Myeloperoxidase-Generated Oxidants Modulate Left Ventricular Remodeling but Not Infarct Size After...

Myeloperoxidase-Generated Oxidants Modulate LeftVentricular Remodeling but Not Infarct Size After

Myocardial InfarctionNikolay Vasilyev, MD; Timothy Williams, MD; Marie-Luise Brennan, PhD; Samuel Unzek, MD;

Xiaorong Zhou, MD; Jay W. Heinecke, MD; Douglas R. Spitz, PhD; Eric J. Topol, MD;Stanley L. Hazen, MD, PhD; Marc S. Penn, MD, PhD

Background—Inflammation after myocardial infarction (MI) heralds worse left ventricular (LV) function and clinicaloutcomes. However, whether inflammation affects LV function by extending myonecrosis and/or altering LVremodeling remains unknown. We hypothesized that cytotoxic aldehydes generated during oxidative stress mayadversely affect remodeling and infarct size. One theoretical source of reactive aldehydes is oxidation of common�-amino acids by myeloperoxidase (MPO) released by leukocytes. However, a role for MPO in formation of aldehydesin vivo and the functional consequences of MPO-generated oxidants in ischemia/reperfusion models of MI have notbeen established.

Methods and Results—In studies with cell types found in vascular tissue, MPO-oxidation products of glycine(formaldehyde) and threonine (acrolein) were the most cytotoxic. Mass spectrometry studies of myocardial tissue frommurine models of acute MI (both chronic left anterior descending coronary artery ligation and ischemia/reperfusioninjury) confirmed that MPO serves as a major enzymatic source in the generation of these cytotoxic aldehydes.Interestingly, although MPO-null mice experienced 35.1% (P�0.001) less LV dilation and a 52.2% (P�0.0001)improvement in LV function compared with wild-type mice 24 days after ischemia/reperfusion injury, no difference ininfarct size between wild-type and MPO-null mice was noted.

Conclusions—The present data separate inflammatory effects on infarct size and LV remodeling and demonstrate thatMPO-generated oxidants do not significantly affect tissue necrosis after MI but rather have a profound adverse effecton LV remodeling and function. (Circulation. 2005;112:2812-2820.)

Key Words: amino acids � infarction � inflammation � remodeling

The inflammatory response to myocardial infarction (MI)has a significant role in determining the size of the

infarct,1 as well as subsequent left ventricular (LV) remodel-ing.1,2 Activated leukocytes can promote myocardial injurythrough multiple mechanisms, including production of reac-tive oxidants and diffusible radical species, activation ofprotease cascades, and expression of proinflammatory cyto-kines.4–9 Myeloperoxidase (MPO), an enzyme released byactivated neutrophils and monocytes, functions as a majorenzymatic source of leukocyte-generated oxidants and ispresent in infarcted myocardium. MPO deposition has beenshown in a murine model of acute myocardial infarction(AMI) to be increased near the sites of myocardial rupture.10

Using mice with a functional deficiency of MPO (ie, MPOknockout mice [MPO�/�]) and a chronic ischemia model ofleft anterior descending coronary artery (LAD) ligation, we

recently identified a significant role for MPO in determiningearly ventricular rupture rates and LV size after AMI.8 Amechanism was proposed for these effects of MPO onventricular remodeling after AMI: activation of the plasmin-ogen/plasmin protease cascade via MPO-mediated oxidationof plasminogen activator inhibitor-1 (PAI-1).8 Whether alter-native processes contribute to the effect of MPO on cell injuryand necrosis in and around infarcted myocardium in thismodel, such as through generation of cytotoxic species, wasnot explored because an ischemia/reperfusion model was notused.

Clinical Perspective p 2820MPO levels are commonly used as quantitative measures

of the extent of inflammation in tissues. In addition to merelyserving as a marker for leukocyte recruitment, this hemopro-

Received February 10, 2005; revision received August 6, 2005; accepted August 11, 2005.From the Departments of Cell Biology (N.V., M.B., S.U., X.Z., S.L.H., M.S.P.) and Cardiovascular Medicine (T.W., E.J.T., S.L.H., M.S.P.) and Center

for Cardiovascular Diagnostics and Prevention (M.B., S.L.H.), Cleveland Clinic Foundation, Cleveland, Ohio; Free Radical and Radiation BiologyProgram, Department of Radiation Oncology, University of Iowa, Iowa City (D.R.S.); and Department of Medicine, University of Washington, Seattle(J.W.H.).

Correspondence to Marc S. Penn, MD, PhD, NC10, Departments of Cardiovascular Medicine and Cell Biology, Cleveland Clinic Foundation, NC10,9500 Euclid Ave, Cleveland, OH 44195. E-mail [email protected]

© 2005 American Heart Association, Inc.

Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.105.542340

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tein can participate in the injury process by virtue of gener-ating reactive species. MPO forms cytotoxic oxidants such ashypochlorous acid (HOCl), molecular chlorine (Cl2), andmonochloramines (RNHCl) that can damage cellular targetsin vitro and in vivo.11,12 Several decades ago, Zgliczynski andcolleagues13,14 suggested the formation of reactive carbonyl-containing products after interaction of amino acids with theMPO-H2O2-Cl� system. In subsequent in vitro studies, westructurally defined a family of aldehydes as the productsformed, as well as their mechanism of generation via theoxidation of common �-amino acids by the MPO-H2O2-Cl�

system of neutrophils.15,16 With the use of multinuclear NMR,high-resolution mass spectrometry, and isotopically labeledprecursor amino acids, the mechanism was shown to involveinteraction of HOCl with the �-amino moiety forming a labileN�-monochloroamine intermediate, which rapidly decom-poses into an aldehyde via a concerted deamination anddecarboxylation reaction.16

Aldehydes identical in structure to those that can begenerated by MPO-mediated oxidation of plasma amino acidsin model systems have been detected in vivo. However, directdemonstration of a role for MPO in formation of reactivealdehyde species in vivo is still lacking. For example,acrolein, a product of MPO-mediated oxidation of threo-nine,15–17 has been detected in vivo. However, the relativecontribution of MPO-catalyzed threonine oxidation to acro-lein formation in vivo and the subsequent effects of MPO-generated acrolein on myocardial necrosis after MI areunknown. A role for MPO in the generation of aldehydes in vivohas been suggested by detection of p-hydroxyphenylacetaldehyde(pHA), an aldehyde formed by exposure of free tyrosine tothe MPO-H2O2-Cl� system, in tissues.18 However, the spec-ificity of pHA as a marker of MPO-catalyzed amino acidoxidation in vivo has not yet been established. Moreover, thephysiological consequences of aldehyde formation duringMPO-catalyzed oxidation of amino acids have not yet beenexplored. The generation of cytotoxic aldehydes by MPOcould have multiple potential implications on the pathophys-iology of cardiovascular disease, including plaque rupture,aneurysm formation, and tissue necrosis after AMI.19,20

In this study we examined the cytotoxic properties ofreactive aldehydes derived from MPO- and neutrophil-mediated oxidation of �-amino acids using cell types repre-sentative of those found in cardiovascular tissues. Further-more, the potential role of MPO in the formation of specificcytotoxic aldehydes in ventricular tissues after AMI wasexamined with the use of MPO�/� mice. The results of thepresent studies demonstrate that MPO is capable of promot-ing oxidation of amino acids to form aldehydes that areextremely cytotoxic to cells of vascular origin. However,detailed studies using a model of ischemia/reperfusion sug-gest that these MPO-derived cytotoxic aldehydes do notsignificantly contribute to myocardial necrosis because in-farct size in MPO-null mice was identical to that measured inwild-type mice. Rather, our data demonstrate markedly im-proved LV function and decreased LV dilation in the MPO-null mice at 24 days after MI. In aggregate, our datademonstrate that leukocyte-derived oxidants do not play a

critical role in myonecrosis but rather have a significantimpact on LV remodeling and ventricular function.

MethodsGeneral ProceduresAll glassware was soaked in NaOCl, extensively rinsed with H2O,and then baked at 500°C overnight before use. H2O was double glassdistilled and stored in glassware with Teflon-lined caps to avoid lowlevels of contaminating short-chain aldehydes shed from plastic anddeionizing cartridges from water purification systems. Buffers andreagents were prepared and stored in pyrolized glassware cappedwith Teflon-lined caps. Buffers were demonstrated to be chlorine-demand free before use, as previously described.15 MPO (A430/A280

ratio �0.8) free of contaminating eosinophil peroxidase was isolatedand stored as previously described.21 H2O2, NaOCl, and 4-hydroxy-2-nonenal (4-HNE) concentrations were determined spectrophoto-metrically (A240�39.4 mol/L�1 · cm�1; A292�350 mol/L�1 · cm�1;A223�13 750 mol/L�1 · cm�1, respectively).22,23

Aldehydes derived from amino acid oxidation were synthesized bydropwise addition of HOCl/ClO� to amino acid solutions (1:1,mol:mol, oxidant:amino acid) at 37°C, as previously described.15

Aldehyde synthesis was confirmed through structural and chromato-graphic characterization by gas chromatography/mass spectrometry(GC/MS) as their oxime derivatives after reaction with pentafluro-benzyl hydroxylamine (PFBHA), as previously described.15 In thecase of pHA synthesis from tyrosine, purity was assessed byreverse-phase high-performance liquid chromatography as de-scribed.24 Throughout sample processing, care was taken to avoidlosses of volatile aldehydes and derivatives through use of gas-tightsyringes, septa-covered reaction vials, and refrigeration or freezingof samples during analyses. Total glutathione (GSH) content (GSHplus glutathione disulfide) was determined with the use of aspectrophotometric assay based on the reaction of GSH with Ell-man’s reagent and the subsequent recycling of glutathione disulfidewith glutathione reductase as described.25

