|AIRWAYS IN HEALTH AND DISEASE
|Year : 2015 | Volume
| Issue : 1 | Page : 20-26
Telomere length in chronic obstructive pulmonary disease
Galal-Eldin M Magd1, Ahmad S Entesar1, Hafez R Manal1, Sobh M Eman1, Alrayes H Mona2
1 Department of Chest Diseases, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt
2 Department of Clinical Pathology, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt
|Date of Submission||09-Jul-2014|
|Date of Acceptance||06-Aug-2014|
|Date of Web Publication||20-Mar-2015|
Sobh M Eman
Department of Chest Diseases, Al-Zahraa University Hospital, 11517 Al-Abbaseya, Cairo
Source of Support: None, Conflict of Interest: None
Background Telomere length (TL) is considered a biomarker of cellular aging. Chronic obstructive pulmonary disease (COPD) is found to be associated with premature aging and the senescence hypothesis is now accepted as a molecular pathway for COPD development.
Purpose The aim of this study was to measure TL in COPD patients and to study its relation to demographic data, spirometric indices, and arterial blood gases parameters.
Participants and methods We measured TL using quantitative PCR in 20 patients with severe to very severe COPD and 11 age-matched and sex-matched nonsmokers.
Results TL was significantly shorter in COPD patients (P < 0.001). Among COPD patients, TL was significantly shorter in current smokers than ex-smokers. In COPD patients, TL was correlated positively with SpO 2 %, pH (P < 0.05), PaO 2 (P < 0.01), FVC% (P < 0.05) and FEV 1 %, and FEF 25-75 % (P < 0.001) and not correlated with pack-year. The BODE (Body mass index, airflow Obstruction, Dyspnea, and Exercise capacity) index was correlated negatively with TL (P < 0.01); among BODE index parameters, the dyspnea score correlated negatively (P < 0.05) with TL. TL was shorter in very severe COPD than severe COPD (P < 0.001).
Conclusion Our data support accelerated cellular senescence in COPD represented by shortening of TL; TL was correlated positively with airflow limitation and it may be related to impaired physical activities in COPD, which is a manifestation of the aging process.
Keywords: Chronic obstructive pulmonary disease, senescence, telomere
|How to cite this article:|
Magd GEM, Entesar AS, Manal HR, Eman SM, Mona AH. Telomere length in chronic obstructive pulmonary disease. Egypt J Bronchol 2015;9:20-6
| Introduction|| |
Although chronic obstructive pulmonary disease (COPD) is primarily a respiratory disease, systemic complications contribute considerably toward the prognosis. Most of these systemic complications, including weight loss, skeletal muscle dysfunction, osteoporosis, and atherosclerosis, are considered age-related abnormalities .
Telomere attrition in circulating white blood cells has been proposed as a marker for cumulative oxidative stress and inflammation and, therefore, as an indicator of the pace of biological aging . Telomeres are DNA sequences and associated proteins that cap and stabilize the ends of linear chromosomes, thereby maintaining genome integrity and stability. Telomere length (TL) is not only related to the basic biology of aging as a trigger of cellular senescence but also reflects the balance between oxidative stress and antioxidant defense mechanisms .
The existing hypothesis for the pathogenetic mechanism of COPD is explained in terms of proteases, oxidants, and inflammation, whereas a new hypothesis is explained in terms of apoptosis, proliferation, and senescence. The two hypotheses can be integrated into a single hypothesis by linking senescence and inflammation .
| Aim of the work|| |
The aim of this work is to measure TL in patients with COPD and to study its relation to demographic data, spirometric indices, and arterial blood gases parameters.
| Participants and methods|| |
This case-control study was carried out at Al-Zahraa University Hospital on 31 age-matched men; they were divided into two groups.
- Group I (control group): It included 11 healthy lifelong nonsmoker volunteers. None of them had any symptoms suggestive of any disease and their spirometric indices and arterial blood gas values were in the normal range.
