Cellular senescence is increased in airway smooth muscle cells of elderly persons with asthma

Abstract

Senescent cells can drive age-related tissue dysfunction partially via a senescence-associated secretory phenotype (SASP) involving proinflammatory and profibrotic factors. Cellular senescence has been associated with a structural and functional decline during normal lung aging and age-related diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Asthma in the elderly (AIE) represents a major healthcare burden. AIE is associated with bronchial airway hyperresponsiveness and remodeling, which involves increased cell proliferation and higher rates of fibrosis, and resistant to standard therapy. Airway smooth muscle (ASM) cells play a major role in asthma such as remodeling via modulation of inflammation and the extracellular matrix (ECM) environment. Whether senescent ASM cells accumulate in AIE and contribute to airway structural or functional changes is unknown. Lung tissues from elderly persons with asthma showed greater airway fibrosis compared with age-matched elderly persons with nonasthma and young age controls. Lung tissue or isolated ASM cells from elderly persons with asthma showed increased expression of multiple senescent markers including phospho-p53, p21, telomere-associated foci (TAF), as well as multiple SASP components. Senescence and SASP components were also increased with aging per se. These data highlight the presence of cellular senescence in AIE that may contribute to airway remodeling.

INTRODUCTION

Lung function gradually declines with aging and often plays an unappreciated role in healthy aging, as a decreased pulmonary reserve is a risk factor for morbidity and mortality (1). Aging is a risk factor for the development of chronic lung diseases such as obstructive pulmonary disease (COPD) (2, 3), and idiopathic pulmonary fibrosis (IPF) (4). There is now increasing recognition that asthma in the elderly (AIE) is also a major health issue with ∼7% prevalence, higher rates of airway hyperresponsiveness, severe presentation, and resistant to standard therapy. Thus, the mechanisms that contribute to AIE become relevant but are not well understood.

Cellular senescence is a well-recognized feature of organismal aging. It is characterized by permanent cell cycle arrest associated with high expression of proinflammatory cytokines, chemokines, and extracellular matrix (ECM)-degrading proteins, collectively known as senescence-associated secretory phenotype (SASP). Although senescence serves important roles in regulating wound repair and suppressing oncogenesis (5), it is now established that senescent cells can also have detrimental influences on the pathogenesis of several age-related diseases. Senescent cells can influence naïve neighboring cells or even have remote effects by secreting SASP (6).

Senescent cells are thought to play an important role in aging and age-related disease by impairing tissue regeneration and inducing chronic inflammation via SASP. Senescent cells have been shown to accumulate during aging in the lung and are associated with diseases such as COPD (2, 3), IPF (4), and bronchiectasis (7). Conversely, clearance of senescent cells has been shown to improve lung compliance, structure, and elasticity in middle-aged mice (4) as well as to improve phenotypes in fibrotic lung disease (4), suggesting that targeting senescent cells may be a strategy to counteract age-related lung pathophysiology (8). Whether senescence plays a role in asthma per se or AIE is not clear.

Beyond senile emphysema, imaging studies show aging bronchial airways are also thicker and fibrotic (9–13) highlighting the idea that bronchial remodeling may be important in both normal aging and AIE, a distinct entity with its own diagnostic and therapeutic challenges (14). Accordingly, a key question becomes what are the cell types in airways that promote remodeling with aging and contribute to AIE? Although multiple cell types within bronchial airway play significant roles in airway hyperreactivity and remodeling, airway smooth muscle (ASM) cells play an integral role in this regard. Particularly, besides changes in the epithelial layer, remodeling can involve ASM hypertrophy (15, 16) and hyperplasia (15–17), and a thickened, fibrotic subepithelial basement membrane (14, 18). Patients with asthma show greater remodeling with changes in collagen subtype deposition, adhesion proteins such as fibronectin and tenascin, and other related ECM proteins (18–28). Importantly, increased ASM proliferation and inflammatory cytokine production are also characteristic of asthma (15, 17). These make ASM potentially significant in aging airways and in AIE. Whether senescent ASM cells accumulate in AIE or contribute to structural and functional changes in this condition is unknown. We tested this hypothesis by investigating the extent of cellular senescence within the ASM layer of bronchial airways and isolated ASM cells from young persons with nonasthma (<45 yr old), elderly persons with nonasthma (>65 yr old), and elderly patients with asthma (>65 yr old).

