas/FasL, declined
at this time point. The degrees of protein induction and
apoptosis were proportional to the TBARS value in oxLDL
preparations (Fig. 6), indicating that
the cytotoxicity of oxLDL was associated with the extent of oxidation.

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Fig. 1.
Apoptosis of vascular smooth muscle cells (VSMC) induced by
oxidized low-density lipoprotein (oxLDL). Cultured rat or human aortic
SMCs at 50-60% confluency were serum deprived for 24 h.
Native or oxLDL at a concentration of 50 µg/ml was added into medium,
and culture continued for 12 h. The apoptotic death of cells
was assessed by TdT-mediated dUTP nick end labeling (TUNEL) staining
(magnification, ×200).
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Fig. 2.
Apoptosis of aortic medial SMCs induced by oxLDL. Rat
aortic tissues were denuded and treated without (C) or with indicated
concentrations of LDL or oxLDL in culture for 24 h. Tissue
sections were then prepared and subjected to TUNEL staining
(magnification, ×400).
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Fig. 3.
Induction of p53, Fas, and Fas ligand (FasL) protein expression by
oxLDL in vascular SMCs. Rat (A) or human (B) SMCs
were serum deprived for 24 h and then treated without (C) or with
indicated amounts of LDL or oxLDL in culture for 12 h. Whole cell
lysates were prepared, and the protein levels of p53, Fas, and FasL
were examined by Western blot analysis (top). The equality
of the protein loading in each lane was demonstrated by Coomasssie blue
stain (bottom). Results shown are representative of 3 independent experiments.
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Fig. 4.
OxLDL-induced FasL mRNA expression in vascular SMCs. Rat vascular
SMCs were treated without (C) or with indicated concentrations of LDL
or oxLDL for 6 and 12 h in culture. Total RNAs were isolated, and
the expression levels of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH; internal control) and FasL were determined by RT-PCR.
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Fig. 5.
Time course of increased expression of death-regulating
proteins and apoptosis in oxLDL-treated rat vascular SMCs. Rat
vascular SMCs were serum deprived for 24 h, followed by treatment
with 50 µg/ml oxLDL for indicated times. A: expression
levels of p53, Fas, and FasL were examined by Western blot analysis.
B: quantitative data were obtained from densitometry
analysis. The basal expression levels for p53, Fas, and FasL at zero
time point are referred to 1. C: apoptotic death of
cells was assessed by TUNEL assay. Data shown are means ± SD of 3 independent experiments.
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Fig. 6.
Effect of extent of oxidation in LDL on induction of
death-regulating proteins and apoptosis in rat SMCs. Human LDL
was incubated with 5 µmol/l CuSO4 at 37°C for indicated
times, and the thiobarbituric acid-reactive substances (TBARS) value
was determined. Rat vascular SMCs were treated with these oxidized LDLs
(50 µg/ml each) for 12 h in culture. A: expression of
p53, Fas, and FasL was examined by Western blot analysis. Three
different preparations of oxLDL were used for the experiments.
B: apoptotic death of cells was assessed by TUNEL assay.
Data shown are means ± SD of 3 independent experiments.
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Oxysterol-induced apoptosis of SMCs.
It has been shown that oxysterols, including 7
-hydroxycholesterol
and 25-hydroxycholesterol, are involved in the cytotoxicity of oxLDL
and act as potent inducers of apoptosis in SMCs (1, 19,
29, 30, 31). To examine whether the oxLDL-induced expression of
death-associated proteins was mediated by oxysterols, the effects of
7
-hydroxycholesterol and 25-hydroxycholesterol on the expression
levels of p53, Fas, and FasL were examined. As shown in Fig.
7A, 25-hydroxycholesterol, but
not 7
-hydroxycholesterol, markedly induced p53 expression.
Nevertheless, both agents upregulated Fas/FasL expression to a similar
extent. In parallel to their abilities in upregulating the expression
of death mediators, TUNEL assay demonstrated that both oxysterols are
potent inducers of apoptosis in vascular SMCs (Fig.
7C).

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Fig. 7.