Cell CultureHuman aortic vascular smooth muscle cells, bovine pulmonaryartery endothelial cells, and the human monocytic cell line desig-nated 28SC were obtained from ATCC, Rockville, Md (cell linesCRL-1999, CRL-1733, and CRL-9855, respectively). Cells weremaintained according to the vendor’s specifications in the followingcell culture media: for human vascular smooth muscle cells: Ham’sF12K medium with 2 mmol/L L-glutamine adjusted to contain 1.5g/L sodium bicarbonate supplemented with 10 mmol/L HEPES,10 mmol/L TES, 0.05 mg/mL ascorbic acid, 0.01 mg/mL insulin,0.01 mg/mL transferrin, 10 ng/mL sodium selenite, 0.03 mg/mLECGS; for bovine pulmonary artery endothelial cells: Ham’s F12Kmedium with 2 mmol/L L-glutamine adjusted to contain 1.5 g/Lsodium bicarbonate); and for the human peripheral blood–derivedmonocytic cell line 28SC: Iscove’s modified Dulbecco’s mediumwith 4 mmol/L L-glutamine adjusted to contain 1.5 g/L, 0.02 mmol/Lthymidine, 0.1 mmol/L hypoxanthine, 0.05 mmol/L mercaptoetha-nol. All media were supplemented with 10% fetal bovine serum, andall cultures were maintained at 37°C in 5% CO2 and air in humidifiedincubators (NuAire). HA1 Chinese hamster ovary fibroblasts weremaintained in Eagle’s MEM as previously described.25 Wild-typeand MPO knockout mice (MPO�/�) were obtained and maintained aspreviously described.26

MPO- and Neutrophil-Mediated OxidationReactionsAmino acid stock solutions (2 mmol/L, except for His and Trp,which were 1 mmol/L) were prepared in Dulbecco PBS with MgCl2

and CaCl2 (Gibco-BRL). Amino acids (100 �mol/L, unless other-wise indicated) were incubated with H2O2 (100 �mol/L) and MPO(25 nmol/L) in sealed reaction vials at 37°C for 1 hour. Catalase wasthen added (30 �g/mL) to scavenge any residual H2O2, and theresulting solutions were incubated with cultured cells for survival

Vasilyev et al Inflammation and LV Remodeling 2813



experiments for 1 hour, unless otherwise indicated. Formation of theanticipated aldehydes in reactions was confirmed before use for allstudies by GC/MS analysis of oxime derivatives after reaction ofaliquots with PFBHA by methods previously described,15 as well asdescribed below.

All protocols for blood donation for neutrophils were approved bythe Cleveland Clinic Foundation institutional review board, andpatients gave written informed consent. Human neutrophils wereisolated by buoyant density centrifugation from plasma anticoagu-lated with EDTA (5 mmol/L) as described.16 Experiments wereperformed in Dulbecco PBS (pH 7.2) with MgCl2 and CaCl2

(Gibco-BRL) supplemented with 100 �mol/L diethylenetriamine-pentaacetic acid (DTPA). Amino acid oxidation experiments withhuman neutrophils were performed in gas-tight vials to prevent lossof volatile aldehydes generated during the prolonged neutrophilincubations. After incubation, formation of reaction products wasconfirmed as their PFBHA oxime derivatives by GC/MS.15

Assays of Cellular InjuryCytotoxicity was monitored by assessing reproductive integrity withthe use of the clonogenic cell survival assay; metabolic viability wasmonitored by trypan blue dye exclusion; and maintenance of cellmembrane integrity was determined by monitoring lactate dehydro-genase (LDH) release, as previously described.25 Briefly, for sur-vival analysis, cells were treated with reaction mixtures containingaldehydes for 1 hour at 37°C and rinsed, and then cells recovered byexposure to trypsin (0.1%) and EDTA (500 �mol/L). Cells insuspension were then diluted in complete media and plated atdifferent densities generated by serial dilutions for clonogenicsurvival for each exposure, and control/sham conditions were used tocalculate the data shown. Thus, each data point arises from multipledishes. Metabolically viable cells were counted by mixing cells withtrypan blue (1:1, vol/vol with 0.4% trypan blue in saline), and �100cells counted under a phase-contrast microscope with blue cells werescored as dead. HA1 control cloning efficiencies were 73% to 84%,and �95% of sham-treated cells excluded trypan blue dye. Allexperimental clonogenic survival data were normalized to therespective sham-treated control cell type. In selected experiments,LDH release from cells was quantified as a measure of cellular injurywith the use of a TOX-7 in vitro toxicology assay kit (SigmaChemical Co). This method uses spectrophotometric monitoring ofLDH-catalyzed reduction of NAD into NADH, coupled to stoichio-metric conversion of a tetrazolium dye.

LAD Ligation/ReperfusionAll animal protocols were approved by the Animal ResearchCommittee, and all animals were housed in the Association forAssessment and Accreditation of Laboratory Animal Care Interna-tional–approved animal facility of the Cleveland Clinic Foundation.Anterior wall MI was performed as recently described.8 Briefly,AMI was induced in �20- to 25-g male littermate wild-type(MPO�/�) (C57BL/6J) or knockout (MPO�/�)26 mice by ligation ofthe LAD.10,27 Animals were intubated and ventilated with room air at100 breaths per minute with the use of a rodent ventilator (HarvardApparatus). Sternotomy was performed, and the proximal LAD wasidentified with the use of a surgical microscope (Leica M500) afterretraction of the left atrium and ligated with 7-0 Prolene. Blanchingand dysfunction of the anterior wall verified LAD ligation. For thoseanimals that subsequently underwent reperfusion, microsurgicalscissors were used to cut the knot in the ligature at the level of themyocardium 30 minutes after LAD ligation. Successful reperfusionwas verified by return of red color to the tissue that was initiallyblanched at the time of LAD ligation, as well as gross evidence ofsome recovery of anterior wall motion.

