 |
INTRODUCTION |
Ample evidence supports the notion that oxidatively modified
low-density lipoprotein
(oxLDL)1 plays a key role in
the onset of atherogenic processes (1-8). Endothelial injury is
considered to be one of the earliest atherogenic events (9-11). The
cytotoxic effects of oxLDL are well established (12-15). Endothelial
injury plays a prominent role in the increased adherence of monocytes
and their migration into the subendothelial space of blood vessels
(16-18). The adhesion of monocytes to the endothelium is a key
atherogenic process (9, 10). Recently it has been shown that the
activation of the cellular suicide pathway of the endothelial cell may
be crucial to the development of atherosclerosis (19-26). Although the
exact mechanism of oxLDL-induced apoptosis in endothelial cells remains
unknown, published reports suggest a role for free radical
intermediates (27-31). It has also been reported that the
up-regulation of endothelial nitric-oxide synthase (eNOS) and
copper-zinc superoxide dismutase and/or manganese superoxide dismutase
protects endothelial cells against oxLDL-induced apoptosis (32).
Collectively, these reports reveal an intriguing link between oxLDL,
apoptosis, and nitric oxide (·NO)/superoxide (O
2)
interaction in endothelial cells.
Nitric oxide has been reported to have a dual effect on
cell-dependent LDL oxidation (33). ·NO acts as a
pro-oxidant in the presence of O
2 and an antioxidant in the
presence of lipid peroxyl radical (34-36). The reaction between
·NO and O
2 to form peroxynitrite (ONOO
)
is one of the most facile radical-radical recombination reactions in
free radical biology (37, 38). ·NO also reacts with lipid
peroxyl radical (LOO·) at a nearly diffusion-controlled rate
(k = 1-3 × 109
M
1 s
1) (39). This rate constant
is ~107 times greater than that reported for the reaction
between LOO· and unsaturated lipid, and 104 times
greater than the rate constant for the reaction of LOO· with
-tocopherol (36). Thus, ·NO can act as a potent
chain-breaking antioxidant. Consequently, the reaction between
·NO and O
2 has the combined effect of removing an
antioxidant such as ·NO, and generating the prooxidant,
ONOO
. The role of these reactions in regulating
downstream apoptosis cell signaling has not been fully investigated.
In vivo immunohistochemical studies in atherosclerotic
tissues indicate the presence of oxidation and nitration marker
products (i.e. lipid hydroperoxides and nitrotyrosine) that
are diagnostic for reactive oxygen and nitrogen species (23, 37-40).
In this study we tested the hypothesis that the propagation of lipid
peroxidation is primarily responsible for oxLDL-induced apoptosis.
Therefore, we investigated the effects of extracellular and
intracellular ·NO donors (NONOates), cell-permeable SOD mimetics
and ONOO
scavengers (MnTBAP and FeTBAP), a
cell-permeable mimetic of glutathione peroxidase and phospholipid
hydroperoxide glutathione peroxidase (ebselen), lipophilic phenolic
antioxidant (probucol), and a nitrone spin trap
-phenyl-tert-butyl nitrone (PBN) (Fig.
1) on endothelial apoptosis induced by
oxLDL. Results show that ·NO inhibits oxLDL-induced apoptosis as
do MnTBAP/FeTBAP, ebselen, Probucol, and PBN. These structurally
diverse antioxidants share a common mechanism that is their ability to
inhibit the propagation of lipid peroxidation. Therefore, we propose
that the major antiapoptotic mechanism of ·NO involves peroxyl
radical scavenging.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one)
and PBN were obtained from Sigma. PBN was also obtained from the
Oklahoma Medical Research Foundation Spin Trap Source as a gift from
Dr. Ronald Mason (NIEHS, National Institutes of Health, Research
Triangle Park, NC). Mn(III)-tetrakis(4-benzoic acid) porphyrin
(MnTBAP), FeTBAP, and S-nitrosoglutathione (GSNO) were
synthesized according to the published methods (42, 43). Probucol was
purchased from Sigma. Diethylenetriamine NONOate (DETA/NO) was obtained from Cayman Chemical Co. Anti-Hsp70 antibody and hamster anti-human Bcl-2 antibody and diethylenetriaminepentaacetic acid (DTPA) were purchased from Pharmingen.
