Potent Modification of Low Density Lipoprotein by Group X Secretory Phospholipase A2 Is Linked to Macrophage Foam Cell Formation
2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês
10.1074/jbc.m202867200
ISSN1083-351X
AutoresKohji Hanasaki, Katsutoshi Yamada, Shigenori Yamamoto, Yoshikazu Ishimoto, Akihiko Saiga, Takashi Ono, Minoru Ikeda, Mitsuru Notoya, Shigeki Kamitani, Hitoshi Arita,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe deposition of cholesterol ester within foam cells of the artery wall is fundamental to the pathogenesis of atherosclerosis. Modifications of low density lipoprotein (LDL), such as oxidation, are prerequisite events for the formation of foam cells. We demonstrate here that group X secretory phospholipase A2 (sPLA2-X) may be involved in this process. sPLA2-X was found to induce potent hydrolysis of phosphatidylcholine in LDL leading to the production of large amounts of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which contrasted with little, if any, lipolytic modification of LDL by the classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in oxidized LDL. The sPLA2-X-modified LDL was efficiently incorporated into macrophages to induce the accumulation of cellular cholesterol ester and the formation of non-membrane-bound lipid droplets in the cytoplasm, whereas the extensive accumulation of multilayered structures was found in the cytoplasm in oxidized LDL-treated macrophages. Immunohistochemical analysis revealed marked expression of sPLA2-X in foam cell lesions in the arterial intima of high fat-fed apolipoprotein E-deficient mice. These findings suggest that modification of LDL by sPLA2-X in the arterial vessels is one of the mechanisms responsible for the generation of atherogenic lipoprotein particles as well as the production of various lipid mediators, including unsaturated fatty acids and lyso-PC. The deposition of cholesterol ester within foam cells of the artery wall is fundamental to the pathogenesis of atherosclerosis. Modifications of low density lipoprotein (LDL), such as oxidation, are prerequisite events for the formation of foam cells. We demonstrate here that group X secretory phospholipase A2 (sPLA2-X) may be involved in this process. sPLA2-X was found to induce potent hydrolysis of phosphatidylcholine in LDL leading to the production of large amounts of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which contrasted with little, if any, lipolytic modification of LDL by the classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in oxidized LDL. The sPLA2-X-modified LDL was efficiently incorporated into macrophages to induce the accumulation of cellular cholesterol ester and the formation of non-membrane-bound lipid droplets in the cytoplasm, whereas the extensive accumulation of multilayered structures was found in the cytoplasm in oxidized LDL-treated macrophages. Immunohistochemical analysis revealed marked expression of sPLA2-X in foam cell lesions in the arterial intima of high fat-fed apolipoprotein E-deficient mice. These findings suggest that modification of LDL by sPLA2-X in the arterial vessels is one of the mechanisms responsible for the generation of atherogenic lipoprotein particles as well as the production of various lipid mediators, including unsaturated fatty acids and lyso-PC. low density lipoprotein apolipoprotein B apolipoprotein E phospholipase A2 secretory PLA2 group IIA sPLA2 phosphatidylcholine group X sPLA2 group IB sPLA2 cyclooxygenase lysophosphatidylcholine fetal calf serum bovine serum albumin lipoxygenase nordihydroguaiaretic acid very low density lipoprotein high density lipoprotein high performance liquid chromatography thiobarbituric acid-reactive substances phosphate-buffered saline antibody. Initiation of atherosclerosis is characterized by the appearance of fatty streaks underlying the endothelium of large arteries. Recruitment of macrophages and their subsequent uptake of low density lipoprotein (LDL)1-derived cholesterol are the major cellular events contributing to fatty streak formation (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10002) Google Scholar, 2Glass C.K. