proatherogenic modification of ldl by surface-bound myeloperoxidase

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Chemistry and Physics of Lipids 180 (2014) 72–80

Contents lists available at ScienceDirect

Chemistry and Physics of Lipids

jou rn al h om epage : www.elsev ier .com/ locate /chemphys l ip

roatherogenic modification of LDL by surface-boundyeloperoxidase

lexej V. Sokolova,b,c,∗, Valeria A. Kostevicha,b, Olga L. Runovaa, Irina V. Gorudkod,adim B. Vasilyeva,c, Sergej N. Cherenkevichd, Oleg M. Panasenkob

Institute of Experimental Medicine of the N-W Branch of the Russian Academy of Medical Sciences, Saint-Petersburg, RussiaResearch Institute of Physico-Chemical Medicine, Moscow, RussiaSaint-Petersburg State University, Saint-Petersburg, RussiaBelarusian State University, Minsk, Belarus

r t i c l e i n f o

rticle history:eceived 16 July 2013eceived in revised form 31 January 2014ccepted 24 February 2014vailable online 11 March 2014

eywords:yeloperoxidase

ow density lipoproteinseactive halogen speciesholesterol accumulation

a b s t r a c t

One of the factors promoting oxidative/halogenating modification of low-density lipoproteins (LDL) ismyeloperoxidase (MPO). We have shown previously that MPO binds to the LDL surfaces. The LDL–MPOcomplex is uncoupled in the presence of peptide EQIQDDCTGDED that corresponds to a fragment ofapoB-100 (445–456). In this paper we studied how this peptide, as well as inhibitors and modulatorsof halogenating activity of MPO such as ceruloplasmin (CP), 4-aminobenzoic acid hydrazide (ABAH) andthiocyanate (SCN−) affect the accumulation of cholesterol and its esters in monocytes/macrophages afterincubation with LDL subjected to different kinds of MPO-dependent oxidative/halogenating modification.In the presence of H2O2 and halides MPO causes stronger proatherogenic modification of LDL than exoge-nous reactive halogen species (HOCl and HOBr). Both monocytes, which differentiate into macrophages,and neutrophils secrete MPO in response to the presence of damaged LDL. The peptide EQIQDDCTGDED

xidative/halogenative stresstherosclerosis

preventing interaction between MPO and LDL reduces the uptake of modified LDL and MPO by mono-cytes/macrophages and thus precludes the accumulation of intracellular cholesterol. Our results indicatethat binding to MPO is important for LDL to become modified and acquire proatherogenic properties. Thepeptide EQIQDDCTGDED, CP, ABAH, and SCN− can play the role of anti-atherogenic factors reducing thedeleterious effect of catalytically active MPO on LDL and accumulation of cholesterol in macrophages.

© 2014 Elsevier Ireland Ltd. All rights reserved.

. Introduction

In recent years, the role of immunologic factors in atherogenesisas been extensively studied. Atherosclerosis is now regardeds chronic immune inflammation. Meanwhile, the key role ofxcess amounts of cholesterol accumulated in the intima haseen recognized. As D. Steinberg put it, hypercholesterolemia

Abbreviations: ABAH, 4-aminobenzoic acid hydrazide; CP, ceruloplasmin;DH, lactate dehydrogenase;LDL, low density lipoproteins; MPO, myeloperoxidase;1–15, peptide EEEMLENVSLVCPKD; P445–456, peptide EQIQDDCTGDED.∗ Corresponding author at: Institute of Experimental Medicine of the Russiancademy of Medical Sciences, Academika Pavlova str., 12, Saint-Petersburg, 197376,ussia. Fax: +7 812 234 94 89.

E-mail address: biochemsokolov@gmail.com (A.V. Sokolov).

ttp://dx.doi.org/10.1016/j.chemphyslip.2014.02.006009-3084/© 2014 Elsevier Ireland Ltd. All rights reserved.

constitutes about 5% (Schultz and Kaminker, 1962) and 0.9% (Deby-Dupont et al., 1999), respectively, of the total protein in these cells.Inflammation in the body is accompanied by respiratory burstof leukocytes. As a result, MPO reacts with hydrogen peroxideand is converted into active compound I. The latter is capable ofoxidizing chloride (Cl−), bromide (Br−), or thiocyanate (SCN−) withformation of HOCl, HOBr, or HOSCN, respectively (Klebanoff, 2005;van Dalen et al., 1997). HOCl and HOBr participate in antimicrobialdefense, but are also capable of damaging biomolecules withincells and tissues of the host organism (Davies et al., 2008). HOSCNis an efficient antimicrobial agent too, but is a comparatively mildoxidant (Chandler and Day, 2012). It should be noted that thespecificity of MPO to the aforementioned substrates follows therelation Cl−:Br−:SCN− = 1:60:730 (van Dalen et al., 1997). Theoxidants formed by MPO can modify both the lipid component

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A.V. Sokolov et al. / Chemistry an

ypical feature of atherosclerosis (Panasenko et al., 1997a; Nichollsnd Hazen, 2009). Biomarkers of halogenating activity of MPO3-chlorotyrosine, 5-chlorouracil, �-chloroaldehydes, chlorohy-rins of lipid acyl chains) and the active enzyme itself were found

n atherosclerotic plaques (Daugherty et al., 1994; Hazell et al.,996; Hazen and Heinecke, 1997; Hazell et al., 2001; Thukkanit al., 2003; Zheng et al., 2004; Takeshita et al., 2006; Messnert al., 2008; Delporte et al., 2013; Panasenko et al., 2013). Studiesf MPO levels in patients with acute coronary syndrome show thathis enzyme is an independent predictor of myocardial infarctionBaldus et al., 2003).

