Healthy Ageing Across the Life Span

Healthy ageing is a critical but often overlooked factor in the investigation of fundamental biology and disease progression in the lung. It has a profound effect on the regenerative capacity of the lung thought to be mediated by tissue-resident stem-progenitor cells [1–5]. Both intrinsic and extrinsic factors conspire to diminish the potential of stem-progenitor cells to self-renew and differentiate, participate in homeostasis, and respond to injuries and disease in the ageing lung. While the changes in tissue architecture and pulmonary function that accompany ageing are well known, the relationships between stem-progenitor cell ageing and organ regeneration in the lung are poorly understood. Studies of ageing have been segmented into life stages. These include lung development, a post-development period where lung regeneration can be reinitiated (e.g., by partial pneumonectomy, “PNX”), a period after which reinitiation is no longer feasible, and a “late ageing” period typified by senescence.

During development, the growth of airways and acini follows a pattern of symmorphosis, optimizing ventilation-perfusion matching, and minimizing convective losses, generating the so-called good lung [6]. This is the only period where structure and function of the lung are in perfect balance in healthy mammals.

In the period immediately following the end of lung development (age 2 years in humans [7] and 4 weeks in rodents), somatic growth of the chest continues in the absence of alveolar multiplication. However, the lung remains competent to reinitiate new growth of acini including respiratory bronchioles, alveolar ducts, and alveoli in response to sufficient mechanical stimuli such as partial pneumonectomy (PNX) [8, 9]. For example, in immature dogs, as little as 45 % PNX is an effective stimulus, initiating the restoration of alveolar volume, surface area, and gas exchange. Importantly, no other lung injury permits the elucidation of age-dependent regenerative mechanisms in the lung as accurately and comprehensively as the PNX model. In contrast, injuries elicited by bleomycin or naphthalene administration, or influenza infection damage the airway and alveolar epithelia and promote re-epithelialization (normal or aberrant), but without demonstrable neo-alveolarization or neo-angiogenesis.

Ageing of the lung is first evident as discrepancies between post-natal lung growth and the regenerative process that occurs after PNX. Firstly, multiple studies show that alveolar multiplication after PNX in rodents is incomplete, with total lung alveolar numbers reaching approximately 80 % (not 100 %) control values [10, 11]. Secondly, regrowth after PNX is non-uniform, with greatest alveolarization in lung lobes (e.g., the cardiac lobe, also called the accessory lobe) [12–16] and lung regions (medial and mediastinal lobes) [17] that experience the greatest mechanical stress. These lobes also display more cellular proliferation [17, 18] and expression of genes encoding growth and differentiation [19]. Thus, it can be quite useful to focus sampling and mechanistic studies on the cardiac (accessory) lobe in this model. Interestingly, non-uniformity of lobar regrowth is present even 1 year after PNX, whether the surgery is performed in the immature (post-natal) or mature (post-development) animal. This implies that reactivation of signals during post-natal lung development does not mitigate or exaggerate structural non-uniformity [14, 20]. Rather this appears to be a pattern established by the topography of mechanical stress placed on the organ after PNX irrespective of age. Growth of alveoli after PNX is accompanied by early bronchiolar lengthening without commensurate bronchial dilation, resulting in dysanaptic growth [9, 21]; therefore, contribution from distal airway epithelium to lung regeneration is important to regeneration per se, but not identical to development. Despite the topographical-functional differences that may exist between alveolarization during late lung development and after PNX, the latter serves as a convenient, minimally injurious model system for the study of reinitiation, in an organ with slow cell and matrix turnover.

