© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Vitamin C Protects Human Vascular Smooth Muscle Cells Against Apoptosis Induced by Moderately Oxidized LDL Containing High Levels of Lipid Hydroperoxides
From the Centre for Cardiovascular Biology and Medicine, GKT School of Biomedical Sciences, King's College, London (R.C.M.S., K.C.P., G.E.M.), and Cell and Molecular Biology Research Division, School of Animal and Microbial Sciences, University of Reading, Reading (J.P.R., D.S.L.), UK.
Correspondence to Dr Richard Siow, Division of Cardiovascular Medicine, Department of Medicine, School of Clinical Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK. E-mail rcms2{at}cam.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Vascular cell death is a key feature of atherosclerotic lesions and may contribute to the plaque "necrotic" core, cap rupture, and thrombosis. Oxidatively modified low-density lipoproteins (LDLs) are implicated in the pathogenesis of atherosclerosis, and dietary antioxidants are thought to protect the vasculature against LDL-induced cytotoxicity. Because LDL oxidative modification may vary within atherosclerotic lesions, we examined the effects of defined, oxidatively modified LDL species on human arterial smooth muscle cell apoptosis and the cytoprotective effects of vitamin C. Moderately oxidized LDL (0 to 300 µg protein/mL), which has the highest content of lipid hydroperoxides, induced smooth muscle cell apoptosis within 6 hours, whereas native LDL and mildly and highly oxidized LDL had no effect. Moderately oxidized LDL increased cellular DNA fragmentation, release of fragmented DNA into the culture medium, and annexin V binding and decreased mitochondrial dehydrogenase activity and expression of the antiapoptotic mediator Bcl-xL. Treatment of cells with native LDL together with the lipid hydroperoxide 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (HPODE, 200 µmol/L, 6 to 24 hours) also induced apoptotic cell death. Pretreatment of smooth muscle cells with vitamin C (0 to 100 µmol/L, 24 hours) attenuated the cytotoxicity and apoptosis induced by both moderately oxidized LDL and HPODE. Our findings suggest that moderately oxidized LDL, with its high lipid hydroperoxide content, rather than mildly or highly oxidized LDL, causes apoptosis of human smooth muscle cells and that vitamin C supplementation may provide protection against plaque instability in advanced atherosclerosis.
Key Words: smooth muscle, vascular • low-density lipoprotein, oxidized • apoptosis • ascorbic acid • Bcl-xL • atherosclerosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Oxidatively modified LDLs are involved in the initiation and progression of atherosclerosis and have been localized within atherosclerotic lesions.1 2 3 4 5 Evidence that oxidized LDL induces vascular cell death by apoptosis6 7 8 and the presence of apoptotic cells in atherosclerotic and restenotic lesions7 9 10 has potential clinical implications for atherogenesis and thrombosis. Although smooth muscle cell death may enhance lesion regression,11 it can also result in the accumulation of necrotic debris within the lesion core and instability of the fibrous plaque cap, leading to its rupture and thrombosis.10 The extent to which LDL becomes oxidized within atherosclerotic lesions has not been well defined, and levels of oxidation may differ within lesions. Because the toxicity of oxidized LDL appears to increase with the degree of oxidative modification,12 13 this may contribute to the impaired vascular reactivity in patients with coronary heart disease.14
Oxidized LDL is cytotoxic to smooth muscle cells,12 15 endothelial cells,12 16 17 and macrophages18 and has been shown to induce apoptosis in several vascular cell types.6 7 8 19 Although a mildly oxidized species of LDL (moxLDL) has been reported to induce endothelial cell toxicity and apoptosis,20 only limited information is available on the effects of moderately oxidized LDL (modLDL), which contains the highest lipid hydroperoxide levels. Dietary antioxidants attenuate the cytotoxic effects of oxidatively modified LDL6 16 20 21 and restore endothelial function in patients with coronary artery disease.22 Moreover, a low plasma vitamin C concentration is implicated as an independent predictor of unstable coronary artery disease.23 In this context, we previously reported that vitamin C protects human vascular smooth muscle cells against oxidized LDL–induced adaptive increases in the synthesis of the key intracellular antioxidant glutathione.24 These results provided the first evidence that vitamin C spares endogenous antioxidant responses in human vascular smooth muscle cells exposed to atherogenic lipoproteins.
In this study, we investigated further the cytoprotective effects of vitamin C by examining LDL-induced apoptosis of human smooth muscle cells. modLDL, which contains the peak level of lipid hydroperoxides, but not highly oxidized LDL (oxLDL), caused smooth muscle cell apoptosis within 6 hours; this apoptosis was attenuated markedly by pretreatment with vitamin C. In addition, cells treated with native LDL (nLDL) and the lipid hydroperoxide 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (HPODE) also underwent apoptosis, which was attenuated by vitamin C. Our findings may have important clinical implications for therapies designed to arrest LDL oxidation, because modLDL appears to be potentially more atherogenic than oxLDL. Moreover, vitamin C supplementation may provide protection against the cytotoxic effects of modLDL and lipid hydroperoxides.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Reagents
Smooth muscle cell culture medium (MCDB 131) was obtained from Clonetics; cellular DNA fragmentation ELISA kit, from Boehringer Mannheim; ApoAlert annexin V apoptosis kit, from Clontech; HPODE, from Biomol Research Laboratories; Bcl-xL polyclonal antibody, from Transduction Laboratories; and the enhanced chemiluminescence kit, from Amersham. All other reagents were of the highest analytical grade and were obtained from Sigma Chemical Co.
