© 2001 American Heart Association, Inc.
Vascular Biology |
Cellular Senescence After Single and Repeated Balloon Catheter Denudations of Rabbit Carotid Arteries
From the Cell Biology Group, Centre for Cardiovascular Biology and Medicine, Department of Medicine (M.F., D.J.K., J.D.E.) and Department of Surgery (S.B.), Royal Free and University College Medical School, University College London, London, UK.
Correspondence to Jorge D. Erusalimsky, PhD, Department of Medicine, University College London, Rayne Institute, Ground Floor, Room G15, 5 University St, London WC1E 6JJ, UK. E-mail j.erusalimsky{at}ucl.ac.uk
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Abstract |
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Abstract—The hypothesis that increased cellular proliferation in the vasculature may lead to replicative senescence has been tested in a model of neointima formation. We have used a biomarker of replicative senescence, senescence-associated ß-galactosidase (SA-ß-gal), to detect senescence in rabbit carotid arteries subjected to single and double balloon denudations. We found an accumulation of senescent cells in the neointima and media of all injured vessels, in contrast to the near absence of such cells in control vessels. The relative area occupied by SA-ß-gal–positive cells was higher in vessels subjected to double denudation than in those subjected to single denudation, both in the neointima (0.99% versus 0.06%, respectively; P<0.001) and in the media (0.11% versus 0.01%, respectively; P<0.02). The majority of SA-ß-gal–positive cells were vascular smooth muscle cells, and a minority were endothelial cells. SA-ß-gal–positive cells showed no evidence of apoptosis by use of terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling. Our results indicate that the proliferative response that follows intraluminal injury to the artery leads to the emergence of senescent endothelial and smooth muscle cells. The demonstration that vascular cell senescence can occur in vivo suggests that this process may be involved in cardiovascular pathologies that have a proliferative component.
Key Words: endothelial cells • vascular smooth muscle cells • senescence • ß-galactosidase • neointima
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Introduction |
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Most normal mammalian cells have a limited potential for proliferation, a property that is classically manifested on serial passage in culture.1 2 When this potential is exhausted, cells enter into a permanent nondividing state referred to as replicative senescence.1 2 3 4 It has been postulated that this process may also occur in vivo in tissues in which cell proliferation continues throughout the entire lifespan of the organism.2 Furthermore, it has been suggested that replicative senescence may contribute to aging of the individual and to the development of age-related diseases.2 5 In tissues such as skin, for which the rates of cell turnover are relatively high, the presence of senescent cells has been confirmed.6 In contrast, in tissues such as the blood vessel wall, for which the rates of cell turnover are lower,7 8 the suggestion that senescent cells might accumulate in vivo has remained controversial.9 10 11 Furthermore, although circumstantial evidence points to the accumulation of senescent cells in pathological conditions involving endothelial and vascular smooth muscle cell proliferation,12 13 14 15 16 17 a definitive demonstration of this phenomenon in vivo is still lacking.
Until recently, the putative identification of individual senescent cells has had to rely mainly on morphological examination. This situation has changed with the discovery that a cytochemical assay of ß-galactosidase can be used to detect senescent cells selectively if the reaction is performed at pH 6.0.6 This endogenous pH 6.0 activity has been termed senescence-associated ß-galactosidase (SA-ß-gal).6 Initially, SA-ß-gal was found in senescent cultures of fibroblasts,6 keratinocytes,6 and epithelial cells18 and in skin biopsies of aged human donors.6 Recently, we have demonstrated the presence of SA-ß-gal in senescent human umbilical vein endothelial cells (ECs) and rabbit vascular smooth muscle cells (VSMCs) grown in culture19 and have elucidated the biological basis of this assay.20 These studies suggest that SA-ß-gal could be a useful marker to obtain direct evidence that vascular cell senescence occurs in vivo.
Previous work has shown that repeated denudation of the rabbit carotid artery leads to successive waves of endothelial and VSMC proliferation.21 We have used this model to investigate whether such pronounced vascular cell replication results in the accumulation of senescent cells. In the present study, we demonstrate for the first time the emergence of senescent ECs and VSMCs resulting from the increase in cell proliferation that follows repeated denudation of the rabbit carotid artery.
