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Temporally and Spatially Coordinated Expression of
Cell Cycle Regulatory Factors After Angioplasty
Geeyeon Laura Wei, BA; Kevin Krasinski, BA; Marianne Kearney, BS; Jeffrey M.
Isner, MD; Kenneth Walsh, PhD; Vicente Andrés, PhD
From the Departments of Medicine (Cardiology) and Biomedical Research, St.
Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135
Running title: Cell cycle control after angioplasty.
Corresponding author:
Vicente Andrés, PhD
St. Elizabeth’s Medical Center
736 Cambridge Street
Boston, MA 02135
Tel: (617) 562-7509
Fax: (617) 562-7506
E-mail: VANDRES@OPAL.TUFTS.EDU
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Abstract
Intimal hyperplasia following angioplasty results in part from the migration and proliferation of
vascular smooth muscle cells (VSMCs). However, the cell cycle regulatory networks underlying
injury-induced VSMC proliferation are largely unknown. In the present study, we examined the
kinetics of expression and activity of cell cycle regulatory factors after angioplasty in rat and
human arteries. Cell lysates were prepared from uninjured rat carotid arteries and at different time
points after balloon denudation. Marked induction of the proliferating cell nuclear antigen (PCNA),
the G1/S cyclin-dependent kinase cdk2, and its regulatory subunits, cyclin E and cyclin A, occurred
between 1-2 d following angioplasty, was sustained up to 10 d post-injury and then declined.
Induction of these factors correlated with increased cdk2-, cyclin E- and cyclin A-dependent kinase
activity, indicating the assembly of functional cdk2/cyclin E and cdk2/cyclin A holoenzymes in the
injured arterial wall. Immunohistochemical analysis revealed early expression of cdk2, cyclin E
and PCNA within the media of injured carotid arteries. At later time points, expression of these
markers declined to basal levels in the media, but was detected within the intimal lesion. Thus,
VSMC proliferation after angioplasty in the rat carotid artery is associated with a temporally and
spatially coordinated expression of cdk2, cyclins E and A, and PCNA. Analysis of human arteries
also revealed expression of these factors in VSMCs within restenotic lesions. Thus, cdk2 and its
regulatory cyclins may be suitable targets to limit human restenosis.
KEY WORDS: angioplasty, restenosis, cell cycle control.
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At homeostasis, vascular smooth muscle cells (VSMCs) are postmitotic and express markers of
the differentiated phenotype. However, mature VSMCs can undergo phenotypic modulation in
response to several environmental stimuli. Pathological VSMC proliferation is thought to play a
central role during atherosclerosis and restenosis [ Owens, 1995 #735; Fuster, 1992 #709; Ross,
1993 #177] . Therefore, understanding the fundamental basis of VSMC proliferation is of obvious
interest. Antisense oligonucleotides against several protooncogenes [ Shi, 1994 #382; Simons, 1992
#192; Bennett, 1994 #376] and positive cell cycle control genes [ Abe, 1994 #852; Morishita, 1993
#763; Morishita, 1994 #762; Morishita, 1994 #847] , or arterial gene transfer of negative cell cycle
control genes [ Chang, 1995 #413; Chang, 1995 #718] and growth stimulatory genes [ Nabel, 1993
#764; Nabel, 1993 #765] affected injury-induced VSMC proliferation in different animal models
of vascular remodeling. However, the kinetics of expression of the endogenous cell cycle
regulatory factors in response to vascular injury remain largely unknown.
Progression through the mammalian mitotic cycle is controlled by multiple holoenzymes
comprised of a catalytic cyclin-dependent protein kinase (cdk) and a cyclin regulatory subunit
[ Heichman, 1994 #713; King, 1994 #714; Morgan, 1995 #453; Motokura, 1993 #730; Nurse, 1994
#399] . Functional cdk/cyclin holoenzymes are presumed to phosphorylate target protein substrates
that facilitate cell cycle progression [ Graña, 1995 #772; Peeper, 1994 #712; Weinberg, 1995
#446] . Different cdk/cyclin complexes are orderly activated at specific phases of the cell cycle.
