Telomere Length Dynamics In Vascular Disease Biology Essay

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Introduction. Telomeres are specialised DNA-protein complexes which cap the ends of linear chromosomes serving to maintain DNA integrity during cell division. Telomere length naturally shortens with successive cell divisions and represents a cellular marker of biological age. This paper aims to provide an overview of telomere biology and review the evidence for any association between vascular surgical conditions and short telomere length.

Methods. A systematic review of the literature was performed using the search terms 'telomere' and 'vascular'.

Results. Considerable association between shorter mean telomere length and vascular risk factors such as age, sex, smoking, obesity, hypertension and diabetes have been observed. Vascular diseases including abdominal aortic aneurysm, peripheral vascular disease and carotid disease were also associated with shorter telomere lengths but evidence was limited to a small number of studies. There were no reports of short telomere length associated with varicose veins or arterio-venous malformations suggesting a novel area for further investigation.

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Conclusion. Multiple associations between short telomere length and vascular disease characterised by atherosclerosis suggest telomere attrition may be linked to disease mechanisms. Further studies are warranted to validate and define the role of telomeres in vascular disease pathogenesis.

Keywords: telomere, telomerase, vascular, cardiac

Telomere Length Dynamics in Vascular Disease: A Review

Introduction

Telomeres are specialised DNA-protein complexes present at the ends of linear chromosomes in eukaryotes. They safeguard gene integrity during mitosis by preventing DNA degradation at the chromosomal terminus. Telomere length can be likened to a 'mitotic clock' since it naturally shortens with each cell division thereby reflecting cellular turnover. Shortening of telomeres to a critical length induces cellular senescence (cessation of mitosis) or apoptosis and causes chromosomal instability. This mechanism has been implicated in the pathogenesis of 'replicative senescence' (cessation of mitosis due to a finite number of cell divisions with age) at the cellular level and ageing at the organism level.

Studies relating to coronary heart disease have demonstrated short telomere length in peripheral blood leukocytes to be an independent risk factor for disease.1,2 Observations suggest that shorter telomere length may represent a superior predictor of disease onset than chronological age. Correlations between telomere length with specific stages of disease progression observed in cancer studies have revealed their potential prognostic and diagnostic value. The aim of this review was to determine whether this evidence exists for vascular surgical conditions including aortic aneurysms and carotid artery disease.

Methods

The Medline, Embase and SCOPUS databases were searched from 1965 to October 2009 using the search terms 'telomere' and 'vascular'. Limits filtered 'English language' and 'Human' papers only. Additional articles were obtained manually from reference lists. Articles were screened and those appropriate included in the qualitative synthesis as per PRISMA guidelines (Figure 1). 3

Results

Fifty-seven remaining articles were included in the final review. Following an overview of telomere biology, we present salient findings from the literature under subheadings pertaining to different cardiovascular conditions.

Telomere Structure

Human telomeres consist of 3 components, (i) tandem repeats of guanine-rich non-coding DNA sequences, predominantly TTAGGG in 5' to 3' direction, (ii) several associated proteins and (iii) telomerase, a DNA polymerase.4-6 Telomeric DNA is arranged in a duplex loop formed by a double-stranded telomere loop (T-loop) and a single-stranded (D-loop) (Figure 2).5,6

Telomeric proteins bind to and protect the duplex loop. They interact with other signaling proteins to achieve telomere-end protection and length control. Removal of TRF2 (telomere repeat-binding factor-2) triggers apoptosis or senescence and can result in fusion of chromosomal ends. TRF1 and RAP1 overexpression results in gradual decrease in telomeric DNA length.5,6,8 Conversely, Tankyrase (Tank-1, Tank-2) may release TRF1 from the telomere and cause DNA elongation. TRF1-interacting protein 2 (TIN2) is thought to mediate TRF1 activity and collectively both can inhibit DNA lengthening by telomerase. The in vivo functions of Ku proteins are unclear however they are thought to offer chromosomal-end protection.9

Telomerase

Telomerase is a reverse transcriptase that synthesizes telomeric DNA and prevents telomere attrition. It comprises an RNA component (hTERC) which serves as a template for DNA synthesis and a reverse transcriptase (hTERT) which catalyses the elongation. Telomerase is absent in most somatic cells but detected in low levels in highly proliferative cell lines such as skin and intestinal mucosa.10,11 Higher levels of telomerase expression in germline, embryonic and stem cells, may account for minimal telomere attrition rates in these cell lines.11

