Key points• Advancing age is the major risk factor for the development of cardiovascular diseases.• Arterial endothelial dysfunction, characterized by impaired endothelium-dependent dilatation (EDD), is a key antecedent to age-associated clinical cardiovascular disease.• We tested the hypothesis that changes in autophagy, the process by which cells recycle damaged biomolecules, may be an underlying cause of the age-related reduction in EDD.• We show that autophagy is impaired in arteries of older humans and mice with reduced EDD, and that enhancing autophagy restores EDD by reducing superoxide-dependent oxidative stress and inflammation, and increasing nitric oxide bioavailability.• Our results identify impaired autophagy as a potential cause of age-related arterial dysfunction and suggest that boosting autophagy may be a novel strategy for the treatment of arterial endothelial dysfunction and prevention of cardiovascular diseases with ageing.Abstract Ageing causes arterial endothelial dysfunction that increases the risk of cardiovascular diseases (CVD), but the underlying mechanisms are incompletely understood. The aim of the present study was to determine the role of autophagy, the cellular process of recycling damaged biomolecules, in endothelial dysfunction with ageing. In older humans, expression of autophagy markers in arterial endothelial cells was impaired by ∼50% (P < 0.05) and was associated with an ∼30% (P < 0.05) reduction in arterial endothelium-dependent dilatation (EDD). Similarly, in C57BL/6 control mice ageing was associated with an ∼40% decrease (P < 0.05) in arterial markers of autophagy and an ∼25% reduction (P < 0.05) in EDD. In both humans and mice, impaired EDD was mediated by reduced nitric oxide (NO) bioavailability and was associated with increased oxidative stress and inflammation (P < 0.05).In old mice, treatment with the autophagy-enhancing agent trehalose restored expression of autophagy markers, rescued NO-mediated EDD by reducing oxidative stress, and normalized inflammatory cytokine expression. In cultured endothelial cells, inhibition of autophagy increased oxidative stress and reduced NO production, whereas trehalose enhanced NO production via an autophagy-dependent mechanism. These results provide the first evidence that autophagy is impaired with ageing in vascular tissues. Our findings also suggest that autophagy preserves arterial endothelial function by reducing oxidative stress and inflammation and increasing

Aging impairs arterial function through oxidative stress and diminished nitric oxide (NO) bioavailability. Life-long caloric restriction (CR) reduces oxidative stress, but its impact on arterial aging is incompletely understood. We tested the hypothesis that life-long CR attenuates key features of arterial aging. Blood pressure, pulse wave velocity (PWV) (arterial stiffness), carotid artery wall thickness and endothelium-dependent dilation (EDD) (endothelial function) were assessed in young (Y: 5–7 mo), old ad libitum (Old AL: 30–31 mo.) and life-long 40% CR old (30–31 mo.) B6D2F1 mice. Blood pressure was elevated with aging (P<0.05) and was blunted by CR (P<0.05 vs. Old AL). PWV was 27% greater in old vs. young AL fed mice (P<0.05), and CR prevented this increase (P<0.05 vs. Old AL). Carotid wall thickness was greater with age (P<0.05), and CR reduced this by 30%. CR effects were associated with amelioration of age-related changes in aortic collagen and elastin. Nitrotyrosine, a marker of cellular oxidative stress, and superoxide production was greater in old AL vs. young (P<0.05) and CR attenuated this increase. Carotid artery EDD was impaired with age (P<0.05); CR prevented this by enhancing NO and reducing superoxide-dependent suppression of EDD (Both P<0.05 vs. Old AL). This was associated with a smaller age-related increase in NADPH oxidase activity and p67 expression, with increases in superoxide dismutase (SOD), total SOD and catalase activities (All P<0.05 Old CR vs. Old AL). Lastly, CR normalized age-related changes in the critical nutrient sensing pathways SIRT-1 and mTOR (P<0.05 vs. Old AL). Our findings demonstrate that CR is an effective strategy for attenuation of arterial aging.

