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Age-dependent increase in vascular smooth muscle cell proliferation, cyclin A
expression and c-fos activity. A potential link between aging and atherosclerosis.
Alain Rivard, Nicole Principe and Vicente Andrés
Department of Medicine (Cardiology) and Department of Biomedical Research, St.
Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135.
Corresponding author:
Vicente Andrés, Ph. D.
Division of Cardiovascular Research
St. Elizabeth's Medical Center
736 Cambridge Street
Boston, MA 02135
Tel: (617) 562-7509
FAX: (617) 562-7506
E-mail: vicente_andres@hotmail.com
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Abstract
Excessive proliferation of vascular smooth muscle cells (VSMCs) is thought to contribute to
atherosclerosis and restenosis. Aging is a major risk factor for atherosclerosis and has been shown to
be associated with a higher level of VSMC proliferation and neointimal formation after balloon
angioplasty in animal models. Accordingly, we investigated potential mechanisms involved in the
age-dependent increase in VSMC proliferation. Primary cultures of VSMCs were isolated from
young (6-8 months old) or old (4-5 years old) rabbits. Results from cell counting assays and FACS
analysis were consistent with a shortening of the cell cycle in old VSMCs. Western blot analysis in
serum stimulated cells showed a significant increase in the level of cyclin A and cyclin-dependent
kinase 2 (CDK2) proteins in the old versus young VSMCs. In marked contrast, expression of cyclin
E in VSMCs was not influenced by aging. Transient transfection assays with a reporter gene
construct showed an age-dependent increase in transcription from the human cyclin A promoter.
Parallel studies demonstrated that the expression of the AP1 transcription factor c-fos, which has
been shown to interact with the cyclin A promoter and stimulate VSMC proliferation, was also
increased in old VSMCs. Consistent with this notion, electrophoretic mobility shift assays
demonstrated an increase in AP1 DNA-binding activity in old VSMCs. These studies suggest that
age-associated increase in c-fos acivity contributes to augmented cyclin A expression and VSMC
proliferation in old animals. These mechanisms might contribute to the higher prevalence and
severity of atherosclerosis in the elderly.
KEY WORDS: Aging, cell cycle control, vascular smooth muscle cell, cyclin A, c-fos.
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Introduction
VSMCs in adult organisms are typically quiescent and display a fully differentiated
phenotype. However, unlike striated myocytes, VSMCs can reenter the cell cycle in response to
different forms of insult to the vessel wall. It is generally accepted that abnormal proliferation and
migration of VSMCs plays an important role during spontaneous atherosclerosis (1-5). Excessive
proliferation of VSMCs is also thought to contribute to restenosis, an accelerated form of
atherosclerosis that limits the long-term success of revascularization in ~25-55% of patients
undergoing percutaneous transluminal coronary angioplasty (6-8). Recent studies have identified
some of the mechanisms implicated in the control of VSMC proliferation in vitro and in vivo (9).
Normal aging leads to changes in the cardiovascular system that are associated with an
increased risk of atherosclerosis (10-12). It has been previously shown that VSMCs isolated from old
rats have a significantly higher mitogen-mediated proliferative response than young cells (13-15).
Moreover, aging in rats was associated with a greater and more prolonged proliferative response of
VSMCs to balloon angioplasty (16), and transplantation experiments have suggested that this
response appears to be a function of the age of the arterial segment rather than the host environment
(17). Taken together, these findings suggest that the age-dependent increase in VSMC proliferation
may contribute to the increased prevalence and severity of atherosclerosis in the elderly. However,
the mechanisms that contribute to augmented proliferation in old VSMCs remain largely unknown.
