Excess reactive oxygen species production by mitochondria is a key mechanism of age-related vascular dysfunction. Our laboratory has shown that supplementation with the mitochondrial-targeted antioxidant MitoQ improves vascular endothelial function by reducing mitochondrial reactive oxygen species and ameliorates arterial stiffening in old mice, but the effects in humans are unknown. Here, we sought to translate our preclinical findings to humans and determine the safety and efficacy of MitoQ. Twenty healthy older adults (60–79 years) with impaired endothelial function (brachial artery flow–mediated dilation <6%) underwent 6 weeks of oral supplementation with MitoQ (20 mg/d) or placebo in a randomized, placebo-controlled, double-blind, crossover design study. MitoQ was well tolerated, and plasma MitoQ was higher after the treatment versus placebo period ( P <0.05). Brachial artery flow–mediated dilation was 42% higher after MitoQ versus placebo ( P <0.05); the improvement was associated with amelioration of mitochondrial reactive oxygen species–related suppression of endothelial function (assessed as the increase in flow-mediated dilation with acute, supratherapeutic MitoQ [160 mg] administration; n=9; P <0.05). Aortic stiffness (carotid–femoral pulse wave velocity) was lower after MitoQ versus placebo ( P 7.60 m/s; n=11). Plasma oxidized LDL (low-density lipoprotein), a marker of oxidative stress, also was lower after MitoQ versus placebo ( P 0.1). These findings in humans extend earlier preclinical observations and suggest that MitoQ and other therapeutic strategies targeting mitochondrial reactive oxygen species may hold promise for treating age-related vascular dysfunction. Clinical Trial Registration—: URL: http://www.clinicaltrials.gov . Unique identifier: NCT02597023.

Cellular senescence is emerging as a key mechanism of age-related vascular endothelial dysfunction, but evidence in healthy humans is lacking. Moreover, the influence of lifestyle factors such as habitual exercise on endothelial cell (EC) senescence is unknown. We tested the hypothesis that EC senescence increases with sedentary, but not physically active, aging and is associated with vascular endothelial dysfunction. Protein expression (quantitative immunofluorescence) of p53, a transcription factor related to increased cellular senescence, and the cyclin-dependent kinase inhibitors p21 and p16 were 116%, 119%, and 128% greater (all P < 0.05), respectively, in ECs obtained from antecubital veins of older sedentary (60 ± 1 yr, n = 12) versus young sedentary (22 ± 1 yr, n = 9) adults. These age-related differences were not present (all P > 0.05) in venous ECs from older exercising adults (57 ± 1 yr, n = 13). Furthermore, venous EC protein levels of p53 ( r = −0.49, P = 0.003), p21 ( r = −0.38, P = 0.03), and p16 ( r = −0.58, P = 0.002) were inversely associated with vascular endothelial function (brachial artery flow-mediated dilation). Similarly, protein expression of p53 and p21 was 26% and 23% higher (both P < 0.05), respectively, in ECs sampled from brachial arteries of healthy older sedentary (63 ± 1 yr, n = 18) versus young sedentary (25 ± 1 yr, n = 9) adults; age-related changes in arterial EC p53 and p21 expression were not observed ( P > 0.05) in older habitually exercising adults (59 ± 1 yr, n = 14). These data indicate that EC senescence is associated with sedentary aging and is linked to endothelial dysfunction. Moreover, these data suggest that prevention of EC senescence may be one mechanism by which aerobic exercise protects against endothelial dysfunction with age.

Background: High‐resistance inspiratory muscle strength training (IMST) is a novel, time‐efficient physical training modality. Methods and Results: We performed a double‐blind, randomized, sham‐controlled trial to investigate whether 6 weeks of IMST (30 breaths/day, 6 days/week) improves blood pressure, endothelial function, and arterial stiffness in midlife/older adults (aged 50–79 years) with systolic blood pressure ≥120 mm Hg, while also investigating potential mechanisms and long‐lasting effects. Thirty‐six participants completed high‐resistance IMST (75% maximal inspiratory pressure, n=18) or low‐resistance sham training (15% maximal inspiratory pressure, n=18). IMST was safe, well tolerated, and had excellent adherence (≈95% of training sessions completed). Casual systolic blood pressure decreased from 135±2 mm Hg to 126±3 mm Hg ( P <0.01) with IMST, which was ≈75% sustained 6 weeks after IMST ( P 0.05). Twenty‐four hour systolic blood pressure was lower after IMST versus sham training ( P =0.01). Brachial artery flow‐mediated dilation improved ≈45% with IMST ( P <0.01) but was unchanged with sham training ( P =0.73). Human umbilical vein endothelial cells cultured with subject serum sampled after versus before IMST exhibited increased NO bioavailability, greater endothelial NO synthase activation, and lower reactive oxygen species bioactivity ( P 0.05). Conclusions: High‐resistance IMST is a safe, highly adherable lifestyle intervention for improving blood pressure and endothelial function in midlife/older adults with above‐normal initial systolic blood pressure. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03266510.