Progression through the first gap-phase (G1) requires both cyclin D-dependent cdk4 and cdk6, and
cdk2/cyclin E holoenzymes. Functional cdk2/cyclin A complexes are required for DNA synthesis
(S-phase) and, subsequently, cdc2/cyclin A and cdc2/cyclin B pairs are assembled and activated
during the second gap-phase (G2) and mitosis (M-phase), respectively. Recent evidence has been
provided suggesting the requirement of cdk2 for entry into mitosis as a positive regulator of
cdc2/cyclin B kinase activity [ Guadagno, 1996 #776] .
In this study, we examined the regulation of cell cycle control proteins following vascular
injury. Our findings demonstrate the temporally and spatially coordinated induction of PCNA,
cdk2 and its regulatory subunits, cyclin E and cyclin A, in balloon-injured rat carotid arteries.
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Upregulation of these proteins was associated with the formation of functional cdk2/cyclin E and
cdk2/cyclin A complexes. Immunohistochemical staining of human restenotic lesions also revealed
the expression of cdk2, cyclin E and PCNA in SM -actin immunoreactive cells. Thus, induction
of cdk2 and its regulatory cyclin subunits in the vessel wall may contribute to intimal thickening
during restenosis.
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Materials and Methods
Antibodies. The following antibodies were used in this study: (a) Rabbit polyclonal antibodies sc397 (anti-p21), sc-163 (anti-cdk2), sc-481 (anti-cyclin E), sc-198 (anti-human cyclin E), and sc-751
(anti-cyclin A) (Santa Cruz Biotechnology); (b) Mouse mAb anti-smooth muscle -actin (SM actin) (Enzo Diagnostics) and anti-SM -actin conjugated to alkaline phosphatase (Sigma
Chemicals); (c) Mouse mAb anti-proliferating cell nuclear antigen (PCNA) (Signet Laboratories).
Human vascular tissue. Coronary and peripheral atherosclerotic lesions were retrieved
percutaneously by therapeutic directional atherectomy as previously described [ Pickering, 1993
#263] . Six coronary lesions were obtained from patients undergoing percutaneous
revascularization for the first time (primary lesions). Another five coronary and four peripheral
lesions were identified at the site of a previous angioplasty and were therefore designated as
restenotic lesions. Tissue specimens were fixed by immersion in 100% methanol overnight and
then embedded in paraffin and cut in 5-m sections. Specimens were estained with
hematoxylin/eosin and elastic tissue trichrome for histopathological examination.
Rat model of balloon injury. Acute endothelial denudation of the left common carotid artery
was performed essentially as described by Clowes et al. [ Clowes, 1983 #312] . Male SpragueDawley rats weighing 400-500 g were anesthetized with sodium pentobarbital (intraperitoneal
injection, 45 mg/kg body wt; Abbot Laboratories). The bifurcation of the left common carotid
artery was exposed through a midline incision and the left common, internal, and external carotid
arteries were temporarily ligated. A 2F embolectomy catheter (Baxter Edwards Healthcare Corp.)
was introduced into the external carotid and advanced to the distal ligation of the common carotid.
The balloon was inflated with saline and drawn towards the arteriotomy site three times to produce
a distending, deendothelializing injury. After withdrawing the catheter, the proximal external
carotid artery was ligated and blood flow was restored to the common carotid artery by release of
the ligatures. The right uninjured carotid artery was used as control tissue. At the indicated times
post-injury, rats were euthanized with sodium pentobarbital (intraperitoneal injection, 100 mg/kg
body wt), and both injured and uninjured common carotid arteries were perfused with saline and
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dissected free of the surrounding tissue. For immunohistochemistry, tissue specimens were fixed
by immersion in 100% methanol overnight and then embedded in paraffin. Arteries for the
preparation of cell extracts were quickly frozen in liquid nitrogen and stored at -80 oC until further
manipulation.
Preparation and analysis of arterial extracts. To minimize animal-to-animal and procedure
variability, whole cell extracts were prepared from the pooled tissue from 8-11 rat carotid arteries
at each time point. Arteries were homogenized in 1ml of ice-cold lysis buffer [50 mmol/L Tris-Cl
(pH 7.6), 100 mmol/L NaCl, 5% NP-40, 30 mmol/L NaF, 1 mmol/L PMSF, 10% glycerol, 2
g/ml leupeptin] using a Tissumizer Mark II homogenizer (Tekmar). Tissue lysates were
centrifuged at 4 oC for 10 minutes in a microfuge set at maximum speed, and the supernatant was
stored at -80 oC in small aliquots.
