Role of the growth suppressor p27Kip1 during vascular remodeling
Antonio Díez-Juan, Claudia Castro, M. D. Edo and Vicente Andrés*
Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and
Therapy, Instituto de Biomedicina de Valencia (IBV-CSIC), Spanish Council for Scientific
Research, Valencia, Spain.
* Corresponding author:
Tel: 96-3391752
FAX: 96-3690800
E-mail: vandres@ibv.csic.es
KEY WORDS: atherosclerosis, restenosis, neovascularization, proliferation, migration,
p27Kip1
List of abbreviations: AngII, angiotensin II; apoE, apolipoprotein E; ASMC, aortic smooth
muscle cell; bFGF, basic fibroblast growth factor; CDK, cyclin-dependent kinase; CKI,
cyclin-dependent kinase inhibitor; EC, endothelial cell; ECM, extracellular matrix; ERK,
extracellular signal-regulated kinase; FSMC, femoral artery smooth muscle cell; HUVEC,
human vein endothelial cell; IMA, internal mammary artery; MAPK, mitogen-activated
protein kinase; PDGF-BB, plateled-derived growth factor-BB; SV, saphenous vein; VSMC,
vascular smooth muscle cell.
1
Abstract
At homeostasis, vascular cells display a very low proliferative rate and scant
migratory activity. However, hyperplastic growth and locomotion of vascular cells are a
hallmark of vascular remodeling during several pathophysiological conditions (e. g.,
neovascularization, arteriosclerosis and restenosis post-angioplasty). Thus, a better
understanding of the molecular mechanisms that control vascular cell proliferation and
migration should facilitate the development of novel therapies to treat cardiovascular
disease. In this review, we will discuss recent studies implicating the cell cycle regulatory
protein p27Kip1 as a key modulator of vascular cell growth and locomotion in vitro and
during vascular remodeling in vivo.
2
Introduction
According to the response-to-injury hypothesis, atherosclerosis is triggered by
different risk factors (i. e., hypercholesterolemia, aging, hypertension, smoking and
diabetes) that cause endothelial dysfunction [1]. Studies in hypercholesterolemic animals
and in human atherosclerotic specimens have identified three processes involved in the
formation of the atherosclerotic plaque once the normal properties of the endothelium have
been altered by the action of cardiovascular risk factors: 1) accumulation of lipid and
mostly free and esterified cholesterol in the surrounding extracellular matrix (ECM) and
the associated cells; 2) proliferation and migration of vascular cells within the injured
arterial wall (Fig. (1)); and 3) formation of a connective tissue matrix comprising elastic
fiber proteins, collagen and proteoglycans. Numerous observations suggest that vascular
smooth muscle cells (VSMCs) in atherosclerotic lesions have changed from a contractile
to a synthetic phenotype, in which they can respond to different growth factors and
synthetize ECM components [2,3]. “Activated” VSMCs migrate toward the arterial lumen
and express abundant levels of novel matrix components and proteases that modify the
surrounding ECM. It is accepted that this “growth and synthetic” response of VSMCs
contributes to neointimal lesion development during atherogenesis.
Excessive VSMC proliferation and migration also contribute to restenosis postangioplasty (Fig. (2)), the recurrence of arterial narrowing at the site of revascularization
that occurs in 20-40% of coronary artery disease patients after successful angioplasty [4-6].
Acute mechanical disruption of the protective endothelial lining at the site of angioplasty
appears to trigger this aggressive form of vascular obstructive disease.
Although arterial cell proliferation occurs in animal models during all phases of
atherogenesis [1,7-9], studies with hyperlipidemic rabbits have shown an inverse
correlation between atheroma size and cellular proliferation within the atheromatous
3
plaque [10-12]. Consistent with the response-to-injury hypothesis [1], medial cell
proliferation at early stages of atherogenesis in fat-fed rabbits increased as a function of
intimal lesion size [1,7-9]. Experimental angioplasty is also characterized by abundant
proliferation of VSMCs (Fig. (2)), followed by the reestablishment of the quiescent
phenotype typically within 2-4 weeks [4-6]. These animal studies suggest that vascular cell
proliferation prevails at the onset of atherogenesis and restenosis.