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar). Oxidative modifications in the lipid and apolipoprotein B (apoB) components of LDL are thought to drive the formation of fatty streaks (2Glass C.K. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar, 3Navab M. Berliner J.A. Watson A.D. Hama S.Y. Territo M.C. Lusis A.J. Shih D.M. Van Lenten B.J. Frank J.S. Demer L.L. Edwards P.A. Fogelman A.M. Arterioscler. Thromb. Vasc. 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Among them, secretory PLA2 (sPLA2) have several characteristics, including a low molecular mass (13–18 kDa) and an absolute catalytic requirement for millimolar concentrations of Ca2+ (10Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1223) Google Scholar, 11Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). At present, nine different groups of sPLA2s have been identified in humans (IB, IIA, IID, IIE, IIF, III, V, X, and XII) (10Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1223) Google Scholar, 12Ishizaki J. Suzuki N. Higashino K. Yokota Y. Ono T. Kawamoto K. Fujii N. Arita H. Hanasaki K. J. Biol. Chem. 1999; 274: 24973-24979Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 13Suzuki N. Ishizaki J. Yokota Y. Higashino K. Ono T. Ikeda M. Fujii N. Kawamoto K. Hanasaki K. J. Biol. 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Res. 2000; 86: 707-714Crossref PubMed Scopus (68) Google Scholar). In addition, sPLA2-IIA was shown to induce the lipolysis of LDL leading to enhanced retention of LDL to human aortic proteoglycans (19Hakala J.K. Oorni K. Pentikainen M.O. Hurt-Camejo E. Kovanen P.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1053-1058Crossref PubMed Scopus (111) Google Scholar, 20Hurt-Camejo E. Camejo G. Peilot H. Oorni K. Kovanen P. Circ. Res. 2001; 89: 298-304Crossref PubMed Scopus (198) Google Scholar), suggesting a potential role of sPLA2-IIA in the accumulation of LDL in the proteoglycan matrix on the subendothelial layer of the arterial intima. However, potent modifications of LDL leading to increased uptake by macrophages were reported with the type III bee venom sPLA2 but not with sPLA2-IIA (21Aviram M. Maor I. Biochem. Biophys. Res. Commun. 1992; 185: 465-472Crossref PubMed Scopus (42) Google Scholar). The differences in the potency of LDL modification might be due to discrepancies in substrate specificity in the mixed micelle assay, because sPLA2-IIA preferably hydrolyzes anionic phospholipids (22Ono T. Tojo H. Kuramitsu S. Kagamiyama H. Okamoto M. J. Biol. Chem. 1988; 263: 5732-5738Abstract Full Text PDF PubMed Google Scholar) such as phosphatidylglycerol and phosphatidylserine and has a very low enzymatic activity toward phosphatidylcholine (PC), a major phospholipid component of LDL (23Hevonoja T. Pentikainen M.O. Hyvonen M.T. Kovanen P.T. Ala-Korpela M. Biochim. Biophys. Acta. 2000; 1488: 189-210Crossref PubMed Scopus (333) Google Scholar). Recently, we and other groups (24Hanasaki K. Ono T. Saiga A. Morioka Y. Ikeda M. Kawamoto K. Higashino K. Nakano K. Yamada K. Ishizaki J. Arita H. J. Biol. Chem. 1999; 274: 34203-34211Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) have shown that, among the endogenous sPLA2s in mammals, group X sPLA2 (sPLA2-X) is one of the enzymes with a potent hydrolyzing activity toward PC. sPLA2-X has 16 cysteine residues located at positions characteristic of the classic types of group IB sPLA2(sPLA2-IB) and sPLA2-IIA