Published online before print May 3, 2007, doi: 10.1161/01.RES.0000269183.13937.e8
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© 2007 American Heart Association, Inc.
Clinical Research |
Direct Evidence of Endothelial Oxidative Stress With Aging in Humans
Relation to Impaired Endothelium-Dependent Dilation and Upregulation of Nuclear Factor-
B
From the Department of Integrative Physiology, University of Colorado, Boulder.
Correspondence to Anthony J. Donato, PhD, Department of Integrative Physiology, University of Colorado at Boulder, 354 UCB, Boulder, CO 80309. E-mail tony.donato{at}colorado.edu
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Abstract |
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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-B p65, a component of the redox-sensitive nuclear transcription factor nuclear factor-B, was elevated in both arterial (0.73±0.07 versus 0.53±0.05 NF-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-B may contribute to endothelial oxidative stress with aging in humans.
Key Words: flow-mediated dilation • NAD(P)H oxidase • xanthine oxidase • antioxidants
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Introduction |
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The arterial vascular endothelium plays an essential role in the initiation and progression of cardiovascular disease (CVD).1 Consistent with this, vascular endothelial dysfunction, as reflected by impaired endothelium-dependent dilation (EDD), is observed in patients with CVD and predicts future events in this population.2 Oxidative stress, defined as increased production of reactive oxygen species relative to antioxidant defenses, is believed to play a key role in the development of endothelial dysfunction in the setting of CVD.1
Older age is a major risk factor for the development of CVD.3 EDD becomes impaired with aging in adult humans4–8 and is thought to contribute to this age-associated increase in CVD risk.3 In experimental animals, age-related reductions in EDD are associated with vascular oxidative stress9 and upregulation of systems supporting the production of reactive oxygen species.10–12 In humans, reductions in EDD with aging are inversely related to plasma markers of oxidative stress5 and are reversed by administration of supraphysiological concentrations of vitamin C, a potent antioxidant.7,13 However, direct evidence supporting the development of oxidative stress with aging in the vascular endothelium per se, its relation to impaired EDD, and the associated molecular mechanisms is lacking.
The primary purpose of the present study was to determine if endothelial oxidative stress develops with age in healthy adult humans and if this is related to age-associated reductions in EDD. We also wished to gain insight into the molecular events that are associated with the development of endothelial oxidative stress and reduced EDD with aging in humans. To do so, we measured EDD and obtained endothelial cells (EC) from peripheral arteries and veins of healthy young and older men. Abundance of nitrotyrosine, an oxidatively modified amino acid and "footprint" of oxidative stress,11,14,15 was determined in ECs using quantitative immunofluorescence.16–18 To explore the associated molecular mechanisms, we also measured EC expression of major cellular oxidant enzymes (nicotinamide adenine dinucleotide phosphate oxidase-p47phox, NAD[P]H oxidase-p47phox; xanthine oxidase) and antioxidant enzymes (catalase; cytosolic copper zinc superoxide dismutase [CuZn SOD]; mitochondrial manganese SOD [Mn SOD]) from venous sampling and the important redox-sensitive, proinflammatory transcription factor, nuclear factor (NF)-B, in ECs obtained from both arterial and venous sampling.
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Subjects and Methods |
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Subjects
A total of 95 healthy men, 51 young (aged 18 to 30 years), and 44 older (55 to 78 years) were studied. Details concerning subject inclusion/exclusion criteria are presented in the online data supplement at http://circres.ahajournals.org. All procedures were approved by the Human Research Committee of the University of Colorado at Boulder. The nature, benefits, and risks of the study were explained to the volunteers, and their written informed consent was obtained before participation.
Study Procedures
All measurements were performed at the University of Colorado at Boulder General Clinical Research Center after an overnight fast and a 24-hour abstention from alcohol and physical activity.
Subject Characteristics
Body mass index was measured from height and body mass. Arterial blood pressure and resting heart rate were measured over the brachial artery during supine rest using a semiautomated device (Dynamap XL, Johnson and Johnson). Leisure time and occupational physical activity were estimated and summed to provide a measure of total physical activity as described previously.19 Fasting plasma metabolic factors were determined by standard assays. Plasma samples were analyzed for oxidized low-density lipoprotein, a marker of systemic oxidative stress, and serum samples were analyzed for total antioxidant status, a measure of systemic antioxidant defenses, as previously described.20 Serum concentrations of C-reactive protein were measured using a high-sensitivity ELISA (Olympus AU400e Chemistry Analyzer and reagents). Dietary intake of macro- and micronutrients was estimated from 4-day diet records as described previously.21
EDD and Endothelium-Independent Dilation
Duplex ultrasonography was used to assess EDD via measurement of brachial artery flow-mediated dilation (FMD) and endothelium-independent dilation via measurement of brachial artery dilation in response to sublingual nitroglycerin, as previously described by our laboratory5–8 (see the online data supplement for more details).