During our study, we had �40% mortality in the MPO�/� andMPO�/� mice on the day of surgery and an additional 10% mortalityduring the first night after surgery. For animals that were used forassessment of aldehyde formation or protease activation, 24 and 72hours after MI, animals were anesthetized and rapidly perfused withsaline to clear blood, and the hearts were removed and flash-frozenin liquid nitrogen.

Quantification of Area at Risk and Infarct SizeSeventy-two hours after MI, animals were anesthetized and placedback on the ventilator. A PE50 catheter was placed into the aorticroute via the right carotid artery. The chest was opened, and aligature was placed around the LAD at the same site as the originalligature was placed 72 hours previously. Once the LAD was ligated,Evan’s blue dye (1 mg/mL) was infused to define the volume ofmyocardium not at risk. After Evan’s blue dye infusion, the heartwas harvested and sectioned into 3 pieces defined as the base, mid,and apex. The base and mid sections were incubated in tetrazoliumfor 30 minutes, rinsed, and then placed in formalin overnight.

Quantification of Amino Acid–Derived Aldehydesby GC/MSAldehydes underwent derivatization with 10 mmol/L final concen-tration of freshly prepared PFBHA (PFBHA stock; 100 mmol/L inH2O). This derivatization method readily reverses Schiff bases andother reversible covalent adducts (eg, with thiol residues) forming astable oxime. NaOH (10N stock) was then added to a final concen-tration of 1N, and the mixture was incubated at 65°C for 1 hour ingas-tight vials. [D8]Phenylacetaldehyde was added to samples im-mediately before GC/MS analyses to correct for potential inter-sample variations in derivatization and extraction efficiencies.[D8]Phenylacetaldehyde was synthesized from L-[D8]phenylalaninewith reagent NaOCl, as previously described.15,16 Samples wereinitially extracted with ethyl acetate (1:1, vol/vol). The remainingaqueous phase was then acidified with concentrated HCl (finalconcentration 2N) and again extracted with ethyl acetate, as above.The combined organic extracts were stored in gas-tight vials untilanalysis.

GC/MS experiments were performed on a Finigin VoyagerGC/MS system in negative ion chemical ionization mode undersplitless conditions with helium and methane as the carrier andreagent gases. Gas chromatographic separations of PFB-oximederivatives of aldehydes were conducted on a Restek RTX-200column (15 m, 0.33-mm ID, 1-�m film thickness) with the use of alinear temperature gradient over 20 minutes from an initial columntemperature of 70°C to a final temperature of 280°C. Identificationand quantification of PFB-oxime derivatives of aldehydes wereaccomplished as described.15,16,28 We have previously extensivelydescribed the mass spectrometric analyses of oxime derivatives ofaldehydes in biological matrices,28 where fragmentation analysesrevealed conserved fragmentation pathways, and the utility ofmonitoring structurally informative ions (eg, the molecular anion[M] · �, molecular ion [M] [M-HF]�, [M-HF-HCN]�, or [M-181]�)and common ions shared by all oxime derivatives of aldehydes(mass-to-charge ratio 178 or 181). Aldehyde identification wastherefore confirmed by demonstrating comigration of at least 2structurally informative ions and 1 common ion, with an identicalretention time to that observed with authentic derivatized material(commercial or generated with HOCl/NaOCl) in the selected ion-monitoring mode, as described.15

Statistical AnalysesAll data are expressed as mean�SD. Statistical analysis was per-formed with use of SPSS software (version 10.0 for Windows, SPSSInc). Continuous variables were compared with the use of nonpara-metric 1- and 2-factor Wilcoxon rank sum tests. A value of P�0.05was considered statistically significant.

ResultsIdentification of MPO-Generated AldehydesProduced During Oxidation of Amino Acids ThatAre CytotoxicTo identify cytotoxic products of MPO-mediated oxidationof amino acids, we exposed cells to culture media condi-tioned by prior incubation of the MPO-H2O2-Cl� systemalone (control) or in the presence of each of the 20

2814 Circulation November 1, 2005



common �-amino acids. The cytotoxic properties of theMPO-generated oxidation products were then initiallyassessed by performing clonogenic cell survival assaysafter 1-hour exposures to cultured HA1 fibroblasts (Figure1). Although no significant toxicity was noted in cellsexposed to low levels of the MPO-H2O2-Cl� system alone,enhanced clonogenic killing was noted if specific aminoacids were included in the MPO-H2O2-Cl� reaction mix-tures, consistent with formation of more stable cytotoxicby-products of amino acid oxidation. The greatest extentsof cytotoxicity were seen after exposure to media contain-ing the MPO-H2O2-Cl� system plus either glycine (alde-hyde derivative: formaldehyde) or threonine (aldehydederivative: 2-hydroxyproprianaldehyde�acrolein).15,17

Mild cytotoxicity was also observed with the oxidation

products of lysine and tryptophan (Figure 1). Glycoalde-hyde, derived from serine oxidation and a precursor ofadvanced glycation end products,15,17 demonstrated mini-mal cytotoxicity. Similarly, exposure to media containingtyrosine plus the MPO-H2O2-Cl� system, which producespHA in high yield,18 demonstrated nominal toxicity.

