To determine whether impaired endothelium-dependent dilation (EDD) in older adults is associated with changes in the expression of major vasoconstrictor or vasodilator proteins in the vascular endothelium, endothelial cells (EC) were obtained from the brachial artery and peripheral veins of 56 healthy men, aged 18-78 yr. Brachial artery EC endothelin-1 (ET-1) [0.99 +/- 0.10 vs. 0.57 +/- 0.10 ET-1/human umbilical vein EC (HUVEC) intensity, P = 0.01] and serine 1177 phosphorylated endothelial nitric oxide synthase (PeNOS) (0.77 +/- 0.09 vs. 0.44 +/- 0.07 PeNOS/HUVEC intensity, P < 0.05) (quantitative immunofluorescence) were greater, and EDD (peak forearm blood flow to intrabrachial acetylcholine) was lower (10.2 +/- 0.9 vs. 14.7 +/- 1.7 ml.100 ml(-1).min(-1), P < 0.05) in older (n = 18, 62 +/- 1 yr) vs. young (n = 15, 21 +/- 1 yr) healthy men. EDD was inversely related to expression of ET-1 (r = -0.39, P < 0.05). Brachial artery EC eNOS expression did not differ significantly with age, but tended to be greater in the older men (young: 0.23 +/- 0.03 vs. older: 0.33 +/- 0.07 eNOS/HUVEC intensity, P = 0.08). In the sample with venous EC collections, EDD (brachial artery flow-mediated dilation) was lower (3.50 +/- 0.44 vs. 7.68 +/- 0.43%, P < 0.001), EC ET-1 and PeNOS were greater (P< 0.05), and EC eNOS was not different in older (n = 23, 62 +/- 1 yr) vs. young (n = 27, 22 +/- 1 yr) men. EDD was inversely related to venous EC ET-1 (r = -0.37, P < 0.05). ET-1 receptor A inhibition with BQ-123 restored 60% of the age-related impairment in carotid artery dilation to acetylcholine in B6D2F1 mice (5-7 mo, n = 8; 30 mo, n = 11; P < 0.05). ET-1 expression is increased in vascular EC of healthy older men and is related to reduced EDD, whereas ET-1 receptor A signaling tonically suppresses EDD in old mice. Neither eNOS nor PeNOS is reduced with aging. Changes in ET-1 expression and bioactivity, but not eNOS, contribute to vascular endothelial dysfunction with aging.

Objective-Reactive hyperemia is the compensatory increase in blood flow that occurs after a period of tissue ischemia, and this response is blunted in patients with cardiovascular risk factors. Key Words: endothelium Ⅲ cardiovascular risk Ⅲ surrogate markers Ⅲ reactive hyperemia Ⅲ flow-mediated dilation R eactive hyperemia is a complex response that occurs after a period of tissue ischemia and primarily depends on local production of adenosine and other non-endothelium-dependent vasodilators that dilate tissue microvessels. 1 Studies in humans have shown that endothelium-derived nitric oxide also contributes to reactive hyperemia. 2,3 Peak brachial artery hyperemic flow velocity after 5-minute cuff occlusion of the arm relates inversely to traditional cardiovascular disease risk factors 4 and to markers of inflammation 5 in the Framingham Heart Study. Smaller scale mechanistic studies suggest that the nitric oxide-dependent component of reactive hyperemia may be particularly affected by risk factors. 3 The relation of reactive hyperemia to the incidence of cardiovascular disease events in atherosclerosis has not been previously studied.