Lung regenerative responses are absent or incomplete in the ageing lung, after a transient period whereby reinitiation of alveolarization can be induced by PNX [22–24]. Even 5 years after PNX in mature dogs, there was no evidence of compensatory lung growth [25]. Furthermore, after extensive lung resections (55 %) which evoke substantial mechanical stretch in adult dogs, there was no evidence of new alveolar development [26, 27]. Retinoic acid treatment which mediates early lung bud formation and branching morphogenesis also failed to induce re-alveolarization or improve lung function in pneumonectomized adult dogs [27–30]. In the absence of alveolar multiplication, alveolar septal cells respond to PNX not by hyperplasia of typical stem-progenitor cells, but by local “adaptation” responses, i.e., accommodation of mechanical stresses by transiently intense remodeling of the alveolar septae including hyperplasia of endothelial, interstitial, and epithelial cells and matrix without formation of secondary septae [31]. Precise in vivo identity of the cells that participate in the adaptive response to stress in mature animals has not been determined. Eventually, these adaptations are remodeled to restore the harmonic mean septal barrier thickness, yet, twofold cell numbers persist in those regions. These data demonstrate that ageing to maturity results in a shift in the nature of responses to stress, rather than the absence of responses in the lung. Given that the lung continues to respond to mechanical stress, it is possible that post-PNX lung growth potential has been underestimated in humans, since most studies have not spanned decades or an equivalent period in animal. For example, one report provided compelling evidence for new acinar development which was discovered 15 years after PNX in a 33-year-old woman [32]. Studies of post-resection patients vary considerably with regard to the concise age range over which compensatory lung growth (including alveolar multiplication) occurs. In animals, the alveolar multiplication is observed for several months after sexual maturity is reached [23]. In humans, it is generally thought that efficient compensatory lung growth is arrested much earlier (e.g., 2–3 years of age) [23]. Knowledge arising from longitudinal study of remnant lung in mature living lung donors and lobectomy patients, using advanced noninvasive imaging modalities [33–35] may refine our understanding of regenerative potential in the adult lung.

Advanced ageing in the lung is characterized by irreversible structural and functional decline including severe enlargement of airspaces, diminution of elastic recoil associated with loss of elastin fibers, accumulation of collagen III (col-III) in alveolar septae and col-IV in basement membranes, reduced forced vital capacity, increased residual volume, impaired gas exchange, and increasing vulnerability to infection, fibrosis, and cancer [36, 37]. Forced vital capacity can be less than 50 % of peak values in elderly >80 year of age [38–40]. In rodents, there is similar loss of elastin, accumulation of collagen, and increased mean chord length [41]. The lung of humans (as well as rodents) adopts the appearance of “senile emphysema,” characterized by greatly dilated alveolar ducts and alveolar spaces without necessarily a reduction in alveolar numbers [42, 43]. One study posits that in women the decline in estrogen and upregulation of estrogen receptors with ageing promotes this phenotype, given that it is reversible by estrogen replacement therapy in mouse models [44]. Intrinsic and extrinsic mechanisms of ageing contribute to this final transformation, including telomere shortening, oxidative stress, DNA damage, mitochondrial dysfunction, and matrix (and thus niche) disruption, resulting in cellular senescence and stem cell exhaustion [45], acknowledging that these mechanisms are inextricably linked. Knowledge concerning the participation of each of these ageing factors in the attrition of lung stem-progenitor cells and their homeostatic functions at the cellular-molecular level are compelling topics of research with great translational significance, yet have been largely neglected in the lung.

Cellular Participants of Lung Regeneration Vulnerable to Ageing

Alveolarization relies on a specialized population of myofibroblasts derived from PDGFRαpos fibroblasts which are dedicated to secondary septation [46]. The subpopulation of precursor PDGFRαpos fibroblasts and PDGFRα expression itself are under the reciprocal control of PPARγ which drives the elastogenic “structural” phenotype, and FGFR2 which drives α-smooth muscle actin expression and the “contractile” phenotype [47]. The magnitude of PDFGRα expression is also associated with differentiation toward myofibroblasts versus preservation of secretory lipofibroblasts that do not participate in secondary septation [48, 49]. Moreover, decreased Sox9 and increased MRTF-A and α-smooth muscle actin in PDGFRαhigh alveolar fibroblasts have been associated with differentiation to myofibroblasts from P8 to P12 [49]; interestingly, loss of Sox9 in respiratory epithelium is inconsequential to lung development [50] implying that developmental pathways that persist specifically in interstitial fibroblasts may be crucial for sustaining post-natal development and beyond. Lipofibroblasts identified as CD166neg reserve the potential to differentiate to CD166pos myofibroblasts and support epithelial stem cells through secretion of FGF10, particularly in the absence of TGFβ1 [51]. Attesting to the role for TGFβ1, induction of the post-natal lung exposed to hyperoxia leads to excessive TGFβ1 and bronchopulmonary dysplasia [52] perhaps reducing lipofibroblast frequency or survival. Lipofibroblast differentiation to myofibroblasts is disrupted by an age-related elevation of Wnt4, through a β-catenin-dependent mechanisms [37] thus factors that secure a proper balance between lipofibroblast secretions (e.g., FGF10, SDF1) and differentiation capacity to myofibroblasts are important to the understanding of alveolarization during lung development and beyond.

