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Originally published In Press as doi:10.1074/jbc.M604039200 on August 14, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31298-31308, October 20, 2006
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Phospholipid Hydroxyalkenals, a Subset of Recently Discovered Endogenous CD36 Ligands, Spontaneously Generate Novel Furan-containing Phospholipids Lacking CD36 Binding Activity in Vivo*

Shengqiang Gao{ddagger}§1, Renliang Zhang{ddagger}§, Michael E. Greenberg{ddagger}§, Mingjiang Sun{ddagger}§, Xi Chen, Bruce S. Levison{ddagger}§, Robert G. Salomon, and Stanley L. Hazen{ddagger}§||2

From the Departments of {ddagger}Cell Biology and ||Cardiovascular Medicine and the §Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, April 27, 2006 , and in revised form, July 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently identified a novel family of oxidized choline glycerophospholipid (oxPC) molecular species enriched in atheroma that serve as endogenous ligands for the scavenger receptor CD36 (oxPCCD36), facilitating macrophage cholesterol accumulation and foam cell formation (Podrez, E. A., Poliakov, E., Shen, Z., et al. (2002) J. Biol. Chem. 277, 38517–38523). A high affinity CD36 recognition motif was defined within oxPCCD36, an oxidatively truncated sn-2 acyl group with a terminal {gamma}-hydroxy (or oxo)-{alpha},beta-unsaturated carbonyl. The fate of these species once formed in vivo is unknown. Here we show that a subset of oxPCCD36, a phosphatidylcholine molecular species possessing sn-2 esterified fatty acyl hydroxyalkenal groups, can undergo a slow intramolecular cyclization and dehydration reaction to form novel oxPC species possessing a sn-2 acyl group that incorporates a terminal furyl moiety (oxPC-furan). Using high performance liquid chromatography with on-line tandem mass spectrometry in combination with unambiguous organic synthesis, we confirm that oxPC-furans, ultimately derived from phospholipids with sn-2 esterified docosahexaenoic, arachidonic, or linoleic acids, are formed during exposure of model membranes and isolated lipoproteins to physiological oxidant systems. In vivo generation of oxPC-furans at sites of enhanced oxidant stress is also demonstrated, such as within brain tissues following cerebral ischemia. Cell binding studies reveal that in contrast to their oxPCCD36 precursors, oxPC-furans lack CD36 binding activity. Taken together, the present studies identify oxPC-furans as a novel family of oxidized phospholipids that are formed in vivo from phospholipid hydroxyalkenals but that lack CD36 binding activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophage recognition of senescent or apoptotic cells and modified lipoproteins represents a critical housekeeping function of the innate immune system. Facilitated by scavenger receptors that function through pattern recognition of common structural motifs, scavenger receptors like CD36 thus play an important role in the engulfment of biological debris, limiting inflammatory responses through coordinated phagocytosis (1, 2). CD36, a prototypic member of the class B scavenger receptor superfamily, is a glycosylated integral membrane protein (3, 4). Expressed on diverse cell types including macrophages, dendritic cells, platelets, adipocytes, microvascular endothelial cells, and specialized epithelial cells, CD36 functions in vivo in the phagocytosis of senescent or apoptotic cells, as well as in fatty acid transport and cell-matrix interactions (3–7). Recent studies have suggested CD36 may serve as a participant in the atherosclerotic process because of its ability to recognize oxidized forms of low density lipoprotein (LDL)3 (7–11). CD36 interactions with oxidized LDL mediate lipid accumulation and macrophage foam cell formation in vitro and in vivo (7, 10, 12, 13), and most animal models employing genetically susceptible atherosclerosis-prone mice demonstrate reductions in atherosclerotic plaque formation when crossed with CD36 null mice (8).

