Alveolar Cell Senescence in Patients with Pulmonary Emphysema

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 snowing 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 p16^sup INK4a^ and p21^sup CIP1/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).

METHODS

The methods are described in detail in the online supplement.

Subjects

Three groups of subjects were evaluated: a group of 13 patients with emphysema (FEV^sub 1^/FVC < 70%), a group of 10 asymptomatic smokers (FEV^sub 1^/FVC = 70%), and a group of 11 asymptomatic nonsmokers (FEV^sub 1^/FVC = 70%). Lung tissue blocks were obtained from the patients with emphysema during lung volume reduction surgery (LVRS), and lung tissue blocks were obtained from the asymptomatic smokers and the nonsmokers during pulmonary resection for localized lung cancer. No patients had a^sub 1^-antitrypsin deficiency. The characteristics of the patients are shown in Table 1. The protocol of the study conformed to the Declaration of Helsinki, and informed consent was obtained from each patient.

Tissue Preparation