EC Protein Expression
The procedures used for collection of ECs and measurement of EC protein expression were described originally by Feng et al22 and Colombo et al16 and more recently by our laboratory5,8,18 (see the online data supplement for more details). Briefly, 2 sterile J-wires were advanced into the brachial artery and/or an antecubital vein (4 cm beyond the tip of the catheter) and retracted through an 18-gauge catheter, then transferred to a dissociation buffer solution, where cells were recovered by washing and centrifugation. Collected cells were fixed with 3.7% formaldehyde and plated on poly-L-lysine coated slides (Sigma Chemical, St Louis, Mo). ECs from arterial samples were available only on 19 of the young and 23 of the older men in the overall study sample (see online data supplement for details).
For immunofluorescence staining ECs were rehydrated with PBS and nonspecific binding sites blocked with 5% donkey serum (Jackson Immunoresearch, West Grove, Pa). Afterward cells were incubated with monoclonal antibodies for one of the following: nitrotyrosine (Abcam; Cambridge, Mass), xanthine oxidase (US Biological; Swampscott, Mass), NAD(P)H oxidase-p47phox (Abcam; Cambridge, Mass), CuZn SOD (Upstate; Lake Placid, NY), Mn SOD (Research Diagnostics; Concord, Mass), catalase (Abcam; Cambridge, Mass) or NF-B p65 (Novus; Littleton, Colo). Cells were next incubated with CY3-conjugated secondary antibodies (Research Diagnostics; Concord, Mass).
For analysis, slides were viewed using a fluorescence microscope (Eclipse 600, Nikon, Melville, NY) and EC images were digitally captured by a Photometrics CoolSNAPfx digital camera (Roper Scientific, Inc, Tucson, Ariz). ECs were documented by cell staining of von Willebrand factor and nuclear integrity was confirmed using DAPI (4',6'-diamidino-2-phenylindole hydrochloride) staining. Once endothelial cells with intact nuclei were identified, they were analyzed using Metamorph Software (Universal Imaging Corp, Downingtown, Pa) to quantify the intensity of CY3 staining (ie, average pixel intensity). Values are reported as ratios of EC protein expression/human umbilical vein EC (HUVEC); this minimizes the possible counfounding effects of differences in intensity of staining among different staining sessions.
Data Analysis
Statistical analyses were performed with SPSS. Group differences were determined by t-tests for independent sample comparisons. Pearson correlation analysis was used to determine relations of interest. Statistical significance for all analyses was set at P<0.05.
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Results |
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Subject Characteristics
Characteristics of the young and older subjects are shown in Table 1. Body mass, fasting plasma total cholesterol, low-density lipoprotein (LDL) cholesterol, triglyceride concentrations, resting blood pressure, and fasting plasma glucose concentrations were higher in the older men (all P<0.05), but all values were within normal ranges. Body mass index, high-density lipoprotein (HDL) cholesterol, physical activity levels and resting heart rate were not different in the 2 groups. Plasma oxidized LDL concentrations were greater in the older men (P<0.05), but the 2 groups did not differ in total antioxidant status. Plasma C-reactive protein concentrations were higher in the older men (P<0.05), although generally within the normal range.
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Information on diet intake is shown in the supplemental Table. Estimated caloric intake was lower in the older men, as were absolute intakes of carbohydrate, fat, protein, folate, niacin and sodium (all P<0.05). The % of caloric intake from carbohydrate, fat, and protein, as well as intake of vitamins C and E, were not different in the 2 groups.
Impaired EDD, but Preserved Endothelium-Independent Dilation With Aging
In the overall sample, brachial artery FMD was 50% lower in the older compared with young subjects (n=42 older/n=51 young, P<0.01, Figure 1A and 1B). In contrast to EDD, brachial artery dilation in response to sublingual nitroglycerin was similar in the 2 groups (Figure 1C and 1D). The age-associated differences in brachial FMD were similar in subjects in whom we obtained endothelial cells from arterial samples compared with the overall sample (percentage of FMD, 3.9±0.3% versus 7.6±0.3%, P<0.01, for overall sample; 4.3±0.7% versus 7.8±0.6%, P<0.01, for arterial EC sample). These observations establish that in the present study, EDD was markedly impaired with aging in the presence of preserved endothelium-independent dilation.
