© 1999 American Heart Association, Inc.
Original Contribution |
Determinants of Vascular Smooth Muscle Cell Apoptosis After Balloon Angioplasty Injury
Influence of Redox State and Cell Phenotype
From the Molecular Vascular Cell Biology Research Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Gary H. Gibbons, MD, Brigham and Women's Hospital, Thorn Cardiovascular Research Laboratories-1326, 75 Francis St, Boston, MA 02115. E-mail ggibbons{at}bustoff.bwh.harvard.edu
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Abstract |
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Abstract—We have observed that acute medial cell loss is an initial event in the response to vascular injury induced by balloon-catheter distention of the rabbit carotid artery. Numerous apoptotic medial cells were observed as early as 30 minutes after balloon inflation, and a 70% loss of cellularity was evident by 90 minutes. Balloon injury was associated with oxidative stress as reflected by a fall in glutathione levels by 63% within 30 minutes after injury. We hypothesized that balloon injury activated a redox-sensitive signaling pathway coupled to the regulation of apoptosis. Indeed, the activity of the proapoptotic signal mediator, stress-activated protein kinase, was increased severalfold within 10 minutes after injury. Moreover, modifying the vascular redox state by the administration of 1 of 2 structurally dissimilar antioxidants, N-acetyl cysteine or pyrrolidine dithiocarbamate, markedly attenuated both stress-activated protein kinase activation and the induction of apoptosis at 30 minutes. We hypothesized further that the induction of vascular smooth muscle cell apoptosis is modulated by phenotype. In contrast to medial cells, we observed that neointimal cells were relatively resistant to apoptotic death induced by angioplasty injury. This resistance to balloon injury–induced death was associated with an upregulation of the antiapoptotic mediator bcl-xL. This study suggests that acute apoptotic cell death after vascular injury is a highly regulated process governed by cellular redox state and the relative expression of antiapoptotic genes. Angioplasty-induced vascular cell apoptosis may be an important determinant of vascular remodeling and restenosis.
Key Words: balloon angioplasty • apoptosis • stress-activated protein kinase • bcl-x • restenosis
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Introduction |
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Although balloon angioplasty is one of the most common cardiovascular procedures performed in the industrialized world, the cellular mechanisms that govern whether the procedure has long-term success or results in restenosis remain poorly characterized. In previous characterizations of balloon-catheter injury models, it has been well documented that mechanical injury promotes an initial loss of cellularity.1 2 This acute cell loss has been presumed to involve cell death by necrosis as a result of traumatic damage induced by mechanical injury. However, recent studies by our laboratory and others have documented that programmed cell death or apoptosis is observed early after balloon distention injury and may contribute to the early medial smooth muscle cell loss.3 The cellular and molecular mechanisms that govern acute apoptosis after balloon injury remain to be characterized. Although the signal transduction pathways involved in regulating apoptosis are poorly defined, emerging evidence suggests that members of the mitogen-activated protein kinase family of mediators may play an important regulatory role. For example, activation of stress-activated protein kinase (SAPK) occurs in response to a variety of extracellular stimuli associated with apoptosis, such as cytokines, Fas ligand, and biomechanical stretch.4 5 6 Moreover, an important mediator role is inferred by the demonstration that blockade of SAPK activation prevents cell death.7 8 One potential mechanism by which SAPK activation promotes apoptosis is suggested by recent reports that indicate that SAPK promotes the activation of the caspases that mediate the process of cellular suicide.9
In addition to mediators in the mitogen-activated protein kinase family, an emerging body of evidence indicates that reactive oxygen species may also modulate the cell suicide program.10 11 12 The potential importance of reactive oxygen species as signaling mediators involved in the response to vascular injury can be inferred from animal studies as well as from clinical trials that have documented modulatory effects of antioxidants on the process of restenosis.13 14 15 Indeed, a recent study has demonstrated that antioxidant vitamins reduce the levels of superoxide anion produced in vessels after injury.16 Accordingly, our study tests the hypothesis that vascular smooth muscle cell (VSMC) death after balloon injury is governed by intrinsic cellular properties coupled to redox-sensitive regulatory mechanisms.
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Materials and Methods |
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Injury Models
Balloon Distention Injury
Normal Carotid Artery
Experiments were performed on 87 male New Zealand White rabbits weighing 3 to 4 kg. All experiments were performed in accordance with a protocol approved by the Standing Committee on Animals, Harvard Medical School. Rabbits were anesthetized with an intramuscular injection of 0.25 mL xylazine (20 mg/mL/) and 2.25 mL ketamine hydrochloride (100 mg/mL). The left common carotid artery was exposed by a 6-cm midline cervical incision. Proximal and distal blood flow was occluded by clamping. Polyethylene 10 tubing was inserted retrograde into the internal carotid artery and advanced into the left common carotid artery. After gentle flushing of the artery with normal saline, the tubing was removed and a 2-French (F) Fogarty embolectomy balloon catheter was inserted. Balloon inflation to 1.5 to 1.8 times the external diameter of the artery was achieved by caliper measurement under stereomicroscopy. After holding the inflation for 30 s, the catheter was removed.