Telomere function

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Telomeres largely confer a protective role for each chromosome. The telomeric duplex loop prevents the chromosomal terminus being falsely recognised as a DNA break by DNA repair complexes. Telomeres also prevent enzymatic erosion, non-homologous recombination and end-end fusion of chromosomal DNA. These can induce cellular apoptosis and death due to chromosomal instability.4 Telomere shortening in humans can induce replicative senescence which halts mitosis. This mechanism appears to prevent genomic instability and development of malignancy in aged cells by limiting the number of cell divisions.10 Malignant cells can bypass this process to become immortalised by telomere extension, mostly due to telomerase activation.7

Telomere length

Each round of DNA replication results in a shorter telomere due to incomplete replication of the telomere terminus. This is due to the 'end replication problem' that arises from the inability of DNA polymerase to replicate the 5' end of the lagging strand after degradation of an RNA primer (Figure 3). This loss of telomeric DNA can be regenerated by telomerase. Mean telomere length varies between tissues from 12 to 16 Kilobase pairs long.5,12

Human telomere length is highly heterogeneous and may vary between chromosomes belonging to the same cell and between individuals of the same age.13,14 During intrauterine life however, telomere length appears to be consistent between various foetal tissues such as heart, liver, lung, muscle and intestine.15 Okuda et al. also demonstrated synchrony in telomere length between white cells, skin and umbilical artery in the newborn, suggesting variability seen in adults is probably influenced by hereditary factors and extraneous environmental factors.13 Loss of this synchrony may be attributable to variable cellular proliferation rates as one matures. It is evident that the rate of telomere attrition varies in different tissues, and accounts to 30 to 150 base pairs (bp) of telomere loss per year (Table 1).12-14,16-21

Age, gender and inheritance

Njajou et al. showed a strong association between mean peripheral blood leukocyte (PBL) telomere length in fathers and their children as well as between PBL telomere length of female children and paternal lifespan, suggesting paternal inheritance.22 Women's telomeres have been reported to be 3.5% longer than men's after adjustment for age (n = 143).23 PBL telomere length is closely similar in newborn boys and girls, suggesting that this sexual dimorphism in adult telomere length arises during extrauterine life.14 Age-dependent telomere attrition occurs at a higher rate in males compared to females.24 However, after the age of 60 there is no difference in the rate of PBL telomere shortening and women tend to maintain longer telomeres than men into old age.23 Others could not find any difference in PBL telomere length between males and females in people aged more than 79 years.22,25

Telomere length assessment

Telomere length is thought to be an emerging marker of cellular ageing and age-related disease and has therefore been measured by an array of techniques. Evidence of shortened tissue telomere length patterns observed in both malignant and benign diseases including colorectal carcinoma, colonic polyps and gastric adenomas has revealed their potential diagnostic and prognostic value.17,26-27

Telomere length has been most commonly measured by Southern Blot analysis of terminal restriction fragments (TRF) but other techniques including hybridization protection assays, fluorescence in situ hybridization, flow cytometry, quantitative-polymerase chain reaction (qPCR) and single-telomere length analysis have also been employed.5

Telomere length and aortic disease

We implemented the Southern Blot technique to perform a case-control study comparing PBL telomere length in abdominal aortic aneurysms (AAA, n=169) vs. controls (n=151) in an age and sex matched cohort.28 We detected significantly shorter mean telomere length in AAA relative to controls, evident by a mean difference of 193bp (95% CI 0.002). Secondly, our data demonstrated a significant negative correlation between AAA diameter and telomere length.28 Wilson et al. compared PBL 'telomere content' from AAA (n = 20, n = 12 controls) in a separate cohort of patients by qPCR.29 Results were expressed as telomere: genomic DNA ratio and revealed significantly reduced telomere content in AAA against controls (0.82, SD 0.06 and 1.27, SD 0.10 respectively, p<0.001). Secondly, the mean telomere content of vascular wall biopsies was compared in the same cases (obtained during elective AAA repair) and controls (macroscopically normal cadaveric aorta). Telomeric DNA was reduced in AAA vs. controls after adjusting for age and gender (2.17, 95% CI 1.77-2.56 vs. 2.80, 95% CI 2.32-3.28, P<0.05). Furthermore, significant positive correlation of PBL telomere content with tissue telomere content was apparent for both cases and controls (partial correlation coefficient 0.62, p<0.001). This correlation suggests that such PBL telomere measurements hold potential as accessible markers of vascular ageing in the aorta, in individuals with or without aortic disease.

