Vascular Smooth Muscle Cells Undergo Telomere-Based
Senescence in Human Atherosclerosis
Effects of Telomerase and Oxidative Stress
Charles Matthews,* Isabelle Gorenne,* Stephen Scott, Nicola Figg, Peter Kirkpatrick,
Andrew Ritchie, Martin Goddard, Martin Bennett
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Abstract—Although human atherosclerosis is associated with aging, direct evidence of cellular senescence and the
mechanism of senescence in vascular smooth muscle cells (VSMCs) in atherosclerotic plaques is lacking. We examined
normal vessels and plaques by histochemistry, Southern blotting, and fluorescence in situ hybridization for telomere
signals. VSMCs in fibrous caps expressed markers of senescence (senescence-associated ␤-galactosidase [SA␤G] and
the cyclin-dependent kinase inhibitors [cdkis] p16 and p21) not seen in normal vessels. In matched samples from the
same individual, plaques demonstrated markedly shorter telomeres than normal vessels. Fibrous cap VSMCs exhibited
markedly shorter telomeres compared with normal medial VSMCs. Telomere shortening was closely associated with
increasing severity of atherosclerosis. In vitro, plaque VSMCs demonstrated morphological features of senescence,
increased SA␤G expression, reduced proliferation, and premature senescence. VSMC senescence was mediated by
changes in cyclins D/E, p16, p21, and pRB, and plaque VSMCs could reenter the cell cycle by hyperphosphorylating
pRB. Both plaque and normal VSMCs expressed low levels of telomerase. However, telomerase expression alone
rescued plaque VSMC senescence despite short telomeres, normalizing the cdki/pRB changes. In vivo, plaque VSMCs
exhibited oxidative DNA damage, suggesting that telomere damage may be induced by oxidant stress. Furthermore,
oxidants induced premature senescence in vitro, with accelerated telomere shortening and reduced telomerase activity.
We conclude that human atherosclerosis is characterized by senescence of VSMCs, accelerated by oxidative
stress-induced DNA damage, inhibition of telomerase and marked telomere shortening. Prevention of cellular
senescence may be a novel therapeutic target in atherosclerosis. (Circ Res. 2006;99:156-164.)
Key Words: atherosclerosis 䡲 smooth muscle 䡲 aging
H
uman atherosclerotic plaques comprise inflammatory
cells (macrophages and T lymphocytes), vascular
smooth muscle cells (VSMCs), and intracellular and extracellular lipids. Plaque disruption results in acute myocardial
infarction and stroke, whereas repeated rounds of subclinical
rupture and repair also promote plaque growth. Although
VSMC proliferation occurs in atherogenesis, most proliferating cells are macrophages, and VSMC mitotic rates are lower
in advanced plaques than early lesions, even after plaque
rupture,1 suggesting that plaque VSMCs may exhibit
senescence.
Cellular senescence can be defined as cell cycle arrest
accompanying the exhaustion of replicative potential.2 Senescent cells display a characteristic morphology (vacuolated,
flattened cells) and gene expression, including markers such
as senescence-associated ␤-galactosidase (SA␤G).3 Senescence may be triggered by 2 broadly different mechanisms. In
most primary cells, the telomeres of chromosomes shorten at
each cell division because of incomplete chromosomal replication. Replicative senescence may be induced at critical
telomere lengths or structures, such as telomeric fusion or
dicentrics or loss of telomere-bound factors.4,5 Cells also
undergo “stress-induced premature senescence” (SIPS), for
example, in response to activated oncogenes (eg, Ha-Ras) and
suboptimal culture conditions.6
Although telomere loss occurs with replication, both premature senescence and telomere breaks may be induced by
oxidative DNA damage. Reactive oxygen species (ROS),
particularly superoxide anions, hydrogen peroxide, and hydroxyl radicals, can produce a large variety of DNA damage,
including DNA strand breaks and DNA base modifications.
ROS can accelerate telomere loss during replication in some
cell types7 but also induces premature senescence independently of telomere shortening.8 Increased levels of ROS are
Original received March 17, 2006; resubmission received May 17, 2006; revised resubmission received June 7, 2006; accepted June 9, 2006.
From the Division of Cardiovascular Medicine (C.M., I.G., S.S., N.F., M.B.) and Department of Surgery (P.K.), University of Cambridge,
Addenbrooke’s Hospital; and the Department of Surgery (A.R.) and Histopathology (M.G.), Papworth Hospital, Cambridge, UK.
*Both authors contributed equally to this study.
Correspondence to Martin Bennett, Division of Cardiovascular Medicine Box 110, ACCI, Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK. E-mail
[email protected]
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000233315.38086.bc
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found in atherosclerosis in all layers of the diseased arterial
wall and, particularly, in the plaque itself.9 These findings
suggest that ROS within plaques may promote cellular
senescence.
Replicative senescence and SIPS converge on the tumor
suppressor genes p53 and rb, the latter being regulated by
cyclin-dependent kinase inhibitors (cdkis), including p16 and
p21. pRB, p53, p21, or p16 expression can induce senescence
and are often increased in both replicative senescence and
SIPS.10 Telomere loss or damage activates p53 via DNA
damage–sensing mechanisms,11 with subsequent transcription of p21, whereas stress-induced activation of p16 accelerates replicative senescence independently of telomere
length.12 In addition, p16 expression effectively renders
telomere-based senescence irreversible,12 in part, by promoting repressive heterochromatin at loci containing targets of E2F transcription factors.13 Although the division
between replicative senescence and SIPS is useful, the
pathways have multiple areas of overlap. Indeed, both
telomere-based DNA damage and stress-induced activation of p16 may occur simultaneously, inducing a growth
arrest with cell cycle regulator expression, reflecting
activation of both pathways.12
We therefore examined both the evidence for senescence in
atherosclerosis and the mechanism of growth arrest in human
plaque VSMCs. We demonstrate that plaque VSMCs show
multiple markers of senescence in vivo and in vitro and
demonstrate marked telomere shortening. We further show
that plaque VSMC senescence is completely rescued by
telomerase but accelerated by oxidative stress.
Atherosclerosis and Telomere Shortening
157
Immunoblotting and Immunoprecipitation
Immunoblotting and immunoprecipitation were performed as previously described.14 (See the online data supplement.)
Virus-Based Expression of hTERT, Cyclin D1,
and CDK4
Retroviruses were used to infect VSMCs as previously described.16
(See the online data supplement.) Adenoviruses encoding cyclin D1
and CDK4 were used to express these products in human plaques
VSMCs (see the online data supplement).
Real-Time Quantitative Telomeric Repeat
Amplification Protocol Assay for
Telomerase Activity
Telomerase activity was measured by real-time quantitative telomeric repeat amplification protocol (RTQ-TRAP) assay as previously described (see the online data supplement).
Materials and Methods
Isolation of VSMCs From Human Tissue
Normal human aortic medial vascular smooth muscle cells (VSMCs)
were isolated from recipients undergoing cardiac transplant (n⫽10)
and plaque-derived VSMCs from carotid atherectomies (n⫽50) with
informed consent using protocols approved by the Cambridge or
Huntingdon Research Ethical Committee. VSMCs were isolated,
cultured, and characterized as previously described14 using immunofluorescence labeling for ␣–smooth muscle actin (␣-SMA), sm
muscle myosin, calponin, desmin, and vimentin (Figure I in the
online data supplement, available at http://circres.ahajournals.org).
