Pharmacological Reprogramming Reverses Endothelial Cell Senescence and Restores Vascular Regenerative Function

Background and purpose: Senescent endothelial cells (EC) are key players in the pathophysiology of cardiovascular diseases and are 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.

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/6J 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), while significantly reducing markers of cellular aging such as p16ink4a, p14arf, TNFα, IL-1b, IL-6, and CD44 (p<0.01 respectively p<0.0001), and enhancing cellular function. Pharmacological reprogramming notably augmented the migratory (p<0.001) and proliferative capacities (p<0.01) of replicative senescence ECs 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 the production of reactive oxygen species decreased (p<0.05). Non-senescent ECs 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, significantly improved blood flow was observed after hind limb ischemia in 22-month-old C57BL/6 mice after 7 and 14 days (p<0.001), alongside a significant increase in capillary density in the gastrocnemius quantified by CD31 staining (p<0.05). In 3-month-old C57BL/6 mice, blood flow was not influenced by the treatment (p>0.05).

Conclusion: In conclusion, our findings demonstrate that a brief induction of OSKM through a pharmacological approach has the potential to effectively reverse senescence in endothelial cells in vitro, resulting in enhanced regenerative and angiogenic capacities. Consequently, this approach holds great promise for enhancing endothelial regenerative capacity in vivo and could represent a highly effective strategy for treating ischemic diseases.