Model of Reinjury
Initial Injury. The left common carotid artery was exposed, and
a 2-F Fogarty embolectomy balloon catheter was inserted retrograde
through the internal carotid artery. The balloon was inflated until
moderate resistance to catheter withdrawal was felt. The catheter was
withdrawn to the carotid bifurcation, deflated, and then readvanced.
The procedure was repeated for a total of 3 times before catheter
removal, ligation of the internal carotid artery, and closure of the
incision. After a 4-week period to allow formation of a
neointima, the left common carotid artery was reexposed,
and a 2-F embolectomy catheter was inserted retrograde through the
facial artery. A balloon distention injury to 1.5 to 1.8 times the
vessel diameter was performed as described above, the facial artery was
ligated, and the incision was closed.
Endothelial Removal
The left carotid artery was exposed, flushed with normal saline,
and cannulated retrograde via the internal carotid artery with a 2-F
Fogarty embolectomy balloon catheter as described above. Under
stereomicroscopic visualization, the balloon was inflated until minimal
resistance to catheter withdrawal was felt without affecting the
external diameter of the vessel. The balloon catheter was withdrawn to
the carotid bifurcation, deflated, and readvanced. The procedure was
repeated for a total of 3 times before removing the catheter. The
extent of denudation achieved by this method (>80%) was assessed in
separate animals by staining with Evans blue.
Local Antioxidant Administration
The left carotid artery was exposed, cannulated, and flushed
with normal saline as described above. After performing the balloon
distention injury described above, polyethylene 10 tubing was
advanced 1 cm into the common carotid artery. The artery was gently
flushed and incubated at a nondistending pressure for 30 minutes with
either PBS (vehicle), 100 µmol/L pyrrolidine dithiocarbamate
(Sigma Chemical), or 50 mmol/L N-acetylcysteine (NAC)
(Sigma). After incubation, the tubing was removed and blood flow was
restored.
Tissue Analyses
Tissue Preparation
At the end of each experimental protocol, animals were
euthanized by intravenous pentobarbital overdose. Carotid
vessels were quickly harvested, pressure fixed for 15 minutes at
100 mm Hg in 10% neutral buffered formalin, and immersed in the
same fixative for 
24 hours.
Analysis of Apoptosis
DNA Chromatin Morphology
After fixation, the vessels were stained for 2 hours at 37°C
with Hoechst 33342 (5 µg/mL in PBS) (Molecular Probes), and the
luminal surface was viewed en face under UV microscopy. Vessels were
analyzed in a blinded fashion for normal versus condensed and
coalesced (apoptotic) nuclear chromatin
morphology.17
DNA Laddering
At the time of vessel harvest, total cellular DNA was extracted
from unfixed vessels, as previously described.17
Five-microgram samples were size fractionated by 2% agarose gel
electrophoresis and stained for 30 minutes with SYBR Green I (1:10 000
dilution in Tris acetate EDTA) (Molecular Probes) before UV
visualization.
Assessment of Cellularity
After fixation, vessels were stained with Hoechst 33342 as
described above, and the luminal surface was viewed en face under UV
microscopy (x100). As an assessment of cellularity, a total
fluorescence index (nuclear staining index) was obtained by
computer-assisted analysis (Sony charge-coupled device camera
and NIH Image software) and averaged over 5 fields of view. The total
fluorescence index represents nuclear staining in the 2
most luminal layers of medial smooth muscle cells. In pilot
experiments, this index correlates with en face manual cell counts.
Glutathione Assay
Total cellular glutathione levels were analyzed
with an enzymatic recycling assay in which glutathione was oxidized by
5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and reduced by NADPH in the
presence of glutathione reductase, as previously
described.18 Carotid artery segments were immediately
frozen in liquid nitrogen, pulverized, homogenized in lysis
buffer (10 mmol/L Tris, 1% SDS), and centrifuged at
10 000 rpm at 4°C for 15 minutes. A small aliquot of the supernatant
was removed for protein determination with a bicinchoninic acid
(BCA) protein assay (Pierce Chemical Co). To assay for total
glutathione, 700 µL of 0.3 mmol/L NADPH (Sigma), 100 µL of
6 mmol/L DTNB (Sigma), and 200 µL of sample
homogenate were warmed to 30°C. Glutathione reductase (50
U) (Sigma) was then added, and the rate of 2-nitro-5-thiobenzoic acid
formation was monitored over a linear time frame at 412 nm. Glutathione
concentration was determined by comparison with a standard curve
generated with glutathione standards. Total cellular glutathione
content was expressed as µg of glutathione/mg protein.
