© 2000 American Heart Association, Inc.
Vascular Biology |
Phosphatidylinositol 3-Kinase Signaling Is Important for Smooth Muscle Cell Replication After Arterial Injury
From the Department of Pathology, University of Washington, Seattle.
Correspondence to Michael A. Reidy, PhD, Department of Pathology, University of Washington, Box 357335, Seattle WA 98195. E-mail mar1{at}u.washington.edu
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Abstract—The phosphoinositide 3-kinase [PI(3)K] pathway is a key signaling pathway important for replication of mammalian cells. In this study, we examined the role of PI(3)K in smooth muscle cell (SMC) replication after balloon catheter injury of rat carotid arteries. Protein kinase B (PKB), a downstream target of PI(3)K, was phosphorylated at 30 and 60 minutes after injury and to a lesser degree after 6 hours and 1 and 2 days but not after 7 days. Wortmannin (10 µg per rat), a PI(3)K inhibitor, given to rats 60 and 5 minutes before and 11 hours after balloon injury, reduced the levels of phosphorylated PKB. SMC replication quantified between 24 to 48 hours was significantly reduced compared with control replication, as were the levels of cyclin D1. Wortmannin was also administered to rats between days 7 and 8 and between days 7 and 9 after balloon catheter injury. A reduction in levels of phosphorylated PKB was detected, but no decrease in the replication of intimal SMCs was observed in either experiment. These data demonstrate that the PI(3)K signal transduction pathway plays an important role in medial but not intimal SMC replication.
Key Words: balloon injury • smooth muscle cell replication • protein kinase B • wortmannin
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Introduction |
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Balloon injury to the rat carotid artery initiates smooth muscle cell (SMC) replication in a predictable manner. Our previous data suggest that the replication of intimal and medial cells is controlled by different mechanisms. For example, the extracellular signal–regulated kinase (ERK)1/2 cascade is involved in the early proliferative response of the artery after mechanical injury, inasmuch as PD98095, a mitogen-activated protein kinase kinase (MEK)1 inhibitor, significantly blocked ERK1/2 phosphorylation and SMC replication 48 hours after injury. In contrast, the MEK1 inhibitor was not able to block intimal SMC replication after 7 days.1 These data have led us to suggest that other signaling pathways may be important for intimal cell replication.
Phosphoinositide 3-kinase [PI(3)K] is activated by many growth factors, and there is good evidence that this pathway is involved in the entry of cells into S phase.2 3 4 Activation of PI(3)K leads to the generation of phosphatidylinositol 3,4-diphosphate and phosphatidylinositol 3,4,5-triphosphate in the cell membrane. These phospholipids form a binding site for proteins with a pleckstrin homology domain, such as protein kinase B (PKB), and in doing so a cryptic phosphorylation site is exposed. These sites are then phosphorylated by phosphoinositide-dependent kinases (PDKs), thus leading to their activation. Once activated, PKB phosphorylates several substrates, including glycogen synthase kinase-3ß5 and glucose transporter 4.6 PKB is also known to be involved in regulating cell survival and cell replication.7 8 9 10
In a previous study, we noted an increase in p70s6k activity in rat arteries after balloon injury, which suggested that the PI(3)K signal transduction pathway may be involved in controlling SMC replication.1 The purpose of the present study was to determine whether the PI(3)K pathway is activated after balloon injury and whether this pathway is necessary for acute and late SMC replication. Our studies show that activation of PI(3)K is critical for the initial medial cell replication but that inhibition of PI(3)K does not affect the replication of intimal SMCs between 7 and 9 days after injury.
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Methods |
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Inhibition of Medial PI(3)K Activity
Male Sprague-Dawley rats (B&K Universal, Kent, Wash) aged 3 to 4 months were used in all experiments, and all surgery was performed with the use of general anesthesia with an intraperitoneal injection of xylazine (Phoenix Pharmaceutical Inc) and ketamine (Phoenix Pharmaceutical Inc). Carotid injury was induced as previously described.11
Wortmannin (Sigma Chemical Co) was dissolved in 4.8% dimethyl sulfoxide to a final concentration of 2.5, 5, or 10 µg per 500 µL. To confirm the ability of wortmannin to inhibit PI(3)K, animals received 2 intravenous injections of wortmannin (2.5, 5, or 10 µg) or vehicle (4.8% dimethyl sulfoxide) at 60 minutes and 5 minutes before injury. Rats were then euthanized 30 minutes after injury, and phosphorylated (phospho)-PKB was measured by Western blot.