The high reactivity of oxidants formed in MPO-dependent reac-ions suggests the existence of several mechanisms that restricthe effect of this enzyme in blood plasma. For example, in addi-ion to some low-molecular-weight antioxidants interacting withOCl and HOBr, ceruloplasmin (CP) binds to MPO in blood plasma,

hus inhibiting the enzyme (Segelmark et al., 1997; Chapman et al.,013). However, only intact CP inhibits the chlorinating activ-

ty of MPO, whereas partially proteolyzed CP (CPpr) loses thisapacity (Panasenko et al., 2008; Sokolov et al., 2008). This effects provided by the contact between the heme pocket of MPOnd the most protease-vulnerable loop in CP that competes withubstrates for the active site of MPO (Samygina et al., 2013). Inlood plasma MPO interacts not only with CP, but also with apoB-00-containing lipoproteins such as LDL and very low-density

ipoproteins (VLDL) (Sokolov et al., 2010). Complexes containingP, MPO, and LDL/VLDL are found in atherosclerotic patients withlevated MPO level. In view of the fact that anti-apoB-100 antibod-es prevented the interaction of MPO with LDL and VLDL (Sokolovt al., 2010) we assumed that the MPO binding site is localized in therotein moiety of lipoprotein particles. The amino acid sequence45EQIQDDCTGDED456 in anionic apoB-100 provides the interac-ion with cationic MPO (pI 9–10) (Sokolov et al., 2011).

The importance of direct LDL–MPO binding for the initial stagesf atherosclerosis has been poorly studied. We assumed that bind-ng of MPO to the LDL surface is the key point in LDL modificationy oxidants formed in MPO-dependent reactions, in LDL transfor-ation to the proatherogenic form, in cholesterol accumulation

n subendothelial cells, and in the development of atheroscleroticesions in arteria. In this study using a model of LDL-consuming

onocytes/macrophages we applied a peptide homologue of thePO-binding site in LDL, capable of uncoupling the LDL–MPO com-

lex, as well as compounds that inhibit and modulate the activity ofPO, to demonstrate the importance of MPO binding to the surface

f LDL for their proatherogenic modification and for intracellularholesterol accumulation.

. Experimental

.1. Reagents

We used goat antibodies against rabbit IgG conjugatedith horseradish peroxidase, skimmed dry milk, and Tween

0 (BioRad, USA), NaCl, NaBr, NaSCN, hydrogen peroxideMerck, Germany), chromatographic resins (Pharmacia, Sweden),holesterol oxidase (Nocardia erythropolis), cholesterol esterasePseudomonas fluorescens), Coomassie G-250, horseradish per-xidase, 3,3′,5,5′-tetramethylbenzidine (TMB) (Serva, Germany),-phenylendiamine, NaOCl, 4-aminobenzoic acid hydrazideABAH), scopoletin, phorbol-12-myristate-13-acetate (PMA)Sigma–Aldrich, USA), reagents for electrophoresis (Laboratory

edigen, Russia), and heparin (SPOFA, Poland). Solid phase synthe-is of peptides EEEMLENVSLVCPKD (P1–15) and EQIQDDCTGDEDP445–456) was accomplished in the Research Institute of Extra-ure Biopreparations (Saint-Petersburg). Peptides were 99.5%

sics of Lipids 180 (2014) 72–80 73

pure as judged by the results of HPLC and mass spectrometry. Allsolutions were prepared using apyrogenic deionized water withresistivity 18.2 M� cm.

A stock solution of NaOCl was kept in the dark at 4 ◦C. Its con-centration was determined at pH 12 using ε290 = 350 M−1 cm−1

(Morris, 1966). Aqueous solution of HOBr was prepared mixingequimolar amounts of HOCl and NaBr (Gazda and Margerum, 1994).Concentration of HOBr solution was determined at pH 12 usingε329 = 332 M−1 cm−1 (Kumar and Margerum, 1987).

2.2. Proteins and LDL isolation

MPO from human leukocytes was purified by successive chro-matography on heparin-Sepharose, phenyl-agarose and SephacrylS-200 HR. A430/A280 (RZ) in purified MPO was 0.85, which is indica-tive of the enzyme’s homogeneity (Sokolov et al., 2007).

Stable at storage monomeric CP was purified from humanblood plasma by chromatography on UNO-Sphere Q andneomycin–agarose. Preparation of purified CP contained more than95% of non-proteolyzed protein and had A610/A280 > 0.050 (Sokolovet al., 2012). Proteolyzed CP (CPpr) was obtained by limited plas-min hydrolysis of intact CP for 1 h at 37 ◦C (CP:plasmin = 1:500,w/w). Hydrolysis was stopped by adding 6-aminocaproic acid to thefinal concentration 10 �M. About 90% of the protein in the prepa-ration of CPpr were fragments with M 116 and 19 kDa as judged bySDS-PAAG electrophoresis.