The Product(s) Formed During MPO-CatalyzedOxidation of Threonine Is a Potent Cytotoxin toCells Found in Cardiovascular TissuesMPO-H2O2-Cl� treatment of L-threonine produced a cyto-toxic species that was significantly more toxic than 4-HNE ona molar basis and comparable in toxicity to authentic acrolein,as monitored by clonogenic cell survival assays in HA1fibroblasts (Figure 2A) and pulmonary artery endothelialcells (Figure 2B). The MPO-H2O2-Cl� oxidation products ofneither tyrosine nor serine (pHA and glycolaldehdye, respec-tively) elicited significant toxicity on exposure to cells, evenat levels as high as 1 mmol/L (Figure 2A). To determine thecytotoxic potencies of L-threonine oxidation products of theMPO-H2O2-Cl� system, we compared the relative toxicity ofproducts formed from the MPO-H2O2-Cl� system aloneversus the MPO-H2O2-Cl� system plus L-threonine, as well asauthentic 4-HNE and glycolaldehyde, using alternative celltypes present in cardiovascular tissues, including vascularsmooth muscle cells and a monocytic cell line. Each of thecell types examined demonstrated significantly enhancedtoxicity on exposure to the MPO-H2O2-Cl� system in thepresence versus absence of L-threonine. The toxicity of theproduct formed by the MPO-H2O2-Cl�–threonine system wasmore toxic on a molar basis than 4-HNE.

Neutrophil Activation in Media Containing theAmino Acid L-Threonine Forms a CytotoxicAldehyde via the MPO-H2O2-Cl� System:Cytoprotective Effects of Intracellular GlutathioneTo demonstrate that activated neutrophils (polymorphonu-clear leukocytes [PMNs]) could mediate the formation ofcytotoxic products of L-threonine oxidation, clonogenic sur-vival was monitored in HA1 cells after a 1-hour exposure tothe L-threonine oxidation products generated by PMNs (Fig-ure 3A). We have previously shown that acrolein is formedby this system.11,17 Similar to the requirements for acroleinformation, production of a cytotoxic product required leuko-cyte activation with phorbol ester and the presence ofL-threonine (Figure 3A). Furthermore, production of a toxicproduct by leukocytes was inhibited by the addition of activecatalase or the heme protein inhibitor aminotriazole, demon-strating a critical role for cell-generated H2O2 and peroxidaseactivity, respectively. The aldehydic nature of the cytotoxicproduct was revealed by the effectiveness of catalyticallyactive aldehyde dehydrogenase (ADH) in partially detoxify-ing the L-threonine oxidation product (Figure 3A). Confirma-tion of a role for the MPO-H2O2-Cl� system in neutrophils forthe generation of cytotoxic products from L-threonine wasachieved by demonstrating that the cell-free system (MPO-H2O2-Cl�) similarly generated oxidation products ofL-threonine that were cytotoxic (Figure 3B). Furthermore, thealdehydic nature of the cytotoxic product was again con-

Figure 1. Relative toxicities of MPO-H2O2-Cl�–generated oxida-tion products alone and in the presence of each common aminoacid. Clonogenic survival assays using HA1 fibroblasts wereperformed after 1-hour exposure to the MPO-H2O2-Cl� systemin the absence (control) vs presence of the indicated aminoacid, as described in Methods. Note that under the conditionsused, the HOCl formed failed to produce significant clonogenictoxicity, whereas addition of some amino acids to reaction mix-tures markedly enhanced the toxicity of the system. The con-centrations of H2O2 and the indicated amino acids used forMPO (40 nmol/L) reactions were 100 �mol/L each. Catalasewas added at the end of each reaction before exposure of thecells to remove residual H2O2. Data are expressed as percenttoxicity relative to surviving fractions obtained from untreatedcells. Data are from a representative experiment performed intriplicate. Data points represent the mean of at least 3 cloningdishes taken from a single treated culture. *P�0.01 comparedwith control.




firmed by showing that catalytically active ADH partiallydetoxified the L-threonine oxidation product (Figure 3B).

These results are consistent with acrolein, an aldehydeproduced during exposure of L-threonine to the MPO-H2O2-Cl� system, as a major cytotoxic species formed during

neutrophil activation in the presence of L-threonine. Acroleinis a known cytotoxin, which induces cell death via pathwayssensitive to intracellular GSH levels.29,30 We therefore soughtto examine the influence of cellular GSH levels on thecytotoxicity of the product formed by exposure of L-threonineto the MPO-H2O2-Cl� system. Bovine endothelial cells werepretreated overnight with 1 mmol/L buthionine sulfoxide(BSO), and depletion of intracellular GSH pools (�95%reduction) was confirmed as described.25 Consistent withprior observations with acrolein, GSH depletion resulted in adramatic sensitization to the toxic effects of the amino acidoxidation products (Figure 3C).

