Leukocyte telomere length is inversely associated with arterial wave reflection in 566 normotensive and never-treated hypertensive subjects

www.aging-us.com 12376 AGING INTRODUCTION Telomeres have several important functions for cell homeostasis, such as preventing chromosomes from attaching to each other and helping deoxyribonucleic acid (DNA) repairing to function properly. Without telomeres, DNA repair mechanisms would misrecognize chromosomes as broken DNA, and this would lead to inappropriately connected chromosomes [1]. Telomere shortening takes place during every life cycle of cells due to the lagging DNA strand that cannot be completely replicated because of the Okazaki fragments [2]. Short telomeres have a more pronounced influence on cell senescence than long telomeres [3], and when the telomeres become critically short, the cell enters replicative senescence and finally apoptosis [4]. www.aging-us.com AGING 2020, Vol. 12, No. 12 Research Paper Leukocyte telomere length is inversely associated with arterial wave reflection in 566 normotensive and never-treated hypertensive subjects Milja Honkonen1, Kati Vääräniemi2, Outi Saijonmaa3, Anna Nyman3, Antti J. Tikkakoski1,4, Jenni Koskela1,5, Terho Lehtimäki1,6, Mika Kähönen1,4, Jukka Mustonen1,5, Frej Fyhrquist3, Ilkka Pörsti1,5 1Faculty of Medicine and Health Technology and Finnish Cardiovascular Research Center Tampere, Tampere University, Tampere, Finland 2Department of Internal Medicine, Central Hospital of Central Finland, Jyväskylä, Finland 3Minerva Institute for Medical Research, Biomedicum U2 Helsinki, Helsinki, Finland 4Department of Clinical Physiology, Tampere University Hospital, Tampere, Finland 5Department of Internal Medicine, Tampere University Hospital, Tampere, Finland 6Department of Clinical Chemistry, Fimlab Laboratories Ltd., Tampere, Finland Correspondence to: Ilkka Pörsti; email: [email protected] Keywords: aging, circulatory system, hemodynamics, telomeres Received: February 17, 2020 Accepted: May 27, 2020 Published: June 23, 2020 Copyright: Honkonen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Telomeres are short segments in chromosome ends, the length of which is reduced during cell lifecycles. We examined the association of mean leukocyte telomere length (LTL) and short telomere proportion (STP) with hemodynamic variables in normotensive and never-treated hypertensive volunteers (n=566, 19-72 years). STP and mean LTL were determined using Southern blotting, and supine hemodynamics recorded using continuous tonometric pulse wave analysis and whole-body impedance cardiography. The analyses were adjusted for age, body mass index (BMI), alcohol use, smoking, plasma chemistry, and estimated glomerular filtration rate (eGFR). In univariate analyses, mean LTL and STP both correlated with age, BMI, eGFR, aortic blood pressure, augmentation index, and pulse wave velocity (p<0.05 for all). Mean LTL also correlated with systemic vascular resistance (p<0.05). In linear regression analyses of all hemodynamic variables, mean LTL was only an independent explanatory factor for augmentation index (Beta -0.006, p=0.032), while STP was not an explanatory factor for any of the hemodynamic variables, in contrast to age, BMI and several cardiovascular risk factors. To conclude, augmentation index was predominantly related with chronological aging, but also with mean LTL, suggesting that this variable of central wave reflection is a modest marker of vascular biological aging. www.aging-us.com 12377 AGING Reduced leukocyte telomere length (LTL) has been associated with cardiovascular risk factors, such as family history of cardiovascular disease, lower endothelial progenitor cell number, and lower highdensity lipoprotein cholesterol (HDL-C) concentration [5]. In addition to aging, lifestyle factors like smoking and alcohol consumption, and several cardiovascular risk factors may accelerate endothelial cell senescence via telomere shortening and damage due to oxidative stress and inflammation [6, 7]. Increased cell turnover and higher hemodynamic stress may wear telomeres down, and shortening of telomeres has been found to be more apparent in the arterial intima than in the media [8]. Shorter LTL was associated