INTRODUCTION

Oxidative modification of low density lipoprotein (LDL)¹ by incubation with iron or copper salts generates a particle with altered chemical properties including elevated levels of lysophosphatidylcholine and oxidized lipids and increased electrophoretic mobility (1). Besides inducing foam cell formation in cultured macrophages (2), oxidized LDL has other properties in vitro consistent with a pro-atherogenic function, e.g., it increases macrophage residence in the vessel wall by chemoattractant activity for monocytes (3), by inducing cellular production of chemoattractants (4), and by enhancing monocyte binding to EC (Ref. 5; see Ref. 6 for review). Oxidized LDL also is chemotactic for SMC (7) and reduces EC wound-healing responses (8) in vitro.

Although the presence of oxidized LDL in atherosclerotic lesions has been established (9, 10), the mechanism(s) of oxidation in vivo is unknown. LDL may be oxidized in the plasma and taken up by the vessel wall, or it may be oxidized within the vessel wall by cell-mediated processes. Oxidation of LDL by cells of the type found in the vessel wall, including EC (11, 12), SMC (11, 13), and monocytic cells (14, 15, 16) has been described. Cell-derived superoxide (O₂) is required for oxidation of LDL by SMC (17), EC (18), and adherent monocytic cells (16, 19), although conflicting results have been reported for the latter two cell types (20, 21). LDL oxidation by EC (12) and SMC (13) also requires exogenous transition metal ions.

Given the absence of measurable free copper and iron in plasma and interstitial fluids (22), recent attention has focused on the identification of physiological metal-containing complexes that promote LDL oxidation (23, 24, 25) and on metal ion-independent mechanisms of oxidation (26, 27). We (28, 29) and others (30, 31) have investigated the role of the copper-containing plasma protein ceruloplasmin in this process. Ceruloplasmin is an acute phase reactant containing 7 copper atoms and carrying about 95% of plasma copper (see Refs. 32 and 33 for review). Ceruloplasmin has a well documented ``oxidase'' activity capable of oxidizing aromatic amines and ferrous ions (32). This activity is thought to be catalyzed by a 4-copper complex within ceruloplasmin and accompanied by 4-electron reduction of O₂ to H₂O (34).

In addition to its oxidase activity, purified human ceruloplasmin oxidatively modifies LDL as measured by multiple determinants of lipid oxidation including formation of thiobarbituric acid reacting-substances (TBARS), conjugated dienes, and lipid hydroperoxides and by increased electrophoretic mobility (29). The oxidant activity of ceruloplasmin is strongly dependent on protein structure; incubation with Chelex-100 (which removes only one of seven copper atoms) and proteolytic cleavage at a single site (into 115- and 19-kDa fragments) both suppress activity (29). We now present evidence that intact human ceruloplasmin stimulates oxidative modification of LDL by vascular cells and that this activity depends on the cellular release of O₂.

EXPERIMENTAL PROCEDURES

Ceruloplasmin was purified from human plasma as described (28). The resulting ceruloplasmin preparation was pure as determined by oxidase specific activity (35), by an absorbance ratio (610 nm/280 nm) greater than 0.045 (32), and by homogeneity according to SDS-polyacrylamide gel electrophoresis and silver staining. Purified ceruloplasmin was primarily the intact 132-kDa monomer but also contained the 115- and 19-kDa proteolytic fragments of ceruloplasmin also present in serum (about 10% of total by SDS-polyacrylamide gel electrophoresis). Catalase, dimethylthiourea, glutathione, sodium formate, superoxide dismutase, xanthine, xanthine oxidase, and all assay reagents were from Sigma, and Chelex-100 was from Bio-Rad.

EC were isolated from adult bovine aortas as described (36), subcultured by trypsinization, and grown to confluence in Dulbecco's modified Eagle's medium and Ham's F-12 medium containing 5% fetal calf serum (Hyclone Laboratories, Logan, UT). SMC were isolated by the explant method using intima-media of adult bovine aortas (37). The cells were made quiescent in Dulbecco's modified Eagle's medium containing 1 mg/ml gelatin for at least 24 h. SMC proliferation was measured as incorporation of [³H]thymidine into trichloroacetic acid-precipitable material (36), the rate of protein synthesis by SMC was measured as incorporation of [³H]leucine into trichloroacetic acid-precipitable material, and EC migration was measured by the razor-wound assay as described previously (8). Human aortic EC cultured on a fibronectin-coated (1 µg/cm²) surface, and human aortic SMC were used in some experiments.

