Low Density Lipoprotein Receptor-related Protein Is Required for Macrophage-mediated Oxidation of Low Density Lipoprotein by 12/15-Lipoxygenase*

The oxidative modification of low density lipoprotein (LDL) has been implicated in the early stage of atherosclerosis through multiple potential pathways, and 12/ 15-lipoxygenase is suggested to be involved in this oxidation process. We demonstrated previously that the 12/15-lipoxygenase overexpressed in mouse macro-phage-like J774A.1 cells was required for the cell-medi-ated LDL oxidation. However, the mechanism of the oxidation of extracellular LDL by the intracellular 12/ 15-lipoxygenase has not yet been elucidated. In the present study, we found that not only the LDL receptor but also LDL receptor-related protein (LRP), both of which are cell surface native LDL-binding receptors, were down-regulated by the preincubation of the cells with cholesterol or LDL and up-regulated by lipoprotein-de-ficient serum. Moreover, 12/15-lipoxygenase-expressing cell-mediated LDL oxidation was decreased by the preincubation of the cells with LDL or cholesterol and increased by the preincubation with lipoprotein-deficient serum. Heparin-binding protein 44, an antagonist of the LDL of “hepatic” LRP in mice Our study implicates a novel function of “macrophage” LRP in the 12/15-lipoxygen-ase-mediated LDL oxidation as the initial trigger of the progression of atherosclerosis. Further investigations are needed to explore the LRP-mediated LDL oxidation in detail in rela-tion to other receptors and cellular factors.

Lipoxygenases are a class of enzymes that incorporate one molecular oxygen into unsaturated fatty acids giving rise to their hydroperoxy derivatives. There are 5-, 8-, 12-, and 15lipoxygenases in mammalian tissues, named according to the number of carbon atoms of arachidonic acid to be oxygenated (1)(2)(3)(4). The 12-lipoxygenase subfamily includes leukocyte, platelet, and epidermal isoforms. 15-Lipoxygenase-1 was first isolated from reticulocytes, and 15-lipoxygenase-2 was cloned from hair follicles (3). Because the leukocyte 12-lipoxygenase and 15-lipoxygenase-1 are highly related in their primary structures and enzymological properties and are abundant in various tissues of many species, these enzymes are called collectively 12/15-lipoxygenases (2,5). Although the pathophysiological functions of the 12/15-lipoxygenases are still a subject of investigation and discussion, recent research progress has revealed the involvement of the enzymes in the development of atherosclerosis (6 -8).
The oxidative modification of low density lipoprotein (LDL) 1 has been implicated in the early stage of atherosclerosis (9,10). Macrophages are likely candidates to mediate in vivo LDL oxidation, because they are accumulated in the atherosclerosis lesions and are capable of in vitro LDL oxidation in culture medium free of metal ion additives (11). A number of evidences suggest that the 12/15-lipoxygenase present in monocyte-macrophage contributes to the cell-mediated LDL oxidation (12,13). Incubation of LDL with 12/15-lipoxygenase led to significant oxidation of LDL (14,15). The 12/15-lipoxygenase and oxidized fatty acids colocalized with oxidized LDL in fatty streak lesions (16 -18). A disruption of the 12/15-lipoxygenase gene diminished atherosclerosis in apoE-deficient mice (19), and overexpression of 12/15-lipoxygenase facilitated atherosclerosis in the LDL receptor-deficient mice (20).
