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The Aging Lung and Idiopathic Pulmonary Fibrosis
REVIEW ARTICLE
Feature Issue: Update on Pulmonary Fibrosis
The Aging Lung and Idiopathic Pulmonary Fibrosis Swati Gulati, MBBS,MS1 and Victor J. Thannickal, MD1,2 1
Division of Pulmonary, Allergy, and Critical Care Medicine and 2 Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama
ABSTRACT Idiopathic pulmonary fibrosis (IPF) is one of many clinical syndromes that are associated with aging, and is increasing in both incidence and prevalence with the rapid rise in aging populations world-wide. There is accumulating data on how the biology of aging may influence the susceptibility to lung fibrosis in the elderly. In this review, we explore some of the known “hallmarks of aging,” including telomere attrition, genomic instability, epigenetic alterations, loss of proteostasis, cellular senescence and mitochondrial dysfunction in the pathobiology of IPF. Additionally, we discuss age-associated alterations in extracellular matrix that may contribute to the development and/or progression of IPF. Key Indexing Terms: Aging; Idiopathic pulmonary fibrosis; Fibrosis. [Am J Med Sci 2019;357(5):384−389.]
INTRODUCTION
T
he increases in life expectancy across most countries world-wide may be considered a landmark achievement over the past century. The proportion of the elderly population, aged 65 and above, has been rising and is projected to more than double to an estimated 21% by the year 2050; this represents over 2 billion people world-wide, outnumbering younger individuals aged 10-24 years.1,2 With these demographic transitions, there has been an unprecedented rise in the incidence and prevalence of agerelated diseases such as atherosclerosis, cancer, Alzheimer’s dementia and idiopathic pulmonary fibrosis (IPF).2,3 IPF is the prototype of an age-related disease as it usually occurs in individuals older than 50 years and its incidence increases drastically with age.3 IPF is characterized by a progressive decline in lung function from a seemingly irreversible destruction of lung architecture. IPF has a median survival of approximately 3 years from the time of diagnosis and the currently approved antifibrotic agents, nintedanib and pirfenidone, only slow the progressive decline in lung function without improving survival or quality of life. Due to the aging population, at least in part, the incidence and prevalence of IPF is on the rise.4 A better understanding of the pathophysiology of this disease and its relationship to the biology of aging will lead to the discovery of more effective therapies (Figure 1).
AGING AND IDIOPATHIC PULMONARY FIBROSIS Aging can be defined as the inevitable time-dependent functional decline, characterized by progressive loss of physiological integrity, reduced homeostatic control, and increased vulnerability to death.5 Evolutionary 384
theories of aging include the concept of “antagonistic pleiotropy,” which supports the selection of genes that benefit early reproductive life even if they have a deleterious effect at later ages. Our group has postulated that fibrosis and fibrosis-promoting genes/pathways may have evolved as a trade-off for early-life fitness and survival over a late-life trait that may result in “pathological” fibrosis due to a loss of cellular plasticity and the capacity to resolve tissue fibrosis.6-8 In addition to the question of “why” we age, the cellular and molecular mechanisms for “how” we age are beginning to be deciphered.9 Lopez-Otin et al proposed nine “hallmarks of aging” including genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intercellular communication.9 Since aging confers the greatest risk for IPF, beyond any known environmental or genetic risk factor, some have suggested that IPF may represent a form of “accelerated” lung aging.10 The mechanistic links between IPF and “aging hallmarks” are just beginning to be unraveled. In this review, we explore common mechanisms by which aging hallmarks may predispose to lung fibrosis.11 Genomic Instability Over the course of life, DNA is exposed to various endogenous and exogenous damaging stimuli. The ability to preserve genetic stability usually declines with age leading to gradual accumulation of damaged DNA, which is a well-recognized feature of aging. Several studies have also reported the presence of such genome instability in IPF patients. Sputum samples from IPF patients display evidence of genomic instability, either THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES VOLUME 357 NUMBER 5 MAY 2019
The Aging Lung and Idiopathic Pulmonary Fibrosis
FIGURE 1. Aging and idiopathic pulmonary fibrosis. Abbreviations: AEC, alveolar epithelial cells; ECM, extracellular matrix; Fbs, fibroblasts; IL, interleukin; NOX4, NADPH oxidase 4; SASP, senescence-associated secretory phenotype.
microsatellite instability (MSI) or loss of heterozygosity (LOH), when compared with matched controls.12,13 LOH has been located in key cell cycle genes such as p16, a marker of cellular senescence.14 Another study found evidence of MSI in the TGF-b receptor II gene in alveolar epithelial cells isolated from honeycomb regions of the IPF lung.15 These findings suggest that genomic instability resulting from DNA damage is not uncommon in these patients and may contribute to disease pathogenesis.
