We assessed the efficacy of oral supplementation with the flavanoid apigenin on arterial function during aging and identified critical mechanisms of action. Young (6 months) and old (27 months) C57BL/6N mice (model of arterial aging) consumed drinking water containing vehicle (0.2% carboxymethylcellulose; 10 young, 7 old) or apigenin (0.5 mg/ml in vehicle; 10 young, 9 old) for 6 weeks. In vehicle-treated animals, isolated carotid artery endothelium-dependent dilation (EDD), bioassay of endothelial function, was impaired in old vs young (70±9 vs 92±1 %, P<0.0001) due to reduced nitric oxide (NO) bioavailability. Old mice had greater arterial reactive oxygen species (ROS) production and oxidative stress (higher nitrotyrosine) associated with greater nicotinamide adenine dinucleotide phosphate oxidase (oxidant enzyme) and lower superoxide dismutase 1 and 2 (antioxidant enzymes); ex vivo administration of TEMPOL (antioxidant) restored EDD to young levels, indicating ROS-mediated suppression of EDD. Old animals also had greater aortic stiffness as indicated by higher aortic pulse wave velocity (PWV, 434±9 vs 346±5 cm/sec, P<0.0001) due to greater intrinsic aortic wall stiffness associated with lower elastin levels and higher collagen, advanced glycation end-products (AGEs) and pro-inflammatory cytokine abundance. In old mice, apigenin restored EDD (96±2%) by increasing NO bioavailability, normalized arterial ROS, oxidative stress and antioxidant expression, and abolished ROS inhibition of EDD. Moreover, apigenin prevented foam cell formation in vitro (initiating step in atherosclerosis) and mitigated age-associated aortic stiffening (PWV 373±5 cm/sec) by normalizing aortic intrinsic wall stiffness, collagen, elastin, AGEs, and inflammation. Thus, apigenin is a promising therapeutic for arterial aging.

Vascular dysfunction: develops progressively with ageing; increases the risk of cardiovascular diseases (CVD); and is characterized by endothelial dysfunction and arterial stiffening, which are primarily mediated by superoxide‐driven oxidative stress and consequently reduced nitric oxide (NO) bioavailability and arterial structural changes. Interventions initiated before vascular dysfunction manifests may have more promise for reducing CVD risk than interventions targeting established dysfunction. Gut microbiome‐derived trimethylamine N‐oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk, and can be suppressed by 3,3‐dimethyl‐1‐butanol (DMB). We investigated whether DMB supplementation could prevent age‐related vascular dysfunction in C57BL/6N mice when initiated prior to development of dysfunction. Mice received drinking water with 1% DMB or normal drinking water (control) from midlife (18 months) until being studied at 21, 24 or 27 months of age, and were compared to young adult (5 month) mice. Endothelial function [carotid artery endothelium‐dependent dilatation (EDD) to acetylcholine; pressure myography] progressively declined with age in control mice, which was fully prevented by DMB via higher NO‐mediated EDD and lower superoxide‐related suppression of EDD (normalization of EDD with the superoxide dismutase mimetic TEMPOL). In vivo aortic stiffness (pulse wave velocity) increased progressively with age in controls, but DMB attenuated stiffening by ∼ 70%, probably due to preservation of endothelial function, as DMB did not affect aortic intrinsic mechanical (structural) stiffness (stress–strain testing) nor adventitial abundance of the arterial structural protein collagen. Our findings indicate that long‐term DMB supplementation prevents/attenuates age‐related vascular dysfunction, and therefore has potential for translation to humans for reducing CV risk with ageing. Key points Vascular dysfunction, characterized by endothelial dysfunction and arterial stiffening, develops progressively with ageing and increases the risk of cardiovascular diseases (CVD). Interventions aimed at preventing the development of CV risk factors have more potential for preventing CVD relative to those aimed at reversing established dysfunction. The gut microbiome‐derived metabolite trimethylamine N‐oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk and can be suppressed by supplementation with 3,3‐dimethyl‐1‐butanol (DMB). In mice, DMB prevented the development of endothelial dysfunction and delayed and attenuated in vivo arterial stiffening with ageing when supplementation was initiated in midlife, prior to the development of dysfunction. DMB supplementation or other TMAO‐suppressing interventions have potential for translation to humans for reducing CV risk with ageing.

