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Aging, Telomeres and Atherosclerosis
María Dolores Edo and Vicente Andrés1
Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy,
Instituto de Biomedicina de Valencia-CSIC, 46010 Valencia, Spain
KEY WORDS: Aging, telomeres, telomerase, atherosclerosis, hypertension, diabetes
WORD COUNT: 8123
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Corresponding autor:
Laboratory of Vascular Biology
Department of Molecular and Cellular Pathology and Therapy
Instituto de Biomedicina de Valencia
C/Jaime Roig 11, 46010 Valencia (Spain)
Tel: +34-96-3391752
FAX: +34-96-3391751
E-mail: vandres@ibv.csic.es
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SUMMARY
Although the level and pace of population aging display high geographical variability,
virtually all countries have been experiencing growth in their elderly population, particularly in
developed nations. The US Census Bureau has estimated that aged population will rise steeply
during the next decades. Because the morbidity and mortality associated with cardiovascular
disease is so profound within the elderly, understanding the physiologic and molecular aspects of
vascular aging is of up most importance. Mounting evidence suggests that the gradual shortening
of telomeres during ageing is an important factor in the pathogenesis of atherosclerosis and
associated disease. Here we review our current knowledge on the molecular mechanisms
controlling telomere length in vascular cells, the importance of telomeres in vascular pathobiology
as learnt from both human and animal studies, and potential applications of these findings to
improve therapeutic angiogenesis.
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1. Introduction
Aging is a major risk factor for atherosclerosis and associated disease. [1,2] Population
aging is decidedly increasing, particularly in developed countries. For example, data from the US
Census Bureau estimate that the population over the age of 65 years will represent 20% in US and
25% in Europe. [3] Thus, a major challenge is to unravel the molecular mechanisms contributing
to ageing to help implement novel preventive strategies and therapeutic approaches to reduce the
impact of cardiovascular disease within the elderly. Because atherosclerosis is the most frequent
cause of myocardial infarction (MI) and stroke, this review will focus on age-dependent structural
and functional alterations of the vessel wall, and on the interrelationships between telomeres and
atherosclerosis. Telomere alterations in hypertensive and diabetic patients will be also discussed,
as these individuals are at higher risk of cardiovascular disease.
During the last decades, human and animal studies have identified cardiovascular risk factors
and molecular networks that control atheroma progression, including both adaptive and innate
immune mechanisms. [1,2,4-7] Endothelial dysfunction induced by a variety of atherogenic stimuli
(i. e., hyperlipemia, hypertension, diabetes, cigarette smoking) is at the onset of atherosclerosis at
sites of predisposition to atheroma formation. Adhesion of circulating leukocytes to the damaged
endothelium leads to the formation of fatty streaks, an early form of atherosclerotic lesion enriched
in highly proliferative macrophages that avidly uptake lipoproteins to become lipid-laden foam
cells. Activated intimal immune cells abundantly secrete inflammatory mediators that promote the
proliferation and migration of the underlying medial smooth muscle cells (SMCs), thus further
contributing to atheroma progression. [1,2,8,9] Rupture or erosion of advanced atherosclerotic
lesions can lead to thrombus formation and myocardial infarction (MI) or stroke.
Whether pathological alterations in the aged vessel wall result from the cumulative damage
imposed by atherogenic stimuli, a consequence of age-dependent intrinsic vascular alterations, or a
combination of both remains controversial. Old rabbits fed a low-dose hypercholesterolemic diet
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(0.25% cholesterol) for 2 and 18 months developed more atherosclerosis than did young
counterparts. [10,11] However, old rabbits exposed for 2 months to 1% cholesterol feeding
displayed reduced aortic atherosclerosis compared with juvenile counterparts. [12] Of note in this
regard, young and old rabbits receiving for 2 months the 1% cholesterol diet averaged plasma
cholesterol levels of 1747-2488 mg/dL, [12] while animals challenged for the same period of time
with 0.25% cholesterol diet averaged 148-178 mg/dL. [11] Thus, age-dependent intrinsic
alterations in the vessel wall appear to increase the susceptibility to atherosclerosis in aged rabbits
subjected to a mildly atherogenic stimulus. In contrast, the predisposition to atherosclerosis upon
extreme dietary hypercholesterolemia is significantly higher in young rabbits. It has been recently
shown that despite showing similar alterations in lipid profile, senescence-accelerated prone mice
(SAM-P), which age at an accelerated rate, are more susceptible to early diet-induced
atherosclerosis than senescence-accelerated resistant mice (SAM-R), which age normally. [13]
Because mice in this study were fed for 17 weeks a Western-type diet containing only 0.15%
cholesterol, it would very informative to investigate the atherogenic response of SAM-P and SAMR mice under a highly atherogenic dietary regime (i. e., 1% cholesterol).
