Published ahead of print on August 3, 2006, doi:10.1164/rccm.200509-1374OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200509-1374OC
Alveolar Cell Senescence in Patients with Pulmonary EmphysemaFirst Department of Medicine, Tokyo Women's Medical University, Tokyo, Japan Correspondence and requests for reprints should be addressed to Atsushi Nagai, M.D., First Department of Medicine, Tokyo Women's Medical University 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: anagai{at}chi.twmu.ac.jp
Rationale and Objectives: The prevalence of chronic obstructive pulmonary disease (COPD) is age-dependent, suggesting an intimate relationship between the pathogenesis of COPD and aging. In this study we investigated whether the senescence of alveolar epithelial and endothelial cells is accelerated in emphysematous lungs. Methods: Samples of lung tissue were obtained from patients with emphysema, asymptomatic smokers, and asymptomatic nonsmokers. Paraffin-embedded lung tissue sections were evaluated for cellular senescence by quantitative fluorescence in situ hybridization to assess telomere shortening, and by immunohistochemistry to assess the expression of senescence-associated cyclin-dependent kinase inhibitors. Tissue sections were also immunostained for proliferating cell nuclear antigen (PCNA), surfactant protein A, and CD31. Main Results: The patients with emphysema had significantly higher percentages of type II cells positive for p16INK4a and p21CIP1/WAF1/Sdi1 than the asymptomatic smokers and nonsmokers. They had also significantly higher percentages of endothelial cells positive for p16INK4a than the asymptomatic smokers and nonsmokers, and higher percentages of endothelial cells positive for p21CIP1/WAF1/Sdi1 than the asymptomatic nonsmokers. Telomere length in alveolar type II cells and endothelial cells was significantly shorter in the patients with emphysema than in the asymptomatic nonsmokers. The level of p16INK4a expression was negatively correlated with the level of PCNA expression. The level of alveolar cell senescence was positively correlated with airflow limitation. Conclusions: These results suggest that the senescence of alveolar epithelial and endothelial cells is accelerated in patients with emphysema. Cellular senescence may explain the abnormal cell turnover that promotes the loss of alveolar cells in emphysematous lungs.
Key Words: cyclin-dependent kinase inhibitors • senescence • telomere Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide, and the age-dependent increase in prevalence of COPD suggests an intimate relationship between the pathogenesis of COPD and aging (1). The pathogenesis of aging-related diseases is unknown, but may include cellular senescence, a state of permanent growth arrest that limits tissue renewal. In fact, recent evidence suggests that cellular senescence contributes not only to the physiologic aging process but to aging-related diseases, such as liver cirrhosis (2) and atherosclerosis (3), since, for example, the accumulation of senescent hepatocytes (2) and endothelial cells (3) in these diseases has been shown to compromise tissue regenerative capacity. The role of cellular senescence in the etiology of COPD, however, is unknown. Emphysematous changes in the lungs are associated with increased levels of apoptosis in alveolar epithelial (4) and endothelial cells (5). To maintain the integrity of the alveolar structure, the alveolar cells lost by apoptosis must be replaced by proliferation, and recent studies showing that emphysema is associated with increased levels of alveolar cell proliferation as well as apoptosis support this premise (4, 6). However, as with as other somatic cells, the ability of alveolar cells to proliferate is limited because repeated cell cycles eventually cause senescence. Once alveolar cells reach the senescence stage, the proliferation that compensates for apoptosis stops, and the lost alveolar cells may not be replaced. Since alveolar cell loss contributes to the pulmonary destruction and reduced lung surface area that are characteristic of emphysema (6, 7), cellular senescence may play a role in the changes that occur in the lungs during the pathogenesis of emphysema. Cellular senescence is induced by telomere shortening (replicative senescence) and by telomere-independent signals, such as DNA damage and oxidative stress (stress-induced premature senescence) (8). Telomeres are structures composed of specialized terminal DNA sequence repeats (TTAGGG/CCCTAA) complexed with telomere-binding proteins, and they are located at the ends of every human chromosome. Telomeres become shorter during each cell division, and cells reach the senescence stage when telomere shortening disrupts telomere structures. The repeated cell cycles of alveolar cells in emphysematous lungs may shorten telomere length, resulting in replicative senescence. Furthermore, environmental stress associated with emphysema, such as caused by exposure to cigarette smoke and oxidants, may induce alveolar cells to undergo premature senescence without telomere shortening. Cellular senescence in tissue can be evaluated by immunohistochemical visualization of the accumulation of senescence-associated cyclin-dependent kinase inhibitors (CDKIs), such as p16INK4a and p21CIP1/WAF1/Sdi1 (9, 10), by using a fluorescence in situ hybridization (FISH)-based method to measure telomere length (11, 12), or by a biochemical method that measures senescence-associated products, such as lipofuscin (13). In this study we hypothesized that alveolar cell senescence is accelerated in emphysematous lungs, and we applied these methods to tissue samples of emphysematous lungs to test this hypothesis. Some of the results of this study have been previously reported in the form of an abstract (14).