with impaired pressurediameter relation of the carotid artery in American Indians [9], while men but not women with shorter LTL were more likely to have increased pulse pressure and pulse wave velocity (PWV) [10]. These findings associate telomere shortening with arterial stiffness. The characteristic aging-related changes in the cardiovascular system are increased large arterial stiffness with subsequent increases in systolic blood pressure (BP) and pulse pressure, and enhanced arterial wave reflections, manifested as higher level of augmentation index (AIx) [11–13]. In 163 hypertensive men treated with BP and lipid lowering medications, shorter LTL was related to the presence of carotid artery plaques [14]. LTL was also shorter in 203 patients with premature myocardial infarction (<50 years of age) than in 180 healthy controls [15]. Patients with coronary heart disease induced cardiac failure had shorter LTL than healthy subjects of the same age [16]. Also 190 patients with abdominal aortic aneurysm had shorter LTL when compared with 183 controls, suggesting a role for telomeres in vascular biological aging [17]. Reduced LTL has even been reported to moderately predict cardiovascular disease in drug-treated patients with hypertension and left ventricular hypertrophy [18]. In contrast, Denil et al. examined 2524 subjects free from cardiovascular disease, aged 35-55 years, and found no correlations between LTL and PWV, pulse pressure, left ventricular mass index, ejection fraction, and peak systolic septal annular motion [19]. However, LTL was associated with variables reflecting left ventricular filling [19]. According to a recent review, we do not have the final evidence whether telomere shortening is a true cause or merely a consequence of cardiovascular disease [20]. Altogether, detailed information about the association of LTL with hemodynamic variables is scarce, especially in subjects not using antihypertensive medications or other compounds with direct influences on cardiovascular function. Here we tested the hypothesis whether mean LTL or short telomere proportion (STP), determined using Southern blotting, were independently associated with hemodynamic variables in 566 normotensive and never-treated hypertensive volunteers. RESULTS Study population The study included 566 subjects, 287 men and 279 women, and their mean age was 45 years (age range 1972 years) (Table 1). In women, body mass index (BMI), use of alcohol, proportion of present and previous smokers, and office BP were lower than in men. The mean LTL was higher, while STP was lower, in women than in men (Table 1). Women also presented with lower blood hemoglobin, plasma potassium, uric acid, glucose, and more favorable lipid profile than men. Although plasma cystatin C and creatinine concentrations were lower in women, estimated glomerular filtration rate (eGFR) calculated from cystatin C plus creatinine concentrations was similar in both sexes (Table 1). Hemodynamic measurements Radial and aortic systolic and diastolic BPs, systemic vascular resistance index (SVRI), and PWV were lower in women than in men (Table 2). Heart rate and the variables of wave reflection, AIx and AIx at heart rate 75/min (AIx@75), were higher in women than in men. Aortic pulse pressure and cardiac index were similar in both sexes (Table 2). Univariate correlations of age, leukocyte telomere length, and short telomere proportion with hemodynamic variables Age directly correlated with aortic systolic BP (rS=0.444), aortic diastolic BP (rS=0.314), AIx@75 (rS=0.569), and PWV (rS=0.653) (Figure 1). Age also correlated with SVRI (rS=0.290, p<0.001), but not with heart rate, ventricular ejection duration, or stroke volume (rS range from -0.016 to 0.020, p value range from 0.636 to 0.884). Of note, the variables BP, PWV, systemic vascular resistance, heart rate, ventricular ejection duration, and stroke volume are all hemodynamic determinants of AIx [21–23]. The factors that are known to influence LTL presented with expected results: age (rS=-0.333) and BMI (rS=-0.243) were inversely correlated, while eGFR (rS=0.174) was directly correlated with mean LTL (Figure 2). Mean LTL inversely correlated with aortic systolic BP (rS=-0.183), aortic