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• W2168897708 abstract "The macrophage scavenger receptor CD36 plays an important role in the uptake of oxidized forms of low density lipoprotein (LDL) and contributes to lesion development in murine models of atherosclerosis. However, the structural basis of CD36 lipoprotein ligand recognition is unknown. We now identify a novel class of oxidized phospholipids that serve as high affinity ligands for CD36 and mediate recognition of oxidized forms of LDL by CD36 on macrophages. Small unilamellar vesicles of homogeneous phosphatidylcholine (PC) molecular species were oxidized by the myeloperoxidase (MPO)-H2O2-NO 2−system, and products were separated by sequential LC/ESI/MS/MS. In parallel, fractions were tested for their ability to bind to CD36. Four major structurally related phospholipids with CD36 binding activity were identified from oxidized 1-palmitoyl-2-arachidonyl-PC, and four corresponding structural analogs with CD36 binding activity were identified from oxidized 1-palmitoyl-2-linoleoyl-PC. Each was then synthetically prepared, its structure confirmed by multinuclear NMR and high resolution mass spectrometry, and shown to possess identical CD36 binding activity and LC/ESI/MS/MS characteristics in both native and derivatized forms. Based upon the structures of the active compounds identified, and structure-function studies with a variety of synthetic analogs, we conclude that the structural characteristics required for high affinity binding of oxidized PC species to CD36 are a phospholipid with an sn-2 acyl group that incorporates a terminal γ-hydroxy(or oxo)-α,β-unsaturated carbonyl (oxPCCD36). LC/ESI/MS/MS studies demonstrate that oxPCCD36 are formed during LDL oxidation by multiple distinct pathways. Formation of this novel class of oxidized PC species contributes to CD36-mediated recognition of LDL oxidized by MPO and other biologically relevant mechanisms. The present results offer structural insights into the molecular patterns recognized by the scavenger receptor CD36 and provide a platform for the development of potential therapeutic inhibitory agents. The macrophage scavenger receptor CD36 plays an important role in the uptake of oxidized forms of low density lipoprotein (LDL) and contributes to lesion development in murine models of atherosclerosis. However, the structural basis of CD36 lipoprotein ligand recognition is unknown. We now identify a novel class of oxidized phospholipids that serve as high affinity ligands for CD36 and mediate recognition of oxidized forms of LDL by CD36 on macrophages. Small unilamellar vesicles of homogeneous phosphatidylcholine (PC) molecular species were oxidized by the myeloperoxidase (MPO)-H2O2-NO 2−system, and products were separated by sequential LC/ESI/MS/MS. In parallel, fractions were tested for their ability to bind to CD36. Four major structurally related phospholipids with CD36 binding activity were identified from oxidized 1-palmitoyl-2-arachidonyl-PC, and four corresponding structural analogs with CD36 binding activity were identified from oxidized 1-palmitoyl-2-linoleoyl-PC. Each was then synthetically prepared, its structure confirmed by multinuclear NMR and high resolution mass spectrometry, and shown to possess identical CD36 binding activity and LC/ESI/MS/MS characteristics in both native and derivatized forms. Based upon the structures of the active compounds identified, and structure-function studies with a variety of synthetic analogs, we conclude that the structural characteristics required for