Using HPLC tandem mass spectrometry (LC/MS/MS) and unambiguous organic synthesis guided by bioassay, we recently identified a novel family of structurally specific oxidized phosphatidylcholine molecular species (oxPCs) derived from 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PL-PC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (PA-PC), and 1-palmitoyl-2-docosahexanoyl-sn-glycero-3-phosphatidylcholine (DHA-PC), that serve as high affinity ligands for CD36 (oxPCCD36) (14–16). In addition to being enriched within plaque-laden aortic tissues (14), oxPCCD36 have been shown to: (i) be formed during LDL oxidation by multiple distinct pathways (15); (ii) function as a phagocytic "eat me" signal, promoting CD36-specific phagocytosis when incorporated into cell membranes or lipoproteins at only a few molecules per particle (14); and (iii) facilitate cholesterol accumulation and macrophage foam cell formation under physiological conditions (14, 15). A common structural motif for oxPCCD36 species was identified through extensive structure-function studies, an oxidatively truncated sn-2 acyl group with a terminal {gamma}-hydroxy(or oxo)-{alpha},beta-unsaturated carbonyl (see Fig. 1A) (14, 15).

Although structural determinants of macrophage CD36 recognition of oxPCCD36 species have been studied along with pathways involved in their formation, little research effort has focused on the metabolic fates of oxPCCD36 species. One subset of oxPCCD36, those possessing hydroxyalkenal moieties, are of interest because of their inherent potential chemical reactivity. Like other hydroxyalkenals, phospholipid-esterified hydroxyalkenals are anticipated to react with nucleophilic groups, forming Schiff base and Michael adducts on proteins, amino lipids, and other biological targets (17, 18).

During the conduct of in vitro LC/MS/MS studies, we noted that vesicles with phospholipid hydroxyalkenals appeared unstable even in the absence of nucleophilic species and under inert atmosphere, slowly decomposing into non-CD36-binding phospholipids. Species structurally similar to oxPCCD36 phospholipid hydroxyalkenals (HODA-PC, HOOA-PC, and HOHA-PC; see Fig. 1 for structures and nomenclature) but representing potential dehydration products (m/z – 18) were observed, providing potential insight into a novel decomposition pathway for this subset of oxPCCD36 under physiologically relevant conditions. We now report that a subset of oxPCCD36, phospholipid hydroxyalkenals, when not consumed through irreversible covalent adduct formation with nucleophilic targets, may slowly form a novel and stable family of oxPC species possessing a sn-2 acyl group that incorporates a terminal furyl moiety (oxPC-furan). Studies confirm clear precursor-product relationships between phospholipid hydroxyalkenals and oxPC-furans, as well as their formation both in model systems and in vivo. Finally, cell binding studies demonstrate that in contrast to their oxPCCD36 precursors, oxPC-furans do not bind with CD36. Thus, conversion of phospholipid hydroxyalkenals into oxPC-furans represent one potential mechanism for turning off the phagocytic eat me signal produced with in vivo generation of this subset of oxPCCD36.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
1,2-Ditridecanoyl-sn-glycero-3-phosphatidylcholine (DT-PC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine (lyso-PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (PO-PC), PL-PC, PA-PC, and DHA-PC were purchased from Avanti%20Polar%20Lipids">Avanti Polar Lipids Inc. (Alabaster, AL). 1,1'-Dioctadecyl-3,3,3',3'-tetramethlyindocarbocyanine perchlorate (Di-I) was obtained from Molecular Probes (Sunnyvale, CA). Antibody to mouse CD36 was bought from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were purchased from either Fisher or Sigma-Aldrich unless otherwise specified. LDL was prepared from human plasma and oxidized by exposure to leukocyte myeloperoxidase in the presence of nitrite as previously described (15).

