Anthony J. Donato

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.

Aging and vascular endothelial function in humans

Advancing age is the major risk factor for the development of CVD (cardiovascular diseases). This is attributable, in part, to the development of vascular endothelial dysfunction, as indicated by reduced peripheral artery EDD (endothelium-dependent dilation) in response to chemical [typically ACh (acetylcholine)] or mechanical (intravascular shear) stimuli. Reduced bioavailability of the endothelium-synthesized dilating molecule NO (nitric oxide) as a result of oxidative stress is the key mechanism mediating reduced EDD with aging. Vascular oxidative stress increases with age as a consequence of greater production of reactive oxygen species (e.g. superoxide) without a compensatory increase in antioxidant defences. Sources of increased superoxide production include up-regulation of the oxidant enzyme NADPH oxidase, uncoupling of the normally NO-producing enzyme, eNOS (endothelial NO synthase) (due to reduced availability of the cofactor tetrahydrobiopterin) and increased mitochondrial synthesis during oxidative phosphorylation. Increased bioactivity of the potent endothelial-derived constricting factor ET-1 (endothelin-1), reduced endothelial production of/responsiveness to dilatory prostaglandins, the development of vascular inflammation, formation of AGEs (advanced glycation end-products), an increased rate of endothelial apoptosis and reduced expression of oestrogen receptor α (in postmenopausal females) also probably contribute to impaired EDD with aging. Several lifestyle and biological factors modulate vascular endothelial function with aging, including regular aerobic exercise, dietary factors (e.g. processed compared with non-processed foods), body weight/fatness, vitamin D status, menopause/oestrogen deficiency and a number of conventional and non-conventional risk factors for CVD. Given the number of older adults now and in the future, more information is needed on effective strategies for the prevention and treatment of vascular endothelial aging.

Habitual exercise and arterial aging

Aging affects the function and structure of arteries and increases the risk of cardiovascular diseases (CVD). In healthy sedentary adults, aging is associated with increased stiffness (reduced compliance) of large elastic arteries; impaired vascular endothelial function, including reductions in endothelium-dependent dilation (EDD), release of tissue-type plasminogen activator (fibrinolytic capacity) and endothelial progenitor cell number and function; increased intima-media wall thickness (IMT); and peripheral vasoconstriction (decreased basal leg blood flow). Habitual physical activity/increased aerobic exercise capacity is associated with reduced risk of CVD. Compared with their sedentary peers, adults who regularly perform aerobic exercise demonstrate smaller or no age-associated increases in large elastic artery stiffness, reductions in vascular endothelial function, and increases in femoral artery IMT. A short-term, moderate-intensity aerobic exercise intervention (brisk daily walking for 12 wk) improves carotid artery compliance and can restore vascular endothelial function in previously sedentary middle-aged and older adults. Reduced oxidative stress may be an important mechanism contributing to these effects. Habitual resistance exercise increases (high-intensity) or does not affect (moderate-intensity) large elastic artery stiffness, and prevents/restores the age-associated reduction in basal leg blood flow independent of changes in leg fat-free mass. Habitual exercise favorably modulates several expressions of arterial aging, thus preserving vascular function and possibly reducing the risk of CVD.

Effects of ageing and exercise training on endothelium‐dependent vasodilatation and structure of rat skeletal muscle arterioles

Ageing reduces endothelium‐dependent vasodilatation in humans and animals, and in humans, exercise training reverses the ageing‐associated reduction in endothelium‐dependent vasodilatation. The purpose of this study was to determine the mechanism(s) by which 10–12 weeks of treadmill exercise enhances endothelium‐dependent vasodilatation in muscles of differing fibre composition from young and old rats. Three‐ and 22‐month‐old male Fischer 344 rats were assigned to young sedentary, young exercise‐trained, old sedentary, or old exercise‐trained groups. Arterioles were isolated from the soleus and gastrocnemius muscles; luminal diameter changes were determined in response to the endothelium‐dependent vasodilator acetylcholine (ACh, 10−9–10−4 mol l−1) alone and in the presence of the nitric oxide synthase (NOS) inhibitor l‐NAME (10−5 mol l−1) or the combination of l‐NAME and the cyclooxygenase inhibitor indomethacin (10−5 mol l−1). Training ameliorated the ageing‐induced reduction in endothelium‐dependent vasodilatation in soleus muscle arterioles. Treatment with l‐NAME alone and in combination with indomethacin abolished differences in ACh vasodilatation occurring with ageing and training. Expression of endothelial NOS (eNOS) mRNA in soleus arterioles was unaltered by ageing, whereas eNOS protein was increased with age; training elevated both eNOS mRNA and protein. In gastrocnemius muscle arterioles, ageing did not alter maximal vasodilatation, but ageing and training increased maximal arteriolar diameter. These results demonstrate that ageing‐induced reductions and training‐induced enhancement of endothelial vasodilatation both occur through the nitric oxide signalling mechanism in highly oxidative skeletal muscle, but ageing and training do not appear to act on the same portion of the signalling cascade.