Human LDL (density = 1.019-1.063 g/ml) was prepared by sequential ultracentrifugation from freshly drawn, citrated plasma to which EDTA was added before centrifugation (38). Before use, LDL was dialyzed at 4 °C against saline and incubated with cell cultures in serum- and transition metal-free RPMI 1640 medium at 37 °C. LDL oxidation was determined as TBARS in the conditioned medium and expressed as nmol malondialdehyde-equivalents/mg LDL cholesterol. LDL oxidation was also measured as the formation of conjugated dienes (39), total lipid peroxide content (40), and conversion of phosphatidylcholine to lysophosphatidylcholine by solvent extraction, thin layer chromatography, and Coomassie Blue staining (41). Lipoprotein-deficient serum (LPDS; density > 1.25) was prepared by ultracentrifugation of human plasma (38).

Superoxide production by cells (or by a xanthine/xanthine oxidase generating system) was measured spectrophotometrically by Cu/Zn-superoxide dismutase (SOD)-inhibited reduction of cytochrome c (42). Cultured cells were incubated in RPMI 1640 medium (without phenol red dye) with 3 × 10⁵ M cytochrome c. O₂-specific reduction of cytochrome c was determined as the difference in absorbance at 550 nm between dishes incubated ± SOD (100 units/ml). Cell-free dishes were used as controls.

RESULTS

The ability of human ceruloplasmin to promote LDL oxidation by bovine aortic SMC and EC was investigated. LDL and purified human ceruloplasmin were added to confluent cell cultures in serum- and transition metal-free RPMI 1640 medium. Ceruloplasmin stimulated SMC-mediated oxidation, measured as TBARS, from less than 1 to about 30 nmol malondialdehyde/mg cholesterol (Fig. 1A). The activation curve was sigmoidal with half-maximal activity at 25-50 µg/ml and maximal oxidation at 100 µg/ml, a concentration below the mean unevoked plasma concentration of 300 µg/ml. Ceruloplasmin at concentrations up to 300 µg/ml did not alter the cellular rate of protein synthesis as measured by [³H]leucine uptake. Ceruloplasmin-stimulated oxidation of LDL was confirmed by three other chemical measures of LDL oxidation. In the presence of SMC, a maximal amount of ceruloplasmin increased: (i) the level of conjugated dienes in LDL from 0.04 to 0.12 absorbance units (A_{234 nm}), (ii) the amount of lipid peroxides from 0.0 to 0.7 µmol/mg LDL cholesterol, and (iii) the lysophosphatidylcholine content of LDL from 10 ± 8 to 260 ± 20 µg of lysophosphatidylcholine/mg LDL cholesterol (not shown). Furthermore, LDL oxidized by the combined presence of ceruloplasmin and bovine SMC had two biological activities that have been described for metal ion-oxidized LDL (8, 43); it stimulated SMC proliferation (measured by [³H]thymidine uptake) by about 75%, and almost completely inhibited EC migration (measured by the razor-wound assay (8)) with a half-maximal inhibition at 100 µg LDL cholesterol/ml (not shown). Ceruloplasmin also stimulated LDL oxidation by bovine aortic EC but to a lesser extent than by SMC (Fig. 1A). In multiple independent experiments, the maximal ceruloplasmin-stimulated oxidant activity of EC was 15-50% of that of SMC. Similar enhancement of oxidation by ceruloplasmin was seen with aortic SMC and EC derived from adult human tissue; the maximal oxidation (at 100 µg/ml of ceruloplasmin) by human SMC was 18-22 nmol malondialdehyde/mg cholesterol, and stimulation by human EC was generally 50-75% of that of SMC (not shown).

