© 2000 American Heart Association, Inc.
Vascular Biology |
Endothelin-1 Inhibits Apoptosis of Vascular Smooth Muscle Cells Induced by Nitric Oxide and Serum Deprivation via MAP Kinase Pathway
From the Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
Correspondence to Masayoshi Shichiri, MD, PhD, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail mshichiri.med2{at}med.tmd.ac.jp
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Abstract—Endothelin (ET)-1, an endothelium-derived vasoconstrictor and mitogen, acts as an antiapoptotic factor against serum deprivation–induced apoptosis of endothelial cells and fibroblasts but enhances apoptosis of some cancer cells. In the present study, we examined whether nitric oxide (NO) and ET-1 modulate apoptosis of rat vascular smooth muscle cells (VSMCs) via the mitogen-activated protein (MAP) kinase pathway. Both serum deprivation and NO donors (FK409 and SNAP) caused apoptosis of VSMCs, as demonstrated by TdT-mediated dUTP-biotin nick end-labeling, appearance of fragmented DNA, and induction of caspase-3 activity. ET-1 dose-dependently antagonized apoptosis induced by serum deprivation and NO donors. A selective ETA receptor antagonist (BQ123) and a nonselective ETA/B receptor antagonist (TAK044), but not a selective ETB receptor antagonist (BQ788), inhibited the antiapoptotic effect of ET-1, indicating that the antiapoptotic effect of ET-1 is mediated via the ETA receptor. ET-1 activated MAP kinase, whose effect was inhibited by FK409. Transfection with an unphosphorylated wild-type MAP kinase kinase-1 (MAPKK-1) or its constitutively activated mutant protected VSMCs against apoptosis induced by serum deprivation and NO donors. Inhibition of MAP kinase activity with PD98059, a specific inhibitor of MAPKK-1, or by transfection of a dominant-negative MAPKK-1 mutant antagonized the antiapoptotic effect of ET-1, suggesting the involvement of MAP kinase in the antiapoptotic effect. The potent inhibitory effect of ET-1 on apoptosis of VSMCs induced by serum deprivation and NO suggests that the counterbalance between the 2 endothelium-derived factors contributes to the process of vascular remodeling by determining VSMC survival and death, respectively, via a common MAP kinase pathway.
Key Words: endothelin • endothelium-derived factors • apoptosis • remodeling
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Apoptosis, a genetically controlled cell death program, is an important determinant of cell number under physiological and certain pathological conditions. Apoptosis is associated with distinct morphological and biological events, such as cellular shrinkage, membrane blebbing, and chromatin condensation and fragmentation. Apoptosis is under the control of environmental stimuli, such as serum deprivation, irradiation, chemotherapeutic agents, and antioxidants,1 whereas certain growth factors and cytokines are known to inhibit apoptosis.2 3 Overexpression of c-Myc protein combined with serum deprivation induces apoptosis in several cell types, including vascular smooth muscle cells (VSMCs).4 5 Using diploid fibroblast cell lines with 1 endogenous c-myc gene copy knocked out by gene targeting, we recently demonstrated that serum deprivation–induced apoptosis is dependent on endogenous c-myc expression,6 suggesting that cells without ectopic oncogene overexpression may also undergo apoptosis in the event of growth factor shortage. To date, 2 general models have been proposed for apoptosis induced by serum deprivation: (1) a conflict of growth-promoting and growth-inhibitory signals triggers apoptosis,7 and (2) c-Myc directly activates an apoptotic pathway whose execution is regulated by the availability of survival factors.2
A potent endothelium-derived relaxing factor, nitric oxide (NO), has also been implicated in the regulation of apoptosis. Whether NO induces or suppresses apoptosis appears to depend on species or cell type, or alternatively, on experimental design. In macrophages,8 thymocytes,9 tumor cells,10 and pancreatic ß-cells,11 NO is proapoptotic, whereas in lymphocytes and lymphoma cells, NO functions as a survival-promoting factor.12 13 In cultured VSMCs, NO donor has been reported to inhibit proliferation14 and induce apoptosis.15 16 We recently confirmed the apoptotic effects of excess NO on VSMCs after transfection of rat inducible NO synthase gene.17
Endothelin (ET)-1, a potent vasoconstrictor peptide originally isolated from the vascular endothelium, regulates vascular remodeling as well as vascular tonus.18 Despite the distinct function of ET-1 as a vasoconstrictor and mitogen, its role as a modulator of apoptosis remained unappreciated until we found that ET-1 suppresses serum deprivation–induced apoptosis of rat fibroblasts6 and endothelial cells19 as well as NO donor–induced apoptosis of rat endothelial cells.20 In contrast, it has been reported that ET-1 enhances apoptosis of human melanoma cell lines.21
Accumulating evidence suggests that both ET-1 and NO are involved in the process of vascular remodeling, migration, proliferation, and extracellular matrix accumulation of VSMCs.22 23 A close interaction between ET-1 and NO is thought to reduce the magnitude of their opposing actions, such as regulation of vascular tonus; ET-1 stimulates NO production,24 whereas NO inhibits ET-1 production in endothelial cells.25 However, little information is available on whether ET-1 and NO interact with each other to affect cell survival or apoptosis of VSMCs. Therefore, the present study was designed to determine whether (1) ET-1 affects apoptosis of VSMCs induced by serum withdrawal and NO and (2) the mitogen-activated protein (MAP) kinase pathway is involved in the mechanism of the cell survival effect of ET-1 in VSMCs.
