In Vivo Induction of Endothelial Apoptosis Leads to Vessel
Thrombosis and Endothelial Denudation
A Clue to the Understanding of the Mechanisms of Thrombotic
Plaque Erosion
E. Durand, MD*; A. Scoazec, PhD*; A. Lafont, PhD; J. Boddaert, MD; A. Al Hajzen, MD;
F. Addad, MD; M. Mirshahi, PhD; M. Desnos, MD; A. Tedgui, PhD; Z. Mallat, MD, PhD
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Background—The mechanisms of thrombosis on plaque erosion are poorly understood. We examined the potential role
of endothelial apoptosis in endothelial erosion and vessel thrombosis.
Methods and Results—Segments of New Zealand White rabbit femoral arteries were temporarily isolated in vivo. One
artery was incubated with staurosporin for 30 minutes, whereas the contralateral artery was incubated with saline and
served as control. Three days later, thrombosis was evaluated angiographically and histologically. TUNEL score in the
endothelial layer was significantly increased in staurosporin-treated arteries compared with controls (2.43⫾0.30 versus
0.93⫾0.44, respectively; P⫽0.001). Large areas of endothelial denudation were detectable in staurosporin-treated
vessels, whereas endothelium integrity was almost preserved in the saline group. Vessel thrombosis occurred in 58% of
staurosporin-treated arteries (7 of 12) but in only 8% of saline-treated segments (P⬍0.01). Immunoreactivities for tissue
factor, platelets, and fibrin were detectable within the thrombus. Addition of ZVAD-fmk (0.1 mmol/L) significantly
reduced the occurrence of thrombosis (1 of 7 arteries or 14%, P⫽0.04). These results were confirmed in balloon-injured
atheromatous arteries.
Conclusions—In vivo induction of endothelial apoptosis leads to both vessel thrombosis and endothelial denudation.
Endothelial apoptosis may be a critical step in the transition from a stable endothelialized plaque to plaque erosion and
thrombosis. (Circulation. 2004;109:2503-2506.)
Key Words: endothelium 䡲 apoptosis 䡲 atherosclerosis 䡲 thrombosis
B
esides thrombotic plaque rupture, Virmani et al1 identified noninflammatory plaque erosion as responsible for
almost 30% to 40% of thrombotic coronary sudden death. In
this case, thrombus is formed on a denuded endothelial
plaque surface and is in direct contact with activated smooth
muscle cells.1 Despite continuous progress in the understanding of the risk factors that predispose to thrombotic plaque
erosion,2 including the potentially important role of blood
thrombogenicity,3 the pathophysiological mechanisms leading to both superficial endothelial denudation and formation
of a platelet/fibrin-rich thrombus remain poorly understood.4
Recently, we have reported a significant increase in the
occurrence of apoptotic death in luminal endothelial cells
located in low-shear-stress areas of advanced human carotid
atherosclerotic plaques.5 Because apoptotic endothelial cells
promote thrombin generation6 and platelet adhesion,7 we
hypothesized that in vivo occurrence of endothelial apoptosis
may initiate thrombus formation and lead, as expected, to
endothelial denudation (by detachment of apoptotic cells),
reproducing the 2 major features of thrombotic plaque erosion. This hypothesis was tested in segments of rabbit femoral
arteries incubated with staurosporin or placebo.
Methods
Experimental Protocol
New Zealand White rabbits (n⫽12; CEGAV, St Mars d’Egrenne,
France) were anesthetized with xylazine (5 mg/kg) and ketamine (35
mg/kg). Ligatures were used to isolate a femoral artery segment (1
cm long) that was punctured proximally by a 27-gauge needle and
incubated randomly with either staurosporin (10⫺5 mol/L) on one
side (n⫽12) or saline on the contralateral side (n⫽12). After 30
minutes of incubation, femoral segments were washed twice with
saline and reexposed to the circulating blood for 3 days. An
additional 4 rabbits were used, and femoral segments (n⫽8) were
preincubated for 10 minutes with ZVAD-fmk (0.1 mmol/L), a broad
inhibitor of caspases, before the 30-minute staurosporin incubation.