and has an amino acid C-terminal extension that is typical of group II sPLA2subtypes (25Cupillard L. Koumanov K. Mattei M.G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). We have shown that sPLA2-X can induce potent release of arachidonic acid leading to cyclooxygenase (COX)-dependent prostaglandin formation, as well as marked production of lysophosphatidylcholine (lyso-PC) in various cell types, including macrophages, spleen cells, and colon cancer cells (26Saiga A. Morioka Y. Ono T. Nakano K. Ishimoto Y. Arita H. Hanasaki K. Biochim. Biophys. Acta. 2001; 1530: 67-76Crossref PubMed Scopus (25) Google Scholar, 27Morioka Y. Saiga A. Yokota Y. Suzuki N. Ikeda M. Ono T. Nakano K. Fujii N. Ishizaki J. Arita H. Hanasaki K. Arch. Biochem. Biophys. 2000; 381: 31-42Crossref PubMed Scopus (78) Google Scholar, 28Morioka Y. Ikeda M. Saiga A. Fujii N. Ishimoto Y. Arita H. Hanasaki K. FEBS Lett. 2000; 487: 262-266Crossref PubMed Scopus (59) Google Scholar). During the process of these cell-based experiments, we found that sPLA2-X elicits potent release of unsaturated fatty acids from the culture medium containing fetal calf serum (FCS) in cell-free systems. These observations prompted us to examine its potential role in lipolysis of human serum lipoproteins. In the present study, we first evaluated the potencies of three types of human sPLA2s (sPLA2-IB, -IIA, and -X) with respect to the release of fatty acids and the contents of PC and lyso-PC in LDL. We then compared the characteristics of sPLA2-X-modified LDL with oxidized LDL in terms of phospholipid composition, negative charge, and apoB aggregation as well as for the efficacy in uptake into macrophages. We found that sPLA2-X induced potent lipolysis of LDL leading to the formation of numerous lipid droplets in the macrophages. Finally, we showed elevated expression of sPLA2-X in the foam cells in the atherosclerotic arterial wall in high fat-fed mice deficient in apolipoprotein E (apoE). Purified recombinant human sPLA2-IB, sPLA2-X, and mouse sPLA2-X proteins were prepared as described previously (24Hanasaki K. Ono T. Saiga A. Morioka Y. Ikeda M. Kawamoto K. Higashino K. Nakano K. Yamada K. Ishizaki J. Arita H. J. Biol. Chem. 1999; 274: 34203-34211Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 27Morioka Y. Saiga A. Yokota Y. Suzuki N. Ikeda M. Ono T. Nakano K. Fujii N. Ishizaki J. Arita H. Hanasaki K. Arch. Biochem. Biophys. 2000; 381: 31-42Crossref PubMed Scopus (78) Google Scholar). Recombinant human sPLA2-IIA was a generous gift from Dr. Ruth Kramer (Eli Lilly, Indianapolis, IN). Rabbit anti-human sPLA2-X Ab was prepared as described previously (24Hanasaki K. Ono T. Saiga A. Morioka Y. Ikeda M. Kawamoto K. Higashino K. Nakano K. Yamada K. Ishizaki J. Arita H. J. Biol. Chem. 1999; 274: 34203-34211Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), and anti-sPLA2-IB and anti-sPLA2-IIA Abs were purchased from Cayman Chemicals. Bovine serum albumin (BSA), indomethacin (COX inhibitor), and nordihydroguaiaretic acid (NDGA, lipoxygenase (LOX) inhibitor) were obtained from Sigma Chemical Co. Indoxam (sPLA2 inhibitor) was synthesized at Shionogi Research Laboratories (29Hagishita S. Yamada M. Shirahase K. Okada T. Murakami Y. Ito Y. Matsuura T. Wada M. Kato T. Ueno M. Chikazawa Y. Yamada K. Ono T. Teshirogi I. Ohtani M. J. Med. Chem. 1996; 39: 3636-3658Crossref PubMed Scopus (205) Google Scholar). Very low density lipoprotein (VLDL, density less than 1.006 g/ml), LDL (d = 1.019–1.063 g/ml), and high density lipoprotein (HDL,d = 1.085–1.210 g/ml) were isolated from plasma of healthy and fasting donors by sequential ultracentrifugation, as described previously (30Hara S. Shike T. Takasu N. Mizui T. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1258-1266Crossref PubMed Scopus (42) Google Scholar). For modification of LDL with sPLA2s, 1 mg/ml LDL was incubated with various concentrations of sPLA2-IB, -IIA, or -X at 37 °C in buffer composed of 1 mm CaCl2, 12.5 mm Tris-HCl (pH 8.0), 0.25 m NaCl, and 0.0125% BSA. The reaction was stopped by addition of EDTA at a final concentration of 5 mm. For oxidative modification, 1 mg/ml LDL was incubated with 20 μm CuSO4 at 37 °C and then dialyzed against 150 mm NaCl containing 0.24 mm EDTA (pH 7.4). LDL prepared by incubation without any modification was used as native LDL. Human LDL (1 mg/ml) was preincubated for 10 min at 37 °C and stimulated with various concentrations of sPLA2 enzymes in a final volume of 40 μl. The reaction was stopped by the addition of 160 μl of Dole's reagent, and the released fatty acids were extracted, labeled with 9-anthryldiazomethane (Funakoshi, Japan), and analyzed by reverse-phase high-performance liquid chromatography (HPLC) on a LiChroCART 125-4 Supersphere 100 RP-18 column (Merck), as described previously (24Hanasaki K. Ono T. Saiga A. Morioka Y. Ikeda M. Kawamoto K. Higashino K. Nakano K. Yamada K. Ishizaki J. Arita H. J. Biol. Chem. 1999; 274: 34203-34211Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 31Tojo H. Ono T. Okamoto M. J. Lipid Res. 1993; 34: 837-844Abstract Full Text PDF PubMed Google Scholar). For measurement of the amounts of PC and lyso-PC in LDL, lipids were extracted with organic solvent as described previously (26Saiga A. Morioka Y. Ono T. Nakano K. Ishimoto Y. Arita H. Hanasaki K. Biochim. Biophys. Acta. 2001; 1530: 67-76Crossref PubMed Scopus (25) Google Scholar). The extracted phospholipids were then separated by normal-phase HPLC on Ultrasphere silica 4.6 × 250 mm (Beckman) with a guard column of 4.6 × 45 mm using a solvent of acetonitrile/methanol/sulfuric acid (100:7:0.05, v/v) with a flow rate of 1 ml/min at room temperature. Fractions corresponding to authentic PC orl-α-lyso-PC (Sigma Chemical Co.), detected at the wavelength of 202 nm, were pooled and subjected to quantitative phosphorus analysis (32Chalvardjian A. Rudnicki E. Anal. Biochem. 1970; 36: 225-226Crossref PubMed Scopus (303) Google Scholar). Following modification with sPLA2s and CuSO4 oxidation, lipid peroxidation was assessed by the following procedures. The peroxides were quantified in terms of thiobarbituric acid-reactive substances (TBARS) according to the method of Nagano et al. (33Nagano Y. Arai H. Kita T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6457-6461Crossref PubMed Scopus (247) Google Scholar). Conjugated dienes were determined by monitoring the changes in absorbance of A 234 at a final concentration of 200 μg/ml LDL, as reported previously (34Esterbauer H. Striegl G. Puhl H. Rotheneder M. Free Radic. Res. Commun. 1989; 6: 67-75Crossref PubMed Scopus (1692) Google Scholar). The electrophoretic mobility of LDL was analyzed by agarose gel electrophoresis (Titan Gel Lipoproteins, Helena Laboratories, Japan), as described previously (35Noble R.P. J. Lipid Res. 1968; 9: 693-700Abstract Full Text PDF PubMed Google Scholar). For analysis of apoB modification, LDL was delipidated and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 4% acrylamide, as described previously (36Tong H. Knapp H.R. VanRollins M. J. Lipid Res. 1998; 39: 1696-1704Abstract Full Text Full Text PDF PubMed Google Scholar). Mouse peritoneal macrophages were obtained from the peritoneal cavity of male C57BL/6J mice (8 weeks) 5 days after injection of 3% thioglycolate (Difco Laboratories). The cells were washed, resuspended in serum-free medium, X-VIVO 15 (BioWhittaker), and plated in 24-well plates (Costar) (5 × 105cells/well). Non-adherent cells were removed by washing and adherent macrophages were incubated with native LDL or modified LDL (200 μg/ml) for 