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Materials
DMEM was purchased from Flow Laboratories, FBS from Cell Culture Laboratories, and synthetic ET-1 from Peptide Institute, Inc. The ETA/B receptor antagonist TAK044 was supplied by Takeda Research Laboratory. The ETA receptor antagonist BQ123 and ETB receptor antagonist BQ788 were generously provided by Banyu Research Laboratory, and the NO donor FK409 [(+)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide, NOR3] by Fujisawa Pharmaceutical. PD98059 was purchased from Amersham International and S-nitroso-N-acetyl penicillamine (SNAP) from Biomol Research Laboratories. All other compounds or reagents were of molecular biology grade. The plasmids MAP kinase kinase-1 (MAPKK1) WT, an unphosphorylated wild-type MAPKK1 expressed from the SV40 early promoter; MAPKK1 S218D/S222D, a constitutively activated form; and MAPKK1 S222A, a dominant-interfering form were kindly provided by Drs Anne Brunet and Jacques Pouyssegur (Centre de Biochime-CNRS, Université de Nice, France).26
Cell Culture
Rat aortic VSMCs were prepared by the explant method as reported
previously27 and cultured in DMEM supplemented with 10%
FBS in 5% CO2 atmosphere at 37°C in a Costar
Polystyrene dish or multiwell plates (Corning Inc), and the cells (6th
to 12th passages) were used in the experiments. To produce
apoptosis by serum deprivation, cells were washed
with PBS, the medium was replaced with serum-free DMEM, and the cells
were incubated for the indicated times. To elicit NO-induced
apoptosis, medium was replaced with DMEM supplemented with 1%
FBS containing FK409 or SNAP, and the cells were incubated for the
indicated times.
Transfection
Transient transfection was performed with the synthetic cationic
liposome
(+)-N,N-[bis(2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl]
ammonium iodide according to instructions provided by the supplier
(Promega) with the following modification using
transferrin-receptor–operated transfer.28 In brief,
3 µL of liposome was added to 100 µL HEPES-buffered saline (20
mmol/L HEPES, pH 7.4, and 100 mmol/L NaCl) containing 16 µg
human holo-transferrin (Sigma Chemical Co), incubated for 20 minutes at
room temperature, mixed with 100 µL HEPES-buffered saline containing
various amounts of DNA mixtures, and further incubated for 15 minutes.
DNA-liposome-transferrin complex was then overlaid on cells that had
been covered with serum-free DMEM and incubated for 2
hours.19 DNA-liposome complex was confirmed to form
multilamellar vesicles known to exert high transfection
efficiency.29 30 To determine the transfection efficiency,
ß-galactosidase–expressing construct was introduced in the same
manner as indicated above. Regardless of the DNA concentrations used
(0.05 to 1 µg/well), nearly 100% of total cells were transfected,
and the intensity of the reporter signal correlated well with the DNA
concentrations used (data not shown).
