Keywords

Introduction

Accelerated ageing plays a key role in several pulmonary diseases, particularly chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), but also may play a role in asthma, bronchiectasis, pulmonary hypertension and lung infections. Although cystic fibrosis (CF) is a disease of childhood, with advances in management patients may survive into middle age so that ageing processes may interact with the disease. In this chapter most focus is on COPD as this is a prevalent disease and most research on lung ageing has been related to this disease.

Lung Ageing

In normal individuals lung functions, such as forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) and increased functional residual capacity (resting lung volume) decline slowly with age after a peak of around 25 years in men (Janssens et al. 1999). There is a change in the flow volume loop that suggests that the loss of lung volumes is largely due to narrowing of peripheral airways. This loss of lung function over time may be associated with loss of lung elasticity and enlargement of alveolar spaces (which has been termed senile emphysema), resulting in a reduction in gas transfer, although this is not associated with alveolar wall destruction as in COPD. None of these changes in lung function with age are sufficient to cause symptoms or impaired oxygenation and do not lead to respiratory symptoms. The aged lung appears to be more susceptible to damage by environmental stresses, such as cigarette smoking and more susceptiblity to infection.

COPD

COPD is a global epidemic, with rising prevalence as populations live longer because of reduced mortality from infectious and cardiovascular diseases. COPD is the third commonest cause of death in developed countries and the fifth ranked cause of disability, but is increasing to a greater extent in developing countries (Lozano et al. 2012). COPD is present in approximately 10% of people over 45 years and in developed countries it as common in women as in men, reflecting the prevalence of smoking in the population (Barnes et al. 2015). The major risk factor for COPD world-wide is cigarette smoking, but in developing countries exposure to biomass smoke and household air pollution, especially in rural areas is also common (Sood et al. 2018). COPD is also linked to low socioeconomic status, which is likely to be explained by a combination of factors, such as smoking, air pollution, poor nutrition and damp housing (Burney et al. 2014).

COPD is characterized by largely irreversible and progressive airway obstruction as a result of fibrosis and obstruction of small airways (chronic obstructive bronchiolitis) and destruction of the lung parenchyma (emphysema) (Hogg and Timens 2009). This results physiologically in air trapping, hyperinflated lungs and dynamic hyperinflation that leads to shortness of breath on exertion, the major symptom of COPD patients. This results in accelerated decline in lung function over time, although only half of the patients diagnosed with COPD show rapid decline, the remainder have a normal decline starting from low peak lung volumes as a result of poor lung growth during childhood (Lange et al. 2015). About 20% of smokers develop COPD but the reasons for this susceptibility have not been fully determined. Although several genes have been implicated in susceptibility, but do not account for most of this susceptibility, other factors such as epigenetic modifications may be important for inherited and environmental influences.

The underlying mechanisms of COPD are poorly understood but it is associated with chronic inflammation that is largely corticosteroid-resistant (Barnes 2016). This inflammation is similar to that found in normal smokers, but appears to be amplified. A significant mechanism of amplification involves a reduction in the nuclear enzyme histone deacetylase-2 (HDAC2), which plays an important role in switching off activated inflammatory genes (Barnes 2009). Many cells and mediators are involved in COPD (Barnes 2014) and increased oxidative stress from inhaled smoke, or endogenously from activated lung inflammatory cells, is a key driving mechanism and results in a reduction in HDAC2 (Kirkham and Barnes 2013).

The global increase in COPD is likely to be related to the ageing population, as this disease predominantly affects the elderly, with peak prevalence around 65 years. Airway obstruction in COPD is slowly progressive and represents an acceleration of the normal decline in lung function with age, so this has suggested that COPD, and especially emphysema, involves acceleration of the normal lung ageing process (Ito and Barnes 2009; Mercado et al. 2015). There is increasing evidence that in COPD there are all of the hallmarks of accelerated ageing, compared to smokers with normal lung function and non-smokers, and this is discussed below (Table 3.1). Age is the most important risk factor for several chronic diseases, including COPD, and drives morbidity and mortality (Kennedy et al. 2014).

Table 3.1 Features of accelerated ageing found in COPD and IPF patients
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Idiopathic Pulmonary Ibrosis

IPF involves slowly progressive fibrosis of the lung parenchyma that is not associated with any causative mechanisms (Sgalla et al. 2018). It is usually diagnosed in people over the age of 70 and has a poor prognosis. The fibrotic process is thought to be due to sustained alveolar microinjury and aberrant repair in genetically predisposed individuals. There is increasing evidence that IPF is due to accelerated ageing and accumulation of senescent cells (Schafer et al. 2017). Removal of senescent cells abrogates the development of fibrosis in animal models of IPF.

Some patients with IPF also have emphysema and this overlap syndrome called combined pulmonary fibrosis and emphysema (CPFE) is increasingly recognized (Cottin and Cordier 2012). It is related to cigarette smoking, which is a defined risk factor for IPF as well as COPD. CPFE is characterized by accelerated ageing and cellular senescence (Chilosi et al. 2013).

Hallmarks of Ageing in Lung Disease

Chronic lung diseases are characterized by increased oxidative stress, which may be a key mechanisms in driving accelerated ageing and cellular senescence in COPD and IPF (Kirkham and Barnes 2013; Fois et al. 2018). Excessive reactive oxygen species (ROS) induce the accumulation of molecular damage, which shortens lifespan and an optimal level of ROS is needed for healthy ageing (Hekimi et al. 2011). Oxidative stress that is greater than that associated with normal ageing, may induce age-related degeneration that favors chronic inflammation.

All of the classical hallmarks of ageing are seen in COPD and IPF, including telomere shortening, genomic instability, epigenetic alterations, loss of proteostasis, mitochondrial dysfunction, deregulated nutrient-sensing and stem cell exhaustion (Table 3.1) (Mercado et al. 2015; Meiners et al. 2015; Sgalla et al. 2018). There is an accumulation of senescent cells, which characteristically have irreversible cell cycle arrest, but are metabolically active and display what is termed a “senescence-associated secretory phenotype” (SASP) or “inflammaging”, with secretion of proinflammatory cytokines, chemokines and matrix metalloproteinases (MMP) (Salama et al. 2014). These proinflammatory mediators may induce further senescence in the cell itself (autocrine) and surrounding cells (paracrine), thus amplifying and spreading cellular senescence (Fig. 3.1). Senescent cells accumulate with age in tissues and the SASP proinflammatory state may be an important driving mechanism in age-related lung diseases.