Contamination with other cell types was excluded using markers for
ECs, macrophages, and T lymphocytes. Separate cultures isolated
from individual patients were studied. Cells were passaged at 80%
confluence and split 1:2. Proliferation rates were determined for cells
at approximately 50% confluence as described previously.14
Coronary endarterectomy plaque and normal artery segments
(aorta and bypass conduit) were isolated at coronary artery bypass
surgery and coronary artery segments from patients undergoing
cardiac transplantation obtained from recipient hearts.
Measurement of Telomere Length
See the online data supplement.
Telomere/Immunostaining Fluorescence In
Situ Hybridization
Telomere/immunostaining fluorescence in situ hybridization (TELIFISH) was performed using a modification of previous methods.15
(See the online data supplement.)
SA␤G Staining and Immunohistochemistry
See the online data supplement.
Figure 1. Senescence of human plaque VSMCs. A, Atherosclerotic plaque stained for SA␤G (blue)/eosin. B, High magnification of area outlined in A, showing SA␤G-positive cells (arrows)
in plaque intima. C and D, Colocalization of SMA-positive cells
(brown) with SA␤G (blue) in advanced plaque fibrous cap
(arrows). D, High magnification of area outlined in C. E, CD3
(brown)/SA␤G (blue) staining demonstrating ⬍5% lymphocytes
express SA␤G. F, CD68 (brown)/SA␤G (blue) staining demonstrating faint SA␤G staining in macrophages in necrotic core. G
and H, Colocalization of p16 (brown)/SA␤G (blue) (H) or p21
(brown)/SA␤G (blue) (H) in fibrous cap VSMCs (arrows). Insets
show enlarged views of areas outlined in G and H. Scale bar
represents 40 ␮m in D and insets shown in G and H; 200 ␮m in
other panels.
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July 21, 2006
Determination of Mitochondrial Mass and
ROS Levels
The fluorescent dyes H2-DCFDA and acridine orange (Molecular
Probes) were used to measure cellular ROS content and mitochondrial mass, respectively. (See the online data supplement).
Statistics
An unpaired 2-tailed Student’s t test was used to examine differences
between 2 groups, when data approximated a normal distribution.
When data did not approximate a normal distribution, Mann–
Whitney U test was used.
Results
Evidence for VSMC Senescence in Atherosclerosis
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We first examined expression of senescence markers in
plaque VSMCs in vivo including SA␤G, coexpression of the
cdkis p16/p21/p27 with SA␤G, and cell-lineage markers in
human carotid atherectomies (Figure 1). Among fibrous cap
VSMCs, 18%⫾2.6 (mean⫾SEM, [n⫽6]) were SA␤G positive, but SA␤G-positive VSMCs were not found in normal
vessels (n⫽6). Macrophages showed faint SA␤G expression
throughout plaques, most likely reflecting their high lysosomal content,17 but ⬍5% of lymphocytes were SA␤G positive.
More than 70% SA␤G-positive VSMCs also expressed p16,
and ⬎90% expressed p21, although neither p16 nor p21 was
expressed in normal vessels. p27 expression was evident in
both normal medial and plaque VSMCs and correlated poorly
with SA␤G, suggesting a limited role for p27 in plaque
VSMC senescence.
We next examined expression of these markers in cultured
plaque or normal VSMCs. First passage plaque VSMCs
demonstrated 80.5%⫾12.0 (mean⫾SEM) SA␤G expression
compared with 5.2%⫾1.7 (mean⫾SEM, P⬍0.01) in normal
VSMCs. Both VSMC types reached near 100% expression
over time, at passage 3 to 4 for plaque VSMCs and 10 to 12
for normal VSMCs. Plaque VSMCs exhibited large, flattened
morphology and low culture density, with lower rates of cell
proliferation on time-lapse videomicroscopy at any time point
(eg, median [interquartile range]: 19.64 [15.4 to 25.2] versus
65.98 [46.2 to 92.6] divisions per 100 cells per 24 hours
[U⫽4, P⬍0.001] at passage 3). Plaque VSMCs underwent
culture senescence at passageⱕ3 compared with passageⱖ8
in normal VSMCs.
Cell Cycle Regulation Is Disrupted in Human
Plaque VSMCs
Expression of G1 regulatory proteins in plaque and normal
VSMCs was examined at passage 1 to 2, when marked
differences in cell proliferation were observed. Plaque
VSMCs demonstrated higher levels of nonphosphorylated
pRB, reduced cyclin D1-3, reduced cyclin E and p27, but
increased p16 and p21 (Figure 2A). No differences were
observed in CDK2 or CDK4. Plaque VSMCs had reduced
E2F-1 expression and a higher proportion of E2F-1 bound
to pRB. E2F-2 and E2F-3 levels were unchanged. To
determine whether the pattern of pRB, p21, and p16
expression was unique to plaque VSMCs, we examined
their expression at replicative senescence of normal
VSMCs (Figure 2B). p16 was elevated with reduced
cyclins D and E and pRB phosphorylation, although p21
Figure 2. Expression of G1/S regulators in VSMCs. Western blot
of G1/S regulators and immunoprecipitation of E2F1–3 with pRB
in passage 1 to 2 normal or plaque VSMCs (A), or control (C) or
senescent (S) normal human VSMCs (B) (n⫽4).
expression was unchanged. Thus, senescence of plaque
VSMCs may be attributable to other pathways interacting
with replication arrest.
To prove that pRB phosphorylation is required for plaque
VSMC senescence, we expressed CDK4 and cyclin D1 in
human plaque VSMCs using adenovirus vectors and examined cell cycle progression by 5-bromodeoxyuridine
(BrdU) incorporation and protein expression by Western blot
(Figure 3). CDK4 and cyclin D1 expression induced robust
phosphorylation of pRB, accompanied by increased expression of E2F-1 and the G2/M cyclin, cyclin A. pRB phosphorylation was sufficient to induce cell cycle entry of plaque
VSMCs, as demonstrated by increased BrdUrd incorporation
(Figure 3).
Telomere Length Is Markedly Shorter in
Plaque VSMCs
To examine whether the premature senescence of plaque
VSMCs reflected telomere loss, we first examined telomere
restriction fragment (TRF) length in cultured normal aortic
VSMCs. VSMC telomeres progressively shortened with increasing passage, by ⬇100 bp per cumulative population
doubling (CPD) (Figure 4A). To examine TRF length in vivo,
we isolated coronary artery segments from 20 patients undergoing cardiac transplantation for ischemic or dilated cardiomyopathy, aged between 40 to 60 years; parallel histology
demonstrated advanced atherosclerosis or no atherosclerosis,
respectively. Despite similar chronological age, TRF length
exhibited large variations between individuals in either group
(Figure 4B). To circumvent this variability, we studied
matched diseased and normal vessels from the same patient,
using coronary endarterectomies and bypass conduits (left
internal mammary artery, aorta, radial artery). Normal vessels
from different vascular beds in the same patient showed
identical telomere length (Figure 4C), allowing comparison
of normal and atherosclerotic arteries from different sites.
Coronary plaque telomeres were ⬇1 kb (range 0.74 to 1.31)
(P⬍0.01) shorter than matched normal vessels (Figure 4D). If
Matthews et al
Atherosclerosis and Telomere Shortening
159
severity. In contrast, telomere signals were significantly
reduced in intimal plaque VSMCs versus medial VSMCs in
the same section (Figure 5C) and were inversely proportional
to disease severity, with signals barely detectable in the most
advanced lesions.