Western immunoblot analysis (50 g protein) was performed as previously described [ Andrés,
1996 #527] . The following dilutions of primary antibodies were used: 1:5 (anti SM a-actin), 1:100
(anti-cyclin A, anti-p21), 1:200 (anti-PCNA), 1:250 (anti-cyclin E), and 1:500 (anti-cdk2). For
histone H1 kinase assays, arterial lysates (75 g protein) were incubated at 4 oC for 1.5-2 h under
constant rotation in 0.5 ml of lysis buffer containing 0.15 g of the indicated antibodies and 25 l
of Protein A/G PLUS-Agarose beads (Santa Cruz). Immunocomplexes were washed three times
with lysis buffer and twice with kinase buffer [40 mmol/L Tris-Cl (pH 7.6), 20 mmol/L MgCl2, 2
mmol/L DTT]. Subsequently, the beads were resuspended in 30 l of kinase buffer containing 2 g
of histone H1 (Boehringer Mannheim), 7 mol/L ATP, and 5 Ci of [-32P]ATP. The reaction
mixtures were incubated at 30 oC for 30 minutes and then separated on 12% SDS/polyacrylamide
gels [ Laemmli, 1970 #620] . Gels were stained with Coomassie Blue (Sigma Chemicals), dried,
and autoradiographed. Quantification of the signal was performed by counting individual histone
H1 bands in a scintillation counter. Background cpm determined from regions of the dried gel that
did not contain protein were substracted.
Immuhistochemistry. Methanol-fixed, paraffin-embedded rat and human specimens were cut in
5-µm sections. Sections were deparaffinized and rehydrated with PBS. Immunohistochemical
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Wei et al. (R96-832/R1)
staining was performed using a HistoMark universal strepatavidin/biotin kit according to the
recommendations of the manufacturer (Kirkegaard & Perry Laboratories, Inc.). The following
dilutions of primary antibodies (in PBS containing 2% normal goat serum, 0.02% sodium azide)
were used: 1:500 (cdk 2), 1:150 (cyclin E sc-481), 1:100 (cyclin E sc-198), 1:50 (PCNA), and 1:30
(SM -actin). Rat carotid and human coronary arteries were immunostained with cyclin E
antibodies sc-481 and sc-198, respectively. Incubation with primary antibodies was performed
either at 37 oC for 1 h, or overnight at 4 oC. For peptide competition, a 10-fold mass excess of
either cdk2 or cyclin E immunogenic peptide was added to the corresponding primary antibody 1-2
h prior to incubation of sections. SM -actin-alkaline phosphatase immunocomplexes were
directly stained with Fast Red substrate (Bio Genex). Other primary immunocomplexes were
detected with species-appropriate biotinylated secondary antibodies and strepatavidine-peroxidase.
For double immunohistochemistry of human specimens, sections were first stained for PCNA,
cyclin E and cdk2 as described above and peroxidase activity was detected with 0.05% (wt/vol) 3,
3'-diaminobenzidine tetrahydrochloride dihydrate substrate. Specimens were then washed with
PBS, incubated with anti SM -actin antibody conjugated to alkaline phosphatase and stained with
Fast Red substrate. The specimens were mounted with glycerol gelatin aqueous mounting media
(Sigma Chemicals) and examined on an Olympus Vanox-T microsocope (Olympus America, Inc.).
Pictures were recorded on Kodak Gold Plus film (Eastman Kodak Co.).