Epigenetic Alterations An epigenetic trait is defined as a stably heritable phenotype resulting from changes in chromatin remodeling and gene expression without alterations in DNA sequence. The three pillars of epigenetic alterations, namely, changes in DNA methylation patterns, posttranslational histone modifications and noncoding RNAs, have been proposed to play critical roles in the biology of aging and pathophysiology of aging-related diseases.9 There is accumulating evidence supporting the role of epigenetic alterations in the pathogenesis of IPF. For instance, altered microRNA profiles have been observed in patients with IPF, including down-regulation of some microRNAs. Experimental loss-of-function of these
specific microRNAs induces fibrosis in mouse models, whereas their overexpression protects from bleomycininduced lung fibrosis.16-18 Aging can also modify the expression of sirtuins (SIRTs), a family of deacetylases that target both histone and nonhistone proteins. SIRT1 suppresses the expression of senescence-associated secretory phenotype (SASP) factors via histone deacetylation of their gene promoter regions.19 The level of SIRT1 decreases with aging, accompanied by reduced mitochondrial biogenesis, which is a common finding in a number of aging-related diseases. In a model of bleomycin-induced lung injury, upregulation of SIRT1 attenuated fibroblast activation and development of fibrosis.20 Interestingly, treatment with the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), also ameliorates bleomycin-induced pulmonary fibrosis in mice in association with induction of fibroblast apoptosis, suggesting that the specific targets of histone acetylation and deacetylation may be critical in determining the effect on fibrosis.21 Reduction in SIRT3 expression has been shown to promote pulmonary fibrosis in human lung fibroblasts and exacerbates inflammation in mice with endotoxin-induced acute lung injury.22,23(p3) SIRT 3 upregulation has also shown to rejuvenate hematopoietic stem cells and in fact, reverse aging-associated degeneration.24
Copyright © 2019 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved. www.amjmedsci.com www.ssciweb.org
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Loss of Proteostasis The autophagy-lysosomal system and the ubiquitinproteosomal system are the two major proteolytic systems, both of which decline with aging, leading to increased accumulation of misfolded proteins, increased endoplasmic reticulum (ER) stress and defective autophagy.9 Recent studies have indicated proteostasis plays an important role in the development of IPF. For instance, familial IPF has been linked to protein misfolding mutations in surfactant C.25,26 Similarly, markers of ER stress are seen in lungs from sporadic IPF, most notably in alveolar epithelial cells (AEC) from areas with dense fibrosis and fibroblast foci.27 ER stress has been shown to influence mitochondrial function in AECs by downregulating PINK1, a critical regulator of mitochondrial homeostasis.28 Autophagy is a catabolic process by which damaged organelles and endogenous proteins are destroyed intracellularly within lysosomes. Decline in autophagic capacity is seen in both aging and IPF.9,29 Inhibition of autophagy accelerate cellular senescence in epithelial cells and promote myofibroblast differentiation of lung fibroblasts.30 In a bleomycin-induced lung fibrosis mouse model, deficiency of the autophagic protein, Atg4, led to higher inflammatory and fibrotic responses.31 The mammalian target of rapamycin (mTOR) pathway is a critical regulator of autophagy. Rats receiving the rapamycin analogue, SDZ RAD, are protected from bleomycin-induced experimental fibrosis, although mechanisms of its action on specific cell types are not well understood.32 Activation of autophagy with the AMPK activator, metformin, is capable of accelerating the resolution of lung fibrosis in mice, in part by promoting collagen turnover.29 Interestingly, the antifibrotic drug, pirfenidone, has been shown to induce noncanonical autophagy in lung fibroblasts which may, in part, contribute to its beneficial effects. These studies indicate that age-associated decline in autophagy may be a therapeutic target to maintain proteostasis in the aging lung. Telomere Attrition IPF is the most common manifestation of telomererelated disorders. Genetic mutations related to telomerase (telomere elongating enzyme) are one of the common genetic defects found in patients with familial IPF.33 In addition to activating DNA damage pathways, shortened telomeres are also thought to contribute to fibrosis when exposed to injurious stimulus, by impairing tissue repair. Chromosomal shortening resulting from telomere attrition activates checkpoint inhibitor p53. Activated p53, in turn, reduces mitochondrial biogenesis, increases ROS production and activates cellular senescent pathways.34 Cellular Senescence Cellular senescence is commonly defined as an irreversible state of stable growth arrest in association with a high metabolic activity and secretory phenotype.9 386