Animal procedures received institutional approval and conformed to
Received June 17, 2003; de novo received February 19, 2004; accepted April 13, 2004.
From Institut National de la Santé et de la Recherche Médicale, INSERM U541, and Institut Fédératif de Recherche Paris 7, Hôpital Lariboisière (S.A.,
B.J., T.A., M.Z.); INSERM EMI-U00-16 (D.E., L.A., A.H.A., A.F., D.M.); and INSERM E99-12 (M.M.), Paris, France.
*These authors contributed equally to this work.
Correspondence to Ziad Mallat, MD, PhD, INSERM U541, Hôpital Lariboisière, 41, Bd de la Chapelle, 75010 Paris, France. E-mail
[email protected]
© 2004 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000130172.62481.90
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Circulation
June 1, 2004
Figure 1. Representative photomicropgraphs of
transversal sections from saline-treated (c) or
staurosporin-treated arteries (a, b, and d through
f). Saline-treated segments (c) showed no fibrin
staining (not shown) and only adventitial TF positivity (arrows in c, red). Staurosporin-treated segments showed fibrin-rich (green staining in b, negative control in a) and platelet-rich (red staining in
e, negative control in f) thrombi, as well as diffuse
thrombus-associated TF staining (d). m indicates
media; i, intima.
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the Guide for the Care and Use of Laboratory Animals published by
the National Institutes of Health.
In additional experiments, the staurosporin-versus-saline studies
were performed on atherosclerotic-like lesions induced in femoral
arteries of rabbits (n⫽16) by the combination of air desiccation and
high-cholesterol diet (30 days), as previously described.8
Statistical Analysis
Assessment of Vessel Thrombosis and
Immunohistochemistry
We first examined the occurrence of thrombus in
staurosporin-treated animals. We found a 7-fold increase in
vessel thrombosis after staurosporin treatment compared with
controls (7 of 12 arterial segments or 58.3% versus 1 of 12 or
8.3%, respectively; P⬍0.01) (Figure 1). Complete occlusion
occurred in 43% of thrombosed vessels (3 of 7). Nonocclusive thrombi occupied 35% to 63% of the lumen vessel area.
Thrombi were rich in fibrin (Figure 1) and stained positive for
GP1b and TF (Figure 1). Staurosporin treatment was associated with an increase in TUNEL positivity in luminal endothelial cells compared with controls (2.43⫾0.30 versus
0.93⫾0.44, respectively; P⫽0.001). In addition,
staurosporin-treated vessels showed large areas of endothelial
denudation, whereas endothelial integrity was preserved in
control segments (Figure 2), indicating a contribution of
apoptosis to endothelial denudation. TUNEL-positive endothelial cells were detected at the interface between the
thrombus and the vessel wall (Figure 2), suggesting a relationship between induction of apoptosis and thrombus formation. TUNEL staining was confirmed by positive staining
against active caspase-3 (data not shown). TF staining in
thrombosed vessels was diffuse within the thrombus but
could also be seen in nondenuded endothelial cells (Data
Supplement Figure). Addition of ZVAD-fmk (0.1 mmol/L;
n⫽8) to staurosporin-treated vessels significantly inhibited
both the extent of endothelial apoptotic score (1.25⫾0.25,
Arteries underwent fixation with 4% buffered paraformaldehyde and
were embedded in OCT medium and stored at ⫺70°C. Each femoral
artery was cut in serial sections (8 to 10 ␮m), at sites 1 to 2 mm apart,
from the proximal to the distal end. At least 8 sections per animal
were used for immunostaining. Thrombus was defined histologically
as an adherent, fibrin-positive intraluminal material responsible for
partial or total lumen occlusion. Sections were stained with the
following antibodies: a monoclonal anti-CD31 antibody (Dako), a
monoclonal anti-rabbit tissue factor (TF) antibody (American Diagnostica), a monoclonal anti-GP1b antibody (Beckman Coulter), a
monoclonal anti-macrophage antibody (RAM-11; Dako), a biotinylated rabbit antibody against human fibrin (Dako),9 or a biotinylated
antibody against mouse caspase-3 (Cell Signaling).
