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Introduction
Aspirin (acetylsalicylic acid, ASA), a traditional
analgesic-antipyretic drug, is now widely used in the prevention of
cardiovascular diseases[1] and may even reduce the risk of
Alzheimer's disease[2] and
cancers[3]. The classic mechanism of aspirin in the prevention of cardiovascular diseases
has been known to inhibit the activity of cyclooxygenase
(COX) in platelets, which leads to the reduction of
prostaglandin and thromboxane A2
production[4]. However, recent studies show that the COX-inhibiting mechanism can not
provide a full explanation of the cardiovascular protective
effects of aspirin. Accumulating experimental evidence have
indicated that many other effects of aspirin contribute to its
cardiovascular protection, such as modulating the activity
of the intracellular metabolism of ATP, inhibiting inducible
nitric oxide synthase, modulating the activity of nuclear
factor (NF)-kappa B and mitogen-activated protein kinases
(MAPK)[5_7].
Apoptosis, or programmed cell death, is an active
progress of cell elimination in physiological or pathological
conditions[8]. Pathological apoptosis of endothelial cells not
only damages the integrity of endothelium, but also affects
the cytokine secretion of endothelial cells. Impaired
endothelium function will then facilitate the formation of thrombus
and promote atherogenesis[9]. However, inducing apoptosis
of endothelial cells is potentially an effective strategy for
blocking the progression of tumor development by
inhibiting angiogenesis[10].
MAPK [extracellular signal-regulated kinase, Jun
N-terminal kinase (JNK) and p38 MAPK] are well-known signal
molecules involved in the regulation of cell proliferation and
apoptosis. Among the 3 MAPK family members, p38 MAPK
is activated by cell stress promoting cell
apoptosis[11]. Aspirin has been shown to modulate MAPK expression in
various cell types, including vascular endothelial
cells[12_14]. In the present study, we investigated the direct effect of
aspirin on the apoptosis of bovine aorta endothelial cells and the
role of p38 MAPK in this process.
Materials and methods
Cell culture Bovine aortic endothelial cells (BAEC) were
harvested as previously described[15] and cultured in
Dulbecco's modified Eagle's medium (DMEM) with 10%
heat-inactivated fetal bovine serum (FBS), 100 kU/L benzyl-
penicillin, and 100 mg/L streptomycin. Confluent cells were
subcultured by trypsin digestion. Experiments were
performed with cells from passages 4 to 10.
Cell viability assay Cell viability was measured through
a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) assay. BAEC were seeded into 24-well plates
and grown to 80% confluence in DMEM with 10% FBS.
After starved in phenol-free M1640 medium for 24 h, the cells
were incubated with the respective test substance. MTT
dissolved in phenol red free M1640 at a concentration of 5
g/L was added to the cell cultures at the end of the experi-ments.
After incubation for 1 h at 37 oC, solubilization solution
containing 10% (v/v) Triton X-100 and 0.1 mol/L HCl in
isopropanol were added into the cell cultures to stop the reaction
and dissolve formazan crystals. Metabolic activity was
quantified by measuring light absorbance at 570 nm.
Morphologic determination and quantification of
apopto-sis After the respective treatment, BAEC were stained with
Hoechst 33258 and then observed under a fluorescence
microscope. Under the fluorescence microscope, apoptotic
cells with condensed or fragmented nuclei were easily
distinguished from normal cells with intact nuclei.
Quantification of apoptosis was routinely determined by counting the
number of apoptotic cells: for each plate, we randomly chose
6 fields of view, and counted the total cells and apoptotic
cells with a minimum number of 500 cells scored.
DNA electrophoresis At the end of experiments, the cells
were harvested and DNA was extracted with standard
phenol-chloroform extraction. Electrophoresis of DNA was
performed in ethidium bromide-stained 1.5% agarose gel and
visualized by exposure under UV light.
Western blotting BAEC cultured in 12-well culture plates
were grown to 80%-90% confluence and then starved for
24 h in serum-free M1640 medium. After different treatment,
the cells were scraped into lysis buffer containing (in
mmol/L) NaCl 50, Na3VO4 2, phenylmethylsulfonyl fluoride 0.5, and
HEPES 10 at pH 7.4, along with 0.01% Triton X-100 and 10
mg leupeptin. The cells were then disrupted by sonication,
after which they were centrifuged for 20 min at
12 000×g to separate particular proteins from soluble fractions. The
amount of proteins in each fraction was determined by the
bicinchoninic acid (BCA) method.
