© 2000 American Heart Association, Inc.
Cellular Biology |
Extracellular Adenosine Induces Apoptosis of Human Arterial Smooth Muscle Cells via A2b-Purinoceptor
From the Unité INSERM 441, Pessac, France.
Correspondence to Dr Claude Desgranges, Unité INSERM 441, avenue du Haut-Lévêque, 33600 Pessac, France. E-mail claude.desgranges{at}bordeaux.inserm.fr
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Abstract—Apoptosis of arterial smooth muscle cells (ASMCs) could play an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that extracellular adenosine induces apoptosis in various cell types. Our aim was to delineate the capacity of this nucleoside to induce ASMC apoptosis in arterial diseases. We demonstrate that adenosine dose-dependently triggers apoptosis of cultured human ASMCs. Apoptotic cell death was quantified by analysis of nuclear chromatin morphology and characterized by DNA laddering. The involvement of adenosine receptors was suggested, because neither an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, nor an inhibitor of cellular nucleoside transport, dipyridamole, was able to inhibit adenosine-induced ASMC apoptosis. In contrast, an A1/A2-adenosine receptor antagonist, xanthine amine congener, totally inhibited adenosine-induced apoptosis. Furthermore, among more selective inhibitors of P1 purinoceptor subtypes, only alloxazine, an antagonist of A1- and A2-adenosine receptors, completely inhibited adenosine-induced ASMC apoptosis, suggesting that adenosine triggers ASMC apoptosis via either 1 or both of these receptors. However, 8-cyclopentyl-1,3-dipropylxanthine, 8-(3-chlorostyryl) caffeine, and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate, which are A1-, A2a-, and A3-adenosine receptor antagonists, did not inhibit adenosine-induced apoptosis, suggesting an involvement of the A2b-receptor in this process. Moreover, the cAMP increase followed by cAMP-dependent protein kinase activation appears essential to mediate adenosine-induced ASMC apoptosis, thus confirming the previous hypothesis. These results indicate that adenosine-induced apoptosis of ASMCs is essentially mediated via A2b-adenosine receptor and involves a cAMP-dependent pathway.
Key Words: apoptosis • adenosine • arterial smooth muscle cell • A2b purinoceptor • cAMP
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Introduction |
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Migration of arterial smooth muscle cells (ASMCs) from the media to the intima and their proliferation in the intimal space is thought to be responsible for intimal ASMC hyperplasia and consequently for intimal atherosclerotic thickenings.1 ASMCs from normal adult artery are quiescent because of the balance between the growth promoters and growth inhibitors locally present.1 2 Disruption of this balance in favor of growth promoters, possibly occurring after endothelial dysfunction, could trigger ASMC proliferation.3 However, in advanced atherosclerotic lesions, the rate of ASMC growth is close to baseline levels.4 In addition, a decrease in cellular density has been described in atherosclerotic lesions, leading to acellular zones.5 This cell decrease can be attributed to cell oncosis of the various cell types constituting the intimal lesions, ie, ASMCs, macrophages, and T lymphocytes, in response to accumulation of toxic factors such as oxidized LDLs.6 Moreover, recent studies have demonstrated that this cell death could also be related to cell apoptosis, although cell oncosis remains possible.5 7 8 9 10 11 Cell apoptosis can be distinguished from cell oncosis by morphological and biochemical criteria.12 Indeed, cells undergoing death by oncosis are characterized by membrane disruption, swollen cell organelles, and lack of chromatin condensation. In contrast, apoptotic cells show a nuclear and cytoplasmic condensation followed by DNA degradation into multiples of 180-bp internucleosomal fragments.13 The major cell types undergoing apoptosis in human atherosclerotic lesions are ASMCs5 7 8 10 and macrophages.9 ASMC apoptosis occurs not only in atherosclerotic lesions but also in human restenotic intimal lesions.8 Furthermore, ASMC apoptosis has also been described in animal models of intimal thickenings.14 The role of apoptosis in intimal thickening is uncertain. Apoptosis of intimal ASMCs may be a normal process involved in the control of hyperplasia in evolving intimal thickenings. In contrast, the high rate of ASMC apoptosis detected in advanced atherosclerosis may contribute to the destabilization of the fibrous lesion and, consequently, promote plaque rupture and its clinical consequences.