diastolic BP (rS=-0.130), AIx@75 www.aging-us.com 12378 AGING Table 1. Characteristics of the study population. All Men Women Number 566 287 279 Age (years) 45 (12) 45 (12) 44 (11) Age range (years) 19-72 19-72 21-72 BMI (kg/m2) 26.8 (4.4) 27.4 (3.9) 25.9* (4.7) Alcohol (standard doses/week) 4.5 (5.8) 6.3 (6.7) 2.7* (3.8) Smoking (present or previous, %) 29 33 25* Office blood pressure (mmHg) Systolic 141 (21) 146 (20) 135* (20) Diastolic 89 (13) 92 (12) 86* (12) Mean telomere length (kb) 8.52 (0.33) 8.49 (0.33) 8.56* (0.32) Short telomere proportion (%) 11.93 (1.55) 12.07 (1.59) 11.79* (1.50) Blood hemoglobin (g/l) 144 (12) 152 (8) 136* (9) eGFR (ml/min per 1.73 m2) 98 (15) 98 (15) 99 (14) Fasting plasma Creatinine (μmol/l) 74 (14) 82 (12) 66* (10) Cystatin C (mg/l) 0.84 (0.15) 0.88 (0.14) 0.80* (0.14) Sodium (mmol/l) 140 (2) 141 (2) 140 (2) Potassium (mmol/l) 3.8 (0.3) 3.9 (0.3) 3.8* (0.3) Uric acid (μmol/l) 302 (77) 348 (67) 255* (55) Glucose (mmol/l) 5.4 (0.6) 5.6 (0.6) 5.3* (0.5) CRP (mg/l) 1.6 (2.9) 1.6 (3.6) 1.7 (1.9) Total cholesterol (mmol/l) 5.1 (1.0) 5.2 (1.1) 5.1 (1.0) Triglycerides (mmol/l) 1.3 (1.0) 1.5 (1.2) 1.1* (0.6) HDL cholesterol (mmol/l) 1.6 (0.4) 1.4 (0.3) 1.8* (0.4) LDL cholesterol (mmol/l) 3.0 (0.96) 3.23 (1.0) 2.8* (0.9) Mean (standard deviation), *P<0.05 men vs. women; BMI, body mass index; eGFR, cystatin C and creatinine based CDK-EPI formula for estimated glomerular filtration rate [59]; CRP, C-reactive protein; HDL, high density lipoprotein; LDL, low density lipoprotein. Table 2. Hemodynamic variables of the study population. All Men Women Radial systolic BP (mmHg) 136 (134-137) 140 (138-142) 131* (128-133) Radial diastolic BP (mmHg) 79 (78-81) 82 (81-84) 77* (75-78) Aortic systolic BP (mmHg) 123 (122-125) 126 (124-128) 121* (118-123) Aortic diastolic BP (mmHg) 80 (79-81) 83 (81-85) 78* (76-79) Aortic pulse pressure (mmHg) 43 (42-44) 43 (42-44) 43 (42-44) Heart rate (1/min) 63 (62.7-64.3) 63 (61-64) 65* (63-66) Cardiac index (l/min per m2) 2.92 (2.87-2.96) 2.95 (2.89-3.01) 2.88 (2.83-2.94) SVRI (dyn*s/cm5*m2) 2654 (2604-2704) 2721 (2654-2788) 2585* (2513-2657) Augmentation index (%) 22.6 (21.6-23.6) 18.6 (17.3-19.9) 26.7* (25.4-28.0) AIx@75 (%) 17.8 (16.8-18.7) 13.3 (12.1-14.6) 22.3* (21.1-23.6) Pulse wave velocity (m/s) 8.5 (8.3-8.6) 8.9 (8.6-9.1) 8.0* (7.8-8.2) Mean (95% confidence interval), *P<0.05 men vs. women; BP, blood pressure; AIx, augmentation index; AIx@75, AIx adjusted to heart rate 75/min; SVRI, systemic vascular resistance index. www.aging-us.com 12379 AGING (rS=-0.181), PWV (rS=-0.286) (Figure 3), and SVRI (rS=-0.108, p=0.010). Mean LTL also inversely correlated with the magnitude of AIx (i.e. variable of wave reflection that was not adjusted for heart rate 75/min; rS=-0.188, p<0.001, Supplementary Figure 1). Mean LTL did not correlate with heart rate (rS=-0.021, p=0.369) or cardiac index (rS=-0.025, p=0.547), but was also inversely correlated with office systolic BP (rS=-0.167, p<0.001) and office diastolic BP (rS=-0.158, p<0.001). Table 3 shows the correlations of STP with demographic and hemodynamic variables and eGFR. Age (rS=0.333), BMI (rS=0.151), aortic systolic BP (rS=0.073), aortic pulse pressure (rS=0.144), AIx (rS=0.164), AIx@75 (rS=0.161), and PWV (rS=0.258) directly correlated, while eGFR inversely correlated (rS=-0.141) with STP (Table 3). Linear regression analyses: explanatory variables for leukocyte telomere length and short telomere proportion In linear regression analysis with age, sex, BMI, smoking status, categorized alcohol intake, central systolic and diastolic BP, eGFR, and fasting plasma concentrations of potassium, uric acid, low-density lipoprotein cholesterol (LDL-C), and glucose as included variables, the significant explanatory variables for LTL were age (beta -0.293, p<0.001), BMI (beta -0.119, p=0.005), and moderate alcohol consumption category (beta -0.093, p=0.021) (R square value 0.139). The significant explanatory variables for STP were age (beta 0.317, p<0.001), male sex (beta 0.083, p=0.041), and low alcohol intake category (beta -0.117, p=0.004) (R square value 0.123). Figure 1. Scatter plots and Spearman correlations (rS) between age and aortic systolic blood pressure (A), aortic