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Results
Kinetics of expression and activity of cell cycle regulators after angioplasty in the rat carotid
artery. To begin to elucidate the molecular mechanisms underlying the proliferative response of
VSMCs to arterial injury, we analyzed the kinetics of expression and activity of cell cycle
regulatory factors in control and balloon-injured rat carotid arteries. Intimal hyperplasia in this
model of vascular remodeling results primarily from an excesive proliferative response of VSMCs,
which also undergo dedifferentiation and migration [ Clowes, 1986 #779; Clowes, 1983 #312;
Clowes, 1985 #777] . Emergence of VSMCs from quiescence involves the transition from G0 to G1
and S-phase of the cell cycle. Since the cdk inhibitory protein p21Cip1 has been involved in
withdrawal from the cell cycle in a variety of differentiated cell types [ Andrés, 1996 #527; Guo,
1995 #433; Halevy, 1995 #422; Parker, 1995 #423; Jiang, 1994 #393; Guo, 1995 #433; Halevy,
1995 #422; Parker, 1995 #423; Zhang, 1995 #788; Andrés, 1996 #527; Liu, 1996 #789; Macleod,
1995 #631; Poluha, 1996 #783] , we hypothesized that downregulation of p21Cip1 might be
involved in VSMC proliferation following angioplasty. However, p21Cip1 was undetectable in
control rat carotid arteries and up to 60 h post-angioplasty under conditions where its expression
was detected in cultures of quiescent skeletal myocytes (Fig. 1A). Thus, these findings provided no
indication that p21Cip1 is involved in the maintenance of the postmitotic state of VSMCs in vivo.
Positive regulators of cellular proliferation that were induced following angioplasty included
cdk2 and its cognate cyclins E and A (Fig. 1B). Following the accumulation of cyclin E within 1 d
after injury, cdk2 and cyclin A expression was upregulated at 2 d post-injury. Western blot analysis
at earlier time points revealed induction of cyclin E within 8h, which also preceded the
upregulation of cdk2 and cyclin A (Fig. 2). Expression of these proteins was sustained up to 10 d
and then declined at 18 d following angioplasty (Fig. 1B). Of note, the temporal pattern of
expression of cdk2 and its cyclin subunits correlated with the expression of PCNA (Fig. 1B), a
marker of cell growth [ Hall, 1990 #826; Bravo, 1980 #821; Bravo, 1986 #820; Bravo, 1987 #814;
Almendral, 1987 #815; Jaskulski, 1988 #816] .
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We next sought to test whether cdk2 and cyclins E and A assembled into functional
holoenzymes in the injured arterial wall. To this end, cdk2-, cyclin E- and cyclin A-containing
protein complexes were harvested from control and injured rat carotid arteries using specific
antibodies, and histone H1 kinase activity was assayed in the immunoprecipitates. A marked
induction of kinase activity at 36 h post-injury was observed in anti-cdk2 (Fig. 2A), anti-cyclin E
(Fig. 2B) and anti-cyclin A (Fig. 2C) immunoprecipitates, which correlated with upregulation of
cdk2, cyclin E and cyclin A protein levels. Analysis at 60 h post-angioplasty disclosed diminished
cdk2-, cyclin E- and cyclin A-dependent kinase activity. Of note, cdk2 and cyclin A protein levels
were similar at 36 and 60 h post-injury. Thus, downregulation of cdk2- and cyclin A-dependent
kinase activity between 36 and 60 h post-injury might result from post-translational inactivation of
cdk2/cyclin A holoenzymes.
Spatially coordinated expression of cdk2, cyclin E and PCNA following vascular injury in the
rat carotid artery. To further characterize the kinetics of expression of cell cycle regulatory
proteins following angioplasty, we performed immunohistochemical analysis in control and injured
rat carotid arteries. Cdk2 expression was upregulated in medial VSMCs at 36 h post-injury (Fig.
3A, 3B). At later time points when intimal hyperplasia was first noted, cdk2 was detected in the
intimal lesion, but was expressed at low or undetectable levels in the media (Fig. 3C). By 2 wk,
when a prominent intimal lesion was formed, cdk2 expression was largely confined to the luminal
surface (Fig. 3D, 3E). As shown in Fig. 4, cyclin E upregulation was also noted in the media at 36
h after angioplasty and declined thereafter. Expression of cyclin E was detected in the emerging
intimal lesion (Fig. 4E), and then was predominantly seen in the luminal surface of the intima at 2
wk (Fig. 4G). The specificity of cdk2 and cyclin E signal in these immunohistochemical analyses
was demonstrated in control experiments, in which preincubation of the primary antibodies with an
excess of the corresponding immunogenic peptide abroggated the signal (Fig. 3F, 4B, 4D, 4F, 4H,
and data not shown).
