doi: 10.1161/hq1201.099423
© 2001 American Heart Association, Inc.
Vascular Biology |
Myeloperoxidase and Hypochlorite, but Not Copper Ions, Oxidize Heparin-Bound LDL Particles and Release Them From Heparin
From the Wihuri Research Institute, Helsinki, Finland.
Correspondence to Petri T. Kovanen, Wihuri Research Institute, Kalliolinnantie 4, 00140 Helsinki, Finland. E-mail petri.kovanen{at}wri.fi
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Abstract |
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A key factor in atherosclerosis is the retention of low density lipoprotein (LDL) in the extracellular matrix of the arterial intima, where it binds to the negatively charged glycosaminoglycan chains of proteoglycans. Oxidation may lead to modification of the lysine residues of apolipoprotein B-100 of LDL, which normally mediate the binding of LDL to glycosaminoglycans. Here, we studied whether various modes of oxidation can release LDL from heparin, a glycosaminoglycan with a strong negative charge, in vitro. We found that copper ions were unable to oxidize heparin-bound LDL particles because of their redox inactivation by the glycosaminoglycans. In contrast, myeloperoxidase and hypochlorite, a product of myeloperoxidase, were able to oxidize heparin-bound LDL, and this oxidation led to the release of the oxidized particles from heparin. When the released LDL particles were compared with the residual heparin-bound LDL particles, the released particles were more electronegative and contained more modified lysine residues than did the particles that remained bound. Because human atherosclerotic lesions contain catalytically active myeloperoxidase and (lipo)proteins modified by hypochlorite, the results suggest that myeloperoxidase-secreting monocytes/macrophages in the arterial intima can oxidize and extract LDL from the extracellular matrix with ensuing uptake by the macrophages of the oxidized and released LDL, with eventual formation of foam cells.
Key Words: LDL • glycosaminoglycans • oxidation • myeloperoxidase • hypochlorite
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Introduction |
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Retention of LDL particles in the arterial intima precedes formation of atherosclerotic lesions.1–3 Increasing evidence suggests that retention of LDL depends on the interaction of LDL particles with glycosaminoglycans (GAGs) of the arterial extracellular matrix. Thus, LDL particles have been shown to interact with GAGs in vitro4 and to colocalize with GAGs in the arterial intima5–8; moreover, complexes of LDL and GAGs have been isolated from the arterial intima.9–11 Most important, Borén et al12 have shown that despite hypercholesterolemia, the development of atherosclerotic lesions is delayed in mice expressing proteoglycan-binding-deficient LDL, suggesting that binding of LDL to GAGs has a causal role in the development of atherosclerosis.
In the arterial intima, LDL particles become oxidized,13 and at present, there is strong evidence that at least lipoxygenase,14,15 peroxynitrite,16 oxygen-centered radicals,17 and hypochlorite/hypochlorous acid (HOCl)17–19 participate in the oxidative modification of LDL in this tissue. A likely source of the HOCl in the arterial intima is myeloperoxidase (MPO), an enzyme secreted by activated macrophages. Indeed, human atherosclerotic lesions have been shown to contain catalytically active MPO,20,21 which, like LDL, may bind to GAGs.22
LDL binds to GAGs via ionic interactions that are formed between positively charged lysine and arginine residues of apoB-100 and negatively charged sulfate and carboxyl groups of the GAGs. Binding of LDL to GAGs induces irreversible changes in the conformation of apoB-100 and in the organization of LDL lipids.23 These changes increase the sensitivity of LDL to oxidation: after binding and subsequent release of LDL from GAGs, the LDL particles are more readily oxidized by copper24,25 and peroxidases.26 On oxidation of LDL, the unsaturated fatty acids of LDL lipids may become decomposed (eg, to malondialdehyde [MDA] and 4-hydroxynonenal). These compounds can react with the lysine residues of apoB-100, thereby neutralizing them and interfering with their ability to interact with GAGs. Thus, various modes of oxidation render LDL particles unable to bind to GAGs.27,28 In the present study, we have analyzed whether LDL particles can be oxidized while bound to heparin GAG and, if so, whether the oxidized LDL particles remain bound or are released from the GAGs.