SAPK Activity Assay
Rabbit carotid arteries were rinsed with PBS and snap frozen in
liquid nitrogen. Samples were homogenized in buffer C
(10 mmol/L Tris HCl, 5 mmol/L EDTA, 50 mmol/L NaF,
50 mmol/L NaCl, 1% v/v Triton X-100, 0.1% wt/vol fatty
acid–free BSA, 20 µg/mL aprotinin, and 2 mmol/L
Na3VO4) and
centrifuged at 14 000g for 20 minutes. Protein
concentrations were determined with a Bio-Rad protein assay. Two
hundred micrograms of protein from each sample was immunoprecipitated
with 20 µg of a SAPK-1 agarose conjugated antibody (sc-474, Santa
Cruz Biotechnology) for 5 hours at 4°C. Samples were then repeatedly
washed with buffer C, followed by buffer D (50 mmol/L Tris HCl,
0.1 mmol/L EGTA, 0.5 mmol/L
Na3VO4, and 0.1%
ß-mercaptoethanol). The pellets were resuspended in kinase buffer
(20 µmol/L ATP, 20 mmol/L HEPES, 20 mmol/L
MgCl2, 20 mmol/L ß-glycerophosphate,
2 mmol/L DTT, and 0.1 mmol/L
Na3VO4), 1 µCi of
[
-32P]ATP, and 2 µg of glutathione
S-transferase c-jun (Upstate Biotechnology), and
incubated at 30°C for 20 minutes. Laemmli sample buffer was added,
and samples were boiled for 3 minutes before being loaded on a 12% SDS
gel. Following SDS-PAGE, the gel was stained overnight, fixed, dried,
and analyzed by autoradiography.
Immunohistochemistry
We assessed immunohistochemical localization of bcl-x within
samples obtained from rabbit carotid artery neointimal
hyperplasia lesions (4 weeks after balloon injury). Rabbit polyclonal
anti-bcl-x antibody (Transduction Laboratories) and a control rabbit
IgG (Sigma) were biotinylated (HTI Bio-Products) and used as
primary antibodies. Acetone-fixed 6-µm cryosections were treated with
peroxidase block (Dako Corp), 10% goat serum and 2% BSA. Samples were
sequentially incubated with primary antibody (25 µg/mL), streptavidin
peroxidase (Dako Corp), and 3-amino-9-ethylcarbazol and
photographed under light microscopy (x200).
Immunoblot
For immunoblot analysis, segments of normal
rabbit carotid arteries or the neointima of balloon
injury–induced lesions were selectively removed by fine dissection,
pulverized in liquid nitrogen, and homogenized in lysis
buffer (10 mmol/L Tris and 1% SDS solution). Protein
concentrations were determined by a BCA assay (Pierce Chemical). Cell
lysates (50 µg) were loaded on a 12% SDS-polyacrylamide gel,
electrophoretically transferred to a nitrocellulose membranes (Hybond
enhanced chemiluminescence, Amersham), and the membrane was stained
with india ink to verify equal loading and transfer efficiency. The
membrane was blocked in PBS, 0.1% Tween, and 5% nonfat dry milk and
probed with a biotinylated polyclonal bcl-x antibody (1 µg/ml)
(Transduction Laboratories), followed by a streptavidin-biotinylated
horseradish peroxidase–linked secondary antibody (1:100 dilution)
(Dako Corp), and detected with electrochemical luminescence detection
reagents (Amersham) by autoradiography.
Statistical Analysis
Tests for significance of differences were made by Student's
t test using the program StatView. Significance was
determined as P<0.05.
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Results |
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Acute Induction of Apoptosis After Vascular Injury
In our initial studies, we performed a time course analysis to define the onset and mode of cell death after injury. As shown in Figure 1
, we observed
evidence of apoptotic nuclei within the media as early as 30
minutes after injury and ongoing cell death over the ensuing 24 hours.
This process of cell death was most readily apparent at the early time
points within the innermost layers of the media and appeared to radiate
outward in a wave of cell death in a time-dependent manner. The cells
undergo the process of shrinkage, nuclear condensation, and
autophagocytosis typical of apoptotic death until the innermost
areas of the vessel are relatively acellular as early as 90 minutes
after injury (Figure 1). It is noteworthy that substantial cell
death occurs without a significant inflammatory cell influx into the
vessel wall.
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The medial smooth muscle cells exhibited the characteristic condensed
and fragmented nucleus typical of apoptotic cell death. To
further verify that the mode of cell deletion involves an
apoptotic process, we analyzed extracted DNA from the
injured vessels to determine the pattern of DNA fragmentation by gel
electrophoresis. These studies confirmed that DNA harvested from
balloon-injured vessels is cleaved into the classical internucleosomal
pattern typical of cell death by apoptosis (Figure 1
).
Taken together, these data indicate that acute cell death after balloon
injury involves the activation of an intrinsic cell death program.
We postulated that the induction of apoptosis may be determined by either the mechanical stimulus of overdistention or blood-borne humoral mediators that come in close proximity to medial cells after denudation of the endothelium. To address this question, we initially examined the effect of gentle denudation of the endothelium in a manner that avoids significant distention injury to the medial layer using methods similar to those previously described.19 Although gentle endothelium denudation results in the exposure of medial cells to blood flow and blood-borne elements, we did not see evidence of medial smooth muscle cell death (data not shown). Thus exposure to blood-borne elements after denudation of the endothelium is not a sufficient stimulus to induce apoptotic cell death of medial smooth muscle cells.