Isolation and Oxidation of LDL
LDL was isolated from the blood of healthy volunteers by
sequential density ultracentrifugation (1.019 to
1.063g/mL) in the presence of EDTA.25 nLDL was diluted to
100 µg protein/mL in sterile PBS. nLDL was oxidized with
CuSO4 (5 µmol/L net above the EDTA carried
over from the LDL) at 37°C, and the conjugated diene content was
determined every 15 minutes by removing an aliquot, measuring its
absorbance at 234 nm (A234), and then returning
it to the LDL sample. When A234 increased by 0.2
above the initial value after 80 to 100 minutes, moxLDL was removed,
1 mmol/L EDTA was added to stop oxidation, and the sample was
stored at 4°C. The remaining LDL was further oxidized for 
3 hours
until A234 reached a peak (an increase of 1.5 to
1.9). We defined this as modLDL, which contains the peak levels of
lipid hydroperoxides.26 The
level of conjugated dienes closely follows the level of lipid
hydroperoxides during the early stages of LDL oxidation.27
A portion was removed, 1 mmol/L EDTA was added, and the sample was
stored at 4°C. The remaining LDL was oxidized at 37°C for 24 hours
before EDTA (1 mmol/L) was added to obtain what we define as
oxLDL. During this time, the lipid hydroperoxides fall, but the later
secondary products of lipid and protein oxidation
increase.26 Solid KBr was then dissolved in the oxidized
LDLs to increase their densities to 1.2 g/mL. Chelex-100 was added as
an extra precaution to inactivate transition metal ions.
Once the KBr had dissolved, the Chelex-100 was removed by
centrifugation at 250g for 10 minutes at
4°C. The oxidized LDL species were then centrifuged at
149 000g for 18 hours at 4°C to concentrate them. The
oxidized LDL layer was removed and dialyzed overnight at 4°C in the
dark against four 1-L volumes of phosphate buffer (140 mmol/L
NaCl, 1.9 mmol/L
NaH2HPO4, 8.1 mmol/L
Na2HPO4, and 100
µmol/L EDTA, pH 7.4). The oxidized LDL species were then
filter-sterilized and stored at 4°C. LDL protein content was
determined by a modified Lowry assay; lipid hydroperoxide content was
determined by an iodometric method28 ; and relative
electrophoretic mobility was compared with nLDL, as an index of protein
modification, using Beckman Paragon Lipo gels. The mobility of the LDL
increased with the degree of oxidative modification, whereas the lipid
hydroperoxide content was maximal in modLDL and significantly lower in
moxLDL and oxLDL (see Table
).
|
Culture of Vascular Smooth Muscle Cells
Human arterial smooth muscle cells (HUASMCs) were
cultured from umbilical artery explants in MCDB131 medium supplemented
with 10% (vol/vol) FCS, penicillin (100 U/mL), and streptomycin (100
µg/mL) at 37°C in a 5% CO2/95% air
atmosphere. Cells were confirmed as smooth muscle by their typical
"hill-and-valley" morphological features and by
immunofluorescent staining for smooth muscle 
-actin. HUASMCs
between passages 3 and 7 were used in all experiments. Confluent,
randomly cycling cells were incubated in the absence or presence of up
to 300 µg protein/mL nLDL, moxLDL, modLDL, or oxLDL for 3 to 24 hours
or nLDL in combination with HPODE (200 µmol/L) for 6 or 24
hours. Exposure to LDL was terminated by gently washing the cells twice
with warmed PBS.
In experiments examining the effects of vitamin C, cells were pretreated with vitamin C (0 to 100 µmol/L) for 24 hours in serum-containing medium, monolayers were washed with warmed PBS, and cells were reincubated for an additional 3 to 24 hours in complete medium containing either defined LDL species or nLDL in combination with HPODE but in the absence of vitamin C.
Measurements of Cytotoxicity
Mitochondrial dehydrogenase activity was used as an index of
cell viability and was assessed using the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
assay.29 Confluent HUASMCs were pretreated in the absence
or presence of vitamin C (100 µmol/L, 24 hours), washed with
PBS, and then incubated with defined LDL species (3 to 24 hours) in the
absence of vitamin C. Culture medium was then replaced with fresh
medium containing MTT (500 µg/mL), and cells were incubated at 37°C
and 5% CO2 for an additional 1 to 2 hours. DMSO
was then added to each well, and the microtiter plates were incubated
at room temperature for another 20 minutes before absorbance was
measured at 540 nm.
Plasma membrane damage in HUASMCs was also assessed by monitoring the leakage of DNA fragments into the culture medium after treatment of confluent HUASMC monolayers with oxidized LDL for defined time periods. Proliferating cells in 96-well microtiter plates were labeled with 10 µmol/L 5-bromo-2'-deoxyuridine (BrdU) for 24 hours, washed with PBS, and treated with LDL in the absence of BrdU for defined time periods. The culture medium was then collected, and labeled DNA fragments released by the cells were assayed using a DNA fragmentation ELISA (see below). Levels of fragmented DNA were expressed as absorbance at 450 nm.