- Group II (COPD group): It included 20 COPD patients with symptoms of chronic airflow limitation and fulfilled the spirometric criteria set out by the GOLD 2011 guideline. The patients were included only if they had a stable course of disease with regular follow-up during the preceding 1 year and no hospitalization for COPD-related illness during the preceding 6 months. All of them had irreversible/partially reversible obstruction of airflow. COPD patients had a postbronchodilator FEV 1 /FVC% of less than 70%. They had an increase in FEV 1 of less than 200 ml or less than 12% of baseline value 20 min after two puffs of inhaled salbutamol (100 μg) administered by a metered-dose inhaler. Most patients were using medications to treat COPD, including β2-agonists (salbutamol or formoterol), theophylline, and inhaled corticosteroids (budesonide); none of them was using oral steroids or long-term oxygen therapy.
Individuals known to have infective or interstitial lung diseases, bronchial asthma, cardiac, hepatic, renal, gastrointestinal, metabolic or endocrine diseases, malignancy, and inflammatory diseases such as diabetes mellitus were excluded from the study.
The study was approved by the ethical committee of Al-Azhar University. An informed written consent was obtained from all participants before their enrollment into the study.
All participants were subjected to the following studies: detailed assessment of history and complete clinical examination were performed, and age, BMI, pack-years of smoking (current smokers and ex-smokers), and mean arterial blood pressure (MAP) were recorded. The BODE (Body mass index, airflow Obstruction, Dyspnea, and Exercise capacity) index score was calculated . Dyspnea was assessed using the modified medical research council dyspnea scale (mMRC scale), whereas exercise was assessed by the 6-min walk test, which was carried out according to the ATS .
Spirometry was carried out using spirometry Spirosift 5000 manufactured for Fukuda Denshi USA, INC. 7102-A 180th Avenue Northeast Redmond, WA, 98052 by Nippon Systemhouse CO., LTD; made in Japan. Spirometric indices were calculated using the best of three technically satisfactory trials in accordance with the recommendations of the ATS.
Telomere length measurement
Fasting venous blood samples (5 ml) were drawn into EDTA-containing tubes in the morning (08:00-09:00 a.m.) and were stored at −70°C. DNA was extracted from white blood cells. Leukocyte TL was measured using the quantitative real-time method described by Cawthon , which measures the relative TL by determining the ratio of telomere repeat copy number (T) to the single-copy gene (S) number (T/S ratio) in experimental samples relative to a reference sample. The target gene in our study was the telomere and the reference gene was the acidic ribosomal phosphoprotein PO (36B4).
The TL of peripheral blood leukocytes were standardized to the reference single-copy gene (S) to yield a T/S ratio using the comparative Ct method
(T/S = 2 -ΔΔCt ).
The ΔC t value for each sample was determined by calculating the difference between the Ct value of the target gene and the Ct value of the endogenous reference gene .
Statistical presentation and analysis of the study data were carried out using Statistical Package for the Social Sciences (SPSS) version 17. Parametric data were expressed as mean ± SD and nonparametric data were expressed as number and percentage of the total. An unpaired Student t-test was used to compare between two groups in quantitative data according to the computer program SPSS for Windows. The analysis of variance test was used for comparison when there were more than two groups in quantitative data. The linear correlation coefficient was used to detect the correlation between two quantitative variables in one group. P value greater than 0.05 was considered nonsignificant, P value less than 0.05 was considered significant, and P value of 0.01 or less was considered highly significant.
| Results|| |
[Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6],[Figure 7] and [Figure 8] indicate that the TL ratio in COPD patients shows a statistically significant positive correlation with FVC%, FEV 1 %, FEF 25-75 %, SpO 2 %, pH, and PaO 2 . Moreover, TL ratio showed a significant negative correlation with the BODE index; among BODE index parameters, it was negatively correlated with the dyspnea score.
