Journal of Surgical Research 138, 156–162 (2007)

Association for Academic Surgery, 2004

Differential Regulation of the Superoxide Dismutase Family inExperimental Aortic Aneurysms and Rat Aortic Explants1

Indranil Sinha, M.D., and Charles G. Pearce, B.S., Brenda S. Cho, M.S., Kevin K. Hannawa, B.S.,Karen J. Roelofs, D.V.M., James C. Stanley, M.D., Peter K. Henke, M.D., and Gilbert R. Upchurch, Jr., M.D.2

Jobst Vascular Research Laboratories, Section of Vascular Surgery, Department of Surgery, University of Michigan, Ann Arbor, Michigan

Submitted for publication July 10, 2006

doi:10.1016/j.jss.2006.07.011

Objective. Oxidative stress has been implicated inabdominal aortic aneurysm pathogenesis. This studysought to characterize the relevance of superoxide dis-mutases (SOD), a family of reactive oxygen catalyzingmetalloenzymes, including manganese SOD (MnSOD),copper-zinc SOD (CuZnSOD), and extracellular SOD(EcSOD), in a rodent aortic aneurysm model.

Methods. Male rat infrarenal abdominal aortas wereperfused with either saline (control) or porcine pan-creatic elastase (6 U/mL). Aortic diameter was mea-sured and aortas harvested on post-operation days 1,2, and 7 (N � 5–6 per treatment group per day). Mn-SOD, CuZnSOD, EcSOD, catalase, MMP-2, MMP-9, and�-actin expression in aortic tissue was determined byquantitative real-time PCR. MnSOD protein levelswere measured using western immunoblotting andimmunohistochemistry. In subsequent experiments,aimed at understanding the mechanism by which SODis involved in AAA pathogenesis, rat aortic explants(RAEs) were incubated in media for 24 h in the pres-ence of interleukin-1� (IL-1�, 2 ng/mL) and TEMPOL(SOD mimetic), catalase, or a combined SOD and cata-lase mimetic. Media MMP-2 and MMP-9 activity wasdetermined by zymography. Data were analyzed byStudent’s t-tests and ANOVA.

Results. Elastase-perfused aortic diameters weresignificantly increased compared to control aortas bypost-perfusion day 7 (P � 0.016). MnSOD mRNA levelsin elastase perfused aortas were 6.0 (P � 0.05) and 7.5times (P < 0.01) greater than control aortas at post-perfusion days 1 and 2, respectively. EcSOD, CuZn-SOD, catalase, and MMP-2 mRNA expression did notstatistically vary between the two groups. MMP-9 ex-

1 Presented at the 38th Annual Meeting of the Association forAcademic Surgery, Houston, TX, November 11–13, 2004.

2 To whom correspondence and reprint requests should be ad-dressed at University of Michigan Hospital, 2210 THCC, 1500 EastMedical Center Drive, Ann Arbor, MI 48109-0329. E-mail: riversu@

umich.edu.

1560022-4804/07 $32.00© 2007 Elsevier Inc. All rights reserved.

pression was 3.5-fold higher in the elastase group onpost-perfusion day 2 (P � 0.04). Western immunoblot-ting confirmed MnSOD protein was up-regulated onday 4 in the elastase-perfused group compared to con-trols (P � 0.02). Immunohistrochemistry demonstratedincreased MnSOD staining in the elastase group on day4. In RAE experiments, TEMPOL increased both MMP-9and MMP-2 activity 2 (P � 0.09) and 3-fold (P � 0.05),respectively, whereas catalase and the combined SOD/catalase mimetic failed to increase MMP-2 or MMP-9activity.

Conclusion. Experimental abdominal aortic aneu-rysm formation is associated with early increases inMnSOD expression and an increase in MMP-9 activity.Strategies aimed at inhibiting oxidative stress duringAAA formation should focus on MnSOD. © 2007 Elsevier Inc.

