Tier 3 — preclinical

Senolytic treatment with fisetin reverses age-related endothelial dysfunction partially mediated by SASP factor CXCL12

Sophia A. Mahoney, Krystyna Mazan-Mamczarz, Dimitrios Tsitsipatis, Nicholas S. VanDongen, Charnae' Henry-Smith, Ada N. Okereke, Rachel Munk, Sanna Darvish, Kevin O. Murray, Supriyo De, Myriam Gorospe, Douglas R. Seals, Matthew J. Rossman, Allison B. Herman, Zachary S. Clayton

bioRxiv (preprint — not peer reviewed) 2025 bioRxiv preprint 2025.08.13.670216, posted 18 August 2025

Bibliography

DOI: 10.1101/2025.08.13.670216
PubMed: PMID 40894771
PubMed Central: PMC7593534
Funding: This research was supported in part by the Intramural Research Program of the National Institute on Aging. Additional support from National Institutes of Health grants F31 HL165885 (SAM), F31 AG087709 (SD), F32 HL167552 (KOM), K01 DK115524 (MJR), K99 HL159241 (ZSC), R21 AG078408 (DRS and ZSC), R01 AG055822 (DRS and ZSC), and R01 AG055822-S1 (DRS and ZSC), and American Heart Association award AHA 23CDA1056582 (MJR).
Competing interests: None declared.

Study snapshot

Design	Multi-modal preclinical: in vivo senolytic dosing with aortic single-cell RNA-seq, ex vivo isolated-artery plasma perfusion bioassay, and in vitro human aortic endothelial cell culture with CXCL12 add-back and pharmacological inhibition rescue
Model	In vivo: young (6-month) and old (27-month) male and female C57BL/6JN mice. Ex vivo: carotid arteries from young intervention-naïve wildtype mice. In vitro: primary human aortic endothelial cells (HAECs) from a single 80-year-old female donor, non-smoker, no known cardiovascular disease
Sample	In vivo: 42 mice total — young vehicle 12 (6M/6F), young fisetin 12 (6M/6F), old vehicle 8 (3M/5F), old fisetin 10 (4M/6F); scRNA-seq performed on 3 aortas per sex per treatment group. Ex vivo and in vitro replicates approximately n=5–11 per condition per assay
Intervention	Fisetin 100 mg/kg/day via oral gavage in vehicle (10% ethanol, 30% PEG400, 60% Phosal 50 PG) on an intermittent senolytic paradigm — one week on, two weeks off, one week on — with sacrifice one to two weeks after final dose (fisetin plasma half-life ~3.1 h). Rescue arms used recombinant mouse CXCL12 added to old-fisetin plasma to match old-vehicle CXCL12 levels (3210 pg/mL), and LIT-927 CXCL12 neutraligand at 1 μg/mL added to old-vehicle plasma
Duration	Approximately 5 weeks total in vivo dosing, with a 1–2 week washout before sacrifice. Ex vivo plasma perfusion: 24 h. In vitro HAEC exposure: 2 h for NO and mitochondrial superoxide, 24 h for SA-β-galactosidase and mRNA endpoints
Endpoints	Aortic single-cell transcriptomics: 11 cell types, 12 endothelial subclusters, EndoSen and SenMayo module scoring; Senescent endothelial subcluster 10 identification, differential RNA analysis, and pathway analysis (KEGG, Reactome); Circulating multiplex SASP panel (CCL4, CCL7, CCL8, CXCL12, FGF Basic, GDF15, IGFBP3, PAI-1, S100A9, TIMP4); Ex vivo carotid endothelium-dependent dilation (acetylcholine dose-response) and endothelium-independent dilation (sodium nitroprusside); Arterial and HAEC senescence-associated β-galactosidase burden; HAEC Cdkn1a and Cdkn2a mRNA; HAEC nitric oxide bioactivity (DAR-4M AM); Arterial mitochondrial superoxide (EPR spectrometry) and HAEC mitochondrial superoxide (MitoSOX); HAEC endothelial-to-mesenchymal transition markers: Cdh5, Pecam1, Tgfb1, Acta2; CXCL12 add-back and LIT-927 pharmacological inhibition rescue across each functional endpoint

What the study showed, in plain terms

Note: this study is currently a bioRxiv preprint and has not been peer reviewed. Findings, numbers, and interpretations may change before final publication.

Senescent cells — cells that have exited the cell cycle but stubbornly refuse to die — accumulate with age and release a cocktail of inflammatory signals known as the senescence-associated secretory phenotype (SASP). In the vasculature, this SASP is one leading candidate explanation for why endothelial function declines as we age and why cardiovascular disease risk climbs. Fisetin has been shown in earlier work by the same team (Mahoney 2023, Aging Cell) to improve arterial function in old mice as a senolytic — a compound that selectively kills senescent cells. What was missing was the molecular chain of causation: which senescent cells, releasing which SASP factor, driving which specific vascular defect?