Rauscher et al. found that, compared with BM-derived progenitor cells from young
atherosclerosis-free apolipoprotein E (apoE)-null mice, cells obtained from old atherosclerotic
counterparts were much less effective at preventing atherosclerosis when transplanted into apoE
recipients. [14] The authors suggested that age-dependent progenitor cell deficits, by impairing
arterial cell repair and rejuvenation, may contribute to the development of atherosclerosis.
Likewise, it has been suggested that aging exacerbates negative remodeling and impairs
endothelial regeneration after balloon injury. [15]
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2. Structural and functional vascular alterations during ageing
A sustained effort has been taken in the Baltimore Longitudinal Study on Ageing (BLSA) in
order to characterize the effects of ageing on multiple aspects of cardiovascular structure and
function in a single population. [16] Relevant conclusions from this, as well other studies in
humans and animal models, will be summarized below. Age-associated changes of the artery wall,
such as luminal dilation, intimal and medial thickening (wall thickening), vascular stiffness and
endothelial dysfunction, are quite similar in humans, rodents and nonhuman primates. [17,18]
1.1. Wall thickening. Wall thickening and dilatation are prominent structural features of aged
large elastic arteries. [19] Postmortem studies have shown that age-dependent aortic wall
thickening consists mainly of intimal thickening, [20] and non invasive measurements in BLSA
individuals showed a thickness increment of 2-to-3-fold between 20 and 90 years of age. Arterial
wall thickening occurs in both aged atherosclerotic and atherosclerosis-free arteries in humans and
experimental animal, suggesting that arterial thickening is an intrinsic age-associated process
independent of atherosclerosis that may predispose to future risk of cardiovascular disease. [20,21]
Cellular, enzymatic and molecular mechanisms underlying progressive intimal and medial
thickening have been studied largely in rodents. The thickened intima in old rats is enriched in
matrix molecules (i. e. collagen, fibronectin and proteoglycans) and SMCs. In addition, the aged
intima exhibits increased immunostaining for transforming growth factor beta (TGF-), interstitial
cell adhesion molecule (ICAM-1), the zinc-dependent endopeptidase type-2 metalloproteinase
(MMP-2) and its activator, membrane type metalloproteinase-1. Remarkably, the results of tissue
culture and rat carotid balloon angioplasty studies suggest that chemotactic invasion of SMCs to
the intimal layer through the basement membrane requires activated MMP-2 and type IV
collagenase activity potentially derived from cytokine stimulated vascular SMC. [22,23] This
reflects a chronically enhanced level of cytokine stimulation and an exaggerated chemotaxis and
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proliferative response to growth factors, such as PDGF. [24,25] On the other hand, although the
antiproliferative actions of TGF- decrease with age, [26] TGF- suppresses the activity of
proteases and activates tissue inhibitors of MMPs as well as the synthesis of extracellular matrix
proteins. Indeed, TGF- accumulation in the intima of older rats may be related to age-associated
increase in arterial fibronectin and collagen. [18,27] Upregulation of angiotensin converting
enzyme (ACE) activity has been observed in the aorta of old rats, and this correlates with increased
level of angiotensin II. [28] Angiotensin II, in turn, is able to regulate TGF- and fibronectin
expression. [29]
1.2. Wall stiffness. Age-associated increase in intima/media thickening is accompanied by both
luminal dilatation and a reduction in compliance or distensibility, resulting in increased vessel
stiffness. [21] Although age-associated increase in arterial stiffness has been observed in
individuals with little or no atherosclerosis, [30] recent data emerging from epidemiological
studies indicate that increased large vessel stiffening also occurs in the context of atherosclerosis
and diabetes. [31,32]
The total mucopolysaccharide content of the interstitial matrix is unaltered with aging,
however aged arteries display increased chondroitin sulphate and heparin sulphate levels, and
reduced hyaluronate and chondroitin content. [33] The distribution of unstretched collagen also
changes with age, and it has been proposed that age-associated changes in collagen involve a
decrease in the coiling and twisting of molecular chains and a reduction in effective chain length.