All rights reserved.

Key Words: abdominal aortic aneurysms; superoxidedismutase; hydrogen peroxide; matrix metalloproteinases.

INTRODUCTION

Abdominal aortic aneurysms (AAAs) are character-ized by significant extracellular matrix (ECM) destruc-tion, leading to subsequent vessel dilation [1]. Matrixmetalloproteinases (MMPs), a family of enzymes in-volved in the degradation of elastin and structuralcollagen, have been implicated in the formation andprogression of AAAs in both humans and rodent mod-els [2–8]. Particularly, it has been demonstrated that agenetic deletion of the MMP-2 or MMP-9 gene attenu-ates aneurysm development in experimental murineAAA models [9, 10]. MMP-2 and MMP-9 expressionand activity have also been found to be up-regulated inhuman AAA tissue [4, 5].

Mechanisms regulating MMP expression remainlargely unknown, but numerous studies now suggestthat reactive oxygen species (ROS) may regulate

MMPs, including MMP-2 and MMP-9 [11–13]. In this

157SINHA ET AL.: DIFFERENTIAL REGULATION OF THE SUPEROXIDE DISMUTASE FAMILY

regard, enzymes that regulate ROS levels may be rel-evant to AAA formation [14–16]. A recent study sug-gests that tamoxifen may inhibit experimental aneu-rysm formation by increasing catalase expression,thereby reducing oxidative stress and subsequent MMPproduction [17]. In addition, multiple studies have sug-gested that vascular smooth muscle cell (SMC) deple-tion and a decrease in extracellular matrix (ECM) pro-duction are relevant to AAA pathogenesis [18–20].Although SMC depletion is already known to occur byapoptosis through induction of the Fas pathway inhumans, [21] recent animal studies have demonstratedthat ROS may mediate mitochondrial dependent apo-ptosis and subsequent vascular SMC depletion in ex-perimental AAAs [22].

Hydrogen peroxide (H2O2), may be of particular rel-evance to AAA formation given that increased localH2O2 associated with endothelial cell dysfunction mayinitiate a number of vascular diseases [16, 23–24].Furthermore, studies have shown that H2O2 increasesMMP-2 activity in vitro in vascular SMCs [11]. Thesuperoxide dismutase (SOD) family of enzymes is in-volved in the formation and regulation of H2O2 andtherefore may be important in AAA formation. Thepresent study investigates the natural history of theSOD enzymes during experimental AAA formation andprovides a possible mechanism by which increased ox-idative stress, seen in AAAs, may increase pathologicMMP production.

MATERIALS AND METHODS

All rats were obtained from Charles River Laboratories (Wilming-ton, MA). All experiments and procedures were approved by theUniversity of Michigan Universal Committee on the Use and Care ofAnimals (UCUCA #8566).

Aneurysm Induction

Aneurysms were induced in male Sprague-Dawley rats, weighing200 to 250 g, by elastase perfusion as previously described [25].Briefly, rats were anesthetized with 1 to 2% isoflurane inhalationand the infrarenal aorta isolated. Temporary control of the aorta wasobtained, lumbar branches were ligated, and an aortotomy was madenear the bifurcation using a 30-gauge needle. The aorta was thenperfused for 30 min with either 2 mL of normal saline or 12 units ofporcine pancreatic elastase (Sigma, St. Louis, MO) in 2 mL of normalsaline.

Aortas were harvested at 1, 2, and 7 days post-perfusion (n � 5–6at each time point for both saline controls and elastase perfusedanimals) and subjected to mRNA extraction. Infrarenal aortic diam-eters were measured before perfusion, immediately post-infusion,and before harvest. This was accomplished using a Spot Insight ColorOptical Camera (Diagnostic Instruments, Inc., Sterling Heights, MI)attached to an operating microscope (Nikon, Melville, NY) using ImagePro Express software (Media Cybernetics, Inc., Silver Springs, MD).

