Cellular Senescence Contributes to the Progression of Hyperoxic Bronchopulmonary Dysplasia

Xigang Jing; Shuang Jia; Maggie Teng; Billy W Day; Adeleye J Afolayan; Jason A Jarzembowski; Chien-Wei Lin; Martin J Hessner; Kirkwood A Pritchard, Jr; Stephen Naylor; G Ganesh Konduri; Ru-Jeng Teng

doi:10.1165/rcmb.2023-0038OC

. 2023 Oct 24;70(2):94–109. doi: 10.1165/rcmb.2023-0038OC

Cellular Senescence Contributes to the Progression of Hyperoxic Bronchopulmonary Dysplasia

Xigang Jing ^1,², Shuang Jia ^1,², Maggie Teng ⁶, Billy W Day ⁷, Adeleye J Afolayan ^1,², Jason A Jarzembowski ^2,³, Chien-Wei Lin ⁴, Martin J Hessner ^1,², Kirkwood A Pritchard Jr ^2,^5,⁷, Stephen Naylor ⁷, G Ganesh Konduri ^1,², Ru-Jeng Teng ^1,^2,^✉

PMCID: PMC12042139 PMID: 37874230

Abstract

Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress sequentially occur in bronchopulmonary dysplasia (BPD), and all result in DNA damage. When DNA damage becomes irreparable, tumor suppressors increase, followed by apoptosis or senescence. Although cellular senescence contributes to wound healing, its persistence inhibits growth. Therefore, we hypothesized that cellular senescence contributes to BPD progression. Human autopsy lungs were obtained. Sprague-Dawley rat pups exposed to 95% oxygen between Postnatal Day 1 (P1) and P10 were used as the BPD phenotype. N-acetyl-lysyltyrosylcysteine-amide (KYC), tauroursodeoxycholic acid (TUDCA), and Foxo4 dri were administered intraperitoneally to mitigate myeloperoxidase oxidant generation, ER stress, and cellular senescence, respectively. Lungs were examined by histology, transcriptomics, and immunoblotting. Cellular senescence increased in rat and human BPD lungs, as evidenced by increased oxidative DNA damage, tumor suppressors, GL-13 stain, and inflammatory cytokines with decreased cell proliferation and lamin B expression. Cellular senescence–related transcripts in BPD rat lungs were enriched at P10 and P21. Single-cell RNA sequencing showed increased cellular senescence in several cell types, including type 2 alveolar cells. In addition, Foxo4-p53 binding increased in BPD rat lungs. Daily TUDCA or KYC, administered intraperitoneally, effectively decreased cellular senescence, improved alveolar complexity, and partially maintained the numbers of type 2 alveolar cells. Foxo4 dri administered at P4, P6, P8, and P10 led to outcomes similar to TUDCA and KYC. Our data suggest that cellular senescence plays an essential role in BPD after initial inducement by hyperoxia. Reducing myeloperoxidase toxic oxidant production, ER stress, and attenuating cellular senescence are potential therapeutic strategies for halting BPD progression.

Keywords: cellular senescence, oxidative stress, bronchopulmonary dysplasia, endoplasmic reticulum stress, myeloperoxidase

Clinical Relevance

This research demonstrates that cellular senescence contributes to bronchopulmonary dysplasia (BPD) progression. Cellular senescence coexists with apoptosis in the BPD lungs and links with myeloperoxidase-mediated oxidant injury, endoplasmic reticulum stress, and autophagy to form a cycle of destruction in BPD.

Bronchopulmonary dysplasia (BPD) is the most common pulmonary morbidity in premature infants, affecting 10,000–50,000 infants annually in the United States (1). BPD survivors frequently experience impaired lung function into adolescence (2) or are at risk for chronic obstructive pulmonary disease because of disrupted lung growth (1). Unfortunately, decades of research have not effectively decreased the incidence of BPD (3). Most premature infants need supplemental oxygen or mechanical ventilation to support metabolic homeostasis. Oxidative stress (OS), inflammation, and impaired angiogenesis after respiratory therapies are the main contributors to BPD. OS generated from respiratory treatments can easily overwhelm the antioxidant capacity of premature lungs. Unopposed OS thus elicits neutrophil infiltration (4), endoplasmic reticulum (ER) stress (5), mitochondrial dysfunction (6), DNA damage, and apoptosis (7) that impair angiogenesis and alveolar formation, which culminates in BPD.

Alveolar formation occurs mainly between Postnatal Day 4 (P4) and P21 in rats. Rat pups exposed to a high-oxygen (HOX) environment (>90%) since birth for 10 days develop a BPD phenotype (5). We recently demonstrated a sequential change from OS- to myeloperoxidase (MPO)-mediated damage to ER stress propagated by the high-mobility group box protein 1 (HMGB1) released from damaged cells in BPD rat pup lungs. We termed this process the BPD “cycle of destruction” (5). We have also reported that N-acetyl-lysyltyrosylcysteine amide (KYC), a novel systems pharmacology agent that inhibits MPO toxic oxidant production (8) and tauroursodeoxycholic acid (TUDCA), a chemical chaperone that decreases ER stress, can effectively reduce the severity of BPD in rat pup lungs (5, 8).