Cell cycle progression in mammalian cells is regulated in part by the balance between
multiple growth suppressor proteins which limit cellular growth, and CDK/cyclin holoenzymes
which promote proliferation (18-23). Among these, CDK2 and its regulatory subunits, cyclin E and
cyclin A, are markedly induced after vascular injury in rat and human arteries (24, 25). We have also
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shown that the AP1 transcription factor c-fos is an important component of the signaling cascade that
links Ras activity to cyclin A transcription and VSMC proliferation (26). To elucidate molecular
mechanisms underlying age-dependent increase in VSMC proliferation, we have analyzed the
expression and activity of cell cycle regulatory proteins following serum stimulation of VSMCs
isolated from the aorta of young and old rabbits. Our findings suggest that augmented cyclin A gene
expression via the action of c-fos contributes to increased VSMC proliferation with advanced age.
Materials and Methods
Isolation of VSMCs and proliferation studies.
VSMCs were isolated from the aorta of young (6-8 months old) and old (4-5 years old) New
Zealand White male rabbits as described by Pickering et al. (27). Cells were incubated at 37 0C in a
humidified 5% CO2-95% O2 atmosphere in DME medium supplemented with 2 mM L-glutamine,
200 units/ml penicillin, 0.25 mg/ml streptomycin, and serum as indicated. To render cells quiescent,
primary cultures were maintained for 3 d in DME supplemented with 0.5% FBS and then stimulated
with 10% FBS/DME for different periods of time. When indicated, cells were starvationsynchronized in serum-free IT medium (28). Cells for flow cytometry analysis were trypsinized,
fixed in 70% methanol and stained with a solution containing 50 g/ml of each propidium iodide and
ribonuclease A (Boehringer Mannheim). Samples were analyzed in triplicate with a BectonDickinson FACScan using the CellFIT Cell-Cycle Analysis software (Becton-Dickinson).
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The CellTiter 96 AQ nonradioactive cell-proliferation assay (Promega) was also used to
assess cell growth. The assay is composed of the tetrazolium compound MTS and the electroncoupling reagent PMS. Viable cells reduce MTS to formazan, which can be measured with a
spectrophotometer by the amount of absorbance at 490-nm. Formazan production is time dependent
and proportional to the number of viable cells. VSMCs from young and old animals were cultured in
10% FBS/DME in 96-well flat-bottomed culture plates (Becton Dickinson). Cultures were seeded at
1000 cells/well and allowed to attach overnight. After the indicated time of culture, 20 l MTS/PMS
(1:0.05) mixture was added per well, and cells were incubated 2 hours before measuring absorbance
at 490 nm. Background absorbance from the control wells (same media, no cells) was substracted.
Eight duplicate measurements were performed for each experimental condition.
Transient tansfections and luciferase assays.
VSMCs from young and old rabbits were seeded into 100-mm dishes and maintained in
DME supplemented with 10% FBS. The next day, cells (~60-80% confluence) were transiently
transfected with 10 g of a reporter construct containing the firefly luciferase gene under the
transcriptional control of the –924/+245 promoter region from the human cyclin A gene (29) and 30
g of Lipofectamine reagent (GIBCO Laboratories). To correct for differences in transfection
efficiency, luciferase activity was normalized relative to the level of alkaline phosphatase activity
produced from cotransfected pSVAPAP plasmid (0.5 g), which contains the reporter gene under the
control of the simian virus 40 enhancer-promoter (30). Cells were incubated with transfection
mixtures for 90 min and then were washed with PBS and fed as indicated for 3 days after
transfection. Cells were restimulated with 10% FBS for different periods of time prior to the
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preparation of cell lysates. Luciferase and alkaline phosphatase activity was measured as previously
described (31). Results represent the mean ± SE of three independent transfections.
Whole cell extracts and Western blot analysis.