That interstitial fibroblasts are critical to lung regeneration is further evidenced by the over-expression in lung of genes encoding proteins associated with mesenchymal cell proliferation (Igf1, Cyr61, Tnfrsf12a, Ctgf, Igfbp3, and Ifgbp2) [53, 54]. Further, in isolated lipofibroblasts (Sca-1pos, EpCAMneg, CD45neg, and CD31neg), genes encoding many important growth factors (Midkine, Fgf10, Vegfb, Tgfb3, Ifgbp2/3, and Gdf1/10) and extracellular matrix (ECM) (Col1, Col III, Eln, Fn, and Fbn1) are over-expressed [51, 55–57]. Overall stromal fibroblasts appear to be critically important to lung development and regeneration, but gain and loss of function and genetic lineage-tracing studies are needed to better define the temporal and spatial role for these cells.

Angiogenesis is implicit to lung development and post-PNX neo-alveolarization, effectively matching perfusion with alveolar ventilation. Peak endothelial cell expansion and neo-angiogenesis occurs approximately 7 days after PNX and is concentrated in subpleural regions notably in lobes with greatest mechanical stress and regrowth (i.e., cardiac lobe) [58–61]. VEGF markedly accelerates compensatory lung growth after PNX through stimulation of endothelial (and quite possibly AECII) proliferation, although bFGF which was effective in augmenting liver regeneration in past studies was ineffective in this respect [62]. Endothelial cell production of VEGFA [60], HIF1α [63, 64], PDGFBB [65], nitric oxide synthetase [66], and epoxyeicosanoids [67] are also shown to be important to post-natal alveolarization or post-PNX lung regeneration. Moreover, pulmonary capillary endothelial cells (PCEC) produce MMP14 under regulatory control by VEGFR2 and FGFR1 that cause the shedding of EGFR ligands from matrix and surrounding cells necessary for expansion of neighboring AECII and BASC after PNX [68]. These data expose additional, potentially age-sensitive mechanisms. Senescence is likely to impact several of these mechanisms; for example, nitric oxide signaling is markedly disturbed with ageing as are several protective antioxidant mechanisms (e.g., SOD3) [69], and the HIF1α-adrenomedullin axis is markedly depressed with age in rats [70].