MPO (7), OS (8), inflammation (9), and ER stress (10) have all been implicated in DNA damage. Tumor suppressors are activated in response to DNA damage to facilitate DNA repair (11). Increased p21 (CKDN1A, p21^cip1/wap1) and p53 (TP53, LFS1), two tumor suppressors for cellular senescence, have been reported in BPD animal lungs (12). Increased p53 activity can induce apoptosis or cellular senescence, depending on its interaction with the Foxo proteins (13). Although p21 initiates cellular senescence, p16 (CDKN2A or INK4A) maintains the senescent state (14). The coexistence of other biomarkers, such as increased lipofuscin (+) cells, DNA damage, decreased cell proliferation, and senescence-associated secretory phenotype (SASP) and others, are required to assess the biology of cellular senescence (15).

Cellular senescence is a state of irreversible cell cycle arrest driven by genotoxic damage and was first described more than 50 years ago (16). Both proliferative and nonproliferative cells can become senescent (17). One way to remove senescent cells is to redirect them into apoptosis, which prompts their removal by phagocytes. Senolytics are compounds that encourage senescent cells to enter apoptosis, whereas senomorphics reduce the damaging effects of SASP (18). Yet, cellular senescence has a complex biological role, as evidenced by knocking out some tumor suppressors, which will aggravate alveolar simplification in mice (19). Studies have shown the essential functions of cellular senescence on tumor suppression, development, organ patterning, and wound healing (20). The SASP exemplifies cellular senescence juxtaposition by secreting growth factors and inflammatory mediators (21). Another study demonstrated that specific p16INK4a expression fibroblasts act as resident sentinels in the stem cell niche of the lung (22). The existence of p16-positive cells in the premature cord blood suggests an unidentified role of cellular senescence during fetal development (19). When cellular senescence is unchecked, its chronic inflammatory and hypermetabolic states can hamper organ growth and function (16). Even worse, senescent cells can turn neighboring cells into senescent cells via a paracrine-type mechanism (23). Therefore, senescent stem cells or progenitor cells can be expected to have long-lasting adverse effects on growing organs (24). In a recent study, cellular senescence was demonstrated to significantly affect lung development in mouse pups (25). These authors determined that cellular senescence plays an essential role during the saccular stage of lung development. HOX exposure during the saccular stage aggravates cellular senescence, which is associated with alveolar simplification. Importantly, senolytics were shown in this report to promote alveolar formation in mouse pups exposed to HOX during the saccular stage (25).

The lung experiences the highest oxygen concentrations of all organs and continuous xenogeneic insults from birth. OS originating from supplemental oxygen is a crucial mediator of BPD (26). All of these insults cause DNA damage that can lead to cellular senescence, a phenomenon implicated in numerous adult lung diseases (27) as well as in childhood cystic fibrosis (28) and asthma (29). Increased tumor suppressor expression has been reported in BPD animal lungs (12), and cellular senescence was seen in fetal lung cells exposed in vitro to HOX (30). However, despite our current knowledge of cellular senescence in neonatal lungs, its effect on BPD and growth potential remains limited.

Type 2 alveolar (AT2) cells are resident lung progenitor cells (31). AT2 cells in neonatal lungs are more susceptible to HOX than AT2 cells in adult lungs, because AT2 cell counts drop precipitously after short exposure to 100% O₂, which explains the poor growth potential of BPD lungs (32). We hypothesize that the cycle of destruction initiated by HOX and the subsequent cellular senescence synergistically decrease AT2 counts in BPD lungs. Persistent low AT2 counts may explain why premature adults often have reduced gas exchange capacity.

This study shows that cellular senescence increases in BPD humans and rat lungs. We demonstrate that Foxo4 dri, a senolytic agent that decreases Foxo4 binding to p53 (33), reduces cellular senescence, increases AT2 cell counts, and attenuates BPD in HOX neonatal rat pups. We also show that targeting MPO and ER stress with KYC and TUDCA results in similar benefits, suggesting that both agents have senotherapeutic properties in neonatal rat lungs under HOX. These data indicate that potential senotherapy approaches hold promise for decreasing BPD severity and improving lung growth potential.

Methods

Detailed methods are available in the data supplement. The Pediatric Pathology Department of Children’s Wisconsin provided human lung tissues under the institutional review board’s approval. BPD was determined clinically (34). Age- and sex-matched human lungs without pulmonary diseases were used as controls. Rat pups were mixed and then randomly allocated, per sex, to nursing dams. The dams and pups were cared for in >90% oxygen (HOX) or room air (NOX) from P1 to P10, then back in NOX. The nursing dams were alternated daily.

Animal Treatment

TUDCA (100 mg/kg/d) and KYC (5 mg/kg twice daily) were administered intraperitoneally daily between P2 and P10. Foxo4 dri 10 mg/kg was administered at P4, P6, P8, and P10. Lungs were obtained at P10 or P21. Blood was collected from the hearts after euthanasia.

Histology

Lung morphometrics were obtained as previously reported (35). Apoptosis was identified by in situ TUNEL. Proliferation was quantified by Ki67 stain. Foxo4-p53 binding was studied by the Duolink proximity ligation assay (PLA) method. GL13 and p16 stains identified senescent cells. RT-40, RT-70, and RECA1 antibodies identified AT1, AT2, and endothelial cells.

Transcriptomics Study

Lung RNA was labeled and hybridized to Affymetrix RG230 2.0 arrays. Principal component analyses and nonparametric rank product tests evaluated gene expression differences. Type I errors in multiple tests were determined by false discovery rate (FDR) (36). Enrichment analyses were done by g:Profiler (37) or gene set enrichment analysis (GSEA) 4.3.2 (38) using the SenMayo gene set (39). Genesis was used for generating hierarchical clustering and heatmaps (40). Data were deposited with the National Center for Biotechnology Information Gene Expression Omnibus (GSE197403 for P10 and GSE223745 for P21).