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Methods |
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Experimental Design
Twelve New Zealand White male rabbits (2.5 to 3.0 kg) were obtained from Charles River UK Ltd (Kent, UK), divided into 2 groups, and maintained on a normal diet. One group (n=6) underwent a single balloon endothelial denudation injury of the right carotid artery, whereas the other group (n=6) underwent 2 successive right carotid artery denudations, performed 6 weeks apart. These groups are referred to as the single-denudation and double-denudation groups, respectively. Animals were culled 6 weeks after the first or second denudation. The left carotid arteries served as control vessels. All experiments were performed under a license granted by the UK Home Office in full compliance with the Animals (Scientific Procedures) Act 1986 and in accordance with Home Office and University College London guidelines.
Surgical Procedures and Tissue
Harvesting
Before surgery, the rabbits were anesthetized
with fentanyl/fluanisone (0.3 mL/kg IM, Janssen Animal Health Ltd)
followed by diazepam (2 mg/kg IV, Phoenix Pharmaceuticals Ltd); then
they were given enrofloxacin (10 mg/kg SC, Bayer AG). Both common
carotid arteries were exposed via a midline incision to the neck. An
arteriotomy was made in the right common carotid artery after this
vessel had been clamped, and a 2F embolectomy catheter (Mantis Surgical
Ltd) was inserted via this opening. The catheter was advanced 5 cm
cephalically, inflated with normal saline, and then withdrawn to the
site of the arteriotomy, before deflation. After repeating the
inflation/deflation sequence twice more, the catheter was removed
completely, and the arterial incision was closed.
Preliminary experiments had demonstrated that this denudation procedure
caused complete removal of the endothelium with minimal
or no damage to the internal elastic lamina. The left carotid artery
was sham-operated. Each rabbit was given buprenorphine (0.03 mg/kg SC,
Reckitt and Colman Products Ltd) for postoperative analgesia. For
the double-denudation group, the entire procedure was repeated in the
same manner, 6 weeks after the first denudation.
Six weeks after 1 or 2 denudation procedures, the carotid arteries were harvested as follows: Each rabbit was anesthetized again, as detailed above. The neck and thorax were opened to expose the carotid arteries, the heart, and the great vessels. The rabbit was then culled with sodium pentobarbital (280 mg/kg IV, Rhône Mérieux Ltd). The descending thoracic aorta was immediately clamped, and 50 mL of PBS (pH 7.4) was pressure-perfused via the left ventricle, followed by 500 mL of 2% paraformaldehyde in PBS, which was delivered over a 20-minute period. To allow excess fluid to be released, the inferior vena cava was divided. After perfusion fixation, the right and left common carotid arteries were excised, trimmed of excess periadventitial tissue, and rinsed briefly in ice-cold PBS.
SA-ß-Gal
Staining
The rinsed carotid arteries were cut transversely
into 3 equal portions, and the central portion from each vessel was
incubated for 24 hours at 37°C in SA-ß-gal staining solution
containing 1 mg/mL 5-bromo-4-chloro-3-indolyl
ß-D-galactopyranoside
(X-Gal, Sigma), 5 mmol/L potassium ferrocyanide, 5 mmol/L
potassium ferricyanide, 150 mmol/L NaCl, 2 mmol/L
MgCl2, and 40 mmol/L trisodium citrate,
titrated with NaH2PO4 to
pH 6.0. Where indicated, acidic ß-galactosidase activity was detected
by use of the same solution, but titration was to pH 4.0. After
incubation in staining solution, the vessels were briefly rinsed in
ice-cold PBS, snap-frozen in liquid nitrogen, and stored at
-80°C.