Abstract-Aging is associated with impaired vascular endothelial function, as indicated in part by reduced endotheliumdependent dilation (EDD). Decreased EDD with aging is thought to be related to vascular endothelial cell oxidative stress, but direct evidence is lacking. We studied 95 healthy men: 51 young (23Ϯ1 years) and 44 older (63Ϯ1 years). EDD (brachial artery flow-mediated dilation) was Ϸ50% lower in older versus young men (3.9Ϯ0.3% versus 7.6Ϯ0.3%, PϽ0.01; nϭ42 older/nϭ51 young). Abundance of nitrotyrosine (quantitative immunofluorescence), an oxidatively modified amino acid and marker of oxidative stress, was higher in endothelial cells (ECs) obtained from the brachial artery (1.25Ϯ0.12 versus 0.61Ϯ0.11 nitrotyrosine intensity/human umbilical vein EC [HUVEC] intensity, Pϭ0.01; nϭ11 older/nϭ11 young) and antecubital veins (0.55Ϯ0.04 versus 0.34Ϯ0.03, PϽ0.05; nϭ19 older/nϭ17 young) of older men. Flow-mediated dilation was inversely related to arterial EC nitrotyrosine expression (rϭϪ0.62, Pϭ0.01; nϭ22). In venous samples, EC expression of the oxidant enzyme NAD(P)H oxidase-p47 phox was higher in older men (0.71Ϯ0.05 versus 0.57Ϯ0.05 NAD[P]H oxidase-p47 phox intensity/HUVEC intensity, PϽ0.05; nϭ19 older/nϭ18 young), whereas xanthine oxidase and the antioxidant enzymes cytosolic and mitochondrial superoxide dismutase and catalase were not different between groups. Nuclear factor-B p65, a component of the redox-sensitive nuclear transcription factor nuclear factor-B, was elevated in both arterial (0.73Ϯ0.07 versus 0.53Ϯ0.05 NF-B p65 intensity/HUVEC intensity, PϽ0.05; nϭ9 older/nϭ12 young) and venous (0.65Ϯ0.07 versus 0.34Ϯ0.05, PϽ0.01; nϭ13 older/nϭ15 young) EC samples of older men and correlated with nitrotyrosine expression (rϭ0.51, PϽ0.05 nϭ16). These results provide direct support for the hypothesis that endothelial oxidative stress develops with aging in healthy men and is related to reductions in EDD. Increased expression of NAD(P)H oxidase and nuclear factor-B may contribute to endothelial oxidative stress with aging in humans. (Circ Res. 2007;100:1659-1666.) Key Words: flow-mediated dilation Ⅲ NAD(P)H oxidase Ⅲ xanthine oxidase Ⅲ antioxidants T he arterial vascular endothelium plays an essential role in the initiation and progression of cardiovascular disease (CVD). 1 Consistent with this, vascular endothelial dysfunction, as reflected by impaired endothelium-dependent dilation (EDD), is observed in patients with CVD and predicts future events in this population. 2 Oxidative stress, defined as increased production of reactive oxygen species relative to antioxidant defenses, is believed to play a key role in the development of endothelial dysfunction in the setting of CVD. 1 Older age is a major risk factor for the development of CVD. 3 EDD becomes impaired with aging in adult humans 4 -8 and is thought to contribute to this age-associated increase in CVD risk. 3 In experimental animals, age-related reductions in EDD are associated with vascular oxidative stress 9 and upregulation of systems supporti...

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Background-Obesity may alter vascular endothelial cell protein expression (VECPE) of molecules that influence susceptibility to atherosclerosis. Methods and Results-Quantitative immunofluorescence was performed on vascular endothelial cells collected from 108 men and women free of clinical disease who varied widely in adiposity (body mass index 18.4 to 36.7 kg/m 2 ; total body fat 5.8 to 55.0 kg; waist circumference: 63.0 to 122.9 cm). All 3 expressions of adiposity were positively associated with VECPE of the oxidant enzyme subunit NAD(P)H oxidase-p47 phox (part correlation coefficient [r part ] 0.22 to 0.24, all PϽ0.05) and the antioxidant enzyme catalase (r part ϭ0.71 to 0.75, all PϽ0.001). Total body fat was positively associated with VECPE of nitrotyrosine (r part ϭ0.36, Pϭ0.003), a marker of protein oxidation, and, in men, with Ser1177-phosphorylated endothelial nitric oxide synthase (r part ϭ0.46, Pϭ0.02), an activated form of endothelial nitric oxide synthase. Overweight/obese subjects (body mass index Ն25 kg/m 2 ) had 35% to 130% higher VECPE of NAD(P)H oxidasep47 phox , nitrotyrosine, catalase, and the cytosolic antioxidant CuZn superoxide dismutase (all PϽ0.05), as well as a 56% greater VECPE of the potent local vasoconstrictor endothelin-1 (Pϭ0.05) than normal-weight subjects (body mass index Ͻ25 kg/m 2 ). Nuclear factor-B protein expression was Ϸ60% to 100% greater in the most obese adults than in the leanest adults (PՅ0.01). These relations were independent of sex but were selectively reduced after accounting for the influence of plasma C-reactive protein, fasting glucose-insulin metabolism, or serum triglycerides. Conclusions-Compared with their normal-weight peers, overweight and obese adults demonstrate increased vascular endothelial expression of NAD(P)H oxidase-p47 phox and evidence of endothelial oxidative stress, with selective compensatory upregulation of antioxidant enzymes and Ser1177-phosphorylated endothelial nitric oxide synthase. Endothelin-1 and nuclear factor-B protein expression also appear to be elevated in obese compared with lean adults. These findings may provide novel insight into the molecular mechanisms linking obesity to increased risk of clinical atherosclerotic diseases in humans. (Circulation. 2007;115:627-637.)