high affinity binding of oxidized PC species to CD36 are a phospholipid with an sn-2 acyl group that incorporates a terminal γ-hydroxy(or oxo)-α,β-unsaturated carbonyl (oxPCCD36). LC/ESI/MS/MS studies demonstrate that oxPCCD36 are formed during LDL oxidation by multiple distinct pathways. Formation of this novel class of oxidized PC species contributes to CD36-mediated recognition of LDL oxidized by MPO and other biologically relevant mechanisms. The present results offer structural insights into the molecular patterns recognized by the scavenger receptor CD36 and provide a platform for the development of potential therapeutic inhibitory agents. oxidized low density lipoprotein apolipoprotein butylated hydroxytoluene Cu2+-oxidized LDL Dulbecco's modified Eagle's medium 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine diethylenetriaminepentaacetic acid fetal calf serum the glutaric and nonanedioic monoesters of 2-lyso-PC the 9-hydroxy-10-dodecenedioic acid and 5-hydroxy-8-oxo-6-octenedioic acid esters of 2-lyso-PC the 9-hydroxy-12-oxo-10-dodecenoic acid and 5-hydroxy-8-oxo-6-octenoic acid esters of 2-lyso-PC hydrogen peroxide the 9-keto-12-oxo-10-dodecenoic acid and 5-keto-8-oxo-6-octenoic acid esters of 2-lyso-PC the 9-keto-10-dodecendioic acid and 5-keto-6-octendioic acid esters of 2-lyso-PC HPLC with on-line electrospray ionization tandem mass spectrometry low density lipoprotein lipoprotein-deficient serum mouse peritoneal macrophage myeloperoxidase multiple reaction monitoring nitrite 1-palmitoyl-sn-glycero-3-phosphocholine LDL modified by the MPO-H2O2-NO 2−system PAPC vesicles modified by the MPO-H2O2-NO 2−system the 5-oxovaleric acid and 9-oxononanoic acid esters of 2-lyso-PC oxidized phosphatidylcholine species that bind with high affinity to CD36 1-hexadecanoyl-2-eicosatetra-5′,8′,11′,14′-enoyl-sn-glycero-3-phosphocholine 1-hexadecanoyl-2-octadec-9′-enoyl-sn-glycero-3-phosphocholine 1-hexadecanoyl-2-octadecadi-9′,12′-enoyl-sn-glycero-3-phosphocholine phosphatidylserine scavenger receptor class A type I high pressure liquid chromatography Chinese hamster ovary phosphate-buffered saline bovine serum albumin 4-hydroxy-2-nonenal peroxisome proliferator-activated receptor γ electrospray ionization CD36 is a heavily glycosylated, single chain, integral plasma membrane protein that belongs to an evolutionarily conserved family of proteins that serve as scavenger and lipid receptors (1Silverstein R.L. Febbraio M. Curr. Opin. Lipidol. 2000; 11: 483-491Crossref PubMed Scopus (108) Google Scholar, 2Daviet L. McGregor J.L. Thromb. Haemostasis. 1997; 78: 65-69Crossref PubMed Scopus (61) Google Scholar). It is expressed on the surface of adipocytes, microvascular endothelial cells, macrophages, platelets, and specialized epithelial cells (1Silverstein R.L. Febbraio M. Curr. Opin. Lipidol. 2000; 11: 483-491Crossref PubMed Scopus (108) Google Scholar, 2Daviet L. McGregor J.L. Thromb. Haemostasis. 1997; 78: 65-69Crossref PubMed Scopus (61) Google Scholar). CD36 functions in vivo in scavenger recognition of oxidized lipoproteins and senescent or apoptotic cells, fatty acid transport, cell-matrix interactions, and anti-angiogenic actions (3Nozaki S. Kashiwagi H. Yamashita S. Nakagawa T. Kostner B. Tomiyama Y. Nakata A. Ishigami M. Miyagawa J. Kameda- Takemura K. J. Clin. Invest. 