Measurements of Apoptosis
DNA Fragmentation
As described previously,30 cellular DNA
fragmentation was measured using a commercially available cellular DNA
fragmentation ELISA kit. Proliferating cells in 96-well microtiter
plates were labeled with 10 µmol/L BrdU for 24 hours, washed
with PBS, and treated in the absence of BrdU with nLDL or oxidized LDL
for defined time periods. Cells were then washed with PBS and incubated
with the kit lysis buffer (BSA, EDTA, and Tween 20) for 30 minutes at
room temperature, and soluble BrdU-labeled DNA fragments present in
the buffer were quantified using the ELISA. Briefly, an anti-DNA
antibody was adsorbed onto the wells of a microtiter plate, and lysis
buffer supernatants containing BrdU-labeled DNA fragments were allowed
to bind the immobilized anti-DNA antibody. After washing,
the immunocomplexed BrdU-labeled DNA fragments were denatured and fixed
to the surface of the microtiter plate by microwave irradiation.
Labeled fragments were detected using an anti-BrdU antibody conjugated
to horseradish peroxidase. The amount of peroxidase bound to the immune
complex was determined by addition of tetramethyl benzidine substrate,
and DNA fragmentation was expressed as absorbance at 450 nm.
Annexin V Binding
Active externalization of phosphatidylserine
to the cell surface is an early event in apoptotic cell death,
and annexin V binding to phosphatidylserine can be
used as a marker of early apoptotic events that precede nuclear
changes.31 In this study, binding of FITC-conjugated
annexin V to HUASMCs treated with nLDL, oxidized LDL, or nLDL combined
with HPODE was assessed using a commercially available kit (ApoAlert
annexin V, Clontech). Cells were also costained with propidium iodide
as a marker of cell membrane permeability, which occurs during the
later stages of apoptosis and in necrosis. Cells were washed
with PBS and incubated with FITC-annexin V (0.5 µg/mL) or propidium
iodide (0.125 µg/mL). Staining was determined by fluorescence
microscopy using dual-filter sets for FITC and rhodamine to visualize
annexin V binding and nuclear propidium iodide staining,
respectively.
Bcl-xL Protein Expression
Changes in protein expression of the antiapoptotic
mediator Bcl-xL32 were determined by
Western blot analysis in cells treated with LDL. In brief,
confluent HUASMCs were treated with oxidized LDL for defined periods,
and incubations were terminated by washing cells with ice-cold PBS.
Cells were lysed in buffer (2% wt/vol SDS, 10% vol/vol glycerol,
50 mmol/L Tris-HCl, and 1 mmol/L PMSF, pH 6.8), and the
lysate was heated at 100°C for 5 minutes. Sample protein content was
determined, and samples were heated at 100°C in a mixture of 1%
2-mercaptoethanol and 0.05% bromophenol blue for an additional 3
minutes. Equal protein amounts were separated by
SDS-polyacrylamide gel electrophoresis, transferred to a
polyvinylidine difluoride membrane, and probed with a
polyclonal rabbit anti-rat Bcl-xL antibody. A
horseradish peroxidase–conjugated goat anti-rabbit secondary antibody
was used in conjunction with enhanced chemiluminescence to visualize
the 26-kDa Bcl-xL bands on
autoradiographic film, and the level of expression was
quantified using image analysis software.
Statistical Analysis
Statistical variance from the mean was determined using the
normal distribution and represented by mean±SE (n=number
of different cell cultures). When 2 values were compared, confidence
limits were established using the unpaired Student's t
test. ANOVA was used to compare multiple groups. Statistical
significance between data sets was established at
P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
LDL-Mediated Cytotoxicity
The activity of mitochondrial dehydrogenases (MTT assay) was significantly decreased after incubation of HUASMCs with modLDL (300 µg protein/mL) for 24 hours, whereas nLDL and moxLDL had negligible effects and oxLDL increased mitochondrial dehydrogenase activity above basal levels (Figure 1
). The
decrease in MTT activity induced by modLDL was time-dependent (0 to 24
hours) and concentration-dependent (0 to 300 µg protein/mL) (Figure 2A and 2B). Pretreatment of cells with
vitamin C (100 µmol/L, 24 hours) before LDL exposure abolished
the changes in MTT activity induced by modLDL and oxLDL (Figure 1), with maximal effects observed at vitamin C concentrations
>50 µmol/L (Figure 2C). Plasma membrane permeability,
assessed by release of fragmented DNA into the culture medium, was
increased in HUASMCs treated with modLDL (300 µg protein/mL, 24
hours) but not in those treated with nLDL, moxLDL, or oxLDL (Figure 3A). This release of DNA induced by
modLDL was abolished after preincubation of cells with vitamin C
(100 µmol/L, 24 hours) (Figure 3A).
|
) or presence (
) of vitamin C (100 µmol/L, 24
hours), cells were exposed to either nLDL, moxLDL, modLDL, or oxLDL
(300 µg protein/mL) for an additional 24 hours in the absence of
vitamin C. Mitochondrial dehydrogenase activity (MTT) was assayed as an
index of cell viability. Values are mean±SE (n=3 to 6 cell cultures).