The factors and mechanisms triggering apoptosis in atherosclerosis essentially remain unknown. However, recent reports have shown that apoptosis of cultured ASMCs can be induced by physical agents15 ; by growth factor deprivation16 ; or by treatment with NO donors,16 oxidized LDL,17 reactive oxygen species,18 or various cytokines.19 Cyclic nucleotides probably participate in the ASMC apoptotic process via protein kinase activation.20 Because extracellular adenosine not only induces apoptosis of various cell types21 22 23 24 but also mediates an increase in NO production and cyclic nucleotide levels in vascular muscle,25 the effect of extracellular adenosine on ASMC apoptosis was evaluated. The various effects of extracellular adenosine were mediated by plasma membrane adenosine receptors of the P1 purinoceptor family. Currently, 4 adenosine receptors, termed A1, A2a, A2b, and A3, have been cloned and characterized by pharmacological studies.26 All of these P1 receptors belong to the family of 7 transmembrane G-protein–coupled receptors. However, A2a and A2b receptors activate adenylate cyclase, whereas A1 and A3 receptors inhibit this enzyme. All 4 P1 receptors have been demonstrated in ASMCs.27 28
In the present work, we show that extracellular adenosine induces apoptosis in cultured human ASMCs and demonstrate that this effect is essentially mediated via the A2b-adenosine receptor present on the ASMC surface membrane. Furthermore, we show that the cAMP-dependent pathway is involved in adenosine-induced apoptosis.
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Materials and Methods |
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Materials
Propidium iodide (PI) and 9-(tetrahydro-2'-furyl)adenine (SQ 22586) were products of Calbiochem. Adenosine, dibutyryl cAMP, forskolin, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), alloxazine, dipyridamole, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), Hoechst 33342 (H33342), and DNase-free RNase were purchased from Sigma. Xanthine amine congener (XAC), N6-cyclopentyladenosine (CPA), 2-(p-[2-carboxyethylphenylethylamino])-5'-N-ethylcarboxamidoadenosine (CGS-21680), N6-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (IB-MECA), 8-(3-chlorostyryl) caffeine (CSC), enprofylline, 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191), and protein kinase inhibitor 5-24 (PKI 5-24) were obtained from Research Biochemicals, Inc. F-10 HAM medium, FCS, and trypsin-EDTA were purchased from GIBCO.
ASMC Isolation and Culture
Human ASMCs were isolated from tunica media of aorta as
previously described.29 The cells were cultured in F-10
HAM medium supplemented with 10% FCS, penicillin (100 U/mL),
streptomycin (100 µg/mL), and 10 mmol/L HEPES buffer. Cells were
identified as ASMCs by their characteristic hills-and-valleys growth
pattern and by immunofluorescence detection of
-actin using an anti–-smooth muscle actin monoclonal antibody
(Sigma). Cells were passaged by trypsinization when they reached
confluence and were plated at a density of 104
cells/cm2 in polystyrene
75-cm2 flasks (Nunc, Inc). Culture media were
changed every 2 days. Cells from passages 5 to 10 were used in this
study.
Determination of Apoptosis
Experimental Protocols
ASMCs (1.6x104) in 250 µL of
F-10 HAM medium supplemented with serum were seeded on coverslips
(14-mm diameter) placed in 12-well culture plates (Falcon). After
adhesion, 2 mL of F-10 HAM medium containing 10% FCS were added to
each well. After 24 hours, the cells were rinsed with serum-free F-10
HAM medium (SFM) and then incubated in 2 mL of this medium with
or without various agents to test for their susceptibility to induce
apoptosis. Generally, ASMCs were incubated for 24 hours.
Quantitative Analysis of Apoptotic Nuclei by
Fluorescent Microscopy
Cell apoptosis was detected after incubation of cells
with the membrane-permeable fluorescent DNA-binding dye H33342.
Indeed, nuclei from normal cells demonstrated a normal uniform
chromatin pattern, clearly different from the characteristic condensed
or fragmented chromatin pattern of apoptotic cells. The
membrane-impermeable DNA-binding dye PI was used to identify nonviable
cells (cells in oncosis) with normal nuclei. At the end of each
incubation period with the tested compounds, H33342 (10 µg/mL) and PI
(1 µg/mL) were added to each well for an additional 30-minute
incubation at 37°C. Coverslips were then placed upside down on a
glass slide and immediately observed by fluorescent microscopy
using a photomicroscope (Nikon Microphot FXA) equipped with an
epifluorescence device. For each experiment, 300 to 400 nuclei
from 5 random fields of each coverslip were examined at high
magnification (x400). The percentage of apoptotic nuclei was
calculated as follows: (number of apoptotic nuclei/total number
of nuclei)x100. Each experiment was repeated 3 times. In these
experiments, apoptosis was quantified on the basis of the
number of adherent cells present after 24 hours of treatment.
