Neal L. WeintraubFrancis J. Miller, Jr, William J. Sharp, Xiang Fang, Larry W. Oberley, Terry D. Oberley and

Aneurysmal RemodelingOxidative Stress in Human Abdominal Aortic Aneurysms : A Potential Mediator of

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Oxidative Stress in Human Abdominal Aortic AneurysmsA Potential Mediator of Aneurysmal Remodeling

Francis J. Miller, Jr, William J. Sharp, Xiang Fang, Larry W. Oberley,Terry D. Oberley, Neal L. Weintraub

Abstract—Abdominal aortic aneurysm (AAA) is an inflammatory disorder characterized by localized connective tissuedegradation and smooth muscle cell (SMC) apoptosis, leading to aortic dilatation and rupture. Reactive oxygen speciesare abundantly produced during inflammatory processes and can stimulate connective tissue–degrading proteases andapoptosis of SMCs. We hypothesized that reactive oxygen species are locally increased in AAA and lead to enhancedoxidative stress. In aortas from patients undergoing surgical repair, superoxide levels (measured by lucigenin-enhancedchemiluminescence) were 2.5-fold higher in the AAA segments compared with the adjacent nonaneurysmal aortic (NA)segments (6638�2164 versus 2675�1027 relative light units for 5 minutes per millimeter squared, respectively; n�7).Formation of thiobarbituric acid–reactive substances and conjugated dienes, 2 indices of lipid peroxidation, wereincreased 3-fold in AAA compared with NA segments. Immunostaining for nitrotyrosine was significantly greater inAAA tissue. Dihydroethidium staining indicated that increased superoxide in AAA segments was localized toinfiltrating inflammatory cells and to SMCs. Expression of the NADPH oxidase subunits p47phox and p22phox andNAD(P)H oxidase activity were increased in AAA segments compared with NA segments. Thus, oxidative stress ismarkedly increased in AAA, in part through the activation of NAD(P)H oxidase, and may contribute to the diseasepathogenesis. (Arterioscler Thromb Vasc Biol. 2002;22:560-565.)

Key Words: aneurysm � aortas � free radicals � antioxidants � NADPH oxidase

Abdominal aortic aneurysms (AAAs) occur in �3% ofhumans aged �65 years and are characterized by

localized structural deterioration of the aortic wall, leading toprogressive aortic dilation.1 The morbidity and mortalityassociated with AAAs are considerable. Surprisingly, how-ever, little is known about the mechanisms responsible foraneurysmal formation and progression.

Infiltration of inflammatory leukocytes is a prominentpathological feature of AAA.2–4 The inflammatory responseis believed to play a key role in the degeneration of the elasticmedia, which, in turn, promotes aneurysmal remodeling ofthe aortic wall.4 Degeneration of the elastic media has beenattributed to proteolytic degradation of structural proteins,such as elastin, consequent to the activation of matrixmetalloproteinases (MMPs) and other proteases.5–7 Recently,apoptosis of smooth muscle cells (SMCs) has also beendetected in AAAs and may contribute to the disease pro-cess.8–10 SMCs are the most abundant cellular component ofthe arterial media and promote structural integrity by synthe-sizing matrix proteins, such as elastin. The emerging conceptis that the increased destruction and decreased production ofmatrix proteins, which are due to the activation of proteasesand apoptosis of SMCs, respectively, act in concert topromote the aneurysmal remodeling process.