Serum Cell-Free dsDNA Measurement

The Quant-iT PicoGreen double-stranded DNA (dsDNA) reagent was used to quantify serum cell-free DNA.

2,2-Diphenyl-1-Picrylhydrazyl Antioxidant Assay

The antioxidant capacity was quantified using the commercial kits described in the data supplement.

Telomere Length Measurement

The telomere length of genomic DNA extracted from formalin-treated lungs was quantified by PCR against a reference telomere.

Immunoprecipitation

Lung lysates were immunoprecipitated with p53 antibody and then eluted by the citrate buffer (pH 2.2). The eluates were neutralized and heated at 95°C for 10 min in Laemmli buffer. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted for Foxo4, p-serine¹⁵-p53, or p53.

Immunoblotting

Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting. Integrated optic density (OD) was processed with ImageJ 1.53t using β-actin as the loading control.

Single-Cell RNA Sequencing

Details of single-cell RNA sequencing (scRNA-seq), clustering, and cell type annotation are described in the data supplement.

Statistical Analysis

Data were analyzed with GraphPad Prism version 9.0.0 (GraphPad Software). The Student t test or a nonparametric test was used to compare the two groups. One-way ANOVA with the Tukey test was used to compare data between more than two groups. Values are expressed as mean ± SD. Differential gene expression between two groups (1.2-fold; P < 0.05) and Benjamini-Hochberg FDR <0.1 were used for g:Profiler analysis, whereas all transcripts were used for GSEA. Cell-free dsDNA was logarithmically transformed before performing the Kolmogorov-Smirnov test. All P values <0.05 were considered significant.

Results

Increased Cellular Senescence in BPD Human Lungs

Ten sex- and age-matched human lung pairs were obtained. The percentages of 8-hydroxydeoxyguanosine (8-OH-dG) (+) nuclei (Figure 1A), p16 (+) cells (Figure 1B), GL13 (+) cells (Figure 1C), phospho-p53 (+) cells (Figure 1D), Foxo4 (+) cells (Figure 1E), and p53 (+) cells (Figure 1F) were significantly increased by 1.4-, 2.9-, 4.7-, 1.8-, 1.7-, and 1.5-fold, respectively, in BPD human lungs, suggesting increased cellular senescence. However, the increased cellular senescence is probably not the result of shortened telomeres, because the average telomere lengths in BPD human lungs were similar to the average telomere lengths in control lungs (Figure 1G).

Increased Cellular Senescence in BPD Rat Lungs

In BPD rats, evidence of increased DNA damage was indicated by higher 8-OH-dG staining (Figure 2A), higher cell-free dsDNA in serum (Figure 2B), and increased γ-H2A histone family member X (γH2AX) expression (Figure 2C) with decreased lamin B1 expression in lung lysates (Figure 2D). Similar to human BPD lungs, no differences were seen in the average telomere length between BPD and control rat lungs (Figure 2E). The decreased cell proliferation shown by Ki67 staining (Figure 2F) and increased GL13 staining at P10 and P21 further supported increased senescence in BPD rat lungs (Figure 2G). No NOXA expression was changed, whereas p53-upregulated modulator of apoptosis (PUMA) expression increased only at P10 (Figure 2H).

Cellular Senescence–related Transcripts Are Increased in BPD Rat Lungs

Transcriptomic studies revealed gene expression differences between BPD and control rat lungs at P10 (Figure 3A). Several senescence-related transcripts were increased, including growth/differentiation factor 15 (GDF15), serpine 1, and p21 (Figure 3B). The p16 transcript increased by 1.3-fold (P = 0.018, n = 6). Serum GDF15 levels increased by 11.8-fold, and the GDF15 transcript increased by 32.7-fold, with the mature GDF15 expression increased by 2.1-fold in BPD rat lungs at P10 (Figure 3C). Unbiased pathway analyses by GSEA using the SenMayo gene set revealed that cellular senescence increased in BPD rat lungs at P10 (Figure 3D) and P21 (Figure 3E). This indicates that increased cellular senescence persists after rat pups have recovered in NOX for 11 days. According to GO:BP, the g:Profiler analysis showed gene enrichment in apoptosis, aging, wound healing, autophagy, inflammation, unfolded protein response, and cellular senescence (see Figure E1A in the data supplement). In addition, transcripts for maintaining telomere length were enriched (Table E1), explaining why telomere length did not shorten in BPD rat lungs. According to the KEGG (Kyoto Encyclopedia of Genes and Genomes) database, g:Profiler identified gene enrichment in p53 signaling, apoptosis, Foxo signaling, autophagy, and cellular senescence in BPD rat lungs at P10 (Figure E1B). Notably, senescence-related genes were enriched in BPD rat lungs at P10 (Figure E1C and Table E2). Because of the apparent differences in gene expression between male and female HOX rat lungs at P21 under the SenMayo gene set, we further analyzed the data by sex and showed male rats did have higher enrichment in cellular senescence than females (Figure E1D). Female HOX rat lungs also enriched more than NOX rat lungs (Figure E1E). The g:Profiler analysis showed transcripts involved in tissue/organ development, Wnt signaling, Hippo signaling, angiogenesis, cell differentiation, tissue/organ development, and others were decreased in BPD rat lungs at P10 (Figure E2). Several pathways involved in cellular senescence were also enriched but did not include the DNA repair pathway (Figure E3).