Cell Culture--
Bovine aortic endothelial cells (BAEC)
harvested from thoracic aortas were maintained (37 °C, 5%
CO2) in Dulbecco's modified eagles medium (1 g/liter of glucose, Life Technologies, Inc.) containing 15% fetal
bovine serum (Sigma) with antibiotics. Cells used in this study were
between passages 5 and 10.
Human umbilical vein endothelial cells were obtained from Clonetics and
cultured in endothelial basal medium (Clonetics) containing 2% fetal
bovine serum, 10 ng/ml human recombinant epidermal growth factor, 1 µg/ml hydrocortisone, 50 µg/ml gentamycin, 50 ng/ml amphotericin-B,
and 3 mg/ml bovine brain extract. Cells cultured between passages 2 and
4 were used in this study.
Preparation of LDL and LDL Lipid Extraction--
LDL was
isolated by sequential ultracentrifugation (d = 1.019-1.063) from freshly drawn, normolipidemic human plasma to which EDTA was added (44). LDL was oxidized by adding ONOO
(1 mM). In control experiments, LDL was added to a phosphate buffer (pH 7.4, 100 mM) containing pre-decomposed
ONOO
. Medium containing 150 µg of the modified LDL was
extracted by adding 2 volumes of ice-cold methanol followed by 2 volumes of chloroform (45). The mixture was centrifuged at 1800 × g for 10 min to separate the phases. The lipid phase was
carefully removed and evaporated under a stream of nitrogen and
re-dissolved in a minimum volume of methanol.
Treatment of BAEC with OxLDL--
For all treatments, cells were
washed twice with Dulbecco's phosphate-buffered saline and incubated
with serum-free Dulbecco's modified Eagle's medium in the presence or
absence of reagents. Either native or oxidized LDL was added to a final
concentration of 150 µg of LDL protein/ml.
Quantification of DNA Fragmentation by Gel
Electrophoresis--
DNA was isolated from BAEC. Culture medium was
removed and centrifuged at 3000 × g for 5 min to
collect any detached cells. Adherent cells were lysed with a hypotonic
lysis buffer (10 mM Tris-HCl, 10 mM EDTA, 0.5%
Triton X-100) and then pooled with the pellet made up of detached
cells. After incubation at 4 °C for 15 min, lysates were incubated
with 10 µl of 10 mg/ml RNase A for 1 h at 37 °C followed by
10 µl of 20 mg/ml proteinase K for 2 h at 50 °C. DNA was
extracted using chloroform:phenol:isoamyl alcohol (25:24:1). It was
then precipitated overnight with 1 volume of isopropyl alcohol at
20 °C, electrophoresed on 2% agarose gel, and then visualized
under UV light after staining with ethidium bromide.
Terminal Deoxynucleotidyltransferase-mediated Nick-end Labeling
Assay--
Apoptosis was detected in BAEC's using terminal
deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) assay
(46). Labeling of 3' free hydroxyl ends of the fragmented DNA with
fluorescein-dUTP, was catalyzed by terminal deoxynucleotidyltransferase
(TdT) using a commercially available kit (ApoAlert,
CLONTECH) following the manufacturer's directions.
The areas of apoptotic cells were then detected by fluorescence
microscopy equipped with rhodamine (for propidium iodide staining) and
fluorescein isothiocyanate filters. The quantification of apoptosis was
performed using Sigma Scan 5.0 Image Analysis package. Propidium
iodide-stained cells (which represents the total number) were counted
under rhodamine filter and the apoptotic (TUNEL positive) cells were
counted under fluorescein isothiocyanate filter. Percentage of
apoptosis was calculated from the ratio of these two measurements.
Caspase-3 Enzyme Activity--
Cells were washed after treating
with appropriate drugs with phosphate-buffered saline and resuspended
in 50 µl of chilled lysis buffer (Caspase-3 assay kit,
CLONTECH) and incubated on ice for 10 min. The cell
lysates were centrifuged in a microcentrifuge at 12,000 rpm for 3 min
at 4 °C to precipitate cellular debris.
