activation of poly(adp-ribose) polymerase by myocardial

INTRODUCTIONOver the last 2 decades, coronary

reperfusion therapy has been estab-lished for the management of acute ST-segment elevation myocardial infarction(STEMI) as an absolute prerequisite forthe survival of ischemic myocardium(1). Paradoxically, reperfusion of ische-mic areas, in particular the readmissionof oxygen, may trigger tissue damageresulting in a spectrum of reperfusion-associated pathologies, collectivelycalled “reperfusion injury” (2,3), withclinical manifestations including myo-

cardial stunning, potentially lethal ar-rhythmias, and endothelial and/or mi-crovascular dysfunction resulting in theno-reflow phenomenon.

Reintroduction of abundant oxygen atthe onset of reperfusion evokes a burstof potent oxygen- and nitrogen-derivedfree radicals and is considered a funda-mental element of reperfusion injury(4,5). Free radicals and oxidants triggermodifications in lipid membranes andproteins and produce DNA damage,culminating in loss of cellular integrity(6). Poly(ADP-ribose) polymerase-1

(PARP-1), activated by single-strandDNA breaks, emerged as a critical regu-latory component of the immediate cel-lular response to DNA damage (7,8). Inphysiological conditions, PARP-1 is anabundant nuclear chromatin-boundDNA repair enzyme, catalyzing transferof ADP-ribose moieties from NAD+ toacceptor DNA binding proteins (7,9).Under pathophysiological conditions,overactivation of PARP leads to con-sumption of the cellular NAD+ and ATPcontent, mitochondrial dysfunction, andultimately, necrotic cell death (7); theenzyme plays a key role in myocardialreperfusion injury (10).

Hypoxia/reperfusion promote explicitPARP-1 activation, mitochondrial dam-age, and dysfunction in myocytes andin endothelial cells by decreasing themitochondrial transmembrane potential

M O L M E D 1 2 ( 9 - 1 0 ) 2 2 1 - 2 2 8 , S E P T E M B E R - O C T O B E R 2 0 0 6 | T O T H - Z S A M B O K I E T A L . | 2 2 1

Activation of Poly(ADP-Ribose) Polymerase by Myocardial Ischemia and Coronary Reperfusion in Human Circulating Leukocytes

Address correspondence and reprint requests to Csaba Szabo M.D., Ph.D., Depart-

ment of Surgery, University of Medicine and Dentistry of New Jersey, 185 South Or-

ange Avenue, University Heights, Newark, NJ 07103-2714 (phone: 973-972-5045; fax:

973-972-6803; e-mail: [email protected]). E.T.-Z. and E.H. contributed equally to this work.

Submitted July 11, 2006; accepted for publication July 31, 2006.

Emese Tóth-Zsámboki,1 Eszter Horváth,2,3 Katarina Vargova,1 Eszter Pankotai,2 Kanneganti Murthy,4

Zsuzsanna Zsengellér,4 Tamás Bárány,2 Tamás Pék,2 Katalin Fekete,2 Róbert Gábor Kiss,1 István Préda,1

Zsombor Lacza,2 Domokos Gerö,3 and Csaba Szabó3,5

1Cardiovascular Research Group of the Hungarian Academy of Sciences and Semmelweis University & National Health Center, Budapest, Hungary; 2Department of Human Physiology and Experimental Research, Semmelweis University Medical School, Budapest, Hungary; 3CellScreen Applied Research Center, Budapest, Hungary; 4Inotek Pharmaceuticals Corporation, Beverly, MA,USA; 5Department of Surgery, University of Medicine and Dentistry of Newark, NJ, USA

Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequentoveractivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARPis activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coro-nary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hy-droxy-2′-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in cir-culating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ri-bose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered byreperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually in-creased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage,which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. Theseresults represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to testwhether pharmacological inhibition of PARP may offer myocardial protection during primary PCI.Online address: http://www.molmed.orgdoi: 10.2119/2006–00055.Toth-Zsamboki