In situ detection of apoptotic cells was performed using TUNEL.
A semiquantitative apoptosis score (0 through 3) evaluating the
extent of apoptosis in the endothelial lining was established by a
blinded investigator, as follows: 0 indicated no or barely detectable
staining; 1, weak positive staining; 2, moderate limited staining; and
3, strong diffuse staining. Areas where endothelial cell lining was
absent (negative CD31 staining) were considered as resulting from
apoptosis-induced endothelial denudation and were counted as apoptotic (score 3). In addition, we have quantified the absolute number
of endothelial cells (total and apoptotic) per arterial section in
staurosporin-treated vessels with (n⫽7) or without (n⫽7) ZVADfmk. A thrombotic score was also used to evaluate the severity of the
thrombotic response, as follows: 0 indicated no thrombosis; 1, a
thrombus with less than 50% of lumen vessel obstruction; 2, between
50% and 75%; and 3, more than 75%.
Results are expressed as mean⫾SEM. Differences in thrombus
formation between groups were compared using the ␹2 test. One-way
ANOVA was used to identify group differences with regard to the
extent of TUNEL staining.
Results
Durand et al
Thrombosis After Endothelial Apoptosis
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Figure 2. Representative photomicropgraphs of
transversal sections stained for CD31 or TUNEL.
Positive CD31 staining (red in c) and negative
TUNEL reaction in saline-treated segments indicate
preservation of endothelial integrity. Large areas of
endothelial denudation were seen in staurosporintreated vessels (a and b). CD31 staining was scant
at the junction between the thrombosed lumen and
the vessel wall (arrows in a and b). These endothelial cells were TUNEL-positive (arrows in d and e),
suggesting ongoing apoptosis. CD31 and TUNEL
double-positive cells (or cell debris) were also
detected within the thrombus (arrowheads).
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P⬍0.05) and thrombus formation (1 of 8 segments or 14.3%,
P⬍0.05) compared with staurosporin-treated vessels without
ZVAD-fmk. Total number of endothelial cells per arterial
section was not different between arterial segments injected
with saline, which served as controls for staurosporin with or
without ZVAD-fmk (186.9⫾17.2 versus 191.1⫾20.1, respectively). The percentage of endothelial cells remaining after
staurosporin treatment was 11.7⫾2.5%. Addition of ZVADfmk markedly increased the percentage of nondenuded endothelial cells (83.2⫾5.6%, P⬍0.0001). In addition,
52.9⫾3.6% of the remaining endothelial cells were TUNEL
positive in staurosporin-treated vessels compared with only
3.9⫾1.0% in segments treated with ZVAD-fmk (P⬍0.0001).
Our observations were confirmed in arteries with
atherosclerotic-like disease. As expected, the intima of these
arteries induced mild luminal narrowing (⬍30%) and were
rich in smooth muscle cells and collagen, with mild infiltration by macrophage foam cells (data not shown). Staurosporin treatment was associated with increased endothelial
TUNEL positivity compared with controls (TUNEL score,
2.58⫾0.22 versus 1.05⫾0.31, respectively; P⬍0.001).
Seventy-five percent (12 of 16) of staurosporin-treated vessels showed thrombosis (9 complete occlusions) compared
with 25% of saline-treated arteries (4 of 16, P⬍0.01), with a
significant correlation between TUNEL score and the severity of thrombosis (r2⫽0.76, P⬍0.001). Addition of ZVADfmk (0.1 mmol/L; n⫽8) to staurosporin-treated vessels significantly inhibited thrombus formation (2 of 8 segments or
25%, P⬍0.05) compared with staurosporin-treated vessels
without ZVAD-fmk.
