Novel role for retinol-binding protein 4 in the regulation of blood pressure.

Elevated levels of serum retinol-binding protein 4 (RBP4) contribute to insulin resistance and correlate with increased prevalence of hypertension and myocardial infarction. We sought to determine whether lowering RBP4 would improve blood pressure (BP) and protect against obesity- or angiotensin (Ang)-II-induced hypertension. Systolic and diastolic BP were lower in the RBP4-knockout (RBP4-KO) mice and higher in the RBP4-overexpressing (RBP4-Tg) mice compared with BP in the wild-type (WT) littermates. Carbachol-induced vasodilatation was increased in arteries from the RBP4-KO compared with the WT mice and was impaired in the RBP4-Tg mice. Aortic eNOS(Ser1177) phosphorylation was enhanced ∼50% in the RBP4-KO mice, with no change in total eNOS protein. Feeding a high-fat diet increased BP in the RBP4-KO mice only to the level in the WT mice fed chow and had no effect on aortic eNOS(Ser1177) phosphorylation. Ang-II infusion resulted in 22 mmHg lower systolic BP in the RBP4-KO than in the WT mice, although the relative BP increase over saline infusion was ∼30% in both. Ang-II treatment decreased aortic eNOS(Ser1177) phosphorylation in the WT and RBP4-KO mice, but phosphorylation remained higher in the RBP4-KO mice. Cardiac hypertrophy with Ang-II treatment was diminished by 56% in the RBP4-KO mice. Thus, elevated serum RBP4 raises BP and lack of RBP4 reduces it, with commensurate changes in aortic eNOS(Ser1177) phosphorylation. Lowering RBP4 may reduce BP through enhanced eNOS-mediated vasodilatation and may be a novel therapeutic approach for hypertension.

Regular aerobic exercise protects against impaired fasting plasma glucose-associated vascular endothelial dysfunction with aging.

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.

Quantitative measurement of full-length and C-terminal proteolyzed RBP4 in serum of normal and insulin-resistant humans using a novel mass spectrometry immunoassay.

Serum retinol-binding protein 4 (RBP4) levels are increased in insulin-resistant humans and correlate with severity of insulin resistance in metabolic syndrome. Quantitative Western blotting (qWestern) has been the most accurate method for serum RBP4 measurements, but qWestern is technically complex and labor intensive. The lack of a reliable, high-throughput method for RBP4 measurements has resulted in variability in findings in insulin-resistant humans. Many commonly used ELISAs have limited dynamic range. Neither the current ELISAs nor qWestern distinguish among full-length and carboxyl terminus proteolyzed forms of circulating RBP4 that are altered in different medical conditions. Here, we report the development of a novel quantitative mass spectrometry immunoaffinity assay (qMSIA) to measure full-length and proteolyzed forms of RBP4. qMSIA and qWestern of RBP4 were performed in identical serum aliquots from insulin-sensitive/normoglycemic or insulin-resistant humans with impaired glucose tolerance or type 2 diabetes. Total RBP4 qMSIA measurements were highly similar to qWestern and correlated equally well with clinical severity of insulin resistance (assessed by clamp glucose disposal rate, r = -0.74), hemoglobin A1c (r = 0.63), triglyceride/high-density lipoprotein (r = 0.55), waist/hip (r = 0.61), and systolic blood pressure (r = 0.53, all P < 0.001). Proteolyzed forms of RBP4 accounted for up to 50% of total RBP4 in insulin-resistant subjects, and des(Leu)-RBP4 (cleavage of last leucine) correlated highly with insulin resistance (assessed by glucose disposal rate, r = -0.69). In multiple regression analysis, insulin resistance but not glomerular filtration rate was the strongest, independent predictor of serum RBP4 levels. Thus, qMSIA provides a novel tool for accurately measuring serum RBP4 levels as a biomarker for severity of insulin resistance and risk for type 2 diabetes and metabolic syndrome.

Habitually exercising older men do not demonstrate age-associated vascular endothelial oxidative stress.