(10-12). In oxidant-challenged myocytes,PARP also regulates the mitochondrial-to-nuclear translocation of cell death fac-tors such as apoptosis-inducing factor(AIF) and cytochrome c (13). In vitro ex-perimental animal studies using per-fused heart systems demonstrated thatPARP inhibitors improve myocardialcontractility and preserve myocardialATP and NAD+ pools during reperfusion(14-16). PARP inhibitors significantly di-minish infarct size and reduce creatinephosphokinase levels and mortality inmouse, rat, pig, and rabbit models (10).Moreover, PARP inhibitors protect en-dothelial cell integrity, preserve endothe-lium-dependent relaxation, and down-regulate multiple pro-inflammatorygenes during reperfusion (17,18). Theseobservations were confirmed in vivo, bydemonstrating that transgenic mice lack-ing the functional PARP-1 gene are re-sistant to myocardial infarction (19). Fur-thermore, activation of PARP andbeneficial effects of PARP inhibition havebeen demonstrated after heart transplan-tation or cardiopulmonary bypass (20).

Recently it was reported in a ratmodel that free radical–mediated activa-tion of PARP is not limited to themyocardium: in response to myocardialischemia, in circulating leukocytes—similarly to myocytes—significant PARPactivation occurs after reperfusion. Thisphenomenon—which is most likelycaused by free radicals as the circulatingcells pass through the reperfused myo-cardial tissue—has been proposed toserve as a potential marker of myocardialoxidative and peroxidative injury (21).

Whereas the above experimental datademonstrated the role of PARP in myo-cardial reperfusion injury in animalmodels, PARP activation in human myo-cardial infarction has not yet been stud-ied. In the present study, circulating pe-ripheral leukocytes were isolated fromcardiovascular patients with STEMI toanalyze coronary reperfusion-relatedpathways in humans. The objective ofthe study was to test whether directDNA damage occurs during STEMI, asindicated by increased levels of serum

8OHdG (8-hydroxy-2′-deoxyguanosine).We also tested whether PARP-1 activitychanges after the primary PCI-mediatedcoronary reperfusion in circulatinghuman leukocytes. Moreover, we testedwhether reperfusion may induce tyrosinenitration (a marker of nitrosative stress)and whether it promotes nuclear translo-cation of AIF, a downstream event ofPARP-1 activation.

METHODS

Patient PopulationWe enrolled 15 cardiovascular patients

with acute ST-segment elevation myocar-dial infarction referred to our institutionfor primary percutaneous coronary inter-vention between October 2004 and Febru-ary 2005. Blood and leukocyte samplesfrom age-matched patients with stable an-gina pectoris undergoing elective coro-nary angiography (n = 6) and elective per-cutaneous intervention (n = 9) were alsocollected and analyzed in parallel as nega-tive controls. The study protocol was ap-proved by the institutional and regionalethics review committee, and written in-formed consent was obtained from allparticipating patients before enrollment.

Blood Sampling and Preparation ofPeripheral Leukocytes

Resting venous blood was taken intonative and heparin- and EDTA-containingtubes from STEMI and elective PCI pa-tients at 4 different time points: (1) beforethe angiography, (2) within 15 min afteropening of the infarct-related or targetcoronary artery, (3) 24 ± 4 h after the PCI,and (4) 96 ± 4 h after the PCI. Peripheralleukocytes were prepared usingHistopaque-1077 (Sigma-Aldrich, St.Louis, MO, USA) and the density gradi-ent centrifugation method from he-parinized blood (6 mL). After isolation(centrifugation at 400g for 30 min) andwashing in PBS, a 100-mL aliquot ofmononuclear cells was used to prepareperipheral smears for immunohistochem-istry. Remaining leukocytes were pelletedand kept at –80 °C until Western blotanalysis.