[34] Regarding elastin fibrils, their glycoprotein component decreases and eventually disappears
with aging. In aged rats, elastin becomes frayed, and its calcium content increases. The increased
mineralization of elastin with aging is associated with an increase in the content of more polar
amino acids. [19]
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As the wall of large arteries become stiffer, central systolic arterial pressure raises, diastolic
arterial pressure decreases, and the pulse pressure increases. These findings suggest that altered
structure/function resulting from stiffness of the vessel wall is a risk factor for future vascular
events, independently of the associated increase in systolic and pulse pressures. [21] In addition to
structural properties, arterial stiffness in vivo is determined by vascular smooth muscle contractile
tonus, which is controlled in part by neurohumoral factors (catecholamines, angiotensin, etc) and
by the SMC calcium balance. [19]
1.3. Endothelial dysfunction. Aging enhances the expression of adhesion molecules in the rat
aorta, [18] and increases monocyte adherence to the endothelium of the rabbit aorta. [35]
Moreover, aged rodents show increased vascular permeability, [36] and the intima of older rabbits
accumulates glycosaminoglycans, [35] which play an important role in regulating vascular
permeability. In addition, aging elevates nitrite and nitrate levels in plasma, [37] and nitricoxidedependent endothelial vasodilatation is attenuated by reduction in nitric oxide in the aged aorta.
[38,39]
3. Telomere biology
Eukaryotic telomeres prevent the recognition of chromosomal ends as double stranded DNA
breaks thus preserving genome integrity and stability. Telomeres are specialized DNA-protein
complexes located at both ends of linear chromosomes. As depicted in Fig. 1A, telomeric DNA
consists of non-coding double-stranded repeats of G-rich tandem sequences (TTAGGG in humans)
that extend several thousand base pairs and end in a 3' single-stranded overhang. Conventional
DNA polymerases are unable to synthesize telomeric DNA, an activity that requires specialized
telomere-associated proteins (i. e., telomerase, TRAF1, TRAF2, Ku86, etc). The enzyme
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telomerase has a catalytic telomerase reverse transcriptase (TERT) component and a telomerase
RNA (Terc) that provides a template for new telomeric DNA synthesis.
Telomere homeostasis depends on several factors, including the precise composition of
telomere-associated proteins, the level of telomerase activity, and telomere length itself. [40,41]
Cells with sufficiently long telomeres do not require telomerase activity, but lack of telomerase
activity in cells with critically short telomeres leads to chromosomal fusions, replicative
senescence, and apoptosis (Fig. 1B). Telomerase expression and activity and telomere length are
tissue and developmentally regulated in several species, including humans. [42-45] In general,
these parameters are greater during embryonic development and become low or undetectable after
birth, although significant differences in adult tissues have been reported. For instance, human
telomeres shorten at an estimated rate of 29-60 bp/year in liver, renal cortex and spleen, but
telomere length is maintained in cerebral cortex. [46] Notably, human premature aging syndromes
(i. e., Werner syndrome, ataxia telangectasia, dyskeratosis congenita) are characterized by an
accelerated rate of telomere attrition (Figure 1B). Progressive telomere shortening in cell culture
and during ageing of the whole organism is a characteristic of most adult somatic cells, which
exhibit low or absent telomerase activity. [47-49] Of note in this regard, human TERT (hTERT) is
alternatively spliced in specific patterns by different tissue types during development, and this
mechanism often leads to the expression of hTERT protein lacking functional reverse transcriptase
domains. [50] In contrast to adult somatic cells, the extended proliferative capacity of germ and
tumor cells correlates with the maintenance of high telomerase activity and long telomeres. Indeed,
ectopic TERT overexpression inhibits replicative senescence and extends the lifespan of numerous
cell types, including SMCs and endothelial cells (ECs) (Table). By comparing the chronological
changes in the expression of cell cycle and apoptosis-related genes in hTERT transduced human
normal fibroblasts and ECs, Kumazaki et al. suggested that cell-type specific differential gene
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expression after telomerase activation may be important to acquire telomere-maintenance capacity
and immortality. [51]
Telomere length displays individual differences in both rodents [44,45] and humans. [46,5254] Since Okuda et al found high variability of telomeric DNA length in white blood cells
(WBCs), umbilical artery and skin from donor newborns independently of gender, they suggested
that genetic and environmental determinants that start exerting their effect during embryonic
development are key determinants of telomere length. [52] Further support to the notion that
telomere size is familial has arisen from human studies in twins. [53,55] By measuring terminal
restriction fragment (TRF) length in WBC DNA taken from individuals from a family-based
cohort, Nawrot et al. concluded that inheritance of telomere length is linked to X chromosome.
[56]
4. The importance of telomeres in vascular pathobiology
In the next sections, we will discuss tissue culture, animal and human studies that highlight
the importance of telomeres in vascular pathobiology, including recent insights into the
mechanisms that alter telomere homeostasis in response to several stimuli with relevance in
atherosclerosis (i. e., estrogens, oxidative stress, hypertension and diabetes) (Table and Fig. 1 and
2). The role of telomeres on cardiac pathobiology has been comprehensively discussed elsewhere.
