From the Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia, Spanish Council for Scientific Research (CSIC), 46010-Valencia, Spain
Received for publication, May 14, 2002, and in revised form, October 24, 2002
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ABSTRACT |
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Excessive proliferation and migration of vascular
smooth muscle cells (SMCs) participate in atherosclerotic plaque
growth. In this study, we investigated whether SMCs from vessels with different atherogenicity exhibit distinct growth and migratory potential and investigated the underlying mechanisms. In fat-fed rabbits, we found increased cell proliferation and atheroma formation in the aortic arch versus the femoral artery. When examined
in culture, SMCs isolated from the aortic arch (ASMCs) displayed a
greater capacity for inducible proliferation and migration than paired
cultures of femoral artery SMCs. Two lines of evidence suggested
that distinct regulation of the growth suppressor p27Kip1
(p27) contributes to establishing these phenotypic dissimilarities. First, p27 expression was comparably lower in ASMCs, which exhibited a
higher fraction of p27 phosphorylated on Thr-187 and ubiquitinated. Second, forced p27 overexpression in ASMCs impaired their proliferative and migratory potential. We found that platelet-derived growth factor-BB-dependent induction of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway was
comparably higher in ASMCs. Importantly, pharmacological inhibition of
MAPKs increased p27 expression and attenuated ASMC proliferation and
migration. In contrast, forced MAPK activation diminished p27
expression and markedly augmented femoral artery SMC proliferation and
migration. We propose that intrinsic differences in the regulation of
MAPKs and p27 play an important role in creating variance in the
proliferative and migratory capacity of vascular SMCs, which might in
turn contribute to establishing regional variability in atherogenicity.
Atherosclerotic cardiovascular disease is the leading
cause of mortality and morbidity in developed countries. Although
percutaneous transluminal angioplasty has become a well established
technique for revascularization of patients with arterial occlusive
disease. The occurrence of restenosis at the site of angioplasty
remains the major limitation despite the successful procedure. The
molecular basis of atherosclerosis and restenosis involves
dedifferentiation of vascular smooth muscle cells
(SMCs)1 to a so-called
"synthetic state" characterized by abundant production of matrix
components and excessive proliferative and migratory activities (1-3).
Therefore, a better understanding of the molecular mechanisms
underlying these processes should help develop novel therapeutic
approaches for the treatment of cardiovascular disease.
Cellular proliferation is regulated by the balance between multiple
cyclin-dependent kinase (CDK)/cyclin holoenzymes and
members of the Cip/Kip and INK4 families of CDK inhibitors (4,
5). Active CDK/cyclin complexes promote cell cycle progression by phosphorylating the retinoblastoma gene product, pRb, and the related
pocket proteins p107 and p130 from mid-G1 to mitosis. CDK
inhibitors associate with and inhibit the activity of CDK/cyclin holoenzymes. Studies arguing for a role of the Cip/Kip protein p27Kip1 (p27) in the pathophysiology of the cardiovascular
system include the following. 1) p27 may contribute to the
reestablishment of the quiescent phenotype after the initial
proliferative response to balloon angioplasty in rat and porcine
arteries, and adenovirus-mediated overexpression of p27 inhibited
neointimal growth in these experimental models (6-8). 2) p27 may
function as a molecular switch that regulates the phenotypic response
of vascular SMCs to both hyperplastic and hypertrophic stimuli (9, 10).
3) p27 is a negative regulator of endothelial cell proliferation and
migration in vitro, and adenovirus-mediated overexpression
of p27 inhibited angiogenesis in vivo (11, 12). 4) p27 may
contribute to integrin-mediated control of vascular SMC proliferation
(13). 5) p27 may limit cardiomyocyte proliferation during early
post-natal development and after injury in adult mice (14, 15). 6)
Changes in p27 expression might regulate human vascular cell
proliferation within atherosclerotic lesions (7, 16), and a causal link
between reduced p27 expression and atherosclerosis has been established in apolipoprotein E-deficient mice (17). It has been established that
the expression of p27 is regulated mainly at the level of translation
and protein turnover (18).
Multiple growth factors and cytokines interact with specific receptors
located in the cytoplasmic membrane of vascular cells in response to a
variety of pathological stimuli, thus triggering a complex signal
transduction cascade, which culminates in changes in gene expression
that execute a proliferative and migratory response (2, 3). The
activation of the mitogen-activated protein kinase (MAPK) signal
transduction pathway is thought to play an important role during
cardiovascular disease (19-23).
It has been well established that different segments of the arterial
tree display significant differences in their susceptibility to
atherosclerosis, both in animal models and humans. In this regard, it
is notable that vascular SMCs display regional phenotypic variance both
when comparing cells obtained from different compartments of the same
vessel or cells isolated from vessels from different vascular beds
(24-30). The findings of this study demonstrate that p27 and MAPKs are
critical regulators of vascular SMC proliferation and migration. Our
results suggest that intrinsic differences in the regulation of p27 and
MAPKs may contribute to the establishment of regional variance in the
proliferative and migratory capacity of SMCs from distinct regions of
the vascular system.