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 years) had higher (P<0.05) physical activity (79±7 vs. 30±6 MET hours per week) and maximal exercise oxygen consumption (42±1 vs. 29±1 mL kg(-1) per minute) vs. sedentary men (n=28, 63±1 years). Brachial artery flow-mediated dilation (FMD), 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 years, 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 (p47(phox) 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(-1) per minute) 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.

Sex-specific effects of habitual aerobic exercise on brachial artery flow-mediated dilation in middle-aged and older adults.

Brachial artery FMD (flow-mediated dilation) is impaired with aging and is associated with an increased risk of CVD (cardiovascular disease). In the present study, we determined whether regular aerobic exercise improves brachial artery FMD in MA/O (middle-aged/older) men and post-menopausal women. In sedentary MA/O adults (age, 55-79 years) without CVD, 8 weeks of brisk walking (6 days/week for approx. 50 min/day; randomized controlled design) increased treadmill time approx. 20% in both MA/O men (n=11) and post-menopausal women (n=15) (P<0.01), without altering body composition or circulating CVD risk factors. Brachial artery FMD increased >50% in the MA/O men (from 4.6±0.6 to 7.1±0.6%; P<0.01), but did not change in the post-menopausal women (5.1±0.8 compared with 5.4±0.7%; P=0.50). No changes occurred in the non-exercising controls. In a separate cross-sectional study (n=167), brachial artery FMD was approx. 50% greater in endurance-exercise-trained (6.4±0.4%; n=45) compared with sedentary (4.3±0.3%; n=60) MA/O men (P<0.001), whereas there were no differences between endurance-trained (5.3±0.7%, n=20) and sedentary (5.6±0.5%, n=42) post-menopausal women (P=0.70). Brachial artery lumen diameter, peak hyperaemic shear rate and endothelium-independent dilation did not differ with exercise intervention or in the endurance exercise compared with sedentary groups. In conclusion, regular aerobic exercise is consistently associated with enhanced brachial artery FMD in MA/O men, but not in post-menopausal women. Some post-menopausal women without CVD may be less responsive to habitual aerobic exercise than MA/O men.

Vascular endothelial dysfunction with aging: endothelin-1 and endothelial nitric oxide synthase.

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.

Modulation of vascular endothelial function by low-density lipoprotein cholesterol with aging: influence of habitual exercise.

BACKGROUND: Aging is associated with reduced endothelium-dependent dilation (EDD) and increased risk for cardiovascular disease (CVD), but the mechanisms are incompletely understood. Clinically elevated plasma low-density lipoprotein cholesterol (LDL-C) is associated with impaired EDD. The purpose of this study was to determine whether circulating LDL-C within the "normal" range modulates EDD in healthy older adults and whether young age or habitual aerobic exercise protects against this adverse effect. METHODS: In 83 healthy men with optimal/near optimal LDL-C (<130 mg/dl) or borderline high LDL-C (130-159 mg/dl), EDD (brachial artery flow-mediated dilation, FMD), and endothelium-independent dilation (sublingual glyceryl trinitrate, GTN) were assessed. RESULTS: FMD was 35% lower in older nonexercising men with borderline high LDL-C vs. optimal/near optimal LDL-C (3.1 +/- 0.5 vs. 4.8 +/- 0.4%Delta, P < 0.05), whereas the GTN response did not differ (P = 0.86). In contrast, FMD was similar between groups of young nonexercising men and between groups of older exercising men differing in LDL-C (P = 0.89-0.95). FMD was inversely related to LDL-C among the older nonexercising men (r = -0.43, P < 0.001), whereas there was no relation in the other groups (P > 0.05). CONCLUSIONS: Borderline high plasma LDL-C is associated with impaired EDD in older sedentary men, but not in young sedentary or older exercising men. Thus, modest elevations in plasma LDL-C within the normal range may contribute to the increased risk of CVD in sedentary older men by exacerbating vascular endothelial dysfunction, whereas resistance to this adverse influence may help explain the enhanced endothelial function and reduced CVD risk associated with young age and regular aerobic exercise.

Cytochrome P-450 2C9 signaling does not contribute to age-associated vascular endothelial dysfunction in humans.