Cellular Senescence Is Seen in Multiple Cell Types of the BPD Rat Lungs

Colocalization of p16 with RT1-40 (Figure 4A), RT2-70 (Figure 4B), and CD31 (Figure 4C) was seen in the BPD rat lungs, indicating that cellular senescence occurred in AT1, AT2, and endothelial cells. The Duolink PLA showed a binding between Foxo4 and p53 in some nuclei of BPD rat lungs (Figure 4D). The 1.8-fold increase in serine 15 phosphorylation of p53 indicated a p53 activation. The 2.3-fold increase in Foxo4-p53 binding suggested that increased interaction between the two proteins contributes to cellular senescence in BPD rat lungs (Figure 4E).

Tumor Suppressors, SASP Markers, and Autophagy Are Increased in BPD Rat Lungs

HOX did not affect the alveolar structure at P4 but did impair alveolar formation at P10 and P21 (Figure 5A). Expression of senescence markers at the corresponding time points showed no change at P4 but increased at P10 and P21 (Figure 5B). We also found increased expression of serpine 1, SPP1, IL-6, transforming growth factor (TGF)-β, and TNF-α at P10 and P21 but not at P4 (Figure 5C). The increased GDF15 (Figure 3C), higher HMGB1 expression in our previous report (8), and increased expression of IL-6, TGF-β, and TNF-α indicated the existence of SASP in BPD rat lungs. The characteristic autophagy marker—LC3BII/LC3BI ratio—increased in BPD rat lungs at P10 and P21, suggesting that autophagy was activated after P4 (Figure 5D).

Foxo4-p53 Binding Suppresses Alveolar Formation and Growth Potential in BPD Rat Lungs

To determine whether Foxo4-p53 binding contributes to BPD progression, we used Foxo4 dri (14) to investigate the effects on the alveolar formation and AT2 counts in BPD rat lungs. The antioxidant activity of Foxo4 dri (1 mg/ml) is extremely low at 2.92 × 10⁻⁴ Trolox equivalent antioxidant capacity (TEAC). No antioxidant protein increased in Foxo4 dri–treated BPD rat lungs (Figure E4A). Alveolar complexity (Figure 6A) and cell proliferation (Figure 6B) improved in BPD rat lungs at P21 after Foxo4 dri treatment. In addition, the treated BPD rat lungs had an increased p-S⁷⁸⁰-Rb with decreased p-S¹⁵-p53, p16, and p21, indicating decreased cellular senescence (Figure E4B). No malondialdehyde or protein carbonyl change was seen in Foxo4 dri–treated BPD rat lungs, further suggesting that the activity of Foxo4 dri is probably not through enhancing antioxidant activity (Figure E4C). The Foxo4 dri treatment also reduced the number of GL13 (+) cells (Figure 6C). These changes strongly indicated an attenuation of cellular senescence in the BPD rat lungs by Foxo4 dri. Interestingly, the increased number of TUNEL (+) cells in BPD rat lungs decreased with Foxo4 dri treatment through a mechanism worth investigating (Figure 6D). However, the decreased AT2 count (∼50%) in BPD rat lungs was partially reversed (∼80%) by the Foxo4 dri treatment (Figure 6E), suggesting that lung growth potential can be improved by targeting Foxo4-p53 binding. The effects of Foxo4 dri were not through NOXA and PUMA, because the levels were not changed (Figure 6F).

Figure 6. — Foxo4 dri attenuates alveolar simplification in BPD rat lungs. (A) Rat pups that received Foxo4 dri treatment during HOX exposure maintain a better alveolar formation and vessel count at P21. No difference in radial alveolar count (RAC) was seen in control rat lungs by Foxo4 dri, but Foxo4 dri does improve the RAC in BPD lungs (16.0 ± 0.7 vs. 16.4 ± 0.7 vs. 11.0 ± 0.9 vs. 12.9 ± 1.0; P < 0.001; n = 12). Similar improvement is also seen in mean linear intercept (54.3 ± 3.2 μm vs. 54.6 ± 5.2 μm vs. 77.0 ± 5.1 μm vs. 57.2 ± 3.8 μm; P < 0.001; n = 12), secondary septation (22.2 ± 1.0/high-power field (HPF) vs. 22.7 ± 0.9/HPF vs. 15.1 ± 1.9/HPF vs. 19.2 ± 1.3/HPF; P < 0.001; n = 5–7) and blood vessel count (27.1 ± 6.5% vs. 25.8 ± 2.8% vs. 12.9 ± 3.4% vs. 20.4 ± 4.8%; P < 0.001; n = 9–10; pictures not shown). The increased Ki67 stain (8.3 ± 1.7/HPF vs. 9.9 ± 1.5/HPF vs. 3.7 ± 1.3/HPF vs. 7.8 ± 2.1/HPF; P < 0.001; n = 9–11) and (C) decreased GL13(+) cells (7.6 ± 0.8% vs. 7.7 ± 1.7% vs. 18.2 ± 2.7% vs. 8.6 ± 2.2%; P < 0.001; n = 12) suggested less cellular senescence in Foxo4 dri–treated BPD lungs. (D) Foxo4 dri treatment also decreases apoptosis (3.0 ± 0.8% vs. 2.8 ± 0.7% vs. 6.1 ± 1.3% vs. 4.1 ± 0.4%; P < 0.001; n = 5–6) and (E) increases AT2 cell counts (8.9 ± 1.1/HPF vs. 13.8 ± 1.9/HPF vs. 4.4 ± 2.4/HPF vs. 7.1 ± 1.6/HPF; P < 0.001; n = 5–6) in BPD rat lungs, suggesting an improved lung growth potential. (F) The effect of Foxo4 dri is not through affecting NOXA or PUMA, because the expression is not changed in the treated BPD rat lungs. Arrows indicate cells with positive stains. Scale bars: A, 500 μm; *B-E*, 50 μm. *P < 0.05.