Aortic Explants

Male Sprague-Dawley rats, weighing 200 to 250 g, were anesthe-tized by isoflurane inhalation (1-2%). Through a midline abdominal

incision, the infrarenal aorta was isolated and exised. Aortic tissue

was washed in PBS and then stored overnight in separate 24-wellculture plates with Dulbecco’s modified Eagle’s media (DMEM) con-taining 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan,UT), 100 units/mL penicillin, and 100 �g/mL streptomycin at 37°C.Media and antibiotics were purchased from GIBCO (Rockville, MD).Aortic explants were then placed in a new well with the experimentalconditions carried out in serum-free media. All conditions involved2 ng/mL IL-1� and either saline control, TEMPOL (10 mM), a gen-eral SOD mimetic (10 mM), catalase (10 mM), or MnTMPyP (10 mM),a generalized SOD and catalase mimetic (all experimental reagentspurchased from Sigma). After 24 h of incubation at 37°C, media wascollected and subjected to gelatin zymography. The explants weresolubilized in 0.1% sodium dodecyl sulfate (SDS) (Sigma) for 24 h andanalyzed for total protein concentration.

Quantitative Real Time PCR

Expression of MnSOD, CuZnSOD, EcSOD, MMP-2, MMP-9, cata-lase, and �-actin were determined using quantitative real-time re-verse transcriptase-polymerase chain reaction (RT-PCR). mRNAwas isolated by treatment of aortic segments with TRIzol reagent(Life Technologies, Inc. Rockville, MD), and cDNA produced by re-verse transcription using Oligo-(dT) primer and M-MLV reversetranscriptase (Life Technologies, Inc.). The resultant cDNA was am-plified by Taq Polymerase (Promega, Madison, WI) in a SmartCyclerquantitative PCR system (Cepheid, Sunnyvale, CA). SYBR interca-lating dye (Roche, Indianapolis, IN) was used to monitor cDNAamplification for each gene. Primer sequences were generated withknown standards (Premier Biosoftware International, Palo Alto, CA)and were as follows:

1. MnSOD: TTA ACG CGC AGA TCA TGC A (sense)CCT CGG TGA CGT TCA GAT TGT (antisense)

2. CuZnSOD: CGG CTT CTG TCG TCT CCT (sense)GTT CAC CGC TTG CCT TCT (antisense)

3. EcSOD: GGC CCA GCT CCA GAC TG A (sense)CTC AGG TCC CCG AAC TCA TG (antisense)

4. Catalase: ACT CAG GTG CGG ACA TTC (sense)GGA GTT GTA CTG GTC CAG AAG AGC C (antisense)

5. MMP-9: TCT TCA AGG ACG GTC GGT AT (sense)TGC GGG CAA TAA GAA AGG (antisense)

6. MMP-2: CAT CGC TGC ACC ATC GCC CAT CAT C (sense)CCC AGG GTA CAC AGC TCA TCA TCA TCA AAG (antisense)

7. �-actin: ATG GGT CAG AAG GAT TCC TAT GTG (sense)CTT CAT GAG GTA GTC AGT CAG GTC (sense)

Quantification data are presented as cycle threshold (Ct) and theresults normalized to the �-actin gene. Quantification of mRNAlevels used �Ct values calculated from the formula �Ct � Cttarget gene –Ct�-actin gene. Expression of the target gene in ratio to �-actin expressionwas calculated with the formula: target gene expression/�-actin ex-pression � 2(-�Ct).