Telomerase Delays VSMC Senescence
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Figure 3. Hyperphosphorylation of pRB induces cell cycle entry
in plaque VSMCs. A, Western blot of G1/S regulators in plaque
VSMCs after infection with adenoviruses encoding empty vector
(Rad 60), cyclin D1, or cyclin D1⫹CDK4. B, Immunocytochemistry of plaque VSMCs for BrdU after infection with Rad60 (left) or
cyclinD1⫹CDK4 (right). BrdUrd-positive nuclei (blue) are indicated by arrows. C, Percentage of BrdUrd-positive plaque
VSMCs after infection with either Rad60 or cyclin D1⫹CDK4
adenoviruses. Data are means⫾SEM (n⫽6).
telomere loss in vivo occurs to the same extent per division to
telomere loss in vitro, and is confined to VSMCs, this
difference represents approximately 7 to 13 CPDs.
Plaques are heterogeneous structures, containing cells not
present in normal arteries. To examine VSMC telomere
lengths, we used quantitative TELI-FISH (qTELI-FISH), a
semiquantitative assay based on binding of fluorescently
labeled peptide nucleic acid complexes complementary to
telomere sequences. We examined plaques with a range of
disease severity (American Heart Association Grades I
through V)18 for telomere length, double-labeled with ␣-SMA
to identify VSMC telomere signals in the fibrous cap (Figure
5A), with VSMCs in the remote, uninvolved arterial media
acting as internal controls. Telomere signals were easily
detected in ␣-SMA negative cells in the plaque and in medial
VSMCs. In contrast, both telomere intensity and average
telomere number were markedly reduced in intimal VSMCs
(Figure 5B and 5C). Total telomere fluorescence (number of
telomeres⫻fluorescence intensity of each telomere) of medial
VSMCs did not change significantly with increasing disease
Although telomerase enzymatic activity requires TERT,
TERC, and other proteins, hTERT expression is a ratelimiting factor in telomerase activity. We therefore examined
telomerase (hTERT) expression in plaques or normal vessels.
Telomerase expression was low in both human plaque and
normal vessel VSMCs in vivo (Figure 6A through 6C). In
contrast, telomerase expression was easily detectable in
macrophages (Figure 6D).
To examine whether telomere maintenance could block
VSMC senescence, we stably reexpressed hTERT or vector
control in human VSMCs using retrovirus vectors. Antibiotic
selection was used to produce cultures whereby all cells
expressed the product. Parallel cultures were maintained in
identical culture conditions and passaging. Normal VSMCs
expressing the vector showed little telomerase expression on
Western blotting; activity measured by RTQ-TRAP, a highly
sensitive and quantitative assay for telomerase activity,19 was
barely above background (heated controls). hTERT VSMCs
expressed detectable telomerase (Figure 6E) and significantly
elevated telomerase activity (0.202⫾0.011 arbitrary units
versus 0.06⫾0.006 in control VSMCs [n⫽3 mean⫾SEM]).
VSMCs expressing the vector senesced around passage 15,
expressing p16 and low levels of phosphorylated pRB, cyclin
D1-3 and cyclin E. hTERT delayed senescence of normal
VSMCs by ⬎30 CPDs and effectively immortalized the
cultures. This was associated with maintained cyclins D1-3 and
E expression and pRB phosphorylation despite high levels of
p21 and p16, indicating that ongoing proliferation occurred
despite normal arrest signals associated with senescence.
hTERT expression significantly elevated telomerase activity
in plaque VSMCs (0.26⫾0.006 versus 0.029⫾0.005 in noninfected plaque cells). Plaque VSMCs expressing the control
vector do not survive selection, as cells undergo senescence
before a culture can be established. In contrast, hTERT
rescued plaque VSMC senescence, completely reversed the
high p21 and p16 expression seen in control plaque VSMCs,
and normalized P-pRB despite having minimal effects on
cyclin D1-3 and cyclin E (Figure 6E). Importantly, hTERT did
not reduce TRF loss/CPD in normal VSMCs, and hTERT
VSMCs showed similar TRF lengths at the CPD that vectorVSMCs underwent senescence (Figure 6F and 6G). However,
hTERT allowed cells to proliferate at telomere lengths far
shorter than those seen at VSMC senescence.
Oxidative DNA Damage Accelerates Telomere
Loss and Induces Early Senescence
To examine the role of oxidative stress in VSMC senescence
in atherosclerosis, we first examined plaques for 8-oxo-G, an
abundant oxidative lesion in mammalian DNA.20 8-Oxo-G
was not detected in medial VSMCs of normal arteries or
uninvolved segments of arteries containing plaques. In contrast, 22%⫾4.8 of fibrous cap VSMCs and 35%⫾6.7
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Figure 4. Telomere lengths of normal arteries or
atherosclerotic plaques. A, Southern blot for TRF
length (as marked) in normal VSMCs in culture.
Three separate aortic VSMC lines are shown at
passage 2 and passage 6 to 8. ␭ HIND III and 1-kb
DNA markers are shown for comparison. B, Variability of TRF length in coronary arteries of patients
with dilated (N) or ischemic cardiomyopathy (P).
Patient age and sex are marked. C, TRF length
(marked) in 3 normal arteries (aorta, radial, left
internal mammary artery [LIMA]) or plaque (coronary endarterectomy) from the same patient. D,
TRF length (marked) for matched plaque (P) vs
normal vessel (N) from the same patient for 4 separate individuals.
(mean⫾SEM, n⫽8) of plaque macrophages expressed
8-oxo-G (Figure 7).
We next examined oxidative stress in plaque and normal
VSMCs, in normal VSMCs at replicative senescence, and
after treatment of VSMCs with tert-butylhydroperoxide (tBHP), an inducer of hydrogen peroxide21 that can induce
DNA single-strand breaks.22,23 Flow cytometric analysis of
fluorescence of the redox-sensitive dye H2-DCFDA showed
significantly increased oxidative stress in plaque versus
normal VSMCs and also in normal VSMCs at replicative
senescence. These changes paralleled a significant increase in
mitochondrial mass in plaque and senescent VSMCs (supplemental Figure II). t-BHP also significantly induced oxidative
stress (supplemental Figure II). A single (acute) administration of 80 mmol/L t-BHP for 1 hour induced growth arrest
(13.2⫾1.4% versus 2.8⫾1.0% Ki67-positive cells, P⬍0.01)
and increased the percentage of SA␤G from 11.9⫾1.3% to
55.5⫾0.4% (P⬍0.01 [mean⫾SEM]), without inducing cell
death, as determined by time-lapse videomicroscopy and
trypan blue exclusion (not shown). To mimic chronic oxidant
stress, human VSMCs were treated every passage with t-BHP
(16 to 80 mmol/L) for 1 hour, then cells returned to medium
without t-BHP. Cell proliferation, passage number at senescence, telomere loss with each CPD, and G1/S regulator
expression were examined, compared with untreated controls.
Chronic t-BHP treatment dose-dependently accelerated senescence and increased telomere loss/CPD (Figure 7E and
7F). t-BHP induced p21, and reduced pRB phosphorylation
and cyclins D1-3 and E, although p16 expression was unchanged (Figure 7G). t-BHP also reduced telomerase activity
in a dose-dependent manner, even at concentrations that did
not accelerate telomere shortening (Figure 7H).
Discussion
We identify human atherosclerosis as a disease characterized
by VSMC senescence. SA␤G-positive VSMCs were readily
detected within advanced human plaques, colocalized with
p16 and p21, confirming a senescence phenotype in vivo.
