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Instituto de Biomedicina de Valencia (IBV-CSIC), Spanish Council for Scientific Research, 46010-Valencia, Spain
1Correspondence: Instituto de Biomedicina de Valencia (IBV-CSIC), C/Jaime Roig, 11, 46010 Valencia, Spain. E-mail: vandres{at}ibv.csic.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fourfold) and accelerates atherogenesis (sixfold) compared with
apolipoprotein E-deficient mice with an intact p27 gene. Analysis of
apolipoprotein E-null mice bearing only one p27 allele
inactivated reveals that a moderate decrease in p27 protein expression
in the setting of hypercholesterolemia is sufficient to predispose to
atherogenesis. Thus, our study establishes a molecular link between
decreased p27 protein expression and atherogenesis in
hypercholesterolemic animals.—Díez-Juan, A., Andrés, V.
The growth suppressor p27Kip1 protects against diet-induced
atherosclerosis.
Key Words: cell cycle • p27 • hypercholesterolemia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Different
CDK/cyclin holoenzymes are activated at specific phases of the cell
cycle. Active CDK/cyclin complexes phosphorylate the retinoblastoma
gene product and the related pocket proteins p107 and p130 from mid-G1
to mitosis, which in turn regulate the activity of members of the E2F
family of transcription factors (2
, 3)
. Cell proliferation
in mammals is negatively regulated by CDK inhibitory proteins (CKIs),
which associate with and inhibit the activity of CDK/cyclin holoenzymes
(1
, 4)
. CKIs of the CIP/KIP family (p21, p27, and p57)
bind to and inactivate a broad range of CDKs, whereas members of the
INK4 family (p15, p16, p18, p19) are specific for CDK4- and
CDK6-containing holoenzymes.
Coronary artery disease (i. e., atherosclerosis, postangioplasty
restenosis, and vein-graft failure) is the leading cause of mortality
and morbidity in the Western population (5)
. The molecular
basis of these disorders involves dysregulated growth of vascular
smooth muscle cells (VSMCs) and macrophages (6
, 7)
.
Accumulating evidence implicates the growth suppressor p27 as an
important regulator of the phenotypic response of VSMCs to mitogenic
and hypertrophic stimuli, both in vitro and in vivo. First,
up-regulation of p27 may limit the growth of VSMCs at late time points
after balloon angioplasty (8
, 9)
. Consistent with this
possibility, overexpression of p27 efficiently blocked mitogen- and
c-fos-dependent induction of cyclin A promoter activity in cultured
VSMCs (8
, 10)
, and adenovirus-mediated overexpression of
p27 inhibited vascular occlusive lesion formation in balloon-injured
arteries (8
, 11)
. Moreover, p27 is hypothesized to serve
as a molecular switch that determines whether VSMCs undergo
hypertrophic or hyperplastic growth (12
, 13)
. Regarding
the role of p27 on hematopoietic cells, it has been suggested that
suppression of p27 enhances hematopoietic progenitor cell proliferation
and facilitates early development of promyeloid cells into macrophages
(14
, 15)
.
Immunohistochemical analysis have suggested an inverse correlation
between p27 expression and proliferation of macrophages and VSMCs
within human atherosclerotic tissue (9
, 16)
. However, a
causal link between p27 and the pathogenesis of atherosclerosis has not
been established. In the present study, we investigated the effect of
p27 inactivation on diet-induced atherosclerosis. Our study establishes
a molecular link between decreased p27 protein expression and
atherosclerosis in hypercholesterolemic animals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(mixed C57BL/6x129Sv background; gift from J. Osada, Zaragoza, Spain)
and p27 (18)
(C57BL/6 background; gift from M. Serrano,
Madrid, Spain) were mated and the double heterozygous F1 offspring were
intercrossed. F2 mice were genotyped by PCR analysis and
brother-sister mating of p27+/-apoE-/- mice was
performed to obtain p27+/+apoE-/-, p27+/-apoE-/-, and
p27-/-apoE-/- mice. After weaning, mice were maintained on a
low-fat standard diet (2.8% fat, Panlab, Barcelona, Spain). At 2
months of age, mice received for 1 month an atherogenic diet containing
15.8% fat, 1.25% cholesterol, and 0.5% sodium cholate (TD 88051;
Harlan/Teklad, Madison, WI). Blood was withdrawn before and after the
high-fat diet to measure plasma cholesterol and triglyceride levels
using enzymatic procedures (Sigma, St. Louis, MO).