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Wei et al. (R96-832/R1)
To investigate whether expression of cdk2 and cyclin E was spatially correlated with VSMC
proliferation following balloon angioplasty, we examined the expression of PCNA by
immunohistochemistry. PCNA expression was undetectable in uninjured vessels (Fig. 5A) and its
expression was upregulated at 36 h post-injury (Fig. 5B). At later time points, expression of PCNA
was detected throughout the emerging neointima (Fig. 5C), and then became limited to the luminal
surface (Fig. 5D). Adjacent sections incubated with control non-immune mouse IgG disclosed lack
of staining (Fig. 5E-H). Taken together, these results indicate that VSMC proliferation is correlated
with the spatially coordinated expression of cdk2 and cyclin E following angioplasty in the rat
carotid artery.
Expression of cell cycle regulators in human atherectomy specimens. The data thus far suggest
that induction and activation of cdk2-containing holoenzymes may be involved in VSMC
accumulation following injury in the rat carotid artery. Since the biology of injury-induced VSMC
proliferation may differ in animals and human arteries, we next sought to examine the expression
of cell-cycle regulatory proteins in human atherosclerotic lesions obtained by directional
atherectomy (Table 1). Each of the primary lesions analyzed were hypocellular. In marked contrast,
extensive foci of hypercellularity were observed in all restenotic lesions. Other histopathological
findings are summarized in Table 1.
Sections were stained with anti SM -actin antibody to identify VSMCs. All restenotic
specimens and 4/6 (67%) primary lesions analyzed disclosed SM -actin immunoreactive cells
(Table 1). Of these, eight restenotic (89%) and one primary (25%) specimen revealed expression of
cdk2 and cyclin E in regions of the plaque that stained positive for SM -actin and PCNA (Table 1,
and Fig. 6A-D). Preincubation of the cdk2 and cyclin E antibodies with an excess of the
corresponding immunogenic peptide abroggated the signal (Fig. 6E, F), demonstrating the
specificity of cdk2 and cyclin E immunostaining. Lack of PCNA staining in human VSMCs
correlated with undetectable levels of cdk2 and cyclin E in 3/4 (75%) primary lesions and 1/9
(11%) restenotic specimen (Table 1, and Fig. 7). Thus, in agreement with our findings in balloon-
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injured rat carotid arteries, abundant expression of cdk2 and cyclin E was detected in regions of
human restenotic lesions that contained SM -actin and PCNA immunoreactive cells. Further
evidence for the expression of proliferation markers within human restenotic VSMCs was provided
using a sequential double immunostaining approach, which disclosed expression of PCNA, cdk2
and cyclin E in SM -actin immunoreactive cells (Fig. 8).
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Discussion
This study demonstrates the temporally and spatially coordinated induction of positive regulators
of cell growth following balloon angioplasty in the rat carotid artery. Induction of cdk2 and its
regulatory subunits, cyclin E and cyclin A, was associated with the formation of functional
cdk2/cyclin E and cdk2/cyclin A holoenzymes in injured arteries. Both the time course and spatial
distribution of these proteins correlated with the expression of the proliferation marker PCNA.
Cdk2 and cyclin E were also detected in regions of human restenotic lesions that contained SM actin and PCNA immunoreactive cells. Thus, cdk2 and its regulatory cyclins may be suitable
targets for restenosis therapy.
Temporally and spatially coordinated induction of positive regulators of cell cycle progression
after angioplasty in the rat carotid artery. The main goal of the present study was to elucidate
regulatory mechanisms underlying the proliferative response of VSMCs to arterial injury.
Endothelial denudation in the rat carotid artery induces a well-characterized proliferative and
migratory response of VSMCs [ Clowes, 1983 #312; Clowes, 1985 #777; Clowes, 1986 #779] . The
first response to injury (0-3 d) consists of VSMCs proliferation in the media. Subsequently,
migration of VSMCs from the media into the intima is observed (3-14 d), and later cell
proliferation appears to account for intimal lesion growth between 1-3 wk after injury. Several
lines of evidence indicate that cdk2 and its regulatory subunits, cyclins E and A, may contribute to
intimal lesion growth in this animal model of arterial injury. First, Western blot analysis and kinase
assays revealed a rapid induction and activation of cdk2 and cyclins E and A between 1 and 2 d
post-injury. Expression of these proteins, which correlated with PCNA expression, was sustained
up to 10 d following angioplasty and then declined by 18 d. Secondly, immunohistochemical
analyses at 36 h post-injury revealed cdk2 and cyclin E expression in medial VSMCs.
