SummaryWe tested the hypothesis that supplementation of nicotinamide mononucleotide (NMN), a key NAD + intermediate, increases arterial SIRT1 activity and reverses age‐associated arterial dysfunction and oxidative stress. Old control mice (OC) had impaired carotid artery endothelium‐dependent dilation (EDD) (60 ± 5% vs. 84 ± 2%), a measure of endothelial function, and nitric oxide (NO)‐mediated EDD (37 ± 4% vs. 66 ± 6%), compared with young mice (YC). This age‐associated impairment in EDD was restored in OC by the superoxide (O2−) scavenger TEMPOL (82 ± 7%). OC also had increased aortic pulse wave velocity (aPWV, 464 ± 31 cm s−1 vs. 337 ± 3 cm s−1) and elastic modulus (EM, 6407 ± 876 kPa vs. 3119 ± 471 kPa), measures of large elastic artery stiffness, compared with YC. OC had greater aortic O2− production (2.0 ± 0.1 vs. 1.0 ± 0.1 AU), nitrotyrosine abundance (a marker of oxidative stress), and collagen‐I, and reduced elastin and vascular SIRT1 activity, measured by the acetylation status of the p65 subunit of NFκB, compared with YC. Supplementation with NMN in old mice restored EDD (86 ± 2%) and NO‐mediated EDD (61 ± 5%), reduced aPWV (359 ± 14 cm s−1) and EM (3694 ± 315 kPa), normalized O2− production (0.9 ± 0.1 AU), decreased nitrotyrosine, reversed collagen‐I, increased elastin, and restored vascular SIRT1 activity. Acute NMN incubation in isolated aortas increased NAD + threefold and manganese superoxide dismutase (MnSOD) by 50%. NMN supplementation may represent a novel therapy to restore SIRT1 activity and reverse age‐related arterial dysfunction by decreasing oxidative stress.

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.

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SummaryThe vascular endothelium may develop a proinflammatory profile with aging, but evidence is limited in humans. Expression of inflammatory proteins was determined in vascular endothelial cells (EC) obtained from peripheral veins of 24 young (23 ± 1 years, mean ± SE) and 36 older (63 ± 1) healthy men and women using quantitative immunofluorescence. The older subjects had lower vascular endothelium‐dependent dilation (forearm blood flow responses to acetylcholine, p < 0.05), and higher plasma concentrations of C‐reactive protein, interleukin‐6 (IL‐6), and oxidized low‐density lipoprotein (all p < 0.05), but not tumor necrosis factor‐α (TNF‐α). Total (O: 0.52 ± 0.04 vs. Y: 0.33 ± 0.05 NFκB/HUVEC intensity, p < 0.05) and nuclear (O: 0.59 ± 0.04 vs. Y: 0.41 ± 0.04) expression of nuclear factor κ B p65 (NFκB), a proinflammatory gene transcription factor, was greater in EC from the older subjects (p < 0.05). EC expression of the inhibitor (of nuclear translocation) of NFκB (IκBα) was lower in the older subjects (O: 0.16 ± 0.02 vs. Y: 0.24 ± 0.03, p < 0.05), whereas IκB kinase (IκK) was not different. EC expression of the proinflammatory proteins IL‐6 (O: 0.42 ± 0.06 vs. Y: 0.29 ± 0.03, p < 0.05), TNF‐α (O: 0.52 ± 0.06 vs. Y: 0.33 ± 0.05, p < 0.05) and monocyte chemoattractant protein 1 (MCP‐1) (O: 0.59 ± 0.06 vs. Y: 0.38 ± 0.02, p < 0.05) was greater in the older subjects, whereas cyclooxygenase 2 and the receptor for advanced glycation end‐products did not differ. These findings indicate that impaired function with aging in healthy adults is associated with the development of a proinflammatory phenotype in the vascular endothelium that could be caused in part by reduced IκB‐mediated inhibition of NFκB.

Reductions in arterial SIRT1 expression and activity with aging are linked to vascular endothelial dysfunction. We tested the hypothesis that the specific SIRT1 activator SRT1720 improves endothelial function [endothelium-dependent dilation (EDD)] in old mice. Young (4-9 mo) and old (29-32 mo) male B6D2F1 mice treated with SRT1720 (100 mg/kg body wt) or vehicle for 4 wk were studied with a group of young controls. Compared with the young controls, aortic SIRT1 expression and activity were reduced (P < 0.05) and EDD was impaired (83 ± 2 vs. 96 ± 1%; P < 0.01) in old vehicle-treated animals. SRT1720 normalized SIRT1 expression/activity in old mice and restored EDD (95 ± 1%) by enhancing cyclooxygenase (COX)-2-mediated dilation and protein expression in the absence of changes in nitric oxide bioavailability. Aortic superoxide production and expression of NADPH oxidase 4 (NOX4) were increased in old vehicle mice (P < 0.05), and ex vivo administration of the superoxide scavenger TEMPOL restored EDD in that group. SRT1720 normalized aortic superoxide production in old mice, without altering NOX4 and abolished the improvement in EDD with TEMPOL, while selectively increasing aortic antioxidant enzymes. Aortic nuclear factor-κB (NF-κB) activity and tumor necrosis factor-α (TNF-α) were increased in old vehicle mice (P < 0.05), whereas SRT1720 normalized NF-κB activation and reduced TNF-α in old animals. SIRT1 activation with SRT1720 ameliorates vascular endothelial dysfunction with aging in mice by enhancing COX-2 signaling and reducing oxidative stress and inflammation. Specific activation of SIRT1 is a promising therapeutic strategy for age-related endothelial dysfunction in humans.