MATERIALS AND METHODS

Human Lung Tissue Specimens

Lung and airway specimens were obtained from patients undergoing thoracic surgery at Mayo Clinic under an IRB-approved protocol and with patient written informed consent obtained during presurgery evaluation. Patients undergoing surgery for pneumonectomies, lobectomies, or noninfectious diseases were included, whereas disseminated cancers or infectious causes were excluded. Normal areas distant from (typically) tumors were identified by the surgical pathologist. Patient medical data were collected, but patient identifiers were not stored (unique numbers assigned to samples). Table 1 lists patient details on samples included in this study.

Table 1.

All patient data

	Young		Old		Old Asthma
	Lung Tissue	ASM Cells	Lung Tissue	ASM Cells	Lung Tissue	ASM Cells
Age, yr	33.7 ± 2.0	31.0 ± 2.3	72.4 ± 0.9	71.7 ± 1.2	71.3 ± 1.2	70.6 ± 1.9
Females, n	7	8	8	5	10	5
Males, n	5	4	4	10	2	5

Values are means ± SE or n. ASM, airway smooth muscle.

ASM Cell Isolation

Techniques for isolating human ASM cells have been described previously (29–32). Briefly, the epithelial layer was separated from bronchial airways, the ASM layer dissected, and ASM cells enzymatically dissociated. Cell pellets were resuspended in DMEM/F12 medium containing 10% FBS (R&D Systems, Minneapolis, MN), 100 U·mL⁻¹ penicillin, 100 µg·mL⁻¹ streptomycin, and 250 ng·mL⁻¹ amphotericin B (Gibco), and cultured under standard conditions. Experiments were limited to cells from two subcultures.

IHC Staining for Collagen and Fibronectin Deposition

Formalin-fixed paraffin-embedded (FFPE) human lung tissues containing airways of different sizes were cut at 6 µm, mounted on glass slides, and processed for immunohistochemistry (IHC) staining [hematoxylin-eosin (H&E) staining for initial evaluation] following a standard deparaffinization, rehydration, and antigen retrieval procedure.

Masson’s trichome (MT) staining was performed following standard protocol. Briefly, tissue sections were fixed using Bouin’s solution at 56°C for an hour, followed by washing in running tap water for 1 min. Next, tissue sections were stained in Weigert’s iron hematoxylin working solution for 10 min followed by washing in running tap water for 5 min and then Biebrich scarlet-acid fuchsin solution for 2 min. Tissue sections were washed using distilled water and incubated for 10 min in a phosphomolybdic-phosphotungstic acid solution. Subsequently, tissue sections were transferred into aniline blue for 20 min, rinsed with distilled water, and placed in 1% acetic acid solution for 4 min. Next, tissue sections were washed and dehydrated in 95% and 100% ethanol (2 times each) and two times xylene for 5 min each. Finally, tissue sections were mounted using Cytoseal 60 mounting media (Thermo Fisher Scientific; Waltham, MA) and covered.

For IHC, primary antibodies used were mouse monoclonal anti-CDKN2A/p16^INK4a (ab54210; Abcam, Waltham, MA; 1:300) (29); rabbit polyclonal anti-fibronectin (ab2413; Abcam; 1:150), and mouse monoclonal anti-smooth muscle actin (M0851; Agilent Dako, Santa Clare, CA; 1:500). Isotype control antibodies were anti-mouse IgG2b (401202, Biolegend, San Diego, CA) for p16 and anti-rabbit IgG (7074; Cell Signaling, Danvers, MA) for fibronectin. Standard overnight primary antibody incubation was done at 4°C. An ImmPRESS Duet Double Staining Polymer Kit was used (Vector Laboratories, Burlingame, CA). Horseradish peroxidase and anti-mouse IgG-brown and alkaline phosphatase enzyme polymers conjugated to anti-rabbit IgG-magenta were used.