Methods
Synthesis of Fatty Acid Furans and Oxidized Phospholipids—Synthetic oxPCCD36 phospholipid hydroxyalkenals (HODA-PC, HOHA-PC, and HOOA-PC) were prepared as previously described (14, 15, 19, 20). Total synthesis of 1-palmitoyl-2-(8-(2-furyl)octanoyl)-sn-glycero-3-phosphatidylcholine (oxPC-furan(7)), 1-palmitoyl-2-(4-(2-furyl) butanoyl-sn-glycero-3-phosphatidylcholine oxPC-furan(3), and 1-palmitoyl-2-(3-(2-furyl)propanoyl-sn-glycero-3-phosphatidylcholine (oxPC-furan(2)) were performed using their corresponding synthetic fatty acid furans and lysophosphatidyl choline as described elsewhere (19, 21). Taking oxPC-furan(2) as an example, a mixture of 3-(2-furyl)propionic acid and 1-palmitoyl-2-lyso-sn-glycero-3-phosphatidylcholine, which were dried on a vacuum pump (0.1 mm Hg) equipped with a dry ice acetone trap for 10 h at room temperature, was dissolved in dry CHCl3 (5 ml, shaken with P2O5 for 0.5 h and distilled). Dicyclohexylcarbodiimide (240 mg, 1.2 mmol) and N,N-dimethylaminopyridine (24 mg, 0.2 mmol) were added. The mixture was stirred for 48 h under nitrogen. The mixture was then concentrated, and the residue was purified by flash chromatography on silica with CHCl3/MeOH/H2O (16/9/1) to produce the furyl phospholipids. Chemical structures of all synthetic lipids were confirmed by multinuclear NMR and high resolution mass spectrometry (15, 19). If any lipid was found to be less than 95% pure, it was reisolated by HPLC prior to use. For HPLC purifications, a reverse phase C18 semipreparative column (250 x 10 mm, 5 µm; LUNA, Phenomenex, Rancho Palos Verdes, CA) was used, and an isocratic mobile phase composed of methanol and H2O (85/15, v/v), each incorporating 0.2% (v/v) formic acid, was employed. All of the lipids and procedures were performed under inert (N2 or Ar) atmosphere, and lipids were always kept in amber or foil-wrapped vials to protect from light. Concentrations of synthesized oxidized phospholipids were determined by microphosphorus assay (22).

Small Unilamellar Vesicles (SUV) Preparation and Oxidation—SUV were prepared as described elsewhere (14, 15). Briefly, 10 mol % of specific native or oxidized phospholipids was mixed in freshly distilled chloroform with PO-PC as lipid carrier and dried down under nitrogen gas flow. SUV were prepared in argon-sparged sodium phosphate buffer (50 mM, pH 7.0, supplemented with 100 µM of the metal chelator diethylenetriamine pentaacetic acid, by extrusion (11 passes) through a 0.1-µm polycarbonate filter using an Avanti Mini-Extruder Set (Avanti%20Polar%20Lipids">Avanti Polar Lipids, Inc., Albaster, AL) protected from light and maintained at 37 °C under argon atmosphere. For two-color flow cytometry binding assay, 1 mol % of the fluorescent dye Di-I was dried down together with the mixture of phospholipids before extrusion. For some studies, oxPC species were quantified by LC/MS/MS following exposure of either LDL or SUV (10 mol percent DHA-PC, PA-PC, or PL-PC within PO-PC carrier; 0.2 mg of total lipids/ml) to an oxidation system comprised of myeloperoxidase (60 nM), an H2O2 generating system composed of glucose (100 µM) and glucose oxidase (100 ng/ml), and NaNO2 (0.5 mM) at 37 °Cas described (15). The reactions were stopped by the addition of butylated hydroxytoluene (40 µM) and catalase (300 nM) and stored under argon atmosphere at –80 °C until cell binding studies and parallel mass spectrometric assays were performed.

Thioglycollate Elicited Mouse Peritoneal Macrophages—4 days after injection of 1 ml of 4% thioglycollate into mouse, peritoneal lavage containing macrophages was harvested. The cells were then pelleted by centrifuging at 1000 rpm for 5 min and taken up in RPMI medium to perform CD36 binding assays.

