Elevated Senescence Markers in Developing Trisomy 21 Human Lungs

Abstract

Human chromosomal anomalies, notably trisomies, disrupt gene expression, leading to diverse cellular and organ phenotypes. Increased cellular senescence (SEN) and oxidative stress in trisomies have gained recent attention. We assessed SEN, SEN-associated secretory phenotype (SASP), and oxidative stress on trisomy 13 (T13), T18, and T21 human fetal lung tissues and isolated primary human fetal lung fibroblasts. Telomerase-associated foci staining showed DNA damage primarily within T21 and T18 lungs. These results were confirmed by real-time quantitative PCR showing an increase of the SEN marker CDKN2B and SASP markers IL-6 and CXCL8. In contrast, lung tissues from T13 showed an upregulation of CDKN2A, whereas no significant changes in SASP marker genes were observed. γ-H2AX (H2A histone family member X) was upregulated in each genotype, particularly in T21. Isolated fibroblasts demonstrated a strong association between T21 and several SEN markers. An increase of γ-H2AX–positive cells were observed in fibroblasts from T21, T18, and T13, but only T21 exhibited an increase in P21 expression. Only T21 fibroblasts displayed a significant increase in reactive oxygen species levels, as indicated by MitoSOX and CellROX. This study provides the first evidence of a link between SEN and trisomy anomalies during prenatal human lung development, particularly in T21.

Clinical Relevance

Our study shows how impactful trisomy 21 (T21) is on lung development. Increased senescence appears to be a contributing factor to the abnormalities observed in the developing T21 lung. This brings to light the possibility of introducing senolytics to potentially alleviate the defects observed in T21 lungs.

Human chromosomal abnormalities, notably trisomies, induce several alterations in gene expression, resulting in various phenotypic abnormalities. The most prevalent chromosomal abnormality worldwide, trisomy 21 (T21), also referred to as Down syndrome, is caused by the presence of an additional complete copy of Hsa21 (human chromosome 21) (1, 2). This multisystem condition is characterized by a spectrum of medical problems, such as craniofacial abnormalities, cognitive impairment, gastrointestinal implications, and autoimmunity. Among these, respiratory and cardiovascular complications remain the primary cause of mortality and morbidity observed in these individuals (3–5). Currently, our understanding of the underlying mechanisms of lung disease in individuals with T21 is limited; however, we have previously demonstrated that T21 lung abnormalities originate during fetal development (6). We observed that >70% of prenatal T21 lungs present with histological abnormalities, as well as alterations in several genes, pathways, and cellular phenotypes. Similarly, T18, also known as Edwards syndrome, is a chromosomal condition resulting in various phenotypic abnormalities such as low birth rate, heart defects, and intellectual disabilities (7–9). Children with T18 often experience a variety of airway complications (10, 11). Furthermore, T18 has been shown to be associated with inadequate congenital development of vessels and alveoli in the lung (10). As a result of the various medical problems, only approximately 5–10% of children with T18 survive past their first year of life (12). Finally, T13, referred to as Patau syndrome, is associated with severe intellectual disabilities and physical abnormalities, resulting in a similar life expectancy to that observed in T18 (13). Despite limited evidence of lung disease in T13 and T18, a recent study described pulmonary complications in patients with T13 or T18 following cardiac surgery (14).

Several genetic syndromes are associated with diseases and pathobiology of aging, including T21. T21 has been shown to present with the highest associations with aging-related pathologies among many other genetic syndromes, thus classifying it as the leading progeria syndrome (15). T21 is also associated with accelerated epigenetic aging (16). Recent studies have demonstrated that dermal fibroblasts derived from individuals with T21, T18, and T13 exhibit characteristics of oxidative stress and premature cellular senescence (SEN) (16). Whether SEN is present in the lungs of patients with trisomies remains to be determined.

Cellular SEN is a process that imposes permanent proliferative cell arrest in response to a variety of factors and is known to be important in development and organogenesis (17, 18). In proliferating human cells, progressive telomere erosion ultimately exposes free double-stranded chromosome ends. Consequently, this process upregulates the tumor suppressor protein p53 and its transcriptional target P21 (CDKN1A) (19). Concurrently, activation of the P16 (CKDN2A) promoter has been shown to increase with aging and inflammation (20). Increased transcript or protein levels of P16 and P21 have been effective in demonstrating the presence of SEN in pathological tissues, including the lung (21). Although SEN can be beneficial in growth and repair (22), it becomes detrimental when SEN cell numbers exceed immune clearance (23). SEN cells are in G1 cell cycle arrest. They secrete factors, known as the SEN-associated secretory phenotype (SASP), that mediate paracrine effects on naive cells (24). Cellular SEN can be induced by DNA damage, oxidative stress, and mitochondrial dysfunction, all of which are increased in T21 (25). In this study, we aimed to characterize cellular SEN in T13, T18, and T21 during human fetal lung development through evaluation of SEN and SASP markers in whole lung tissue and isolated mesenchymal cells from developing lung samples with these conditions.