Whole cell extracts were prepared as previously described (32). After separation on SDSpolyacrylamide gels (33), proteins were transferred by semidry blotting to Immobilon-P (0.45 m,
Millipore). Membranes were blocked for 1 h with PBS containing 0.05% Tween-20 (TPBS) and 4%
nonfat dry milk, and then incubated for 1 h with primary antibodies diluted in TPBS containing 2%
nonfat dry milk. The following rabbit polyclonal antibodies were purchased from Santa Cruz
Biotechnology and were diluted as follows: anti-cdk2 (sc-163, 1:500), anti-cyclin A (sc-751, 1:100),
anti-cyclin E (sc-451, 1:250), and anti-c-fos (sc-052, 1:200). After washes with TPBS, membranes
were incubated for 30 min. with anti-rabbit horseradish peroxidase-conjugated secondary antibodies
(Amersham), washed with TPBS and finally with PBS. Visualization of the immune complexes was
carried out with an enhanced chemiluminescent system (Amersham).
Electrophoretic mobility shift assay.
Electrophoretic mobility shift assays were carried out in buffer containing 10 mM Tris-HCl
(pH 7.5), 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, and 0.05 mg/ml
poly(dI-dC)/poly(dI-dC). Radiolabeled double-stranded oligonucleotide probes spanning either the
AP1 consensus binding site (5’-CGCTTGATGACTCAGCCGGAA-3’; AP1 site underlined) or the
CRE from the human cyclin A promoter region (position -84 to -63; 5'-
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TTGAATGACGTCAAGGCCGCG-3'; CRE underlined) were used. Whole cell extracts were
preincubated in binding buffer for 10 min on ice. Then probe was added for an additional 30 min.
Competition assays were performed by adding a 20-fold molar excess of unlabeled oligonucleotide
to the preincubation mixture prior to the addition of radiolabeled probe. The CRE mut
oligonucleotide contains mutations within the CRE sequence (5'TTAAATGAATTCAAGGCCGCG-3') (34). Binding reactions were separated at 4oC in
nondenaturing 4% acrylamide gels in 0.5X TBE running buffer.
Results
Aging causes increased serum-induced VSMC proliferation.
We isolated VSMCs from the aorta of young and old rabbits and analyzed their kinetics of
proliferation. Cells were plated at a density of 60,000 cells/well, maintained in serum-rich medium
and the number of cells was counted every other day over a period of 4 days. As seen on Figure 1A,
the old VSMCs showed a 2.1-fold increase in cell number when compared to the young VSMCs 4
days after plating (old = 175,000 ± 6,600, young = 82,500 ± 8700, p = 0.001). Consistent with these
findings, in cultures maintained for 5 days in high-serum medium, the MTS cell-proliferation assay
showed a 1.7-fold increase in activity in the old compared to the young VSMCs (old = 1.0 ± 0.03
versus young = 0.6 ± 0.03, p < 0.001) (Fig. 1B). These results suggested that old VSMCs proliferate
faster than young cells when stimulated with serum-rich medium. To test whether old cells proceed
through the cell cycle faster than young cells, starvation-synchronized VSMCs were serum
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restimulated and their cell cycle profile was analyzed at different time points after addition of serum.
As shown by the flow cytometry analysis of Fig. 1C, old VSMCs disclosed maximal DNA synthesis
(S phase) around 18 hours after serum restimulation, whereas increased DNA replication in young
cells occurred at later time points. Collectively, these findings indicate that aortic VSMCs isolated
from old rabbits proliferate at a higher rate than young cells when stimulated with serum.
Age-dependent increase in VSMC proliferation is associated with augmented cyclin A protein
expression and promoter activity.
CDK2 and its regulatory subunits, cyclins E and A are essential for progression through the
G1 and S phase of the mammalian cell cycle (18, 20, 22, 23). We previously showed that VSMC
proliferation in response to mechanical acute injury in rat and human arteries correlates with the
induction of CDK2, cyclin E and cyclin A (24, 25). To investigate whether age-dependent increase in
VSMC proliferation is associated with increased expression of these cell cycle regulators, Western
blot analysis was performed following serum restimulation of starvation–synchronized young and
old VSMCs. As shown in Fig. 2, serum restimulation resulted in the induction of cyclin A and CDK2
protein levels in both young and old VSMCs. However, increased cyclin A and CDK2 expression
appeared more robust in old VSMCs. Of note, induction of cyclin A in old VSMCs preceded the
induction observed in young cells, in agreement with our flow cytometry analysis (see Fig. 1C). In
marked contrast, cyclin E protein levels did not appear to be regulated by serum and aging (Fig. 2).