Previous studies have demonstrated SA␤G activity in human
plaques, in both ECs and VSMCs.24 –26 However, although
SA␤G is a useful marker, its activity is critically dependent
on detection conditions, and it is also expressed in nonsenescent cells with a high lysosomal content (Kurz et al17 and
shown here). Multiple markers of senescence are therefore
recommended to demonstrate senescence in vivo, together
with cell lineage markers, to identify cell type undergoing
senescence. Although there were minor differences in expression of specific markers in vitro and in vivo, mostly likely
reflecting culture conditions, atherosclerotic plaque VSMCs
in vitro manifest characteristic morphological features of
senescence, impaired proliferation and early culture senescence, and express multiple senescence markers (SA␤G, p21,
p16). Increased p16 and p21 coupled with low cyclin D and
E expression lead to reduced pRB phosphorylation and
increased E2F-1 bound to pRB, preventing E2F-1–mediated
gene activation required for S-phase progression. We show
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Figure 5. Telomere signals in atherosclerotic plaques. A, SMA
(green) and DAPI stain (blue) identifying VSMCs in fibrous cap of
plaque (arrows). B, qTELI-FISH of same section in a, demonstrating loss of telomere signals in SMA-positive cells (white
arrows), compared with SMA-negative cells (white arrowheads).
C, Mean qTELI-FISH signals (⫾SD) (n⫽8 to 10) of intimal and
medial VSMCs in the same section of separate human plaques
of different American Heart Association Grade.
that impaired pRB phosphorylation induces plaque VSMC
senescence, as pRB phosphorylation (by ectopic CDK4 and
cyclin D1 expression) is sufficient to induce plaque VSMC
cell cycle transition. We show that p16 (but not p21)
expression occurs in replicative senescence of normal
VSMCs in culture, whereas p21 (but not p16) occurs after
oxidative stress. Our data therefore suggest that plaque
VSMC senescence is attributable to a combination of (oxidative) DNA damage accelerating replicative senescence,
thereby activating both p21 and p16 and inducing pRB
hypophosphorylation (Figure 8).
To determine whether atherosclerosis is accompanied by
telomere shortening in vivo, we examined matched normal
arteries and plaques from the same patient. Telomeres in
whole human plaques were markedly shorter than in matched
normal vessels, by approximately 1 kb in advanced lesions. If
only VSMCs have shortened telomeres, this represents at
least 7 to 13 additional CPDs. Although ECs25 and peripheral
leukocytes27 may show telomere attrition in atherosclerosis,
this calculation may underestimate VSMC telomere loss, as
non-VSMCs retained telomere signals even in advanced
Atherosclerosis and Telomere Shortening
161
Figure 6. Telomerase prevents VSMC senescence in vitro. A
through C, Double-labeling for SMA (red) and telomerase (blue)
in fibrous cap of plaque (A and B) or media (C). A single
telomerase-positive VSMC is indicated (arrow), although most
plaque and medial VSMCs are negative. D, Double-labeling for
macrophages (red) and telomerase (blue) indicates telomerase
expression in necrotic core macrophages underlying the plaque
(purple). Scale bar represents 200 ␮m in A; 100 ␮m in C and D;
50 ␮m in B, which represents a high-power image of area outlined in A. E, Western blot for TERT and G1/S regulator expression in VSMCs expressing either hTERT (T) or control vector (C)
or noninfected (U) at senescence of control cells. F, Southern
blot for TRF length. G, Graph showing change in TRF length
(⌬TL) in hTERT or vector VSMCs with increasing CPD (n⫽3).
lesions (Figure 5). Intimal VSMC telomere signals were
inversely correlated with disease status, with even early
lesions associated with significant telomere shortening.
Previous studies have found that age-dependent telomere
attrition is higher in both intima and media of the distal versus
proximal abdominal aorta,28 the intima of iliac artery versus
internal thoracic artery,29 or endothelial cells from plaque
versus normal arteries.30 However, the first 2 studies did not
identify which cells showed telomere loss, and VSMCs have
not been studied previously. In addition, any relationship
between telomere length and atherosclerosis was lost after
adjustment for age.28 In contrast, using qTELI-FISH to
provide the first detailed mapping of telomere signals in
atherosclerosis, we show that plaque VSMCs within the
fibrous cap undergo marked telomere loss compared with
medial VSMCs of the same lesion. Telomere signals are
barely detectable in advanced plaque VSMCs, whereas nonVSMCs demonstrate robust signals. Importantly, it is relative
rather than absolute telomere length that correlates with
plaque development. Plaques at the same stage of development show similar telomere loss compared with matched
normal vessels, despite marked heterogeneity of telomere
length in normal vessels or plaques between individuals of
the same chronological age.
The telomere loss in plaque VSMCs most likely represents
additional replications involved in lesion development.
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Figure 7. Effects of oxidative stress on VSMCs. A and B, Immunohistochemistry for 8-oxo-G (brown) and SMA (blue), demonstrating 8-oxo-G–positive VSMCs in plaque fibrous caps
(arrows). C and D, Immunohistochemistry for 8-oxo-G (brown)
and CD68 (blue), demonstrating 8-oxo-G–positive macrophages
in plaque necrotic cores (arrows). B and D, High-power magnifications of areas outlined in A and C, respectively. Scale bar
represents 200 ␮m in A and C; 50 ␮m in B and D. E, Southern
blot for TRF length in VSMCs before treatment (C) or after 1
hour of treatment with t-BHP (80 mmol/L) or control with
increasing CPD (indicated). Mean TRF lengths are as marked. F,
Graph showing change in telomere length (⌬TL) with CPD for
increasing t-BHP concentrations (n⫽2). G, Western blot for G1/S
regulator expression in control VSMCs (C) or treated with
80 mmol/L t-BHP. H, Telomerase activity (arbitrary units) in normal VSMCs with increasing doses of t-BHP. Means⫾SEM
(n⫽3).
VSMCs of fibrous caps in plaques are monoclonal in origin,31,32 a phenomenon not seen in ECs or inflammatory
cells.32 Clonal expansion may represent proliferation in a
developmentally determined “patch” of intimal cells, selective recruitment of VSMCs of a specific phenotype, or
genetic alteration in a small number of cells. Although our
data cannot determine which of these possibilities is occurring, the 7 to 13 CPD required for 1-kb telomere loss favors
selective expansion of a small number of cells. The telomere
loss in intimal VSMCs of early (type I and type II) lesions,
which demonstrate the highest rates of cell proliferation,
suggests that such selection occurs early in atherosclerosis.
Apoptosis of VSMCs, which is also a feature of advanced
atherosclerosis, could also promote telomere shortening by
reducing the number of cells left in the cap that can replicate.
Figure 8. Model of VSMC senescence in atherosclerosis.
Plaque VSMC senescence occurs by a combination of replicative senescence and SIPS. Replication in VSMCs (which have
low telomerase activity) induces telomere dysfunction and
expression of p16. ROS induce DNA damage in both nuclear
and mitochondrial DNA. DNA damage activates a damage
response pathway involving activation of p53, with subsequent
p21 transcription. p16 and p21 induce pRB hypophosphorylation and senescence. ROS also accelerate telomere loss during
replication, in part, by damage to telomeric DNA and reduction
in telomerase activity.