Fig. 3.1
figure 1

Senescence-associated secretory phenotype (SASP). Senescent cells that express p16 and p21 are metabolically active, with activation of nuclear factor-kappa B (NF-κB) and mitogen- activated protein kinase p38, resulting in the increased expression of multiple inflammatory proteins, including cytokines, chemokines, proteases and growth factors, all of which are increased in chronic lung diseases. This inflammatory response has paracrine effects inducing senescence in adjacent cells and senescent cells also produce reactive oxygen species that further drive the senescence process

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Telomere Shortening in Lung Disease

Telomere shortening is greater in COPD and IPF (Birch et al. 2017). Telomere shortening is found in circulating leukocytes from patients with COPD to a greater extent than in smokers with normal lung function and appears to be related to a greater risk of lung cancer (Lee et al. 2012; Houben et al. 2009; Savale et al. 2009; Rutten et al. 2016). Short telomeres increase the risks of developing emphysema amongst smokers (Houben et al. 2009). In a large observational study short telomeres in circulating leukocytes have been linked to reduced lung function, although the independent effect of COPD, after correction for age and smoking, is small. Shorter telomeres have also been described in alveolar epithelial and endothelial cells from patients with emphysema (Tsuji et al. 2006), although this may also be found in smokers with normal lung function (Tomita et al. 2010). Telomere-associated DNA damage is found in small airway epithelial cells from COPD patients compared to age-matched controls (Birch et al. 2015). A family with a genetic defect in telomerase has been linked to early onset emphysema (Alder et al. 2011). Approximately 1% of patients (mainly female) with severe emphysema have mutations of telomere reverse transcriptase (TERT) resulting in short telomeres (Stanley et al. 2015). Telomerase null mice with short telomeres showed increased susceptibility to develop emphysema and increased cellular senescence after chronic cigarette exposure (Alder et al. 2011). Knock-out of telomerase in mice results in replicative senescence of alveolar cells and a low grade lung inflammation, with increased interleukin(IL)-1, IL-6, KC (murine IL-8) and CCL2, indicating how telomere shortening can result in COPD-like lung disease (Chen et al. 2015). Telomerase knockout mice also show increased emphysema after cigarette smoke exposure (Birch et al. 2015). Genetic determinants of telomere length may increase susceptibility to COPD but also to risk of developing comorbidities, such as cardiovascular and metabolic diseases.

The mechanisms leading to telomere shortening in association with chronic diseases such as COPD are not yet understood. Increased oxidative stress impairs telomerase activity and telomerase DNA is particularly susceptible to DNA damage (Passos et al. 2007). Telomere shortening in turn results in activation of p21, resulting in cellular senescence and the release of proinflammatory mediators, such as IL-6 and CXCL8 (IL-8). Cultured pulmonary endothelial cells from COPD patients have reduced telomerase activity, which is associated with shorter telomeres with increased cellular senescence and a SASP response compared to age-matched non-smoking control subjects (Amsellem et al. 2011).

Telomere shortening is also an important feature in IPF. Telomerase defiance mice develop pulmonary fibrosis (Povedano et al. 2015). IPF patients have shorter telomeres in circulating leukocytes and in alveolar type 2 cells (Alder et al. 2008) and there is an increase in telomere DNA damage foci (Schafer et al. 2017). Mutation in telomerase genes is commonly found in patients with IPF, particularly if there is a family history (Armanios 2012). Shortened telomeres and telomere damage have also been described in airway epithelial cells from patients with bronchiectasis (Birch et al. 2016). Shorter telomeres have also been described in circulating leukocytes of patients with life-long chronic persistent asthma, possibly as a result of chronic inflammation and oxidative stress (Belsky et al. 2014).

Cellular Senescence

Diseases of accelerated ageing are characterized by the accumulation of senescent cells in tissues, which cells enter a state of irreversible cell cycle arrest (Fig. 3.2) (Munoz-Espin and Serrano 2014; van Deursen 2014). Mammalian cells have a limited number of divisions and cells enter cellular senescence and subsequently death by apoptosis once DNA damage can no longer be repaired effectively. The accumulation of senescent cells results in age-related loss of function and removal of senescent cells prolongs lifespan of mice and delays the development of organ failure and development of cancers (Baker et al. 2011; Baker et al. 2016a). Cellular senescence results from the activation of the tumor suppression pathways p53 and p21CIP1/WAF1 and the p16INK4a/retinoblastoma protein pathways, which are activated by the DNA damage response (DDR) in response to critical telomere shortening and telomeric and non-telomeric DNA damage. Deletion of cells that express p16INK4a increases the lifespan of normal mice (by up to 30%) and delays several age-related diseases and the onset of cancer (Baker et al. 2011, 2016a). In COPD and IPF there is an accumulation of senescent cells (Tsuji et al. 2006; Rutten et al. 2016; Schafer et al. 2017).

Fig. 3.2
figure 2

Cellular senescence pathways in lung disease. Repeated cell division leads to progressive telomere shortening (replicative senescence) or DNA (particularly telomeres) is damaged by reactive oxygen species (ROS), activating DNA damage pathways, including the DNA repair kinase ataxia-telangectasia mutated kinase (ATM) which phosphorylates histone 2AX (H2AX), leading to activation of p53 and the activation of p21CIP1/WAF1 which results in cell cycle arrest and senescence. Oxidative stress and other stresses acvtivate p16INK4a , which is a cyclin inhibitor and phosphorylates retinoblastoma protein (pRb) which also activates the cyclin dependent kinase inhibitor p21. Senescent cells secrete cytokines and chemokines – the senescence-activated secretory phenotype (SASP), which amplifies and spreads cellular senescence