To confirm the correlation between protease activity and signal
detection, we performed a control reaction as follows. An induced
sample was incubated with 0.5 µl of 1 mM DEVD-fmk
(caspase inhibitor) at 37 °C for 30 min during which time the other
tubes consisting of lysis buffer were kept on ice. Subsequently, 50 µl of reaction buffer (containing 7 µl of 1 mM
dithiothreitol/ml of reaction buffer) was added, followed by the
addition of 5 µl of 1 mM conjugated substrate
(DEVD-p-nitroanilide, 50 µM final concentration) to each sample. After incubating at 37 °C for 1 h, absorptions were monitored at 405 nm in a spectrophotometer (46).
Analysis of Hsp70 and Bcl-2 Protein Levels--
BAEC were washed
with ice-cold phosphate-buffered saline and resuspended in 100 µl of
RIPA buffer (20 mM Tris-HCl, pH 7.4, 2.5 mM
EDTA, 1% Triton-X 100, 1% sodium deoxycholate, 1% SDS, 100 mM NaCl, 100 mM sodium fluoride. To a 10-ml
solution of the above, the following agents were added: 1 mM sodium vanadate, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 10 µg/ml pepstatin inhibitors.) Cells were homogenized
by passing the suspension through a 25-gauge needle (10 strokes). The
lysate was centrifuged at 750 × g for 10 min at
4 °C to pellet out the nuclei. The remaining supernatant was
centrifuged for 15 min at 12,000 × g. Protein was
determined with the Lowry method (47), and 25 µg were used for the
Western blot analysis. Proteins were resolved on polyacrylamide gels
(8% for Hsp70 and 18% for Bcl-2) and blotted onto nitrocellulose membranes. Sheets were washed twice with TBS (140 mM NaCl,
50 mM Tris-HCl, pH 7.2) containing 0.1% Tween 20 before
blocking the nonspecific binding with TBS, 5% skim milk, 1%
fetal calf serum. Filters were incubated with either mouse anti-Hsp70
antibody (Pharmingen) or hamster anti-human Bcl-2 antibody (Pharmingen) 1 µg/ml in TBS, 2% skim milk, 0.7% fetal calf serum for 2 h at room temperature. Sheets were washed 5 times and detected by
horseradish peroxidase-conjugated goat anti-mouse monoclonal antibody
(1:1000) for Hsp70 and anti-hamster IgG (1:1000) for Bcl-2 for 1.5 h at room temperature using the ECL method (Amersham Pharmacia Biotech).
 |
RESULTS |
The Effect of Small Molecular Weight Peroxyl Radical
Scavengers, Cell-permeable Antioxidant Enzyme Mimetics, and Antioxidant
Enzymes on OxLDL-induced Apoptosis--
After incubating BAEC for
24 h with native LDL, no DNA fragmentation was observed (Fig.
2A, lane 2). However, if the
LDL was pre-oxidized, either with copper (II) sulfate (100 µM) or with ONOO
(1 mM),
significant DNA laddering occurred (Fig. 2A, lanes 3 and
4). DNA laddering did not occur in the presence of
decomposed ONOO
(not shown). In addition, the dialysis of
LDL after treatment with ONOO
did not alter the
ability of LDL to fragment cellular DNA (not shown). This indicates
that ONOO
caused the oxidation of a component of LDL to
an intermediate or product that stimulated DNA fragmentation, and that
this intermediate remained associated with the LDL particle during
dialysis. This suggests that apoptosis is not caused by a decomposition
product of ONOO
, nor by a low-molecular weight, soluble,
LDL oxidation product, such as malondialdehyde or 4-hydroxynonenal in
cells exposed to ONOO
-modified LDL. OxLDL-induced DNA
laddering was significantly inhibited in the presence of Probucol (Fig.
2B, lane 4), nitrone trap, PBN (Fig. 2B, lane 6),
and metalloporphyrin antioxidant, FeTBAP (Fig. 2B, lane
7).

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of oxLDL on DNA fragmentation in BAEC
and the effect of free radical scavengers. BAEC were incubated for
16 h with and without additions. Cells were harvested and DNA was
extracted for fragmentation analysis. A, the lanes
correspond to: 1, cells alone; 2, native LDL (150 µg/ml); 3, copper (II)-modified oxLDL; and 4, ONOO -modified oxLDL. B, the lanes correspond
to: 1, native LDL; 2, oxLDL (150 µg/ml); and
oxLDL with the addition of: 3, probucol (10 µM); 4, probucol (100 µM);
5, PBN (100 µM); 6, PBN (1 mM); and 7, FeTBAP (10 µM).
|
|
In addition to DNA fragmentation, the effect of oxLDL on the activation
of caspase-3 was also investigated. Caspase-3, one of the downstream
members of this enzyme family, is activated by proteolysis and is
considered to be a committed step in several apoptotic pathways (46).
