Role of p44/p42 MAP Kinase in the Age

Role of p44/p42 MAP Kinase in the Age-Dependent Increase
in Vascular Smooth Muscle Cell Proliferation and
Neointimal Formation
Giuseppa Gennaro, Catherine Ménard, Edith Giasson, Sophie-Élise Michaud, Maria Palasis,
Sylvain Meloche, Alain Rivard
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Objective—Age-dependent increase in vascular smooth muscle cell (VSMC) proliferation is thought to contribute to the
pathology of atherosclerotic diseases. In this study, we investigated the role of mitogen-activated protein kinases
(MAPKs) on VSMC proliferation and neointimal formation in the context of aging.
Methods and Results—VSMCs were isolated from the aorta of young and old rabbits. The proliferative index after serum
stimulation was significantly increased in old versus young VSMCs. This was associated with a significant and specific
age-dependent increase in p44/p42 MAPK activation. Treatment with MEK inhibitor PD98059 successfully inhibited
p44/p42 MAPK activities and VSMC proliferation. These results were confirmed in vivo using a model of balloon injury
in rabbit iliac arteries. p44/p42 MAPK activities were rapidly induced by angioplasty in young and old animals.
However, the levels of p44/p42 MAPK activities achieved in arteries of old rabbits were significantly higher than those
of young rabbits. This was associated with a higher cellular proliferative index and a significant increase in neointimal
formation in old animals. Local delivery of PD98059 in old rabbits successfully inhibited p44/p42 MAPK activities after
angioplasty, which led to a significant reduction in cellular proliferation and neointimal formation in treated animals.
Conclusions—Our study suggests for the first time that increased p44/p42 MAPK activation contributes to augmented
VSMC proliferation and neointimal formation with aging. p44/p42 MAPK inhibition could represent a novel therapeutic
avenue against atherosclerotic diseases. (Arterioscler Thromb Vasc Biol. 2003;23:204-210.)
Key Words: astherosclerosis 䡲 aging 䡲 vascular smooth muscle cell proliferation
䡲 mitogen-activated protein kinase
A
signaling pathway are presently unknown. Mitogen-activated
protein kinases (MAPKs) are a family of protein-serine/
threonine kinases that include at least 4 distinctly regulated
groups in mammals: extracellular signal–related kinases
(ERK) 1/2 (or p44/p42 MAPK), Jun amino-terminal kinases
(JNK1/2/3), p38 proteins, and ERK5.13 These enzymes phosphorylate different intracellular proteins and play important
roles in regulating gene expression, cell proliferation, cell
survival and death, cell motility, and cell differentiation.
MAPKs are activated by a variety of stimuli, including
growth factors, cytokines, antigens binding to T- and B- cell
receptors, hormones that bind to G protein– coupled receptors, lipid compounds, and mechanical forces such as fluid
shear stress and stretch. Moreover, MAPKs (p44/p42 MAPK
and JNK) and MAP kinase phosphatase (MKP-1) have been
reported to be activated in the arterial wall after balloon injury
in different animal models.14 –18 This suggests that MAPKs
could represent a link between arterial injury and VSMC
proliferation in atherosclerotic diseases.
bnormal proliferation and migration of vascular smooth
muscle cells (VSMCs) play important roles in the
physiopathology of atherosclerotic diseases.1,2 Previous studies have shown that, in marked contrast to most cell types,
VSMCs isolated from old animals replicate more actively
than corresponding cultures from young animals.3– 6 Moreover, aging has been associated with an increased proliferative response of VSMCs after balloon angioplasty,7 and this
response seems to be a function of the arterial segment rather
than the host environment.8 Taken together, these findings
suggest that age-dependent increase in VSMC proliferation
may contribute to the increased prevalence and severity of
atherosclerosis in the elderly.9 –11
The cellular and molecular mechanisms responsible for the
enhanced proliferation of old VSMCs remain largely undefined. We have recently shown that augmented cyclin A
expression via the action of the AP1 transcription factor c-fos
contributes to the age-dependent increase in VSMC proliferation.12 However, the upstream elements involved in that
Received November 5, 2002; accepted December 9, 2002.