Stained tissue sections were digitally scanned. Borders of bronchial airways were identified (29) and quantified using Orbit Image Analysis software (Idorsia Pharmaceuticals Ltd.; Allschwil, Switzerland) for collagen and fibronectin analysis following protocols established by the software manufacturer (33, 34). Briefly, tissue sections were differentiated using a pixel-based classification model to differentiate tissue from the background (exclusion) and quantify the amount of collagen or fibronectin staining (inclusion). Subsequently, the model was trained and used to categorize stained tissue. The region of interest [ROI; bronchial airways (29–32)] was measured, and the result of the ratio was multiplied by 100. All airways within a section were quantified regardless of shape or size to minimize bias.

ASM mass area was measured (35–38) using Aperio ImageScope (12.4.3; Leica Biosystems, Deer Park, IL) following protocols available within the software. Briefly, ASM mass area (µm²) of each airway was measured and divided by the total area of the airway (µm²); the result of the ratio was then multiple by 100. All airways within the tissue section for each patient were measured regardless of shape or size to minimize bias.

Quantitative Pathology and Bioimage Analysis software (QuPath; Center for Cancer Research and Cell Biology, Queen’s University Belfast) was used for p16^INK4a quantification according to software specifications; see Supplemental Material for script codes (1a and 1b; all Supplemental material is available at https://doi.org/10.6084/m9.figshare.21215183) (39). ASM versus epithelial cells were identified by morphology and relative location within the airway as previously described (29–32). All bronchial airways were quantified regardless of shape or size to minimize bias.

Telomere-Associated DDR Foci Staining

Telomere-associated DNA damage response (DDR) foci (TAF) staining techniques were previously developed by our group to identify and quantify senescent cells in aging and senescence, for use in vitro and in vivo (40). Briefly, TAF staining involves immunostaining for γH2A.X (marker of DNA damage) followed by in situ hybridization of telomere-specific markers that provide a more specific indication of cellular senescence specific to DNA damage pathways. Lung tissue sections were stained and quantified as previously described (7, 41). Briefly, FFPE tissue sections were deparaffinized, rehydrated, and followed by antigen retrieval. Tissue sections were blocked using normal goat serum (1:60 prepared in BSA/PBS) for 30 min and incubated overnight at 4°C with rabbit anti-γH2A.X (mAb 9718, Cell Signaling; 1:200). A biotinylated secondary antibody (BA-1000-1.5, Vector Laboratories; 1:200) was then incubated for 45 min and followed by a tertiary Cy5 streptavidin for 20 min (SA-1500-1, Vector Laboratories; 1:500 in PBS). Sections were washed and cross-linked using 4% paraformaldehyde in PBS and dehydrated in a series of cold ethanol solutions. Hybridization solution (10 µL) containing 70% deionized formamide (Sigma), 25 mM MgCl₂, 1 M Tris pH 7.2, 5% blocking reagent (Roche), 25 µg/mL Cy-3-labeled telomere-specific (C-rich probe repeats CCCTAA; F1002 PNAprobe; Panagene) was added and placed in the oven at 80°C for 10 min. Tissue sections were washed in a series of 70% formamide in 2× saline-sodium citrate (SSC) for 10 min, 2× SSC for 10 min, and PBS for 10 min. Tissue sections were mounted using Prolong mounting media with DAPI to counterstain cell nuclei (Thermo Fisher Scientific). Slides were allowed for 2 days to dry and then imaged using Nikon confocal microscope with optical sectioning and Z stacking (minimum of 30 optical slices using a ×100 lens). FIJI-ImageJ (42, 43) was used for TAF quantifications within ASM layer of bronchial airways; see Supplemental Materials (1c).

Western Blot Analysis

Lung tissues were homogenized using 1× cell lysis buffer (Cell Signaling) supplemented with a pierce protease inhibitor tablet (Thermo Fisher). Protein concentrations were measured using a DC protein assay kit (Bio-Rad), loaded at 25 µg per sample into Criterion TGX Precast Gels. Gels were transferred onto 0.2-µm nitrocellulose membrane (Trans-Blot TurboTM). Intercept Blocking Buffer (LI-COR Biosciences, Lincoln, NE) was used. Primary antibodies were: rabbit polyclonal anti-phospho-p53 (Ser15) (53 kDa, 9284; Cell Signaling), rabbit monoclonal anti-p21 (21 kDa, ab109520; Abcam), and rabbit monoclonal anti-GAPDH (37 kDa, 2118; Cell Signaling). Primary antibodies were diluted according to company recommendations. Secondary antibody IRDaye 800CW goat anti-rabbit (926–32211; LI-COR; 1:10,000) was used, and membranes were imaged and bands quantified using a LI-COR Odyssey XL system. All biological replicates were run on the same gel.