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Cellular senescence may be enhanced by extraneous stressful stimuli, such as oxidative stress and ultraviolet radiation in the case of skin. Cigarette smoke exposure in mice increases the number of p16 expressing cells in the lung, indicating that smoking can accelerate lung ageing (Sorrentino et al. 2014). Unlike apoptotic cells, senescent cells remain metabolically active and therefore may influence other cells through the SASP response (Salama et al. 2014; Correia-Melo et al. 2014)., The SASP response is triggered by p21, which activates p38 mitogen-activated protein (MAP) kinase and Janus-activated kinases (JAK). This results in the activation of the proinflammatory transcription factor nuclear factor-κB (NF-κB), resulting in secretion of cytokines, such as IL-1β, IL-6, TNF-α, growth factors, such as transforming growth factor (TGF)-β and chemokines, such as CXCL1, CXCL8 and CCL2, and elastolytic matrix metalloproteinases (MMP), such as MMP-2 and MMP-9, all of which are increased in diseases of accelerated ageing including COPD. Plasminogen activator inhibitor-1 (PAI-1) is another characteristic SASP protein and is increased in the sputum of COPD patients (To et al. 2013). This makes it likely that the chronic inflammation seen in COPD is a result of cellular senescence. CXCL8 acts via the chemokine receptor CXCR2, which induces cellular senescence and DNA damage, whereas blocking CXCR2 reduces both replicative and stress-induced senescence (Acosta et al. 2008). The SASP response itself mediates cellular senescence thus amplifying and spreading senescence. Activation of the p16INK4a pathway also activates NADPH oxidases, resulting in increased oxidative stress, which further activates NF-κB (Takahashi et al. 2006). JAK inhibitors inhibit the SASP response and thereby reduce frailty in ageing mice (Xu et al. 2015). SASP is also inhibited by rapamycin through inhibition of MK2, which is downstream of p38 MAPK (Herranz et al. 2015). The SASP also includes activation of the NRLP3 inflammasome and secretion of IL-1β (Acosta et al. 2013).

There is an accumulating of senescent cells in murine lungs with age, which is associated with emphysematous changes and deposition of collagen (Calhoun et al. 2016). Elimination of senescent cells (positive for p19ARF) appears to abrogate the decline in lung elasticity in ageing mice (Hashimoto et al. 2016). Cellular senescence is found in emphysematous lungs with enhanced expression of p21CIP1/WAF1, p16INK4a and senescence-associated β-galactosidase (SA-βGal) activity (Chilosi et al. 2013; Amsellem et al. 2011). Lung macrophages from COPD patients also express the senescence marker p21CIP1/WAF1 (Tomita et al. 2002). Furthermore, in COPD there is an increase expression of SASP components, including IL-1, IL-6, TNF-α, CXCL8, CCL2, TBG-β, MMP2 and MMP9 (Barnes 2004) (Fig. 3.1). Similarly lung fibroblasts and alveolar epithelial cells from IPF patients also show cellular senescence, with increased expression of SA-βGal, p16, p21, p53 and the SASP response, that may important in the fibrotic process (Alvarez et al. 2017; Schafer et al. 2017; Minagawa et al. 2011).

Increased number of senescent airway epithelial cells that express p16 are also seen in cystic fibrosis (Fischer et al. 2013) and in bronchiectasis (Birch et al. 2016).

Senolytic Therapy

Removal of senolytic cells expressing p16INK4a using a caspase-activated system extends the lifespan of mice and delays the development of organ failure (Baker et al. 2016a). Using this approach pulmonary fibrosis induced by bleomycin and its associated inflammatory response is prevented (Schafer et al. 2017). Senolytics are drugs that mimic this effect by inducing apoptosis in senescent cells and have little or no effect on proliferating cells (Kirkland et al. 2017). Several senolytic drugs have been identified by screening and include the naturally occurring polyphonic quercitin and the cytotoxic agent navitoclax (ABT263) which inhibits the Bcl2 family of anti-apoptotic proteins (Zhu et al. 2016). A combination of quercitin and navitoclax is effective in protecting against bleomycin-induced pulmonary fibrosis in mice (Schafer et al. 2017) and navitoclax also reduces the senescence associated with lung irradiation by selectively eliminating senescent type 2 pneumocytes (Pan et al. 2017). A senolytic combination of quercitin and dasitinib also reduced the numbers of senescent type 2 pneumocytes in bleomycin-induced fibrosis in mice, with reduced p16 and SASP proteins (Lehmann et al. 2017). Another approach is to target FOXO4, which is important in maintaining senescent cell viability through binding to p53. A cell penetrant D-retro-inverso peptide FOXO4-DRI interferes with this interaction so that cells enter apoptosis and in vivo it counteracts chemotherapy-induced senescence in normal and a strain of fast ageing mice (Baar et al. 2017). Several biotechnology and pharmaceutical companies are now searching for safe and effective senolytic therapies.

PI3K-mTOR Signaling

Signaling though the phosphoinositide-3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway plays a key role in inducing cellular senescence and ageing and inhibiting this pathway extends the lifespan of many species, from yeast to mammals (Johnson et al. 2013). Rapamycin Inhibits mTOR and increases longevity in mice (Harrison et al. 2009). This may partly be due to a reduction in SASP, which reduces the paracrine spread of senescence (Laberge et al. 2015; Herranz et al. 2015). mTOR comprises two complexes, mTORC1 and mTORC2, with mTORC1 playing the most well defined role on growth and inhibited by rapamycin through its binding to the FK506-binding protein FKB12. mTORC1 is activated by growth factors, stress (including oxidative stress) and caloric nutrients, whereas mTORC2 is activated by growth factors. Inhibition of mTORC1 by rapamycin may results in up-regulation of mTORC2, which may reduce its efficacy so that dual inhibitors of mTORC1 and 2 might be more effective in chronic therapy. mTORC1 activation by oxidative and other cellular stresses and by nutrients results in increased protein synthesis through ribosomal S6 kinases with secretion of growth factors, such as TGF-β and vascular-endothelial growth factor (VEGF).

The mTOR pathway has multiple effects, including inhibition of FOXO transcription factors that are linked to longevity. There is evidence for PI3K activation in the lungs and cells of COPD patients as shown by increased expression of the downstream kinase phosphorylated AKT, which in turn activates mTOR (To et al. 2010) (Fig. 3.3). The mTORC1 complex is activated in COPD peripheral lung and peripheral blood mononuclear cells (PBMC), as shown by increased phosphorylated S6 kinase and is effectively inhibited by rapamycin (Mitani et al. 2016).