From the Department of Cardiovascular Research, Centre Hospitalier de l’Université de Montréal (G.G., C.M., S-É.M., A.R.) and Institut de
Recherches Cliniques de Montréal (S.M., E.G.), Montréal, Québec, Canada and Boston Scientific Corporation (M.P.), Natick, Mass.
Correspondence to Alain Rivard, MD, Centre Hospitalier de l’Université de Montréal, 1560 Sherbrooke Est, Suite Y-3605, Montréal, Que, H2L 4 M1,
Canada. E-mail [email protected]
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
DOI: 10.1161/01.ATV.0000053182.58636.BE
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Gennaro et al
Aging and Vascular p44/p42 MAP Kinase Activation
205
In the present study, we investigated the effect of aging on
MAPK activation after serum stimulation of VSMCs in vitro
and in response to arterial injury in vivo. In both cases, we
found that the induction of p44/p42 MAPK activities was
significantly enhanced in old compared with young animals.
In contrast, activities of MAPKs p38 and JNK were similar in
young and old animals. We also demonstrate that the inhibition of p44/p42 MAPK activities can prevent the agedependent increase in VSMC proliferation and neointimal
formation. Thus, our study illustrates for the first time a
specific signal transduction pathway by which increased
p44/p42 MAPK activity contributes to greater VSMC proliferation and neointimal formation with aging. Our results
suggest that p44/p42 MAPK could play a significant role in
the physiopathology of atherosclerotic diseases, especially in
the context of aging.
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Methods
The Methods section can be found online at http://www.
atvb.ahajournals.org.
Results
Effect of Aging on Rabbit VSMC Proliferation
and Apoptosis
We compared the proliferative capacity of VSMCs isolated
from the aorta of young or old rabbits using cellular counts
and the MTS assay. The standard curves for the MTS assay
were almost identical in young and old VSMCs (see Figure
IA, available online at http://www.atvb.ahajournals.org). Using these curves to convert optical density measurements to
cell number, we found a 80% increase in the proliferative
activity of old versus young VSMCs after 24 hours of serum
stimulation (Figure 1A, P⬍0.001). Importantly, the agedependent increase in cellular proliferation was not found in
other cell types, such as rabbit fibroblasts (Figure 1A) and
rabbit myoblasts (data not shown), suggesting that this is a
specific characteristic of VSMCs. We also investigated the
effect of aging on the rate of apoptosis in VSMCs using
FITC-Annexin V/PI dual color flow cytometry (see Figure
IB, available online at http://atvb.ahajournals.org). Although
a few cells (6% to 8%) were found to be apoptotic after 24
hours of serum stimulation, the level of apoptosis was similar
in young and old VSMCs. This indicates that the agedependent increase in cell number is not attributable to a
decreased rate of apoptotic cell death in old VSMCs.
Comparison of MAPK Activation in Young and
Old VSMCs
We used phospho-specific antibodies to compare the activation of MAPKs in serum-stimulated VSMCs harvested from
young or old rabbits (Figure 1B). p44/p42 MAPK activities
were very low or undetectable in quiescent VSMCs. After
serum stimulation, maximal p44/p42 MAPK activities were
seen at 30 minutes both in young and old VSMCs. However,
p44/p42 MAPK activities were significantly higher in old
compared with young VSMCs at 5 and 30 minutes after
serum stimulation. In contrast, although both p38 and JNK
activities were shown to be induced by serum, no significant
difference in activities could be detected between young and
Figure 1. Effect of aging on VSMC proliferation and MAPK activation after serum stimulation. A, Quantification of cellular proliferation after 24 hours of serum stimulation in young and old
rabbit VSMCs and fibroblasts was performed using the MTS
assay, n⫽4 per group. *P⬍0.001 vs young VSCMs. B, Cellular
extracts were prepared from VSMCs (or fibroblasts used as
controls) isolated from young and old rabbits after serum stimulation for the time indicated (in minutes). Western blot analysis
was performed using either a phospho-specific p44/p42 antibody, phospho-specific p38 antibody, or phospho-specific JNK
antibody. C, Kinase assay. p44/p42 MAPK was purified by
immunoprecipitation from young and old VSMC lysates, and
activity was determined by an in vitro kinase assay using myelin
basic protein as substrate. Quantification of p44/p42 MAPK
activity was performed by liquid scintillation counting. Bars are
percent of maximal p44/p42 MAPK activity obtained in old
VSMCs at 30 minutes; mean⫾SEM, n⫽8 per group. *P⬍0.05 vs
young VSCMs; **P⬍0.005 vs young VSCMs.