We previously demonstrated that 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was involved essentially in the oxidation of LDL based upon the stereospecific oxygenation of esterified unsaturated fatty acid in LDL (21). This fact suggests direct interaction of the enzyme with LDL or the transfer of cellular lipids oxygenated by the enzyme to LDL. Secretion or leakage of the 12/15-lipoxygenase to the medium was ruled out (21). As the mechanism of the cell-mediated oxidation of extracellular LDL, we postulate that binding of native LDL to cell surface receptors is the first step in the 12/15-lipoxygenase-expressing cells. Among such receptors, the LDL receptor plays an important role in LDL metabolism in liver and steroidogenic tissues. However, the LDL receptor is not expressed in the intima of normal or atherosclerotic arteries (22). Importantly, the LDL receptor is not required in the cell-mediated LDL oxidation as shown by in vitro experiments (23) and LDL receptor-deficient mice studies (20,24). The native LDL also binds to LDL receptor-related protein (LRP) and scavenger receptor BI (22). The former is also referred to as ␣ 2 -macroglobulin receptor or CD91 and the latter as high density lipoprotein receptor. It was demonstrated that the both receptors were expressed on the cell surface of macrophages in atherosclerotic lesions (22,25), and LRP internalized the ligand into the cell by endocytosis (26). It is of importance to identify the receptor involved in the initial step of LDL oxidation that leads to the development of atherosclerosis. In the present study, we demonstrate for the first time the essential requirement of LRP for the cell-mediated oxidation of LDL by 12/15-lipoxygenase.
Cell Culture-J774A.1 cells transfected with the pEF-BOS vector carrying porcine leukocyte 12/15-lipoxygenase cDNA and mock-transfected cells were established as described previously (21). The cells were maintained in a humidified incubator at 37°C with 5% CO 2 in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 g/ml streptomycin sulfate. The cells were subcultured every 2-3 days using a standard trypsin protocol.
LDL Isolation-Human LDL (relative density ϭ 1.019 -1.063) was prepared from fresh plasma of healthy volunteers by sequential floating ultracentrifugation in potassium bromide density gradient (21,30). Approximately 20 mg of protein of LDL was obtained from 50 ml of plasma. LDL was stored at 4°C in the dark and used within 2 weeks. EDTA was removed from the LDL by dialysis against phosphate-buffered saline at 4°C for 24 h before each experiment.
Antibody against the LDL Receptor-A synthetic peptide, CDSDRD-CLDGSDE, was conjugated to keyhole limpet hemocyanin (Calbiochem, San Diego, CA). The peptide represents 106 -118 amino acid residues in the third cysteine-rich repeat of the ligand binding domain of the human LDL receptor and shares 92% identity with the mouse receptor. Rabbits were immunized with the conjugate in Freund's complete adjuvants and subsequently received six booster injections at 2-week intervals. IgG was purified from rabbit serum by ammonium sulfate precipitation and protein A-Sepharose chromatography according to manufacturer protocol. Specificity of the anti-LDL receptor antibody was confirmed by Western blotting using a membrane fraction from Chinese hamster ovary cells expressing the human LDL receptor and HepG2 cells.
Thiobarbituric Acid Reactive Substance (TBARS) Assay-The 12/15lipoxygenase-expressing cells (2 ϫ 10 5 ) were incubated with 400 g/ml of LDL in 100 l of DMEM without serum for 12 h, and the culture medium was subjected to TBARS assay as described previously (21). The protein concentration was determined by the method of Lowry et al. (31) using bovine serum albumin as a standard.
RT-PCR Analysis-Total RNA was isolated from 12/15-lipoxygenaseexpressing cells using Sepasol according to manufacturer instructions. cDNA was synthesized using oligo(dT) 12-18 as a primer and SuperScript II reverse transcriptase. PCR was carried out with ExTaq DNA polymerase and primers as shown in Table I under the following conditions: denaturation at 94°C for 1 min, annealing at 55°C for 30 s, and extension at 72°C for 1 min. An equal amount of aliquots from 15 and 20 thermocycles was electrophoresed in 2% agarose gel. The amplified DNAs were transferred to a nylon membrane and fixed by UV irradiation. The membrane was hybridized in QuikHyb hybridization solution with cDNA for the LDL receptor, LRP, scavenger receptor BI, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which had been labeled with [␣-32 P]dCTP using the Megaprime DNA labeling system. After washing the membrane, radioactivity was analyzed by a Fujix BAS1000 imaging analyzer (Tokyo, Japan). Nucleotide sequences of the LDL receptor, LRP, scavenger receptor BI, GAPDH, and heparin-binding protein 44 (see below) were determined by dRhodamine terminator cycle sequencing kit using an automated DNA sequencer ABI PRISM 310 (PerkinElmer Life Sciences).
