Cellular senescence is associated with trisomies during human lung development

Abstract

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.