Fig. 3.3
figure 3

Reduced sirtuins and senescence. Sirtuin-1 is reduced by reactive oxygen species (ROS) through the activation of phosphoinositide-3-kinase (PI3K), mammalian target of rapamycin (mTOR) and microRNA-34a (MiR-34a). This accelerates ageing through increased acetylation of several proteins, including Ku70 that is important in double-stranded DNA repair, peroxisome proliferator gamma coactivator-1 (PGC-1), resulting in mitochondrial dysfunction, nuclear factor-kappa B (NF-κB), which orchestrates the senescence-associated secretory phenotype (SASP) and matrix metalloproteinase-9 (MMP-9), forkhead transcription factor-3a (FOXO3a) which reduces antioxidants and further increases oxidative stress. Sirtuin-6 is also reduced by oxidative stress through the same pathway, including miR-34a and this leads to activation of NF-κB, reduced β-catenin leading to reduced vascular-endothelial growth factor (VEGF), reduced telomere length and reduced Nrf2, further increasing oxidative stress. The coordinated reduction of these sirtuins leads to cellular senescence, accelerated aging and emphysema

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Not surprisingly, there are important endogenous inhibitory mechanisms to limit the activation of this pathway. PI3K is inhibited endogenously by the membrane tyrosine phosphatases phosphatase and tensin homologue (PTEN) and SH2-containing inositol-5'-phosphatase-1 (SHIP-1). Both have oxidation-susceptible cysteine residues in the active site that is readily modified by oxidative stress, and thus excessive oxidative stress reduces their catalytic activity (Worby and Dixon 2014). PTEN polymorphisms have been shown to be a genetic risk factor for COPD (Hosgood et al. 2009). PTEN activity and expression are markedly reduced in COPD lungs and this is likely to be due to oxidative stress and leads to activation of PI3K (Yanagisawa et al. 2017a).

mTOR is inhibited endogenously by 5’-adenosine monophosphate activating kinase (AMPK) which is activated by low ATP levels in the cell. AMPK is activated by the biguanide metformin, which therefore inhibits mTOR signaling and has been shown to extend the lifespan of several species, including mammals (Anisimov et al. 2008). AMPK is also activated by caloric restriction, also extends lifespan (Colman et al. 2014). This suggests that activation of the mTOR pathway may play an important role in COPD and inhibition of this pathway offers a future therapeutic opportunity (Lamming et al. 2013).

Mitochondrial Dysfunction

Mitochondrial dysfunction is an important feature of ageing and mitochondria are an important intracellular source of ROS (Correia-Melo and Passos 2015). There is a gradual accumulation of mutations in mitochondrial DNA during ageing, with a reduced resistance to damage by oxidative stress (Zheng et al. 2012). Mitochondrial function is regulated by the transcription factors peroxisome proliferator-activated receptor-gamma co-activator PGC-1α and PGC-1β, which are inhibited by telomere shortening and by mTOR activation so that with senescence there is a reduction in mitochondrial membrane potential, fragmentation and reduced mitochondria numbers. In turn mitochondria derived ROS may maintain DNA damage, thus driving further senescence through feedback loops. Damaged mitochondria are removed by autophagy (mitophagy) which is impaired in senescent cells (Garcia-Prat et al. 2016). Mitochondria play an important role in senescence and in driving the SASP response in lung disease (Birch et al. 2017).

COPD is associated with increased mitochondrial ROS production, decreased intracellular anti-oxidants and reduced numbers of mitochondria (Kirkham and Barnes 2013; Sureshbabu and Bhandari 2013). COPD cells show increased mitochondrial ROS generation (Wiegman et al. 2015). Mitochondrial dysfunction is also found in skeletal muscle of COPD patients with muscle weakness, with increased mitochondrial ROS production and decreased numbers (Meyer et al. 2013). Prohibitins (PHB), which are localized to the inner membrane of mitochondria, play a role in mitochondrial biogenesis and maintaining normal function (Artal-Sanz and Tavernarakis 2009). However, PHB1 expression is reduced in epithelial cells of smokers and to a greater extent in COPD patients, suggesting a mechanism for mitochondrial dysfunction in COPD patients (Soulitzis et al. 2012). Mitochondrial fragmentation with increased expression of mitochondrial fission/fusion markers, oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V) are found in epithelial cells from COPD patients to a greater extent than cells from normal smokers, along with SASP expression with increased IL-1β, IL-6, CXCL8 secretion (Hoffmann et al. 2013). The mitochondrial stress markers Parkin and PTEN-induced protein kinase-1 (PINK1) are also increased in COPD patients (Hoffmann et al. 2013; Mizumura et al. 2014). Knockdown of the PINK1 protects mice against mitochondrial oxidative stress induced by cigarette smoke (Mizumura et al. 2014). Damaged mitochondria are normally efficiently removed by mitophagy through highly regulated pathways, including PTEN, which may be reduced by oxidative stress and PINK1. Failure of mitophagy leads to generation of intracellular ROS and eventually cell death. PGC-1α, which is a key regulator of mitochondrial biogenesis and generation of mitochondrial ROS is increased in epithelial cells of mild COPD patients but reduced with increasing COPD severity (Li et al. 2010).

Mitochondrial dysfunction is also evident in IPF and type 2 pneumocytes show the accumulation of abnormal and dysfunctional mitochondria associated with reduced PINK1 and increased expression of profibrotic mediators (Bueno et al. 2015). PINK1 gene knockout in mice is linked to increased susceptibility to lung fibrosis.

