Jessica R Durrant

Voluntary wheel running restores endothelial function in conduit arteries of old mice: direct evidence for reduced oxidative stress, increased superoxide dismutase activity and down‐regulation of NADPH oxidase

Habitual aerobic exercise is associated with enhanced endothelium‐dependent dilatation (EDD) in older humans, possibly by increasing nitric oxide bioavailability and reducing oxidative stress. However, the mechanisms involved are incompletely understood. EDD was measured in young (6–8 months) and old (29–32 months) cage control and voluntary wheel running (VR) B6D2F1 mice. Age‐related reductions in maximal carotid artery EDD to acetylcholine (74 vs. 96%, P < 0.01) and the nitric oxide (NO) component of EDD (maximum dilatation with ACh and l‐NAME minus that with ACh alone was −28%vs.−55%, P < 0.01) were restored in old VR (EDD: 96%, NO: −46%). Nitrotyrosine, a marker of oxidative stress, was increased in aorta with age, but was markedly lower in old VR (P < 0.05). Aortic superoxide dismutase (SOD) activity was greater (P < 0.01), whereas NADPH oxidase protein expression (P < 0.01) and activity (P= 0.05) were lower in old VR vs. old cage control. Increasing SOD (with 4‐hydroxy‐2,2,6,6‐tetramethylpiperidine 1‐oxyl) and inhibition of NADPH oxidase (with apocynin) improved EDD and its NO component in old cage control, but not old VR mice. VR increased endothelial NO synthase (eNOS) protein expression (P < 0.05) and activation (Ser1177 phosphorylation) (P < 0.05) in old mice. VR did not affect EDD in young mice. Our results show that voluntary aerobic exercise restores the age‐associated loss of EDD by suppression of oxidative stress via stimulation of SOD antioxidant activity and inhibition of NADPH oxidase superoxide production. Increased eNOS protein and activation also may contribute to exercise‐mediated preservation of NO bioavailability and EDD with ageing.

Arterial stiffening with ageing is associated with transforming growth factor-β1-related changes in adventitial collagen: reversal by aerobic exercise

We tested the hypothesis that carotid artery stiffening with ageing is associated with transforming growth factor‐β1 (TGF‐β1)‐related increases in adventitial collagen and reductions in medial elastin, which would be reversed by voluntary aerobic exercise. Ex vivo carotid artery incremental stiffness was greater in old (29–32 months, n= 11) vs. young (4–7 months, n= 8) cage control B6D2F1 mice (8.84 ± 1.80 vs. 4.54 ± 1.18 AU, P < 0.05), and was associated with selective increases in collagen I and III and TGF‐β1 protein expression in the adventitia (P < 0.05), related to an increase in smooth muscle α‐actin (SMαA) (myofibroblast phenotype) (P < 0.05). In cultured adventitial fibroblasts, TGF‐β1 induced increases in superoxide and collagen I protein (P < 0.05), which were inhibited by Tempol, a superoxide dismutase. Medial elastin was reduced with ageing, accompanied by decreases in the pro‐synthetic elastin enzyme, lysyl oxidase, and increases in the elastin‐degrading enzyme, matrix metalloproteinase 2. Fibronectin was unchanged with ageing, but there was a small increase in calcification (P < 0.05). Increased incremental stiffness in old mice was completely reversed (3.98 ± 0.34 AU, n= 5) by 10–14 weeks of modest voluntary wheel running (1.13 ± 0.29 km day−1), whereas greater voluntary wheel running (10.62 ± 0.49 km day−1) had no effect on young mice. The amelioration of carotid artery stiffness by wheel running in old mice was associated with reductions in collagen I and III and TGF‐β1, partial reversal of the myofibroblast phenotype (reduced SMαA) and reduced calcification (all P < 0.05 vs. old controls), whereas elastin and its modulating enzymes were unaffected. Adventitial TGF‐β1‐related oxidative stress may play a key role in collagen deposition and large elastic artery stiffening with ageing and the efficacious effects of voluntary aerobic exercise.

SIRT‐1 and vascular endothelial dysfunction with ageing in mice and humans

Non‐technical summary  Advancing age is a major risk factor for the development of cardiovascular disease. A key characteristic of older arteries that may lead to cardiovascular disease is reduced endothelial function, characterized by blunted endothelium‐dependent dilatation. Sirtuins, specifically sirtuin‐1, are proteins linked to increases in lifespan and lower incidence of age‐related diseases. We hypothesized that diminished sirtuin‐1 with advancing age may alter regulation of a key endothelium dilatory enzyme, nitric oxide synthase. Our findings provide novel translational evidence that sirtuin‐1 expression and activity contribute to arterial endothelial dysfunction with ageing and that this may be due to altered nitric oxide synthase activation. Importantly, our results provide further compelling support for sirtuin‐1 as a potential therapeutic target for lifestyle and pharmacological interventions aimed at the prevention and treatment of arterial ageing and age‐associated cardiovascular diseases.