Quantification of Injury Score, Intimal
Thickening, and SA-ß-Gal
Staining
The frozen carotid arteries were mounted in OCT
compound (Merck Ltd), and serial transverse cryostat sections
(10 µm) were cut. Alternate sections were immersed in 3%
paraformaldehyde in PBS for 5 minutes at room
temperature and, unless otherwise indicated below, counterstained with
eosin. The remaining sections were stored at -80°C for
immunohistochemical analysis and terminal
deoxynucleotidyl transferase–mediated dUTP nick
end-labeling (TUNEL). Morphometric analysis was performed by
video microscopy (Nikon) and a computerized color image
analysis software system (LUCIA M, Laboratory Imaging Ltd). The
intima/media ratios, the percentages of intimal and medial areas
covered by blue-stained cells, and counts of adventitial blue-stained
cells were scored in 10 cross sections spaced at 100-µm intervals and
averaged for each vessel. These averages were then used to derive
mean±SD values for each treatment group. Results were compared
statistically by use of a nested ANOVA model after logarithmic
transformation of the data to achieve normal distributions. Statistical
analysis was performed with use of the Intercooled Stata
software package (release 6.0), and a value of
P<0.05 was considered
statistically significant.
The degree of injury in each denuded vessel was measured by use of a score devised by Schwartz et al22 as adapted by Burchenal et al23 for balloon angioplasty of rabbit arterial tissue. Grade 0 refers to vessels showing an intact internal elastic lamina, media, and external elastic lamina. A Grade 1 injury describes laceration of the internal elastic lamina, with an intact media and external elastic lamina. No vessels in the present study scored grade 2 (medial injury) or grade 3 (laceration of the external elastic lamina).
Immunohistochemistry
Sections were fixed for 20 minutes in acetone at
4°C, except in the case of proliferating cell nuclear antigen (PCNA)
detection, for which fixation was performed in 4%
paraformaldehyde for 2 minutes, followed by ethanol for
10 minutes at room temperature. Sections were pretreated with protein
blocking reagent (DAKO) and then incubated with primary antibodies
diluted in Tris-buffered saline (pH 7.4) for 1 hour at room
temperature. The following primary mouse monoclonal antibodies (all
from DAKO, unless otherwise stated) were used at the indicated
dilutions: anti-CD31 (clone JC/70A, 1:20), anti–smooth muscle
-actin (clone 1A4, 1:100), anti-CD45 (clone L12/201 [Serotec],
1:50), anti-rabbit macrophage (clone RAM11, 1:50), and
anti-PCNA (clone PC10, 1:50). Sections incubated with equivalent
amounts of appropriate isotype-matched antibodies raised against
Aspergillus niger
glucose oxidase (DAKO) were used as negative controls. A biotinylated
F(Ab')2 fragment of rabbit anti-mouse
immunoglobulin (DAKO) was used as the secondary reagent. Visualization
was performed by using either streptavidin–alkaline phosphatase in
combination with the New Fuchsin Chromogenic Substrate
System or streptavidin–horseradish peroxidase with diaminobenzidine
(all from DAKO). After they were mounted, the sections were viewed
under bright-field illumination with use of an Axiophot 2 microscope
(Carl Zeiss) and were photographed with Kodak Ektachrome 160T color
positive film.
Detection of Apoptotic Cells
Sections were fixed for 1 hour at room temperature in
4% paraformaldehyde and then
permeabilized in 0.1% Triton X-100/0.1% sodium
citrate for 2 minutes at 4°C. Apoptotic cells were identified
by the TUNEL method with use of terminal
deoxynucleotidyl transferase and
fluorescein-conjugated dUTP (Roche Molecular Biochemicals).
Sections processed with labeling solution without terminal transferase
were used as negative controls. Samples of
staurosporine-treated rabbit VSMCs grown on coverslips were
used as positive controls. Nuclei were counterstained with
4',6-diamidino-2-phenylindole (DAPI) in Vectashield mounting medium for
fluorescence (Vector Laboratories). Specimens were examined by
fluorescence microscopy with a x63/1.4 numerical aperture Plan
Apochromat objective and fluorescence filter sets appropriate
for fluorescein and DAPI (Carl Zeiss). Bright-field and
epifluorescence images of representative fields
were photographed with Kodak Ektachrome 400X color reversible
film.
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Results |
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Morphology and Morphometry of Neointimal Lesions
Four of the 6 denuded arteries from each group of animals were found to be patent at the time of culling. The other 2 injured vessels in each group were completely occluded by neointima, and because these vessels showed a marked degeneration of the vessel wall architecture, they were excluded from further study. Of the 4 patent vessels in each group, 3 had an injury score of grade 0, and 1 had an injury score of grade 1.