Advancing age is the primary risk factor for the development of cardiovascular diseases (CVD), driven largely by age‐related arterial dysfunction. Two key manifestations of arterial dysfunction with advancing age are aortic stiffening and vascular endothelial dysfunction. Excessive reactive oxygen species (ROS)‐induced oxidative stress is a major macro‐mechanistic process causing arterial dysfunction during aging; however, the upstream mechanistic event(s) driving oxidative stress are incompletely understood. A rapidly emerging candidate is cellular senescence, a state of irreversible cell‐cycle arrest that can be cleared with senolytics. Purpose We tested the hypotheses that late‐life treatment with the senolytic ABT‐263 (ABT) would: 1) reverse age‐related aortic stiffening by reducing aortic intrinsic mechanical wall stiffness (elastic modulus [EM]); and 2) increase endothelial function by increasing nitric oxide (NO) bioavailability and reducing oxidative stress. Methods Old (27 mo) male C57BL6/N mice were treated with vehicle ([V]; 10% EtOH, 30% PEG400 and 60% Phosal 50 PG; n = 7) or ABT (50 mg/kg/day in [V]; n = 6) by oral gavage using a 1 week on – 2 weeks off – 1 week on dosing paradigm. A cohort of young adult mice (6 mo; n = 5) served as a young control (YC) reference group. Aortic pulse wave velocity (PWV), an in vivo measure of aortic stiffness, was measured pre‐ and post‐treatment. Aortic elastic modulus was assessed by performing stress‐strain testing in excised aortic rings. Endothelial function was assessed via ex vivo carotid artery endothelial‐dependent dilation (EDD) with increasing concentrations of acetylcholine (ACh). NO bioavailability (ACh in the presence of the NO‐synthase inhibitor, L‐NAME) and the role of excessive ROS in regulating EDD (ACh with the addition of the ROS scavenger, TEMPOL) were assessed as potential mechanisms. Results Aortic stiffness. ABT reversed aortic PWV in old mice (pre: 456 ± 7 vs post: 375 ± 11 cm/sec, P = 0.0003), to levels of YC (348 ± 13 cm/sec; P = 0.14 vs. ABT), whereas no effect was observed in the [V]‐treated group. Reduced aortic PWV with ABT was accompanied by lower aortic EM (V: 2738 ± 152 kPa, ABT: 2228 ± 155 kPa, YC: 2120 ± 362 kPa; P = 0.03). Endothelial function. ABT‐treated mice had greater peak EDD relative to [V]‐treated animals and comparable to YC (ABT: 94 ± 3% vs. V: 78 ± 5%, P = 0.01; YC: 91 ±2). Group differences in peak EDD were abolished in the presence of L‐NAME, suggesting that ABT rescued EDD by restoring NO bioavailability. TEMPOL restored peak EDD in [V]‐treated old mice to YC levels (92 ±5%, P = 0.003 vs. ACh alone), while having no effect in ABT or YC animals, suggesting that ABT selectively ameliorated the tonic ROS‐related suppression of EDD with aging. Conclusion Cellular senescence is mechanistically implicated in age‐related arterial dysfunction, and treatment with the senolytic compound ABT‐263 may be a therapeutic strategy for improving arterial function, with the potential for reducing CVD risk with aging.