|Figure 1: Correlation between telomere length (T/S ratio) and FVC% in the COPD group. COPD, chronic obstructive pulmonary disease; FVC, forced vital capaci ty.|
Click here to view
|Figure 2: Correlation between telomere length and FEV1% in the COPD group. COPD, chronic obstructive pulmonary disease; FEV1, forced expiratory volume in 1 s.|
Click here to view
|Figure 3: Correlation between telomere length and FEF25– 75% in the COPD group. COPD, chronic obstructive pulmonary disease; FEF, forced expiratory flow.|
Click here to view
|Figure 4: Correlation between telomere length and SpO2% in the COPD group. COPD, chronic obstructive pulmonary disea se.|
Click here to view
|Figure 5: Correlation between telomere length and PaO2 in the COPD group. COPD, chronic obstructive pulmonary disea se.|
Click here to view
|Figure 6: Correlation between telomere length and pH in the COPD group. COPD, chronic obstructive pulmonary disea se.|
Click here to view
|Figure 7: Correlation between telomere length and the BODE index in the COPD group. BODE, Body mass index, airfl ow Obstruction, Dyspnea, and Exercise capacity; COPD, chronic obstructive pulmonary disease.|
Click here to view
|Figure 8: Correlation between telomere length and dyspnea score in the COPD group. COPD, chronic obstructive pulmonary disease.|
Click here to view
TL was not correlated with age, smoking/year, smoking duration, MAP, vital capacity, FEV 1 /FVC%, PaCO 2 , HCO 3 , BMI, and six-min walk distance (6MWD).
In addition, TL was significantly shorter in patients with very severe COPD (FEV 1 <30) than those with severe COPD (FEV 1 >30) (P = 0.00).
| Discussion|| |
There is a growing realization that COPD involves several processes present in aging and cellular senescence . Some have suggested that COPD is a disease of accelerated aging .
In the current study, peripheral blood leukocytes were assessed for TL not only because they were readily available, easily obtained, and processed, but also because they reflect the amount of stress on immune cells because of cigarette smoke and/or other environmental stresses . Several studies have shown that TL in peripheral blood mononuclear cells is representative of that of many tissues; the intraindividual correlation between TL in different tissues is high .
The real-time PCR approach was chosen for TL measurement as a method based on PCR requires a lesser amount of DNA and can be completed in a short time. In addition, this method targets only the telomeric region and does not include the subtelomeric region as the TL restriction fragments method does . For practical reasons, quantitative PCR has become the favored technique in telomere research over the past few years . We have chosen the 36B4 gene as a single-copy gene for normalization because it has already been validated for gene-dosage studies .
Patients with diseases other than COPD were excluded from the study as many diseases were associated with TL shortening. Also, COPD patients who were receiving systemic steroids were excluded from the study because of the immune modulating effect of steroids . Exposure of human T lymphocytes to cortisol is associated with a significant reduction in telomerase activity .
In the current study, the COPD patients and control participants selected were men to avoid TL variation with sex. Many studies confirmed that in adulthood the age-adjusted TL is shorter in men than in women [15-18]. TL in adults may have potential differences because of sex differences and exposures to oxidative stress ; also, it may be attributed to potential telomerase upregulation by estrogens .
In the current study, there was no significant correlation between TL and BMI in both groups (P > 0.05). McGrath et al.  and Lee et al.  did not find a significant relationship between TL and BMI.
TL showed no significant correlation with MAP. Jeanclos et al.  reported that TL correlated inversely with pulse pressure. This difference may be explained by the fact that all participants in our study had normal blood pressure.
