Vascular Smooth Muscle Cells Undergo Telomere-Based Senescence in Human Atherosclerosis
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Charles Matthews, Isabelle Gorenne, Step
参见附件。
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.
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 [SAG] 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 SAG 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.
Key Words: atherosclerosis smooth muscle aging
Introduction
Human 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 (SAG).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 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.
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 (TELI-FISH) was performed using a modification of previous methods.15 (See the online data supplement.)
SAG Staining and Immunohistochemistry
See the online data supplement.
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).
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
We first examined expression of senescence markers in plaque VSMCs in vivo including SAG, coexpression of the cdkis p16/p21/p27 with SAG, and cell-lineage markers in human carotid atherectomies (Figure 1). Among fibrous cap VSMCs, 18%±2.6 (mean±SEM, [n=6]) were SAG positive, but SAG-positive VSMCs were not found in normal vessels (n=6). Macrophages showed faint SAG expression throughout plaques, most likely reflecting their high lysosomal content,17 but <5% of lymphocytes were SAG positive. More than 70% SAG-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 SAG, 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) SAG 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 passage3 compared with passage8 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 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 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 telomeresxfluorescence intensity of each telomere) of medial VSMCs did not change significantly with increasing disease 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
Although telomerase enzymatic activity requires TERT, TERC, and other proteins, hTERT expression is a rate-limiting 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 vector-VSMCs 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 (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 (t-BHP), 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 SAG 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. SAG-positive VSMCs were readily detected within advanced human plaques, colocalized with p16 and p21, confirming a senescence phenotype in vivo. Previous studies have demonstrated SAG activity in human plaques, in both ECs and VSMCs.24–26 However, although SAG 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 (SAG, 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 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 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 non-VSMCs 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. 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.
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 ROS-induced 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 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.
Acknowledgments
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.).
Disclosures
None.
Footnotes
Both authors contributed equally to this study.
Original received March 17, 2006; resubmission received May 17, 2006; revised resubmission received June 7, 2006; accepted June 9, 2006.
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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.
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 [SAG] 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 SAG 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.
Key Words: atherosclerosis smooth muscle aging
Introduction
Human 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 (SAG).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 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.
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 (TELI-FISH) was performed using a modification of previous methods.15 (See the online data supplement.)
SAG Staining and Immunohistochemistry
See the online data supplement.
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).
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
We first examined expression of senescence markers in plaque VSMCs in vivo including SAG, coexpression of the cdkis p16/p21/p27 with SAG, and cell-lineage markers in human carotid atherectomies (Figure 1). Among fibrous cap VSMCs, 18%±2.6 (mean±SEM, [n=6]) were SAG positive, but SAG-positive VSMCs were not found in normal vessels (n=6). Macrophages showed faint SAG expression throughout plaques, most likely reflecting their high lysosomal content,17 but <5% of lymphocytes were SAG positive. More than 70% SAG-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 SAG, 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) SAG 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 passage3 compared with passage8 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 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 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 telomeresxfluorescence intensity of each telomere) of medial VSMCs did not change significantly with increasing disease 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
Although telomerase enzymatic activity requires TERT, TERC, and other proteins, hTERT expression is a rate-limiting 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 vector-VSMCs 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 (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 (t-BHP), 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 SAG 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. SAG-positive VSMCs were readily detected within advanced human plaques, colocalized with p16 and p21, confirming a senescence phenotype in vivo. Previous studies have demonstrated SAG activity in human plaques, in both ECs and VSMCs.24–26 However, although SAG 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 (SAG, 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 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 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 non-VSMCs 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. 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.
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 ROS-induced 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 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.
Acknowledgments
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.).
Disclosures
None.
Footnotes
Both authors contributed equally to this study.
Original received March 17, 2006; resubmission received May 17, 2006; revised resubmission received June 7, 2006; accepted June 9, 2006.
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