diastolic blood pressure (B), augmentation index at heart rate 75/min (C), and pulse wave velocity (D) in the 566 study subjects. www.aging-us.com 12380 AGING Figure 2. Scatter plots and Spearman correlations (rS) between age (A), body mass index (B), and estimated glomerular filtration rate (C), and mean leukocyte telomere length in the 566 study subjects. Linear regression analyses: explanatory factors for hemodynamic variables In addition to mean LTL or STP, the included variables in these analyses were age, sex, BMI, smoking status, categorized alcohol intake, plasma C-reactive protein (CRP), sodium, potassium, uric acid, LDL-C, HDL-C, triglycerides, glucose, and eGFR (Tables 4 and 5). In the analyses of explanatory factors for AIx@75 and AIx, also PWV and SVRI were included in the model. In the analyses of explanatory factors for PWV, aortic systolic BP and heart rate were included in the model. Mean LTL was not an explanatory factor for aortic systolic BP, aortic diastolic BP, or PWV, in contrast to age, and several cardiovascular risk factors (Table 4). However, mean LTL was a moderate but independent explanatory factor for AIx@75 (beta -0.065, p=0.032; Table 4) and AIx (beta -0.063, p=0.033; Supplementary Table 1). In contrast, STP was not an explanatory factor for any of the hemodynamic variables (Table 5). Finally, neither mean LTL (beta 0.008, p=0.765) nor STP (beta -0.037, p=0.167) were explanatory variables for SVRI. DISCUSSION In this study we examined whether mean LTL or STP were associated with hemodynamic variables in 566 normotensive and never-treated hypertensive volunteers. None of the subjects used medications with direct influences on cardiovascular function. We found multiple significant correlations between mean LTL or STP and hemodynamic variables in univariate analyses. However, in the regression analyses AIx, a marker of wave reflections, was the only hemodynamic variable that was moderately but independently related with mean LTL, while none of the hemodynamic variables were independently related with STP. The most important task of telomeres is to prevent chromosome heads from attaching to each other and to protect chromosomes from inadequate DNA repair mechanisms [1]. After telomeres reach a critically short stage, the cells enter apoptosis [7]. The enzyme telomerase, found e.g. in stem cells and cancer cells, can maintain telomere length and enable almost limitless number of cell replication cycles [24]. A multitude of extrinsic and intrinsic factors like oxidative stress and inflammation accelerate telomere shortening [7, 25]. In concert with the concept that telomere shortening takes place during aging [6, 7, 26], age was the most important determinant of mean LTL and STP in the linear regression analyses of the present study. www.aging-us.com 12381 AGING We found that in addition to age, the explanatory variables for mean LTL and STP in regression analyses were BMI and alcohol consumption category, and in the case of STP also male sex. Benetos et al. suggested that for a given chronological age, biological aging in men is more advanced than in women [10]. The finding that male sex was an independent explanatory variable for STP in our study corresponds to this view. A previous meta-analysis found that higher BMI was associated with shorter telomeres, especially in younger individuals [27], while shorter LTL was also found to predict the development of insulin resistance, a condition is closely associated with higher BMI [28]. Moderate alcohol consumption has been suggested to be beneficial for health, and low and moderate alcohol intake might have some protective effect on all-cause and cardiovascular mortality [29]. Moderate beer consumption may even provide some benefit against cardiovascular disease [30]. While a meta-analysis reported that the results concerning alcohol consumption and LTL are unclear [31], a disorder with heavy alcohol use was associated with telomere shortening [32]. In the present study, moderate alcohol consumption category was associated with shorter telomeres, while low alcohol intake category was associated with lower proportion of short telomeres. These findings do not support the view that moderate alcohol