Thus, increased proliferation in older VSMCs correlates with higher levels of CDK2 and cyclin A
protein expression.
We next examined the kinetics of induction of cyclin A promoter activity following serum
restimulation in young and old VSMCs. To this end, cells were transiently transfected with a
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luciferase reporter construct driven by the human cyclin A promoter region from –924 to +245
relative to the main transcription initiation site (29). In agreement with previous studies in fibroblasts
(29, 35-38) and pulmonary arterial VSMCs (26), transcription from the cyclin A promoter was
induced by serum refeeding in both young and old VSMCs (Fig. 3A, B), although maximum seruminducible cyclin A promoter activity was higher in old than in young VSMCs (Fig. 3C). Moreover,
increased transcription from the cyclin A promoter in old versus young VSMCs was also seen in
serum-starved cells. Indeed, old VSMCs maintained in low serum (0.5% FBS/DME) or in serumfree IT media disclosed a 6-fold increase in cyclin A promoter activity when compared to young
VSMCs (Fig. 3D). These findings indicate that basal and maximal serum-induced cyclin A promoter
activity is higher in old VSMCs. Moreover, these results suggest that increased cyclin A gene
transcription contributes to augmented cyclin A protein expression in older VSMCs.
Age-dependent increase in cyclin A expression in VSMCs is associated with augmented c-fos
expression and DNA-binding activity.
Members of the AP-1 family of transcription factors have been implicated in the control of
cellular growth (39) and their expression correlates with VSMC proliferation in response to balloon
angioplasty (26, 40, 41). We have recently shown that the AP1 transcription factor c-fos links Rasdependent mitogenic signaling to cyclin A transcription and VSMC growth (26). Therefore, we
sought to investigate whether increased c-fos expression and DNA-binding activity might contribute
to age-related augmented cyclin A gene expression. Western blot analysis disclosed higher levels of
c-fos in old VSMCs maintained in low serum medium and at 12 hours after serum restimulation
(Fig. 4A). Moreover, electrophoretic mobility shift assays using a radiolabeled probe containing the
AP1 consensus binding site disclosed higher levels of DNA-binding activity in serum-restimulated
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old VSMCs (Fig. 4B; lanes 2-5 versus 7-10). Competition experiments using an excess of unlabeled
AP1 binding site demonstrated the specificity of the retarded bands (Fig. 4B; lane 5 versus 6, lane 9
versus 11, and lane 10 versus 12). These results indicate that aging leads to an increased seruminducible binding of c-fos to its consensus target sequence.
VSMC proliferation induced by serum in vitro and balloon angioplasty in vivo is associated
with increased binding of c-fos to the cAMP-responsive element (CRE) in the cyclin A promoter,
and this interaction is essential for c-fos-dependent induction of cyclin A gene expression (26).
Therefore, we next investigated the DNA-binding activity associated with the cyclin A CRE in
young and old VSMCs. As shown in Fig. 4C, serum stimulation caused a transient increase in CREdependent binding activity in both young (lanes 1-3) and old (lanes 8-10) VSMCs. However,
maximum activity at 12 hours after addition of serum was more pronounced in old VSMCs (Fig. 4C,
compare lanes 2 and 9). Incubation with an excess of unlabeled wild-type CRE oligonucleotide was
efficient at reducing binding (Fig. 4C; lanes 1-3 versus 4-6, and 8-10 versus 11-13), whereas an
oligonucleotide containing a mutated CRE sequence did not compete (Fig. 4C; lane 3 versus 7, and
lane 10 versus 14). Collectively, these results suggest that age-dependent increase in the expression
and DNA-binding activity of c-fos contribute to augmented cyclin A expression in old VSMCs.