Detection of Apoptosis
For demonstration of nucleosomal ladders, cellular fragmented
DNA was extracted by the NP-40 lysis method, which efficiently
eliminates intact chromatin.31 Floating and/or adherent
VSMCs in 10-cm dishes were collected, and apoptotic DNA
fragments were extracted with NP-40, fractionated on 1.6% agarose gel
electrophoresis, and stained with ethidium bromide as reported
previously.6 19 32
Both floating and adherent cells were stained for free 3'-hydroxyl ends of DNA fragments with dUTP-FITC with an APO-DIRECT apoptosis detection kit (PharMingen) and counterstained for total DNA content with propidium iodide. Stained cells were then analyzed with a FACS Calibur flow cytometer (Becton-Dickinson).33
Apoptotic cells were also detected in situ by the terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL) method using an in situ cell death detection kit (Takara Biomedicals) as described previously.6 In brief, cells grown on LAB-TEK Chamber Slides (Nalge Nunc International) were fixed for 15 minutes in 4% paraformaldehyde in PBS, blocked for 15 minutes with 0.3% H2O2 in methanol, washed, and permeabilized for 2 minutes with 0.1% sodium citrate in PBS, followed by sequential exposure to the enzymatic reaction mixture for 60 minutes at 37°C, anti-FITC horseradish peroxidase conjugate for 30 minutes at 37°C, and 0.05% diaminobenzidine in 1% nickel sulfate and 0.01% H2O2. To quantify the extent of apoptosis, we calculated the percentage of TUNEL-positive cells relative to total cell population by counting all cells from 5 random microscopic fields at a magnification of x100.
Enumeration of Viable and Floating Apoptotic Cells
Rat VSMCs plated in 24-well dishes (104
cells per well) were incubated in DMEM containing 10% FBS for 48
hours. The cells were extensively washed with PBS, then incubated in
fresh DMEM supplemented or unsupplemented with 1% FBS, in the presence
of the compounds tested. After 24 hours, all floating cells were
collected after 2 washes with PBS. All adherent cells were also
collected after trypsinization for quantitative analysis of
total apoptotic events in a given cell
population,6 19 and the numbers of the floating and
adherent cells were determined with a Sysmex CDA-500
Autoanalyzer (Toa Medical Electronics).
Determination of MAP Kinase Activity
An extracellular signal-regulated kinase (ERK)/MAP
kinase–dependent reporter system (PathDetect Trans-Reporting System,
Stratagene) was used to measure MAP kinase activity. The system
includes the fusion activator plasmids that consist of the
DNA-binding domain of the yeast GAL4 fusion activator
(pFR-Luc) and the activation domain of the Elk1 transcription factor
(pFC2-Elk1). Cells plated in 96-well plates were cotransfected with
pFC2-Elk1 (50 ng each per well), pFR-Luc reporter plasmid (1 µg each
per well), pRL-TK vector (1 µg each per well) (Promega) expressing
Renilla luciferase as an internal control, and with or
without MAPKK mutants as indicated in the text. The cells were
incubated for 24 hours after transfection in DMEM containing 10% FBS,
switched to serum-free medium for an additional 24 hours, pretreated or
untreated with PD98059 for 1 hour, and then treated with ET-1 for the
indicated time, after which firefly and Renilla luciferase
activities were measured with the Dual Luciferase Reporter Assay System
(Promega) in a single-tube assay format using
MicroLumatPlus (EG&G
Berthold).34 The firefly luciferase activity of each
sample was normalized to an internal reference standard of
Renilla luciferase activity.
Determination of Caspase-3 Activity
Caspase activity was assayed with the CaspACE Assay System,
Colorimetric (Promega). Treated cells in 6-cm dishes
were washed twice with PBS and centrifuged, and the pellets
were resuspended in precooled cell lysis buffer. Lysates were
centrifuged at 15 000 rpm at 4°C for 20 minutes, and protein
concentrations were determined (Pierce Chemical Co). Extracts were
stored at -80°C until assayed. Aliquots of protein (25 µg) were
incubated with 2 µmol/L of the caspase-3 substrate
Ac-DEVD-p-nitroaniline in a total volume of 100
µmol/L at 37°C for 4 hours. The colorimetric
release of p-nitroaniline from the Ac-DEVD-pNA substrate was
recorded at 405 nm. Enzymatic activity for caspase-3 was linear
over the range of protein concentrations used to calculate the specific
activity.
Statistical Analysis
Data are expressed as mean±SEM. Statistical analysis
was performed by unpaired Wilcoxon’s t test. A
value of P<0.05 was considered statistically
significant.