Quantitative Real-Time PCR

Quantitative real-time PCR (qRT-PCR) was performed using standard methods (29). Briefly, RNA was extracted using a Qiagen kit, and concentrations were measured using a Thermo Scientific NanoDrop. qRT-PCR was completed in duplicate for each sample with normalization to S16 (LightCycler 96, Roche Diagnostics Corporation, Indianapolis, IN). All primers were obtained from IDT (Newark, NJ) or Qiagen. Fold changes were calculated using ΔΔCT method (2^−ΔΔCT). For homogenized lung tissue, young, old, and old persons with asthma were normalized to young age groups. For ASM cell culture, young, old, and old persons with asthma on day 7 were normalized to the young age groups on day 0.

Senescence-Associated β-Galactosidase Activity

ASM cells from different groups were cultured in 24-well plates at a final seeding density of 2 × 10⁴ mL⁻¹. On day 3, cells were washed with 1× Dulbecco’s phosphate-buffered saline (DPBS; Gibco), followed by senescence-associated β-galactosidase activity (SA-βGal) staining according to the manufacturer’s protocol (Cell Signaling), and counterstained with DAPI (Thermo Fisher Scientific). A Cytation 5 imaging system (BioTek Instruments, Winooski, VT) was used to count the number of SA-βGal positive cells, normalized to total cell count.

ELISA and Gene Expression

On day 7 of ASM cell culture, the supernatant was collected to assess SASP profile (IL-6 and IL-8) using ELISA kits (R&D Systems); and ASM cells were then processed for qRT-PCR.

Statistical Analysis

Three groups including both females and males in each group were used: young (<45 yr old), old (>65 yr old), and old persons with asthma (>65 yr old); see Table 1. Experiments were performed using a set of 12 patient samples, although not every patient sample was used for all protocols. Details regarding the patient samples used for specific protocols are provided in the Supplemental Tables S1–S5. Data analysis was performed using GraphPad Prism (8.4.3) with one-way ANOVA with Holm–Sidak correction for multiple comparisons. For a nonparametric test, Kruskal–Wallis analysis for multiple comparisons was performed. Robust regression and outlier removal method (ROUT) was used to identify and remove outliers (44). Values are expressed as means ± SE; P values <0.05 were considered significantly different.

RESULTS

Airway Remodeling Is Increased in Aging Lungs and Exacerbated in AIE

Bronchial collagen deposition was quantified in MT-stained lung sections using Orbit Image Analysis. Greater percentages of bronchial collagen were detected in elderly persons with nonasthma and elderly persons with asthma compared with young age group; P < 0.05 and P < 0.01, respectively (Fig. 1A).

Figure 1.

Airway remodeling in aging and asthma in the elderly (AIE). A: representative images of Masson’s trichrome staining of bronchial airway sections from young, elderly persons with nonasthma, and elderly patients with asthma (see methods and Table 1 for details). More abundant collagen (yellow arrow) was seen within bronchial airways particularly in the airway smooth muscle (ASM) layer (black arrow) of elderly persons with nonasthma and further increased in elderly patients with asthma compared with young (n = 5 patients/group). B: representative images of chromogenic double staining for fibronectin (magenta) and smooth muscle actin (dark brown) with cell nuclei counterstained with hematoxylin (blue). Higher fibronectin (yellow arrow) was seen within the ASM layer (black arrow) of elderly patients with asthma (n = 4–6 patients/group). C: greater ASM mass (brown) was seen in airways of elderly person with nonasthma and asthma (n = 4–6 patients/group). Scale bar = 60 µm. *P < 0.05; **P < 0.01. Means ± SE.