Oxidative stress impairs endothelium-dependent dilation (EDD) with aging in healthy sedentary adults. Increased cytochrome P-450 2C9 (CYP 2C9) signaling can contribute to oxidative stress-mediated suppression of EDD, but its role in aging is unknown. We hypothesized that inhibition of CYP 2C9 signaling with sulfaphenazole would improve EDD in older, but not young, healthy sedentary adults. At baseline, increases in forearm blood flow (FBF; venous occlusion plethysmography) in response to brachial artery infusions of ACh (1, 2, 4, and 8 microg.100 ml forearm volume(-1).min(-1)), an endothelium-dependent dilator, were smaller in older [n = 14, 63 +/- 1 (SE) yr] than in young (n = 11, 23 +/- 2 yr) adults (P < 0.05), with a reduction in peak FBF of 32% (11.8 +/- 1.7 vs. 17.3 +/- 2.3 ml.100 ml tissue(-1).min(-1)). Infusion of sulfaphenazole at doses that block CYP 2C9 signaling in humans did not affect the FBF responses to ACh in the older (peak FBF = 13.0 +/- 4.3 ml.100 ml tissue(-1).min(-1), P = 0.41) or the young (peak FBF = 17.1 +/- 1.9 ml.100 ml tissue(-1).min(-1), P = 0.55) adults. Coadministration of the nitric oxide inhibitor l-NMMA and sulfaphenazole decreased the FBF response to ACh in young and older subjects (P < 0.05); the effect was smaller in the older subjects, but group differences in EDD remained (P < 0.05). Endothelium-independent dilation assessed with sodium nitroprusside was not different in the young and older subjects. These results provide the first support for the concept that increased CYP 2C9 signaling does not contribute to impairments in EDD with aging in healthy adults.

High-dose ascorbic acid infusion abolishes chronic vasoconstriction and restores resting leg blood flow in healthy older men.

Resting whole leg blood flow and vascular conductance decrease linearly with advancing age in healthy adult men. The potential role of age-related increases in oxidative stress in these changes is unknown. Resting leg blood flow during saline and ascorbic acid infusion was studied in 10 young (25 +/- 1 yr) and 11 older (63 +/- 2 yr) healthy normotensive men. Plasma oxidized LDL, a marker of oxidative stress, was greater in the older men (P < 0.05). Absolute resting femoral artery blood flow at baseline (iv saline control infusion) was 25% lower in the older men (238 +/- 25 vs. 316 +/- 38 ml/min; P < 0.05), and it was inversely related to plasma oxidized LDL (r = -0.56, P < 0.01) in all subjects. Infusion of supraphysiological concentrations of ascorbic acid increased femoral artery blood flow by 37% in the older men (to 327 +/- 52 ml/min; P < 0.05), but not in the young men (352 +/- 41 ml/min; P = 0.28), thus abolishing group differences (P = 0.72). Mean arterial blood pressure was greater in the older men at baseline (86 +/- 4 vs. 78 +/- 2 mmHg; P < 0.05), but it was unaffected by ascorbic acid infusion (P >/= 0.70). As a result, the lower baseline femoral artery blood flow in the older men was mediated solely by a 32% lower femoral artery vascular conductance (P < 0.05). Baseline femoral vascular conductance also was inversely related to plasma oxidized LDL (r = -0.65, P < 0.01). Ascorbic acid increased femoral vascular conductance by 36% in the older men (P < 0.05) but not in the young men (P = 0.31). In conclusion, ascorbic acid infused at concentrations known to scavenge reactive oxygen species restores resting femoral artery blood flow in healthy older adult men by increasing vascular conductance. These results support the hypothesis that oxidative stress plays a major role in the reduced resting whole leg blood flow and increased leg vasoconstriction observed with aging in men.

Direct evidence of endothelial oxidative stress with aging in humans: relation to impaired endothelium-dependent dilation and upregulation of nuclear factor-kappaB.

