Effects of Reagent and Enzymatically Generated Hypochlorite on ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 272, No. 47, Issue of November 21, pp. 29711–29720, 1997 Printed in U.S.A.

Effects of Reagent and Enzymatically Generated Hypochlorite on Physicochemical and Metabolic Properties of High Density Lipoproteins* (Received for publication, March 21, 1997, and in revised form, September 21, 1997)

Ute Panzenboeck, Sabine Raitmayer, Helga Reicher, Helmut Lindner‡, Otto Glatter‡, Ernst Malle, and Wolfgang Sattler§ From the University Graz, Department of Medical Biochemistry, Harrachgasse 21, and ‡Department of Physical Chemistry, Heinrichstrasse 28, A-8010 Graz, Austria

Myeloperoxidase (MPO), a protein secreted by activated phagocytes, may be a potential candidate for the generation of modified/oxidized lipoproteins in vivo via intermediate formation of HOCl, a powerful oxidant. During the present study, the effects of reagent NaOCl and OCl2 generated by the MPO/H2O2/Cl2 system on physicochemical and metabolic properties of high density lipoprotein (HDL) subclass 3 (HDL3) were investigated. Up to a molar oxidant:lipoprotein ratio of approximately 30:1, apolipoprotein A-I (apoA-I), the major HDL3 apolipoprotein component, represented the preferential target for OCl2 attack (consuming 35–76% of the oxidant), thereby protecting HDL3 fatty acids (consuming between 17 and 30% of the oxidant) against OCl2mediated modification. At molar oxidant:HDL3 ratios > 60:1, we have observed pronounced consumption of HDL3 unsaturated fatty acids with concomitant formation of fatty acid chlorohydrins. Modification of HDL3 in the presence of the MPO/H2O2/Cl2 system resulted in amino acid oxidation in a manner comparable with that found with reagent NaOCl only. Treatment of HDL3 with reagent NaOCl as well as modification by the MPO/H2O2/ Cl2 system resulted in significantly enhanced turnover rates of HDL3 by mouse peritoneal macrophages, an effect that was not a result of HDL3 aggregation as judged by dynamic and static light-scattering experiments. In comparison with native HDL3, the degradation by macrophages was enhanced by 4- and 15-fold when HDL3 was modified with reagent NaOCl or the MPO/ H2O2/Cl2 system. Finally, the ability of HDL3 to promote cellular cholesterol efflux from macrophages was significantly diminished after modification with reagent NaOCl. Collectively, these results demonstrate that the modification of HDL3 by hypochlorite (added as reagent or generated by the MPO/H2O2/Cl2 system) transformed an antiatherogenic lipoprotein particle into a modified lipoprotein with characteristics similar to lipoproteins commonly thought to initiate foam cell formation in vivo.

* This work was supported by Austrian Research Foundation Grants P12000 MED (to W. S.) and P11276 MED (to E. M.) and by FranzLanyar Stiftung and Austrian National Bank Projects 6232 (to W. S.) and 5677 (to E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed: Karl-Franzens University Graz, Dept. of Medical Biochemistry, Harrachgasse 21, A-8010 Graz, Austria. Tel.: 43-316-380-4188; Fax: 43-316-380-9615; E-mail: [email protected]. This paper is available on line at http://www.jbc.org

In contrast to low density lipoproteins (LDL),1 high plasma concentrations of high density lipoproteins (HDL) are associated with a decreased risk for the development of coronary artery disease, an effect commonly attributed to their central role in reverse cholesterol transport (1). During this process, HDL is able to promote efflux of cholesterol from peripheral tissues, and the accepted cholesterol is (at least in part) esterified by the action of lecithin-cholesterol acyltransferase (2). Cholesteryl esters (CEs) formed by the lecithin-cholesterol acyltransferase reaction are then transferred from HDL to other lipoproteins mediated by the cholesterol ester transfer protein or are delivered to the liver for bilary secretion or reutilization during lipoprotein assembling (3). Within the oxidative theory, HDL appears to be Janus-faced (4 – 6). The majority of lipid hydroperoxides (the first detectable products of lipoprotein oxidation) in plasma are transported in the HDL fraction (7), and HDL was suggested to act as a sink for preformed lipid hydro(pero)xides. HDL-associated cholesteryl ester hydroperoxides, which are probably transferred to HDL by the action of the cholesterol ester transfer protein (8), are preferentially catabolized over nonoxidized CEs; this was shown in HepG2 cells (9), in situ perfused rat liver (10), and intact rats (11). HDL was also shown to protect LDL from lipid peroxidation, inhibiting the formation of lipid hydroperoxides but not the formation of conjugated dienes (12). On the other hand, HDL is more easily oxidized than LDL (7), HDLassociated lecithin-cholesterol acyltransferase is modified by reactive short and long chain aldehydes (13), and the ability of oxidatively modified HDL to promote cellular cholesterol efflux is diminished (14 –16), most probably due to alterations in its apolipoprotein moiety. During the acute phase response, up to 80% of apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, is displaced by serum amyloid A (17), and due to this exchange in the HDL apolipoprotein domain, native, anti-inflammatory HDL became proinflammatory during the acute phase response (18). The above mentioned results imply that HDL can protect LDL against oxidative modifications and could serve as a vehicle for detoxification of potentially (cyto)toxic lipid hydroperoxides. However, under certain circumstances many of the important physiological properties of HDL can be lost during oxidation/modification, transforming HDL into a proatherogenic lipoprotein particle. 1 The abbreviations used are: LDL, low density lipoprotein(s); apo, apolipoprotein; CE, cholesteryl ester; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; GC-MS; gas chromatography-mass spectrometry; HDL, high density lipoprotein(s); MPO, myeloperoxidase; NaOCl, sodium hypochlorite; LPDS, lipoprotein-deficient serum; PBS, phosphate-buffered saline; RH, hydrodynamic radius; TBS, Tris-buffered saline; TMS, trimethylsilyl; [3H]Ch18:2, [cholesteryl-1,2,6,73 H]linoleate.

29711

29712

Hypochlorite Modification of High Density Lipoproteins

Within the wide spectrum of oxidants available for the organism, hypochlorous acid (HOCl) is thought to play an important role during microbial killing and inflammatory tissue injury by neutrophils and monocytes (19). Hypochlorite (OCl2), formed in vivo via the myeloperoxidase (MPO)/H2O2/halide system from activated neutrophils and/or monocytes, may react with a wide range of biological target molecules, including lipids, antioxidants, and proteins (20). Modification of LDL with HOCl in vitro has generated a modified particle, which was avidly taken up by macrophages (21). The importance of MPO as a potential in vivo oxidant is further underlined by the presence of enzymatically active MPO in human atherosclerotic lesions (22). Accordingly, the presence of HOCl-modified (lipo)proteins was recently demonstrated in advanced human atherosclerotic lesions (23) and inflammatory kidney tissues rich in MPO (24). In the current study, we examined the consequences of NaOCl modification on the apolipoprotein and lipid domain of HDL3, the concomitant changes in HDL3-associated CE and holoparticle metabolism in macrophages, and the cholesterol efflux properties. We found that HDL3-associated amino acids, unsaturated fatty acids, and cholesterol were modified by HOCl, either added as reagent or generated by the MPO/H2O2/ Cl2 system. Presumably as a result of apolipoprotein modification, binding, internalization, and degradation of NaOCl and MPO modified HDL3 by mouse peritoneal macrophages were greatly enhanced. We demonstrate further that NaOCl treatment of HDL3 resulted in impaired ability to promote cholesterol efflux from the cellular plasma membrane of macrophages. EXPERIMENTAL PROCEDURES

Materials NaOCl, BF3/MeOH, egg yolk lecithin, hexamethyldisilazane, trimethylchlorosilane, organic solvents, and potassium bromide were obtained from Sigma. MPO (isolated from human leukocytes) was from Alexis Biochemicals. Radiochemicals were purchased from NEN Life Science Products. Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were obtained from Boehringer Ingelheim Bioproducts. Plasticware used for tissue culture was obtained from Costar. All other chemicals were obtained from Merck, except where indicated.