Damage accumulation caused by processes such as telomere attrition, oxidative stress and oncogenic stress stimulate the activity of cyclin-dependent kinase inhibitors p16Ink4a and/or p53-p21Cip1/Waf1, which then antagonize cyclin-dependent kinases to block cell cycle progression.35 Despite being in replicative arrest, these cells are metabolically active, and display a SASP consisting of cytokines, growth factors and proteases. Cellular senescence serves a protective function to halt the proliferation of damaged cells and to trigger their own destruction by the immune system.36 Alveolar epithelial cells and fibroblasts have been shown to assume senescent identities in IPF, contributing to development of fibrosis.28,37,38 Established senescent markers such as p21, p16 and senescenceassociated b-galactosidase activity (SA-b-gal) are increased in both fibroblasts and epithelial cells in human IPF lung tissue as compared with controls.38-40 IPFderived fibroblasts exhibit accelerated replicative cellular senescence and increased resistance to oxidative stress-induced cytotoxicity as compared to normal lung fibroblasts. The secretome of both, IPF fibroblasts and AECs, exhibits increased profibrotic and inflammatory markers and is thought to have profound effect on the lung microenvironment by secretion of these mediators which perpetuate fibrotic scarring.37,40 Cellular senescence is being increasingly recognized as a therapeutic target in IPF. Based on the apoptosisresistant phenotype of senescent fibroblasts and myofibroblasts, strategies to increase their sensitivity to apoptosis by inhibition of antiapoptotic factors have been suggested. The antiapoptotic and prosenescent enzyme, NADPH oxidase 4 (NOX4), has been targeted in an aging murine model of persistent lung fibrosis with induction of apoptosis of interstitial myofibroblasts and marked improvement in lung fibrosis and survival.38,41,42 A senolytic cocktail of Dasatinib and Quercetin was effective in eliminating senescent AECs and alleviating fibrosis in a bleomycin-injury model of lung fibrosis.37 A similar observation was made using the same senolytic combination in a murine model of radiation-induced lung fibrosis.40 Cellular senescence offers a potentially effective therapeutic target in IPF and there is active research to determine the safety and efficacy of this approach.
Mitochondrial Dysfunction Mitochondria are tightly regulated in their life-cycle of biogenesis, networking/dynamics, repair, and recycling/ mitophagy. These organelles are responsible for energy (ATP) production and function as bidirectional signaling platforms communicating with the nucleus and other cellular organelles. Altered mitochondrial homeostasis such as reduced mitochondrial biogenesis, increased reactive oxygen species (ROS) production, increased mitochondrial DNA damage and defective mitophagy are found in healthy aging lung.43,44 Many such changes are also THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES VOLUME 357 NUMBER 5 MAY 2019
The Aging Lung and Idiopathic Pulmonary Fibrosis
seen in fibrotic lungs and are hence thought to contribute to IPF.28,43,45 Mitochondrial biogenesis, the process of producing additional mitochondria, is controlled by master regulators PPARg coactivator-1a (PGC-1a) and PGC1b, and nuclear respiratory factors (NRFs), NRF-1 and NRF-2.46 PGC-1 expression is lower in IPF lungs and also in bleomycin exposed wild mice lung models.47 Telomere attrition and DNA damage have been shown to reduce mitochondrial biogenesis by activating p53 signaling mechanisms which subsequently reduce PGC-1.48,49 AECs from human IPF lungs accumulate dysfunctional mitochondria with upregulation of markers of ER stress compared with age-matched controls.28 These dysfunctional mitochondria are associated with low expression of the regulator of mitochondrial homeostasis PTEN-induced putative kinase 1 (PINK1).50(p3) As a result, PINK1-deficient mice exhibit increased susceptibility to apoptosis, lung injury and TGF-b-driven lung fibrosis.51 PINK1-PARK2 signaling pathway also mediates mitophagy, a process by which dysfunctional mitochondria are removed to maintain cellular health and integrity.52 Insufficient mitophagy caused by PARK2 deficiency activates platelet-derived growth factor receptor (PDGFR) and mammalian target of rapamycin (mTOR) signaling, which results in increased myofibroblast differentiation and proliferation.52 Pirfenidone has been shown to augment mitophagy by enhancing PARK-2 expression and inhibiting myofibroblast differentiation. Although these pathways have been demonstrated to play a role in age- and disease-related alterations in cellular bioenergetics, additional translational investigations in IPF lungs are needed to firmly establish their role.