Western Blot Analysis

Electrophoresis and Western Blot Materials were purchased fromBio-Rad (Hercules, CA). MnSOD Western blotting was performed onintracellular protein extraction using 1% sodium dodecyl sulfate(SDS). Proteins were separated electrophoretically on a 10% SDS-polyacrymalide gel and blotted onto a nitrocellulose membrane. Non-specific binding was blocked by incubating the membrane for 1 h in20 mM Tris-HCl (pH 7.5) containing 0.5 M NaCl, 0.1% Tween 20, and5% nonfat milk. MnSOD antibodies were obtained from Santa CruzBiotechnologies (Santa Cruz, CA). Immunoreactive bands were vi-sualized using peroxidase-coupled secondary antibodies and a chemi-luminescence kit from Amhersham (Piscataway, NJ). Each protein

band was measured by densitometry, as later described.

158 JOURNAL OF SURGICAL RESEARCH: VOL. 138, NO. 2, APRIL 2007

Substrate Gel Zymography

MMP activity from experimental media was determined by zy-mography using reagents from Novex (San Diego, CA). Gelatinsubstrate zymograms were prepared using precast 10% SDS-polyacrylamide gels containing 1 mg/mL gelatin. Experimental samplesof equal volume were diluted into 2X Tris-glycine SDS sample bufferand separated electrophoretically under non-reducing conditions. Pro-teins were renatured in 2.7% Triton X-100 and the gels developed forvarying times to best quantify the desired MMP band at 37°C in50 mM Tris-HCl containing 5 mM CaCl2 and 0.2% Brij 35. Afterstaining with Coomassie Blue R-250 and destaining with 10% aceticacid, gelatinase activity was evident as a clear band against a darkblue background.

The relative molecular weight of each band was determined bycomparison of the bands against MMP-2 (72 kDa) and MMP-9(92 kDa) standards (Oncogene, Cambridge, MA). Semiquantitativemeasurements of MMP activity were performed by densitometry, aslater described.

Immunohistochemistry

Aortic tissue deparaffinization and rehydration were performedusing xylene and graded alcohols. Heat induced epitope retrieval wasperformed using 10 mM sodium citrate, pH 6.0, for 10 min in themicrowave. Blocking was performed using 1.5% horse serum and0.2% Triton X-100 in PBS for 60 min. Sections were then incubatedwith a 1:100 dilution of the primary antibody monoclonal mouse antiMnSOD (Chemicon International, Temecula, CA) in PBS for up to48 h at 4 degrees C. This was followed by staining with Mouse IgGVectastain ABC-AP Kit (Vector Laboratories, Burlingame, CA) witha 1:200 dilution of secondary antibody for 60 min. Red AlkalinePhosphatase Substrate Kit was then used followed by HematoxylinQS counterstain (Vector Laboratories).

Total Protein Assay

Total cellular protein was determined by a bicinchoninic acidprotein assay (Pierce, Rockville, IL) in RAEs after they had beensolubilized 24 h at 37°C in 1% SDS.

Densitometry

Zymography and Western blots were imaged using a FOTO/Analyst charge coupled device camera (Fotodyne, Hartland, WI) andband strength was quantified using GEL-Pro analyzer software ver-sion 3.1 (Media Cybernetics, Silver Spring, MD). Optical densitieswere normalized to protein levels from the corresponding proteinassay.

Statistics

Data analysis was performed using non-paired Student’s t-Testsand analysis of variance (ANOVA), with significance set as P � 0.05.All data are expressed as the mean � standard error of the mean(SEM). Statistical analysis was performed using SigmaStat (Version2.03; SPSS Inc., Chicago, IL).

RESULTS

Elastase Perfusion Results in Aneurysm Formation

Abdominal aortic aneurysm diameter increased frompre-perfusion by a mean (� SEM) of 69% � 4%, 76% �7%, and 263 � 66% following elastase perfusion onpost-perfusion days 1, 2, and 7, respectively (Fig. 1).

There was 100% (6/6) of elastase-perfused rodents

formed AAAs, defined as an increase in aortic diameterof 100%, by post-perfusion day 7. In contrast, no salinecontrols (0/6) formed aneurysms by post-perfusion day 7.