[57,58]
4.1. Telomeres and estrogens. Indirect effects on lipoprotein metabolism and direct actions on
vascular ECs and SMCs are thought to contribute to the cardioprotective effects of estrogens in
premenopausal women. [59-62] It is noteworthy that human and animal studies have shown higher
telomerase activity and a decelerated rate of age-dependent telomere exhaustion resulting in
greater telomere lengths in females than in males, [43,45,55,56,63,64] and these sex differences
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might be directly related to estrogen-dependent activation of endothelial telomerase via
phosphoinositol 3-kinase (PI3K)/Akt and nitric oxide signaling. First, estrogen upregulates hTERT
mRNA, and this correlates with direct and indirect effects on the hTERT promoter (specific
binding of 17-estradiol to an imperfect palindromic estrogen-responsive element, and 17estradiol-dependent induction of c-Myc/Max binding to E-boxes, respectively). [65] Second, nitric
oxide production may contribute to telomerase activation in vascular ECs. [66,67] Third, treatment
of human ECs with estrogen activates the PI3K/Akt pathway, [66] which in turn leads to hTERT
phosphorylation and activation. [68] In contrast, either overexpression of dominant negative Akt or
PI3K inhibition attenuate telomerase activity in ECs, [69] and Akt inactivation by proatherogenic
oxidized low density lipoproteins impairs telomerase activity in ECs. [69] Collectively, these
studies suggest that a positive effect of PI3K/Akt on telomerase expression and activity may
contribute to the maintenance of EC integrity and function. Contrary to this notion, Miyauchi et al.
recently reported that constitutive activation of Akt promotes senescence-like arrest of human EC
growth via a p53/p21-dependent pathway. [70] Additional studies are thus required to clarify the
links between telomere function, PI3K/Aht signaling and EC pathobiology.
4.2. Telomeres and oxidative stress. Accumulation of oxidative damage is thought to play an
important role in aging and associated diseases. [71] Mounting evidence implicating oxidative
stress in telomere dysfunction include the following: 1) Chronic oxidative stress induces a rapid
and sustained decrease in TERT activity and accelerates telomere attrition in human umbilical vein
ECs (HUVECs); [72] 2) maximum levels of glutathione coincide with a peak of telomerase
activity in proliferating 3T3 fibroblasts; moreover, glutathione depletion decreases by 60%
telomerase activity, and restitution of glutathione levels restores telomerase activity; [73] 3) agedependent telomere shortening in HUVECs is slowed down by Asc2P, and oxidation-resistant
derivative of vitamin C which reduced by 53% the level of proatherogenic reactive oxygen
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intermediates; [74] 3) oxidized low density lipoproteins inhibit EC telomerase activity; [69] 4)
oxidative stress induced in human ECs by exposure to H2O2 leads to translocation of both
endogenous and overexpressed hTERT from the nucleus into the cytosol via Src kinase familydependent phosphorylation of hTERT tyrosine 707, which reduces the antiapoptotic capacity of
TERT; [75] furthermore, reduction of intracellular reactive oxygen species formation by the
antioxidant N-cetylcysteine prevented mitochondrial damage and delayed nuclear export of TERT
protein, loss of TERT activity and the onset of replicative senescence; [76] and 5) formation of 8oxo-7,8-dhydro-2’-deoxyguanosine at the GGG triplet in the telomeric DNA sequence may be a
mechanism facilitating telomere shortening induced by oxidative stress. [77]
4.3. Role of telomeres in vascular pathobiology: Evidence from human studies
4.3.1. Atherosclerosis. By inducing replicative senescence, age-dependent telomere erosion is
likely to contribute to progressive endothelial dysfunction and atherosclerosis. Indeed, a
characteristic senescent phenotype is observed in the endothelium of atherosclerotic lesions. [78]
Of note, overexpression of a dominant-negative mutant of telomere repeat binding factor 2 (TRF2)
induces senescence in human aortic EC cultures, and TERT transduction can prevent replicative
senescence of these cells. [78] Both telomere shortening and increased frequency of aneuploidy is
observed in ECs from the aged abdominal aorta. [79] Remarkably, endothelial telomere erosion
occurs at a greater rate in the iliac arteries versus iliac veins (102 bp/yr vs. 47 bp/yr, respectively),
and age-dependent intimal telomere attrition is greater in the iliac artery compared to the internal
thoracic artery (147 bp/yr vs. 87 bp/yr, respectively), [80] a vessel subjected to a reduced amount
of hemodynamic stress. Okuda et al. also reported increased age-dependent telomere ablation in
both the intima and media of the distal versus proximal abdominal aorta, and found an inverse
correlation between atherosclerotic grade and telomere length (although this relationship was not
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statistically significant after adjustment for age). [81] Taken together, these studies suggest a
higher rate of telomere shortening in aged vascular beds with increased shear wall stress and
enhanced cellular turnover.