Effects of oxysterols and antioxidants on apoptosis of rat
vascular SMCs. A: rat SMCs were treated without (C) or with
50 µg/ml oxLDL, 25 µg/ml 7 -hydroxycholesterol (7-OH Chol), or 25 µg/ml 25-hydroxycholesterol (25-OH Chol) in culture for 12 h.
Whole cell lysates were prepared, and the expression levels of p53,
Fas, and FasL were examined by Western blot analysis. B:
cells were treated without (C) or with 50 µg/ml oxLDL in the absence
or presence of 100 µM -tocopherol (Vit E), 100 µM deferoxamine
(Def), or 5 mM N-acetylcysteine (NAC) in culture for 12 h. The expression levels of p53, Fas, and FasL were then examined by
Western blot. C: cells incubated with 50 µg/ml oxLDL, 25 µg/ml 7 -hydroxycholesterol, or 25 µg/ml 25-hydroxycholesterol in
the absence or presence of 100 µM -tocopherol, 100 µM
deferoxamine, or 5 mM NAC in culture for 12 h were processed for
TUNEL assay. Data shown are means ± SD of at least 3 independent
experiments. Significant difference vs. cells without treatment with
antioxidants: *P < 0.01, **P < 0.025, ***P < 0.05.
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Effects of antioxidants on oxLDL- or oxysterol-induced
apoptosis in SMCs.
To examine whether the reactive oxygen intermediates are involved in
mediating the protein expression and apoptotic death, the effects
of antioxidants, including
-tocopherol, deferoxamine (iron
chelator), and N-acetylcysteine (NAC) on oxLDL- or
oxysterol-induced cell death were assessed. As shown in Fig. 7,
B and C, the induction of p53, Fas, and FasL as
well as apoptosis in rat vascular SMCs exposed to oxLDL or
oxysterols was significantly inhibited by coincubation of cells with
-tocopherol or deferoxamine. NAC did not exhibit the beneficial
effect. Conversely, the apoptotic death appeared to be slightly
increased by NAC treatment under these experimental conditions.
Effect of FasL neutralizing antibody on oxLDL-induced
apoptosis.
To assess the biological importance of Fas/FasL interaction in
oxLDL-induced apoptosis, rat vascular SMCs were subjected to oxLDL treatment in the presence of FasL neutralizing antibody. As shown
in Fig. 8, the oxLDL-induced cell death
was significantly inhibited by the incubation of cells with
rabbit-specific antibodies against FasL, but not with rabbit control
IgG. The extent of inhibition was proportional to the amounts of FasL
antibody used, and a maximal inhibition (~50%) was achieved by 1 ug/ml of FasL antibody. Higher concentrations of antibody did not
result in less cell death.

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Fig. 8.
Inhibition of oxLDL-induced apoptosis by anti-FasL
antibody. Rat SMCs were treated with 50 µg/ml oxLDL in the absence or
presence of indicated amounts of rabbit IgG or rabbit anti-rat Fas
ligand IgG in culture for 12 h. A: extent of
apoptotic death of cells was assessed by TUNEL. Data shown are
means ± SD of 3 independent experiments. Significant difference
vs. cells treated with the same concentration of control rabbit IgG:
*P <0.05, **P <0.005. B: TUNEL
staining of cells treated without (C) or with 50 µg/ml oxLDL in the
absence or presence of 2 µg/ml rabbit IgG or anti-FasL IgG for
12 h. Ab, antibody; Ab Conc, antibody concentration.
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Activation of caspase 8 and caspase 3 in oxLDL-treated SMCs.
It has been shown that upon Fas/FasL interaction, procaspase 8 is
recruited to the receptor death-signaling complex and subsequently activated, leading to a cascade of proteolytic events, such as the
activation of caspase 3, and ultimately execution of apoptosis (2). As shown in Fig. 9,
oxLDL, but not LDL, induced significant increases in caspase 8 and
caspase 3 activities in rat vascular SMCs time dependently. The
proteolytic specificity of caspase was further confirmed by the
inhibition experiment using the specific substrate inhibitor to the
corresponding caspase.

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Fig. 9.
Activation of caspase 8 (A) and caspase 3 (B) in rat vascular SMCs upon exposure to oxLDL. Rat SMCs
were incubated without (control) or with 50 µg/ml of LDL or oxLDL in
culture for indicated times. Cell lysates were prepared, and caspase 8 and caspase 3 activities were assayed in the presence or absence of
specific inhibitor as described in MATERIALS AND METHODS.