MPO Serves as a Primary Source for GeneratingCytotoxic Aldehydes in Ventricular Tissues After AMITo determine whether amino acid–derived aldehydes areformed in a MPO-dependent fashion in vivo, we harvestednormal and ischemic ventricular tissues from wild-type andMPO�/� mice that were sham operated versus those after MI.AMI was induced in mice by direct LAD ligation. The LADwas either chronically ligated (hearts collected 72 hours afterAMI) or ligated for 30 minutes followed by reperfusion(hearts collected 24 hours after AMI). Mass spectrometryanalyses of these hearts revealed that low levels of aminoacid–derived aldehydes were observed in normal myocardialtissue (ie, sham operated) from wild-type and MPO�/� ani-

Figure 2. Dose dependence of toxicity from 1-hour exposure toamino acid oxidation products. A, Clonogenic survival of HA1fibroblasts after 1-hour exposure to the indicated concentrations ofauthentic acrolein, the product of threonine oxidation by the MPO-H2O2-Cl� system, 4-HNE, authentic glycolaldehyde, and pHA. B,Comparison of cytotoxicity of 4-HNE and the product formed byL-threonine oxidation by the MPO-H2O2-Cl� system on pulmonaryartery endothelial cells. Concentrations shown indicate those usedfor aldehyde exposures. In the case of L-threonine oxidation, con-centrations noted on the x axis correspond to levels of both H2O2

and L-threonine included in reaction mixtures, as described inMethods. Note the different x-axis scales and logarithmic y axes inA and B, done to illustrate the potent cytotoxicity of both 4-HNEand acrolein, the product of L-threonine oxidation by the MPO-H2O2-Cl� system, relative to pHA and glycoaldehyde. Data arefrom a representative experiment performed in triplicate. Datapoints represent the mean of at least 3 cloning dishes taken from asingle treated culture. At all concentrations �0 �mol/L, acrolein,threonine, and 4-HNE treatment led to statistically significantdecreases in clonal survival (P�0.001) compared with pHA andglycolaldehyde. At all concentrations studied, treatment withL-threonine oxidized by MPO-H2O2-Cl� led to statistically signifi-cant decreases in clonal survival compared with 4-HNE (P�0.01).

Figure 3. Activated neutrophils use the MPO-H2O2-Cl� systemto generate cytotoxic products of L-threonine (Thr) oxidation.Toxicity (percentage relative to sham exposure) is indicated, asmeasured by clonogenic survival with the use of HA1 cells after1-hour exposure to the L-threonine (100 �mol/L) oxidation prod-ucts generated by activated PMNs (1�106/mL) (A) or the MPO-H2O2-Cl� system (B) with the indicated additions or deletions. C,Clonogenic survival of bovine aortic endothelial cells after1-hour exposure to the L-threonine oxidation products gener-ated with and without BSO pretreatment. ATZ indicates amin-otriazole; cofactor, NADH; hkCatalase, heat-inactivated catalase;and PMA, phorbol ester. Data points represent the mean of trip-licate determinations. *P�0.01 compared with complete system.




mals (P�0.50 for all comparisons between wild-type andMPO�/� mice). In both AMI models, significant increases inthe level of all aldehydes monitored were noted in wild-typecompared with MPO�/� mice (Figure 4). Interestingly, asignificant amount of aldehyde generation in wild-type mice(72% formaldehyde and 42% acrolein relative to 72-hourchronic ligation model) occurred within the first 24 hoursafter AMI, during early neutrophil recruitment.

MPO-Generated Oxidants Do Not Significantly AffectVentricular Infarct Size After Ischemia/ReperfusionInjury but Dramatically and Adversely Affect LVRemodeling and Function After MITo determine whether MPO-generated cytotoxic aldehydeshave a role in myonecrosis after AMI, we compared infarctsize using an ischemia/reperfusion model. After 30-minuteischemia and 72-hour reperfusion, we found no significantdifference in infarct size whether expressed directly or as apercentage of the area at risk (Figure 5A). Although these

results suggest that MPO may not directly participate inmyonecrosis after AMI, we further examined whether MPOmight participate in long-term LV remodeling and cardiacfunction in a model of ischemia/reperfusion using wild-typeand MPO�/� mice. Echocardiographic studies 24 days afterischemia/reperfusion revealed a significant increase in ante-rior wall thickness (0.96�0.07 mm in wild-type versus1.18�0.07 mm in MPO�/� mice; P�0.017), marked de-creases in LV end-systolic (decreased by 35.1%) and end-di-astolic dilation (decreased by 16.6%), and a significantincrease in LV function as measured by shortening fraction(increased by 52.2%) in MPO�/� mice (Figure 5B and 5C).

DiscussionInflammation and the subsequent oxidant generation afterAMI are correlated with a worse clinical outcome.2,31,32 Thewhite blood cell count at presentation in patients with acutecoronary syndrome has been shown to predict mortality at 6months.31 Similarly, the peak monocyte count after MI wasshown to predict LV size2 and ultimately the development ofcongestive heart failure.32 Although there is compellingevidence that inflammation and oxidant generation are linkedwith worse outcomes in patients with MI, the mechanism(s)

Figure 4. MPO generates aldehydic oxidation products afterAMI in vivo. The reduced Schiff base adduct formed betweenpHA and the �-amino moiety of protein lysine residues, pHA-lysine, was quantified by stable isotope dilution GC/MS analy-ses. Formaldehyde, acrolein, and glycoaldehyde were quantifiedas their PFBHA oximine derivatives by GC/MS analyses. Shownare analyses performed 3 days after MI induced by ischemia/reperfusion in wild-type (black bars; n�6) and MPO-null (openbars; n�6) mice or chronic ligation of the LAD in wild-type (graybars; n�4) and MPO-null (open bars; n�7) mice. Data representmean�SD. In all cases, generation of the reactive aldehyde spe-cies was markedly less in tissues recovered from MPO knock-out mice compared with wild-type mice (P�0.001 each).