Expression of a variety of proliferation markers in human primary atheroma and
restenotic lesions has been well documented [13-22]. However, controversy exists
regarding the magnitude of the proliferative response during human atherosclerosis and
restenosis, ranging from a very low index of cell proliferation [14,15,17,19,21] to
abundance of dividing cells [16,23]. Aside from methodological issues (e. g., differences in
the fixatives used for tissue preservation, antigen accessibility, analysis of different
proliferation markers), some of the reported variance with regard to the issue of cell
proliferation might relate to differences in the arteries being analyzed (e. g., peripheral,
coronary and carotid arteries) and variance in the stage of atherogenesis at the time of
tissue harvesting [24].
Proliferating cells within human atherosclerotic tissue include VSMCs, leukocytes
and endothelial cells (ECs) [13-15,17-19,21]. Histological examination in 20 patients
undergoing antemorten coronary angioplasty revealed that the extent of intimal
proliferation was significantly greater in lesions with evidence of medial or adventitial
tears than in lesions with no or only intimal tears [25]. Regarding the relative magnitude of
intimal and medial cell proliferation, analysis of human carotid plaques revealed more
proliferative activity in the intimal lesion versus the underlying media [19]. This study also
disclosed differential distribution of proliferating cells in the intima versus the media;
while the prevailing proliferative cell type in the intima was the monocyte/macrophage
4
(46% versus 9.7% -actin immunoreactive VSMCs, 14.3% ECs, 13.1% T lymphocytes),
VSMCs were the preponderant proliferating cell type in the media (44.4% versus 20%
ECs, 13.0% monocyte/macrophages, and 14.3% T lymphocytes). It is noteworthy that
studies in human peripheral and coronary lesions have suggested more prominent
proliferation in restenotic compared to primary lesions [17,23]. Moreover, cultured
VSMCs from human advanced primary stenosing disclosed lower proliferative capacity
than cells from fresh restenosing lesions [26]. Thus, similar to the situation in animal
models, proliferation during human atherosclerosis and restenosis might peak at the onset
of these pathologies and then progressively decline.
Molecular control of cell proliferation in mammalian cells
Mammalian cell cycle progression requires the activation of cyclin-dependent
kinases (CDKs) through their association with regulatory subunits called cyclins [27].
Different CDK/cyclin holoenzymes are orderly activated at specific phases of the cell
cycle. Active CDK/cyclin complexes are presumed to hyperphosphorylate the
retinoblastoma gene product and the related pocket proteins p107 and p130 from mid G1
to mitosis. VSMC proliferation in the balloon-injured rat carotid artery is associated with a
temporally and spatially coordinated expression of CDK2 and its regulatory subunits,
cyclin E and cyclin A [22]. Induction of these factors correlated with increased CDK2-,
cyclin E- and cyclin A-dependent kinase activity, indicating that functional CDK2/cyclin E
and CDK2/cyclin A holoenzymes are assembled in the injured arterial wall. Expression of
CDK2 and cyclin E was also detected in VSMCs within human atherosclerotic and
restenotic tissue [16,22,28].
CDK activity is negatively regulated by the interaction with specific CDK inhibitory
proteins (CKIs) [29]. CKIs of the Cip/Kip family (for CDK interacting protein/Kinase
5
inhibitory protein) (p21Cip1, p27Kip1 and p57Kip2) bind to an inhibit a wide spectrum of
CDK/cyclin holoenzymes, while members of the Ink4 family (for inhibitor of CDK4)
(p16Ink4a, p15Ink4b, p18Ink4c, p19Ink4d) are specific for cyclin D-associated kinase activity
(CDK4 and CDK6). In the next sections, we will discuss in vitro and in vivo studies that
implicate p27Kip1 as an important regulator of restenosis and atherosclerosis.
Coordinate control of cell proliferation and migration by p27Kip1.