Expression of Heparin-binding Protein 44 -The full-length cDNA for hexahistidine-tagged (underlined below) heparin-binding protein 44 (32) was prepared with total RNA from J774A.1 cells by RT-PCR using the following primers: upstream, 5Ј-TCTAGAATGGGGGGTTCTCAT-CATCATCATCATCATTACTCGCGAGAGAAGAACGAGCC-3Ј, and downstream, 5Ј-TCTAGATCAGAGCTCATTGTGCCGAGCCC-3Ј. The cDNA was subcloned in pCR2.1 and then ligated to the pEF-BOS vector. The resultant plasmid was introduced into COS-7 cells by the DEAE-dextran method (33). After incubation for 48 h, the cells were harvested and sonicated on ice in 50 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , and 1 mM phenylmethanesulfonyl fluoride. ␤-Octylglucopyranoside at 50 mM was added, and the lysate was incubated on ice for 1 h (34). After centrifugation at 250,000 ϫ g at 4°C for 30 min, the supernatant was subjected to nickel-nitrilotriacetate agarose column chromatography using manufacturer instructions. The histidine-tagged heparin-binding protein 44 was eluted with 0.2 M histidine in phosphate-buffered saline at pH 7.4.
Antisense and Sense Oligodeoxyribonucleotides-Sequences of sense and antisense oligodeoxyribonucleotides for the LDL receptor, LRP, and scavenger receptor BI are shown in Table II. To destroy secondary structure the oligodeoxyribonucleotides were heated at 95°C for 5 min and then chilled on ice. 12/15-Lipoxygenase-expressing cells were incubated with 25 M of the oligodeoxyribonucleotides in DMEM supplemented with 1% fetal bovine serum for 7 days (27). The cells were incubated further for 12 h with 400 g/ml LDL followed by TBARS assay. The oligodeoxyribonucleotides-treated cells were also subjected to RT-PCR analysis for the LDL receptor, LRP, and scavenger receptor BI.
DiI-LDL Uptake-The 12/15-lipoxygenase-expressing cells were incubated at 37°C for 12 h in DMEM supplemented with 10% fetal bovine serum. DiI-LDL at 5 g of protein/ml was added, and the cells were incubated further for 2 h. After washing the cells three times at 4°C, photographs were taken using an Olympus BX-50/BX-FLA fluorescent microscope (Tokyo, Japan) equipped with an appropriate excitation filter (35). For flow-cytometric analysis, the cells were dispersed with trypsin, washed twice with Hanks' balanced salt solution, and suspended in phosphate-buffered saline. The 6 ϫ 10 3 cells were analyzed in each experiment using a FACScalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ) equipped with an argon ion laser that emits at 488 nm. Forward and side scatter were adjusted to exclude debris and dead cells. The mean fluorescence intensity was determined after subtracting the auto fluorescence obtained from the cells incubated in the absence of DiI-LDL.