Quantification of atherosclerosis, histomorphometry, and
immunohistochemistry
Fat-fed mice were killed and their aortas were fixed in situ
with 4% paraformaldehyde. After carefully removing the adventitia, the
aorta was excised, fixation was continued overnight, and vessels were
opened longitudinally and stained with Oil Red O solution (0.2% in
80% MetOH) (Sigma). Specimens were pinned onto a flat surface and
photographed with a Sony DKC-CM30 camera (Tokyo, Japan) mounted on a
Zeiss Stemmi 2000-C dissecting scope (Jena, Germany). To determine the
extent of atherosclerosis in the aortic arch region (from the aortic
root up to 1.5 mm beyond the left subclavian artery), digital images
were analyzed by computer-assisted quantitative morphometry using Sigma
Scan Pro v5.0 (Jandel Scientific, San Rafael, CA). For each animal, the
area stained with Oil Red O was divided by total investigated area
(average 14 mm2). Differences in lesion area
between males and females were not significant, so data from both sexes
were included in these analyses.
The intima-to-media ratio was determined as an independent measure of
atheroma formation. In another set of animals, the heart and the
proximal aorta were fixed with 100% methanol. Specimens were
paraffin-embedded and mounted in a Microm microtome (Heidelberg,
Germany). Once the three valve cusps were reached, sections throughout
the first 2 mm of the ascending aorta were discarded. Then, 25
consecutive sections (5 µm thickness) were taken from two or three
regions of the aortic arch separated by 60 µm. Three cross
sections from each region were stained with hematoxylin/eosin. Images
were captured with the digital camera mounted on a Zeiss Axiolab
stereomicroscope and the area occupied by atherosclerotic lesions
(intima) and the area of the media were determined by computer-assisted
quantitative morphometry to calculate the intima-to-media ratio. The
results for each animal were calculated by averaging all independent
values.
Histological examination of methanol fixed cross sections included
quantification of cellularity and fibrous cap thickness in the
lesions of p27-/-apoE-/- and p27+/+apoE-/- mice. Lesion
cellularity was estimated by dividing the number of cells per
mm2 of plaque. The relative fibrous cap thickness
was calculated by dividing fibrous cap thickness at the center of the
lesion by lesion thickness (19)
in 66 fibrous caps from 5
mice of each genotype.
Methanol-fixed cross sections were also analyzed by
immunohistochemistry. Proliferation was estimated with anti-PCNA
antibodies (1/50, sc-7907, Santa Cruz Biotechnology, Santa Cruz, CA), a
biotin/streptavidin-peroxidase detection system (Signet Laboratories,
Dedham, MA), and 0.05% (w/v) 3,3'-diaminobenzidine tetrahydrochloride
dihydrate substrate (Vector Laboratories; Burlingame, CA). For each
mouse, PCNA-positive cells were counted in at least three sections from
different regions of the aortic arch and results were averaged.
Macrophages were identified using anti-F4/80 antibody (1/10, MCAP497,
Serotec, Raleigh, NC) after pretreatment with 0.25% trypsin (3 min,
37°C). Immunocomplexes were detected using biotinylated secondary
antibody (sc-2041, Santa Cruz Biotechnology) and alkaline
phosphatase-conjugated Streptavidin (Biogenex, San Ramon, CA). VSMCs
were identified with alkaline phosphatase-conjugated anti-smooth muscle
-actin (SM-actin) antibody (1/200, a-5691, Sigma). Alkaline
phosphatase activity was detected with Fast Red (Sigma). VSMC content
in atherosclerotic lesions was determined morphometrically by dividing
the SM-actin-positive area by total plaque area.
Statistical analysis
Results are reported as mean ± SE. In
experiments with two groups, differences were evaluated using a
2-tailed, unpaired t test. Analysis involving more than two
groups were done using ANOVA and Fisher’s post hoc test (Statview, SAS
institute, Cary, NC).