consumption would be beneficial for LTL. Figure 3. Scatter plots and Spearman correlations (rS) between mean leukocyte telomere length and aortic systolic blood pressure (A), aortic diastolic blood pressure (B), augmentation index at heart rate 75/min (C), and pulse wave velocity (D) in the 566 study subjects. www.aging-us.com 12382 AGING Table 3. Correlation of short telomere proportion with age, body mass index (BMI), estimated glomerular filtration rate (eGFR), and hemodynamic variables. Variable rS P-value n Age 0.333 <0.001 566 BMI (kg/m2) 0.151 0.001 566 eGFR (ml/min/1.73 m2) -0.141 0.001 558 Radial systolic BP (mmHg) 0.073 0.085 565 Radial diastolic BP (mmHg) 0.056 0.182 564 Aortic systolic BP (mmHg) 0.109 0.010 565 Aortic diastolic BP (mmHg) 0.055 0.192 564 Aortic pulse pressure (mmHg) 0.144 0.001 564 Heart rate (1/min) 0.012 0.769 565 Cardiac index (l/min/m2) 0.005 0.899 564 SVRI (dyn*s/cm5*m2) 0.044 0.301 564 Augmentation index (%) 0.164 <0.001 563 AIx@75 (%) 0.161 <0.001 563 Pulse wave velocity (m/s) 0.258 <0.001 564 rS, Spearman’s rank correlation; eGFR, cystatin C and creatinine based CDK-EPI formula for eGFR [59]; BP, blood pressure; SVRI, systemic vascular resistance index; AIx@75, augmentation index adjusted to heart rate 75/min. Table 4. Linear regression analyses with backward elimination for the hemodynamic variables including leukocyte telomere length in the explanatory factors. Aortic systolic BP (R square 0.337) Aortic diastolic BP (R square 0.290) B Beta P B Beta P (Constant) -24.599 (Constant) 77.353 Age 0.278 0.158 0.001 BMI 0.641 0.196 <0.001 BMI 0.991 0.209 <0.001 High alcohol intake category 10.941 0.117 0.001 High alcohol intake category 13.398 0.099 0.007 eGFR -0.269 -0.276 <0.001 eGFR -0.276 -0.195 <0.001 LDL cholesterol 3.447 0.235 <0.001 LDL cholesterol 5.186 0.243 <0.001 Male sex 3.347 0.118 0.002 Plasma sodium 0.842 0.080 0.025 Leukocyte telomere length 0.017 0.663 Leukocyte telomere length 0.006 0.877 Augmentation index@75 (R square 0.571) Lg10 of Pulse wave velocity (R square 0.595) B Beta P B Beta P (Constant) 9.704 (Constant) 0.409 Age 0.470 0.473 <0.001 Age 0.004 0.466 <0.001 BMI -0.288 -0.107 0.002 Low alcohol intake category -0.012 -0.062 0.025 Male sex -11.563 -0.498 <0.001 Present smoker -0.027 -0.095 0.001 Present smoker 3.127 0.089 0.002 Aortic systolic BP 0.001 0.209 <0.001 PWV 0.522 0.089 0.021 Heart rate 0.002 0.178 <0.001 SVRI 0.004 0.186 <0.001 HDL cholesterol -0.019 -0.086 0.007 LDL cholesterol 0.925 0.077 0.023 Uric acid 0.0002 0.165 <0.001 Leukocyte telomere length -2.334 -0.065 0.032 Glucose 0.013 0.080 0.010 Leukocyte telomere length -0.002 0.936 BP, blood pressure; BMI, body mass index; eGFR, cystatin C and creatinine based CDK-EPI formula for eGFR [59]; LDL, low density lipoprotein; PWV, pulse wave velocity; SVRI, systemic vascular resistance index; HDL, high density lipoprotein. www.aging-us.com 12383 AGING Table 5. Linear regression analyses with backward elimination for the hemodynamic variables including short telomere proportion in the explanatory factors. Aortic systolic BP (R square 0.337) Aortic pulse pressure (R square 0.229) B Beta P B Beta P (Constant) -24.599 (Constant) -52.148 Age 0.278 0.158 0.001 Age 0.296 0.336 <0.001 BMI 0.991 0.209 <0.001 BMI 0.295 0.124 0.003 High alcohol intake category 13.398 0.099 0.007 Male sex -1.862 -0.090 0.025 eGFR -0.276 -0.195 <0.001 Low alcohol intake category 1.645 0.075 0.049 LDL cholesterol 5.186 0.243 <0.001 LDL cholesterol 1.548 0.145 0.001 Plasma sodium 0.842 0.080 0.025 Plasma sodium 0.491 0.094 0.018 Short telomere proportion -0.049 0.196 Short telomere proportion -0.008 0.851 Augmentation index@75 (R square 0.568) Lg10 of Pulse wave velocity (R square 0.595) B Beta P B Beta P (Constant) -11.620 (Constant) 0.409 Age 0.489 0.492 <0.001 Age 0.004 0.466 <0.001 BMI -0.261 -0.098 0.005 Low alcohol intake category -0.012 -0.062 0.025 Male sex -11.413 -0.492 <0.001 Present smoker -0.027 -0.095 0.001 Present smoker 3.268 0.093 0.001 Aortic systolic BP 0.001 0.209 <0.001 PWV 0.528 0.090 0.020 Heart rate 0.002 0.178 <0.001 SVR 0.004 0.183 <0.001 HDL cholesterol -0.019 -0.086 0.007 LDL cholesterol 0.934 0.078 0.022 Uric acid 0.0002 0.165 <0.001 Short telomere proportion 