LDL were isolated by gel-filtration of EDTA-stabilized bloodplasma (5 ml) on a column with BioGel A-5 m (100 × 2.5 cm) equil-ibrated with PBS (10 mM phosphate buffer containing 145 mMNaCl, pH 7.4) at 4 ◦C. LDL-containing fraction was pooled and con-centrated by VivaSpin 20 centrifuge-cell. LDL isolation took about7–8 h. Homogeneity of purified LDL was analyzed by electrophore-sis in agarose gel (1%, m/v) with Coomassie G-250 and SudanBlack staining. Formation of the complex ‘LDL–MPO’ was evidencedby disk-electrophoresis in PAAG (Davis, 1964). MPO activity wasrevealed after soaking PAAG in solution containing 4-chloro-1-naphtol and H2O2 (Sokolov et al., 2007).

2.3. Oxidative/halogenating modification of LDL and peptides

To treat LDL (0.5 �M apoB-100) with oxidants we used HOCl(1:100 mol/mol), HOBr (1:100 mol/mol), or a mixture of 5 nM MPOwith 50 �M H2O2 in PBS (150 mM NaCl, 10 mM Na-phosphatebuffer, pH 7.4) to which 100 mM NaCl, or 5 mM NaBr, or 0.2 mMNaSCN were added at option. P1–15 and P445–456 (100 �M) hadbeen mixed with HOCl (1:2 mol/mol) in advance. 5 �M ABAH,6 �M CP/CPpr, or 200 �M NaSCN were used to inhibit the oxida-tive modification of LDL. Intact and HOCl-modified peptides P1–15and P445–456 (0.1–1 �M), the latter preventing the interactionbetween MPO and LDL, were used. After 30 min of incubation atroom temperature the reaction was stopped by adding 50 �M cys-teine. Modified LDL were used for studying their proatherogenicproperties (see Section 2.6).

To assay the affinity of modified LDL for MPO the former weredialyzed for 12 h at +4 ◦C against 500-fold excess of PBS (150 mMNaCl, 10 mM Na-phosphate buffer, pH 7.4).

2.4. Analytical methods

Protein content in fractions was assayed in micro-reaction withCoomassie G-250, maximum sensitivity being 0.5 �g/ml (Bradford,1976). Cholesterol and its esters were assayed in highly sensi-

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4 A.V. Sokolov et al. / Chemistry an

etermination) or 0.05 U of cholesterol esterase (for cholesterolnd its esters determination), 0.5 U of horseradish peroxidase, and.5 mM TMB in 50 mM sodium-phosphate buffer, pH 6.5, with 0.01%odium desoxycholate and 0.05% Triton X-100. After 30 min at 37 ◦C630 was measured and the content of cholesterol and its esters wasalculated. MPO was assayed by ELISA (Sokolov et al., 2010).

.5. Determination of kinetic parameters of MPO activity

Affinity of MPO for modified LDL was judged by their effect onhe inhibition of MPO peroxidase activity by CP (Sokolov et al.,010). The mixture was composed of 10 nM MPO, 0.1–0.5 mMMB in 0.1 M sodium-phosphate buffer, pH 7.4, and 100 �M H2O2.ydrogen peroxide was added to initiate reaction. The reaction

ate was registered at 20 ◦C on SF 2000-02 spectrophotometer asA650/min. Enzymatic activity was assayed both in the absence of

roteins interacting with MPO and in the presence of CP (0.2 �M),ifferent forms of LDL (0.5 nM) and P445–456 (10 nM). The respec-ive KM and Vmax were rated from the graphs in Hanes–Woolfoordinates using least squares fitting (Microsoft Excel). Along withi, characterizing the effect of CP on MPO-catalyzed reaction in theresence and in the absence of LDL, we estimated the “inhibitiononstants” for the effect of LDL on suppression of MPO activity byP (K∗

i ). These values were calculated using the formula for non-ssociative inhibition (Krupyanko, 2007)

∗i = [I]√

((K ′M/KM) − 1)2 + ((Vmax/V ′max) − 1)2

,

here [I] is the concentration of LDL, while KM and Vmax are thealues of kinetic parameters of the substrates’ oxidation measuredhen MPO was inhibited by CP alone, and K ′

M and V ′max are the

ame parameters obtained in the presence of LDL.

.6. Isolation and culturing monocytes with LDL

Monocytes were isolated from the blood of healthy donorsy centrifugation on ficoll-verographin (1.077) density gradientPanasenko et al., 1991). Cells (107 cell/ml) were cultured in 12-wellat bottom tissue culture plates in RPMI-1640 medium with 10%

etal calf serum, 1% l-glutamine, 50 U/ml penicillin, 100 mM strep-omycin in humidified air (5% CO2 at 37 ◦C). After 4 h a new mediumupplemented with 50 nM PMA and 40 nM LDL (native or modi-ed by HOCl, HOBr, or MPO/H2O2/Cl−/Br−/SCN−) was introduced.amples of LDL modified in the presence of P1–15 or P445–456ontained 8–80 nM of these peptides. Incubation medium wasollected after 12 h and 72 h, admixtures of cells were removedy centrifugation (5 min at 3000 × g) and the total protein, MPO,holesterol and its esters were analyzed in the supernatant. Choles-erol accumulation by cells was determined as the differenceetween its content in a sample under study and samples withoutells.