Methods

Human Fetal Lung Tissues and Study Approval

Deidentified human fetal lungs with or without T13, T18, and T21 at 12.5–26.2 weeks of gestation were collected under institutional review board approval (The Lundquist Institute 18CR-32223-01) (Tables E1–E3 in the data supplement) and provided to the laboratory by the University of Washington Birth Defects Research Laboratory. A segment of tissue was fixed as previously described for histological analyses, another segment (6, 26) of tissue was snap-frozen in RNALater for gene expression analyses, and a third was used for fibroblast isolation (27).

Primary Fibroblast Isolation

To obtain primary fibroblasts, tissues were mechanically and enzymatically dissociated and allowed to grow by differential adhesion as described in the data supplement.

Real-Time PCR Analyses

RNA from human fetal lung tissues, including an equal distribution of proximal and distal airways, or human fetal lung fibroblasts (HFLFs) at passage 3 were extracted using an easy-spin Total RNA Extraction Kit (Intron Bio., Boca Scientific; cat. no. 17221). cDNA was synthesized using a Tetro cDNA synthesis kit (cat. no. BIO-65043; Bioline). cDNA (12.5 ng) was amplified using specific TaqMan gene expression assays (listed in Table E4; Applied Biosystems) and the TaqMan Universal PCR Master Mix II (Applied Biosystems). PCR products were detected using QuantStudio 3 (Applied Biosystems). Each sample was run in triplicate.

Fluorescence in Situ Hybridization and Immunofluorescence Staining

Human fetal lung tissues were fixed overnight in 4% paraformaldehyde at 4°C, paraffin-embedded, sectioned, and processed for staining as previously described (28). Human fetal lung fibroblasts were fixed in 4% paraformaldehyde for 30 minutes and immunofluorescence-stained as previously described (27). Antibodies are detailed in Table E5. Ten images were captured with a 40× objective and quantified using the HALO Image Analysis Platform (version 3.4.2986; Highplex FL and area quantification module; Indica Labs Inc). Each pair was individually assessed to assure accurate quantification of protein expression.

Telomerase-associated Foci Staining

Progressive loss of telomere repeats and uncapping results in association of destabilized, damaged telomeres with DNA damage response factors, e.g., phospho-H2A.X and ATM (29). Colocalizing γ-H2AX (H2A histone family member X) and telomeres by combining immunostaining and fluorescence in situ hybridization (FISH) allows for identification and quantification of SEN (30). We used the telomerase-associated foci (TAF) staining technique developed by Aghali and coworkers and previously validated for quantification of SEN in adult lung disease (31). Immuno-FISH was performed as previously described (31). Briefly, lung tissue sections were fixed and blocked using normal goat serum and incubated overnight with anti-γH2AX (see Table E5 and the figures in the data supplement) followed by secondary antibody. They were washed and re–cross-linked for subsequent hybridization using Cy-3–labeled telomere-specific peptide nucleic acid probe (C-rich probe repeats CCCTAA; F1002 PNAprobe; Panagene). DAPI was used for nuclear localization and imaged using a Nikon confocal microscope with optical sectioning and Z-stacking. FIJI-ImageJ was used for TAF quantification.

Reactive Oxygen Species Assay

CellROX (green; C1044; Thermo Fisher Scientific) and MitoSOX (red; M36008; Thermo Fisher Scientific) assays were performed on trisomy and control fibroblasts following the manufacturer’s instructions. Pictures were acquired on a Leica Thunder inverted microscope, and the mean fluorescence intensity was quantified using HALO software (area deconvolution module).

Statistical Analyses

Statistical analyses were performed using GraphPad Prism (GraphPad Software Inc). Normality was assessed for each group using the Shapiro-Wilk test. If a group passed assumptions for parametric testing, a paired parametric t test was employed to compare each trisomy to sex- and age-matched non-trisomy lung samples. The resulting P values were corrected using the Dunnett test. If data did not pass the Shapiro-Wilk test, a Wilcoxon matched-pairs test was conducted. The resulting P values were adjusted using Dunn’s test. The results were considered significant if the P value was 0.05 or lower.