0.018 0.542 Glucose 0.013 0.080 0.010 Short telomere proportion 0.011 0.703 BP, blood pressure; BMI, body mass index; eGFR, cystatin C and creatinine based CDK-EPI formula for eGFR [59]; LDL, low density lipoprotein; PWV, pulse wave velocity; SVR, systemic vascular resistance; HDL, high density lipoprotein. In the course of aging, the amounts of elastin and smooth muscle cells decrease in arteries whereas the quantity of collagen increases [33]. These changes result in increased arterial stiffness, manifested as higher PWV, increased wave reflections, and elevated pulse pressure [13, 34]. Another factor contributing to arterial stiffness is atherosclerosis, characterized by inflammation and increased turnover of the intimal and medial cells in the vascular wall [33]. Previously, changes in telomere length have been related with increased arterial stiffness. Among 120 men and 73 women, men but not women with shorter LTL were more likely to have increased PWV and higher pulse pressure [10]. In a population of patients with cardiovascular disease, hypertension, diabetes, and chronic kidney disease, shorter telomeres were associated with increased arterial stiffness [9]. Although these results were adjusted for the use of antihypertensive medications, diabetes, and hyperlipidemia, these factors remain a potential source of confounding. In contrast, Denil et al. found no significant correlations between LTL and the hemodynamic variables PWV, pulse pressure, and left ventricular mass in 2524 subjects of the Asklepios study [19]. Furthermore, longer LTL was even associated with higher left ventricular mass in 334 Flemish participants, a finding that was related to the role of telomere length in determining cardiomyocyte replication [35]. Recently, ageand sex-adjusted LTL, but not muscle telomere length, was found to be shorter in patients with atherosclerotic cardiovascular disease than in controls [36]. Increased LTL attrition due to genetic and environmental factors was considered as the likely explanation for shorter LTL in atherosclerotic cardiovascular disease, and shorter LTL was suggested www.aging-us.com 12384 AGING as the predisposing factor for atherosclerosis [36]. In that study, the mean subject age (67 vs. 58 years), and proportions of women (17% vs. 47%), smokers (present 74% vs. previous 30%), and patients with hypertension (61% vs. 30%), diabetes (26% vs. 13%), and dyslipidemia (38% vs. 8%) were different between the study groups (atherosclerotic cardiovascular disease vs. controls, respectively), and such differences make a potential source of confounding [36]. In the present study, AIx was the only hemodynamic variable with a small independent influence on LTL in the regression analyses. Therefore, our results suggest that the influences of chronological and biological aging in the vasculature are reflected in the magnitude of the AIx. The level of AIx is influenced by arterial stiffness, heart rate, ventricular ejection duration, body height, BP, systemic vascular resistance, and stroke volume [21–23, 37, 38]. From these variables, BP and arterial stiffness are known to increase in the course of aging [23, 34], corresponding to the findings of the present study. The present results also showed that the level of SVRI was directly related with age (rS=0.290). In contrast, heart rate, ventricular ejection duration, and stroke volume were not associated with age in our study population. Aging, obesity and cardiovascular risk factors like smoking and heavy alcohol use impair cardiovascular function and are associated with telomere shortening [6, 7]. Although life style factors have a strong impact on telomere shortening [6], socioeconomic status that is strongly associated with health-related behavior [39], was not associated with LTL in the large West of Scotland Coronary Prevention Study comprising 1542 men [40]. Corresponding to the view of the inheritance of shorter telomeres, LTL was shorter in 45 healthy offspring of subjects with premature coronary artery disease than in 59 offspring from families without such history [41]. The present study was carried out in normotensive and never-treated hypertensive subjects, and the multivariable linear regression