The methods are described in detail in the online supplement.
Subjects
Tissue Preparation Tissue blocks (  2 x 2 x 2 cm) were collected from the subpleural pulmonary parenchyma during surgery, transferred into a 50-ml plastic syringe containing saline, inflated by exposure to negative pressure created by pulling up the plunger of the syringe, and fixed in 10% formalin for 24 h. After fixation, each tissue block was embedded in paraffin and cut into serial sections 4 µm thick.
Immunohistochemistry A single observer (T.T.) who was not informed of the clinical data examined 10 randomly selected microscopic fields of each slide. The percentages of the total number of cells positive for SP-A that were positive for both SP-A and p16INK4a, positive for both SP-A and p21CIP1/WAF1/Sdi1, or positive for both SP-A and PCNA, and the percentages of the total number of cells positive for CD31 that were positive for both CD31 and p16INK4a, positive for both CD31 and p21CIP1/WAF1/Sdi1, or positive for both CD31 and PCNA were calculated for each field, and the mean value was calculated for each patient.
FISH for Telomeres Digitized video images of 20–30 fields randomly selected in each section were analyzed with a computer running WinRoof semiautomated image analysis software (WinRoof Version 3.5; Mitani Corporation, Fukui, Japan). A single observer (T.T.) who was uninformed of the clinical data calculated the mean telomere signal intensity in alveolar type II cells and alveolar endothelial cells.
Schmorl Reaction
Statistical Analysis
Clinical Characteristics of the Subjects As shown in Table 1, the patients with emphysema had lower FEV1 and FEV1%predicted values than the asymptomatic smokers (p < 0.01) or asymptomatic nonsmokers (p < 0.01). The difference in cigarette pack-years between the patients with emphysema and the asymptomatic smokers was not statistically significant (p = 0.16). Both the patients with emphysema and asymptomatic smokers had quit smoking at least 1 mo before undergoing surgery. No significant differences in age were observed among the groups.
Analysis of p16INK4a, p21CIP1/WAF1/Sdi1, and Lipofuscin Expression by Alveolar Cells
Analysis of the Telomere Length of Alveolar Cells Figure 3 shows representative FISH results for telomeres in lung tissue sections obtained from the asymptomatic smokers. As shown in Figure 3, telomeres hybridized with a Cy3-labeled telomere specific probe were identified as multiple, red-colored, fine spots within in the cell nuclei of type II cells and endothelial cells that immunostained green for SP-A and CD31, respectively. To determine whether telomere length in the alveolar cells in the emphysematous lungs was altered, we used computer-assisted image analysis software to evaluate telomere signal intensity in type II cells and endothelial cells in the lungs of the patients with emphysema, asymptomatic smokers, and nonsmokers (Figure 4). Previous studies had shown a linear correlation between telomere signal intensity and telomere length (11, 12). Telomere signal intensity in the type II cells and endothelial cells in the patients with emphysema and the asymptomatic smokers was significantly lower than in the nonsmokers (Figures 4A and 4B). No significant differences in telomere signal intensity in type II cells or endothelial cells were observed between the patients with emphysema and the asymptomatic smokers (Figures 4A and 4B). These results suggest that the telomeres in type II cells are shorter in patients with emphysema patients than in asymptomatic nonsmokers.