Compared with young, a higher percentage of bronchial fibronectin was detected in elderly persons with asthma (P < 0.05), although there was no statistical difference between elderly persons with nonasthma versus young (Fig. 1B). ASM mass area within bronchial airways was also increased in elderly persons with asthma and nonasthma compared with the young age group (Fig. 1C).

Senescent Cell Markers Are Increased in Aging Lungs and Exacerbated in AIE

Senescence markers were selected on their broad expression in senescent cells in different organs or having been identified in other lung diseases such as COPD and IPF (45). Although there is no gold standard marker for cellular senescence, two tumor suppressors are often used since they represent interacting but independent signaling pathways: p53-p21^CIP1 and p16^Ink4a-Rb (45). Downstream, these pathways promote permanent cell cycle arrest at the G₁/S transitional phase (45).

In whole lung tissue, Western blot analysis of tumor suppressor protein phospho-p53 and cyclin-dependent kinase inhibitor p21^CIP1 showed higher p53 phosphorylation in lungs of elderly persons with asthma compared with elderly persons with nonasthma or young age group (P < 0.01 and P < 0.01, respectively; Fig. 2A). p21^CIP1 was also higher in elderly persons with asthma compared with old persons with nonasthma and young age groups (P < 0.05 and P < 0.05, respectively; Fig. 2A). qRT-PCR analyses of mRNA for senescence-associated markers revealed higher expression in lungs of elderly persons with asthma for p16 and p21, compared with elderly persons with nonasthma or younger age group (P < 0.05 and P < 0.05 respectively; Fig. 2B). Interestingly, no substantial change in p53 mRNA expression was observed in elderly persons with asthma compared with elderly persons with nonasthma or young (Fig. 2B).

Figure 2.

Senescent cell burden in bronchial airways with normal aging and asthma in the elderly (AIE). A: Western blot analysis of lungs of elderly patients with asthma show higher phospho-p53 (53 kDa) and p21^CIP1 (21 kDa) protein expression compared with persons with nonasthma age-matched and young age groups (n = 9–12 patients/group), each patient was normalized to its loading control, GAPDG (37 kDa). All biological replicates (per group, young, old, and old asthma) were run on the same gel. B: qRT-PCR analysis showed significantly higher expression of senescent markers p21 and p16 in lungs of elderly person with asthma compared with young age groups. C: similarly, higher levels of SASP-related gene expression (PAI-1, TNFα, MMP1, and CCL2) were observed in lungs of elderly persons with asthma compared with nonasthma age-matched and young (n = 4–10 patients/group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Means ± SE.

The senescence-associated secretory proteins (SASP) profile is cell- and context-specific, but there are common elements (45) including inflammatory mediators such as TNFα, IL-6 and IL-8, ECM proteins, and modulators such as plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinases (MMPs) (45). Analysis of SASP-related genes in homogenized lung tissues shows substantial changes in PAI-1, MMP1, and CCL2 expression in elderly persons with asthma compared with elderly persons with nonasthma and young age group while TNF-α was significantly higher in elderly persons with nonasthma and asthma compared with young (Fig. 2C).

ASM Cells Show Increased Senescence with Aging, Exacerbated in AIE

Based on the data that senescence-associated markers increase in the whole lung with aging, especially in elderly persons with asthma, we sought to investigate if ASM cells are a contributing cell type, given their role in airway hyperreactivity and remodeling. IHC chromogenic staining of senescence-associated marker p16^INK4A showed a higher number of p16^INK4A positive ASM nuclei in elderly persons with nonasthma (P < 0.05) and patients with asthma (P < 0.001) compared with young (Fig. 3A).

Figure 3.

