Phillip E. Gates

Direct Evidence of Endothelial Oxidative Stress With Aging in Humans: Relation to Impaired Endothelium-Dependent Dilation and Upregulation of Nuclear Factor-κB

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 ≈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-p47phox was higher in older men (0.71±0.05 versus 0.57±0.05 NAD[P]H oxidase-p47phox 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-&kgr;B p65, a component of the redox-sensitive nuclear transcription factor nuclear factor-&kgr;B, was elevated in both arterial (0.73±0.07 versus 0.53±0.05 NF-&kgr;B 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-&kgr;B may contribute to endothelial oxidative stress with aging in humans.

Dietary Sodium Restriction Rapidly Improves Large Elastic Artery Compliance in Older Adults With Systolic Hypertension

We determined the temporal effects of dietary sodium restriction on large elastic artery compliance and systolic blood pressure (SBP) in 12 untreated, older (64±2 years) men and women (6 each) with stage 1 systolic hypertension. After baseline measurements subjects were assigned to 4 weeks of low or normal sodium intake (randomized, crossover design). Urinary sodium excretion was reduced by 60% by the end of week 1 of sodium restriction (54±11 mmol/d, P < 0.01) versus baseline (135±14). Compared with baseline (0.11±0.01 mm/mm Hg), carotid artery compliance was increased by 27% (to 0.14±0.02, P < 0.05) at the end of week 1 of sodium restriction, attaining peak levels by week 2 (+46%, to 0.16±0.02, P < 0.01). Similarly, supine resting brachial artery SBP was reduced by >5 mm Hg by week 1 of sodium restriction, attaining peak reductions by week 2 (−12 mm Hg, P < 0.01 versus baseline). The 24-hour ambulatory SBP was ≈3 mm Hg lower at week 1 of sodium restriction and ≈6 mm Hg lower by week 2 (P < 0.01 versus baseline). The reductions in resting SBP from baseline to week 2 of sodium restriction were strongly related to the corresponding increases in carotid compliance (r = 0.80, P < 0.01). Urinary sodium excretion, carotid artery compliance, and SBP were not different during normal sodium intake versus baseline. Other subject characteristics were not different across conditions. Sodium restriction rapidly improves large elastic artery compliance in older adults with stage 1 systolic hypertension. These improvements in central arterial compliance appear to be a key mechanism in the rapid normalization of SBP by sodium restriction in these patients.

Greater Age-Related Reductions in Central Arterial Compliance in Resistance-Trained Men

Reductions in the compliance of central arteries exert a number of adverse effects on systemic cardiovascular function and disease risk. Using the cross-sectional study design, we determined the relation between chronic resistance training and carotid arterial compliance. A total of 62 healthy normotensive men, 20 to 39 years of age (young) and 40 to 60 years of age (middle-aged), who were either sedentary or resistance-trained, were studied. In both activity groups, carotid arterial compliance (simultaneous ultrasound and applanation tonometry) was lower ( P P P r =−0.35; P

Overweight and Obese Humans Demonstrate Increased Vascular Endothelial NAD(P)H Oxidase-p47phox 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-p47phox (part correlation coefficient [rpart] 0.22 to 0.24, all P<0.05) and the antioxidant enzyme catalase (rpart=0.71 to 0.75, all P<0.001). Total body fat was positively associated with VECPE of nitrotyrosine (rpart=0.36, P=0.003), a marker of protein oxidation, and, in men, with Ser1177-phosphorylated endothelial nitric oxide synthase (rpart=0.46, P=0.02), an activated form of endothelial nitric oxide synthase. Overweight/obese subjects (body mass index ≥25 kg/m2) had 35% to 130% higher VECPE of NAD(P)H oxidase-p47phox, 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-&kgr;B protein expression was ≈60% to 100% greater in the most obese adults than in the leanest adults (P≤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-p47phox 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-&kgr;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.