1995; 96: 1859-1865Crossref PubMed Scopus (285) Google Scholar, 4Febbraio M. Abumrad N.A. Hajjar D.P. Sharma K. Cheng W. Pearce S.F. Silverstein R.L. J. Biol. Chem. 1999; 274: 19055-19062Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 5Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Nat. Med. 2000; 6: 41-48Crossref PubMed Scopus (860) Google Scholar). Its deficiency in humans has been correlated with alterations in myocardial fatty acid uptake, hypertrophic cardiac myopathy, and insulin resistance (6Kashiwagi H. Tomiyama Y. Nozaki S. Honda S. Kosugi S. Shiraga M. Nakagawa T. Nagao N. Kanakura Y. Kurata Y. Matsuzawa Y. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1026-1032Crossref PubMed Scopus (42) Google Scholar, 7Tanaka T. Okamoto F. Sohmiya K. Kawamura K. Jpn. Circ. J. 1997; 61: 724-725Crossref PubMed Scopus (45) Google Scholar, 8Miyaoka K. Kuwasako T. Hirano K. Nozaki S. Yamashita S. Matsuzawa Y. Lancet. 2001; 357: 686-687Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Recent studies (3Nozaki S. Kashiwagi H. Yamashita S. Nakagawa T. Kostner B. Tomiyama Y. Nakata A. Ishigami M. Miyagawa J. Kameda- Takemura K. J. Clin. Invest. 1995; 96: 1859-1865Crossref PubMed Scopus (285) Google Scholar, 4Febbraio M. Abumrad N.A. Hajjar D.P. Sharma K. Cheng W. Pearce S.F. Silverstein R.L. J. Biol. Chem. 1999; 274: 19055-19062Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 9Febbraio M. Podrez E.A. Smith J.D. Hajjar D.P. Hazen S.L. Hoff H.F. Sharma K. Silverstein R.L. J. Clin. Invest. 2000; 105: 1049-1056Crossref PubMed Scopus (831) Google Scholar, 10Podrez E.A. Febbraio M. Sheibani N. Schmitt D. Silverstein R.L. Hajjar D.P. Cohen P.A. Frazier W.A. Hoff H.F. Hazen S.L. J. Clin. Invest. 2000; 105: 1095-1108Crossref PubMed Scopus (366) Google Scholar, 11Boullier A. Gillotte K.L. Horkko S. Green S.R. Friedman P. Dennis E.A. Witztum J.L. Steinberg D. Quehenberger O. J. Biol. Chem. 2000; 275: 9163-9169Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) have focused attention on CD36 as a participant in the atherosclerotic process because of its ability to recognize oxidized forms of LDL (oxLDL).1 CD36 mediates lipid accumulation and macrophage foam cell formation in vitro andin vivo (3Nozaki S. Kashiwagi H. Yamashita S. Nakagawa T. Kostner B. Tomiyama Y. Nakata A. Ishigami M. Miyagawa J. Kameda- Takemura K. J. Clin. Invest. 1995; 96: 1859-1865Crossref PubMed Scopus (285) Google Scholar, 12Huh H.Y. Pearce S.F. Yesner L.M. Schindler J.L. Silverstein R.L. Blood. 1996; 87: 2020-2028Crossref PubMed Google Scholar, 13Nakata A. Nakagawa Y. Nishida M. Nozaki S. Miyagawa J. Nakagawa T. Tamura R. Matsumoto K. Kameda-Takemura K. Yamashita S. Matsuzawa Y. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1333-1339Crossref PubMed Scopus (153) Google Scholar). It is heavily expressed in lipid-rich atheroma, and CD36 knockout mice demonstrate a dramatic decrease in lesion progression (9Febbraio M. Podrez E.A. Smith J.D. Hajjar D.P. Hazen S.L. Hoff H.F. Sharma K. Silverstein R.L. J. Clin. Invest. 2000; 105: 1049-1056Crossref PubMed Scopus (831) Google Scholar). In addition, uptake of oxLDL through CD36 plays a role in differentiation of monocytes and in the induction of nuclear receptors such as the peroxisome proliferator-activated receptor γ (PPARγ), a receptor that participates in lipid and carbohydrate metabolism (12Huh H.Y. Pearce S.F. Yesner L.M. Schindler J.L. Silverstein R.L. Blood. 