Type II alveolar epithelial cells (AECII) are widely considered to be stem cells of the alveolar compartment, which amplify and differentiate to type I alveolar epithelial cells (AECI) following injury to alveolar epithelial cells [71, 72]. A subpopulation of AECII possesses telomerase expression, further evidence of their long-term self-renewal potential and “stemness” [73]. In the event that AECII are depleted as a consequence of epithelial injury, e.g., injuries due to bleomycin or influenza [74], data assert that non-AECII cells including bronchioalveolar stem cells [75], integrin α6β4 expressing cells [76], c-kitpos multi-lineage potential lung stem cells [77], CD90pos AECII progenitors [78, 79], CD45neg CD31neg EpCAMhigh integrin α6pos CD104pos CD24low lung cells [80], or low density epithelial precursors [81] from healthy rodent or human lungs may reconstitute AECII. AECII can also arise from distal airway Scgbln1 expressing cells (Club cells) in bleomycin [82, 83] or influenza injury [83, 84] consistent with the idea that damage to AECII promotes cytokine driven autocrine repair and generates an inductive milieu for differentiation of cells to the AECII phenotype [85]. AECII transplantation augments post-PNX compensatory lung growth in rats whose compensatory lung growth was limited suggesting a significant paracrine role for AECII [86]. In this regard, AECII produce paracrine signals that control elastogenesis through FGFR3/4 on interstitial fibroblasts [87] and during development, FGFR3 and FGFR4 double-null mice fail to undergo alveologenesis [88]. Signaling of HGF from endothelial cells to c-Met on the surface of AECII is required for normal lung development; knockdown of c-Met in AECII of adult lungs reduces AECII frequency (consistent with HGF improvement of AECII survival), airspace dilation, oxidative stress and inflammation, as well as pruning of the vascular bed [89], reminiscent of the aged lung. Lung mesenchymal stromal cells (“l-MSC”: Sca-1pos, EpCAMneg, CD31pos, CD45pos) supports by co-culture epithelial stem-progenitor cell colony formation and self-renewal, features that can be substituted by l-MSC derived paracrine factors (HGF, FGF10) [80]. Furthermore, Lgr6pos multipotent epithelial stem-progenitor cells [90] under the control of p38α expression secrete SDF-1 which activates stromal fibroblasts in the lung [91]. Hence, epithelial-mesenchymal and epithelial-endothelial crosstalk through PPARγ, FGFR/FGF, HGF/c-Met, and SDF-1/CXCR4 signaling pathways elaborate bidirectional mechanisms that with ageing, are presumed to disrupt alveolar homeostasis and regenerative capacity. Indeed, Paxson [55] was able to show that FGFR1, a cornerstone in lung development and upregulated after PNX, is dramatically downregulated (>600-fold) in isolated lung fibroblasts (CD45neg CD31neg EpCAMneg Sca-1pos) from 12-month-old mice when compared to 3-month-old mice. Significant work needs to be done to refine our understanding of signaling pathways during late development that are most vulnerable to age.

Ageing Transition Between Post-Natal and Post-PNX Lung

The question whether lung regeneration is a recapitulation of development remains critical to our understanding of ageing in the lung [23, 92, 93]. Early studies showed that the post-PNX lung is distinct with regard to several molecular pathways, including erythropoietin receptor processing [94], epidermal growth factor and receptor expressions, and surfactant protein A and C expression [95]. This has led to further genome-wide investigations of transcriptional regulation, comparing lung development in adulthood before or after PNX. Wolff et al. [53] compared whole lung gene expression arrays (G4122A, Agilent) between post-natal (days 1 or 3 after birth) and post-PNX adult (10 week) lungs of C57BL6 mixed gender mice. The post-natal lung (vs. post-PNX) exhibited greater numbers of upregulated growth and differentiation genes, and substantially lower expression of immune dense genes. Genes encoding enzymes and enzyme inhibitors and mitochondrial respiratory chain-related proteins were also markedly downregulated in the post-PNX lung but not in the post-natal lung, suggesting that growth potential depends on mitochondrial energetics, which is repressed in the adult lung even after PNX. Importantly, none of the conflicts between gene expression in the post-natal versus adult or adult post-PNX lung encompassed embryonic gene expression, consistent with the lack of embryonic gene expression in whole gene analyses. Likewise, Paxson [54] found that PNX in adult C57BL6 female (12 weeks) mice influenced many growth-differentiation genes found during post-natal alveolarization, but not those which are activated during embryonic or saccular lung development. Present in both post-natal lung development and post-PNX alveolarization includes Igf signaling, but absent in post-PNX lung was upregulation of Fgf, Pdgf signaling, and HoxA5 [54]. Alpha-smooth muscle actin which is abundantly expressed in the secondary septae of the post-natal lung was conspicuously absent after PNX in young mice [54] but present in older mice after PNX [96]. These data imply that the phenotype and function of myofibroblasts associated with alveolarization at various life stages are diverse and require further exploration.

Kho et al. [19] sought to clarify the developmental context of gene expression after PNX in adult C57BL6 mice within the entire spectrum of gene expression from embryonic (E9.5) through lung development, adulthood, and from 1 to 56 days after PNX, a more prolonged period than previously attempted. The 3–7 days post-PNX transcriptome was concordant with that of early post-natal alveolarization; therefore, the authors contended that PNX induce a sort of “de-differentiation,” which resolved into a period of later developmental gene regulation, referred to by the authors as “redevelopment.” Accordingly, de-differentiation and re-development are cardinal features of limb regeneration in salamanders [97], considered a classical form of organ regeneration. Thus, the bulk of evidence supports that post-PNX gene expression corroborates events during late, not early lung development. This paradigm is consistent with liver and kidney regeneration, for which gene expression in the adult organ similarly aligns with cellular-molecular events that transpire during late development (hyperplasia and hypertrophy of the functional units, respectively).