CD36 Binding Assay—CD36 cell binding assays used two-color flow cytometry, permitting simultaneous quantification of CD36 surface expression and CD36 specific vesicle binding, as recently described.4 Briefly, the open reading frame encoding wild type human CD36 protein was subcloned into pCGCG (24) bicistronic expression vector containing green fluorescent protein (GFP) under translational control of a picornavirus encephalomyocarditis virus internal ribosome entry site. This plasmid is referred to as CD36,GFP (bicistronic). K562 cells were transfected with 20 µg of DNA in medium composed of 150 mM NaCl supplemented with 10 mM HEPES, pH 7.4, using a Bio-Rad electroporator at 950 microfarads and 230 V. Time constants were generally in the range of 44–48 ms. The GFP only transfected cells (control), CD36,GFP (bicistronic) cells, or mouse peritoneal macrophages (2 x 105 cells in 200 µl of medium) were incubated with 50 µl 500 µg/ml Di-I-labeled liposomes containing specific oxidized phosphatidylcholine (PC) at 4 °C for 1 h. The cells were then pelleted by centrifuging at 1000 rpm for 5 min, and unbound liposomes were removed by three-time washing with phosphate-buffered saline containing 0.1% bovine serum albumin and then resuspended in 400 µl of phosphate-buffered saline for immediate analysis. Flow cytometry studies were performed on a Becton Dickinson FacsScan instrument. The binding data were analyzed using Flow-jo software analyses.

Stroke Model of Cerebral Ischemia—All of the animal studies were performed using approved protocols from the Animal Research Committee of the Cleveland Clinic Foundation. C57BL/6J mice (18 to 20 g) were first anesthetized by isoflurane anesthesia (1.2% for induction and 0.8% for maintenance) in 70% N2O and 30% O2 with a face mask. Focal cerebral ischemia was produced by ligation of the left middle cerebral artery. Twenty-four hours later, the mice were sacrificed, infarcted region surgically excised, rinsed of free blood, and submerged in argon-sparged 50 mM sodium phosphate buffer (pH 7.4) supplemented with butylated hydroxytoluene (100 µM) and diethylenetriaminepentaacetic acid (2 mM, pH 7.4). Control tissues were similarly harvested from sham-operated mice. The specimens were immediately snap frozen in liquid nitrogen in cryovials overlaid with argon and stored at –80 °C until analysis. For analysis, frozen tissue and antioxidant mixture together were pulverized into a fine powder in a high grade stainless steel mortar and pestle under liquid nitrogen. Pulverized tissue was transferred to a threaded glass test tube with a polytetrafluoroethylene (PTFE)-lined cap, and then lipids were extracted under argon atmosphere by the method of Bligh and Dyer (25) as described below.

Identification and Quantification of Phospholipids by Mass Spectrometry—After the addition of a known amount of DT-PC as internal standard, the lipids were extracted three sequential times by the method of Bligh and Dyer (25). The combined chloroform extracts were then dried under a stream of nitrogen, resuspended in 200 µl of 85% methanol/water, and injected onto an HPLC interfaced with the mass spectrometer for quantification. All of the procedures were performed in Teflon-lined amber glass vials under inert atmosphere to prevent any significant intrapreparative oxidation from occurring and artificially forming oxPC species. In control studies, spiking specimens with a large molar excesses of HPLC-purified parent phospholipids (PL-PC, PA-PC, and DHA-PC) failed to significantly increase absolute levels of the oxPC molecular species quantified.

Individual phospholipids were quantified by LC/MS/MS by methods similar to those recently described for oxPCCD36 (14–16). Briefly, reconstituted lipid extracts (20 µl) were injected onto a reverse phase C18 HPLC column (2 x 150 mm, 5 µm, ODS; Phenomenex, Rancho Palos Verdes, CA) at a flow rate of 0.2 ml/min generated by a Waters Alliance 2690 HPLC (Waters, Wilmington, DE). Phospholipids were resolved using a ternary gradient system comprised of mobile phase A (water containing 0.2% formic acid), mobile phase B (methanol containing 0.2% formic acid), and mobile phase C (isopropanol). The column was equilibrated with 85% mobile phase B/mobile phase A mixture and held at this composition for 6 min after the injection. A linear gradient was then run from 85 to 88% mobile phase B (versus A) over 12 min, and then in 2 min, the mobile phase B was linearly changed to 100%. After holding solvent composition at 100% mobile phase B for 11 min, the mobile phase was linearly (over 1 min) changed to 100% mobile phase C and held for 6 min (to clean the column). The column was then recycled to the initial mobile phase composition (85% mobile phase B, 15% mobile phase A) over 3 min, and the mixture was held for at least an additional 7 min prior to the next injection.