1996; 87: 2020-2028Crossref PubMed Google Scholar, 14Tontonoz P. Nagy L. Alvarez J.G. Thomazy V.A. Evans R.M. Cell. 1998; 93: 241-252Abstract Full Text Full Text PDF PubMed Scopus (1617) Google Scholar, 15Nagy L. Tontonoz P. Alvarez J.G. Chen H. Evans R.M. Cell. 1998; 93: 229-240Abstract Full Text Full Text PDF PubMed Scopus (1599) Google Scholar, 16Han J. Hajjar D.P. Tauras J.M. Nicholson A.C. J. Lipid Res. 1999; 40: 830-838Abstract Full Text Full Text PDF PubMed Google Scholar). We recently described a pathway for oxidative modification of LDL by the myeloperoxidase (MPO)-H2O2-NO 2−system of monocytes (17Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (424) Google Scholar). LDL modified by MPO-generated reactive nitrogen species (NO2-LDL) is avidly taken up and degraded by macrophages in vitro, leading to cholesterol deposition and foam cell formation. The macrophage scavenger receptor CD36 is responsible for recognition of NO2-LDL and is essential for foam cell formation in this model (10Podrez E.A. Febbraio M. Sheibani N. Schmitt D. Silverstein R.L. Hajjar D.P. Cohen P.A. Frazier W.A. Hoff H.F. Hazen S.L. J. Clin. Invest. 2000; 105: 1095-1108Crossref PubMed Scopus (366) Google Scholar). The pathway appears physiologically plausible for several reasons. First, a number of studies demonstrated that enzymatically active MPO accumulates in subendothelial space, leading to MPO-dependent nitration and chlorination of targets (18Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1134) Google Scholar, 19Hazen S.L. Heinecke J.W. J. Clin. Invest. 1997; 99: 2075-2081Crossref PubMed Scopus (755) Google Scholar, 20Hazell L.J. Arnold L. Flowers D. Waeg G. Malle E. Stocker R. J. Clin. Invest. 1996; 97: 1535-1544Crossref PubMed Scopus (541) Google Scholar, 21Baldus S. Eiserich J.P. Mani A. Castro L. Figueroa M. Chumley P., Ma, W. Tousson A. White C.R. Bullard D.C. Brennan M.L. Lusis A.J. Moore K.P. Freeman B.A. J. Clin. Invest. 2001; 108: 1759-1770Crossref PubMed Scopus (298) Google Scholar, 22Brennan M.-L., Wu, W., Fu, X. Shen Z. Song W. Frost H. Vadseth C. Narine L. Lenkiewicz E. Borchers M.T. Lusis A.L. Lee J.J. Lee N.A. Abu-Soud H.M. Ischiropoulos H. Hazen S.L. J. Biol. Chem. 2002; 277: 17415-17427Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Second, recent genetics and clinical studies further suggest a role for MPO in development of atherosclerosis in human subjects. A cross-sectional analysis of nearly 100 individuals with MPO deficiency showed that MPO-deficient subjects have a reduced rate of cardiovascular disease (23Kutter D. Devaquet P. Vanderstocken G. Paulus J.M. Marchal V. Gothot A. Acta Haematol. (Basel). 2000; 104: 10-15Crossref PubMed Scopus (218) Google Scholar). Similarly, decreased prevalence of atherosclerosis was recently reported for subjects containing a single nucleotide polymorphism in the promoter region of the MPO gene that results in decreased expression in reporter constructs in vitro (24Nikpoor B. Turecki G. Fournier C. Theroux P. Rouleau G.A. Am. Heart J. 2001; 142: 336-339Crossref PubMed Scopus (197) Google Scholar). Third, we have demonstrated that MPO-generated reactive nitrogen species convert LDL into a form recognized by CD36 at pathophysiological concentrations of nitrite and in the presence of serum constituents, in contrast to LDL oxidation by free transition metal ions (e.g. Cu2+) (10Podrez E.A. Febbraio M. Sheibani N. Schmitt D. Silverstein R.L. Hajjar D.P. Cohen P.A. Frazier W.A. Hoff H.F. Hazen S.L. J. Clin. Invest. 2000; 105: 1095-1108Crossref PubMed Scopus (366) Google Scholar). Moreover, studies examining peroxidation of endogenous plasma lipids by activated leukocytes isolated from normal and MPO-deficient subjects strongly supports a role for the MPO-H2O2system of human leukocytes as a physiological mechanism for initiating lipid peroxidation in vivo (25Zhang R. Shen Z. Nauseef W.M. Hazen S.L. Blood. 