Proteins (10 mg) were subjected to SDS-PAGE and
electrophoretically transferred to a nitrocellulose membrane. The
membranes were blocked with Tris-buffered saline
Tween-20 (TBST) containing 5% bovine serum albumin
(BSA) and immunoblotting was performed using either an antitotal-p38
MAPK antibody or antiphospho-p38 MAPK antibody. The
membranes were then incubated with a second antibody
conjugated to horseradish peroxidase and the blot was
visualized by the Phototope Western Detection System (New
England Biolabs, Ipswich, MA, USA) To control equal
protein concentration in the experiments, 2 gels for each group
were loaded parallely with the same protein samples and
blotted for activated, phosphorylated p38 MAPK or total
p38 MAPK. Bands of protein were quantitatively determined
by thin-layer chromatography with Shimadzu
Dual-Wavelength Chromato-Scanner (Model CS-930, Tokyo, Japan).
Reagents BSA, DMEM medium, SB203580, and Hoechst
33258 were purchased from Sigma Chemical Co (St Louis,
MO, USA). phospho-p38 MAPK monoclonal antibody, horseradish
peroxidase (HRP)-conjugated anti-rabbit secondary antibody, and a Phototope-HRP Western Detection kit
were purchased from New England Biolabs Inc (New England Biolabs, Ipswich, MA, USA).
Statistical analysis Values were expressed as mean±SD
and assessed by one-way ANOVA and Student's
t-test. Values of P<0.05 were considered to be statistically significant.
Results
Cell viability Low doses of aspirin protected BAEC from
apoptosis induced by H2O2. Co-incubation of aspirin
1×10-10_1×10-8 mol/L reversed the decrease of BAEC viability induced
by H2O2 at 200 µmol/L. Interestingly, aspirin at
1×10-9 mol/L exhibited a stronger protective effect
than 1×10-10 and
1×10-8 mol/L (Figure 1).
Unexpectedly, at relatively high concentrations, aspirin
from 1×10-7 mol/L to
1×10-4 mol/L, showed no protection
effect against the apoptosis of BAEC (data not shown), and
it by itself decreased the viability of BAEC. After incubation
with 1×10-4 mol/L aspirin for 24 h, cell viability decreased to
78.0% of the control (absorbance:
0.401±0.011 vs 0.514±0.019 of the control, P<0.01). SB203580 (10 µmol/L), a
specific p38 MAPK inhibitor, markedly reduced the toxic
effect of aspirin and increased cell viability by about
12.7% (absorbance: 0.452±0.012 vs 0.401±0.011 of
1×10-4 mol/L aspirin, P<0.01; Figure 2).
Morphologic changes After 24 h incubation of the
indicated treatments, BAEC showed typical morphologic changes
of apoptosis, such as reduction of cell volume and
condensation and fragmentation of chromosomes; normal cells
showed uniformed nuclei (Figure 3).
Quantification of apoptosis To further determine that
the viability change of BAEC was mainly caused by apoptosis rather than necrosis, the apoptotic rate was
directly measured under fluorescence microscope as described
in Materials and methods.
At low concentration, aspirin from
1×10-10 mol/L to
1×10-8 mol/L reduced the apoptotic rate induced by 200
µmol/L H2O2 (Table 1). Incubation with aspirin
(1×10-4 mol/L) for 24 h increased the apoptotic rate of BAEC to
26.0%±1.8%, while pretreatment with SB203580 reduced the
apoptotic rate to 13.9%±2.0% (Table 2). These results were
consistent with the results in the cell viability assay.
DNA electrophoresis Following analyses with 1.5%
agarose gel, DNA isolated from BAEC treated with 200 µmol/L
H2O2 for 24 h showed a typical DNA ladder which represents
integer multiples of the internucleosomal DNA length (about
180_200 base pair). Aspirin at
1×10-9 mol/L greatly reduced DNA fragmentation (Figure 4). Incubation with aspirin (1×
10-4 mol/L) for 24 h also induced a typical DNA ladder which
was reversed by 10 µmol/L SB203580, a specific inhibitor of
p38 MAPK (Figure 5).