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SUMMARY We tested the hypothesis that older men who perform habitual aerobic exercise do not demonstrate age-associated vascular endothelial oxidative stress compared with their sedentary peers. Older exercising men (n=13, 62±2 y) had higher (P<0.05) physical activity (79±7 vs. 30±6 MET h/wk) and maximal exercise oxygen consumption (42±1 vs. 29±1 ml/kg/min) vs. sedentary men (n=28, 63±1 y). Brachial artery flow-mediated dilation, a measure of vascular endothelial function, was greater (P<0.05) in the exercising vs. sedentary older men (6.3±0.5 vs. 4.9±0.4 %Δ) and not different than young controls (n=20, 25±1 y, 7.1 ± 0.5 %Δ). In vascular endothelial cells sampled from the brachial artery, nitrotyrosine, a marker of oxidative stress, was 51% lower in the exercising vs. sedentary older men (0.38±0.06 vs. 0.77±0.10 AU). This was associated with lower endothelial expression of the oxidant enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (p47phox subunit, 0.33±0.05 vs. 0.61±0.09 AU) and the redox-sensitive transcription factor nuclear factor kappa B (NFκB) (p65 subunit, 0.36±0.05 vs. 0.72±0.09 AU). Expression of the antioxidant enzyme manganese superoxide dismutase (SOD) (0.57±0.13 vs. 0.30±0.04 AU) and activity of endothelium-bound extracellular SOD were greater (6.4±0.5 vs. 5.0±0.6 U/ml/min) in the exercising men (both P<0.05), but differences no longer were significant after correcting for adiposity and circulating metabolic factors. Overall, values for the young controls differed with those for the sedentary, but not the exercising older men. Older men who exercise regularly do not demonstrate vascular endothelial oxidative stress and this may be a key molecular mechanism underlying their reduced risk of cardiovascular diseases.

Aging is associated with a decline in vascular endothelial function, manifesting in part as impaired flow-mediated arterial dilation (FMD), but the underlying mechanisms are uncertain. Impaired FMD may be mediated in part by a decrease in synthesis of nitric oxide by endothelial nitric oxide synthase, and in clinical populations this has been attributed to competitive inhibition of l-arginine binding sites by asymmetric dimethylarginine (ADMA). If this mechanism is involved in the age-associated decline in FMD, increasing l-arginine concentration may swing the competitive balance in favor of l-arginine binding, restoring nitric oxide synthesis, and enhancing FMD in older humans. To test this hypothesis, we measured FMD (brachial ultrasound) in 10 younger (21 +/- 1 yr) and 12 older healthy men and women (60 +/- 2 yr) following infusion of vehicle or vehicle + l-arginine. Baseline FMD in the older subjects was only approximately 60% of that in the younger subjects (P = 0.002). l-Arginine did not significantly increase FMD in either group despite 23-fold (older) and 19-fold (younger) increases in plasma l-arginine concentrations (P < 0.0001 vs. control). Protein expression (immunofluorescence) in vascular endothelial cells showed that ADMA and the enzyme isoform that controls its degradation, dimethylarginine dimethylaminohydrolase II, were not different in the younger and older subjects. Endothelium-independent vasodilation (sublingual nitroglycerine) was not different between age groups or conditions. We conclude that acutely increasing plasma concentrations of l-arginine do not significantly improve brachial artery FMD in healthy older subjects and thus does not restore the age-associated loss of FMD. Together with the finding that endothelial cell ADMA protein expression was not increased in older adults, these findings suggest that competitive inhibition of l-arginine binding sites on endothelial nitric oxide synthase by ADMA is not an important mechanism contributing to impaired conduit artery endothelium-dependent dilation with aging in healthy humans.