Rehan, Anika

Abstract Rationale Respiratory disease is a major cause of mortality in Down syndrome (DS; trisomy 21, T21). DS is considered a progeroid syndrome, with elevated cellular senescence (SEN) and oxidative stress. Because SEN shapes organogenesis, its dysregulation may impair lung morphogenesis. We previously found SEN/SASP (senescence-associated secretory phenotype) abnormalities in prenatal human T21 lungs. Here, we test whether these alterations persist and change after birth. Methods We analyzed human lung single-nuclear RNA (snRNA-seq) datasets (prenatal and postnatal) to quantify epithelial and mesenchymal SEN/SASP signatures. Sex- and age-matched T21 and non-T21 prenatal and postnatal lungs were studied; fibroblasts were isolated as prenatal human lung fibroblasts (HFLF) and postnatal human lung fibroblasts (HLF). SEN/SASP were measured by RT-qPCR and immunofluorescence (IF) in tissue and HFLF/HLF. HFLF/HLF were assayed for SA-β-gal, oxidative stress (CellROX/MitoSOX), and Ca²+ dynamics (Fura-2 AM). 48h treatments with tunicamycin (1µM, SEN inducer), 4-phenylbutyrate (4-PBA, 2 mM, ER Stress inhibitor), or pyrroloquinoline quinone (PQQ, 20 μM, antioxidant) were performed on T21 and non-T21 HFLF/HLF. Conditioned media (CM) were collected after 48h from untreated cultures and then applied to cells of the opposite genotype for 24, 48, and 72h. Results Overall SEN profile was similar in epithelium and mesenchyme across ages, but pathway signatures differed. Epithelium shows an inflammatory SASP, while mesenchyme shows a less-inflammatory, remodeling/growth-factor SASP. These lineage patterns were more evident postnatally. Whole-tissue IF showed increased γ-H2AX and p21 in T21 vs non-T21 both prenatally and postnatally (p < 0.05). At baseline, T21 HFLF/HLF had higher CDKN1A, CDKN2A, TP53, CDKN2B, and SERPINE1 (p < 0.05; n = 5-6), with increased γ-H2AX/p21 IF (p < 0.01; n = 6), SA-β-gal (p < 0.05; n = 5-6), and ER/mitochondrial stress (p < 0.05; n = 5-6). Fura-2 AM showed greater Ca²+ release in T21 HFLF/HLF at baseline and after acetylcholine (10 μM, p < 0.01; n = 5). Mitochondrial-morphology genes (FIS1, DNM1L, MFN1) were elevated in T21 HFLF/HLF (p < 0.05; n = 6). In T21 HFLF, 4-PBA and PQQ each reduced SA-β-gal (p < 0.01; n = 6). In postnatal HLF, PQQ had the stronger effect (p < 0.05; n = 5). T21 CM induced progressively greater SEN/SASP over 24/48/72h in non-T21 cells (p < 0.05; n = 5-6), while non-T21 CM partially rescued T21-HLF phenotypes at 24h only (p < 0.05; n = 5-6). Conclusion These data indicate that lung senescence in T21 initiates in utero and intensifies after birth and reflects lineage-specific pathways. ER-stress-related mechanisms are more prominent prenatally, while mitochondrial/redox mechanisms are predominant postnatally. This evidence suggests age and lineage-dependent drivers of senescence that may help explain DS lung disease and highlight potential new mechanistic targets. This abstract is funded by: NIH/NHLBI

Rationale: Respiratory disease is a major cause of mortality in Down syndrome (DS; trisomy 21, T21). DS is considered a progeroid syndrome, with elevated cellular senescence (SEN) and oxidative stress. Because SEN shapes organogenesis, its dysregulation may impair lung morphogenesis. We previously found SEN/SASP (senescence-associated secretory phenotype) abnormalities in prenatal human T21 lungs. Here, we test whether these alterations persist and change after birth. Methods: We analyzed human lung single-nuclear RNA (snRNA-seq) datasets (prenatal and postnatal) to quantify epithelial and mesenchymal SEN/SASP signatures. Sex- and age-matched T21 and non-T21 prenatal and postnatal lungs were studied; fibroblasts were isolated as prenatal human lung fibroblasts (HFLF) and postnatal human lung fibroblasts (HLF). SEN/SASP were measured by RT-qPCR and immunofluorescence (IF) in tissue and HFLF/HLF. HFLF/HLF were assayed for SA-β-gal, oxidative stress (CellROX/MitoSOX), and Ca 2 + dynamics (Fura-2 AM). 48h treatments with tunicamycin (1µM, SEN inducer), 4-phenylbutyrate (4-PBA, 2 mM, ER Stress inhibitor), or pyrroloquinoline quinone (PQQ, 20 μM, antioxidant) were performed on T21 and non-T21 HFLF/HLF. Conditioned media (CM) were collected after 48h from untreated cultures and then applied to cells of the opposite genotype for 24, 48, and 72h. Results: Overall SEN profile was similar in epithelium and mesenchyme across ages, but pathway signatures differed. Epithelium shows an inflammatory SASP, while mesenchyme shows a less-inflammatory, remodeling/growth-factor SASP. These lineage patterns were more evident postnatally. Whole-tissue IF showed increased γ-H2AX and p21 in T21 vs non-T21 both prenatally and postnatally (p< 0.05). At baseline, T21 HFLF/HLF had higher CDKN1A, CDKN2A, TP53, CDKN2B, and SERPINE1 (p< 0.05; n=5–6), with increased γ-H2AX/p21 IF (p< 0.01; n=6), SA-β-gal (p< 0.05; n=5–6), and ER/mitochondrial stress (p< 0.05; n=5–6). Fura-2 AM showed greater Ca 2 + release in T21 HFLF/HLF at baseline and after acetylcholine (10 μM, p< 0.01; n=5). Mitochondrial-morphology genes (FIS1, DNM1L, MFN1) were elevated in T21 HFLF/HLF (p< 0.05; n=6). In T21 HFLF, 4-PBA and PQQ each reduced SA-β-gal (p< 0.01; n=6). In postnatal HLF, PQQ had the stronger effect (p< 0.05; n=5). T21 CM induced progressively greater SEN/SASP over 24/48/72h in non-T21 cells (p< 0.05; n=5-6), while non-T21 CM partially rescued T21-HLF phenotypes at 24h only (p< 0.05; n=5-6). Conclusion: These data indicate that lung senescence in T21 initiates in utero and intensifies after birth and reflects lineage-specific pathways. ER-stress-related mechanisms are more prominent prenatally, while mitochondrial/redox mechanisms are predominant postnatally. This evidence suggests age and lineage-dependent drivers of senescence that may help explain DS lung disease and highlight potential new mechanistic targets. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

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.