Figure 5. No significant difference in infarct size but a significantdifference in cardiac size and function was observed between wild-type (WT) and MPO-null mice. A, After 30 minutes of LAD ligationand 72 hours of reperfusion, the left anterior artery was religated atthe same site. Evan’s blue dye was infused to define the area ofthe ventricle not at risk. The heart was harvested and stained withthe use of tetrazolium to identify viable and dead cardiac tissuein the area at risk (AAR) as a percentage of total LV area. Infarctsize (IS) as a percent area at risk was quantified as the area ofdead myocardium divided by the area at risk�100%. Data repre-sent mean�SD. No significant difference in any of these measureswas observed between groups. LV end-diastolic (LVEDD) and end-systolic (LVESD) dimensions (B) and LV shortening fraction (C) 24days after 30 minutes of LAD ischemia in wild-type (n�9) andMPO-null (n�8) mice as measured by 2-dimensional M-modeechocardiography are shown. At 24 days, the anterior wall wasthicker (*P�0.05), the ventricle was less dilated (*P�0.001), andshortening fraction was significantly greater (*P�0.001) in theMPO-null mice. Data represent mean�SD.




of this effect is still unclear. Whether inflammation after MIis linked to infarct expansion, altered LV remodeling, or bothis unknown.

One mechanism by which inflammatory infiltrate couldlead to infarct expansion or adverse ventricular function isthrough the generation of cytotoxic aldehydes. Aldehydesderived from lipid peroxidation have long been implicated asmediators of cell injury and oxidant stress during inflamma-tion and cardiovascular diseases. Several years ago, analternative pathway for aldehyde formation involving MPO-dependent oxidation of common plasma �-amino acids wasproposed.15,16 The pathway was initially identified duringincubation of free L-tyrosine with either HOCl alone, theMPO-H2O2-Cl� system, or activated phagocytes, all of whichgenerated an aldehyde, pHA, in high yield.24 Because of thehigh yield of aldehyde formation and the central role reactivealdehydes are thought to play in tissue injury at sites ofinflammation and vascular disease, the mechanisms involvedin aldehyde formation and the possibility that other aminoacids might similarly be converted to their correspondingaldehydes were explored. Subsequent studies demonstratedthe generality of aldehyde formation from oxidation ofcommon �-amino acids by the MPO-generated oxidantHOCl.15,16

In the present study the cytotoxic potential for MPO-generated aldehydes during amino acid oxidation was exam-ined with the use of a variety of toxicity assays includingclonogenic cell survival, a sensitive measure of cellularreproductive integrity. Oxidation products of several aminoacids markedly enhanced the cytotoxic potential of theMPO-H2O2-Cl� system in multiple cell types. The mostpotent species were produced during incubation of the MPO-H2O2-Cl� system with glycine and threonine, reactions knownto produce formaldehyde and acrolein, respectively, invitro.15,16 Furthermore, studies with both isolated humanleukocytes and cell-free MPO-H2O2-Cl� systems confirmedthe aldehydic nature of the cytotoxic species formed, asevidenced by its detoxification in the presence of catalyticallyactive aldehyde dehydrogenase. Although acrolein and itsanalogue, 4-HNE, both possess reactive �-�–unsaturated,�-hydroxy aldehyde moieties that give rise to the chemicalreactivity of these species, acrolein is �200-fold more potentas an electrophile.33 Enhanced cytotoxicity of MPO-generated acrolein, relative to less reactive aldehydes includ-ing 4-HNE, is thus consistent with the toxic potential of thesespecies depending on their chemical reactivity with criticalintracellular nucleophilic targets. Thus, GSH depletion, acommon feature of oxidant stress attendant with inflamma-tion, enhanced the cytotoxic potential of MPO-generatedreactive aldehydes (Figure 3). Prior studies have demon-strated that 4-HNE induces cytotoxicity in target cells25,34 thatis dependent on GSH conjugation of 4-HNE for cytoprotec-tion.35,36 The present studies suggest that MPO-generatedaldehydes similarly promote cytotoxicity, as well as contrib-ute to the enhanced cytotoxicity seen at sites of GSHdepletion, such as during inflammation.

To determine the in vivo relevance of aldehydes producedduring amino acid oxidation and their dependence on MPOfor generation, the levels of the cytotoxic species formalde-

hyde and acrolein, as well as the relatively noncytotoxicspecies pHA and glycoaldehyde, were determined after MI inwild-type and MPO�/� mice. Aldehydes were quantified withthe use of sensitive and specific GC/MS-based methodspreviously developed for monitoring aldehydes as theiroxime derivatives in complex biological tissues.15,16,28 Thedata clearly show that there is a significant increase in thegeneration of all aldehydes monitored in postinfarcted tissuesof wild-type mice. Furthermore, by comparing the levels ofaldehydes in tissues of wild-type and MPO�/� mice, asignificant role for MPO in the formation of these species wasclearly demonstrated. Finally, the levels of MPO-dependentcytotoxins formed in vivo, such as acrolein, were in excess ofthe levels required to promote potent toxic effects.