Antibodies--
The following antibodies were purchased from
Santa Cruz Biotechnology: cyclin D1 (sc-450), cyclin A (sc-751), cyclin
E (sc-198), p27 (sc-1641), Rabbit Studies--
Male white New Zealand rabbits
(4-5-month-old) were fed either control chow (n = 5)
or received for 2 months a high fat diet (n = 10)
containing 10 g of cholesterol (Sigma) and 60 ml of peanut oil/kg
control chow (1% cholesterol). Animals received four intraperitoneal injections of 5-BrdUrd (20 mg/kg each, Sigma) at 12-h intervals starting 48 h before sacrifice. Rabbits were killed with an
overdose of pentobarbital. A cut was made in the cava vein, and the
systemic circulation was thoroughly perfused with saline through the
heart. The aortic arch and the right femoral artery were fixed in
situ with 100% methanol. Arteries were removed, fixation was
continued overnight, and tissues were paraffin-embedded and cut in
5-µm cross-sections. Immunohistochemistry using mouse monoclonal
anti-BrdUrd antibody (1:50) was done with a
biotin/streptavidin-peroxidase detection system (Signet Laboratories)
and 3,3'-diaminobenzidine tetrahydrochloride substrate (Sigma).
Cell Culture and Retroviral Infection--
The aortic arch, the
common carotid artery, and the femoral artery of 4-month-old male New
Zealand White rabbits were extracted to prepare primary cultures (SMCs
isolated from the aortic arch (ASMCs), carotid artery SMCs (CSMCs), and
femoral artery SMCs (FSMCs), respectively). Arteries were dissected
free from surrounding tissue and adventitia and cut into small pieces.
Aortic tissue was digested with collagenase (2 mg/ml, Worthington) in
Dulbecco's modified Eagle's medium-F12 supplemented with 5% fetal
bovine serum (FBS) for 3 h in a shaking bath at 37 °C. Cells
were incubated at 37 °C in a humidified 5% CO2, 95%
O2 atmosphere in Dulbecco's modified Eagle's medium-F12
supplemented with 10% FBS, 100 units/ml penicillin, 0.1 mg/ml
streptomycin, and 2 mmol/liter L-glutamine. All studies
were carried out with primary cultures between passages 2 and 8. Pharmacological inhibition of MAPK kinase (MEK) was achieved by
exposing ASMC cultures to PD98059 (Tocris) as indicated in the figure legends.
Recombinant retrovirus were generated using the retroviral vectors
pBabePuro-p27wt (31) and pBabePuro-MEKE, which encode for
wild-type p27 and a constitutively active MEK1 mutant (32),
respectively. pBabePuro-MEKE was generated by digesting
pcDNAIII-MEKE (a gift of C. Caelles) with BamHI and
XhoI and subcloning the MEKE cDNA into pBabePuro. The
infection of asynchronously growing cells was performed as suggested by
the supplier of the PT67-packaging cells
(Clontech). Infected cells were selected in the
presence of puromycin (2.5 µg/ml, Sigma).
Immunofluorescence Labeling of Vascular SMC Differentiation
Markers and TUNEL Assay--
Cells were plated onto glass coverslips.
To examine the expression of differentiation markers, cells were grown
until reaching confluence and then were maintained in mitogen-free
insulin-transferrin-selenium (Invitrogen) supplemented with 250 µmol/liter ascorbic acid (ITC, Sigma) medium (33) for 2 days.
Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline
(PBS) at room temperature for 1 h and permeabilized with 0.1%
Triton X-100/PBS. Cells were blocked with 1% bovine serum albumin/PBS,
and expression of smooth muscle
For TUNEL assays, cells were grown to ~60% confluence and were
maintained in mitogen-free ITC medium for 2 days. For UV light irradiation, cell culture medium was removed and the cells were washed
twice with PBS. The cultures then were placed in the tissue culture
hood and exposed to UV light for 45 min (UV G-30-watt lamp, Sylvania,
Japan). Control (not irradiated) and UV-irradiated cells were fixed and
permeabilized as indicated above, and TUNEL assay was performed using
an in situ cell death detection kit as suggested by the
manufacturer (Roche Molecular Biochemicals).
Proliferation Assays--
Cells for
[3H]thymidine incorporation assays were plated in
10% FBS/Dulbecco's modified Eagle's medium-F12 at a density of 4 × 104 cells/well in 12-well plates. When ~80%
confluence was reached, cells were rendered quiescent by maintaining
cultures for 48-72 h in mitogen-free ITC medium (33).
Starvation-synchronized cultures were stimulated with platelet-derived
growth factor-BB (PDGF-BB) (10 ng/ml) to induce cell cycle reentry, and
cells were pulsed with 1 mCi/liter [3H]thymidine
(Amersham Biosciences) during the last 4 h of incubation. After
washes with cold PBS, DNA was precipitated with 15% trichloroacetic acid and solubilized with 0.2 mol/liter NaOH. Radioactivity
incorporated into DNA was measured in a scintillation counter (Wallac).
Migration Assays--
Migration of cultured cells labeled with
the fluorescent dye calcein-AM (Molecular Probes) was assessed with the
FALCON HTS FluoroBlock system as suggested by the manufacturer (BD
Biosciences). Labeled cells were placed in the inserts (8.0-µm pore
size, 5 × 104 cells/insert) in serum-free media. The
lower chamber contained either serum-free media (unstimulated cells) or
the chemotactic agent (10% FBS or 10 ng/ml PDGF-BB) (induced cells).
Serum-free medium was supplemented with 0.1% bovine serum albumin.
Chemotaxis at different times after plating the cells was assessed by
detecting the fluorescence in the lower chamber using a Victor 4120 multilabel counter (Wallac). Results represent the average fluorescence
of induced cells (n = 3) after subtracting the
fluorescence of unstimulated cells (n = 2-3).