Effect of Up-regulation and Down-regulation of LDL-binding
Receptors on LDL Oxidation-We previously reported that the 12/15-lipoxygenase of porcine leukocytes overexpressed in macrophage-like J774A.1 cells was responsible for oxidative modification of LDL in the medium (21). As shown in the control of Fig. 1, the oxidation of LDL by the 12/15-lipoxygenase-expressing cells was 3.7 times higher than that by mock-transfected cells as determined by TBARS generation. To explore mechanisms of the LDL oxidation caused by intracellular 12/15lipoxygenase, we examined the possible involvement of the LDL receptor expressed on the surface of 12/15-lipoxygenaseexpressing cells. It is known that expression of the LDL receptor is down-regulated by the incubation of the cells with LDL or cholesterol and up-regulated by the incubation of lipoproteindeficient serum (36). In fact, as shown in Fig. 2, incubation of the 12/15-lipoxygenase-expressing cells with LDL or cholesterol decreased the mRNA level of the LDL receptor by 77 and 58%, respectively, as determined by RT-PCR using each set of primers shown in Table I. In contrast, incubation of the cells with lipoprotein-deficient serum increased the mRNA of the LDL receptor by 2.6 times under our experimental conditions. We examined whether the LDL oxidation would be changed by the above-mentioned preincubations. As shown in Fig. 1, TBARS generation by the 12/15-lipoxygenase-expressing cells was reduced by 39 and 19% after the preincubation of the cells with LDL and cholesterol, respectively. On the other hand, an ϳ2-fold increase in TBARS generation was observed after the preincubation of the cells with lipoprotein-deficient serum. There were no apparent changes of TBARS generation in mocktransfected cells preincubated with LDL, cholesterol, or lipoprotein-deficient serum (Fig. 1, open bars). These results suggest that the LDL oxidation by the 12/15-lipoxygenaseexpressing cells is mediated by either the LDL receptor or other cell surface proteins that bind to native LDL and are down-and up-regulated by the culture conditions described above. It was reported that LRP and scavenger receptor BI bound to native LDL and expressed in J774A.1 cells (22,37). We then examined whether expression levels of these receptors would be altered by the different culture conditions. RT-PCR was carried out to analyze the mRNA level of these receptors in the 12/15-lipoxygenase-expressing cells preincubated with LDL, cholesterol, or lipoprotein-deficient serum. Primers for GAPDH were also included as internal control for RNA quantity and integrity. As shown in Fig. 2A, aliquots from 15 and 20 cycles were analyzed by agarose gel electrophoresis to verify the linearity of PCR amplification. The LRP mRNA level of the 12/15-lipoxygenaseexpressing cells was decreased by preincubation with LDL and cholesterol to 65 and 62% of the control, respectively. The LRP expression was up-regulated 1.3-fold after the preincubation of the cells with lipoprotein-deficient serum (Fig. 2B). On the other hand, scavenger receptor BI mRNA expression apparently did not change under the same preincubation conditions.
Effect of Heparin-binding Protein 44 on LDL Oxidation-Heparin-binding protein 44, a mouse homologue of human LRP receptor-associated protein, is a universal antagonist of the LDL receptor family including the LDL receptor and LRP and inhibits the binding of LDL (38 -40). To examine the effect of heparin-binding protein 44 on the 12/15-lipoxygenase-mediated LDL oxidation, the hexahistidine-tagged heparin-binding protein 44 was expressed in COS-7 cells and purified by affinity chromatography. The 12/15-lipoxygenase-expressing or mocktransfected cells were incubated with LDL in the presence of the purified heparin-binding protein 44. As shown in Fig. 3A, heparin-binding protein 44 inhibited TBARS generation in a concentration-dependent fashion, and a maximal inhibition was observed at a concentration as low as 2 g/ml. These results suggest that the LDL receptor and/or LRP are involved in the LDL oxidation by intracellular 12/15-lipoxygenase.