Aging is associated with impaired vascular endothelial function, as indicated in part by reduced endothelium-dependent 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 (23+/-1 years) and 44 older (63+/-1 years). EDD (brachial artery flow-mediated dilation) was approximately 50% lower in older versus young men (3.9+/-0.3% versus 7.6+/-0.3%, P<0.01; n=42 older/n=51 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 (1.25+/-0.12 versus 0.61+/-0.11 nitrotyrosine intensity/human umbilical vein EC [HUVEC] intensity, P=0.01; n=11 older/n=11 young) and antecubital veins (0.55+/-0.04 versus 0.34+/-0.03, P<0.05; n=19 older/n=17 young) of older men. Flow-mediated dilation was inversely related to arterial EC nitrotyrosine expression (r=-0.62, P=0.01; n=22). In venous samples, EC expression of the oxidant enzyme NAD(P)H oxidase-p47(phox) was higher in older men (0.71+/-0.05 versus 0.57+/-0.05 NAD[P]H oxidase-p47(phox) intensity/HUVEC intensity, P<0.05; n=19 older/n=18 young), whereas xanthine oxidase and the antioxidant enzymes cytosolic and mitochondrial superoxide dismutase and catalase were not different between groups. Nuclear factor-kappaB p65, a component of the redox-sensitive nuclear transcription factor nuclear factor-kappaB, was elevated in both arterial (0.73+/-0.07 versus 0.53+/-0.05 NF-kappaB p65 intensity/HUVEC intensity, P<0.05; n=9 older/n=12 young) and venous (0.65+/-0.07 versus 0.34+/-0.05, P<0.01; n=13 older/n=15 young) EC samples of older men and correlated with nitrotyrosine expression (r=0.51, P<0.05 n=16). 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-kappaB may contribute to endothelial oxidative stress with aging in humans.

Overweight and obese humans demonstrate increased vascular endothelial NAD(P)H oxidase-p47(phox) expression and evidence of endothelial oxidative stress.

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/m2; 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 P < 0.05) and the antioxidant enzyme catalase (r(part) = 0.71 to 0.75, all P < 0.001). Total body fat was positively associated with VECPE of nitrotyrosine (r(part) = 0.36, P = 0.003), a marker of protein oxidation, and, in men, with Ser1177-phosphorylated endothelial nitric oxide synthase (r(part) = 0.46, P = 0.02), an activated form of endothelial nitric oxide synthase. Overweight/obese subjects (body mass index > or = 25 kg/m2) had 35% to 130% higher VECPE of NAD(P)H oxidase-p47(phox), nitrotyrosine, catalase, and the cytosolic antioxidant CuZn superoxide dismutase (all P < 0.05), as well as a 56% greater VECPE of the potent local vasoconstrictor endothelin-1 (P = 0.05) than normal-weight subjects (body mass index < 25 kg/m2). Nuclear factor-kappaB protein expression was approximately 60% to 100% greater in the most obese adults than in the leanest adults (P < or = 0.01). 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-kappaB 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.

Modulatory influences on ageing of the vasculature in healthy humans.

Increased arterial stiffness and impaired vascular endothelial function are the two most clinically important events that occur with vascular ageing in humans. Together they contribute to age-associated increases in systolic hypertension, left ventricular remodeling and diastolic dysfunction, coronary artery and other atherosclerotic vascular diseases, congestive heart failure, and the attendant cardiac events such as myocardial infarction. However, there is marked individual variability in arterial stiffness and endothelial function with advancing age, which suggests modulation by one or, more likely, several biological and/or lifestyle factors. Consistent with this idea, habitual aerobic exercise appears to attenuate or completely prevent these adverse changes. Other factors including sex hormone status, circulating total and low-density lipoprotein-cholesterol levels, total body and abdominal fatness, and dietary sodium intake also appear to influence arterial stiffening and endothelial dysfunction with ageing. It is now clear that a number of physiological factors and lifestyle behaviors collectively determine how much and, perhaps in some cases, if functionally or clinically significant vascular ageing occurs in adult humans. Of these, the existing evidence indicates that habitual aerobic exercise may be the single most important modulatory influence.

Xanthine oxidase does not contribute to impaired peripheral conduit artery endothelium-dependent dilatation with ageing.