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Okuda et al. assessed the telomere length in biopsies from non-aneurysmal aorta against the severity of any atherosclerotic lesions present.30 Although telomere length in distal aortic biopsies shortened as atherosclerotic grade increased, this association was not significant after adjustment for age (Partial Spearman rank correlation coefficient=0.28, p=0.06).30

Peripheral vascular disease

Van der Harst et al. measured leukocyte telomere length in patients with chronic heart failure, with or without other atherosclerotic manifestations including peripheral vascular disease (age 40-80, cases n=620, controls n=183).31 The presence or past history of peripheral vascular disease, defined as 'claudication', was an independent predictor of a short telomere length (age-adjusted regression analysis, p=0.001). The ambiguous definition of 'peripheral vascular disease' however, requires cautious interpretation of results. These findings failed to be replicated by the prospective Cardiovascular Health Study (age >65, n=419), where peripheral vascular disease (PVD) was defined as an ankle-brachial pressure index of <0.9 by Doppler.32 Twenty-two cases of PVD were identified (over 7.9 year follow-up) and did not correlate with a shorter leukocyte telomere length (p=0.09).

Telomeres and carotid disease

The Cardiovascular Health Study (n=419) included patient assessment of common carotid and internal carotid intima-media thickness by B-mode ultrasound.32 Although shorter leukocyte telomere length was observed with increasing carotid stenosis, the association failed to reach significance (internal carotid, linear regression p=0.07).

Benetos et al. had explored the relationship of leukocyte telomere length to the presence of carotid plaques in an exclusively hypertensive cohort (n=163).33 B-mode ultrasound detected carotid plaques in 73 patients (44%), whom collectively exhibited a significantly shorter telomere length compared to those without plaques (age-adjusted TRF length 8.43kb vs. 8.21kb respectively, p=0.03). There was no difference in mean lipid levels (HDL, LDL, total cholesterol) between the two groups and statistical significance persisted after excluding 55 patients on lipid-lowering drugs. This suggests that a shorter leukocyte telomere length may be a superior predictor of carotid plaque development in hypertensive patients and raises questions about the role of telomeres in plaque pathogenesis.

Subsequently, Adaikalakoteswari et al. studied a cohort of type 2 diabetics, and compared leukocyte telomere length in those, with (n=30) or without evidence of carotid plaques and femoral plaques on ultrasound (n=30).34 Telomere length was shorter in diabetics with plaques (5.39, SEM 0.2kb) compared to those without (6.21, SEM 0.2 kb) and collectively these were significantly shorter than those of non-diabetic controls (8.7, SEM 0.5 kb). However, they have failed to specify what proportion of the group of 'diabetics with plaques' had either femoral or carotid plaques.

Telomeres and venous disease

Zee et al. have conducted the only study to relate telomere length to venous disease.35 Mean leukocyte telomere length in male physicians who developed an incident venous thromboembolism (n=108, DVT 60%, PE 40%) was compared with an equal number of age-matched controls. There was no significant difference in telomere length detected between cases and controls before and after multi-variate adjusted analysis (p=0.31, p=0.62).

Telomeres and vascular risk factors

Hypertension and pulse pressure

In the Framingham Heart Study, hypertensive male subjects (n=171) exhibited significantly shorter age-adjusted leukocyte telomere length compared with their normotensive peers (n=156).36 The Cardiovascular Health Study also demonstrated this correlation in a mixed male and female cohort, although not reaching significance (p=0.82).

Arterial pulse pressure widens with age since systolic blood pressure gradually rises and diastolic blood pressure plateaus or even declines in older individuals. This prompted Jeanclos et al. to hypothesize that it may represent a phenotype of biological age. They consequently investigated leukocyte telomere length against pulse pressures in a twin population (n=98 pairs).37 They observed a significant inverse correlation of leukocyte telomere length with pulse pressure, suggesting those with shorter telomeres exhibit a wider pulse pressure (p=0.0032).