Effect of Antibody against the LDL Receptor or LRP-To examine whether the LDL receptor and LRP are involved in the cell-mediated LDL oxidation, we employed antibodies against the LDL receptor (this study) and LRP (29), both of which blocked the LDL binding to the cells. The LDL binding to the LDL receptor was inhibited almost completely by 10 g/ml IgG against the LDL receptor (data not shown). The 12/15lipoxygenase-expressing cells and mock-transfected cells then were incubated with 400 g/ml LDL in the serum-free DMEM in the presence of the anti-LDL receptor IgG at 37°C for 12 h. As shown in Fig. 3B, the 12/15-lipoxygenase-mediated TBARS generation was not inhibited by the antibody at concentrations up to 50 g/ml. It should be noted that the anti-LRP antibody suppressed TBARS generation by the 12/15-lipoxygenaseexpressing cells in a dose-dependent manner (Fig. 3C). The TBARS generation was inhibited by more than 90% with 10 l/ml antiserum. The anti-LRP antibody had no effects on the LDL oxidation by CuSO 4 or mock-transfected cells. On the other hand, the nonimmunized rabbit serum was without effect on the TBARS generation. The results strongly suggest that LRP is at least one of the receptors required for the LDL oxidation by 12/15-lipoxygenase-expressing cells.
Effect of Antisense Oligodeoxyribonucleotides-To confirm the role of LRP in cell-mediated LDL oxidation, antisense oligodeoxyribonucleotides complementary to the 5Ј region of the mouse LRP, LDL receptor, and scavenger receptor BI mRNA containing the initiator AUG codon were synthesized as well as the corresponding sense oligodeoxyribonucleotides (Table II). After incubation of the 12/15-lipoxygenase-expressing cells with the antisense or sense oligodeoxyribonucleotides for 7 days, RT-PCR analysis was carried out. As shown in Fig. 4A, the antisense oligodeoxyribonucleotides inhibited the mRNA expression of respective receptors, whereas the sense oligodeoxyribonucleotides did not inhibit the receptor expression. We performed Western blotting for LRP and the LDL receptor, but the specific bands were not observed probably because of the low expression of these receptors. We then examined the TBARS generation by the 12/15-lipoxygenase cells after the treatment with the antisense or sense oligodeoxyribonucleotides. As shown in Fig. 4B, the antisense oligodeoxyribonucleotides against LRP suppressed TBARS generation in the culture medium by the 12/15-lipoxygenase cells by 67% as compared with control incubation, whereas sense oligodeoxyribonucleotides did not inhibit the TBARS generation. As anticipated from the data in Fig. 3B, antisense oligodeoxyribonucleotides against the LDL receptor or scavenger receptor BI did not affect the TBARS generation by the 12/15-lipoxygenase cells. These results taken together indicate that LRP is responsible for the 12/15-lipoxygenase cell-mediated LDL oxidation, and neither the LDL receptor nor scavenger receptor BI is involved in this process.
Specific Binding of LDL-To confirm the specific binding of native LDL to LRP, fluorescence-labeled LDL, DiI-LDL, was incubated with the 12/15-lipoxygenase-expressing cells. After a 2-h incubation, bright fluorescence was observed in most of the cells (Fig. 5A), whereas little or no fluorescence in the presence of a 100-fold excess of unlabeled LDL (Fig. 5D). When the LRP (Fig. 5B) or the LDL receptor (Fig. 5C) was blocked by coincubation of the antibodies with DiI-LDL, much less fluorescence was observed in the 12/15-lipoxygenase-expressing cells as compared with the control incubation. The means of the fluorescence intensity as determined by the flow-cytometric analysis was decreased by 39, 41, and 78% in the presence of anti-LRP antibody at 10 l/ml, anti-LDL receptor IgG at 50 g/ml, and a 100-fold excess of unlabeled LDL, respectively. This result indicates that LRP as well as the LDL receptor is responsible for the specific binding of LDL to the 12/15-lipoxygenase-expressing cells. DISCUSSION LRP was shown to be expressed in the 12/15-lipoxygenaseexpressing J774A.1 cells and responsible for cell-mediated LDL oxidation by these cells. The LRP expression was down-regulated by incubations with LDL or cholesterol and up-regulated by lipoprotein-deficient serum (Fig. 2). A previous study showed that LRP expression was not changed significantly by cholesterol loading in the LDL receptor-deficient human fibro- FIG. 2. Changes of expression level of LDL-binding receptors under different culture conditions. A, the 12/15lipoxygenase-expressing cells were incubated at 37°C for 48 h, and abbreviations are as described in the Fig. 1 legend. Total RNA from the treated cells was subjected to RT-PCR analysis for the LDL receptor (LDLR), LRP, and scavenger receptor BI (SR-BI); bp, base pairs. GAPDH (339 base pairs) was also amplified as an internal standard. B, radioactivity was quantified and normalized with that of GAPDH. The ratios to control are shown.