We find that oxidative stress accelerates telomere shortening in vitro, suggesting that oxidative DNA damage in
plaques in vivo may accelerate telomere shortening with each
replication. ROS (particularly hydroxyl radicals) damage
DNA, forming a series of adducts including 8-oxo-G; senescent cells also produce high levels of ROS and contain higher
levels of oxidatively damaged DNA.33 We find that 8-oxo-G
is expressed highly in both macrophages and VSMCs in
plaques, and oxidative stress is increased in plaque and
senescent VSMCs. Whereas acute oxidative stress induces
growth arrest, chronic stress accelerates senescence and
increases telomere loss per CPD. There are numerous mechanisms by which ROS can promote telomere damage or loss.
Increased telomere loss per division can occur in individual
cells because of a telomere-specific deficiency in base excision repair, leading to preferential accumulation of ROSinduced single-stranded DNA breaks34 preventing replication
of distal telomeres when cells divide. ROS also promote the
nuclear export of telomerase in ECs, reducing telomerase
activity and promoting senescence.35,36 Although this mechanism has not been shown in VSMCs, we find that ROS
inhibit telomerase activity in VSMCs in addition to causing
Matthews et al
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DNA damage. Alternatively, repeated stress increases the
proportion of cells undergoing growth arrest,21 so that only a
subset of VSMCs undergoes replication in vivo.
Our data do not exclude the possibility that changes in
telomere structure or function or telomerase activity also
contribute to atherosclerosis. Similar to previous studies,37,38
we find that ectopic hTERT expression prolongs the lifespan
of normal VSMCs. Furthermore, telomerase also rescues
early plaque VSMC senescence, reversing their typical G1/S
regulator pattern. However, in contrast to earlier studies,38
telomerase maintained VSMC proliferation despite no measurable effect on TRF length, allowing cells to proliferate
despite very short telomeres. Low levels of exogenous
hTERT can increase proliferative lifespan and reduce chromosome fusions in fibroblasts while telomere length continues to shorten,39,40 possibly via actions on chromatin maintenance and DNA damage responses.41 Telomerase can also
promote proliferation independent of its DNA synthesis
capacity.42
Currently, the role of senescence in atherosclerosis is
controversial. Senescence-accelerated mice develop increased atherosclerosis, suggesting that cellular senescence
ultimately promotes atherogenesis.43 In contrast, apoE⫺/⫺
mice also lacking TERT show less aortic atherosclerosis than
control apoE⫺/⫺ mice, suggesting that TERT deficiency protects against atherogenesis.44 Although these studies demonstrate that accelerated senescence can affect atherogenesis,
the role of senescence in established plaques or specifically in
VSMCs was not examined. Both studies resulted in early
senescence of all cells, and the effects of TERT deficiency
were mostly manifest in inflammatory cells.43,44
In humans, VSMC senescence may exert profound effects
on atherogenesis and stability of advanced plaques. Most
patients with Hutchison–Gilford progeria syndrome (HGPS),
an accelerated aging syndrome, die of atherosclerosis.45
VSMC depletion is a major feature in progeria, and normal
aging,46 and likely represents replicative senescence, telomere shortening, and decreased capacity for repair, as HGPS
VSMCs are more susceptible to hemodynamic and ischemic
stress.46 Replicative senescence and ongoing apoptosis in the
fibrous cap would result in cap thinning, frequently seen in
advanced human lesions, predisposing to plaque rupture.
Senescent cells may also promote plaque instability by
overexpressing proteins such as adhesion molecules,47 regulators of hemostasis,48 and matrix metalloproteinases.49
In conclusion, we demonstrate that human atherosclerosis
is characterized by VSMC senescence and marked telomere
shortening and that telomerase expression can delay senescence. Oxidative DNA damage is seen in vivo, and chronic
oxidative stress accelerates telomere loss and VSMC senescence. This suggests that human VSMCs undergo a replicative senescence that is accelerated by oxidative stress. Prevention of cellular senescence may be a novel therapeutic
target in atherosclerosis.
Sources of Funding
This study was supported by British Heart Foundation grants
PG98/009 and 04/107/17742 and a British Heart Foundation PhD
studentship (to S.S.).
Atherosclerosis and Telomere Shortening
163
Disclosures
None.
References
1. Lutgens E, de Muinck ED, Kitslaar PJ, Tordoir JH, Wellens HJ, Daemen
MJ. Biphasic pattern of cell turnover characterizes the progression from
fatty streaks to ruptured human atherosclerotic plaques. Cardiovasc
Res. 1999;41:473– 479.
2. Hayflick L. The limited in vitro lifetime of human cell strains. Exp Cell
Res. 1965;37:614 – 636.
3. Campisi J. The biology of replicative senescence. Eur J Cancer. 1997;
33:703–709.
4. Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of
human fibroblasts. Nature. 1990;345:458 – 460.
5. Blackburn E. Switching and signaling at the telomere. Cell. 2001;106:
661– 673.
6. Sherr CJ, DePinho RA. Cellular senescence: mitotic clock or culture
shock? Cell. 2000;102:407– 410.
7. von Zglinicki T, Saretzki G, Docke W, Lotze C. Mild hyperoxia shortens
telomeres and inhibits proliferation of fibroblasts: a model for
senescence? Exp Cell Res. 1995;220:186 –193.
8. Chen QM, Prowse KR, Tu VC, Purdom S, Linskens MH. Uncoupling the
senescent phenotype from telomere shortening in hydrogen peroxidetreated fibroblasts. Exp Cell Res. 2001;265:294 –303.
9. Hathaway CA, Heistad DD, Piegors DJ, Miller FJ Jr. Regression of
atherosclerosis in monkeys reduces vascular superoxide levels. Circ Res.
2002;90:277–283.
10. Campisi J. Senescent cells, tumor suppression, and organismal aging:
good citizens, bad neighbors. Cell. 2005;120:513–522.
11. d’Adda di Fagagna F, Reaper PM, Clay-Farrace L, Fiegler H, Carr P, Von
Zglinicki T, Saretzki G, Carter NP, Jackson SP. A DNA damage
checkpoint response in telomere-initiated senescence. Nature. 2003;426:
194 –198.
12. Beausejour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P,
Campisi J. Reversal of human cellular senescence: roles of the p53 and
p16 pathways. EMBO J. 2003;22:4212– 4222.
13. Narita M, Nunez S, Heard E, Lin AW, Hearn SA, Spector DL, Hannon
GJ, Lowe SW. Rb-mediated heterochromatin formation and silencing of
E2F target genes during cellular senescence. Cell. 2003;113:703–716.
14. O’Sullivan M, Scott S, McCarthy N, Shapiro LM, Kirkpatrick PJ, Bennett
MR. Differential cyclin E. Expression in human in stent stenosis vascular
smooth muscle cells identifies targets for selective anti-restenotic therapy.
Cardiovasc Res. 2003;60:673– 683.
15. Meeker AK, Gage WR, Hicks JL, Simon I, Coffman JR, Platz EA, March
GE, De Marzo AM. Telomere length assessment in human archival
tissues: combined telomere fluorescence in situ hybridization and immunostaining. Am J Pathol. 2002;160:1259 –1268.
16. Garton KJ, Ferri N, Raines EW. Efficient expression of exogenous genes
in primary vascular cells using IRES-based retroviral vectors. Biotechniques. 2002;32:830, 832, 834 passim.
17. Kurz DJ, Decary S, Hong Y, Erusalimsky JD. Senescence-associated
beta-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci. 2000;113:
3613–3622.
18. Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W Jr, Rosenfeld
ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW. A definition
of initial, fatty streak, and intermediate lesions of atherosclerosis. A report
from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1994;89:2462–2478.
19. Hou M, Xu D, Bjorkholm M, Gruber A. Real-time quantitative telomeric
repeat amplification protocol assay for the detection of telomerase
activity. Clin Chem. 2001;47:519 –524.
20. Beckman KB, Ames BN. Oxidative decay of DNA. J Biol Chem. 1997;
272:19633–19636.
21. Toussaint O, Houbion A, Remacle J. Aging as a multi-step process
characterized by a lowering of entropy production leading the cell to a
sequence of defined stages. II. Testing some predictions on aging human
fibroblasts in culture. Mech Ageing Dev. 1992;65:65– 83.
22. Latour I, Demoulin JB, Buc-Calderon P. Oxidative DNA damage by
t-butyl hydroperoxide causes DNA single strand breaks which is not
linked to cell lysis. A mechanistic study in freshly isolated rat hepatocytes. FEBS Lett. 1995;373:299 –302.
23. Aherne SA, O’Brien NM. Mechanism of protection by the flavonoids,
quercetin and rutin, against tert-butylhydroperoxide- and menadione-
164
24.
25.
26.
27.
28.
29.
30.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
31.
32.
33.
34.
35.
36.
Circulation Research
July 21, 2006
induced DNA single strand breaks in Caco-2 cells. Free Radic Biol Med.
2000;29:507–514.
Vasile E, Tomita Y, Brown LF, Kocher O, Dvorak HF. Differential
expression of thymosin beta-10 by early passage and senescent vascular
endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis. FASEB J. 2001;15:
458 – 466.
Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I.
Endothelial cell senescence in human atherosclerosis: role of telomere in
endothelial dysfunction. Circulation. 2002;105:1541–1544.
Minamino T, Yoshida T, Tateno K, Miyauchi H, Zou Y, Toko H, Komuro
I. Ras induces vascular smooth muscle cell senescence and inflammation
in human atherosclerosis. Circulation. 2003;108:2264 –2269.
Samani NJ, Boultby R, Butler R, Thompson JR, Goodall AH. Telomere
shortening in atherosclerosis. Lancet. 2001;358:472– 473.
Okuda K, Khan MY, Skurnick J, Kimura M, Aviv H, Aviv A. Telomere
attrition of the human abdominal aorta: relationships with age and atherosclerosis. Atherosclerosis. 2000;152:391–398.
Chang E, Harley CB. Telomere length and replicative aging in human
vascular tissues. Proc Natl Acad Sci U S A. 1995;92:11190 –11194.
Ogami M, Ikura Y, Ohsawa M, Matsuo T, Kayo S, Yoshimi N, Hai E,
Shirai N, Ehara S, Komatsu R, Naruko T, Ueda M. Telomere shortening
in human coronary artery diseases. Arterioscler Thromb Vasc Biol. 2004;
24:546 –550.
Benditt EP, Benditt JM. Evidence for a monoclonal origin of human
atherosclerotic plaques. Proc Natl Acad Sci U S A. 1973;70:1753–1756.
Murry CE, Gipaya CT, Bartosek T, Benditt EP, Schwartz SM. Monoclonality of smooth muscle cells in human atherosclerosis. Am J Pathol.
1997;151:697–705.
Chen Q, Fischer A, Reagan JD, Yan LJ, Ames BN. Oxidative DNA
damage and senescence of human diploid fibroblast cells. Proc Natl Acad
Sci U S A. 1995;92:4337– 4341.
Petersen S, Saretzki G, von Zglinicki T. Preferential accumulation of
single-stranded regions in telomeres of human fibroblasts. Exp Cell Res.
1998;239:152–160.
Haendeler J, Hoffmann J, Brandes R, Zeiher A, Dimmeler S. Hydrogen
peroxide triggers nuclear export of telomerase reverse transcriptase via
Src kinase family dependent phosphorylation of tyrosine 707. Mol Cell
Biol. 2003;23:4598 – 4610.
Haendeler J, Hoffmann J, Diehl F, Vasa M, Spyridopoulos I, Zeiher A,
Dimmeler S. Antioxidants inhibit nuclear export of telomerase reverse
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
transcriptase and delay replicative senescence of endothelial cells. Circ
Res. 2004;94:768 –775.
McKee JA, Banik SS, Boyer MJ, Hamad NM, Lawson JH, Niklason LE,
Counter CM. Human arteries engineered in vitro. EMBO Rep. 2003;4:
633– 638.
Minamino T, Mitsialis SA, Kourembanas S. Hypoxia extends the life
span of vascular smooth muscle cells through telomerase activation. Mol
Cell Biol. 2001;21:3336 –3342.
Zhu J, Wang H, Bishop JM, Blackburn EH. Telomerase extends the
lifespan of virus-transformed human cells without net telomere
lengthening. Proc Natl Acad Sci U S A. 1999;96:3723–3728.
Masutomi K, Yu EY, Khurts S, Ben-Porath I, Currier JL, Metz GB,
Brooks MW, Kaneko S, Murakami S, DeCaprio JA, Weinberg RA,
Stewart SA, Hahn WC. Telomerase maintains telomere structure in
normal human cells. Cell. 2003;114:241–253.
Masutomi K, Possemato R, Wong JM, Currier JL, Tothova Z, Manola JB,
Ganesan S, Lansdorp PM, Collins K, Hahn WC. The telomerase reverse
transcriptase regulates chromatin state and DNA damage responses. Proc
Natl Acad Sci U S A. 2005;102:8222– 8227.
Sarin KY, Cheung P, Gilison D, Lee E, Tennen RI, Wang E, Artandi MK,
Oro AE, Artandi SE. Conditional telomerase induction causes proliferation of hair follicle stem cells. Nature. 2005;436:1048 –1052.
Fenton M, Huang HL, Hong Y, Hawe E, Kurz DJ, Erusalimsky JD. Early
atherogenesis in senescence-accelerated mice. Exp Gerontol. 2004;39:
115–122.
Poch E, Carbonell P, Franco S, Diez-Juan A, Blasco MA, Andres V.
Short telomeres protect from diet-induced atherosclerosis in apolipoprotein E-null mice. FASEB J. 2004;18:418 – 420.
Brown WT. Progeria: a human-disease model of accelerated aging. Am J
Clin Nutr. 1992;55:1222S–1224S.
Stehbens WE, Wakefield SJ, Gilbert-Barness E, Olson RE, Ackerman
J. Histological and ultrastructural features of atherosclerosis in progeria.
Cardiovasc Pathol. 1999;8:29 –39.
Saito H, Papaconstantinou J. Age-associated differences in cardiovascular
inflammatory gene induction during endotoxic stress. J Biol Chem. 2001;
276:29307–29312.
Yamamoto K, Takeshita K, Shimokawa T, Yi H, Isobe K, Loskutoff DJ,
Saito H. Plasminogen activator inhibitor-1 is a major stress-regulated
gene: implications for stress-induced thrombosis in aged individuals.
Proc Natl Acad Sci U S A. 2002;99:890 – 895.
Zeng G, McCue HM, Mastrangelo L, Millis AJ. Endogenous TGF-beta
activity is modified during cellular aging: effects on metalloproteinase
and TIMP-1 expression. Exp Cell Res. 1996;228:271–276.