TL was significantly shorter in COPD [1.1 ± 1.127 (0.001-4.427)] in comparison with the control group [4.352 ± 2.611 (1.0-8.143)] [Table 1]. Thériault et al.  and Mui et al.  found that COPD patients had significantly shorter TL compared with healthy controls. Savale et al.  reported decreased TL in peripheral leukocytes from COPD patients compared with both healthy nonsmoker and smoker participants. Houben et al.  documented that TL was significantly shorter in peripheral blood lymphocytes of COPD patients than controls. Morlα et al.  found shorter TL in circulating lymphocytes from smoker COPD compared with healthy controls. Tomita et al.  found reduced TL of alveolar macrophages (AM) from bronchoalveolar of smokers than nonsmokers, whereas there was no difference between healthy smokers and smokers with COPD and shorter TL in smokers (with and without COPD) than nonsmokers. Tsuji et al.  reported that TL in alveolar type II cells and endothelial cells was significantly shorter in patients with emphysema than in asymptomatic nonsmokers, but no difference was detected between COPD and non-COPD smokers [Table 2] and [Table 3].
We studied TL in smoker COPD and healthy controls and found that TL was significantly shorter in smokers (COPD) [1.1 ± 1.127 (0.001-4.427)] than in nonsmoker controls [4.352 ± 2.611 (1.0-8.143)] [Table 1]. TL was shorter in current smokers (0.631 ± 0.416) compared with ex-smokers (1.484 ± 1.385); the difference was statistically nonsignificant, but when compared with healthy controls, it showed significant shortening [Table 4]. However, TL was not correlated with cigarette smoke exposure (pack-year smoking) (P = 0.87). Although smoking status was not associated with a significant difference in TL, the TL from COPD patients, who all had significant smoking history (on average more than 10 pack-years), was significantly shorter than that of the control participants of the same age. These data suggest that previous cigarette exposure or COPD accelerates telomere attrition, leading to short telomeres. Similar results have been reported by many investigators; Houben et al.  concluded that shorter TL was not related to smoking exposure in COPD. Mui et al.  found no association between TL and pack-year, and no difference was found between current and ex-smokers. However, Valdes et al.  recorded a dose-dependent TL correlation with smoking, and each pack-year smoked was equivalent to an additional five base pair loss of TL. Their results emphasize the proaging effects of cigarette smoking. McGrath et al.  reported significantly shorter TL in healthy smokers than healthy nonsmokers. They did not observe an association between smoking status (current vs. former; never vs. ever) and TL among control participants. Similarly, Morlα et al.  have reported previously that TL was reduced in peripheral blood lymphocytes in smokers with and without COPD; they observed a dose-response relationship between cumulative lifetime exposure to tobacco smoking and TL. Lee et al.  found no significant difference in TL between the quitter group and the smoker groups (sustained quitters vs. intermittent quitters and sustained quitters vs. continuous smokers); however, a significant difference existed when adjusted for age. TL of the control group was significantly longer than all three smoker groups. Tomita et al.  reported evidence that smoking induces shortening of TL in AMs from both young and old individuals. Their findings suggested that smoking might induce AMs senescence and COPD might be associated with premature aging. Tsuji et al.  reported no significant differences in TL between patients with emphysema and asymptomatic smokers. Shen et al.  reported that TL was associated inversely with pack-years of smoking among controls. The significant relationship found between a history of smoking and TL reported in some studies, but not in others, including our study, may be because of differences in smoking history, age, and stages of COPD.
|Table 4: Comparison of TL among control participants, current smokers COPD, and ex-smokers COPD|
Click here to view
In the present study, no significant correlation was found between TL and years of smoking; however, Shen et al.  found a significant positive interaction between TL and years of smoking. McGrath et al.  reported that age-adjusted TL was five base pair shorter for every pack-year smoked and observed a significant correlation between TL and pack-years of smoking. Hou et al.  reported that TL tended to decrease with increasing pack-years of cigarette smoking. Several factors may explain this discrepancy, such as different white blood cells that were studied, the fact that our study and that of Morlα et al.  included only men, and the relatively small population sizes of both studies.
