FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2073645536> ?p ?o ?g. }

• W2073645536 endingPage "32752" @default.
• W2073645536 startingPage "32746" @default.
• W2073645536 abstract "Accumulating evidence indicates that secretory phospholipase A2 (sPLA2) enzymes promote atherogenic processes. We have previously showed the presence of Group V sPLA2 (GV sPLA2) in human and mouse atherosclerotic lesions, its hydrolysis of low density lipoprotein (LDL) particles, and the ability of GV sPLA2-modified LDL (GV-LDL) to induce macrophage foam cell formation in vitro. The goal of this study was to investigate the mechanisms involved in macrophage uptake of GV-LDL. Peritoneal macrophages from C57BL/6 mice (wild type (WT)), C57BL/6 mice deficient in LDL receptor (LDLR–/–), or SR-A and CD36 (DKO) were treated with control LDL, GV-LDL, oxidized LDL (ox-LDL) or LDL aggregated by vortexing (vx-LDL). As expected, ox-LDL induced significantly more cholesterol ester accumulation in WT and LDLR–/– compared with DKO macrophages. In contrast, there was no difference in the accumulation of GV-LDL or vx-LDL in the three cell types. 125I-ox-LDL exhibited high affinity, saturable binding to WT cells that was significantly reduced in DKO cells. Vx-LDL and GV-LDL showed low affinity, non-saturable binding that was similar for both cell types, and significantly higher compared with control LDL. GV-LDL degradation in WT and DKO cells was similar. Analyses by confocal microscopy indicated a distinct intracellular distribution of Alexa-568-labeled GV-LDL and Alexa-488-labeled ox-LDL. Uptake of GV-LDL (but not ox-LDL or vx-LDL) was significantly reduced in cells preincubated with heparin or NaClO3, suggesting a role for proteoglycans in GV-LDL uptake. Our data point to a physiological modification of LDL that has the potential to promote macrophage foam cell formation independent of scavenger receptors. Accumulating evidence indicates that secretory phospholipase A2 (sPLA2) enzymes promote atherogenic processes. We have previously showed the presence of Group V sPLA2 (GV sPLA2) in human and mouse atherosclerotic lesions, its hydrolysis of low density lipoprotein (LDL) particles, and the ability of GV sPLA2-modified LDL (GV-LDL) to induce macrophage foam cell formation in vitro. The goal of this study was to investigate the mechanisms involved in macrophage uptake of GV-LDL. Peritoneal macrophages from C57BL/6 mice (wild type (WT)), C57BL/6 mice deficient in LDL receptor (LDLR–/–), or SR-A and CD36 (DKO) were treated with control LDL, GV-LDL, oxidized LDL (ox-LDL) or LDL aggregated by vortexing (vx-LDL). As expected, ox-LDL induced significantly more cholesterol ester accumulation in WT and LDLR–/– compared with DKO macrophages. In contrast, there was no difference in the accumulation of GV-LDL or vx-LDL in the three cell types. 125I-ox-LDL exhibited high affinity, saturable binding to WT cells that was significantly reduced in DKO cells. Vx-LDL and GV-LDL showed low affinity, non-saturable binding that was similar for both cell types, and significantly higher compared with control LDL. GV-LDL degradation in WT and DKO cells was similar. Analyses by confocal microscopy indicated a distinct intracellular distribution of Alexa-568-labeled GV-LDL and Alexa-488-labeled ox-LDL. Uptake of GV-LDL (but not ox-LDL or vx-LDL) was significantly reduced in cells preincubated with heparin or NaClO3, suggesting a role for proteoglycans in GV-LDL uptake. Our data point to a physiological modification of LDL that has the potential to promote macrophage foam cell formation independent of scavenger receptors. A critical event in early atherogenesis is the formation of lipid-laden macrophages (“foam cells”) (1Goldstein J.L. Ho Y.K. Basu K. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 333-337Crossref PubMed Scopus (1948) Google Scholar, 2Yla-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. J. Clin. Investig. 1989; 84: 1086-1095Crossref PubMed Google Scholar, 3Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9989) Google Scholar, 4Tabas I. J. Clin. Investig. 2002; 110: 905-911Crossref PubMed Scopus (528) Google Scholar). According to the “response-to-retention” hypothesis (5Williams K.J. Tabas I. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar), conditions leading to enhanced LDL 2The abbreviations used are: LDL, low density lipoprotein; PL, phospholipid; CE, cholesterol ester; GV sPLA2, group V secretory phospholipase A2; GV-LDL, LDL hydrolyzed by Group V sPLA2; mock-LDL, LDL incubated in hydrolysis buffer in the absence of enzyme; ox-LDL, Cu2+-oxidized LDL; vx-LDL, LDL aggregated by vortexing; HSPG, heparan sulfate proteoglycan; BSA, bovine serum albumin; WT, wild type, DKO, double knock-out; LRP, LDL receptor-related protein. entrapment in the subendothelium trigger this process. Once retained in the vessel wall, LDL undergoes various modifications in both protein and phospholipid (PL) moieties that promote retention and lead to enhanced macrophage uptake. Several types of modifications of LDL, such as oxidation (6Henriksen T. Mahoney E.M. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6499-6503Crossref PubMed Scopus (818) Google Scholar, 7Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3883-3887Crossref PubMed Scopus (1418) Google Scholar, 8Heinecke J.W. Rosen H. Chait A. J. Clin. Investig. 1984; 74: 1890-1894Crossref PubMed Scopus (432) Google Scholar), depletion of sphingomyelin by secretory sphingomyelinase (9Xu X.X. Tabas I. J. Biol. Chem. 1991; 266: 24849-24858Abstract Full Text PDF PubMed Google Scholar), hydrolysis of glycero-PLs by sPLA2 enzymes (10Hanasaki K. Yamada K. Yamamoto S. Ishimoto Y. Saiga A. Ono T. Ikeda M. Notoya M. Kamitani S. Arita H. J. Biol. Chem. 2002; 277: 29116-29124Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 11Wooton-Kee C.R. Boyanovsky B.B. Nasser M.S. de Villiers W.J.S. Webb N.R. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 762-767Crossref PubMed Scopus (97) Google Scholar, 12Murakami M. Kudo I. Curr. Opin. Lipidol. 2003; 14: 431-436Crossref PubMed Scopus (71) Google Scholar), and aggregation (13Aviram M. Maor I. Keidar S. Hayek T. Oiknine J. Barel Y. Adler Z. Kertzman V. Milo S. Biochem. Biophys. Res. Commun. 1995; 216: 501-513Crossref PubMed Scopus (75) Google Scholar, 14Guyton J.R. Klemp K.F. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 4-11Crossref PubMed Scopus (207) Google Scholar, 15Hoff H.F. Morton R.E. Ann. N. Y. Acad. Sci. 1985; 454: 183-194Crossref PubMed Scopus (63) Google Scholar, 16Khoo J.C. Miller E. McLoughlin P. Steinberg D. Arteriosclerosis. 1988; 8: 348-358Crossref PubMed Google Scholar, 17Suits A.G. Chait A. Aviram M. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 868: 2713-2717Crossref Scopus (162) Google Scholar, 18Tabas I. Annu. Rev. Nutr. 1999; 19: 123-139Crossref PubMed Scopus (113) Google Scholar) have been implicated in lipid accumulation in the vessel wall. The sPLA2 family comprises a group of enzymes that hydrolyze the acyl-ester bond at the 2 position (sn-2) of glycero-PLs. Of the 10 sPLA2 isozymes that have been described in mammals, three members (Group IIA, Group V, and Group X) have been detected in human and/or mouse atherosclerotic lesions (10Hanasaki K. Yamada K. Yamamoto S. Ishimoto Y. Saiga A. Ono T. Ikeda M. Notoya M. Kamitani S. Arita H. J. Biol. Chem. 2002; 277: 29116-29124Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 11Wooton-Kee C.R. Boyanovsky B.B. Nasser M.S. de Villiers W.J.S. Webb N.R. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 762-767Crossref PubMed Scopus (97) Google Scholar, 19Hurt-Camejo E. Olsson U. Wiklund O. Bondjers G. Camejo G. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1011-1017Crossref PubMed Scopus (137) Google Scholar). Accumulating evidence indicates that sPLA2 hydrolysis of LDL-PL results in structural alterations of the particles that promote lipid accumulation in the vessel wall and enhances macrophage uptake. Hydrolysis of LDL by sPLA2 in vitro results in an increased affinity for proteoglycans, which would be expected to increase retention in the subendothelium (20Hakala J.K. Oorni K. Pentikainen M.O. Hurt-Camejo E. Kovanen P.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21Crossref PubMed Scopus (111) Google Scholar, 21Hurt-Camejo E. Camejo G. Sartipy P. Curr. Opin. Lipidol. 2000; 11: 465-471Crossref PubMed Scopus (85) Google Scholar). Lipolysis of LDL by sPLA2 leads to conformational changes in apoB-100 and reorganization of lipids that induce particle aggregation (22Kleinman Y. Krul E. Burnes M. Aronson W. Pfleger B. Schonfeld G. J. Lipid Res. 1988; 29: 729-743Abstract Full Text PDF PubMed Google Scholar, 23Gorshkova I.N. Menschikowski M. Jaross W. Biochim. Biophys. Acta. 1996; 1300: 103-113Crossref PubMed Scopus (39) Google Scholar, 24Flood C. Gustafsson M. Pitas R.E. Arnaboldi L. Walzem R.L. Boren J. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 564-570Crossref PubMed Scopus (96) Google Scholar). We recently reported that in vitro hydrolysis of LDL by Group V sPLA2 in the presence of physiological concentrations of albumin leads to spontaneous particle aggregation, and the extent of aggregation is proportional to the degree of LDL hydrolysis (11Wooton-Kee C.R. Boyanovsky B.B. Nasser M.S. de Villiers W.J.S. Webb N.R. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 762-767Crossref PubMed Scopus (97) Google Scholar). When incubated with mouse peritoneal macrophages, this modified LDL induces foam cell formation. Group X sPLA2 modification of LDL has also been shown to promote macrophage foam cell formation, although extensive modification by Group X sPLA2 is not reported to induce LDL aggregation (10Hanasaki K. Yamada K. Yamamoto S. Ishimoto Y. Saiga A. Ono T. Ikeda M. Notoya M. Kamitani S. Arita H. J. Biol. Chem. 2002; 277: 29116-29124Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The structural alterations of the LDL particles brought about by sPLA2 hydrolysis that are responsible for enhancing macrophage lipid accumulation and the pathway(s) by which macrophages take up this modified LDL have not been defined. In the present study, we investigated the mechanisms involved in macrophage uptake of GV sPLA2-modified LDL. Using mouse peritoneal macrophages deficient in LDLR or SR-A and CD36, we demonstrated that GV-LDL uptake and degradation is not dependent on LDLR or scavenger receptors SR-A and CD36. Analysis by confocal microscopy revealed differences in the rate of uptake and subsequent intracellular trafficking of GV-LDL and ox-LDL by macrophages. Treatments that inhibited apoB-proteoglycan interaction or proteoglycan assembly significantly decreased GV-LDL accumulation by macrophages but had no effect on uptake of ox-LDL or LDL aggregated by vortexing. Taken together, our results indicate that Group V sPLA2-hdyrolyzed LDL promotes macrophage foam cell formation through a pathway that is distinct from other modified forms of LDL and involves cell-surface proteoglycans. Isolation, Modification, and Labeling of LDL—LDL (density 1.019–1.063) was isolated from the plasma of healthy volunteers by sequential ultracentrifugation and stored at 4 °C under argon gas. LDL was subjected to hydrolysis by GV sPLA2, as previously described (11Wooton-Kee C.R. Boyanovsky B.B. Nasser M.S. de Villiers W.J.S. Webb N.R. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 762-767Crossref PubMed Scopus (97) Google Scholar). Briefly, LDL particles (1 mg/ml) were incubated in hydrolysis buffer (0.1 m HEPES, 0.1 m NaCl, 1 mm CaCl2, 10 mg/ml fatty acid-free BSA, and 0.01% butylated hydroxytoluene) under argon gas at 37 °C for 24 h in the presence (GV-LDL) or absence (mock-LDL) of 500 units/ml GV sPLA2. LDL-PL hydrolysis was quantified by measuring the amount of free fatty acids released in the solution (11Wooton-Kee C.R. Boyanovsky B.B. Nasser M.S. de Villiers W.J.S. Webb N.R. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 762-767Crossref PubMed Scopus (97) Google Scholar). For the experiments described here, incubation with GV sPLA2 in the absence of EDTA resulted in the hydrolysis of >80% of LDL-PL. For some experiments, a portion of GV-LDL was filtered through a 0.1-μm filter (Whatman, Clifton, NJ), and the total cholesterol content of samples before and after filtration was used to estimate the amount of GV-LDL retained by the filter. We also analyzed hydrolyzed particles before and after filtration by electron microscopy. LDL preparations were stained with 2% uranyl acetate solution and then viewed and photographed in a Philips Tecnai 12 transmission electron microscope at the Electron Microscopy and Imaging Facility, University of Kentucky. The diameters of aggregates were measured from the electron micrographs using Scion Image software (Scion Corporation, Frederick, MD). For other experiments, LDL was incubated with GV sPLA2 in hydrolysis buffer supplemented with 10 mm EDTA to prevent LDL-PL hydrolysis (25Chen Y. Dennis E.A. Biochim. Biophys. Acta. 