.7. Isolation, activation and degranulation of neutrophils

Neutrophils were isolated by centrifugation in the Lymphoprepensity gradient (Timoshenko et al., 1998). The percentage of neu-rophils in cell preparations was >95%. The viability was >95% asetermined by the ability to exclude trypan blue. To estimate the

nfluence of LDL on viability of human neutrophils, the lactateehydrogenase (LDH) activity in cell supernatants was measuredsing a Lactate Dehydrogenase Kit (001.010.01) from Analiz-Plus

sics of Lipids 180 (2014) 72–80

component of specific granules) as described previously (Gorudkoet al., 2010). MPO activity in the supernatants was measuredusing o-dianisidine and H2O2 as substrate. MPO concentration insupernatants was measured by ad-hoc ELISA elaborated previously(Gorudko et al., 2009). Lactoferrin concentration in the super-natants was determined using commercial ELISA kit (“Vector-Best”,Russia).

2.8. Statistical analysis

Experiments were repeated at least three times (n ≥ 3) andthe mean values were calculated as Xm = (1/n)

∑Xi, where Xi is a

value of each successive sample. The standard error was expressed

as S*/n, where S∗ =√

(∑

(Xi − Xm)2)/(n − 1), and the confidence

interval was calculated as Xm ± (S*/n1/2)tn−1,1−˛/2, for which t wasfound in the table of values on condition that in our experiments

= 0.05.

3. Results

3.1. Affinity of MPO for modified LDL

The affinity of MPO for lipoproteins can be assayed with highsensitivity by measuring their effect on kinetics of inhibition by CPthe chromogenic substrates’ peroxidation by MPO (Sokolov et al.,2010). This approach does not require solid-phase immobiliza-tion of the interacting components and makes it possible to studythe LDL–MPO interaction in the presence of a physiologic MPOinhibitor. Table 1 summarizes the data on the influence of LDL onKM and Vmax of MPO in the presence of CP. As shown in controlexperiments, neither unmodified LDL nor all types of modified LDLinhibited the activity of MPO without CP. The effects of native LDLand modified LDL on inhibition of the MPO activity by CP were alsosimilar; each of them caused a decrease of KM and Vmax. For all formsof LDL, Ki was close to 0.2 nM. Thus, modification of LDL by HOCl andHOBr or using the MPO/H2O2/(Cl− or Br−) system does not affecttheir affinity for MPO. It is interesting that the P445–456 peptidethat prevents the MPO–LDL interaction, completely abrogated theeffect of LDL on inhibition of MPO activity by CP. This observationconfirmed the relevance of the method used to estimate the LDLaffinity for MPO.

3.2. Accumulation of cholesterol and its esters inmonocytes/macrophages after their incubation with modified LDL

Monocytes isolated from peripheral blood were incubatedwith LDL modified under various conditions, and accumulation ofcholesterol and its esters in the cells was measured (Figs. 1 and 2).To evaluate the effect of the LDL–MPO interaction on accumulationof intracellular cholesterol we accomplished parallel tests with thepeptide P445–456 that precludes MPO and LDL from interacting.Unmodified LDL per se or in the presence of MPO, CP, or CPpr (datanot presented) did not cause accumulation of cholesterol and itsesters.

In contrast, LDL modified using HOCl or HOBr caused significantaccumulation of cholesterol and its esters in the cells. Even strongereffect was achieved when LDL were modified by the MPO/H2O2/Cl−

and MPO/H2O2/Br− systems, which caused, respectively, 4- and7-fold increase in accumulation of cholesterol and its esters.Uncoupling the LDL–MPO interaction by P445–456 diminishedaccumulation of cholesterol and its esters in the cells. Indeed, the

Thiocyanate (SCN−) is a substrate of the MPO halogenating cycle.The enzyme catalyzes conversion of thiocyanate into a relatively

A.V. Sokolov et al. / Chemistry and Physics of Lipids 180 (2014) 72–80 75

Table 1Changes of kinetic parameters of MPO-catalyzed oxidation of TMB in the presence of CP and LDL.

KM, �M Vmax, A650/min Ki(CP), nM Ki(LDL), nM

MPO 258 ± 12 0.833 ± 0.020 – –MPO + CP 479 ± 14 0.812 ± 0.017 233 ± 16 –MPO + LDL 236 ± 16 0.808 ± 0.014 – –MPO + CP + LDL 70 ± 3 0.353 ± 0.015 84 ± 7 0.21 ± 0.02MPO + CP + LDL + P445–456 490 ± 15 0.822 ± 0.015 222 ± 14 –MPO + LDL/HOCl 219 ± 12 0.795 ± 0.017 – –MPO + LDL/HOBr 234 ± 13 0.818 ± 0.016 – –MPO + LDL/MPO/H2O2/Cl− 242 ± 11 0.825 ± 0.016 – –MPO + LDL/MPO/H2O2/Br− 235 ± 12 0.817 ± 0.016 – –MPO + CP + LDL/HOCl 69 ± 4 0.353 ± 0.012 84 ± 6 0.21 ± 0.02MPO + CP + LDL/HOBr 70 ± 2 0.353 ± 0.012 84 ± 6 0.21 ± 0.01MPO + CP +LDL/MPO/H2O2/Cl− 68 ± 3 0.356 ± 0.011 84 ± 7 0.21 ± 0.02MPO + CP + LDL/MPO/H2O2/Br− 70 ± 2 0.