Methods Preparation of Human, ApoE-free HDL3 Human apoE-free HDL3 was prepared by discontinuous density ultracentrifugation of plasma obtained from normolipemic donors in a TL120 Beckman tabletop ultracentrifuge using a TLA100.4 rotor (Beckman) as described (25).

NaOCl Modification of HDL3 Modification of HDL3 with OCl2 was performed as described previously (21, 26). One mg of HDL3 protein/ml of phosphate-buffered saline (PBS) was incubated with NaOCl solution (added as single addition and gentle vortexing) at molar ratios of NaOCl to HDL3 ranging from 7.5 (90 mM NaOCl) to 400 (5.3 mM NaOCl; 60 min, 37 °C, under argon) with the final pH adjusted to 7.4. The modified HDL3 preparations were passed over a PD10 column to remove unreacted NaOCl. The concentration of the reagent NaOCl was determined spectrophotometrically using a molar absorption coefficient for OCl2 of 350 cm21 at 292 nm (27). Depending on the type of experiment performed after NaOCl treatment, the modified HDL3 preparations were used between 2 and 24 h after exposure to the oxidant.

MPO Modification of HDL3 HDL3 modification in the presence of the MPO/H2O2/Cl2 system was performed at pH 7.4 or 4.5 to generate HOCl or Cl2 as the oxidant (28, 29). Briefly, to the substrates (1 mg of HDL3 protein/ml in PBS (50 mM, pH 7.4) or in sodium phosphate (50 mM, 100 mM diethylenetriamine pentaacetic acid, pH 4.5)) additions of 20 mM H2O2 were made at 10-min intervals at 37 °C until reaching a total of 15 additions (final concentration 300 mM; assuming quantitative conversion, a molar oxidant: lipoprotein ratio of ;20:1 could be expected). MPO (13 nM) was added at the start and subsequently at every second addition of H2O2. At alter-

nate additions of H2O2, 2 mM ascorbate was added. To blank incubations, only H2O2 and ascorbate were added in the same concentrations and sequence as described above. The reaction mixture was incubated for 1 h (37 °C) and subsequently dialyzed against PBS (10 mM, 4 °C).

Lipoprotein Labeling Procedures HDL3 Labeling with 125INa—Iodination of HDL3 was performed as described by Sinn et al. (30) using N-Br-succinimide as the coupling agent. Routinely, 1 mCi of 125INa (Amersham Corp.) was used to label 5 mg of HDL3 protein. This procedure resulted in specific activities between 300 and 450 dpm/ng of protein with less than 3% lipid-associated activity. No cross-linking or fragmentation of apoA-I due to the iodination procedure could be detected by SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. HDL3 Labeling with [cholesteryl-1,2,6,7-3H]Linoleate ([3H]Ch18:2)— HDL3 was labeled with [cholesteryl-1,2,6,7-3H]linoleate (NEN Life Science Products) by cholesterol ester transfer protein-catalyzed transfer from donor liposomes essentially as described (9). [3H]Ch18:2-labeled HDL3 was reisolated in a TLX120 benchtop ultracentrifuge using a TLA100.4 rotor as described (25). This labeling procedure resulted in specific activities of 5–7 cpm/ng HDL3 protein.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting SDS-polyacrylamide gel electrophoresis of HDL3 apolipoproteins was performed using 5–15% polyacrylamide gradient gels with electrophoresis at 150 V for 90 min in a Bio-Rad miniprotean chamber (BioRad, Austria) (26). For Western blotting experiments, proteins were electrophoretically transferred to nitrocellulose membranes (150 mA, 4 °C, 90 min). Immunochemical detection of NaOCl-modified apolipoproteins was performed with a monoclonal antibody clone 2D10G9 (Ref. 26; dilution 1:50) followed by peroxidase-conjugated goat anti-mouse IgG (Bio-Rad, 1:5000). Immunochemical detection of apoA-I in the same lipoprotein samples was performed with rabbit polyclonal anti-human apoA-I (Behring, Germany) as a primary antibody, followed by peroxidase-conjugated goat anti-rabbit IgGs as secondary antibodies. Detection was performed by the ECL method.

Dynamic Light Scattering Dynamic light-scattering experiments were performed with native and NaOCl-modified HDL3. The laboratory-built goniometer was equipped with an Argon1 laser (Spectra Physics, model 2060-55, Pmax 5 5 watts, l 5 514.5 nm), single mode fiber detection and an ALV-5000 correlator (ALV, Germany). Measurements were carried out at a scattering angle of 90° at 25 °C and a laser power of 200 milliwatts. The time dependence of the scattering intensity, represented by its correlation function, provides information on diffusional motion of the scatterer. The diffusion coefficient is related to the apparent hydrodynamic radius, RH, by the Stokes-Einstein equation, i.e. an equivalent sphere with radius RH shows the same diffusion behavior as the particle under investigation and serves as a size parameter. Polydisperse systems give rise to a correlation function with a spectrum of different decay constants. A series expansion called cumulant fit (31) results in a mean value for the diffusion coefficient (first cumulant, c1) and its variance (second cumulant, c2). The ratio c2/c12 is called the polydispersity index. Any aggregation of HDL3 particles will lead to an increase of the measured RH values and the polydispersity index.

Static Light Scattering and Integrated Intensity The integrated intensity (averaged over long times compared with the time scale of fluctuations in the dynamic experiment) is another, completely independent, parameter for aggregation phenomena. For particles that are small compared with the wavelength of the scattered radiation, this intensity is directly related to the mean mass or aggregation number of the particles and independent of its diffusion dynamics. Any increase in this intensity relative to the native control is equivalent to the degree of aggregation, i.e. dimerization would lead to a 2-fold increase of the integrated intensity.

Amino Acid Analysis Aliquots of native and NaOCl-modified HDL3 (450 mg of protein) were lyophylized in 5-ml ampules and purged with nitrogen before hydrolysis in constant boiling 6 N HCl (24 h, 120 °C). Amino acid analysis was performed as described by Parks and Rudel (32) on a Biotronics analyzer.

Hypochlorite Modification of High Density Lipoproteins Fatty Acid and Cholesterol Analysis Fatty acid analysis of HDL3 lipids was performed as described (33). Separation of fatty acid methylesters (2-ml samples) was performed on a fused silica 25-m FFAP-CB column (0.32-mm inner diameter; Chrompack) using an HP 5890 gas chromatograph equipped with a flame ionization detector and a split/splitless injector (Hewlett-Packard Co.). Cholesterol analysis of HDL3 lipids was performed as described by Sattler et al. (25).