1998; 1394: 57-64Crossref PubMed Scopus (74) Google Scholar). To prepare oxidized LDL, native LDL was dialyzed against 5 μm CuSO4 overnight at 4 °C. Oxidation was stopped by adding EDTA at a final concentration of 1 mm. Ox-LDL was then dialyzed against 150 mm saline and stored under argon gas. Relative electrophoretic mobility was assayed by running the ligands for 90 min at 100 V on 2% agarose gel (26Noble R.P. J. Lipid Res. 1968; 9: 693-700Abstract Full Text PDF PubMed Google Scholar). Mock-LDL, GV-LDL, and ox-LDL were labeled with 125I according to Bilheimer's modification (27Bilheimer D.W. Eisenberg S. Levy R.I. Biochim. Biophys. Acta. 1972; 260: 212-221Crossref PubMed Scopus (1185) Google Scholar) of the procedure described by Goldstein et al. (1Goldstein J.L. Ho Y.K. Basu K. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 333-337Crossref PubMed Scopus (1948) Google Scholar). Native LDL or 125I-LDL was aggregated by vortexing at maximal speed (Fisher, vortex model Genie 2) for 1 min. Native LDL and ox-LDL were labeled with Alexa-Fluor 568 and Alexa-Fluor 488, respectively (Molecular Probes, Eugene, OR), according to manufacturer's instructions. Native LDL was then hydrolyzed with GV-sPLA2, as already described. Isolation of Peritoneal Macrophages—C57BL/6 mice and C57BL/6 mice lacking the LDL receptor were originally obtained from The Jackson Laboratory (Bar Harbor, ME). To generate SR-A/CD36-deficient mice, SR-A–/– mice (28Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Honda M. Kurihara H. Aburatani H. Doi T. Matsumoto A. Azuma S. Noda T. Toyoda Y. Itakura H. Yazaki Y. Horiuchi S. Takahashi K. Kruijt J.K. van Berkel T.J.C. Steinbreher U.P. Ishibashi S. Maeda N. Gordon S. Kodama T. Nature. 1997; 386: 292-296Crossref PubMed Scopus (1010) Google Scholar) backcrossed at least six times in C57Bl/6 background were crossed with CD36–/– mice (29Febbraio M. Abumrad N.A. Hajjar D.P. Sharma K. Cheng W. Pearce S.F.A. Silverstein R.L. J. Biol. Chem. 1999; 274: 19055-19062Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar) backcrossed at least four times in the C57BL/6 strain. Animals were injected intraperitoneally with a sterile solution (1 ml) of 1% Biogel 100 (Bio-Rad) in phosphate-buffered saline. After 96 h, the animals were anesthetized, and peritoneal macrophages were harvested by lavage with 5 ml of ice-cold phosphate-buffered saline. All procedures were in accordance with the guidelines of the Veterans Affairs Institutional Animal Care and Use Committee. Macrophages were seeded in 12-well dishes at a density of ∼2.0 × 106 cells/well in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, 2 mm l-glutamine, and 25 ng/ml macrophage colony-stimulating factor (Calbiochem) and allowed to attach for 4 h. Non-attached cells were then removed by washing the dishes with phosphate-buffered saline. Macrophages were incubated overnight at 37 °C. When indicated, cells were preincubated for 3 h with 100 units/ml heparin (Sigma) or 5 mg/ml lactoferrin (Sigma) or overnight with 10 mm NaClO3 prior to the addition of ligands. Lipid Extraction and Cholesterol Ester (CE) Measurements—After incubation with LDL ligands, cells were solubilized in 0.25 ml of 0.1 n NaOH for 2 h on a shaking platform, and 50-μl aliquots were used for protein determinations. Lipids were extracted from the remaining cell lysate by adding 3 ml of methanol:chloroform 2:1 (v/v) and incubating at 37 °C for 1 h. Phases were separated by adding 2 ml of 0.05% H2SO4 followed by vigorous vortexing and centrifugation at 3,000 revolutions/min for 10 min. The organic phase was collected, and 1 ml of 1% Triton X-100 in chloroform was added. The samples were dried in a Freezedry system (Freezone 4.5, Labconco, Kansas City, MO) and dissolved in H20. Aliquots were assayed for total and free cholesterol content using a colorimetric kit (Wako, Richmond, VA). CE was calculated as the difference between the total and free cholesterol. Binding and Degradation Assay—For binding studies, cells were cooled to 4 °C and after washing with ice-cold phosphate-buffered saline, were incubated with ice-cold medium containing the indicated concentrations of 125I-labeled ligands. After a 2-h incubation at 4 °C, the medium was removed and cells were washed rapidly three times with washing buffer (50 mm Tris, 150 mm NaCl, and 2 mg/ml