As shown in Table I, native LDL did not enhance caspase-3 activity, whereas
oxLDL stimulated caspase-3 activity by ~5-fold. This suggests that
the oxLDL-mediated apoptosis acts through the activation of
caspase-3.
View this table:
[in this window]
[in a new window]
|
Table I
Effect of antioxidants and antioxidant enzymes on oxLDL-induced
caspase-3 activation
OxLDL was prepared by adding ONOO (100 µM) to
native LDL (150 µg/ml) in 50 mM phosphate buffer after
2 h, the LDL particle was extensively dialyzed to remove excess
ONOO and other oxidants. BAEC were treated with oxLDL for
16 h. Values are mean ± S.D. of three independent
experiments.
|
|
In order to examine the mechanism of oxLDL-induced apoptosis, the
effects of a range of antioxidants were examined (Table I). These
compounds were chosen based on their wide range of targets and on their
differential compartmentalization. BHT, a peroxyl radical scavenger,
and DTPA, a metal chelator, both inhibited caspase-3 activation.
This suggests that metal ion-dependent lipid peroxidation
propagation reactions, involving the breakdown of LOOH and the
formation of LO·/LOO· are important steps in mediating
the apoptotic cascade. Ebselen, a selenium-containing glutathione
peroxidase mimetic abolished the activation of oxLDL-mediated
caspase-3. Ebselen is able to access both the intracellular and
extracellular compartments and cause degradation of both intracellular
and extracellular hydroperoxides (48). However, the availability of
thiols (which are required to mediate ebselen-dependent
peroxide decomposition) may be limited in the extracellular
environment. The antioxidant enzymes, SOD and catalase, had little
effect on caspase-3 activation. As the actions of these enzymes are
limited to the extracellular environment, this observation implies that
neither superoxide nor hydrogen peroxide present in the extracellular
compartment is involved in the initiation of apoptosis. In contrast,
the cell-permeable metalloporphyrin SOD mimetics, MnTBAP and FeTBAP,
inhibited caspase-3 activation to almost control levels (49-51). It is
noteworthy that FeTBAP was effective at a 10-fold lower concentration
than MnTBAP. Although the exact reasons for this differential effect
are not understood, it is consistent with the increased activity of
FeTBAP as a catalyst of superoxide dismutation or ONOO
decomposition. The metal-free porphyrin, TBAP, had little effect on
caspase-3 activation. Nitrone trap caused a 70% inhibition in
caspase-3 activation as did desferral, a chelator of redox-active iron
(Table I). The present data suggest that oxLDL-induced intracellular superoxide or chain-propagating lipid peroxyl radicals mediate the
intracellular signal transduction mechanism leading to caspase-3 activation.
The Effect of Extracellular and Intracellular Nitric Oxide Donors
on OxLDL-induced Apoptosis--
We previously reported that ·NO
donor compounds and S-nitrosothiols will inhibit the
toxicity of oxLDL to endothelial cells in culture (52). We ascribed
this effect to the ability of ·NO to scavenge LOO·
radicals and so to prevent lipid hydroperoxide-mediated oxidation of
the membranes (35, 36, 53). In the present study we examined whether
nitric oxide donors and S-nitrosothiols could inhibit oxLDL-mediated apoptosis. Fig. 3 confirms
the pro-apoptotic effect of oxLDL on BAEC, as treatment of cells with
ONOO
-modified oxLDL for 18 h results in significant
TUNEL positive staining (Fig. 3A). Both the nitric oxide
donor, DETA/NO, and the S-nitrosothiol, GSNO, significantly
prevented the TUNEL positive staining (Fig. 3, C and
D), suggesting that these compounds inhibited DNA
fragmentation. These results are shown quantitatively in Fig. 3E.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of NO donors on DNA
fragmentation. A, BAEC were treated for 16 h with native LDL (150 µg/ml); B, with oxLDL (150 µg/ml); C, with oxLDL and DETA/NO (100 µM);
D, with oxLDL and 1 mM GSNO. Cells were
harvested, stained for TUNEL-positive cells, and examined by
fluorescence microscopy, and E, the percent apoptosis was
quantitated by image analysis.