*P<0.05 relative to control cells in the absence of
vitamin C.
|
) or modLDL (•). C, Effects
of vitamin C (0 to 100 µmol/L, 24 hours) pretreatment on
nLDL-induced (
|
LDL- and HPODE-Mediated Apoptosis
modLDL (300 µg protein/mL, 24 hours) caused an increase in
cellular DNA fragmentation (Figure 3B
) that was both
time-dependent (Figure 4A) and
concentration-dependent (Figure 4B). nLDL, moxLDL, and oxLDL had
negligible effects on cellular DNA fragmentation (Figure 3B).
Pretreatment of cells with vitamin C for 24 hours attenuated DNA
fragmentation induced by modLDL (Figure 3B), with vitamin C
providing protection at concentrations >50 µmol/L (Figure 4C). Binding of annexin V, a marker of early apoptosis,
was observed in cells treated for 6 or 24 hours with modLDL (300 µg
protein/mL) (Figure 5A and 5C), whereas
no binding was detected in HUASMCs treated with nLDL, moxLDL, or oxLDL
(300 µg protein/mL) (data not shown). Moreover, cells treated with
modLDL for 24 hours, but not for 6 hours, were permeable to propidium
iodide (Figure 5C), suggesting that cells progressed to a late
stage of apoptosis after prolonged exposure to modLDL. Vitamin
C pretreatment (100 µmol/L, 24 hours) abolished the early (6
hours) annexin V binding (Figure 5B) and attenuated propidium
iodide staining (Figure 5D) in cells exposed to modLDL for 24
hours. Moreover, binding of annexin V was also observed when smooth
muscle cells were treated for 6 or 24 hours with nLDL (300 µg
protein/mL) together with 200 µmol/L HPODE (Figure 6A and 6C), a level comparable to the
lipid hydroperoxide content in 300 µg protein/mL modLDL. In addition,
cells treated with nLDL and HPODE for 24 hours, but not for 6 hours,
were also permeable to propidium iodide (Figure 6C), indicating
late-stage apoptosis. Pretreatment with vitamin C (100
µmol/L, 24 hours) abolished the early (6 hours) annexin V binding
(Figure 6B) and propidium iodide staining (Figure 6D) in
cells exposed to HPODE for 24 hours.
|
|
|
Bcl-xL Expression
Basal expression of the antiapoptotic mediator
Bcl-xL was unaltered in HUASMCs treated for up to
24 hours with nLDL, moxLDL, or oxLDL (300 µg protein/mL, data not
shown), whereas treatment with modLDL for 24 hours significantly
decreased Bcl-xL expression (Figure 7A and 7B). Pretreatment of cells with
vitamin C (100 µmol/L, 24 hours) prevented the decrease in
Bcl-xL expression induced by modLDL and appeared
to slightly elevate Bcl-xL expression, although
this increase was not significant. The lower-molecular-weight band
observed may reflect nonspecific cross-reactivity of the antibody with
the closely related Bcl-xS protein, but this was
not investigated further.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This study established that modLDL and the lipid hydroperoxide HPODE, but neither nLDL, moxLDL, nor oxLDL, induced apoptosis of human vascular smooth muscle cells. Our findings suggest that the high lipid hydroperoxide content of modLDL is more cytotoxic to smooth muscle cells than secondary lipid oxidation products, such as aldehydes, ketones, and lipid alcohols, generated during extended copper oxidation of LDL.27 33 Moreover, pretreatment of cells with physiologically relevant vitamin C concentrations attenuated the cytotoxicity induced by modLDL and HPODE. The protection against smooth muscle cell apoptosis provided by vitamin C may reflect that this dietary antioxidant maintained cellular levels of the antiapoptotic mediator Bcl-xL.32
Increased apoptotic cell death within atherosclerotic lesions
and its contribution to plaque rupture and accumulation of the necrotic
lipid core has been established.7 34 LDL is oxidized
within lesions35 and contributes to the impaired vascular
cell function observed in atherogenesis.3 4 Although the
toxicity of LDL increases with the extent of LDL
oxidation,13 the methods used to oxidize LDL vary
considerably between different laboratories and are often not well
defined.36 In this study, we used a well-defined protocol
for LDL oxidation24 36 to obtain mildly, moderately, and
highly oxidized LDL species. The method relies on oxidizing a low
concentration of LDL (100 µg protein/mL) with copper; the kinetics of
LDL oxidation under these conditions have been characterized in detail
elsewhere27 37 (see Table). In agreement with our
observations in human smooth muscle cells, Kuzuya et al38
reported that the toxicity of LDL to endothelial cells
initially increases and then decreases with time of LDL oxidation. The
enhanced mitochondrial enzyme activity observed in human smooth muscle
cells treated with oxLDL may have arisen from increased mitochondrial
metabolism of oxysterols present in higher amounts in
oxLDL.39 Although some studies have reported that oxidized
LDL enhances smooth muscle cell proliferation,13 17 this
seems an unlikely explanation for our findings because protein
concentrations were similar in cell monolayers treated with the
different LDL species.