Results

Lung Tissues from Trisomies Are Associated with DNA Damage

Telomere shortening and DNA damage are recognized as significant contributors to cellular SEN and the aging process. Accelerated telomere dysfunction is associated with various age-related human conditions, including trisomies (32, 33). However, it remains unclear whether this process initiates in utero. To investigate this, we analyzed human fetal lung tissue sections from four or five independent samples with T21, T18, and T13, alongside sex- and age-matched controls via TAF staining (Figure 1A) and quantification of colocalization of γ-H2AX in telomeres (Figure 1B). The data demonstrated a significant increase in colocalization for T18 (32.92% ± 13.65; P = 0.0113) that was exaggerated in T21 lungs (46.50% ± 16.37; P = 0.0006) compared with controls (7.2% ± 5). However, the trend in T13 samples did not reach significance for the colocalization of γ-H2AX in telomeres (25.4% ± 11.45; P = 0.0765) compared with controls. These findings indicate that DNA damage occurs primarily during the development of human T18 and T21 fetal lungs.

Figure 1.

DNA damage is associated with trisomies in human lung development. (A) Fluorescence in situ hybridization staining for TAF (telomere; red) and immunofluorescent (IF) staining for γ-H2AX (H2A histone family member X; green) on formalin-fixed, paraffin-embedded human fetal lung sections from patients with trisomy 13 (T13), T18, and T21 syndrome and sex- and age-matched controls (n = 4–5 patients per group) and associated quantification of telomerase-associated foci staining (B). Results are shown as individual data points and mean ± SEM; *P < 0.05 and ***P < 0.001. Scale bars: large inset, 50 μm; small inset, 10 μm. ns = not significant; TAF = telomerase-associated foci.

Prenatal Human T21 Lungs Display an Altered Expression of SEN Markers

To better assess cellular SEN in T21 fetal lung tissue, we evaluated the expression of SEN and SASP markers in human fetal lungs between 12.5 and 26.2 weeks of gestation (Figure 2). Real-time quantitative PCR (RT-qPCR) analyses demonstrated significantly increased expression of CDKN2B in T21 compared with non-T21 tissue (0.001 ± 0.0001 vs. 0.0006 ± 0.0001; n = 6; P = 0.033; Figure 2D). However, no difference was noted for the other SEN markers tested (CDKN1A, CDKN2A, TP53, and CDKN2B; Figures 2A–2C). Moreover, expression of the SASP marker CXCL8 was significantly increased in whole T21 fetal lung tissue compared with sex- and age-matched control tissue (0.05 ± 0.01 vs. 0.01 ± 0.004; n = 6; P = 0.0260; Figure 2G), whereas no changes were observed for IL1β, IL-6, and TGFβ (Figures 2E, 2F, and 2H).

Figure 2.

T21 prenatal lung tissues display significant alterations in senescence (SEN) markers. Relative gene expression of CDKN1A, CDKN2A, TP53, CDKN2B, IL1β, IL-6, CXCL8, and TGFβ (A–H) within human fetal T21 lung tissues and sex- and age-matched control tissue (n = 6). IF staining on T21 human fetal tissue and sex- and age-matched control tissue for γ-H2AX (I) and P21 (M). Quantification by HALO software shown by dot plots: the percentage of total γ-H2AX– and P21-positive cells in whole tissue (J and N; n = 6), in the epithelium compartment (K and O), and in the mesenchyme (L and P). Results are shown as individual data points and mean ± SEM; *P < 0.05 and **P < 0.01. Scale bars: large inset, 50 μm; small inset, 15 μm.