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Methods |
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Materials
1,1,3,3-Tetramethoxypropane was from Aldrich; A-5m chromatography medium was from Bio-Rad; PD-10 columns, HiTrap heparin columns, heparin-Sepharose beads, [3H]cholesteryl linoleate, NHS-activated Sepharose, and Sepharose CL-4B were from Amersham Pharmacia Biotech; MPO was from Calbiochem; Alcian blue 8GX was from Fluka; PBS was from GIBCO; cellulose acetate plates were from Helena Laboratories; the CHOD kit was from Merck; hydrogen peroxide (H2O2) was from Riedel de-Haën; catalase (from human erythrocytes) and heparin were from Sigma; and NaOCl was from YA-Chemie.
Isolation and Labeling of LDL
LDL was isolated from plasma obtained from healthy volunteers by sequential ultracentrifugation.29 LDL was labeled with [3H]cholesteryl linoleate to yield [3H]cholesteryl linoleate-LDL ([3H]CL-LDL) as described.30 For the experiments, the buffer of the LDL was changed to PBS by gel filtration chromatography over a PD-10 column. The amounts and concentrations of LDL are expressed in terms of its protein content, which was determined by the method of Lowry et al,31 with BSA as a standard.
Isolation of Proteoglycans From Human Aorta and Preparation of Proteoglycan-Sepharose
Proteoglycans from the intima-media of human aortas were obtained at autopsy within 24 hours of accidental death and were prepared essentially by the method of Hurt-Camejo et al,32 as described previously.28 GAGs were determined by the method of Bartold and Page,33 and the amounts of the proteoglycans are expressed in terms of their GAG content. Proteoglycan-Sepharose was prepared by coupling 5 mg of human aortic proteoglycans to a 1-mL NHS-activated HiTrap column according to the manufacturer’s instructions.
Preparation of LDL-Heparin Complexes
In a typical experiment, 500 µL of heparin-Sepharose beads were first washed with PBS and then incubated with 1 mg [3H]CL-LDL in 2 mL PBS for 20 minutes at room temperature. Unbound LDL was removed by washing the beads twice with PBS by centrifugation (1000g for 2 minutes). LDL-heparin-Sepharose complexes were resuspended in 1 mL PBS, and the amount of LDL bound to heparin-Sepharose was determined by scintillation counting.
Modification of Heparin-Bound LDL
MDA was generated by acid hydrolysis of 1,1,3,3-tetramethoxypropane.34 LDL-heparin-Sepharose complexes (containing 0.5 mg/mL LDL) were prepared as described above and then incubated (1) with the indicated concentrations of MDA for 1 hour at 37°C, which had been generated by acid hydrolysis of 1,1,3,3-tetramethoxypropane,34 (2) with the indicated concentrations of HOCl for 15 minutes on ice,35 or (3) with the indicated concentrations of CuSO4 in PBS for 24 hours at 37°C. The concentration of HOCl in the NaOCl solution was determined by using the CHOD-iodide reagent with H2O2 as standard.36 LDL-heparin-Sepharose complexes (50 µL containing 0.5 mg/mL LDL) were also modified with MPO (0.5 µg) by repeated additions of 1.3 nmol H2O2 at 1-minute intervals,35 and in some experiments, catalase (1 µg) was added to these incubations. After the various incubations, the LDL-heparin-Sepharose complexes were sedimented by centrifugation (1000g for 5 minutes), and the unbound LDL in the supernatant was removed and quantified by scintillation counting. In some experiments, the LDL remaining bound to the heparin-Sepharose beads was released by incubation in buffer containing 0.5 mol/L NaCl. LDL was modified with CuSO4 in a heparin column by injecting 1 mg [3H]CL-LDL into a 1-mL HiTrap heparin column equilibrated in PBS, followed by equilibration of the column with 10 mL PBS supplemented with 50 µmol/L CuSO4. The column was incubated for 24 hours at 37°C and then eluted at a flow rate of 1 mL/min with a 150
500 mmol/L NaCl gradient in a fast-performance protein liquid chromatography system (SMART system from Amersham-Pharmacia Biotech). Fractions were collected and analyzed for radioactivity and thiobarbituric acid-reactive substances (TBARS).37
Analysis of LDL and Heparin After Modification
Unmodified lysine residues were quantified by the trinitrobenzenesulfonic acid method.38 The amount of TBARS in LDL was measured as described previously.37 The electrophoretic mobility of LDL was analyzed by electrophoresis on cellulose acetate plates. To find out whether HOCl causes fragmentation of heparin, 100 µg of heparin was incubated in the absence and presence of 3.2 mmol/L HOCl for 15 minutes on ice and analyzed by gel filtration chromatography on an A-5m (40x1-cm) column. Fractions (1 mL) were collected and analyzed for heparin by Alcian blue staining. To find out whether the charge of heparin is altered during incubation with HOCl, 100 µg heparin was incubated with various concentrations of HOCl for 15 minutes on ice and electrophoresed on cellulose acetate plates, and the plates were stained with Alcian blue. After modification with the indicated concentrations of HOCl for 15 minutes on ice, the ability of heparin-Sepharose to bind LDL was studied by incubation with an excess of [3H]CL-LDL for 1 hour at room temperature and quantifying the LDL sedimented with the beads after centrifugation.