Subsequently, cdk2 and cyclin E expression was detected in VSMCs within the emerging intimal
lesion, and became low or undetectable within the media. At later time points when a prominent
hyperplastic response was noted, expression of cdk2 and cyclin E was largely confined to the
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luminal surface of the intima. That expression of cdk2 and cyclin E was spatially correlated with
VSMC proliferation following angioplasty was suggested by PCNA immunohistochemistry, which
displayed essentially the same spatial pattern of expression. These findings are consistent with
previous [3H]thymidine and 5-bromodeoxyuridine pulse-labeling experiments in rats [ Belknap,
1996 #782; Clowes, 1983 #312; Majesky, 1987 #865] demonstrating a peak of VSMC
proliferation in the media of balloon-injured carotid arteries between 33-48 h, which then declined
rapidly thereafter to return to baseline levels. Proliferative activity was noted throughout the newly
formed intimal lesion, and subsequently became limited to the luminal surface. Taken together,
these studies demonstrate a striking temporal and spatial correlation between the kinetics of VSMC
proliferation and the kinetics of expression and activity of cdk2, cyclin E and cyclin A during
injury-induced vascular remodeling in the rat carotid artery. In this regard, antisense cdk2
oligonucleotides have been shown to inhibit intimal hyperplasia in the rat carotid model of balloon
angioplasty [ Morishita, 1994 #762; Abe, 1994 #852] .
Induction of cyclin E preceded the upregulation of cdk2 and cyclin A in balloon-injured rat
carotid arteries. Thus, assuming that emergence of VSMCs from quiescence in this model of
angioplasty is synchronous [ Majesky, 1987 #865] , it is conceivable that, on a cell-by-cell basis,
cdk2 associates first with cyclin E and subsequently with cyclin A. This conclusion is consistent
with cell culture studies demonstrating that cdk2/cyclin E pairs assemble first during G1, and then
cdk2/cyclin A complexes associate early in S phase [ Peeper, 1994 #712; Graña, 1995 #772; Sherr,
1994 #403] .
Recent evidence has been presented indicating the involvement of the cdk inhibitor p21Cip1 in
promoting permanent growth arrest during terminal differentiation in several cell lineages,
including skeletal myocytes [ Andrés, 1996 #527; Guo, 1995 #433; Halevy, 1995 #422; Parker,
1995 #423] , hematopoietic cells [ Jiang, 1994 #393; Liu, 1996 #789; Macleod, 1995 #631; Zhang,
1995 #788] , and neuronal cells [ Poluha, 1996 #783] . We show here a lack of basal p21Cip1
expression in uninjured vessels, which contain mostly postmitotic cells and did not display
detectable levels of cdk2 kinase activity. Moreover, induction of p21Cip1 in primary cultures of rat
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VSMCs did not correlate with diminished cdk2 kinase activity and, unlike postmitotic skeletal
myotubes [ Guo, 1995 #433] , no evidence for heat-stable cdk2 inhibitory activity was found in
cultures of quiescent rat VSMCs (data not shown). Thus, these data support the notion that basal
expression of p21Cip1 is not necessary for the maintenance of the quiescent state in uninjured
arteries. Perhaps the lack of expression of p21Cip1 may contribute to the reversibility of cell cycle
exit that distinguishes VSMCs from terminally differentiated cells.