Western Blot Analysis, Immunoprecipitation, and CDK
Assays--
Cell lysates were prepared with either ice-cold lysis
buffer A or buffer B supplemented with protease inhibitor
CompleteTM Mini-mixture (Roche Molecular Biochemicals).
Buffer A contained 50 mmol/liter Hepes (pH 7.5), 1% Triton X-100, 150 mmol/liter NaCl, 1 mmol/liter dithiothreitol, 0.1 mM
orthovanadate, 10 mM
CDK activity in cell lysates (100 µg of protein) was determined as
described previously (6) with the exception that CDK/cyclin holoenzymes
were immunoprecipitated with 0.2 µg of each of the anti-cyclin E and
anti-cyclin A antibodies.
Statistical Analysis--
Results are reported as the mean ± S.E. Differences were evaluated using either two-tail unpaired
Student's t test or ANOVA and Fisher's post hoc test
(Statview, SAS Institute).
Arterial Cell Proliferation and Atherogenesis in Different Vascular
Beds of Hypercholesterolemic Rabbits--
We investigated
atherogenesis in fat-fed New Zealand White rabbits, which rapidly
develop atheromas in response to dietary manipulation (34). To examine
arterial cell proliferation, animals received four injections of BrdUrd
prior to sacrifice. Although aortic atherosclerosis and BrdUrd
immunoreactivity were essentially undetectable in rabbits fed with
control chow (n = 5, data not shown), all of the
fat-fed rabbits included in our study displayed atheromatous lesions in
the aortic arch and exhibited abundant BrdUrd immunoreactivity in both
intimal and medial cells (n = 10, Fig.
1A). In marked contrast, only
3 of 10 fat-fed rabbits displayed small atherosclerotic lesions in the
femoral artery (Fig. 1B). Moreover, the number of
BrdUrd-positive cells in femoral arteries was negligible in the media
and was lower within the lesions as compared with the aortic arch (Fig.
1B). These findings are consistent with previous rabbit
studies demonstrating that the aortic arch is highly susceptible to
diet-induced atherosclerosis (34-37).
ASMCs and FSMCs Display Dissimilar Migratory and Proliferative
Activity in Vitro--
Having demonstrated distinct proliferative
response and atherogenicity in the aortic arch and femoral artery, we
isolated SMCs from these vessels (ASMCs and FSMCs, respectively) to
ascertain whether their phenotypic dissimilarities were maintained
in vitro. In primary cultures grown to confluence in
serum-free media, ASMCs exhibited an epithelioid shape (Fig.
2A), whereas FSMCs disclosed a
bipolar, spindle-shaped morphology (Fig. 2B). We next
performed indirect immunofluorescence experiments in passage 2 cultures to examine the expression of SMC differentiation markers. Both ASMCs
and FSMCs revealed abundant SM
We next compared the migratory and proliferative capacity of cultured
ASMCs and FSMCs. Although FSMCs did not migrate in response to 6 h
of stimulation with either PDGF-BB or FBS, both agents elicited a
robust migratory response in paired cultures of ASMCs (Fig.
2G). Likewise, [3H]thymidine incorporation in
starvation-synchronized cultures restimulated with PDGF-BB was lower in
FSMCs (Fig. 2H). For example, compared with starved
cultures, maximum [3H]thymidine incorporation at 24 h post-stimulation increased by 16- and 42-fold in FSMCs and ASMCs,
respectively. The proliferative response toward 10% FBS was also
stronger in ASMCs (data not shown). In contrast, as determined by the
TUNEL assay, apoptosis was similar in ASMCs and FSMCs, both under
control conditions and after UV irradiation (Fig. 2I).
Lineage analysis experiments have suggested that neural crest-derived
(ectoderm) SMCs prevail in arterial segments proximal to the heart
(i.e. aortic arch and great vessels of the head and neck),
whereas arteries located more distally to the heart contain mainly
mesoderm-derived SMCs (i.e. abdominal aorta and hind limb arteries) (1, 27, 38). Thus, dissimilar behavior and morphology of
ASMCs and FSMCs raised the possibility that adult SMC phenotypic properties are related, at least in part, to their primary embryonic lineage. Consistent with this notion, we found that CSMCs (also of
neural crest origin) behaved in a similar fashion as the ASMCs in
proliferation and migration assays (Fig.
3).
Role of p27 in the Establishment of Phenotypic Variance between
ASMCs and FSMCs--
Differences in proliferation and migration
between ASMCs and FSMCs prompted us to investigate the underlying
molecular mechanisms. Consistent with the results of Fig. 2H
showing greater PDGF-BB-dependent proliferation in ASMCs
than in FSMCs, CDK activity was higher in PDGF-BB-stimulated ASMCs
(Fig. 4A). Likewise, the
up-regulation of the positive cell cycle regulators cyclin D1 and
cyclin A, whose expression is induced as starvation-synchronized cells
resume progression through G1 and S-phase upon mitogen
restimulation (4, 5), occurred earlier and was more prominent in
PDGF-BB-stimulated ASMCs versus FSMCs (Fig. 4B).
The expression of the PDGFR-
We next investigated the expression of the growth suppressor p27 in the
same confluent cultures of ASMC and FSMC used for the PDGFR-
We next investigated the effect of p27 overexpression on ASMC
proliferation and migration by infecting these cells with retroviral vectors encoding for p27 (Rev-p27). Rev-p27-infected ASMCs disclosed a
3-fold increase in p27 expression, which caused a reduction in
[3H]thymidine incorporation (Fig.
5A) and migration (Fig.
