Advancing age is associated with reductions in nitric oxide bioavailability and changes in metabolic activity, which are implicated in declines in motor and cognitive function. In preclinical models, sodium nitrite supplementation (SN) increases plasma nitrite and improves motor function, whereas other nitric oxide-boosting agents improve cognitive function. This pilot study was designed to translate these findings to middle-aged and older (MA/O) humans to provide proof-of-concept support for larger trials. SN (10 weeks, 80 to 160 mg/day capsules, TheraVasc, Inc.) acutely and chronically increased plasma nitrite and improved performance on measures of motor and cognitive outcomes (all p<0.05 or better) in healthy MA/O adults (62 ± 7 years). Untargeted metabolomics analysis revealed that SN significantly altered 33 (160 mg/day) to 45 (80 mg/day) different metabolites, 13 of which were related to changes in functional outcomes; baseline concentrations of 99 different metabolites predicted functional improvements with SN. This pilot study provides the first evidence that SN improves aspects of motor and cognitive function in healthy MA/O adults, and that these improvements are associated with, and predicted by, the plasma metabolome. Our findings provide the necessary support for larger clinical trials on this promising pharmacological strategy for preserving physiological function with aging.

Insufficient nitric oxide (NO) bioavailability plays an important role in endothelial dysfunction and arterial stiffening with aging. Supplementation with sodium nitrite, a precursor of NO, ameliorates age-related vascular endothelial dysfunction and arterial stiffness in mice, but effects on humans, including the metabolic pathways altered, are unknown. The purpose of this study was to determine the safety, feasibility, and efficacy of oral sodium nitrite supplementation for improving vascular function in middle-aged and older adults and to identify related circulating metabolites. Ten weeks of sodium nitrite (80 or 160 mg/day, capsules, TheraVasc; randomized, placebo control, double blind) increased plasma nitrite acutely (5- to 15-fold, P < 0.001 vs. placebo) and chronically ( P < 0.10) and was well tolerated without symptomatic hypotension or clinically relevant elevations in blood methemoglobin. Endothelial function, measured by brachial artery flow-mediated dilation, increased 45-60% vs. baseline ( P < 0.10) without changes in body mass or blood lipids. Measures of carotid artery elasticity (ultrasound and applanation tonometry) improved (decreased β-stiffness index, increased cross-sectional compliance, P < 0.05) without changes in brachial or carotid artery blood pressure. Aortic pulse wave velocity was unchanged. Nitrite-induced changes in vascular measures were significantly related to 11 plasma metabolites identified by untargeted analysis. Baseline abundance of multiple metabolites, including glycerophospholipids and fatty acyls, predicted vascular changes with nitrite. This study provides evidence that sodium nitrite supplementation is well tolerated, increases plasma nitrite concentrations, improves endothelial function, and lessens carotid artery stiffening in middle-aged and older adults, perhaps by altering multiple metabolic pathways, thereby warranting a larger clinical trial.

After feeding, Burmese pythons (Python molurus bivittatus) undergo physiological cardiac remodeling associated with increased cardiac output. Because the left ventricle (LV) of the heart and aorta are anatomically and mechanically coupled, we tested the hypothesis that chronic overfeeding causes aortic remodeling. Nine‐month old snakes consumed 25% of their total body weight every 2 weeks (normal fed, NF, n=5) or once every 3 days (overfed, OF, n=7) for 12 weeks. Compared with NF, OF had a greater aortic lumen diameter (D, 2.42 ± 0.11 vs. 1.47 ± 0.04 mm, p<0.01) and total wall thickness (WT, 353 ± 12 vs. 227 ± 12 μm, p<0.001), with unchanged WT:D ratio (p=0.67). This remodeling was associated with increased intrinsic mechanical stiffening (elastic modulus: 6,457 ± 670 vs. 3,667 ± 501 kPa, p=0.01; stiffness: 18,811 ± 4,318 vs. 7,093 ± 1,187 kPa*m, p<0.05) and expression of collagen I (2.27 ± 0.34 vs. 1.00 ± 0.15 AU, p<0.05) and advanced glycation end‐products (1.84 ± 0.33 vs. 1.00 ± 0.35 AU, p<0.05). However, no difference in the oxidative stress marker nitrotyrosine was observed between groups, and OF actually had greater expression of the antioxidant enzyme CuZnSOD (2.45 ± 0.06 vs. 1.00 ± 0.25 AU, p=0.05). Chronic overfeeding induces a physiological remodeling of the aorta in Burmese pythons that is not mediated by oxidative stress, but may be an adaptive response to increased blood flow and cardiac remodeling.