Recent studies have identified the presence of epitopes of LDL, oxidized by copper to increasing extents, within animal and human atherosclerotic lesions and in circulating plasma LDL.40 41 Chisholm et al42 proposed that the cytotoxicity of copper-oxidized LDL to cells is mainly due to cholesterol hydroperoxides, which may be more cytotoxic than hydroxycholesterols or ketocholesterols. We showed that oxLDL, which contains a higher level of hydroxycholesterols and ketocholesterols than modLDL,33 does not induce human smooth muscle cell apoptosis. Our findings contrast with those of Nishio and Watanabe43 and Harada-Shiba et al,8 who reported that oxidized LDL species induced smooth muscle and endothelial cell apoptosis. This discrepancy may reflect the differences in the degree of LDL oxidation and the criteria used to define oxidized LDL. Nevertheless, it is interesting that 7-ketocholesterol and 25-hydroxycholesterol have been shown to induce endothelial and smooth muscle cell apoptosis.8 43 On the basis of the results of our study, using standardized methods for oxidizing LDL,36 we conclude that cholesterol, cholesteryl esters, or phospholipids with a hydroperoxyl group may be more cytotoxic to human smooth muscle cells than those with a hydroxyl or ketone group. Although studies in rat pheochromocytoma cells44 and human T cell lines45 have demonstrated that lipid hydroperoxides can induce apoptotic cell death, similar studies in vascular smooth muscle cells are lacking. To our knowledge, our experiments provide the first evidence that HPODE can induce apoptosis of human smooth muscle cells.
Apoptosis of vascular endothelial and smooth
muscle cells induced by oxidized LDL species involves activation of
caspase-like proteases6 43 and downregulation of the
antiapoptotic protein Bcl-2.43 46 The latter
protein is known to act as an endogenous antioxidant,
inhibiting lipid peroxidation in apoptosis.47 We
here report the first evidence that modLDL, in contrast to moxLDL and
oxLDL, decreases expression of the closely related
antiapoptotic mediator
Bcl-xL32 in human smooth muscle
cells (Figure 7). These findings are consistent with the
observed apoptosis of smooth muscle cells induced by modLDL
(Figures 2, 3, and 4). Vitamin C pretreatment abolished the
decrease in Bcl-xL protein levels and markedly
inhibited cellular DNA fragmentation and annexin V binding to smooth
muscle cells treated with modLDL. Our results thus provide further
mechanistic evidence of the cytoprotective effects of vitamin C against
oxidized LDL–induced smooth muscle dysfunction in atherogenesis.
Dimmeler et al6 previously reported that a combination of
vitamins C and E prevented the endothelial cell
apoptosis induced by oxidized LDL by inhibiting CPP32
activation. Moreover, these vitamins also prevent
endothelial cell apoptosis induced by
lipopolysaccharide by preserving Bcl-2 protein
levels.48 Thus, vitamin C could protect against
apoptosis induced by oxidative stress and lipid hydroperoxides
by modulating mediators of apoptosis. It was recently shown
that enhanced expression of Bcl-xL protects
against atherosclerosis and endothelial
cell activation,49 so maintained or enhanced expression of
antiapoptotic genes by vitamin C supplementation may be
protective in atherogenesis.
The extent of LDL oxidation within atherosclerotic lesions remains a controversial issue, yet our findings suggest that protein modifications associated with oxLDL are not necessarily required for LDL-induced cytotoxicity. Although antioxidant dietary vitamins protect against vascular dysfunction and plaque instability22 23 and reduce the oxidative modification of LDL,26 it is also possible that LDL modification may be arrested at a more atherogenic "moderately oxidized" level by vitamin supplementation. Thus, modLDL could induce more smooth muscle cells within the lesion to undergo apoptosis than oxLDL. Prolonged dietary antioxidant supplementation may possibly have different effects on advanced atherosclerotic lesions, because initially antioxidants would inhibit oxidative modification of LDL in the arterial wall and limit the formation of lesions. It is beyond the scope of this study to determine whether antioxidants such as vitamin C are proatherogenic through the arrest of LDL oxidation and inhibition of neointimal smooth muscle cell apoptosis. Our study supports the current consensus that antioxidant consumption reduces coronary heart disease,50 although additional clinical trials seem warranted to establish the efficacy of vitamin C against accumulation of necrotic debris within the lesion core and instability of the fibrous plaque in atherosclerosis.
|
Acknowledgments |
|---|
This work was supported in part by the British Heart Foundation (PG95148), Ministry of Agriculture Fisheries and Food (ANO413), and a Wellcome Trust Research Leave Fellowship (D.S.L.). We thank Dr John Murphy for helpful comments on the manuscript and for providing the Bcl-xL antibody, Dr V. Moens for taking blood samples, and Dr E. Ruiz for assistance with some of the experiments.
Received February 9, 1998; accepted October 3, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low-density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086–1095.
2. Witztum JL, Steinberg D. Role of oxidized low-density-lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785–1792.
3. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
4.
Steinberg D. Low density lipoprotein oxidation and its
pathobiological significance. J Biol Chem. 1997;272:20963–20966.
5.
Hajjar DP, Haberland ME. Lipoprotein trafficking in
vascular cells: molecular Trojan horses and cellular saboteurs.
J Biol Chem. 1997;272:22975–22978.
6.