Cdk2 and cyclin E are expressed in proliferating vascular myocytes within human restenotic
lesions. The contribution of several protooncogenes, cell cycle control genes, and growth
stimulatory genes to intimal hyperplasia has been suggested in several animal models of vascular
remodeling [ Abe, 1994 #852; Bennett, 1994 #376; Chang, 1995 #413; Chang, 1995 #718;
Morishita, 1993 #763; Morishita, 1994 #762; Morishita, 1994 #847; Nabel, 1993 #764; Nabel,
1993 #765; Shi, 1994 #382; Simons, 1992 #192] . However, information regarding the factors
underlying VSMC proliferation in human atherosclerotic lesions is limited. Since both PCNA
mRNA and protein are detectable in proliferating cells, but not in quiescent cells [ Hall, 1990 #826;
Bravo, 1980 #821; Bravo, 1986 #820; Bravo, 1987 #814; Almendral, 1987 #815; Jaskulski, 1988
#816] , PCNA immunostaining has been used to evaluate cellular proliferation in tissue sections as
an alternative to 5'-bromodeoxyuridine incorporation [ Sanders, 1993 #827; Galand, 1989 #797;
García, 1989 #819; Hall, 1990 #817; Kawakita, 1992 #818; Pickering, 1993 #263] . Our
immunohistochemical analyses in methanol-fixed human atherectomy specimens demonstrate the
expression of both cdk2 and cyclin E in 8/9 (89%) restenotic and 1/4 (25%) primary lesions that
contained cells expressing SM -actin. Expression of cdk2 and cyclin E was seen in regions of the
lesion that disclosed PCNA and SM -actin immunoreactivity. In contrast, cdk2 and cyclin E were
undetectable in regions of primary plaques that contained SM -actin immunoreactive cells that did
not express PCNA. Thus, these results indicate a correlation between VSMC proliferation and the
expression of cdk2 and cyclin E in human restenotic tissue. In this regard, it is noteworthy to point
out that overexpression of cyclin E and D-type cyclins has been reported in several types of solid
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tumors as well as leukemia [ Dou, 1996 #813; Dutta, 1995 #812; Keyomarsi, 1993 #811;
Keyomarsi, 1994 #810; Keyomarsi, 1995 #809] , suggesting that deregulated cyclin expression may
be a common theme in pathological -nonmalignant and neoplastic- cell growth. Future studies
aimed to elucidating the mechanisms underlying the upregulation of cdk2, cyclin E and cylin A
expression in VSMCs should not only provide insight into the pathobiology of restenosis, but
might also have important implications for developing approaches to limit or prevent restenosis.
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Acknowledgments
This study was supported in part by research grants from the National Institutes of Health,
Bethesda. We are grateful to Jihong Yang for tissue sectioning, and María J. Andrés for the
preparation of figures.
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References
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Table 1. Expression of cell cycle regulatory proteins in human atherectomy specimens.
-actin
SM
PCNA
cdk2
cyclin E
Calcific
deposits
Foam
cells
Cholesterol
clefts
Thrombus
+
+
+
+
-
+
-
+
-
+
-
+
+
+
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
-
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
+
+
+
+
a) Primary *
(Coronary)
1
2
3
4
5
6
b) Restenotic *
Coronary
1 (6 mo)†
2 (3 mo)†
3 (6 mo)†
4 (6 mo)†
5 (6 mo)†
Peripheral
1 (11 mo)†
2 (6 mo)†
3 (8 mo)†
4 (7 mo)†
* Specimens were retrieved at the time of percutaneous revascularization by directional
atherectomy. Adjacent sections were examined by immunohistochemistry. Histopathology was
evaluated by hematoxylin/eosin and elastic tissue trichrome staining.
† Months between primary percutaneous revascularization and directional atherectomy.
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Figure 1
Temporally coordinated induction of cdk2, cyclin E and cyclin A following
angioplasty in the rat carotid artery. Whole cell extracts (50 g protein) prepared from
uninjured (C=control) and injured rat carotid arteries were examined by Western blot analysis. Cell
extracts from irreversibly quiescent murine C2C12 skeletal myocytes (SKM) were prepared as
previously described [ Andrés, 1996 #527] and used as positive control for p21Cip1 expression in
(A). The molecular weight of protein markers is shown (kD).
Figure 2
Upregulation of cdk2-, cyclin E-, and cylin A-dependent histone H1 kinase
activity following angioplasty in the rat carotid artery. Cdk2- (A), cyclin E- (B) and cyclin Adependent (C) histone H1 kinase activity was assayed in immunoprecipitates harvested from
uninjured (C=control) and injured rat carotid arteries. Bars represent the mean ± SEM of two
independent assays. Representative autoradiographs show phosphorylation of the histone H1
substrate. Shown below are Western immunoblot analysis using the same extracts. (A) Lysates
were immunoprecipitated with anti-cdk2 antibodies. Activity at 36 h post-injury is defined as
100%. For the assays in anti-cyclin E (B) and anti-cyclin A (C) immunoprecipitates, histone H1
kinase activity was also assayed in anti-cdk2 immunoprecipitates at 60 h post-injury (not shown).
