Summary We tested the hypothesis that short‐term nitrite therapy reverses vascular endothelial dysfunction and large elastic artery stiffening with aging, and reduces arterial oxidative stress and inflammation. Nitrite concentrations were lower (P < 0.05) in arteries, heart, and plasma of old (26–28 month) male C57BL6 control mice, and 3 weeks of sodium nitrite (50 mg L−1 in drinking water) restored nitrite levels to or above young (4–6 month) controls. Isolated carotid arteries of old control mice had lower acetylcholine (ACh)‐induced endothelium‐dependent dilation (EDD) (71.7 ± 6.1% vs. 93.0 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (P < 0.05 vs. young), and sodium nitrite restored EDD (95.5 ± 1.6%) by increasing NO bioavailability. 4‐Hydroxy‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (TEMPOL), a superoxide dismutase (SOD) mimetic, apocynin, a nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH) inhibitor, and sepiapterin (exogenous tetrahydrobiopterin) each restored EDD to ACh in old control, but had no effect in old nitrite‐supplemented mice. Old control mice had increased aortic pulse wave velocity (478 ± 16 vs. 332 ± 12 AU, P < 0.05 vs. young), which nitrite supplementation lowered (384 ± 27 AU). Nitrotyrosine, superoxide production, and expression of NADPH oxidase were ∼100–300% greater and SOD activity was ∼50% lower in old control mice (all P < 0.05 vs. young), but were ameliorated by sodium nitrite treatment. Inflammatory cytokines were markedly increased in old control mice (P < 0.05), but reduced to levels of young controls with nitrite supplementation. Short‐term nitrite therapy reverses age‐associated vascular endothelial dysfunction, large elastic artery stiffness, oxidative stress, and inflammation. Sodium nitrite may be a novel therapy for treating arterial aging in humans.

Key pointsr The development of age-related arterial endothelial dysfunction, a key antecedent of increased cardiovascular disease (CVD) risk, is mediated largely by reduced nitric oxide bioavailability as a consequence of oxidative stress.r Mitochondria are critical signalling organelles in the vasculature, which, when dysregulated, become a source of excessive reactive oxygen species; the role of mitochondria-derived oxidative stress in age-related vascular dysfunction is unknown.r We show that a mitochondria-targeted antioxidant, MitoQ, ameliorates vascular endothelial dysfunction in old mice and that these improvements are associated with the normalization of mitochondria-derived oxidative stress and markers of arterial mitochondrial health.r These results indicate that mitochondria-derived oxidative stress is an important mechanism underlying the development of age-related vascular endothelial dysfunction and therefore may be a promising therapeutic target.r Mitochondria-targeted antioxidants represent a novel strategy for preserving healthy vascular endothelial function in primary ageing and preventing age-related CVD in humans.Abstract Age-related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age-related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria-targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ß30% in old (ß27 months) compared with young (ß8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium-independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age-related increases in total and mitochondria-derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age-related increase in endothelial susceptibility to acute mitochondrial damage (rotenone-induced impairment in EDD). Our results suggest that mitochondria-derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria-targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age-rela...

Age-related vascular endothelial dysfunction is a major antecedent to cardiovascular diseases. We investigated whether increased circulating levels of the gut microbiome-generated metabolite trimethylamine-N-oxide induces endothelial dysfunction with aging. In healthy humans, plasma trimethylamine-N-oxide was higher in middle-aged/older (64±7 years) versus young (22±2 years) adults (6.5±0.7 versus 1.6±0.2 µmol/L) and inversely related to brachial artery flow-mediated dilation ( r 2 =0.29, P <0.00001). In young mice, 6 months of dietary supplementation with trimethylamine-N-oxide induced an aging-like impairment in carotid artery endothelium-dependent dilation to acetylcholine versus control feeding (peak dilation: 79±3% versus 95±3%, P <0.01). This impairment was accompanied by increased vascular nitrotyrosine, a marker of oxidative stress, and reversed by the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. Trimethylamine-N-oxide supplementation also reduced activation of endothelial nitric oxide synthase and impaired nitric oxide-mediated dilation, as assessed with the nitric oxide synthase inhibitor L-NAME (N G -nitro-L-arginine methyl ester). Acute incubation of carotid arteries with trimethylamine-N-oxide recapitulated these events. Next, treatment with 3,3-dimethyl-1-butanol for 8 to 10 weeks to suppress trimethylamine-N-oxide selectively improved endothelium-dependent dilation in old mice to young levels (peak: 90±2%) by normalizing vascular superoxide production, restoring nitric oxide-mediated dilation, and ameliorating superoxide-related suppression of endothelium-dependent dilation. Lastly, among healthy middle-aged/older adults, higher plasma trimethylamine-N-oxide was associated with greater nitrotyrosine abundance in biopsied endothelial cells, and infusion of the antioxidant ascorbic acid restored flow-mediated dilation to young levels, indicating tonic oxidative stress-related suppression of endothelial function with higher circulating trimethylamine-N-oxide. Using multiple experimental approaches in mice and humans, we demonstrate a clear role of trimethylamine-N-oxide in promoting age-related endothelial dysfunction via oxidative stress, which may have implications for prevention of cardiovascular diseases.