However, for high concentrations of adenosine (
500
µmol/L), some apoptotic cells were detached from the glass
coverslips. Indeed, for 1 mmol/L adenosine, 
10% of
total cells became nonadherent. Therefore, for these concentrations,
the percentage of apoptotic nuclei was slightly
underestimated.
Analysis of Internucleosomal DNA Fragmentation: DNA
Laddering
ASMCs grown at confluence in 10% FCS in
75-cm2 flasks were incubated for an additional 24
hours in SFM in the presence or absence of adenosine. DNA
fragmentation was determined using an adaptation of a previously
described technique.30 Briefly, after shaking the flasks,
weakly adherent and nonadherent apoptotic cells were collected
by centrifugation of the cell culture medium. Cells of
the pellet were incubated at 37°C for 3 hours in a lysis buffer
consisting of, in mmol/L, Tris-HCl (pH 8.0) 10, EDTA 5, and NaCl
100, as well as 0.5% (vol/vol) SDS and 10 µg/mL proteinase K (Sigma)
under agitation. This incubation was followed by dropwise addition of 5
mol/L NaCl to a final concentration of 1 mol/L and an incubation at
4°C for 1 hour. After centrifugation at 12 000 rpm
for 30 minutes at 4°C, supernatants were recovered, and DNAs were
extracted with an equal volume of 25:24:1 phenol/chloroform/isoamyl
alcohol (vol/vol/vol) and precipitated in the presence of an equal
volume of isopropanol at -20°C overnight. After
centrifugation at 12 000 rpm for 10 minutes at 4°C,
the pellets were washed in 75% ethanol, resuspended in water, and
digested with 1 mg/mL DNase-free RNase for 30 minutes at 37°C. DNA
electrophoresis was carried out in 1.5% agarose gels containing 0.5
µg/mL ethidium bromide, and DNA fragments were visualized under UV
light.
Western Blot Analysis
Protein extracts were obtained from cultures by rinsing the
cells with ice-cold NaCl 9% (wt/vol) once. Cells were resuspended in
cold lysis buffer containing, in mmol/L, Tris-HCl (pH 7.5) 50,
NaCl 150, EDTA 3, and AEBSF (4-(2-aminoethyl)benzenesulfonyl
fluoride; Interchim) 0.1; 1% (vol/vol) Triton X-100; 0.25%
-amino-n-caproic acid; and 25 mg/L
1-antitrypsin. The cells were then incubated
for 45 minutes on ice and centrifuged at 12 000 rpm for 10
minutes at 4°C in a microcentrifuge. The supernatants were
stocked at 4°C. The protein concentrations were measured by the
method of Bradford31 with a Bio-Rad protein assay kit
according to the manufacturer’s instructions. BSA was used as a
protein assay standard.
Samples (80 µg protein) were denatured with SDS loading buffer at 95°C for 5 minutes and then separated under reducing conditions on a SDS/9% polyacrylamide gel with a 5% stacking gel in SDS/Tris/glycine running buffer.32 The protein was electrophoretically transferred to an Immobilon-P membrane (Millipore), which was then blocked with 5% (wt/vol) nonfat milk in TTBS buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, and 0.1% [vol/vol] Tween 20) for 1 hour at room temperature under agitation. The membrane was then incubated overnight with specific rabbit antiserum raised against human A2b-receptor at 2 µg/mL (Chemicon) in TTBS buffer supplemented with 1% (wt/vol) nonfat milk. The blot was incubated with biotin-labeled donkey anti-rabbit antibody (1/2000, Amersham) followed by streptavidin-biotin horseradish peroxidase detection (1/1000, Amersham) for 1 hour each at room temperature in TTBS buffer supplemented with 1% nonfat milk. The anti–A2b-receptor antibodies were then detected using an enhanced chemiluminescence detection system (Amersham) and exposed to x-ray film.