HPLC column effluent was introduced into a Quattro Ultima triple quadrupole mass spectrometer (Micromass, Manchester, UK) after a 4-min delay, diverting the solvent flow-through. The mass spectrometer was configured with capillary voltage at 3.0 kV, cone voltage at 40 V, collision energy at 20 V, source temperature at 120 °C, and a desolvation temperature at 250 °C. The flow rate for the nitrogen in the cone gas and desolvation gas was 80 and 600 liters/h, respectively. Collision-induced dissociation was obtained using argon gas. Mass spectrometric analyses were performed on-line using electrospray ionization tandem mass spectrometry (ESI/MS/MS) in the positive ion mode with multiple reaction monitoring mode. The multiple reaction monitoring transitions used to identify and quantify individual PC molecular species were the m/z for the molecular cation [MH]+ and the daughter ion m/z 184 (the phosphocholine group). Calibration curves were constructed with a fixed amount of DT-PC internal standard and varying mol % of each authentic synthetic phospholipid prior to extraction and LC/MS/MS analysis.


Figure 1
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  		FIGURE 1.
oxPCCD36 and formation of oxPC-furan species. A, structures of phospholipids generated from oxidation of DHA-PC, PA-PC, and PL-PC that possess high affinity CD36 binding activity (oxPCCD36). The compounds generated from different precursors are similar except for the number of methylene groups on the truncated oxidized fatty acid esterified to the sn-2 positions of lyso-PC: n = 2, DHA-PC; n = 3, PA-PC; and n = 7, PL-PC. B, suspected cyclization and dehydration steps through which oxPC-furan species are formed from their respective oxPCCD36 precursor.

 
In some studies, lipid extracts from infarcted brain tissues were injected onto reverse phase HPLC, and eluent was split post column with one portion directed to the mass detector for MS/MS analysis as above, and the other portion diverted into a fraction collector. Fractions corresponding to those where oxPC-furans eluted were extracted by the method of Bligh and Dyer (25) and then incubated in argon-sparged and sealed reaction vials with Naja naja naja phospholipase A2 in phosphate-buffered saline supplemented with 10 mM CaCl2. The reaction mixtures were then re-extracted by the Bligh and Dyer method in the presence of 1 mM acetic acid to protonate fatty acids and improve extraction efficiency, divided into three parts, and dried under a nitrogen stream. One part was resuspended in 85% methanol and injected onto a reverse phase C18 HPLC column for LC/MS/MS analysis for detection of nonderivatized fatty acid furans. The other two parts were immediately derivatized to either their corresponding pentafluorobenzyl esters or their tert-butyldimethylsilyl (TBDMS) esters and then analyzed by reverse phase HPLC with on-line tandem mass spectrometry analysis using a chromatographic system similar to that described above. For standards, synthetic fatty acid furan(2), furan(3), and furan(7) were prepared, and their structures were confirmed by both NMR and mass spectrometry as described elsewhere (19, 21). Each synthetic fatty acid furan standard was individually analyzed in parallel to the brain tissue-derived lipid extracts in native (nonderivatized) form, as well as in their corresponding TBDMS or pentafluorobenzyl ester forms.