Vascular endothelial dysfunction develops with aging as a result of insufficient nitric oxide (NO) bioavailability. Supplementation with nitrite, a precursor to NO, may improve NO bioavailability. We tested the safety, dose (80 vs. 160 mg/d), duration (4 and 10 weeks), and efficacy of oral sodium nitrite for improving endothelial dysfunction in middle‐aged and older (MA/O) adults (n=28, 61±1 yr) in a randomized, placebo‐controlled, double‐blind pilot intervention study. Both doses of nitrite acutely increased plasma nitrite (10 to 20‐fold, p<0.05) and were well‐tolerated without symptomatic hypotension or clinically‐relevant elevations in blood methemoglobin (maximal value=1.2%). Ten weeks of nitrite capsules chronically increased plasma nitrite (p<0.05, both doses) without altering blood pressure. Endothelial function, measured by brachial artery flow‐mediated dilation (FMD), was increased 55‐65% vs. baseline (p<0.05) after 10, but not 4 weeks of nitrite vs. placebo. The reduction in FMD with 20 min of forearm cuff occlusion (ischemia‐reperfusion [I/R] stress) was only half as great (‐50%, p<0.05) after nitrite treatment vs. placebo (‐100%). These results indicate that sodium nitrite supplementation for 10 weeks at 80 or 160 mg/d is well‐tolerated, improves vascular endothelial function, and provides protection from endothelial I/R injury in MA/O adults.Grant Funding Source: Supported by NIH HL107105, RR025780, AG000279

Reduced nitric oxide (NO) bioavailability contributes to physiological dysfunction with aging. Oral sodium nitrite supplementation (SN) increases circulating nitrite, a precursor of NO, and improves some physiological functions in middle‐aged and older (MA/O) adults. To gain insight into the underlying molecular mechanisms, the metabolome was assessed by liquid chromatography‐mass spectrometry in plasma samples from MA/O adults before and after SN (80 mg/d capsules, TheraVasc, Inc.) (n=8, 59±2 yrs) or placebo (P, n=7, 61±3 yrs) (randomized, double blind). Of ~4,000 metabolites detected, significant changes in 37 intermediary metabolites were observed in SN vs. P groups (minimum 1.5 fold‐change, p<0.05). Several metabolites associated with a pro‐aging phenotype changed with SN, including pro‐inflammatory mediators and molecules related to sphingolipid, glycan, glycerophospholipid, fatty acyl, polyketide, and choline metabolism. Importantly, these SN‐induced changes occurred in the absence of changes in clinical characteristics including body composition and blood lipid profiles (all p>0.05). These preliminary results suggest that SN influences human plasma metabolome pathways linked to aging. Further targeted analysis will allow the identification of novel biomarkers in metabolic pathways modulated by SN and offer insight into the mechanisms by which SN improves specific domains of physiological function in MA/O adults.Grant Funding Source: Supported by NIH HL107105, RR025780, AG000279

Declines in exercise capacity with aging are associated with increased risk of disability and mortality. Dietary interventions that increase circulating nitrite may improve exercise capacity. We performed a pilot study to assess the effects of chronic oral sodium nitrite supplementation (NS) on incremental treadmill exercise performance (modified Balke protocol) in sedentary and recreationally‐active middle‐aged and older (MA/O) healthy adults (n=26, 62±2 yrs). Subjects abstained from dosing for >12 hr before exercise. NS increased plasma nitrite and nitrate (p<0.01). Peak exercise respiratory exchange ratio (RER), heart rate (HR) and ratings of perceived exertion (RPE), markers of voluntary effort, did not differ pre‐post treatment in any group after 10 weeks of NS (80 or 160 mg/d capsules, TheraVasc, Inc., randomized, placebo‐control, double‐blind). NS did not influence maximal oxygen consumption (VO2) (Pl: ‐0.55 ± 2.01; 80mg: 0.41 ± 1.69; 160mg: ‐0.11 ± 1.19 ml/kg/min, p=0.63), although there was a trend for improvement in exercise duration (Pl: ‐0.3 ± 20.0 s; 80mg: +40.9 ± 65.5 s; 160mg: +18.6 ± 28.4 s, p=0.16). There were no significant effects of NS on VO2, HR, RER, RPE or minute ventilation during standardized submaximal workloads. These preliminary results suggest that NS may not increase aerobic exercise capacity or modify responses to submaximal exercise in healthy normally active MA/O adults. It is possible, however, that NS may improve exercise capacity or favorably modify submaximal exercise responses in adults with greater baseline nitrite deficiency (e.g., patients with clinical disease).Grant Funding Source: Supported by NIH HL107105, RR025780, AG000279

