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.
Age‐related vascular endothelial dysfunction is mediated by excess reactive oxygen species (ROS)—mitochondria (mito) being a key source—which can reduce nitric oxide (NO) bioavailability. Cellular senescence, a fundamental mechanism of aging, may exacerbate mito ROS and be a potential therapeutic target to combat age‐related vascular dysfunction. Purpose To determine if treatment with the natural flavonoid fisetin improves endothelial function with aging by suppressing cellular senescence, scavenging excess whole‐cell and mito ROS, and increasing NO bioavailability. Methods & Results Old (27 mo) male C57BL/6 mice were treated with vehicle (V; 10% EtOH, 30% PEG400 and 60% Phosal 50 PG; n = 9) or fisetin (Fis; 50 mg/kg/day in V; n = 10) by oral gavage following a 1 wk on – 2 wk off – 1 wk on dosing paradigm. Endothelial function Endothelial function was assessed by ex vivo carotid artery endothelium‐dependent dilation (EDD) and endothelium‐independent dilation (EID) to increasing doses of acetylcholine and sodium nitroprusside, respectively. EDD was greater in Fis vs V treated mice (Peak EDD [%]: 97 ± 1 vs 84 ± 3, P < .05) and no differences were observed among groups in EID (P = .54). Cellular senescence Fis‐treated mice had lower vascular abundance of p16, an established marker of cellular senescence (Fis, .12 ± .01 vs V, .19 ± .02 chemiluminescence units [CU], P < .05). Next, we administered Fis or V to old (27 mo) p16‐3MR mice (a model that allows for clearance of senescent cells with ganciclovir [GCV]; n = 6/group). Fis‐treated p16‐3MR mice had greater EDD (Peak EDD [%]: Fis, 93 ± 2 vs V, 74 ± 5, P < .05). Ex vivo carotid artery incubations with GCV eliminated group differences in EDD, suggesting that Fis reduced senescent cells to improve EDD. Whole‐cell ROS Electron paramagnetic resonance (EPR) spectroscopy was used to assess whole‐cell vascular (aortic) ROS. Fis‐treated mice had lower whole‐cell ROS (Fis, 4703 ± 455 vs V, 8173 ± 1243 amplitude units [AU], P < .05). Incubation with the ROS scavenger TEMPOL eliminated group differences in EDD, implying that Fis ameliorated ROS‐related suppression of EDD. Mito ROS EPR was used to assess vascular mito ROS. Fis‐treated mice had lower mito ROS (Fis, 2527 ±440 vs V, 6603 ± 1956 AU, P < .05). Further, Fis‐treated mice had lower abundance of vascular p‐p66SHC, a recognized marker of mito oxidative stress (Fis, .029 ± .003 vs V, .049 ± .008 CU, P < .05) and greater abundance of Mn superoxide dismutase, a mito antioxidant enzyme (Fis, .41 ± .1 vs V, .20 ± .02 CU, P < .05). Incubation with the mito‐specific antioxidant, MitoQ, eliminated group differences in EDD, implying that Fis ameliorated mito ROS‐related suppression of EDD. NO Addition of the NO‐synthase inhibitor, L‐NAME, abolished group differences in EDD suggesting Fis improved age‐related EDD impairments by increasing NO bioavailability. Conclusion Fisetin supplementation may be a novel strategy to target excess cellular senescence and thereby reduce mito ROS to improve NO‐mediated endothelial function with ag...
The aorta stiffens with aging in both men and women, which predicts cardiovascular mortality. Aortic wall structural and extracellular matrix (ECM) remodeling, induced in part by chronic low-grade inflammation, contribute to aortic stiffening. Male mice are an established model of aortic aging. However, there is little information regarding whether female mice are an appropriate model of aortic aging in women, which we aimed to elucidate in the present study. We assessed two strains of mice and found that in C57BL/6N mice, in vivo aortic stiffness (pulse wave velocity, PWV) was higher with aging in both sexes, whereas in B6D2F1 mice, PWV was higher in old vs. young male mice, but not in old vs. young female mice. Because the age-related stiffening that occurs in men and women was reflected in male and female C57BL/6N mice, we examined mechanisms of stiffening in this strain. In both sexes, aortic modulus of elasticity (pin myography) was lower in old mice, occurred in conjunction with and was related to higher plasma levels of the elastin-degrading enzyme matrix metalloproteinase-9 (MMP-9), and was accompanied by higher numbers of aortic elastin breaks and higher abundance of adventitial collagen-1. Plasma levels of the inflammatory cytokines interferon-γ, interleukin 6 and monocyte chemoattractant protein-1 were higher in both sexes of old mice. In conclusion, female C57BL/6N mice exhibit aortic stiffening, reduced modulus of elasticity, and structural/ECM remodeling, and associated increases in MMP-9 and systemic inflammation with aging, and thus are an appropriate model of aortic aging in women.
The peer review history is available in the Supporting Information section of this article (https://doi.org/10.1113/ JP282581#support-information-section).Advancing age is a primary, non-modifiable risk factor for the development of cardiovascular diseases (CVDs), which are the leading cause of morbidity and mortality worldwide. CVDs are largely driven by age-related vascular dysfunction which is characterized by two key manifestations: large elastic artery (e.g. aorta) stiffening and vascular endothelial dysfunction. These processes are largely caused by excess reactive oxygen species (ROS)-induced oxidative stress and inflammation which are mutually reinforcing and can reduce the bioavailability of the vasodilatory molecule nitric oxide (NO) (Trott et al. 2021). However, there have been an insufficient number of studies examining the cell type(s) that may induce this age-related increase in inflammation and in turn influence oxidative stress. In a recent publication in The Journal of Physiology, Trott et al. (2021) aimed to determine the role of T-cells in mediating age-related changes in vascular inflammation and associated aortic stiffening and vascular endothelial dysfunction.
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