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Vascular Smooth Muscle Cells Undergo Telomere-Based Senescence in Human
Atherosclerosis: Effects of Telomerase and Oxidative Stress
Charles Matthews, Isabelle Gorenne, Stephen Scott, Nicola Figg, Peter Kirkpatrick, Andrew
Ritchie, Martin Goddard and Martin Bennett
Circ Res. 2006;99:156-164; originally published online June 22, 2006;
doi: 10.1161/01.RES.0000233315.38086.bc
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Online Supplement
C Matthews, et al.
Atherosclerosis and telomere shortening
Methods
Immunohistochemistry
Formalin-fixed, paraffin embedded sections were de-waxed in Histoclear and
hydrated through graded methanol solutions to water. Primary antibodies were mouse
anti-α-smooth muscle actin antibody (1:50, DAKO), anti-CD68 antibody (1:150,
DAKO), anti-CD3 antibody (Biocarta), anti-p21 (1:600, Pharmingen), -p16 and -p27
(1:150, Pharmingen), -Telomerase (1:200, Santa Cruz) or -8-oxo-G (1:5000, Japan
Institute of Aging) or isotype-matched control. Sections were washed twice in PBS,
incubated for 30 minutes in horse anti-mouse biotin-conjugated antibody, washed in
PBS and incubated in Avidin-Biotin complex, washed in PBS and incubated with
diaminobenzidine (DAB).
For quantification of VSMC expression of SAβG, p21 and p16 in Figure 1, and
qTELI-FISH signal in Figure 5, the fibrous cap was defined as the distinct layer of
connective tissue completely covering the lipid core on the luminal side, usually
consisting of smooth muscle cells in a collagenous-proteoglycan matrix, with varying
degrees of infiltration by macrophages and lymphocytes.
Immunoblotting/immunoprecipitation
Primary antibodies used were: mouse anti-human CDK2, rat anti-CDK4, anti-human
cyclin D1-3, anti-human cyclin E, anti-human E2F-1, anti-human E2F-2, anti-human
E2F-3, anti-human p16INK4, anti-p21, anti-p27, anti-human pRB, anti-human p53,
anti-human cyclin A (all from Pharmingen), and rabbit anti-human TERT (Rockland
Inc., Gilbertsville, PA).
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C Matthews, et al.
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Infection of human plaque VSMCs with adenoviruses encoding CDK4 and cyclin D1.
Plaque-derived VSMCs were cultured in slide-flasks. When cells were approximately
70% confluent they were infected with either Rad-empty adenovirus (RAD 60
1600pfu/cell) or a combination of both nuclear localised cyclin D1 adenovirus
(800pfu/cell) and CDK4 adenovirus (800pfu/cell) 1 (generous gifts from Dr Ikeda,
Tokyo University). Parallel infection with a β-galactosidase-expressing virus
(800pfu/cell) demonstrated that >98% of cells expressed the gene product after
infection. After incubation of cells with adenovirus for 24 hours the medium was
replaced with fresh HEPES-buffered medium and the cells observed by time lapse
videomicroscopy for 72 hours. Protein lysates were taken from these filmed cells and
blotted for expression of cyclinD1, CDK4, pRB, E2F-1and cyclin A as above.
To examine the ability of CDK4 and cyclin D1 to promote cell cycle entry, plaquederived VSMCs were cultured in 4-well chamber slides, and cells infected with Rad
60 or both cyclin D1 and CDK4 adenoviruses. After 2 hours 10µM BrDU was added
and cells incubated for a further 22 hours. Cells were then stained for BrDU
incorporation by immunocytochemistry.
Measurement of telomere length
High-molecular weight DNA (> 23 Kbp) was isolated from tissues and cultured cells
using the DNeasy Kit (Qiagen, UK). DNA quality was assessed by electrophoresis
prior to restriction digestion. 4-5 µg DNA was then digested with HinFI and RsaI,
separated on a 0.5% TAE agarose gel overnight and transferred to Hybond N+
(Amersham, UK) nylon membrane. Southern blots underwent two rounds of
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C Matthews, et al.
Atherosclerosis and telomere shortening
hybridization, the first performed at 650C with probes specific to DNA ladders, the
second at 370C with a telomere specific oligonucleotide probe (5’-CCCTAA-3’)
labelled with
3
32
P using PNK (Promega, UK). Radioactive blots and gels were
analysed and quantified by phosphorimaging (Packard Cyclone phosphorimager and
Optiquant software, Packard Biosciences, USA). Mean telomere length was
calculated using the formula: L=ε(ODi)/ε(ODi/Li), where ODi is the integrated signal
intensity and Li is DNA length at position i.
Telomere/Immunostaining-FISH (TELI-FISH)
Briefly, 7µM tissue sections were deparaffinised in xylene, hydrated in graded
ethanol and washed in deionised water containing 0.1% Tween-20. Slides were
microwaved in citrate buffer, washed in water and PBST for 5 minutes each, rinsed in
deionised water, dehydrated in 95% ethanol and air-dried. 35-40µl Cy3-labelled
telomere-specific peptide nucleic acid (PNA, Applied Biosystems, UK). 0.3 µg/ml
PNA in 75% formamide, 10mmol/l Tris (pH 7.5), 0.5% Boehringer Mannheim
blocking reagent was applied to each slide, the section cover-slipped, slides denatured
by incubation at 850C for 7.5 minutes. Hybridisation was performed in the dark at
room temperature for 2h. Coverslips were removed and slides washed twice in PNA
wash solution (70 % formamide, 10mmol/l Tris, pH 7.5, 0.1 % BSA), three times in
TBS (plus 0.1 % Tween-20), rinsed in PBST and blocked in 5% goat serum in PBS,
1% BSA for 30 min at room temperature. Slides were washed in PBS, incubated in
primary antibody (mouse anti α-sm-actin, 1:100, DAKO) for 1h at room temperature,
rinsed in PBST followed by application of secondary antibody, Alexa Fluor 488
(Molecular Probes, UK) diluted 1:1000 in PBS for 30min. Slides were washed in
3
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C Matthews, et al.
Atherosclerosis and telomere shortening
PBST, once in deionised water, and mounted in Vectashield Mounting Medium
containing DAPI (Vector Laboratories, USA).
QTELI-FISH signals were quantified using computer controlled fluorescence
acquisition. Sampling areas within the media were used to determine integration
times, and background and threshold levels were equalised between slides. Gain and
exposure times were established within a linear range of signal intensity. Typically,
integration times of 500–2000 ms for Cy3 signals, 2–5 ms for DAPI nuclear staining
and 100-500 ms for Alexa Fluor conjugated anti-smooth muscle actin were used. For
each slide all Cy3 signals utilized the same integration and exposure times. For
normal tissue sections both medial and intimal nuclei were chosen randomly as long
as the Cy3 signals were in sharp focus. In diseased sections, the nuclei within the
media were chosen randomly, but within the thickened intima nuclei with the
brightest signals were selected. This was necessary as most nuclei in the diseased
intima contained few and faint Cy3 signals hardly observable over the background.
For each nuclear image, fluorescent signals were measured using ImageJ
(http://rsb.info.nih.gov/ij/). Briefly, for each telomeric signal within the area of the
nucleus (as determined by DAPI staining) the mean Cy3 signal and the area of the
Cy3 spot was determined. Similarly background fluorescence was determined for an
area of the nucleus containing no noticeable Cy3 signal. Following background
fluorescence subtraction, the sum of the mean fluorescence and the area of the Cy3
signal represented the total fluorescence for each given telomere. The individual
telomere signals for the whole nucleus were summated and then divided by the total
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Atherosclerosis and telomere shortening
area of the nucleus. This corrected for differences in the cutting plane of each nucleus
and so different nuclei on the same slide could be directly compared.