Hydrogen Peroxide and 8OHdG LevelMeasurements

Using the OxyStat assay (BiomedicaGruppe, Wien, Austria), we determinedthe total hydrogen peroxide concentra-tion in patient EDTA plasma samples(detection limit 7 μmol/L). Serum levelsof 8OHdG were measured using a com-petitive enzyme-linked immunosorbentassay based on a 8OHdG monoclonal an-tibody (Gentaur, Brussels, Belgium).Serum samples were purified using anultrafilter according to the instructions ofthe manufacturer.

Poly(ADP-Ribose) Western Blot AnalysisIsolated peripheral mononuclear cell

pellets were resuspended in SDS-containing loading buffer (20 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS,100 mM β-mercaptoethanol, and100 μg/mL bromophenol blue) to aconcentration of 2.5 × 107 cells/mL.Twenty microliters of the samplewas separated on a 4% to 20% SDS-polyacrylamide gel. Blots were probedwith anti-PAR (poly-ADP-ribose) poly-clonal antibody (EMD Biosciences) andwith an HRP-conjugated secondary anti-body. Bound enzyme was detected withthe enhanced chemiluminescence system(ECL, Amersham). Densitometric analy-sis of Western blots was performed usingAlphaImager (Alpha Innotech Corpora-tion, San Leandro, CA, USA); arbitrarydensitometric units were corrected tobackground intensity.

ImmunohistochemistryAnti-nitrotyrosine rabbit polyclonal

antibody (Upstate Biotechnology, LakePlacid, NY, USA) (1:80, 4 °C, overnight)was used to stain 3-nitro-tyrosine, themarker of tyrosine nitration. Poly(ADP-ribose) detection was performed usingthe mouse monoclonal anti-PAR anti-body (Tulip Biolabs, West Point, PA,USA) (1:100, 4 °C, overnight) after anti-gen retrieval (0.1 M citrate buffer, pH 3,cooked in microwave oven for 15 min).Anti-AIF rabbit polyclonal antibody(Chemicon International, Temecula, CA,USA) (1:100, 4 °C, overnight) was used to

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P A R P A C T I VA T I O N D U R I N G P R I M A R Y P C I I N A C U T E M I

label AIF. A specific labeling wasavoided by incubating the smears in 15%normal goat/horse serum for 1 h at roomtemperature. Secondary labeling wasachieved using biotinylated anti-mousehorse or anti-rabbit goat antibody (VectorLaboratories, Burlingame, CA, USA) (30min, room temperature). Horseradishperoxidase–conjugated avidin (30 min,room temperature) and diaminobenzi-dine (6 min, room temperature) wasused to visualize the labeling (VectorLaboratories). Smears were counter-stained with hematoxylin.

To determine the number of NT- andAIF-positive cells, at least 300 cells werecounted on each smear. SemiquantitativePAR-positivity score was establishedfrom 1 to 10 and used to score the slidesby an investigator who was blinded tothe identity of the individual slides: score1, no staining; 2, light cytoplasmic stain-ing; 3, strong cytoplasmic staining; 4, cy-toplasmic staining with a few positivenuclei; 5, approximately 50% of the nu-clei positive; 6, approximately 75% of thenuclei positive; 7, general nuclear stain-ing with a few negative cells; 8, all nucleipositive; 9, strong nuclear staining in allcells; 10, very strong general nuclearstaining in all cells.

Statistical AnalysisResults are expressed as mean ± SEM

and SD by box plots. Data were not nor-mally distributed; therefore, nonparamet-ric statistic tests were performed usingStatistica 6.0 software (Stat Soft, Tulsa,OK, USA). Mann-Whitney U test wasconducted to investigate the associationbetween independent parameters of dif-ferent patient groups. To analyze depen-dent variables, the Wilcoxon matched-pairs test was used. P values less than0.05 were considered significant.