Fatness Is a Better Predictor of Cardiovascular Disease Risk Factor Profile Than Aerobic Fitness in Healthy Men

Background—The prevalence of cardiovascular disease (CVD) is partly attributable to an inactive and/or overweight population. However, the independent association of body fatness and aerobic fitness with CVD risk factors is uncertain. We sought to determine whether fatness or fitness better predicted traditional CVD risk factors in men with broad fatness, aerobic fitness, and age ranges using 3 expressions of adiposity. Methods and Results—In 135 carefully screened healthy men, we measured 18 established CVD risk factors, body mass index, total percent body fat, waist circumference, and maximal aerobic capacity. Body mass index, percent body fat, and waist circumference were consistently associated with all metabolic risk factors (r=−0.44 to 0.51, P<0.05) after partialling out the effects of aerobic fitness and age. Body mass index and waist circumference were also independently associated with selective hemodynamic risk factors (r=0.20 to 0.30, P≤0.01). In contrast, aerobic fitness was independently associated with only selective metabolic risk factors (r=−0.21 to 0.19, P<0.05) and was not associated with any hemodynamic risk factors (P>0.05). Both aerobic fitness and body fatness were independently associated with selective hemostatic risk factors (r=−0.22 to −0.26, P≤0.01; r=−0.32 to 0.48, P<0.05, respectively). Overall, fatness was more strongly and consistently associated with CVD risk factors than aerobic fitness. Conclusions—Body fatness is a better predictor of CVD risk factor profile than aerobic fitness in healthy men. Although habitual physical activity is an effective strategy for preventing CVD, elevated body fatness is associated with an adverse CVD risk factor profile independently of aerobic fitness.

Human endothelial function and microvascular ageing

Age is a primary risk factor for cardiovascular disease, and this is an increasingly important public health concern because of an increase in the absolute number and proportion of the population at an older age in many countries. A key component of cardiovascular ageing is reduced function of the vascular endothelium, and this probably contributes to the impaired microvessel function observed with ageing in multiple vascular beds. In turn, impaired microvessel function is thought to contribute to the pathophysiology of cardiovascular and metabolic diseases. Here we review evidence that the first signs of altered endothelial and microvessel function can appear in childhood and at all stages of the human lifespan; low‐birth‐weight babies have reduced endothelial function in skin microvessels at 3 months, and by age 10 years their brachial artery endothelial function is reduced in comparison with normal‐birth‐weight babies. In overweight/obese adolescent children with clustering of traditional cardiovascular disease risk factors, endothelial function is reduced compared with normal‐weight children, and this appears to persist into early adulthood. Adult ageing is associated with impaired microvessel endothelial function and an increase in capillary blood pressure. Biological and lifestyle factors that influence microvessel function include body fat and visceral adiposity, sex hormone status, diet and physical activity. The mechanisms underlying age‐associated changes in microvessel function are uncertain but may involve alterations in nitric oxide, prostanoid, endothelium‐derived hyperpolarizing factor(s) and endothelin‐1 pathways.

IN VITRO AND PRELIMINARY IN VIVO VALIDATION OF ECHO PARTICLE IMAGE VELOCIMETRY IN CAROTID VASCULAR IMAGING

Noninvasive, easy-to-use and accurate measurements of wall shear stress (WSS) in human blood vessels have always been challenging in clinical applications. Echo particle image velocimetry (Echo PIV) has shown promise for clinical measurements of local hemodynamics and wall shear rate. Thus far, however, the method has only been validated under simple flow conditions. In this study, we validated Echo PIV under in vitro and in vivo conditions. For in vitro validation, we used an anatomically correct, compliant carotid bifurcation flow phantom with pulsatile flow conditions, using optical particle image velocimetry (optical PIV) as the reference standard. For in vivo validation, we compared Echo PIV-derived 2-D velocity fields obtained at the carotid bifurcation in five normal subjects against phase-contrast magnetic resonance imaging (PC-MRI)-derived velocity measurements obtained at the same locations. For both studies, time-dependent, 2-D, two-component velocity vectors; peak/centerline velocity, flow rate and wall shear rate (WSR) waveforms at the common carotid artery (CCA), carotid bifurcation and distal internal carotid artery (ICA) were examined. Linear regression, correlation analysis and Bland-Altman analysis were used to quantify the agreement of different waveforms measured by the two techniques. In vitro results showed that Echo PIV produced good images of time-dependent velocity vector maps over the cardiac cycle with excellent temporal (up to 0.7 ms) and spatial (∼0.5 mm) resolutions and quality, comparable with optical PIV results. Further, good agreement was found between Echo PIV and optical PIV results for velocity and WSR measurements. In vivo results also showed good agreement between Echo PIV velocities and phase contrast MRI velocities. We conclude that Echo PIV provides accurate velocity vector and WSR measurements in the carotid bifurcation and has significant potential as a clinical tool for cardiovascular hemodynamics evaluation.