Western blot analysis
For each genotype, snap-frozen arteries from three animals were
pooled and lysed in ice-cold 50 mM Tris-HCl buffer (pH 7.5) containing
1% Triton X-100, 150 mM NaCl, 1 mM DTT, and protease inhibitor
Complete Mini mixture (Roche, Mannheim, Germany) using an Ultraturrax
T25 basic (IKA Labortechnik, Staufen, Germany). Western blot analysis
was performed as described previously (8)
using the
following primary antibodies (Santa Cruz Biotechnology): rabbit
polyclonal anti-p27 (1/200, sc-776) and mouse monoclonal anti-tubulin
(1/200, sc-3035). The relative intensity of protein bands was
determined by densitometry.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, we performed our
studies in apoE-deficient mice, which develop atherosclerotic lesions
resembling those observed in humans (17
, 21)
. By
intercrossing mice deficient for p27 and apoE, we generated apoE-null
mice bearing various levels of p27 (p27+/+apoE-/-, p27+/-apoE-/-,
and p27-/-apoE-/-). Our studies also included p27-null mice with an
intact apoE gene (p27-/-apoE+/+). We first examined the plasma
triglyceride and cholesterol levels in mice fed either a control diet
or a high-fat diet (Fig. 1
). All mice maintained on control chow exhibited normolipidemia.
Likewise, p27-/-apoE+/+ animals challenged with the high-fat diet for
1 month remained normolipidemic. In contrast, fat-fed apoE-deficient
mice developed comparable hyperlipidemia regardless of their status for
p27. Thus, in agreement with previous studies (17
, 21)
,
mice with an intact apoE gene did not display hyperlipidemia when
exposed to an atherogenic diet. Moreover, reduction or ablation of p27
did not affect the hyperlipidemic response normally observed in fat-fed
apoE-null mice.
|
We next analyzed atherogenesis in fat-fed mice by examining whole
arteries stained with Oil Red O (Fig. 2A
). Based on gross morphological inspection, p27-null mice
with intact apoE revealed essentially no visible atheromas. As
expected, p27+/+apoE-/- mice developed atherosclerosis in the aorta,
which prevailed within the aortic arch. All double nullizygous
p27-/-apoE-/- mice revealed augmented atherosclerosis compared
with p27+/+apoE-/- mice, and p27 heterozygous mice with
inactivated apoE (p27+/-apoE-/-) appeared to have an
intermediate phenotype. To rigorously determine the extent of
atherosclerosis in these mice, we performed computer-assisted
quantitative morphometry of Oil Red O-stained arteries. When compared
with p27+/+apoE-/- mice (n=12), the area of lesions
positive for Oil Red O was enhanced by threefold in
p27+/-apoE-/- mice (n=10, P<0.002) and
by sixfold in p27-/-apoE-/- mice (n=10,
P<0.0001) (Fig. 2B
). The above findings were
confirmed by examination of arterial cross sections stained with
hematoxylin/eosin (Fig. 3A
). These analyses disclosed an intima-to-media ratio of
0.25 ± 0.02 in p27+/+apoE-/- mice (n=8),
0.43 ± 0.06 in p27+/-apoE-/- mice
(n=5), and 0.96 ± 0.20 in p27-/-apoE-/- mice
(n=8; P<0.0015 vs. p27+/+apoE-/-;
P<0.025 vs. p27+/-apoE-/-) (Fig. 3B
).
Collectively, these findings indicate that p27 disruption alone is not
sufficient to induce atherosclerosis in mice with an intact apoE gene,
which remain normolipidemic despite the exposure to a high-fat diet.
However, the severity of atherosclerosis in fat-fed apoE-null mice,
which develop acute hyperlipidemia, is incrementally exacerbated by
inactivation of one or two p27 alleles. As expected, immunoblot
analysis revealed the absence of p27 expression in the aorta of
p27-/-apoE-/- mice (Fig. 2C
). Densitometric analysis of
two independent blots revealed an average 42% decrease of p27
protein expression in the aorta of p27+/-apoE-/- compared with
p27+/+apoE-/- mice (Fig. 2C
). Thus, reduction or ablation
of p27 expression enhances atherosclerosis in hyperlipidemic mice.