|
|
Caspase-3 is a cysteine protease that is synthesized in an inactive
"pro" form. The apoptotic cascade results in the proteolytic activation of pro-caspase to generate active caspase-3. It has been
demonstrated that caspase-3 can be inactivated by
S-nitrosation of the active site thiol (54, 55). In addition
to DNA fragmentation, ONOO
-treated LDL caused an almost
5-fold induction in caspase-3 activity, as measured by following the
formation of p-nitroanilide (Table I). Both DETA/NO and GSNO
substantially inhibited caspase-3 activation (Fig.
4A). This inhibition of
caspase activity was not due to S-nitrosation as the
addition of dithiothreitol, which will remove any
S-nitrosothiols, did not alter the caspase activity (data not shown).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 4.
The effect of NO donors on oxLDL-induced
caspase activation. A, the effects of native LDL (150 µg/ml), oxLDL (150 µg/ml), GSNO (1 mM), and DETA/NO
(100 µM) on caspase-3 activity measured in BAEC. Cells
were treated 16 h with the respective agents and incubated.
B, time course of caspase-3 activation induced by oxLDL (150 µg/ml) in the presence and absence of 100 µM
DETA/NO.
|
|
The time course of caspase activation by oxLDL is shown in Fig.
4B. Caspase-3 activity was stimulated between 4 and 8 h
after adding oxLDL, and remained high for up to 24 h. In the
presence of an ·NO donor, DETA/NO, only a slight increase in
caspase-3 activity was observed over 24 h, but the large increase
in activity between 4 and 8 h was abolished. DETA/NO, an
extracellular ·NO donor, spontaneously decays within the cell
culture medium. Consequently, ·NO is generated in the solution
above the cells. To examine the effect of intracellular ·NO
production, we used a novel set of compounds that pass through the cell
membrane and are de-esterified inside the cell to give the active
·NO donor (56). Consequently, ·NO released from these
compounds is generated within the intracellular environment. As shown
in Fig. 5, these compounds were able to suppress oxLDL-mediated caspase-3 activation at concentrations that
were 50-100-fold lower than the extracellular ·NO donor,
DETA/NO. With 1 µM of these esterase-sensitive
intracellular ·NO donors, robust inhibition was observed,
whereas 5 µM AcOM-DEA/NO or AcOM-PYRRO/NO completely
suppressed caspase-3 activation (Fig. 5).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
The effect of cell-permeable ·NO
donors on oxLDL-induced caspase 3 activation. BAEC were treated
with ONOO -oxLDL (150 µg/ml) for 16 h in the
presence and absence of cell-permeable ·NO donors. For
structures of ·NO donors, see Fig. 1.
|
|
To determine the component of the LDL particle that was responsible for
apoptosis, we isolated the lipid component of oxidized and native LDL
and examined the effects of the extracts on caspase-3 activation. As
shown in Fig. 6, exposure of cells to the
lipid extract of native LDL resulted in only a minor increase in
caspase activity, which was inhibited by DETA/NO and GSNO. In contrast, the lipid fraction of oxLDL caused a significant activation of caspase-3 activity to a level almost equivalent to that observed with
whole oxLDL particle. This indicates that the lipid component of the
LDL particle is largely responsible for the activation of caspase-3.
Both DETA/NO and GSNO inhibited caspase-3 activation by oxLDL extract,
indicating that the pro-apoptotic intermediate, that is inhibited by
·NO, is present in the lipid component of oxLDL. The influence of lipid hydroperoxides on caspase-3 activation was assessed by reducing the peroxides to alcohols using potassium iodide or sodium borohydride. Treatment of oxLDL lipid extract with these agents, before
addition to the cells, ablated the ability of this lipid mixture to
activate caspase-3 (Fig. 6). These results suggest that the lipid
hydroperoxide component of oxLDL is responsible for caspase
activation.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
The effect of ·NO donors and
reductants on caspase-3 activation induced by LDL lipid extracts.