Aortic Wall MnSOD is Up-Regulated in ExperimentalAneurysm Formation

MnSOD mRNA expression was significantly up-reg-ulated on post-perfusion days 1 and 2 in elastase-perfused aortas compared to saline controls (23.3 �6.9 versus 3.9 � 1.3, P � 0.05 and 6.2 � 0.57 versus0.83 � 0.11, P � 0.003, on days 1 and 2, respectively)(Fig. 2A). CuZnSOD and EcSOD did not significantlydiffer between the two groups at any time point tested(Figs. 2B, 2C). Similarly, catalase expression did notvary between the two groups at any time point tested(data not shown). Western immunoblotting confirmedMnSOD protein up-regulation on day 4 only in theelastase treated group compared to saline controls(1.02 � 0.34 OD/protein versus 0.15 � 0.06 OD/protein,P � 0.02) (Fig. 3A, 3B). Imunohistochemistry con-firmed subjectively increased MnSOD staining in theelastase-perfused group diffusely in the adventitia com-pared to saline-perfused controls at day 4 (Fig. 4A, 4B).

Aortic Wall MMP-9 Up-Regulation Occurs After Increase inMnSOD Expression

MMP-9 mRNA was also up-regulated on post-operative day 2 in elastase treated rodents compared tosaline controls (0.14 � 0.03 versus 0.04 � 0.01, P �0.04) (Fig. 5A, 5B). No significant difference was foundin MMP-9 expression in the elastase-perfused group ondays 1 and 7. MMP-2 did not vary significantly be-

FIG. 1. Increased aortic diameter following elastase perfusion.By post-operative day 7, elastase-perfused aortas were significantlylarger than saline-perfused control aortas (n � 5-6 per group at eachday of harvest). All elastase perfused aortas formed aneurysms byday 7. In contrast, no saline perfused aortas became aneurysmal, asdefined by a 100% increase in aortic diameter. *P � 0.05, n � 5-6 pergroup at each day of harvest.

tween the two groups (data not shown).

159SINHA ET AL.: DIFFERENTIAL REGULATION OF THE SUPEROXIDE DISMUTASE FAMILY

TEMPOL, a General SOD Mimetic, Increases MMPActivity in RAEs

All RAEs demonstrated MMP activity only in the zy-mogen form. TEMPOL administration increased MMP-9(0.47 � 0.12 absorbance (OD)/protein versus 0.23 �0.06 OD/protein, P � 0.09) and MMP-2 (1.06 � 0.31OD/protein versus 0.37 � 0.09 OD/protein, P � 0.05)activity 2-fold and 3-fold, respectively. The addition ofcatalase to IL-1� treated RAEs attenuated MMP-9 ac-tivity completely (0 � 0 OD/protein versus 0.23 � 0.06OD/protein, P � 0.03) and reduced MMP-2 (0.14 � 0.03OD/protein versus 0.37 � 0.09 OD/protein, P � 0.006).The addition of a combined SOD and catalase mimeticto IL-1� induced RAEs did not significantly alterMMP-2 and MMP-9 activity (Figs. 6A, 6B).

DISCUSSION

In the present study, aortic wall MnSOD expressionis significantly up-regulated early following aortic elas-tase perfusion. Interestingly, there was no statisticallysignificant difference in CuZnSOD or EcSOD expres-sion in any of the time points tested. Catalase expres-

sion also was not significantly different between the two

groups. Increased MnSOD expression precedes that ofMMP-9, which becomes significantly up-regulated onpost-operative day 2. In vitro experiments aimed at bet-ter understanding the mechanism(s) by which SODeffects AAAs revealed that a general SOD mimeticincreased MMP-2 and MMP-9 activity.