Chang et al. suggest that telomere shortening may occur in arterial segments susceptible to increased hemodynamic stress. They showed iliac artery endothelial cells have shorter telomeres compared to those from the iliac vein and internal thoracic arteries.19 In addition, Okuda et al. noted a higher telomere attrition rate was apparent in infra-renal aortic specimens relative to the proximal abdominal aorta.13 These data collectively suggest that shorter telomere length, associated with increased endothelial cell turnover, may be a consequence of higher shear stresses in the vasculature. Replacement of these cells with senescent endothelial cells, a process accelerated by exposure to reactive oxygen species, have been implicated in the pathogenesis of atherosclerosis.19,36,38

Smoking and obesity

Shorter telomere lengths observed in association with smoking and obesity suggest possible links between biological ageing and risk of atherosclerotic vascular disease. Valdes et al. reported that the mean leukocyte telomere length of obese women (BMI≥30, n=119) was found to be 240bp shorter than in lean women (BMI≤20, n=85, p=0.026).39 In turn, a cohort study of both sexes (n=70, 57% female) demonstrated yearly leukocyte telomere length to significantly fall in those with an increasing BMI, independent of age over a 10-year period (p=<0.001).40

Valdes et al. demonstrated a dose-dependent relationship between leukocyte telomere shortening and smoking.39 Each pack year smoked equated to a loss of an additional 5bp or 18% of the average annual loss in age-adjusted telomere length. The dose-dependent effects of smoking were replicated by Morla et al. in a cohort of male smokers with and without COPD (50 smokers, 26 'never-smokers').41 Telomere length shortening was seen with cumulative exposure to tobacco, independent of concomitant COPD.

These results collectively implicate smoking and obesity with pro-ageing effects. However, these findings were not reproducible by two studies of elderly individuals which could not correlate a shorter leukocyte telomere length with number of cigarettes smoked per day, or total number of cigarettes smoked.42,43 The mean ages of each cohort were considerably older in these studies, at 81 and 79. Recent evidence of telomere instability in individuals over 85 may explain the discrepancies between these studies with different demographics.43

Diabetes

Studies on South Asian populations have shown shorter leukocyte telomere lengths in individuals with both non-insulin dependent diabetes (NIDDM) and impaired glucose tolerance compared to healthy controls.34,44 Jeanclos et al. previously demonstrated the same findings for insulin dependent diabetes mellitus (IDDM) but not for NIDDM in Caucasian men.45 Others have shown association between short leukocyte telomere length and diabetes, fasting insulin and fasting glucose and higher attrition rates in increasing insulin resistance.32,36,40 Conversely, Sampson et al. could only demonstrate shorter telomere length specifically in monocytes, not lymphocytes, in those with NIDDM or insulin resistance.46 The immunopathological role of circulating white cells and those which infiltrate the pancreas in IDDM, may suggest a putative link between higher telomere attrition rates and the disease.

Telomeres and coronary heart disease

Several studies have described association between coronary heart disease and short telomere length in both tissue specimens and peripheral blood DNA. We describe recent examples and subsequently discuss studies relating to telomere length as a predictor of cardiovascular disease-specific mortality.

i) Evidence from tissue studies

Telomere length studies in coronary endothelium have been limited due to difficulty obtaining sufficient DNA from inherently small target vessels. However, Ogami et al. were able to compare telomeric DNA content in diseased coronary arteries against normal arteries from autopsy specimens (11 cases, 22 controls). They used Southern Blot analysis to show significantly lower telomere content in the group with diseased arteries.47

ii) Evidence from peripheral blood leukocytes (PBL)

A prospective study following healthy individuals aged 60-97 years assessed the impact of baseline leukocyte telomere length on subsequent cause-specific mortality (n=143).23 Individuals in the lower half of telomere length distribution displayed a 3.18-fold higher risk of heart disease-specific mortality than those in the higher distribution. Comparable results have arisen from a ten-year prospective analysis of 419 individuals (aged up to 73) by Fitzpatrick et al. This revealed a 3-fold risk of myocardial infarction in individuals with short telomere length. 32. Most recently (2008), replication of these results has been achieved by a prospective case-control study with larger numbers (337 cases, 337 controls), limited however, by an entirely male cohort.48

Starr et al. aimed to limit the impact of age as a confounding factor for telomere shortening by studying a cohort with a narrow age-range.49 Telomere lengths were significantly shorter in patients reporting ischaemic heart disease symptoms and in those with ischaemic changes on ECG.