analyses were adjusted for several factors that influence cardiovascular function. Our results may be generalized to the Finnish population, as the demographic characteristic of the study participants were similar to the results of a recent population study in our country [42]. The study subjects equally represented both sexes with an age range of 1972 years, which probably reduced a possible selection among the participants. We chose Southern blot measurements for this study as they are more accurate than quantitative polymerase chain reaction (qPCR) – based assessments, although the latter method has been more widely applied [43]. A methodological advantage of Southern blotting over qPCR was that both mean LTL and STP could be determined. Regardless of mean LTL, short telomeres have a more pronounced influence on cell senescence than long telomeres. Therefore, the inclusion of STP gives valuable additional information about the biological influences of telomeres [3]. If we had applied two different methods for LTL determinations that would have yielded similar associations with hemodynamic variables, the conclusions of the study would be stronger. Two previous studies that compared Southern blotting and qPCR for LTL analyses found correlations exceeding 0.8 between these two methods [44, 45], but in the report by Elbers et al. the correlation was lower 0.52 [43]. Thus, the outcome of the analyses may differ depending on the method of LTL determination. Southern blot measures mean telomere restriction fragment length containing both the canonical and noncanonical components of telomeres, not absolute canonical length [44, 46]. The qPCR technique measures only the canonical component of telomeres, and the outcome of qPCR is normalized to a single gene to provide a mean telomere length for the examined leukocytes [44, 45]. However, the measurement error defined by coefficient of variation is significantly higher for qPCR than for Southern blotting [43, 44]. Aviv et al. concluded that measurement error should be the primary consideration in cross-sectional studies that examine LTL over a wide age range [44], a view that strongly supports the use of Southern blotting in the present study with participant ages ranging 19-72 years. Altogether, the qPCR method is better suited for large genetic epidemiological studies [45, 47]. The other limitations of this study originate from the hemodynamic measurements that were implemented using non-invasive methods, which require mathematic equations and physiological simplifications [48]. However, the present methods have been found to be reliable and repeatable [48–51]. PWV was recorded by the use of the bioimpedance signal and not the tonometric method, which is considered to be the gold standard for estimating arterial stiffness [52]. However, PWV measured using impedance cardiography correlates well with the values measured using either Doppler ultrasound or the tonometric method [22, 53]. Also, PWV measured by the present method independently predicted incident hypertension in 1183 Finnish adults aged 30-45 years [54]. Naturally, the cross-sectional study design of this study does not allow conclusions about causality between LTL and cardiovascular variables. A significant association between short LTL and increased odds ratio for stroke and myocardial infarction was reported in a meta-analysis [55]. Another meta-analysis comprising 24 studies found ~50% www.aging-us.com 12391 AGING SUPPLEMENTARY MATERIALS Supplementary Figure Supplementary Figure 1. Scatter plots and Spearman correlations (rS) between mean leukocyte telomere length and augmentation index. www.aging-us.com 12392 AGING Supplementary Table Supplementary Table 1. Linear regression analyses with backward elimination of the explanatory factors for the magnitude of augmentation index. Augmentation index (R square 0.604) B Beta P (Constant) -4.175 Age 0.532 0.530 <0.001 Male sex -11.747 -0.502 <0.001 SVRI 0.007 0.378 <0.001 Stroke index 0.270 0.164 <0.001 BMI -0.339 -0.126 0.002 Uric acid 0.014 0.091 0.017 Heart rate -0.098 -0.079 0.029 Present smoker 2.661 0.075 0.007 Leukocyte telomere length -2.253 -0.063 0.033 SVRI, systemic vascular resistance index; BMI, body mass index.