|
|
Histological and immunohistochemical characterization of
atherosclerotic lesions in p27+/+apoE-/- and p27-/-apoE-/-
mice
We then examined the effect of p27 inactivation on the cellular
composition of atherosclerotic lesions. Histomorphometric analysis
revealed higher cellularity in the lesions of p27-/-apoE-/- mice
compared with p27+/+apoE-/- mice (9.7±0.1 cells
mm-2 vs. 6.6±0.6 cells
mm-2, respectively, n=7,
P<0.05) (Fig. 4A
, C
). In both groups, immunohistochemical analysis using rat
monoclonal F4/80 antibody demonstrated the presence of macrophages
throughout the intimal lesions (Fig. 5C
and data not shown). We also examined the presence of VSMCs
using a mouse monoclonal anti-SM-actin antibody (Fig. 4B
). As indicated by the ratio SM-actin-positive
area/total plaque area, VSMCs were more abundant within the lesions of
p27-/-apoE-/- mice (0.47±0.05 vs. 0.22±0.08 in
p27+/+apoE-/- mice, n=7, P<0.04) (Fig. 4D
). Moreover, relative fibrous cap thickness was greater in
p27-/-apoE-/- mice compared with control animals (0.35±0.02
vs. 0.18±0.02, n=66 fibrous caps, P<0.0001
(Fig. 4A
, E
). Thus, lesions in the p27-/-apoE-/- mice
appeared more advanced, consistent with accelerated atherogenesis in
these animals.
|
|
We reasoned that enhanced cell proliferation might contribute to
augmented atherosclerosis and hypercellularity in the lesions of
p27-/-apoE-/- mice. To test this possibility, we performed
immunohistochemical analysis using specific antibodies for
proliferating cell nuclear antigen (PCNA) as a measure of cell
proliferation (22
23
24
25)
. Quantitative analysis revealed
that PCNA immunoreactivity in the aortic arch of p27-/-apoE-/- mice
was significantly higher than in p27+/+apoE-/- mice, both in the
media (fivefold increase, P<0.0015) and within
atherosclerotic lesions (threefold increase, P<0.035) (Fig. 5A
). To investigate the identity of proliferating cells, we
performed immunohistochemical analysis. By double immunohistochemistry,
we found that the majority of PCNA-positive cells in the media of
p27+/+apoE-/- and p27-/-apoE-/- mice were also
SM-actin immunoreactive (Fig. 5B
). In contrast,
colocalization of PCNA and SM-actin was rarely observed within
atheromatous plaques (Fig. 5B
), where PCNA immunoreactivity
was seen mainly in macrophage-rich areas (Fig. 5C
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 26
, 27)
.
This phenotype has been attributed to increased cell proliferation,
most clearly in tissues that express higher levels of p27. It is
accepted that p27 functions as a tumor suppressor gene
(28)
, and p27 also safeguards against excessive cell
proliferation during glomerulonephritis and ureteral obstruction
(29)
. Given that excessive proliferation of macrophages
and VSMCs underlies the atherogenic process (6)
, we sought
to investigate the effect of p27 inactivation on diet-induced
atherosclerosis. These studies were performed in apoE-null mice, a
well-characterized animal model of atherosclerosis that recapitulates
important features of the human disease (17
, 21)
. Our
results demonstrate that a moderate (42%) reduction in p27 protein
expression in the aorta of hyperlipidemic p27+/-apoE-/- mice is
sufficient to augment diet-induced atherosclerosis compared with
p27+/+apoE-/- mice, and this phenotype is exacerbated in apoE-null
mice lacking both p27 alleles (p27-/-apoE-/-). Fero et al.
(30)
have demonstrated that p27 is haplo-insufficient for
tumor suppression. These authors showed that p27 nullizygous mice are
predisposed to tumor formation induced by ionizing radiation or a
chemical carcinogen, and p27 heterozygous mice showed intermediate
rates of tumorigenesis. Collectively, these studies indicate that a
moderate reduction in p27 expression might be sufficient to increase
the risk of cancer and atherosclerosis. Indeed, p27 expression is
frequently down-regulated in human tumors and a reduced level of p27
correlates with poor prognosis (28
, 31
32
33)
.
Our results suggest that enhanced atherosclerosis in fat-fed
p27-/-apoE-/- mice results, at least in part, from increased VSMC
and macrophage proliferation. Consistent with our findings, previous
studies have shown that p27 is a potent inhibitor of VSMC proliferation
(8
, 9)
and that suppression of p27 expression might
promote the proliferation of hematopoietic progenitor cells and their
differentiation into macrophages (14
, 15)
. Augmented cell
proliferation by disruption of the growth suppressor p53 has been also
associated with enhanced atherogenesis in fat-fed apoE-deficient mice
(34)
. Given that the CKI p21 has been detected in human
atherosclerotic tissue (9)
, it will be instructive to test
the effect of p21 inactivation on atherogenesis.