One limitation of these whole lung transcriptomic studies is a lack of lineage specificity and thus granularity of the findings. While studies of whole lung gene expression have failed to show that PNX reactivates certain embryonic and pseudoglandular (Ttf1, Rarα/β, Gli2/3, Wnt2/2b), canalicular (FoxA1/A2, Foxfla, HoxA5, Mycn, Ttf1, Tcf2l, Sox2l, Foxjl, Alk3), or saccular stage (Klf2, Nf1B, Gata6, Erm) genes [98], studies of respiratory epithelium provide histologic evidence for over-expressed genes marking definitive endodermal specification and morphogenesis (Sox17, Foxa2, Foxj1, β-catenin in bronchial epithelium; Ttf-1 in alveolar ducts) after PNX [99] implying that reactivation of embryonic signaling pathways is possible, specifically in epithelium [100]. Ttf-1 was found to be expressed in alveolar duct proSP-Cpos cells after PNX, and knockdown of Ttf-1 resulted in transient delay (although not a reduction in magnitude) of compensatory lung growth in mice, consistent with reactivation of Ttf-1 and participation of this factor in AECII proliferation or differentiation during post-PNX lung regeneration [101]. Conditional knockdown of other developmental genes or ablation of specific subsets of progenitor cells is warranted to further our understanding of the ageing regenerative capacity of the lung.

Ageing Effects on Competence of the Post-PNX Lung to Regenerate

Following PNX in young mice, progenitor cells including PCEC [62, 102], bronchiolar epithelial cells (Club cells, bronchioalveolar stem cells [17, 18]), AECII [17, 18, 58, 103], interstitial fibroblasts [55, 96], pleural lining cells [58], and macrophages [103, 104] show marked proliferation consistent with their participation in regeneration. l-MSC in particular reach their maximal rate of proliferation early after PNX (days 0–3) while proliferative activity of endothelial and bronchial and alveolar epithelial progenitor cells peak later (days 4–7), implying a key role for MSCs to drive re-alveolarization. The impact of age on l-MSC frequency was more profound than on endothelial or epithelial (bronchial, AECII) progenitor cells [55]. At 9 months (vs. young, or 3 months) of age, there was a dramatic decline in the abundance of l-MSC (prior to PNX) that was not evident for endothelial or epithelial progenitor cells. Shortly after PNX (0–3 days) the initial rise in l-MSC, endothelial cells, or bronchial epithelial progenitor cells is virtually absent in older mice (9 months, 17 months), consistent with arrest of cell division (i.e., early senescence) in these populations. In contrast, baseline abundance and proliferative responses of AECII to PNX were unaffected by age, implying that AECII populations are sustained by unique mechanisms.

The mechanism by which age alters l-MSC was further investigated ex vivo; the CFU and growth rate of l-MSC was inferior in 12-month-old (middle age) mice compared to young (3 months) old mice [55]. Moreover, the phenotype of older l-MSC was significantly more myofibroblastic, implying that attrition of precursors (i.e., lipofibroblasts) is a significant event underlying the restricted regenerative response at the tissue level after PNX. Under-expressed in ageing l-MSC were genes associated with retinoic acid signaling (Aldh1a3, Rbp4), Fgfr1/Wnt signaling (Fgfr1, Sfrp1, Ctnnb1, and Wnt2), and ECM formation (Col1a1, Eln, Fbn1, and Sdc2). In particular, Fgfr1 expression was markedly downregulated (>600-fold) at middle age (12 months) vs. young (3 months) consistent with the abrupt cessation of key regulatory mechanisms essential for alveolarization. Mechanistically, Wnt4 and Wnt5a expressions increase lipofibroblast (PPARγpos) differentiation to myofibroblasts in the aged mouse lung [37]; similarly, activation of Wnt3a causes differentiation of bone marrow MSCs to fibroblasts and myofibroblasts with increased expression of col1, α-smooth muscle actin in vitro, and reduced the ability to control HCl-induced lung fibrosis in vivo [105]. This implies that loss of lipofibroblast by differentiation may limit post-PNX re-alveolarization or lung injury repair, as similar losses impair lung development.

