Background and purpose: Endothelial dysfunction is an important aspect in the pathogenesis of cardiovascular disease and can be casually linked to the accumulation of senescent endothelial cells (EC) in the vasculature. These cells are characterized by a limitation of physiological functions, enhanced inflammation and a decreased regenerative and angiogenic capacity. Therefore, targeting EC senescence might represent a promising therapeutic strategy to improve vascular function. 

Here, we established a pharmacological, partial reprogramming strategy to induce the activation of the Yamanaka-factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) to reverse EC senescence and restore the regenerative potential of the cells. Further, the functional recovery of the EC should enhance the revascularization and tissue regeneration after tissue damage.

Methods: Methods to characterize the effects of the pharmacological reprogramming included the quantification of gene and protein expression as well as the analysis of EC function by proliferation, migration, tube and sprouting formation in vitro. In addition, the regenerative capacity of EC was evaluated in a hind-limb ischemia model in vivo.

Results: The application of the pharmacological reprogramming cocktail resulted in a robust but timely restricted activation of OSKM in replicative senescent ECs (P<0.0001), associated with a significant reduction of senescence markers p16ink4a, p14arf, TNFα, IL-1b, IL-6 and CD44 (P<0.001). Additionally, telomere length was stabilized. We further observed significant enhanced functional properties in the treated senescent EC, such as proliferation, migration, sprouting and tube formation. Furthermore, the reprogramming cocktail did not negatively affect non-senescent cells. Continuous cultivation of treated EC for up to 28 days indicated that expression of p16ink4a and p14arf remained low (P < 0.05, respectively P < 0.01), and the migratory capacity was preserved.  In vivo, a significantly improved blood flow and capillary density was observed after hind limb ischemia in 22 months old C57BL/6 mice after 7 and 14 days.

Conclusion: In summary, we demonstrate that a short induction of OSKM via a pharmacological approach of FDA approved drugs is sufficient to positively influence characteristics of senescent EC and restore their functional activity. Further, angiogenic capacity was enhanced by the treatment in vivo.