Aging is the major risk factor for cardiovascular diseases (CVD). This is attributable primarily to adverse changes in arteries, notably, increases in large elastic artery stiffness and endothelial dysfunction mediated by inadequate concentrations of the vascular-protective molecule, nitric oxide (NO), and higher levels of oxidative stress and inflammation. Inorganic nitrite is a promising precursor molecule for augmenting circulating and tissue NO bioavailability because it requires only a one-step reduction to NO. Nitrite also acts as an independent signaling molecule, exerting many of the effects previously attributed to NO. Results of recent studies indicate that nitrite may be effective in the treatment of vascular aging. In old mice, short-term oral sodium nitrite supplementation reduces aortic pulse wave velocity, the gold-standard measure of large elastic artery stiffness, and ameliorates endothelial dysfunction, as indicated by normalization of NO-mediated endothelium-dependent dilation. These improvements in age-related vascular dysfunction with nitrite are mediated by reductions in oxidative stress and inflammation, and may be linked to increases in mitochondrial biogenesis and health. Increasing nitrite levels via dietary intake of nitrate appears to have similarly beneficial effects in many of the same physiological and clinical settings. Several clinical trials are being performed to determine the broad therapeutic potential of increasing nitrite bioavailability on human health and disease, including studies related to vascular aging. In summary, inorganic nitrite, as well as dietary nitrate supplementation, represents a promising therapy for treatment of arterial aging and prevention of age-associated CVD in humans.

In the present study, we tested the hypothesis that age-associated vascular endothelial dysfunction is exacerbated by IFG (impaired fasting plasma glucose) and that regular aerobic exercise prevents this effect. Data were analysed from a cohort of 131 non-smoking men and women without overt clinical disease. Compared with young adult controls (age=24±1 years, n=29; values are means±S.E.M.), brachial artery FMD (flow-mediated dilation), a measure of conduit artery EDD (endothelium-dependent dilation), was 33% lower [7.93±0.33 against 5.27±0.37%Δ (% change), P<0.05] in MA/O (middle-aged/older) adults with NFG (normal fasting plasma glucose) (≤99 mg/dl, 62±1 years, n=35). In MA/O adults with IFG (100-125 mg/dl, 64±1 years, n=28), FMD was 30% lower (3.37±0.35%Δ) than in their peers with NFG and 58% lower than young controls (P<0.05). Brachial artery FMD was greater (6.38±0.35%Δ) in MA/O adults with NFG who regularly performed aerobic exercise (>45 min/day for ≥5 days/week, 62±1 years, n=23) compared with their non-exercising peers and only slightly less than young controls (P<0.05). Most importantly, FMD was completely preserved in MA/O adults with IFG who regularly performed aerobic exercise (6.99±0.69%Δ, 65±1 years, n=16). In the pooled sample, fasting plasma glucose was inversely related to FMD (r=-0.42, P<0.01) and was the strongest independent predictor of FMD (R(2)=0.32). Group differences in FMD were not affected by other subject characteristics or brachial artery properties, including brachial artery dilation to sublingual NTG (nitroglycerine, i.e. endothelium-independent dilation). IFG exacerbates age-associated vascular endothelial dysfunction and this adverse effect is completely prevented in MA/O adults who regularly perform aerobic exercise.

DeVan et al. [1] undertook a comprehensive cross-sectional study involving the independent variables of IFG (impaired fasting glucose) and exercise training status. In keeping with the effects of many other independent variables, including age [2,3], exercise [4], red wine [5] and cardiovascular disease [6], both FMD% [percentage FMD (flow-mediated dilation)] and D base (baseline artery diameter) were found to be different between the study samples. For example, D base was 0.46 mm larger in non-exercising older adults with IFG than the trained older adults with IFG, whereas FMD% was 3.6% higher in the latter sample. Brachial FMD% is mathematically equivalent to the ratio of D peak (peak diameter) divided by D base, i.e. D peak/ D … DeVan et al. [1] undertook a comprehensive cross-sectional study involving the independent variables of IFG (impaired fasting glucose) and exercise training status. In keeping with the effects of many other independent variables, including age [2,3], exercise [4], red wine [5] and cardiovascular disease [6], both FMD% [percentage FMD (flow-mediated dilation)] and D base (baseline artery diameter) were found to be different between the study samples. For example, D base was 0.46 mm larger in non-exercising older adults with IFG than the trained older adults with IFG, whereas FMD% was 3.6% higher in the latter sample. Brachial FMD% is mathematically equivalent to the ratio of D peak (peak diameter) divided by D base, i.e. D peak/ D …