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Endothelial Oxidative Stress Develops With Aging and Is Related to EDD
Abundance of nitrotyrosine was 105% greater in ECs obtained from the brachial artery (1.25±0.12 versus 0.61±0.11 nitrotyrosine intensity/HUVEC intensity, P<0.01, n=11 older/n=11 young) and 60% higher in ECs obtained from antecubital veins (0.55±0.04 versus 0.34±0.03, P<0.05, n=19 older/n=17 young) of the older compared with the young men (Figure 2A and 2B). In the overall study sample, brachial artery FMD was inversely related to the abundance of nitrotyrosine in ECs obtained from brachial artery (r=–0.62, P<0.01, Figure 3, n=22) and peripheral venous (r=–0.44, P<0.01, n=29) samples. Nitrotyrosine measured in ECs obtained from venous samples was positively related to plasma (venous) concentrations of oxidized LDL (r=0.44, P<0.05, n=29). These observations suggest that endothelial oxidative stress develops with aging in healthy men and is related to age-associated reductions in EDD, as well as to a common marker of systemic oxidative stress.
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Selective Upregulation of Endothelial Oxidant Enzyme Protein Expression and Unchanged Antioxidant Enzyme Protein Expression With Aging in Venous Cell Samples
NAD(P)H oxidase-p47phox EC protein expression was 20% higher in the older men (0.71±0.05 versus 0.57±0.05 NAD[P]H oxidase-p47phox intensity/HUVEC intensity, P<0.05, Figure 4A, n=19 older/n=18 young), whereas xanthine oxidase was not different in the 2 groups (Figure 4B, n=15 older/15 young). EC expression of the antioxidant enzymes CuZn SOD, Mn SOD, and catalase were not different between groups (Table 2). These observations suggest that in venous cell samples, EC expression of the oxidant enzyme NAD(P)H oxidase is increased with aging in healthy men in the absence of changes in the oxidant enzyme xanthine oxidase and antioxidant enzymes.
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Endothelial Protein Expression of NF-B Increases With Aging and Is Related to Increases in Endothelial Nitrotyrosine
NF-B p65 was elevated by 38% and 91%, respectively, in ECs obtained from arterial (0.73±0.07 versus 0.53±0.05 NF-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/15 young) samples of the older men (Figure 5). NF-B p65 protein expression determined in ECs from arterial samples (r=0.51, P<0.05, n=16; Figure 6) was positively related to arterial EC nitrotyrosine. NF-B p65 protein expression determined in ECs from venous, but not arterial, samples was inversely related to brachial artery FMD (r=–0.40, P=0.03, n=22). These results suggest that expression of the redox-sensitive, proinflammatory transcription factor NF-B increases with aging in healthy men and is related to age-associated increases in nitrotyrosine.
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Discussion |
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The key novel findings of the present study are as follows. First, nitrotyrosine, a cellular marker of oxidative stress, is increased in ECs from older compared with young healthy men. Second, these increases in EC nitrotyrosine are significantly related to age-associated reductions in EDD. Third, NAD(P)H oxidase-p47phox, a component of the oxidant producing enzyme NAD(P)H oxidase, is increased in venous ECs from older men in the absence of upregulation of xanthine oxidase, another key oxidant producing enzyme, or changes in antioxidant enzymes. Finally, NF-
B p65, a primary component of the redox sensitive, proinflammatory NF-B transcription factor complex, is increased in ECs from older men and is positively related to age-associated increases in EC nitrotyrosine. Collectively, these results suggest that oxidative stress develops in ECs with aging in healthy men and is related to reductions in EDD. Our observations also suggest that increases in NAD(P)H oxidase and NF-B, but not reductions in antioxidant enzymes, may be among the molecular mechanisms contributing to EC oxidative stress with aging in humans. To our knowledge, these observations represent the first direct evidence that endothelial oxidative stress develops with aging and is related to impairments in vascular endothelial function. Moreover, we have provided insight into the molecular events that may be involved.