Senescence in airway smooth muscle (ASM) cells in aging and asthma in the elderly (AIE). A: representative images of chromogenic staining for p16^INK4A (dark brown) with cell nuclei counterstained with hematoxylin (blue). Red and orange arrows show p16^INK4A positive cells while green and black arrows show p16^INK4A negative cells within ASM and epithelial layers, respectively (n = 5 or 6 patients/group). Scale bar = 60 µm. B: representative images of quantitative fluorescence in situ hybridization (FISH) combined with immunofluorescence staining for γH2AX (red) and telomeres (green); cell nuclei counterstained with DAPI (blue). Accumulation of cell nuclei containing telomere-associated foci (TAF) within ASM layer was significantly higher in elderly asthmatic bronchial airway compared with elderly patients with nonasthma and young. White arrows point to TAF of colocalization at a single plane of Z-stack at higher magnification (top right, elderly and elderly asthma) (n = 3 or 4 patients/group). C: SA–βGal staining was increased in ASM cells from aging airways of elderly persons with asthma and elderly persons with nonasthma compared with young (n = 5–10 patients/group); Scale bar = 200 µm. D: qRT-PCR analysis showed significant changes in senescence-associated genes p53, p21, and p16 in ASM cells of elderly persons with asthma compared with young (n = 6–10 patients/group). E: ELISA of supernatants from cultured ASM cells of elderly persons with asthma showed increased IL-6 and IL-8 (n = 3–9). (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Means ± SE.

Telomere shortening and DNA damage response (DDR) are hallmarks of cellular senescence (40). Fluorescence in situ hybridization (FISH) of telomerase combined with immunofluorescence staining of γ-H2AX allows for localization of telomere-associated foci (TAF), and has been used to identify senescent cells (3, 45): a technique developed by the Passos group (40). Critically short telomeres induce senescence via the activation of a DNA damage response (DDR), and telomere/DNA damage is irreparable and leads to a persistent DDR during cellular senescence (40). We, therefore, investigated telomere dysfunction in ASM cells using Immuno-FISH to quantify colocalization between DDR proteins γH2A.X and telomeres, i.e., TAF. Increased ASM nuclei containing TAF were observed in elderly persons with asthma compared with elderly persons with nonasthma and young (P < 0.05 and P < 0.01, respectively; Fig. 3B).

ASM cells isolated from elderly patients with asthma also showed elevated levels of senescence markers. Senescence-associated β-galactosidase (SA-βgal) is widely utilized as a marker to distinguish senescent cells in vitro and in vivo (45, 46). The blue chromagenic assay relies on lysosomal enzyme activity at a pH of 6.0 present in senescent cells (46). We observed a greater percentage of SA-βGal positive ASM cells in elderly persons with asthma compared with elderly persons with nonasthma or young (P < 0.001 and P < 0.0001, respectively; Fig. 3C). qRT-PCR analysis of senescence-associated genes revealed higher expression for p53 and p21 in ASM cells of elderly persons with asthma compared with elderly persons with nonasthma cells or young (Fig. 3D). Higher expression of p16 was also observed in ASM of elderly persons with asthma (P < 0.001) compared with nonasthma or young (Fig. 3D).

ELISA analysis of the supernatants from ASM cell culture showed significantly higher levels of IL-6 in elderly persons with asthma compared with nonasthma or young (Fig. 3E). A similar pattern for IL-8 was observed in elderly persons with asthma compared with elderly persons with nonasthma or young (P < 0.01; Fig. 3E).

A Pearson correlation was performed to explore the strength and direction of association between senescence markers or ECM deposition. Interestingly, although we found no correlation between p16^INK4A positive cells within ASM layer and collagen deposition, we found a significant correlation between the percentage of fibronectin and p16^INK4A positive cells within the ASM layer of bronchial airways, suggesting that senescent cells may be drivers of airway fibrosis (Fig. 4).

Figure 4.

Correlation between senescence and extracellular matrix (ECM). The percentage of fibronectin staining within bronchial airways and p16 positive airway smooth muscle (ASM) cells was higher in elderly patients with asthma. Pearson’s correlation coefficient (r) and P value for <0.05 significance are shown.