On the basis of these findings, we hypothesized that the mechanical
distention of the vessel wall may be a critical determinant of cell
death after balloon inflation injury. To further test this hypothesis,
we performed experiments in which we induced balloon injury in the
usual manner but prevented subsequent contact with blood-borne elements
by ligating the vessel to prevent blood flow into the carotid artery
after injury. Assessment of vessel cellularity at 24 hours after injury
in the absence of blood flow revealed significant apoptotic
cell death and acellular regions within the medial layer (Figure 2). In contrast, uninjured control
vessels subjected to a similar occlusion of blood flow manifested
preserved medial smooth muscle cell viability during this time
interval. Thus, distention injury created by balloon inflation appears
to be a sufficient stimulus for the induction of cell death.
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Although we established that endothelial
denudation and exposure to blood-borne elements is not sufficient to
induce medial cell death, we hypothesized that exposure to blood-borne
elements may modulate the kinetics of the response to the mechanical
distention. Indeed, in a time-course analysis, we observed that
the induction of cell death after vascular injury was substantially
delayed in the absence of blood flow. As shown in Figure 2, medial cell viability is maintained at 2 hours after vascular injury in
the absence of flow, whereas there is substantial cell death in injured
vessels exposed to blood flow. Hence, blood flow and exposure to
blood-borne elements markedly accelerated the time-course kinetics of
apoptotic cell death induced by balloon distention injury.
Molecular Modulators of Acute Apoptosis: Role of Cellular
Redox State
To further elucidate the regulatory mechanisms that may lead to
acute cell death after balloon distention injury, we examined the role
of SAPK. Activation of SAPK occurs in response to a variety of
extracellular stimuli associated with apoptosis such as
cytokines, Fas ligand, and biomechanical
stretch.4 5 6 It has recently been established that
stimulation of SAPK activation promotes activation of the caspases that
mediate apoptotic cell death.9 Moreover, blockade
of SAPK activation results in the prevention of cell
death.7 8 We postulated that SAPK activation may be
associated with cell death after balloon injury. As shown in Figure 3, a normal, uninjured vessel exhibits
low levels of SAPK activity. In contrast, there is a dramatic increase
in SAPK activity within 10 minutes after balloon distention injury and
blood reflow.
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We were intrigued by the observation that blood flow appears to
accelerate the induction of apoptosis after vascular injury.
Accordingly, we hypothesized that exposure to blood flow and
blood-borne elements may promote vascular injury and subsequent cell
death by promoting oxidative stress. To test this hypothesis, we
measured vascular tissue levels of glutathione, an
endogenous antioxidant that serves as a marker of the
cellular redox state. Glutathione levels in normal vessels were
compared with vessels harvested 30 minutes after balloon distention
injury. As shown in Figure 4, normal
vessels had glutathione levels of 7.0±1.0 µg glutathione/mg protein.
However, in response to vascular injury, glutathione levels fell
rapidly by 63% to a mean value of 2.6±0.4 µg glutathione/mg protein
(n=6; P<0.001). Thus, balloon distention injury and the
subsequent induction of cell death were associated with oxidative
stress and a depletion of vascular glutathione content.
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An emerging body of evidence indicates that reactive oxygen species may
modulate the cell fate programs regulating cell growth20
as well as apoptosis.10 11 12 We hypothesized that
reactive oxygen species generated in response to balloon distention and
blood-borne elements may activate a redox-sensitive pathway
linked to SAPK activity and the induction of apoptosis. To test
this postulate, we examined the effect of treating vessels immediately
following balloon distention and before blood reflow with the
antioxidant NAC. As shown in Figure 5, NAC treatment significantly reduced SAPK activation in response to
balloon distention injury. These findings suggest that SAPK is part of
a redox-sensitive pathway associated with the acute induction of medial
smooth muscle cell apoptosis in response to balloon injury. To
further confirm this working hypothesis, we examined whether blockade
of oxidative stress and inhibition of SAPK activity in response to
antioxidants would also prevent apoptotic cell death after
balloon injury. In vessels treated with vehicle, we observed cell loss
of 70±9% 90 minutes after injury (Figure 6). In contrast, treatment of the vessel
immediately following balloon distention and before blood reflow with
the antioxidant NAC markedly reduced the induction of cell loss from
70±9% to 15±5%. To further verify the specificity of this response,
we assessed the effect of a mechanistically distinct antioxidant,
pyrrolidine dithiocarbamate (PDTC). Indeed, treatment of the vessel
with PDTC also resulted in a similar reduction in the incidence of cell
death in response to balloon distention vascular injury (70±9% to
23±7%). The results obtained by computer-assisted analysis of
total fluorescence indices (nuclear staining indices)
correlated with manual cell counts (615±27 cells/field [control
uninjured], 158±17 cells/field [injured], 512±27 cells/field
[NAC], and 457±29 cells/field [PDTC]; n=3;
P<0.001 for NAC and PDTC, compared with injured vessels).