Aging and EC Oxidative Stress
Oxidative stress is believed to be an important process contributing to the development of CVD.1,23 Aging is associated with a marked increase in risk of CVD,3 and this has been postulated to be caused in part by oxidative stress.3,24 Findings in experimental animals9–12 and indirect observations in humans support the concept that systemic and vascular oxidative stress develop with aging, even in healthy subjects.5–7,13,25 Our finding that plasma oxidized LDL concentrations, a systemic marker of oxidative stress,26 were greater in older men is in agreement with previous observations from our laboratory in similarly healthy, well-screened men and postmenopausal women.5,20 The present results extend these findings by providing direct evidence of oxidative stress in ECs from older adults. Specifically, we found that nitrotyrosine, which reflects nitration of tyrosine residues on proteins27 and, thus, serves as an indicator of oxidative stress,28 is elevated in ECs collected from the brachial artery and peripheral veins of older compared with young healthy men. These findings are consistent with previous observations of increased nitrotyrosine in arteries of old rats.9,12 However, our results provide the first evidence for oxidative stress in the vascular endothelium per se with aging and further establish that such changes occur in older adult humans in the absence of overt clinical vascular disease. We also found that EC nitrotyrosine was positively related to plasma concentrations of plasma oxidized LDL. This association may or may not reflect an influence of systemic, circulating levels of oxidative stress on the vascular endothelium.
Age-Associated EC Oxidative Stress and Impaired EDD
The results of the present study confirmed our previous findings that brachial FMD, a measure of conduit artery EDD, is reduced by 50% in healthy older adults compared with young adults.5–8 The present results extend these observations by showing that age-associated reductions in brachial FMD are related to increases in EC nitrotyrosine. These data support the idea that the development of oxidative stress in the vascular endothelium with aging in humans is associated with impaired endothelial function.25,29 The present results linking EC oxidative stress to age-associated reductions in EDD are consistent with earlier observations that administration of vitamin C restores EDD in older healthy adults7,13 and that EDD is inversely related to systemic markers of oxidative stress among healthy adults of increasing age.5 Because human conduit artery FMD is dependent in part on nitric oxide bioavailability,30 the link between EC oxidative stress and impaired EDD with aging likely involves reduced nitric oxide bioavailability, as reported previously by Taddei et al.13
Age-Associated EC Oxidative Stress: Oxidant and Antioxidant Enzyme Expression
NAD(P)H oxidase and xanthine oxidase are major sources of superoxide (O2
) in the vasculature and have been linked to CVD.1,31 NAD(P)H oxidase-p47phox is an obligatory cytosolic accessory protein, which when translocated to membrane-bound NAD(P)H oxidase catalytic subunits (eg, p22phox), stimulates O2 production.31,32 In the present study, the older men exhibited elevated venous EC NAD(P)H oxidase-p47phox compared with young subjects. In contrast, there were no age group differences in venous EC expression of xanthine oxidase, in agreement with recent observations from our laboratory on smaller groups of young and older adults.5 These results support the concept that endothelial expression of NAD(P)H oxidase-p47phox, but not xanthine oxidase, is upregulated with aging in ECs obtained from antecubital veins of healthy men. Our findings are consistent with previous studies in rodents reporting enhanced vascular NAD(P)H oxidase O2 production10–12 with unchanged xanthine oxidase O2 production12 in arteries from older compared with young adult animals. The present results also are in agreement with recent findings from our laboratory that administration of the xanthine oxidase antagonist allopurinol does not improve EDD in older adults.5
Mn and CuZn SOD convert O2 to hydrogen peroxide (H2O2) in the mitochondrial and cytosolic regions of the ECs, respectively, whereas catalase catalyzes the conversion of H2O2 to water and oxygen. In the present study, there were no significant age-associated differences in expression of these antioxidant enzymes in ECs obtained from antecubital veins. These findings indicate that the development of EC oxidative stress with aging in healthy men is not associated with reductions in these key antioxidants. However, increased bioavailability of reactive oxygen species induces the expression of antioxidant enzymes in ECs in vitro.33 Thus, it might be expected that in the presence of EC oxidative stress, the older men would demonstrate greater antioxidant enzyme expression than the young men. As such, the absence of an obvious, consistent upregulation of venous EC antioxidant enzymes may represent an inappropriate compensatory response that contributes to the development of EC oxidative stress with aging. Indeed, in rodents both Mn SOD and CuZn SOD have been reported to be increased in large arteries with aging,34 although unchanged or reduced SOD activity and protein expression have been observed in smaller resistance arteries of older animals.35,36 It also is possible that the activity of these antioxidant enzymes was increased in the older men in the present study in the absence of increases in protein.