Because leukocytes are key players during atherosclerosis, [1,2,6,7] several studies have
compared telomere length in WBC from healthy controls and cardiovascular patients. Patients with
vascular dementia, a disorder that is frequently associated with cerebrovascular atherosclerosis and
stroke, have significantly shorter telomeres in WBCs compared with 3 age-matched control
groups, namely cognitively competent patients suffering from cerebrovascular or cardiovascular
disease alone, patients with probable Alzheimer's dementia, and apparently healthy control
subjects. [82] After adjustment for age and sex, leukocyte average telomere length in 10 patients
suffering severe coronary artery disease (CAD) was significantly reduced compared with 20
controls with normal coronary angiograms. [83] In a larger study including 180 controls and 203
cases of premature MI, age- and sex-adjusted mean TRF length of controls was significantly larger
than that of patients, and subjects with shorter than average telomeres had between 2.8- and 3.2fold higher risk of MI. [84] Likewise, analysis of telomere length in blood DNA from 143 normal
unrelated individuals over 60 years of age disclosed an association between shorter telomeres and
poorer survival that was partly attributed to a 3.18-fold mortality rate from heart disease and a
8.54-fold higher mortality rate from infectious disease. [85]
4.3.2. Hypertension and diabetes. Hypertensive patients are more prone to atherosclerotic lesions
and acute ischemic events that normotensive individuals. [5] Analysis of 49 twin pairs (38 males
and 60 females within 18 and 44 years of age) revealed a positive correlation between WBC TRF
and diastolic blood pressure. [55] Because TRF length and systolic blood pressure were inversely
associated, telomere length seems to negative correlate with pulse pressure. Notably, the link
between TRF length and pulse pressure was independent of gender and both parameters appeared
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highly heritable. Benetos et al. also investigated WBC telomere length and blood pressure
parameters that are associated with stiffness of large arteries (pulse pressure and pulse wave
velocity) in individuals who were not under antihypertensive medications (120 men, 73 women,
mean age of 56±11 years). [64] While telomere length negatively correlated with age in both
sexes, multivariate analysis showed that telomere shortening significantly contributes to increased
pulse pressure and pulse wave velocity only in men. In both studies, women showed age-adjusted
longer telomeres, suggesting that biological aging is slowed down in women. More recently,
Benetos et al. examined the relationship between WBC telomere length and carotid artery
atherosclerosis in 163 treated hypertensive men who were volunteers for a free medical
examination. [86] They found that telomere length was shorter in hypertensive men with carotid
artery plaques versus hypertensive men without plaques (8.17+/-0.07 kb versus 8.46+/-0.07 kb;
p<0.01), and multivariate analysis revealed that, in addition to age, telomere length significantly
predicts the presence of carotid artery atherosclerosis.
The prevalence of both microvascular and macrovascular disease is significantly increased in
diabetic patients. [4] Telomere length in WBCs from insulin-dependent diabetes mellitus patients
is reduced compared with age-matched nondiabetic subjects. [87] This parameter was similar in
non insulin-dependent diabetes mellitus patients and nondiabetic controls, suggesting that telomere
erosion only occurs in a subset of WBCs that play a role in the pathogenesis of insulin-dependent
diabetes mellitus. Further support implicating telomere exhaustion as a mechanism contributing to
coronary atherosclerosis under some circumstances of metabolic disorders arises from the
observation that hypercholesterolemic and diabetic CAD patients display shorter telomeres in
peripheral blood mononuclear cells than healthy controls. [88]
4.3.3. Neovascularization. Proliferation of vasa vasorum promotes atherosclerosis. [89] On the
other hand, therapeutic neovascularization is essential for the restoration of blood flow into
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ischemic territories in the adult organism, which depends on the development of new collateral
vessels from established vascular networks (angiogenesis) and on de novo vessel formation by
endothelial progenitor cells (vasculogenesis). [90] Using an experimental model of limb ischemia,
Rivard et al demonstrated and impairment of angiogenesis in old versus young rabbits. [91] They
also showed diminished expression of the angiogenic cytokine vascular endothelial growth factor
(VEGF) with ageing, and old rabbits exposed to exogenous VEGF exhibited a similar increase in
blood pressure ratio, angiographic score, and capillary density than did young counterparts. Thus,
VEGF downregulation might be critical for age-dependent impairment of angiogenesis in response
to ischemia, and this may result from reduced activity of the transcription factor hypoxia-inducible
1 (HIF-1). [92] Remarkably, whereas both angiogenic cytokines VEGF and fibroblast growth
factor-2 (FGF-2) elicited a mitogenic response in cultured HUVECs, only FGF-2 induced the
upregulation of hTERT mRNA and enzymatic activity and delayed the appearance of a senescent
phenotype. [93,94] Because of these differences, it would be interesting to compare the angiogenic
response elicited by exogenous FGF-2 and VEGF in old rabbits.