Regular exercise, hormone replacement therapy and the age-related decline in carotid arterial compliance in healthy women

OBJECTIVE Carotid arterial compliance is reduced with age in sedentary estrogen-deficient women, contributing to the development of cardiovascular disorders. We determined the effects of regular aerobic exercise, hormone replacement therapy (HRT), and their interaction on carotid arterial compliance using a combination of cross-sectional and intervention study designs. METHODS Cross-sectionally, we studied three groups of healthy postmenopausal women (50-80 years): 20 sedentary not taking HRT; 24 sedentary taking HRT; and 14 endurance-trained not taking HRT; and 11 sedentary premenopausal controls (20-37 years). In the intervention study, 12 sedentary postmenopausal women (58+/-3 years) who were taking HRT were studied before and after participation in a 3-month aerobic exercise (walking) program. Carotid arterial compliance was measured via simultaneous common carotid artery ultrasound imaging and applanation tonometry. RESULTS Cross-sectional study. Carotid arterial compliance was lower (P<0.001) in all three postmenopausal groups compared with premenopausal women. Among the postmenopausal groups, arterial compliance was 33-43% higher in the sedentary HRT and endurance-trained women than in their sedentary estrogen-deficient peers. Intervention study. Arterial compliance increased (P<0.05) by approximately 40% to levels that were no longer different than premenopausal women. CONCLUSIONS HRT use and regular aerobic exercise are associated with augmented carotid arterial compliance in healthy postmenopausal women. Moderate, short-term aerobic exercise can restore carotid arterial compliance in previously sedentary postmenopausal women taking HRT.

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.

Voluntary wheel running restores endothelial function in conduit arteries of old mice: direct evidence for reduced oxidative stress, increased superoxide dismutase activity and down‐regulation of NADPH oxidase

Habitual aerobic exercise is associated with enhanced endothelium‐dependent dilatation (EDD) in older humans, possibly by increasing nitric oxide bioavailability and reducing oxidative stress. However, the mechanisms involved are incompletely understood. EDD was measured in young (6–8 months) and old (29–32 months) cage control and voluntary wheel running (VR) B6D2F1 mice. Age‐related reductions in maximal carotid artery EDD to acetylcholine (74 vs. 96%, P < 0.01) and the nitric oxide (NO) component of EDD (maximum dilatation with ACh and l‐NAME minus that with ACh alone was −28%vs.−55%, P < 0.01) were restored in old VR (EDD: 96%, NO: −46%). Nitrotyrosine, a marker of oxidative stress, was increased in aorta with age, but was markedly lower in old VR (P < 0.05). Aortic superoxide dismutase (SOD) activity was greater (P < 0.01), whereas NADPH oxidase protein expression (P < 0.01) and activity (P= 0.05) were lower in old VR vs. old cage control. Increasing SOD (with 4‐hydroxy‐2,2,6,6‐tetramethylpiperidine 1‐oxyl) and inhibition of NADPH oxidase (with apocynin) improved EDD and its NO component in old cage control, but not old VR mice. VR increased endothelial NO synthase (eNOS) protein expression (P < 0.05) and activation (Ser1177 phosphorylation) (P < 0.05) in old mice. VR did not affect EDD in young mice. Our results show that voluntary aerobic exercise restores the age‐associated loss of EDD by suppression of oxidative stress via stimulation of SOD antioxidant activity and inhibition of NADPH oxidase superoxide production. Increased eNOS protein and activation also may contribute to exercise‐mediated preservation of NO bioavailability and EDD with ageing.

Aging is Associated with Greater Nuclear NFκB, Reduced IκBα and Increased Expression of Proinflammatory Cytokines in Vascular Endothelial Cells of Healthy Humans

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

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.

Short-term calorie restriction reverses vascular endothelial dysfunction in old mice by increasing nitric oxide and reducing oxidative stress.

To determine if short‐term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n = 30) and young (Y, n = 10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, approximately 30%) for 8 weeks. Ex vivo carotid artery endothelium‐dependent dilation (EDD) was impaired in old ad libitum (OAL) vs. young ad libitum (YAL) (74 ± 5 vs. 95 ± 2% of maximum dilation, P < 0.05), whereas old calorie‐restricted (OCR) and YCR did not differ (96 ± 1 vs. 94 ± 3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P < 0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P < 0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P < 0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97 ± 2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P < 0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P < 0.05), but unaffected by CR. Carotid artery endothelium‐independent dilation did not differ among groups. Short‐term CR initiated in old age reverses age‐associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NADPH oxidase–mediated superoxide production and stimulation of anti‐oxidant enzyme activity), and upregulation of sirtuin‐1.