48 h. The lipid extracts of macrophages were prepared, evaporated, dissolved with isopropanol and the cholesterol mass was quantified by enzyme fluorometry (37Gamble W. Vaughan M. Kruth H.S. Avigan J. J. Lipid Res. 1978; 19: 1068-1070Abstract Full Text PDF PubMed Google Scholar). The amount of esterified cholesterol was calculated by subtracting the free cholesterol from total cholesterol. The amounts of cellular proteins were quantified with BCA Protein Assay reagent (Pierce) after dissolving the cells in 0.2 n NaOH. Mouse peritoneal macrophages were prepared as described above and cultured in four-well tissue culture chambers (Iwaki, Japan, 1.25 × 105cells/well). The adherent macrophages were incubated with either native LDL or modified LDL (200 μg/ml) for 48 h at 37 °C. After incubation, the cells were washed three times with phosphate-buffered saline (PBS) and fixed with 11% formaldehyde in PBS for 15 min. They were then stained with oil red O for 30 min and counterstained with Meyer's hematoxylin for 1 min. The stained cells were examined by light microscopy. All procedures were performed at room temperature. Macrophages were prepared as described above and cultured in four-well glass slides (Lab-Tek II chamber slide, Nalge Nunc International Corp.). Acetylation of LDL was performed as described previously (38Zhang H. Yang Y. Steinbrecher U.P. J. Biol. Chem. 1993; 268: 5535-5542Abstract Full Text PDF PubMed Google Scholar). After incubation with native LDL or modified LDL (200 μg/ml) for 24 or 48 h at 37 °C, the cells were fixed with 0.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mphosphate buffer, pH 7.2. Samples were rinsed in 7% sucrose in 0.1m phosphate buffer, post-fixed in 1% OsO4, dehydrated, and then embedded in epoxy resin. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined with a Jeol 1200EX microscope. ApoE-deficient mice (8 weeks) were obtained from The Jackson Laboratory, and age-matched C57BL/6J mice were obtained from Clea Japan. They were fed a high fat and high cholesterol diet (15.8% cocoa butter, 1.25% cholesterol, 0.5% sodium cholate) for 9 weeks then flushed with PBS via the abdominal aorta under pentobarbital anesthesia and perfused with 4% paraformaldehyde in PBS. Segments of the proximal aorta and the portions of the heart containing the aortic arches were swiftly removed and cut into small pieces, which were then immersed in the same fresh fixative at 4 °C overnight. Fixed samples were thoroughly rinsed with PBS and subsequently dehydrated by passage through an alcohol series diluted with double-distilled water followed by n-butyl alcohol. The tissue preparations were then passed into paraffin at 56 °C. Transversal tissue sections (6-μm thickness) were cut from embedded paraffin blocks and mounted on slides freshly thin-coated with 3-aminoprophyltriethoxysilane. Immunohistochemistry was performed after paraffin dewaxing. The tissue slides were incubated in methanol containing 0.3% H2O2 for 30 min and then treated with 5% normal rabbit serum for 20 min. The slides were incubated with anti-sPLA2-X Ab (6 μg/ml), anti-sPLA2-IB Ab (5 μg/ml), or anti-sPLA2-IIA Ab (7 μg/ml) in PBS containing 0.1% BSA for 1 h at room temperature. After rinsing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG for 30 min followed by treatment with horseradish peroxidase avidin-biotin complex reagent (Vector Laboratories). After washing, the peroxidase activity was visualized by 10-min incubation in 50 mm Tris-HCl (pH 7.6) containing 200 μg/ml 3,3′-diaminobenzidine and 0.006% H2O2. After counterstaining of the nuclei with 0.4% hematoxylin, the preparations were mounted in Malinol resinous medium (Muto Pure Chemicals, Japan). Positive signals were detected as dark brown diaminobenzidine deposits. Neutralization