Superoxide dismutase is known to be important inacute inflammation and may regulate the expression ofMMPs mediated by ROS [14, 26–29]. As the SOD fam-ily of enzymes is important in catalyzing the formationof H2O2, it may be speculated that H2O2 mediatesincreased MMP expression. Further supporting thishypothesis is that MMP activity in the RAEs was at-tenuated with catalase administration, an enzyme thatcatalyzes degradation of H2O2. A mechanism by whichROS up-regulates MMP expression and activity is byincreased activity of transcription factors, includingAP-1 and nuclear factor kappa B (NF-�B) [14, 26]. TheMnSOD isoform may be especially important in the acuteinflammatory reaction accompanying AAA formation, asmultiple cytokines involved in AAA formation modulateits activity. IL-1�, TNF-�, and IL-6, known proinflamma-tory cytokines involved in AAA pathogenesis, increase

FIG. 2. Increased MnSOD expression following elastase perfu-sion. (A) MnSOD/�-actin mRNA expression by real-time PCR dem-onstrates significantly elevated expression in elastase-perfused aor-tas compared to saline-perfused controls on post-perfusion days 1and 2. *P � 0.05, n � 5-6 per group at each day of harvest. Real-timePCR demonstrates no significant difference in either EsSOD (B) orCuZnSOD (C) mRNA expression on any of the time points tested (n �5-6 per group at each day of harvest).

MnSOD expression and lead to vascular wall increases

160 JOURNAL OF SURGICAL RESEARCH: VOL. 138, NO. 2, APRIL 2007

in H2O2 with subsequent increases in MMP expressionand activation [1, 30–32]. Although it may seem counter-intuitive to consider SOD as pro-oxidative in certain sit-uations, it can indeed contribute to increased local ROS,specifically H2O2. This is especially true if catalase levelsdo not increase similarly to MnSOD. The flux betweenH2O2 formation by SOD and breakdown by catalase thenbecomes biased toward H2O2 production.

Multiple lines of evidence suggest that H2O2 is partic-ularly important in MMP regulation. Yoshida et al. dem-onstrated increased H2O2 levels correlate with increasedMMP-3, MMP-9, and MMP-12 production in alveolarmacrophages [33]. In addition, H2O2 can modulate MMPexpression by activation of the MAP kinase family oftranscription factors, including p38, ERK 1/2, and JNK[26, 34, 35]. It is also known that ROS, including H2O2,mediate activation of multiple MMPs, causing auto-cleavage from their latent (or pro-) to activated forms[11]. Grigoryants et al. demonstrated attenuation of ro-dent experimental aneurysm development with catalasesupplementation, further supporting the theory thatH2O2 plays a pathologic role in AAA formation [17]. Col-lectively, these data suggest ROS are important regula-tors of both MMP expression and activity.

Miller et al. demonstrated increased oxidative stressdiffusely in human AAAs compared to controls by com-paring lipid peroxidation and superoxide (O2

.�) stain-ing compared to normal, healthy aortas [16]. It was

FIG. 3. Increased aortic wall MnSOD protein expression follow-ing elastase perfusion. (A) Representative western blot demonstrat-ing increased MnSOD protein in elastase-perfused aortas. (B) AorticMnSOD protein quantity is significantly upregulated in elastase-perfused aortas compared to saline controls on post-operative day 2.*P � 0.05, n � 5-6 per group at each day of harvest.

speculated that H2O2 was up-regulated as well. Yajima

et al. studied oxidative stress in experimental aneu-rysms using DNA microarrays and demonstrated mul-tiple increased genes associated with increased oxida-tive stress, particularly inducible nictric oxide synthase

FIG. 4. Increased MnSOD staining in elastase perfused aortas.Immunohistochemistry (40X) at post-operative day 4 revealed littledetectable MnSOD in the saline perfused aortas (A), compared withprominent and diffuse MnSOD staining in the adventitia of elastaseperfused aortas (B). L � lumen, A � adventitia, arrows point to

positive MnSOD staining.