To investigate the effects of wortmannin on PKB phosphorylation, animals were given injections of 10 µg wortmannin and euthanized at 5 and 30 minutes and 1 and 6 hours after injury. To test the effects on medial SMC proliferation, animals received injections 60 minutes and 5 minutes before injury with an additional injection 11 hours after injury; for intimal SMC proliferation, animals received 2 injections 1 hour apart on day 7 and a further injection 11 hours later. Animals were euthanized at 48 hours for medial cell replication and 8 days for intimal cell replication. The study on intimal SMC replication was repeated, and animals were given 2 additional wortmannin injections 12 hours apart on day 8 and were killed on day 9.
Wortmannin was also administered intra-arterially to the left carotid artery. The common carotid and external carotid arteries were exposed, the proximal common carotid artery was clamped with a vessel clip, and a PE-10 tube (Clay Adams) was introduced into the common carotid artery through the external carotid artery. After a flushing with Ringer’s solution, 200 µL wortmannin solution (100 nmol/L wortmannin and 1% dimethyl sulfoxide in Ringer’s solution) was gently infused into the common carotid artery. The distal part of common carotid artery was then clamped, and the common carotid artery was left for 1 hour. After these clamps were removed, the carotid artery was injured with a balloon catheter.
Quantification of Cell Proliferation
Arterial SMC proliferation was evaluated by
bromodeoxyuridine (BrdU) labeling as previously
described.1 Proliferation was measured by calculating the
BrdU index as follows: (labeled nuclei)/(total nuclei). At least 4
sections were counted from each of 5 animals. Significance between the
average of the 2 groups was calculated by Student t
test.
Western Blot Analysis
For Western blot analysis, a minimum of 3 carotid
arteries were pooled in each group. Tissue samples were ground to a
fine powder under liquid nitrogen and prepared as previously
described.1 Equal amounts of protein were run out on a
10% SDS-polyacrylamide gel and transferred to nitrocellulose.
Nonspecific binding was blocked with 5% nonfat dry milk for 1 hour.
The blots were incubated with anti-ERK (New England Biolabs),
Akt(PKB) (New England Biolabs), phospho-ERK1/2 (Thr202/Tyr204,
New England Biolabs), phospho-Akt(PKB) (Ser473, New England BioLabs),
cleaved caspase-3 antibodies (New England BioLabs), and cyclin D
(Upstate Biotechnology) for 1 hour. Horseradish peroxidase–labeled IgG
secondary antibodies (Amersham) was used in conjunction with an
enhanced chemiluminescence kit (Amersham) to visualize the bands. An
identical blot was incubated with normal IgG as a control. Blots were
washed 3 times with TBS-T (25 mmol/L Tris [pH 7.6], 150
mmol/L NaCl, and 0.1% Tween 20) between incubations. All incubations
were carried out with agitation at room temperature in TBS-T containing
2.5% nonfat dry milk. Each gel was repeated at least twice, and
similar results were obtained. Gels composed of 3 pooled carotids for
each time point are presented. For quantification, images were
analyzed by use of NIH Image software.
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Results |
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PI(3)K Signaling Pathway Is Activated After Balloon Injury
Activation of the PI(3)K pathway was assessed by measuring the phosphorylation of a downstream target, PKB. Balloon injury of rat arteries stimulated a marked increase in PKB phosphorylation after 30 and 60 minutes, with a smaller increase still observed after 1 and 2 days. No PKB phosphorylation was detected 7 days after injury (Figure 1
).