The histological appearances of representative patent vessels in the control, single-denudation, and double-denudation groups are shown in Figure IA (please see online data supplement available at http://atvb.ahajournals.org). Although after a single denudation, neointimal smooth muscle cells were aligned in the direction of blood flow, after a second denudation, the neointima appeared to be stratified, consisting of 2 layers of smooth muscle fibers oriented perpendicularly to each other. Morphometric analysis (online Figure IB) revealed that neointimal hyperplasia was significantly greater in the double-denudation than in the single-denudation group (mean intima/media ratios 1.79 and 0.29, respectively; P<0.01). In contrast, the thickness of the media was similar in the 3 groups (data not shown). Immunohistochemical staining for CD31 (Figure II; see online data supplement available at http://atvb.ahajournals.org) revealed that 6 weeks after a single denudation, the endothelium was essentially completely regenerated (online Figure IIB). In contrast, 6 weeks after a second denudation, reendothelialization was noticeably incomplete (online Figure IIC).
Endogenous
ß-Galactosidase in
Sham-Operated Arteries
Most mammalian cells express an acidic lysosomal
ß-galactosidase whose activity is detected at pH 4.0 and that is
normally demonstrable in cells of all replicative
ages.6 In agreement with this
notion, sections of a control artery that had been incubated with the
ß-galactosidase substrate at pH 4.0 showed positive (blue) staining
of cells throughout the entire vessel wall (Figure IIIA; see online
data supplement available at http://atvb.ahajournals.org). In contrast,
when control vessels were incubated at pH 6.0 to detect the SA-ß-gal
activity, positive staining was seen only occasionally, mostly in the
adventitia (online Figure IIIB, arrow) and more rarely in the media
(not shown).
SA-ß-Gal
in Injured Arteries
Figure 1
shows SA-ß-gal staining in
representative sections from control and denuded
arteries. In contrast to control vessels, in which SA-ß-gal–positive
cells were virtually absent
(Figure 1A and 1D), denuded arteries consistently
showed an accumulation of these cells in the neointima and
the media. In single-denudation arteries, blue cells were found in
small numbers
(Figure 1E, arrow). In double-denudation arteries, blue cells
were more conspicuous
(Figure 1C and 1F); in the neointima, they often
occurred in clusters distributed throughout its depth and sometimes
also in a discontinuous layer near or at the luminal border.
Characteristically, at high magnification, the blue precipitate in
SA-ß-gal–positive cells had a punctate appearance
(Figure 1E and 1F; see also
Figure 4D).
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Computer-assisted image analysis was used to
quantify the SA-ß-gal–positive cell accumulation in denuded vessels
(Figure 2). Repeated denudation resulted in a progressive
increase in the proportion of neointima covered by blue
cells (0.06% versus 0.99% for single- and double-denudation groups,
respectively; P<0.001;
Figure 2, left). Changes in the media
(Figure 2, right) were also statistically significant (0.01%
versus 0.11% for single- and double-denudation groups, respectively;
P<0.02), although blue cells
were less prevalent in the media than in the neointima. In
contrast to the accumulation of SA-ß-gal–positive cells in the
neointima and media, the number of blue cells in the
adventitia did not rise after injury (data not
shown).
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Identification of
SA-ß-Gal–Positive Cell
Types
The identity of the SA-ß-gal–positive cells in
denuded vessels was determined by immunohistochemistry
(Figure 3). In areas in which the endothelium
had been partially regenerated
(Figure 3A), some of the cells bordering the lumen were
costained for CD31 and SA-ß-gal
(Figure 3B). The remaining SA-ß-gal–positive cells
throughout the neointima
(Figure 3C and 3D) and also those in the media (not shown)
were stained positively for smooth muscle -actin. In contrast,
SA-ß-gal–positive cells were not stained positively for CD45, a
marker for leukocytes. Indeed, some CD45-positive cells were found on
the luminal surface of the vessel
(Figure 3E and 3F), apparently adhering to blue
neointimal cells (already identified as VSMCs on an
adjacent section,
Figure 3C). Similarly, SA-ß-gal–positive cells showed no
costaining with RAM11, an antibody that detects rabbit
macrophages (data not shown).