To test the hypothesis that the antioxidant enzyme superoxide dismutase (SOD) mimetic TEMPOL improves arterial aging, young (Y, 4–6 mo) and old (O, 26–28 mo) male C57BL6 mice received regular or TEMPOL-supplemented (1mM) drinking water for 3 weeks (n=8/group). Aortic superoxide was 65% greater in O (p<0.05 vs. Y), which was normalized by TEMPOL. O had large elastic artery stiffening, as indicated by greater aortic pulse wave velocity (aPWV, 508 ± 22 vs. 418 ± 22 AU), which was associated with increased adventitial collagen I expression (p<0.05 vs. Y). TEMPOL reversed the age-associated increases in aPWV (434 ± 21 AU) and collagen in vivo, and SOD reversed increases in collagen I in adventitial fibroblasts from older rats in vitro. Isolated carotid arteries of O had impaired endothelial function as indicated by reduced acetylcholine-stimulated endothelium-dependent dilation (EDD) (75.6 ± 3.2 vs. 94.5 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (L-NAME) associated with decreased endothelial NO synthase (eNOS) expression (p<0.05 vs. Y). TEMPOL restored EDD (94.5 ± 1.4%), NO bioavailability and eNOS in O. Nitrotyrosine and expression of NADPH oxidase were ~100–200% greater, and MnSOD was ~75% lower in O (p<0.05 vs. Y). TEMPOL normalized nitrotyrosine and NADPH oxidase in O, without affecting MnSOD. Aortic pro-inflammatory cytokines were greater in O (p<0.05 vs. Y) and normalized by TEMPOL. Short-term treatment of excessive superoxide with TEMPOL ameliorates large elastic artery stiffening and endothelial dysfunction with aging, and this is associated with normalization of arterial collagen I, eNOS, oxidative stress and inflammation.

Key pointsr Age-related arterial dysfunction, characterized by oxidative stress-and inflammation-mediated endothelial dysfunction and arterial stiffening, is the primary risk factor for cardiovascular diseases.Vienna E. Brunt received her PhD in Human Physiology from the University of Oregon in 2016. She is currently a postdoctoral fellow in Dr D. R. Seals' Integrative Physiology of Aging Laboratory at the University of Colorado Boulder. The studies described in the present study represent work carried out as part of an NIH T32 fellowship through the Division of Cardiology at the University of Colorado Denver. Her long-term research goals are to investigate the efficacy of novel interventions for preserving vascular function with ageing, thereby preventing and/or delaying the progression of cardiovascular diseases. Rachel A. Gioscia-Ryan completed her PhD in the Integrative Physiology of Aging Laboratory at the University of Colorado Boulder in 2016 and is currently in medical school at the University of Michigan. She is pursuing a career as a clinician-scientist conducting integrative physiological studies with the aim of improving human health and patient care. * These authors contributed equally to this work.Abstract Oxidative stress-mediated arterial dysfunction (e.g. endothelial dysfunction and large elastic artery stiffening) is the primary mechanism driving age-related cardiovascular diseases. Accumulating evidence suggests the gut microbiome modulates host physiology because dysregulation ('gut dysbiosis') has systemic consequences, including promotion of oxidative stress. The present study aimed to determine whether the gut microbiome modulates arterial function with ageing. We measured arterial function in young and older mice after 3-4 weeks of treatment with broad-spectrum, poorly-absorbed antibiotics to suppress the gut microbiome. To identify potential mechanistic links between the gut microbiome and age-related arterial dysfunction, we sequenced microbiota from young and older mice and measured plasma levels of the adverse gut-derived metabolite trimethylamine N-oxide (TMAO). In old mice, antibiotics reversed endothelial dysfunction [area-under-the-curve carotid artery dilatation to acetylcholine in young: 345 ± 16 AU vs. old control (OC): 220 ± 34 AU, P < 0.01; vs. old antibiotic-treated (OA): 334 ± 15 AU; P < 0.01 vs. OC] and arterial stiffening (aortic pulse wave velocity in young: 3.62 ± 0.15 m s −1 vs. OC: 4.43 ± 0.38 m s −1 ; vs. OA: 3.52 ± 0.35 m s −1 ; P = 0.03). These improvements were accompanied by lower oxidative stress and greater antioxidant enzyme expression. Ageing altered the abundance of gut microbial taxa associated with gut dysbiosis. Lastly, plasma TMAO was higher with ageing (young: 2.6 ± 0.4 μmol L −1 vs. OC: 7.2 ± 2.0 μmol L −1 ; P < 0.0001) and suppressed by antibiotic treatment (OA: 1.2 ± 0.2 μmol L −1 ; P < 0.0001 vs. OC). The results of the present study provide the first evidence for the gut microbiome being an important mediator of age-related arterial dysfunction and ...