A time course experiment using aortic SMC showed a 2-3-h lag followed by a nearly linear increase in ceruloplasmin-stimulated oxidation of LDL for at least 24 h (Fig. 1B). The lag before the appearance of detectable amounts of TBARS in EC was longer, about 10 h, and the rate of oxidation during the linear phase was less than half of that seen with SMC. The longer lag phase in EC may be due to its lower rate of production of reactive oxygen intermediates (see below), and consequently slower destruction of the antioxidants within LDL believed to be responsible for the observed lag period (39). Ceruloplasmin by itself, i.e., in the absence of cells, only slightly increased LDL oxidation (Fig. 1, A and B). This observation was consistent with our earlier results showing that ceruloplasmin oxidized LDL in phosphate-buffered saline but that proteins and amino acids blocked its activity (29). The ability of cultured cells to overcome the inhibition by amino acids in the culture medium indicated that SMC and EC cells may supply an additional factor(s) required for ceruloplasmin-stimulated LDL oxidation under these conditions. The well known nonspecific antioxidant activity of proteins (44, 45) raises questions on the physiological relevance of the stimulation of cellular oxidation of LDL by ceruloplasmin. However, LPDS (used instead of serum to minimize the complicating presence of lipoproteins), at concentrations as high as 10%, inhibited the oxidant activity of 100 µg/ml ceruloplasmin by only 30%, and no inhibition of 500 µg/ml ceruloplasmin was observed (Fig. 1C).

Treatment of ceruloplasmin with Chelex-100 removes one of seven copper atoms and suppresses oxidant activity under cell-free conditions (29). The results in Fig. 1D showed that pre-treatment of ceruloplasmin with Chelex-100 totally blocked its ability to support SMC-mediated (and EC-mediated, not shown) LDL oxidation. Ceruloplasmin was subjected to proteolytic modification by incubation with a partially purified preparation of the metalloproteinase separated from ceruloplasmin during its purification (28). The treatment modified the intact 132-kDa ceruloplasmin molecule to a complex of 115- and 19-kDa degradation fragments. Proteolytically modified ceruloplasmin did not stimulate SMC-mediated (and EC-mediated, not shown) LDL oxidation (Fig. 1D).

Several laboratories have shown evidence that cellular release of O₂ is required for metal ion-dependent, EC- and SMC-mediated LDL oxidation (18, 42). In those studies transition metals were supplied either as free iron or copper in the culture medium. We therefore examined the role of O₂ in ceruloplasmin-stimulated cell oxidation processes. The rate of O₂ production by confluent bovine EC and SMC cultures was measured at 15 min intervals by spectrophotometric measurement of superoxide dismutase-inhibited reduction of cytochrome c. The generation rate by SMC cultures was linear for 60 min with a production rate of 2.30 nmol/well/h. This rate was 85% higher than that by EC, which had a rate of 1.25 nmol/well/h. The level of LDL oxidation (measured as TBARS) by SMC was about 105% higher than that by EC providing correlative evidence for a relationship between O₂ production and LDL oxidation by these cell types. A similar relationship was seen for human aortic EC and SMC (not shown) and has been described in multiple isolates of monkey SMC (42).

To further address the role of O₂ production in ceruloplasmin- and cell-mediated oxidation, inhibition by scavengers of O₂ and other reactive oxygen species was examined. Cu/Zn-SOD blocked about 80% of ceruloplasmin-stimulated oxidation by SMC cultures (Fig. 2A). Because dismutation of O₂ releases H₂O₂, we also added catalase to eliminate the latter species. Although catalase alone was not an effective inhibitor, SOD and catalase in combination blocked all LDL oxidation. Essentially identical results were seen in human aortic SMC and in human and bovine aortic EC (not shown). Heat treatment completely inactivated SOD (as shown by measuring O₂ production by the xanthine/xanthine oxidase system), and the inactivated enzyme did not reduce ceruloplasmin-stimulated oxidation by SMC, even in the presence of catalase. Jessup et al. (21) have suggested that heat inactivation of SOD may release free metal ions that exert oxidant activity and thereby mask antioxidant activity. To test this possibility, heat-inactivated SOD was subsequently treated with Chelex-100 to remove free divalent metal ions and was found to give the same results as heat-inactivated SOD. Three hydroxyl radical scavengers, mannitol, glutathione, and sodium formate, did not significantly reduce ceruloplasmin-stimulated LDL oxidation by SMC. However, dimethylthiourea, also a hydroxyl radical scavenger, was effective. At least two possibilities are consistent with these data: (i) hydroxyl radical is required for LDL oxidation under these conditions, but only dimethylthiourea has sufficient scavenging activity, or (ii) hydroxyl radical is not required for oxidation, and dimethylthiourea exerts its inhibitory activity by a distinct pathway. The second scenario is supported by the observation that dimethylthiourea covalently interacts with O₂ (46). We confirmed that dimethylthiourea blocked O₂ production by xanthine and xanthine oxidase (not shown).