Gas Chromatography-Mass Spectrometry

TABLE I Amino acid analysis of native and NaOCl-modified HDL3 apolipoproteins ApoE-free HDL3 was prepared by discontinuous density ultracentrifugation and modified with increasing concentrations of NaOCl (7.5–300 molecules/HDL3 particle). Amino acid analysis was performed on hydrolysates (900 mg of total lipoprotein mass of native or NaOCl-modified HDL3) prepared in sealed tubes under vacuum in 6 N HCl at 105 °C for 24 h. Data shown represent mean values from one experiment performed in triplicate. Results are expressed in mol of amino acids/mol of HDL3.

Gas chromatography-mass spectrometry (GC-MS) analysis of NaOCl-modified HDL3 lipids was performed essentially as described by van den Berg et al. (28, 34). The extracted HDL3 lipids were transmethylated with BF3/MeOH as described above. The fatty acid and chlorohydrin fatty acid methylesters were converted to the corresponding trimethylsilyl (TMS)-ether derivatives with pyridine/hexamethyldisilazane/trimethylchlorosilane (9:3:1; v/v/v) at 65 °C for 1 h. One-ml samples of the O-methylester-O-TMS-ether derivatives were analyzed by GC-MS without further extraction. Separation was performed on a DB-5 capillary column (15 m, 0.25-mm inner diameter, 0.25-mm film thickness from J & W Scientific, Folsom, CA) using helium as carrier gas. The column was kept at 120 °C (3 min) and programmed to 180 °C at a rate of 8 °C/min. A second ramp was programmed from 180 °C at 5 °C/min to 260 °C with an isothermal hold at 260 °C for 5 min. The quadrupole was operated in the electron impact mode at 70 eV and a source temperature of 200 °C.

Amino acid

Cholesterol Efflux Experiments Thioglycollate-stimulated mouse peritoneal macrophages were harvested and plated as described above. The cells were incubated in the presence of DMEM containing LPDS (10%, v/v) and [3H]cholesterol (0.05 mCi/ml) for 2 days (39). Prior to the cholesterol efflux experiments the [3H]cholesterol-containing medium was aspirated, and the cells were washed twice in TBS (containing 5% (w/v) bovine serum albumin) and twice in TBS. Efflux experiments were then initiated by the addition of DMEM containing LPDS (10%, v/v) and HDL3 or NaOCl-HDL3 (0.5 mg of protein/ml). At the indicated time points, the medium was collected, and the cells were lysed in 0.3 N NaOH to estimate both the remaining radioactivity and the cellular protein content. The efflux of radioactive label to the medium was calculated as the percentage of radioactivity present in the cells prior to the addition of NaOCl-HDL3or native HDL3-containing medium.

NaOCl:HDL3 (ratio)

Native 7.5

15

30

60

150

300

mol/mol

Cell Culture Studies Thioglycollate-elicited mouse peritoneal macrophages were plated on 6- or 12-well trays in DMEM containing 10% (v/v) FCS as described recently by Panzenboeck et al. (35). After 4 h, cells were washed three times with PBS and kept in DMEM (containing FCS) for 2 or 3 days. One day prior to the experiments, cells were preincubated in DMEM containing lipoprotein-depleted serum (LPDS, 10%, v/v). Lipid loading experiments of macrophages were performed with radioactively labeled (125INa or [3H]Ch18:2) native HDL3 and NaOCl-HDL3 in the presence or absence of a 20-fold excess of unlabeled HDL3. Radioactively labeled native or NaOCl-modified HDL3 was added to a final concentration of 50 mg of protein/well, and cells were incubated in DMEM (10% FCS) at 37 °C for 6 h. Following this incubation, the cells were washed twice in Tris-buffered saline (TBS) containing bovine serum albumin (5%, w/v), followed by three washes in TBS. To release cell membrane-bound HDL3, cells were incubated at 37 °C for 10 min in the presence of trypsin (0.05%, Cytosystems). The trypsin-releasable fraction is referred to as “bound” fraction. Cells were then lysed in NaOH (1 ml, 0.3 N, 1 h at 4 °C) to determine both the non-trypsin-releasable fraction (“internalized” fraction) and the cell protein in the lysate. Specific binding/internalization was calculated as the difference between total and nonspecific binding/internalization. Protein measurement was performed according to Lowry (36). On average, the protein content per well was 300 –350 mg. Degradation of 125I-HDL3 or 125I-NaOCl-HDL3 by mouse peritoneal macrophages was estimated by measuring the nontrichloroacetic acid-precipitable radioactivity in the medium after precipitation of free iodine with AgNO3 (37). To facilitate the comparison of results obtained with 125I-HDL3/125I-NaOCl-HDL3 and [3H]Ch18:2HDL3 /[3H]Ch18:2-NaOCl-HDL3, selective uptake of HDL3-CE is expressed as apparent HDL3 particle uptake (i.e. as the amount of apoA-I that would deliver the observed amount of tracer if uptake were solely mediated by holoparticle uptake) as suggested by Pittman et al. (38).

29713

Asxa Thr Ser Glxb Pro Gly Ala Val Cys Met Ile Leu Tyr Phe Lys His Trp Arg

45.7 31.1 39.0 110.1 24.2 22.3 40.5 32.5 1.3 1.9 4.7 78.2 22.7 20.3 55.3 8.7 NDc 29.0

46.0 30.3 39.1 110.4 24.4 22.5 40.4 33.3 0.6 1.7 4.6 78.9 21.6 20.0 53.7 8.5 ND 28.7

44.9 29.7 39.0 109.9 24.5 21.4 40.9 32.7 1.1 1.4 4.3 78.2 20.0 19.4 52.5 8.4 ND 28.5

45.1 28.7 38.2 110.7 24.5 21.8 41.8 32.8 NTd 1.2 4.0 79.3 16.9 19.4 48.9 7.8 ND 27.9