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ET-1 Inhibits Serum Deprivation–Induced Apoptosis of VSMCs
After serum deprivation, a fraction of VSMCs started to show morphological features characteristic of apoptosis, such as membrane blebbing, cellular shrinkage, nuclear condensation and fragmentation, followed by detachment from the culture plates. By the NP-40 lysis method, which efficiently eliminates intact chromatin,31 fragmented DNA was extracted from all floating and adherent VSMCs after serum deprivation for 6 to 24 hours and subjected to agarose gel electrophoresis. Nucleosomal ladders in DNA samples extracted from serum-deprived VSMCs were visible as early as 6 hours, whereas the addition of ET-1 in higher concentrations (10-9 to 10-6 mol/L), but not in lower concentrations (10-12 to 10-10 mol/L), resulted in a dose-dependent inhibition of DNA fragmentation (Figure 1A
). VSMCs were analyzed by flow
cytometry using cells stained for free 3'-hydroxyl ends of DNA with
FITC. When floating and adherent cells were analyzed separately
24 hours after serum deprivation, adherent cells contained
a limited number of positive cells, whereas floating cells were
apoptotic (Figure 1B). Few apoptotic cells were
detected among growing VSMCs incubated in DMEM containing 10% FBS.
Apoptosis increased markedly after serum
deprivation, whereas ET-1 (10-7
mol/L) markedly suppressed the fraction of apoptotic cells.
These results indicate that serum deprivation induces
apoptosis of VSMCs and ET-1 inhibits serum
deprivation–induced apoptosis.
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To determine which receptor subtype (ETA,
ETB) is responsible for the antiapoptotic
effect of ET-1, we examined the effects of several ET receptor
antagonists on serum deprivation–induced VSMC
apoptosis (Figure
IA; Figures I through IV can be found online
at http://atvb.ahajournals.org/cgi/content/full/20/4/989/DC1).
Both an ETA receptor antagonist,
BQ123 (10-7 mol/L), and a nonselective
ETA/B receptor antagonist, TAK044
(10-7 mol/L), abrogated the
inhibitory effect of ET-1 (10-8
mol/L) on apoptosis, whereas BQ788 (10-6
mol/L), an ETB receptor antagonist,
did not have any effect; neither ET receptor antagonists
added alone nor an ETB receptor agonist, BQ3020
(10-6 mol/L), showed any appreciable effect. DNA
strand breaks caused by endonuclease were detected in situ by the TUNEL
method using adherent VSMCs (Figure IB, online). In contrast to
negative staining in cells cultured with 10% FBS, positive staining
was visible in many nuclei of adherent cells deprived of serum for 6
hours (19.7±4.5%, n=5), whereas ET-1 (10-8
mol/L) markedly reduced the number of TUNEL-positive cells
(1.1±0.6%). Coincubation with BQ123 (10-7
mol/L) abrogated the antiapoptotic effect of ET-1
(18.8±4.3%), whereas coincubation with BQ788
(10-7 mol/L) did not show any effect
(1.0±0.7%) (Figure IB, online). These results indicate that
the ET-1–induced antiapoptotic effect is mediated via the
ETA receptor.
ET-1 Inhibits NO-Induced Apoptosis of VSMCs
We next examined whether ET-1 inhibits NO-induced
apoptosis of VSMCs. Minimal nucleosomal laddering was noted in
DNA samples extracted from VSMCs incubated with 1% FBS during 24
hours. In contrast, the addition of a potent NO donor, FK409
(10-4 mol/L), caused a distinct nucleosomal
ladder, whose effect was abrogated by coincubation with ET-1
(10-7 to 10-6 mol/L)
(Figure IIA, online). These findings were further confirmed by flow
cytometric analysis (Figure IIB, online): compared with
negative apoptotic cells before NO donor addition, FK409
(10-4 mol/L) increased the apoptotic
fraction; however, its effect was reduced by coincubation with ET-1
(10-7 mol/L). Similar results were obtained when
another potent NO donor, SNAP (10-4 mol/L), was
used (Figure II, online). When VSMCs were incubated in a medium
to which FK409 (10-4 mol/L) or SNAP
(10-4 mol/L) had been added for 24 hours to
allow NO donors to decay spontaneously, no distinct nucleosomal
laddering was observed, suggesting that the effect was not due to any
of their decomposition products. The in situ TUNEL method (Figure
IIIA, online) further showed that FK409 (10-4
mol/L) caused the appearance of many TUNEL-positive cells (22.6±5.4%,
n=5), which were reduced by coincubation with ET-1
(10-7 mol/L) (5.3±2.6%). BQ123
(10-6 mol/L) abolished the cell-protective
effect of ET-1 (25.2±6.3%), whereas BQ788
(10-6 mol/L) did not show any effect
(6.8±2.6%). Because most VSMCs undergoing apoptosis were
detached from culture plates and floated, the number of floating
apoptotic cells was measured (Figure IIIB, online).