Age‐related vascular dysfunction (e.g., large elastic artery [aorta] stiffening and endothelial dysfunction) is mediated by excess reactive oxygen species (ROS) leading to lower nitric oxide (NO) bioavailability. The upstream mechanisms mediating excess ROS are mostly unknown. Cellular senescence is a principal mechanism of aging and the senescence associated secretory phenotype (SASP) may exacerbate ROS. Purpose To: 1) determine if senescent cell clearance (senolysis) lowers aortic stiffness (pulse wave velocity [PWV]) and increases endothelial function (endothelium‐dependent dilation [EDD]), and if these effects are mediated by reduced ROS and increased NO bioavailability; and 2) isolate the influence of the circulating SASP on age‐related vascular dysfunction. Methods and Results Young (6 mo) and old (27 mo) adult male and female p16‐3MR mice were treated with vehicle ([V]; saline) or a p16‐3MR senolytic, ganciclovir (GCV; 25 mg/kg/day) injected intraperitoneally for 5 days. This resulted in 4 groups/sex (Young‐V [YV], n = 23; Young‐GCV [Y‐GCV]; n = 16, Old‐V [OV]; n = 22, Old‐GCV [O‐GCV]; n = 21). No sex differences were observed, so results were combined. Aortic Stiffness. Aortic PWV (aPWV) was assessed pre and post V and GCV treatment. Old mice had higher aPWV at baseline vs young (aPWV [cm/sec]: O‐GCV, 450 ± 16; OV, 441 ± 13; YV, 352 ± 7; Y‐GCV, 351 ± 12; P<.05). Following GCV treatment, old mice had reduced aPWV (pre: 441 ± 13 vs post: 375 ± 5 cm/sec, P< .05), which was not different from YV (P= .63) or Y‐GCV (P= .72). These data suggest that cellular senescence mediates aortic stiffening with advancing age. Endothelial function. OV animals had impaired ex vivocarotid artery EDD to acetylcholine (an established assay of endothelial function) relative to young and GCV treatment resulted in greater peak EDD (Peak EDD [%]: O‐GCV, 95 ± 1; OV, 83 ± 4; YV, 93 ± 4; Y‐GCV, 95 ± 1; P<.05). Addition of the NO‐synthase inhibitor, L‐NAME, abolished group differences suggesting the age‐related increase in cellular senescence reduced EDD by lowering NO bioavailability. Administration of the ROS scavenger TEMPOL eliminated group differences in EDD, implying that cellular senescence causes endothelial dysfunction with aging by amplifying ROS. SASP. To determine the effect of the circulating SASP on aortic stiffness, we incubated aortic rings from young adult (6 mo) male and female C57BL/6 mice, with plasma from sex‐matched OV and O‐GCV mice, or under fetal bovine serum (control). Following a 48h incubation, we assessed intrinsic mechanical wall stiffness (elastic modulus [EM]), an established ex vivomeasure of aortic stiffness. OV plasma resulted in 1.5 ± .01‐fold greater EM vs control (P< .05), which did not occur in O‐GCV plasma (P= 0.84 vs control), suggesting the SASP may increase age‐related aortic stiffening. Conclusion . The circulating SASP factors may be a mechanism by which cellular senescence induces age‐related vascular dysfunction, and as such, these processes represent novel therapeutic targets.

Consumption of a Western-style diet (WD; high fat, high sugar, low fiber) is associated with impaired vascular function and increased risk of cardiovascular diseases (CVD), which could be mediated partly by increased circulating concentrations of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO). We investigated if suppression of TMAO with 3,3-dimethyl-1-butanol (DMB; inhibitor of microbial TMA lyase) in mice could prevent: 1) WD-induced vascular endothelial dysfunction and aortic stiffening; and 2) WD-induced reductions in endurance exercise tolerance and increases in frailty, as both are linked to WD, vascular dysfunction, and increased CVD risk. C57BL/6N mice were fed standard chow or WD (41% fat, ~25% sugar, 4% fiber) for 5 months beginning at ~2 months of age. Within each diet, mice randomly received (n=11-13/group) normal drinking water (control) or 1% DMB in drinking water for the last 8 weeks (from 5-7 months of age). Plasma TMAO was increased in WD-fed mice but suppressed by DMB. WD induced endothelial dysfunction, assessed as carotid artery endothelium-dependent dilation to acetylcholine, and progressive increases in aortic stiffness (measured serially in vivo as pulse wave velocity), both of which were fully prevented by supplementation with DMB. Endurance exercise tolerance, assessed as time to fatigue on a rotarod test, was impaired in WD-fed mice but partially recovered by DMB. Lastly, WD-induced increases in frailty (31-point index) were prevented by DMB. Our findings indicate DMB or other TMAO-lowering therapies may be promising for mitigating the adverse effects of WD on physiological function, and thereby reducing risk of chronic diseases.