RESULTS

Patient DemographicsDetailed patient demographics, clini-

cal parameters, and angiography andlaboratory test results are summarizedin Tables 1 and 2. The enrolled STEMI

R E S E A R C H A R T I C L E


Table 1. Patient baseline characteristics.

Acute ST-segment

Stable angina pectoriselevation

myocardial infarction Coronarography Elective PCI

n 15 6 9Age, y 68.13 ± 2.93 57.0 ± 2.47 61.22 ± 3.6Sex, M/F 9/6 2/3 5/4Body mass index, kg/m2 28.11 ± 0.86 30.83 ± 1.32 30.3 ± 1.28Risk factors

Family history of IHD 6 (40) 4 (80) 4 (66)History of tobacco use 7 (46.7) 5 (100) 9 (100)Hypertension 15 (100) 4 (80) 7 (77)Diabetes mellitus 5 (33) 2 (40) 4 (44)

Previous evidence of IHD 7 (46.7) 5 (100) 9 (100)Angina 1-6 days before admission 4 (26.7) 4 (80) 6 (66)Previous PCI 0 1 (20) 8 (88)Diagnosis on admission

Stable angina pectoris — 5 (100) 9 (100)Acute myocardial infarction — —

Inferior 3 (20)Infero-posterior 6 (40)Anterior 2 (13.4)Extensive anterior 3 (20)Posterior 1 (6.7)

Time from event to balloona

<3 h 3 (20)3-6 h 8 (53.3)>6 h 4 (26.6)

Coronarography resultsLM stenosis/occlusion 3/0 1/0 1/0LAD stenosis/occlusion 9/4 3/1 7/1RCA stenosis/occlusion 2/10 2/2 6/0LCx stenosis/occlusion 8/1 2/0 2/0R. diagonalis stenosis/occlusion 6/0 1/0 2/0RRV stenosis/occlusion 1/1 0/0 0/0OM stenosis/occlusion 6/0 0/0 1/0

Number of coronary stenoses per 2.27 ± 0.32 1.8 ± 0.58 2.11 ± 0.2patient

Number of coronary occlusions per 1.06 ± 0.12 0.6 ± 0.24 0.11patient

Number of implanted stents per 1.53 ± 0.27 — 0.88 ± 0.26patient

TIMI flow grade of the target vessel after PCI 2.77 ± 0.12 — 2.75 ± 0.16

ComplicationsAtrialflutter/fibrillation 2 (13.3) — —Ventricular tachycardia/fibrillation 4 (26.6) — —AV block 3 (20) — —Pacemaker therapy 2 (13.3) — —Cardiogenic shock 3 (20) — —IABP 2 (13.3) — —Gastrointestinal/femoral bleeding 4 (26.6) — —Exithus lethalis 2 (13.3) — —

Values are mean ± SEM or n (%). Percentages are compared to the entire patientpopulation. aTime between the onset of persisting chest pain and the beginning of thePCI. IHD indicates ischemic heart disease; PCI, percutaneous coronary intervention; LM,left main coronary artery; LAD, left anterior descending artery; RCA, right coronary artery;LCx, left circumflex artery; RRV, right retroventricularis; OM, obtuse marginal; TIMI,thrombolysis in myocardial infarction; IAB, intra-aortic balloon pump.

patients were predominantly men, withmultiple cardiovascular risk factors intheir medical history. All of them hadpermanent chest pain (maximum 12 h)and showed ST elevation (≥ 2 mm) in atleast 2 consecutive ECG leads on ad-mission. In each case, coronary angiog-raphy revealed subtotal or total coro-nary artery occlusion, and successfulrecanalization was confirmed by highTIMI flow rate values after PCI. Defini-tive acute myocardial damage wasdemonstrated by elevated CK/CK MBvalues.