BAEC were treated with lipid extracts of oxLDL and native LDL in the
presence and absence of DETA/NO (100 µM), GSNO (500 µM), KI (1 mM), and NaBH4 (1 mM). After 16 h, caspase activity was measured.
|
|
It has been previously reported that 7-ketocholesterol (7-KC), a known
oxidation product of cholesterol, is pro-apoptotic (57-59). In Fig.
7, the effect of oxLDL was compared with
7-KC. Both agents enhanced caspase-3 activation, whereas only
oxLDL-dependent caspase-3 activation was inhibited by
DETA/NO. This suggests that 7-KC enhances caspase-3 activity, and hence
apoptosis, by a mechanism that is distinct from oxLDL. However, the
level of 7-KC (e.g. 40-100 µg/ml), required to promote
apoptosis far exceeds the levels of 7-KC expected to be present in
oxLDL used in these experiments.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 7.
The effect of ·NO donor on
7-ketocholesterol-induced apoptosis. BAEC were treated with 10, 40, and 100 µg/ml 7-KC for 16 h in the presence and absence of
DETA/NO (100 µM). For the sake of comparison, data using
oxLDL alone and oxLDL in the presence of DETA/NO are also
included.
|
|
The Effect of Hsp70 and Bcl-2 Induction on OxLDL-induced
Apoptosis--
It has previously been reported that the
anti-apoptotic effects of ·NO can be correlated to the
stimulation of heat shock protein 70 (Hsp-70) synthesis (60, 61). Fig.
8A demonstrates that control
cells showed little or no Hsp-70 content as determined by the Western
blot analysis. However, when cells with DETA/NO, GSNO, or native LDL
were incubated, Hsp-70 synthesis was induced. We further observed that
oxLDL induced a much lower level of protein expression, which was
increased in the presence of DETA/NO or GSNO. This data suggested an
inverse correlation between Hsp-70 content and caspase-3 activation. To
examine if preinduction of Hsp-70 could protect against oxLDL, we
pretreated cells for 6 h with DETA/NO, GSNO, or native LDL and
then exposed these cells to oxLDL. After 16 h these cells showed a
robust expression of Hsp-70 with a high level of caspase activation
(Fig. 8B). Under these conditions we found no correlation
between caspase-3 activation and Hsp-70 induction. Therefore the
induction of Hsp-70 is not inhibitory to oxLDL-mediated apoptosis, but
is a parallel process that is stimulated by exposure of cells to
DETA/NO, GSNO, or native LDL.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 8.
The effect of Hsp70 induction on
oxLDL-induced apoptosis in BAEC. A, cells were
treated with 100 µM DETA/NO or 500 µM GSNO
for 16 h in the absence and presence of LDL (150 µg/ml).
Increase in Hsp70 expression was shown by the Western blot analysis.
The lanes correspond to: 1, control; 2, DETA/NO;
3, GSNO; 4, oxLDL; 5, oxLDL and GSNO;
6, oxLDL and DETA/NO; and 7, nLDL. B,
cells were treated with ·NO donors for 6 h then cells were
washed free of ·NO donors and treated with oxLDL for 16 h.
Caspase-3 activity was measured subsequently.
|
|
Results described in Fig. 8B demonstrate that DETA/NO and
GSNO must be present at the same time as the oxLDL in order to prevent caspase activation. Hence, these agents affect the cell directly and
instantly rather than through the sustained and mediated induction of
antioxidant or antiapoptotic enzymes in the system.
In search of the molecular mechanism(s) responsible for the protective
effects, we also measured the relative amounts of Bcl-2 protein as
described in Fig. 9. Bcl-2 was
significantly inhibited in the oxLDL-treated group (62). OxLDL-induced
depletion of Bcl-2 was restored in the presence of ·NO donors
and other antioxidants (Fig. 9). Similar results were obtained using
human umbilical vein endothelial cells (data not shown). This indicates
that the antiapoptotic effect of ·NO in human umbilical vein
endothelial cells is likely to be mediated by the same mechanism as
occurs in BAEC.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 9.