Vascular oxidative stress is the key mechanism involved in the age-related decline in endothelium-dependent dilatation (EDD). We tested the hypothesis that xanthine oxidase (XO), a major vascular source of reactive oxygen species, contributes to the impairment in EDD with ageing. At baseline, brachial artery flow-mediated dilatation (FMD) was 55% lower in older (n = 9, 64 +/- 2 years, 8M/1F, mean +/- S.E.M.) versus young (n = 9, 26 +/- 1 years, 8M/1F) healthy adults (3.41 +/- 0.44 versus 7.53 +/- 0.67%, P < 0.001), whereas endothelium-independent dilatation (EID; sublingual nitroglycerin) did not differ between groups. Plasma oxidized low-density lipoprotein (oxi-LDL), a measure of systemic oxidative stress, was greater at baseline in the older subjects (58.3 +/- 5.9 versus 46.8 +/- 2.4 U l(-1), P < 0.05) and inversely correlated with baseline FMD (r = - 0.54; P < 0.05). Acute administration of allopurinol, a competitive inhibitor of XO, reduced plasma uric acid concentrations similarly in both groups (P < 0.001), but did not affect FMD, EID, or oxi-LDL in either group. Vascular endothelial protein expression of XO (immunofluorescence) was not different in antecubital venous cells from the young and older subjects (0.56 +/- 0.12 versus 0.68 +/- 0.19 XO intensity/human umbilical vein endothelial cell intensity, P = 0.49). We conclude that XO does not contribute to oxidative stress-associated reductions in peripheral conduit artery EDD with ageing in humans, possibly due to an absence of age-associated up-regulation of endothelial XO.

Tetrahydrobiopterin augments endothelium-dependent dilatation in sedentary but not in habitually exercising older adults.

Endothelium-dependent dilatation (EDD) is impaired with ageing in sedentary, but not in regularly exercising adults. We tested the hypotheses that differences in tetrahydrobiopterin (BH(4)) bioactivity are key mechanisms explaining the impairment in EDD with sedentary ageing, and the maintenance of EDD with ageing in regularly exercising adults. Brachial artery flow-mediated dilatation (FMD), normalized for local shear stress, was measured after acute oral placebo or BH(4) in young sedentary (YS) (n = 10; 22 +/- 1 years, mean +/- s.e.m.), older sedentary (OS) (n = 9; 62 +/- 2), and older habitually aerobically trained (OT) (n = 12; 66 +/- 1) healthy men. At baseline, FMD was approximately 50% lower in OS versus YS (1.12 +/- 0.09 versus 0.57 +/- 0.09 (Deltamm (dyn cm(-2))) x 10(-2), P < 0.001; 1 dyn = 10(-5) N), but was preserved in OT (0.93 +/- 0.08 (Deltamm (dyn cm(-2))) x 10(-2)). BH4 administration improved FMD by approximately 45% in OS (1.00 +/- 0.10 (Deltamm (dyn cm(-2))) x 10(-2), P < 0.01 versus baseline), but did not affect FMD in YS or OT. Endothelium-independent dilatation neither differed between groups at baseline nor changed with BH4 administration. These results suggest that BH4 bioactivity may be a key mechanism involved in the impairment of conduit artery EDD with sedentary ageing, and the EDD-preserving effect of habitual exercise.

Ascorbic acid does not affect large elastic artery compliance or central blood pressure in young and older men.