of sPLA2-X-specific signals was performed by incubating anti-sPLA2-X Ab with purified mouse sPLA2-X protein (60 μg/ml) for 2 h at room temperature followed by addition to the slides. In separate experiments, the slides were incubated with rat anti-mouse macrophage F4/80 Ab (Serotec, UK) and then incubated with biotin-conjugated rabbit anti-rat IgG Ab followed by treatment with peroxidase avidin-biotin complex reagent. We first examined the potency of three types of human sPLA2 for the release of fatty acids in human plasma at a concentration of 50 nm and found that sPLA2-X induced the most potent release of unsaturated fatty acids (data not shown). We then prepared three types of lipoprotein fraction (VLDL, HDL, and LDL) from freshly isolated human plasma and then examined the potencies of sPLA2s for the release of fatty acids. As shown in Fig.1, sPLA2-X elicited marked release of various types of unsaturated fatty acids from human LDL in the following order: linoleic acid (C18:2) > arachidonic acid (C20:4) > oleic acid (C18:1) ≈ docosahexaenoic acid (C22:6), whereas sPLA2-IB and -IIA caused little release. In contrast, there was little, if any, release of saturated fatty acids, including myristic acid, palmitic acid, and stearic acid, from LDL after sPLA2-X treatment. The profiles of free fatty acids released by sPLA2-X were almost identical among LDL, HDL, and VLDL. In addition, there were no significant changes in the contents of sphingomyelin in LDL and HDL (data not shown). As shown in Fig. 2 A, sPLA2-X induced time-dependent release of arachidonic acid from LDL. In contrast, no significant release was observed during treatment with sPLA2-IB or -IIA for 4 h. Fig. 2 Bshows the dose-dependent release of arachidonic acid by three types of sPLA2 during 1-h incubation. sPLA2-X induced significant release at 5 nm, whereas sPLA2-IB evoked slight but significant release at 500 nm. There was little, if any, release with sPLA2-IIA treatment even at 500 nm, demonstrating that sPLA2-X elicits more potent release of unsaturated fatty acids from human LDL as compared with sPLA2-IB and -IIA.Figure 2Time- and dose-dependent release of arachidonic acid by sPLA2s in LDL. A, time-dependent release of arachidonic acid by human sPLA2s. LDL (1 mg/ml) was incubated with 50 nmpurified human sPLA2s for various times at 37 °C.B, dose-dependent release of arachidonic acid by human sPLA2s. LDL (1 mg/ml) was incubated with various concentrations of purified human sPLA2s for 1 h at 37 °C. The released arachidonic acid was quantified as described under "Experimental Procedures." The results are shown after subtracting the values obtained with incubation in the absence of sPLA2s at each time point. Each point represents the mean ± S.D. of triplicate measurements. The data are representative of three experiments.View Large Image Figure ViewerDownload (PPT) Because PC is a major component of phospholipids in LDL (23Hevonoja T. Pentikainen M.O. Hyvonen M.T. Kovanen P.T. Ala-Korpela M. Biochim. Biophys. Acta. 2000; 1488: 189-210Crossref PubMed Scopus (333) Google Scholar), we next examined the PC contents in LDL after treatment with sPLA2s or CuSO4. As shown in Fig.3 A, PC contents were time-dependently decreased after treatment with 50 nm sPLA2-X and oxidation. Over half of the PC was diminished in LDL by sPLA2-X within 3 h, and PC was completely degraded after 24-h treatment. Corresponding to the reduction of PC contents, the amounts of lyso-PC in LDL was increased up to 24 h after sPLA2-X treatment (Fig.3 B). Incubation with the sPLA2-specific inhibitor indoxam (10 μm) or anti-sPLA2-X Ab (100 μg/ml), both of which have been shown to block the enzymatic activity of sPLA2-X (24Hanasaki K. Ono T. Saiga A. Morioka Y. Ikeda M. Kawamoto K. Higashino K. Nakano K. Yamada