2002; 85: 950-958Google Scholar). Finally, a recent clinical study (26Zhang R. Brennan M.L., Fu, X. Aviles R.J. Pearce G.L. Penn M.S. Topol E.J. Sprecher D.L. Hazen S.L. J. Am. Med. Assoc. 2001; 286: 2136-2142Crossref PubMed Scopus (771) Google Scholar) identified MPO levels in blood and leukocytes as strong independent predictors of coronary artery disease in angiographically defined subjects. Although the scavenger receptor functions of CD36 are well documented, the exact molecular structure(s) of the ligand(s) recognized by CD36 remain unknown. Oxidized lipids were first suggested to participate in recognition of oxLDL by mouse peritoneal macrophages (MPM) based on studies using liposomes generated from lipid extracts of LDL that was extensively oxidized by Cu2+ (Cu2+-oxLDL) (27Terpstra V. Bird D.A. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1806-1811Crossref PubMed Scopus (76) Google Scholar). A role for CD36 as the receptor responsible for recognition of oxidized lipids extracted from Cu2+-oxLDL was subsequently shown (10Podrez E.A. Febbraio M. Sheibani N. Schmitt D. Silverstein R.L. Hajjar D.P. Cohen P.A. Frazier W.A. Hoff H.F. Hazen S.L. J. Clin. Invest. 2000; 105: 1095-1108Crossref PubMed Scopus (366) Google Scholar, 11Boullier A. Gillotte K.L. Horkko S. Green S.R. Friedman P. Dennis E.A. Witztum J.L. Steinberg D. Quehenberger O. J. Biol. Chem. 2000; 275: 9163-9169Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Oxidized phospholipids covalently linked to apolipoprotein B-100 (apoB) in extensively oxidized LDL (e.g.Cu2+-oxLDL) have also been suggested to serve as ligands for CD36 based upon indirect competition studies using a monoclonal antibody to oxidized PAPC-protein adducts and either reconstituted apoB from Cu2+-oxLDL or adducts of BSA with the aldehydic phospholipid 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (OV-PC), an oxidation product of PAPC (11Boullier A. Gillotte K.L. Horkko S. Green S.R. Friedman P. Dennis E.A. Witztum J.L. Steinberg D. Quehenberger O. J. Biol. Chem. 2000; 275: 9163-9169Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 28Horkko S. Bird D.A. Miller E. Itabe H. Leitinger N. Subbanagounder G. Berliner J.A. Friedman P. Dennis E.A. Curtiss L.K. Palinski W. Witztum J.L. J. Clin. Invest. 1999; 103: 117-128Crossref PubMed Scopus (474) Google Scholar, 29Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). We have shown that the lipid portion of LDL and PAPC vesicles that are mildly oxidized by the MPO-H2O2-NO 2−system serve as ligands for CD36 using stable transfected cells and MPM from wild type versus CD36 knockout mice (10Podrez E.A. Febbraio M. Sheibani N. Schmitt D. Silverstein R.L. Hajjar D.P. Cohen P.A. Frazier W.A. Hoff H.F. Hazen S.L. J. Clin. Invest. 2000; 105: 1095-1108Crossref PubMed Scopus (366) Google Scholar). Conversion of LDL into a ligand for CD36 is a very early event during LDL oxidation in this system, occurring before substantial modification of apoB, as monitored by loss of free lysine residues and alteration in relative electrophoretic mobility (10Podrez E.A. Febbraio M. Sheibani N. Schmitt D. Silverstein R.L. Hajjar D.P. Cohen P.A. Frazier W.A. Hoff H.F. Hazen S.L. J. Clin. Invest. 