We tested the hypothesis that regular aerobic exercise reverses arterial inflammation with aging. When compared with young controls (6.2 ± 0.4 mo; n = 7), old (31.3 ± 0.5 mo; n = 11) male B6D2F1 cage-restricted mice demonstrated increased arterial activation of the proinflammatory transcription factor NF-κB, as indicated by greater aortic phosphorylation of both the inhibitor of NF-κB kinase (IKK) and the p65 subunit of NF-κB (both P < 0.05). Similarly, aortic expression of the proinflammatory cytokines IL-1 and IL-6, IFN-γ, and TNF-α were greater in the old mice (all P < 0.05). Macrophage and T lymphocyte abundance was unchanged with age in the aortic intima and media but was markedly increased in the adventitia and perivascular fat tissue of old mice (all P < 0.05). This proinflammatory arterial phenotype with aging was associated with vascular dysfunction, as reflected by impaired nitric oxide-mediated endothelium-dependent dilation. Voluntary wheel running (10-14 wk) normalized aortic IKK-NF-κB activation, cytokine expression, adventitial and perivascular macrophage infiltration, and vascular function in old mice (32.4 ± 0.3 mo; n = 8) while having no consistent effects in young mice. Short-term voluntary wheel running started late in life reverses arterial inflammation with aging in mice possibly via outside-in actions. These anti-inflammatory effects may play an important role in the amelioration of age-associated vascular dysfunction by regular aerobic exercise.

SummaryInhibition of mammalian target of rapamycin, mTOR, extends lifespan and reduces age‐related disease. It is not known what role mTOR plays in the arterial aging phenotype or if mTOR inhibition by dietary rapamycin ameliorates age‐related arterial dysfunction. To explore this, young (3.8 ± 0.6 months) and old (30.3 ± 0.2 months) male B6D2F1 mice were fed a rapamycin supplemented or control diet for 6–8 weeks. Although there were few other notable changes in animal characteristics after rapamycin treatment, we found that glucose tolerance improved in old mice, but was impaired in young mice, after rapamycin supplementation (both P < 0.05). Aging increased mTOR activation in arteries evidenced by elevated S6K phosphorylation (P < 0.01), and this was reversed after rapamycin treatment in old mice (P < 0.05). Aging was also associated with impaired endothelium‐dependent dilation (EDD) in the carotid artery (P < 0.05). Rapamycin improved EDD in old mice (P < 0.05). Superoxide production and NADPH oxidase expression were higher in arteries from old compared to young mice (P < 0.05), and rapamycin normalized these (P < 0.05) to levels not different from young mice. Scavenging superoxide improved carotid artery EDD in untreated (P < 0.05), but not rapamycin‐treated, old mice. While aging increased large artery stiffness evidenced by increased aortic pulse‐wave velocity (PWV) (P < 0.01), rapamycin treatment reduced aortic PWV (P < 0.05) and collagen content (P < 0.05) in old mice. Aortic adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation and expression of the cell cycle‐related proteins PTEN and p27kip were increased with rapamycin treatment in old mice (all P < 0.05). Lastly, aging resulted in augmentation of the arterial senescence marker, p19 (P < 0.05), and this was ameliorated by rapamycin treatment (P < 0.05). These results demonstrate beneficial effects of rapamycin treatment on arterial function in old mice and suggest these improvements are associated with reduced oxidative stress, AMPK activation and increased expression of proteins involved in the control of the cell cycle.

Stiffening of large elastic arteries with age increases the risk of cardiovascular diseases (CVD), but the underlying mechanisms are incompletely understood. We investigated the role of mitochondrial quality control (QC, i.e., mitophagy and biogenesis) in arterial stiffening with aging. In C57BL6 mice, aging was associated with impaired aortic expression of mitochondrial QC mediators, greater activation of the mitochondrial redox/stress sensor p66shc, elevated superoxide production and increased arterial stiffness—as indicated by ~20% higher aortic pulse wave velocity (aPWV). In old mice, supplementation with trehalose, a nutraceutical reported to enhance mitophagy, normalized mitochondrial QC markers, p66shc activation and superoxide production, and reduced aPWV and aortic collagen I (a structural protein that confers stiffness). In vitro experiments indicated that mitochondrial QC processes were enhanced in aorta from old trehalose-treated mice, and in aortic rings studied ex vivo, both aging and treatment with the mitochondrial stressor rotenone were associated with increases in p66shc activation and intrinsic mechanical stiffness, whereas co-incubation with trehalose prevented these effects. Taken together, these findings suggest that mitochondrial stress/dysfunction as a result of impaired mitochondrial QC contributes to large elastic artery stiffening with age. Enhancing mitochondrial QC with agents such as trehalose may be a novel strategy for reducing age-associated arterial stiffness and CVD.