The team dosed old (27-month-old) and young (6-month-old) mice with fisetin on an intermittent schedule (one week on, two weeks off, one week on), then took the aortas apart cell by cell using single-cell RNA sequencing. Endothelial cells — the inner lining of the blood vessel — turned out to be the most affected by senescence with age and the most responsive to fisetin, which cleared a specific senescent endothelial cluster and returned the transcriptome toward youthful levels. One SASP factor stood out: Cxcl12, a chemokine, was the most strongly elevated with age and the most reduced by fisetin. Circulating CXCL12 protein followed the same pattern.

The team then tested whether CXCL12 was actually causing the endothelial dysfunction, not just travelling alongside it. When plasma from old vehicle-treated mice was perfused through young carotid arteries, endothelial dilation dropped by 17%. Plasma from old fisetin-treated mice preserved dilation. Adding recombinant CXCL12 back to old fisetin plasma reversed the benefit; inhibiting CXCL12 with a small-molecule neutraligand rescued the old vehicle plasma. The same pattern appeared for nitric oxide production, mitochondrial oxidative stress, senescence induction, and endothelial-to-mesenchymal transition in cultured human endothelial cells. The findings map a mechanistic path from senescent endothelial cells to circulating CXCL12 to endothelial dysfunction — and identify fisetin's ability to break that chain as the likely reason older mice on fisetin end up with better arteries. A human trial of fisetin for endothelial function in mid-life and older adults is underway (NCT06133634).

Key findings

Preprint status: this study has not been peer reviewed. Effect sizes and interpretations may change on publication.

In aged mouse aortas, endothelial cells carried a disproportionately high senescence burden compared with other vascular cell types; fisetin dosed on an intermittent senolytic paradigm cleared this burden and returned the aortic transcriptome toward youthful patterns.
A specific senescent endothelial subcluster expanded 10.7-fold with age (p<0.0001) and was reduced 0.02-fold by fisetin (p<0.0001); this subcluster was enriched for the canonical senescence marker Cdkn1a and multiple SASP factors.
Cxcl12 mRNA was the most highly upregulated SASP factor in senescent endothelial cells with age. Circulating CXCL12 protein rose to ~3210 pg/mL in old vehicle mice and was restored toward young levels by fisetin.
Plasma from old vehicle mice perfused into young carotid arteries reduced peak endothelium-dependent dilation by 17% (p<0.0001); plasma from old fisetin mice preserved dilation. Adding recombinant CXCL12 back to old-fisetin plasma re-established dysfunction (–22%, p<0.0001).
In cultured human aortic endothelial cells, old plasma raised SA-β-galactosidase 5.6-fold, raised Cdkn1a 45-fold, cut NO production by 24%, and induced endothelial-to-mesenchymal transition markers. Each effect was blunted by fisetin treatment of donor mice; each was rescued in either direction by CXCL12 add-back or the CXCL12 neutraligand LIT-927.
The mechanistic thread — senescent endothelial cells → circulating CXCL12 → endothelial dysfunction — provides a molecular explanation for the arterial-function improvements this team previously reported in Mahoney 2023 (Aging Cell) using the same intermittent fisetin dosing.

What this study can and cannot tell us

This is a bioRxiv preprint and has not undergone peer review. Numerical results, statistical approach, and conclusions may change before final publication. Tier assignment and interpretation should be reviewed at that point.
Preclinical only — in vivo mouse work, ex vivo mouse artery bioassay, and in vitro human endothelial cell culture. Direct human clinical evidence is pending trial NCT06133634.
In vitro work used primary human aortic endothelial cells from a single 80-year-old female donor. Findings may not generalise across sex, donor age, ethnicity, or vascular bed.
Fisetin dosing (100 mg/kg/day oral gavage, on/off/on paradigm) used a specific formulation vehicle. Consumer-scale extrapolation to oral fisetin capsules is not supported by this study — allometric scaling to a nominal ~500 mg/day human-equivalent dose has been circulated in commentary but is not validated in humans.
The ex vivo plasma perfusion assay exposes young intervention-naïve arteries to old plasma for 24 hours, which does not reproduce chronic in vivo exposure of aged vessels to their own aged circulating milieu.
CXCL12 was pursued as a candidate after multiplex analysis; other SASP factors elevated with age (CCL8, FGF Basic, S100A9, PAI-1) were not individually rescued and may contribute independently.
LIT-927 (the CXCL12 neutraligand used) has been characterised in preclinical models but is not a validated therapeutic in humans. Its use here is a mechanistic tool, not a clinical intervention.
Fisetin plasma half-life is short (~3.1 h). The 1–2 week washout between final dose and sacrifice is designed to isolate senolytic effects from acute pharmacology, but does not fully rule out lingering non-senolytic effects.

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