5Ј-TGCACCCAGCATACGGTCTC-3Ј
Scavenger receptor BI-1F blasts (29). The reasons for apparently different response of LRP expression to cholesterol are not known and may be the difference of cell types used in the experiments. It should be emphasized that changes of both LDL oxidation and LRP expression of the 12/15-lipoxygenase-expressing cells coincided under the above culture conditions (Figs. 1 and 2). The change of LDL oxidation was not caused by the alteration of 12/15lipoxygenase expression, because the enzyme activity did not change significantly under these culture conditions (data not shown).

5Ј-CTCGGCGTTGTCATGATCCTCAT-3Ј
Involvement of LRP in the LDL oxidation by 12/15-lipoxygenase-expressing cells is also supported by the experiment with heparin-binding protein 44. This protein is a mouse homologue of the human LRP receptor-associated protein, is a universal antagonist of the LDL receptor family, and inhibits the binding of LDL to the LDL receptor and LRP (38 -41). The LDL oxidation caused by the 12/15-lipoxygenase-expressing cells was blocked by heparin-binding protein 44, confirming that LDL binding to cell surface receptors was necessary for the cellmediated LDL oxidation (Fig. 3A).
The LDL oxidation by 12/15-lipoxygenase-expressing cells was blocked by an anti-LRP antibody in a dose-dependent manner (Fig. 3C). Furthermore, antisense oligodeoxyribonucleotides against LRP that suppressed the mRNA expression of LRP also inhibited the LDL oxidation (Fig. 4). We tested whether LRP could mediate LDL oxidation by thioglycollateinduced peritoneal macrophages of C57BL/6 mice (42). The anti-LRP serum at 10 l/ml inhibited LDL oxidation by 56.3%.
The LDL oxidation as determined by TBARS of 1 ϫ 10 5 peritoneal macrophages was 0.39 Ϯ 0.03 (n ϭ 4) nmol of malondialdehyde/mg of LDL, and that of 2 ϫ 10 5 12/15-lipoxygenaseexpressing J774A.1 cells was 0.26 Ϯ 0.07 (n ϭ 4) nmol of malondialdehyde/mg of LDL. When the 12/15-lipoxygenase activity of these cells was determined using 25 M exogenous arachidonic acid as substrate (21), it was 74.8 and 37.2 nmol/10 min/mg of protein, respectively. Our results taken together suggest that LRP, at least in part, mediates the LDL oxidation not only by 12/15-lipoxygenase-expressing J774A.1 cells but also by normal macrophages, which express 12/15-lipoxygenase at a high level and accumulate in atherosclerotic lesions (16,22). The very low level of LDL oxidation by mock-transfected cells that hardly expressed 12/15-lipoxygenase was not affected by heparin-binding protein 44 or an anti-LRP antibody, suggesting 12/15-lipoxygenase-independent LDL oxidation, if any, was not mediated by LRP.
We could not detect specific bands of LRP or the LDL receptor by Western blotting, suggesting that the expression level of these receptors in the 12/15-lipoxygenase-expressing cells was not high. However, antibodies against these two receptors significantly inhibited binding of DiI-LDL to these cells as assessed by flow-cytometric analysis, indicating that functional receptors were definitely expressed in these cells. Small fluorescence was still observed even when both antibodies were added at the same time to the cells incubated with DiI-LDL (data not shown). The results agree with the expression of scavenger receptor BI in these cells and also suggest the presence of other LDL-binding receptors.
