Lisa A Lesniewski

Nuclear Factor-κB Activation Contributes to Vascular Endothelial Dysfunction via Oxidative Stress in Overweight/Obese Middle-Aged and Older Humans

Background— We tested the hypothesis that nuclear factor-&kgr;B (NF-&kgr;B) activity contributes to vascular endothelial dysfunction with aging and obesity in humans. Methods and Results— We conducted a randomized, double-blind, placebo-controlled crossover study in 14 nondiabetic overweight or obese (body mass index ≥25 kg/m2) middle-aged and older (age 52 to 68 years) adults. Salsalate (nonacetylated salicylate, 4500 mg/d), a compound that inhibits NF-&kgr;B activity, or placebo was administered for 4-day periods. Plasma salicylate concentrations reached the midtherapeutic range (21.8±1.1 mg/100 mL, P≤0.0001 versus placebo) by day 4 of salsalate treatment. Salsalate increased expression of the inhibitor of NF-&kgr;B and reduced total and nuclear expression of NF-&kgr;B in endothelial cells obtained from the subjects (all P<0.05). Salsalate increased brachial artery flow-mediated dilation by 74% (from 4.0±0.4% to 6.6±0.5%, P<0.001) but did not affect endothelium-independent dilation (P=0.83). The change in brachial artery flow-mediated dilation with salsalate was inversely related to baseline flow-mediated dilation (r=−0.77, P<0.01). Infusion of vitamin C increased brachial artery flow-mediated dilation during placebo (P<0.001) but not after salsalate (P=0.23). Salsalate reduced nitrotyrosine (P=0.06) and expression of NADPH oxidase p47phox (P<0.05) in endothelial cells obtained from the subjects but did not influence circulating or endothelial cell inflammatory proteins. Conclusions— Our findings provide the first direct evidence that NF-&kgr;B, in part via stimulation of oxidative stress, plays an important role in mediating vascular endothelial dysfunction in overweight and obese middle-aged and older humans.

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.

Habitual exercise and vascular ageing.

Age is the major risk factor for cardiovascular diseases (CVD) and this is attributable in part to stiffening of large elastic arteries and development of vascular endothelial dysfunction (e.g. impaired endothelium‐dependent dilatation, EDD). In contrast, regular aerobic exercise is associated with reduced risk of CVD. Endurance exercise‐trained middle‐aged/older adults demonstrate lower large elastic artery stiffness and greater EDD than their sedentary peers. With daily brisk walking, previously sedentary middle‐aged/older adults show reduced stiffness and improved EDD. The mechanisms underlying the effects of regular aerobic exercise on large elastic artery stiffness with ageing are largely unknown, but are likely to include changes to the composition of the arterial wall. Enhanced EDD in older adults who exercise is mediated by increased nitric oxide (NO) bioavailability associated with reduced oxidative stress. Arteries from old rodents that perform regular aerobic exercise demonstrate increased expression and activity of endothelial NO synthase, reduced oxidative damage associated with reduced expression and activity of the oxidant enzyme NADPH oxidase, and increased activity of the antioxidant enzyme superoxide dismutase. Aerobic exercise also may protect arteries with ageing by increasing resistance to the effects of other CVD risk factors like LDL‐cholesterol. Habitual aerobic exercise is an effective strategy to combat arterial ageing.

Short-term calorie restriction reverses vascular endothelial dysfunction in old mice by increasing nitric oxide and reducing oxidative stress.

To determine if short‐term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n = 30) and young (Y, n = 10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, approximately 30%) for 8 weeks. Ex vivo carotid artery endothelium‐dependent dilation (EDD) was impaired in old ad libitum (OAL) vs. young ad libitum (YAL) (74 ± 5 vs. 95 ± 2% of maximum dilation, P < 0.05), whereas old calorie‐restricted (OCR) and YCR did not differ (96 ± 1 vs. 94 ± 3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P < 0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P < 0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P < 0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97 ± 2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P < 0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P < 0.05), but unaffected by CR. Carotid artery endothelium‐independent dilation did not differ among groups. Short‐term CR initiated in old age reverses age‐associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NADPH oxidase–mediated superoxide production and stimulation of anti‐oxidant enzyme activity), and upregulation of sirtuin‐1.

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.

Life-Long Caloric Restriction Reduces Oxidative Stress and Preserves Nitric Oxide Bioavailability and Function in Arteries of Old Mice

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 month), old ad libitum (Old AL: 30–31 month) and life‐long 40% CR old (30–31 month) 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 were greater in old AL vs. young (P < 0.05) and CR attenuated these 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 blunted 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.

Life-long caloric restriction elicits pronounced protection of the aged myocardium: a role for AMPK.

Short-term caloric restriction (CR) protects the young myocardium against ischemia/reperfusion (I/R) injury through a mechanism involving AMP-activated protein kinase (AMPK). Here we ask whether a life-long CR intervention can extend this protection to the aged myocardium, and whether AMP-activated protein kinase (AMPK) plays a role in that protection. Hearts from ad libitum fed (AL) and life-long calorically restricted (LCR) mice were examined at 30 months of age by 25/90min global I/R, with and without AMPK inhibition (AraA). LCR hearts were protected from infarction (AL, 28±4% vs. LCR, 10±1%, p<0.01) and post-ischemic functional deficit (LVDP recovery: AL, 65±8% vs. LCR, 93±7%, p<0.01). Pre-ischemic AraA impaired both of these protective effects (Infarct size: LCR+AraA, 22±4%; LVDP recovery: LCR+AraA, 82±9%, both p vs. AL >0.1). AMPKα phosphorylation was dramatically increased in LCR hearts prior to I/R (AL, 1.18±0.01 vs. LCR, 1.68±0.04, ratio, p<0.0001), and accompanied by a more modest increase in total AMPKα (AL, 2.18±0.03 vs. LCR, 2.39±0.08 ratio, p<0.05). These results indicate that life-long caloric restriction profoundly protects the aged heart against I/R injury, and suggest that AMPK may play a role in that protection.