One potential mechanism by which inflammatory re-sponses could promote aneurysm formation is through en-hanced production of reactive oxygen species (ROS), such assuperoxide (O2

·�) and hydrogen peroxide (H2O2). These toxicsubstances are produced by inflammatory leukocytes andcontribute to the tissue destruction observed in a variety ofimmunologic and infectious disorders. In addition, O2

·� isproduced by the 3 major types of cells resident in the aorticwall: SMCs, fibroblasts, and endothelial cells. Importantly,recent studies indicate that ROS activate MMPs and induceapoptosis of vascular SMCs.11,12

In the present study, we measured levels of O2·� and lipid

peroxidation products in segments of AAA and in adjacentnonaneurysmal aortic (NA) tissue removed from patients under-going elective AAA repair. We examined the cellular productionof O2

·� in situ and the expression and activity of NAD(P)Hoxidase. Our results indicate that levels of ROS are increasedlocally in AAAs, partially because of NAD(P)H oxidase activ-ity, and lead to marked increases in oxidative stress.

MethodsSegments of infrarenal AAA and adjacent NA tissues were obtainedfrom 12 patients undergoing elective aneurysm repair at the Univer-sity of Iowa Hospitals and Clinics and at the Department of VeteransAffairs Medical Center in Iowa City, Iowa. The patients ranged in

Received November 8, 2001; revision accepted February 13, 2002.From the Departments of Internal Medicine (F.J.M., N.L.W.), Surgery (W.J.S.), Biochemistry (X.F.), and Radiation Research Laboratory (L.W.O.),

University of Iowa College of Medicine, Iowa City, and the Department of Pathology (T.D.O.), University of Wisconsin, Madison.Consulting Editor for this article was Elizabeth Nabel, National Institutes of Health, Bethesda, Md.Correspondence to Francis J. Miller, Jr, MD, Department of Internal Medicine, E314-4 GH, University of Iowa, Iowa City, IA 52242. E-mail

francis-miller@uiowa.edu© 2002 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000013778.72404.30

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age from 60 to 90 years, and all but 3 were male. Removal of thesetissues was accomplished as part of the normal operative procedure,in which AAA and adjacent NA segments were trimmed in prepa-ration for graft anastomosis. Care was taken to ensure that none ofthe tissues was subjected to surgical manipulation or cross-clamping.Aortic tissues were obtained from 7 additional patients without AAA(aged 16 to 70 years). These included organ donors (n�4), individ-uals with aortic dissection (n�2), and individuals with Marfan’ssyndrome (n�1). Immediately after procurement, segments wereplaced in 0.9% saline (4°C) and transported to the laboratory. Theprotocol was approved by the Human Subjects Office at theUniversity of Iowa.

Histological AnalysesSegments were snap-frozen in Tissue-Tek OCT mounting media forhistological analysis. Cryosections (8 �m thick) were stained withhematoxylin and eosin or Verhoeff–van Gieson stain.

Immunohistochemical staining for nitrotyrosine was performed byusing Vectastain ABC-AP kit (Vector Laboratories). Anti-nitrotyrosine (Upstate Biotechnology Inc) was used at a dilution of1:100. After they were washed, the slides were incubated withbiotinylated anti-mouse antibody and then washed and incubatedwith Vectastain ABC-AP reagent. Visualization was accomplishedafter treating the slides with alkaline phosphatase substrate solutionand counterstaining with nuclear fast red.

The presence of macrophages was assessed by immunohistochem-istry with the mouse monoclonal anti-human macrophage antibodyNCL-MACRO (1:80 dilution). Immunostaining for the NADPHoxidase subunits p47phox (1:20, Santa Cruz Biochemicals) and 22phox

(1:100, monoclonal antibody 44.1, obtained by permission from DrA.J. Jesaitis, Department of Microbiology, Montana State Univer-sity, Bozeman) was also performed.

Assessment of O2·� Levels

Aortic segments were prepared for in situ imaging of O2·� generation

with the fluorescent dye dihydroethidium (DHE, Molecular Probes,Inc), as previously described.13 Unfixed frozen aortic segments werecut into 30-�m-thick sections and placed on a glass slide. DHE (2�mol/L) was topically applied, and slides were incubated for 30minutes in a humidified chamber at 37°C. Images were obtained witha Bio-Rad laser scanning confocal microscope. AAA and NAsections were processed and imaged in parallel (excitation at 488 nmand detection of fluorescence at 585 nm with use of a long-passfilter) at identical laser settings.