2000; 105: 1095-1108Crossref PubMed Scopus (366) Google Scholar). Although studies thus far clearly support the notion that oxidized phospholipids play a major role in the binding of oxLDL forms (including NO2-LDL) to CD36, particularly early in the oxidation process, the precise nature of the lipid ligand or ligands within the lipid phase of oxLDL species have not yet been identified. We now report the first systematic study aimed at directly identifying the structures of specific oxidized phospholipids that serve as high affinity ligands for the scavenger receptor CD36. Tissue culture media and additives were purchased from Invitrogen. Na125I and [14C]oleate were supplied by ICN Pharmaceutical, Inc. (Costa Mesa, CA). [14C]Cholesterol, [14C]PAPC, and [3H]DPPC were from American Radiolabeled Chemicals (St. Louis, MO), and [3H]cholesteryl linoleate was from PerkinElmer Life Sciences. C57BL/6 mice (16–20 weeks of age) were purchased from the Trudeau Institute (Saranac Lake, NY). 1-Hexadecanoyl-2-eicosatetra-5′,8′,11′,14′-enoyl-sn-glycero-3-phosphocholine (PAPC), 1-hexadecanoyl-2-octadec-9′-enoyl-sn-glycero-3-phosphocholine (POPC), and 1-hexadecanoyl-2- octadec-9′,12′-dienoyl-sn-glycero-3-phosphocholine (PLPC), phosphatidylserine (PS), and DPPC were purchased from Avanti Polar Lipids (Alabaster, AL). Anti-CD36 monoclonal antibody, FA6-152, was purchased from Immunotech (Westbrook, ME). All other reagents were obtained from Sigma unless otherwise specified. Human myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and LDL were isolated and quantified as described (17Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (424) Google Scholar). All buffers were treated with Chelex-100 resin (Bio-Rad) and supplemented with diethylenetriaminepentaacetic acid (DTPA) to remove trace levels of transition metal ions that might catalyze LDL oxidation during incubations. LDL was labeled with Na125I to a specific activity between 100 and 250 dpm/ng protein, as described (30Hoppe G. O'Neil J. Hoff H.F. J. Clin. Invest. 1994; 94: 1506-1512Crossref PubMed Scopus (100) Google Scholar). Extraction of cellular lipids and thin layer chromatography separation of radiolabeled cholesterol esters were performed as described (17Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (424) Google Scholar). Incorporation of [14C]oleate into cholesteryl esters by cells following incubation with the indicated lipoproteins (50 μg/ml) was determined as described (17Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (424) Google Scholar). Total syntheses of the γ-hydroxy-α,β-unsaturated aldehydic phospholipids, the 9-hydroxy-12-oxo-10-dodecenoic acid, and 5-hydroxy-8-oxo-6-octenoic acid esters of 2-lyso-PC (HODA-PC and HOOA-PC, respectively) (31Deng Y.H. Salomon R.G. J. Org. Chem. 1998; 63: 7789-7794Crossref Scopus (13) Google Scholar), the γ-keto-α,β-unsaturated aldehydic phospholipids, the 9-keto-12-oxo-10-dodecenoic acid, and 5-keto-8-oxo-6-octenoic acid esters of 2-lyso-PC (KODA-PC and KOOA-PC, respectively), as well as the analogous carboxylic phospholipids, the 9-hydroxy-10-dodecenedioic acid, and 5-hydroxy-8-oxo-6-octenedioic acid esters of 2-lyso-PC (HDdiA-PC and HOdiA-PC, respectively), and the 9-keto-10-dodecendioic acid and 5-keto-6-octendioic acid esters of 2-lyso-PC (KDdiA-PC and KOdiA-PC, respectively) were performed as described elsewhere (32Sun M. Deng Y. Batyreva E. Sha W. Salomon R.G. J. Org. Chem. 2002; 67: 3575-3584Crossref PubMed Scopus (67) Google Scholar). Saturated aldehydic phospholipids, the 5-oxovaleric acid and 9-oxononanoic acid esters of 2-lyso-PC (OV-PC and ON-PC, respectively), were synthesized from stable phospholipid precursors containing a dimethylacetal-protected carbonyl that was deprotected in the presence of catalytic amounts of acidic ion exchange resin Amberlyst-15 (29Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). Their carboxylic analogs, the glutaric and nonanedioic monoesters of 2-lyso-PC (G-PC and