Brouilette et al. detected shorter telomere lengths in patients who developed coronary heart disease compared to controls in a 4.9 year follow up study.50 The risk of developing coronary heart disease was doubled in patients with telomere lengths in the middle and lowest tertiles. Use of statins significantly attenuated the risk of coronary heart disease associated with shorter telomere length, independently from their effects on lipid levels and markers of inflammation. Brouilette et al. have subsequently confirmed these findings in a larger case-control study (cases 484, controls 1058) and conclude that telomere length is a predictor of future coronary events. 1

Discussion

Multiple studies support an association between short telomere length and increased predisposition to clinical and sub-clinical cardiovascular morbidity such as myocardial infarction and hypertension. Short telomeres have also been associated with vascular disease risk factors including obesity, smoking and diabetes. However, only a small sub-set of studies show direct association with vascular surgical conditions including aortic aneurysm, carotid atherosclerosis and peripheral vascular disease (Table 2). The few studies refuting such associations involved elderly cohorts (>85 years) suggesting telomere attrition may increase cardiovascular risk up to a certain point, beyond which other risk factors including chronological age may invoke a greater additional risk. It is debatable that telomere instability may play a role in the latter findings.43 Aside from one study relating to deep vein thrombosis, no studies were retrieved concerning telomere length in venous disorders such as varicose veins and arterio-venous malformations suggesting a novel area for further investigation.

The associations reported above suggest that premature vascular ageing and associated disease may be discernable by a shorter telomere length, which arises as a cause or consequence of pathogenesis. Although a causal relationship between short telomere length and disease is yet to be determined, the evidence of cellular senescence being a major feature of atherosclerotic plaques, lends support to the hypothesis that shorter telomeres could contribute to the pathogenesis of conditions characterised by atherosclerosis. Evidence of short telomeres and differential expression of senescence-associated markers exists in vascular smooth muscle cells (VSMC) derived from atherosclerotic plaque specimens compared to normal VSMC.51

Accelerated replacement of healthy functional tissue with senescent tissue may link telomere attrition to disease development since the senescent phenotype has been linked to endothelial dysfunction and plaque instability.52,53 Other proposed mechanisms linking telomere attrition to vascular disease include; (1) oxidative DNA damage- particularly in the presence of smoking and hypertension (known states of high oxidative stress) with or without synchronous telomere dysfunction; (2) telomere attrition due to higher rates of leukocyte proliferation as part of a global inflammatory response accompanying atherosclerotic disease; (3) Reduced telomerase activity; and (4) association of short leukocyte telomere length with elevated plasma homocysteine (a risk factor for vascular disease).52-56

We suggest that future work aims to validate previous studies correlating leukocyte telomere length with that in vascular tissue to confirm reproducibility as a potential biomarker.29 For example, objective telomere measurement from tissue specimens such as diseased peripheral vessels obtained during open lower-limb bypass surgery, may be compared with paired leukocyte DNA. The ease of access of venous tissue from elective venous surgery should encourage studies establishing concordance of tissue telomere length with that in leukocytes.

Accurate delineation of true changes in telomere length will require prospective longitudinal studies where serial measurement of telomere length may be scaled against incidence and progression of vascular disease over time e.g. AAA incidence and rate of expansion or intermittent claudication progression to critical ischaemia. This will require however, pre-defined and standardised outcome measures of vascular morbidity. For example, the use of objective angiographic evidence of PVD as opposed to subjective reports of claudication symptoms in previous studies. One such study in progress includes the Asklepios Study, a prospective population study intending to monitor leukocyte telomere length and onset of cardiovascular events in an initially disease-free cohort (n=2524).57 We look forward to the results since outcome measures include detection of phenotypes including carotid and femoral stenosis as well as haemodynamic measures of vascular function.

Conflict of interest statement

None declared