IF staining was performed on whole lung tissue for γ-H2AX (Figures 2I–2L) and P21 (CDKN1A; Figures 2M–2P). Whole tissue, epithelial, and mesenchymal compartments were analyzed separately. T21 whole fetal lung tissue displayed a significant increase in γ-H2AX total positive cells compared with control (27.30% ± 2.40 vs. 8.37% ± 1.93; n = 6; P = 0.0042; Figure 2J). This upregulation was observed in the epithelial (Figure 2K; P = 0.0034) and mesenchymal (Figure 2L; P = 0.0026) compartments (37.22% ± 5.5 vs. 10.50% ± 2.34 and 26.91% ± 4.14 vs. 8.17% ± 1.62, respectively; n = 6). Analysis of P21 IF staining on T21 fetal tissue showed no significant changes in the whole tissue (Figure 2N), epithelial compartment (Figure 2O), or mesenchymal compartment (Figure 2P) compared with sex- and age-matched controls. Given the association between cellular SEN and fibrosis, we performed Picrosirius red staining on whole lung tissue of T21, T18, T13, and sex- and age-matched controls (Figures E1A, E1D, and E1G). Our data suggest no difference in staining in any of the trisomy conditions compared with controls, suggesting the absence of fibrosis and airway remodeling.

Isolated Human Prenatal T21 Lung Fibroblasts Present Distinct SEN Markers

Previous studies showed increased cellular SEN in dermal T21 fibroblasts (16). Because SEN can be cell- and context-specific, we sought to investigate SEN in isolated fibroblasts from human fetal T21 lungs. Fibroblasts within passages 2–5 were analyzed to avoid replicative SEN. The same passage was used to compare T21 versus non-T21 control fibroblasts to assure consistency. In contrast to our findings in whole tissue, RT-qPCR analysis for the SEN markers CDKN1A (0.13 ± 0.01 vs. 0.10 ± 0.01; P = 0.0328; Figure 3A), CDKN2A (0.025 ± 0.003 vs. 0.012 ± 0.0007; P = 0.0061; Figure 3B), TP53 (0.072 ± 0.01 vs. 0.037 ± 0.008; P = 0.008; Figure 3C) and CDKN2B (0.006 ± 0.001 vs. 0.003 ± 0.001; P = 0.0096; Figure 3D) were shown to be significantly upregulated in isolated T21 lung fibroblasts compared with sex- and age-matched controls (n = 6). Similar to whole lung tissue, the relative expression of the SASP marker CXCL8 was significantly increased in T21 fetal lung fibroblasts (0.0008 ± 0.00005 vs. 0.0005 ± 0.00008; P = 0.0088; Figure 3G), whereas no differences were observed for IL1β, IL-6, or TGFβ expression (Figures 3E, 3F and 3G). Furthermore, IF staining on isolated fetal fibroblasts for γ-H2AX (Figure 3I) confirmed the results observed in whole tissue, demonstrating significantly more γ-H2AX–positive T21 human fetal fibroblasts compared with non-T21 (29.25% ± 2.86 vs. 8.45% ± 1.65; n = 6; P < 0.0001; Figure 3K). Interestingly, although no changes were observed in P21 IF staining between T21 and non-T21in whole lung tissue, isolated T21 fibroblasts exhibited significantly more P21-positive cells (Figure 3J) compared with non-T21, suggesting a potential epithelial regulation of P21 expression (34.03% ± 5.02 vs. 9.80% ± 2.03; n = 6; P = 0.0057; Figure 3L). To gain insight into the functional implications of these changes, we examined the extracellular matrix (ECM) components in T21 HFLFs and matched controls by assessing the expression of VIM (vimentin) and FN (fibronectin) (see Figures E1B and E1C). These genes were described to be linked to SEN and reactive oxygen species (ROS) (34–37). Our results showed a significant upregulation of these two genes in T21 compared with non-T21 tissue (1.092 ± 0.02 vs. 1.007 ± 0.01 [P = 0.0252] and 1.075 ± 0.08 vs. 0.936 ± 0.07 [P = 0.0133]; n = 5).

Figure 3.

Isolated prenatal T21 lung fibroblasts present distinct SEN markers. Relative gene expression of CDKN1A, CDKN2A, TP53, CDKN2B, IL1β, IL-6, CXCL8, and TGFβ (A–H) in T21 and sex- and age-matched control human fetal lung fibroblasts (HFLFs; n = 6). Representative image for γ-H2AX (I) and P21 (J) IF staining on T21 HFLFs compared with non-T21 and associated quantification by HALO software (K and L; n = 6). Representative images of CellROX (M) and MitoSOX (N) staining performed on T21 and sex- and age-matched control HFLFs. (O and P) Dot plots of mean fluorescence intensity (MFI) quantification of the CellROX (M) and MitoSOX (N) (MFI; O and P; n = 6). Results are shown as individual data points and mean ± SEM; *P < 0.05, **P < 0.01, and ****P < 0.0001. Scale bars: large inset, 50 μm; small inset, 10 μm.