44.6 28.1 34.5 107.9 24.2 21.2 41.4 31.8 NT NT 4.0 77.5 13.4 19.4 38.1 7.6 ND 27.4

42.5 27.6 35.1 106.3 23.5 20.3 39.3 32.4 NT NT 4.0 76.4 4.9 19.1 30.6 4.8 ND 23.2

44.4 27.4 34.5 109.0 23.6 20.5 39.5 31.8 NT NT 3.9 77.8 0.3 6.7 25.2 2.9 ND 10.8

Total Lost

567.6

564.7 3

556.7 11

549.1 19

521.2 46

490.0 78

458.3 109

Asp 1 Asn. Glu 1 Gln. ND, not determined. d NT, not detectable. a b c

RESULTS

Effect of NaOCl Modification on HDL3 Apolipoproteins—To investigate the effect of NaOCl on the amino acid composition of HDL3-associated apoA-I, HDL3 aliquots (containing a total HDL3 mass of 900 mg) were treated with increasing concentrations of NaOCl (pH 7.4). The molar oxidant:lipoprotein ratio ranged between 7.5 and 300. The results of the amino acid analyses are shown in Table I. An increasing degree of NaOClmodification of HDL3 was paralleled by an increasing loss of total amino acids from 3 to 109 mol/mol HDL3. Following the loss of individual amino acids as a function of increasing NaOCl concentration, three groups of amino acids with respect to their sensitivity toward NaOCl reactivity could be discriminated. The first and most sensitive group was composed of Cys, Met, and Tyr. Cys and Met were completely consumed at a molar NaOCl:HDL3 ratio of 30 and 60, respectively. Tyr was consumed in a dose-dependent fashion by 5–95% at NaOCl:HDL3 ratios of 7.5:1 and 300:1. The second group (less reactive toward NaOCl modification) is constituted by Phe, Lys, His, and Arg, which were consumed by 67, 55, 67, and 63%, respectively, at the highest NaOCl concentration used. Finally, the third group, relatively insensitive to NaOCl treatment, is composed of Asx, Thr, Ser, Glx, Pro, Gly, Ala, Val, Ile, and Leu. As can be seen from data shown in Table I, between 36 and 78% of NaOCl was consumed by reactions with HDL3 apolipoprotein amino acids. In line with these findings, the relative electrophoretic mobility of NaOCl-modified HDL3 as assessed by agarose gel electrophoresis increased to 1.02 (15:1), 1.1 (30:1), 1.2 (60:1), 1.6 (150:1), and 1.9 (300:1). The effects of the MPO/H2O2/Cl2 system on the amino acid composition of HDL3-associated apolipoproteins are shown in Table II. Incubations were performed at pH 7.4 and 4.5 to

29714


TABLE II Amino acid analysis of native and MPO-modified HDL3 apolipoproteins HDL3 was modified in the presence of the MPO/H2O2/Cl2 system at pH 7.4 and 4.5 to generate OCl2 and Cl2, respectively. MPO modification at pH 7.4 and 4.5 was performed as described under “Experimental Procedures.” Amino acid analysis was performed on hydrolysates (1000 mg of total lipoprotein mass of native or MPO-modified HDL3) prepared in sealed tubes under vacuum in 6 N HCl at 105 °C for 24 h. Data shown represent mean values from one experiment performed in duplicate. Results are expressed in mol of amino acids/mol of HDL3. Amino acid

Native

MPO, pH 7.4

MPO, pH 4.5

Asx Thr Ser Glxb Pro Gly Ala Val Cys Met Ile Leu Tyr Phe Lys His Trp Arg

45.9 31.3 40.9 112.7 25.9 25.1 44.8 35.3 1.2 4.7 6.0 82.7 21.8 19.9 55.9 8.8 NDc 29.1

45.4 27.4 41.4 109.9 25.1 26.6 42.4 33.0 0.6 2.8 5.3 82.3 18.9 18.4 53.8 8.4 ND 29.3

45.8 29.3 40.1 112.0 24.4 26.3 42.2 33.8 0.5 2.9 6.1 82.5 18.2 18.9 53.4 8.6 ND 28.5

Total Lost

591.8

571.0 21

573.5 18

a

Asp 1 Asn. Glu 1 Gln. c ND, not determined. a b

generate either HOCl or Cl2 as the oxidant (28, 29). Assuming quantitative conversion of H2O2 by MPO, a molar oxidant: HDL3 ratio of ;20 –23 would be expected. Under these experimental conditions, we have observed pronounced Cys and Met consumption (60 and 40%), in line with data presented in Table I (NaOCl:HDL3 $ 15:1). Tyr, Phe, and Lys were also modified by the MPO/H2O2/Cl2 system, although to a lesser extent (17, 7, and 5%, respectively). Comparable modification rates were observed for Thr, Ala, and Val (Table II). With the exception of Thr, the modification rates for individual amino acids at pH 7.4 and 4.5 were quite similar. In summary, both HOCl and Cl2 generated by the MPO/H2O2/Cl2 system resulted in modification of HDL3-associated apolipoproteins. Next the modification of native apoA-I during treatment of HDL3 with increasing NaOCl concentrations was followed by Western blotting experiments. Detection of apoA-I and NaOClmodified apolipoproteins was performed with polyclonal rabbit anti-human apoA-I antiserum and a monoclonal antibody, specifically recognizing HOCl-modified proteins (26). An increasing molar oxidant:lipoprotein ratio resulted in a gradual loss of monomeric, 28-kDa apoA-I, with the concomitant formation of high molecular mass aggregates of apoA-I (Fig. 1A). At a molar oxidant:lipoprotein ratio of 50:1, immunoreactive bands with apparent molecular masses of ;45, 60, 90, and 140 kDa were formed. The appearance of high molecular mass apoA-I products (Fig. 1A, lanes 2–5) increased as a function of increasing NaOCl concentrations. The observed molecular masses suggest the formation of dimeric up to pentameric apoA-I products. While no NaOCl-modified epitopes were present in native apoA-I (Fig. 1B, lane 1), modification of HDL3 with increasing NaOCl concentrations resulted in the generation of NaOClmodified epitopes (Fig. 1B, lanes 2–5). Detection of immunoreactive bands by monoclonal antibody 2D10G9 revealed the formation of NaOCl-modified apoA-I cross-linked products with apparent molecular masses of approximately 40, 80, and 120 –

FIG. 1. Detection of native and NaOCl-modified apoA-I by Western blots. Native and NaOCl-HDL3 (5 mg of protein) were separated on 5–15% polyacrylamide gradient gels under denaturing conditions. Proteins were transferred to nitrocellulose as described under “Experimental Procedures.” Unmodified apoA-I (lane 1) and apoA-I of HDL3 modified with molar oxidant:HDL3 ratios of 50 (lane 2), 100 (lane 3), 200 (lane 4), and 400 (lane 5) were detected using rabbit anti-human apoA-I and visualized with goat anti-rabbit IgG with an ECL-Western blotting detection system (A). NaOCl-modified apolipoproteins were detected using monoclonal antibody 2D10G9 (26), and immunoreactive bands were visualized with goat anti-mouse IgG (B). The arrow indicates the position of the native apoA-I.

FIG. 2. Static and dynamic light-scattering analysis of native and NaOCl-modified HDL3. Static and dynamic light-scattering experiments were performed on a laboratory-built goniometer equipped with an Argon1 laser with single fiber detection and an ALV-5000 correlator as described under “Experimental Procedures.” Measurements were carried out at a scattering angle of 90° at 25 °C and a laser power of 200 milliwatts. RH values were obtained from the dynamic scattering experiments, and intensities were from static scattering experiments. Measurements were performed in PBS (10 mM, pH 7.4) at 25 °C and an HDL3 concentration of 1 mg of protein/ml.