Inhibition of Copper-Mediated Oxidation by GAGs
Control Sepharose was prepared by blocking an NHS-activated HiTrap column with ethanolamine according to the manufacturer’s instructions. The effects of control Sepharose and heparin-Sepharose on copper-mediated LDL oxidation were studied by incubating LDL (50 µg) in 300 µL PBS containing 5 µmol/L copper sulfate in the presence 25 µL of the Sepharose preparations for 3 hours at 37°C and then analyzing the amount of TBARS in LDL. Binding of copper sulfate to heparin and human proteoglycans was studied by passing copper sulfate solution through control Sepharose, heparin-Sepharose, and proteoglycan-Sepharose columns and studying the oxidation of LDL in the eluate. For this purpose, 1-mL columns of Sepharose, heparin-Sepharose, and proteoglycan-Sepharose were preequilibrated to PBS, and 2 mL PBS containing 5 µmol/L copper sulfate was injected into the columns. The first milliliter of the eluate was discarded, and LDL oxidation in the second milliliter of the eluate was studied by incubating LDL (50 µg) in 300 µL of the eluate for 3 hours at 37°C and then analyzing the amount of TBARS in LDL. The effects of Sepharose and heparin-Sepharose on oxidation of ascorbic acid were studied by incubating ascorbic acid (0.1 mmol/L) in 500 µL PBS containing 2 µmol/L copper sulfate in the absence and presence of 50 µL Sepharose or heparin-Sepharose. After incubation for 45 minutes at 37° with continuous agitation, the samples were centrifuged for 1 minute at 5000g, and absorbances of the supernatants at 265 nm were measured to determine ascorbic acid concentrations in the samples.
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Results |
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To determine whether LDL is released from LDL-GAG complexes by oxidation, LDL-GAG complexes were prepared by incubating LDL with heparin-Sepharose beads. The heparin-Sepharose beads used were found to have high capacity to bind LDL (3 mg LDL/mL heparin-Sepharose containing 10 mg heparin), and there was little spontaneous release of the bound LDL during the incubations. First, we studied the ability of the oxidation product MDA (which is known to modify lysine residues of LDL)39 to release LDL from heparin. We found that increasing the concentration of MDA in the incubation mixture resulted in progressive release of LDL from heparin (Figure 1). When LDL release was analyzed as a function of time, it was found that most of the LDL was released from heparin by 25 mmol/L MDA within 15 minutes. These results are consistent with those of Haberland et al,40 who found that the lysine residues of heparin-bound LDL are susceptible to modification by MDA.
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Next, we studied oxidation of heparin-bound LDL by copper. When LDL-heparin-Sepharose complexes were incubated with up to 50 µmol/L copper concentrations for 24 hours at 37°C, no significant release of LDL from the heparin-Sepharose was observed (Figure 2A). Although the electrophoretic mobility of the LDL was increased by 10% on cellulose acetate, no formation of TBARS was observed. Moreover, when 1 mg LDL was injected into a 1-mL heparin-Sepharose column, which was subsequently equilibrated with 10 mL of PBS containing 50 µmol/L CuSO4 and incubated at 37°C for 24 hours, almost no release of LDL from the column was detected (Figure 2B). Some TBARS were generated, a fraction of which eluted from the column with PBS and was not associated with LDL; the rest eluted with LDL during the NaCl-dependent release of LDL. Most important, the LDL eluted from the column in a manner like that of native LDL, revealing that no changes in its affinity for heparin had occurred.