The effect ·NO donors and antioxidants
on Bcl-2 levels during oxLDL-induced apoptosis. BAEC were treated
with: 1, no treatment; 2, nLDL (150 µg/ml);
3, oxLDL (150 µg/ml); 4, oxLDL + 500 µM GSNO; 5, oxLDL + 100 µM
DETA/NO; 6, oxLDL + 1 mM PBN; and 7, oxLDL + 100 µM MnTBAP for 16 h and Bcl-2 protein
levels were measured by the Western analysis.
|
|
 |
DISCUSSION |
Mechanism of OxLDL-induced Endothelial Apoptosis--
The
pathophysiological effects of oxLDL in vascular cells have previously
been investigated using oxLDL as a whole (1-10). It is also well known
that cells are more vulnerable to oxLDL-induced toxicity if serum or
other proteins are excluded from the media. Reports also indicate that
oxLDL-induced endothelial apoptosis is markedly diminished in the
presence of added serum (63). As our objective was to elucidate the
mechanism of oxLDL-induced cellular injury, experiments were performed
in the absence of serum.
The individual components of oxLDL responsible for mediating apoptosis
are not known. OxLDL is a mixture of several cytotoxic components
consisting of lipid hydroperoxides (e.g. 9- and
13-hydroperoxyoctadecadienoic acid, cholesteryl
hydroperoxyoctadecadienoate, aldehydes such as 4-hydroxynonenal and
malondialdehyde, and oxysterols (7-ketocholesterol, 7
-hydroxycholesterol)). Individually these components are potent inducers of apoptosis in several cell types including bovine and human
endothelial cells. In the present work, we have shown that the lipid
extract of oxLDL containing lipid hydroperoxides induces endothelial
apoptosis in BAEC or human umbilical vein endothelial cells exposed to
ONOO
-modified LDL. Possible proapoptotic candidates
present in ONOO
-modified LDL include hydroperoxy
derivatives of cholesteryl linoleate, linoleate, and cholesteryl
hydroperoxides. Pretreatment of endothelial cells with ebselen, a
synthetic glutathione peroxidase/phospholipid hydroperoxide glutathione
peroxidase mimic, has been shown to afford protection against copper
oxLDL (64). Scheme 1 summarizes the
reactions of pro-apoptotic reactive oxygen and nitrogen species and the
anti-apoptotic mechanism(s) of several antioxidants including ·NO.

View larger version (21K):
[in this window]
[in a new window]
|
Scheme 1.
A hypothetical model describing the
inhibitory role of ·NO and other antioxidants in oxLDL-mediated
apoptosis. Nitric oxide and antioxidants could inhibit apoptosis
by chelating redox-active metal ions and by scavenging or decomposing
reactive oxygen and nitrogen species at various points as indicated by
the dotted arrow. The mechanism by which various agents
inhibit cellular lipid oxidation and apoptsis is proposed as follows:
DTPA and desferral (redox-metal ion chelation); PBN (trapping of
lipid or lipid-derived peroxyl radical); BHT and probucol (scavenging
of the lipid peroxyl radical); ebselen (decomposing lipid
hydroperoxides and hydrogen peroxide); MnTBAP/FeTBAP (dismutating
superoxide and hydrogen peroxide) and FeTBAP (decomposing
peroxynitrite).
|
|
Based on the effects of PBN, ebselen, desferral, SOD mimetics, phenolic
antioxidants, and ·NO donors as shown in the present study, we
postulate that lipid peroxyl radicals trigger the apoptotic cascade
(Scheme 1). This is linked to membrane lipid peroxidation which is
presumably one of the earliest upstream apoptotic signaling events
(65). Agents that inhibit this process are likely to negatively
influence the apoptotic signaling process. Literature data also
indicate that the metalloporphyrin class of SOD mimetics are efficient
inhibitors of lipid peroxidation (66). The antioxidant effect of
·NO is linked to its ability to scavenge lipid peroxyl radicals (35, 36). Thus we propose that ·NO can potentially inhibit
formation of intracellular reactive oxygen species. This antiapoptotic
mechanism of ·NO will compete with the proapoptotic
peroxynitrite-inducing reaction.