Extracellular Matrix Although not considered one of the hallmarks of aging, alterations to the extracellular matrix (ECM) with aging may have profound effects on susceptibility of aging and lung fibrosis. ECM of aging lungs expresses a profibrotic phenotype characterized by increased Type I and III collagen and TGF-b1 expression along with enhanced fibronectin extracellular domain A (EDA).53 Old lungs also have a more oxidizing extracellular environment as compared to young lungs, which has been shown to induce intracellular signals that stimulate proliferation and matrix expression in fibroblasts via upregulation of TGF-b 1 expression.54,55 Similarly, ECM of IPF lung fibroblasts have also shown to be rich in collagen and fibronectin EDA. The ability of Type I collagen to limit fibroblast proliferation during physiological repair is shown to be lost in IPF fibroblasts, which potentially contributes to pathological fibrosis.56 Further research to study the alterations in IPF lung EMC, matrix-cell interactions and to
explore the role of matrix-directed therapeutics is required. Impaired Mucociliary Clearance There is an emerging concept that impaired mucociliary clearance may represent a lung aging phenotype and contribute to IPF pathogenesis.
FUTURE DIRECTIONS The progress in our understanding of IPF has greatly advanced in recent years, and has accelerated in part due to the incorporation of aging biology concepts in disease pathogenesis. Critical gaps in understanding the pathogenesis include the role of epigenetic modifications, metabolic sensing, ECM alterations, and airway host defense mechanisms such as impaired mucociliary clearance. Further insights into these mechanisms will lead to the development of novel diagnostics and therapeutics for IPF and other age-associated lung diseases. Advances in imaging modalities and biomarkers that define/detect changes in the metabolic and inflammation-fibrosis related pathways will facilitate clinical translation.
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34. Sui B, Hu C, Jin Y. Mitochondrial metabolic failure in telomere attritionprovoked aging of bone marrow mesenchymal stem cells. Biogerontology. 2016;17(2):267–279. https://doi.org/10.1007/s10522-015-9609-5. 35. van Deursen JM. The role of senescent cells in ageing. Nature. 2014;509 (7501):439–446. https://doi.org/10.1038/nature13193. 36. Hoenicke L, Zender L. Immune surveillance of senescent cells−biological significance in cancer- and non-cancer pathologies. Carcinogenesis. 2012;33(6):1123–1126. https://doi.org/10.1093/carcin/bgs124. 37. Lehmann M, Korfei M, Mutze K, et al. Senolytic drugs target alveolar epithelial cell function and attenuate experimental lung fibrosis ex vivo. Eur Respir J. 2017;50(2). https://doi.org/10.1183/13993003.023672016. 38. Hecker L, Logsdon NJ, Kurundkar D, et al. Reversal of persistent fibrosis in aging by targeting Nox4-Nrf2 redox imbalance. Sci Transl Med. 2014;6(231):231ra47. https://doi.org/10.1126/scitranslmed.3008182. 39. Lomas NJ, Watts KL, Akram KM, et al. Idiopathic pulmonary fibrosis: immunohistochemical analysis provides fresh insights into lung tissue remodelling with implications for novel prognostic markers. Int J Clin Exp Pathol. 2012;5(1):58–71. 40. Schafer MJ, White TA, Iijima K, et al. Cellular senescence mediates fibrotic pulmonary disease. Nat Commun. 2017;8:14532. https://doi.org/ 10.1038/ncomms14532. 41. Sanders YY, Liu H, Liu G, et al. Epigenetic mechanisms regulate NADPH oxidase-4 expression in cellular senescence. Free Radic Biol Med. 2015;79:197–205. https://doi.org/10.1016/j.freeradbiomed.2014. 12.008. 42. Hecker L, Vittal R, Jones T, et al. NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury. Nat Med. 2009;15(9):1077–1081. https://doi.org/10.1038/nm.2005. 43. Mora AL, Bueno M, Rojas M. Mitochondria in the spotlight of aging and idiopathic pulmonary fibrosis. J Clin Invest. 2017;127(2):405–414. https:// doi.org/10.1172/JCI87440. 44. Rangarajan S, Bernard K, Thannickal VJ. Mitochondrial dysfunction in pulmonary fibrosis. Ann Am Thorac Soc. 2017;14(Supplement_5):S383– S388. https://doi.org/10.1513/AnnalsATS.201705-370AW. 45. Kim S-J, Cheresh P, Jablonski RP, et al. Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage. Free Radic Biol Med. 2016;101:482–490. https://doi.org/10.1016/j.freeradbiomed.2016.11.007. 46. Lehman JJ, Barger PM, Kovacs A, et al. Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis. J Clin Invest. 2000;106(7):847–856. https://doi.org/10.1172/ JCI10268. 47. Yu G, Tzouvelekis A, Wang R, et al. Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function. Nat Med. 2018;24(1):39–49. https://doi.org/10.1038/nm.4447. 48. Sahin E, Colla S, Liesa M, et al. Telomere dysfunction induces metabolic and mitochondrial compromise. Nature. 2011;470(7334):359–365. https://doi.org/10.1038/nature09787. 49. Fang EF, Scheibye-Knudsen M, Brace LE, et al. Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction. Cell. 2014;157(4):882–896. https://doi.org/10.1016/j.cell.2014.03.026. 50. Bueno M, Brands J, Voltz L, et al. ATF3 represses PINK1 gene transcription in lung epithelial cells to control mitochondrial homeostasis. Aging Cell. 2018;17(2). https://doi.org/10.1111/acel.12720. 51. Patel AS, Song JW, Chu SG, et al. Epithelial cell mitochondrial dysfunction and PINK1 are induced by transforming growth factor-beta1 in pulmonary fibrosis. PloS One. 2015;10(3) e0121246. https://doi.org/10.1371/ journal.pone.0121246. 52. Kobayashi K, Araya J, Minagawa S, et al. Involvement of PARK2mediated mitophagy in idiopathic pulmonary fibrosis pathogenesis. J Immunol Baltim Md 1950. 2016;197(2):504–516. https://doi.org/10.4049/ jimmunol.1600265. 53. Sueblinvong V, Neujahr DC, Mills ST, et al. Predisposition for disrepair in the aged lung. Am J Med Sci. 2012;344(1):41–51. https://doi.org/ 10.1097/MAJ.0b013e318234c132. 54. Watson WH, Burke TJ, Zelko IN, et al. Differential regulation of the extracellular cysteine/cystine redox state (EhCySS) by lung fibroblasts
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from young and old mice. Oxid Med Cell Longev. 2016;2016:1561305. https://doi.org/10.1155/2016/1561305. 55. Ramirez A, Ramadan B, Ritzenthaler JD, et al. Extracellular cysteine/ cystine redox potential controls lung fibroblast proliferation and matrix expression through upregulation of transforming growth factor-beta. Am J Physiol Lung Cell Mol Physiol. 2007;293(4):L972–L981. https://doi.org/ 10.1152/ajplung.00010.2007. 56. Xia H, Diebold D, Nho R, et al. Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis. J Exp Med. 2008;205(7):1659–1672. https://doi.org/ 10.1084/jem.20080001.
Submitted January 8, 2019; accepted February 10, 2019. Conflict of interest: Swati Gulati and Victor J. Thannickal have no conflicts of interest. Victor J Thannickal has received grants from the NIH (P01 HL114470 and R01 AG046210) and the VA Merit Award I01BX003056. Funding: The author(s) received no specific funding for this work. Correspondence: Swati Gulati, M.D., Division of Pulmonary, Allergy, and Critical Care, Department of Medicine, University of Alabama at Birmingham, 1900 University Blvd THT 541E, Birmingham, AL 35294-2180. (E-mail: [email protected]).
Copyright © 2019 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved. www.amjmedsci.com www.ssciweb.org
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Feature Issue: Update on Pulmonary Fibrosis
The Aging Lung and Idiopathic Pulmonary Fibrosis Swati Gulati, MBBS,MS1 and Victor J. Thannickal, MD1,2 1
Division of Pulmonary, Allergy, and Critical Care Medicine and 2 Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama
ABSTRACT Idiopathic pulmonary fibrosis (IPF) is one of many clinical syndromes that are associated with aging, and is increasing in both incidence and prevalence with the rapid rise in aging populations world-wide. There is accumulating data on how the biology of aging may influence the susceptibility to lung fibrosis in the elderly. In this review, we explore some of the known “hallmarks of aging,” including telomere attrition, genomic instability, epigenetic alterations, loss of proteostasis, cellular senescence and mitochondrial dysfunction in the pathobiology of IPF. Additionally, we discuss age-associated alterations in extracellular matrix that may contribute to the development and/or progression of IPF. Key Indexing Terms: Aging; Idiopathic pulmonary fibrosis; Fibrosis. [Am J Med Sci 2019;357(5):384−389.]
INTRODUCTION
T
he increases in life expectancy across most countries world-wide may be considered a landmark achievement over the past century. The proportion of the elderly population, aged 65 and above, has been rising and is projected to more than double to an estimated 21% by the year 2050; this represents over 2 billion people world-wide, outnumbering younger individuals aged 10-24 years.1,2 With these demographic transitions, there has been an unprecedented rise in the incidence and prevalence of agerelated diseases such as atherosclerosis, cancer, Alzheimer’s dementia and idiopathic pulmonary fibrosis (IPF).2,3 IPF is the prototype of an age-related disease as it usually occurs in individuals older than 50 years and its incidence increases drastically with age.3 IPF is characterized by a progressive decline in lung function from a seemingly irreversible destruction of lung architecture. IPF has a median survival of approximately 3 years from the time of diagnosis and the currently approved antifibrotic agents, nintedanib and pirfenidone, only slow the progressive decline in lung function without improving survival or quality of life. Due to the aging population, at least in part, the incidence and prevalence of IPF is on the rise.4 A better understanding of the pathophysiology of this disease and its relationship to the biology of aging will lead to the discovery of more effective therapies (Figure 1).