Data shown are means ± SD of 5 independent experiments.
Significant difference vs. control cells: *P < 0.005.
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DISCUSSION |
Induction of apoptosis by oxLDL.
Considerable evidence has supported the possibility that LDL oxidation
is one of the crucial events leading to the formation of
atherosclerotic lesion in the vascular wall (6). In
addition to the primary role in foam cell formation, oxLDL has been
shown to exhibit a broad spectrum of biological effects, including the induction of apoptosis in vascular cells (10, 12, 13, 19, 20, 25, 34-36). Consistent with previous reports by other
investigators (25, 34), here we show that oxLDL induced
apoptosis in cultured human and rat vascular SMCs. Experiments
performed with rat aortic explants also demonstrated the apoptotic
cell death in medial SMCs upon exposure to oxLDL, although it required
longer incubation with higher concentrations of oxLDL, which was likely
caused by the inefficient diffusion of oxLDL into tissues. However,
these observations support the idea that oxLDL is a potent inducer of apoptosis for vascular SMCs in vivo.
Upregulation of apoptosis-associated proteins by oxLDL.
Although a number of studies have been conducted to disclose the
mechanisms underlying the oxLDL-induced apoptosis in
endothelial cells and macrophages (10, 12, 19, 26, 39),
the death-regulating pathways involved in the apoptotic death of
SMCs induced by oxLDL are less explored. Nevertheless, histological
assessments on human atherosclerotic lesions and aneurysms have
revealed that the expression of death mediators, including tumor
suppressor p53 and Fas antigen, is associated with the death of SMCs in
the disease states (15, 22, 23, 32). It is intriguing to
know whether these death mediators are involved in the
apoptosis of SMCs induced by oxLDL. Our results clearly show
that oxLDL induced p53 and Fas/FasL expression in human and rat
vascular SMCs. In the experiment with human SMCs, it was noted that the
expression of the death mediators induced by 50 µg/ml oxLDL was less
than that induced by 25 µg/ml oxLDL. Since the cell death induced by
the higher dose of oxLDL was much more severe, we speculate that the
lower levels of mediators detected resulted from the proteolytic
degradation occurring in the dead cells. Treatment of cells with high
concentrations of LDL also led to the elevated expression of these
death regulators. Nevertheless, the degree of induction by LDL was much
less than that by oxLDL at the same concentration. Since a previous
study has demonstrated that incubation of LDL with vascular SMCs would
lead to the oxidation of LDL in culture (21), we speculate
that the induction of death mediators by LDL is likely a secondary
event resulting from the oxidation of LDL under the present
experimental conditions.
The role of Fas/FasL-mediated death pathway.
A previous study by Bennett et al. (5) has demonstrated
that apoptosis of vascular SMCs may occur via p53-dependent and -independent pathways. Although we did not perform additional experiments to elucidate the role of p53 in the oxLDL-induced cell
death in SMCs, an earlier study by Kinscherf et al. (26) demonstrated that the p53 induction is involved in the
apoptosis of macrophages induced by oxLDL. It is envisioned
that p53 may be a common transducer of oxLDL-induced apoptosis
in different cell types. The expression of Fas/FasL, which is
originally identified in activated T cells and natural killer
cells, has been shown to play a role to induce apoptosis of
infiltrating inflammatory cells and prevent immune attack in some
immune-privileged tissues and tumors (2, 17). More
recently, studies on endothelial cells also revealed that the
Fas/FasL-dependent pathway mediates the oxLDL-induced apoptosis
(39). It has been found that endothelial cells express
both Fas and FasL, but refractive to the Fas-mediated apoptosis. The oxLDL-induced apoptotic death of endothelial
cells is due to the increase in the responsiveness of endothelial cells to Fas activation, but not upregulation, of the Fas/FasL expression (39). This situation appears to be quite different from
that observed in SMCs. Similar to previous reports by others (15, 37), we found that cultured vascular SMCs express Fas but that FasL is barely detectable. It is conceivable that the increase in FasL
expression is crucial for the initiation of apoptosis through
the Fas-mediated pathway in vascular SMCs. This notion was supported by
a recent study showing that adenovirus-mediated FasL expression leads
to apoptosis in vascular SMCs (37). Our data
showed that the FasL expression was upregulated by oxLDL in SMCs.