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Wortmannin Suppresses PKB Phosphorylation in
Injured Arteries
To document the importance of PI(3)K for SMC replication,
wortmannin (100 nmol/L) was infused into an isolated segment of the
carotid artery for 60 minutes before balloon catheter injury; in this
way, the drug was limited to the artery. The addition of wortmannin in
this manner caused a significant decrease in medial SMC replication,
22.5±3.4% versus 7.2±2.8% (P<0.0001) at 48 hours after
balloon injury, and significantly blocked PKB
phosphorylation (data not shown).
Local administration of wortmannin to replicating intimal SMCs (ie,
after 7 days) required additional surgery to reexpose the artery, which
increased phosphorylation of PKB and ERK1/2. Therefore,
we decided to administer wortmannin by intravenous
injections at 60 and 5 minutes before balloon injury. Initially, we
measured the effect of differing concentrations of wortmannin by
measuring any decrease in PKB. A dose of 10 µg, but not 2.5 or 5
µg, suppressed the levels of phospho-PKB as early as 5 minutes, which
remained suppressed for at least 6 hours after balloon catheter injury
(Figure 2). By 12 hours, the levels of
phospho-PKB were again similar to control, and so another injection (10
µg per rat) was given 11 hours after balloon injury. This again
suppressed phosphorylation of PKB (data not shown). In
all future studies, therefore, 3 doses of wortmannin (10 µg per rat)
were given 60 and 5 minutes before balloon injury and a further 11
hours after surgery. Wortmannin given in this manner did not decrease
phosphorylation of ERK1/2 at any of the times studied
(data not shown).
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PI(3)K Inhibition Blocks Cyclin D1 Expression and
SMC Replication
The PI(3)K pathway is thought to permit the entry of cells into S
phase through the interaction on downstream targets of the cell cycle
and, in particular, cyclin D1. At 36 and 48
hours, wortmannin (3 times at 10 µg per rat ) not only blocked PKB
phosphorylation but also inhibited the steady-state
expression of cyclin D1 after injury (Figure 3). Treatment with wortmannin also
significantly inhibited SMC replication 24 to 48 hours after balloon
injury (Figure 4).
Histological examination of these arteries showed the
presence of replicating SMCs in the media (brown nuclear stain) and
revealed that zones close to the lumen were often devoid of SMCs (see
arrows). Quantification of the number of SMCs in these arteries showed
a small but significant decrease in the wortmannin-treated animals
(Figure 4B).
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This reduction in cell number may have been caused by an increase in programmed cell death. Therefore, we evaluated apoptosis by measuring the cleavage of caspase-3 in the extracts of these injured arteries. Up to 2 days after balloon injury, no cleaved caspase-3 was detected, but an increase was noted in injured arteries after 7 days (data not shown). This study was repeated with the use of arteries from wortmannin-treated rats. No cleavage of caspase-3 was noted up to 2 days after injury, and at 7 days, no further increase in the levels of cleaved caspase-3 was noted (data not shown). Therefore, the loss of cells from the wortmannin-treated arteries does not appear to be associated with an increase in apoptosis.
Inhibition of PI(3)K Does Not Block Injury-Induced Intimal
Cell Replication
The next experiments examined the effect of wortmannin on intimal
cell replication. First, we ensured that wortmannin could suppress
PI(3)K activity in these arteries. Phospho-PKB levels 7 days after
injury are very low and are approximately equivalent to the levels
detected in uninjured arteries (Figure 1
). To visualize PKB
phosphorylation in these samples, the Western blots
(Figure 5) were exposed for a longer time
than for Figures 1 and 2. The addition of wortmannin (3
times at 10 µg per rat) starting on day 7 decreased PKB
phosphorylation at 30 minutes and 6 and 12 hours later,
and a decrease was also detected at 24 hours. A reduction in
phospho-PKB was also noted in arteries from animals that received
wortmannin (5 times at 10 µg per rat) over the last 48 hours (Figure 5). No change in total PKB levels was noted at any time.
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Once we established that phospho-PKB levels were decreased, a similar
group of animals was subjected to balloon injury, and 7 days later,
wortmannin (3 times at 10 µg per rat) was administered over the last
24 hours or over the last 48 hours (5 times at 10 µg per rat). In
both experiments, SMC replication was measured over the last 24 hours,
and no significant change in intimal cell replication or in the level
of cyclin D1 was detected (Figure 6).