In the present study, TL was shorter in COPD patients than in the healthy controls [Table 1]; among COPD patients, it was shorter in those with FEV 1 % less than 30% than those with FEV 1 % greater than 30%, and it was correlated positively with FVC%, FEV 1 %, and FEF 25-75 %. The same results were obtained by Tsuji et al.  as they reported that TL in type II pneumocytes and endothelial cells was correlated positively with FEV 1 %. Schulz et al.  reported that FEV 1 , FVC, but not flow rates, were correlated positively with TL. Mui et al.  reported a strong, but nonsignificant, correlation between TL and FEV 1 % in COPD, whereas a significant positive relationship was found between TL and FEV 1 /FVC in COPD, but no association was found between TL and FVC% in COPD. However, Savale et al.  found no relationship between TL and FEV 1 , and FVC, and reported no significant difference in TL between patients with FEV 1 less than or greater than 50%. Lee et al.  found no significant relationship between TL and FEV 1 %. This discrepancy may be explained by the different stages of airflow limitation, which was severe to very severe in our study and moderate to severe in Savale et al.  and mild to moderate in Lee et al. .
In the current study, there was a significant positive correlation between TL and SpO 2 , pH, and PaO 2 , whereas PaCO 2 showed an inverse nonsignificant correlation [Figure 4],[Figure 5] and [Figure 6]; the same results were obtained by Savale et al. , who found a strong positive correlation of TL with both PaO 2 and SpO 2 as well as a negative correlation with PaCO 2 .
In the current study, TL was not correlated with age (P > 0.05). The same result was obtained by Lee et al. , Shen et al. , and McGrath et al. ; they found no significant relationship between TL and age. However, many studies observed that TL shortened linearly with age ,,,,. Morlα et al.  found that TL significantly decreased with age in smokers, but no correlation was found in never-smokers. The exact reason for this discrepancy is unclear; it may be explained by the fact that smoking also reduces TL and attenuates the relationship between chronological age and TL and the narrow age spectrum in our study. It may also be because TL is considered to be a biomarker of biological age rather than chronological age .
In the current study, in COPD, there was a significant negative correlation between TL and the BODE index (r = −0.594; P = 0.006); among the BODE components, the dyspnea score was correlated negatively with TL (P = 0.011) [Figure 7] and [Figure 8]. Savale et al.  reported no relationship between TL and the BODE index; among the BODE components, 6MWD was correlated positively with TL. Mui et al.  found no significant relationship between TL and BMI.
The present study has some limitations that deserve comments. First, the sample size of the present study is relatively small. Second, all patients had severe to very severe COPD; thus, the relationship of TL across the full range of COPD severity is unknown.
| Conclusion and recommendations|| |
The results of this study support the link between TL shortening and COPD, which confirms accelerated cellular senescence in COPD.
TL was correlated positively with airflow limitations and it may be related to impaired physical activities in COPD patients. Early smoking cessation for all COPD patients to decrease the rate of cellular senescence is mandatory. Further study of TL involving a large number of patients and different stages of COPD to assess the effect of disease severity is required. A cohort study to detect the rate of telomere attrition in COPD and its relation to morbidity and mortality is required.
| Acknowledgements|| |
The authors thank Professor Abeer Mostafa El-Sayed Ashmawey, Department of Cancer Biology, National Cancer Institute, Cairo University, for her valuable support and continuous help throughout this work.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Sabit R, Bolton CE, Edwards PH, et al.
Arterial stiffness and osteoporosis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med
Savale L, Chaouat A, Bastuji-Garin S, et al.
Shortened telomeres in circulating leukocytes of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med
Matthews C, Gorenne I, Scott S, et al.
Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress. Circ Res
Aoshiba K, Nagai A. Senescence hypothesis for the pathogenetic mechanism of chronic obstructive pulmonary disease. Proc Am Thorac Soc
Celli BR, MacNee W, Agusti A, et al.