Age-Associated EC Oxidative Stress: NF-B Expression
NF-B is an important nuclear transcription factor involved in the regulation of genes that encode for a large number of proteins involved in inflammatory responses, including oxidant-producing enzymes such as NAD(P)H oxidase.37,38 Increased NF-B expression is associated with CVD.39 NF-B is found in atherosclerotic plaques in humans,40 and expression of NF-B is increased in ECs located in areas of the aorta susceptible to plaque development.41
The present results are the first to demonstrate that NF-B, as measured by NF-B p65 subunit expression, is increased in ECs with aging in humans. Moreover, the age-associated increase in EC NF-B was positively related to the increase in arterial EC nitrotyrosine. This is consistent with the idea that increased expression of NF-B may contribute to the development of oxidative stress in the vascular endothelium with aging in humans. Alternatively, increased EC expression of NF-B could be the result of oxidative stress. Because NF-B both stimulates the production of and is stimulated by reactive oxygen species,37,38 the exact cause and effect relation underlying its association with EC oxidative stress cannot be discerned. It is also possible that the increased EC NF-B expression in the older men was related to a greater inflammatory state. Inflammatory stimuli can induce NF-B gene transcription and protein expression,42–44 and plasma concentrations of C-reactive protein, a circulating marker of systemic inflammation, were greater in the older men in the present study, although still within a normal CVD risk range.45
Limitations
We recognize several limitations of our study. First, we had a limited number of ECs obtained from arterial samples. Although we were able to use arterial cell samples to document EC oxidative stress with aging, as well as its relation to EC NF-B expression, our other measurements of protein expression were restricted to ECs obtained from venous sampling. We do not believe, however, that this limitation affected our key results and conclusions because we found a highly significant positive relationship between expression of nitrotyrosine measured in cells obtained from venous compared with arterial samples collected from the same subjects on the same day (r=0.86, P=0. 003; n=9); other proteins studied also demonstrated positive correlations (mean r=0.71, A.E. Silver, D. Christou, D.R. Seals, unpublished results, 2006). Moreover, expected differences in protein expression between healthy controls and patients with CVD are observed in ECs obtained from veins using the same procedures as in the present study.16 Nevertheless, although ECs obtained from both arterial and venous cell samples showed increased levels of nitrotyrosine and NF-B protein expression in older men in the present study, the age-associated differences were not the same in ECs from arterial versus venous samples.
Second, we measured the expression of NAD(P)H oxidase-p47phox,46 but not its phosphorylation status, which determines enzyme activation.47 In addition, the translocation of NF-B p65 to the nucleus was not measured in the present study.
Third, we studied only healthy men. As such, the results may not reflect differences with aging in healthy females or adults with clinical disease or CVD risk factors.
Fourth, as discussed recently,18 we acknowledge the semiquantitative nature of the immunofluorescence technique. However, quantitative immunofluorescence has been validated against immunoblotting by our laboratory and others.16,18 Moreover, all techniques for measuring protein expression are semiquantitative, and the number of cells that can be obtained in vivo from our human subjects does not yield enough total protein or mRNA to use alternative techniques such as Western blotting or real-time RT-PCR. It is important to emphasize that despite this limitation, we were able to identify several expected differences in EC protein expression with aging. Moreover, the limited number of endothelial cells available also does not allow for measurements of enzyme activity, which clearly would complement and extend the insight provided by our analyses of protein expression.
Finally, we recognize the variability in the relationships reported between brachial FMD and endothelial cell nitrotyrosine, and between endothelial cell nitrotyrosine and NF-B (Figures 3 and 6
). This indicates, for example, that brachial artery FMD varies significantly among individuals at a particular level of endothelial cell nitrotyrosine. One factor that likely contributes to this variability is measurement error. The error associated with each of the measurements adds to the variability and acts to reduce the correlation, thus underestimating the true physiological association between the variables. However, individual differences in the physiological relationship between the variables also likely plays a role in this variability. At a given level of endothelial nitrotyrosine, brachial FMD may differ as a consequence of individual differences in other factors known to modulate endothelial function (eg, endothelial production of autocrine and paracrine molecules such as endothelin-1, circulating vasoactive hormones, sympathetic nervous system activity, etc).
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Conclusions |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionConclusionsReferences |
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The results of the present study are consistent with the idea that endothelial oxidative stress develops with aging in healthy men and is related to reductions in EDD. Our findings also suggest that increased expression of NAD(P)H oxidase and NF-
B may contribute to endothelial oxidative stress with aging in humans. Collectively, our findings provide the first direct insight into the molecular mechanisms by which aging leads to vascular endothelial dysfunction and increased risk of CVD in humans.
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Acknowledgments |
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We thank Rhea Chiang, Andrew Radford, Cassandra Roeca, and Brooke Lawson for technical assistance.
Sources of Funding
This work was supported by NIH grants AG006537, AG013038, AG022241, AG000279, HL007851, and RR00051.
Disclosures
None.
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Footnotes |
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Original received January 16, 2007; revision received April 16, 2007; accepted April 25, 2007.
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References |
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