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Discussion |
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In a recent report, we showed that the ERK1/2 signaling pathway was activated within 30 minutes after balloon catheter injury, peaked by 6 hours, and remained slightly elevated for up to 2 days. At later times, only minimal activation, similar to that of control arteries, was observed.1 Evidence for the importance of the ERK1/2 pathway in SMC replication was provided by the use of a MEK1 inhibitor, PD98059, which blocked phosphorylation of ERK1/2 and also inhibited medial SMC proliferation after balloon injury.1 However, this inhibitor had no effect on intimal SMC proliferation after 7 days. Thus, we concluded that other signaling pathways play an important role in intimal SMC replication.
PI(3)K Signaling Pathway Is Activated by Balloon
Injury
In the present study, we chose to examine PI(3)K because we
had already noted that p70s6k, a downstream target of PI(3)K signaling,
was activated by balloon catheter injury.1 The
formation of phosphatidylinositol triphosphate and
phosphatidylinositol diphosphate by action of PI(3)K recruits PKB to
the cell membrane and thus allows its phosphorylation
by PDK 1 and PDK 2 (for a review, see References 10
and 12 ). Active PKB then targets a variety of
substrates, including ribosomal protein S6 kinase,15
glucose transporter 4,16 glycogen synthase kinase
3,17 18 and cell survival factors.19 Of
interest to us was the strong relationship between the PI(3)K pathway
and cell proliferation.7 8 9 Our data show that PKB was
phosphorylated within 30 minutes after balloon catheter
injury and remained active for up to 48 hours. In this respect, the
activation of the PI(3)K pathway followed a pattern similar to that
observed for the ERK pathway.1
Inhibition of PI(3)K Blocks Medial SMC Replication After
Injury
To determine whether PI(3)K activity is necessary for SMC
proliferation, we used a specific PI(3)K inhibitor,
wortmannin, to block its activity.20 21 Incubation of an
arterial segment with wortmannin significantly inhibited
PKB phosphorylation and significantly decreased SMC
replication while having no effect on ERK1/2
phosphorylation. However, we discontinued infusing
wortmannin directly into the artery because at later times after
injury, addition surgery would be required to reexpose the artery, and
this activated the ERK and PI(3)K pathways. Thus, to compare
the effects of wortmannin on the early and 7-day SMC replication, we
opted to use an intravenous route for wortmannin
administration. The administration of wortmannin
intravenously also allowed us the opportunity to add
multiple doses of the inhibitor if required. Two injections
of 10 µg wortmannin, given 1 hour apart before arterial
injury, inhibited PKB phosphorylation for up to 6
hours, but by 12 hours, the levels of phosphorylated
PKB levels were again elevated (data not shown). Therefore, we
administered a third dose of wortmannin 11 hours after injury to ensure
a more prolonged inhibition of this pathway. By use of this protocol,
PKB phosphorylation was depressed for up to 24 hours,
and medial SMC proliferation, measured between 24 to 48 hours, was
markedly inhibited.
Wortmannin binds to and irreversibly inhibits the p110 catalytic subunit of PI(3)K,21 22 but there are data showing that high concentrations of wortmannin (>100-fold higher) can interfere with other signals, such as myosin light chain kinase.22 23 Perhaps more important is the finding that wortmannin can partially inhibit platelet-derived growth factor–dependent Raf1 and ERK activation in certain cells.24 Thus, there is the possibility that wortmannin might block the ERK pathway and thus influence SMC replication. To determine whether wortmannin was acting in this way, we asked whether other signaling pathways associated with entry into the cell cycle were affected by this treatment. The administration of wortmannin had no effect on ERK1/2 phosphorylation at any time studied or on other mitogen-activated protein kinases, including c-Jun N-terminal kinase, p38, and big mitogen-activated protein kinase 1 (BMK1) (data not shown). We believe that perhaps the best evidence that intravenous wortmannin did not block other pathways necessary for SMC replication is the finding that this inhibitor had no effect on SMC replication 7 days after injury (see below). If wortmannin acted by blocking a non-PI(3)K pathway required for cell replication, then the administration of this inhibitor should have blocked replication regardless of the time after injury. This was clearly not the case.
