Abstract

Abstract Funding Acknowledgements None. Background Senescent endothelial cells (EC) are key players in the pathophysiology of cardiovascular diseases and characterized by a reduced angiogenic and regenerative potential. Therefore, reversing EC senescence represents a promising therapeutic strategy to restore vascular integrity and increase health and lifespan. Purpose Here, we show a reversal of EC senescence following the application of a pharmacological, non-genetic, partial reprogramming strategy to induce a timely restricted induction of the Yamanaka-factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM). Methods Methods to characterize the effects of pharmacological reprogramming included the quantification of gene expression as well as functional analysis of EC in vitro. In addition, the regenerative capacity of EC was evaluated in a hind-limb ischemia model in vivo in young and old C57BL/6 mice. Results The application of a pharmacological reprogramming cocktail led to a strong but timely restricted activation of the Yamanaka-factors Oct3/4, Sox2, Klf4 and c-Myc in replicative senescent ECs. This activation occurred on both mRNA and protein levels (p<0.0001), concurrently significantly reducing markers of cellular aging as p16ink4a, p14arf, TNFα, IL-1b, IL-6 and CD44 (p<0.01 respectively p<0.0001), while enhancing their cellular function. Pharmacological reprogramming notably augmented the migratory (p<0.001) and proliferative capacities (p<0.01) of the replicate senescence EC and positively influenced their angiogenic functions such as sprouting (p<0.0001) and tube formation (p<0.01). Telomere length was stabilized (p<0.05) and production of reactive oxygen species decreased (p<0.05). Non-senescent EC were not influenced by the pharmacological treatment (p>0.05). Over prolonged periods, the treated ECs maintained consistently low expression of aging markers (p16ink4a and p14arf, p<0.01) while preserving their migratory capabilities (p<0.05). In vivo, a significantly improved blood flow was observed after hind limb ischemia in 22 months old C57BL/6 mice after 7 and 14 days (p<0.001), alongside a significant increase in capillary density in the gastrocnemius muscle quantified by CD31 staining (p<0.05). In 3 months old C57BL/6 mice blood flow was not influenced by the treatment (p>0.05). Conclusion In conclusion, we demonstrate that a short induction of OSKM via a pharmacological approach holds the potential to reverse cellular senescence in EC in vitro with improved regenerative and angiogenic capacities and thus to enhance endothelial regenerative capacity in vivo. This might present a promising strategy for ischemic diseases.