ND-PC, respectively), were prepared by coupling 2-lyso-PC with the corresponding acid anhydride (29Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 32Sun M. Deng Y. Batyreva E. Sha W. Salomon R.G. J. Org. Chem. 2002; 67: 3575-3584Crossref PubMed Scopus (67) Google Scholar). Purification was achieved by flash silica column chromatography or HPLC, as described elsewhere (29Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 32Sun M. Deng Y. Batyreva E. Sha W. Salomon R.G. J. Org. Chem. 2002; 67: 3575-3584Crossref PubMed Scopus (67) Google Scholar). The structures of all synthetic lipids were confirmed by multinuclear NMR and high resolution mass spectrometry prior to use (29Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 31Deng Y.H. Salomon R.G. J. Org. Chem. 1998; 63: 7789-7794Crossref Scopus (13) Google Scholar, 32Sun M. Deng Y. Batyreva E. Sha W. Salomon R.G. J. Org. Chem. 2002; 67: 3575-3584Crossref PubMed Scopus (67) Google Scholar). Synthetic lipids were routinely analyzed by HPLC with on-line electrospray ionization tandem mass spectrometry. If lipids were found to be less than 98% pure, they were re-isolated prior to use. Stock solutions (2 mg/ml) of small unilamellar vesicles composed of PLPC, POPC, or PAPC with varying mol % of specific oxidized phospholipids were prepared in argon-sparged sodium phosphate buffer by extrusion (10 times) through a 0.1-μm polycarbonate filter using an Avanti Mini-Extruder Set (Avanti Polar Lipids, Inc., Alabaster, AL) at 37 °C. For direct binding experiments, [3H]DPPC (25 μCi/mg of phospholipids) or 1 mol % of the fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate was added to phospholipids. CD36 ligands were isolated from PAPC or PLPC vesicles (0.2 mg lipid/ml) following incubation with MPO (30 nm), an H2O2-generating system (constant flux of 0.80 μm/min) composed of glucose (100 μm) and glucose oxidase (100 ng/ml), and NaNO2 (0.5 mm) at 37 °C for 20 h. Reactions were stopped by the addition of BHT (50 μm) and catalase (300 nm) and stored under argon atmosphere at −80 °C. LDL modified by MPO-generated nitrating intermediates (NO2-LDL) was formed by incubating LDL (0.2 mg of protein/ml) at 37 °C in 50 mm sodium phosphate, pH 7.0, 100 μm DTPA, 30 nm MPO, 100 μg/ml glucose, 20 ng/ml glucose oxidase, and 0.5 mmNaNO2 for 8 h. Oxidation reactions were terminated by addition of 40 μm BHT and 300 nm catalase to the reaction mixture. LDL acetylation was performed as described earlier (17Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (424) Google Scholar). Oxidation of LDL (0.2 mg protein/ml) by copper was performed by dialysis versus 5 μmCuSO4 in PBS for 24 h at 37 °C. Oxidation was terminated by addition of BHT (40 μm) and DTPA (100 μm) and dialysis against PBS containing DTPA (100 μm). Oxidation of LDL by ceruloplasmin-bound copper was performed as described (33Ehrenwald E. Fox P.L. J. Clin. Invest. 1996; 97: 884-890Crossref PubMed Scopus (71) Google Scholar). Thioglycollate-elicited MPMs from wild type (C57BL/6) were isolated and cultured as described (17Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (424) Google Scholar). Human foreskin fibroblasts were cultured as described previously (17Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (424) Google Scholar). CHO cells expressing mouse scavenger receptor class A, type I (CHO-mSR-AI), and control vector-transfected parental LDL receptor-negative CHO cells were a generous gift from Dr. M. Krieger (Massachusetts Institute of Technology, Boston) (34Ashkenas J. Penman M. Vasile E. Acton S. Freeman M. Krieger M. J. Lipid Res. 1993; 34: 983-1000Abstract Full Text PDF PubMed Google Scholar). Experiments with CHO-mSR-AI were performed on confluent cell monolayers in Ham's F-12 medium containing 3% lipoprotein-deficient fetal calf serum, BHT (20 μm), DTPA (100 μm), and catalase (300 nm). 