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Discussion
Aging is a major risk factor for atherosclerosis (10-12). According to the response-to-injury
hypothesis, abnormal VSMC proliferation plays an important role in the pathogenesis of
atherosclerosis and restenosis (1-5). In this regard, several in vitro and in vivo studies have suggested
that age-dependent increase in VSMC proliferation may contribute to the increased risk of
atherosclerosis seeing with aging (13-17). In the present study, we sought to investigate age-related
mechanisms causing enhanced VSMC growth. We hypothesized that vascular aging is associated
with increased expression of positive regulators of VSMC proliferation. CDK2 and its regulatory
subunits, cyclin E and A have been shown to be essential for progression through G1 and S phase of
the cell cycle (18, 20, 22, 23), and their expression is induced in human and rat arteries following
vascular injury (24, 25). Therefore, we analyzed the expression of these cell-cycle regulators in
VSMCs isolated from young and old rabbits. Our results demonstrate that age-dependent increased
proliferation following serum restimulation of starvation-synchronized VSMCs is associated with
higher cyclin A and CDK2 protein levels. Of note, disruption of CDK2 (42) and cyclin A (43-45)
function inhibits S phase entry and overexpression of cyclin A accelerates the G1-to-S transition (46,
47), suggesting that cyclin A expression can be rate limiting for cellular proliferation. Combined,
these studies indicate that age-dependent increase in cyclin A and CDK2 expression contributes to
enhanced proliferation in old VSMCs.
To elucidate the molecular mechanisms underlying age-dependent increase in cell-cycle gene
expression, we analyzed the activity of reporter genes containing a luciferase expression cassette
under the transcriptional control of the human cyclin A promoter region extending from –924 to
+245. Previous studies in fibroblasts (29, 35-38) and pulmonary arterial VSMCs (26) have
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demonstrated that the cyclin A gene is transcribed in a cell cycle-dependent manner, starting in late
G1 and increasing until G2 phase. As expected, our results show a marked induction of cyclin A
promoter activity in both young and old VSMCs, although maximum activity was significantly
increased in old versus young VSMCs. Thus, age-associated augmented cyclin A gene transcription
might contribute to increased cyclin A protein expression.
Balloon angioplasty has been shown to induce several members of the AP1 transcription
factor family, including fos and jun proteins (26, 40, 41, 48). We have recently shown that c-fos,
through its interaction with the CRE site in the cyclin A promoter, contributes to the induction of
cyclin A gene expression and VSMC growth (26). Increased binding of c-fos to the cyclin A CRE
preceded the onset of DNA replication in VSMCs induced by serum in vitro and by angioplasty in
vivo. Taken together, these findings suggest that c-fos is a critical component of the signaling
cascade that links Ras activity to VSMC proliferation and neointimal lesion formation. The present
study demonstrates a marked age-associated increase in AP1 DNA-binding activity after serum
restimulation of starvation-synchronized cells (Fig. 4B). Likewise, old VSMCs disclosed increased
DNA-binding to the cyclin A CRE site (Fig. 4C), consistent with a role of c-fos in age-dependent
increase of cyclin A expression and VSMC proliferation. Aging was also associated with higher
levels of c-fos and cyclin A promoter activity in serum-starved VSMCs (Fig. 3D, 4A). In this regard,
McCaffrey et al have demonstrated that the older VSMCs display increased proliferation even in the
absence of mitogens (14). Thus, this predisposition of old VSMCs to proliferate faster than young
cells may be related to higher basal levels of c-fos. Other transcription factors whose expression
and/or activity have been shown to change during aging include stimulatory protein-1 (Sp1), agedependent factor (ADF), heat shock factor-1 (HSF1), and NF-kB (49).
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In summary, our results suggest that augmented cyclin A expression via the action of AP1
transcription factors contributes to increased VSMC proliferation with advanced age. To the best of
our knowledge, these findings illustrate for the first time a transcriptional regulatory network that
might contribute to the increased prevalence and severity of atherosclerosis in the elderly.