Discussion
The mechanisms leading to the 2 major characteristics of
plaque erosion, ie, endothelial denudation and platelet/fibrinrich thrombotic occlusion, are still poorly understood. The
fibrin component of the thrombus is intriguing. Indeed, in the
absence of deep injury, subendothelial active TF is barely
detectable and would not lead to the formation of a fibrin-rich
adherent thrombus.4,10,11 The second enigma is endothelial
denudation per se. It is unlikely that this process occurs
before thrombus formation, because large areas of spontaneous endothelial denudation, such as those reported in thrombosed plaque erosion, are not seen even in advanced human
atherosclerotic plaques.4,12 Therefore, one has to assume the
presence of a process leading to the formation of a platelet/
fibrin-rich thrombus on a nondenuded luminal endothelium.
We hypothesized that endothelial apoptosis may be a clue to
the understanding of both thrombus formation and endothelial denudation.
Several authors have shown that apoptotic cells become
procoagulant in part because of increased expression of
phosphatidylserine.6 In addition, apoptotic endothelial cells
show a marked increase in the binding of nonactivated
platelets and could contribute to platelet incorporation within
the thrombus.7 In the present study, we have shown that
induction of vascular endothelial cell apoptosis led to platelet/fibrin-rich thrombus formation and endothelial denudation. Both processes were significantly inhibited by addition
of a broad caspase inhibitor. Although caspase activation may
be involved in processes other than apoptosis, our findings
that ZVAD-fmk reduced both endothelial TUNEL positivity
and denudation suggest a role for endothelial apoptosis in the
complex pathophysiological process leading to thrombotic
erosion in the present study.
Kolodgie et al13 recently reported a specific accumulation
of hyaluronan and CD44 at sites of plaque erosion. This
finding fits well with our hypothesis given the increased
propensity of endothelial cells to apoptosis when cultured on
hyaluronan substrates. Moreover, significant apoptosis of
luminal endothelial cells has been reported in advanced
human atherosclerotic plaques,5 a finding compatible with an
initiating role for endothelial apoptosis in plaque erosion.
Apoptotic endothelial cells and microparticles exposing phosphatidylserine could play a significant role in the activation of
circulating TF,3 which has been identified as a major contributor to blood thrombogenicity.3 Interestingly, increased levels
of circulating endothelial-derived microparticles have been
reported in patients with unstable angina or myocardial
infarction,14 suggesting a contribution of endothelial apoptotic injury to these acute processes. Taken together, these
results provide evidence for a central role of endothelial
apoptosis in the process leading to plaque erosion. It should
be noted, however, that our results do not exclude the
contribution of other nonapoptotic forms of endothelial injury13,15 or distinct prothrombotic mechanisms to plaque erosion (ie, activation/apoptosis of circulating blood cells, a
prothrombogenic subendothelial surface, or hyperlipidemia).
Moreover, because ZVAD-fmk was added before apoptosis
induction, no therapeutic implications can be inferred from
our results. Finally, caution is needed before extrapolating
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June 1, 2004
these results, obtained in a rabbit model, to a more complex
chronic atherosclerotic disease.
In conclusion, in vivo induction of endothelial cell apoptosis leads to both thrombus formation and endothelial denudation and may be a critical factor in the transition from a
stable plaque phenotype to thrombotic plaque erosion.
Acknowledgments
This work was supported by grants from Fondation de France and
Fondation pour la Recherche Médicale, France, and by grant OPAL
from Sanofi-Sythelabo.
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In Vivo Induction of Endothelial Apoptosis Leads to Vessel Thrombosis and Endothelial
Denudation: A Clue to the Understanding of the Mechanisms of Thrombotic Plaque
Erosion
E. Durand, A. Scoazec, A. Lafont, J. Boddaert, A. Al Hajzen, F. Addad, M. Mirshahi, M.
Desnos, A. Tedgui and Z. Mallat
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Circulation. 2004;109:2503-2506; originally published online May 17, 2004;
doi: 10.1161/01.CIR.0000130172.62481.90
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