Aerobic exercise reverses arterial inflammation with aging in mice.

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.

B6D2F1 Mice Are a Suitable Model of Oxidative Stress–Mediated Impaired Endothelium-Dependent Dilation With Aging

To determine if B6D2F1 mice represent a suitable model of oxidative stress-mediated impaired endothelium-dependent dilation (EDD) with aging, mice were studied at 6.9 +/- 0.3 and 31.9 +/- 0.6 months. EDD to acetylcholine (ACh) was 26% (p < .001) and 12% (p < .001) lower, respectively, in isolated carotid (n = 10-11) and femoral (n = 10) arteries from older mice, and reductions in arterial pressure to systemic ACh infusion were smaller in older mice (n = 6-10; p < .01). Nitrotyrosine was marked in aorta of older mice (p < .05, n = 4). Superoxide production in carotid arteries was greater (p < .05), and TEMPOL restored dilation in carotid arteries and systemically in older mice. N(G)-nitro-l-arginine methyl ester (l-NAME) reduced carotid artery dilation in young more than older mice, whereas TEMPOL restored the effects of l-NAME in older mice. Carotid artery stiffness was increased in older compared with young mice (p = .04). Our results provide the first comprehensive evidence that B6D2F1 mice are a useful model for investigating mechanisms of reduced nitric oxide-dependent, oxidative stress-associated EDD and increased arterial stiffness with aging.

Salicylate Treatment Improves Age-Associated Vascular Endothelial Dysfunction: Potential Role of Nuclear Factor κB and Forkhead Box O Phosphorylation

We hypothesized that I kappa B kinase (IKK)-mediated nuclear factor kappa B and forkhead BoxO3a phosphorylation will be associated with age-related endothelial dysfunction. Endothelium-dependent dilation and aortic protein expression/phosphorylation were determined in young and old male B6D2F1 mice and old mice treated with the IKK inhibitor, salicylate. IKK activation was greater in old mice and was associated with greater nitrotyrosine and cytokines. Endothelium-dependent dilation, nitric oxide (NO), and endothelial NO synthase phosphorylation were lower in old mice. Endothelium-dependent dilation and NO bioavailability were restored by a superoxide dismutase mimetic. Nuclear factor kappa B and forkhead BoxO3a phosphorylation were greater in old and were associated with increased expression/activity of nicotinamide adenine dinucleotide phosphate oxidase and lower manganese superoxide dismutase expression. Salicylate lowered IKK phosphorylation and reversed age-associated changes in nitrotyrosine, endothelium-dependent dilation, NO bioavailability, endothelial NO synthase, nuclear factor kappa B and forkhead BoxO3a phosphorylation, nicotinamide adenine dinucleotide phosphate oxidase, and manganese superoxide dismutase. Increased activation of IKK with advancing age stimulates nuclear factor kappa B and inactivates forkhead BoxO3a. This altered transcription factor activation contributes to a pro-inflammatory/pro-oxidative arterial phenotype that is characterized by increased cytokines and nicotinamide adenine dinucleotide phosphate oxidase and decreased manganese superoxide dismutase leading to oxidative stress-mediated endothelial dysfunction.

Sustained activation of AMPK ameliorates age-associated vascular endothelial dysfunction via a nitric oxide-independent mechanism.

Exercise restores endothelium-dependent dilation (EDD) in old mice by reducing oxidative stress and increasing nitric oxide (NO) bioavailability. Adenosine monophosphate protein kinase (AMPK) activation mimics some effects of exercise. Old (28-30 months) B6D2F1 mice had reduced arterial AMPK expression and superoxide-mediated suppression of EDD vs. young (3-6 months) controls. Pharmacological activation of AMPK by aminoimidazole carboxamide ribonucleotide (AICAR) for 2 weeks increased arterial AMPK and reversed this superoxide-induced impairment of EDD. The improvement in EDD was independent of NO or prostaglandin signaling, suggesting enhanced endothelium-dependent hyperpolarizing factor-related dilation. AMPK activation may represent a novel therapy for treating age-associated vascular dysfunction.