Overexpression of hTERT in human dermal microvascular ECs (HDMECs) augments their
capacity to form more durable microvascular structures when subcutaneously xenografted in
severe combined immunodeficiency mice. [95] Similarly, hTERT gene transfer into human
endothelial progenitor cells improves their proliferative and migratory activity, enhances survival,
and augments neovascularization when applied in a murine hindlimb ischemia model. [96]
Recruitment of SMCs and pericytes into new capillaries composed by a monolayer of ECs is
important for their stabilization and maturation into fully functional vessels. Hypoxia is a major
angiogenic stimulus that induces TERT protein expression and phosphorylation in cultured SMCs.
[97] Telomerase inhibition shortened the life span of hypoxic cultures, and constitutive TERT
expression extended life span under normoxia, suggesting that hypoxia-mediated telomerase
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activation promotes long-term SMC growth. It remains to be established if chronic hypoxia can
induce endothelial telomerase activity.
The importance of telomerase in angiogenesis is further emphasized by the progressive
induction of hTERT mRNA expression in human ECs of newly formed vessels within tumors. [98]
Endothelial hTERT expression was observed in 29%, 56% and 100% of low-grade astrocytomas,
anaplastic astrocytomas and advanced glioblastomas multiforme, respectively. While the
proliferation rate and hTERT mRNA expression are dissociated in human ECs from low-grade and
anaplastic astrocytomas, high proliferation rate and hTERT mRNA level positively correlated in
glioblastomas multiforme. Remarkably, hTERT mRNA and protein expression and telomerase
activity are induced in EC cultures exposed to diffusible factor(s) produced by glioblastoma cells.
[99]
4.4. Role of telomeres in vascular pathobiology: Lessons learnt from animal studies
Terc-deficient mice have been a valuable tool to investigate at the organismal level the role
of telomere homeostasis. [100-107] In spite of Terc inactivation, the breeding of successive
generations of Terc-null mice is needed to reach critically short telomeres leading to abnormal
chromosome end-to-end fusions and symptoms of premature aging and disease, such as infertility,
graying of hair, alopecia, impaired wound healing, small intestine and spleen atrophy, reduced
proliferation of T and B lymphocytes, and hematopoietic disorders. Furthermore, late-generation
Terc-null mice display reduced lifespan. We will review in the next sections studies in Terc-null
mice and spontaneously hypertensive rats that link telomeres and vascular pathobiology (Fig. 2).
4.4.1. Atherosclerosis. Because age-dependent telomere shortening is more prominent in human
arteries subjected to a higher atherogenic hemodynamic stress, and individuals with shorter
telomere length in WBC present a higher prevalence of arterial lesions and higher risk of
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cardiovascular disease mortality (see above), it is tempting to speculate that telomere exhaustion
may be a primary abnormality that renders the organism more susceptible to cardiovascular risk
factors. Alternatively, reduced telomere length in these scenarios may simply reflect augmented
cell turnover induced by the chronic inflammatory response underlying atherosclerosis. Our recent
studies using genetically-modified mice seem to support the latter. [107] We found that lategeneration mice doubly deficient for Terc and apoE (Terc/apoE-KO) have shorter telomeres and
are protected from diet-induced atherosclerosis compared with atherosclerosis-prone apoE-null
counterparts with an intact Terc gene. This beneficial effect of telomere exhaustion could not be
attributed to reduced hypercholestrolemia, and was correlated with a significant inhibition in the
proliferative capacity of lymphocytes and macrophages. It is worth recognizing the limitations of
these animal models, therefore prospective epidemiological studies are warranted to assess
whether WBC telomere length at birth can predict the risk of developing CAD in adulthood
independently of known cardiovascular risk factors. Another important issue when interpreting the
atheroprotective role of telomere exhaustion in hypercholesterolemic Terc/apoE-KO mice is that
human ageing is associated with telomere erosion in most somatic cells, [48,49] yet atherosclerosis
is more prevalent within the elderly. These seemingly conflicting findings might be reconciled
accepting that accumulation of cellular damage imposed by prolonged exposure to cardiovascular
risk factors may ultimately prevail over protective mechanisms such as telomere shortening.