Acknowledgments
This work was supported by Public Health Service grant AG15227 from the National
Institutes of Health to V. Andrés. A. Rivard is supported by a grant from the Heart and Stroke
Foundation of Canada.
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Figure legends
Fig. 1 Age-dependent increase in VSMC proliferation. (A) VSMCs were isolated from the aortas
of old and young rabbits and cultured in 10% FBS/DME. Cultures were seeded at 60,000 cells/well
and counted with an hemacytometer every other day over a period of 4 days. Three duplicate
measurements were performed for each experimental condition. (B) Cells were seeded at 1,000
cells/well in 96-well flat-bottomed culture plates to perform the CellTiter 96 AQ cell-proliferation
assay (see methods). After the indicated time in culture, 20 l of MTS/PMS (1:0.05) mixture was
added per well, and cells were incubated 2 hours before measuring absorbance at 490 nm. Eight
duplicate measurements were performed for each experimental condition. (C) The cells were
rendered quiescent by maintaining them for 3 days in DME medium supplemented with 0.5% FBS
and then stimulated with 10% FBS. After the indicated periods of time, the cells were trypsinized,
fixed with 70% ethanol and stained with a solution containing 50 g/ml of each propidium iodine
and ribonuclease A. Samples were analyzed in triplicate with a Becton-Dickinson FACScan using
the CellFIT Cell-Cycle Analysis software.
Fig. 2 Expression of cell cycle regulatory proteins in serum restimulated old and young
VSMCs. Whole cell extracts were prepared from VSMCs isolated from old and young rabbits after
serum stimulation for the indicated periods of time (in hours). Western blot analysis were performed
for different regulatory proteins implicated in the cell cycle. Serum stimulation resulted in the
induction of cyclin A and CDK2 protein levels in both young and old VSMC, although the ultimate
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level was higher in old VSMCs. In marked contrast, cyclin E protein levels did not appear to be
regulated by serum or aging.
Fig. 3 Age-dependent increase in cyclin A promoter activity. VSMCs from young and old rabbits
were transiently transfected with 10 µg of a reporter construct containing the firefly luciferase gene
under the transcriptional control of the human cyclin A gene promoter. To correct for differences in
transfection efficiency, luciferase activity was normalized relative to the level of alkaline
phosphatase activity produced from cotransfected pSVAPAP plasmid containing the reporter gene
under the control of the SV40 promoter. Results are expressed as the ratio of luciferase over alkaline
phosphatase activity (mean ± SEM of three independent assays). Following transfection, young (A)
and old (B) VSMCs were kept in 0.5% FBS/DME for 3 days and then serum restimulated for the
indicated periods of time (in hours). (C) The results shown in A and B are plotted together to point
out the marked increase in maximal cyclin A promoter activity in the serum-stimulated old VSMCs.
(D) After 3 days incubation in serum-free medium (IT) or in 0.5% FBS/DME (DME), transfected
cells were harvested without serum restimulation. The results for DME are the same as shown in C
(0 hour time point).
Fig. 4 Age-dependent increase in AP1 and CRE DNA-binding activity. VSMCs isolated from old
and young rabbits were serum-starved and then serum restimulated for the indicated periods of time
(in hours). (A) Western blot analysis for the AP1 transcription factor c-fos showed higher expression
in old VSMCs in serum-starved cells (0) and 12 hours after serum restimulation. (B) Electrophoretic
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mobility shift assays was carried out with a radiolabeled double-stranded oligonucleotide probe
spanning the AP1 consensus binding site. Competition assays shown in lanes 18c and 24c were
performed by preincubating the cell lysates with a 20-fold molar excess of unlabeled oligonucleotide
(lanes 6, 11 and 12). (C) Electrophoretic mobility shift assays was carried out with a radiolabeled
probe containing the CRE sequence from the human cyclin A gene promoter. For competition
assays, cell lysates were preincubated with a 20-fold molar excess of unlabeled CRE wild type (wt)
or mutant (mt) oligonucleotide. Only the specific retarded bands are shown.
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