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.

Background-We tested the hypothesis that nuclear factor-B (NF-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 Ն25 kg/m 2 ) middle-aged and older (age 52 to 68 years) adults. Salsalate (nonacetylated salicylate, 4500 mg/d), a compound that inhibits NF-B activity, or placebo was administered for 4-day periods. Plasma salicylate concentrations reached the midtherapeutic range (21.8Ϯ1.1 mg/100 mL, PՅ0.0001 versus placebo) by day 4 of salsalate treatment. Salsalate increased expression of the inhibitor of NF-B and reduced total and nuclear expression of NF-B in endothelial cells obtained from the subjects (all PϽ0.05). Salsalate increased brachial artery flow-mediated dilation by 74% (from 4.0Ϯ0.4% to 6.6Ϯ0.5%, PϽ0.001) but did not affect endotheliumindependent dilation (Pϭ0.83). The change in brachial artery flow-mediated dilation with salsalate was inversely related to baseline flow-mediated dilation (rϭϪ0.77, PϽ0.01). Infusion of vitamin C increased brachial artery flow-mediated dilation during placebo (PϽ0.001) but not after salsalate (Pϭ0.23). Salsalate reduced nitrotyrosine (Pϭ0.06) and expression of NADPH oxidase p47 phox (PϽ0.05) 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-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. Key Words: endothelium Ⅲ brachial artery Ⅲ obesity Ⅲ inflammation Ⅲ aging V ascular endothelial dysfunction plays an important role in the increased risk of cardiovascular diseases with aging, 1 particularly in overweight and obese adults. 2,3 Inflammation and oxidative stress are thought to be important processes by which aging and increased adiposity lead to development of vascular endothelial dysfunction 4 -7 ; however, the molecular mechanisms involved, particularly in humans, are poorly understood. Clinical Perspective p 1292The nuclear transcription factor nuclear factor-B (NF-B) and its inhibitory protein, inhibitor of NF-B (IB), compose a tightly controlled system that regulates the inflammatory and redox states of vascular endothelial cells. 8,9 Data from animal and cell culture models implicate NF-B in both age-and obesity-associated vascular endothelial dysfunction. 10,11 Recently, we found that endothelial cells obtained from overweight/obese 12 and older 13,14 nondiabetic adults have greater protein expression of NF-B than normal-weight and young adults, respectively. Moreover, NF-B was positively related to nitrotyrosine, a cellular marker of oxidative stress. 13 Endothelial cell expression of the oxidant-producing enzyme NADPH oxidase also was greater in overweight/obese 12 and older 13 adults. Taken together, these ...

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.

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.Abstract We tested the hypothesis that reductions in the cellular deacetylase, sirtuin-1 (SIRT-1), contribute to vascular endothelial dysfunction with ageing via modulation of endothelial nitric oxide synthase (eNOS) acetylation/activation-associated nitric oxide (NO) production. In older (30 months, n = 14) vs. young (5-7 months, n = 16) B6D2F1 mice, aortic protein expression of SIRT-1 and eNOS phosphorylated at serine 1177 were lower (both P < 0.05), and acetylated eNOS was 6-fold higher (P < 0.05), whereas total eNOS did not differ (P = 0.65). Acetylcholine (ACh)-induced peak endothelium-dependent dilatation (EDD) was lower in isolated femoral arteries with ageing (P < 0.001). Incubation with sirtinol, a SIRT-1 inhibitor, reduced EDD in both young and older mice, abolishing age-related differences, whereas co-administration with L-NAME, an eNOS inhibitor, further reduced EDD similarly in both groups. Endothelium-independent dilatation to sodium nitroprusside (EID), was not altered by age or sirtinol treatment. In older (64 ± 1 years, n = 22) vs. young (25 ± 1 years, n = 16) healthy humans, ACh-induced forearm EDD was impaired (P = 0.01) and SIRT-1 protein expression was 37% lower in endothelial cells obtained from the brachial artery (P < 0.05), whereas EID did not differ. In the overall group, EDD was positively related to endothelial cell SIRT-1 protein expression (r = 0.44, P < 0.01). Reductions in SIRT-1 may play an important role in vascular endothelial dysfunction with ageing. SIRT-1 may be a key therapeutic target to treat arterial ageing.

A senescent phenotype in endothelial cells is associated with increased apoptosis, reduced endothelial nitric oxide synthase (eNOS) and inflammation, which are implicated in arterial dysfunction and disease in humans. We tested the hypothesis that changes in microRNAs are associated with a senescent phenotype in human aortic endothelial cells (HAEC). Compared with early-passage HAEC, late-passage HAEC had a reduced proliferation rate and increased staining for senescence-associated beta-galactosidase and the tumor suppressor p16INK4a. Late-passage senescent HAEC had reduced expression of proliferation-stimulating/apoptosis-suppressing miR-21, miR-214 and miR-92 and increased expression of tumor suppressors and apoptotic markers. eNOS-suppressing miR-221 and miR-222 were increased and eNOS protein and eNOS activation (phosphorylation at serine1177) were lower in senescent HAEC. Caveolin-1 inhibiting miR-133a was reduced and caveolin-1, a negative regulator of eNOS activity, was elevated in senescent HAEC. Inflammation-repressing miR-126 was reduced and inflammation–stimulating miR-125b was increased, whereas inflammatory proteins were greater in senescent HAEC. Development of a senescent arterial endothelial cell phenotype featuring reduced cell proliferation, enhanced apoptosis and inflammation and reduced eNOS is associated with changes in miRNAs linked to the regulation of these processes. Our results support the hypothesis that miRNAs could play a critical role in arterial endothelial cell senescence.