Large elastic artery compliance is reduced and arterial blood pressure (BP) is increased in the central (cardiothoracic) circulation with aging. Reactive oxygen species may tonically modulate central arterial compliance and BP in humans, and oxidative stress may contribute to adverse changes with aging. If so, antioxidant administration may have beneficial effects. Young (Y; 26 +/- 1 yr, mean +/- SE) and older (O; 63 +/- 2 yr, mean +/- SE) healthy men were studied at baseline and during acute (intravenous infusion; Y: n = 13, O: n = 12) and chronic (500 mg/day for 30 days; Y: n = 10, O: n = 10) administration of ascorbic acid (vitamin C). At baseline, peripheral (brachial artery) BP did not differ in the two groups, but carotid artery compliance was 43% lower (1.2 +/- 0.1 vs. 2.1 +/- 0.1 mm(2)/mmHg x 10(-1), P < 0.01) and central (carotid) BP (systolic: 116 +/- 5 vs. 101 +/- 3 mmHg, P < 0.05, and pulse pressure: 43 +/- 4 vs. 36 +/- 3 mmHg, P = 0.16), carotid augmentation index (AIx; 27.8 +/- 7.8 vs. -20.0 +/- 6.6%, P < 0.001), and aortic pulse wave velocity (PWV; 950 +/- 88 vs. 640 +/- 38 cm/s, P < 0.01) were higher in the older men. Plasma ascorbic acid concentrations did not differ at baseline (Y: 71 +/- 5 vs. O: 61 +/- 7 micromol/l, P = 0.23), increased (P < 0.001) to supraphysiological levels during infusion (Y: 1240 +/- 57 and O: 1,056 +/- 83 micromol/l), and were slightly elevated (P < 0.001 vs. baseline) with supplementation (Y: 96 +/- 5 micromol/l vs. O: 85 +/- 6). Neither ascorbic acid infusion nor supplementation affected peripheral BP, heart rate, carotid artery compliance, central BP, carotid AIx, or aortic PWV (all P > 0.26). These results indicate that the adverse changes in large elastic artery compliance and central BP with aging in healthy men are not 1). mediated by ascorbic acid-sensitive oxidative stress (infusion experiments) and 2). affected by short-term, moderate daily ascorbic acid (vitamin C) supplementation.

Effect of acute and chronic ascorbic acid on flow-mediated dilatation with sedentary and physically active human ageing.

Peripheral conduit artery flow-mediated dilatation decreases with ageing in humans. The underlying mechanisms and efficacy of preventive strategies are unknown. Brachial artery flow-mediated dilatation was determined at baseline and after ascorbic acid (vitamin C) intravenous infusion and chronic supplementation (500 mg day(-1) for 30 days) in three groups of healthy men: young sedentary (n= 11; 25 +/- 1 years, mean +/-s.e.m.), older sedentary (n= 9; 64 +/- 2), and older endurance-exercise trained (n= 9; 64 +/- 2). At baseline, flow-mediated dilatation (normalized for the hyperaemic stimulus) was approximately 45% lower in the older (0.015 +/- 0.001) versus young (0.028 +/- 0.004) sedentary men (P < 0.01), but was preserved in older exercising men (0.028 +/- 0.004). Ascorbic acid infusion increased plasma concentrations > 15-fold in all groups and restored flow-mediated dilatation in the sedentary older men (to 0.023 +/- 0.002; P > 0.1 versus other groups), with no effects in the other two groups. Oral ascorbic acid supplementation did not affect flow-mediated dilatation in any group. Brachial artery endothelium-independent dilatation (sublingual nitroglycerin) did not differ among the groups at baseline nor change with ascorbic acid administration. These results provide the first evidence for an important role of oxidative stress in both the impairment in peripheral conduit artery flow-mediated dilatation with sedentary human ageing and the preservation of flow-mediated dilatation with physically active ageing.

Ascorbic acid increases cardiovagal baroreflex sensitivity in healthy older men.