Cellular senescence and the senescence‐associated secretory phenotype (SASP) contribute to age‐related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP‐related inflammation were lower 1 week after the final dose of oral intermittent (1 week on—2 weeks off—1 weeks on dosing) fisetin supplementation. Old fisetin‐supplemented mice had higher endothelial function. Leveraging old p16‐3MR mice, a transgenic model allowing genetic clearance of p16^INK4A‐positive senescent cells, we found that ex vivo removal of senescent cells from arteries isolated from vehicle‐ but not fisetin‐treated mice increased endothelium‐dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular‐ and mitochondrial‐related oxidative stress. Arterial stiffness was lower in fisetin‐treated mice. Ex vivo genetic senolysis in aorta rings from p16‐3MR mice did not further reduce mechanical wall stiffness in fisetin‐treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age‐related arterial dysfunction.

BACKGROUND: Here, we assessed the role of cellular senescence and the senescence associated secretory phenotype (SASP) in age-related aortic stiffening and endothelial dysfunction. METHODS: We studied young (6-8 mo) and old (27-29 mo) p16-3MR mice, which allows for genetic-based clearance of senescent cells with ganciclovir (GCV). We also treated old C57BL/6N mice with the senolytic ABT-263. RESULTS: In old mice, GCV reduced aortic stiffness assessed by aortic pulse wave velocity (PWV; 477±10 vs. 382±7 cm/s, P <0.05) to young levels (old-GCV vs. young-vehicle, P =0.35); ABT-263 also reduced aortic PWV in old mice (446±9 to 356±11 cm/s, P <0.05). Aortic adventitial collagen was reduced by GCV ( P <0.05) and ABT-263 ( P =0.12) in old mice. To show an effect of the circulating SASP, we demonstrated that plasma exposure from Old-vehicle p16-3MR mice, but not from Old-GCV mice, induced aortic stiffening assessed ex vivo (elastic modulus; P <0.05). Plasma proteomics implicated glycolysis in circulating SASP-mediated aortic stiffening. In old p16-3MR mice, GCV increased endothelial function assessed via peak carotid artery endothelium-dependent dilation (EDD; Old-GCV, 94±1% vs. Old-vehicle, 84±2%, P <0.05) to young levels (Old-GCV vs. young-vehicle, P =0.98), and EDD was higher in old C57BL/6N mice treated with ABT-263 vs. vehicle (96±1% vs. 82±3%, P <0.05). Improvements in endothelial function were mediated by increased nitric oxide (NO) bioavailability ( P <0.05) and reduced oxidative stress ( P <0.05). Circulating SASP factors related to NO signaling were associated with greater NO-mediated EDD following senescent cell clearance. CONCLUSIONS: Cellular senescence and the SASP contribute to vascular aging and senolytics hold promise for improving age-related vascular function.

Aging is associated with a decline in physiological function and exercise performance. These effects are mediated, at least in part, by an age-related decrease in the bioavailability of nitric oxide (NO), a ubiquitous gasotransmitter and regulator of myriad physiological processes. The decrease in NO bioavailability with aging is especially apparent in sedentary individuals, whereas older, physically active individuals maintain higher levels of NO with advancing age. Strategies which enhance NO bioavailability (including nutritional supplementation) have been proposed as a potential means of reducing the age-related decrease in physiological function and enhancing exercise performance and may be of interest to a range of older individuals including those taking part in competitive sport. In this brief review we discuss the effects of aging on physiological function and endurance exercise performance, and the potential role of changes in NO bioavailability in these processes. We also provide a summary of current evidence for dietary supplementation with substrates for NO production - including inorganic nitrate and nitrite, l-arginine and l-citrulline - for improving exercise capacity/performance in older adults. Additionally, we discuss the (limited) evidence on the effects of (poly)phenols and other dietary antioxidants on NO bioavailability in older individuals. Finally, we provide suggestions for future research.