Aside from its well established growth suppressive function, recent studies suggest a
role for p27Kip1 as a negative regulator of cell migration. First, we have shown that
overexpression of p27Kip1 can reduce human vein endothelial cell (HUVEC) and rabbit
aorta VSMC migration in culture, and impair angiogenesis in vivo [30,31]. Second,
p27Kip1-null VSMCs were more resistant than wild-type cells to the antimigratory
properties of rapamycin [32], a bacterial macrolide that attenuates experimental restenosis
[33] and has shown promising results in preventing human in-stent restenosis [34]. In view
of these results, we investigated whether the dual function of p27Kip1 as a cell-cycle and
migration inhibitor is achieved via common or independent molecular pathways (DíezJuan and Andrés, unpublished work - submitted for publication). Using retroviral vectors,
we found that physiologically high level of p27Kip1 expression inhibits CDK activity and
attenuates both proliferation and migration of VSMCs and fibroblasts. Mutations that
rendered p27Kip1 unable to abrogate CDK activity also prevented p27Kip1-induced growth
arrest and migration blockade. Collectively, these findings suggest that p27Kip1
coordinately modulates cell proliferation and locomotion via regulation of CDK activity
(Fig. (3)). It is noteworthy that the related Cip/Kip protein p21Cip1, which reduced
neointimal thickening in normocholesterolemic [35,36] and hypercholesterolemic animals
[37], also inhibited VSMC migration in vitro [38]. Thus, p21Cip1 and p27Kip1 might
attenuate neointimal growth by blocking both cell proliferation and chemotaxis.
6
Role of p27Kip1 in establishing regional phenotypic variance in VSMCs from different
vascular beds
Internal mammary artery (IMA) bypass grafts have a higher patency than saphenous
vein (SV) grafts. Yang et al. investigated the growth properties of human VSMCs isolated
from IMA and SV [39]. Cell outgrowth from explants over a 20-day period and seruminduced increase in cell number over an 8-day period were more pronounced in SV than in
IMA of the same patient. Both types of VSMCs displayed functional growth factor
receptor expression and activation of the mitogen-activated protein kinase (MAPK)
pathway. Interestingly, platelet-derived growth factor-BB (PDGF-BB) markedly
downregulated p27Kip1 protein level in SV, but this was much less pronounced in IMA.
Thus, sustained p27Kip1 expression in spite of growth stimuli may contribute to the
resistance to growth of VSMCs from IMA and to the longer patency of arterial versus
venous grafts.
While basic fibroblast growth factor (bFGF or FGF2) is a potent stimulator of medial
VSMC proliferation after balloon angioplasty [40-42], neutralizing antibodies to FGF2
failed to inhibit intimal VSMC proliferation after balloon angioplasty [43], and only a
small increase in proliferation was seen when bFGF was added to arteries with existing
intimal lesions [40,42]. Attenuated bFGF-dependent proliferation of intimal VSMCs
occurred despite a robust activation of the MAPK/extracellular signal-regulated kinase
(ERK) pathway and induction of positive cell cycle regulators (e. g., cyclin D, cyclin E,
CDK2 and CDK4) [42]. Interestingly, intimal VSMCs expressed high levels of p15 Ink4b
and p27Kip1 compared with medial VSMCs, and bFGF infusion did not reduce the level of
these inhibitors in arteries with established intimal lesions. Collectively, these studies
suggest that high level of expression of p15Ink4b and p27Kip1 can attenuate VSMC
7
proliferation in cultures displaying MAPK activation and expression of positive cell cycle
regulators.
We have investigated whether VSMCs from vessels with different atherogenicity
exhibit distinct growth and migratory potential, and investigated the underlying
mechanisms [31]. We found increased cell proliferation and atheroma formation in the
aortic arch versus the femoral artery of fat-fed rabbits. When examined in culture, VSMCs
isolated from the aortic arch (ASMCs) displayed a greater capacity for inducible
proliferation and migration than paired cultures of VSMCs isolated from the femoral artery
(FSMCs). Two lines of evidence suggested that distinct regulation of p27Kip1 contributes to
establishing these phenotypic dissimilarities. First, p27Kip1 expression was comparably
lower in ASMCs. Importantly, ASMCs exhibited a higher fraction of p27Kip1
phosphorylated on threonine 187 and ubiquitinated, two posttranslational modifications
implicated in proteasome-dependent degradation of p27Kip1 [44]. Second, forced p27Kip1
overexpression in ASMCs impaired their proliferative and migratory potential. We found
that PDGF-BB-dependent induction of the ERK pathway was comparably higher in
ASMCs. Importantly, pharmacological inhibition of ERKs increased p27Kip1 expression
and attenuated ASMC proliferation and migration. In contrast, forced ERK activation
diminished p27Kip1 expression and markedly augmented FSMC proliferation and
migration. These findings suggest that intrinsic differences in the regulation of MAPKs
and p27Kip1 play an important role in creating variance in the proliferative and migratory
capacity of VSMCs, which might in turn contribute to establishing regional variability in
atherogenicity (Fig. (4)).