Dimmeler S, Haendeler J, Galle J, Zeiher AM. Oxidized
low-density lipoprotein induces apoptosis of human
endothelial cells by activation of CPP32-like
proteases: a mechanistic clue to the `response to injury' hypothesis.
Circulation. 1997;95:1760–1763.
7.
Jovinge S, Crisby M, Thyberg J, Nilsson J. DNA
fragmentation and ultrastructural changes of degenerating cells in
atherosclerotic lesions and smooth muscle cells exposed to oxidized LDL
in vitro. Arterioscler Thromb Vasc Biol. 1997;17:2225–2231.
8.
Harada-Shiba M, Kinoshita M, Kamido H, Shimokado K.
Oxidized low density lipoprotein induces apoptosis in cultured
human umbilical vein endothelial cells by common and
unique mechanisms. J Biol Chem. 1998;273:9681–9687.
9. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest. 1995;95:2266–2274.
10. Bjorkerud S, Bjorkerud B. Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. Am J Pathol. 1996;149:367–380.[Abstract]
11. Pollman MJ, Hall JL, Mann MJ, Zhang LN, Gibbons GH. Inhibition of neointimal cell bcl-x expression induces apoptosis and regression of vascular disease. Nature Med. 1998;4:222–227.[Medline] [Order article via Infotrieve]
12.
Thorne SA, Abbot SE, Winyard PG, Blake DR, Mills PG.
Extent of oxidative modification of low-density-lipoprotein determines
the degree of cytotoxicity to human coronary-artery cells.
Heart. 1996;75:11–16.
13.
Bjorkerud B, Bjorkerud S. Contrary effects of lightly
and strongly oxidized LDL with potent promotion of growth versus
apoptosis on arterial smooth muscle cells,
macrophages, and fibroblasts. Arterioscler Thromb Vasc
Biol. 1996;16:416–424.
14. Cox DA, Cohen ML. Effects of oxidized low-density lipoprotein on vascular contraction and relaxation: clinical and pharmacological implications in atherosclerosis. Pharmacol Rev. 1996;48:3–19.[Abstract]
15. Chatterjee S. Role of oxidized human plasma low-density lipoproteins in atherosclerosis: effects on smooth-muscle cell-proliferation. Mol Cell Biochem. 1992;111:143–147.[Medline] [Order article via Infotrieve]
16. Schmitt A, Salvayre R, Delchambre J, Negre-Salvayre A. Prevention by alpha-tocopherol and rutin of glutathione and ATP depletion induced by oxidized LDL in cultured endothelial cells. Br J Pharmacol. 1995;116:1985–1990.[Medline] [Order article via Infotrieve]
17. Auge N, Pieraggi MT, Thiers JC, Negresalvayre A, Salvayre R. Proliferative and cytotoxic effects of mildly oxidized low-density lipoproteins on vascular smooth-muscle cells. Biochem J. 1995;309:1015–1020.
18. Marchant CE, Law NS, Vanderveen C, Hardwick SJ, Carpenter KLH, Mitchinson MJ. Oxidized low-density lipoprotein is cytotoxic to human monocyte-macrophages: protection with lipophilic antioxidants. FEBS Lett. 1995;358:175–178.[Medline] [Order article via Infotrieve]
19. Nishio E, Arimura S, Watanabe Y. Oxidized LDL induces apoptosis in cultured smooth muscle cells: a possible role for 7-ketocholesterol. Biochem Biophys Res Commun. 1996;223:413–418.[Medline] [Order article via Infotrieve]
20. Vieira O, Escargueil-Blanc I, Meilhac O, Basile JP, Laranjinha J, Almeida L, Salvayre R, Negre-Salvayre A. Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL. Br J Pharmacol. 1998;123:565–573.[Medline] [Order article via Infotrieve]
21. Guyton JR, Lenz ML, Mathews B, Hughes H, Karsan D, Selinger E, Smith CV. Toxicity of oxidized low-density lipoproteins for vascular smooth muscle cells and partial protection by antioxidants. Atherosclerosis. 1995;118:237–249.[Medline] [Order article via Infotrieve]
22.
Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JF
Jr, Vita JA. Ascorbic acid reverses endothelial
vasomotor dysfunction in patients with coronary artery disease.
Circulation. 1996;93:1107–1113.
23.
Vita JA, Keaney JF, Raby KE, Morrow JD, Freedman JE,
Lynch S, Koulouris SN, Hankin BR, Frei B. Low plasma ascorbic acid
independently predicts the presence of an unstable coronary
syndrome. J Am Coll Cardiol. 1998;31:980–986.
24.
Siow RCM, Sato H, Bannai S, Leake DS, Pearson JD, Mann
GE. Vitamin C protects human arterial smooth muscle cells
against atherogenic lipoproteins: effects of antioxidant vitamins on
oxidized LDL-induced increases in cystine transport and glutathione.
Arterioscler Thromb Vasc Biol. 1998;18:1662–1670.