Senescence associated β-Galactosidase staining
Cells were washed twice in PBS, fixed for 10 minutes at 37˚C in 4%
paraformaldehyde, washed twice in PBS and incubated for 24h at 37˚C in SAβG
staining solution (1mg/ml X-Gal, 5mmol/l potassium ferrocyanide, 5mmol/l
potassium ferricyanide, 150mmol/l NaCl, 2mmol/l MgCl2, 40mmol/l citrate (titrated
to pH 6.0 with NaH2PO4)). Slides were washed in PBS, water and mounted in DAPI
fluorescence mounting medium (DAKO). Tissue specimens were washed twice in
PBS, incubated for 24h at 37˚C in SAβG staining solution, washed twice in PBS and
fixed in 10% formalin for 24h.
Retrovirus-based expression of hTERT in human VSMCs
Human TERT cDNA was cloned into the retroviral vector pBMN IRES puro 2 and
transfected into Bosc23 ecotropic packaging cells using Superfect (Qiagen). Virus
was recovered 48 hours later and used to infect GPenv Am12 amphotropic packaging
cells for 3h in medium containing 8µg/ml polybrene. Virus was harvested at 48 hours
and used to infect human VSMCs for 16 hours in medium containing 8µg/ml
polybrene. VSMCs were selected in medium containing 0.5µg/ml puromycin,
resistant cells pooled, and cultures maintained in puromycin-containing media
thereafter.
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RTQ-TRAP assay for telomerase activity
Telomerase activity was measured by RTQ-TRAP assay
3, 4
using TS (5’-
AATCCGTCGAGCAGAGTT-3’) and ACX (5’-GCGCGG(CTTACC)3CTAACC-3’)
primers3. Cell pellets were washed in ice-cold PBS, resuspended in lysis buffer
(TRAPeze 1x CHAPS lysis buffer, Chemicon Int., Temecula, CA) containing 1U/µl
of ribonuclease inhibitor (RNasin, Promega Corp., Madison WI) and incubated for 30
min at 4oC. Lysates were centrifuged at 12,000g for 20min at 4oC and supernatants
collected. Protein content of supernatants was measured using the Dc Protein Assay
(BioRad). Total protein per reaction (2-200ng) was in the linear range of the assay.
Extension of TS primers was performed at 30oC for 30 min. PCR amplification was
performed as previously described
4
using the HotStarTaq DNA polymerase
(0.62U/reaction, Qiagen GmBH, Hilden, Germany) and a Rotor Gene 3000 real time
thermocycler (Corbett Research, Australia). Negative controls were generated by
heating cell lysates at 80oC for 10 min prior to extension. Dilutions of Jurkat cell
lysates were used to generate standard curves. Telomerase activity in VSMCs lysates
was extrapolated from the standard curve. Telomerase activity from control and BHPtreated VSMCs was expressed as percent of values in untreated samples. Negative
controls were generated by heating cell lysates at 80oC for 10 min prior to extension,
in order to inactivate telomerase. Residual signal in heated lysates was less than 0.1%
of telomerase activity generated signal.
Flow cytometric analysis for determination of ROS and mitochondrial mass
ROS content in normal VSMCs (passage 2-5), SAβG-positive replicatively senescent
cells (passage 12-16), plaque VSMCs (passage 2-4) or t-BHP treated VSMCS (40
µM, 1 h), was measured in triplicates using the probe 2’,7’-dichlorofluorescein
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C Matthews, et al.
Atherosclerosis and telomere shortening
diacetate (H2-DCFDA; Molecular Probes). The cells were incubated for 1 h with 2
µM H2-DCFDA, rinsed in PBS and trypsinized for flow cytometric analysis.
Alternatively, cells were rinsed in PBS and photographed under a fluorescence
microscope. The fluorescent dye 10-n-Nonyl-Acridine Orange (NAO; Molecular
Probes) was used to measure mitochondrial mass as previously described5.
Autofluorescence values from each cell group (no fluorophore control) were
subtracted from total fluorescence.
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C Matthews, et al.
Atherosclerosis and telomere shortening
References
1.
Tamamori-Adachi M, Ito H, Sumrejkanchanakij P, Adachi S, Hiroe M,
Shimizu M, Kawauchi J, Sunamori M, Marumo F, Kitajima S, Ikeda MA.
Critical role of cyclin D1 nuclear import in cardiomyocyte proliferation. Circ
Res. 2003;92:e12-19.
2.
Garton KJ, Ferri N, Raines EW. Efficient expression of exogenous genes in
primary vascular cells using IRES-based retroviral vectors. Biotechniques.
2002;32:830-834.
3.
Kim NW, Wu F. Advances in quantification and characterization of
telomerase activity by the telomeric repeat amplification protocol (TRAP).
Nucleic Acids Res. 1997;25:2595-2597.
4.
Hou M, Xu D, Bjorkholm M, Gruber A. Real-time quantitative telomeric
repeat amplification protocol assay for the detection of telomerase activity.
Clin Chem. 2001;47:519-524.
5.
Haendeler J, Hoffmann J, Diehl F, Vasa M, Spyridopoulos I, Zeiher A,
Dimmeler S. Antioxidants inhibit nuclear export of telomerase reverse
transcriptase and delay replicative senescence of endothelial cells. Circ Res.
2004;94:768-775.
8
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Atherosclerosis and telomere shortening
Legends to Online Figures
Online Figure 1
Identification of plaque-derived cells as VSMCs by immunocytochemical
staining.
Plaque derived VSMCs were incubated with antibodies to α-smooth muscle actin (A),
calponin (B), smooth muscle myosin (C), vimentin (D), desmin (E), von Willebrand
factor (F), irrelevant antibody (G) and no primary antibody (H), prior to labelling with
FITC-conjugated secondary antibody. DAPI was used to label nuclei.
Online Figure 2
t-BHP and replicative arrest induces oxidant stress in VSMCs
ROS levels (A) or mitochondrial mass (B) in low passage normal VSMCs (CTR),
plaque VSMCs (PLQ) and normal VSMCs at replicative senescence (SEN).
Fluorescence levels were analysed by flow cytometry using the redox sensitive dye
H2-DCFDA
(ROS)
or
10-n-nonyl-Acridine
Orange
(mitochondrial
mass).
Fluorescence values are expressed as arbitrary units (AU) and results are means±SEM
of 4 independent experiments. C) ROS levels in normal VSMCs either untreated
(CTR) or treated with 40 µM BHP analysed by fluorescence microscopy (left panels)
or quantified by flow cytometry (right panel). Fluorescence values are expressed as
arbitrary units (AU). Results are means ± SEM of at least 4 independent experiments.
*p<0.05 vs control (CTR), **p<0.01 vs control (CTR).
9
A
B
C
D
E
F
G
H
Online Figure 1
A
**
B 1000
**
900
800
*
120
100
NAO fluorescence (AU)
DCFDA fluorescence (AU)
140
*
80
60
40
20
700
600
500
400
300
200
100
0
0
CTR
PLQ
SEN
CTR
PLQ
SEN
**
CTR
BHP
DCFDA fluorescence (AU)
C
240
220
200
180
160
140
120
100
80
60
40
20
0
CTR
Online Figure 2
BHP