FK409 (10-4 mol/L) significantly increased the
number of floating apoptotic cells. The effect of FK409 was
markedly suppressed by ET-1 (10-7 mol/L); both
BQ123 (10-6 mol/L) and TAK044
(10-6 mol/L) abrogated the antiapoptotic
effect of ET-1, whereas BQ788 (10-6 mol/L)
failed to show any effect. These results indicate that ET-1 also
inhibits NO-induced apoptosis of VSMCs via the
ETA receptor.
Antiapoptotic Effect of ET-1 Is Mediated by MAP Kinase
Activation
Because the intracellular signaling mechanism that mediates
antiapoptosis involves the MAP kinase pathway in PC-12
cells,35 we examined whether ET-1 activates MAP
kinase in rat VSMCs. ET-1 (10-7 mol/L)
stimulated MAP kinase activity in VSMCs deprived of serum for 48 hours.
Further experiments showed that this effect was concentration-dependent
(10-8 to 10-6 mol/L,
Figure IVA, online). The results were confirmed by
phosphorylation of p42 and p44 as demonstrated by
immunoblot analysis using anti-phosphotyrosine
antibody and by enzyme activity data obtained with an in vitro p42/p44
kinase assay system (Amersham) using
[
-32P]ATP (data not shown).6
Moreover, FK409 dose-dependently (10-5 to
10-4 mol/L) reduced MAP kinase activity
stimulated by ET-1 (10-7 mol/L) (Figure
IVA, online). In contrast to quiescent VSMCs, a higher dose of
FK409 was necessary to antagonize MAP kinase activation induced by ET-1
(10-7 mol/L) in cells maintained in 1% FCS
(data not shown).
We next examined whether the MAPKK-1/MAP kinase pathway is involved in
the ET-1–induced survival effect. Pretreatment with PD98059
(10-5 mol/L), a specific inhibitor
of MAPKK-1, blocked the increase in MAP kinase activation induced by
ET-1 (10-7 mol/L) (Figure IVA, online)
and antagonized the survival effect of ET-1 on apoptosis
induced by both serum deprivation and FK409 as evaluated by
nucleosomal laddering (Figure 2A).
Transfection of VSMCs with a constitutively activated form of
MAPKK-1 (MAPKK-1 S218D/S222A) and an unphosphorylated
wild-type MAPKK-1 expressing DNA construct (MAPKK-1 WT) to a lesser
degree rescued VSMCs from apoptosis induced by serum
deprivation and by FK409 (10-4
mol/L) (Figure 3), suggesting the
involvement of MAP kinase in serum deprivation–induced
apoptosis. Transfection of VSMCs with MAPKK-1 S222A, a
dominant-negative construct, resulted in a complete blockade of MAP
kinase activation induced by ET-1 (Figure IVB, online) and
abolished the preventive effect of ET-1 from both serum
deprivation– and FK409-induced apoptosis, whereas
empty vector was without effect (Figure 2B). These results
suggest the important role of the MAPKK-1/MAP kinase pathway in the
ETA receptor–mediated survival effect in both
serum deprivation– and NO-induced apoptosis of
VSMCs.
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Regulation of Caspase-3 Activation by Serum
Deprivation, NO, and ET-1
To exclude the possibility that apoptosis may be
secondarily triggered by cell detachment from substrate (anoikis), we
measured caspase-3 activity using adherent VSMCs after serum
deprivation and addition of NO donors. Both serum
deprivation (Figure 4) and NO
donors (FK409, SNAP) (Figures 5 and 6) increased caspase-3 activity, reaching
a peak level at 30 to 60 minutes; the effects of FK409 and SNAP were
dose-dependent (10-6 to
10-4 mol/L) (data not shown). FK409 and SNAP
decayed spontaneously within 24 hours and did not induce caspase-3
activity. Coincubation of ET-1 (10-7 mol/L)
suppressed the increase in caspase-3 activity, and this effect was
antagonized by pretreatment with PD98059 (10-5
mol/L) (Figures 4, 5, and 6). These results
confirm that apoptosis induced by serum deprivation
and NO is not a consequence of cell detachment but rather their direct
effects via activation of caspase-3 pathway.