fatty acid-free BSA) followed by two washes with washing buffer without BSA. All washes were performed at 4 °C with prechilled solutions. The cells were solubilized in 0.25 ml of 0.1 n NaOH for 2 h on a rotary platform at room temperature. The total radioactivity was measured on a γ counter (Cobra II, Packard Instrument Co., Minneapolis, MN). Kd values were determined by nonlinear regression analysis of receptor-specific cell association values using Prism® software (GraphPad Software, San Diego, CA). For apoB degradation assays, cell-free supernatants were analyzed for trichloroacetic acid-soluble, chloroform-unextractable radioactivity (30Bierman E.L. Stein O. Stein Y. Circ. Res. 1974; 35: 136-150Crossref PubMed Scopus (307) Google Scholar). Background for degradation assays was determined by performing ligand incubations in wells containing no cells. Oil Red O Staining—Peritoneal macrophages from C57BL/6 mice were seeded on glass coverslips in 12-well dishes and incubated for 48 h with 0.2 mg/ml mock-LDL, GV-LDL, or GV-LDL prefiltered through a 0.1-μm filter, as described under “Isolation, Modification, and Labeling of LDL.” After incubations, the cells were fixed for 10 min in 10% formalin, washed with 60% isopropyl alcohol, and then stained with Oil Red O (60% in isopropyl alcohol; Sigma) for 30 min. After washing twice with 60% isopropyl alcohol, the cells were counterstained with hematoxylin and mounted on slides for light microscopy. Confocal Microscopy—Peritoneal macrophages from WT or DKO mice were seeded on glass coverslips and incubated with 0.2 mg/ml Alexa-fluor 488-labeled ox-LDL and Alexa-fluor 568-labeled GV-LDL. After incubations, the cells were mounted on slides using fluorescence protecting medium (Vectashield, Vector Laboratories, Burlingame, CA). Confocal microscopy was performed at the University of Kentucky Imaging Facility using a Leica laser scanning confocal microscope with argon (488 nm) and krypton (568 nm) lasers. Statistical Analysis—Data are expressed as mean ± S.E. Results were analyzed by Student's t test and one-way analysis of variance followed by Bonferroni's post-test. Values of p < 0.05 were considered statistically significant. Hydrolysis by GV sPLA2 Promotes Macrophage LDL Uptake—We reported previously that LDL hydrolyzed by GV sPLA2 (GV-LDL) is susceptible to aggregation and promotes the accumulation of neutral lipids in mouse peritoneal macrophages (11Wooton-Kee C.R. Boyanovsky B.B. Nasser M.S. de Villiers W.J.S. Webb N.R. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 762-767Crossref PubMed Scopus (97) Google Scholar) (Fig. 1B). In the present study, we investigated the mechanisms involved in macrophage uptake of GV-LDL. Because aggregated LDL is a potent inducer of foam cell formation (16Khoo J.C. Miller E. McLoughlin P. Steinberg D. Arteriosclerosis. 1988; 8: 348-358Crossref PubMed Google Scholar), we considered the possibility that GV-LDL is taken up by macrophages because of its highly aggregated state. To determine the size of the aggregates formed after LDL hydrolysis, we analyzed mock-LDL and GV-LDL by electron microscopy (Fig. 1A). Our data revealed that the mean size of the aggregates was 123 ± 19 nm. To remove these aggregates from GV-LDL preparations, we filtered GV-LDL through a 0.1-μm filter, which appeared to efficiently remove the large aggregates (Fig. 1A). Based on the cholesterol content of the filtered fraction, >75% of the hydrolyzed LDL was recovered in the flow-through. We then assessed the ability of the filtered fraction to induce foam cell formation. Mouse peritoneal macrophages were incubated for 48 h with equivalent amounts of GV-LDL or GV-LDL-filtered fraction. For comparison, cells were also incubated with a control LDL, which was treated similarly to GV-LDL, except that the enzyme was omitted from the reaction mixture (mock-LDL). Compared with mock-LDL, incubation with GV-LDL did not lead to increased cellular free cholesterol content in macrophages (Fig. 1C), whereas CE accumulation in macrophages treated with GV-LDL was significantly higher compared with cells treated with mock-LDL (Fig. 1, B and C). Our data indicated a slight and insignificant decrease in CE accumulation in cells incubated with the filtered fraction compared with cells treated with unfiltered GV-LDL. Thus, increased CE