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To better understand why copper ions were unable to oxidize LDL when LDL was complexed with heparin-Sepharose, we compared the ability of Sepharose (HiTrap column blocked with ethanolamine) and heparin-Sepharose to inhibit LDL oxidation. We found that heparin-Sepharose but not Sepharose alone (control Sepharose) inhibited LDL oxidation, showing that heparin was the component in the beads responsible for the antioxidant effect (39±5.3 [mean±SD] nmol TBARS/mg LDL protein in the presence of Sepharose and 5.1±3.3 nmol TBARS/mg LDL protein in the presence of heparin-Sepharose after 3 hours of oxidation, n=4). To test whether the observed antioxidant effect of heparin depends on its ability to bind copper, we passed 5 µmol/L copper sulfate solution through Sepharose or heparin-Sepharose columns and studied oxidation of LDL in the eluate. As shown in Figure 3, allowing the copper-containing solution to flow through the column resulted in loss of the ability of the solution to oxidize LDL. Most important, the column to which proteoglycans isolated from the human aortic intima-media were coupled was also able to eliminate the LDL-oxidizing ability from the copper sulfate solution (Figure 3). The above results suggested that the copper ions were bound to GAGs. To test whether copper was redox active when bound to heparin-Sepharose, we tested effect of Sepharose and heparin-Sepharose on the oxidation of ascorbic acid. We found that heparin-Sepharose inhibited oxidation of ascorbic acid when 0.1 mmol/L was incubated for 45 minutes with 2 µmol/L copper sulfate, whereas Sepharose alone had no effect. Thus, it appears that heparin can bind copper ions, and this binding leads to redox inactivation of copper.
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We then studied the effect of hypochlorite, which has previously been shown to efficiently oxidize the lysine residues of LDL.35,41 To induce oxidation of the protein moiety of LDL with minimal lipid peroxidation, only short incubation times (15 minutes) were used.42 We found that addition of hypochlorite dose-dependently released LDL from heparin (Figure 4A). When the LDL fraction released from heparin and the LDL fraction remaining bound to heparin after HOCl oxidation were compared, the released LDL was found to be more electronegative and to contain less unmodified lysine residues (Figure 4B and 4C). Thus, the results show that when present in limited amounts, HOCl rapidly oxidizes a fraction of the heparin-bound LDL particles and that the oxidized particles are then released from heparin.
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A number of studies have shown that GAGs can also undergo oxidative degradation when exposed to strong oxidants. Thus, hypochlorite has been shown to attack N-acetylglucosamine rings in chondroitin-4 sulfate, which can lead to fragmentation of the GAG polymer.43 Moreover, heparin has been shown to be cleaved into fragments by hydroxyl radicals produced either by Fenton’s reagent or by the Haber-Weiss reaction.44 To verify that the observed oxidation-dependent release of LDL from heparin was not due to fragmentation of the heparin chains, we performed the following experiments. First, HOCl was added to heparin in amounts maximally used to release LDL from the heparin-Sepharose, as shown in Figure 4A. When analyzed by gel filtration chromatography, the treated heparin eluted as a single peak identical to that of untreated heparin, indicating that no fragmentation of the heparin had occurred. Second, when the charge of heparin was analyzed by electrophoresis on cellulose acetate plates after the addition of HOCl, no change was observed. Third, when HOCl (1.6 µmol/L final concentration) was added to heparin-Sepharose beads in the absence of LDL, it was found that preoxidation decreased the capacity of the heparin beads to bind LDL by only 13% but that when LDL was bound to heparin-Sepharose during oxidation, 75% of the LDL was released (see Figure 4A). Thus, it appears that under the conditions used, the release of LDL was caused by oxidation of LDL and not by oxidation of heparin.
Next, we studied whether treatment of LDL-heparin-Sepharose complexes by the enzyme MPO can lead to the release of LDL from heparin. Because MPO is readily inactivated by the presence of high concentrations of its substrate H2O2,35 the experiments were accomplished by multiple small additions of H2O2. As shown in Figure 5, H2O2 alone did not release any LDL from heparin. In contrast, in the presence of MPO, LDL was dose-dependently released from heparin after the additions of H2O2. Finally, this oxidation could be completely inhibited by catalase, an enzyme that catalyzes dissociation of H2O2 to O2 and H2O. Consistent with the results obtained with hypochlorite, LDL released from heparin by MPO was found to be more electronegative and to contain less unmodified lysine residues than the LDL remaining bound to heparin (not shown). Thus, MPO, by generating hypochlorite, could release LDL from heparin.