Determination of the OxidativeImbalance: Plasma Total PeroxideConcentration

Myocardial reperfusion-related ox-idative/peroxidative stress is known tolead to cellular damage by lipid peroxi-dation (10). Therefore, total plasma hy-drogen peroxide concentration as an es-tablished marker of oxidative injuryand lipid peroxidation was determined.As shown in Figure 1A, total hydrogenperoxide concentration was not affectedby coronary reperfusion directly; pre-and post-PCI values were not differentstatistically. A significant increase wasobserved in total peroxide levels 24 and96 h after the acute cardiovascularevent. Baseline values in patients withstable angina pectoris and acute MIwere not different. Similar to the pri-mary PCI group, in control stable an-gina patients hydrogen peroxide con-centrations were identical before andafter coronarography (420 ± 90 vs. 431 ±104 μmol/L, respectively, n = 5, datanot shown).

Verification of PCI-Related DNADamage: Serum 8OHdG LevelMeasurements

DNA is a major target of constantoxidative damage from endogenousoxidants (4). In contrast to peroxide levels,primary PCI, representing a successfulmyocardial reperfusion, led to an explicit,rapid 8OHdG level increase (P < 0.005),

indicating systemic, immediate, reperfu-sion-related DNA damage in the STEMIpatients in response to PCI (Figure 1B).Furthermore, unlike peroxide concentra-tions, 8OHdG levels were normalizedwithin 96 h. Serum 8OHdG concentra-tions in control stable angina patientswere not different statistically from pre-PCI STEMI values.

Analysis of PARP-1 Activation inCirculating Leukocytes

Immunohistochemistry and densitom-etry analysis of the buffy-coat cell pelletWestern blots (n = 15) confirmed a sig-nificant immediate PARP-1 activation incirculating leukocytes due to myocardialreperfusion achieved by primary PCI(Figure 2A,C). Similar to the kinetics of8OhdG serum levels, the increase ofPARP-1 activity in the isolated leuko-



Table 2. Laboratory results in the STEMI group

Pre-PCI Post-PCI After 24 h After 96 h

CK, U/L 230.6 ± 64.9 2152.8 ± 914.3 854.2 ± 194.42 275.5 ± 58.7CKMB, U/L 53.3 ± 17.1 254.4 ± 108.7 121.1 ± 35.46 33.25 ± 6.3LDH, U/L 366.1 ± 35.8 — 1339.21 ± 207.2 1703 ± 488.6AST, U/L 40.13 ± 8.29 — 174.1 ± 35.84 157.63 ± 91.53ALT, U/L 27.2 ± 5.51 — 48.07 ± 9.61 56.18 ± 16.03

Values are mean ± SEM. CK indicates creatine kinase; LDH, lactate dehydrogenase; AST,aspartate aminotransferase; ALT, alanine aminotransferase.

Figure 1. Determination of the oxidative imbalance in patients with stable anginapectoris and acute ST-segment elevation myocardial infarction. (A) Total plasma hy-drogen peroxide concentration measurements. (B) Serum 8OHdG level measure-ments. Results are expressed as mean (represented by squares) ± SEM (representedby boxes) and ± SD (represented by bars). Lane 1 indicates peroxide levels in stableangina patients; lanes 2-5 show peroxide concentration in patients with acute myo-cardial infarction before coronarography (lane 2), just after the successful primary PCI(lane 3), 24 ± 4 h after reperfusion of the ischemic myocardium (lane 4), and 96 ± 4 hafter PCI (lane 5). Primary PCI itself did not affect total peroxide levels; gradual in-crease of hydrogen peroxide concentration was observed at 24- and 96-h time pointsafter myocardial infarction. In contrast, serum 8OHdG levels showed a significant,rapid increase after the primary PCI, and were normalized by 96 h. *P < 0.05, **P <0.005; NS, nonsignificant.

cytes occurred rapidly after myocardialreperfusion and decreased over time.Importantly, in patients undergoing elec-tive percutaneous intervention (n = 9),no PARP-1 activation occurred after thePCI. These results indicate that signifi-cant myocardial ischemia is required be-fore reperfusion to induce PARP-1 acti-vation. Baseline initial PARP-1 activity instable angina and STEMI patients wascomparable.