293 cells (embryonic kidney epithelial cells transformed with adenovirus) were obtained from the ATCC (Manassas, VA) and maintained in DMEM with 5% FCS. CD36 expressing 293 cells were a generous gift from Dr. W. Frazier (Washington University, St. Louis, MO) (35Sheibani N. Frazier W.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6788-6792Crossref PubMed Scopus (150) Google Scholar). 293 transfected cells were grown in the presence of G418 (500 μg/ml), and clones were isolated, and expression of CD36 was confirmed by FACS analysis using the monoclonal antibody FA6-152. Experiments with 293 cells and CHO cells were performed on confluent cell monolayers in the appropriate culturing media containing 200 μg/ml LDL, BHT (20 μm), DTPA (100 μm), and catalase (300 nm). Lipids were maintained under inert atmosphere (argon or nitrogen) at all times. Lipids from either oxidized PAPC or PLPC vesicles, or from NO2-LDL, were extracted three times sequentially by the method of Bligh and Dyer (36Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43112) Google Scholar) immediately after adding an equal volume of saturated NaCl solution (to enhance lipid extraction). The combined chloroform extracts were evaporated under nitrogen, and lipids were then resuspended in methanol (at ∼200 μg/0.1 ml), filtered through an Acrodisc CR PTFE filter, and applied on a reverse-phase column (Luna C18, 250 × 10 mm, 5 μm, Phenomenex, Torrance, CA). Lipids were resolved at a flow rate of 3 ml/min using a ternary (acetonitrile/methanol/H2O) gradient (Gradient I, see below) generated by a Waters 600 E Multisolvent delivery system HPLC (Waters, Milford, MA) and monitored using an evaporative light scattering detector (Sedex 55, Sedere, Alfortville, France). Time(min)012203070Acetonitrile(%)6570707070MeOH(%)2020233030H2O(%)1510700 GRADIENTI The ability of lipids within collected fractions to block125I-NO2-LDL binding to CD36 was then examined as follows. Lipids were rapidly extracted into chloroform, dried under N2, resuspended in 20 μl of PBS with 10% ethanol, and further diluted (final ethanol concentration ≤0.5%) in DMEM containing 125I-NO2-LDL (5 μg/ml), 5% FCS, 200 μg/ml LDL, BHT (20 μm), DTPA (100 μm), and catalase (300 nm) (10Podrez E.A. Febbraio M. Sheibani N. Schmitt D. Silverstein R.L. Hajjar D.P. Cohen P.A. Frazier W.A. Hoff H.F. Hazen S.L. J. Clin. Invest. 2000; 105: 1095-1108Crossref PubMed Scopus (366) Google Scholar). The resulting mixture was incubated with CD36-transfected 293 cells for 3 h at 4 °C, and then unbound125I-NO2-LDL was removed by washing with ice-cold PBS. The amount of bound 125I-NO2-LDL was then determined. CD36 independent binding of125I-NO2-LDL was assessed in vector-transfected 293 cells and subtracted from binding to CD36-transfected 293 cells as a background. The binding to vector-transfected cells was typically less than 10–20% of that to CD36 transfected cells. Each measurement was performed in triplicate, and the percent of inhibition was calculated as follows: 10" @default.
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• W2168897708 title "Identification of a Novel Family of Oxidized Phospholipids That Serve as Ligands for the Macrophage Scavenger Receptor CD36" @default.
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