140 kDa, data similar to those shown in Fig. 1A. To investigate whether HDL3 particles aggregated during NaOCl modification, native and NaOCl-modified lipoproteins were analyzed by dynamic and static light scattering. As can be seen from Fig. 2, NaOCl modification led to a dose-dependent increase in RH values from 6.7 6 0.3 nm (native) to 9.0 6 0.8 nm (NaOCl:HDL3 5 300:1). The polydispersity index was independent of the molar NaOCl:HDL3 ratio, with a value of 0.1 6 0.02. The mean scattering intensity increased in a dosedependent fashion from 53.3 6 1.2 (native HDL3) to 75.7 kcps


29715

TABLE III Fatty acid composition of native and NaOCl-modified HDL3 lipids Apo E-free HDL3 was modified with NaOCl at a molar ratio of 7.5–300 molecules/HDL3 particle. Fatty acid analysis was performed by gas chromatography of fatty acid methylesters as described under “Experimental Procedures.” Cholesterol was analyzed by high pressure liquid chromatography. Results represent the mean 6 S.D. from two experiments performed in triplicate and are expressed in mol/mol of HDL3. Fatty acid

NaOCl:HDL3 (ratio)

Native 7.5

15

30

60

150

300

mol/mol

14:0 16:0 16:1 18:0 18:1 (n-7) 18:1 (n-9) 18:2 18:3 20:4 22:6

1.0 6 0.08 36.3 6 1.54 2.7 6 0.11 14.1 6 0.58 22.0 6 0.97 1.9 6 0.10 39.9 6 1.56 1.1 6 0.04 18.9 6 0.70 5.0 6 0.11

1.0 6 0.16 35.9 6 2.06 2.6 6 0.16 13.6 6 0.77 21.5 6 1.08 1.9 6 0.13 39.5 6 2.29 1.1 6 0.05 18.0 6 1.03 1.5 6 0.10

1.0 6 0.05 35.1 6 0.23 2.6 6 0.02 13.6 6 0.07 21.4 6 0.12 1.9 6 0.07 38.7 6 0.03 1.3 6 0.07 17.6 6 0.10 1.5 6 0.02

0.9 6 0.02 34.5 6 1.18 2.6 6 0.11 13.3 6 0.37 21.0 6 0.64 1.8 6 0.05 37.9 6 1.10 1.2 6 0.04 17.0 6 0.50 1.4 6 0.07

0.9 6 0.01 34.3 6 0.35 2.5 6 0.12 13.2 6 0.10 20.9 6 0.26 1.8 6 0.07 36.4 6 0.58 1.1 6 0.06 15.1 6 0.06 1.3 6 0.02

0.9 6 0.01 36.7 6 0.36 2.1 6 0.09 13.0 6 0.13 14.6 6 0.09 2.6 6 0.01 19.6 6 0.14 0.6 6 0.06 3.8 6 0.05 NT

0.9 6 0.10 36.5 6 0.33 NTa 12.8 6 0.01 2.0 6 0.09 NT NT NT NT NT

Total UFAsb UFAs lost Cholc

139.2 6 5.8 88.0 6 3.62 0 6

136.6 6 7.8 86.1 6 4.84 2 6

134.4 6 0.8 84.9 6 0.43 3 6

131.6 6 4.1 82.9 6 2.51 5 5.5

127.5 6 1.6 79.1 6 1.17 9 5

93.9 6 0.9 43.4 6 0.44 45 4

52.1 6 0.5 2.0 6 0.09 86 3

a

NT, not detectable. UFAs, unsaturated fatty acids. c Unesterified cholesterol. b

TABLE IV Fatty acid composition of native and MPO-modified HDL3 lipids HDL3 was modified in the presence of the MPO/H2O2/Cl2 system at pH 7.4 and 4.5 to generate OCl2 and Cl2, respectively. Incubations at pH 7.4 and 4.5 were performed as described under “Experimental Procedures.” HDL3 lipids were extracted and transmethylated, and fatty acid methyl esters were analyzed by gas chromatography as described under “Experimental Procedures.” Results represent the mean 6 S.D. from one experiment performed in triplicate and are expressed in mol/mol of HDL3. Fatty acid

Native

H2O2 blank

MPO (pH 7.4)

MPO (pH 4.5)

mol/mol

14:0 16:0 16:1 18:0 18:1 (n-7) 18:1 (n-9) 18:2 18:3 20:4 22:6

0.6 6 0.12 31.5 6 2.31 2.6 6 0.17 13.4 6 0.79 18.8 6 1.85 2.0 6 0.19 39.9 6 2.2 0.7 6 0.13 13.6 6 1.94 2.5 6 0.16

0.6 6 0.08 29.5 6 2.17 2.5 6 0.09 13.0 6 0.51 16.1 6 1.12 2.1 6 0.07 37.3 6 1.3 NT NT NT

0.8 6 0.09 29.3 6 2.75 2.5 6 0.22 12.8 6 2.31 17.0 6 0.21 1.7 6 0.12 32.7 6 0.29 NT NT NT

0.6 6 0.14 30.9 6 1.91 2.0 6 0.12 13.0 6 1.15 15.3 6 0.93 1.6 6 0.08 32.6 6 2.91 NT NT NT

Total UFAsa UFAs lost Cholb

125.6 6 9.86 80.1 6 6.64 0 6

101.1 6 5.34 58.0 6 2.58 22 6

96.8 6 5.99 53.9 6 5.99 26 6

96.0 6 7.24 51.5 6 4.99 28 4

a

UFAs, unsaturated fatty acids. Unesterified cholesterol. c NT, not detectable. b

(NaOCl:HDL3 5 300:1). This moderate increase indicates that only a few HDL3 particles aggregate even at the highest NaOCl concentrations used. The mean aggregation number at a molar ratio of 30:1 was 1.1 (calculated from the integrated intensities shown in Fig. 2), and even at a 300-fold molar excess of NaOCl we have calculated aggregation numbers of only 1.4. Summarizing the light-scattering results shown in Fig. 2, we were able to demonstrate that NaOCl modification of HDL3 does not lead to essential interparticle aggregation. NaOCl-mediated Loss of Fatty Acids in HDL3—The fatty acid composition of HDL3 lipids before and after NaOCl treatment is shown in Table III. The fatty acid composition of the native sample is in good agreement with previous results (40). Treatment of HDL3 by increasing concentrations of NaOCl resulted in gradual loss of unsaturated fatty acids, i.e. C16:1, C18:1, C18:2, C18:3, C20:4, and C22:6. At a 300-fold molar excess of NaOCl, unsaturated fatty acids were almost completely consumed. Under the experimental conditions described in Tables III and IV, no formation of lipid peroxidation

products (measured as thiobarbituric acid-reactive substances) could be detected (data not shown). MPO/H2O2/Cl2-mediated Loss of Fatty Acids in HDL3— These experiments were performed to clarify whether oxidants generated by the MPO/H2O2/Cl2 system could inflict fatty acid modification in the lipid moiety of HDL3. Incubations of HDL3 in the presence of H2O2 and the complete MPO/H2O2/Cl2 system were performed at pH 7.4 and 4.5, and the resulting fatty acid composition is presented in Table IV. It is evident that the addition of H2O2 in the absence of MPO led to complete oxidation of 18:3, 20:4, and 22:6. When HDL3 was modified in the presence of the complete MPO/H2O2/Cl2 system at either pH 7.4 or 4.5, we observed a small increase in fatty acid consumption as compared with the H2O2 blank. The resulting fatty acid modification is very similar to data presented in Table III at NaOCl:HDL3 ratios of 15:1. In line with data reported for LDL (29), the modification of cholesterol in HDL3 exposed to the MPO/H2O2/Cl2 system occurred only at pH 4.5 (Table IV). In the next series of experiments, we investigated whether