Despite our attempts to ensure that wortmannin specifically blocked the PI(3)K pathway, we acknowledge that such chemical inhibitors could have unknown effects in vivo and that, as such, it will be important to validate this result by other means. This may not be easily accomplished. The other commonly used PI(3)K inhibitor, LY294002, is very insoluble, and we have been unable to inhibit PKB phosphorylation after injury, presumably because an effective concentration could not be achieved in the artery (data not shown). Another approach would be to infect SMCs at the time of injury with dominant-negative PKB by use of an adenoviral vector. This approach is not without problems, the major drawback being that the transgene expression declines within days after infection; this appears to be related to the extent of cell replication.25 As such, this approach would not be suitable to determine whether inhibition of the PI(3)K pathway is important for intimal SMC replication.
How the PI(3)K regulates entry into the cell cycle is not fully understood, but recent studies suggest that this pathway is required for mitogen-induced cyclin D1 expression.26 27 Cyclin D binds to and activates cdk4/6, and its expression is essential for cell cycle progression (for a review, see Reference 28 ). The PI(3)K pathway is also involved in stabilizing the cyclin D1 protein.29 Our results show that blockade of this pathway with wortmannin reduced the levels of cyclin D1 after balloon catheter injury; however, we cannot conclude whether this was due to a decrease in transcription or a change in protein stability. However, in other studies in vitro, we have found that PI(3)K activation is necessary for cyclin D1 transcription (data not shown).
Inhibition of PI(3)K Does Not Block Intimal SMC Replication 7 Days
After Injury
When wortmannin was given to animals whose arteries had been
injured with a balloon catheter 7 days previously, intimal SMC
proliferation was not significantly inhibited. That wortmannin was
delivered in an effective dose to these replicating cells was
ascertained by the fact that the background levels of PKB
phosphorylation were blocked. Why this signaling
pathway is necessary for acute SMC replication but not replication at
later times cannot not readily be explained. One reason for this result
may lie in the difference between these 2 SMC populations with respect
to their positions in the cell cycle at the time of wortmannin
treatment. The proliferation induced by balloon injury between 24 and
48 hours will be synchronized. In contrast, at 7 days after injury, the
intimal SMCs are likely to be proliferating asynchronously. Thus,
inhibition of PI(3)K by wortmannin in asynchronously cycling cells over
24 hours may have affected only a subgroup of proliferating cells. To
compensate for this possibility, we administered wortmannin every 12
hours over a 48-hour period (days 7 to 9 after injury) and measured
proliferation over the final 24 hours. This treatment reduced the level
of PKB phosphorylation but still had no effect on the
proliferation rate of the intimal SMCs. Thus, even prolonged inhibition
of PI(3)K over 48 hours does not influence the replication of intimal
SMCs.
The above data suggest that PI(3)K is not necessary for the replication of SMCs after 7 days. This finding is similar to the ERK pathway, in which SMC replication can be blocked only with a MEK1 inhibitor immediately after injury and not after 7 days.1 Presumably, therefore, other signaling pathways are important for the entry of these SMCs into S phase. Interestingly, intimal SMCs show strong expression of cyclin D1, and recent studies have suggested that several pathways, other than ERK1/2 and PI(3)K, may upregulate cyclin D1 expression and thus entry into the cell cycle (for a review, see Reference 30 ). These pathways include activation of Rho GTPase and Ral.26 31 32 Studies are now under way to determine whether these factors play a role in cyclin D1 expression in the growing neointima.