Free amino acids are collectively present at concentra-tions of 4 to 5 mmol/L in plasma.37,38 On a molar basis,they serve as abundant targets for interaction with haloge-nating oxidants produced by the MPO-H2O2-Cl� system ofactivated phagocytes. The present studies demonstrate thataldehydes derived from MPO-catalyzed oxidation of com-mon �-amino acids represent a highly relevant and rapidmechanism for generation of cytotoxic species at sites ofinflammation. The ability of aldehydes to react withnucleophilic moieties on proteins, lipids, and DNA alsosuggests that the generation of such species might repre-sent an important mechanism for covalent modification ofbiological targets at sites of inflammation.

One of the surprising findings of the present study is thesuggestion that MPO-generated oxidants do not play a sig-nificant role in myonecrosis after myocardial ischemia be-cause infarct sizes in wild-type and MPO�/� mice weresimilar. These data, taken in the context of those of Lefer andcolleagues39 that showed no significant decrease in infarctsize in the NADPH-null compared with wild-type mice andrecent clinical trials that showed no change in infarct size inpatients treated with an anti-CD18 antibody,40 collectivelyindicate that leukocyte-mediated oxidation in the inflamma-tory tissue after AMI does not have a significant role indetermining infarct size. Rather, the present results demon-strate that MPO-generated oxidants produced by leukocytesincrease LV dilation and worsen cardiac function in thepost-AMI setting. MPO may contribute to impairment ofmyocardial function and adverse ventricular remodeling afterMI via numerous potential mechanisms. MPO generates anarray of reactive oxidants and cytotoxic species, including thealdehydes demonstrated in the present studies. It is temptingto speculate that MPO-generated aldehydes or alternativereactive species may covalently modify critical residues onkey ion channels or transporters as a potential mechanismcontributing to impairment in myocardial contractile functionafter ischemia/reperfusion injury. Another potential determi-nant of LV dysfunction may involve MPO-dependent con-sumption of nitric oxide. Finally, yet another potential mech-anism through which MPO may affect ventricular remodelingis via activation of protease cascades after MI.3–8 Consistentwith our previous observations in a chronic LAD model ofMI,8 we observed a significant increase in plasmin activity intissue lysates from wild-type compared with MPO�/� mice 24hours after ischemia/reperfusion (data not shown). MPO-




catalyzed oxidative inactivation of PAI-1, ultimately resultingin enhanced plasmin activity, may thus serve as an additionalpotential mechanism by accelerating matrix degradation, arequisite process for ventricular wall thinning and chamberdilation.

In summary, the present studies serve to clarify the role ofinflammation after MI and identify MPO as a rational targetfor the development of pharmacological inhibitors to opti-mize LV remodeling and attenuate development of heartfailure after MI.

AcknowledgmentThis work was supported by National Institutes of Health grants P01HL076491, P01 HL77107, HL70621, HL51469, HL74400, andHL61878 and the American Heart Association. Mass spectrometrystudies were performed within the Mass Spectrometry Core Facilityof the Cleveland Clinic Foundation.

DisclosureDr Heinecke has served on the speakers’ bureaus of and/or receivedhonoraria from Merck and diaDexus and has served as a consultantto Oxis International.

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CLINICAL PERSPECTIVEInflammation and oxidant generation after acute myocardial infarction (AMI) have correlated with a worse clinicaloutcome in multiple studies. The white blood cell count at presentation in patients with acute coronary syndrome has beenshown to predict mortality at 6 months. Similarly, the peak monocyte count after AMI predicts left ventricular (LV) sizeand ultimately the development of congestive heart failure. Whether inflammation after myocardial infarction is linked toinfarct expansion, altered left ventricular remodeling, or both is under active investigation. Myeloperoxidase (MPO), anenzyme released by activated neutrophils and monocytes, functions as a major enzymatic source of leukocyte-generatedoxidants. MPO-generated oxidants have been shown to produce cytotoxic aldehydes and lipid oxidation products and toinhibit the activity of protease inhibitors. We hypothesized that in an ischemia/reperfusion model, MPO-null mice wouldhave significantly smaller infarcts because of the decreased generation of cytotoxic aldehydes like acrolein andformaldehyde. Contrary to our hypothesis, we found no difference in infarct size between the 2 groups despite a dramaticdecrease in cytotoxic aldehyde generation in the MPO-null group. Interestingly, we observed significantly less LV dilationand improved cardiac function in the MPO-null group. Our results indicate that worse outcome associated with an increasedwhite blood cell count and/or inflammation at the time of AMI is due to adverse LV remodeling and not increased infarctsize. These findings suggest that LV remodeling and functional parameters, but not infarct size, are the appropriate endpoints for clinical trials focused on modulating inflammation in patients with AMI.




Jay W. Heinecke, Douglas R. Spitz, Eric J. Topol, Stanley L. Hazen and Marc S. PennNikolay Vasilyev, Timothy Williams, Marie-Luise Brennan, Samuel Unzek, Xiaorong Zhou,

Infarct Size After Myocardial InfarctionMyeloperoxidase-Generated Oxidants Modulate Left Ventricular Remodeling but Not

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