DISCUSSION

With an increasing elderly population (47–49), understanding aging-associated structural and functional changes in airways and lung parenchyma becomes important toward improving health (50–52) as well as understanding diseases such as asthma (53–56), COPD, and IPF (57–61) that disproportionately affect the elderly. Although aging is usually associated with “senile emphysema” and thus the alveolar compartment, imaging studies show that aging bronchial airways are also thicker and more fibrotic (9–13), highlighting the idea that bronchial remodeling may be important in both normal aging and AIE. Accordingly, key questions in the field become what are the cell types in airways that promote remodeling with aging and contribute to AIE, and what are the mechanisms at play? In this study, we report novel data on cellular senescence and remodeling changes that occur with AIE by comparing three groups, young (<45 yr of age), elderly persons with nonasthma, and elderly persons with asthma (>65 yr of age), toward demonstrating clinical significance of our studies. Our results indicate that aging results in increased expression of senescence-associated markers and SASP components in ASM, with greater ECM that could contribute to the thicker and stiffer airways found in the aging lung. Importantly, we find that AIE involves exacerbated senescence, SASP, and ECM, suggesting that cellular senescence may play a role in the enhanced fibrosis of the aging asthmatic airway (Fig. 5).

Figure 5.

Schematic summarizing increased senescent-associated markers and extracellular matrix (ECM) in aging airways, exacerbated in asthma in the elderly (AIE). ASM, airway smooth muscle; SA-Gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory phenotype; TAF, telomere-associated foci. [Image created with BioRender.com and published with permission.]

Although multiple cell types usually contribute to structural and functional changes in the bronchial airway that occur in asthma, ASM cells are recognized to be important given their role in contractility as well as in remodeling via cell proliferation and fibrosis as occurs in diseases such as asthma (62–64), COPD (65), and even IPF (66). However, there is very little known about ASM cells and ECM deposition with aging or in AIE. Most data investigating aging and ECM deposition demonstrate a role for lung fibroblasts during aging (4, 67–70). We previously showed that aging increases ASM deposition of collagen III and fibronectin, whereas the ECM modifying proteins MMP2 and MMP9 are decreased, suggesting reduced ECM turnover (71). In the present study, we also find aging-associated increases in collagen and fibronectin in the bronchial airways as well within the ASM layer, along with exacerbated increases in AIE, underlining the potential role of ASM cells in the pathogenesis of fibrosis in elderly persons with asthma. Indeed, airway remodeling in elderly patients with asthma involves a thickened, fibrotic subepithelial basement membrane (14, 18) with increased ASM mass. The latter can occur via ASM hypertrophy (15, 16) and hyperplasia (15–17). We previously showed that aging is associated with increased ASM proliferation (71). Whether senescent cells within the aging airway contribute to such proliferation, exacerbated in AIE, is unknown.

Cellular senescence contributes to aging-associated structural and functional changes within organs (1, 72). Senescent cells release SASP that can include proinflammatory and profibrotic elements, including IL-6 and IL-8 that we explored, leading to altered cell proliferation, upregulation of endoplasmic reticulum stress, disruption of the unfolded protein response, mitochondrial dysfunction (73–75), fibrosis (4, 8, 76, 77), and inflammation (4, 8, 29, 77). The presence of senescent cells in the lung or in lung diseases is recognized (78–82). In pulmonary fibrosis, epithelial cells express p21^CIP1 and p53 (83) and fibrotic foci show p16^INK4A (84). Fibroblasts in pulmonary fibrosis produce SASP that enhances ECM production by naïve fibroblasts (4). In mice, removal of senescent cells reverses bleomycin-induced injury, p21^CIP1, and SASP (85). Senescent fibroblasts and epithelial cells (86–88) are also increased in COPD (1, 3, 89). Compared with these findings, senescence in adult asthma has not been well studied, although senescence is suggested by increased p16^INK4A, p21^CIP1, and SA-βgal-staining in the asthmatic epithelium (90, 91). The contribution of ASM cells in this regard is entirely unknown. Accordingly, our data showing increased senescence within airways and particularly in ASM with aging become significant. ASM cells show an upregulation of proteins associated with activation of senescent pathways such as p21^CIP1, p16^INK4A, and SA-βgal. This increase in senescence-associated markers expression is accompanied by changes in SASP components such as IL-6 and IL-8. Overall, these novel data highlight the presence of ASM senescence in aging airways and in AIE.