Superoxide levels were also measured by lucigenin-enhanced chemi-luminescence as described previously.13 Aortic segments were placed in

PBS and lucigenin (5 �mol/L), and after 2 minutes of dark adaptation,relative light units (RLU) emitted were measured for 5 minutes in anFB12 luminometer (Zylux Corp). To assess NAD(P)H oxidase activity,NADH (0.1 mmol/L) or NADPH (0.1 mmol/L) was added to thePBS-lucigenin containing the vessel segment, and O2

·� levels weremeasured. Some segments were preincubated with polyethylene-glycolated superoxide dismutase (SOD, 250 U/mL) or the flavininhibitor diphenylene iodonium (DPI, 0.1 mmol/L). Surface areas weremeasured for each segment to allow normalization for tissue size.

Determination of Lipid PeroxidationSegments of tissues were washed with cold PBS to remove red bloodcells and homogenates (10% [wt/vol] in PBS) prepared by using aTempest homogenizer (Vertis). Lipid peroxidation products in ho-mogenates were determined by measuring thiobarbituric acid–reac-tive substances (TBARS) with the use of a fluorometric assay.14 Aseparate test for the formation of conjugated dienes was alsoperformed by using a spectrophotometric assay (234 nm).14 Concen-tration of conjugated dienes was calculated by using the molarextinction coefficient of 28 500 and is expressed as nanomoles pergram of tissue. Values of TBARS are expressed as nanomolarequivalents of malondialdehyde per gram of tissue.

Statistical AnalysesAll data are expressed as mean�SEM. Differences between meanvalues were analyzed by Student t tests or by one-way ANOVAcombined with the Student-Newman-Keuls multiple comparisontest. Values of P�0.05 were considered to be statistically significant.

ResultsAtherosclerotic changes (ie, neointimal proliferation andfoam cell formation) were observed in AAA and adjacent NAsegments, but extensive medial degeneration was observedpredominately in the aneurysmal tissues. In addition, local-ized inflammatory cell infiltrates were frequently observed inthe AAA tissues.

To examine the distribution and cellular sources of O2·�,

AAAs and adjacent NAs were incubated with the oxidativefluorescent dye DHE. There were diffuse increases in O2

·�

levels, as measured by DHE fluorescence, throughout thewall of AAA segments, particularly within the medial layer(Figure 1, n�7). In addition, AAA tissues frequently con-tained regions of inflammatory cell infiltration associatedwith increased levels of O2

·� (Figure 2). Immunohistochem-

Figure 1. Superoxide levels are increased in AAA.Segments of aorta procured from the same patientundergoing elective AAA repair were sectioned, incu-bated with the oxidative fluorescent dye DHE, andexamined by confocal microscopy. Cellular O2

·� lev-els are increased in AAA tissues (right) comparedwith adjacent NA tissue (left). Ad indicates adventitia;M, media; and IEM, internal elastic membrane. Rela-tive differences in fluorescence between AAA and NAsegments are representative of studies performed in7 patients.

Figure 2. Association of O2·� with inflammatory infiltrate

and disruption of medial SMCs and elastin in AAA.Adjacent sections of AAA were examined for histology(hematoxylin and eosin staining, left), for O2

·� (DHEstaining, bottom inset), or for elastin (Verhoeff–van Gie-son stain, right). A higher magnification of the inflamma-tory infiltrate is also shown (top inset). Althoughincreases in O2

·� were observed throughout the AAAtissues, focal inflammatory infiltrates were associatedwith localized increases in O2

·�, disruption of medialSMC organization, and loss of elastic fibers. The arrowdenotes the inflammatory infiltrate. M indicates mediallayer; V, vasa vasorum within the adventitial layer.Images are representative of those obtained from 4patients.