TUDCA Reduces Cellular Senescence and Autophagy in BPD Rat Lungs

Previously, we reported that ER stress is critical to BPD development (5). TUDCA effectively maintained the alveolar complexity of HOX rat lungs. In addition, markers for cellular senescence (Figures 7A and 7B) and autophagy (Figure 7C) in BPD rat lungs decreased by TUDCA treatment with increased AT2 counts (Figure 7D; from ∼42% to ∼86%), suggesting a senotherapeutic activity of TUDCA.

Figure 7. — Reducing endoplasmic reticulum stress and myeloperoxidase-induced oxidative stress attenuate cellular senescence in BPD rat lungs. Rat lungs are obtained at P10. (A) Tauroursodeoxycholic acid (TUDCA) treatment decreases cellular senescence–related tumor suppressors, H2A histone family member X, and two SASP proteins in BPD lungs. TUDCA increases the expression of phosphorylated RB protein, indicating less cellular senescence in BPD lungs. (B) The percentage of GL13(+) cells is dramatically decreased in BPD lungs by TUDCA (1.2 ± 0.1% vs. 1.2 ± 0.1% vs. 24.7 ± 10.4% vs. 2.6 ± 0.5%; P < 0.001; n = 6–9). Scale bars, 50 μm. (C) TUDCA decreases the expression of all autophagy markers in BPD lungs. (D) TUDCA increases AT2 cell counts (11.3 ± 3.0/HPF vs. 11.9 ± 2.2/HPF vs. 4.8 ± 2.1/HPF vs. 0.7 ± 1.1/HPF; P < 0.001; n = 6) in BPD lungs. Scale bars, 50 μm. (E) N-acetyl-lysyltyrosylcysteine-amide (KYC) treatment decreases all cellular senescence markers like TUDCA. (F) The percentage of GL13(+) cells decreases by KYC. (1.8 ± 0.6% vs. 2.1 ± 1.6% vs. 33.4 ± 2.4% vs. 3.6 ± 2.2%; P < 0.001; n = 6). Scale bars, 50 μm. (G) KYC decreases most of the autophagy markers in BPD lungs except ATG7. Scale bars, 50 μm. (H) KYC treatment also increases the AT2 cell counts (15.2 ± 1.2/HPF vs. 15.3 ± 2.8/HPF vs. 5.2 ± 1.5/HPF vs. 11.9 ± 2.6/HPF; P < 0.001; n = 6) in BPD lungs. Arrows indicate cells with positive stains. *P < 0.05.

Decreasing MPO Toxic Oxidant Production Reduces Cellular Senescence and Autophagy in BPD Rat Lungs

MPO-related OS is a significant player in BPD development (7). Increased MPO expression occurred earlier than P4 in BPD rat lungs (Figure E5) before evidence of cellular senescence was detected and was associated with increased autophagy/mitophagy (Figure E6), which has also been implicated in cellular senescence (41). We previously reported that KYC decreases OS-induced DNA damage in BPD rat lungs (7). Therefore, we used KYC to investigate MPO’s role in cellular senescence (8). KYC induces effects similar to TUDCA for attenuating cellular senescence (Figures 7E and 7F) and autophagy (Figure 7G) in BPD rat lungs. KYC also improved AT2 counts in BPD rat lungs (from ∼34% to ∼78%) (Figure 7H).

Increased Cellular Senescence in BPD Rat Lungs

Totals of 6,839 and 3,942 cells from BPD and control rat lungs, respectively, passed the quality assurance for scRNA-seq (GSE242310). Fourteen clusters were identified (Figure E7A) with six (clusters 0, 1, 3, 8, 9, and 11) showing increased cellular senescence (Figure E7B). Cell annotation of clusters with increased cellular senescence identified several cell types, including AT2 (Figures E7C and E7D, cluster 8), AT1, endothelial cells, fibroblasts, macrophages, and lymphocytes (Figure E7E). Transcripts of typical AT2 markers (Sftpa1, Sftpb, Sftpc, Slc34a2, Napsa, Muc1, Lamp3, and Lpcat1) were all enriched in cluster 8.

Discussion

Cellular senescence is a state of irreversible cell cycle arrest driven mainly by genotoxic insults mediated primarily by OS (14, 16). It is detrimental to rapidly growing organs because of its hypermetabolism, proinflammation, and cell-cycle arrest properties. Senescent cells negatively impact organ function further by transforming neighboring cells into a senescent state through SASP paracrine-dependent signaling (23). Although it is the major player in aging, its involvement in tumor suppression, development, organ patterning, and wound healing makes it a vital essential biological function (20). The lungs are susceptible to OS-induced insult as the first organ to face continuous oxygen and xenogeneic insults since birth. Therefore, it is likely that cellular senescence can manifest in premature lungs with immature antioxidative capacity. Cellular senescence has been implicated in several lung diseases in adults (27) and children (28, 29). Mouse pups are born with a high percentage of cellular senescence in the saccular stage of the lungs, then the percentage decreases toward the beginning of the alveolar stage (∼P4). HOX during the saccular stage aggravates cellular senescence and alveolar simplification in mice (25). We have already reported increased oxidative DNA damage, MPO-mediated OS damage, HMGB1 levels, and ER stress in the BPD rat pup model that can lead to apoptosis or cellular senescence (5, 7). Previously, a few BPD animal models described increased tumor suppressor(s) in the lungs (12). However, our knowledge of the mechanistic role of cellular senescence in BPD remains limited.