Fig. 1. Effect of P445–456 (80 nM) on cholesterol accumulation in mono-cytes/macrophages incubated for 12 and 72 h with native or modified LDL.LDL were pre-modified by 30-min incubation with HOCl, HOBr, MPO/H2O2/Cl− ,MPO/H2O2/Br− or MPO/H2O2/SCN− . To a number of samples MPO inhibitors or mod-ulators, such as ABAH (5 �M), CP (6 �M), CPpr (6 �M), SCN− (200 �M), were addedalong with peptide P445–456. Cells incubated without LDL served as control.

Fig. 2. Effect of P445–456 (80 nM) on cholesterol esters’ accumulation in mono-cytes/macrophages incubated for 12 and 72 h with native or modified LDL (40 nM).LDL were pre-modified by 30-min incubation with HOCl, HOBr, MPO/H2O2/Cl− ,MPO/H2O2/Br− or MPO/H2O2/SCN− . To a number of samples MPO inhibitors or mod-ulators, such as ABAH (5 �M), CP (6 �M), CPpr (6 �M), SCN− (200 �M), were addedalong with peptide P445–456. Cells incubated without LDL served as control.

354 ± 0.012 84 ± 7 0.21 ± 0.02

mild oxidant, HOSCN (van Dalen et al., 1997). LDL modified in thepresence of the MPO/H2O2/SCN− system did not cause accumula-tion of cholesterol or its esters in the cells. Competing with Cl− andBr−, the SCN− ion can alter the halogenating activity of MPO, reduc-ing or preventing the yield of HOCl/HOBr. Indeed, LDL modified byMPO/H2O2/(Cl− or Br−) in the presence of SCN− did not cause accu-mulation of cholesterol and its esters in monocytes/macrophages.The P445–456 peptide preventing the interaction between MPOand LDL, had virtually no effect on the antiatherogenic propertiesof SCN− (Figs. 1 and 2). LDL treated with MPO/H2O2/(Cl− or Br−)systems in the presence of ABAH, a synthetic inhibitor of MPO,caused no accumulation of cholesterol and its esters in the cells(Figs. 1 and 2). With this respect the effect of ABAH was similar tothat of SCN−.

When LDL were modified by MPO/H2O2/Cl− system, adding6 �M CP reduced accumulation of intracellular cholesterol and itsesters by 62%, but had no effect on cholesterol accumulation if LDLwere modified by the bromide-containing system (MPO/H2O2/Br−)(Figs. 1 and 2). Adding CPpr had no effect on proatherogenic prop-erties of MPO. This is in line with the notion that inhibition of MPOby non-fragmented CP is needed to reduce the accumulation ofcholesterol and its esters in the cells.

3.3. Exo- and endocytosis of MPO by leukocytes stimulated bymodified LDL

A component of monocytes and neutrophils, MPO is secretedinto the extracellular space upon activation of these cells. One ofthe likely mechanisms of MPO release is activation of leukocytes byproteins, lipids, and lipoproteins modified by oxidants (HOCl, HOBr,etc.), which is in agreement with the positive feedback principle(Kopprasch et al., 1998; Witko-Sarsat et al., 2003; Gorudko et al.,2010, 2014).

Treatment of human neutrophils with LDL did not affect cellviability as evidenced by trypan blue test. Moreover, no LDH activityin extracellular space was detected.

Fig. 3 shows the effect of HOCl- and HOBr-modified LDL ondegranulation of human blood neutrophils. Adding HOCl- andHOBr-modified LDL to a suspension of neutrophils was followedby an increase of MPO concentration and activity in the extracel-lular space, indicating that MPO-containing azurophilic granulesof neutrophils became degranulated (Fig. 3A and B). Lactofer-rin, a component of specific granules in neutrophils was alsoreleased, that supports our notion that HOCl- and HOBr-modifiedLDL provoke degranulation of these cells (Fig. 3C). The capability of

76 A.V. Sokolov et al. / Chemistry and Physics of Lipids 180 (2014) 72–80

Fig. 3. Effect of LDL (50 �g/ml) modified by HOCl (LDL/HOCl) and HOBr (LDL/HOBr) on neutrophil degranulation, evaluated as MPO activity (A, expressed in ng/ml of pureM tants,

ihrtbL

ua1omcsemfiMecbttcPtibc

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2008), intact CP reduced the release of MPO if LDL were modifiedby the products of chlorinating reaction catalyzed by MPO. In caseof MPO-catalyzed bromination both CP forms were inefficient.

Fig. 4. Effect of P445–456 (80 nM) on MPO concentration in the medium withmonocytes/macrophages incubated for 12 and 72 h in the presence of the native

PO), MPO concentration (B) and lactoferrin concentration (C) in cell-free superna

ntensity of scopoletin fluorescence. Thus LDL modified with hypo-alous acids (HOCl and HOBr) that are formed in MPO-dependenteactions, stimulate the egress of this enzyme from neutrophils andhe production of H2O2. In the presence of hydrogen peroxide MPOecomes catalytically active, which favors further modification ofDL.

