Novel Method to Observe Endothelial Cell Telomere Dynamics in Regions Exposed to Lifelong Disturbed Flow in Murine Aorta

• Published in: The FASEB journal 2021-05, Vol.35 (S1), p.n/a, Article fasebj.2021.35.S1.04213
• Main Authors: Tucker, Jordan, Bloom, Samuel, Lesniewski, Lisa, Donato, Anthony
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2021.35.S1.04213

Senescent cells accumulate in arteries with advancing age and may be responsible for age‐associated vascular dysfunction. Telomeres protect chromosome ends. However, when telomeres become uncapped or their length becomes critically short through replicative processes or oxidative damage, cells become senescent. Understanding telomere dynamics is essential because telomere dysfunction is a biomarker of biological aging that may contribute to the process of aging and associated diseases. We hypothesize that compared to regions of laminar flow (atheroprotective), such as the descending thoracic aorta, endothelial cells (ECs) in atheroprone regions that experience turbulent flow, such as the minor aortic arch, will demonstrate greater DNA damage and telomere uncapping that is characteristic of cellular senescence. Additionally, ECs in atheroprone areas will be more susceptible to DNA damage and telomere uncapping with advancing age. To test this, we developed a novel method to examine the colocalization of a DNA double‐stranded break (DSB) marker, 53BP1, and telomeric DNA, using immunofluorescence and fluorescence in situ hybridization (IF‐FISH), respectively, in en face aorta tissue samples. The application of IF‐FISH on an en face aortic preparation is advantageous because it provides a comprehensive analysis of telomere dynamics in individual ECs and enables the comparison between arterial regions that experience differential shear stress patterns. Young (~6 mo, N=4) and old (~26 mo, N=3‐4) C57BL6 mice were euthanized and perfused via the left ventricle with saline (0.9% NaCl) followed by 50 ml of 4% paraformaldehyde. Aortas were then cleaned, IF‐FISH was performed, and samples were segmented (thoracic region and minor arch) and mounted using an en face method. ECs were imaged using a confocal microscope, and DSBs and telomere uncapping were quantified (Img. 1). Contrary to our hypothesis, there was no difference in the percentage of ECs with DSBs between regions in young animals (p=0.4, fig. 1A). Additionally, there were no regional differences in telomere uncapping in young animals (p=0.3, fig. 1B). However, when we compared atheroprone regions to areas that experience laminar flow in old animals, DSBs were more prevalent in the atheroprone regions (p=0.008, fig. 1A). Likewise, the percentage of cells with uncapped telomeres was elevated in the minor arch compared to the thoracic aorta (p=0.005, fig. 1B). When examining the effect of lifelong exposure to