Oxidative stress induces myeloperoxidase expression in endocardial endothelial cells from patients with chronic heart failure

Giampiero La RoccaAntonino Di StefanoErmanno EleuteriRita AnzaloneFrancesca MagnoSimona CorraoTiziana LoriaAnna MartoranaClaudio Di GangiMarilena ColomboFabrizio SansoneFrancesco PataneFelicia FarinaMauro RinaldiFrancesco CappelloPantaleo GiannuzziGiovanni Zummo

Oxidative stress induces myeloperoxidaseexpression in endocardial endothelial cellsfrom patients with chronic heart failure

Received: 3 December 2007Returned for 1. Revision: 7 January 20081. Revision received: 18 June 2008Returned for 2. Revision: 17 July 20082. Revision received: 17 October 2008Accepted: 27 October 2008Published online: 22 November 2008

j Abstract Increased oxidative stress has been implicated in thepathogenesis of a number of cardiovascular diseases. Recent findingssuggest that myeloperoxidase (MPO) may play a key role in the initiationand maintenance of chronic heart failure (CHF) by contributing to thedepletion of the intracellular reservoir of nitric oxide (NO). NOconsumption through MPO activity may lead to protein chlorination ornitration, leading to tissue damage. Primary cultures of humanendocardial endothelial cells (EEC) obtained at heart transplantation ofpatients with CHF and human umbilical vein endothelial cells (HUVEC)were subjected to oxidative stress by incubation with hydrogen peroxideat non lethal (60 lM) dose for different exposure times (3 and 6 h).Treated and control cells were tested by immunohistochemistry and RT-PCR for MPO and 3-chlorotyrosine expression. Both endothelial celltypes expressed myeloperoxidase following oxidative stress, with higherlevels in EEC. Moreover, 3-chlorotyrosine accumulation in treated cellsalone indicated the presence of MPO-derived hypochlorous acid.Immunohistochemistry on sections from post-infarcted heart confirmedin vivo the endothelial positivity to MPO, 3-chlorotyrosine and, to aminor extent, nitrotyrosine. Immunohistochemical observations wereconfirmed by detection of MPO mRNA in both stimulated EEC andHUVEC cells. This study demonstrates for the first time that EEC canexpress MPO after oxidative stress, both in vitro and in vivo, followed byaccumulation of 3-chlorotyrosine, an end product of oxidative stress.Deregulation of endothelial functions may contribute to the developmentof a number of cardiovascular diseases, including CHF. The results alsohighlight the notion that endothelium is not only a target but also a keyplayer in oxidative-driven cardiovascular stress.

j Key words 3-chlorotyrosine – endocardium – endothelial cells –myeloperoxidase – oxidative stress

ORIGINAL CONTRIBUTIONBasic Res Cardiol 104:307–320 (2009)DOI 10.1007/s00395-008-0761-9

BR

C761

G. La Rocca and A. Di Stefano contributedequally to the current work.

Electronic supplementary material: Theonline version of this article (doi:10.1007/s00395-008-0761-9) contains sup-plementary material, which is available toauthorized users.

Dr. G. La Rocca (&) Æ R. AnzaloneF. Magno Æ S. Corrao Æ T. Loria Æ F. FarinaF. Cappello Æ G. ZummoSezione di Anatomia UmanaDipto. di Medicina SperimentaleUniversita degli Studi di PalermoVia del Vespro 12990127 Palermo, ItalyTel.: +39-091/7655-3576Fax: +39-091/655-3580E-Mail: [email protected]

A. Di Stefano Æ M. ColomboLaboratorio di CitoimmunopatologiaApparato Cardio-RespiratorioFondazione S. Maugeri, IRCCSVeruno (NO), Italy

E. Eleuteri Æ P. GiannuzziDivisione di CardiologiaFondazione S. Maugeri, IRCCSVeruno (NO), Italy

A. MartoranaDipto. di Patologia UmanaUniversita degli Studi di PalermoPalermo, Italy

C. Di GangiIstituto di Ostetricia e GinecologiaUniversita degli Studi di PalermoPalermo, Italy

F. Sansone Æ F. Patane Æ M. RinaldiDivisione di CardiochirurgiaOspedale S. Giovanni BattistaTurin, Italy

http://dx.doi.org/10.1007/s00395-008-0761-9

Introduction

Myeloperoxidase is a heme protein which useshydrogen peroxide and chloride to generate the po-tent microbicidal hypochlorous acid (HOCl) [16].This molecule is prominently expressed in polymor-phonuclear neutrophils as well as in monocytes, butrecent data show that its expression is not restrictedto the myeloid cells. In fact, it is expressed even ingranule-containing and pyramidal neurons of thehippocampus, as well as in several neuronal cell lines[18]. When monocytes differentiate into tissue mac-rophages, MPO protein is no longer present, but it hasbeen reported that the gene can be reactivated incertain subsets of macrophages [24, 40].