old VSMCs. Moreover, the age-dependent increase in p44/
p42 MAPK was specific for VSMCs, because a similar level
of p44/p42 MAPK activation was found in young and old
rabbit fibroblasts (Figure 1B) and myoblasts (data not
shown). To confirm the effect of aging on p44/p42 MAPK
activities in VSMCs, we performed specific kinase assays on
cell lysates obtained at different time points after serum
stimulation. As shown in Figure 1C, there was a significant
increase in p44/p42 MAPK activities in old versus young
VSMCs at 5 minutes (55% increase, P⫽0.02) and 30 minutes
(47% increase, P⫽0.003) after serum stimulation.
206
Arterioscler Thromb Vasc Biol.
February 2003
activities after treatment with PD98059 were still significantly higher in old versus young VSMCs at all the time
points studied (100% increase at 5 minutes, 61% increase at
30 minutes, and 29% increase at 2 hours after serum stimulation, P⬍0.001). We then evaluated the effect of p44/p42
MAPK inhibition on VSMC proliferation (Figure 2B). Treatment with PD98059 led to a 60% and 63% inhibition of the
proliferative index of young and old VSMCs, respectively
(P⬍0.001). However, the residual proliferative activity after
treatment with PD98059 was still significantly higher in old
versus young VSMCs (P⬍0.001). Treatment with PD98059
resulted in a 6% to 9% increase in the absolute rate of
apoptosis in young and old VSMCs (see Figure IIB online at
http://atvb.ahajournals.org). Our results suggest that the reduced cell number with PD98059 treatment is mostly attributable to decreased cellular proliferation and, to a lesser
extent, increased rate of apoptosis.
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Effect of Aging on p44/p42 MAPK Activation
After Angioplasty
Figure 2. In vitro inhibition of p44/p42 MAPK activity by
PD98059. Cellular extracts were prepared from VSMCs isolated
from young and old rabbits after serum stimulation for the time
indicated (in minutes). A, Kinase assay. p44/p42 MAPK was
purified by immunoprecipitation from young, PD98059-treated
young, old, and PD98059-treated old VSMC lysates, and activity
was measured by in vitro kinase assay. Bars are percent of
maximal p44/p42 MAPK activity obtained in old VSMCs at 30
minutes; mean⫾SEM; n⫽8 per group. *P⬍0.001 vs same-age/
untreated VSCMs; †P⬍0.001 vs young VSMCs treated with
PD98059. B, Quantification of young and old VSMC proliferation
was performed using the MTS assay. n⫽4 per group. *P⬍0.001
vs same-age/untreated VSCMs; †P⬍0.001 vs young VSMCs
treated with PD98059.
Inhibition of p44/p42 MAPK Activity in Cultured
VSMCs by PD98059
We used the MEK inhibitor PD98059 to suppress p44/p42
MAPK activation after serum stimulation of young and old
VSMCs. Treatment with 50 ␮mol/L of PD98059 resulted in
significant (although incomplete) inhibition of p44/p42
MAPK activation in young and old VSMCs, as assessed by
immunoblotting with phospho-specific antibody (see Figure
IIA, available online at http://atvb.ahajournals.org) and specific p44/p42 MAPK kinase assays (Figure 2A). Higher
concentrations of PD98059 did not additionally inhibit p44/
p42 MAPK inhibition compared with the 50-␮mol/L dosage
(data not shown). At 5 minutes after serum stimulation,
treatment with PD98059 resulted in a 64% and 54% reduction
of MAPK activities in young and old VSMCs, respectively
(P⬍0.001). Similar results were obtained at 30 minutes (54%
and 46% reduction of MAPK activities in young and old
VSMCs, P⬍0.001) and 2 hours (48% and 47% reduction of
MAPK activities in young and old VSMCs, P⬍0.001) after
serum stimulation. However, the residual p44/p42 MAPK
To evaluate the effect of aging on p44/p42 MAPK activation
after arterial injury in vivo, we used a rabbit model of iliac
artery angioplasty. Arteries harvested at different time points
after angioplasty showed a rapid induction of p44/p42 MAPK
activity, as revealed by immunoblotting with phosphospecific antibody and specific p44/p42 MAPK kinases assays
(Figure 3A). p44/p42 MAPK activity was maximal at 30
minutes, decreased by 4 hours, and remained at low levels at
days 3 and 7 after angioplasty (data not shown). However,
p44/p42 MAPK activities induced by arterial injury were significantly higher in old versus young rabbits. At 30 minutes after
angioplasty, old animals showed a 85% increase in p44/p42
MAPK activity compared with young animals (P⬍0.001).
