Abstract Body (Do not enter title and authors here): Endothelial cell (EC) senescence has been identified as a significant contributor to the development of cardiovascular disease, resulting in impaired angiogenesis and tissue regeneration. Consequently, the reversal of EC senescence emerges as a promising therapeutic strategy to restore vascular function and promote healthy aging. In this study, we demonstrate that pharmacological, non-genetic partial reprogramming via transient activation of the Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc (OSKM) can rejuvenate senescent ECs.
Gene expression profiling and functional assays were performed in vitro, and the regenerative capacity of the murine hind-limb ischemia model was assessed using young and aged C57BL/6J mice.
Treatment with the reprogramming cocktail resulted in a robust, transient induction of OSKM at both the mRNA and protein levels (p<0.0001) in replicatively senescent ECs. This was accompanied by a marked reduction in senescence-associated markers, including p16Ink4a, p14Arf, TNFα, IL-1β, IL-6, and CD44 (p<0.01 to p<0.0001) and restoration of EC function. Specifically, the treated cells demonstrated enhanced migration (p<0.001), proliferation (p<0.01), sprouting (p<0.0001), and tube formation (p<0.01). Telomere length was shown to be stabilized (p<0.05) and there was a decrease in reactive oxygen species levels (p<0.05). Furthermore, non-senescent ECs demonstrated no response to the treatment (p>0.05). Notably, following treatment, senescent ECs exhibited diminished expression of senescence markers and maintained their migratory capacity also over time. In vivo, the aged (22-month-old) mice demonstrated significantly enhanced perfusion recovery at 7 and 14 days following the induction of ischaemia (p<0.001), accompanied by an augmentation in capillary density within the gastrocnemius muscle (CD31-staining, p<0.05). Conversely, no effects were observed in young mice (3 months old) (p>0.05).
In conclusion, the transient pharmacological induction of OSKM has been demonstrated to reverse endothelial senescence and restore regenerative function in vitro and in vivo. This approach offers a novel and clinically relevant strategy to enhance vascular repair and combat age-related ischemic diseases.