The demographics of ageing are changing dramatically such that there will be many more older adults in the near future. This setting is projected to produce a new boomer-driven epidemic of physiological dysfunction, disability and risk of chronic degenerative disorders, including cardiovascular diseases. Standing out against this dreary biomedical forecast are Masters athletes, a group of middle-aged and older adults who engage in regular vigorous physical training and competitive sport. Compared with their sedentary/less active (untrained) peers, Masters athletes who perform endurance training-based activities demonstrate a more favourable arterial functionstructure phenotype, including lower large elastic artery stiffness, enhanced vascular endothelial function and less arterial wall hypertrophy. As such, they may represent an exemplary model of healthy or successful vascular ageing. In contrast, Masters athletes engaged primarily/exclusively in intensive resistance training exhibit less favourable arterial functionstructure than their endurance-trained peers and, in some instances, untrained adults. These different arterial properties are probably explained in large part by the different intravascular mechanical forces generated during endurance versus resistance exercise-related training activities. The more favourable arterial functionstructure profile of Masters endurance athletes may contribute to their low risk of clinical cardiovascular diseases.

Flow-mediated dilation (FMD) is a non-invasive index of endothelial function. In an attempt to standardize FMD for shear stimulus, shear rate (velocity/diameter), rather than shear stress (viscosity*velocity/diameter), is commonly used as a surrogate measure, although it is limited by individual differences in blood viscosity. The purpose of this study was to determine the contribution of whole blood viscosity to FMD and other key measures of vascular function. Blood viscosity, FMD, carotid artery compliance, and carotid-femoral pulse wave velocity (cfPWV) were measured in 98 apparently healthy adults varying widely in age (18-63 years). Whole blood viscosity was not significantly correlated with FMD, cfPWV, or carotid artery compliance. Shear rate was a stronger correlate with FMD than shear stress that takes blood viscosity into account (r = 0.43 vs 0.28). No significant differences were observed between whole blood viscosity and traditional risk factors for cardiovascular disease. Age was positively correlated with cfPWV (r = 0.65, p < 0.001) and negatively correlated with FMD (r = -0.24, p < 0.05) and carotid artery compliance (r = -0.45, p < 0.01). Controlling for viscosity did not reduce the strength of these relations. These results indicate that whole blood viscosity does not significantly impact measures of vascular function and suggests that the common practice to use shear rate, rather than shear stress, in the adjustment of FMD is valid.

BACKGROUNDWe performed a pilot study to test the hypothesis that acute oral ingestion of tetrahydrobiopterin (BH4), a key cofactor modulating vascular nitric oxide (NO) synthase activity, improves large elastic artery stiffness with aging in men.METHODSHealthy older (63 +/- 2 years; n = 8) and young (age 25 +/- 1 years; n = 6) men were studied 3 h after ingestion of BH4 (10 mg.kg(-1) body weight) or placebo on separate days in a randomized, placebo-controlled, double-blind study.RESULTSBaseline carotid artery compliance was 37% lower (0.17 +/- 0.02 vs. 0.22 +/- 0.02 mm/mm Hg.10(-1)) and beta-stiffness was 42% higher (7.3 +/- 1.1 vs. 4.2 +/- 0.5 AU) in the older men (both P < 0.05). BH4 ingestion markedly increased circulating BH4 concentrations in both groups (17-19-fold, P < 0.05), but increased compliance (+39% to 0.23 +/- 0.02 mm/mm Hg.10(-1), P < 0.01) and decreased beta-stiffness index (-27% to 5.3 +/- 0.7 AU, P < 0.01) only in the older men. BH4 also reduced carotid systolic blood pressure (SBP) in the older men (P < 0.05).CONCLUSIONSThese preliminary results support the possibility that limited BH4 bioavailability contributes to impaired carotid artery compliance in healthy older men. Further studies are needed to determine if increasing BH4 bioavailability though oral BH4 supplementation may have therapeutic efficacy for improving large elastic artery compliance and reducing central SBP with aging.