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Absence of Markers for Proliferation or
Apoptosis in SA-ß-Gal–Positive Cells
In agreement with previous
findings,21 PCNA staining of
sections derived from double-denudation vessels showed the presence of
proliferating cells in the neointima and the media 6 weeks
after injury. No clear pattern between the localization of the
PCNA-positive cells and the SA-ß-gal–positive cells was apparent
(Figure IV; see online data supplement available at
http://atvb.ahajournals.org). Thus, in some sections, proliferating
cells were found in areas devoid of SA-ß-gal–positive cells (online
Figure IVA), whereas in others, these 2 different cell types were found
in the same vicinity (online Figure IVC and IVD). In all cases,
however, SA-ß-gal–positive cells were not costained for
PCNA.
The occurrence of apoptosis during the weeks after
balloon injury of the vessel wall has been widely
documented.24 25 26
To obtain a preliminary assessment of the relationship between
apoptosis and senescence, sections from each of the
double-denuded vessels were examined for the occurrence of TUNEL and
SA-ß-gal staining. In all arterial cross sections
studied, sparse TUNEL-positive nuclei were detected in the
neointima and the media, ranging from 1 to 4 per section.
In most cases, these TUNEL-positive nuclei showed a marked degree of
fragmentation
(Figure 4B
and 4C; see also Figure VB and VC in the online
data supplement available at http://atvb.ahajournals.org), suggesting
that these corresponded to late apoptotic cells. No
apoptotic cells were found at the luminal border of the
neointima. Notably, no SA-ß-gal–positive cells showed
TUNEL fluorescence
(Figure 4D through 4F; see also online Figure VD through
VF).
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Discussion |
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Replicative senescence during cell growth in culture is a well-documented phenomenon (see reviews3 4 ). However, it is not entirely clear whether in vitro senescence has a parallel in vivo. To investigate the occurrence of in vivo senescence in vascular tissue, we have used a well-established model of neointima formation. In this model, endothelial denudation of the rabbit carotid artery induces EC and VSMC proliferation within the injured area.9 21 27 Hence, we have hypothesized that this proliferation would gradually deplete the cellular replicative capacity within the vessel wall, leading to the emergence of senescent cells. Because a repeated denudation of the carotid artery is known to cause a further wave of cell proliferation,21 we have similarly hypothesized that a second injury would accentuate the accumulation of senescent cells. The results in the present study confirm these predictions. Although SA-ß-gal–positive cells were virtually absent from the wall of noninjured carotid arteries, we found that balloon denudation resulted in a marked accumulation of senescent cells. Furthermore, the magnitude of this accumulation was significantly greater after a second denudation. These findings are consistent with the possibility that the wave of proliferation that follows a second injury includes cells whose replicative capacity has been partially depleted after a first injury.
The senescent cells were found predominantly in the
neointima, in agreement with the notion that cells forming
this layer are the product of a higher number of cumulative
divisions than are those found in the media. The area of
neointima covered by senescent cells was relatively small
(
1% in double-denuded vessels). However, because the
neointima is also composed of extracellular matrix, this
measure may underrepresent the actual number of cells that have
undergone senescence. This possible discrepancy, though, would not
affect our overall finding that compared with single-denudation
vessels, double-denudation vessels had a significantly greater
proportion of senescent cells. In the neointima,
SA-ß-gal–positive cells were predominantly VSMCs, although some
senescent ECs were also detected. In the media, the senescent cells
were identified as VSMCs. Although some SA-ß-gal–positive cells were
found in the adventitia, their numbers did not increase with an
increasing number of denudations.
In the present study, the extent of the proliferative response was evaluated by the degree of neointimal hyperplasia and reendothelialization present 6 weeks after denudation. We consistently found that a second denudation resulted in increased neointimal thickening. This concurred with the findings of Azuma et al,21 who in addition demonstrated that a second injury led to a more extensive and persistent wave of cell proliferation within the neointima and the media, a phenomenon that would underlie the marked increase in the accumulation of senescent cells found in the present study. Azuma et al also found that significant numbers of proliferating cells were still present 6 weeks after a second denudation. Our observation of SA-ß-gal–positive cells in the vicinity of PCNA-positive cells indicates that senescence and proliferation can coexist within the same tissue. Furthermore, our findings confirm that SA-ß-gal–positive cells are not themselves proliferating.