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In the present study, apoptosis of cultured rat VSMCs was induced by serum deprivation, potent NO donors, FK409, and SNAP. Occurrence of apoptosis was evidenced by (1) nucleosomal ladders on agarose gel electrophoresis by the NP-40 lysis method, (2) positive staining for free 3'-hydroxyl ends of DNA fragments by flow cytometry, (3) positive staining with the in situ TUNEL method, and (4) increased caspase-3 activity. Because the NP-40 lysis method very efficiently eliminates intact chromatin, the final extracted sample before agarose gel electrophoresis should contain only fragmented DNA, whereas samples free of apoptotic cells result in very low DNA yield. FK409, (±)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide, is a novel and potent NO donor that spontaneously releases NO under neutral aqueous conditions with a half-life of 46 minutes.36 Addition of media to which FK409 or SNAP had been added and has decayed spontaneously did not induce apoptosis. These data are compatible with those of recent studies from our laboratory showing that FK409 induced apoptosis of rat endothelial cells without any effect via its decomposition product(s).20 We have also shown that transfection of the inducible NO synthase gene into rat VSMCs caused massive apoptosis, whose effect was blocked by a nonselective NO synthase inhibitor, NG-monomethyl-L-arginine.17 Considered together, these observations suggest that massive NO and serum deprivation may function as important determinants of the number of VSMCs by inducing apoptosis. Lower doses of FK409 or SNAP induced limited apoptosis, implying limited contributions of small amounts of NO in negative vascular remodeling.
The phenomenon that apoptosis is triggered by detachment of cells from the supportive substrates (anoikis) is an established feature of endothelial cells.19 37 Therefore, we examined whether serum deprivation and NO primarily induced cell detachment and that the observed apoptosis was a secondary feature rather than the direct effect of NO on the cell death mechanism. We found that many adherent cells remained TUNEL-positive when fixed without being washed (Figures IB and IIIA, online), whereas the number of TUNEL-positive cells diminished when extensively washed before fixation. Moreover, serial phase-contrast microscopic examination revealed that cells first showed morphological features of apoptosis and then detached later. These observations were further confirmed by the findings that both serum deprivation and NO induced caspase-3 activity in adherent VSMCs as early as 30 to 60 minutes before the development of apoptosis. Considered together, the results indicate that apoptosis observed in the present study is not due to cell detachment but rather to the direct effect on cell death mechanisms. Therefore, calculation of floating/total cell ratio by counting both floating and adherent cell numbers after extensive washes truly represents the approximate percentage of apoptotic cells in a given VSMC population.
The antiapoptotic effect of ET-1 against serum deprivation– and NO-induced apoptosis of rat VSMCs was confirmed by the following experiments: (1) suppression of nucleosomal ladders, (2) reduction in the number of in situ TUNEL-positive cells among adherent cells, (3) reduction of positively stained cells by flow cytometry analysis, (4) reduced number of floating dead cells, and (5) suppression of caspase-3 activity in adherent cells. The dose-dependent inhibition of apoptosis by ET-1 (10-11 to 10-8 mol/L) is almost comparable to that in rat fibroblasts6 and endothelial cells, as recently described.19 Because ET-1 suppressed the increase in caspase-3 activity induced by serum deprivation and NO, the antiapoptotic effect of ET-1 is not due to its indirect influence on suppression of cell detachment but rather represents a direct antagonism against cell death. Given the paracrine/autocrine action of ET-1 secreted mainly by endothelial cells on neighboring VSMCs, our findings suggest a novel role for endothelium-derived ET-1 as a potent survival factor for VSMCs against apoptosis.