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consumption of HDL3 fatty acids by NaOCl is accompanied by the formation of fatty acid chlorohydrins. Chlorohydrin formation was followed by GC-MS, essentially as described by van den Berg et al. (28, 34). The total ion current (TIC) trace of NaOCl-modified HDL3 lipid extracts revealed the occurrence of two peak clusters with retention times of 28.02 and 28.51 min not present in native HDL3 lipids (Fig. 3A, trace II). The mass spectrum of the peak eluting at 28.51 min (Fig. 3B) showed characteristic fragment ions at m/z values of 259 and 263 (relative abundance of 25 and 1.3%, respectively; base peak at m/z 73), corresponding to fragmentation next to the -O-TMS group of the [9-O-TMS,10-Cl] derivative of the chlorohydrin stearic acid methylester. As a comparison, the mass spectrum of a chlorohydrin stearic acid methylester obtained by NaOCl modification of oleic acid is shown in Fig. 3C. The fragment ions at m/z 215 and 307 (relative abundance of 17.9 and 0.96%, respectively; base peak at m/z 73) correspond to fragmentation next to the -O-TMS group of the 9-Cl,10-O-TMS stearic acid methylester. The peak cluster eluting at 28.02 min (Fig. 3A) contained several diagnostic mass fragments (m/z 173, 221, 259, 268, 307, and 403) indicative of the presence of 18:1 monochlorohydrins, which are formed by modification of linoleic acid with NaOCl (42). Although we could detect the Mz1-CH3 fragment (m/z 403, Fig. 3A, trace VI), we consistently failed to detect the molecular ion at m/z 418 of 18:1 monochlorohydrins. Turnover of NaOCl-modified HDL3 by Mouse Peritoneal Macrophages—The next sets of experiments were designed to study the turnover of native and NaOCl-modified HDL3 by mouse peritoneal macrophages with emphasis on holoparticle and HDL3-CE turnover. Holoparticle, total HDL3-CE, and selective HDL3-CE uptake was measured as described under “Experimental Procedures.” In Fig. 4, a comparison of holoparticle (A), total HDL3-CE (B), and selective HDL3-CE uptake (C) of native and NaOCl-modified HDL3 preparations is presented. In these experiments, we have not discriminated between bound and internalized fraction, i.e. uptake refers to the sum of bound and internalized fraction. The values for maximal uptake of native HDL3 were 364 6 54, 3554 6 207, and 3191 6 217 ng of HDL3 protein/mg of cell protein for holoparticle, total [3H]Ch18:2, and selective [3H]Ch18:2 uptake, respectively. When HDL3 was modified with increasing concentrations of NaOCl, we observed increased holoparticle, total [3H]Ch18:2, and selective [3H]Ch18:2 uptake. Holoparticle uptake was increased 1.2- and 2.4-fold (150 and 300 mol of NaOCl/mol of HDL3; Fig. 4A). Total and selective [3H]Ch18:2 uptake was increased 2-fold (Fig. 4, B and C). Selective uptake exceeded particle uptake by a factor of 8.7 and 9.3 in native and NaOClHDL3 (molar oxidant:lipoprotein ratio of 150), while the capacity for selective uptake decreased to 7.3 in HDL3 treated with a molar NaOCl:lipoprotein ratio of 300 (Fig. 4C). Taken together, the results shown in Fig. 4 indicate that NaOCl modification of HDL3 converts this lipoprotein particle into a “high uptake” form for mouse peritoneal macrophages, leading to intracellular cholesterol(ester) accumulation in these cells in further consequence. The next series of experiments was designed to identify mechanisms responsible for increased uptake of NaOCl-modified HDL3 over native HDL3. During binding experiments at 4 °C, we have observed a pronounced increase in HDL3 binding with increasing modification rates. In parallel, the ability of native HDL3 to compete for binding of NaOCl-modified HDL3 was gradually lost with increasing modification rates (data not shown), indicating that native and NaOCl-modified HDL3 are bound by different receptors and/or binding proteins on mouse peritoneal macrophages. To test whether NaOCl treatment of lipoprotein particles re-

sulted in altered binding, internalization, and degradation rates by mouse peritoneal macrophages, the cells were incubated at 37 °C in the presence of 20 mg of protein/ml of iodinated (native and NaOCl-modified) HDL3 for 6 h. Results of these experiments are shown in Fig. 5. NaOCl treatment of HDL3 (NaOCl:HDL3 5 300) increased the amount of trypsin-releasable material (i.e. bound HDL3 particles) 1.6-fold. Lower NaOCl:HDL3 molar ratios were without effect on steady state binding of HDL3 holoparticles at 37 °C. As can be seen in Fig. 5, the effect of NaOCl modification on internalization and degradation was even more pronounced; internalization was increased in a dose-dependent manner 1.1-, 1.2-, and 1.7-fold (75, 150, and 300 mol of NaOCl/mol of HDL3, respectively). Degradation rates for NaOCl-modified HDL3 were also significantly enhanced over base-line values; while mouse peritoneal macrophages degraded 180 6 9.0 ng of native HDL3/mg of cell protein, these values increased to 287 6 14.3, 765 6 38, and 699 6 35 ng/mg of cell protein (75-, 150-, and 300-fold molar excess of NaOCl, respectively). The data presented above demonstrated that modification of HDL3 by NaOCl enhanced binding, internalization, and degradation of modified HDL3 by mouse peritoneal macrophages up to 1.6-, 1.7-, and 4.3-fold in comparison with native HDL3. These findings were also confirmed by fluorescence microscopy of macrophages incubated in the presence of fluorescently labeled native and NaOClmodified HDL3.2 Turnover of MPO-modified HDL3 by Macrophages—In this set of experiments, we have modified HDL3 in the presence of MPO/H2O2/Cl2 (see “Experimental Procedures”) at pH 7.4 and pH 4.5 to generate OCl2 and Cl2 as the oxidant. Results of these experiments are shown in Fig. 6. Binding of MPO-modified HDL3 at 4 °C was enhanced 2.5- and 1.5-fold (pH 7.4 and 4.5). At 37 °C, the bound and internalized fraction of MPO/ H2O2/Cl2-modified HDL3 was 1.9 –2.4-fold higher as compared with controls. MPO modification led to strongly increased degradation, exceeding control values by 14.7-fold (302 6 7.7 versus 4429 6 380 ng/mg of cell protein, modified at pH 7.4) and 17.7-fold (302 6 7.7 versus 5352 6 520.7 ng/mg cell protein, modified at pH 4.5). Incubation of HDL3 in the presence of H2O2 alone did not cause increased degradation (253 6 10.4 ng of HDL3/mg of cell protein). Taken together, these results demonstrate that enzymatically active MPO can convert high density lipoproteins into a “high uptake form” for macrophages, ultimately resulting in lipid decomposition in these cells. It is important to note that despite relatively small modifications in the lipid and protein domain of HDL3 particles, degradation of MPO-modified HDL3 was approximately 4-fold higher as observed for HDL3 modified in the presence of the highest molar NaOCl:HDL3 ratios. Effect of NaOCl Modification on Cholesterol Acceptor Properties of HDL3—We have been interested in whether modification of apoA-I by NaOCl may alter the cholesterol efflux efficiency of HDL3. Results of a representative experiment are shown in Fig. 7. During the incubation with the radioactive tracer, cells acquired 66.000 6 2.784 dpm/mg of cell protein (n 5 3, 60% of the initial radioactivity added). Following a 24-h incubation period of macrophages in the presence of native HDL3, the cellular cholesterol content decreased to 30 6 2.7%. However, 39 6 0.5% cholesterol remained in the cells when the efflux experiment was performed in the presence of NaOClmodified HDL3 (NaOCl:HDL3 5 300). Accordingly, the radioactivity present in the medium (after a 24-h incubation) was 74 6 0.8 and 61 6 2.1% of the initial radioactivity when cells were cultivated in the presence of native and NaOCl-modified