Isolated Human Prenatal T21 Lung Fibroblasts Show ROS Differences

CellROX and MitoSOX staining were performed on isolated fetal lung fibroblasts (Figures 3M and 3N) for characterization of oxidative stress and ROS. CellROX and MitoSOX mean fluorescence intensities were significantly higher in isolated T21 fetal lung fibroblasts compared with sex- and age-matched controls (62.52 ± 6.43 vs. 49.11 ± 6.51 [P = 0.0074] and 20.19 ± 1.24 vs. 13.48 ± 1.33 [P = 0.023]; n = 6; Figures 3O–3P), indicative of increased levels of ROS.

To validate these results, we performed RT-qPCR for genes associated with ROS and mitochondria regulation (Figure E2). Interestingly, T21 HFLFs displayed a significantly higher expression of APP (0.2823 ± 0.05 vs. 0.1843 ± 0.04; P = 0.008; n = 6; Figure E1A), SOD1 (0.042 ± 0.007 vs. 0.030 ± 0.003; P = 0.0358; n = 6; see Figure E1B), KEAP1 (0.012 ± 0.001 vs. 0.009 ± 0.0008; P = 0.0164; n = 6; see Figure E1C), and MFN1 (0.008 ± 0.0006 vs. 0.005 ± 0.0003; P = 0.005; n = 6; see Figure E1D), whereas a downregulation of FIS1 was observed (0.2823 ± 0.05 vs. 0.1843 ± 0.04; P = 0.008; n = 6; Figure E1E).

Taken together, these results suggest that the increase in cellular SEN in T21 fetal lung fibroblasts is associated with increased endoplasmic reticulum and mitochondrial ROS.

Limited Correlation between SEN and T18 in Human Fetal Lung Tissue

The study that reported increased cellular SEN in T21 dermal fibroblasts also reported increased cellular SEN in T18 dermal fibroblasts (16). We did find increased TAF staining in T18 fetal lung tissues (Figure 1). Therefore, we sought to further assess cellular SEN markers in T18 human fetal lung tissues (Figure 4). RT-qPCR results demonstrated an increase of CDKN2A and CDKN2B expression (Figures 4A–4D) in T18 versus control tissues (0.0016 ± 0.0003 vs. 0.0010 ± 0.0001; n = 6 [P = 0.049] and 0.0016 ± 0.0002 vs. 0.0008 ± 0.0001 [P = 0.026]; Figures 4A and 4D). We also observed a significant increase in the expression of the SASP marker IL-6 (0.004 ± 0.001 vs. 0.0012 ± 0.0003; n = 6; P = 0.025; Figure 4F) in T18 compared with control. No changes were observed for the other markers. Whole tissue staining for γ-H2AX (Figures 4I–4L) showed an increase in the total percentage of positive cells in fetal lung tissues from T18 versus controls (7.55% ± 1.42 vs. 5.35% ± 0.0003; n = 4; P = 0.46; Figure 4J). Although these results were not significantly associated with a specific compartment, it does appear to be primarily epithelial-driven (P = 0.10 for epithelium vs. P = 0.99 for the mesenchyme; Figures 4K–4L). P21 IF staining showed no differences between T18 and control (Figures 4M–4P). As for T21 tissues, no fibrosis was detected within T18 tissue compared with non-T18 tissue (see Figure E1D).

Figure 4.

Increase in specific SEN markers is observed in T18 human fetal lungs. Relative gene expression of CDKN1A, CDKN2A, TP53, CDKN2B, IL1β, IL-6, CXCL8, and TGFβ (A–H) within human fetal T18 lung tissues and sex- and age-matched control tissues (n = 6). IF staining on T18 human fetal tissue and sex- and age-matched control tissues for γ-H2AX (I) and P21 (M). Quantification by HALO software shown by dot plots: the percentage of total γ-H2AX and P21 positive cells in whole tissue (J and N; n = 4), in the epithelium compartment (K and O), and in the mesenchyme (L and P). Results are shown as individual data points and mean ± SEM; *P < 0.05. Scale bars: large inset, 50 μm; small inset, 15 μm.

