Cherry, Caroline

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.

Rationale: Lung associated diseases are one of the leading causes of death in Down syndrome/Trisomy21 (DS/T21). DS is often considered a progeroid syndrome, associated with increased cellular senescence and oxidative stress in dermal T21 fibroblasts. Senescence plays a crucial role in development and organogenesis. We recently demonstrated that T21 lung anomalies are initiated in utero. We aim to better understand the role of senescence in lung disease in T21. Methods: Fresh T21 and, age- and sex-matched non-T21 human fetal lungs were collected, dissociated, and human fetal lung fibroblast (HFLF) cultures were established using differential adhesion. Baseline senescence levels were determined in whole tissue and isolated fibroblasts using TAF staining for tissue only and RT-qPCR and immunofluorescence (IF) for both. β-galactosidase, CellROX/MitoSOX, and calcium release (FURA2/AM imaging) assays were performed on T21 and non-T21 fibroblasts, cultured in the absence or presence of an ER stress inhibitor (4-PBA, 2mM) or a mitochondriogenesis stimulator (PQQ, 20μM). Results: Whole tissue sections demonstrated increased TAF and γ-H2AFX staining in T21 vs non-T21. RT-qPCR demonstrated significantly increased expression of several senescence markers including CDKN1A, CDKN2A, TP53, CDKN2B and PAI1 (p<0.05; n=6) at baseline in T21 HFLF as compared to non-T21. IF for γ-H2AFX and P21 were also significantly increased in T21 cultures vs non-T21 (p<0.01, n=6). Upregulated senescence was further confirmed in T21 HFLF by increased β-galactosidase activity (6-fold vs non-T21, p<0.05, n=4) as well as increased ER and mitochondria stress markers (CellROX and mitoSOX, p<0.05, n=6) in T21 cultures. Fura2/AM assay revealed that T21 HFLF exhibit greater intracellular calcium release (p<0.01, n=3) than non-T21 HFLF at both baseline and post Acetylcholine (10uM) challenge, an indicator of increased ER stress. In addition, several mitochondria morphology genes ( FIS1, DNML1 and MFN1) increased in T21 HFLF (p<0.05, n=6). Both 4-PBA or PQQ treatments reversed senescence in T21 HFLF as shown by decreased β-galactosidase staining (p<0.01; n = 4). No differences were observed in non-T21 HFLF after treatment. However, gene expression analyses of CDKN1A, CDKN2A, TP53 and CDKN2B demonstrated attenuated expression in T21 HFLF only following PQQ treatment but not 4-PBA (p<0.05, n=4). Conclusion: In this study, we established that fibroblasts from T21 lungs present with both ER and mitochondria-associated senescence. However, only PQQ, the mitochondriogenesis stimulator, was able to completely reverse the senescence observed at baseline in T21 HFLF. Understanding this increase in senescence in the lungs of individuals with Down Syndrome may help address the respiratory issues that arise after birth. CIRM (EDUC4-12837) Postdoctoral Training Grant (RB) NIH/NHLBI R01HL141856 (DA); NIH/NHLBI Offce of The Director, National Institutes of Health (OD) R01HL155104 (SD); NIH/NHLBI R21HL165411 (SD and DA). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rationale Human chromosomal anomalies, notably trisomies, induce several alterations in gene expression, leading to various disease phenotypes. Recent studies showed that dermal fibroblasts from patients with trisomy 13 (T13), T18 or T21 present features of increased senescence and oxidative stress. We recently demonstrated that T21 lungs present abnormalities prenatally. Senescence is a state of cell division arrest that plays an important role in organogenesis. However, little is known about cellular senescence in fetal human lung with T13, T18 and T21. We therefore sought to characterize cellular senescence in these lung tissues. Methods Fresh human fetal lung tissues with T13, T18 and T21 were collected along with euploid age and sex matched controls. RT-qPCR and immunofluorescence staining (IF) were performed to assess the expression of senescence markers as well as Senescence-Associated Secretory Phenotype (SASP) elements in the various lung compartments (epithelial and mesenchymal). In addition, fibroblasts were isolated and cultured from each sample to determine the extent of senescence on isolated mesenchyme by IF and RT-qPCR. Staining for oxidative stress detection and Reactive oxygen species were assessed by CellROX and MitoSOX. Results RT-qPCR analysis in T13 whole lung tissues demonstrated no significant differences in the expression of either senescence or SASP markers as compared to control ( P16/ P21/ TP53 and IL6/ IL1 β/ TGF β/ CXCL8 respectively; n=5). Interestingly however, IF staining in T13 tissues showed a significant increase of γ-H2AFX, a marker of senescence, in the mesenchyme (n=4) as well as significantly decreased P21 staining in both the epithelial and mesenchymal compartments. Additionally, isolated fibroblasts from T13 lungs expressed significantly more IL6 as compared to control (n=3), with IF staining demonstrating a significant increase of γ-H2AFX. T18 lungs displayed significantly increased expression of P16 as compared to control (n=6), whereas IF staining revealed an increase of γ-H2AFX in whole tissue (n=4). Fibroblasts isolated from T18 lungs revealed no significant difference in any markers (gene or protein). Lastly, T21 lungs showed a significant increase of CXCL8 expression as compared to control (n=6). IF staining of T21 lungs demonstrated an increase γ-H2AFX in both the epithelium and mesenchyme (n=8). However, isolated T21 lung fibroblasts displayed significantly increased expression of P16, P21, TP53, and CXCL8. Furthermore, IF staining of T21 lung fibroblasts displayed significantly more γ-H2AFX and P21 positive cells as compared to controls (n=5). Interestingly, only cells from T21 lungs presented a significant increase of CellROX and MitoSOX staining (n=5). Conclusion In this study we established that expression of both senescence and SASP markers differ depending on the trisomy. Our results suggest that cellular senescence is more pronounced in lungs from individuals with trisomy 21. These authors acknowledge funding from NIH/NHLBI R01HL141856 (to DAA); NIH/ Office Of The Director, National Institutes Of Health (OD) R01HL155104 (SD); NIH/NHLBI R21HL165411 (SD and DAA); CIRM training grant EDUC4-12837 (RB) and NIH/NICHD 2R24HD000836-52 (IG). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.