2 U. Panzenboeck, S. Raitmayer, H. Reicher, H. Lindner, O. Glatter, E. Malle, and W. Sattler, unpublished data.


FIG. 3. GC-MS analysis of native and NaOCl-modified HDL3 lipids. 500 mg of HDL3 protein were modified with NaOCl at a molar oxidant:lipoprotein ratio of 150. Lipid extraction and derivatization to the corresponding O-methylester-OTMS-ether derivatives and subsequent GC-MS analysis was performed as described under “Experimental Procedures.” A, GC-MS profile of HDL3 lipids obtained from native (TIC, trace I) and NaOClmodified HDL3 (TIC, trace II). Ions monitored at m/z 221 (trace III), 307 (trace V), and 403 (trace VI) atomic mass units are diagnostic fragments for the detection of 18:1 monochlorohydrins, while fragments at m/z 263 (trace IV), 307 (trace V), and 405 (trace VI) are indicative for the presence of 18:0 chlorohydrins. Peak assignments (according to retention times) are as follows: 18.26 min, palmitic acid; 20.26 min, heptadecanoic acid (internal standard); 21.42 min, linoleic acid; 21.58 min, oleic acid; 22.16 min, stearic acid; 24.32 min, arachidonic acid; 24.68 min, docosahexaenoic acid; 28.02–28.07 min, 18:1 monochlorohydrins; 28.51–28.62 min, 18:0 chlorohydrins. B, EI1 mass spectrum of the peak eluting at 28.51 min. C, EI1 mass spectrum of O-methylester-O-TMSether 18:0 chlorohydrins prepared by reaction of oleic acid micelles with NaOCl (molar ratio of NaOCl to 18:1 was 2:1).

29717

29718


FIG. 5. Effect of NaOCl modification of HDL3 on binding, internalization, and degradation rates by mouse peritoneal macrophages. Cells were preincubated in DMEM containing 10% LPDS for 12 h prior to the uptake experiments and then incubated with 20 mg of 125I-HDL3 protein of either native or NaOCl-modified 125IHDL3 preparations (molar oxidant:lipoprotein ratio was 75, 150, and 300, respectively). Following incubation (6 h at 37 °C), the cellular supernatant was collected to determine the non-trichloroacetic acidprecipitable radioactivity as described under “Experimental Procedures” (Degraded). The cells were washed and treated with trypsin (0.05%, 37 °C, 10 min) to release bound HDL3 holoparticles (Bound), while the remaining cells were lysed in 0.3 N NaOH to determine the internalized fraction. Data shown represent the mean 6 S.D. from triplicate dishes from one experiment.

HDL3, respectively. This became even more pronounced when the time necessary to remove 50% of the cell-associated [3H]cholesterol from the plasma membrane (t/2) was calculated by nonlinear regression analysis; the calculated t/2 values were 11.4 6 0.5 and 19 6 1.2 h for native and NaOCl-modified HDL3, approaching t/2 values observed with J774 macrophages (41), cells with particularly long efflux times. Taken together, the above mentioned results demonstrate that the efficacy of NaOCl-modified HDL3 to promote cholesterol efflux was significantly diminished in comparison with native HDL3. DISCUSSION

FIG. 4. HDL3 holoparticle (A), total HDL3-CE (B), and selective HDL3-CE (C) uptake of native HDL3 and two different NaOClmodified HDL3 preparations (molar NaOCl:HDL3 ratio was 150 and 300) by mouse peritoneal macrophages. Macrophages were obtained from thioglycollate-elicited mice as described under “Experimental Procedures.” Cells were seeded in DMEM containing 10% FCS and cultivated for 4 h. 12 h prior to the uptake experiments, the medium was switched to DMEM containing 10% LPDS. Cells were then incubated in the presence of the indicated concentrations of native and NaOCl-modified 125I-labeled and [3H]Ch18:2-labeled HDL3 preparations for 6 h at 37 °C. Subsequently, the cells were washed, and the total (i.e. bound and internalized) radioactivity was measured. 125I uptake represents HDL3 holoparticle cell association, while uptake of

One important question arising from our study is whether HOCl concentrations that favor HDL3 modification exist in vivo. Based on in vitro experiments (42, 43), the HOCl concentrations at sites of acute inflammation were calculated to be about 340 mM or greater (44). HDL3 plasma concentrations of ;6 –12 mM (and it is conceivable that subendothelial concentrations would be lower) would yield a minimum estimate of HOCl:HDL3 ratios of approximately 30:1 to 50:1. Even at these low oxidant:lipoprotein ratios, we have observed apolipoprotein and fatty acid modification in the HDL3 particle. In addition to these theoretical considerations, our results obtained with the MPO/H2O2/Cl2 system support the assumption that enzymatically active MPO, as expressed in atherosclerotic lesions (22), could transform HDL into a proatherogenic form that is avidly

[3H]Ch18:2 represents cell association of HDL3-associated CEs. Selective uptake was calculated as the difference between [3H]Ch18:2 and 125 I-labeled HDL3 cell association. To allow the comparison of cellular uptake of HDL3 tracers, uptake is shown in terms of apparent HDL3 particle uptake (expressed as HDL3 protein that would be necessary to account for the observed tracer uptake; see “Experimental Procedures”). Values shown (mean 6 S.D. from triplicate dishes from one representative experiment) represent specific cell association calculated as the difference of activities measured in the absence or presence of a 20-fold excess of unlabeled HDL3.


FIG. 6. Effect of MPO-mediated modification of HDL3 on binding, internalization, and degradation rates by mouse peritoneal macrophages. Cells were cultivated as described in the legend to Fig. 5 and incubated with 10 mg of 125I-HDL3 protein of either native or MPO-modified 125I-HDL3 preparations. MPO modification of HDL3 was performed at pH 7.4 to generate OCl2 or pH 4.5 to generate Cl2 as the oxidizing agent. 125I-HDL3 incubated in the presence of H2O2 but in the absence of MPO served as a control. Bound, internalized, and degraded fraction was measured as described in the legend to Fig. 5. Data shown represent the mean 6 S.D. from triplicate dishes from one experiment.