Defective Autophagy

Autophagy is a highly regulated process that removes degraded proteins, damaged organelles (including mitochondria, as discussed above) and foreign organisms (such as bacteria), in order to maintain normal cellular function. Defective autophagy is a key characteristic of ageing cells and age-related diseases, including COPD (Mizumura et al. 2012; Mizushima et al. 2008). Autophagy removes damaged proteins, organelles and pathogens via lysosomal degradation. However, ageing cells accumulate damaged and misfolded proteins through a decline in autophagy, leading eventually to cellular senescence. Although autophagy plays a protective role in response to exogenous stress, prolonged and excessive autophagy has been associated with cell death when cytosol and organelles are destroyed irreparably. Inhibition of autophagy increases susceptibility to oxidative damage and apoptosis, whereas activation of autophagy leads to inhibition of apoptosis (Murrow and Debnath 2013). Cigarette smoke and oxidative stress activate autophagy, indicating that autophagy may be a protective response designed to remove damaged proteins and organelles. Alveolar macrophages from smokers show defective autophagy and this could contribute to accumulation of aggregates, abnormal mitochondrial function and defective clearance of bacteria (Monick et al. 2010). Patients and mice with emphysema show increased autophagy markers in lung tissue, with increased activation of autophagic proteins, such as light chain-3 (LC3-II), and autophagy may be contributory to apoptosis and alveolar cell destruction (Chen et al. 2010). Other studies have demonstrated increased autophagic vacuoles (autophagosomes) in COPD. However, it is possible that completion of autophagy may not occur (defective autophagic flux). Macrophages from smokers show defective autophagic flux, resulting in accumulation of the substrate of autophagy, p62, and misfolded proteins due to dysfunctional lysosomal digestion of the autophagosomal load caused by a reduction in the lysosomal protein LAMP2 (Monick et al. 2010). Inhibition of autophagy after cigarette smoke exposure leads to accumulation of p62 and ubiquitinated proteins in airway epithelial cells, resulting in increased cellular senescence and SASP with secretion of CXCL8, thereby mimicking the changes seen in COPD cells (Fujii et al. 2012). Loss of autophagy might account for reduced mitophagy and also contribute to defective phagocytosis of bacteria in COPD (Donnelly and Barnes 2012). Autophagy may be impaired through the activation of PI3K-mTOR signaling, resulting in inhibition of unc-51 like autophagy-activating kinase-1 (ULK1) complex that normally activates autophagy, thereby linking defective autophagy to the accelerated ageing mechanisms (Dunlop and Tee 2014). Defective autophagy and mitochondrial function play a key role in IPF and the generation of profibrotic mediators, such as TGF-β (Mora et al. 2017).

Stem Cell Exhaustion

The clearance of senescent cells means that progenitor cells are needed to maintain cell numbers, but this may become less efficient or may exhaust the regenerative capacity of stem cells. Stem cell exhaustion and depletion are characteristic feature of ageing (Lopez-Otin et al. 2013).

Senescence of mesenchymal stem cells (fibroblast, endothelial cells) may be a mechanism of emphysema and the failure to repair lung injury. Damaged alveolar cells may be replaced by progenitor cells, particularly alveolar type 2 (AT2) cells (Kubo 2012). However, once the alveolar architecture is destroyed by elastases, such as MMP9, progenitors cannot rebuild the appropriate functional lung structure. In stem cells, ROS force cells out of quiescence and into a more proliferative state by activating PI3K/AKT signaling and further promoting the production ROS, thus repressing FOXO-mediated stress response and autophagy. AT2 cells are progenitors for type I alveolar cells and show evidence of senescence in patients with emphysema (Tsuji et al. 2006).

Circulating endothelial progenitor cells (EPC) and specifically blood outgrowth endothelial cells are made in the bone marrow and are important in maintaining endothelial integrity (Fadini et al. 2012). EPC from smokers and COPD patients show cellular senescence and increased DNA double-strand breaks compared to cells from non-smokers and this is correlated with reduced expression of sirtuin-1 (Paschalaki et al. 2013). These senescent stem cells are poorly effective in repairing endothelial damage and provide a link between COPD and vascular ageing that leads to ischaemic heart disease.

In diseases of accelerated ageing, stem cells, including AT2 cells and EPCs, show features of cellular senescence, oxidative stress-induced DNA damage. This leads to loss of regenerative capacity and eventually to organ failure, including lungs (Sousounis et al. 2014). Understanding the molecular mechanisms of stem cell ageing is critical to elucidating accelerated ageing in the lungs (Oh et al. 2014). Senescent stem cells characteristically show increased generation of mitochondrial ROS, mitochondrial damage and dysfunction, activation of the PI3K-mTOR signaling, DNA damage with double-stranded DNA breaks and a defect in the DNA damage-sensing kinase ATM (ataxia telangiectasia mutated), defective proteostasis and deficiency of anti-ageing molecules, such as sirtuin-1 and FOXO transcription factors. EPCs from COPD patients show activation of PI3K, reduced ATM and reduced sirtuin-1, all of which may be reversible by resveratrol, a sirtuin activator (Paschalaki et al. 2013). Accumulation of various types of DNA damage in stem cells with age may be a major mechanism leading to progressive failure of stem cells to repair damage of organs and thus to slowly progressive organ failure (Behrens et al. 2014). Epigenetic changes in stem cells, including DNA methylation and histone acetylation, also play an important role in stem cell senescence.

MicroRNA and Lung Ageing

MicroRNA (miRNA) are small non-coding single-stranded RNAs of 18-22 nucleotides that regulate the post-transcriptional expression of genes by inhibiting their translation or inducing degradation of mRNAs by binding to complimentary sequences in the 3’-UTR of mRNAs. There is increasing evidence that miRNAs play an important role in in the ageing process and may regulate several key proteins involved in cellular senescence, such as p16 (miR-24), p53 (miR-885-5p), SASP (miR-146) and reduced sirtuin-1 expression (miR-34a) (Gorospe and Abdelmohsen 2011; Munk et al. 2017). Genome-wide assessment of miRNA expression in monocytes of elderly compared to young individuals show that miRNA tend to decrease with age and regulate PI3K-mTOR signaling and DNA repair (Noren Hooten et al. 2010). MiRNAs that have been implicated in cellular senescence are also increased in diseases of accelerated ageing, including COPD (Dimmeler and Nicotera 2013).

MiR-34a, which is known to down-regulate sirtuin-1, shows increased expression in peripheral lungs and epithelial cells of COPD patients and is correlated with increased markers of cellular senescence in lung cells (Baker et al. 2016b). MiR-34a also regulates sirtuin-6, which is reduced in COPD along with sirtuin-1 (Nakamaru et al. 2009). MiR-34a is increased by oxidative stress through activation of PI3K-mTOR signaling pathways and this leads to a parallel reduction in sirtuin-1 and sirtuin-6, whereas other sirtuins are unchanged. Importantly, an antagomir of miR-34a restores sirtuin-1 and -6 in small airway epithelial cells from COPD patients, reduces markers of cellular senescence (p16, p21, p53), reduces the SASP response (IL-1β, IL-6, TNF-α, CCL2, MMP9) and increases the growth of senescent epithelial cells (Baker et al. 2016b). This suggests that blocking specific miRNA may result in rejuvenation of these cells. MiR-34a is also increased in macrophages of COPD patients and this may be associated with the impaired phagocytosis and uptake of apoptotic cells (efferocytosis) that is seen in COPD patients (McCubbrey et al. 2016). MiR-126, which plays a key role in maintain vascular integrity and endothelial cell function, is reduced in endothelial progenitor cells and airway epithelial cells and from COPD patients and plays a key role in regulating the DNA damage response pathway that is linked to cellular senescence (Paschalaki et al. 2018).