Isolated Human Prenatal T18 Fibroblasts Display Minor Alterations in SEN Markers

RT-qPCR of SEN and SASP markers on isolated mesenchymal cells from T18 fetal lung tissues (Figures 5A–5H) showed a significant decrease in CDKN2A gene expression compared with control (0.003 ± 0.0009 vs. 0.024 ± 0.005; n = 4; P = 0.037; Figure 5B). Additionally, IF staining for γ-H2AX on T18 and control fibroblasts did not exhibit the increase observed in whole tissue (Figures 5I and 5K). Moreover, no difference was observed in the number of P21-positive cells (Figures 5J and 5L). We also investigated ECM genes by assessing VIM and FN expression. Although no change was noted for FN expression (see Figure E1F), VIM expression was significantly downregulated in T18 versus non-T18 controls (0.815 ± 0.02 vs. 0.948 ± 0.01; n = 4; P = 0.03; see Figure E1E).

Figure 5.

Absence of changes in SEN markers in T18 fetal lung fibroblasts. Relative gene expression of CDKN1A, CDKN2A, TP53, CDKN2B, IL1β, IL-6, CXCL8, and TGFβ (A–H) within T18 and sex- and age-matched control HFLFs (n = 4). Representative image for γ-H2AX (I) and P21 (J) IF staining on T18 HFLFs compared with non-T18 HFLFs and associated quantification by HALO software (K and L; n = 4). Representative images of CellROX (M) and MitoSOX (N) staining performed on T18 and sex- and age-matched control HFLFs and respective MFI quantification (O and P; n = 4). Results are shown as individual data points and mean ± SEM; *P < 0.05. Scale bars: large inset, 50 μm; small inset, 10 μm.

Finally, unlike what was observed in T21, the assessment of endoplasmic reticulum and mitochondrial ROS levels (CellRox and MitoSox) did not show any differences between fibroblasts from T18 human fetal lungs compared with fibroblasts from control lungs (Figures 5M–5P). These results were confirmed by RT-qPCR for genes associated with ROS and mitochondria (see Figure E2). APP and SOD1 expression were downregulated (0.05 ± 0.006 vs. 0.12 ± 0.008 [n = 5; P = 0.0063] and 0.03 ± 0.007 vs. 0.05 ± 0.01 [n = 5; P = 0.0257]; see Figures E2F and E2G), whereas there were no significant differences in KEAP1, MFN1, and FIS1 (see Figures E2H and E2J) compared with non-T18 controls.

Minor Alterations in SEN Markers in Human Fetal T13 Lung Tissue

Although we did not detect a significant increase in TAF staining within the T13 fetal lungs, a previous study reported increased SEN in T13 dermal fibroblasts (16). Therefore, we sought to further analyze the expression of SEN and SASP markers in our cohort of fetal T13 lung tissues (n = 6). RT-qPCR of the SEN and SASP markers showed an increase solely in CDKN2A expression compared with sex- and age-matched controls (0.003 ± 0.001 vs. 0.0009 ± 0.0002; n = 6; P = 0.026; Figure 6B). Interestingly, our results from IF staining for the SEN marker γ-H2AX (Figures 6I–6L) showed a significant increase in the total number of γ-H2AX–positive cells in T13 versus control tissues (10.19% ± 1.68 vs. 4.38% ± 1.07; n = 4; P = 0.026; Figure 6J). However, when assessing the compartments separately, we noted a significant increase exclusively in the mesenchyme, whereas only a trend was observed in the epithelium (P = 0.09; Figure 6L; 5.35% ± 1.01 vs. 1.17% ± 0.30; n = 4; P = 0.009, Figure 6K). In contrast, IF for P21 (Figures 6M–6P) displayed a significant downregulation of the total number of P21-positive cells in T13 versus control (2.22% ± 0.52 vs. 13.09% ± 2.71; n = 4; P = 0.042; Figure 6N). Picrosirius red staining did not show any difference between T13 and non-T13 lung tissue (see Figure E1G).

Figure 6.

No significant changes were observed in SEN or oxidative stress markers in T13 lung fibroblasts. Relative gene expression of CDKN1A, CDKN2A, TP53, CDKN2B, IL1β, IL-6, CXCL8, and TGFβ (A–H) within human fetal T13 lung tissues and sex- and age-matched control tissues (n = 6). IF staining on T13 human fetal tissue and sex- and age-matched control tissue for γ-H2AX (I) and P21 (M). Quantification by HALO software shown by dot plots: the percentage of total γ-H2AX– and P21-positive cells in whole tissue (J and N; n = 4), in the epithelium compartment (K and O), and in the mesenchyme (L and P). Results are shown as individual data points and mean ± SEM; *P < 0.05. Scale bars: large inset, 50 μm; small inset, 15 μm.