Apart from its well characterised role followingneutrophil activation in the oxidative burst, MPO hasbeen recently viewed as a key player in the destabil-isation of intimal homeostasis in vascular diseases. Infact, one of the better characterised processes of MPOintervention in endothelial homeostasis is the deple-tion of intracellular NO, to produce nitrogen dioxide,which can in turn be converted to nitrogen radical,causing nitration in tyrosine residues of proteins.Moreover, since MPO is the only enzyme capable ofgenerating HOCl at plasma halide concentrations, it isalso responsible for the formation of chlorinatedaminoacids, of which 3-chlorotyrosine is a stable anddetectable end-product [20, 27, 36]. It has been re-ported that endothelial cells are able to performtranscytosis of MPO, from the vessel lumen to thebasement membrane, where the enzyme specificallytriggers extracellular matrix protein nitration, as re-cently shown for fibronectin [1, 3]. Recent in vivofindings have also shown that the levels of circulatingmyeloperoxidase inversely correlate with brachialartery dilation, suggesting an active role for themolecule in interfering with NO-mediated vasodila-tion [46]. Endothelial dysfunction caused by animbalance between oxidative and nitrosative stresshas also been related to myocyte hypertrophy andultimately to the development of CHF [13, 15, 17, 19,28, 37].

Our study originated from the hypothesis thatendothelial cells may intervene in myeloperoxidasemetabolism not only as a target of the neutrophil-derived enzyme, or through their role in the translo-cation of the molecule to the inner vessel wall, butalso by reacting to oxidative stress with the produc-tion of a number of active molecules. Hydrogen per-oxide is a reactive oxygen specie which is normallypresent in endothelial cells at higher concentrationsthan in other cell types. Moreover, at the low micro-molar range (under 50 lM) it exerts a proliferativeeffect on human umbilical vein endothelial cells(HUVEC) [6]. Several reports in the literature suggest

that hydrogen peroxide may act as a mediator ofactivation of endothelial gene expression, causing forexample the well known increase in vascular perme-ability and adhesiveness for leukocytes [5]. Moregenerally, reactive oxygen species have been impli-cated in the pathophysiology of a number of cardio-vascular diseases [5, 6].

The present study shows for the first time thatoxidative stress induces the expression of MPO byprimary human endothelial cells in culture. SinceMPO is the only enzyme capable of generating HOClat physiological halide concentrations, under thesame conditions we were able to demonstrate theintracellular accumulation of 3-chlorotyrosine, one ofthe stable compounds derived from myeloperoxidase-derived HOCl oxidation. Oxidative and nitrosativerelated end products were also found in post-infarctendocardial endothelial cells (EEC).

Methods

j Tissue sampling

For EEC isolation, soon after transplantation, tissueslices were sampled from the left ventricle, stored incold transport buffer, and cell isolation was performedwithin 6 h. Samples were obtained from seven patientswith post-infarct chronic heart failure (CHF). Con-cerning HUVEC isolation, umbilical cords were ob-tained from twelve subjects immediately after thepartum, and stored in transport buffer [31]. Isolationof cells was performed within 2 h of tissue collection.The investigation conformed to the principles outlinedin the Declaration of Helsinki. The patients gave theirinformed consent prior to be included in this study.

j Cells isolation and culturing: Human endocardialendothelial cells

The isolation method for EEC is based on the use ofcollagenase to detach cells from the sub-endocardialbasement membrane. Heart wall slices sampled fromthe left ventricle were washed extensively with Ca2+

and Mg2+ free HBSS (GIBCO, Milan, Italy) in order toremove residual blood cells. Then the slices wereplaced in sterile Petri dishes filled with Type II colla-genase (GIBCO) diluted in Ca2+ and Mg2+ free HBSS.The dishes were placed at 37�C for 15 min, withoutagitation, and then, after a brief rinse with collagenasesolution, the reaction was stopped by adding an equalvolume of Isolation Medium (same as for HUVEC, seebelow) and centrifuging the cells. After centrifugation,the cells were resuspended in the complete CultureMedium (same as for HUVEC, see below) and plated

308 Basic Research in Cardiology, Vol. 104, No. 3 (2009)� Steinkopff Verlag 2008

on gelatin-coated 6-well culture plates (Corning,Milan, Italy). EEC were used for experiments at pas-sages 2–4, as single primary cell lines.

j Cells isolation and culturing: HUVEC

The method for HUVEC isolation is based on theseparation of endothelial cells from vessel wall usingcollagenase to digest the subendothelial basementmembrane, according to previously published meth-ods [17, 24, 25] with slight modifications regardingthe composition of culture and isolation media andantibiotic treatments. Briefly, for each cord (totalnumber = 24), the vein was cannulated and perfusedwith a heparin solution (250 U/ml), followed by Ca2+

and Mg2+ free HBSS (GIBCO) in order to removeblood residue from the vessel. Then the vein wasperfused with collagenase (Type II, GIBCO) and, afterclamping of the ends, incubated for 15 min at 37�C.Collagenase solution was then collected in a sterilefalcon container, and the vein was thoroughly flushedwith Isolation Medium (IM) (M-199 supplementedwith 200 U/ml penicillin, 200 lg/ml streptomycin,0.5 lg/ml amphotericin B and 2 Mm L-glutamine,10% foetal bovine serum (FBS), all from GIBCO), inorder to detach all endothelial cells. After centrifu-gation, the cells were resuspended in complete Cul-ture Medium (CM) (M-199 with 100 U/ml penicillin,100 lg/ml streptomycin, 0.25 lg/ml amphotericin Band 2 mM L-glutamine, 10% FBS), supplemented withthe appropriate growth supplements for endothelialcells: 20 lg/ml endothelial cell growth factor (ECGF,Roche, Milan, Italy), 14 U/ml sodium heparin (Sigma-Aldrich, Milan, Italy) [21, 30, 31]. Cells were seeded ingelatin-coated culture flasks at a concentration of30,000/cm2. Primary HUVEC were used for theexperiments at passages 2–4. Four experimental setswere made on endothelial cells pooled from threedifferent cords on each occasion.