AGING AND IDIOPATHIC PULMONARY FIBROSIS Aging can be defined as the inevitable time-dependent functional decline, characterized by progressive loss of physiological integrity, reduced homeostatic control, and increased vulnerability to death.5 Evolutionary 384
theories of aging include the concept of “antagonistic pleiotropy,” which supports the selection of genes that benefit early reproductive life even if they have a deleterious effect at later ages. Our group has postulated that fibrosis and fibrosis-promoting genes/pathways may have evolved as a trade-off for early-life fitness and survival over a late-life trait that may result in “pathological” fibrosis due to a loss of cellular plasticity and the capacity to resolve tissue fibrosis.6-8 In addition to the question of “why” we age, the cellular and molecular mechanisms for “how” we age are beginning to be deciphered.9 Lopez-Otin et al proposed nine “hallmarks of aging” including genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intercellular communication.9 Since aging confers the greatest risk for IPF, beyond any known environmental or genetic risk factor, some have suggested that IPF may represent a form of “accelerated” lung aging.10 The mechanistic links between IPF and “aging hallmarks” are just beginning to be unraveled. In this review, we explore common mechanisms by which aging hallmarks may predispose to lung fibrosis.11 Genomic Instability Over the course of life, DNA is exposed to various endogenous and exogenous damaging stimuli. The ability to preserve genetic stability usually declines with age leading to gradual accumulation of damaged DNA, which is a well-recognized feature of aging. Several studies have also reported the presence of such genome instability in IPF patients. Sputum samples from IPF patients display evidence of genomic instability, either THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES VOLUME 357 NUMBER 5 MAY 2019
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FIGURE 1. Aging and idiopathic pulmonary fibrosis. Abbreviations: AEC, alveolar epithelial cells; ECM, extracellular matrix; Fbs, fibroblasts; IL, interleukin; NOX4, NADPH oxidase 4; SASP, senescence-associated secretory phenotype.
microsatellite instability (MSI) or loss of heterozygosity (LOH), when compared with matched controls.12,13 LOH has been located in key cell cycle genes such as p16, a marker of cellular senescence.14 Another study found evidence of MSI in the TGF-b receptor II gene in alveolar epithelial cells isolated from honeycomb regions of the IPF lung.15 These findings suggest that genomic instability resulting from DNA damage is not uncommon in these patients and may contribute to disease pathogenesis.
Epigenetic Alterations An epigenetic trait is defined as a stably heritable phenotype resulting from changes in chromatin remodeling and gene expression without alterations in DNA sequence. The three pillars of epigenetic alterations, namely, changes in DNA methylation patterns, posttranslational histone modifications and noncoding RNAs, have been proposed to play critical roles in the biology of aging and pathophysiology of aging-related diseases.9 There is accumulating evidence supporting the role of epigenetic alterations in the pathogenesis of IPF. For instance, altered microRNA profiles have been observed in patients with IPF, including down-regulation of some microRNAs. Experimental loss-of-function of these
specific microRNAs induces fibrosis in mouse models, whereas their overexpression protects from bleomycininduced lung fibrosis.16-18 Aging can also modify the expression of sirtuins (SIRTs), a family of deacetylases that target both histone and nonhistone proteins. SIRT1 suppresses the expression of senescence-associated secretory phenotype (SASP) factors via histone deacetylation of their gene promoter regions.19 The level of SIRT1 decreases with aging, accompanied by reduced mitochondrial biogenesis, which is a common finding in a number of aging-related diseases. In a model of bleomycin-induced lung injury, upregulation of SIRT1 attenuated fibroblast activation and development of fibrosis.20 Interestingly, treatment with the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), also ameliorates bleomycin-induced pulmonary fibrosis in mice in association with induction of fibroblast apoptosis, suggesting that the specific targets of histone acetylation and deacetylation may be critical in determining the effect on fibrosis.21 Reduction in SIRT3 expression has been shown to promote pulmonary fibrosis in human lung fibroblasts and exacerbates inflammation in mice with endotoxin-induced acute lung injury.22,23(p3) SIRT 3 upregulation has also shown to rejuvenate hematopoietic stem cells and in fact, reverse aging-associated degeneration.24
