Cellular senescence mediates doxorubicin‐induced arterial dysfunction via activation of mitochondrial oxidative stress and the mammalian target of rapamycin

Abstract

The mechanisms underlying arterial dysfunction (large elastic artery [e.g., aorta] stiffening and vascular endothelial dysfunction) caused by the common chemotherapeutic agent doxorubicin (DOXO) are incompletely understood. Purpose To determine if clearance of senescent cells (senolysis) following DOXO would: 1) prevent aortic stiffening via reduced aortic intrinsic mechanical wall stiffness (elastic modulus [EM]), in part due to inhibition of the mammalian target of rapamycin (mTOR); and 2) prevent endothelial dysfunction by preserving nitric oxide (NO) bioavailability and inhibiting excess mitochondrial reactive oxygen species (mtROS) production. Results Young adult (4 mo) p16-3MR male (n = 5-9) and female (n = 3-5) mice received a single IP injection of DOXO (10 mg/ml in saline) or vehicle (V; saline). One week after DOXO, a p16+ senolytic used in p16-3MR mice, ganciclovir (GCV; 25 mg/kg/day x 5 days), or vehicle (V; saline x 5 days) were administered IP, resulting in 4 groups/sex (DOXO-V; DOXO-GCV; V-V; V-GCV) that were studied 3 weeks later. No sex differences were observed, so results were combined. Aortic Stiffness No pre-treatment differences in aortic stiffness (aortic pulse wave velocity [PWV]) were found. DOXO increased aortic PWV by 17% in DOXO-V (P = 0.03), which was prevented by senolysis (DOXO-GCV, Pre vs Post treatment: P = 0.88). These group differences in aortic PWV were explained by differences in aortic EM, which was higher in DOXO-V mice vs. all groups (P = 0.02), suggesting senolysis prevented the increased EM with DOXO. Next, we assessed the mTOR-mediated contribution to aortic EM by incubating (48h) aortic rings from all groups with the mTOR inhibitor, rapamycin, or sham (DMSO). EM was higher in sham-incubated DOXO-V vs. V-V rings (2579±200 vs. 1900±54 kPa, P = 0.04). Rapamycin lowered aortic EM in the DOXO-V group (rapamycin vs. sham: 1604±115 vs. 2579±200 kPa, P = 0.02), which eliminated group differences. This suggests senolysis prevents aortic stiffening caused by DOXO via mTOR inhibition. Endothelial function DOXO impaired isolated carotid artery endothelium-dependent dilation (EDD) to acetylcholine, an established bioassay of endothelial function (DOXO-V vs V-V: 62±5 vs 93±2 %, P < 0.0001), which was fully prevented with senolysis (DOXO-GCV: 95±1%). Addition of the NO-synthase inhibitor, L-NAME, abolished group differences, suggesting senolysis following DOXO preserves EDD by maintaining NO bioavailability. Administration of the mt-targeted antioxidant MitoQ eliminated group differences in EDD, suggesting excess mtROS impaired endothelial function in the DOXO-V group, which was prevented with senolysis. Conclusion Arterial dysfunction with DOXO is mediated by cellular senescence. mTOR and mtROS may be therapeutic targets to prevent/treat DOXO-mediated arterial dysfunction.