Gary L. Pierce

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.

25-Hydroxyvitamin D Deficiency Is Associated With Inflammation-Linked Vascular Endothelial Dysfunction in Middle-Aged and Older Adults

We tested the hypothesis that vascular endothelial function, assessed by endothelium-dependent dilation, is related to serum vitamin D status among middle-aged and older adults without clinical disease, and that this is linked to inflammation. Brachial artery flow-mediated dilation, a measure of endothelium-dependent dilation, was lower (P<0.01) in vitamin D–insufficient (3.7±0.2%; serum 25-hydroxyvitamin D [25(OH)D]: 20 to 29 ng/mL; 62±1 years of age; n=31; mean± SE) and vitamin D–deficient (3.2±0.3%; 25(OH)D: <20 ng/mL; 63±2 years of age; n=22) versus vitamin D–sufficient (4.6±0.4%; 25(OH)D: >29 ng/mL; 61±1 years of age; n=22) subjects, whereas endothelium-independent dilation (brachial dilation to sublingual nitroglycerine) did not differ (P=0.45). Among all subjects, brachial flow-mediated dilation was positively related to serum 25(OH)D (%&Dgr;: r=0.35; P<0.01) but not 1,25-dihydroxyvitamin D (r=−0.06; P=0.61), the active form of vitamin D. Vascular endothelial cell expression of the proinflammatory transcription factor nuclear factor &kgr;B was greater in deficient versus sufficient subjects (0.59±0.07 versus 0.44±0.05; P<0.05), and inhibition of nuclear factor &kgr;B (4 days oral salsalate) improved flow-mediated dilation to a greater extent in subjects with lower versus higher 25(OH)D (+3.7±0.6 versus +2.0±0.2%; P<0.05). Endothelial cell expression of the downstream proinflammatory cytokine interleukin-6 also was higher in deficient versus sufficient subjects (0.67±0.08 versus 0.47±0.05; P<0.01) and inversely related to serum 25(OH)D level (r=−0.62; P<0.01), whereas vitamin D receptor and 1-&agr; hydroxylase, the 25(OH)D to 1,25-dihydroxyvitamin D converting enzyme, were lower (P<0.05). Inadequate serum 25(OH)D is associated with vascular endothelial dysfunction among healthy middle-aged/older adults, and this is mediated in part by nuclear factor &kgr;B–related inflammation. Reduced vitamin D receptor and 1-&agr; hydroxylase may be molecular mechanisms linking vitamin D insufficiency to endothelial dysfunction.

Nuclear Factor-κB Activation Contributes to Vascular Endothelial Dysfunction via Oxidative Stress in Overweight/Obese Middle-Aged and Older Humans

Background— We tested the hypothesis that nuclear factor-&kgr;B (NF-&kgr;B) activity contributes to vascular endothelial dysfunction with aging and obesity in humans. Methods and Results— We conducted a randomized, double-blind, placebo-controlled crossover study in 14 nondiabetic overweight or obese (body mass index ≥25 kg/m2) middle-aged and older (age 52 to 68 years) adults. Salsalate (nonacetylated salicylate, 4500 mg/d), a compound that inhibits NF-&kgr;B activity, or placebo was administered for 4-day periods. Plasma salicylate concentrations reached the midtherapeutic range (21.8±1.1 mg/100 mL, P≤0.0001 versus placebo) by day 4 of salsalate treatment. Salsalate increased expression of the inhibitor of NF-&kgr;B and reduced total and nuclear expression of NF-&kgr;B in endothelial cells obtained from the subjects (all P<0.05). Salsalate increased brachial artery flow-mediated dilation by 74% (from 4.0±0.4% to 6.6±0.5%, P<0.001) but did not affect endothelium-independent dilation (P=0.83). The change in brachial artery flow-mediated dilation with salsalate was inversely related to baseline flow-mediated dilation (r=−0.77, P<0.01). Infusion of vitamin C increased brachial artery flow-mediated dilation during placebo (P<0.001) but not after salsalate (P=0.23). Salsalate reduced nitrotyrosine (P=0.06) and expression of NADPH oxidase p47phox (P<0.05) in endothelial cells obtained from the subjects but did not influence circulating or endothelial cell inflammatory proteins. Conclusions— Our findings provide the first direct evidence that NF-&kgr;B, in part via stimulation of oxidative stress, plays an important role in mediating vascular endothelial dysfunction in overweight and obese middle-aged and older humans.

Weight Loss Alone Improves Conduit and Resistance Artery Endothelial Function in Young and Older Overweight/Obese Adults

Obesity is associated with vascular endothelial dysfunction, as indicated by impaired endothelium-dependent dilation. Presently there is no direct evidence that energy intake–restricted weight loss alone improves conduit or resistance artery endothelium-dependent dilation, the mechanisms involved, or whether improvements differ with patient age. A total of 40 overweight or obese (body mass index: ≥25<40 kg/m2) nondiabetic men and women aged 21 to 69 years completed 12 weeks of reduced energy intake (n=26; 15 male) or attention control (n=14; 9 male) and 4 weeks of weight maintenance (randomized trial). Energy intake restriction reduced estimated total energy intake (33%), body weight (10.5%), total and abdominal body fat, plasma leptin, oxidized low-density lipoprotein, and improved several metabolic risk factors. Brachial artery flow-mediated dilation was increased by 30% (6.0±0.7% versus 7.9±0.7%&Dgr;; P=0.01; n=17). Peak forearm blood flow during intrabrachial artery infusion of acetylcholine was increased by 26% (16.8±1.4 versus 21.1±1.9 mL/100 mL per minute; P<0.05; n=15); this was inversely related to the reduction in the abdominal visceral:subcutaneous fat ratio (r=−0.46; P<0.05) and was abolished by inhibition of NO synthesis with NG-monomethyl-l-arginine. Improvements in endothelium-dependent dilation were not related to age: mean increases in subjects >50 years of age were similar to or greater than those <50 years of age. Energy intake–restricted weight loss alone is an effective intervention for improving peripheral conduit and resistance artery endothelial function in young and older overweight/obese adults. The improvements in resistance artery function are mediated by an increase in NO bioavailability and are related to reductions in abdominal visceral fat.