We tested the hypothesis that peripheral blood mononuclear cells (PBMC) of older adults demonstrate a proinflammatory/-oxidative gene expression profile that can be improved by regular aerobic exercise. PBMC were isolated from young (n ϭ 25, 18 -33 yr) and middle-aged/older (n ϭ 40, 50 -76 yr) healthy adults. The older adults had greater mRNA expression (real-time RT-PCR) of the proinflammatory/-oxidant transcription factor nuclear factor-B (1.58-fold, P Ͻ 0.05) and receptor for advanced glycation end products (1.12-fold, P Ͻ 0.05), the proinflammatory cytokines tumor necrosis factor-␣ (1.90-fold, P Ͻ 0.05) and monocyte chemoattractant protein-1 (1.47-fold, P Ͻ 0.05), and the oxidant-producing enzymes nicotinamide adenine dinucleotide phosphate-oxidase (0.91-fold, P Ͻ 0.05) and inducible nitric oxide synthase (2.60-fold, P Ͻ 0.05). In 11 subjects (58 -70 yr), maximal oxygen consumption (ϩ11%) and exercise time (ϩ19%) were increased (both P Ͻ 0.001), and expression of the above proinflammatory/-oxidative genes was or tended to be decreased in PBMC after vs. before 2 mo of aerobic exercise (brisk walking ϳ6 days/wk, 50 min/day, 70% of maximal heart rate). Expression of interleukin-6 was not different with age or exercise intervention. Age group-and exercise intervention-related differences in gene expression were independent of other factors. PBMC of healthy older adults demonstrate increased expression of several genes associated with inflammation and oxidative stress, which is largely ameliorated by habitual aerobic exercise. This proinflammatory/-oxidative gene signature may represent a therapeutic target for lifestyle and pharmacological prevention and treatment strategies. aging; oxidative stress; inflammation; cytokines; immune cells AGE IS THE MAJOR RISK FACTOR for cardiovascular diseases (CVD), largely as the result of vascular dysfunction (18). Chronic low-grade inflammation and oxidative stress are believed to play important roles in the development of vascular disorders with age (1, 29, 31). Plasma markers of inflammation and oxidative stress often are elevated in older adults (9,11,13). Proinflammatory changes to the intimal layer of arteries with aging have been reported on autopsy (34). Moreover, we recently found increased expression of the redox-sensitive proinflammatory nuclear transcription factor nuclear factor B (NF-B), the proinflammatory cytokines tumor necrosis factor-␣ (TNF-␣), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), and the oxidant enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase in vascular endothelial cells obtained from older compared with young adults (9, 10). The proinflammatory/redox-sensitive proteins receptor for advanced glycation end products (RAGE) and inducible nitric oxide synthase (iNOS) also have been found to increase with age (2, 15).Although evidence is accumulating for inflammation and oxidative stress in plasma and vascular cells/tissues with advancing age in humans, relatively little is known about the possibility of such...

Ketogenic diets (KDs) are increasingly utilized as treatments for epilepsy, other neurological diseases, and cancer. Despite their long history in suppressing seizures, the distinct molecular mechanisms of action of KDs are still largely unknown. The goal of this study was to identify key metabolites and pathways altered in the hippocampus and plasma of rats fed a KD versus control diet (CD) either ad libitum or calorically restricted to 90% of the recommended intake. This was accomplished using a combination of targeted methods and untargeted MS-based metabolomics analyses. Various metabolites of and related to the tryptophan (TRP) degradation pathway, such as kynurenine (KYN), kynurenic acid as well as enzyme cofactors, showed significant changes between groups fed different diets and/or calorie amounts in plasma and/or the hippocampus. KYN was significantly downregulated in both matrices in animals of the CD-calorically restricted, KD-ad libitum, and KD-calorically restricted groups compared with the CD-ad libitum group. Our data suggest that the TRP degradation pathway is a key target of the KD.

We tested the hypothesis that vascular endothelial function and oxidative stress are related to dietary niacin intake among healthy middle-aged and older adults. In 127 men and women aged 48-77 yr, brachial artery flow-mediated dilation (FMD) was positively related to dietary niacin intake [%change (Δ): r = 0.20, P < 0.05; mmΔ: r = 0.25, P < 0.01]. In subjects with above-average dietary niacin intake (≥ 22 mg/day, NHANES III), FMD was 25% greater than in subjects with below-average intake (P < 0.05). Stepwise linear regression revealed that dietary niacin intake (above vs. below average) was an independent predictor of FMD (%Δ: β = 1.8; mmΔ: β = 0.05, both P < 0.05). Plasma oxidized low-density lipoprotein, a marker of systemic oxidative stress, was inversely related to niacin intake (r = -0.23, P < 0.05) and was lower in subjects with above- vs. below-average niacin intake (48 ± 2 vs. 57 ± 2 mg/dl, P < 0.01). Intravenous infusion of the antioxidant vitamin C improved brachial FMD in subjects with below-average niacin intake (P < 0.001, n = 33), but not above-average (P > 0.05, n = 20). In endothelial cells sampled from the brachial artery of a subgroup, dietary niacin intake was inversely related to nitrotyrosine, a marker of peroxynitrite-mediated oxidative damage (r = -0.30, P < 0.05, n = 55), and expression of the prooxidant enzyme, NADPH oxidase (r = -0.44, P < 0.01, n = 37), and these markers were lower in subjects with above- vs. below-average niacin intake [nitrotyrosine: 0.39 ± 0.05 vs. 0.56 ± 0.07; NADPH oxidase: 0.38 ± 0.05 vs. 0.53 ± 0.05 (ratio to human umbilical vein endothelial cell control), both P < 0.05]. Our findings support the hypothesis that higher dietary niacin intake is associated with greater vascular endothelial function related to lower systemic and vascular oxidative stress among healthy middle-aged and older adults.