Dietary sodium restriction (DSR) improves carotid artery compliance (CC) in middle‐aged and older adults (MA/O) with moderately elevated systolic blood pressure (SBP), but the mechanisms of action are unknown. Reduced tetrahydrobiopterin (BH4) bioactivity contributes to age‐associated vascular dysfunction. We hypothesized that increased BH4 bioavailability may contribute to improvements in CC with DSR. SBP was reduced following 4 weeks of a low sodium (LS; 56±11 mmol/day; 125±2 mmHg) vs. normal sodium (NS; 160±7 mmol/day; 141±4 mmHg) diet (p<0.001; randomized, cross‐over design; 5M/4F; 61±3 yrs). Baseline CC was 43% greater during the LS vs. NS condition (0.14±0.02 vs. 0.097±0.02 mm/mmHg, p=0.05). Acute oral BH4 administration (10 mg/kg) improved CC vs. placebo during the NS condition (0.13±0.02 mm/mmHg, p<0.05) to levels not significantly different than the LS placebo state, but had no effect on CC during the LS condition (0.14±0.02 mm/mmHg). BH4 had no significant influence on SBP in either condition (LS, 127±3 mmHg, p=0.30; NS, 138±3 mmHg; p=0.13). Results were similar when presented as the β‐stiffness index, a more BP‐independent expression of carotid artery distensibility. These findings are consistent with the hypothesis that DSR improves CC in MA/O with moderately elevated SBP in part by enhancing BH4 bioavailability.NIH AG013038, AG006537, AG03114, AG033994, RR025780

One of the most commonly evaluated features of the arterial pressure waveforms is augmentation index (AI). Multiple devices have been developed and marketed that measure AI at peripheral arteries. Currently, it is not known if and how these measures of AI are related. Aortic and radial AI (using SphygmoCor), radial AI (Omron), and finger AI (Itamar) were measured in 40 apparently healthy subjects. All the AI values were correlated with each other with Pearson r-values ranging from 0.78 to 0.94. The coefficients of variation ranged from 3.4 to 20.0%. We concluded that even though the absolute values derived by each technique were different, there were high and significant correlations between AI values.

We determined if brachial artery flow‐mediated dilation (FMD), a measure of vascular endothelial function, is related to systemic markers of oxidative modification (stress), antioxidant bioavailability and inflammation in adults without clinical disease (n = 399, 18 – 79 yr, 252 men). Markers of oxidative stress: urinary 15‐isoprostane F2t (r = −0.15, P = 0.05, n = 120, ELISA) and plasma thiobarbituric acid reactive substances (r = −0.21, P = 0.02, n = 110) were weakly related to FMD, whereas serum oxidized low‐density lipoprotein was unrelated to FMD (r = −0.07, P = 0.11, n = 340). Antioxidant markers: whole blood glutathione peroxidase activity (r = 0.02, P = 0.42, n = 181), plasma glutathione reductase activity (r = −0.16, P = 0.09, n = 72), serum total antioxidant status (r = −0.02, P = 0.37, n = 332) and whole blood superoxide dismutase activity (r = 0.12, P = 0.12, n = 99) were not related to FMD. Log‐transformed serum interleukin‐6 (r = −0.08, P = 0.16, n = 156), serum high sensitivity C‐reactive protein (r = −0.03, P = 0.31, n = 298) and plasma TNF‐α (r = 0.02, P = 0.41, n = 127) were unrelated to FMD. Most of these markers were unrelated to brachial artery dilation to sublingual nitroglycerin. Common markers of systemic oxidative modification, antioxidant status and inflammation are unrelated or weakly related to vascular endothelial function among healthy men and women varying in age.

Resistance exercise involves muscular contractions that can render downstream tissues ischemic and may precondition the vasculature against ischemia–reperfusion (IR) injury, but it is unknown if habitual resistance exercise protects against IR injury in humans. We determined the magnitude and recovery from endothelial IR injury induced by forearm occlusion in 22 healthy young sedentary and resistance-trained adults. After IR injury, brachial artery flow-mediated dilation (FMD) significantly decreased by 36% in sedentary, but not resistance-trained subjects and fully recovered within 45 min. Though HDL-cholesterol, handgrip strength and systolic blood pressure were significantly associated with FMD 15 min after IR injury, the change in FMD from before to 15 min after IR injury was not associated with any subject characteristics. These results are consistent with the notion that habitual resistance exercise may protect against endothelial IR injury in young adults, presumably through effects analogous to ischemic preconditioning.