These results and the observation of HOCl-modified LDL stim-lating the respiratory burst of monocyte-like cell lines THP-1nd U937, differentiating into macrophages (Nguyen-Khoa et al.,999), allowed us suggesting that in response to the presencef modified LDL human blood monocytes (that differentiate intoacrophages) release MPO. Subsequent endocytosis by mono-

ytes/macrophages of the enzyme within the LDL–MPO complexeems possible. To verify this assumption we used solid-phasenzyme-linked immunoassay to measure MPO concentration in theedium after incubating cells with LDL. Fig. 4 shows that within the

rst 12 h of modified LDL incubation with monocytes/macrophagesPO was released into the extracellular space, as judged by its

levated concentration. However, after 72 h the extracellular con-entration of MPO returned to the normal level (Fig. 4), this processeing accompanied by accumulation of cholesterol and its esters inhe cells (Figs. 1 and 2). It seems likely that MPO disappears fromhe extracellular space concomitantly with the uptake of LDL parti-les by monocytes/macrophages. Moreover, on account that adding445–456 resulted in several fold lesser reduction of MPO con-ent in the incubation medium after 72 h, one can conclude thatts disappearance from the medium is at least partially providedy the cellular uptake of both LDL and MPO within the LDL–MPOomplex.

It should be noted that adding P445–456 to LDL incubated withatalytically active MPO gave rise to LDL that closely resembledOCl- or HOBr-modified LDL in their capacity to stimulate choles-

erol accumulation in monocytes/macrophages or to cause MPOelease. Thus, on the one hand, interaction of LDL with catalyticallyctive MPO (forming LDL–MPO) serves as an additional stimulus ofPO exocytosis. On the other hand, the level of released MPO after

2 h is significantly decreased in the presence of modified LDL, inontrast to the experiments with P445–456. That is, abrogation ofnteraction between MPO and LDL prevented endocytosis of MPOy the cells.

and the rate of neutrophil H2O2 production (per 106 cells) (D).

Studying the effect of SCN− and ABAH on MPO release andthe uptake of modified LDL by monocytes/macrophages showedthat the presence of ABAH and SCN− per se had virtually no effecton MPO egress from the cells (data not shown). Moreover, whenSCN− was added as a substrate during LDL modification by theMPO/H2O2/SCN− system or it was present during LDL modifica-tion by MPO/H2O2/(Cl− or Br−) systems, no significant additionalrelease of MPO (Fig. 4) or accumulation of cholesterol and its estersin the cells were noticed (Figs. 1 and 2).

In contrast to CPpr which is unable to inhibit MPO (Sokolov et al.,

or modified LDL (40 nM). LDL were pre-modified by 30-min incubation with HOCl,HOBr, MPO/H2O2/Cl− , MPO/H2O2/Br− or MPO/H2O2/SCN− . To a number of samplesMPO inhibitors or modulators, such as ABAH (5 �M), CP (6 �M), CPpr (6 �M), SCN−

(200 �M), were added along with peptide P445–456. Cells incubated without LDLserved as control.

A.V. Sokolov et al. / Chemistry and Physics of Lipids 180 (2014) 72–80 77

Fig. 5. PAAG stained with 4-chloro-1-naphtol and H2O2 after disk-electrophoresis ofLDL (4 �g) mixed with MPO (0.2 �g). 1 – LDL + MPO (sample 1); 2 – sample 1 + 0.5 �gP5

3d

attwr(mmtm4cdttsP

oPmttPtbp

4

tasThi(ue1soCm

Fig. 6. Effect of 8–80 nM P1–15 (squares) and P445–456 (triangles) on cholesteroland cholesterol esters’ accumulation in monocytes/macrophages incubated for 12 h(solid line) and 72 h (dashed line) with MPO-modified LDL. LDL were pre-modified

1–15; 3 – sample 1 + 0.5 �g HOCl-modified P1–15; 4 – sample 1 + 0.5 �g P445–456; – sample 1 + 0.5 �g HOCl-modified P445–456; 6-MPO.

.4. Anti-atherogenicity of P445–456 is conditioned byissociation of the LDL–MPO complex

Anti-atherogenicity of P445–456 can be caused both by dissoci-tion of the LDL–MPO complex and by direct scavenging of HOCl byhe cysteine residue which is the preferred target for HOCl amonghe amino acid residues composing this peptide. To clarify this issuee synthesized another anionic cysteine-containing peptide bor-

owed from the structure of apoB-100, i.e. 1EEEMLENVSLVCPKD15

P1–15) with the aim of comparing its effect on MPO-dependentodification of LDL and the following uptake of such LDL byonocytes/macrophages. It appeared that under conditions of elec-

rophoresis both the intact and the HOCl-treated P445–456 (1:2,ol/mol) displaced MPO from its complex with LDL (Fig. 5, lines

and 5). Under the same conditions P1–15, in contrast, was notapable of displacing MPO (Fig. 5, lines 2 and 3). Irrespective of oxi-ation by HOCl, in course of gel-filtration of MPO mixed with any ofhe peptides tried, P445–456 was eluted from Sephacryl S-200 HRogether with MPO, while P1–15 noticeably lagged behind (data nothown), which favors the notion that both intact and HOCl-oxidized445–456 bind to MPO while P1–15 does not.

The dependence of accumulation in monocytes/macrophagesf cholesterol and its esters on the concentration of P1–15 and445–456 (both intact and previously oxidized by HOCl during LDLodification by the system MPO/H2O2/Cl−) was compared. From

he plots in Fig. 6 it can be deduced that accumulation of choles-erol and its esters by cells does not depend on the presence of1–15. On the contrary, P445–456 prevents cholesterol accumula-ion in a dose-dependent manner. Oxidation of P445–456 causedy HOCl did not alter its capacity to prevent the MPO-dependentro-atherogenic modification of LDL.