ATS/ERS TASK FORCE Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J
ATS Committee on Proficiency Standards for Clinical Pulmonary Function Laboratories. ATS statement: guidelines for the six-minute walk test. Am J Respir Crit Care Med
Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res
Tuder RM, Kern JA, Miller YE. Senescence in chronic obstructive pulmonary disease. Proc Am Thorac Soc
Lee J, Sandford AJ, Connett JE, et al.
The relationship between telomere length and mortality in chronic obstructive pulmonary disease (COPD). PLoS One
Rufer N, Brummendorf TH, Kolvraa S, et al.
Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. J Exp Med
Saretzki G, von Zglinicki T. Replicative aging, telomeres, and oxidative stress. Ann NY Acad Sci 2002; 959
Willeit P, Willeit J, Mayr A, et al.
Telomere length and risk of incident cancer and cancer mortality. JAMA
Haussmann MF, Longenecker AS, Marchetto NM, et al.
Embryonic exposure to corticosterone modifies the juvenile stress response, oxidative stress, and telomere length. Proc R Soc
Choi J, Fauce SR, Effros RB. Reduced telomerase activity in human T lymphocytes exposed to cortisol. Brain Behav Immun
Mayer S, Brüderlein S, Perner S, et al.
Sex-specific telomere length profiles and age-dependent erosion dynamics of individual chromosome arms in humans. Cytogenet Genome Res
Aviv A, Valdes AM, Spector TD. Human telomere biology: pitfalls of moving from the laboratory to epidemiology. Int J Epidemiol 2006; 35
Adaikalakoteswari A, Balasubramanyam M, Ravikumar R, et al.
Association of telomere shortening with impaired glucose tolerance and diabetic macroangiopathy. Atherosclerosis
McGrath M, Wong JY, Michaud D, et al.
Telomere length, cigarette smoking, and bladder cancer risk in men and women. Cancer Epidemiol Biomarkers Prev
Bischoff C, Graakjaer J, Petersen HC, et al.
The heritability of telomere length among the elderly and oldest-old. Twin Res Hum Genet
Edo MD, Andrés V. Aging, telomeres, and atherosclerosis. Cardiovasc Res
Jeanclos E, Schork NJ, Kyvik KO, et al.
Telomere length inversely correlates with pulse pressure and is highly familial. Hypertension
Thériault M-E, Paré M-È, Maltais F et al.
Satellite cells senescence in limb muscle of severe patients with COPD. PLoS One
Mui TS, Man JM, McElhaney JE, et al.
Telomere length and chronic obstructive pulmonary disease: evidence of accelerated aging. J Am Geriatr Soc
Houben JM, Mercken EM, Ketelslegers HB, et al.
Telomere shortening in chronic obstructive pulmonary disease. Respir Med
Morlá M, Busquets X, Pons J, et al.
Telomere shortening in smokers with and without COPD. Eur Respir J
Tomita K, Caramori G, Ito K, et al.
Telomere shortening in alveolar macrophages of smokers and COPD patients. Open Pathol J
Tsuji T, Aoshiba K, Nagai A. Alveolar cell senescence in patients with pulmonary emphysema. Am J Respir Crit Care Med
Valdes AM, Andrew T, Gardner JP et al.
Obesity, cigarette smoking, and telomere length in women. Lancet
Shen M, Cawthon R, Rothman N, et al.
A prospective study of telomere length measured by monochrome multiplex quantitative PCR and risk of lung cancer. Lung Cancer
Hou L, Savage SA, Blaser MJ, et al.
Telomere length in peripheral leukocyte DNA and gastric cancer risk, Cancer Epidemiol Biomarkers Prev
Schulz H, Albrecht E, Behr J, et al.
Are spirometric lung function indices associated with telomere length of circulating leukocytes? Pneumologie
O′Sullivan J, Risques RA, Mandelson MT, et al.
Telomere length in the colon declines with age: a relation to colorectal cancer?. Cancer Epidemiol Biomarkers Prev
Epel ES, Blackburn EH, Lin J, et al.
Accelerated telomere shortening in response to life stress. Proc Natl Acad Sci USA
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4]