j Hydrogen peroxide treatment

In order to expose cells to oxidative stress, bothendothelial cells were plated either in 8-well chamberslides to perform immunocytochemistry, or 6-wellculture plates to extract total RNA. Cells were seededat a density of 30,000/cm2, and, when subconfluent,subjected to a 24 h starvation (CM with 2% FBS), andthen exposed to oxidative stress (60 lM H2O2 in thesame culture medium) for 3 and 6 h.

j Cell cytotoxicity assay (LDH release and activity)

LDH release and activity from both HUVEC andEEC was assessed by the Cytotox 96 kit (Promega),

following manufacturer’s instructions. Briefly, cellswere exposed to hydrogen peroxide in 24-well cultureplates, for 3 or 6 h. Released LDH in culture super-natants of both treated and control cells was mea-sured with a coupled enzymatic assay, which resultsin the conversion of a tetrazolium salt (INT) into ared formazan product. The amount of formazanwas determined spectrophotometrically by reading at490 nm. Triton X-100 (0.8%v/v, provided with the kit)was used as positive control for cytotoxicity.

j Immunocytochemistry

Treated and control cells, grown in chamber slides (BDFalcon), were washed with PBS and fixed in methanolfor 20 min at )20�C. Dried slides were then stored at)20�C until use. For the immunocytochemical proce-dure, cells were permeabilised with 0.1% TritonX-100in PBS. After a subsequent rinse with PBS, slides wereexposed to 0.3% H2O2 in PBS, were then blocked with1% FBS in PBS, and incubated for 2 h with the primaryantibody. The detection was performed using an avi-din-biotin complex kit (LSAB2, DAKO, Milan, Italy);3.3¢-diaminobenzidine (DAB chromogenic substratesolution, DAKO) was used as developer. Nuclearcounterstaining was obtained using haematoxylin(DAKO). Myeloperoxidase was identified by the use ofa polyclonal antibody (MYELOp, Novocastra, Milan,Italy) and further confirmed using a monoclonalantibody (M0748, DAKO). Endothelial cells wereimmunostained with a monoclonal antibody antiCD31 antigen (M0823, DAKO) and using also amonoclonal antibody for wWF antigen (M0616,DAKO). 3-nitrotyrosine and 3-chlorotyrosine wereidentified using a mouse monoclonal (AB7048, Ab-cam, Cambridge, UK) and a rabbit polyclonal anti-body (HP5002, Cellsciences, Canton, MA, USA),respectively. iNOS (inducible Nitric oxide Synthase)expression was assessed using a mouse monoclonalantibody (Sc-7271, Santa Cruz Milan, Italy), whileeNOS (endothelial Nitric oxide Syntase) was identifiedusing a rabbit polyclonal antibody (Sc-654, SantaCruz). Standard negative control was routinely per-formed in each experiment by omitting the first anti-body. Immunopositivity on HUVEC and EEC wasscored using a semiquantitative approach (0 = 0% to3 = 100% of cells positively stained). Three indepen-dent observers evaluated the immunocytochemicalresults and semiquantified the percentage of positivecells for each specimen. The mean value of the threepercentages was considered in this study. The con-version between direct percentage and score was madein a directly proportional fashion. Ten high-powerfields were examined in each culture slide and count-ing of the cells was performed at ·40 magnification. As

G. La Rocca et al. 309Oxidative-driven endothelial expression of myeloperoxidase

an additional control experiment, 3-chlorotyrosine(Sigma) and 3-nitrotyrosine (Sigma) were used in di-rect competition assay by excess co-incubation withthe primary antibody.

j Immunohistochemistry

At least three tissue blocks (1 cm · 1 cm) were cutfrom left ventricles of four patients who underwentheart transplantation, immediately frozen in liquidnitrogen and stored at )80�C until analysis. Cryostatsections were cut and immunostained for identifica-tion of CD31 antigen, MPO, nitrotyrosine and chlo-rotyrosine using the same primary antibodies aspreviously described for immunocytochemistry.Primary antibodies were revealed as previouslydescribed [35]. Immunopositivity on EEC was scoredusing a semiquantitative approach (0 = 0% to3 = 100% endothelial cells positively stained). Threeindependent observers evaluated the immunohisto-chemical results and semiquantified the percentage ofpositive cells for each specimen. The mean value ofthe three percentages was considered in this study.Ten high-power fields were examined in each tissueslide and counting of the cells was performed at ·40magnification. Standard negative control was rou-tinely performed in each experiment by omitting thefirst antibody. As an additional control experiment, 3-chlorotyrosine (Sigma) and 3-nitrotyrosine (Sigma)were used in direct competition assay by excess co-incubation with the primary antibody.

Double immunostaining (immunofluorescence) wasperformed by a mouse anti NT (1:50) Abcam, ab-7048and a rabbit anti prostacyclin synthase (1:50), SantaCruz, sc-20933 revealed by the use of a goat anti rabbit(1:100), Sigma, F 9887 and a goat anti mouse, 1:100(Sigma T 6528). Appropriate negative controls wereperformed by replacement of the primary antibody withunspecific isotype-matched control antibodies.

j Total RNA extraction

Total cellular RNA was isolated using the Quick PrepTotal RNA Extraction Kit (GE Healthcare, Milan,Italy) following the manufacturer’s instructions. RNAyield was evaluated spectrophotometrically (A 260/280) and RNA aliquots were stored at )80�C until use.Total RNA fractions were used for subsequentexperiments only if the A260/A280 ratio exceeded 1.6.

j RT-PCR

Qualitative RT-PCR was performed using the Jump-Start RED HT RT-PCR kit (Sigma-Aldrich).