5B) as compared with control cultures infected with
Rev-LacZ. These findings demonstrate that increased p27 expression is
sufficient to attenuate the growth and migratory capacity of ASMCs.
Thus, distinct regulation of p27 expression might contribute to
establishing differences in the proliferative and migratory capacity of
ASMCs and FSMCs.
Differential Regulation of MAPKs in ASMCs and FSMCs and Role in the
Regulation of Vascular SMC Proliferation and Migration--
Because
the MAPK pathway plays a pivotal role in transducing environmental
signals required for both cellular proliferation and migration (40), we
examined the kinetics of expression and activation of individual MAPKs
in ASMCs and FSMCs. Western blot analysis using an antibody specific
for the phosphorylated (active) form of the MAPK isoforms of 44 and 42 kDa (dubbed ERK1 and ERK2, respectively) revealed a rapid activation of
these proteins upon PDGF-BB stimulation of mitogen-depleted ASMCs and
FSMCs (Fig. 6, top blot).
However, the maximum level of ERK1/2 activation was higher in ASMCs
than in FSMCs. Moreover, ERK1/2 activation was more prolonged in ASMCs.
These differences occurred despite similar level of total ERK1/2 in
ASMCs and FSMCs (Fig. 6, bottom blot).
To determine whether dissimilar MAPK regulation might contribute to
phenotypic differences between ASMCs and FSMCs, we performed loss- and
gain-of-function experiments. Treatment of ASMCs with PD98059, a
selective inhibitor of MEK, impaired PDGF-BB-dependent ERK1/2 activation (Fig. 7A)
and up-regulated p27 expression (Fig. 7B). Importantly, the
exposure of asynchronously growing ASMCs to PD98059 inhibited
[3H]thymidine incorporation in a
dose-dependent manner (Fig. 7C), and
preincubation of starvation-synchronized ASMCs with PD98059 blocked
de novo DNA synthesis upon mitogen stimulation (Fig.
7D). Moreover, the exposure of ASMCs to PD98059 inhibited
migration (Fig. 8A).
We also examined the effect of forced ERK1/2 activation on FSMC
proliferation and migration by infecting cultures with a retroviral vector encoding for a constitutively active MEK1 mutant (Rev-MEKE). As
compared with control cultures, Rev-MEKE-infected FSMCs disclosed constitutive activation of ERK1/2 (Fig.
9A), which markedly reduced p27 expression (Fig. 9B), increased
[3H]thymidine incorporation (Fig. 9C), and
augmented cell migration (Fig. 8B). Collectively, the above
studies suggest that differential regulation of ERK1/2 in ASMCs and
FSMCs plays an important role in the establishment of intrinsic
differences in the proliferative and migratory potential of these
cells.
Vascular SMCs undergo dedifferentiation and excessive
proliferation and migration during atherosclerosis and restenosis
post-angioplasty (1-3). Up-regulation of the growth suppressor p27 in
the arterial wall might limit SMC proliferation at late time points
after balloon angioplasty in rat and porcine arteries (6, 7), and
adenovirus-mediated overexpression of p27 inhibited neointimal
thickening in these animal models (8, 41). Regarding the role of p27 on
atherosclerosis, genetic disruption of p27 increased arterial cell
proliferation and accelerated atheroma formation in
hypercholesterolemic apolipoprotein E-deficient mice (17). Moreover,
p27 might mediate transforming growth factor- Our studies with fat-fed rabbits showed that aortic arch tissue
displays increased cell proliferation and atherogenicity as compared
with femoral artery. We found that primary cultures of ASMCs and FSMCs
maintained marked differences in their growth and migratory potential,
which might be related, at least in part, to their distinct primary
embryonic lineage (neural crest and mesoderm, respectively) (1, 27,
38). Indeed, ASMCs and CSMCs, which are thought to derive from neural
crest ectoderm, behaved similarly in our proliferation and migration
assays. We chose to examine ASMCs and FSMCs as an in vitro
model to elucidate molecular mechanisms involved in the establishment
of dissimilar atherogenicity in distinct vessel segments. Greater ASMC
proliferation and migration correlated with the lower expression of p27
when compared with FSMCs, and retrovirus-mediated overexpression of p27
attenuated the growth and migratory potential of ASMCs. Previous studies also support the notion that distinct regulation of p27 expression plays an important role in establishing differences in the
phenotypic response of vascular SMCs toward a variety of stimuli.
First, Yang et al. (29) reported reduced proliferation of
human internal mammary artery compared with saphenous vein SMCs.
Importantly, PDGF-BB markedly down-regulated p27 protein level in
saphenous vein, but this response was much less pronounced in internal
mammary artery. Thus, sustained p27 expression despite growth stimuli
may contribute to the resistance to growth of SMCs from internal
mammary artery and to the longer patency of arterial versus
venous grafts. Second, p27 may regulate the proliferative response of
vascular SMCs toward fibroblast growth factor 2 (FGF2 or basic FGF).
Whereas FGF2 plays a critical role in the induction of medial SMC
proliferation after balloon angioplasty (30, 43, 44), neutralizing
antibodies to FGF2 failed to inhibit neointimal SMC proliferation in
balloon-injured arteries (45). Moreover, only a small increase in
growth was observed when arteries with existing neointimal lesions were
exposed to FGF2 (30, 43). Attenuated FGF2-dependent
proliferation of neointimal SMCs occurred despite a robust induction of
positive cell cycle regulators (30). Interestingly, neointimal SMCs
expressed high levels of p27 compared with medial SMCs, and FGF2
infusion did not reduce the level of this inhibitor in arteries with
established neointimal lesions.