These findings indicate that the process of cell death after vascular
injury is regulated by a redox-sensitive signaling pathway associated
with SAPK activation.
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Acute Induction of Apoptosis: Influence of Vascular
Cell Phenotype
An abundance of experimental evidence indicates that VSMCs that
accumulate within the neointima after vascular injury are
phenotypically distinct from medial smooth muscle cells.21
We postulated that the susceptibility to the induction of
apoptosis may be modified by the intrinsic properties of
neointimal cells that are distinct from medial cells. To
test this hypothesis, we examined the effect of balloon injury in
vessels with preexistent neointimal lesions. In the
single-injury model described above, we noted that the innermost layer
of medial vascular cells appeared to be the most vulnerable population
of cells to undergo acute apoptotic cell death after balloon
injury. We predicted that intimal cells would experience the greatest
mechanical stress, as well as the highest exposure to blood-borne
elements, and would therefore exhibit a more rapid and marked induction
of cell death in response to vascular injury.
As shown in Figure 7, the response of
medial smooth muscle cells to balloon distention in vessels with
preexistent neointimal lesions was remarkably similar to
the response in normal vessels. Balloon distention injury induced
medial smooth muscle cell apoptosis within hours after vascular
injury. However, in contrast to the response of medial smooth muscle
cells, neointimal smooth muscle cells proved to be
remarkably resistant to the induction of cell death by
distention injury. At 24 hours after vascular injury, large acellular
regions were clearly evident within the medial layer, whereas the
cellularity of the neointima remained intact.
Apoptotic cells were readily apparent within the medial layer
but were rarely observed within the neointima in response
to acute injury. These data indicate that a component of the
distinctive intimal VSMC phenotype appears to involve an
intrinsic antiapoptotic mechanism that renders them
resistant to the proapoptotic effects of balloon
distention injury.
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Previous studies in nonvascular cells have indicated that the
regulation of apoptosis involves the balance in expression of
proapoptotic mediators such as bax and antiapoptotic
mediators such as bcl-2 or bcl-x.22 Accordingly, we
postulated that the relative resistance of intimal smooth muscle cells
to the activation of apoptosis after vascular injury may
involve an alteration in the expression of antiapoptotic genes
as a component of the intimal cell phenotype. In our initial
pilot studies, we detected very little expression of bcl-2 in the
medial VSMCs (data not shown). Similarly, we observed relatively low
levels of the antiapoptotic mediator bcl-x in the medial layer
of normal uninjured vessels. However, immunohistochemical
analysis of vessels at 4 weeks after vessel injury documented a
relative increase in the expression of bcl-x within
neointimal smooth muscle cells as compared with the low
levels observed in the medial smooth muscle cells (Figure 8). Furthermore, this differential
pattern of expression was confirmed by immunoblot
analysis of normal medial cells compared with
neointimal cells isolated from vascular lesions (Figure 8). Additionally, through immunoblot
analysis we confirmed that it is the long form splice variant,
bcl-xL, that is predominantly upregulated within
neointimal cells. It is the bcl-xL
splice variant that is an effective endogenous
inhibitor of apoptosis. Overall, these observations
are consistent with the hypothesis that the upregulation of an
antiapoptotic gene such as bcl-xL by
intimal smooth muscle cells may confer a relative resistance to the
induction of cell death by balloon injury.
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Discussion |
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The process of restenosis after balloon angioplasty is a complex and vexing problem. The relative role of pathobiological processes such as cell growth, cell migration, matrix modification, thrombus formation, and vascular remodeling remain areas of controversy and ongoing investigation. The characterization of the initiating factors involved in the response to balloon angioplasty injury has been the focus of drug-discovery programs designed to prevent restenosis. It has been postulated that the process of vascular lesion formation and vascular remodeling involve the parallel activation of cell growth and apoptotic cell death.23 Recent reports suggest that cell death is observed during the latter stages of vascular lesion formation in animal models of vascular injury as well as human restenotic specimens.24 25 26 27 During the preparation of the manuscript for this article, Perlman et al3 reported that the induction of apoptosis is one of the first cellular events induced by balloon angioplasty injury. However, the cellular signaling events regulating cell fate after vascular injury remained to be further defined.
The present study extends the observations by Perlman et al3 by indicating that the acute induction of apoptotic death in response to balloon distention injury appears to involve a redox-sensitive signaling pathway associated with SAPK activation that is modulated by the expression of endogenous antiapoptotic genes such as bcl-xL.