Control reactions included omission of the anti–human A2b-receptor antibody and neutralization of this antibody with a 40-fold (wt/wt) excess of the 16–amino acid peptide (ATNNSTEPWDGTTNES) used to raise the A2b-receptor antibody.33 This competitor peptide corresponds to a portion of the deduced amino acid sequence34 from the putative second extracellular loop of the human A2b receptor.
cAMP Measurements
Cells (1x105/mL) were plated in
24-well dishes in F-10 HAM medium containing 10% FCS for 24 hours.
Subsequently, they were incubated with the tested compounds or with SFM
for 10 minutes. The incubation was terminated by the addition of 0.5 mL
of 0.5N HClO4. Cells were scraped from the
dishes, collected into 1.5-mL tubes, and sonicated. The cell suspension
was then centrifuged at 14 000 rpm for 10 minutes at 4°C.
The supernatants were decanted and 18.8 µL of 9 mol/L KOH–0.25 mol/L
EDTA was added to 100 µL of supernatants. After precipitation, the
solution was centrifuged at 14 000 rpm for 10 minutes at
4°C, and the supernatants were stored at -20°C. The cAMP level of
the supernatant was determined by radioimmunoassay using a cAMP assay
kit (Immunotech). The values were normalized to the cell number of each
well and expressed as pmol cAMP/106 cells.
Statistical Analysis
Data are expressed as mean±SEM of 3 independent experiments.
Paired data were evaluated by Student t test. A 1-way ANOVA
was used for multiple comparisons. Significance was established when
the probability value was <0.01.
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Results |
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Induction of ASMC Apoptosis by Extracellular Adenosine
In controls, the majority of cells demonstrated a uniformly stained nucleus after staining with the membrane-permeable DNA-binding dye H33342 (Figure 1A
). A 24-hour
exposure of ASMCs to 1 mmol/L adenosine induced
morphological changes typical of apoptosis in 50% of the
cells, such as membrane blebbing, cytoplasm condensation, and nucleus
fragmentation with condensed chromatin, detected by staining with
H33342 (Figure 1B). The nucleus of the remaining cells was
uniformly stained with H33342. The nucleus of apoptotic cells
was not stained by PI, a membrane-impermeable DNA-binding dye,
demonstrating that these cells had not undergone a necrotic process.
However, in ASMCs treated with high concentrations of adenosine
(500 µmol/L), some cells had become permeable to PI. These
necrotic cells represented only a low percentage (<2%) of
total cells.
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Induction of ASMC apoptosis by adenosine was confirmed
by the presence of internucleosomal DNA fragmentation of
adenosine-treated cells into multimers of 180-bp
nucleosomal units (Figure 1C
). In control cells, only a slight
DNA fragmentation, probably due to growth factor
deprivation, was detected.
Adenosine-induced ASMC apoptosis, measured by detection
of chromatin condensation, was strongly dependent on the extracellular
adenosine concentration (Figure 2A). A faint but significant increase in
the percentage of apoptotic nuclei was demonstrated for an
adenosine concentration of 1 µmol/L (9±1% versus
7±0.1% in control cultures, n=3; P<0.01). The number of
apoptotic cells regularly increased up to 250 µmol/L
adenosine and then dramatically increased for higher
adenosine concentrations. An ED50 of
14 µmol/L can be deduced from the first part of the curve
corresponding to lower adenosine concentrations (1 to 250
µmol/L). Maximal apoptosis induction, reached for 1
mmol/L adenosine (49±1% of apoptotic nuclei at 24
hours), was maintained for higher concentrations.
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from the control
value without adenosine at the same time of incubation.
To assess the onset of adenosine-induced apoptosis, a
time-course study was performed (Figure 2B). Addition of 1
mmol/L adenosine to human ASMCs resulted in the onset of
apoptosis within 8 hours. Maximal apoptosis induction
was reached within 28 hours. At this time, 60% to 65% of ASMCs were
in apoptosis. A significant increase in the number of
apoptotic cells was detected in ASMCs incubated with SFM
(2.3±0.6% at 4 hours versus 8.7±1.2% at 8 hours [n=3];
P<0.01). However, this percentage remained low in
comparison with that obtained with 1 mmol/L adenosine.