RT-PCR was performed mixing 2 lg of total RNA,0.5 lg of pd(T)23, with RNAse free water. Tubes wereplaced in thermal cycler at 70�C for 10 minutes. Thereaction comprised a reverse transcription step of 50minutes (42�C), followed by inactivation of the en-zyme at 95�C (5 min). Then 10 pM of specific primerswere added and the reactions were cycled for 94�C/2 min, then 35 cycles of 94�C/15 s, 60�C/30 s, 72�C/60 s, with a final extension at 72�C/10 min. Primersused in this study were as follows:

GAPDH Forward: 5¢-AAGGTGAAGGTCGGAGTCAA-3¢;GAPDH Reverse: 5¢-AAGTGGTCGTTGAGGGCAAT-3¢; product size 914 bp;Beta Actin Forward: 5¢-AAACTGGAACGGTGAAGGTG-3¢;Beta Actin Reverse: 5¢-TCAAGTTGGGGGACAAAAAG-3¢: product size 350 bp;YWHAZ Forward: 5¢-TTGGCAGCTAATGGGCTCTT-3¢;YWHAZ Reverse: 5¢-TCTGTGGGATGCAAGCAAAG-3¢; product size 515 bp;MPO Forward: 5¢-TGAACATGGGGAGTGTTTCA-3¢;MPO Reverse: 5¢-CCAGCTCTGCTAACCAGGAC-3¢;product size 382 bp;vWF Forward: 5¢-GGGGTCATCTCTGGATTCAA-3¢;vWF Reverse: 5¢-CAGGTGCCTGGAATTTTCAT-3¢;product size 317 bp.

Beta Actin was preferred as housekeeping gene, toGAPDH and YWHAZ, for its better linearity ofexpression in all the experimental conditions (datanot shown). The identity of PCR products was con-firmed by incubation with the appropriate restrictionenzyme and subsequent visualization of the cleavageproducts on 2% agarose gel.

j Statistical analyses

Group data were expressed as mean with indication ofstandard error. Differences between control and H2O2

treated groups and between different treatment regi-mens were evaluated by the use of a non parametrictest, the Mann–Whitney-U-test; values of P < 0.05were considered as significant.

j Western blotting

Western blotting was performed as described previ-ously [7, 25]. Briefly, MPO primary antibody was amouse monoclonal from Santa Cruz (clone 266-6K1).Secondary antibody was a rabbit anti-mouse (Amer-sham Biosciences). Antibody binding was revealed bychemiluminescence (Immobilion substrate, Milli-pore). Positive control was a HL-60 cell lysate (SantaCruz).


Results

j Cellular extraction and culturing: immunotypingof primary endothelial cells

EEC and HUVEC were isolated and cultured as de-scribed above, and subjected to morphological andimmunological analyses in order to check the theirconformity with the endothelial phenotype, as wellas the homogeneity of the cellular populations.Freshly isolated EEC and HUVEC showed an elon-gated morphology (Fig. 1a, b), appearing as small

islets of cells on the bottom of the culture flask 24 hafter isolation. At subsequent passages (Fig. 1c, d)the cells acquired the classical ‘‘cobblestone’’ mor-phology, becoming more rounded and forming atconfluence a continuous monolayer. Immunocyto-chemistry for CD31 and vWF endothelial antigenswas performed on cells grown on chamber slides.Almost all cells resulted positive to both markers,showing that the isolation procedure resulted inhomogeneous primary cultures, with endothelialphenotype. Figure 1e–h shows the results of thisanalysis.

HUVEC

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EECFig. 1 Morphology and immunophenotyping of endo-thelial cells. HUVEC (a, c) and endocardial endothelial(EEC) cells (b, d) showed the classical cobblestonemorphology of endothelial cells. While at 1 day afterisolation (a, b) they feature a more elongated cell shape,at subsequent passages (c, d) they become morerounded, establishing lateral cell–cell contacts andgrowing as a continuous monolayer. Both HUVEC (e,g) and endocardial endothelial cells (f, h) used for thepresent study express the typical markers of theendothelial phenotype: CD31 (e, f) and vWF (g, h).Magnifications: 20·, bar 100 l


j Hydrogen peroxide exposure: effects on cellularviability and endothelial markers expression