PI(3)K Inhibition Does Not Promote Cell Death
Inhibition of the PI(3)K pathway has been linked to
apoptosis. For example, overexpression of PKB prevents
apoptosis in primary neurons induced by survival factor
withdrawal.19 Further activated PI(3)K and PKB
have been shown to protect canine kidney (MDCK) cells from
apoptosis induced by detachment from their extracellular
matrix.33 In this experiment, wortmannin decreased SMC
number 48 hours after injury, which might suggest an increase in
apoptosis due to PI(3)K inhibition. In experiments using
caspase-3 cleavage as a marker of apoptosis, we found no
evidence of cell death until day 7 day after injury, and wortmannin did
not affect this result. This finding agrees with the data of Han et
al,34 who first noted SMC apoptosis in rat
arteries 9 days after balloon injury. One likely explanation for the
decrease in SMC cell number seen after 48 hours of treatment is that
the mechanically induced cell loss was accompanied by a decrease in
cell replication caused by wortmannin. Under these conditions, a
reduction in cell number 48 hours after the initial injury would be
expected. In contrast, no significant change in SMC numbers was
detected 8 and 9 days after injury, when wortmannin had no effect on
SMC replication.
In summary, the present study shows that the PI(3)K pathway is
increased for 
48 hours after balloon catheter injury. The
administration of wortmannin, a PI(3)K inhibitor, reduced
PKB phosphorylation with a concomitant reduction in
medial SMC proliferation and cyclin D1 48 hours
after balloon injury. This inhibitor did not inhibit ERK1/2
phosphorylation. Administration of wortmannin did not
reduce intimal SMC proliferation despite the inhibition of PKB
phosphorylation. These data suggest that PI(3)K
activation is required for the early replication of medial SMCs but not
for the replication of SMCs in the developing intimal lesions.
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Acknowledgments |
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This work was funded by a grant from the National Institutes of Health (HL-59908) and from the American Heart Association (9750171).
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Footnotes |
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1 Drs Shigematsu, Koyama, and Olson contributed equally to this study.
Received July 31, 2000; accepted August 1, 2000.
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  W.-F. Chiou, C.-C. Chen, and B.-L. Wei 3,4-Di-O-Caffeoylquinic Acid Inhibits Angiotensin-II-induced Vascular Smooth Muscle Cell Proliferation and Migration by Downregulating the JNK and PI3K/Akt Signaling Pathways Evid. Based Complement. Altern. Med., September 14, 2009; (2009) nep140v1. [Abstract] [Full Text] [PDF]
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 R. A. Nemenoff, P. A. Simpson, S. B. Furgeson, N. Kaplan-Albuquerque, J. Crossno, P. J. Garl, J. Cooper, and M. C.M. Weiser-Evans Targeted Deletion of PTEN in Smooth Muscle Cells Results in Vascular Remodeling and Recruitment of Progenitor Cells Through Induction of Stromal Cell-Derived Factor-1{alpha} Circ. Res., May 9, 2008; 102(9): 1036 - 1045. [Abstract] [Full Text] [PDF]
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 S. Albinsson and P. Hellstrand Integration of signal pathways for stretch-dependent growth and differentiation in vascular smooth muscle Am J Physiol Cell Physiol, August 1, 2007; 293(2): C772 - C782. [Abstract] [Full Text] [PDF]
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Y.-J. Wu, M. Bond, G. B. Sala-Newby, and A. C. Newby Altered S-Phase Kinase-Associated Protein-2 Levels Are a Major Mediator of Cyclic Nucleotide-Induced Inhibition of Vascular Smooth Muscle Cell Proliferation Circ. Res., May 12, 2006; 98(9): 1141 - 1150. [Abstract] [Full Text] [PDF]
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 T. Kozai, M. Eto, Z. Yang, H. Shimokawa, and T. F. Luscher Statins prevent pulsatile stretch-induced proliferation of human saphenous vein smooth muscle cells via inhibition of Rho/Rho-kinase pathway Cardiovasc Res, December 1, 2005; 68(3): 475 - 482. [Abstract] [Full Text] [PDF]