Adenosine induced apoptosis even in the presence of low
percentages (0.1% and 0.5%) of serum in the culture medium, ie, in
culture conditions adequate for a good survival of cultured SMCs, as
demonstrated by the presence of cell divisions and the decrease in
basal apoptosis (Table 1). Indeed,
in the presence of 0.5% FCS, there was a significant decrease in the
percentage of apoptotic nuclei of untreated ASMCs (4.3±0.5%
versus 7.1±0.5% in SFM). In these conditions, increases in SMC
apoptosis induced by 1 to 100 µmol/L adenosine
were close to those induced in the absence of serum. The presence of
platelet-derived growth factor (20 ng/mL), an inhibitor
of growth factor depletion–induced apoptosis,16
in SFM decreased the basal level of SMC apoptosis.
Adenosine (1 µmol/L)–induced apoptosis was also
faintly decreased (Table 2). In contrast,
platelet-derived growth factor did not inhibit the
apoptosis induced by higher doses of adenosine (10 and
100 µmol/L).
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Cellular Mechanisms of Adenosine-Induced ASMC
Apoptosis
Adenosine-induced ASMC apoptosis might occur in 1
of the following 3 different ways: (1) via a catabolic product of
adenosine, (2) intracellularly after adenosine uptake
by ASMCs, and (3) after adenosine binding to 1 or more of its
P1-specific receptors.
As shown in Figure 3A, the
adenosine deaminase inhibitor EHNA, which blocked
the catabolism of adenosine to inosine, was unable to inhibit
adenosine-induced ASMC apoptosis. Indeed, percentages
of apoptotic nuclei after treatment of ASMCs in the presence of
adenosine (100 µmol/L), and adenosine (100
µmol/L) plus EHNA (10 µmol/L), were 21±2% for
adenosine and 21±1% for adenosine plus EHNA, a
condition in which adenosine degradation was entirely
inhibited. This result suggests that adenosine induces ASMC
apoptosis by itself, but not via one of its catabolic
products, as confirmed by the fact that the first adenosine
catabolic product (inosine) only induced a faint apoptosis
in comparison with adenosine (21±2% for 100 µmol/L
adenosine versus a mean of 10±1% for 100 µmol/L
inosine and 8±1% in control).
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To determine whether adenosine-induced apoptosis
requires adenosine entry into the cells, the action of
dipyridamole, an inhibitor of facilitated
intracellular transport of adenosine, was studied. As shown in
Figure 3B, coadministration of 1 µmol/L
dipyridamole with 100 µmol/L adenosine
had no effect on adenosine-induced ASMC apoptosis
compared with 100 µmol/L adenosine alone. Indeed, the
percentages of apoptotic nuclei in ASMCs incubated for 24 hours
with 100 µmol/L adenosine in the presence and absence of
dipyridamole were 20±0.1% with 1 µmol/L
dipyridamole and 19.7±0.6% without (n=3,
P>0.01).
Because adenosine did not induce apoptosis of human
ASMCs either via adenosine catabolic products or by an
intracellular pathway, the role of
P1-adenosine receptors was considered. To
study this pathway, we used XAC, an
A1/A2-adenosine
receptor antagonist. Adenosine-induced
apoptosis was dose-dependently inhibited by the
simultaneous application of XAC (Figure 3C). At
10 µmol/L, XAC entirely inhibited apoptosis induced by
100 µmol/L adenosine in human ASMCs. These findings
suggest that adenosine induces apoptosis via a
P1-purinoceptor.
Role of A2b Purinoceptor in Human ASMC
Apoptosis
To determine the adenosine receptor subtypes involved in
ASMC apoptosis induction, we first studied the effects of
various adenosine receptor agonists. The partially selective
agonists used in our experiments were CPA, an
A1-adenosine receptor agonist; CGS-21680,
an A2a-adenosine receptor agonist; and
IB-MECA, an A3-adenosine receptor
agonist. As shown in Figure 4, these
different adenosine agonists induced ASMC apoptosis in
the same range, so the P1-purinoceptor
responsible for the adenosine apoptotic effect could
not be established. This result is probably due to the fact that at the
concentrations used in this study, these agonists are not selective for
the targeted receptor.35 36
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Secondly, the effects of various adenosine receptor
antagonists were studied to characterize the
P1-purinoceptor(s) involved in
adenosine-induced ASMC apoptosis. DPCPX (an
A1-adenosine receptor
antagonist), CSC (an
A2a-adenosine receptor
antagonist), and MRS 1191 (an
A3-adenosine receptor
antagonist) were unable to inhibit ASMC apoptosis
induced by adenosine (data not shown). In contrast, 10
µmol/L of alloxazine, an
A1/A2-adenosine
receptor antagonist, entirely inhibited ASMC
apoptosis induced by 100 µmol/L adenosine
(Figure 5A). Furthermore, 10
µmol/L enprofylline, another adenosine receptor
antagonist demonstrating a rather high specificity for
A2b receptors37 (A2bRs), blocked
ASMC apoptosis triggered by 100 µmol/L adenosine
(20±1% for 100 µmol/L adenosine versus 9±0.6% for
adenosine plus 10 µmol/L enprofylline, 8±0.5% in
controls and 9±1% for 10 µmol/L enprofylline). The
inhibitory effect of alloxazine was confirmed by DNA
laddering (Figure 5B). Indeed, coincubation of alloxazine
(10 µmol/L) with adenosine (100 µmol/L) abolished
the internucleosomal DNA fragmentation induced by adenosine
alone (100 µmol/L). These results suggest that
adenosine-induced human ASMC apoptosis may involve
mediation via the A2bR, given that A1-,
A2a- and A3-receptor
antagonists did not block the proapoptotic effect
of adenosine, whereas nonspecific A2bR antagonists
inhibited this effect.