Cultured endothelial cells were subjected to oxidativestress by exposure to 60 lM H2O2 for a duration of 3and 6 h. The choice of this dose was made on thebasis of the existing literature reports showing thatdoses between 100–200 lM are apoptotic for bothHUVEC and aortic endothelial cells [5, 6, 44].Moreover, since our main aim was to evaluate theeffects of oxidative stimulation on enzyme synthesisand end-products formation of endothelial cells, wechose to perform exposure times as long as 3 and6 h. Since different groups have used exposure timesranging from 2–6 to 18–24 h [5, 12, 41], theseincubation times were in agreement with previousreports of oxidative stress evaluation on endothelialcells. We performed the LDH release and activityassay as a cytotoxicity test. For HUVEC, the 3 htreatment induced a slight and unsignificant cyto-toxicity (0.53%) (P = 0.2000 as determined by theMann–Whitney test); following 6 h treatment, theincrease in cellular cytotoxicity (2.68%) reachedstatistical significance (P = 0.0286) (Fig. 2a, b). ForEEC, the same treatment resulted in a slight andunsignificant increase of cytotoxicity at both condi-tions (0.82%, P = 0.1143 for 3 h treatment; 1.10%,P = 0.0571 for 6 h treatment) (Fig. 2c, d). Therefore,the concentration of H2O2 used was non-lethal.Moreover, we performed immunocytochemistry toassess variations in the expression levels of CD31and vWF. These experiments showed that the

expression of the two markers was not affected bythe applied stress (not shown).

j Hydrogen peroxide exposure results in de novoexpression of myeloperoxidase in both EEC andHUVEC

Immunocytochemistry for MPO expression in pri-mary endothelial cells exposed to 60 lM H2O2 showedthat in most cases untreated cells did not express themolecule (Fig. 3a, e for HUVEC and Fig. 3b, f forEEC). In contrast, EEC and HUVEC were positivelystained for MPO protein after 3 h (Fig. 3c, g) and 6 h(Fig. 3d, h) treatments. It should be noted that in twoof the four cell lines of EEC a few cells positive tomyeloperoxidase (score: 0.02 ± 0.01) were found alsoin untreated cells (not shown). On the contrary, wedid not find MPO-positive cells in any of the un-treated HUVEC specimens. This result is most prob-ably related to the fact that EEC were from CHF-affected hearts, in which the altered microenviron-mental conditions may severely modify endothelialphenotype. This results in the maintenance of theectopic expression of MPO in some cells also afterprimary culture. In addition, as shown below, this lowexpression rate after three culture passages is inagreement with the relatively low expression of MPOin endocardium in vivo as assessed by immunohis-tochemistry. Furthermore, as depicted in supple-mental figure 1, 6 h treatment showed a significantMPO increase compared to 3 h treatment for HUVEC

HUVEC 3h

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p=0.0571

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p=0.1143

p=0.028610

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Fig. 2 Effects of exposure to hydrogen peroxide onviability of endothelial cells. LDH assay was used toevaluate the cytotoxic effect of 60 lM H2O2. Data arerepresented as means, with the indication of standarderror of three replicate experiments. Significance ofdifferences with respect to untreated cells wascalculated using the Mann–Whitney U-test. Theanalysis was performed for both HUVEC (a, b) andendocardial endothelial (EEC) cells (c, d), subjected tooxidative stress for 3 h (a, c) and 6 h (b, d). A slightlybut significantly decreased viability was observed in3 h H2O2 exposed HUVEC cells. On the contrary, 6 hH2O2 exposure significantly increased proliferation ofEE cells


alone (score: 0.32 ± 0.06 vs. 0.16 ± 0.6 for HUVEC,P = 0.002; 0.28 ± 0.07 vs. 0.25 ± 0.1 for EEC,P = 0.588). These results showed that endothelial cellswere able to produce endogenous MPO in a neutro-phil-free system, following exposure to non-lethaldoses of H2O2. To assess the effect of heparin onmobilisation of endothelial-derived MPO, suggested‘‘in vivo’’ by recent studies [11], endothelial cells wereincubated in a modified CM without heparin, starvedand exposed to H2O2 for both 3 and 6 h. Immuno-cytochemistry showed that heparin removal did not

cause any variation to the number of MPO-positivecells (not shown).

j Hydrogen peroxide exposure results in theformation of end products of tyrosine oxidation:3-chlorotyrosine in HUVECs and endocardialendothelial cells

Since MPO is the only enzyme able, in the presence ofhydrogen peroxide, to produce HOCl at physiological

HUVEC

Control3h

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EEC

A B

C D

E F

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Fig. 3 Oxidative stress causes the expression of myelo-peroxidase by endothelial cells. HUVEC (a, c, e, g, on theleft) and endocardial endothelial cells (b, d, f, h, on theright) were stimulated with hydrogen peroxide for 3 h(a–d) or 6 h (e–h). MPO was not detectable in untreatedendothelial cells - either HUVEC (a, e) or EE cells (b,f)—at both 3 or 6 h experiments. When HUVECs weresubjected to oxidative stress, MPO was detectable with anet cytoplasmatic positivity after 3 h (c) and 6 h (g)stimulation (arrows). The same occurred for EE cells, atboth 3 h (d) and 6 h (h) treatments (arrows).Magnifications: 20·, bar 100 l


halide concentrations [20], we checked for the pres-ence of 3-chlorotyrosine, one of the early and stableend products of MPO activity, which is used as amolecular fingerprint of the enzyme activity [35].Immunocytochemistry for 3-chlorotyrosine showedthat untreated cells, after three passages in culture,were negative, while a strong cytoplasmatic positivitywas observed in both HUVEC (Fig. 4c, g) and EEC(Fig. 4d, h). As depicted in supplemental figure 2,there was a a five to sevenfold increase in 3-chlo-

rotyrosine accumulation after 6 h compared with 3 htreatments, for both cell types (score: 1.62 ± 0.3 vs.0.22 ± 0.07 for HUVEC, P = 0.002; 1.5 ± 0.36 vs.0.28 ± 0.13 for EEC, P = 0.002). In the same condi-tions, no positive cells were detected after nitrotyro-sine immunostaining following H2O2 exposure byEEC or HUVEC (not shown).