MiRNAs are exported from cells in extracellular vesicles, which include microvesicles and exosomes and then may be taken up by other cells (Fujita et al. 2015; Turturici et al. 2014). This might provide a means of spreading senescence from cell to cell within the lung, but also via the circulation to other organs, resulting in accelerated ageing in other systems or multimorbidity (Barnes 2015; Kadota et al. 2018). Thus, targeting miRNAs may be a future therapeutic strategy for treating and preventing cellular senescence and delivery of miRNA via extracellular vesicles and nanoparticles might be a novel way to deliver there therapies (Jiang and Gao 2017).

Epigenetic Mechanisms

There is little evidence that genes have important effects on the ageing process, but increasing evidence that epigenetic mechanisms may be important, with long-term changes in gene expression due to environmental exposures. These epigenetic changes include DNA methylation and modifications in histone proteins (for example, by acetylation, methylation, phosphorylation), which result in increased or decreased gene transcription (Song and Johnson 2018). Chromatin structure changes with age and becomes more “open”, which means it is more transcriptionally active (Feser and Tyler 2011). There is a decline in histone proteins with ageing and consistent changes in histone modifications and associated modifying enzymes, with increased histone acetylation and either increased or decreased histone methylation. Changes in histone methylation with age are likely to be important in accelerated ageing and changes in trimethylation of lysine 4 on histone-3 (H3K4me3) occur during ageing and regulate cellular senescence (Zhang et al. 2014). At present there is little information about histone methylation status in diseases of accelerated ageing, such as COPD. Histone methylation plays an important role in regulating the various proteins involved in autophagy, DNA repair and SASP. The role of histone methyltransferases and demethylases in the regulation of cellular senescence is under intense investigation now that selective enzymes inhibitors are in development. Coactivator-associated arginine methyltransferase-1 (CARM1) deficiency causes cellular senescence in type 2 pneumocytes and accelerates the development of emphysema in mice, indicating a key role for histone methylation in protecting against lung ageing (Sarker et al. 2015). Changes in DNA methylation have been described in COPD cells and may play a role in cellular senescence (Clifford et al. 2018). For example, DNA methylation of the FOXA2 and SPDEF genes in airway epithelial cells of COPD patients has been linked to mucus hypersecretion (Song et al. 2017).

Epigenetic changes associated with ageing are of particular relevance in stem cell populations and lead to dysfunction and depletion (Armstrong et al. 2014). Mesenchymal stem cells from elderly people appear to have less potential for differentiating into different cells types and this is linked to changes in DNA methylation (Horvath 2013).

Immunosenescence

The immune system becomes less effective during ageing, with impaired immune responses to new antigens, a greater susceptibility to infection and an increased tendency to develop autoimmunity. Immunosenescence affects both innate and adaptive immunity resulting in loss of function and has been implicated in chronic inflammatory diseases. Immunosenescence is important in several pulmonary diseases, including COPD, severe asthma and IPF (Murray and Chotirmall 2015). Ageing of innate immunity leads to defective function in innate cells, with impaired cell migration and signaling through pattern recognition receptors, such as Toll-like receptors (TLR) (Hazeldine and Lord 2015). Neutrophils show decreased phagocytosis, chemotaxis and apoptosis and have reduced directional function that is corrected by PI3K inhibition (Sapey et al. 2014). This defect is exaggerated in patients with COPD (Sapey et al. 2011). Macrophages show reduced phagocytosis with age and this is increased to a greater extent in COPD macrophages (Donnelly and Barnes 2012). Dendritic cells produce less interferon and reduced TLR signaling, which is associated with decreased T-cell-mediated immunity, resulting in reduced ability to protect against pathogens in the elderly and an increased risk of carcinogenesis (Mahbub et al. 2011). However in patients with severe COPD there is greater expression of TLR4, which is associated with an amplified inflammatory response to colonizing bacteria (Di Stefano et al. 2017).

Defects in adaptive immunity are also commonly seen in COPD and may be related to ageing. There is a loss of naïve T cells and B cells as the thymus involutes with age, as well as telomere shortening, with consequent reduced responses to new antigens. A characteristic of T cells from elderly individuals is a decrease in CD4+/CD8+ ratios and a loss of the co-stimulatory molecule CD28, with an increase in both CD4+CD28null and CD8+CD28null cells, which have reduced immune and vaccine responses (Arnold et al. 2011). In COPD CD8+CD28null cells have reduced HDAC2 expression and are corticosteroid-resistant (Hodge et al. 2015).

Autoimmunity is also a manifestation of immunosenescence with increased production of autoantibodies that may lead to further tissue damage. In COPD there are increased autoantibodies directed against endothelial cells and against carbonylated proteins formed by exposure to oxidative stress in severe disease (Feghali-Bostwick et al. 2008; Karayama et al. 2010; Kirkham et al. 2011). This increase in autoimmunity has been associated with an imbalance between Th17 and regulatory T cells (Treg). An increased ratio of Th17 to Treg cells is found in sputum In COPD patients (Maneechotesuwan et al. 2013). Treg cells may even transform into Th17 cells under disease conditions with loss of the key Treg transcription factor Foxp3 (Komatsu et al. 2014).

There is evidence for activation of the PI3K signaling pathway. In ageing neutrophils the impaired chemotaxis response is associated with increased PI3K and restored by a PI3K inhibitor (Sapey et al. 2014). A rapamycin analog, everolimus, reduces immunosenescence in normal elderly individuals, with an increased antigenic response to influenza vaccination and a reduction in T cells expressing programmed death-1 (PD-1) receptors that are increased in senescent CD4+ and CD8+ cells (Mannick et al. 2014).