Protein turnover is thought to play a major role in the regulation of
p27 expression. Phosphorylation of p27 on Thr-187 triggers its
ubiquitination and rapid turnover in the proteasome (18). Our Western
blot assays demonstrate that the majority (90%) of p27 in ASMCs
corresponds to a slow migrating form that undergoes phosphorylation on
Thr-187 and ubiquitination. In marked contrast, ~96% p27 in FSMCs
corresponded to a faster migrating p27 band that was not recognized by
the phospho-specific antibody and did not contain ubiquitinated
protein. Thus, the relative amount of p27 phosphorylated on Thr-187 and
ubiquitinated appears higher in ASMCs compared with FSMCs, which might
account for the lower level of p27 detected in ASMCs. Of note,
ubiquitinated p27 in the faster migrating band that does not contain
phosphorylated Thr-187 was also detected in ASMCs (cf. Fig.
4E). This finding is in agreement with recent studies
demonstrating an additional pathway for p27 ubiquitination and
proteolysis independent of phosphorylation of p27 on Thr-187 (46,
47).
We investigated additional regulatory networks involved in the
establishment of vascular SMC-phenotypic variance. A wealth of evidence
implicates the rapid activation of the MAPK signal transduction pathway
during the pathogenesis of cardiovascular disease (19, 21). For
example, it has been suggested that persistent activation and
hyperexpression of ERK1/2 are critical elements to initiate and
perpetuate cell proliferation during diet-induced atherogenesis in the
rabbit (48). Moreover, ERK1/2 activation occurs rapidly after
angioplasty of porcine and rat arteries, (20, 22), and all three MAPKs
are activated in human-failing hearts (49). Our results indicate that
ERK1/2 contribute to establishing phenotypic differences between ASMCs
and FSMCs. First, mitogen-dependent activation of ERK1/2
was more robust in ASMCs than in FSMCs. Second, reduced ERK1/2
activation by exposure of ASMCs to PD98059 impaired their growth and
migratory capacity. By contrast, forced activation of ERK1/2 greatly
increased FSMC proliferation and migration. We observed increased p27
expression upon ERK1/2 blockade in ASMCs and diminished p27 expression
upon forced ERK1/2 activation in FSMCs. Thus, in agreement with
previous studies in NIH 3T3 fibroblasts and cancer cells (50-53), our
findings suggest an important role for the MAPK pathway in the control of p27 expression in ASMCs and FSMCs. Solid ERK1/2 activation in
mitogen-stimulated ASMC cultures might facilitate p27 degradation, thus
favoring proliferation and migration of these cells. In contrast, weaker ERK1/2 activation might contribute to comparably higher expression of p27 in FSMCs, thus hindering their proliferative and
migratory responses. In consideration of this model, it is noteworthy
that PDGF-BB induced similar MAPK activation in cultures of saphenous
vein and internal mammary artery despite distinct regulation of p27 in
these cells (29), suggesting that MAPK-independent mechanisms of p27
regulation might operate in the SMCs of different vascular beds.
In conclusion, we propose that intrinsic differences in
MAPK-dependent signaling and p27 expression in rabbit ASMCs
and FSMCs contribute to establishing variance in their proliferative
and migratory potential. These dissimilarities might be attributable, at least in part, to their distinct primary embryonic origin. Further
clarification of the molecular networks underlying vascular SMC-phenotypic variance should shed significant insight into the mechanisms leading to regional variability in the susceptibility to
intimal lesion development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin (sc-8035), CDK2 (sc-163-G), PDGF
receptor isoform
(PDGFR-
) (sc-432), P-ERK1/2 (sc-7383, reactive
with Tyr-204-phosphorylated ERK1 and ERK2), and ERK2 (sc-154, reactive with ERK2 and, to a lesser extent, ERK1). Other antibodies were purchased from Calbiochem (anti-p27 phospho-specific Thr-187 (catalog number 506128) and anti-ubiquitin (catalog number 662099)), Dako (anti-5-bromodeoxyuridine (BrdUrd)), and Master Diagnostica
(anti-smooth muscle
-actin (clone 1A4) and anti-desmin (clone ZC18)).
-actin (SM
-actin) and desmin was
examined by indirect immunofluorescence. Microscopic images were
digitally recorded on an Axioscope II microscope (Zeiss).
-glycerophosphate, and 10 mM sodium fluoride. Buffer B contained 20 mmol/liter
Tris-HCl (pH 7.5), 0.5% Triton X-100, 0.5% deoxycholate, 150 mmol/liter NaCl, 10 mmol/liter EDTA, and 1 mmol/liter dithiothreitol. 50 µg of protein was electrophoresed on 12% SDS-PAGE to perform Western blot analysis as described previously (6). Antibody dilutions
were 1:100 (cyclin D1, cyclin A, cyclin E, P-ERK1/2, and p27), 1:200
(
-tubulin and CDK2), 1:250 (PDGFR-
), 1:500 (anti-p27 phospho-specific Thr-187), and 1:700 (ERK2). For
immunoprecipitation/Western blot assays, cell lysates were incubated
with anti-ubiquitin antibody (0.5 µg) and protein A/G Plus-agarose
(Santa Cruz Biotechnologies) for 4 h at 4 °C under rotation.