accumulation in macrophages incubated with GV-LDL is not dependent on large (>0.1 μm) LDL aggregates produced as a result of GV sPLA2 hydrolysis. We considered the possibility that GV sPLA2 mediates LDL uptake through a non-enzymatic function, whereby the enzyme itself promotes the interaction of the particle with the cell through a mechanism that does not involve LDL hydrolysis. To investigate this, we used the fact that hydrolytic activity of GV sPLA2, similar to other members of the sPLA2 family, is dependent on millimolar concentrations of Ca2+ (25Chen Y. Dennis E.A. Biochim. Biophys. Acta. 1998; 1394: 57-64Crossref PubMed Scopus (74) Google Scholar). Thus, to inhibit hydrolysis, LDL was incubated with GV sPLA2 in the presence of 10 mm EDTA. Quantification of the amount of free fatty acids released in the reaction mixture confirmed that LDL-PL hydrolysis was completely inhibited under these reaction conditions (data not shown). Incubation of macrophages with LDL treated with GV sPLA2 in the presence of EDTA failed to induce foam cell formation (Fig. 1C). Thus, particle hydrolysis appears to be a prerequisite for increased uptake of GV-LDL by macrophages, suggesting that GV sPLA2 promotes foam cell formation by altering the structure of the LDL particle. LDL Receptor, SR-A, and CD36 Are Not Involved in the Uptake of GV-LDL—We next examined the role of LDLR and scavenger receptors SR-A and CD36 in GV-LDL-mediated foam cell formation. To assess the role of these receptors in GV-LDL uptake, our approach was to compare CE accumulation in peritoneal macrophages isolated from C57BL/6 (WT), LDLR–/–, and SR-A/CD36–/– (DKO) mice after incubations with LDL hydrolyzed with GV sPLA2 in the presence of BSA (1%). For comparison, WT, LDLR–/–, and DKO cells were also incubated with mock-LDL, ox-LDL, or vx-LDL. As expected, macrophage uptake of ox-LDL was dependent on CD36 and SR-A expression (Fig. 2A). Incubation of WT and LDLR–/– cells with ox-LDL induced a significant 2.5-fold increase in intracellular CE content compared with WT and LDLR–/– cells incubated with mock-LDL, and this increased uptake was totally absent in DKO cells. In contrast, WT, LDLR–/–, and DKO macrophages incubated with GV-LDL had significantly more CE accumulation (∼2.2-fold) compared with cells treated with mock-LDL. Large LDL aggregates produced by vortexing also induced massive CE accumulation in mouse peritoneal macrophages (6–7-fold more compared with mock-LDL) that was not dependent on LDLR, SR-A, or CD36 expression. These data indicate that the intracellular accumulation of GV-LDL, like vx-LDL, is not mediated by LDLR, SR-A, or CD36. Because recognition by scavenger receptors is believed to be mediated through interactions with polyanionic ligands, to explain our findings it was important to determine whether GV sPLA2 hydrolysis results in an altered charge of the LDL particle. To assess whether GV sPLA2 hydrolysis alters LDL charge similarly to oxidation, the relative electrophoretic mobility of ox-LDL and GV-LDL on agarose gels was determined. As expected, modification of LDL by copper oxidation resulted in increased anodic migration compared with native LDL (Fig. 2B). In contrast, hydrolysis by GV sPLA2 in the presence of albumin (GV-LDL + BSA) had no effect on LDL electrophoretic mobility. This finding differs from the results of Hanasaki et al. (10Hanasaki K. Yamada K. Yamamoto S. Ishimoto Y. Saiga A. Ono T. Ikeda M. Notoya M. Kamitani S. Arita H. J. Biol. Chem. 2002; 277: 29116-29124Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), who reported that extensive modification by Group X sPLA2 significantly increased the anodic migration of LDL. It is likely that this discrepancy is because of differences in hydrolysis conditions for the two studies. In the case of Group X sPLA2, LDL hydrolysis was carried out in the presence of subphysiological concentrations of BSA (0.0125%), whereas in the current study, Group V sPLA2 hydrolysis was performed in buffer containing 1% BSA. As shown in Fig. 2B, LDL hydrolyzed by GV sPLA2 in the absence of BSA had markedly increased mobility compared with native LDL or GV-LDL hydrolyzed in the presence of BSA. Our results are consistent with the results of Kleinman et al. (22Kleinman Y. Krul E. Burnes M. Aronson W. Pfleger B. Schonfeld G. J. Lipid Res. 1988; 29: 