In addition to providing several lines of evidence that indicate that balloon injury induces acute cell death by apoptosis (nuclear chromatin morphology and DNA fragmentation), we were intrigued by our observations that the process of cell loss does not reflect a passive response to a noxious stimulus typical of necrotic cell death. Indeed, the induction of cell death is subject to regulatory processes and could be modified by interventions that influence cellular behavior. For example, the observation that balloon distention injury in the absence of blood flow failed to induce significant cell death within 2 hours suggests that the mechanical trauma alone is not sufficient to promote the acute cell death and that other cellular signals derived from blood-borne elements must be integrated by the cell to acutely activate the cell death machinery. It is conceivable that blood reflow may be necessary to supply the cellular energy needs (ie, ATP production) to undergo the energy-dependent process of apoptosis. However, our observation that distention injury is sufficient to induce apoptosis in the absence of blood reflow argues against this hypothesis. Similarly, the finding that the induction of cell death could be modified by altering the cellular redox state or inhibited by alterations in vascular cell phenotype are consistent with the characterization of the acute cell deletion after balloon injury as a highly regulated form of programmed cell death. Although it is likely that cell necrosis also occurs after injury, several lines of evidence indicate that the acute induction of apoptosis is a prominent initial feature of the process of lesion formation and remodeling after vascular injury.
Although the cellular mediators that govern cell fate remain to be further elucidated, a growing body of evidence indicates that reactive oxygen species may be important signaling molecules in the regulation of cell growth20 as well as cell death.10 11 12 The observation that exposure of medial smooth muscle cells to blood flow and blood-borne elements after vascular injury accelerated the induction of cell death suggested that alterations in the cellular milieu such as the redox state may be a critical determinant of cell fate in this context. Indeed, we documented that balloon injury is associated with a marked reduction in glutathione levels, a marker of oxidative stress within tissue. These findings are consistent with recent reports that have documented increased generation of reactive oxygen species in response to vascular injury16 or mechanical stretch.28 Furthermore, we demonstrated that treatment with antioxidants such as NAC and PDTC inhibits the acute induction of cell death. Taken together, these findings suggest that balloon injury induces acute cell death via a redox-sensitive mechanism. This observation is consistent with a number of reports involving nonvascular cells that have indicated that the cellular redox state is an important determinant in the activation of the cell death program.10 11 12 The focus of the current study was on the effect of acute administration of antioxidants on the initial wave of cell death induced by vascular injury. It remains to be determined whether chronic administration of potent antioxidants that modulate vascular redox state exert a long-term influence on subsequent waves of cell death that occur after vascular injury. It is intriguing that studies in large-animal models,13 16 as well as recent clinical trials, indicate that antioxidant drugs may have particular efficacy in preventing restenosis in response to balloon injury.14 15 Further studies are needed to determine whether the salutary effects of antioxidants are mediated by alterations in cell growth, cell death, or matrix modulation.
To further elucidate the regulatory mechanisms that may lead to acute cell death after balloon distention injury, we examined the role of SAPK activation. Previous studies have determined that SAPK activation occurs in response to stimuli associated with apoptosis such as Fas and tissue necrosis factor, as well as biomechanical forces such as stretch.4 5 6 It is also noteworthy that SAPK activation appears to be a necessary condition for apoptotic cell death associated with the disruption of integrin-mediated cell-matrix interactions or anoikis.29 Furthermore, SAPK activation is a prerequisite for cell death under various conditions, and blockade of SAPK activation can abort apoptotic cell death.7 8 A potential central role for SAPK as a mediator of apoptosis can also be inferred from recent studies that link SAPK stimulation with activation of the caspase proteolytic cascade involved in cell execution.9 Taken together, these findings suggest that SAPK may play an important mediator role as an intracellular signaling pathway leading to cell death. We were intrigued by the possibility that balloon distention injury may involve a similar disruption of integrin-mediated cell matrix interactions. Indeed, we documented for the first time that balloon injury is associated with a marked SAPK activation in association with medial cell death. Furthermore, we demonstrated that treatment with antioxidants such as NAC and PDTC inhibits both SAPK activation and the acute induction of cell death. Overall, these data are consistent with the hypothesis that SAPK activation is part of the redox-sensitive pathway regulating the acute induction of medial smooth muscle cell apoptosis in response to balloon angioplasty injury. However, it is important to note that SAPK activation is neither necessary nor sufficient for apoptosis in some cell types in response to certain stimuli.30 Clearly, further studies need to be performed to establish a direct causal link between SAPK activation and medial smooth muscle cell apoptosis following balloon angioplasty injury.
On the basis of our observations that alterations in the cellular redox state modulate the activation of the cell death program, we postulated that alterations in vascular cell phenotype may also regulate cell fate in response to vascular injury. A wealth of experimental data has established that intimal cells appear to have a differential regulation of cell growth and differentiation that is quite distinct from medial cells.21 Recent studies in animal models as well as and human specimens have documented both cell proliferation and apoptotic death within neointimal lesions.24 25 26 27 The relatively high prevalence of apoptotic cells within the neointima may involve factors including increased expression of proapoptotic factors (eg, Fas ligand or oxidized LDL cholesterol) within the neointima, the influence of cell cycle progression, and/or impairment of apoptotic cell phagocytosis. Although the quantification of apoptosis in neointimal lesions remains an area of controversy, a recent study examining the frequency of apoptosis in restenotic versus primary atherosclerotic lesions suggests that restenotic intimal cells in vivo may be more resistant to apoptosis.31 The report documented that restenotic lesions contain a higher density of VSMCs and significantly reduced levels of apoptosis as compared with primary atherosclerotic lesions. The differential regulation of apoptosis in intimal versus medial cells is poorly understood. In vitro studies by Bennett et al32 inferred that plaque-derived VSMCs were more susceptible to apoptosis induced by serum withdrawal or forced overexpression of oncogenes than were medial VSMCs in a cell culture model. Although these data are provocative, it is important to recognize that the regulation of apoptosis is highly contextual and is influenced by the nature of the apoptotic stimulus, as well as the aspects of the microenvironment, such as the nature of integrin-matrix interactions and the balance of proapoptotic versus antiapoptotic cytokines within the milieu.