The immune complexes were extensively washed and subjected to Western
blot analysis using anti-p27 antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Arterial cell proliferation and atherogenesis
in the aortic arch and femoral artery of hypercholesterolemic
rabbits. Rabbits received either control chow (n = 5) or a high fat diet (n = 10) for 2 months. Prior to
sacrifice, animals were injected with BrdUrd to assess arterial cell
proliferation. The photomicrographs show representative examples of
BrdUrd immunoreactivity in cross-sections of the aortic arch
(A) and femoral arteries (B) of fat-fed rabbits.
Specimens were counterstained with eosin. Two different magnifications
are shown for each specimen as indicated in each photomicrograph.
Arrows in the ×200 photomicrograph of the femoral artery
indicate two BrdUrd-positive cells within the intimal lesion.
White arrowheads point to the internal elastic
lamina.
-actin immunoreactivity in a
prominent stress fiber pattern (Fig. 2, C and D).
In contrast, desmin expression appeared more abundant in FSMCs (Fig. 2,
E and F). These phenotypes were stable at least
up to passage 8 (data not shown).
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Fig. 2.
Phenotypic differences between ASMCs and
FSMCs. Phase-contrast microscopic view of primary cultures of
ASMCs (A) and FSMCs (B) (×200 magnification).
C-F, indirect immunofluorescence analysis of passage 2 primary cultures of ASMCs (C and E) and FSMCs
(D and F) (×400 magnification). ASMCs and FSMCs
disclosed similar expression of SM -actin (C and
D). By contrast, desmin expression was low in ASMCs
(E) and high in FSMCs (F). The right
panel in E and F shows nuclear staining
(Hoechst 33258) in the same field shown for desmin staining
(left). Phenotypic differences between ASMCs and FSMCs were
maintained up to passage 8. G, primary cultures (passage 6)
were labeled with the fluorescent dye calcein-AM and were placed in
serum-free media in the upper chamber of FALCON HTS FluoroBlock
inserts. The lower chamber contained either serum-free media, 10 ng/ml
PDGF-BB (upper panel) or 10% FBS (lower panel).
Chemotaxis was assessed by detecting the fluorescence of cells
migrating to the lower chamber at the indicated time points after
plating the cells. Results represent the average fluorescence of
PDGF-BB-induced or 10% FBS-stimulated cells after subtracting the
fluorescence of unstimulated cells (n = 3). Differences
were evaluated using ANOVA and Fisher's post hoc test. Only
comparisons versus t = 0 are shown. *,
p < 0.05; **, p < 0.005; and ***,
p < 0.0001. H, cells were maintained for
72 h in mitogen-free ITC medium and then were exposed to 10 ng/ml
PDGF-BB for the indicated time. Cultures were pulsed with
[3H]thymidine. Results represent the average of three
experiments using passage 3, 4, and 6 cultures. Differences were
evaluated using ANOVA and Fisher's post hoc test. Comparisons
versus t = 0 are shown: *, p < 0.025; **, p < 0.015; and ***, p < 0.0001; comparisons between ASMC and FSMC at each time point:
,
p < 0.0001; n = 6 each time point.
I, percentage of TUNEL-positive cells in
starvation-synchronized cultures. Analysis included control and
UV-irradiated cells. The total number of cells analyzed in 10 high
power fields (×400) is indicated below each bar.
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Fig. 3.
ASMCs and CSMCs display similar migratory and
proliferative capacity. Statistical analysis was performed using
ANOVA and Fisher's post hoc test. A, migration was assayed
as described in Fig. 2G using 10% FBS as the chemotactic
agent. *, p < 0.0001 versus
t = 0. B, [3H]thymidine
incorporation was assayed as indicated in Fig. 2H
(n = 4 each time point). Comparisons between ASMC and
CSMC at each time point: , p < 0.001; comparisons
versus corresponding t = 0; *, p < 0.0001.
was similar in ASMCs and FSMCs, both
under mitogen-free conditions and upon PDGF-BB stimulation (Fig.
4C), suggesting that dissimilar PDGF-BB-dependent proliferation and migration in ASMCs and
FSMCs were not a consequence of distinct regulation of PDGFR-
expression. Down-regulation of PDGFR-
9 h after PDGF-BB
stimulation is consistent with the notion that binding of PDGF to its
receptor leads to internalization and degradation of the
ligand-receptor complex in endosomes (39).
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Fig. 4.
ASMCs and FSMCs display dissimilar cell cycle
regulatory protein expression and CDK activation. Confluent
cultures were maintained for 72 h in mitogen-free ITC medium and
then exposed to 10 ng/ml PDGF-BB as indicated. Cell extracts were
prepared in lysis buffer A containing phosphatase inhibitors
(A, B, D, and E) or buffer B
(C), which did not contain phosphatase inhibitors. The
analysis of lysates included cyclin A/cyclin E-associated CDK activity
using histone H1 and [ -32P]ATP substrates
(A), Western blot with the indicated antibodies
(B-D), and immunoprecipitation with an
anti-ubiquitin antibody followed by Western blot of the
immunoprecipitated material using anti-p27 antibodies (E).
A, kinase reactions were analyzed by SDS-PAGE and
autoradiography. Relative activity was estimated after densitometric
analysis (0 h is set as 1 for each cell type). B,
densitometric analysis was performed to estimate the relative level of
cyclin D1 and A. Each cyclin value was divided by its corresponding
CDK2-loading control. Shown below is the PVDF membrane stained with
Ponceau prior to incubation with antibodies. C,
densitometric analysis was performed to estimate the relative p27
level. Each p27 value was divided by its corresponding tubulin-loading
control (ASMC at 0 h = 1; nd, not detected).