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Involvement of cAMP-Dependent Pathway in Adenosine-Induced
ASMC Apoptosis
Activation of A2bRs, which are positively coupled with
adenylate cyclase, results in a significant increase in
intracellular cAMP levels. In contrast, stimulation of
A1-receptor induces a cAMP decrease. Therefore,
to confirm the role of A2bR in adenosine-induced
apoptosis, we examined the role of the cAMP-dependent
cell-signaling pathway in this process.
First, we found that adenosine concentrations from 1 to
100 µmol/L triggered a dose-dependent increase in ASMC cAMP
levels (Table 3). The SMC cAMP content
was increased by 3.4-fold after 100 µmol/L adenosine
treatment.
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Second, we demonstrated that the cAMP elevation induced ASMC
apoptosis by the following 3 experimental approaches: (1) using
a stable cAMP analogue, (2) by stimulation of adenylate
cyclase, and (3) by inhibition of cAMP-dependent phosphodiesterase.
As shown in Figure 6A, the administration
of dibutyryl cAMP, a membrane-permeable cAMP analogue, induced ASMC
apoptosis in a manner similar to that observed with
adenosine. The apoptotic effects of adenosine
on ASMC were also mimicked in a concentration-dependent manner by
forskolin38 (Figure 6B), an activator
of adenylate cyclase, and by rolipram39 (data
not shown), an inhibitor of cAMP-dependent
phosphodiesterase. Forskolin at 5 µmol/L induced a cAMP increase
comparable with that triggered by 100 µmol/L adenosine
(Figure 6C). In both cases, the percentage of apoptotic
cells was in the range of 20%.
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Because the increase in cAMP levels triggered ASMC death, we tested the
hypothesis that adenosine-induced apoptosis is mediated
by a cAMP-dependent pathway. Therefore, we investigated the effect of
blocking cAMP accumulation by inhibition of adenylate
cyclase. The adenylate cyclase inhibitor SQ
2253640 (5 µmol/L) had minimal effects on baseline
cell viability in the absence of adenosine. Coadministration of
SQ 22536 with adenosine (100 µmol/L) significantly
abolished adenosine-induced ASMC apoptosis (Figure 7A). Furthermore, we tested the
hypothesis that the induction of ASMC apoptosis by
adenosine was related to the cAMP-dependent activation of
cAMP-dependent protein kinase. The cAMP-dependent protein kinase
inhibitor PKI 5-2441 alone failed to trigger
ASMC apoptosis (Figure 7B). However, coincubation of PKI
5-24 with adenosine (100 µmol/L) blocked
adenosine-induced ASMC apoptosis. These findings
confirm the role of A2bR in adenosine-induced apoptosis
and suggest that this induction is essentially mediated by a
cAMP-dependent pathway.
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A2b Expression in Cultured ASMCs
Western analysis of cultured ASMCs using an anti-A2bR
antibody identified a protein band of 52 kDa (Figure 8), corresponding to the molecular mass
expected for this receptor (50 to 55 kDa).33 To confirm
that the 52-kDa band was due to the A2bR, we preincubated the anti-A2bR
antibody with a 40-fold excess of the A2bR competitor peptide. In this
condition, the 52-kDa immunoreactive band was not detected; nor was it
detectable when blots were developed with secondary antibody alone
(data not shown).