Given this limitation of the in vitro model, we sought to further define the differential regulation of VSMC apoptosis in an in vivo model system. To our knowledge, this study is the first to directly compare the relative sensitivity of intimal cells versus medial cells to an acute proapoptotic stimulus in the in vivo context. This study demonstrates that neointimal VSMCs are remarkably resistant to the induction of acute apoptosis in response to balloon injury as compared with medial cells. This response is particularly striking, because we originally postulated that these cells situated near the vessel lumen would be exposed to the most potent activators of the cell death program, which are the mechanical stress and blood-borne elements. Moreover, we characterized a potential molecular basis of this resistance to cell death by demonstrating that intimal smooth muscle cells express higher levels of the antiapoptotic mediator bcl-xL compared with medial cells. These data suggest that a novel property of the intimal cell phenotype may involve the upregulation of antiapoptotic genes to enhance cell viability in the context of lesion formation. Although there may be multiple mechanisms by which bcl-x prevents cell death, it is noteworthy that expression of related genes is associated with a resistance to oxidative stress and the inhibition of SAPK activation.29 33 We postulate that the induction of intrinsic mechanisms (eg, upregulated bcl-xL expression) that prevent activation of the apoptotic death program may be a necessary condition for the accumulation of viable cells within the neointima. Indeed, recent studies from our laboratory support the notion that the upregulation of the antiapoptotic mediator bcl-xL may be a necessary condition for neointima formation.34
It has become clear that balloon injury is a complex stimulus capable of activating a variety of cellular functions. It remains to be determined whether the induction of acute apoptosis promotes deleterious sequelae such as neointima formation and constrictive remodeling or whether the induction of vascular cell death ameliorates the course of disease by reducing cellularity of the lesion and promoting enlargement remodeling. The recent clinical trials indicating that antioxidant drugs may have particular efficacy in preventing restenosis14 15 are intriguing in light of our findings. Future studies are necessary to determine whether there is therapeutic value in either preventing or enhancing this process of cell death after injury. Nevertheless, the findings of this study are consistent with the postulate that acute cell death after vascular injury is a regulated process that is governed by the intrinsic properties of VSMCs such as the cellular redox state and the relative expression of antiapoptotic genes. These results have important implications for understanding the process of lesion formation after vascular injury in animal models as well as the causes of restenosis after balloon angioplasty in the clinical context.
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Acknowledgments |
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This work was supported by the Baxter Foundation, the American Heart Association, and the National Institutes of Health. G.H.G. is a Pew Biomedical Research Scholar. M.J.P. is a recipient of a California Affiliate American Heart Association Postdoctoral Fellowship Award.
Received September 11, 1998; accepted October 13, 1998.
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N. Sudoh, K. Toba, M. Akishita, J. Ako, M. Hashimoto, K. Iijima, S. Kim, Y.-Q. Liang, Y. Ohike, T. Watanabe, et al. Estrogen Prevents Oxidative Stress-Induced Endothelial Cell Apoptosis in Rats Circulation, February 6, 2001; 103(5): 724 - 729. [Abstract] [Full Text] [PDF]
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M. Fenton, S. Barker, D. J. Kurz, and J. D. Erusalimsky Cellular Senescence After Single and Repeated Balloon Catheter Denudations of Rabbit Carotid Arteries Arterioscler Thromb Vasc Biol, February 1, 2001; 21(2): 220 - 226. [Abstract] [Full Text] [PDF]
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R. Shibata, H. Kai, Y. Seki, S. Kato, M. Morimatsu, K. Kaibuchi, and T. Imaizumi Role of Rho-Associated Kinase in Neointima Formation After Vascular Injury Circulation, January 16, 2001; 103(2): 284 - 289. [Abstract] [Full Text] [PDF]
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C.-C. Hsieh, M.-H. Yen, C.-H. Yen, and Y.-T. Lau Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells Cardiovasc Res, January 1, 2001; 49(1): 135 - 145. [Abstract] [Full Text] [PDF]
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 E. Rodriguez, E. H. Lambert, M. G. Magno, and J. D. Mannion Contractile smooth muscle cell apoptosis early after saphenous vein grafting Ann. Thorac. Surg., October 1, 2000; 70(4): 1145 - 1152. [Abstract] [Full Text] [PDF]