Statistics—The data represent the means ± S.D. of the indicated number of samples. The statistical analyses were made using a paired Student's t test. For all of these hypotheses, the significance level was 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Proposed Mechanism of Formation of oxPC-furans from Select oxPCCD36 and Demonstration of Precursor-Product Relationship between Phospholipid Hydroxyalkenals and oxPC-furans—As mentioned, during the conduct of studies with oxPCCD36 possessing hydroxyalkenal groups (HODA-PC, HOOA-PC, and HOHA-PC), we noted that CD36 binding activity of SUV in the absence of nucleophiles and under argon atmosphere was slowly lost over time (days), along with the concurrent appearance of new phospholipids possessing –18 atomic mass units. In parallel, we noted that species possessing similar retention times and parent -> daughter ion transitions were seen in fresh lipid extracts from in vivo specimens (see below). Aldehydes can reversibly react with alcohols to form hemiacetals. We therefore hypothesized that phospholipid hydroxyalkenals might slowly undergo a trans-cis isomerization permitting formation of a cyclic hemiacetal intermediate, which could then readily form a stable oxPC-furan through loss of water (Fig. 1B). Before being able to test this hypothesis we needed to first generate the presumptive oxPC-furans and develop specific quantitative assays for their presence in specimens. Each of the anticipated oxPC-furans (Fig. 1B) was synthesized, and their chemical structures were unambiguously confirmed by NMR and high resolution mass spectrometry as described under "Methods." Analytical methods for the simultaneous quantification of each individual oxPC-furan were then developed using HPLC with on-line tandem mass spectrometry. Fig. 2 illustrates standard curves produced for quantifying each oxPC-furan species (A) and both the presumed precursor oxPCCD36 phospholipid hydroxyalkenals and parent phospholipids (B) utilizing a nonphysiological PC species, DT-PC, as internal standard.


Figure 2
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  		FIGURE 2.
LC/ESI/MS/MS calibration curves of oxPC-furans and their precursors. Calibration curves for quantitative analyses of oxPC-furans (A), their corresponding oxPCCD36 precursors, and their parent (unoxidized) PC species (B) were constructed by adding a constant amount of internal standard DT-PC into various amounts of the indicated authentic synthetic phospholipids prior to extraction and LC/MC/MC analysis. The data points are the means ± S.D. of three independent experiments.

 


Figure 3
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  		FIGURE 3.
Time course experiment demonstrating oxPCCD36 phospholipid hydroxyalkenals generate oxPC-furan species. Fixed amounts of the indicated synthetic oxPCCD36 (10 mol % HOHA-PC, HOOA-PC, or HODA-PC) were individually incorporated into model membranes composed of PO-PC (90 mol %) as SUV. SUV (50 µmol of total lipid) were then incubated in chelex-treated 50 mM sodium phosphate buffer, pH 7.0, at 37 °C. At the indicated times, the aliquots were removed, and the lipids were extracted and then analyzed by LC/MS/MS as described under "Methods." A shows data from multiple experiments: decomposition of HODA-PC was accompanied by concomitant generation of oxPC-furan(7); decomposition of HOOA-PC was accompanied by concomitant generation of oxPC-furan(3); and decomposition of HOHA-PC was accompanied by concomitant generation of oxPC-furan(2). Independent experiments analyzing the stability of PO-PC SUV containing either 10 mol % PL-PC, PA-PC, or DHA-PC demonstrated only nominal decay of these polyunsaturated fatty acid containing phospholipids under the conditions employed. B–D demonstrate the clear product-precursor relationship between the indicated individual oxPCCD36 and oxPC-furan. Dashed lines shown in D illustrate the product-precursor relationship between oxPC-furan(2) and HOHA-PC in the presence of 40 mg/ml bovine serum albumin. The results represent the means ± S.D. for three independent experiments.