Correlations between Telomere Shortening and Level of p16INK4a and p21CIP1/WAF1/Sdi1 Expression All subjects were included in the analysis to test for a correlation between telomere shortening and expression of p16INK4a and p21CIP1/WAF1/Sdi1. As shown in Figure 5, telomere signal intensity was negatively correlated with both the level of p16INK4a and p21CIP1/WAF1/Sdi1 expression in type II cells and with the level of p16INK4a expression in endothelial cells. No significant correlation was observed between telomere signal intensity and the level of endothelial expression of p21CIP1/WAF1/Sdi1.
Correlation between Cellular Senescence and Cell Proliferation Since we had previously found that the turnover of alveolar wall cells, particularly of epithelial cells, is increased in emphysematous lungs (4), we attempted to determine whether there was a correlation between cellular senescence and cell proliferation. A correlation analysis that included patients with emphysema and asymptomatic smokers revealed a negative correlation between p16 expression and PCNA expression in epithelial cells (r = –0.61, p < 0.01) and in endothelial cells (r = –0.44, p < 0.05) (Figure 6). The above results suggested an inverse correlation between senescence and proliferation.
Correlation between Cellular Senescence and Airflow Limitation To investigate whether there was a correlation between alveolar cell senescence and the progression of emphysema, we tested the parameters of alveolar cell senescence for correlations with FEV1%predicted. As shown in Figure 7, telomere signal intensity in type II cells and endothelial cells was positively correlated with FEV1%predicted values (Figures 7A and 7B), and the levels of p16INK4a and p21CIP1/WAF1/Sdi1 expression by type II cells and endothelial cells were negatively correlated with FEV1%predicted values (Figures 7C–7F).
The results of our study demonstrate that alveolar type II epithelial and endothelial cell senescence is accelerated in patients with emphysema, in comparison with asymptomatic smokers and nonsmokers. This conclusion was drawn from the results of careful evaluation of evidence of cellular senescence in lung tissue samples that was obtained by different methods. We used quantitative FISH to assess telomere shortening, a marker of replicative senescence, immunohistochemistry to assess the expression of senescence-associated CDKIs, markers of both replicative and premature senescence, and staining for accumulation of lipofuscin, a biomarker of senescence (9–16). The results showed that the lungs of patients with emphysema are characterized by telomere shortening, overexpression of p16INK4a and p21CIP1/WAF1/Sdi1, and lipofuscin accumulation in alveolar type II epithelial and endothelial cells. Although type II cells constitute only 8% of the total number of lung cells, they are thought to contribute to the regeneration of type I cells, which occupy 96% of the gas-exchange surface of the lung (17). We therefore hypothesized that the increased senescence of alveolar type II cells as well as of endothelial cells compromises lung regenerative capacity, thereby accelerating the loss of alveolar architecture (2, 3). Our finding of a significant correlation between decreased FEV1%predicted values and the level of alveolar cell senescence supports this hypothesis. Recent evidence indicates that pulmonary emphysema is associated with accelerated alveolar cell turnover. For example, we and other investigators exploring cell-based pathogenetic mechanisms of emphysema have recently shown increased levels of alveolar cell proliferation that compensate for the apoptotic cell loss (4, 6). Given the results of the current study, we hypothesize that once alveolar cells reach the senescence stage, apoptosis is no longer compensated for by proliferation, and the lack of compensation results in progressive loss of alveolar architecture. This hypothesis is supported by our finding of an inverse correlation between the level of alveolar cellular senescence and proliferation, which suggests that increased turnover is followed by senescence, which may impair alveolar cell regeneration. Impairment of alveolar cell regeneration as a result of senescence may progress during the natural course of emphysema, since Calabrese and colleagues have recently shown a marked imbalance between alveolar cell apoptosis and proliferation in the lungs of patients with end-stage emphysema, suggesting that apoptotic cells are not adequately replaced by new alveolar cells (18). Different mechanisms may contribute to the alveolar cell senescence associated with emphysema. First, the promoted turnover of alveolar cells in emphysematous lungs may cause replicative senescence. Our findings showing telomere shortening in the alveolar epithelial and endothelial cells of the patients with emphysema imply that replicative senescence is a mechanism of the alveolar cell senescence. Second, premature senescence independent of telomere shortening may also contribute to senescence associated with