Role of p27Kip1 as a regulator of the phenotypic response of VSMCs to mitogenic and
hypertrophic stimuli.
8
VSMC hypertrophy is associated with cardiovascular disease in the elderly and in
hypertensive patients. Angiotensin II (Ang II) stimulates hypertrophy but not hyperplasia
of quiescent VSMCs in serum-free media, in spite of increased expression of several
protooncogenes and autocrine growth factors [45-48]. Braun-Dullaeus et al. reported that
while both serum and Ang II treatment of quiescent VSMCs led to upregulation of
positive cell-cycle regulators (e. g., proliferating cell nuclear antigen, cyclin D1, CDK2
and CDC2), only serum-treated VSMCs induced CDK2 and CDC2 activity [49]. These
authors provided compelling evidence implicating p27Kip1 as a molecular switch that
regulates the phenotypic response of VSMCs to mitogenic and hypertrophic stimuli.
Indeed, Ang II-induced hypertrophy of quiescent VSMCs correlated with sustained
expression of p27Kip1, unlike serum-dependent cell-cycle reentry of starvationsynchronized cells, which correlated with a marked downregulation of p27Kip1 protein
level. Importantly, forced overexpression of p27Kip1 inhibited serum-stimulated
proliferation and induced VSMC hypertrophy. Moreover, p27Kip1 inactivation increased
[3H]-thymidine incorporation and the percentage of S-phase cells in Ang II-treated VSMC
cultures. These results demonstrate that Ang II treatment of quiescent VSMCs is
associated with cell-cycle entry, but hypertrophic rather than hyperplastic growth may
prevail by the failure of cells to downregulate p27Kip1. In another study, Servant et al.
compared the effects of Ang II and PDGF-BB on cultured VSMCs [50]. While both
factors stimulated the accumulation of G1 cyclins and CDKs, only PDGF-BB activated
CDK2 in late G1. Lack of CDK2 activity in Ang II-treated cells correlated with sustained
p27Kip1 protein level. In contrast, PDGF-BB downregulated p27Kip1 expression due to a
reduced rate of synthesis and an increased rate of protein degradation. Moreover, PDGFBB-dependent downregulation of p27Kip1 synthesis correlated with diminished p27Kip1
gene transcription and decreased mRNA accumulation. Collectively, these studies identify
9
p27Kip1 as an important regulator of the phenotypic response of VSMCs to mitogenic and
hypertrophic stimuli.
Role of p27Kip1 in the control of VSMC growth by extracellular matrix components.
Specific ECM components and integrins have been implicated in cell cycle control
during atherosclerosis and restenosis [51]. VSMCs within atherosclerotic lesions
synthesize novel ECM components and induce the expression of matrix-degrading
proteases that remodel the surrounding ECM. For example, matrix-degrading
metalloproteinase expression is induced within atherosclerotic plaques and after balloon
angioplasty
[52-55].
Moreover,
metalloproteinase
inhibitors
repressed
VSMC
proliferation in vitro and after angioplasty in vivo [56-58]. Accordingly, these ECM
enzymes have been implicated in the induction of neointimal VSMC hyperplasia during
atherosclerosis and restenosis.
Several animal models of atherosclerosis and angioplasty have demonstrated
significant changes in collagen content within the arterial wall [59-61]. To investigate
whether these alterations may regulate VSMC proliferation, Koyama et al. studied the
growth properties of VSMCs cultured on monomer collagen fibers and on polymerized
collagen [62]. The rationale for these studies is that polymerized collagen may resemble
the scenario of a normal artery composed of quiescent VSMCs, and monomer collagen
might mimic the ECM surrounding proliferating VSMCs within atherosclerotic and
restenotic plaques. Consistent with this notion, mitogen-stimulated VSMCs proliferated
when grown on monomer collagen, but were arrested in G1 when seeded on polymerized
collagen. The inhibitory effect of polymerized collagen on VSMC growth appeared to be
mediated by 2 integrins, and was associated with suppression of p70S6K and upregulation
of p27Kip1 (and to a lesser extent induction of the related protein p21Cip1). Thus, regulation
10
of CKIs in response to changes in specific ECM components might regulate the ability of
VSMCs to respond to growth signals in vitro. Interestingly, the quiescent phenotype of
nonadherent NRK fibroblasts correlated with an increased association of p27Kip1 and
p21Cip1 to cyclin E-containing holoenzymes [63]. Further studies are required to determine
whether cell cycle control in the arterial wall is linked in vivo to integrins and ECM
components through changes in CKI expression.