 
We next tested the hypothesis that individual structurally defined phospholipid hydroxyalkenals (HODA-PC, HOOA-PC, and HOHA-PC) could give rise to the anticipated oxPC-furan under physiological conditions as schematically illustrated in Fig. 1B. The indicated phospholipid hydroxyalkenals were individually incorporated into model membranes comprised of lamellar phase-preferring phospholipid (SUV made of 90 mol % PO-PC and 10 mol % of the indicated oxPCCD36) within phosphate buffer at neutral pH supplemented with metal ion chelator and then incubated at 37 °C under argon atmosphere. At the indicated times, the aliquots were removed, and the content of individual PC molecular species was determined by LC/MS/MS (Fig. 3). Fig. 3A demonstrates the overall decay of HODA-PC, HOOA-PC, and HOHA-PC over time, and the concomitant respective formation of oxPC-furan(7), oxPC-furan(3), and oxPC-furan(2) over time. SUV instead comprised of PO-PC, and the indicated parent (nonoxidized) phospholipid (PL-PC, PA-PC, and DHA-PC) remained stable with little decomposition when incubated under similar conditions (Fig. 3A). Yields of formation of the oxPC-furans from the oxPCCD36 precursors ranged between ~21 and 50% (Figs. 3, B, C, and D, for HODA-PC, HOOA-PC, and HOHA-PC, respectively). Parallel experiments performed under mildly acidic (pH 6.0) conditions, which are thought to exist within human atheroma, resulted in increased overall yield (32–65%, depending upon the oxPC-furan; data not shown).

In additional studies we examined the impact of high protein concentrations on oxPC-furan generation by SUV containing oxPCCD36 hydroxylakenals. We hypothesized that because formation of Schiff bases and some Michael adducts are chemically reversible, yet the dehydration step between hemiacetal and furan was likely irreversible, the inclusion of protein in oxPC hydroxyalkenal reaction mixtures might not totally inhibit oxPC-furan production (i.e. a portion of protein tethered oxPC hydroxyalkenals should exist in equilibrium with free oxPC hydroxyalkenals, permitting ultimate formation of the oxPC-furans and pulling of the overall reaction to the right). The results of these studies are illustrated in Fig. 3D. Remarkably, even in the presence of physiological concentrations of albumin (40 mg/ml), oxPC-furans were formed at levels nearly the same as in the absence of albumin. Of note, although oxPC-furan production was only modestly blunted, the oxPC hydroxyalkenal precursors decayed at a markedly higher rate in the presence of albumin, consistent with their reversible covalent adduction to the protein, and continued availability for generation of the oxPC-furans. Collectively, these experiments directly demonstrated that oxPC-furan(7), oxPC-furan(3), and oxPC-furan(2) are immediate products of HODA-PC, HOOA-PC, and HOHA-PC, respectively, and that formation of these novel furyl-containing phospholipids occurs under physiological conditions.


Figure 4
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  		FIGURE 4.
Demonstration that oxPC-furans are formed during exposure of model membranes or isolated lipoproteins to physiological oxidation systems. SUV comprised of 90 mol % PO-PC and 10 mol % of either PL-PC, PA-PC or DHA-PC (A) or isolated human LDL (B) were incubated in chelex-treated 50 mM sodium phosphate buffer, pH 7.0, at 37 °C in the presence of myeloperoxidase, a H2O2-generating system, and NaNO2 as described under "Methods." At the indicated times, the aliquots were removed, and the lipids were extracted and then nonoxidized and oxPC quantified by LC/MS/MS. The top panels show the decline of parent (nonoxidized) PC versus time of incubation under the experimental conditions. The middle panels demonstrate concomitant formation phospholipid hydroxylakenal oxPCCD36 species. The bottom panels demonstrate concomitant generation of the corresponding oxPC-furans. The results represent the means ± S.D. for three independent experiments.

 
Demonstration that oxPC-furans Are Formed by Oxidation of Both Model Membranes and Native LDL Using Physiological Oxidation Systems—To test the hypothesis that oxPC-furans might form during oxidation of model membranes or lipoproteins, either SUV of defined lipid composition (Fig. 4A) or isolated human LDL (Fig. 4B) were exposed to the myeloperoxidase Formula system as described under "Methods." SUV were comprised of PO-PC carrier and 10 mol % of PL-PC, PA-PC, or DHA-PC. At the indicated times, aliquots of reaction mixtures were removed and stopped by the addition of antioxidant (butylated hydroxytoluene) and H2O2 scavenger (catalase), and the phospholipids were extracted and then quantified by LC/MS/MS as described under "Methods." As anticipated, the content of parent unoxidized PC species (PL-PC, PA-PC, and DHA-PC) in both SUV and LDL decreased over time in proportion to the length of exposure to the oxidation system (Fig. 4). In parallel, formation of the indicated phospholipid hydroxyalkenals and their derivative oxPC-furans was noted during oxidation of both the model membranes and LDL (Fig. 4).