4. 4.2. Neovascularization. Telomere exhaustion in late generation Terc-null mice leads to a sharp
decrease in angiogenesis in both Matrigel implants and murine melanoma grafts, and diminished
tumor cell proliferation, increased tumor cell apoptosis, and a lower tumor growth rate. [104]
Given the data implicating neointimal angiogenesis as a mechanism contributing to atheroma
growth, [89] it will be instructive to examine the effect of telomere shortening on neointimal vasa
vasorum density.
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4.4.3. Hypertension. In the aorta of spontaneously hypertensive rats, but not in other tissues,
telomerase is activated before the onset of hypertension, and telomeres are lengthened both in vivo
and in cultured SMCs from these animals. [108] Downregulation of telomerase by TERT antisense
RNA delivery arrested the increased proliferation of spontaneously hypertensive rat vascular
SMCs and induced apoptosis by a mechanism that can be reversed by p53 overexpression and
worsened by lowering p53. The authors concluded that selective TERT activation and subsequent
telomere lengthening in aortic medial SMCs is the driving force for the imbalance between cell
proliferation and apoptosis that ultimately results in the vascular remodeling seen in genetic
hypertension. Hamet et al. observed in the kidney of spontaneously hypertensive rats a transient
hyperplasic response during the first 2 weeks of postnatal life that was absent in age-matched
normotensive controls. [109] Because shorter telomeres are detected in the kidney of
spontaneously hypertensive rats at all ages examined, the authors suggested that kidney cells from
these animals are subjected to increased turnover, potentially leading to their accelerated aging.
5. Telomerase gene transfer for therapeutic revascularization
By analizing the offspring obtained by mating heterozygous Terc+/- mice and late-generation
Terc-null mice, which have short telomeres, unstable chromosomes and signs of premature aging,
Samper et al. demonstrated that critically short telomeres can become fully functional by
restoration of telomerase. [110] Because age-dependent telomere erosion may compromise the
reestablishment of adequate blood supply into ischemic territories by limiting de novo vessel
formation, telomerase gene transfer appears an attractive strategy to boost therapeutic
neovascularization. It has been shown that ex vivo expanded human endothelial progenitor cells
enhance therapeutic neovascularization in animals, [111] and in vivo transplantation of hTERTtransduced endothelial progenitors increased capillary density and limb salvage in a murine model
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of hindlimb ischemia. [96] In contrast, although TERT overexpression in murine hematopoietic
stem cells prevented telomere loss in these cells during serial transplantation, this strategy did not
extend their transplantation capacity, [112] suggesting that telomere-independent barriers may
limit the transplantation capacity of hematopoietic stem cells.
McKee et al. have provided evidence that hTERT transduction may be an appropriate
strategy for the production of tissue-engineered human arteries for bypass surgery. [113]
Compared with control cells, passaged human aortic SMCs overexpressing hTERT maintained
telomere length, disclosed extended lifespan, and retained a normal morphology and a
differentiated, non-malignant phenotype at late-passage. Remarkably, engineered vessels
containing HUVECs and hTERT-transduced SMCs showed improved cellular viability and were
mechanically and architecturally and superior to vessels generated from non-transduced SMCs.
6. Concluding remarks
Several mechanisms contribute to age-dependent telomere exhaustion, including low
telomerase activity, an imbalance in the relative level of telomere-associated proteins, or a
combination of these factors. By imposing replicative senescence, apoptosis, and chromosome
abnormalities, telomere dysfunction is thought to contribute to aging and associated disorders,
including cardiovascular disease. A high degree of individual variability is observed in telomere
length, which seems to be controlled by both genetic and environmental factors. It is remarkable
that telomere exhaustion is delayed in women compared to age-matched men, and this gender
difference may be related to estrogen-dependent induction of telomerase activity. Thus,
comparably longer telomeres by estrogen action may contribute to the lower incidence of
cardiovascular disease in premenopausal women.
Recent human studies have shown shorter telomeres in human arterial tissue from
atherosclerosis-prone vascular beds and in WBCs from hypertensive, diabetic and CAD patients,
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and this is associated with an increased risk of cardiovascular disease mortality. However, it
remains to be established whether telomere erosion is cause or consequence of these disorders.
Indeed, studies in genetically-modified mice demonstrate that telomere erosion protects against
diet-induced atherosclerosis. Thus, prospective epidemiological studies are necessary to ascertain
whether telomere shortening is an independent cardiovascular risk factor. Moreover, further basic
research is needed to gain mechanistic insight into the role of telomerase and additional telomereassociated proteins in cardiovascular pathobiology and to apply this knowledge for therapeutic
applications.
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Acknowledgements
We thank M. J. Andrés-Manzano for preparing the figures. Work in the laboratory of V.A. is
supported in part by the Ministry of Education and Science of Spain and Fondo Europeo de
Desarrollo Regional (grants SAF2001-2358, SAF2002-1443), and from Instituto de Salud Carlos
III (Red de Centros C03/01). M.D.E. is the recipient of a predoctoral fellowship from Generalitat
Valenciana (Spain).