The reason why ageing lung fibroblasts dissipate over time has not been completely elucidated. Multiple lines of evidence exist that differentiation to, as well as senescence of lung myofibroblasts (as well as pulmonary fibrosis) may be driven by activation of canonical Wnt/β-catenin signaling [37, 105]. Vaidya et al. [106] found in young mice that non-homologous end-joining (NHEJ) as a DNA repair mechanism was higher in lung fibroblasts than fibroblasts of other tissues, but declined significantly in lung fibroblasts between 5 and 24 months of age. Age was also associated with larger deletions, and greater reliance on micro-homology mediated end joining (MMEJ) which is more error-prone sub-pathway of NHEJ. This may further contribute to the attrition of lipofibroblasts and/or myofibroblasts in the lung.

Immune effector cells are critical for organ regeneration, for example, liver regeneration after partial hepatectomy [107]. In the post-PNX lung, blood born macrophages (CD11b) migrate into the interstitial and alveolar spaces, and express putative pro-regenerative factors (e.g., Angpt1, Egf, Mmp9, Tgfb2) [104]. Type M2 macrophages are known to be important for post-natal lung development, releasing growth factors (e.g., Igf1) [108] suggesting that M2 macrophages specifically are critical for post-PNX re-alveolarization. In a recent study, it was observed that large numbers of interstitial macrophages were found in close association with AECII in zones of sprouting and intussusceptive angiogenesis and secondary septation after PNX [61], implying that these macrophages directly participate in remodeling associated with neo-angiogenesis. It has not been established whether this role is an essential one for alveolarization after PNX. With respect to ageing, there is an accumulation of macrophages and B cells with age in the lung [37], and these may contribute to “inflammageing.”

Ageing and the Extracellular Matrix Control of Lung Regeneration

The ECM is a complex mixture of secreted products originating from resident lung cells that imparts a profound inductive influence on fetal development and stem-progenitor cell capacity for self-renewal and differentiation [109, 110]. These include growth factors, cytokines (i.e., matrikines), cryptic peptides, glycosaminoglycans, and glycoproteins that support cell division, migration, recruitment, quiescence, angiogenesis, antimicrobial functions, preservation of phenotype, self-renewal, and/or differentiation, and protect the lung from intense environmental challenges. As well, ECM delivers mechanical cues that evoke outside-in signaling pathways that facilitate survival of anchorage-dependent cells (“tensegrity”) such as endothelial cells and fibroblasts [111]. Indeed, mechanical forces transferred through the ECM are undoubtedly the most important initiation factor in lung regeneration after PNX [112].

Following PNX, desmosine and hydroxyproline are deposited in the lung and resultant elastin and collagens are distributed anatomically in a manner that mirrors development [113]. With ageing, there is significant reduction in elastic recoil and thus FEV1, FVC, and FEV1/FVC [114]. Similarly, lungs in aged mice show an increase in lung compliance and lung volume at pressures equivalent to total lung capacity [115] which is accentuated in certain inbred strains of mice such as DBA/2J [41]. These structural–functional changes in mice coincide with decreases in lung tissue elastin fiber content and Eln gene expression. In humans, elastin life span based on mean carbon residence time is ~74 years, consistent with the absence of new elastin synthesis over the human life span [116]. Despite the apparent longevity of lung elastin, it is evident that in the elderly, elastin in alveolar walls is thin, fragmented, and bundled [36], commensurate with the reduced recoil properties of the organ.