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ical analyses indicated that many of these inflammatory cellsstained positively for NCL-MACRO, a marker for humanmacrophages and monocytes (see online Figure, which can beaccessed at http://atvb.ahajournals.org). Areas of inflamma-tory infiltrate demonstrating increased O2

·� levels were alsoassociated with disruption of the medial SMC architecture, areduction in the density of medial elastic fibers, and disrup-tion of the elastic membrane (Figure 2). Not unexpectedly,there was marked heterogeneity among the diseased aortas,with a spectrum of cellularity and SMC disruption in thesamples studied.

Increased O2·� levels in AAA tissue were confirmed by

performing lucigenin-enhanced chemiluminescence. Levelsof O2

·� obtained from AAA segments were 2.5�0.6-foldhigher than the levels obtained from adjacent NA segments(n�7, Figure 3A). Because the NA segments also undergoatherosclerotic changes, we examined O2

·� levels in aortas

from patients without AAA. Superoxide levels were �10-fold higher in AAA segments compared with NA segments(n�6, Figure 3A).

To determine whether the observed levels of O2·� were

sufficient to induce oxidative stress in aneurysmal tissues,segments of AAA and NA tissues were assayed for theformation of TBARS. Compared with levels in NA tissues,levels of TBARS in AAA tissues were increased �3-fold(Figure 3B). The formation of conjugated dienes, anothermarker of lipid peroxidation, was similarly increased nearly3-fold in AAA compared with NA tissues (Figure 3B). Inaddition, we performed immunostaining with an antibodyagainst nitrotyrosine, which is a marker for amino acidoxidation induced by several oxidant species, includingperoxynitrite, the highly reactive product of O2

·� and NO.Nitrotyrosine immunostaining was markedly enhanced inAAA compared with NA segments (Figure 4). Collectively,these results suggest that tissue damage due to oxidativestress is enhanced in AAA compared with NA tissues.

A plasma membrane–associated NAP(P)H oxidase hasbeen proposed as a major source of O2

·� in vascular cells.15–18

An NAD(P)H oxidase inhibitor, DPI (0.1 mmol/L), reducedO2

·� levels in AAA tissue by �60%, as measured bylucigenin-enhanced chemiluminescence (2103�895 RLU · 5min�1 · mm�2, n�4). In addition, generation of O2

·� inresponse to NADH (0.1 mmol/L) or to NADPH (0.1 mmol/L)was 3-fold greater in AAA segments compared with NAsegments (n�5, Figure 5A). Furthermore, the increases inlucigenin-enhanced chemiluminescence in AAA in responseto NADH and NADPH were attenuated by treatment withDPI. Next, we examined protein expression of p47phox andp22phox, 2 subunits of the phagocyte and vascular NAD(P)Hoxidase.19,20 Immunostaining demonstrated markedly in-creased expression of p47phox and p22phox throughout the AAAsegments (Figure 5B). These findings suggest that NAD(P)Hoxidase activity was increased in AAA and contributed to theincreased production of O2

·�.Increased expression of NAD(P)H oxidase and increased

O2·� levels were not limited to regions of inflammatory cell

infiltration but were present diffusely throughout the mediallayer (Figure 6, and online Figure).

DiscussionThe major finding of the present study is that O2

·� levels areincreased in AAA, thereby leading to a generalized increasein oxidative stress within the vessel wall. The increasedoxidative stress could, in turn, promote remodeling of theextracellular matrix and apoptosis of SMCs. These findings

Figure 3. O2·� levels and lipid peroxidation products in AAA and

NA tissues. A, O2·� levels were measured by lucigenin-enhanced

chemiluminescence in segments of AAA and adjacent NAobtained from patients undergoing elective AAA repair. In addi-tion, a separate group of aortic segments was obtained frompatients without AAA (NL). Increased chemiluminescence in AAAtissues was inhibited by polyethylene-glycolated SOD (250U/mL). There were 5 to 7 segments per group. B, Homogenatesof AAA and NA tissues were prepared and subjected to assaysfor TBARS or conjugated diene formation, as described in Meth-ods (n�6 to 7 per group). MDA indicates malondialdehyde.*P�0.05 vs NA, NL, or AAA�SOD.