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Discussion |
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The present results show that oxidation of LDL by HOCl or MPO and modification of LDL by the oxidation product MDA are able to release LDL from heparin. Given that epitopes of HOCl-modified proteins colocalize with MPO and with peroxidase activity in human atherosclerotic lesions21 and that LDL isolated from atherosclerotic lesions contains epitopes of HOCl-modified proteins18 and MDA adducts,34 we postulate that the present results can be extrapolated to the actual events in the arterial intima, which is a tissue that is rich in GAGs and in which oxidation of LDL takes place. Indeed, because the interaction of LDL is much weaker with the arterial GAGs than with heparin,4 it is likely that even a milder degree of lysine modification than observed in the present study in vitro can induce significant release of LDL from the arterial proteoglycans in vivo.
Copper ions failed to release LDL from heparin because of the minimal oxidation of LDL in the heparin column despite the presence of a high concentration of copper ions. This was found to be due to sequestration of copper by heparin and, interestingly, also by human arterial proteoglycans. Heparin has previously been shown to bind copper ions with a stoichiometry of 1 Cu2+ ion per heparin tetrasaccharide unit or 1 Cu2+ ion per heparin disaccharide unit.45 Thus, because of the high capacity of heparin to bind copper ions, it is likely that heparin effectively competes with LDL in the binding of copper ions and thereby inhibits LDL oxidation. This is in agreement with previous results showing that histidine is able to extract copper ions from LDL and to inhibit LDL oxidation even though the copper ions are still redox active.46 In contrast to histidine, we found that heparin caused redox inactivation of the bound copper ions. With regard to intimal GAGs, it is interesting that in addition to heparin, which is synthesized and secreted by intimal mast cells,47 chondroitin-4 sulfate and dermatan sulfate, but not chondroitin-6 sulfate, have also been shown to inhibit copper-induced oxidation of LDL.48 Although oxidation of LDL by transitional metal ions has been extensively studied in vitro and although the atherosclerotic arterial intima has been suggested to contain catalytically active transitional metal ions, transitional metal ions have been shown to have only a minor role in LDL oxidation, especially in early atherosclerosis.49 We suggest that one explanation for the lack of metal ion-dependent oxidation, at least in the early stages of atherosclerosis, could be the proteoglycan-rich extracellular environment of the arterial intima.
There is convincing evidence that LDL does interact with proteoglycans in the arterial intima. Interaction between LDL and GAG chains leads to conformational changes of the apoB-100 of the LDL particles and may expose hydrophobic domains, making them more accessible for oxidative agents. Thus, such interaction can promote oxidative modifications of LDL.24,40 However, inasmuch as different GAGs are expected to cause conformational changes to a different extent in LDL, we cannot extrapolate the information regarding LDL-heparin complexes to the actual oxidative events occurring in the LDL-containing extracellular matrix of the arterial intima in vivo. Yet, the results of the present in vitro experiments with heparin suggest that any oxidation-dependent neutralization of the positively charged residues of apoB-100 that are involved in the LDL-GAG interaction in vivo tends to weaken the interaction and so promotes release of the bound particles.
It is not known whether LDL in the arterial intima is oxidized while being bound to proteoglycans or while being in the fluid phase. Our previous study showed that oxidized LDL does not bind to arterial proteoglycans,28 and the present study shows that oxidation releases LDL from heparin. Thus, oxidation tends to prevent LDL from interacting with proteoglycans, whether or not LDL is bound to proteoglycans when the oxidative modification starts.
What is the fate of LDL oxidized and released from GAGs in the arterial intima? Oxidized LDL particles are susceptible to self-aggregation.50 Therefore, it is possible that oxidized and subsequently released LDLs aggregate in the intimal fluid and remain deposited in the tight meshwork of the extracellular matrix in the arterial intima. Moreover, oxidized LDL has been shown to bind to lipoprotein lipase with high affinity and, therefore, could interact with cell surface-bound or extracellular matrix-bound lipoprotein lipase.51–54 Finally, oxidized LDL has been shown to be taken up by macrophages via scavenger receptor(s) or, if aggregated, by phagocytotic mechanisms, either of which can lead to foam cell formation. In fact, in our previous study,28 we showed that the degree of oxidation that prohibited the binding of LDL to proteoglycans allowed uptake of LDL by macrophages. Thus, it is an attractive hypothesis that intimal macrophages (by secreting MPO, for instance) not only prevent binding of LDL to the extracellular matrix but also extract LDL from it.
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Acknowledgments |
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The Wihuri Research Institute is maintained by the Jenny and Antti Wihuri Foundation. This study was also supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, and the Federation of Finnish Insurance Companies. We thank Päivi Ihamuotila and Laura Vatanen for expert technical assistance.
Received August 22, 2001; accepted September 10, 2001.
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