Cardiovagal baroreflex sensitivity (BRS) declines with advancing age in healthy men. We tested the hypothesis that oxidative stress contributes mechanistically to this age-associated reduction. Eight young (23 +/- 1 yrs, means +/- SE) and seven older (63 +/- 3) healthy men were studied. Cardiovagal BRS was assessed using the modified Oxford technique (bolus infusion of 50-100 microg sodium nitroprusside, followed 60 s later by a 100- to 150-microg bolus of phenylephrine hydrochloride) in triplicate at baseline and during acute intravenous ascorbic acid infusion. At baseline, cardiovagal BRS (slope of the linear portion of the R-R interval-systolic blood pressure relation during pharmacological changes in arterial blood pressure) was 56% lower (P < 0.01) in older (8.3 +/- 1.6 ms/mmHg) compared with young (19.0 +/- 3.1 ms/mmHg) men. Ascorbic acid infusion increased plasma concentrations similarly in young (62 +/- 9 vs. 1,249 +/- 72 micromol/l for baseline and during ascorbic acid; P < 0.05) and older men (62 +/- 4 vs. 1,022 +/- 55 micromol/l; P < 0.05) without affecting baseline blood pressure, heart rate, carotid artery compliance, or the magnitude of change in systolic blood pressure in response to bolus sodium nitroprusside and phenylephrine hydrochloride infusion. Ascorbic acid (vitamin C) infusion increased cardiovagal BRS in older (Delta58 +/- 16%; P < 0.01), but not younger (Delta - 4 +/- 4%) men. These data provide experimental support for the concept that oxidative stress contributes mechanistically to age-associated reductions in cardiovagal BRS in healthy men.

Declines in physiological functional capacity with age: a longitudinal study in peak swimming performance.

We followed up swimming performance times of 321 women and 319 men who participated in the US Masters Swimming Championships over a 12-yr period. All swimmers placed in the top 10 in their age group over 3 yr (mean = 5 yr). A random coefficients model for repeated measures was used to derive a line of best fit from a group of regression lines for each subject. Both 50- and 1,500-m swimming performance declined modestly until approximately 70 yr of age, where a more rapid decline was observed in both men and women. Compared with 1,500-m swimming, the 50-m freestyle declined more modestly and slowly with age. The rate and magnitude of declines in swimming performance with age were greater in women than in men in 50-m freestyle; such sex-related differences were not observed in 1,500-m freestyle. Overall, the variability along a population regression line increased markedly with advancing age. The present longitudinal findings indicate that 1) swimming performance declines progressively until age 70, where the decrease becomes quadratic; 2) the rates of the decline in swimming performance with age are greater in a long-duration than in a short-duration event, suggesting a relatively smaller loss of anaerobic muscular power with age compared with cardiovascular endurance; 3) the age-related rates of decline are greater in women than in men only in a short-duration event; and 4) the variability of the age-related decline in performance increases markedly with advancing age.

Changes in maximal aerobic capacity with age in endurance-trained women: 7-yr follow-up.

On the basis of cross-sectional data, we previously reported that the absolute, but not the relative (%), rate of decline in maximal oxygen consumption (VO(2 max)) with age is greater in endurance-trained compared with healthy sedentary women. We tested this hypothesis by using a longitudinal approach. Eight sedentary (63 +/- 2 yr at follow-up) and 16 endurance-trained (57 +/- 2) women were reevaluated after a mean follow-up period of 7 yr. At baseline, VO(2 max) was ~70% higher in endurance-trained women (48.1 +/- 1.7 vs. 28.1 +/- 0.8 ml. kg(-1). min(-1). yr(-1)). At follow-up, body mass, fat-free mass, maximal respiratory exchange ratio, and maximal rating of perceived exertion were not different from baseline in either group. The absolute rate of decline in VO(2 max) was twice as great (P < 0.01) in the endurance-trained (-0.84 +/- 0.15 ml. kg(-1). min(-1). yr(-1)) vs. sedentary (-0.40 +/- 0.12 ml. kg(-1). min(-1). yr(-1)) group, but the relative rates of decline were not different (-1.8 +/- 0.3 vs. -1.5 +/- 0.4% per year). Differences in rates of decline in VO(2 max) were not related to changes in body mass or maximal heart rate. However, among endurance-trained women, the relative rate of decline in VO(2 max) was positively related to reductions in training volume (r = 0.63). Consistent with this, the age-related reduction in VO(2 max) in a subgroup of endurance-trained women who maintained or increased training volume was not different from that of sedentary women. These longitudinal data indicate that the greater decrease in maximal aerobic capacity with advancing age observed in middle-aged and older endurance-trained women in general compared with their sedentary peers is due to declines in habitual exercise in some endurance-trained women. Endurance-trained women who maintain or increase training volume demonstrated age-associated declines in maximal aerobic capacity not different from healthy sedentary women.