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Loss of Proteostasis The autophagy-lysosomal system and the ubiquitinproteosomal system are the two major proteolytic systems, both of which decline with aging, leading to increased accumulation of misfolded proteins, increased endoplasmic reticulum (ER) stress and defective autophagy.9 Recent studies have indicated proteostasis plays an important role in the development of IPF. For instance, familial IPF has been linked to protein misfolding mutations in surfactant C.25,26 Similarly, markers of ER stress are seen in lungs from sporadic IPF, most notably in alveolar epithelial cells (AEC) from areas with dense fibrosis and fibroblast foci.27 ER stress has been shown to influence mitochondrial function in AECs by downregulating PINK1, a critical regulator of mitochondrial homeostasis.28 Autophagy is a catabolic process by which damaged organelles and endogenous proteins are destroyed intracellularly within lysosomes. Decline in autophagic capacity is seen in both aging and IPF.9,29 Inhibition of autophagy accelerate cellular senescence in epithelial cells and promote myofibroblast differentiation of lung fibroblasts.30 In a bleomycin-induced lung fibrosis mouse model, deficiency of the autophagic protein, Atg4, led to higher inflammatory and fibrotic responses.31 The mammalian target of rapamycin (mTOR) pathway is a critical regulator of autophagy. Rats receiving the rapamycin analogue, SDZ RAD, are protected from bleomycin-induced experimental fibrosis, although mechanisms of its action on specific cell types are not well understood.32 Activation of autophagy with the AMPK activator, metformin, is capable of accelerating the resolution of lung fibrosis in mice, in part by promoting collagen turnover.29 Interestingly, the antifibrotic drug, pirfenidone, has been shown to induce noncanonical autophagy in lung fibroblasts which may, in part, contribute to its beneficial effects. These studies indicate that age-associated decline in autophagy may be a therapeutic target to maintain proteostasis in the aging lung. Telomere Attrition IPF is the most common manifestation of telomererelated disorders. Genetic mutations related to telomerase (telomere elongating enzyme) are one of the common genetic defects found in patients with familial IPF.33 In addition to activating DNA damage pathways, shortened telomeres are also thought to contribute to fibrosis when exposed to injurious stimulus, by impairing tissue repair. Chromosomal shortening resulting from telomere attrition activates checkpoint inhibitor p53. Activated p53, in turn, reduces mitochondrial biogenesis, increases ROS production and activates cellular senescent pathways.34 Cellular Senescence Cellular senescence is commonly defined as an irreversible state of stable growth arrest in association with a high metabolic activity and secretory phenotype.9 386
Damage accumulation caused by processes such as telomere attrition, oxidative stress and oncogenic stress stimulate the activity of cyclin-dependent kinase inhibitors p16Ink4a and/or p53-p21Cip1/Waf1, which then antagonize cyclin-dependent kinases to block cell cycle progression.35 Despite being in replicative arrest, these cells are metabolically active, and display a SASP consisting of cytokines, growth factors and proteases. Cellular senescence serves a protective function to halt the proliferation of damaged cells and to trigger their own destruction by the immune system.36 Alveolar epithelial cells and fibroblasts have been shown to assume senescent identities in IPF, contributing to development of fibrosis.28,37,38 Established senescent markers such as p21, p16 and senescenceassociated b-galactosidase activity (SA-b-gal) are increased in both fibroblasts and epithelial cells in human IPF lung tissue as compared with controls.38-40 IPFderived fibroblasts exhibit accelerated replicative cellular senescence and increased resistance to oxidative stress-induced cytotoxicity as compared to normal lung fibroblasts. The secretome of both, IPF fibroblasts and AECs, exhibits increased profibrotic and inflammatory markers and is thought to have profound effect on the lung microenvironment by secretion of these mediators which perpetuate fibrotic scarring.37,40 Cellular senescence is being increasingly recognized as a therapeutic target in IPF. Based on the apoptosisresistant phenotype of senescent fibroblasts and myofibroblasts, strategies to increase their sensitivity to apoptosis by inhibition of antiapoptotic factors have been suggested. The antiapoptotic and prosenescent enzyme, NADPH oxidase 4 (NOX4), has been targeted in an aging murine model of persistent lung fibrosis with induction of apoptosis of interstitial myofibroblasts and marked improvement in lung fibrosis and survival.38,41,42 A senolytic cocktail of Dasatinib and Quercetin was effective in eliminating senescent AECs and alleviating fibrosis in a bleomycin-injury model of lung fibrosis.37 A similar observation was made using the same senolytic combination in a murine model of radiation-induced lung fibrosis.40 Cellular senescence offers a potentially effective therapeutic target in IPF and there is active research to determine the safety and efficacy of this approach.