In order to determine the potential contributionsof NO synthases in this in vitro system, we performedimmunocytochemistry to localize iNOS and eNOS

HUVEC

Control3h

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A B

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Fig. 4 Endothelial cells exposed to hydrogen peroxideexpressed 3-chlorotyrosine concomitantly to MPOexpression. HUVEC (a, c, e, g, on the left) andendocardial endothelial cells (b, d, f, h, on the right)were stimulated with hydrogen peroxide for 3 h (a–d) or6 h (e–h). 3-chlorotyrosine was not detectable inuntreated endothelial cells—either HUVEC (a, e) or EEcells (b, f)—at either exposure time. When HUVECs weresubjected to oxidative stress, 3-chlorotyrosine wasrecognisable with a strong cytoplasmatic positivityafter 3 h (c) and 6 h (g) stimulation (arrows). Thesame was true for endocardial endothelial cells, at both3 h (d) and 6 h treatments (h) (arrows). Magnifications:20·, bar 100 l


expression following oxidative stress. iNOS expres-sion was not detectable in either HUVEC or EECfollowing hydrogen peroxide exposure, nor it wasdetected in untreated cells (not shown). It is possiblethat this is due to the absence, in our in vitro system,of proinflammatory mediators capable of inducingiNOS expression. On the other hand, and confirmingpast literature results from other groups [5, 12], eNOSwas amply expressed by untreated HUVEC and EEC,and its expression increased still more after oxidativestress (data not shown).

j Detection of the mRNA for MPO in H2O2 exposedendothelial cells

To demonstrate the synthesis of MPO in EEC andHUVEC following H2O2 exposure, we extracted totalRNA from cells, and, following reverse transcription,end-point PCR was performed using gene-specificprimers. Endothelial cells expressed the mRNAs forb-Actin and vWF both in treated and control cells(Fig. 5). The presence of MPO mRNA was observedonly in treated endothelial cells (both EEC and

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Fig. 5 Endothelial cells exposed to oxi-dative stress express MPO mRNA after 3 htreatment. RT-PCR experiments wereperformed on total RNA extracted fromHUVEC (a–c) and EEC (d–f). HUVECexpressed beta-actin (a) and vWF (c)both in untreated and hydrogenperoxide-exposed cells. MPO mRNA(382 bp) (b) was only detectable afterexposure to oxidative stress. The resultswere confirmed in EEC, where expressionof beta actin (d) and vWF (f) mRNAs wasdetected in both untreated and stressedcells. Moreover, as shown in e, also EECexpressed MPO, but only after exposureto hydrogen peroxide. M DNA ladder,C control cells; 60 lM: cells exposedto oxidative stress. The expression ofMPO has been evaluated by a semi-quantitative densitometric analysis ofthe expression in HUVEC (g) and EEC(h). Data are represented as mean, withthe indication of standard deviation, ofthree replicate experiments. Values werenormalized for beta actin expression


HUVEC), while the 382 bp product was not detect-able in control cells (Fig. 5). Further experimentswere made using 40 and 45 cycles, but MPO mes-senger was never detectable in untreated cells. Semi-quantitative determination of MPO expression levelswas performed as reported previously [25]. Figure 5shows the graph of levels of MPO messenger RNA inHUVEC and EEC after 3 h hydrogen peroxideincubation, determined after normalization withbeta-actin.

j Immunohistochemistry of endocardial tissue frompatients with post-infarct chronic heart failure

In the endocardial heart tissue, immunopositivity of EECwas evaluated and scored for CD31 antigen, myeloper-oxidase (MPO), 3-nitrotyrosine and 3-chlorotyrosine.A mean length of 4,800 ± 577 l of endotheliallayer was analysed for each quantification. CD 31antigen was uniformly expressed by all EE cells (score:3 ± 0 (mean ± SD) (Fig. 6a). Myeloperoxidase wasexpressed occasionally by EE cells (score: 0.15 ± 0.1;Fig. 6b). 3-nitrotyrosine was frequently expressed byEE cells (score: 0.9 ± 0.48; Fig. 6c). 3-chlorotyrosinewas amply expressed by EE cells (score: 2 ± 0.4;Fig. 6d). Endocardial capillary vessels and heartmuscle were also frequently immunostained for3-nitrotyrosine and more extensively, for 3-chloroty-rosine. Inflammatory cells around capillary vesselsand infiltrating heart muscle were also occasionallyimmunostained for 3-nitrotyrosine and more fre-quently, for 3-chlorotyrosine.

Moreover, since prostacyclin synthase is one of themost prevalent nitrated proteins [34, 50], we aimed todetect, by co-staining in immunofluorescence exper-iments, if this protein should be one of the potentialtargets of nitration in post-infarct CHF hearts.Supplemental figure 3 shows the panels of immuno-localization of prostacyclin synthase and 3-nitroty-rosine in a representative sample of human heart. Themerged image (panel B) shows that, even if prosta-cyclin synthase should not be considered the solenitrated protein, in almost three areas of the endo-cardial surface (arrows) there is a clear colocalization.