Excessive VSMC proliferation is also thought to contribute to
restenosis postangioplasty (6
, 7)
. It is hypothesized that
induction of endogenous p21 and p27 may limit the hyperplastic growth
of VSMCs at late time points after angioplasty in rat and porcine
arteries
(8
,
9
,
35
). Conversely, p53 inactivation after
cytomegalovirus infection may increase the risk of human coronary
restenosis (36
, 37)
. Ectopic overexpression of these
growth suppressors has proved efficient at limiting neointimal lesion
development after experimental angioplasty. These include
adenovirus-mediated (8
, 11)
and rapamycin-dependent
(38)
overexpression of p27, adenovirus-mediated
overexpression of p21 (35
, 39
, 40)
, and p53 arterial gene
transfer (41)
. Thus, human studies are warranted to test
the efficacy of p27, p21, and p53 overexpression for the inhibition of
restenosis after percutaneous interventions.
It is noteworthy that inactivation of either p27 (this study) or p53
(34)
by itself is not sufficient to promote atherogenesis
in mice challenged with an atherogenic diet for 4–10 wk. Fat-fed
p27-/- and p53-/- 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 p27 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 (6)
. Another example of a
synergistic pathological response involving p27 concerns tumor
development. Indeed, spontaneous tumorigenesis in p27-deficient mice
appears to be limited to pituitary adenomas (18
, 26
, 27)
.
However, when challenged with tumorigenic agents (i. e., chemical
carcinogens or ionizing radiation), p27-null mice display increased
tumor predisposition in multiple tissues (30)
. These
studies suggest that p27 safeguards against the hyperproliferative
response triggered by a variety of pathological stimuli.
By the use of genetically modified mice, we have shown that reduced p27
protein expression increases the severity of atherosclerosis induced by
hypercholesterolemia. Whether diminished p27 expression may be a common
event during atherogenesis remains to be established. Tanner et al.
(9)
found that p27 protein was abundantly expressed in
intimal and medial VSMCs of nonatherosclerotic human coronary arteries.
Likewise, p27 expression was commonly observed in regions of coronary
atherosclerotic plaques not undergoing proliferation and was absent
within proliferating plaque cells. As indicated by double-labeling
techniques, the majority of cells expressing p27 in early and advanced
human atheromas were VSMCs and macrophages. Ihling et al.
(16)
also reported p27 immunoreactivity in human
atherosclerotic coronary and carotid artery specimens, which localized
to nuclei of macrophages, VSMCs, T lymphocytes, and endothelial cells.
These authors provided evidence that TGF-ß1 present in human
atherosclerotic tissue may mediate its growth-suppressive function
through p27. Bearing in mind these expression studies and our results
demonstrating a causal link between diminished p27 expression and
atherogenesis, it is appropriate to consider the role of p27 in
an atherosclerotic plaque in influencing both its initial response to
mitogenic stimuli and its progression to more advanced stages. Hence,
future studies in animal models and human tissue should thoroughly
investigate the temporal and spatial pattern of expression of p27
during atherogenesis and elucidate molecular mechanisms underlying the
regulation of p27 expression in vascular cells. Such 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 p27 expression (28
, 31
32
33)
.
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ACKNOWLEDGMENTS |
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Received for publication February 21, 2001.
Revision received May 29, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C. Castro, A. Diez-Juan, M. J. Cortes, and V. Andres Distinct Regulation of Mitogen-activated Protein Kinases and p27Kip1 in Smooth Muscle Cells from Different Vascular Beds. A POTENTIAL ROLE IN ESTABLISHING REGIONAL PHENOTYPIC VARIANCE J. Biol. Chem., February 7, 2003; 278(7): 4482 - 4490. [Abstract] [Full Text] [PDF]
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D. GOUKASSIAN, A. DIEZ-JUAN, T. ASAHARA, P. SCHRATZBERGER, M. SILVER, T. MURAYAMA, J. M. ISNER, and V. ANDRES Overexpression of p27Kip1 by doxycycline-regulated adenoviral vectors inhibits endothelial cell proliferation and migration and impairs angiogenesis FASEB J, September 1, 2001; 15(11): 1877 - 1885. [Abstract] [Full Text] [PDF]
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P < 0.0001 vs. p27+/-apoE-/-.
C) Immunoblot analysis of cell lysates prepared from the
aorta of fat-fed apoE-null mice with the indicated genotype for p27.
Primary antibodies were anti-p27 (top) and anti-tubulin (bottom).
Relative to p27+/+apoE-/- mice, aortic p27 expression in
p27+/-apoE-/- was 0.58 ± 0.01 (n=2
densitometric analysis normalized by tubulin content).