Reduced Anti-ageing Molecules

Many endogenous anti-ageing molecules that counteract the mechanisms of senescence and a reduction in their expression may accelerate the ageing process. Defective anti-ageing molecules have been suggested as a mechanism for accelerated lung ageing in COPD and other pulmonary diseases (Ito and Barnes 2009; Faner et al. 2012; Mercado et al. 2015).

Sirtuins

Silent information regulators or sirtuins are well recognized as anti-ageing molecules that regulate lifespan. Sirtuins are highly conserved NAD+-dependent enzymes that play a role in resistance to stress, genomic stability and energy metabolism (Finkel et al. 2009; Watroba et al. 2017). Of the 7 sirtuins found in mammals most attention has centered on sirtuin-1 and sirtuin-6, as both are associated with prolongation of lifespan in many species, including mammals (Guarente 2011). Sirtuin-1 deacetylates many key regulatory proteins and transcription factors that are known to be involved in DNA repair, inflammation, antioxidant gene expression and cellular senescence, including the PI3K-AKT-mTOR pathway and autophagy (Fig. 3.3). Sirtuin-1 deacetylates the transcription factor FOXO3a, which enhances antioxidant responses (particularly superoxide dismutases) and inhibits p53-induced cellular senescence, inhibits NF-κB leading to suppression of inflammation and activates PGC-1α, which maintains mitochondrial function. Sirtuin-1 is markedly reduced in peripheral lung and circulating PBMC of patients with COPD (Nakamaru et al. 2009; Rajendrasozhan et al. 2008; Baker et al. 2016b). Sirtuin-1 is reduced by oxidative stress via reduction in PTEN and activation of the PI3K-mTOR pathway and in turn sirtuin-1 inhibits mTOR signaling. Sirtuin-1 also activates autophagy by inhibiting mTOR (Lee et al. 2008). The polyphenol resveratrol, found in the skin of red fruits and in red wine, activates sirtuin-1, but is poorly bioavailable, so this has led to the development of more potent sirtuin-1 activating compounds (STACs), which are now in clinical development for the treatment of age-related diseases (Hubbard and Sinclair 2014; Bonkowski and Sinclair 2016). Sirtuin-6 is an ADP-ribosylase as well as protein deacetylase and plays a key role in regulating DNA repair, telomere maintenance, inflammation and metabolic homeostasis and, like sirtuin-1, is linked to extension of lifespan (Kugel and Mostoslavsky 2014). Sirtuin-6 expression is reduced in the lungs of patients with COPD (Nakamaru et al. 2009; Takasaka et al. 2014). Reduced sirtuin-6 is found in airway epithelial cells of COPD patients and is reduced by cigarette smoke exposure, resulting in cellular senescence and impaired autophagy (Takasaka et al. 2014). As discussed above, oxidative stress activates PI3K-mTOR signaling, which increases the expression of miR-34a that directly inhibits sirtuin-1 and sirtuin-6 mRNA and protein expression without any effects on the other sirtuins (Baker et al. 2016b). Circulating sirtuin-1 concentrations are also reduced in patients with COPD (Yanagisawa et al. 2017b).

The role of sirtuins in other lung diseases is less well defined. Reduced sirtuin-1 is found in PBMC from elderly patients with severe asthma and appears to increase IL-4 secretion though increased acetylation of the transcription factor GATA-3 (Colley et al. 2016). Sirtuin-1 knockout in mice enhances lung fibrosis, and sirtuin-1 inhibits TGF-β fibroblast activation. Paradoxically sirtuin-1 is increased in the lungs of IPF patients, perhaps as a compensatory mechanisms (Zeng et al. 2017).

Klotho

Animal models of accelerated ageing have identified other key molecules involved in senescence, Klotho is defective in a mouse model of premature ageing, which has a substantially decreased lifespan, with features of accelerated ageing, including emphysema (Dermaku-Sopjani et al. 2013). Klotho is a transmembrane protein that is a co-receptor of fibroblast growth factor (FGF23) and regulates insulin/IGF signaling, phosphate homeostasis, cell survival and proliferation. It is protective against oxidative stress and decreased expression of Klotho is found in epithelial cells of COPD patients and is reduced by oxidative stress, resulting in increased release of inflammatory cytokines from these cells (Gao et al. 2015). Circulating concentrations of Klotho are also reduced in patients with COPD (Kureya et al. 2016).

SMP-30

Senescence marker protein-30 (SMP30) is an anti-ageing molecule that was identified in a mouse model of ageing, which regulates calcium homeostasis and is also sensitive to oxidative stress (Feng et al. 2004). Reduced expression of SMP30 is found in aged tissues, including lung. In SMP30 knockout mice there is a increased susceptibility to development of emphysema after exposure to cigarette smoke (Sato et al. 2006).

Implications for Therapy

Considerable progress has been made in understanding the molecular mechanisms involved in accelerated ageing in chronic lung diseases. Although it is unrealistic (but not impossible) to expect reversal of the normal ageing process, it may be possible to reduce the mechanisms that accelerate senescence in these diseases. Several novel therapeutic targets that have been identified leading to the development of geroprotectors (Moskalev et al. 2017). Existing drugs (such as metformin and rapamycin) may be repurposed and novel drugs developed by screening and rational drug design. The potential for these drugs is that they may treat multimorbidities, which are diseases of ageing that occur together in the elderly, as there appear to be common pathways (Barnes 2015). Future therapies may also involve lifestyle interventions, such as diet and increased physical activity (de Cabo et al. 2014).