Inhibition of p44/p42 MAPK Activity After
Angioplasty by Local Delivery of PD98059
To confirm the role of p44/p42 MAPK on the age-dependent
increase in VSMC proliferation and neointimal formation in
vivo, we used a channel balloon to locally deliver 500 ␮g of
PD98059 or diluent alone immediately after iliac artery
angioplasty in old rabbits. In preliminary experiments, this
dose was shown to maximally inhibit p44/p42 MAPK activity
without being toxic to the animal. Local treatment with
PD98059 successfully inhibited p44/p42 MAPK activation
after arterial injury (Figure 3B). At 30 minutes after angioplasty (time of maximal p44/p42 MAPK activation), local
treatment of old animals with PD98059 resulted in a significant 50% reduction in p44/p42 MAPK activity (P⫽0.004)
compared with injured arteries locally treated with the diluent
alone.
Effect of p44/p42 MAPK Inhibition on VSMC
Proliferation and Neointimal Formation
After Angioplasty
VSMC proliferation was evaluated in young and old
rabbits by BrdU incorporation at 7 days after angioplasty,
a time point associated with high proliferative activity in
that model. At that early time point, however, the neointimal layer was often very thin or even absent, especially
Gennaro et al
Aging and Vascular p44/p42 MAP Kinase Activation
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Figure 3. Age-dependent increase in
p44/p42 MAPK activation in vivo is inhibited by local delivery of PD98059 after
angioplasty. A, p44/p42 MAPK phosphospecific Western blots and kinase assays
(using myelin basic protein as substrate)
were performed on arterial extracts at
different time points after angioplasty in
young and old rabbits. Representative
blots are shown. Bars are percent of
maximal p44/p42 MAPK activity (as
assessed by the kinase assay) obtained
in old arteries at 30 minutes;
mean⫾SEM, n⫽4 per group. *P⬍0.001
vs young animals. B, p44/p42 MAPK
phospho-specific Western blots and
kinase assays performed on arterial
extracts from old rabbits locally treated
with either PD98059 (500 ␮g/artery) or
diluent alone immediately after angioplasty. Bars are percent of maximal p44/
p42 MAPK activity obtained at 30 minutes; mean⫾SEM, n⫽4 per group.
*P⬍0.005 vs diluent. LD indicates local
delivery.
in animals locally treated with PD98059. Accordingly, the
BrdU analysis was restricted to the media for adequate
comparison between the different animal groups. As
shown in Figure 4, aging was associated with a significant
increase in VSMC proliferative index (25⫾1% versus
11.5⫾0.5% in old and young animals, respectively, P⬍0.01).
Local treatment with PD98059 in old animals resulted in a 46%
decrease in the VSMC proliferative index (P⬍0.05), with a
residual level that was comparable to that of young rabbits
(13.5⫾1.5% versus 11.5⫾0.5%, P⫽NS).
Neointimal formation was evaluated in young and old
rabbits 14 days after angioplasty (Figure 5A). As shown in
Figure 5B, the intima to media (I/M) ratio was significantly higher in old compared with young rabbits
(0.55⫾0.06 versus 0.31⫾0.03, P⫽0.02). Local delivery of
PD98059 in old animals led to a 40% decrease in the I/M
ratio at 14 days after angioplasty (P⫽0.004). The amount
of neointimal formation in old rabbits locally treated with
PD98059 was similar to that of young rabbits (0.33⫾0.05
versus 0.31⫾0.03, P⫽0.87). Figure 5C demonstrates that
the differences in I/M ratios observed were attributable to
the specific effect of aging and PD98059 treatment on
neointimal areas, because medial areas were found to be
similar in the different animal groups.