Immunohistochemical Studies ofNitrotyrosine Production andTranslocation of AIF

Immunohistochemical staining demon-strated that tyrosine nitration of the iso-lated cells significantly increased afterPCI, compared with pre-PCI values (Fig-ures 3 and 4A). Again, when nitrotyro-sine-positive cells were counted, rapid ki-netics were observed: tyrosine nitrationwas maximal just after PCI and decreasedby 96 h (Figure 4A). The number of ni-trotyrosine-positive cells did not differbetween control and STEMI patientsprior to surgery (Figure 4A).

In contrast to these parameters,translocation of AIF to the nuclei showeda gradual tendency to increase (Figure 3),a difference that became significant com-pared with the pre-PCI values by day 4(23 ± 2% vs. 33 ± 5% positive cells, P <0.05, n = 8, Figure 4B).

DISCUSSIONDespite the fact that myocardial reper-

fusion therapy is often called a double-edged sword (2), the aim of the clinicianis to reperfuse the ischemic myocardiumas soon as possible to recover contractilefunction and to avoid irreversible myo-cardial damage. Previous studies havedemonstrated oxidative DNA injury andconsequent PARP-1 activation in car-diomyocytes, endothelial cells, andcirculating peripheral leukocytes in ani-mal models of acute myocardial infarc-tion (for review, see Jagtap and Szabo[22]). The current study investigatedmultiple aspects of human myocardialischemia/reperfusion-related pathologiesby analyzing serum, plasma, and iso-



Figure 2. Rapid activation of PARP-1 in peripheral leukocytes induced after recanalizationof the infarct-related coronary artery by primary PCI. (A) Densitometry analysis of PARWestern blots performed on patient leukocytes. (C) Immunohistochemical PAR-scoreanalysis of patient leukocytes. In both figures, lane 1 indicates densitometry units (A) orPAR-score values (C) in stable angina patients before coronarography; lanes 2-5 showPAR content in control patients with elective PCI before coronarography (lane 2), imme-diately after the successful PCI (lane 3), 24 ± 4 h after (lane 4), and 96 ± 4 h after PCI(lane 5); lanes 6-9 indicate PAR content in patients with acute myocardial infarction be-fore coronarography (lane 6), immediately after the successful PCI (lane 7), 24 ± 4 h afterreperfusion of the ischemic myocardium (lane 8), and 96 ± 4 h after PCI (lane 9). PrimaryPCI leads to a significant increase in leukocyte cellular PAR content, reflecting rapid acti-vation of PARP during reperfusion. A gradual decrease of PARP activity can be observedat 24- and 96-h time points after myocardial infarction. PARP activity is not affected dur-ing elective PCI. Results are expressed as mean (represented by squares) ± SEM (repre-sented by boxes) and ± SD (represented by bars). (B) Representative examples of PARWestern blots from 3 stable angina patient leukocytes as controls (first panel) and 3 STEMIpatients (second panel). Time points are indicated. Commercially available PARP en-zyme served as a positive control. *P < 0.05, NS, nonsignificant.

lated peripheral leukocyte samples fromcardiovascular patients with acute ST-segment elevation myocardial infarctionand successful primary PCI. Our resultsprovide evidence for (1) general oxidative/peroxidative imbalance (elevated totalplasma peroxide concentration and aug-mented nitrotyrosine production), (2) di-rect, PCI-generated DNA damage (evi-denced by increased levels of serum8OHdG), (3) rapid post-PCI activation ofPARP-1 in circulating human peripheralleukocytes (shown by immunohisto-chemistry and Western blotting), and(4) translocation of AIF from mitochon-dria to nuclei (which may be a down-stream signaling event triggered byPARP-1 activation). These results providethe first clinical evidence for PARP acti-vation in patients with myocardial in-farction and are consistent with the con-cept that local myocardial hypoxia/reperfusion triggered by percutaneousinterventions in acute myocardial infarc-tion is able to trigger systemic oxidativeresponses in humans.