p27Kip1 and neovascularization
De novo vessel formation (neovascularization) plays a critical role during
physiological and pathological processes. Several studies have demonstrated the presence
of microvessels within human atherosclerotic plaques [64-67]. Arteries with higher degree
of stenosis and more predisposition to plaque rupture displayed more neovascularization
within the intima [68]. Moreover, microvessels appeared absent in 97% of human
coronary atheromas displaying an intima-to-media ratio less than 0.54, whereas 98% of
arteries with larger lesions (intima-to-media ratio higher than 0.54) disclosed evidence of
neovascularization [67]. Importantly, the angiogenesis inhibitors endostatin and TNP-470
significantly reduced atheroma growth in a murine model of atherosclerosis [69].
Collectively, these studies suggest that neovascularization of the atherosclerotic plaque
plays an important role during experimental and human atherosclerosis.
Neovascularization requires ECM remodeling and abundant cell proliferation and
migration. We have demonstrated that ectopic overexpression of p27Kip1 inhibits both
proliferation and migration of VSMCs and HUVECs [30,31] (also Díez-Juan and Andrés,
unpublished work - submitted for publication). Moreover, p27Kip1 overexpression inhibited
the formation of tube-like structures by HUVECs growing on Matrigel substrate, and
impaired neovascularization in a murine model of ischemia-induced neovascularization [30].
11
p27Kip1 and restenosis
Balloon angioplasty in rat and porcine arteries resulted in the induction of p27Kip1 in
VSMCs at late time points that correlated with reduced CDK2 activity and the decline in
VSMC proliferation [20,70]. Moreover, overexpression of p27Kip1 efficiently blocked
mitogen- and c-fos-dependent induction of cyclin A promoter activity in cultured VSMCs
[70,71]. Thus, upregulation of p27Kip1 may contribute to the reestablishment of the quiescent
phenotype that normally occurs at late time points after angioplasty. In agreement with this
hypothesis, we have shown that local delivery of adenovirus encoding for p27Kip1 reduced
neointimal hyperplasia in the rat carotid artery when applied at the time of angioplasty [70].
Likewise, adenovirus-mediated overexpression of p27Kip1 attenuated neointimal thickening
in a porcine model of balloon angioplasty [72]. Of note in this regard, chimaeric p27Kip1p16Ink4a proteins appear to have a more potent antiproliferative activity than p27Kip1 and
p16Ink4a [73].
p27Kip1 and arteriosclerosis
We have recently established a causal relationship between p27Kip1 and
atherosclerosis in apolipoprotein E (apoE)-null mice [8]. p27Kip1–deficient mice challenged
with a high-fat diet for 1 month remained normocholesterolemic and had essentially no
visible atheromas. However, when generated in an apoE-null genetic background that leads
to severe hypercholesterolemia in response to the atherogenic diet, deletion of p27Kip1
enhanced arterial cell proliferation (~4-fold) and accelerated atherogenesis (~6-fold) as
compared to apoE-deficient mice with an intact p27Kip1 gene. Analysis of apoE-null mice
bearing only one p27Kip1 allele inactivated revealed that a moderate decrease in p27Kip1
protein expression in the setting of hypercholesterolemia is sufficient to predispose to
atherogenesis. These studies establish a molecular link between diminished p27Kip1 protein
12
expression and atherogenesis in hypercholesterolemic animals. Regarding human
atherogenesis, Tanner et al. reported abundant p27Kip1 expression in nonproliferating
VSMCs and macrophages within coronary arteries ranging from normal to advanced
atherosclerosis, suggesting an inverse correlation between arterial cell proliferation and
expression of p27Kip1 [20]. Moreover, TGF- present in human atherosclerotic tissue might
mediate its growth suppressive activity through p27Kip1-dependent blockade of cyclin ECDK2 activity [28].