. Discussion

Study of interaction between MPO and LDL has shown that nei-her modification by MPO, nor adding HOCl/HOBr to LDL alters theirffinity for MPO. It can be concluded that the oxidants used in thistudy do not affect the site in MPO that interacts with apoB-100.his effect is totally different from the interaction of MPO withigh-density lipoproteins (HDL), modification of which under sim-

lar conditions results in 100% disappearance of cysteine residuesMarsche et al., 2000). In view that only 20% cysteine disappearpon LDL treatment with HOCl in the molar ratio 1:100 (Mallet al., 2006), it can be suggested that modification of LDL with00-fold molar excess of HOCl does not affect the MPO-binding

by 30-min incubation with MPO/H2O2/Cl− in the presence of peptides P1–15 (darksquares), P1–15-HOCl (white squares), P445–456 (dark triangles) and P445–456-HOCl (white triangles). Cells incubated without LDL served as control.

dissociation of LDL–MPO complex by P445–456 is not altered (seeTable 1, Figs. 5 and 6).

It was observed that the affinity of MPO for HDL becomes higheras the extent of lipoproteins’ oxidative modifications increases(Marsche et al., 2008). Moreover, some data indicate that MPOinteracts with a certain site on the surface of apoA-I and thus causesoxidative alteration of tyrosines (Zheng et al., 2004). This suggeststhe occurrence of a vicious circle, i.e. the more pronounced is theMPO-mediated damage of HDL, the higher is their affinity for MPO.

Our observation that P445–456 completely blocked the effectof LDL on inhibition of MPO by CP allows regarding the evaluationof MPO affinity for modified LDL by assaying the enzyme’s activityas an adequate approach. The Ki value of 0.2 nM, which character-izes the affinity of LDL to MPO, is close to 0.13 nM obtained earlier(Sokolov et al., 2010). Considering this Ki it was reasonable to expectefficient uncoupling of LDL–MPO interaction upon adding LDL andP445–456 to the cells at final concentrations 40 nM and 80 nM,respectively.

We studied the effect of P445–456 on LDL uptake by mono-cytes/macrophages and accumulation of intracellular cholesterol.The peptide had virtually no effect when LDL modified by HOCl orHOBr were incubated with cells (Figs. 1 and 2). However, 72 h later,the concentration of MPO in the medium in the presence of such LDL

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8 A.V. Sokolov et al. / Chemistry an

y the cells, but the chosen concentration of P445–456 ensuredissociation of the LDL–MPO complex as monocytes/macrophages

n the presence of P445–456 took up LDL cholesterol, but notPO. Comparing cholesterol and its esters accumulation by mono-

ytes/macrophages in the presence of HOCl/HOBr-modified LDLith those affected by MPO functioning in the presence of Cl−/Br−,

ne can see that P445–456 brought cholesterol accumulation downo the level practically congruent with that in case of HOCl/HOBr-

odified LDL, when P445–456 becomes inefficient (Figs. 1 and 2).imilar effect is observed when comparing the MPO levels in theedium. After 12 h adding P445–456 reduces MPO exocytosis

aused by LDL modified with MPO/H2O2/(Cl− or Br−) to the levelomparable to that caused by the effect of HOCl/HOBr (Fig. 4). Inontrast, after 72 h the concentration of MPO in the presence of445–456 does not decrease, its value being virtually the sameor LDL modification either with HOCl and MPO/H2O2/Cl−, or withOBr and MPO/H2O2/Br−. A slightly smaller egress of MPO after2 h in the presence of MPO/H2O2/Br− and the peptide as comparedo HOBr may indicate that uncoupling of the LDL–MPO reduces the

odifying effect of the enzyme on LDL and thus diminishes thetimulation of MPO secretion by monocytes (Fig. 4).

The fact that monocytes and neutrophils secrete MPO in theresence of modified LDL agrees with the results of experimentsn the increase of luminol-dependent chemiluminescence of theseells in the presence of oxidized LDL (Panasenko et al., 1991).ccumulation of thiobarbituric acid-reactive substances in LDLpon incubation with monocytes and neutrophils was inhibited,espectively, by 88% and 65% in the presence of catalase, whichpeaks in favor of MPO involvement in the oxidative modificationf LDL (Panasenko et al., 1991). When SCN− or ABAH was addedo the catalytically active MPO, we observed neither accumulationf cholesterol and its esters in monocytes/macrophages nor MPOecretion by these cells. Thus, both inhibition of MPO activity andts switch to production of a less reactive oxidant species OSCN−

educe the oxidative modification of LDL, which, in all probability,s a stimulus for MPO secretion by monocytes.

It was shown earlier that when SCN− is used by MPO as aubstrate, it causes accumulation of lipid peroxidation productsn the LDL (Exner et al., 2004), despite that in the presence ofCN− modification of LDL caused by HOCl is reduced. Moreover,nhibitory analysis shows that cysteine (but not methionine) pre-ents neutrophil-induced lipid peroxidation in LDL in the presencef SCN− (Exner et al., 2004). Given our duration of incubation30 min), SCN− prevented the damaging effect of reactive halogenpecies formed in the halogenating cycle of MPO, but did not itselfrovoke noticeable lipid peroxidation (unpublished data).