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 V. Hampl and J. Herget Role of Nitric Oxide in the Pathogenesis of Chronic Pulmonary Hypertension Physiol Rev, October 1, 2000; 80(4): 1337 - 1372. [Abstract] [Full Text] [PDF]
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K. K. Griendling, D. Sorescu, B. Lassegue, and M. Ushio-Fukai Modulation of Protein Kinase Activity and Gene Expression by Reactive Oxygen Species and Their Role in Vascular Physiology and Pathophysiology Arterioscler Thromb Vasc Biol, October 1, 2000; 20(10): 2175 - 2183. [Abstract] [Full Text] [PDF]
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J. L. Hall, C. M. Matter, X. Wang, and G. H. Gibbons Hyperglycemia Inhibits Vascular Smooth Muscle Cell Apoptosis Through a Protein Kinase C-Dependent Pathway Circ. Res., September 29, 2000; 87(7): 574 - 580. [Abstract] [Full Text] [PDF]
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R. Charles, L. Sandirasegarane, J. Yun, N. Bourbon, R. Wilson, R. P. Rothstein, S. W. Levison, and M. Kester Ceramide-Coated Balloon Catheters Limit Neointimal Hyperplasia After Stretch Injury in Carotid Arteries Circ. Res., August 18, 2000; 87(4): 282 - 288. [Abstract] [Full Text] [PDF]
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L. C.P. Azevedo, M. d. A. Pedro, L. C. Souza, H. P. de Souza, M. Janiszewski, P. L. da Luz, and F. R.M. Laurindo Oxidative stress as a signaling mechanism of the vascular response to injury: The redox hypothesis of restenosis Cardiovasc Res, August 18, 2000; 47(3): 436 - 445. [Abstract] [Full Text] [PDF]
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K. Walsh, R. C. Smith, and H.-S. Kim Vascular Cell Apoptosis in Remodeling, Restenosis, and Plaque Rupture Circ. Res., August 4, 2000; 87(3): 184 - 188. [Full Text] [PDF]
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 R. Safirstein, L. Andrade, and J. M. Vieira Acetylcysteine and Nephrotoxic Effects of Radiographic Contrast Agents -- A New Use for an Old Drug N. Engl. J. Med., July 20, 2000; 343(3): 210 - 212. [Full Text]
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F. J. Miller Jr AIF-1 in the Activated Smooth Muscle Cell : Spectator or Participant? Arterioscler Thromb Vasc Biol, July 1, 2000; 20(7): 1701 - 1703. [Full Text] [PDF]
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W.-G. Li, F. J. Miller Jr, M. R. Brown, P. Chatterjee, G. R. Aylsworth, J. Shao, A. A. Spector, L. W. Oberley, and N. L. Weintraub Enhanced H2O2-Induced Cytotoxicity in "Epithelioid" Smooth Muscle Cells : Implications for Neointimal Regression Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1473 - 1479. [Abstract] [Full Text] [PDF]
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G. H. Gibbons and M. J. Pollman Death Receptors, Intimal Disease, and Gene Therapy : Are Therapies That Modify Cell Fate Moving too Fas? Circ. Res., May 26, 2000; 86(10): 1009 - 1012. [Full Text] [PDF]
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 J. Galle, C. Lehmann-Bodem, U. Hubner, A. Heinloth, and C. Wanner CyA and OxLDL cause endothelial dysfunction in isolated arteries through endothelin-mediated stimulation of O2- formation Nephrol. Dial. Transplant., March 1, 2000; 15(3): 339 - 346. [Abstract] [Full Text] [PDF]
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M. MAYR, C. LI, Y. ZOU, U. HUEMER, Y. HU, and Q. XU Biomechanical stress-induced apoptosis in vein grafts involves p38 mitogen-activated protein kinases FASEB J, February 1, 2000; 14(2): 261 - 270. [Abstract] [Full Text]
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N. J. McCarthy and M. Bennett The regulation of vascular smooth muscle cell apoptosis Cardiovasc Res, February 1, 2000; 45(3): 747 - 755. [Abstract] [Full Text] [PDF]
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K. Walsh and J. M. Isner Apoptosis in inflammatory-fibroproliferative disorders of the vessel wall Cardiovasc Res, February 1, 2000; 45(3): 756 - 765. [Abstract] [Full Text] [PDF]
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X. Wang, Y. Xiao, Y. Mou, Y. Zhao, W. M. Blankesteijn, and J. L. Hall A Role for the {beta}-Catenin/T-Cell Factor Signaling Cascade in Vascular Remodeling Circ. Res., February 22, 2002; 90(3): 340 - 347. [Abstract] [Full Text] [PDF]
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J. L. Hall, X. Wang, Van Adamson, Y. Zhao, and G. H. Gibbons Overexpression of Ref-1 Inhibits Hypoxia and Tumor Necrosis Factor-Induced Endothelial Cell Apoptosis Through Nuclear Factor-{kappa}B-Independent and -Dependent Pathways Circ. Res., June 22, 2001; 88(12): 1247 - 1253. [Abstract] [Full Text] [PDF]
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