Fisetin supplementation targets senescent endothelial cells in old mice and prevents plasma-induced endothelial dysfunction with aging

CU Anschutz Medical Campus Endothelial cells (ECs) line the vascular network and form the interface between the circulation and the vasculature to govern vascular health and vasodilation. With advancing age, ECs readily undergo cellular senescence – a stress response defined by cell cycle arrest – which in excess promotes endothelial dysfunction, a key precursor of age-related cardiovascular (CV) disease. The senescence-associated secretory phenotype (SASP) is a collection of pro-inflammatory factors by which senescent cells may transduce their detrimental effects within tissues and in circulation (e.g., plasma). Fisetin is a natural senolytic – a compound which clears excess senescent cells – that has demonstrated efficacy in improving endothelial function in old mice. However, the mechanisms-of-action by which fisetin acts on senescent ECs and the circulating SASP to improve endothelial function with aging remain to be fully elucidated. Purpose: To determine the effects of aging and fisetin on EC senescence in vivo and identify EC-derived SASP factors that change the circulating milieu to modulate endothelial function. Methods: Old (27 mo) wildtype mice were treated with either a vehicle (10% Ethanol, 30% PEG400, & 60% Phosal 50 PG) or fisetin (100 mg/kg/day in vehicle) using an intermittent 1 week on – 2 weeks off – 1week on dosing paradigm, in addition to a young (6 mo) control group. Following the dosing period, mice were sacrificed, and aortas were collected for single-cell RNA sequencing (scRNAseq) and plasma was collected to determine the role of the circulating SASP on endothelial function. Endothelial function was measured as endothelium-dependent dilation (EDD) in carotid arteries isolated from young, intervention-naïve mice following 24h ex vivo perfusion of plasma from the experimental 3 groups. Results: Aortic scRNAseq. Merged aorta analysisrevealed 11 distinct cell types in the aorta. SenMayo, a general senescence gene signature, was used to identify senescent cells. Although differences were observed in other cell types, ECs demonstrated the greatest age-related shifts in genes associated with cellular senescence, which were mitigated with fisetin supplementation. EC scRNAseq. EndoSen, an EC-specific senescent signature, identified a putative senescent EC subcluster. Differential expression analysis testing of this EC subcluster revealed an upregulation of genes pertaining to the SASP. Reactome pathway analysis confirmed that changes in cellular senescence, SASP, and CV pathology-related pathways were upregulated with aging and normalized (to young ECs) with fisetin supplementation. SASP-Mediated Endothelial Function. We next sought to assess the effects of fisetin on circulating SASP-mediated endothelial dysfunction with aging. Peak EDD was lower in young arteries incubated with old vehicle v. young plasma (old, 76±3% v. young, 92±1%; p<0.001). Old fisetin plasma prevented age-related impairments in peak EDD (old fisetin, 87±2%; p=0.001 v. old vehicle) and demonstrated similar responses to the young plasma (p=0.186 v. old fisetin). Conclusion: These findings suggest that suppression of excess senescent ECs and favorable modulation of the circulating SASP are putative mechanisms by which fisetin supplementation may transduce its beneficial effects on the vasculature with aging. Funding: F31 HL165885, AHA 23CDA1056582, R01 AG055822, R21 AG078408, and K99 HL159241 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.