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 F. Li, C. Zhang, S. Schaefer, A. Estes, and K. U. Malik ANG II-induced neointimal growth is mediated via cPLA2- and PLD2-activated Akt in balloon-injured rat carotid artery Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2592 - H2601. [Abstract] [Full Text] [PDF]
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M. Jonas, E. R. Edelman, A. Groothuis, A. B. Baker, P. Seifert, and C. Rogers Vascular Neointimal Formation and Signaling Pathway Activation in Response to Stent Injury in Insulin-Resistant and Diabetic Animals Circ. Res., September 30, 2005; 97(7): 725 - 733. [Abstract] [Full Text] [PDF]
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J. Shen, S. P. Halenda, M. Sturek, and P. A. Wilden Cell-Signaling Evidence for Adenosine Stimulation of Coronary Smooth Muscle Proliferation via the A1 Adenosine Receptor Circ. Res., September 16, 2005; 97(6): 574 - 582. [Abstract] [Full Text] [PDF]
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F. B. Mehrhof, R. Schmidt-Ullrich, R. Dietz, and C. Scheidereit Regulation of Vascular Smooth Muscle Cell Proliferation: Role of NF-{kappa}B Revisited Circ. Res., May 13, 2005; 96(9): 958 - 964. [Abstract] [Full Text] [PDF]
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 E. V. Gerasimovskaya, D. A. Tucker, and K. R. Stenmark Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation J Appl Physiol, February 1, 2005; 98(2): 722 - 731. [Abstract] [Full Text] [PDF]
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 J. Huang, X.-L. Niu, A. M. Pippen, B. H. Annex, and C. D. Kontos Adenovirus-Mediated Intraarterial Delivery of PTEN Inhibits Neointimal Hyperplasia Arterioscler Thromb Vasc Biol, February 1, 2005; 25(2): 354 - 358. [Abstract] [Full Text] [PDF]
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P. Zahradka, G. Harding, B. Litchie, S. Thomas, J. P. Werner, D. P. Wilson, and N. Yurkova Activation of MMP-2 in response to vascular injury is mediated by phosphatidylinositol 3-kinase-dependent expression of MT1-MMP Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2861 - H2870. [Abstract] [Full Text] [PDF]
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M. Mifune, H. Ohtsu, H. Suzuki, G. D. Frank, T. Inagami, H. Utsunomiya, P. J. Dempsey, and S. Eguchi Signal transduction of betacellulin in growth and migration of vascular smooth muscle cells Am J Physiol Cell Physiol, September 1, 2004; 287(3): C807 - C813. [Abstract] [Full Text] [PDF]
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 H.-M. Yang, H.-S. Kim, K.-W. Park, H.-J. You, S.-I. Jeon, S.-W. Youn, S.-H. Kim, B.-H. Oh, M.-M. Lee, Y.-B. Park, et al. Celecoxib, a Cyclooxygenase-2 Inhibitor, Reduces Neointimal Hyperplasia Through Inhibition of Akt Signaling Circulation, July 20, 2004; 110(3): 301 - 308. [Abstract] [Full Text] [PDF]
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 S.-F. Yan, R. Ramasamy, L. G Bucciarelli, T. Wendt, L. K Lee, B. I Hudson, D. M Stenr, E. Lalla, S. Du Yan, L. L. Rong, et al. RAGE and its ligands: a lasting memory in diabetic complications? Diabetes and Vascular Disease Research, May 1, 2004; 1(1): 10 - 20. [Abstract] [PDF]
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M. E. Mabrouk, Q. N. Diep, K. Benkirane, R. M. Touyz, and E. L. Schiffrin SAM68: a downstream target of angiotensin II signaling in vascular smooth muscle cells in genetic hypertension Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1954 - H1962. [Abstract] [Full Text] [PDF]
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 D. Zohlnhofer, T. G. Nuhrenberg, F.-J. Neumann, T. Richter, A. E. May, R. Schmidt, K. Denker, M. A. Clauss, A. Schomig, and P. A. Baeuerle Rapamycin Effects Transcriptional Programs in Smooth Muscle Cells Controlling Proliferative and Inflammatory Properties Mol. Pharmacol., April 1, 2004; 65(4): 880 - 889. [Abstract] [Full Text]