D and E, the phospho-specific anti-p27 antibody
only recognizes p27 phosphorylated on Thr-187. Open and
closed arrowheads point to the slow and faster migrating
p27-immunoreactive band, respectively. Note that the slow migrating
band that undergoes phosphorylation on Thr-187 and ubiquitinilation
prevailed in ASMCs. By contrast, the faster migrating p27 band that
does not contain protein phosphorylated on Thr-187 and does not undergo
ubiquitination predominated in FSMCs.
immunoblot. Of note, the lysis buffer used in these assays did not
contain phosphatase inhibitors (buffer B). Both under mitogen-free
conditions and at different time points after PDGF-BB stimulation, p27
was detected as a single band that was more abundant in confluent
cultures of FSMCs versus ASMCs (Fig. 4C). For
example, whereas p27 was not detected in ASMC after 9 h of
stimulation, FSMCs expressed more p27 at this time point than did
unstimulated ASMCs. An analysis of subconfluent cultures also disclosed
higher level of p27 expression in FSMCs (data not shown). We next
examined cell lysates prepared in the presence of phosphatase
inhibitors (buffer A), which also disclosed higher p27 expression in
FSMCs versus ASMC (Fig. 4D, top blot).
Notably, these experiments demonstrated the presence of two
p27-immunoreactive bands of different electrophoretic mobility and
distinct relative abundance in these cells. Averaged over four
experiments, the slower migrating band (open arrowhead)
predominated in ASMCs (89.7% ± 8.0), whereas the faster migrating
band (closed arrowhead) prevailed in FSMCs (95.7% ± 1.5).
Western blot analysis using a phospho-specific antibody identified the
slower migrating band as p27 phosphorylated on Thr-187 (Fig.
4D, middle blot). This phosphorylation event is
thought to initiate the major pathway for p27 proteolysis via a
mechanism involving its ubiquitination and subsequent turnover in the
proteasome (18). Consistent with this notion, immunoprecipitation experiments using an anti-ubiquitin antibody followed by Western blot
analysis revealed the presence of ubiquitinated p27 in the slower
migrating p27-immunoreactive band in both ASMCs and FSMCs (Fig.
4E). It is noteworthy that the faster migrating
p27-immnuoreactive band in ASMCs but not in FSMCs also contained
ubiquitinated p27 (see "Discussion"). Collectively, these results
suggest that the majority of p27 in ASMCs undergoes phosphorylation on
Thr-187 and ubiquitination, whereas these post-translational
modifications are detected only in a small fraction of p27 in
FSMCs.
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Fig. 5.
Inhibition of ASMC proliferation and
migration by retrovirus-mediated p27 overexpression. ASMCs were
infected with control retrovirus (Rev-LacZ) or a retrovirus
encoding for p27 (Rev-p27). Infected cells were selected
with puromycin. A, cells were maintained in 10%
FBS/Dulbecco's modified Eagle's medium and pulsed for 4 h with
[3H]thymidine, and radioactivity incorporated into DNA
was quantified. Differences were evaluated using two-tail unpaired
Student's t test (*, p < 0.0015;
n = 5). Puromycin-resistant cells were also lysed in
buffer A to perform Western blot analysis using anti-p27 and
anti- -tubulin antibodies. Densitometric analysis was performed to
estimate the relative level of p27. Each p27 value was divided by its
corresponding tubulin-loading control (Rev-LacZ; n = 1). B, migration of ASMCs infected with Rev-LacZ or Rev-p27
was measured as indicated in Fig. 2G using 10% FBS as the
chemotactic agent. Differences were evaluated using ANOVA and Fisher's
post hoc test. Comparisons versus t = 0: *,
p < 0.005; **, p < 0.0001;
comparisons between Rev-LacZ and Rev-p27 at each time point:
,
p < 0.02;
, p < 0.0001.
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Fig. 6.
ASMCs and FSMCs display dissimilar MAPK
regulation. Western blot analysis of cells maintained for 72 h in mitogen-free ITC medium and then exposed to 10 ng/ml PDGF-BB for
the indicated time. Cell lysates were prepared in lysis buffer
A, P-ERK1/2 and ERK1/2 indicate phosphorylated (active) and
total ERK1/2, respectively. Densitometric analysis was performed to
estimate the relative level of P-ERK1/2. Each P-ERK value was divided
by its corresponding ERK-loading control (ASMC at 0 h = 1).
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Fig. 7.
MAPK inhibition up-regulates p27 expression
and inhibits PDGF-BB-dependent ASMC proliferation.
Differences were evaluated using ANOVA and Fisher's post hoc test.
A and B, ASMCs were maintained for 72 h in
ITC medium and then were exposed to 10 ng/ml PDGF-BB for short
(A) or long (B) periods of time (8 h). Cell
lysates were prepared in lysis buffer A to perform immunoblot analysis
with the indicated antibodies. Treatment with 50 µM
PD98059 was initiated 1 h before the addition of PDGF-BB. P-ERK1/2
and ERK1/2 indicate phosphorylated (active) and total ERK1/2,
respectively. Densitometric analysis was performed to estimate the
relative level of P-ERK1/2 and p27. Each P-ERK or p27 value was divided
by its corresponding loading control (total ERK or tubulin,
respectively; nd, not detected). For p27, results are shown
relative to control (set as 1). C, asynchronously growing
ASMCs were treated for 1 h in mitogen-free ITC medium supplemented
with PD98059 or vehicle, and then cells were incubated for 24 h
with 10 ng/ml PDGF-BB. Cultures were pulsed with
[3H]thymidine during the last 4 h (n = 4). Comparisons versus absence of PD98059: *,
p < 0.0001; comparisons versus 2 µM PD98059: , p < 0.04;
,
p < 0.02. D, cells were maintained for
72 h in ITC media and then were exposed to 10 ng/ml PDGF-BB. When
indicated, mitogen-depleted ASMCs were pretreated with 50 µM PD98059 for 1 h prior to PDGF-BB stimulation.