Phospho-p38 MAPK expression We previously showed
that H2O2 activated p38 MAPK in
BAEC[16]. In this study, aspirin from
1×10-10 to 1×10-8 mol/L significantly decreased
p38 MAPK phosphorylation induced by 200 µmol/L
H2O2 (Figure 6). Interestingly, aspirin at
1×10-9 mol/L exhibited the strongest effect, which paralleled well with its anti-apoptotic
effect.
Aspirin from 1×10-7 mol/L to
1×10-4 mol/L dose-dependently increased the phosphorylation of p38 MAPK in
BAEC, while SB203580 at 10 µmol/L blocked such change
(Figure 7). Aspirin (1×10-4 mol/L) stimulated the
phosphorylation of phospho-p38 MAPK in a time-dependent manner
with a peak effect at 10 min, returning back to baseline at 1 h
(Figure 8).
Discussion
Aspirin was introduced as an anti-inflammatory and
analgesic drug in 1892, and the inhibition of COX has been
thought to be the major mechanism mediating this effect.
During the past decades, novel actions of aspirin have been
discovered, which therefore largely expands the clinical use
of aspirin. Experimental evidence has shown that aspirin
can protect endothelial cells from oxidant damage through
the NO_cGMP pathway, which provides a base for its use as
a cardiovascular protective drug to reduce the occurrence of
heart attack and stroke[7]. Aspirin has also been found to
promote the apoptosis of tumor cells, and can be used to
protect against the development of colon cancer and other
digestive system cancers[5,17,18]. In the present study, we
demonstrate that aspirin exhibits a biphasic effect on the
apoptosis of bovine endothelial cells; at low concentrations,
it protects BAEC from apoptosis induced by
H2O2, while at relatively high concentrations, aspirin by itself induces
apoptosis in BAEC. The endothelium protective effect of
aspirin observed at low concentrations is consistent with its
clinical practice that only a low dose of aspirin is required for
its preventive action on cardiovascular diseases. The
observed apoptotic effects on endothelial cells at high
concentrations sheds new light on its clinical use as a
preventive drug for the carcinogenesis of digestive tract cancers
because apoptosis of endothelial cells is a very effective
way of blocking angiogenesis, a pivotal biological aspect in
tumor development[10]. Our findings may also explain that
the preventive effect of aspirin on carcinogenesis is mostly
limited in the digestive tract, because a relatively high local
concentration of aspirin may only be reached in the
digestive tract.
We next investigated the mechanisms mediating the
biphasic effect of aspirin on BAEC apoptosis. Aspirin has
been shown to induce apoptosis of oesophageal cancer cells
and MCF-7 cells in a COX-dependent and independent
manner, respectively[5,19]. However, aspirin has also been
shown to inhibit endothelial cell apoptosis via its
antioxidant effects[20]. MAPK signal cascades have been
extensively studied in the regulation of cell proliferation and
apoptosis. There are mainly 3 subfamilies, of which, p44/42
MAPK is activated by growth factors and considered to be
related to cell growth and survival. In contrast, p38 MAPK
and stress-activated protein kinase (SAPK)/JNK are usually
activated by stress and pro-inflammatory cytokines, and are
closely associated with apoptosis. Recent studies indicate
that MAPK signal cascades mediate the effect of aspirin on
the proliferation or apoptosis of different cell
types[12,21,22]. In this study, we found that aspirin at low concentrations
inhibited H2O2-induced apoptosis as well as the
phosphorylation of p38 MAPK in BAEC. In contrast, at relatively high
concentrations, aspirin directly triggered apoptosis and p38
MAPK activation in BAEC. By using a specific p38 MAPK
inhibitor, SB203580, we provide convincing evidence that
p38 MAPK is involved in mediating the biphasic effect of
aspirin on endothelial cells. Previous studies reported in our
lab or by others have shown that SB203580 blocks p38
MAPK-mediated apoptosis in a stimulus-dependent
manner[16,23]. Recent studies have shown that there are at least 4
different subfamily members of p38 MAPK: α, β, γ, and
δ; SB203580 can only inhibit member α and
β[24]. Our data suggest that the member
α and/or β of p38 MAPK are involved in mediating the effects of aspirin on the apoptosis
of BAEC. However, we can not exclude the involvement
of the other 2 p38 MAPK sub-family members. Further
investigation is required to clarify this.
In conclusion, aspirin has a biphasic effect on the
apoptosis of BAEC. It can induce apoptosis at high
concentrations and inhibit apoptosis at low concentrations. p38 MAPK is an important signal molecule mediating the
effects of aspirin on endothelial cell apoptosis.
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