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Table 1: Cell culture studies implicating telomere length and telomerase in vascular
pathobiology
Cell Type
Main Findings
Hypoxia-induced telomerase activation in human SMCs correlates
with increased cellular proliferation
Smooth
Telomerase activity correlates with proliferation of primary SMCs,
muscle cell and its inhibition attenuates hyperplastic growth
(SMC)
Endothelial
cell (EC)
Refs.
[97]
[108,114]
hTERT forced overexpression extends the lifespan of rat SMCs
[113]
Telomere attrition correlates with limited proliferative capacity of
passaged human ECs
[80,115]
Telomerase ectopic overexpression or inhibition affect the lifespan
of human aortic ECs
[78,116]
Constitutive hTERT expression enhances the regenerative capacity
of endothelial progenitor cells
[96]
FGF-2, but not VEGF, upregulates telomerase activity and delays
the onset of senescence in HUVECs
[93,94]
Estrogen activates the PI3K/Akt pathway in human ECs, and Akt
activation upregulates human telomerase activity; in contrast, PI3K
inhibition and dominant negative Akt mutant significantly reduce
telomerase activity in EC cultures
[66,68,69]
Telomerase activity and nitric oxide production are induced by
estrogen in vascular ECs
[66,67]
Oxidized low density lipoproteins diminish Akt and telomerase
activity in ECs
[69]
Chronic oxidative stress compromises telomere integrity and
accelerates the onset of senescence in human ECs
[72]
Antioxidants inhibit nuclear export of TERT and delay replicative
senescence of human ECs
[76]
22
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34
A
TELOMERE-ASSOCIATED PROTEINS
p-arms
TERT, hPOT1, TRAF1, TRAF2, TANK1,
TANK2, TIN2, hRAP1, RAD50, NBS1,
MRE11, Ku86, DNA-PKcs
q-arms
TERT
Human: ~10 Kb
Mouse: ~40 Kb
C
TTAGGG TTAGGGTTAGGG
CAAUCCCAAUC
AATCCC
Telomeric DNA
Telomerase
activity
Telomere length
Germ cell line
Tumors
Immortal cells
High
Somatic tissues
Primary cells
Human premature
aging syndromes
Telomerase
RNA (Terc)
TELOMERE MAINTENANCE
Genomic stability
High proliferative capacity
TELOMERE DYSFUNCTION
Low or
absent
Chromosomal abnormalities
Genomic instability
Growth arrest, apoptosis
Number of cell divisions or Age
Figure 1: Telomere integrity and cellular homeostasis. A. Left: Schematic showing
telomeres (black circles) at both ends of chromosomes. Right: Enlargement of the
telomere showing the human telomeric DNA tandem repeat sequence, the telomeric
RNA component (Terc), the catalytic telomerase reverse transcriptase (TERT) subunit,
and
additional
telomerase-associated
proteins.
B.
Telomere
attrition
occurs
progressively in somatic cells with each mitotic cycle during normal aging and passage
in culture, due in part to low or absent telomerase activity. In contrast, high telomerase
activity in germ and tumor cells allows the maintenance of telomere integrity and an
extended proliferative capacity. Accelerated telomere erosion is a characteristic of
several human premature aging syndromes (i. e., Werner syndrome, ataxia
telangectasia, dyskeratosis congenita).
35
HUMAN STUDIES
• Coronary and carotid
arteriosclerosis 83,86
• Premature myocardial
infarction 84
• Hypercholesterolemia and
diabetes mellitus 87,88
• Hypertension 55,64,86
BLOOD CIRCULATING
LEUKOCYTES
Telomere
shortening
Vessels subjected to
higher atherogenic
hemodynamic
stress 78,80
ARTERIAL TISSUE
Greater age-related
telomere shortening
ANIMAL STUDIES
Spontaneously
hypertensive
rats
AORTA / KIDNEY
• Activation of
telomerase and
telomere lengthening
in aorta before onset
of hypertension 108
• Telomere attrition in
the kidney 109
Lategeneration
Terc-null mice
(with critically
short telomeres)
ARTERIAL TISSUE
• Reduced atherogenesis
when combined with
apoE-null genotype 107
• Attenuated angiogenesis 104
Figure 2: Telomeres and telomerase in atherosclerosis and hypertension. Human (left) and
animal (right) studies that have implicated telomere length and telomerase activity in
atherosclerosis and hypertension are summarized. Successive generations of Terc-null mice are
necessary to reach critically short telomeres and associated diseases. Reference numbers are shown
in parenthesis.