Reduction in elastin content in haploinsufficient mice is also associated with airspace dilation [117]. Following PNX, elastin insufficiency is associated with a suppression of epithelial progenitor cell proliferation and compensatory growth [17], implying an indirect effect of lowering mechanical stress after PNX. Similarly, direct interference with mechanical stress after PNX, using plombage, also reduces epithelial (BASC, AECII) responses and alveolarization in mice [17]. In vitro studies corroborate the importance of mechanical stretch to mechanotransduction [118], secretory function [119], and DNA synthesis in AECII [120]. Insufficient mechanical stress on cells presumably contributes to apoptosis and senescence of stem-progenitor cells (AECII, endothelial cells, and interstitial fibroblasts) in disease processes with disrupted matrix.

In addition to extrinsic alterations produced by age-related modifications of ECM, it has recently been reported that ageing impairs intrinsic mechanotransduction through YAP/TAZ signaling including failure of YAP nuclear translocation in breast epithelial progenitor cells [121]. One might speculate that similar mechanisms fail with age in the lung, thus contributing to failure of PNX to reinitiate growth-related signals that are activated upon mechanostimulation.

The inductive properties of exogenous ECM might be effective to mitigate or stabilize the age phenotype of cells. In the lung, fetal (amniotic) basement membrane sustains AECII morphology and phenotype whereby adult lung-derived basement membrane matrix does not [122]. Fetal lung tissues contain more glycosaminoglycans (chondroitin sulfate 4) that support stem-progenitor cell growth and differentiation [123, 124], compatible with the inductive properties of mesenchyme during branching morphogenesis. Hence, a combination of specific mechanical (extrinsic), mechanotransductive (intrinsic), and inductive signals that are derived from ECM appear to be involved in age-related modifications of stem-progenitor cell behavior in the lung.

Ageing of the Lung with Respect to Telomere Length

Evidence is mounting that telomere shortening is responsible for multiple mechanisms leading to an ageing phenotype including stem cell failure, mitochondrial dysfunction, genotoxic stress, and epigenetic changes [125]. Telomerase is significantly reduced after post-natal day 9 in mice, but can be reactivated by hyperoxia, notably within PCNA positive cells suggesting that telomerase is a stem-progenitor cell marker in the lung [73]. Progressive reduction in telomere length in AECII over multiple generations of Terc null mice is evidence that oxidative damage is sustained by lung cells even under normoxic conditions [126]. In that study, Terc null F4 mice showed reduced abundance of AECII, reduced interstitial collagen, and dilated airspaces similar to the biochemical and morphological hallmarks of ageing in mice [96]. Similar to hyperoxia, PNX induces telomerase in wild type and Terc null AECII, but proliferative responses of AECII and BASC as well as re-alveolarization are markedly reduced in Terc null mice consistent with replicative senescence [127], paving the way for our understanding of how senescence and reinitiation of alveolarization are interrelated.

Disease-Induced Premature Ageing of the Lung Phenotype

Cigarette exposure induces genotoxic senescence in alveolar [128] and bronchial [129] epithelia, and this premature senescence of epithelial cells exacerbates the course of COPD [130]. In human emphysema, similar findings of higher than expected senescence in endothelial and AECII, including higher p16Ink4a and p21Waf1 expression, shorter telomeres, and proportional lower pulmonary function are evident [131]. Exposure of epithelial cells or fibroblasts from human lungs to cigarette smoke extract (CSE) results in an increased expression of β-galactosidase, a marker of cellular senescence, and decreased cell proliferation [132]. Moreover, there is evidence that CSE induces mitochondrial fragmentation furthering senescence [121, 133, 134]. As well, cigarette exposure in telomerase null mice induces an emphysema phenotype that is associated with accentuated DNA damage, shorter telomeres, AECII senescence, and limited proliferative capacity [135]. While CSE induced senescence markers, advanced age did not augment CSE-induced senescence, despite shorter telomeres in response to CSE [136]. Thus, the connection between senescence, telomere length, telomerase, and CSE exposure in mice, and the species specificity of these findings requires further clarification. Strong parallels have been made between the senescence associated secretory phenotype (SASP) secondary to DNA damage and damage responses, and COPD phenotype [132] based on similarities in cytokine profiles (e.g., IL-6, IL8). This paradigm has enormous implications for progression of lung disease, in that smoking, genetics, and ageing synergistically act to degrade lung function [137].