Smoking is regarded as a risk factor of atherosclerosis ands reported to be associated with an increased SCN− level inlood plasma. However, chronic activation of neutrophils in smok-rs seems a more likely factor promoting atherogenesis ratherhan an increase of the SCN− concentration. It was shown thatruciferous-containing diet, rich in SCN−, reduces the risk ofardiovascular diseases in laboratory animals and patients withype II diabetes mellitus (Bahadoran et al., 2012; Tomofuji et al.,012). ABAH, an MPO inhibitor, also prevented the proathero-enic effect of LDL modified by reactive halogen species, whichs in line with its anti-inflammatory properties with regard to

onocytes (van der Does et al., 2012). However, using it forrevention of atherosclerosis is hardly possible, considering theomparatively high toxicity of this compound (LD50 180 mg/kg)http://datasheets.scbt.com/sc-204107.pdf).

The degree to which the LDL proatherogenic effect is decreased

sics of Lipids 180 (2014) 72–80

to the competitive inhibition by CP of the chlorinating activity ofMPO. A stronger inhibitory effect of CP on chlorinating, but notbrominating activity of MPO is likely to result from its incapacity tocompete with the higher affinity of MPO for Br− in comparison tothat for Cl− (our unpublished data). The absence of any inhibitoryeffect in case of CPpr is in line with our results showing that it isunable to inhibit the chlorinating activity of MPO either (Panasenkoet al., 2008; Sokolov et al., 2008).

To prove that the revealed anti-atherogenic effect of P445–456 isprovided by dissociation of the LDL–MPO complex, but is not causedby direct scavenging of HOCl by the cysteine (C) residue present inthe peptide, we first of all showed that P445–456 oxidized by HOClin advance, displaces MPO from its complex with LDL, as does thenative P445–456. The fact that oxidation of C residue has no effectupon the affinity of the anionic P445–456 to MPO is not surprisingon account of a relatively weak negative charge of C as comparedto the six carboxylic amino acids present in P445–456. Moreover,another synthetic peptide with a structure repeating that of the N-terminal sequence in apoB-100 (P1–15) also carrying a C residuedid not displace MPO from its complex with LDL (Fig. 5). It is worthnoting that P1–15, like P445–456, contains carboxylic amino acids,but also does not interact with MPO (Fig. 5). This is another evi-dence of the high specificity of the interaction between P445–456and MPO. Our study of a possible dependence of accumulation ofcholesterol and its esters in monocytes/macrophages on the con-centration of P445–456 and of its oxidized form did not revealdifferences between their anti-atherogenic effects (Fig. 6). Mean-while, the control peptide P1–15 had no effect on accumulationof cholesterol and its esters, regardless whether it was oxidized ornot. Therefore, our results allow concluding that anti-atherogenicfeatures of P445–456 are connected with its competition withLDL for binding to MPO, but not for the interaction withHOCl.

Thus, on the basis of the data obtained, we can propose asummarized scheme of proatherogenic modification of LDL andformation of foam cells, accounting for participation in that pro-cess of MPO and MPO-produced reactive halogen species (Fig. 7).Binding of MPO to LDL results in their site-specific modification,which mostly consists in protein damage and also in lipid halo-genation/peroxidation caused by HOCl and HOBr formed in thehalogenating cycle of MPO. Modified LDL serve as a stimulusfor leukocytes, provoking a respiratory burst and MPO exocy-tosis by neutrophils and monocytes (in Fig. 7 this is shownonly for monocytes). The secreted MPO binds to LDL and, usingthe leukocyte-produced H2O2 as the substrate, increases theirdamage, thus closing the “vicious circle” of modified LDL forma-tion. Modified LDL in their complex with MPO are taken up bymonocytes/macrophages, which leads to cholesterol accumula-tion in these cells and to their transformation into foam cells.Inhibitors/modulators of MPO halogenating activity (CP, ABAH andSCN−), along with the compounds uncoupling the LDL–MPO com-plex (P445–456), prevent oxidative/halogenating modification ofLDL. On the one hand, this process reduces the uptake of LDL bycells and accumulation of intracellular cholesterol and its esters; onthe other hand, it breaks the vicious circle of monocyte (neutrophil)activation and formation of modified LDL.

This study opens new prospects for investigating the effect ofuncoupling of the LDL–MPO complex using models of atheroscle-rotic damage, as well as for searching for analogs of P445–456 thatcan just as efficiently compete for MPO in its interaction with LDL,with the aim of creating fundamentally new approaches to prophy-laxis and treatment of atherosclerosis. The indisputable advantageof interventions that involve suppression of MPO–LDL binding andcause the switch of the enzyme to SCN− oxidation, is maintenance

A.V. Sokolov et al. / Chemistry and Physics of Lipids 180 (2014) 72–80 79

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ig. 7. A scheme outlining participation of MPO and the MPO-produced reactive haf inhibitors/modulators (ABAH, CP, SCN−) of halogenating activity in proatherogenell formation.

cknowledgements

This study was supported by RFBR grants 11-04-01262, 12-4-90003, 12-04-00301, 14-04-00807, 14-04-90007, BRFBR grant12R-036 and the RAMS Program “Human Proteome”. Authorsre grateful to Prof. V.N. Kokryakov (Institute of Experimentaledicine) for the generously donated human buffy coat.

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