Our recent report demonstrated that an OS response occurs before P4 and slowly returns to baseline level after HOX rat pups recover in NOX (5). In the present work, we first show increased 8-OH-dG, p16 stain, p53 stain, phospho-p53 stain, Foxo4 stain, and GL13 (+) cells in BPD human lungs that indicate increased cellular senescence compared with age- and sex-matched control lungs (Figure 1). The change cannot be explained as replicative senescence, because there is no shortening of the telomere length (Figure 1). Although experts in senescence research have raised the question about what constitutes the specific SASP or even something such as senescence (Professor Peter L. J. de Keizer, Ph.D., personal communication via e-mail on August, 2023), we determined to use multiple markers commonly adopted by researchers to determine cellular senescence in this study. We identified multiple cellular senescence markers in BPD rat lungs (Figures 2 and 3), including DNA damage (circulating cell-free dsDNA, 8-OH-dG, and γH2AX), tumor suppressors (p53-p21 and p16-Rb axes), GL13 stain, decreased cell proliferation, and the SASP secretome (GDF15, serpine 1, SPP1, HMGB1, IL-6, TNF-α, and TGF-β) (42). The temporal changes of the cellular senescence markers differed from the OS response we previously reported (5), with cellular senescence occurring after OS response. We were intrigued by the lack of gene enrichment in the DNA repair pathway (Figure E3), which might indicate an ineffective DNA repair, which makes cellular senescence even more important in rat pups under OS. The lack of telomere shortening (Figure 2E) also argues against telomere-dependent replicative senescence supporting OS-induced premature senescence. Although we did not see the change in NOXA levels, the increased proapoptotic PUMA level at P10 might assist the p53-mediated apoptosis (Figure 2H), which conforms to the increased apoptosis we previously reported (5) and explains the concomitant increase in cellular senescence and apoptosis in BPD rat lungs. The increased cellular senescence in BPD lungs corroborates the recent report by Yao and colleagues (25). Furthermore, these changes persist even after pups have recovered to NOX, indicating they are not transient phenomena. The array-based transcriptomic study identified 109 senescence-related transcripts that significantly increased in BPD rat lungs. Pathway analyses by g:Profiler and GSEA show that upregulated transcripts are enriched in the biological process of cellular senescence and several cellular senescence-related pathways (Figure E3). Interestingly, we could see a sex difference at P21 in the cellular senescence genes’ enrichment (Figure 3E), with male HOX rat lungs sustaining more than females (Figure E1D) but female HOX rat lungs still having more gene enrichment than the whole NOX group (Figure E1E).

Premature neonatal lungs are in a rapid alveolar formation stage without a fully functional antioxidant defense system. The alveolar formation depends on an intimate interaction between endothelial cells, epithelial cells, and fibroblasts (43). The process occurs exponentially from birth for 2 years in humans, and between P4 and P14, then slows down until P21 in rats (44). Alveolar formation after this critical period is minimal and occurs mainly after lung injury. The hypermetabolic and nonproliferative state of cellular senescence in neonates can be expected to have a much higher and longer impact on the lungs than in adults (45). The clinical implication was demonstrated by a recent study showing that infants born at less than 34 weeks of gestation have threefold odds of having chronic obstructive pulmonary disease and low gas exchange capacity, implying a low alveolar count (46). Cellular senescence also plays a critical role in wound healing and organ patterning, making cellular senescence in BPD development a unique and complex biological activity.

Several mechanisms result in cellular senescence. In our work, the lack of telomere length differences in BPD human lungs (Figure 1E) and BPD rat lungs (Figure 2F) versus controls indicates that telomere shortening is not the cause of cellular senescence in BPD lungs. Our transcriptomic results further support this notion by showing that the transcripts for maintaining telomere length are upregulated in BPD rat lungs. The increased Foxo4-p53 binding in BPD rat lungs suggests that this interaction is at least one possible contributor to cellular senescence (Figures 4D and 4E). Furthermore, cellular senescence and autophagy start after P4 but before P10 in BPD rat lungs (Figure 5). The time sequence between ER stress and autophagy in BPD rat lungs can be reasonably explained by the fact that autophagy is a method to restore cell function under ER stress (47).

Although we show strong evidence of increased cellular senescence in BPD lungs, we were unsure whether senotherapeutics could lead to a better alveolar formation of BPD rat lungs. One potential mechanism that leads to cellular senescence is the increased interaction between p53 and Foxo4. Using the Duolink PLA and coimmunoprecipitation (Figures 4D and 4E), we demonstrated an increased p53–Foxo4 interaction in BPD rat lungs. Because the binding between Foxo4 and p53 increases in the BPD rat lungs, we used Foxo4 dri as an investigative probe (33). The improved lung morphometry and cell proliferation and decreased percentage of senescent cells by Foxo4 dri indicate that Foxo4-p53 binding does contribute to cellular senescence in BPD rat lungs, and senolysis can attenuate the severity of BPD (Figures 6 and E4). The negligible antioxidative activity, no increase of antioxidative protein expression, and lack of change in malondialdehyde and protein carbonyl levels indicate that the senolytic activity of Foxo4 dri is not through the attenuation of OS. Interestingly, Foxo4 dri also increases AT2 cell counts in the BPD rat lungs, suggesting that senolysis may hold therapeutic potential in BPD management. Previously, we have reported persistently increased apoptosis in BPD rat lungs (5). Theoretically, Foxo4 dri treatment directs senescent cells into apoptosis, but, on the contrary, we see a reduction in the in situ TUNEL in BPD rat lungs. This result may suggest an effective removal of the senescent cells by immune phagocytes, which will need further investigation.