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P. M. Mourani, P. J. Garl, J. M. Wenzlau, T. C. Carpenter, K. R. Stenmark, and M. C.M. Weiser-Evans Unique, Highly Proliferative Growth Phenotype Expressed by Embryonic and Neointimal Smooth Muscle Cells Is Driven by Constitutive Akt, mTOR, and p70S6K Signaling and Is Actively Repressed by PTEN Circulation, March 16, 2004; 109(10): 1299 - 1306. [Abstract] [Full Text] [PDF]
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P. J. Garl, J. M. Wenzlau, H. A. Walker, J. M. Whitelock, M. Costell, and M. C.M. Weiser-Evans Perlecan-Induced Suppression of Smooth Muscle Cell Proliferation Is Mediated Through Increased Activity of the Tumor Suppressor PTEN Circ. Res., February 6, 2004; 94(2): 175 - 183. [Abstract] [Full Text] [PDF]
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X. Su, E. M. Smolock, K. N. Marcel, and R. S. Moreland Phosphatidylinositol 3-kinase modulates vascular smooth muscle contraction by calcium and myosin light chain phosphorylation-independent and -dependent pathways Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H657 - H666. [Abstract] [Full Text] [PDF]
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 D. L. Williams, C. Li, T. Ha, T. Ozment-Skelton, J. H. Kalbfleisch, J. Preiszner, L. Brooks, K. Breuel, and J. B. Schweitzer Modulation of the Phosphoinositide 3-Kinase Pathway Alters Innate Resistance to Polymicrobial Sepsis J. Immunol., January 1, 2004; 172(1): 449 - 456. [Abstract] [Full Text] [PDF]
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S. F. Yan, R. Ramasamy, Y. Naka, and A. M. Schmidt Glycation, Inflammation, and RAGE: A Scaffold for the Macrovascular Complications of Diabetes and Beyond Circ. Res., December 12, 2003; 93(12): 1159 - 1169. [Abstract] [Full Text] [PDF]
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R.-H. Zhou, T.-S. Lee, T.-C. Tsou, F. Rannou, Y.-S. Li, S. Chien, and J. Y.-J. Shyy Stent Implantation Activates Akt in the Vessel Wall: Role of Mechanical Stretch in Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, November 1, 2003; 23(11): 2015 - 2020. [Abstract] [Full Text] [PDF]
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 C. C. L. Wang, I. Gurevich, and B. Draznin Insulin Affects Vascular Smooth Muscle Cell Phenotype and Migration Via Distinct Signaling Pathways Diabetes, October 1, 2003; 52(10): 2562 - 2569. [Abstract] [Full Text] [PDF]
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H. S. Shin, H. J. Lee, M. Nishida, M.-S. Lee, R. Tamura, S. Yamashita, Y. Matsuzawa, I.-K. Lee, and G. Y. Koh Betacellulin and Amphiregulin Induce Upregulation of Cyclin D1 and DNA Synthesis Activity Through Differential Signaling Pathways in Vascular Smooth Muscle Cells Circ. Res., August 22, 2003; 93(4): 302 - 310. [Abstract] [Full Text] [PDF]
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 C. Dugourd, M. Gervais, P. Corvol, and C. Monnot Akt Is a Major Downstream Target of PI3-Kinase Involved in Angiotensin II-Induced Proliferation Hypertension, April 1, 2003; 41(4): 882 - 890. [Abstract] [Full Text] [PDF]
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 E. A. Goncharova, A. J. Ammit, C. Irani, R. G. Carroll, A. J. Eszterhas, R. A. Panettieri, and V. P. Krymskaya PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L354 - L363. [Abstract] [Full Text] [PDF]
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J. Huang and C. D. Kontos Inhibition of Vascular Smooth Muscle Cell Proliferation, Migration, and Survival by the Tumor Suppressor Protein PTEN Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 745 - 751. [Abstract] [Full Text] [PDF]
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 H. Kimura, K. Sasaki, T. Meguro, and J. H. Zhang Phosphatidylinositol 3-Kinase Inhibitor Failed to Reduce Cerebral Vasospasm in Dog Model of Experimental Subarachnoid Hemorrhage Stroke, February 1, 2002; 33(2): 593 - 599. [Abstract] [Full Text] [PDF]
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C. M. Reynolds, S. Eguchi, G. D. Frank, and E. D. Motley Signaling Mechanisms of Heparin-Binding Epidermal Growth Factor-Like Growth Factor in Vascular Smooth Muscle Cells Hypertension, February 1, 2002; 39(2): 525 - 529. [Abstract] [Full Text] [PDF]
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