K. Ishizaki J. Arita H. J. Biol. Chem. 1999; 274: 34203-34211Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), resulted in significant suppression of sPLA2-X-induced lipolysis of LDL (data not shown). In contrast, treatment with sPLA2-IB or -IIA caused little change in either PC or lyso-PC contents in LDL. Oxidation of LDL with CuSO4 also caused significant production of lyso-PC. However, the amount of lyso-PC produced during 24-h oxidation was about 30% of that induced by sPLA2-X, although PC was degraded similarly by both treatments. In addition, there was no significant release of long chain unsaturated fatty acids examined during oxidation of LDL (data not shown). Treatment of LDL with CuSO4 caused an increase in TBARS (Table I) as well as the production of conjugated dienes (data not shown), whereas treatment with three types of sPLA2 did not alter these oxidative parameters. Taken together, these findings demonstrate that sPLA2-X induces PC hydrolysis in LDL leading to the production of large amounts of lyso-PC and unsaturated fatty acids without any oxidative modification.Table IAmounts of TBARS in LDL treated with human sPLA2s or CuSO4TreatmentTBARSnmol/mg proteinNone5.70 ± 1.40sPLA2-IB8.09 ± 3.08sPLA2-IIA4.15 ± 0.89sPLA2-X5.60 ± 2.40CuSO436.5 ± 8.561-ap < 0.01 compared to no treatment.Human LDL was incubated with 50 nm human sPLA2s or 20 μm CuSO4 for 3 h at 37 °C, and the amounts of TBARS were measured as described under "Experimental Procedures." Each value for TBARS represents the mean ± S.D. of triplicate measurements. The data are representative of three experiments. Statistical significance was determined by Student'st test.1-a p < 0.01 compared to no treatment. Open table in a new tab Human LDL was incubated with 50 nm human sPLA2s or 20 μm CuSO4 for 3 h at 37 °C, and the amounts of TBARS were measured as described under "Experimental Procedures." Each value for TBARS represents the mean ± S.D. of triplicate measurements. The data are representative of three experiments. Statistical significance was determined by Student'st test. Modifications of apolipoproteins or surface lipids in LDL were shown to affect the cellular uptake of LDL and hence the formation of foam cell macrophages (2Glass C.K. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar). We then examined the effects of sPLA2-X treatment on the electronic charge of LDL by agarose gel electrophoresis. As shown in Fig.4, oxidized LDL was characterized by increased anodic migration compared with native LDL. Treatment with sPLA2-X also caused enhanced mobility of LDL. Small but significant migration was detected after 3 h of treatment, and marked migration was observed after 24 h of treatment with sPLA2-X. In contrast, the mobility of LDL after treatment with sPLA2-IB or -IIA was not changed during 24-h incubation. Addition of anti-sPLA2-X Ab (100 μg/ml) resulted in complete blockade of sPLA2-X-induced mobilization (data not shown), demonstrating that the increase of negative charge in LDL is dependent on the enzymatic activity of sPLA2-X. Next, we examined the effects of sPLA2-X on the modification of apoB by SDS-PAGE analysis. As shown in Fig.5, excessive aggregation and proteolytic fragmentation of apoB was detected in oxidized LDL even at 3 h of incubation. In contrast, apoB in the sPLA2-X-treated LDL was almost intact with slight aggregation at 24 h of treatment compared with native LDL. There were no changes in apoB of LDL treated with sPLA2-IB and -IIA even at 500 nm (data not shown). These findings demonstrate that sPLA2-X induced an increase in the negative charge of LDL with little modification of apoB. Next, we examined the potency of sPLA2-X-treated LDL for uptake into macrophages. After exp
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