729-743Abstract Full Text PDF PubMed Google Scholar), who concluded that, in the absence of lipid binding proteins such as albumin, free fatty acids liberated by sPLA2 accumulate in the LDL particle, resulting in increased anodal migration. When hydrolysis is carried out in the presence of physiological concentrations of BSA, free fatty acids are transferred from LDL to albumin, and electrophoretic mobility is consequently diminished. GV-LDL Binding to Macrophages Is Independent of SR-A and CD36—Equilibrium binding studies were performed using WT and DKO macrophages to compare the binding properties of 125I-labeled mock-LDL, GV-LDL, ox-LDL, and vx-LDL. Compared with WT cells, ox-LDL binding was significantly reduced in macrophages lacking SR-A and CD36 (Fig. 3A). A dependence on SR-A and CD36 was not observed for vx-LDL (Fig. 3B). Hydrolysis of LDL by GV sPLA2 significantly enhanced LDL binding to macrophages compared with mock-LDL, and this increased binding was not dependent on SR-A or CD36 expression (Fig. 3, C and D). Although ox-LDL exhibited saturable, high affinity binding (apparent Kd = 15 μg/ml), vx-LDL and GV-LDL binding to both cell types appeared to be with lower affinity and did not saturate up to 100 μg/ml ligand. These data are consistent with the conclusion that GV-LDL, similar to vx-LDL, associates with macrophages in a high capacity receptor-independent manner. GV-LDL Degradation Is Independent of SR-A and CD36—To determine the extent of GV-LDL internalization and degradation by macrophages, WT and DKO cells were incubated at 37 °C with 0.2 mg/ml 125I-labeled mock-LDL, GV-LDL, ox-LDL, or vx-LDL, and the amount of trichloroacetic acid-soluble, chloroform-unextractable radioactivity in the medium was quantified at selected intervals. In the case of ox-LDL, there was a time-dependent increase in the degradation of apoB in WT macrophages that was significantly higher compared with DKO cells (Fig. 4A). In contrast, for the other three ligands, there was no significant difference in apoB degradation in WT and DKO macrophages. In both vx-LDL- and GV-LDL-treated cells, the amount of degraded apoB was significantly higher compared with cells treated with mock-LDL (Fig. 4, B–D). These findings confirmed our previous data that GV sPLA2 hydrolysis promotes macrophage LDL uptake and that SR-A and CD36 are not involved in the internalization of such modified LDL. Although it appeared that macrophages degraded significantly more vx-LDL compared with the other modified LDL ligands, our data indicate that only 2% of the total amount of vx-LDL associated with cells was degraded after 8 h. This contrasts to ox-LDL and GV-LDL, where ∼40 and 19%, respectively, of the total amount of ligand taken up was degraded after 8 h (data not shown). GV-LDL and ox-LDL Internalized by Macrophages Have Distinct Intracellular Localization—As another approach to comparing macrophage uptake of GV-LDL and ox-LDL, we incubated WT and DKO cells simultaneously with 0.2 mg/ml Alexa-fluor 568-labeled GV-LDL (Fig. 5, red) and Alexa-fluor 488-labeled ox-LDL (green) and then visualized the cells at selected intervals by confocal microscopy. The analysis of DKO macrophages after 10 min of incubation confirmed the finding that macrophage binding of GV-LDL (red) does not require CD36 or SR-A expression (Fig. 5A), unlike ox-LDL (green) (Fig. 5, B versus A). Interestingly, after 10 min of incubation, the majority of the cell-associated GV-LDL appeared to be at or near the cell surface of both WT and DKO cells (Fig. 5, A and B). This contrasts to oxLDL, where a large amount of ligand internalization by WT cells was evident after a 10-min incubation. The analysis of WT cells at later time points showed that, although GV-LDL was eventually taken up, its intracel" @default.
• W2073645536 created "2016-06-24" @default.
• W2073645536 creator A5001864326 @default.
• W2073645536 creator A5058833764 @default.
• W2073645536 creator A5082058844 @default.
• W2073645536 date "2005-09-01" @default.
• W2073645536 modified "2023-10-15" @default.
• W2073645536 title "Group V Secretory Phospholipase A2-modified Low Density Lipoprotein Promotes Foam Cell Formation by a SR-A- and CD36-independent Process That Involves Cellular Proteoglycans" @default.
• W2073645536 cites W126816316 @default.
• W2073645536 cites W1495390974 @default.