161SINHA ET AL.: DIFFERENTIAL REGULATION OF THE SUPEROXIDE DISMUTASE FAMILY

early and heme oxygenase late [15]. The study also dem-onstrated some decreased antioxidative stress genes,including SOD and NADPH reductase. However, thisstudy failed to differentiate the different isoforms ofSOD, which may serve to explain their conclusion thatSOD decreases with aneurysm formation, in contraryto the present findings, which demonstrates increasedMnSOD.

Reactive oxygen species may contribute to AAA patho-genesis through multiple mechanisms. First, ROS up-regulate both MMP expression and activity, known tobe critical in the extracellular matrix (ECM) break-down seen in AAA pathogenesis. Secondly, ROS arealso likely involved in pathologic SMC depletion in theaortic wall. Indirect evidence suggests ROS may medi-ate SMC apoptosis both in vitro and in vivo by extra-cellular and mitochondrial-dependent apoptotic path-ways [21, 22, 31]. Finally, proinflammatory cytokinesmay also induce vascular SMC apoptosis mediatedthrough a mechanism requiring ROS [37–38]. It ishowever important to note that MnSOD may be in-creasing in response to the acute inflammatory effect ofelastase perfusion in the experimental model, a featurethat may not be present in human AAAs, which aremore chronic in nature.

This present study suggests the SOD family of en-zymes, particularly the MnSOD isoform, may be rele-vant in AAA formation. A limitation of the presentstudy is that local oxidative stress, especially H2O2 andO2

.� levels, in the vascular wall was not quantified andtherefore it can only be assumed that an increase inlocal ROS concentration is associated with increasedMnSOD activity. Also, there is no study which docu-ments that elevated ROS definitively leads to AAAprogression. In addition, the quantity of ROS, andH2O2 in particular, also depends on multiple enzymesin a complex regulatory circuit. It was not in the scope

FIG. 5. Increased MMP expression following elastase perfusion.MMP-9/�-actin mRNA expression by real-time PCR demonstratessignificantly elevated expression in elastase-perfused aortas com-pared to saline-perfused controls on post-perfusion day 2. *P � 0.04,

n � 5-6 per group at each day of harvest.

of this study to monitor the multiple genes involved inregulating ROS. Further studies in experimental mod-els using MnSOD gene heterozygous and homozygousknock-outs may also serve to more clearly elucidate itsrole in AAA formation. Finally, MnSOD expression inhuman AAAs has not yet been determined. Therefore,the relevance of these observations in an experimentalmodel compared to human AAAs is at best speculative.Perhaps the clinically more relevant question is whetherantioxidants may reverse the deleterious effects of anacute increase in ROS seen during inflammatory set-tings, such as that which occurs during AAA initiationand formation.[36]

ACKNOWLEDGMENT

Supported by: NIH KO8 (HL67885-02) (GRU), VonLeibig Award-Lifeline Foundation (GRU), the Lifeline Medical Student ResearchAward (IS, CGP, KKH), the Griswold and Margery H. Ruth AlphaOmega Alpha Medical Student Research Fellowship (IS), and the

FIG. 6. SOD mimetics increased aortic explant MMP-2 andMMP-9 activity. (A) TEMPOL, a SOD mimetic, increased MMP-9activity in stimulated RAEs compared to control (P � 0.09). Con-versely, catalase significantly decreased MMP-9 activity (P � 0.05).The addition of a combined SOD and catalase mimetic did not alterMMP-9 activity. *P � 0.05 as compared to IL-1� treated control, n �5-6 per group. (B) TEMPOL significantly increased MMP-2 activity(P � 0.03), where as catalase decreased MMP-2 activity (P � 0.006)in comparison to control explants. The addition of a combined SODand catalase mimetic did not alter pro-MMP-2 activity from RAEtreatment with IL-1� alone. *P � 0.05 as compared to IL-1� treatedcontrol, n � 5-6 per group.

Jobst Foundation.

162 JOURNAL OF SURGICAL RESEARCH: VOL. 138, NO. 2, APRIL 2007

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