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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, 7beta -hydroxycholesterol and 25-hydroxycholesterol, were also effective to induce the expression of death mediators and apoptosis. alpha -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta -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. 7beta -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

Oxysterol-induced apoptosis of SMCs. It has been shown that oxysterols, including 7beta -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 7beta -hydroxycholesterol and 25-hydroxycholesterol on the expression levels of p53, Fas, and FasL were examined. As shown in Fig. 7A, 25-hydroxycholesterol, but not 7beta -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 7beta -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 alpha -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 7beta -hydroxycholesterol, or 25 µg/ml 25-hydroxycholesterol in the absence or presence of 100 µM alpha -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.

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 alpha -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 alpha -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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 7beta - 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, 7beta -hydroxycholesterol induces Fas/FasL expression without much effect on p53. Whether the differential effect on p53 induction accounts for the lower potency of 7beta -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 alpha -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)-kappa B activation (29). Previous studies by some investigators demonstrated that the NF-kappa B activation is essential for the proliferation of SMCs (3, 28). Recently, a report by Erl et al. (11) showed that increased NF-kappa B activity protects SMCs from apoptosis. We speculate that the failure of NAC to inhibit apoptosis may be associated with its effect on NF-kappa 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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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