Senolytic Treatment With Fisetin Reverses Age‐Related Endothelial Dysfunction Partially Mediated by SASP Factor CXCL12

ABSTRACT Background Advancing age is the strongest risk factor for cardiovascular diseases (CVDs), primarily due to progressive vascular endothelial dysfunction. Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related endothelial dysfunction by promoting mitochondrial oxidative stress and inflammation, which reduce nitric oxide (NO) bioavailability. However, the molecular changes in senescent endothelial cells and their role in endothelial dysfunction with aging remain incompletely unclear. As such, in this study we sought to identify the endothelial cell senescence-related signalling pathways, endothelial-derived SASP factors, and their impact on endothelial function with aging. Methods Single-cell transcriptomics was performed on aortas from young (6 months) and old (27 months) mice with and without in vivo senolytic treatment with fisetin (100 mg/kg/day administered in an intermittent dosing paradigm) to characterize endothelial cell senescence and transcript expression changes. Circulating levels of SASP factors were measured to validate transcriptional changes. Plasma exposure and protein addition and inhibiton experiments were conducted in isolated mouse arteries and cultured human endothelial cells to determine the causal role of the circulating SASP milieu and specific SASP factors in mediating endothelial dysfunction and underlying mechanisms-of-action. Results Senescent endothelial cells exhibited elevated expression of SASP factors, particularly Cxcl12 , which was reversed by fisetin supplementation, with responses also reflected in circulating CXCL12 concentrations. Plasma from old mice impaired endothelial function by inducing vascular cell senescence, reducing NO, increasing mitochondrial oxidative stress, and promoting endothelial-to-mesenchymal transition—effects partially driven by CXCL12 and prevented by fisetin. Conclusions These results identify the SASP and CXCL12 as drivers of age-related endothelial dysfunction and establish mechanisms of senolytic intervention with fisetin supplementation. NOVELTY AND SIGNFICANCE What is known? Advancing age is the primary risk factor for cardiovascular disease, in part due to progressive endothelial dysfunction. Cellular senescence contributes to age-related endothelial impairment through the secretion of a pro-inflammatory milieu known as the senescence-associated secretory phenotype (SASP), which can affect neighboring cells and tissue function. Senolytic compounds selectively eliminate senescent cells and improve vascular function in preclinical models of aging. What new information does this article contribute? Endothelial cells are highly susceptible to senescence with aging in vivo and are selectively cleared by senolytic treatment with the natural compound fisetin. Single-cell transcriptomic profiling identifies CXCL12 as the most highly upregulated SASP factor in senescent endothelial cells and in circulation with aging, both of which are reversed by senolytic treatment with fisetin. We identify a specific circulating SASP factor, CXCL12, as a partial mediator of endothelial dysfunction by inducing mitochondrial oxidative stress, impairing nitric oxide bioavailability, and promoting endothelial-to-mesenchymal transition (Endo, which is restored by senolytic treatment with fisetin. Summary This study provides novel mechanistic insight into how senescent endothelial cells and their secretory products—particularily CXCL12—contribute to age-related endothelial dysfunction. It further demonstrates that senolytic treatment with fisetin reverses these effects, highlighting a promising translational strategy for targeting vascular aging and preserving endothelial health. GRAPHICAL ABSTRACT