25. Wilkins GM, Leake DS. The effect of inhibitors of free radical generating-enzymes on low density lipoprotein oxidation by macrophages. Biochim Biophys Acta.. 1994;1211:69–78.[Medline] [Order article via Infotrieve]
26. Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med. 1992;13:341–390.[Medline] [Order article via Infotrieve]
27. Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low-density lipoprotein. Free Radic Res Commun. 1989;6:67–75.[Medline] [Order article via Infotrieve]
28. El-Saadani M, Esterbauer H, El-Sayed M, Goher M, Nassar AY, Jurgens GA. Spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res. 1989;30:627–630.[Abstract]
29. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival: modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986;89:271–277.[Medline] [Order article via Infotrieve]
30. Ortiz EM, Dusetti NJ, Vasseur S, Malka D, Bödeker H, Dagorn JC, Iovanna JL. The pancreatitis-associated protein is induced by free radicals in AR4–2J cells and confers cell resistance to apoptosis. Gastroenterology.. 1998;114:808–816.[Medline] [Order article via Infotrieve]
31.
Martin SJ, Reutelingsperger CPM, McGahon AJ, Rader JA,
Vanschie RC, LaFace DM, Green DR. Early redistribution of
plasma-membrane phosphatidylserine is a general
feature of apoptosis regardless of the initiating stimulus:
inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995;182:1545–1556.
32.
Minn AJ, Boise LH, Thompson CB.
Bcl-xS antagonizes the protective effects of
Bcl-xL. J Biol Chem. 1996;271:6306–6312.
33. Carpenter KLH, Wilkins GM, Fussell B, Ballantine JA, Taylor SE, Mitchinson MJ, Leake DS. Production of oxidized lipids during modification of low-density lipoprotein by macrophages or copper. Biochem J. 1994;304:625–633.
34. Mitchinson MJ, Hardwick SJ, Bennett MR. Cell death in atherosclerotic plaques. Curr Opin Lipidol. 1996;7:324–329.[Medline] [Order article via Infotrieve]
35.
Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC,
Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum
JL. Low-density lipoprotein undergoes oxidative modification in vivo.
Proc Natl Acad Sci U S A. 1989;86:1372–1376.
36. Rice-Evans CA, Leake DS, Bruckdorfer KR, Diplock AT. Practical approaches to low density lipoprotein oxidation: whys, wherefores and pitfalls. Free Radic Res. 1996;25:285–311.[Medline] [Order article via Infotrieve]
37.
Jessup W, Rankin SM, de Whalley CV, Hoult JRS, Scott J,
Leake DS. -Tocopherol consumption during low
density lipoprotein oxidation. Biochem J. 1990;265:399–405.[Medline]
[Order article via Infotrieve]
38. Kuzuya M, Naito M, Funaki C, Hayashi T, Asai K, Kuzuya F. Lipid peroxide and transition metals are required for the toxicity of oxidized low-density lipoprotein to cultured endothelial cells. Biochim Biophys Acta. 1991;1096:155–161.[Medline] [Order article via Infotrieve]
39.
Axelson M, Larsson O. Low density lipoprotein (LDL)
cholesterol is converted to
27-hydroxycholesterol in human fibroblasts: evidence that
27-hydroxycholesterol can be an important intracellular
mediator between LDL and the suppression of cholesterol
production. J Biol Chem. 1995;270:15102–15110.
40.
Yla-Herttuala S, Palinski W, Butler SW, Picard S,
Steinberg D, Witztum JL. Rabbit and human atherosclerotic lesions
contain IgG that recognizes epitopes of oxidized LDL.
Arterioscler Thromb. 1994;14:32–40.
41. Palinski W, Horkko S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK, Witztum JL. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice: demonstration of epitopes of oxidized low-density lipoprotein in human plasma. J Clin Invest. 1996;98:800–814.[Medline] [Order article via Infotrieve]
42.
Chisolm GM, Ma GP, Irwin KC, Martin LL, Gunderson KG,
Linberg LF, Morel DW, Dicorleto PE.
7-Beta-hydroperoxycholest-5-en-3-beta-ol, a component of human
atherosclerotic lesions, is the primary cytotoxin of oxidized human
low-density lipoprotein. Proc Natl Acad Sci U S A. 1994;91:11452–11456.
43. Nishio E, Watanabe Y. Oxysterols induced apoptosis in cultured smooth muscle cells through CPP32 protease activation and bcl-2 protein downregulation. Biochem Biophys Res Commun. 1996;226:928–934.[Medline] [Order article via Infotrieve]
44. Aoshima H, Satoh T, Sakai N, Yamada M, Enokido Y, Ikeuchi T, Hatanaka H. Generation of free radicals during lipid hydroperoxide-triggered apoptosis in PC12h cells. Biochim Biophys Acta. 1997;1345:35–42.[Medline] [Order article via Infotrieve]
45. Sandstrom PA, Pardi D, Tebbey PW, Dudek RW, Terrian DM, Folks T, Buttke TM. Lipid hydroperoxide-induced apoptosis: lack of inhibition by Bcl-2 overexpression. FEBS Lett. 1995;365:66–70.[Medline] [Order article via Infotrieve]
46. Harada K, Ishibashi S, Miyashita T, Osuga J, Yagyu H, Ohashi K, Yazaki Y, Yamada N. Bcl-2 protein inhibits oxysterol-induced apoptosis through suppressing CPP32-mediated pathway. FEBS Lett. 1997;411:63–66.[Medline] [Order article via Infotrieve]
47. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993;75:241–251.[Medline] [Order article via Infotrieve]
48. Haendeler J, Zeiher AM, Dimmeler S. Vitamin C and E prevent lipopolysaccharide-induced apoptosis in human endothelial cells by modulation of Bcl-2 and Bax. Eur J Pharmacol. 1996;317:407–411.[Medline] [Order article via Infotrieve]
49. Hancock WW, Buelow R, Sayegh MH, Turka LA. Antibody-induced transplant atherosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes. Nature Med. 1998;4:1392–1396.[Medline] [Order article via Infotrieve]
50.