Azuma et al21 have reported total reendothelialization of the arterial wall 6 weeks after either a single or a double denudation of rabbit carotid arteries. Although we have also demonstrated virtually total reendothelialization after a single injury, we have found that reendothelialization remained incomplete after a double denudation. This discrepancy may be attributable to differences between the denudation procedures used in the 2 studies. For example, our use of saline (rather than air) to fill the embolectomy balloon may lead to a more stringent removal of the endothelium and, hence, affect the extent of the subsequent reendothelialization. Thus, if in our experiments fewer ECs remain adherent after the injury, more cell doublings would be required to cover the denuded area. These extra doublings could advance the onset of EC senescence and, hence, lead to the attenuation of the ability of the endothelium to repair itself. The fact that in our experiments only a relatively low number of senescent ECs were detected in the regenerated endothelium might in principle argue against this scenario. On the other hand, the ultimate fate of senescent ECs is not known. For this reason, we cannot discount the possibility that senescence was relatively prevalent in the regenerated endothelium but that most senescent cells had already been lost by the time of analysis. Senescent ECs could simply have been shed into the lumen. Furthermore, although we have not detected EC apoptosis 6 weeks after injury, our experiments cannot rule out the possibility that some senescent ECs had undergone apoptosis before this time point.
VSMC apoptosis is known to occur during neointima formation after denudation injuries.24 25 26 In our experiments, no SA-ß-gal–positive apoptotic cells were found. On the other hand, the number of apoptotic cells present in injured vessels was low. This finding is consistent with previous similar studies suggesting that the wave of apoptosis that follows luminal injury subsides considerably within 4 weeks24 25 and with the finding that neointimal cells are relatively resistant to apoptotic death induced by a second angioplasty injury.28 Given the fact that residual apoptosis could be detected in our experiments and that none of the senescent cells in the neointima or media showed TUNEL staining, our results suggest that, at least for VSMCs, apop-tosis is not an inevitable consequence of senescence.
Telomere shortening has been implicated as one mechanism underlying replicative senescence.3 4 Thus, in tissues from donors of different ages, measurement of telomere length by terminal restriction fragment analysis has been used as a surrogate marker of this process.15 In our model, however, this technique is unlikely to yield meaningful results because of the low number of senescent cells present in the lesions and the inherent heterogeneity of telomere length among chromosomes.29
Our finding of senescent cells in vascular tissue may have significant pathophysiological implications. The occurrence of EC senescence may be of importance during the development of postangioplasty restenosis. It is believed that early reendothelialization limits the degree of VSMC proliferation during neointima formation.30 Thus, if EC senescence impairs such reendothelialization, this may result in more severe neointimal thickening and luminal stenosis. The present findings may also have significant implications in terms of our understanding of the mechanisms involved in atherogenesis and plaque progression. A possible link between replicative senescence and atherosclerosis has been inferred from studies in progeroid syndromes.31 32 Morphological studies12 13 14 and telomere length analysis15 in normal individuals have also suggested the presence of senescent ECs covering the surface of atherosclerotic lesions at various stages of disease progression. Furthermore, senescent ECs overexpress in vitro certain proatherogenic and prothrombotic molecules.33 34 As for VSMCs, proliferation studies with cells derived from atherosclerotic plaques suggest that they may have a diminished replicative capacity.16 35 Given these possible associations between replicative senescence and atherosclerosis, our findings suggest that SA-ß-gal could be used to investigate the presence of senescent cells in atherosclerotic lesions.
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Acknowledgments |
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Mark Fenton and Jorge D. Erusalimsky were supported by grants (FS/97008 and RG/98011, respectively) awarded by the British Heart Foundation. David J. Kurz was supported by a fellowship from the Swiss National Science Foundation. We gratefully acknowledge the staff of the UCL Biological Services Unit for their expert technical assistance.
Received March 29, 2000; accepted September 26, 2000.
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