Time-course experiments further demonstrated that the increases in Fas
and FasL protein levels were accompanied with the concomitant induction
of apoptosis following oxLDL treatment, suggesting that the
Fas/FasL-mediated pathway is implicated in the apoptotic signaling.
This idea was further confirmed by the inhibition experiment showing
that the FasL-neutralizing antibody was effective to inhibit the cell
death induced by oxLDL. Nevertheless, the antibody treatment only
caused maximally 50% inhibition, implying that other death signaling,
such as p53-dependent pathway, may also take part in the oxLDL-induced
apoptosis in vascular SMCs. Further experiments revealed that
oxLDL treatment led to the activation of caspase 8, which is the first
downstream caspase turned on upon Fas stimulation (2).
Collectively, these data strongly support the implication of
Fas/FasL-mediated signaling in apoptosis of SMCs induced by oxLDL.
Involvement of oxysterols and reactive oxygen species.
Oxidation of LDL by copper resulted in the formation of a number of
oxysterols, particularly 7-hydroxycholesterol, 7-ketocholesterol, and
25-hydroxycholesterol (9). Earlier studies have shown that oxysterols are involved in the cytotoxicity of oxLDL (9).
This study shows that both 7
- and 25-hydroxycholesterols also
upregulate the expression of Fas/FasL and induce apoptosis in
vascular SMCs to various degrees. In contrast to 25-hydroxycholesterol,
which induces p53 and Fas/FasL, 7
-hydroxycholesterol induces
Fas/FasL expression without much effect on p53. Whether the
differential effect on p53 induction accounts for the lower potency of
7
-hydroxycholesterol to induce apoptosis in SMCs remains to
be clarified. Nevertheless, these results support the possibility that
the cytotoxic effect of oxLDL is mediated by these oxidized lipid
components. When cells were pretreated with
-tocopherol or
deferoxamine (iron chelator), the induction of death mediators as well
as apoptosis was significantly inhibited, indicating that the
oxidative event is involved in death signaling by oxLDL or oxysterols.
However, the other antioxidant, NAC, was not effective in protecting
cells from oxLDL- or oxysterol-induced apoptosis. When SMCs
were treated with NAC alone, ~4% of cells underwent apoptotic
death after a 6-h incubation in culture (data not shown). This
observation is consistent with a previous report by Tsai et al.
(40), who showed that NAC induces apoptosis in
SMCs. It has been shown that NAC is a potent inhibitor for nuclear
factor (NF)-
B activation (29). Previous studies by some
investigators demonstrated that the NF-
B activation is essential for
the proliferation of SMCs (3, 28). Recently, a report by
Erl et al. (11) showed that increased NF-
B activity
protects SMCs from apoptosis. We speculate that the failure of
NAC to inhibit apoptosis may be associated with its effect on
NF-
B activity. The detailed mechanism requires further investigation.
In conclusion, the present study clearly demonstrates that the
induction of Fas/FasL as well as p53 is associated with the apoptotic death of vascular SMCs upon exposure to oxLDL. It was interesting to learn that the oxLDL-induced apoptosis in
endothelial cells and SMCs is mediated by a common Fas/FasL death
pathway, although the underlying mechanisms responsible for the
activation of this pathway are distinct in these two cell types. Since
Fas, but not FasL, is abundantly expressed in SMCs, the induction of FasL appears to be crucial for initiating the death signaling via the
Fas-mediated pathway. The observation that antioxidants effectively
inhibit the induction of death mediators and the subsequent death
process provokes a speculation that the potential benefit of
antioxidant therapy in atherosclerosis may attribute in part to the
protection of vascular cells from apoptotic death in the plaques.
 |
ACKNOWLEDGEMENTS |
This study was supported by Grant NSC-88-2316-B-001-011-M26
from the National Science Council of Taiwan.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: L.-Y.
Chau, Div. of Cardiovascular Research, Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of
China (E-mail: lyc{at}mail.ibms.sinica.edu.tw).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 April 2000; accepted in final form 16 October 2000.
 |
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