It is noteworthy that inactivation of either p27Kip1 [8] or p53 [74] by itself is not
sufficient to promote atherosclerosis in mice challenged with an atherogenic diet for 4-10
weeks. Importantly, fat-fed p27Kip1- and p53-null mice remained normocholesterolemic at
the end of the experimental protocol. However, when generated in an apoE-null genetic
background that leads to severe hypercholesterolemia in response to the atherogenic diet,
ablation of either p27Kip1 or p53 accelerated the underlying atherogenic process triggered
by hypercholesterolemia. These findings support the notion that excessive vascular cell
proliferation is subsidiary to injury to the vessel wall initiated by atherogenic stimuli (i. e.,
hypercholesterolemia) rather than the cause of this process [1]. Another example of a
synergistic pathological response involving p27Kip1 concerns tumor development. Indeed,
spontaneous tumorigenesis in p27Kip1-deficient mice appears limited to pituitary adenomas
[75-77]. However, when challenged with tumorigenic agents (e. g., chemical carcinogens
or ionizing radiation), p27Kip1-null mice displayed increased tumor predisposition in
multiple tissues [78]. These studies suggest that p27Kip1 safeguards against the
hyperproliferative response triggered by a variety of pathological stimuli. Hence, future
studies in animal models and human tissue should thoroughly investigate the temporal and
spatial pattern of expression of p27Kip1 during atherogenesis, and elucidate molecular
mechanisms underlying the regulation of p27Kip1 expression in vascular cells. Such
13
information would find application not only in vascular proliferative diseases, but also in
human neoplastic disorders in which tumor progression and patient mortality might be
associated with reduced p27Kip1 expression [44].
Acknowledgements
This work is dedicated to the memory of Dr. Jeffrey M. Isner, whose intellectual
stimulation was enjoyed by all who were fortunate to work with him. Work in the
laboratory of V. Andrés is supported by the Spanish Ministry of Science and Technology
and Fondo Europeo de Desarrollo Regional (FEDER) (grants SAF2001-2358 and
SAF2002-1443) and by Generalitat Valenciana (grants GV01-488 and CTGCA/2002/04).
C. Castro and M. D. Edo are recipients of a fellowship from Agencia Española de
Cooperación Internacional and Generalitat Valenciana, respectively. A. Díez-Juan was
partially supported from the Spanish DGESIC and FEDER (grant 1FD97-1035-C02-02),
and from Fondo Social Europeo (CSIC-Programa I3P fellowship).
14
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FIGURE LEGENDS
FIGURE 1: Arterial cell proliferation in the aortic arch of fat-fed rabbits. Male
New Zealand rabbits received either control chow or a high-cholesterol diet for 2 months
[31]. Prior to sacrifice, animals were injected with BrdU to assess arterial cell proliferation.
The photomicrographs show representative examples of cross-sections of the aortic arch of
rabbits fed the cholesterol-rich or control diet. Specimens were counterstained with eosin.
Arrowheads point to the internal elastic lamina. While cell proliferation was negligible in
control arteries, abundant nuclear BrdU incorporation was detected in aortic tissue from fatfed rabbits, particularly in cells within the atheromatous lesion.
FIGURE 2: Arterial cell proliferation after balloon angioplasty in the rat carotid
artery. Angioplasty and BrdU in vivo labeling was performed as described [71]. The
photomicrographs show representative cross-sections of control vessels (left) and 10 days
after balloon angioplasty (right). Black and white arrowheads point to the internal and the
external elastic lamina, respectively. Note abundant nuclear BrdU incorporation in ballooninjured arteries.
FIGURE 3: Coordinate regulation of cell proliferation and migration by p27Kip1.
High level of p27Kip1 expression in cultured VSMCs, ECs, and fibroblasts is associated with
reduced cell proliferation and migration [30,31]. Mutations that rendered p27Kip1 unable to
abrogate CDK activity prevented both growth arrest and migration blockade (Díez-Juan And
Andrés, unpublished work – submitted for publication). These findings suggest that changes
in p27Kip1 expression can modulate vascular cell proliferation and locomotion in a
coordinated manner via regulation of CDK activity.
FIGURE 4: Differential regulation of MAPKs and p27Kip1 is associated with
distinct proliferative and migratory capacity of VSMCs isolated from vessels with
24
different atherogenicity. The schematic summarizes the phenotypic profile of rabbit
VSMCs isolated from vessels that responded differently to a high-fat diet [31].
25