Although the pathomechanism of reper-fusion injury has been extensively studied,relatively limited therapeutic applications



Figure 3. Immunohistochemical analysis of tyrosine nitration, PAR content, and AIF translocation. Representative examples indicating gradualincrease of the NT positive cell numbers (arrows) in peripheral leukocyte preparations after STEMI. The second row demonstrates increasedPAR content in leukocytes immediately after the primary PCI. In the third row, AIF staining was performed and positive cells are depicted byarrows; AIF translocation increased by 96 h. Leukocytes from a stable angina patient are shown as negative controls in the first column.

Figure 4. Tyrosine nitration (A) and AIF translocation (B) determined by immunohisto-chemistry scores. After staining, NT-positive cells were counted in peripheral leukocytesmears. Results are expressed as mean (represented by squares) ± SEM (representedby boxes) and ± SD (represented by bars). Lane 1 indicates control samples from sta-ble angina patients; lanes 2-5 show NT-positive cell counts or AIF translocation–positivecell counts in patients with acute myocardial infarction before coronarography (lane2), immediately after the successful primary PCI (lane 3), 24 ± 4 h after reperfusion ofthe ischemic myocardium (lane 4), and 96 ± 4 h after PCI (lane 5). Primary PCI in-duced an immediate increase in tyrosine nitration, whereas a gradual increase of AIFtranslocation was observed at 24- and 96-h time points after reoxygenation of the is-chemic myocardium. *P < 0.05; NS, nonsignificant.

have been developed to date (6,23). So far,therapeutic attempts to prevent reperfu-sion injury are limited to relatively small-scale studies testing the administration ofvitamin E (24), calcium antagonists (25),early use of ACE inhibitors, sulfhydryl-rich reagents such as N-acetylcysteine,magnesium, and various free radical scav-engers (26). However, accelerated myocytenecrosis and destruction related to reoxy-genation of the ischemic myocardium con-tinues to represent a clinically relevantquestion. It is generally accepted that thecombination of the reopening of the coro-nary artery with therapeutic approachesthat protect the reperfused myocardiummay improve the outcome of the PCI. Asdemonstrated by analysis of circulatingleukocytes passing through the reperfusedmyocardium, consequent PARP-1 activa-tion with abrupt kinetics also occurs. Al-though the specific cell types were notidentified with flow cytometry, it is wellknown from the literature that neutrophilsdo not contain the PARP enzyme (7).Therefore, the cells likely to be responsiblefor the observed increase in PARP activityare lymphocytes and/or monocytes.Taken together with previous data fromexperimental myocardial ischemia models(reviewed in Jagtap and Szabo [22]), onecan speculate that, as with leukocytes, my-ocyte PARP activation is likely to developin human reperfusion injury, leading tomyocyte necrotic cell death.

The present observations may also havedirect therapeutic implications. Potentnovel PARP inhibitors have been devel-oped in recent years, and these agentswere shown to be beneficial in in vitroand in vivo ischemia/reperfusion modelsas attenuating reperfusion injury by act-ing at several levels (prevention of ener-getic failure, inflammatory mediator pro-duction, neutrophil infiltration, andendothelial dysfunction) (22,27,28). There-fore, in theory—as is supported by ourhuman data confirming rapid PARP acti-vation due to primary PCI—PARP inhibi-tion before the planned reperfusion mightprovide multiple benefits, most impor-tantly, myocyte salvage and therefore im-proved survival (22). In this context, anal-

ysis of PARP activity in human circulatingleukocytes during PARP inhibitor treat-ment might serve as a useful marker andprovide evidence for the ability of PARPinhibitors to block the activation of thetarget enzyme in clinical trials in vivo.