Mitochondrial Dysfunction Mitochondria are tightly regulated in their life-cycle of biogenesis, networking/dynamics, repair, and recycling/ mitophagy. These organelles are responsible for energy (ATP) production and function as bidirectional signaling platforms communicating with the nucleus and other cellular organelles. Altered mitochondrial homeostasis such as reduced mitochondrial biogenesis, increased reactive oxygen species (ROS) production, increased mitochondrial DNA damage and defective mitophagy are found in healthy aging lung.43,44 Many such changes are also THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES VOLUME 357 NUMBER 5 MAY 2019
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seen in fibrotic lungs and are hence thought to contribute to IPF.28,43,45 Mitochondrial biogenesis, the process of producing additional mitochondria, is controlled by master regulators PPARg coactivator-1a (PGC-1a) and PGC1b, and nuclear respiratory factors (NRFs), NRF-1 and NRF-2.46 PGC-1 expression is lower in IPF lungs and also in bleomycin exposed wild mice lung models.47 Telomere attrition and DNA damage have been shown to reduce mitochondrial biogenesis by activating p53 signaling mechanisms which subsequently reduce PGC-1.48,49 AECs from human IPF lungs accumulate dysfunctional mitochondria with upregulation of markers of ER stress compared with age-matched controls.28 These dysfunctional mitochondria are associated with low expression of the regulator of mitochondrial homeostasis PTEN-induced putative kinase 1 (PINK1).50(p3) As a result, PINK1-deficient mice exhibit increased susceptibility to apoptosis, lung injury and TGF-b-driven lung fibrosis.51 PINK1-PARK2 signaling pathway also mediates mitophagy, a process by which dysfunctional mitochondria are removed to maintain cellular health and integrity.52 Insufficient mitophagy caused by PARK2 deficiency activates platelet-derived growth factor receptor (PDGFR) and mammalian target of rapamycin (mTOR) signaling, which results in increased myofibroblast differentiation and proliferation.52 Pirfenidone has been shown to augment mitophagy by enhancing PARK-2 expression and inhibiting myofibroblast differentiation. Although these pathways have been demonstrated to play a role in age- and disease-related alterations in cellular bioenergetics, additional translational investigations in IPF lungs are needed to firmly establish their role.
Extracellular Matrix Although not considered one of the hallmarks of aging, alterations to the extracellular matrix (ECM) with aging may have profound effects on susceptibility of aging and lung fibrosis. ECM of aging lungs expresses a profibrotic phenotype characterized by increased Type I and III collagen and TGF-b1 expression along with enhanced fibronectin extracellular domain A (EDA).53 Old lungs also have a more oxidizing extracellular environment as compared to young lungs, which has been shown to induce intracellular signals that stimulate proliferation and matrix expression in fibroblasts via upregulation of TGF-b 1 expression.54,55 Similarly, ECM of IPF lung fibroblasts have also shown to be rich in collagen and fibronectin EDA. The ability of Type I collagen to limit fibroblast proliferation during physiological repair is shown to be lost in IPF fibroblasts, which potentially contributes to pathological fibrosis.56 Further research to study the alterations in IPF lung EMC, matrix-cell interactions and to
explore the role of matrix-directed therapeutics is required. Impaired Mucociliary Clearance There is an emerging concept that impaired mucociliary clearance may represent a lung aging phenotype and contribute to IPF pathogenesis.
FUTURE DIRECTIONS The progress in our understanding of IPF has greatly advanced in recent years, and has accelerated in part due to the incorporation of aging biology concepts in disease pathogenesis. Critical gaps in understanding the pathogenesis include the role of epigenetic modifications, metabolic sensing, ECM alterations, and airway host defense mechanisms such as impaired mucociliary clearance. Further insights into these mechanisms will lead to the development of novel diagnostics and therapeutics for IPF and other age-associated lung diseases. Advances in imaging modalities and biomarkers that define/detect changes in the metabolic and inflammation-fibrosis related pathways will facilitate clinical translation.
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Submitted January 8, 2019; accepted February 10, 2019. Conflict of interest: Swati Gulati and Victor J. Thannickal have no conflicts of interest. Victor J Thannickal has received grants from the NIH (P01 HL114470 and R01 AG046210) and the VA Merit Award I01BX003056. Funding: The author(s) received no specific funding for this work. Correspondence: Swati Gulati, M.D., Division of Pulmonary, Allergy, and Critical Care, Department of Medicine, University of Alabama at Birmingham, 1900 University Blvd THT 541E, Birmingham, AL 35294-2180. (E-mail: [email protected]).
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