Figure 4. Peroxynitrite is increased in AAA. Im-munostaining for nitrotyrosine is increased inAAA tissue (right, blue stain, arrow) comparedwith adjacent NA tissue (left) from the samepatient. Tissues are counterstained with nuclearfast red. There is reduced cellularity of the AAAsegment. Images are representative of thoseobtained in 4 patients.

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may have important implications regarding the mechanismsof aneurysmal formation and progression.

Numerous studies published over the past decade supportthe view that inflammation plays a key role in the pathogen-esis of AAA.2–7 On the basis of previous reports, inflamma-tory responses may promote aneurysmal formation throughseveral potential mechanisms. First, infiltrating leukocytesare major sources of MMPs and serine proteases, such astissue plasminogen activator. These proteases degrade struc-tural proteins, such as elastin, collagen, and laminin, therebyweakening the aortic wall.5,7,21 Inhibitors of MMPs preventaneurysmal degeneration and rupture.22 Second, infiltrating

immune cells can induce the death of vascular SMCs inAAAs through induction of the Fas and perforin pathways9

and through cyclooxygenase-2.10,23,24 Induction of cyclooxy-genase-2 leads to augmented production of prostaglandin E2,which, in turn, reduces SMC proliferation and viabilitythrough direct25 and cytokine-mediated10 mechanisms. En-hanced destruction of structural proteins and a reducedsynthetic capacity resulting from SMC apoptosis likely acttogether to progressively weaken the vessel wall, resulting indilatation. Our findings indicate that O2

·� is locally increasedin AAA segments, associated with inflammatory infiltratesand disruption of SMCs and elastin fibers, suggesting thatROS could contribute to these processes.

The possibility that levels of ROS might be locally ele-vated in AAA and contribute to the pathogenesis of thedisease was suggested from several observations. First, infil-trating inflammatory cells produce ROS and generateproatherogenic factors, which augment the production ofROS by SMCs.26,27 Second, ROS have been reported toactivate MMPs11 and to inhibit plasminogen activator inhib-itor-1, which, in turn, could promote extracellular matrixdegradation.26 Moreover, ROS induce the apoptosis of vas-cular SMCs,12,28 which is a recently described feature ofhuman AAA.8–10 In fact, generation of ROS is required forthe induction of apoptosis by diverse proinflammatory fac-tors, including cytokines and p53.28,29 Third, human AAAshave reduced levels of ascorbic acid and antioxidant enzymeactivities.30 Furthermore, plasma levels of vitamin E werefound to be markedly reduced in patients with AAA but notin patients with coronary artery disease in the absence ofAAA.31 These intriguing observations are consistent with ourdata, which demonstrate that the magnitude of oxidativestress is considerably greater in AAA compared with athero-sclerotic, but nonaneurysmal, aorta.

Although our studies focused on O2·�, it is likely that

elevated levels of H2O2, as well as secondary radical speciessuch as hydroxyl radicals, are also present in AAA. Further-more, expression of the inducible form of NO synthase bymacrophages and cytokine-activated SMCs in AAA couldgenerate large quantities of NO, which might also contributeto tissue damage. NO synthase is also a potential source ofO2

·�.32 Enhanced nitrotyrosine immunostaining in AAA seg-ments suggests that reactive nitrogen species are locallyincreased in AAA. Therefore, the milieu of AAA wouldappear to be optimal for the formation and propagation ofinjurious oxidant species. However, it must be emphasizedthat the present study was performed on AAA at an advanced

Figure 5. NAD(P)H oxidase activity and expression is increasedin AAA. A, NADH (0.1 mmol/L)–stimulated and NADPH(0.1 mmol/L)–stimulated production of O2

·� is greater in AAAthan in adjacent NA segments. *P�0.05 for AAA vs NA orAAA�DPI (n�5). B, Immunostaining for the NAD(P)H oxidasecomponents p47phox (top) and p22phox (bottom) are increased inAAA (right) compared with adjacent NA (left). Sections are coun-terstained with nuclear fast red. Images are representative ofthose from 4 patients.