Activities in the anti-cyclin E and A immunoprecipitates are expressed relative to that of the anticdk2 internal control (defined as 100%).
Figure 3
Spatial expression of cdk2 following balloon angioplasty in the rat carotid
artery. Cdk2 immunostaining in control rat carotid arteries (A), and after the indicated time points
following balloon angioplasty (B-F). No signal was detected in control experiments where the cdk2
anti-serum was preincubated with an excess of immunogenic peptide prior to immunostaining (F,
and data not shown). Black and white arrows point to the internal and external elastic lamina,
respectively. med, media; int, intimal lesion. For all pictures, the arterial lumen is on top and the
adventitia is on bottom. Cdk2 expression in the media was noted at 36 h, and then declined
thereafter (C, E). At later time points, cdk2 expression was detected throughout the emerging
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intimal lesion (C), and then became largely confined to the luminal surface (D, E). Magnification is
200x (A-D; bar= 10 m), and 50x (E, F; bar= 50 m).
Figure 4
Spatial expression of cyclin E following balloon angioplasty in the rat carotid
artery. Cyclin E immunostaining in control rat carotid arteries (A, B), and after the indicated time
points following balloon injury (C-H). For peptide competition, the cyclin E anti-serum was
preincubated with an excess of immunogenic peptide prior to immunostaining (B, D, F, H). Black
and white arrows point to the internal and external elastic lamina, respectively. adv, adventitia;
med, media; int, intimal lesion. For all pictures, the arterial lumen is on top and the adventitia is on
bottom. Note that cyclin E expression in medial cells was upregulated within 36 h after injury, and
then declined at later time points. Cyclin E expression was abundant throughout the emerging
intimal lesion (E). By 2 wk, expression of cyclin E was confined to the luminal surface of the
intima (G). Magnification is 200x (A-F; bar= 10 m), and 50x (G, H; bar= 50 m).
Figure 5
Spatial expression of PCNA following balloon angioplasty in the rat carotid
artery. (A-D) PCNA immunostaining in control uninjured and injured rat carotid arteries. (E-F)
Adjacent section were incubated with non-immune mouse IgG (as control). Black and white
arrows point to the internal and external elastic lamina, respectively. adv, adventitia; med, media;
int, intimal lesion. Note that PCNA expression in medial cells was upregulated within 36 h after
injury, and then declined at later time points. Expression of PCNA was abundant throughout the
emerging intimal lesion (C) and then became confined to the luminal surface of the intima (D).
Figure 6
Expression of cdk2, cylin E and PCNA in human coronary restenotic lesions.
(A-D) Immunohistochemical staining was performed on nine methanol-fixed human restenotic
atherectomy specimens (see Table 1). Representative pictures of adjacent sections from coronary
restenotic patient 1 are shown. Note that both cdk2 and cyclin E were abundantly expressed in
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regions of the lesion that contained PCNA and SM -actin immunoreactive cells. (E, F) Incubation
of the cdk2 and cyclin E antibodies with an excess of the corresponding immunogenic peptide
abolished the signal. All panels show the same magnification. Inserts in A-D show high power
fields (Bar = 50 m).
Figure 7
Lack of cdk2, cyclin E and PCNA expression in VSMCs within human coronary
primary lesions. Immunohistochemical staining was performed on six methanol-fixed human
coronary primary atherectomy specimens (see Table 1). Representative pictures of adjacent
sections from coronary primary patient 1 are shown. Note the lack of expression of PCNA, cdk2,
and cyclin E in regions of the lesion that contained SM -actin-positive cells. Bar = 50 m.
Figure 8
Expression of cdk2, cyclin E and PCNA in SM -actin immunoreactive cells
within human restenotic lesions. Double immunohistochemical staining for the indicated pairs of
antigens was performed on five methanol-fixed human pheripheral restenotic specimens.
Representative pictures of adjacent sections from one patient are shown. PCNA, cdk2 and cyclin E
were detected with peroxidase-conjugated secondary antibodies using 3, 3'-diaminobenzidine
tetrahydrochloride dihydrate substrate (brown nuclear staining). SM -actin was subsequently
detected with a monoclonal antibody conjugated to alkaline phosphatase using Fast Red substrate
(red cytoplasmic staining).