We recently reported the causative roles of ER stress and MPO-generated reactive oxidants in the onset and progression of BPD rat lungs (5, 7). Our present results further support that MPO deposition in BPD lungs occurs within days (4), because the increased MPO expression (Figure E5) and ER stress (5) are both detected at P4 and precede cellular senescence and autophagy. Our findings agree with reports that increased ER stress contributes to cellular senescence (48). The relationship between autophagy and cellular senescence is more complicated because both pro- and antisenescent effects have been ascribed to autophagy (46). Still, the two biological processes frequently occur together (48). Our present study suggests how these two biological activities are related. Because our data show a parallel change between autophagy and cellular senescence, we believe autophagy is a survival mechanism in neonatal lungs under chronic HOX exposure to mitigate cellular senescence.

Because autophagy and cellular senescence are preceded by ER stress, we used TUDCA to evaluate the contribution of ER stress to the two biological processes in BPD rat lungs. The reduction in cellular senescence and autophagy with increased AT2 count by early TUDCA treatment (Figure 7) strongly supports the contributing role of ER stress to cellular senescence and autophagy in BPD rat lungs. Myeloid cell infiltration and MPO deposition are observed very early in BPD lungs. The HOCl generated by MPO is a potent reactive oxidant, which damages DNA and elicits ER stress (5). Like the observed effect of TUDCA, KYC, which inhibits toxic oxidant production by MPO (8) and attenuates ER stress (7), also mitigates cellular senescence and autophagy in BPD lungs. All these data indicate that MPO-mediated OS contributes to cellular senescence in BPD rat lungs. Because both MPO-mediated OS and ER stress occur before cellular senescence in BPD, the protective effect of TUDCA and KYC in our results suggests that they have senomorphic properties (18). Although we demonstrate a short-term benefit of senotherapeutic, or senolytic, treatment to BPD rat lungs, a study of its long-term impact must be considered before any clinical application, because cellular senescence has tumor suppression properties by removing the risk of malignant transformation.

To explore what type(s) of lung cells sustained cellular senescence, we implemented scRNA-seq (25), which revealed that macrophages, AT1, AT2, endothelial cells, and fibroblasts are all involved (Figure E7). Our transcriptomic analysis showed a sex difference in cellular senescence at P21 (49). The senescent change of AT2 cells conformed to the recent report by Yao and colleagues (25) and may explain the impaired lung growth trajectory in HOX-exposed neonatal lungs.

Conclusions

In conclusion, our study shows that MPO-mediated OS and ER stress amplify DNA damage after the first wave of OS induced by HOX, resulting in an upregulation of tumor suppressor activity in neonatal lungs. Cellular senescence coexists with apoptosis in the early stage of BPD. Increased binding between Foxo4 and p53 preferentially progresses into cellular senescence in BPD lungs, contributing to AT2 cell depletion. Because AT2 cells are considered residential progenitor cells, any loss in AT2 cells should severely reduce neonatal lung growth and development. Our data indicate that cellular senescence contributes to the impaired alveolar formation caused by HOX and can be part of our previously reported BPD “cycle of destruction” (10). The cycle of destruction in BPD has been updated with these new findings and is shown in the data supplement (Figure E8). This new cycle of destruction in BPD can be summarized as follows. OS elicited by oxygen therapy recruits myeloid cell infiltration to the neonatal lungs. MPO released from the myeloid cells generates HOCl that can damage DNA and induces ER stress. ER stress activates autophagy and aggravates DNA damage, leading to either apoptosis or cellular senescence through p53 signaling. The regenerative activity of the AT2 is suppressed by the neighboring senescent cells. Foxo4 binding to p53 facilitates cellular senescence in the neonatal lung, which the Foxo4 dri can attenuate by directing senescent cells into apoptosis that is removed by phagocytic cells. Reducing the MPO-mediated reactive oxidant generation by KYC or ER stress by TUDCA can mitigate cellular senescence, suggesting that both MPO-mediated secondary and ER stress contribute to cellular senescence in oxygen-exposed neonatal lungs. Because KYC and TUDCA decrease the characteristic markers of cellular senescence, our findings suggest that these two agents have senomorphic properties. Although senotherapy protects the neonatal lung against HOX, we cannot ignore that cellular senescence is essential in wound healing (21, 50) and suppressing tumor transformation (50). The potential long-term side effects of senotherapies, such as chronic wounding and tumor transformation, should be carefully studied before implementing senotherapies to treat BPD. Finally, to our knowledge, this work demonstrates the association and role of cellular senescence, specifically in the progression of BPD.

Acknowledgments

Acknowledgment

The authors thank Mark F. Roethle, M.S., for acquiring the Affymetrix data. The authors also thank Dr. Paytsar Topchyan and Professor Weiguo Cui, M.D., Ph.D., for performing the scRNA-seq.

Footnotes

Supported by the Children’s Wisconsin Foundation, American Diabetes Association grants 1-19-ICTS-129, DK125014, and DK121528 (M.J.H.); Children’s Research Institute – Program Support, Research Unit Leader, and grant HL128371 (K.A.P.); grants HL136597 and HL144519 (G.G.K.); Children’s Research Institute 2021 Pilot Innovative Research Grant, Department of Pediatrics Internal Support, and Advancing a Healthier Wisconsin grant UL1TR001436 (R.-J.T.).

