Iratxe Eskurza

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.

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 ∼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.

Regular endurance exercise induces expansive arterial remodelling in the trained limbs of healthy men

1 In experimental animals chronic elevations in arterial blood flow increase the lumen diameter and reduce the intima‐media thickness (IMT) of the arterial segment involved. We determined whether intermittent elevations in active muscle blood flow associated with regular aerobic leg exercise induced such expansive arterial remodelling in the common femoral artery of humans. 2 In the cross‐sectional study 53 sedentary (47 ± 2 years) and 55 endurance exercise‐trained (47 ± 2 years) men were studied. Common femoral artery lumen diameter (B‐mode ultrasound) was 7 % greater (9.62 ± 0.12 vs. 9.03 ± 0.13 mm), and femoral IMT (0.46 ± 0.02 vs. 0.55 ± 0.02 mm) and IMT/lumen ratio were 16‐21 % smaller in the endurance‐trained men (all P < 0.001). Basal femoral artery blood flow (duplex ultrasound) was not different, shear stress tended to be lower (P = 0.08), and mean femoral tangential wall stress was 30 % higher in the endurance‐trained men (P < 0.001). 3 In the intervention study 22 men (51 ± 2 years) were studied before and after 3 months of regular aerobic leg exercise (primarily walking). After training, the femoral diameter increased by 9 % (8.82 ± 0.18 vs. 9.60 ± 0.20 mm), and IMT (0.65 ± 0.05 vs. 0.56 ± 0.05 mm) and the IMT/lumen ratio were ≈15‐20 % smaller (all P < 0.001). Basal femoral blood flow and shear stress were not different after training, whereas the mean femoral tangential wall stress increased by 31 %. The changes in arterial structure were not related to changes in risk factors for atherosclerosis. 4 Our results are consistent with the concept that regular aerobic leg exercise induces expansive arterial remodelling in the femoral artery of healthy men. This adaptive process is produced by even a moderate training stimulus, is not obviously dependent on corresponding improvements in risk factors for atherosclerosis, and is robust, occurring in healthy men of different ages.

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.

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 (BH4) 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 BH4 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 ∼50% lower in OS versus YS (1.12 ± 0.09 versus 0.57 ± 0.09 (Δmm (dyn cm−2)) × 10−2, P < 0.001; 1 dyn = 10−5 N), but was preserved in OT (0.93 ± 0.08 (Δmm (dyn cm−2)) × 10−2). BH4 administration improved FMD by ∼45% in OS (1.00 ± 0.10 (Δmm (dyn cm−2)) × 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.

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.

Pharmacologic versus flow-mediated assessments of peripheral vascular endothelial vasodilatory function in humans

We have a long-standing interest in the effects of primary aging on cardiovascular function, including vascular endothelial control. In clinical investigations of humans, 2 approaches primarily have been used to study peripheral vascular endothelial vasodilatory responsiveness: intrabrachial artery infusion of endothelium-dependent vasodilators such as acetylcholine with measurement of whole forearm blood flow using venous occlusion plethysmography, and ultrasound-derived brachial artery dilation following sustained occlusion of blood flow to the forearm. Recently, we noticed that the results of previous investigations using these 2 techniques reported different findings related to the effects of aging. Specifically, advancing age was strongly and inversely related to vasodilatory capacity (r = −0.86) as determined by acetylcholine infusion, whereas no such correlation with age was observed (r = −0.11) using brachial artery reactive hyperemia. These discordant findings suggested that these 2 procedures may not be providing similar information regarding the ability of the vascular endothelium to produce vasodilation upon stimulation among individual subjects and/or populations. Accordingly, the aim of the present study was to determine the relation between peripheral vascular endothelial vasodilatory responsiveness assessed by these 2 techniques.

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.

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.

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.