Introduction

 

Among the hallmarks of aging is senescence, accompanied by chronic inflammation, and disruption of the circadian system. Senescent endothelial cells accumulate in blood vessels where they contribute to the development of cardiovascular diseases through chronic inflammation and vascular dysfunction.

 

Aims

The aim of this study was to investigate the relationship between senescence and circadian rhythms in Endothelial Colony Forming Cells (ECFCs) obtained from umbilical cord blood.

 

Methods

ECFCs were isolated from umbilical cord blood of full-term pregnancies with written consent from mothers and ethical approval REC 15/YH/0281. Senescence was induced by the treatment with Etoposide. Cells were treated with 100 nM Dexamethasone for circadian clock synchronisation followed by sample collection every 3 hours for two periods of two 24 hours cycles. Expression of circadian clock genes was examined by RT-qPCR. ECFCs were stably transduced with lentiviral luciferase reporter for PER2, subjected to etoposide induced senescence, and luminescence was monitored by a real time luminometer system. siRNA was used to silence BMAL1 (ARNTL1) gene and the expression of senescence and inflammatory markers was examined by RT-qPCR. Cosinor analysis and Two-way Anova (senescence, time, P<0.05) were performed using GraphPad Prism.

 

Results

Analysis of our previously published single cell sequencing data revelled differential expression of circadian genes ARNT2, CRY1, PER1 and RORA between senescencet and young cells. Expression of clock genes including ARNT1, PER2 and CRY1 displayed phase shifts and changes in expression levels, compared to the young cells. Luminometer recording of PER2 expression revealed significant differences (p<0.001) between young and senescent ECFCs.  On the other hand, knock down of BMAL1 in senescent cells led to upregulation of β-gal staining. Several interferon related genes were upregulated, including ISG15 and BST2, but no differences were detected in the common senescence markers CDKN1A and SASP genes IL8 and IL6.

   

Conclusion

We demonstrate an interplay between senescense and the circadian clock which could affect vascular function. Enhancing the clock may provide a strategy to delay senescence in vascular cells and preserve their function in ageing.