Author Contributions: B.W.D., S.N., and R.-J.T. conceived the study. X.J., M.T., C.-W.L., and R.-J.T. performed the experiments and analyzed the data. S.J., M.J.H., and C.-W.L. analyzed RNA-sequencing data and prepared figures. J.A.J. provided human samples and interpreted data. K.A.P., M.J.H., A.J.A., and R.-J.T. drafted the manuscript, interpreted data, and commented on the manuscript. M.T., B.W.D., K.A.P., S.N., G.G.K., and R.-J.T. finalized the manuscript. All authors read and reviewed the manuscript.

This article has a related editorial.

This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2023-0038OC on October 24, 2023

Author disclosures are available with the text of this article at www.atsjournals.org.

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[bib1] 1. Collaco JM, McGrath-Morrow SA. Respiratory phenotypes for preterm infants, children, and adults: bronchopulmonary dysplasia and more. Ann Am Thorac Soc . 2018;15:530–538. doi: 10.1513/AnnalsATS.201709-756FR. [DOI] [PubMed] [Google Scholar]

[bib2] 2. Harris C, Morris S, Lunt A, Peacock J, Greenough A. Influence of bronchopulmonary dysplasia on lung function in adolescents who were born extremely prematurely. Pediatr Pulmonol . 2022;57:3151–3157. doi: 10.1002/ppul.26151. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] 3. Villosis MFB, Barseghyan K, Ambat MT, Rezaie KK, Braun D. Rates of bronchopulmonary dysplasia following implementation of a novel prevention bundle. JAMA Netw Open . 2021;4:e2114140. doi: 10.1001/jamanetworkopen.2021.14140. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4. Rogers LK, Tipple TE, Nelin LD, Welty SE. Differential responses in the lungs of newborn mouse pups exposed to 85% or >95% oxygen. Pediatr Res . 2009;65:33–38. doi: 10.1203/PDR.0b013e31818a1d0a. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5. Pritchard KA, Jr, Jing X, Teng M, Wells C, Jia S, Afolayan AJ, et al. Role of endoplasmic reticulum stress in impaired neonatal lung growth and bronchopulmonary dysplasia. PLoS One . 2022;17:e0269564. doi: 10.1371/journal.pone.0269564. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6. Ratner V, Starkov A, Matsiukevich D, Polin RA, Ten VS. Mitochondrial dysfunction contributes to alveolar developmental arrest in hyperoxia-exposed mice. Am J Respir Cell Mol Biol . 2009;40:511–518. doi: 10.1165/rcmb.2008-0341RC. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib7] 7. Teng RJ, Jing X, Martin DP, Hogg N, Haefke A, Konduri GG, et al. N-acetyl-lysyltyrosylcysteine amide, a novel systems pharmacology agent, reduces bronchopulmonary dysplasia in hyperoxic neonatal rat pups. Free Radic Biol Med . 2021;166:73–89. doi: 10.1016/j.freeradbiomed.2021.02.006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8. Zhang H, Jing X, Shi Y, Xu H, Du J, Guan T, et al. N-acetyl lysyltyrosylcysteine amide inhibits myeloperoxidase, a novel tripeptide inhibitor. J Lipid Res . 2013;54:3016–3029. doi: 10.1194/jlr.M038273. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib9] 9. Kawanishi S, Ohnishi S, Ma N, Hiraku Y, Murata M. Crosstalk between DNA damage and inflammation in the multiple steps of carcinogenesis. Int J Mol Sci . 2017;18:1808. doi: 10.3390/ijms18081808. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib16] 16. Kumari R, Jat P. Mechanisms of cellular senescence: cell cycle arrest and senescence associated secretory phenotype. Front Cell Dev Biol . 2021;9:645593. doi: 10.3389/fcell.2021.645593. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Cellular Senescence Contributes to the Progression of Hyperoxic Bronchopulmonary Dysplasia

Xigang Jing

Shuang Jia

Maggie Teng

Billy W Day

Adeleye J Afolayan

Jason A Jarzembowski

Chien-Wei Lin

Martin J Hessner

Kirkwood A Pritchard Jr

Stephen Naylor

G Ganesh Konduri

Ru-Jeng Teng

Abstract

Clinical Relevance

Methods

Animal Treatment

Histology

Transcriptomics Study

Serum Cell-Free dsDNA Measurement

2,2-Diphenyl-1-Picrylhydrazyl Antioxidant Assay

Telomere Length Measurement

Immunoprecipitation

Immunoblotting

Single-Cell RNA Sequencing

Statistical Analysis

Results

Increased Cellular Senescence in BPD Human Lungs

Figure 1.

Increased Cellular Senescence in BPD Rat Lungs

Figure 2.

Cellular Senescence–related Transcripts Are Increased in BPD Rat Lungs

Figure 3.

Cellular Senescence Is Seen in Multiple Cell Types of the BPD Rat Lungs

Figure 4.

Tumor Suppressors, SASP Markers, and Autophagy Are Increased in BPD Rat Lungs

Figure 5.

Foxo4-p53 Binding Suppresses Alveolar Formation and Growth Potential in BPD Rat Lungs

Figure 6.

TUDCA Reduces Cellular Senescence and Autophagy in BPD Rat Lungs

Figure 7.

Decreasing MPO Toxic Oxidant Production Reduces Cellular Senescence and Autophagy in BPD Rat Lungs

Increased Cellular Senescence in BPD Rat Lungs

Discussion

Conclusions

Acknowledgments

Acknowledgment

Footnotes

References

ACTIONS

PERMALINK

RESOURCES

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