Our data show that senescent ASM cells accumulate in elderly patients with as well as without asthma, as demonstrated within stained lung tissues and in isolated ASM cells. Mechanistically, senescence can involve multiple pathways, and there are cell- and context-specific differences in the contribution of these pathways. We found that the tumor suppressor protein p16^INK4A, which regulates the cell cycle and is often used as a senescence-associated marker (92) to be increased in ASM nuclei of aging airways and AIE compared with young age. Quantitative analysis of TAF staining revealed a significant increase in ASM cells from aging and in elderly patients with asthma highlighting the accumulation of senescent cells containing dysfunctional telomeres. This novel finding is consistent with the evidence of increased p53 and p21^CIP1 elements in lung and ASM since this pathway plays a substantial role in stress-associated DNA damage response (93–96). The transcriptional factor p16 is also significantly upregulated in elderly patients with asthma, as are several SASP-related genes (TNFα, PAI-1, CCL2, and MMP1). These results suggest that classic senescence pathways including telomere shortening to DNA damage, p53 phosphorylation, p21^CIP1 (97, 98), and p16^INK4A (98) activation following downstream inhibition of cell cycle progression are involved in AIE. These pathways are also elevated in ASM in this group suggesting a role for this cell type: a functional aspect that remains to be explored. Our results are also consistent with previous reports suggesting increased p21^CIP1 and p16^INK4A expression in the asthmatic epithelium of adult patients (90, 91).

Our studies demonstrate that cellular senescence occurs in bronchial airways with aging, exacerbated in AIE, and that senescence of ASM cells follows this pattern by age and disease. Furthermore, we show that increased airway fibrosis occurs with aging and AIE. However, our studies do not functionally or mechanistically link increased ASM senescence to the increased airway fibrosis. Nonetheless, previous data and current understanding of senescent cell biology suggest this is likely the case. Senescent cells are now known to exert their detrimental effects by releasing proinflammatory and profibrotic factors, including TNFa, IL-6, and IL-8. Such factors have been previously implicated in ASM remodeling and contractility per se (29, 99). Thus, an increase in these SASP modulators, produced by senescent ASM cells, for example, could lead to increased fibrosis in aging or AIE. We have previously demonstrated that aging is associated with increased ASM proliferation, and thus it is possible that senescent cells are one driver for these changes in the aging airway, with increased effects in AIE. Exploring these causal links will necessitate approaches to removing senescent cells via novel drugs (senolytics) (45) and transgenic mouse models where INK4A positive senescent cells can be selectively eliminated (45): topics for future studies.

Our current study focused on senescence in ASM cells. However, airway remodeling certainly involves other cell types including epithelium and fibroblasts. And in this regard, senescence in these cell types can also be expected. Indeed, epithelial cell senescence has been shown in pulmonary fibrosis (83, 84), as has been fibroblast senescence and their SASP effects (4). Senescent fibroblasts and epithelial cells in COPD have also been shown (1, 3, 89). We also find p16-positive cells within the airway epithelial layer with aging and asthma (Fig. 3A). Thus, it is possible that in aging bronchial airways, paracrine effects of multiple senescent cell types on multiple cell types occur and overall lead to the observed remodeling. Thus, our study is the first to introduce the idea of ASM as one cell type that could play a role in remodeling in the context of aging and asthma. Future studies, potentially involving cell-type-specific transgenic mouse models of senescence can help address the relative roles of different cell types in aging and asthma.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL DATA

Supplemental script codes (1a, 1b, and 1c) and Supplemental Tables S1–S5: https://doi.org/10.6084/m9.figshare.21215183.

GRANTS

This work was supported by NIH Grants T32 HL105355 (A. Aghali), HL088029 and HL158532 (Y. S. Prakash), HL142061 (C. M. Pabelick), 1R01AG068048-01 (to J. F. Passos), and 1UG3CA268103-01 (to J. F. Passos).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.A., J.F.P., and Y.S.P. conceived and designed research; A.A., L.K., A.B.L., and L.Y.D. performed experiments; A.A., L.K., J.J.T., and Y.S.P. analyzed data; A.A., J.F.P., and Y.S.P. interpreted results of experiments; A.A., A.B.L., J.F.P., and Y.S.P. prepared figures; A.A. drafted manuscript; A.A., J.F.P., and Y.S.P. edited and revised manuscript; A.A., C.M.P., J.F.P., and Y.S.P. approved final version of manuscript.