Habitual exercise and vascular ageing.

Age is the major risk factor for cardiovascular diseases (CVD) and this is attributable in part to stiffening of large elastic arteries and development of vascular endothelial dysfunction (e.g. impaired endothelium‐dependent dilatation, EDD). In contrast, regular aerobic exercise is associated with reduced risk of CVD. Endurance exercise‐trained middle‐aged/older adults demonstrate lower large elastic artery stiffness and greater EDD than their sedentary peers. With daily brisk walking, previously sedentary middle‐aged/older adults show reduced stiffness and improved EDD. The mechanisms underlying the effects of regular aerobic exercise on large elastic artery stiffness with ageing are largely unknown, but are likely to include changes to the composition of the arterial wall. Enhanced EDD in older adults who exercise is mediated by increased nitric oxide (NO) bioavailability associated with reduced oxidative stress. Arteries from old rodents that perform regular aerobic exercise demonstrate increased expression and activity of endothelial NO synthase, reduced oxidative damage associated with reduced expression and activity of the oxidant enzyme NADPH oxidase, and increased activity of the antioxidant enzyme superoxide dismutase. Aerobic exercise also may protect arteries with ageing by increasing resistance to the effects of other CVD risk factors like LDL‐cholesterol. Habitual aerobic exercise is an effective strategy to combat arterial ageing.

Translational evidence that impaired autophagy contributes to arterial ageing

•  Advancing age is the major risk factor for the development of cardiovascular diseases. •  Arterial endothelial dysfunction, characterized by impaired endothelium‐dependent dilatation (EDD), is a key antecedent to age‐associated clinical cardiovascular disease. •  We tested the hypothesis that changes in autophagy, the process by which cells recycle damaged biomolecules, may be an underlying cause of the age‐related reduction in EDD. •  We show that autophagy is impaired in arteries of older humans and mice with reduced EDD, and that enhancing autophagy restores EDD by reducing superoxide‐dependent oxidative stress and inflammation, and increasing nitric oxide bioavailability. •  Our results identify impaired autophagy as a potential cause of age‐related arterial dysfunction and suggest that boosting autophagy may be a novel strategy for the treatment of arterial endothelial dysfunction and prevention of cardiovascular diseases with ageing.

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 (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−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.

Pulse Wave Analysis of the Aortic Pressure Waveform in Severe Left Ventricular Systolic Dysfunction

Background—The effect of moderate left ventricular systolic dysfunction (LVSD) on ventricular/vascular coupling and the aortic pressure waveform (AoPW) has been well described, but the effect of severe LVSD has not. Methods and Results—We used noninvasive, high-fidelity tonometry of the radial artery and a mathematical transfer function to generate the AoPW in 25 treated patients with LVSD (mean LV ejection fraction, 24±8.8%; range, 11% to 40%; 21 patients <30%). Pulse wave analysis of the AoPW was used to characterize ventricular/vascular coupling and compared with pulse wave analysis performed in 25 normal subjects matched for age, gender, height, body mass index, and heart rate. Measurements obtained using pulse wave analysis in LVSD patients indicated features of poor LV stroke performance and also reduced indices of arterial stiffness: increased travel time of the pressure wave (147±10 ms versus 132±21 ms; P<0.001); decreased systolic duration of reflected wave (134±24 ms versus 167±26 ms; P<0.001); ejection duration (277±22 ms versus 299±25 ms; P<0.008); percent systolic duration (32±5.3% versus 35±4.0%; P<0.02); aortic systolic pressure (100±16 mm Hg versus 121±16 mm Hg; P<0.001); unaugmented pressure (24±6.3 mm Hg versus 32±6.4 mm Hg; P<0.001); augmented pressure (4.8±3.1 mm Hg versus 9.6±4.5 mm Hg; P<0.001); pulse pressure (28±7.4 mm Hg versus 42±9.5 mm Hg; P<0.001); augmentation index (12±6.6% versus 23±7.6%; P<0.006); wasted LV effort (5.3±2.8×102 dyne sec/cm2 versus 17±10×102 dyne sec/cm2; P<0.001); systolic pressure time index (17±4.1×102 mm Hg-sec/min versus 23±4.2×102 mm Hg sec/min; P<0.001); and pressure systolic area (383±121 mm Hg sec/min versus 666±150 mm Hg sec/min; P<0.001). Conclusions—Severe LVSD causes measurable changes in the AoPW. Standardization of AoPW findings in LVSD patients may allow for the clinical use of radial artery pulse wave analysis to noninvasively determine the severity of dysfunction and aid in logical therapy.

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.

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.