Extracellular and Intracellular ·NO Donors: Mechanism of
Action--
In this study we used both intracellular and extracellular
·NO donors to investigate the antiapoptotic mechanism of
·NO. We used slow-releasing NONOates from which ·NO was
released at defined rates with known stoichiometry from a thermolytic
decomposition (53). The disadvantage of using extracellular NONOates is
that a substantial proportion of ·NO generated extracellularly
is oxidized before it enters the cell. As a result, we used relatively
higher concentrations (~100 µM) of these compounds. In
contrast, the advantage of using cell-permeable pro-·NO donors
is that they require activation by cytoplasmic esterases in order to
release ·NO. Consequently ·NO will be released
intracellularly (see Scheme 2). Thus,
relatively low concentrations (1-5 µM) of
esterase-specific ·NO donors are needed for maximal
results in apoptosis experiments.
Scavenging of Lipid Peroxyl Radical by ·NO: A Potential
Antiapoptotic Mechanism?--
The collective works of our group (35,
52, 53) and other's (36, 67) have demonstrated the following: (i)
·NO is a highly sensitive inhibitor of copper- and
azo-initiator-dependent LDL oxidation, (ii) ·NO
outcompetes vitamin E as an inhibitor of LDL lipid oxidation, (iii) the
release rate of ·NO from ·NO donors is a critical
determinant of the extent of inhibition of lipid oxidation, (iv)
·NO inhibits cell-dependent LDL oxidation, and (v)
·NO inhibits the toxicity of oxLDL to endothelial cells by a
peroxyl radical scavenging mechanism. The oxLDL itself is unreactive to ·NO. Published reports indicate that ·NO does not
directly react with lipid hydroperoxides, aldehydes, or with other
associated products of oxLDL, nor with
-carotene and
-and
-tocopherols (68). ·NO, however, reacts with the lipid
peroxyl radical associated with oxidizing LDL at a diffusion controlled
rate (39). ·NO is a potent inhibitor of lipid peroxidation and
LDL oxidation. Lipid peroxidation, the central process in most
mechanisms of LDL modification, is controlled by the steady-state
concentrations of lipid peroxyl radicals. Any compound that scavenges
peroxyl radicals (e.g.
-tocopherol, Probucol) to give a
stable end product will inhibit lipid peroxidation. The peroxyl radical
can be thought of as an organic analog of superoxide in the same way
that lipid hydroperoxide is an analog of hydrogen peroxide.
Accordingly, the chemistry between ·NO and peroxyl radical is
similar to that between ·NO and superoxide in that they react at
a diffusion-limited rate through radical-radical recombination (38,
39). Differences occur due to the fact that the product of this
reaction, unlike ONOO
, decomposes to stable products that
do not re-initiate the peroxidation reaction. Because the endothelium
and subendothelial space (the physiological locus of LDL modification)
are exposed to a constant supply of ·NO due to basal NOS
activity, these observations are of critical importance in determining
the oxidative propensity of the artery wall. In addition, ·NO is
a highly sensitive controller of cellular signaling processes that
require lipid oxidation. Published data suggest that oxLDL exposure to
endothelial cells impairs eNOS activation through displacement of eNOS
from caveolae and attenuates the capacity of the endothelium to produce
NO (69). Exposure of endothelial cells to oxLDL was shown to cause a
decrease in the expression of eNOS (70). We propose that lipid peroxyl
radicals or lipid hydroperoxides are responsible for initiating the
apoptotic cell signaling and that this cascade of events is regulated
by ·NO.
The increased formation of intracellular reactive oxygen species was
suggested to be one of the mechanisms of endothelial apoptosis induced
by oxLDL components (71). The intracellular source of ROS, however,
remains to be established. OxLDL-induced cell membrane signaling
leading to ROS generation is an active area of research. ROS can
originate from endothelial nitric-oxide synthase, mitochondrial
electron transport chain, xanthine oxidase, cyclooxygenase, and NADPH
oxidases (72). Pritchard et al. (73), reported that
endothelial cells incubated with atherogenic concentrations of native
LDL released superoxide from eNOS. Wever et al. (41), also
proposed an uncoupling of NOS activity as a mechanism of increased
formation of superoxide and ONOO
under similar
conditions. Clearly, the identification of the intracellular source of
ROS in oxLDL-induced endothelial apoptosis will remain a challenging
task in future studies.
In conclusion, we have shown that ·NO can effectively inhibit
endothelial apoptosis mediated by oxLDL. It is likely that the antiapoptotic mechanism of ·NO is probably linked to its ability
to scavenge the lipid peroxyl radicals that are presumably responsible
for the apoptotic signaling cascade.