O₂ may be necessary for initiating lipid peroxidation, in which case it may be needed only during the beginning of the reaction period; alternatively, O₂ may be necessary for subsequent propagation reactions, which may require its continuous presence. To examine the temporal requirement for O₂, SOD or SOD plus catalase were added to bovine aortic SMC after initiation of oxidation by the addition of ceruloplasmin (Fig. 2B). After an incubation interval totaling 24 h, ceruloplasmin-stimulated LDL oxidation was measured. SOD was found to block subsequent LDL oxidation, independent of its time of addition. This result shows that the early presence of O₂ is not sufficient to initiate oxidation at maximal levels, but rather that the continuous generation of O₂ is required.

A cell-free reconstitution strategy was used to determine if the cell contribution of O₂ can account quantitatively for cellular oxidation of LDL by SMC in the presence of ceruloplasmin. Xanthine and xanthine oxidase were used under aerobic conditions to generate O₂. In this system, xanthine is oxidized to urate by xanthine oxidase with consequent reduction of O₂ to O₂. We first developed an appropriate protocol that mimicked the cellular environment. Concentrations of xanthine and xanthine oxidase were chosen such that the rate of release of O₂ closely approximated that by cultured SMC (about 6 nmol in 3 h). Under these conditions, the rate of O₂ production by the generating system was nearly constant for up to 2-3 h and then gradually dropped off, presumably due to enzyme inactivation under these conditions. The rate of O₂ production by cultured SMC was constant for at least 24 h. A single addition of xanthine/xanthine oxidase to SMC in the presence of ceruloplasmin increased LDL oxidation during a 24-h interval almost 3-fold, a stimulation much less than the 9-fold increase by ceruloplasmin-treated SMC (Fig. 3A). When the O₂ generating system was repeatedly added at 3-h intervals for 24 h, LDL oxidation was increased about 8-fold to an extent closely approximating SMC-mediated oxidation. This amount of O₂ (given according to the same regimen) was completely inactive in the absence of ceruloplasmin. Application of a large ``burst'' of O₂ (by a single addition of the same amount of xanthine and xanthine oxidase used in the multiple addition regimen) did not increase LDL oxidation beyond that found with the single small dose. Thus early addition of O₂, even at high levels, is not sufficient to induce maximal, long-term oxidation (confirming the results of Fig. 2B). More importantly, the results show quantitatively that the amount of O₂ produced by SMC is sufficient to account for the observed ceruloplasmin-dependent oxidation by the cells.

Although these studies show the continuous requirement for the short-lived O₂ species, we also investigated the role of long-lived cellular products on LDL oxidation. Conditioned medium taken from SMC after a 24-h incubation did not cause significant oxidation of LDL either by itself or in the presence of ceruloplasmin, indicating that stable cell products do not contribute significantly to cell-mediated oxidation by ceruloplasmin (Fig. 3B). Likewise, the addition of xanthine and xanthine oxidase to conditioned medium did not enhance LDL oxidation. Simultaneous addition of ceruloplasmin and the O₂ generating system (added at 3-h intervals) to SMC-conditioned medium resulted in oxidation to nearly the same extent as that induced by ceruloplasmin in the presence of SMC cultures. Thus inhibitory agents are not present in SMC-conditioned medium, and oxidant activity of SMC is effectively reconstituted by ceruloplasmin and O₂ under conditions very similar to those present in cultured cells.