emphysema. This possibility is supported by our previous findings showing that exposure of alveolar epithelial cells to cigarette smoke induces premature senescence as a result of increased oxidative stress (15). We therefore speculate that the alveolar cell senescence in emphysematous lungs is induced by different mechanisms. One possible mechanism is replicative senescence as a result of the repeated cell cycles associated with the continual alveolar cell death and regeneration during emphysema. Another possible mechanism is increased exposure to oxidative stress, such as caused by cigarette smoke and oxidants, which is thought to cause telomere-independent premature senescence (15, 16, 19). The results of this study showed no significant differences in telomere shortening between the patients with emphysema and asymptomatic smokers. Recent studies have shown that telomere length in leukocytes is shorter in smokers than in nonsmokers (20), and that it decreases with age in smokers (21). Thus, cellular senescence may be a characteristic of smokers and not be peculiar to emphysema. In other words, alveolar cell senescence may progress during smoking. Based on the close association between smoking and emphysema, we propose the senescence hypothesis to explain emphysema in smokers. Chronic alveolar damage and concomitant regeneration accelerate telomere shortening in the alveolar cells of smokers, and smoking also causes telomere-independent premature senescence. When alveolar cells reach the senescence stage, the alveolar regeneration ceases, but the chronic damage continues. Continuous alveolar damage at this stage triggers the loss of alveolar structure and progression of emphysema (Figure 8). This model provides a plausible explanation for the long latency of the emphysema that develops after several decades of smoking as well as for the high prevalence of emphysema in the elderly, who have consumed telomeres during the normal cell turnover associated with aging.
The results of the accumulation of senescent cells in emphysematous lungs may not be limited to the altered cell turnover that compromises tissue regeneration. In fact, recent evidence suggests that senescent cells have altered cell functions that affect inflammation and tissue remodeling. For example, senescent cells have been shown to produce higher levels of interleukin (IL)-6, IL-1  , nuclear factor- B, and tumor necrosis factor- than young cells (22, 23). Thus, functional alterations associated with cellular senescence may also contribute to perpetuating the abnormal inflammatory response that occurs in patients with emphysema who have quit smoking. Several questions are unanswered by this study. The first unanswered question is whether alveolar cell senescence is correlated with apoptosis. Although a weak correlation was found between the level of p16 expression and the level of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive signals in alveolar type II cells, it was not statistically significant (r = 0.36, p = 0.09; data not shown). This may have been due to a type II error as a result of the relatively small sample size. The second unanswered question is whether markers of senescence are preferentially localized in emphysematous lungs. Our use of LVRS-resected lung tissue samples precluded a morphometric analysis to localize senescent cells in relation to the degree of emphysema, because not fixing them by positive pressure resulted in different lung volumes, which would have made morphometric analysis of emphysema impossible. It was also impossible because the samples were largely composed of severe to very severe emphysematous lesions, since surgeons do not wish to remove normal lung tissues, thereby weakening the statistical power to detect differences in the distribution of senescent cells according to the severity of emphysema. The third question unanswered by this study is whether our conclusions can be generalized to all populations of patients with emphysema, because no female patients with emphysema were included in this study. In conclusion, the senescence of alveolar epithelial and endothelial cells is accelerated in emphysematous lungs. The pathologic cellular mechanisms responsible for emphysema include alterations in apoptosis, proliferation, and senescence. More detailed investigation of the mechanisms of the altered cell dynamics may identify new targets for the treatment of emphysema associated with loss of regenerative capacity.
The authors are very grateful to Masayuki Shino and Yoshimi Sugimura for their technical assistance.
This work was supported by a grant to the Respiratory Failure Research Group from the Ministry of Health, Labor and Welfare, Japan. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200509-1374OC on August 3, 2006 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form September 4, 2005; accepted in final form August 1, 2006
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ABSTRACT
METHODS
70%), and a group of 11 asymptomatic nonsmokers (FEV1/FVC 