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.

Low dietary sodium intake is associated with enhanced vascular endothelial function in middle-aged and older adults with elevated systolic blood pressure:

Background: Age and increasing systolic blood pressure (BP) are associated with vascular endothelial dysfunction, but the factors involved are incompletely understood. We tested the hypothesis that vascular endothelial function is related to dietary sodium intake among middle-aged and older adults (MA and O) with elevated systolic BP. Methods: Data were analyzed on 25 otherwise healthy adults aged 48—73 years with high normal systolic BP or stage I systolic hypertension (130—159 mmHg). Self-reported sodium intake was <100 mmol/d in 12 (7 M) subjects (low sodium, 73±6 mmol/d) and between 100 and 200 mmol/d in 13 (9 M) subjects (normal sodium, 144±6 mmol/d). Results: Groups did not differ in other dietary factors, age, body weight and composition, BP, metabolic risk factors, physical activity and maximal aerobic capacity. Plasma concentrations of norepinephrine, endothelin-1, oxidized low-density lipoproteins (LDL), antioxidant status and inflammatory markers did not differ between groups. Brachial artery flow-mediated dilation (FMD) was 42% (mm Δ) to 52% (% Δ) higher in the low versus normal sodium group (p < 0.05). In all subjects, brachial artery FMD was inversely related to dietary sodium intake (FMD mm Δr =—0.40, p < 0.05; %Δr =—0.53, p < 0.01). Brachial artery FMD was not related to any other variable. In contrast, endothelium-independent dilation did not differ between groups (p ≥ 0.24) and was not related to sodium intake in the overall group (p ≥ 0.29). Conclusions: Low sodium intake is associated with enhanced brachial artery FMD in MA and O with elevated systolic BP. These results suggest that dietary sodium restriction may be an effective intervention for improving vascular endothelial function in this high-risk group.

Dietary Sodium Restriction and Association with Urinary Marinobufagenin, Blood Pressure, and Aortic Stiffness

BACKGROUND AND OBJECTIVES Systolic BP and large elastic artery stiffness both increase with age and are reduced by dietary sodium restriction. Production of the natriuretic hormone marinobufagenin, an endogenous α1 Na+,K+-ATPase inhibitor, is increased in salt-sensitive hypertension and contributes to the rise in systolic BP during sodium loading. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS The hypothesis was that dietary sodium restriction performed in middle-aged/older adults (eight men and three women; 60 ± 2 years) with moderately elevated systolic BP (139 ± 2/83 ± 2 mmHg) would reduce urinary marinobufagenin excretion as well as systolic BP and aortic pulse-wave velocity (randomized, placebo-controlled, and crossover design). This study also explored the associations among marinobufagenin excretion with systolic BP and aortic pulse-wave velocity across conditions of 5 weeks of a low-sodium (77 ± 9 mmol/d) and 5 weeks of a normal-sodium (144 ± 7 mmol/d) diet. RESULTS Urinary marinobufagenin excretion (weekly measurements; 25.4 ± 1.8 versus 30.7 ± 2.1 pmol/kg per day), systolic BP (127 ± 3 versus 138 ± 5 mmHg), and aortic pulse-wave velocity (700 ± 40 versus 843 ± 36 cm/s) were lower during the low- versus normal-sodium condition (all P<0.05). Across all weeks, marinobufagenin excretion was related with systolic BP (slope=0.61, P<0.001) and sodium excretion (slope=0.46, P<0.001). These associations persisted during the normal- but not the low-sodium condition (both P<0.005). Marinobufagenin excretion also was associated with aortic pulse-wave velocity (slope=0.70, P=0.02) and endothelial cell expression of NAD(P)H oxidase-p47phox (slope=0.64, P=0.006). CONCLUSIONS These results show, for the first time in humans, that dietary sodium restriction reduces urinary marinobufagenin excretion and that urinary marinobufagenin excretion is positively associated with systolic BP, aortic stiffness (aortic pulse-wave velocity), and endothelial cell expression of the oxidant enzyme NAD(P)H oxidase. Importantly, marinobufagenin excretion is positively related to systolic BP over ranges of sodium intake typical of an American diet, extending previous observations in rodents and humans fed experimentally high-sodium diets.