Pharmacological Therapies

Better understanding of cellular senescence mechanisms has identified several potential therapies for inhibiting accelerated ageing in lung disease (Fig. 3.4) (Ito et al. 2012). As discussed above, the PI3K- mTOR pathway plays a key role in cellular senescence and inhibition of autophagy, and inhibitors of this pathway may extend lifespan in several species, including mammals. Rapamycin induces autophagy, increases sirtuin-1 and extends the lifespan of all organisms, including mammals (Bjedov and Partridge 2011; Lamming et al. 2013). Rapamycin is very effective in inhibiting activated mTOR in COPD cells in vitro (Mitani et al. 2016). However, rapamycin and its analogues (rapalogs) have several adverse effects, including mouth ulceration, anaemia, pneumonitis and delayed wound healing, making it unsuitable for long-term use. A pilot study in elderly humans demonstrated its potential in lower doses in reversing immunosenescence and this was not associated with significant adverse effects (Mannick et al. 2014). Metformin is widely used to treat type 2 diabetes and indirectly activates AMPK, resulting in inhibition of mTOR and extension of lifespan in mice, possibly though increasing Nrf2-induced antioxidant gene expression (Anisimov et al. 2008; Martin-Montalvo et al. 2013). Metformin is relatively well-tolerated, it might be a suitable therapy for treating accelerated ageing in COPD and other pulmonary diseases. It has been shown to be well-tolerated in COPD patients (Hitchings et al. 2016). PI3K signaling may also be inhibited by activators of the endogenous inhibitor SHIP-1 (Stenton et al. 2013) and these drugs have already entered into clinical trials for treatment of allergic disease (Leaker et al. 2014).

Fig. 3.4
figure 4

Targeting cellular senescence in lung disease. The PI3K-mTOR pathway driven by reactive oxygen species (ROS), which activate phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR), which activate microRNA-34a (miR-34a) to inactivate and reduce sirtuin-1 activity, which accelerates the ageing process. There are several endogenous and exogenous inhibitors of this pathway. Increased ROS may be the result of reduced activity of the antioxidant regulating transcription factor Nrf2. There are several ways to modulate this pathway (green boxes). Nrf2 may be activated by small molecule activators, ROS may also be counteracted by antioxidants. PI3K is inhibited by the phosphatases PTEN (phosphatase and tensin homolog) and SHIP-1 (SH2-containing inositol-5'-phosphatase-1). SHIP-1 activators are now in development and there are several PI3K inhibitors. mTOR is inhibited by rapamycin and its less toxic analogs and endogenously by AMP-kinase (AMPK), which may be activated by caloric restriction and by metformin. Increased miR-34a may be targeted specifically by antagomirs. Sirtuin-1 may be activated by resveratrol and novel sirtuin-activating compounds (STACs). Finally senescent cells may be removed by senolytic drugs

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Naturally occurring molecules may also be effective and may be obtained via dietary supplementation (neutraceuticals). Quercitin, a polyphenol found in apples, is also an activator of AMPK (Mitani et al. 2017) as well as a senolytic (Schafer et al. 2017). Resveratrol, found in the skins of red fruit and in red wine and increases lifespan in several species, including mice, through the activation of sirtuin-1. In vitro resveratrol inhibits the SASP response in COPD epithelial cells (Culpitt et al. 2003) and is effective against neutrophilic inflammation induced by lipopolysaccharide in mice (Birrell et al. 2005). However, resveratrol and related dietary polyphenols have poor oral bioavailability, rapid metabolism and low potency, which has led to the development of novel potent synthetic analogs known as STACs, which work on an allosteric mechanism to stimulate sirtuin-1 activity (Hubbard and Sinclair 2014; Bonkowski and Sinclair 2016). In mice exposed to cigarette smoke the STAC SRT-2171 prevents the increase in MMP-9 that is associated with neutrophilic inflammation and improves lung function (Nakamaru et al. 2009). In addition, resveratrol reduces senescence in EPCs from COPD patients through an increase in sirtuin-1, indicating that exhausted stem cells may be an important target of STACs (Paschalaki et al. 2013). A resveratrol derivative isorhapontigenin, which is found in Chinese herbal remedies used to treat inflammatory diseases, is more potent than resveratrol at inhibiting the SASP response in COPD cells and has a better oral bioavailability (Yeo et al. 2017).

Oxidative stress is an important driving mechanism leading to accelerated ageing in lung disease, suggesting that antioxidants should be effective therapies. However, existing antioxidants, such as N-acetyl cysteine (NAC), are poorly effective as they are thiol derivatives that become inactivated by oxidative stress. Novel antioxidants include NADPH oxidase (NOX) inhibitors, superoxide dismutase mimetics and activators of Nrf2 (Saso and Firuzi 2014). Since there is strong evidence for mitochondria-derived oxidative stress in age-related lung diseases an intracellular antioxidant may be more effective. The mitochondrial antioxidant SkQ1 reverses ageing-related biomarkers in rats, whereas NAC is ineffective (Kolosova et al. 2012). Nrf2 activators are of particular interest as Nrf2 may be defective in several disease models of accelerated ageing, including COPD. Sulforaphane, which occurs naturally in broccoli, is an Nrf2 activator but is non-specific and toxic in high concentrations, leading to a search for new drugs. Bardoxelone methyl is more potent but also has shown toxicity in clinical studies.

Lifestyle Interventions

Caloric restriction (CR) has been shown to prolong lifespan in species from yeast to mammals, including primates (Colman et al. 2014). This leads to inhibition of PI3K-AKT-mTOR signaling though activation of AMPK and reduces the release of insulin and insulin-like growth factor(IGF)-1 and increases sirtuin-1 (Fontana et al. 2010). Several intermittent fasting regimes that give sufficient CR to activate anti-ageing pathways are now being explored. Periodic fasting has been found to be effective in several animal models of age-related diseases but has not been explored in age-related lung diseases. The Mediterranean diet, is rich in fruit, vegetables, red wine and olive oil, contains flavones, polyphenols and stilbenes that may activate sirtuin-1 and increases healthy life, with reduced incidence of neurodegenerative, cardiovascular and metabolic diseases and cancer (Perez-Lopez et al. 2009). For example, a Mediterranean diet may improve the function of EPCs from elderly patients and thus improve endothelial function in cardiovascular disease (Marin et al. 2013). Several compounds that occur in foodstuffs, as outlined above, may also reduce senescence.

Physical inactivity is a risk factor for the development of diseases of ageing, such as COPD, and is an important determinant of mortality (Gulsvik et al. 2012). Aerobic exercise training provides significant clinical benefits in several age-related biomarkers, including lipid profiles, blood pressure, glucose tolerance, bone density, depression, loss of skeletal muscle (sarcopenia) and quality of life (Fleg 2012). Exercise is the major component of pulmonary rehabilitation in COPD, which results in improved lung function, reduced exacerbations and improved quality of life, although it is not certain whether it is able to reduce mechanisms of accelerated ageing (Casaburi and ZuWallack 2009).