No Significant Alteration of SEN Markers in T13 Isolated Fibroblasts

RT-qPCR analyses performed on isolated fibroblasts from T13 fetal human lung tissues did not show differences in SEN marker expression compared with sex- and age-matched controls (Figures 7A–7D). However, expression of the SASP marker IL-6 was upregulated in T13 fibroblasts compared with control (0.005 ± 0.001 vs. 0.001 ± 0.0005; n = 6; P = 0.03; Figure 7F), whereas no differences were observed for the other SASP markers studied (IL1β, CXCL8, and TGFβ; Figures 7E, 7G, and 7H). γ-H2AX staining confirmed the results observed in the whole lung tissue (Figures 7I and 7K), with an increase of γ-H2AX in T13 fibroblasts compared with age- and sex-matched controls (5.83% ± 1.07 vs. 2.87% ± 0.71; n = 4; P = 0.032; Figure 7K), whereas no differences were noted for P21 (Figures 7J and 7L). No differences were observed in the expression of genes related to the ECM (see Figures E1H and E1I).

Figure 7.

No significant changes observed in senescence or oxidative stress markers in T13 lung fibroblasts. Relative gene expression of CDKN1A, CDKN2A, TP53, CDKN2B, IL1β, IL-6, CXCL8, and TGFβ (A–H) within T18 and sex- and age-matched control HFLFs (n = 4). Representative image for γ-H2AX (I) and P21 (J) IF staining on T13 HFLFs compared with non-T13 HFLFs and associated quantification by HALO software (K and L; n = 4). Representative images of CellROX (M) and MitoSOX (N) staining performed on T13 and sex- and age-matched control HFLFs and respective MFI quantification (O and P; n = 4). Results are shown as individual data points and mean ± SEM; *P < 0.05. Scale bars: large inset, 50 μm; small inset, 10 μm.

Finally, fibroblasts from T13 lungs showed no change in ROS level compared with control (Figures 7M–7P). This was further supported by the lack of changes in the expression of genes associated with ROS and mitochondrial function (see Figures E2K–E2O). Altogether, these results suggest that T13 fetal lung tissues display minor alterations in SEN-associated makers.

Discussion

The interplay between cellular aging and chromosomal abnormalities, particularly trisomies, has significant implications for understanding disease complications in individuals with T21, also known as Down syndrome (38, 39). Individuals with T21 often exhibit signs of premature aging, such as early onset of aging-associated conditions like Alzheimer’s disease (39, 40). However, little is known about these processes in other organ systems such as lung. Furthermore, even less is known about the relationship between SEN and other trisomies such as T18 and T13 (16). Moreover, the time of initiation for such SEN and the underlying mechanisms involved in the progeria phenotype associated with chromosomal abnormalities is not well understood. This study aimed to describe the relationship between different trisomies, SEN, and associated oxidative stress using a larger number of human fetal lung samples with T21, T18, and T13 compared with previous studies conducted in other organs (16, 41). Our findings suggest that T21 lungs exhibit early SEN and associated markers (i.e., SASP) more prominently than T18 and T13 lungs, which was observed in whole tissue and isolated fibroblasts (Table 1).

Table 1.

Trisomy and Senescence: Key Findings

Trisomy	Fetal Lung Tissues	Isolated Fibroblasts
T21	DNA damage: TAF-positive, γ-H2AX↑	DNA damage: γ-H2AX↑
	Senescence: CDKN2B↑	Senescence: CDKN1A↑, CKDN2A↑, TP53↑, CDKN2B↑
	SASP: CXCL8↑	Mitochondria: ROS↑ (MitoSOX, CellROX), APP↑, KEAP1↑, SOD1↑, MFN1↑, FIS1↑
T18	DNA damage: TAF-positive, γ-H2AX↑	DNA damage: no change
	Senescence: CDKN2A↑, CDKN2B↑	Senescence: no change
	SASP: IL-6↑	Mitochondria: no change
T13	DNA damage: TAF-positive, γ-H2AX↑	DNA damage: γ-H2AX↑
	Senescence: CDKN2A↑	Senescence: no change
	SASP: no change	SASP: IL-6↑
		Mitochondria: no change

Definition of abbreviations: ROS = reactive oxygen species; SASP = senescence-associated secretory phenotype; TAF = telomerase-associated foci.