Discussion
Although it is well established that aging is associated with an
increased risk of atherosclerotic diseases,9 –11 little is known
about age-related mechanisms causing cardiovascular dysfunction and enhanced atherosclerosis. There is much evi-
dence suggesting that abnormal VSMC proliferation could
contribute to the physiopathology of atherosclerosis in the
elderly.19 However, the mechanisms involved in this agedependent increase in VSMC proliferation are unknown. To
our knowledge, our study is the first to identify a specific
signal transduction pathway (p44/p42 MAPK), which enhanced activity is contributing to the age-dependent increase
in VSMC proliferation and neointimal formation.
We used a rabbit model of arterial injury and evaluated
vascular responses in young and old animals. For in vitro
studies, VSMCs were isolated from the aorta of young or old
rabbits and used at early passages. We found that aging was
associated with a significant increase in VSMC proliferation,
both in vitro (cell counts/MTS assay) and in vivo (BrdU
incorporation, neointimal formation). We also demonstrated
that the age-dependent increase in cell number was not
attributable to a decreased rate of apoptosis in old VSMCs.
These results extend those previously reported in the rat
model of vascular injury.3– 8 We then compared MAPK
activation in young and old rabbits, because these enzymes
are important mediators of cell proliferation13 and have been
shown to be rapidly activated after arterial injury.14 –18 Our
results indicate that the activation of p44/p42 MAPK after
serum stimulation and arterial injury is significantly enhanced
in old compared with young animals. This age-dependent
increase in MAPK activation seems to be specific for p44/p42
MAPK, because similar p38 and JNK activities were found in
both groups of animals. Moreover, age-dependent increase in
p44/p42 MAPK activation and cellular proliferation were not
found in other cell types, such as rabbit fibroblasts and rabbit
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Arterioscler Thromb Vasc Biol.
February 2003
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Figure 4. Effect of p44/p42 MAPK inhibition on VSMC proliferation in vivo. Old rabbits were locally treated with either PD98059
(500 ␮g/artery) or diluent alone immediately after angioplasty.
After 7 days, the rabbits received 2 intraperitoneal infusions of
BrdU 24 and 12 hours before euthanasia. Immunohistochemistry was performed using anti-BrdU biotinylated antibody. A,
Photomicrographs of histological sections (H&E staining, magnification ⫻400) are shown. Arrows indicate BrdU-labeled nuclei.
B, VSMC proliferation in the media was measured by calculating
the BrdU index as follows: (BrdU-labeled nuclei)/(total
nuclei)⫻100. Bars are BrdU index, mean⫾SEM, n⫽4 per group.
*P⬍0.005 vs young; **P⬍0.05 vs old controls. LD indicates local
delivery.
myoblasts, suggesting that these are specific characteristics of
VSMCs.
To confirm the role of p44/p42 MAPK in the agedependent increase in VSMC proliferation and neointimal
formation, we used the MEK inhibitor PD98059 to suppress
p44/p42 MAPK activation in vitro and in vivo. Interestingly,
it was not possible to completely abolish p44/p42 MAPK
activation in VSMCs isolated from old rabbits (Figure 2A),
even when higher doses of PD98059 were used (data not
shown). This suggests that aging is associated with a partial
resistance to PD98059 in rabbit VSMCs. Nevertheless, p44/
p42 MAPK activities in old VSMCs treated with PD98059
were reduced to levels well inferior to those of untreated
young VSMCs. This resulted in a dramatic inhibition of
cellular proliferation after serum stimulation in VSMCs
isolated from old animals. We then evaluated the possibility
of using this strategy to prevent the age-dependent increase in
neointimal formation after arterial injury in vivo. Using a
local delivery technique with the channel balloon, direct
infusion of PD98059 in the arterial wall of old animals after
angioplasty led to a 50% reduction of p44/p42 MAPK
activities. This early inhibition of p44/p42 MAPK activities
after angioplasty resulted in a significant decrease in VSMC
proliferation and neointimal formation at days 7 and 14 after
Figure 5. Effect of p44/p42 MAPK inhibition on neointimal formation. Young and old rabbits were locally treated with
PD98059 (500 ␮g/artery) or diluent alone immediately after
angioplasty and were killed 14 days later. A, Photomicrographs
of representative histological sections (H&E staining, magnification ⫻400) are shown. Arrows indicate internal and external
elastic membranes. B, Neointimal formation was evaluated by
calculating the I/M ratio. Mean⫾SEM, n⫽8 to 11 per group.