Figure 6. Adjacent sections of AAA tissue are immu-nostained for macrophages (left) or p47phox (right).Sections are counterstained with nuclear fast red.Arrows denote the endothelium. The images are rep-resentative of those obtained from 3 patients.

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stage of dilation. Whether oxidative stress is also increased insmaller subclinical aneurysms is unknown.

The cellular sources of O2·� in AAA appear to be resident

vascular cells and infiltrating inflammatory cells. AnNAD(P)H oxidase functions in SMCs and phagocytes togenerate O2

·�. That SMCs are contributing to increased O2·�

levels in AAA is based on 3 observations. First, O2·� levels

are diffusely increased throughout the medial layer in AAA,as detected by DHE (Figure 1), and are not limited to areas ofmacrophages or monocytes (see online Figure). Second,NADH-stimulated O2

·� is increased in AAA (Figure 5A). Thephagocyte form of the oxidase predominantly uses NADPH,whereas the vascular oxidase uses NADH and NADPH assubstrates.33,34 Third, increased expression of NAD(P)H ox-idase subunits in AAA was not confined to infiltratinginflammatory cells but was observed diffusely throughout themedia (Figures 5B and 6).

A number of factors could contribute importantly to theproduction of ROS by SMCs in AAA. For example, angio-tensin II has a proinflammatory effect in the aortas ofhypercholesterolemic mice and induces the formation ofAAA.35 This effect appears to be independent of increases inblood pressure, suggesting a direct action on the vascularwall.36 In vascular cells, angiotensin II stimulates the produc-tion of ROS via NAD(P)H oxidase17 and has proapoptoticeffects.37

A unique feature of the present study is the comparison ofhuman AAA with adjacent NA tissue, thus controlling for thevariables inherent to studies of patients with vascular disease.Previous studies of human AAA have obtained aortas fromautopsy or organ donor patients for comparison. Patients withAAA have a very high incidence of hypertension and athero-sclerosis, which can independently induce alterations in aorticstructure and function. Such confounding factors were elim-inated in the present study, which, in turn, enabled us toestablish with certainty that O2

·� production and oxidativestress are locally enhanced in AAA. To our knowledge, thisis the first study in which AAA segments were directlycompared with adjacent NA segments obtained from thesame patients.

Although the present findings clearly indicate that ROSand oxidative stress are locally enhanced in AAA, they do notprove a pathogenic role in the initiation or progression of thedisease process. Investigation of the role of oxidative stress inthe pathogenesis of AAA would require a large clinical trialusing effective antioxidant therapy to be conducted overseveral years. Considering the magnitude of oxidative stressin AAA and the limited efficacy of nutrient antioxidants, suchas vitamins E and C, the latter agents might be of little or nobenefit in treating AAA. Moreover, our demonstration of anincreased expression of NAD(P)H oxidase in the aneurysmaltissues suggests that targeted inhibition of this enzyme maybe a promising approach in the treatment of AAA. Thus, thepresent findings suggest that additional studies to evaluate therole of oxidative stress in the pathogenesis of AAA arewarranted.

AcknowledgmentsThis work was supported by grants HL-03669 (F.J.M.), HL-49264(N.L.W.), HL-62984 (F.J.M. and N.L.W.), CA-41267 (L.W.O.), and

DE-10758 (L.W.O.) from the National Institutes of Health and grant97-50259N (F.J.M.) from the American Heart Association.

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