Discussion

This study shows for the first time that EEC frompatients with CHF, as well as HUVEC, followingexposure to H2O2 express the enzyme myeloperoxi-dase together with 3-chlorotyrosine, an unphysiologicand specific end product of oxidative stress. These ‘‘invitro’’ data were confirmed in ‘‘in vivo’’ experimentswhere MPO, 3-nitrotyrosine and 3-chlorotyrosinewere identified in endocardium from patients withCHF.

Myeloperoxidase is a key enzyme in the immunedefense system against bacterial pathogens, on ac-count of its capability to generate powerful oxidantssuch as HOCl by using hydrogen peroxide and Cl)

ion at physiological levels [20, 23, 29, 35]. Moreover,protein nitration is also possible through activationof heme peroxidases, such as myeloperoxidase, by

Fig. 6 Photomicrographs showing endocardial tissuefrom a patient with chronic heart failureimmunostained for identification of a CD31 antigen,costitutively expressed by endothelial cells (arrows), bmyeloperoxidase, randomly expressed by endothelialcells (arrows), c nitrotyrosine, moderately expressedby endothelial cells (arrows) and d 3-chlorotyrosine,more frequently expressed by endothelial cells(arrows). Results are representative of those fromfour patients who underwent heart transplantationafter development of post-infarctual chronic heartfailure. Magnification, 20·, bar 50 l


H2O2 promoting oxidation of NO2). Other oxidants

that can oxidise NO2) to nitrating intermediates in-

clude HOCl, the product of MPO-catalyzed Cl) oxi-dation [36, 45]. MPO has been implicated in thedevelopment of a number of cardiovascular diseasesas CHF [11, 37, 40]. Moreover, several reports pro-vide evidence of a significant association betweencirculating MPO levels and the risk of adverse car-diovascular events [42]. In addition, MPO has beendirectly linked to the NO homeostasis in the vesselwall since it is able to interact with NO to formnitrated species, thus depleting the intracellularreservoir of NO, leading to vasoconstriction or tissuedamage [2, 45, 46]. Nitrosative stress due to tyrosinenitration may be exacerbated by oxidative stress. Infact, stimuli that lead to iNOS induction may alsoup-regulate oxidases (Xanthine Oxidase, NADPHoxidase), and concomitant elevations of NO andsuperoxide may lead to formation of peroxynitrite[4, 8, 14]. Nitration of tyrosine in proteins is relevantin heart failure, since this mechanism may interferewith NO redistribution in the sarcolemma alteringthe NO/redox balance [9, 10, 22, 38, 39]. Endothelialcells can actively interact with endogenous (e.g.neutrophil-derived) MPO, as demonstrated recentlyby the molecule’s binding on the surface of endo-thelial cells as well as by transcytosis towards thesubintimal side [1, 42, 44].

In the present study, we isolated primary humanendothelial cells from two different sources, e.g. EECfrom the left ventricle of post infarcted CHF diseasedpatients and HUVEC cells from the umbilical vein.The former represent organ-specific primary celllines, coming from a diseased organ targeted by theinflammatory processes characteristic of the disease.As reported in the Results, the isolation protocol usedallowed to obtain homogeneous populations ofendothelial cells. They were subjected to oxidativestress, using a non-lethal dose (60 lM) which was inthe range of doses previously used in oxidative stressstudies on endothelial cells. [6, 12]. Concerning theexposure times we used (3 and 6 h), this choice wasmade based on the rationale of measuring not simplythe de novo expression of MPO in endothelial cells,but also the ‘‘long-term’’ effects of oxidative stressdue, for example, to the accumulation of detectableend products as 3-chlorotyrosine. Previous studiessuggest that exposure times ranging between 2–24 hshould be used to investigate the pathways related tooxidative stress in endothelial cells [5, 12, 41]. Cyto-toxicity tests confirmed that only in HUVEC, after 6 hexposure to hydrogen peroxide, but not in EEC, wasthere a significant increase of cellular cytotoxicityfollowing H2O2 exposure. This is also in agreement

with the recently reported evidences that endothelialcells do not show a significant increase in cell deathwhen exposed to doses of hydrogen peroxide less than100 lM [6, 43].

The innovative finding of this paper is that endo-thelial cells subjected to oxidative stress showed theyare able to produce endogenous MPO in an in vitrosystem deprived of neutrophils and other potentialsources of MPO. This result was also achieved not-withstanding starvation with lowered serum medium.In fact, since both control and treated cells wereincubated (and starved) in a reduced-serum culturemedium, uptake of MPO from the bovine serum canbe excluded. As reported recently, untreated endo-thelial cells are able to bind MPO [44], but the lack ofimmunopositivity in untreated control cells suggeststhat H2O2 exerts a direct regulatory effect on theexpression of MPO in the endothelium.

As shown by immunocytochemistry and succes-sively confirmed by RT-PCR, human endothelial cellsfrom both umbilical vein and endocardium expressedMPO when exposed to 60 lM H2O2. The number ofimmunocytochemistry-positive cells was higher inendocardial cells than HUVEC.

The sole presence of myeloperoxidase is not suf-ficient, per se, to provoke the important oxidativephenomena of proteins and lipids in the cardiovas-cular system. MPO plays its fundamental role in themetabolism of H2O2, which is used to produce morepowerful oxidants such as HOCl [20]. Then HOCl isable to oxidise different substrates, leading forexample to the formation of chlorinated amino acids,of which 3-chlorotyrosine constitutes a detectable andspecific nonphysiologic end-product [35, 47]. There-fore, we looked for the presence of 3-chlorotyrosinein endothelial cells exposed to oxidative stress, usinga novel commercial anti-chlorotyrosine antibody.Immunocytochemistry revealed the presence of astrong positivity only in treated cells, while no posi-tivity was detectable in untreated cells, either EEC or,HUVEC demonstrating that in our ‘‘in vitro’’ model,MPO upregulation is associated to a specific endproduct of MPO-driven oxidative stress such as 3-chlorotyrosine.