*P⬍0.001 vs young; **P⬍0.05 vs old controls. C, Intimal and
medial areas by planimetric analyses. Mean⫾SEM, n⫽8 to 11
per group. *P⬍0.001 vs young; **P⬍0.01 vs old controls. LD
indicates local delivery.
balloon injury, respectively. In fact, local arterial treatment
with PD98059 in old animals reduced VSMC proliferation
and neointimal formation to levels similar to those of young
animals. To our knowledge, the present study is the first to
demonstrate the efficacy of local MEK inhibitor (PD98059)
administration to prevent neointimal formation after arterial
injury. Because p44/p42 MAPK activity is rapidly induced
after angioplasty, early inhibition with PD98059 could be
necessary to influence cellular proliferation and neointimal
formation. This would be consistent with the fact that in a
previous study using the rat model of carotid injury, local
administration of PD98059 at the time of arterial injury
reduced medial cell replication at 2 days, whereas a similar
treatment at day 6 after arterial injury did not block intimal
cell replication.18 It is interesting to note that the inhibition of
Gennaro et al
Aging and Vascular p44/p42 MAP Kinase Activation
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p44/p42 MAPK activities that we obtained using PD98059
and the channel balloon was not complete. This could be
attributable to inhomogeneous distribution of the drug administered via the channel balloon or to a relative resistance of
old VSMCs to PD98059, as described in vitro. Development
of novel strategies to maximally inhibit p44/p42 MAPK
activities after arterial injury could potentially lead to a more
robust effect on VSMC proliferation and neointimal
formation.
The age-dependent increase in p44/p42 MAPK activities
that we describe in VSMCs is in marked contrast to what is
found in other cell types.20 For instance, reductions in MAPK
activities or defects in MAPK signaling with aging have been
described in lymphocytes,21,22 enterocytes,23 neural cells,24
hepatocytes,25 melanocytes,26 and fibroblasts.27 The exact
mechanisms by which VSMCs behave differently from other
cell types remain to be determined. Heterogeneous populations of VSMCs have been described in the vessel wall and
within the media.28 –34 Because p44/p42 MAPK activation has
been shown to protect against apoptosis in certain situations,35 it is possible that aging is associated with a natural
selection of VSMC populations (or clones) that can express
higher levels of p44/p42 MAPK activities. In these VSMC
populations, higher levels of p44/p42 MAPK activities would
not only promote cell survival during aging but would also be
at least partly responsible for the enhanced cellular proliferation and neointimal formation in response to injury. Interestingly, p44/p42 MAPK activation has recently been shown
to be involved in the determination of VSMC phenotype.36,37
Therefore, it is possible that the enhanced activation of
p44/p42 MAPK in old VSMCs contributes to the phenotypic
modulation from the differentiated to the dedifferentiated
one, a phenotype that has been associated with cell proliferation and migration and the progression of atherosclerosis.1,2
In summary, our results suggest that augmented p44/p42
MAPK activation is at least in part responsible for the
increase in VSMC proliferation and neointimal formation
with advanced age. This signaling pathway could contribute
to explain the increased prevalence and severity of atherosclerosis in the elderly. We propose that p44/p42 MAPK
inhibition could represent a novel therapeutic target against
atherosclerotic diseases, especially in the context of aging.
Acknowledgments
This study was supported by grants from the Canadian Institutes of
Health Research to A. Rivard (No. 57767) and S. Meloche (No.
14168). A. Rivard is a scholar from the Fédération de Recherche en
Santé du Québec. S. Meloche is an investigator of the Canadian
Institutes of Health Research. The authors are grateful to Guylaine
Gévry for technical assistance with the manuscript.
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Role of p44/p42 MAP Kinase in the Age-Dependent Increase in Vascular Smooth Muscle
Cell Proliferation and Neointimal Formation
Giuseppa Gennaro, Catherine Ménard, Edith Giasson, Sophie-Élise Michaud, Maria Palasis,
Sylvain Meloche and Alain Rivard
Arterioscler Thromb Vasc Biol. 2003;23:204-210; originally published online December 19,
2002;
doi: 10.1161/01.ATV.0000053182.58636.BE
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