Ageing is also associated with a predisposition toward a fibrotic phenotype [138]. Lung fibroblasts with ageing of donor also exhibit more myofibroblastic features including greater α-smooth muscle actin, tenascin, fibronectin, Col I, Col III, lower CFU, larger cell size, and loss of clonogenicity and self-renewal in vitro [96]. Pulmonary fibrosis may thus be a manifestation of telomere shortening syndrome even in the absence of identifiable Terc or TR mutations. The relationship between ageing and fibrotic lung disease has been addressed recently by Thannickal et al. [139], Armanios et al. [125], and Chilosi et al. [140]. Briefly, there are a significant percentage of patients with sporadic IPF that harbor genetic mutations in telomerase components (e.g., reverse transcriptase, RT) [140]. Similarly, patients with sporadic IPF have shorter leukocyte telomeres than age-matched controls [141]. Consistent with this observation, a large fraction of patients with identifiable telomere dysfunction exhibit IPF or related liver cirrhosis [125, 142]. The AECII is thought to be a major target for familial IPF mutations, and AECII are often infected with latent Herpesviridae that induce the unfolded protein response, contributing to DNA damage. In familial IPF (~1–2 % IPF patients) mutations specifically target SP-C and SP-A2 in addition to TERT and TERC, resulting in misfolded proteins which contribute to endothelial reticulum (ER) stress in AECII [143]. As a consequence of ER stress, AECII may contribute to epithelial-mesenchymal transition in the lung of IPF patients. In sporadic IPF, there is also evidence of ER stress within AECII implying that familial and sporadic IPF converge on age-related mechanisms [143]. How these findings implicate AECII in the age-related changes observed in lung regeneration is unclear.

In ageing humans, common cancers express markers associated with regenerative stem-progenitor cells in the lung, thus suggesting cell of origin for squamous cell carcinoma (p63, CK5, Sox2 basal cells) or adenocarcinoma (CK14, TTF1, and SP-C AECII cells) [144]. In Kras mice, both BASC and AECII are considered likely cells of origin for adenocarcinomas [75, 145], thus positing an important role for stem-progenitor cell ageing, genomic instability, and transformation to lung cancer.

Epigenetic Ageing of the Lung

Healthy ageing and longevity are strongly associated with changes in cellular DNA methylation [146]. Epigenome-wide association scans (EWAS) were used to extract a significant relationship between age-related phenotype and FVC [146]. Similarly in mice, Lui et al. [147] compared epigenetic and transcriptomic data at 4 weeks vs. 1 week of age, and found a close relationship between ageing, decreased gene expression of growth regulatory genes, and a decrease in H3K4me3 implying the cessation of post-natal growth in lung and kidney may be epigenetically determined. Epigenetic dysregulation is a common feature of ageing in humans and animals, culminating in significant deterioration of stem, immune, and stromal cells [148], so it can be assumed that cells that are critical to reinitiation of lung regeneration are impacted to varying degrees. For example, human diploid lung fibroblasts undergo senescence concomitant with marked alterations to epigenetic regulators such as SUV39H1 that repress genomic instability, demonstrating the close relationship between mechanisms of ageing and transformation of cells [149].

Conclusion

Very few studies have addressed the cellular-molecular biology of healthy ageing in the lung. Given the similarities in mechanisms that echo from post-natal development through the various life stages, ageing and rejuvenation research in the future may address the entire life span of mammals, taking into account environmental, genetic, and epigenetic factors to answer questions. Strategies for better understanding of ageing of regenerative stem-progenitor cells might include great emphasis on genetic lineage tracing, studies of clonality, conditional ablation, epigenetic mechanisms, ECM inductive properties for, and changes to intrinsic mechanotransduction in stem-progenitor cells. As well, the roles for telomere length, genomic instability, and DNA damage in stem-progenitor cells must be further elucidated to understand the link between ageing and stem cell exhaustion. Indeed, the explanation for the decline in lung stem-progenitor cell abundance and differentiation and self-renewal properties is another mystery that needs to be solved given the importance of this population to regenerative capacity. Finally, more studies that focus on rejuvenation in the lung using heterochronic parabiosis, in vivo reprogramming, and transplantation of stem-progenitor cells or their derivatives will pave the way for translation.