Another interesting point is the absence of ni-trotyrosine, in cultured endothelial cells exposed toH2O2; this molecular specie should be formed in thepresence of H2O2 and MPO via a two step reaction ofNO oxidation. Since macrophage-derived iNOS ratherthan eNOS is the main enzyme involved in NO pro-duction during the oxidative burst, it could be arguedthat in our in vitro system MPO action resulted in theformation of 3-chlorotyrosine rather than nitrotyro-sine, also for the presence of lower levels of NO with


respect to those achievable during inflammation [28,33].

‘‘In vivo’’ immunohistochemistry experimentsshowed that endocardium from left ventricle of post-infarct CHF patients, expressed a small amount ofMPO together with moderate levels of nitrotyrosine,as already reported in the literature [1]. Interestingly,abundant levels of 3-chlorotyrosine were detected,thus confirming the ‘‘in vitro’’ observations reportedfor primary EEC. In order to better clarify and sum-marize our findings, we propose a model (depicted inFig. 7) describing the possible pathway of couplingbetween endogenous MPO production and 3-chlo-rotyrosine accumulation in endothelial cells. In par-ticular, the formation of HOCl due to MPO actionshould result also in L-arginine chlorination, thereforeuncoupling eNOS and favouring the production ofsuperoxide and the alteration of NO/ROS balance.Interestingly, eNOS uncoupling should also be aconsequence of BH4 depletion, a process in whichDHFR (dihydrofolate reductase) regulation plays akey role: downregulation of DHFR by hydrogen per-oxide limits recycling of oxidized BH4, thereforelimiting the presence of the essential cofactor in NObiosynthesis [26, 32].

Finally, prostacyclin synthase appeared in vivo asone of the prominently nitrated proteins, as shown byimmunofluorescence experiments. This datum shouldbe of great clinical relevance, since tyrosine nitrationof prostacyclin synthase by peroxynitrite might be animportant pathophysiological event, since it not onlydecreases the production of prostacyclin but canfurther modify endothelial features, leading to in-creased endothelial cell apoptosis and adhesion mol-ecule expression [34, 50].

In conclusion, our study highlights that EEC, aswell as HUVEC, in the presence of non-lethal dosesof hydrogen peroxide similar to those achievable invivo, can synthesize endogenous myeloperoxidase,together with an end product of oxidative stress suchas 3-chlorotyrosine. This response to oxidative stressmay actively contribute to the vascular and cardiacdamage in patients with various vascular and cardiacdiseases including post-infarct CHF. We can onlyspeculate on the mechanism underlying MPOinduction in endothelial cells following hydrogenperoxide exposure. Further experiments are neededto establish if a direct regulatory mechanism (suchas that implied in upregulation of VEGF-A) or anindirect one (such as NFkB-induced eNOS upregu-

OxidativeStress(H2O2)

H2O2

SODO2

-

O2- + NO •

MPOinduction

MPO

CI-

eNOSincreaseDHFR

BH4“uncoupled”

eNOS

3-Cl-tyraccumulation

Tyrosinechlorination

HOCI

L-argchlorination

ONOO-

Tyrosinenitration

3-N-tyraccumulation

Fig. 7 Proposed working model in endothelial cellsof MPO action following oxidative stress. Oxidativestress (H2O2) upregulates MPO which, in the presenceof hydrogen peroxide and chloride ions, catalyzeshypochlorous acid formation. HOCl-dependentoxidation may result in tyrosine chlorination ofproteins, leading to 3-chlorotyrosine formation. Inaddition, HOCl may chlorinate L-arginine, thus‘‘uncoupling’’ eNOS. Uncoupled eNOS may becomea source of superoxide ion (O2

)), which can be readilydismutated by SOD to produce again hydrogenperoxide and close the circuit. Oxidative-driven eNOSincrease may further contribute to the ‘‘uncoupling’’process. Moreover, eNOS uncoupling should also besecondary to BH4 (tetrahydrobiopterine) depletion,due to the inhibitory effect of hydrogen peroxide onDHFR activity. Finally, the levels of NO should berapidly lowered by the interaction with superoxide,with generation of peroxynitrite (ONOO)) which canrapidly cause protein nitration at tyrosine residues.Graph proposed on the basis of our present data andreferences [6, 26, 27, 32, 35, 44, 48]


lation) are in action in endothelial cells exposed tooxidative stress [37, 49]. The uncovering of thisregulatory mechanism should open new ways inclinical intervention to reduce the MPO-driven CHFburden.

j Acknowledgments We thank Rosemary Allpress for her revision ofthe English language. We thank Antonella Chiara, Santina Di Gangiand Giusy Scaduto for the valuable support during the experimentalwork. This work was supported by Fondazione S. Maugeri, IRCCS,Ricerca corrente, and Italian Ministry of University (Ex 60% to GZ, FC,FF) grants. The authors declare no conflict of interests.

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Oxidative stress induces myeloperoxidase expression in endocardial endothelial cells from patients with chronic heart failure