Clearing Senescent Endothelial Cells with Senolytics Restores Replicative Capacity

Aging is a major risk factor for cardiovascular diseases (CVD) and is marked by inflammation, oxidative stress, and cellular senescence. Cellular senescence, a persistent cell cycle arrest, is characterized by the secretion of inflammatory cytokines known as the Senescence Associated Secretory Phenotype (SASP). Accumulation of senescent endothelial cells (ECs) is associated with CVDs such as heart failure, coronary artery disease, and atherosclerosis. However, the potential of senescent cell removal to improve EC proliferation is still unknown. Here, we hypothesize that the senolytics rescue the EC replication capacity and prevent premature senescence in aged ECs. To test this, we utilized senolytics, drugs that selectively induce cell death in senescent cells and improve physiological function and lifespan in old mice. Human umbilical vein endothelial cells (HUVECs) were cultured until replicative senescence. Cells were then treated with DMSO (vehicle), REC-4249 (a novel senolytic from Recursion Pharmaceuticals) at 10uM, or ABT-263 (a BCL-2 inhibitor and established senolytic) at 1.0uM to remove senescent cells. Young HUVECs (passage 4) were used as a non-senescent control. To confirm senescence induction and clearance by senoltyics, we evaluated the senescence markers senescence-associated beta-galactosidase (SA-β-gal), mRNA expression of the tumor suppressors p16, p21, and p53, as well as proliferation rates using population doubling (PD) curve analysis and Bromodeoxyuridine (BrdU) incorporation. Compared to vehicle-treated ECs, senolytics reduced EC senescence as demonstrated by SA-β-gal positive cells 48 hours post-treatment (fig.2, A and B, p<0.001) and mRNA expression of p16, p21, and p53 five days post-treatment. (fig 2 C, p<0.001). To determine if senolysis increased EC proliferation capacity, we calculated the net number of doublings by analyzing the PD difference between 5 days before treatment (DBT) and 5 days after treatment (DAT), and between 5 DAT and 29 DAT. The net number of doublings increased by approximately 3 and 3.8 times in ABT-246 and REC-4249, respectively (fig 3 A, p<0.001). Likewise, BrdU incorporation was elevated, indicative of higher proliferation capacity at 5 DAT (fig 3 B, ABT-263, (p=0.044, and REC-4249, p=0.015). Interestingly, restored proliferation was sustained by 29 DAT (fig 3 C, ABT-263, p=0.040, and REC-4249, p=0.005). Overall, these data suggest that the removal of the senescent ECs with senolytics is sufficient to restore their replicative capacity. Highlighting a promising therapeutic avenue for enhancing endothelial function and mitigating aging-related CVD. Future studies will aim to elucidate whether this restoration is mediated through SASP factors or juxtacrine signaling to determine the mechanism underlies the beneficial effects of senolytics observed in vivo. NIH, R01 AG060395, R01 AG048366, T32 HL139451, F31 AG076312, R01 AG077751, R01 AG076748, Nora Eccles Treadwell Foundation (NETF) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.