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The present study tests the potentiality of extracellular adenosine to trigger ASMC death by apoptosis. The addition of adenosine to the culture medium of human ASMCs causes an extensive cell death due mainly to apoptosis, as suggested by chromatin condensation and internucleosomal DNA fragmentation. An ASMC apoptosis increase was detected with a 1 µmol/L adenosine concentration, and a maximal value was reached at 1 mmol/L. However, high adenosine concentrations (up to 500 µmol/L) could also trigger ASMC death by oncosis, but only in a low percentage of dying cells.
Among the various mechanisms that could be involved in adenosine-induced apoptosis (ie, activation of adenosine membrane receptors, intracellular action of adenosine, or generation of an active adenosine catabolic product), it appears that adenosine acts through its binding to a specific adenosine membrane receptor of ASMCs. Indeed, neither the blockade of adenosine catabolism nor that of adenosine intracellular uptake inhibited adenosine-induced apoptosis. Consequently, it seems that these apoptotic effects of adenosine result from an extracellular action that is presumably receptor mediated. As a confirmation of this hypothesis, complete inhibition of adenosine-induced apoptosis was achieved by XAC, an antagonist of A1- and A2-adenosine receptors. Moreover, apoptosis induced by adenosine was entirely inhibited by 10 µmol/L alloxazine or enprofylline. At this concentration, alloxazine presents a higher specificity for A1- and A2-adenosine receptors42 43 but was not effective on A3-adenosine receptor, which has been demonstrated to mediate adenosine-induced apoptosis in other cell types.21 22 23 24 Similarly, enprofylline was identified as a selective antagonist of recombinant A2bR.37 The involvement of A2bR is suggested by the fact that alloxazine and enprofylline inhibited adenosine-induced ASMC apoptosis, whereas DPCPX, CSC, and MRS 1191, the respective antagonists of A1-, A2a-, and A3-receptors, were unable to block it.
Furthermore, adenosine activation led to an increase in cellular cAMP content, a characteristic of the A2bR that stimulates adenylate cyclase via Gs protein activation.26 Consequently, we hypothesize that adenosine-induced apoptosis is mediated, at least in part, via a cAMP increase. We observed that the apoptotic effect of adenosine was mimicked by a membrane-permeable cAMP analogue or by an increase in intracellular cAMP, by stimulating adenylate cyclase or inhibiting phosphodiesterase. The role of the cAMP-dependent cell-signaling pathway in adenosine-induced ASMC apoptosis was evidenced, demonstrating that the apoptotic effects of adenosine were almost entirely abolished both when cAMP elevation was blocked by adenylate cyclase inhibition and when protein kinase A activity was specifically inhibited. Moreover, a clear parallelism was demonstrated between the cAMP level increase and the percentage of apoptotic cells after SMC treatment with adenosine concentrations ranging from 1 to 100 µmol/L, ie, for concentrations around the EC50 value found for adenosine-induced ASMC apoptosis (14 µmol/L) and for adenosine-induced cAMP level (7 µmol/L) in ASMCs and in cells expressing the recombinant adenosine low-affinity A2bR (10 µmol/L).44 Taken together, these findings provide the first evidence that extracellular adenosine may trigger ASMC apoptosis via the A2bR and suggest that this nucleoside induces ASMC apoptosis by stimulation of the cAMP/PKA signal transduction pathway. The role of A2bR is strengthened by the demonstration of A2bR on the surface membrane of cultured human ASMCs. These results confirm the role of adenosine in cell death but also the variability of the mechanisms involved in adenosine-induced apoptosis according to the cell type. Indeed, it has been shown that adenosine may induce apoptosis via its internalization23 or by binding to specific P1 receptor(s).21 22 24 In this case, the intracellular mechanisms are poorly understood, although cAMP seems to be involved in some cases.21 Our study clearly demonstrates that the apoptotic effect of adenosine on ASMC depends on binding on the A2bR and on the cAMP pathway.