The LDL receptor did not seem to be involved in the cellmediated LDL oxidation (Figs. 3B and 4B). This is in good agreement with the previous reports showing that macrophages prepared from the LDL receptor-deficient mouse could oxidize LDL to the same extent as the wild-type mouse (24). In fact, the LDL receptor processes native LDL via receptormediated endocytosis in which the LDL particle is delivered to lysosomes, in which cholesteryl ester is hydrolyzed to free cholesterol for use by the cells (43). LRP is a multiligand receptor, and the binding of ligands to the receptor is usually followed by receptor-mediated endocytosis and degradation of

5Ј-ATGGGCGGCAGCTCCAGGGC-3Ј
Scavenger receptor BI anti-sense 5Ј-GCCCTGGAGCTGCCGCCCAT-3Ј FIG. 4. Effect of antisense oligodeoxyribonucleotides on cellmediated LDL oxidation. A, antisense oligodeoxyribonucleotides blocked expression of the LDL receptor (LDLR), LRP, and scavenger receptor BI (SR-BI). The 12/15-lipoxygenase-expressing cells (2 ϫ 10 5 / well) were incubated at 37°C for 7 days in DMEM supplemented with 1% fetal bovine serum in the presence of 25 M sense or antisense oligodeoxyribonucleotides. Total RNA was subjected to RT-PCR analysis using primers of the LDL receptor, LRP, and scavenger receptor BI. GAPDH (339 base pairs) was also amplified as an internal standard. bp, base pairs. B, the cells were treated as described for A with either sense or antisense oligodeoxyribonucleotides. Control incubation was carried out in the absence of oligodeoxyribonucleotides. After washing, the cells were incubated further for 12 h with 400 g/ml LDL in DMEM without serum followed by measurement of TBARS in the medium. The data are shown as mean Ϯ S.E. of quadruplicate experiments after the substraction of no-cell control. the ligands in lysosomes (26). However, a recent paper reported that LRP also mediated the selective uptake of cholesteryl ester in LDL, which is transferred to the plasma membrane without internalization and degradation of LDL particles (44). It is possible that the cholesteryl ester in the plasma membrane is oxygenated directly by the intracellular 12/15-lipoxygenase followed by reincorporation to the LDL particles (45,46). Scavenger receptor BI is an 82-kDa protein that binds high density lipoprotein, LDL, modified LDL, and very low density lipoprotein (47). The receptor is expressed on the surface of macrophage in atherosclerotic lesions (22). It was reported that scavenger receptor BI, similar to LRP, mediates the selective uptake of cholesteryl ester in LDL and high density lipoprotein (48 -50). However, mRNA expression of scavenger receptor BI was not altered by the preincubation with LDL, cholesterol, or lipoprotein-deficient serum under our experimental conditions (Fig. 2). Furthermore, no inhibition of LDL oxidation by antisense oligodeoxyribonucleotides against scavenger receptor BI suggests a trivial role of this receptor (Fig. 4). The reason that scavenger receptor BI is not responsible for the cell-mediated LDL oxidation is not known, but this receptor could be involved in the LDL oxidation in the other experimental systems (48).
LRP was reported to bind to a variety of ligands including LDL, ␣ 2 -macroglobulin, very low density lipoprotein remnants, plasminogen activator, and so on (41). Thus, the receptor has been postulated to participate in a number of diverse physiological and pathological processes such as the homeostasis of plasma lipoproteins and atherosclerosis, fibrinolysis, and neuronal regeneration (41). A recent study revealed the role of the receptor in clearance of chylomicron remnants by inducible disruption of "hepatic" LRP in mice (51). Our study implicates a novel function of "macrophage" LRP in the 12/15-lipoxygenase-mediated LDL oxidation as the initial trigger of the progression of atherosclerosis. Further investigations are needed to explore the LRP-mediated LDL oxidation in detail in relation to other receptors and cellular factors.