Tribble DL. Antioxidant consumption and risk of
coronary heart disease: emphasis on vitamin C, vitamin E,
and ß-carotene. Circulation. 1999;99:591–595.
This article has been cited by other articles:
 |
|
 |
|
  A. B. Gerry and D. S. Leake A moderate reduction in extracellular pH protects macrophages against apoptosis induced by oxidized low density lipoprotein J. Lipid Res., April 1, 2008; 49(4): 782 - 789. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-P. Hsu, M.-S. Wu, C.-C. Yang, K.-C. Huang, S.-Y. Liou, S.-M. Hsu, and C.-T. Chien Chronic green tea extract supplementation reduces hemodialysis-enhanced production of hydrogen peroxide and hypochlorous acid, atherosclerotic factors, and proinflammatory cytokines Am. J. Clinical Nutrition, November 1, 2007; 86(5): 1539 - 1547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li and H. E. Schellhorn New Developments and Novel Therapeutic Perspectives for Vitamin C J. Nutr., October 1, 2007; 137(10): 2171 - 2184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Zorn-Pauly, P. Schaffer, B. Pelzmann, E. Bernhart, G. Wei, P. Lang, G. Ledinski, J. Greilberger, B. Koidl, and G. Jurgens Oxidized LDL induces ventricular myocyte damage and abnormal electrical activity-role of lipid hydroperoxides Cardiovasc Res, April 1, 2005; 66(1): 74 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Rosenson-Schloss, E. Chnari, T. A. Brieva, A. Dang, and P. V. Moghe Glutathione Preconditioning Attenuates Ac-LDL-Induced Macrophage Apoptosis via Protein Kinase C-Dependent Ac-LDL Trafficking Experimental Biology and Medicine, January 1, 2005; 230(1): 40 - 48. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-T. Chien, W.-T. Chang, H.-W. Chen, T.-D. Wang, S.-Y. Liou, T.-J. Chen, Y.-L. Chang, Y.-T. Lee, and S.-M. Hsu Ascorbate Supplement Reduces Oxidative Stress in Dyslipidemic Patients Undergoing Apheresis Arterioscler. Thromb. Vasc. Biol., June 1, 2004; 24(6): 1111 - 1117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, Y. Higashi, H. Itabe, Y.-H. Song, J. Du, and P. Delafontaine Insulin-Like Growth Factor-1 Receptor Activation Inhibits Oxidized LDL-Induced Cytochrome C Release and Apoptosis via the Phosphatidylinositol 3 Kinase/Akt Signaling Pathway Arterioscler. Thromb. Vasc. Biol., December 1, 2003; 23(12): 2178 - 2184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Earnest, K. A. Wood, and T. S. Church Complex Multivitamin Supplementation Improves Homocysteine and Resistance to LDL-C Oxidation J. Am. Coll. Nutr., October 1, 2003; 22(5): 400 - 407. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-J. Geng and P. Libby Progression of Atheroma: A Struggle Between Death and Procreation Arterioscler. Thromb. Vasc. Biol., September 1, 2002; 22(9): 1370 - 1380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Jenner, J. E. Ruiz, C. Dunster, B. Halliwell, G. E. Mann, and R. C.M. Siow Vitamin C Protects Against Hypochlorous Acid-Induced Glutathione Depletion and DNA Base and Protein Damage in Human Vascular Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., April 5, 2002; 22(4): 574 - 580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kataoka, N. Kume, S. Miyamoto, M. Minami, M. Morimoto, K. Hayashida, N. Hashimoto, and T. Kita Oxidized LDL Modulates Bax/Bcl-2 Through the Lectinlike Ox-LDL Receptor-1 in Vascular Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., June 1, 2001; 21(6): 955 - 960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Block, A. R. Mangels, E. P. Norkus, B. H. Patterson, O. A. Levander, and P. R. Taylor Ascorbic Acid Status and Subsequent Diastolic and Systolic Blood Pressure Hypertension, February 1, 2001; 37(2): 261 - 267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-C. Hsieh, M.-H. Yen, C.-H. Yen, and Y.-T. Lau Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells Cardiovasc Res, January 1, 2001; 49(1): 135 - 145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. NAPOLI, O. QUEHENBERGER, F. DE NIGRIS, P. ABETE, C. K. GLASS, and W. PALINSKI Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells FASEB J, October 1, 2000; 14(13): 1996 - 2007. [Abstract] [Full Text]
|
|
|
|
|
|
A. M. Jenner, J. E. Ruiz, C. Dunster, B. Halliwell, G. E. Mann, and R. C.M. Siow Vitamin C Protects Against Hypochlorous Acid-Induced Glutathione Depletion and DNA Base and Protein Damage in Human Vascular Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., April 5, 2002; 22(4): 574 - 580. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||