• W2073645536 cites W1502667717 @default.
• W2073645536 cites W1508962214 @default.
• W2073645536 cites W1515028535 @default.
• W2073645536 cites W1516777352 @default.
• W2073645536 cites W1541426877 @default.
• W2073645536 cites W1578786377 @default.
• W2073645536 cites W1893943578 @default.
• W2073645536 cites W1966907812 @default.
• W2073645536 cites W1968171440 @default.
• W2073645536 cites W1968567802 @default.
• W2073645536 cites W1969349787 @default.
• W2073645536 cites W1969352719 @default.
• W2073645536 cites W1991172636 @default.
• W2073645536 cites W2005230846 @default.
• W2073645536 cites W2011069780 @default.
• W2073645536 cites W2012276590 @default.
• W2073645536 cites W2017315967 @default.
• W2073645536 cites W2018445977 @default.
• W2073645536 cites W2023390812 @default.
• W2073645536 cites W2029819414 @default.
• W2073645536 cites W2032731208 @default.
• W2073645536 cites W2033546828 @default.
• W2073645536 cites W2037466306 @default.
• W2073645536 cites W2040579726 @default.
• W2073645536 cites W2042667997 @default.
• W2073645536 cites W2043945957 @default.
• W2073645536 cites W2045600823 @default.
• W2073645536 cites W2045868118 @default.
• W2073645536 cites W2049401036 @default.
• W2073645536 cites W2057124401 @default.
• W2073645536 cites W2058194924 @default.
• W2073645536 cites W2059237817 @default.
• W2073645536 cites W2066352142 @default.
• W2073645536 cites W2077576735 @default.
• W2073645536 cites W2084116516 @default.
• W2073645536 cites W2087757733 @default.
• W2073645536 cites W2093272247 @default.
• W2073645536 cites W2104573407 @default.
• W2073645536 cites W2105204727 @default.
• W2073645536 cites W2107611361 @default.
• W2073645536 cites W2109738937 @default.
• W2073645536 cites W2118877270 @default.
• W2073645536 cites W2125539464 @default.
• W2073645536 cites W2129970089 @default.
• W2073645536 cites W2131162885 @default.
• W2073645536 cites W2137570167 @default.
• W2073645536 cites W2152188478 @default.
• W2073645536 cites W2161491104 @default.
• W2073645536 cites W2163682706 @default.
• W2073645536 cites W2170583921 @default.
• W2073645536 cites W2319573935 @default.
• W2073645536 cites W2473455258 @default.
• W2073645536 cites W4241939777 @default.
• W2073645536 doi "https://doi.org/10.1074/jbc.m502067200" @default.
• W2073645536 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16040605" @default.
• W2073645536 hasPublicationYear "2005" @default.
• W2073645536 type Work @default.
• W2073645536 sameAs 2073645536 @default.
• W2073645536 citedByCount "74" @default.
• W2073645536 countsByYear W20736455362012 @default.
• W2073645536 countsByYear W20736455362013 @default.
• W2073645536 countsByYear W20736455362014 @default.
• W2073645536 countsByYear W20736455362015 @default.
• W2073645536 countsByYear W20736455362016 @default.
• W2073645536 countsByYear W20736455362017 @default.
• W2073645536 countsByYear W20736455362018 @default.
• W2073645536 countsByYear W20736455362019 @default.
• W2073645536 countsByYear W20736455362020 @default.
• W2073645536 countsByYear W20736455362021 @default.
• W2073645536 countsByYear W20736455362022 @default.
• W2073645536 countsByYear W20736455362023 @default.
• W2073645536 crossrefType "journal-article" @default.
• W2073645536 hasAuthorship W2073645536A5001864326 @default.
• W2073645536 hasAuthorship W2073645536A5058833764 @default.
• W2073645536 hasAuthorship W2073645536A5082058844 @default.
• W2073645536 hasBestOaLocation W20736455361 @default.
• W2073645536 hasConcept C111919701 @default.
• W2073645536 hasConcept C12554922 @default.
• W2073645536 hasConcept C170493617 @default.
• W2073645536 hasConcept C185592680 @default.
• W2073645536 hasConcept C2777895019 @default.
• W2073645536 hasConcept C2778163477 @default.
• W2073645536 hasConcept C2779828298 @default.
• W2073645536 hasConcept C2780072125 @default.
• W2073645536 hasConcept C41008148 @default.
• W2073645536 hasConcept C55493867 @default.
• W2073645536 hasConcept C86803240 @default.
• W2073645536 hasConcept C95444343 @default.

• next