DISCUSSION

Lipoprotein oxidation occurs by both metal ion-dependent and -independent pathways in vitro (47). Given the virtual absence of free copper and iron in plasma and other biological fluids (22), the elucidation of protein-bound metal ions that catalyze LDL oxidation represents an important goal in understanding mechanisms of metal ion-dependent oxidation processes in vivo. Our results indicate that human ceruloplasmin at subphysiological concentrations can substitute for free metal ions in stimulating LDL oxidation by vascular cells. Ceruloplasmin copper is required for the activity because pretreatment with Chelex-100 is completely inhibitory. The treatment removes only one of the seven ceruloplasmin copper atoms (29), indicating that this specific copper is required for cell-mediated oxidation. Because treatment with Chelex-100 does not diminish ceruloplasmin amine oxidase activity (29), these results also indicate that oxidase activity is not sufficient to induce cell-mediated LDL oxidation. Cleavage of ceruloplasmin into 115- and 19-kDa fragments completely inactivates the protein. The requirement for intact protein may explain the reported inability of ceruloplasmin to stimulate LDL oxidation by monocytic cells unless ceruloplasmin was pretreated with acid (and copper released, presumably by denaturation) (30). Because about 10% of the ceruloplasmin in plasma is present as the proteolytically modified isoform, it is tempting to speculate that proteolysis represents a physiological mechanism regulating oxidant activity.

Under the conditions of these experiments, ceruloplasmin did not significantly oxidize LDL in the absence of cells. This result contrasts with previous results showing that ceruloplasmin by itself is sufficient to oxidize LDL (when co-incubated in phosphate-buffered saline) (29). This apparent discrepancy is explained by our previous results that show that several proteins and also a mixture of amino acids inhibit ceruloplasmin oxidant activity (29). Thus the inhibition of LDL oxidation by the amino acids in RPMI 1640 (and perhaps other medium components) is consistent with our previous observations using ceruloplasmin and with observations by others using free metal ion systems (44, 48). Because oxidation does occur in the presence of either SMC or EC, these results also suggest that a critical factor(s) provided by the cells is essential for ceruloplasmin-stimulated LDL oxidation under these conditions. The high rate of LDL oxidation in the presence of 10% LPDS contrasts with a recent observation that 6% serum blocked almost 90% of the oxidation induced by murine macrophages in the presence of 10 µM iron complexed to nitriloacetic acid (45). We have not yet resolved whether the different results are due to higher oxidant activity of ceruloplasmin compared with the iron complex, higher activity of EC and SMC compared with murine macrophages, or lower antioxidant activity of LPDS compared with heat-inactivated serum. That ceruloplasmin-stimulated oxidation occurs in the presence of 10% LPDS (and a fibronectin matrix for human EC) suggests that this process may also occur under physiological conditions.

Three experimental avenues indicate that cell-derived O₂ is a critical factor necessary for ceruloplasmin-stimulated LDL oxidation by vascular cells. First, the rate of production of O₂ by SMC was twice that of EC, consistent with their differential rates of LDL oxidation. This result agrees with previous results showing that cells that oxidize LDL also secrete O₂ and that the rate of O₂ production by a particular cell isolate correlates with its ability to oxidize LDL (18, 42). Second, the addition of SOD effectively blocked ceruloplasmin-stimulated LDL oxidation by both SMC and EC. This result is also consistent with published reports of inhibition of cell-mediated LDL oxidation by SOD or by inhibition of O₂ production by elimination of L-cystine from the medium (49). We now add a third line of evidence in support of the role of O₂ in cell-mediated oxidation, namely, that the measured rate of O₂ production by cells can quantitatively account for the level of oxidation observed (in the presence of ceruloplasmin). Repeated addition of xanthine and xanthine oxidase to simulate continuous O₂ production by SMC in the presence of ceruloplasmin and LDL completely reconstituted the oxidation rate of SMC.