Extracellular adenosine might thus be involved in the ASMC apoptosis arising in atherosclerotic and restenotic intimal lesions5 7 8 10 and consequently might play an important role both in the control of hyperplasia and in plaque weakening. Moreover, extracellular adenosine has been described as an inhibitor of ASMC proliferation in vitro45 46 and in vivo.47 The A2bR seems particularly involved both in adenosine-induced apoptosis (this study) and in adenosine-mediated inhibition of proliferation of ASMCs,45 46 probably by increasing the cAMP concentration. However, other intracellular pathways might be involved in adenosine-inhibited ASMC growth. Indeed, adenosine enhances the NO or the cytokine-induced NO synthesis in rat aortic ASMCs,48 49 and it has been demonstrated that NO is also able to induce ASMC apoptosis16 and to inhibit ASMC growth.50 It seems that NO, which may be generated during adenosine treatment, does not intervene significantly in apoptosis induction by adenosine, given that NG-nitro-L-arginine (NO synthase inhibitor) did not block adenosine-induced apoptosis (data not shown). Moreover, although adenosine induces NO production, this activation does not depend on the cAMP/PKA pathway,48 whereas adenosine-induced apoptosis is greatly dependent on it.
The minimal concentration of adenosine required to induce ASMC apoptosis was 1 µmol/L. Adenosine concentrations of 1 to 100 µmol/L increase the percentage of apoptosis in the range of 2% to 13% of apoptotic cells, a value compatible with the apoptosis rate found in atherosclerotic plaque or in restenotic lesions.8 9 10 The adenosine concentration in the interstitial space within the arterial space or intimal thickening has not yet been evaluated. However, interstitial adenosine concentrations have been evaluated in some tissues. In skeletal muscle, this concentration was 0.44 µmol/L during normoxia and 0.85 to 1.03 µmol/L during hypoxia.51 The 0.2 to 1 µmol/L basal concentration found in myocardial interstitial fluid increases to 0.7 to 6 µmol/L during ischemia.52 53 54 55 For the arterial wall, several in vitro studies demonstrate that both endothelial cells and ASMCs release adenosine and its precursor ATP on stimulation by various physicochemical agents such as hypoxia, shear stress, or free radicals,56 57 thus suggesting that extracellular adenosine concentrations comparable with those found in other tissues may occur. Extracellular concentrations of ATP and adenosine released by endothelial cells or ASMCs are dependent of the volume of the local extracellular compartment. However, recent studies have demonstrated that the concentration of released ATP could be higher in the vicinity of the releasing cells because of a concentration gradient58 or the release in membrane structures also containing the effector system, particularly purinergic receptors.59 It is also noticeable that ASMCs can metabolize cAMP to generate releasable adenosine via the cAMP-adenosine pathway.25 Therefore, the cAMP increase induced by A2bR stimulation not only directly triggers ASMC apoptosis, but also promotes the regeneration of adenosine, hence allowing the maintenance of the adenosine proapoptotic effect. In addition to the release from intact cells, adenosine and its precursors can be released in large quantities from dying cells found within intimal thickenings.5 6 7 8 9 10 So, adenosine concentrations in the range 1 to 10 µmol/L could be generated within the intimal thickenings and participate in the ASMC apoptosis found in these lesions.
Therefore, adenosine could play a dual role in the evolution of intimal thickening. First, it could restrict intimal hyperplasia not only via its antiproliferative45 46 and apoptotic effects on ASMCs, but also by promoting re-endothelialization via its mitogenic effect on endothelial cells.60 Secondly, it could play an important role in the formation of the necrotic core in advanced atherosclerotic lesions by triggering not only the death of ASMCs but also that of macrophages via the NO pathway.61 62 Indeed, an initial event such as hypoxia63 could induce a first wave of cell death due to adenosine release. Adenosine generated from ATP64 released from dead cells might in turn lead to the lysis of surrounding cells and, by a cascade of events, to necrotic core spread. Then, in synergy with other events such as matrix degradation, this might lead to plaque rupture. Furthermore, adenosine could also participate in the decrease in the number of cells in the fibrous cap and thus promote plaque weakening.
Therefore, according to the status of arterial intimal lesions, adenosine can be considered either as a beneficial factor controlling intimal thickening formation and even regression14 or as a deleterious one leading to plaque rupture and its dramatic clinical consequences. Consequently, if these opposing effects were confirmed in vivo, different strategies favoring the role of adenosine during intimal lesion development, or in contrast inhibiting its effects in advanced plaques, might be considered.
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Acknowledgments |
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This work was supported by grants from INSERM, from Etablissement Public Régional 96.0301302, and from Fondation de France p8002218.
Received August 23, 1999; accepted October 12, 1999.
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