The diverse effects of ET isopeptides (ET-1, ET-2, ET-3) are mediated by 2 distinct receptor subtypes (ETA, ETB) of G protein–coupled receptors expressed in a wide variety of tissues.38 39 VSMCs mainly express ET-1–selective ETA receptors, which mediate contraction, whereas vascular endothelial cells express non–isopeptide-selective ETB receptors, which mediate vasodilation via generation of NO.24 In the present study, both ETA receptor antagonist (BQ123) and nonselective ETA/B receptor antagonist (TAK044), but not ETB receptor antagonist (BQ788), completely blocked the protective effect of ET-1 against apoptosis induced by serum deprivation and NO. Furthermore, BQ3020, another ETB receptor agonist, failed to suppress apoptosis of VSMCs. These results indicate that the cell survival effect of ET-1 is mediated via the ETA receptor.
ET receptor antagonists have been reported to prevent vascular hypertrophy40 and postangioplasty-induced neointima formation in rats.41 42 However, because of the apparent discrepancy between inhibition of ET-1–induced mitogenic activity and the protective effect against neointima formation by ET receptor antagonists, a mechanism(s) other than mitogenic activity of ET-1 has been strongly suggested.42 In this regard, induction of apoptosis by blockade of ETA receptor may be an important mechanism that explains the potent inhibitory action of ET receptor antagonists in experimental vascular diseases, considering that elimination of apoptotic VSMCs may determine the course of atherosclerosis.43 We have recently shown that endogenous ET-1 secretion by rat endothelial cells protects these cells against serum deprivation–induced apoptotic death via the ETB receptor in an autocrine/paracrine fashion,19 whereas ET-1 suppresses apoptosis via the ETA receptor in rat fibroblasts.6 Because endothelial cells actively synthesize and secrete ET-1, it is most likely that endogenous ET-1 protects not only endothelial cells but also VSMCs from apoptosis in an autocrine/paracrine manner.
The MAP kinase pathway plays a central role in cell proliferation and differentiation. In the present study, ET-1 stimulated MAP kinase activity in quiescent VSMCs in a dose-dependent manner. The effect was significantly suppressed by cotreatment with FK409, suggesting counterregulatory functions of ET-1 and NO in activation/suppression of MAP kinase in VSMCs. Activation of MAP kinase has been shown to inhibit apoptosis in PC-12 cells deprived of nerve growth factor,35 in ceramide-treated HL60 cells,44 and in potassium-deprived cerebellar neurons.45 Raf-1 activation has been shown to protect Rat-1 fibroblasts against c-Myc–induced apoptosis,46 whereas a contrasting report suggests the proapoptotic role of a Raf-MAP kinase pathway in fibroblasts.47 Furthermore, it was recently reported that ET-1 enhances apoptosis via ETB receptor–mediated p53 induction in human melanoma.21 In contrast, ETB receptors mediate suppression of endothelial apoptosis induced by serum deprivation not involving the MAP kinase pathway,19 48 whereas ETA receptors protect fibroblasts against serum deprivation–induced apoptosis via activation of MAP kinase.6 Thus, the involvement of the MAP kinase pathway in ET-1–induced apoptosis/antiapoptosis may depend on cell type and receptor subtype. The present study demonstrated that inhibition of MAP kinase by a specific MAPKK-1 inhibitor (PD98059) as well as transfection of a dominant-interfering MAPKK-1 mutant abrogated the antiapoptotic effect of ET-1, whereas overexpression of MAPKK1 potently antagonized apoptosis. These results strongly suggest the involvement of the MAPKK-1/MAP kinase pathway in ETA receptor–mediated protection against apoptosis of VSMCs. However, it remains to be determined whether NO inhibits Raf-1/MAPKK-1/MAP kinase and/or inactivates MAP kinase via MAP kinase phosphatase-1.
In conclusion, we have demonstrated for the first time that the counterbalance between an endothelium-derived relaxing factor, NO, and an endothelium-derived vasoconstrictor peptide, ET-1, determines VSMC elimination by apoptosis and cell survival, respectively. Imbalances between these 2 endothelium-derived vasoactive factors may occur under certain pathological conditions, such as in atherosclerosis, hypertension, and restenosis after vascular injury, thereby leading to dysregulation of cell survival.
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Acknowledgments |
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This study was supported in part by Grants-in-Aid from the Ministry of Education, Science, and Culture (M.S., Y.H.) and the Ministry of Health and Welfare, Japan (Y.H.), and by the Cardiovascular Research Fund (M.S.). The authors are grateful to Shinobu Yamaguchi for her excellent technical assistance.
Received August 16, 1999; accepted December 21, 1999.
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