taken up by macrophages. During the present study, we have observed modification of apoA-I amino acids and the formation of high molecular mass aggregates of intact apoA-I. ApoA-I cross-links were apparently not mediated by disulfide bridging, and in light of the observed tyrosine modification and other investigations it is conceivable that the formation of immunoreactive high molecular mass apoA-I could proceed via dityrosine formation (45, 46). In principle, apoA-I cross-linking can occur by intra- or interparticle cross-linking. Results obtained from our static and dynamic light-scattering experiments suggest that high molecular mass apoA-I products are formed predominantly by intraparticle reactions. This is supported by a rather moderate increase in RH and aggregation number (from 1 to 1.4), even at the highest NaOCl concentrations used (Fig. 2). In line with results published for LDL modification (21), we have observed pronounced modification of apoprotein amino acids, independent of whether reagent NaOCl or HOCl formed via the MPO/H2O2/ Cl2 system was used in these experiments. The varying sensitivities of individual amino acids toward NaOCl modification could be a reflection of their different location within amphipathic apoA-I helices (47). One remarkable feature of HDL3 modification was the fact that also fatty acids were modified by the MPO/H2O2/Cl2 system and by NaOCl. Fatty acid consumption occurred even at low and probably physiologically occurring NaOCl concentrations. These results are different from observations made during NaOCl-mediated LDL modification, where the majority of the oxidant was consumed by the apolipoprotein domain (21). These findings suggest that apoA-I protects the HDL3 lipid domain against excessive modifications by NaOCl. However, in contrast to LDL, HDL3 apolipoproteins could not provide absolute protection toward modification of cholesterol and fatty acids by NaOCl. GC-MS analyses performed during the present study (Fig. 3) have demonstrated the formation of new compounds with diagnostic ions compatible with the formation of 18:0 chlorohydrins and 18:1 monochlorohydrins as described for the reaction of fatty acid micelles with NaOCl (48). The chlorinating intermediate in the MPO/H2O2/Cl2 system is generally believed to be HOCl, although chlorine (Cl2) production was demonstrated at acidic pH

29719

FIG. 7. Efflux of [3H]cholesterol from the plasma membrane of mouse peritoneal macrophages in the presence of native and NaOCl-modified HDL3. The plasma membrane cholesterol pool of mouse peritoneal macrophages was labeled during a 48-h incubation in the presence of [3H]cholesterol (50 nCi/ml). At time 0, the cells were washed, and three dishes were immediately lysed by the addition of NaOH (100% of control). The efflux experiment was initiated by the addition of DMEM containing native or NaOCl-modified HDL3 (NaOCl: lipoprotein ratio was 300:1; 500 mg of protein/ml). At the indicated time points, the medium was removed and counted, the cells were washed and lysed in 0.3 N NaOH to measure the cellular protein and the fraction of cholesterol retained by the cells. Data shown represent the mean 6 S.D. of one experiment performed in triplicate.

(29), and the formation of a dichlorinated cholesterol derivative was reported upon incubation of LDL with the MPO/H2O2/Cl2 system at pH 4.5 (49). In line with results reported in Ref. 29, cholesterol consumption was only observed when HDL3 was modified in the presence of the MPO/H2O2/Cl2 system at pH 4.5, where Cl2 is generated as the oxidant. It was repeatedly demonstrated by in vitro and in vivo experiments that (oxidative) modification of HDL results in altered metabolic properties of either the protein or lipid constituents, e.g. preferential removal of HDL-associated oxidized CEs over nonoxidized CEs (9 –11). During that part of our study, we have observed up to 4-fold increased degradation rates for HDL3 modified with reagent NaOCl. However, when HDL3 was modified in the presence of MPO/H2O2/Cl2, the increase in degradation rates was enhanced 15- (oxidant generated at pH 7.4) and 17-fold (oxidant generated at pH 4.5), indicating that enzymatically active MPO can convert HDL3 into a high uptake form for macrophages in vitro, a process that could also occur in vivo at local sites of inflammation, where high levels of MPO are expressed. The increase in degradation rates of NaOCl- or MPO/H2O2/Cl2-modified HDL3 suggested a diversification of the intracellular routing of HDL3 holoparticles upon modification. While lipids associated with native HDL are preferentially taken up via the selective uptake pathway and hydrolyzed in an extralysosomal compartment (50), the strongly increased degradation rates of NaOCl and MPO/H2O2/Cl2 125I-HDL3 holoparticles suggested preferential routing to lysosomes. Although the possibility of such routing was not further studied in the present investigation, these observations would be compatible with the metabolism of modified HDL3 via scavenger receptor-mediated pathways, as demonstrated for malondialdehyde-modified HDL in rat sinusoidal hepatic cells (51) and rat liver endothelial cells (52). Taken together, our results suggest that catabolism of HOCl-modified HDL3 led to pronounced accumulation of intracellular lipids and to significantly enhanced degradation rates of HDL3.

29720


HDL or subclasses of HDL are believed to be the physiologically acceptors of excess cellular cholesterol from peripheral tissues (1). Studies with isolated lipoproteins have identified biochemical and physical factors that influence the efficacy of acceptor particles to promote cholesterol efflux from cells (53), among them the functional integrity of the apoA-I molecule (14, 16, 54). Banka et al. (55) have demonstrated that two distinct regions within apoA-I (amino acids 74 –105 and 96 –111) are apparently involved in cholesterol efflux from monocytes to apoA-I proteoliposomes. Another epitope on apoA-I promoting cellular cholesterol efflux (residues 137–144) was identified in pre-b1-HDL (56). A common feature of all of these epitopes is their ability to remove cholesterol located in the plasma membrane. It is important to note that as much as 30% of amino acids located from 96 to 111 could be modified by NaOCl (3 Lys, 1 Tyr, and 1 Trp), while residues 140 –150 (responsible for the promotion of intracellular cholesterol efflux; Ref. 57) contain one Lys and one Arg residue potentially susceptible to OCl2 modification. Taken together, all epitopes apparently involved in the promotion of cholesterol efflux contain amino acids that can be modified by NaOCl. Modification(s) of functionally important amino acid residues could lead to the impaired cholesterol acceptor properties of NaOCl-modified HDL3 as observed during the present study. The presence of HOCl-modified proteins in vivo by specific antibodies raised against HOCl-modified LDL (26) has been demonstrated in advanced atherosclerotic lesions (23) and in inflammatory and degenerative human kidney diseases (24), tissues where high concentrations of MPO are expressed at local sites of inflammation (22). Although much effort has been concentrated to identify other oxidants responsible for (lipo)protein modification in vivo, the MPO/H2O2/Cl2 system has turned out as a likely candidate for HOCl modification of (lipo)proteins. Colocalization of apoA-I and apoB-100 in lipidrich cores and in lysosomal structures of macrophages in human atherosclerotic plaques has been demonstrated (58, 59). Interestingly apoA-I was present in approximately 2-fold excess over apoB-100 in lysosomal structures of macrophages (58), and in this context it is noteworthy that phagocytosis is a potent stimulus for the secretion of MPO and H2O2 into the phagolysosome (60). In vitro studies have demonstrated that different plasma lipoproteins including HDL (61) represent targets for the MPO/H2O2/Cl2 system, and the probability for subendothelial HOCl modification of HDL might be at least as high as for LDL. If occurring in vivo, modification of HDL by the MPO/H2O2/Cl2 system could lead to comparable effects, as observed during the present in vitro study, resembling another facet of a chameleon-like lipoprotein particle (5), displaying pro- rather than antiatherogenic properties. Acknowledgments—We thank Guenther Radspieler for performing the amino acid analyses and Dr. Roland Stocker for stimulating discussions. REFERENCES 1. 2. 3. 4. 5.

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