Cells were pulsed with [3H]thymidine (n = 4; *, p < 0.0001 versus mitogen-depleted
cells;
, p < 0.0001 versus 50 µM PD98059).
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Fig. 8.
Altered MAPK signaling affects ASMC and FSMC
migration. Migration assays were performed as indicated in Fig.
2G using 10 ng/ml PDGF-BB (A) or 10% FBS
(B) as the chemotactic agent. Statistical analysis was done
using ANOVA and Fisher's post hoc test. A, ASMCs were
untreated or exposed to 50 µM PD98059 during labeling
with calcein-AM. PD98059 treatment was maintained in both the upper and
lower chambers. Comparisons versus t = 0: *,
p < 0.05; **, p < 0.01; comparisons
between control and PD98059 at each time point: , p < 0.05;
, p < 0.02;
, p < 0.006. B, migration of FSMCs infected with Rev-LacZ or
Rev-MEKE. Comparisons versus t = 0: *,
p < 0.02, **, p < 0.002, ***,
p < 0.0001; comparisons between Rev-MEKE and Rev-LacZ
at each time point:
, p < 0.04,
,
p < 0.0001.
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Fig. 9.
Forced MAPK activation down-regulates p27
expression and stimulates PDGF-BB-dependent FSMC
proliferation. FSMCs were infected with control retrovirus
(Rev-LacZ) or a retrovirus encoding a constitutively active
MEK1 mutant (Rev-MEKE). Infected cells were selected with
puromycin. Puromycin-resistant cells were maintained for 72 h in
mitogen-free ITC medium, and then cultures were stimulated with 10 ng/ml PDGF-BB for the indicated time. A and B,
cells were lysed in buffer A to perform immunoblot analysis. P-ERK1/2
and ERK1/2 indicate phosphorylated (active) and total ERK1/2,
respectively. Densitometric analysis was performed to estimate the
relative level of P-ERK1/2 and p27. Each P-ERK and p27 value was
divided by its corresponding loading control (total ERK and tubulin,
respectively; nd, not detected). For p27, results are shown
relative to Rev-LacZ at 0 h (set as 1). C,
[3H]thymidine incorporation in mitogen-depleted cells (0 h) and 24 h upon PDGF-BB stimulation. Differences were evaluated
using ANOVA and Fisher's post hoc test. Comparisons among
mitogen-depleted cells: *, p < 0.005 versus
Rev-MEKE; comparisons among PDGF-BB-stimulated cells: **,
p < 0.0001 versus Rev-MEKE;
n = 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dependent
inhibition of cell growth in human atheromas (16), and proliferating
cells within human coronary atheromas appear to express a low level of
p27 (7). Consistent with the observation that p27 overexpression
attenuated human vascular endothelial cell migration in
vitro (12) and that p27 inactivation reduced
rapamycin-dependent inhibition of vascular SMC migration (42), we found that retrovirus-mediated overexpression of p27 inhibited
vascular SMC migration. Thus, p27 might control neointimal thickening
through the regulation of both cell proliferation and migration.
![]() |
ACKNOWLEDGEMENT |
---|
We thank C. Caelles for providing pcDNAIII- MEKE.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants PM97-0136 and SAF2001-2358 from the Spanish Government and Fondo Europeo de Desarrollo Regional.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work is dedicated to Dr. Jeffrey M. Isner.
Received salary support from Agencia Española de
Cooperación Internacional.
§ Partially supported by the Spanish DGESIC and Fondo Europeo de Desarrollo Regional Grant 1FD97-1035-C02-02) and Fondo Social Europeo CSIC-Programa I3P Fellowship.
¶ Present address: Depts. of Medicine and Biology, University of California, San Diego, La Jolla, CA 92093-0665.
To whom correspondence should be addressed: Instituto de
Biomedicina de Valencia, CSIC, C/Jaime Roig, 11, 46010-Valencia, Spain.
Tel.: 34-96-3391752; Fax: 34-96-3690800; E-mail:
vandres@ibv.csic.es.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M204716200
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ABBREVIATIONS |
---|
The abbreviations used are:
SMC, smooth
muscle cells;
CDK, cyclin-dependent kinase;
MAPK, mitogen-activated protein kinase;
p27, p27Kip1;
PDGF, platelet-derived growth factor;
PDGFR, platelet-derived growth factor
receptor;
ERK, extracellular signal-regulated kinase;
P-ERK, phosphorylated ERK;
BrdUrd, bromodeoxyuridine;
ASMCs, SMCs isolated
from the aortic arch;
CSMCs, carotid artery SMCs;
FSMCs, femoral artery
SMCs;
FBS, fetal bovine serum;
ITC, insulin-transferrin-selenium medium
with 250 µmol/liter ascorbic acid;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
PBS, phosphate-buffered saline;
FGF, fibroblast growth factor;
Rev, retroviral vector;
SM-actin, smooth muscle
-actin.
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