However, even though our results did not show any association between SEN, mitochondrial defects, and fibrosis, it appears that ECM-related genes are upregulated in T21 lung whole tissue and isolated fibroblasts, consistent with our previous reports (6). Further research is needed to understand the impact of SEN on lung development and growth, especially concerning early-life exposures that could predispose T21 individuals to lung diseases later in life.

SEN, particularly when prematurely initiated, can disrupt several embryological processes and lead to developmental abnormalities (42). Lung development, in particular, is susceptible to premature SEN-induced abnormalities, such as impairment of epithelial–mesenchymal interactions, improper alveolar formation, reduced lung growth, and altered lung architecture (24, 39). Senescent cells may secrete factors that alter ECM composition, leading to improper alveolar structure and reduced lung growth. These abnormalities can affect the formation of alveoli and development of lung vasculature, potentially leading to compromised gas exchange and respiratory function (43). These developmental abnormalities can manifest in several functional deficits later in life, such as age-related pulmonary diseases like chronic obstructive pulmonary disease, emphysema, and fibrosis, while also playing a crucial role in organ development through pathways like P21 (17, 18). However, its role in lung development is not fully understood. Our study shows that the P21 pathway is activated in fetal lungs regardless of genotype, with heightened activation in T21 lungs, particularly in fibroblasts. This P21-driven SEN in T21 may well contribute to later-life pulmonary diseases. Additionally, T21 has been associated with oxidative stress–related SEN, potentially having a negative influence on lung development. Studies have noted premature SEN in T21 fetal dermal fibroblasts, suggesting that increased P21 expression may explain the premature aging phenotype in T21 individuals (44). Early programmed SEN is essential for postnatal lung development, whereas hyperoxia-induced SEN can lead to lung injury through different mechanisms (45).

Consistent with other trisomies, T18 and T21 show an upregulation of γ-H2AX, which is known to be associated with DNA damage pathways. T21 demonstrated significant increases in γ-H2AX staining in epithelial and mesenchymal cells compared with age- and sex-matched controls, akin to diseases affecting lung development and SEN (46, 47). We also observed increased levels of CXCL8, a SASP marker linked to SEN in lung cells by promoting DNA damage (48, 49). Although CXCL8 is commonly increased in adult lungs during inflammation and SEN-related diseases, its role in developmental lung conditions requires further investigation (50). In T18 and T21 whole lung samples, we found increased expression of CDKN2B (p15), a protein associated with developmental SEN (18, 51). T18 also showed increased CDKN2A and IL-6, known for its role in tissue repair and as a SASP marker, affecting lung development positively but potentially leading to gestational disorders when altered (52–54). Understanding these markers in T18 and T21 can shed light on aging phenotypes and premature lethality. Investigations into T13 fetal lungs revealed increased CDKN2A expression and γ-H2AX staining, primarily in the mesenchymal compartment, consistent with TAF staining. Despite being rare and associated with short life expectancy, T13 cases are increasing, highlighting the need for improved monitoring and treatment strategies (55).

Increased ROS levels associated with SEN in T21 fetal dermal fibroblasts support our findings in T21 lung fibroblasts (16, 44). As a hallmark of SEN, heightened ROS levels can negatively impact lung development and function. In the embryonic lung, excessive oxidative stress can damage cellular components, disrupting normal cell function and development. This oxidative damage may impair lung maturation, leading to long-term functional deficits such as reduced respiratory capacity and increased susceptibility to lung diseases (56). ROS are implicated in aging by causing macromolecular and organelle damage (57–59). Chromosome aneuploidy, as seen in T21, leads to oxidative stress and mitochondrial damage, as indicated by altered mitochondrial markers in fibroblasts (16). Protein oxidation is also increased in T21, akin to senescent cells (60). The use of senolytic drugs has gained traction during the past decade. Accelerated removal of senescent cells might alter the pathophysiology by reducing the burden of SEN-associated damage and inflammation. This could potentially mitigate some developmental or functional impairments associated with SEN. However, the effectiveness of this treatment would depend on timing and efficacy of removal, as well as the impact on normal homeostasis and tissue repair processes.

Despite limitations such as the smaller sample sizes for T13 and T18 cases compared with T21, our study includes the largest sample number to date, focusing on SEN primarily in fibroblasts but highlighting the need for further research in epithelial and other mesenchymal cell types like airway smooth muscle. Overall, this study illuminates the connection between trisomy and SEN during lung development, which is particularly pronounced in T21, necessitating further investigation into the mechanisms and implications for T21-associated pathologies.