Although our results clearly indicate a role for O₂ in ceruloplasmin-stimulated oxidation by EC and SMC, its reported role in free metal ion-stimulated cell oxidation processes is controversial (see Ref. 50 for review). The role of O₂ in SMC oxidation of LDL has not been extensively investigated, but Heinecke et al. (42) have shown that LDL oxidation is inhibited by SOD and that a correlation exists between O₂ production and LDL oxidation by several isolates of monkey SMC. The role played by O₂ in EC oxidation of LDL is not clear because oxidation has been reported to be not inhibited (51), slightly inhibited (20), and completely inhibited (18) by SOD. The role of O₂ in monocytic cell oxidation of LDL is also controversial (16, 19, 21). One limitation of most of these studies is the dependence on a single approach, i.e., inactivation of O₂ by SOD. Thus the reported discrepancies may be due to variable activities of SOD preparations (which upon partial denaturation exhibit oxidant activity (21)) or alternatively may be due to differences in the cell isolates used, in the specific media components, or in the preparation of LDL.

The mechanism(s) of superoxide- and metal ion-dependent oxidation is not known with certainty and has been an area of intense investigation for many years. By analogy to previous work, multiple mechanisms of superoxide- and ceruloplasmin-dependent oxidation should be considered including: (i) the Fenton reaction in which O₂ and H₂O₂ causes metal ion-dependent production of highly reactive OH·, (ii) recycling of the oxidation state of the oxidant copper by reduction by O₂, (iii) O₂-dependent release of copper from ceruloplasmin analogous to the release of Fe²⁺ from ferritin (52, 53), (iv) formation of a putative ``percupryl ion'', i.e., a Cu²⁺/O₂ radical analogous to the proposed perferryl ion (54), or (v) O₂-mediated oxidation of Cu²⁺ to the powerful oxidant Cu³⁺ (55). The first of these mechanisms is unlikely because our studies with catalase indicate that hydrogen peroxide is not required for LDL oxidation. Additional work will be necessary to determine if one of the remaining mechanisms is operative. The possible requirement for interaction of ceruloplasmin with receptors on vascular cells or with LDL also remain key unanswered issues.

According to one theory of atherosclerosis, oxidation of LDL within the wall of major arteries has a key and causative role in the onset and progression of the disease (6). According to our results, co-localization of O₂, ceruloplasmin, and LDL in the same region of the vessel wall would be consistent with the oxidation of the latter. Studies in Watanabe hyperlipemic rabbits show that oxidized LDL is found in macrophage-rich early lesions and is associated with intimal and medial SMC, as well as the necrotic core, in advanced lesions (56). Recent data from several laboratories suggest that O₂ is produced by the endothelium in hypercholesterolemic rabbit aorta (57) and by medial and adventitial SMC in normal rabbit aorta (58). Detectable levels of ceruloplasmin in human atherosclerotic lesions have been reported (59, 60). However, the structural integrity, localization, and source of ceruloplasmin in the vessel wall are not known. EC-mediated uptake from the plasma compartment is one possible source, as is synthesis and secretion by vascular cells. Consistent with the latter mechanism, Fleming et al. (61) have shown that cytokine-stimulated pulmonary macrophages in vitro express the ceruloplasmin gene and synthesize and secrete the protein. There are not any published reports showing production of ceruloplasmin by EC or SMC, and the presence of ceruloplasmin in vessel wall regions adjacent to these cell types has not been examined.

There is no direct evidence that ceruloplasmin expresses oxidant activity in vivo. However, according to a recent report comparing ``oxidation-related'' serum constituents with serum lipid oxidation levels by multivariate analysis, the strongest association was found for ceruloplasmin (62). Furthermore, plasma ceruloplasmin levels are elevated in patients with vascular diseases including abdominal aortic aneurysms, vasculitis, and peripheral arterial disease (63, 64). Although these correlations may be a noncausative result of an accompanying acute phase response, case-control studies also show serum ceruloplasmin is a risk factor for coronary heart disease (65, 66). The recent discovery of patients with mutations in the ceruloplasmin gene and consequent ``aceruloplasminemia'' (67, 68, 69) may provide insights into the role of ceruloplasmin in vascular disease onset and progression. However, severe complications including pathologic iron accumulation and diabetes may obfuscate any direct role of ceruloplasmin. Studies using transgenic animals lacking specific functional domains of ceruloplasmin, e.g., the putative ``oxidant activity domain,'' may be necessary to elucidate the role of the protein in lipid and lipoprotein oxidation processes in vivo.