In the present study, circulating leuko-cytes were used to analyze oxidative im-balance. Nevertheless, other circulatingcells such as circulating endothelial cellsmay also be investigated in future studies.These measurements might confirm in-volvement of oxidative/nitrosative stresspathways in such processes as pathophys-iological endothelial cell function and de-tachment from the vessel wall and mayexplore the role of PARP-1 in endothelialdysfunction related to reperfusion.

Besides acute myocardial infarction,various degrees of myocardial reperfu-sion injury may occur under such com-mon clinical conditions as elective percu-taneous coronary interventions. Our dataindicate that in the case of elective PCIwithout complications, PARP-1 activa-tion did not develop in circulating leuko-cytes. It would be important to testwhether in the case of periproceduralmyocardial necrosis—which is a negativeprognostic factor in patients with electivePCI (29-31)—the oxidative/nitrosativebalance triggers the PARP-1 pathway.

What, then, is the molecular trigger ofPARP activation in human myocardialinfarction? Based on animal studies, ox-idative and nitrosative stress (namely,hydrogen peroxide, peroxynitrite, andhydroxyl radical) are pathophysiologi-cally relevant triggers of PARP activa-tion (32). In the present study, tyrosinenitration (a relatively specific marker ofperoxynitrite production) but not hydro-gen peroxide levels showed a close cor-relation with the degree of PARP activa-tion, possibly implicating the role ofreactive nitrogen species, such as perox-ynitrite. We must point out, neverthe-less, that the likely trigger of PARP acti-vation is within the reperfusedmyocardial tissue, and peripheral bloodparameters may not necessarily correlatewith the changes within the myocardialtissue itself.

It is interesting to note that the cur-rent study demonstrated that AIFtranslocation occurs 4 days after PCIduring myocardial infarction. Thetranslocation of AIF can be triggered bymultiple factors, one of them beingPARP activation. It is unlikely thatPARP is the only contributor of AIFtranslocation in the current study, as thetime course of PAR staining and AIFtranslocation are quite different.

Although our observations provideevidence for primary PCI-relatedoxidative injury and subsequent DNAdamage–induced PARP-1 activation in ahuman cardiovascular patient cohort,our case numbers are limited. A subse-quent large-scale study would be neededto link and correlate these biochemicalmarkers to patient outcome and clinicalparameters, such as major cardiac eventsor cardiovascular death. Analysis of8OHdG or PARP-1 activity levels in con-text to serum troponin, CK MB values, orejection fraction would be particularlyimportant. Also, in future clinical studieswith PARP inhibitors, peripheral leuko-cyte PAR content or PAR immunostain-ing may serve as useful sentinel markersfor the efficacy of the PARP inhibitor toblock its enzymatic target in vivo. In fu-ture studies, using antioxidants or PARPinhibitors, one may also be able to assesswhether oxidant stress contributes to AIFtranslocation, whether there are alter-ations in the viability and lifetime of pe-ripheral blood cells, and whether thesechanges are related to oxidant stress andPARP activation.

In conclusion, our data provide evi-dence for PARP activation for the firsttime in humans suffering from myocar-dial infarction. In the present popula-tion of cardiovascular patients withST-segment elevation myocardial infarc-tion, primary percutaneous interventionis accompanied by significant systemicDNA damage, PARP-1 activation, andconsequent AIF translocation. PARP ac-tivation in circulating cells may serve asa sentinel pharmacodynamic marker inongoing or future clinical trials (22) uti-lizing PARP inhibitors.



ACKNOWLEDGMENTSThe study was supported by research

grants from the Hungarian Science Foun-dation (OTKA T042605, F046711, andK49488), the Hungarian Ministry ofHealth (ETT 086/2003, ETT 583/2003),and from the National Institutes of Health(RO1 GM60915 to CS). E.T.-Z. is a recipi-ent of the “Bolyai” Scholarship Grant ofthe Hungarian Academy of Sciences. E.H.was supported by the Hungarian Na-tional Eötvös Fellowship.

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