oxPC-furan Species, in Contrast to Their oxPCCD36 Precursors, Have Poor CD36 Binding Activity—To examine the CD36 binding activity of oxPC-furans, a CD36 binding assay that permits simultaneous monitoring of CD36 surface expression and extent of SUV binding was used. The cells were transfected with a bicistronic CD36,GFP reporter plasmid containing an internal ribosome entry signal, permitting simultaneous expression of intact human CD36 and GFP. Use of this construct results in GFP expression in approximate constant molar ratio with intact native human CD36 within transfected cells, permitting cellular mean GFP fluorescence to be used as a semi-quantitative measure of CD36 (Fig. 5A). Demonstration that surface CD36 is functional in the transfected cells was achieved by showing that cells only bind appreciable levels of Di-I labeled oxidized LDL in the presence of CD36 (data not shown). Two-color flow cytometry analyses with the above system (GFP fluorescence monitored for measure proportional to cell CD36 expression, and Di-I fluorophore for labeled SUV) permitted simultaneous monitoring of CD36-specific increases in vesicle binding across a range of CD36 surface expression levels (Fig. 5A). Mean SUV binding values were obtained at different CD36 expression levels to generate binding curves of the phospholipid hydroxyalkenal oxPCCD36 (HODA-PC, HOOA-PC, and HOHA-PC) and their corresponding derived oxPC-furans (oxPC-furan(7), oxPC-furan(3), and oxPC-furan(2)) within PO-PC SUV (Fig. 5B). Data are also shown for individual oxPC binding at a single arbitrary selected CD36 expression level in bar graph format (Fig. 5C) using cells transfected in the presence versus absence (GFP alone) of CD36. Under all of the experimental conditions examined, the SUV containing the oxPC-furans failed to demonstrate any binding activity to CD36, whereas SUV containing oxPCCD36 (HODA-PC, HOOA-PC, and HOHA-PC) readily bound specifically to the scavenger receptor transfected cells.


Figure 5
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  		FIGURE 5.
oxPC-furans, in contrast to their oxPCCD36 precursors, do not bind to CD36. K562 cells transfected with plasmids containing intact human CD36 and GFP (bicistronic) were cultured overnight and then incubated with SUV (90 mol % oleic acid ester of 2-lyso-PC) containing 10 mol % of the indicated oxPCCD36 or oxPC-furan and tracer (<1 mol % of the lipophilic dye Di-I) at 4 °C for 60 min. After extensive washing to remove unbound SUV, CD36 expression and oxPC binding were analyzed simultaneously by two-color flow cytometry. A shows a representative flow cytometry dot plot. B, the mean SUV binding (Di-I fluorescence) is plotted against the relative CD36 expression levels as monitored by the mean GFP fluorescence intensity for the various SUV examined. C and D, data shown represent oxPC binding for the single relative CD36 expression level (rectangle in B) in K562 cells transfected with either intact human CD36 and GFP (bicistronic) or GFP only (controls). E, binding studies with mouse peritoneal macrophages and Di-I-labeled SUV comprised of PO-PC only, or PO-PC carrier (90 mol %) and 10 mol % of either oxPC-furan(2) or HOHA-PC. The data shown represent the means ± S.D. for three independent experiments.

 
In additional studies binding assays were performed using PO-PC vesicles containing either 10 mol % oxPC-furan(2), 10 mol % HOHA-PC, or PO-PC vesicles containing both 10 mol % oxPC-furan(2) and 10 mol % HOHA-PC. Vesicles containing both oxPC-furan and the oxPCCD36 precursor demonstrated similar CD36 binding activity as PO-PC vesicles containing just the oxPCCD36 precursor at similar surface mol % (Fig. 5D). Thus, because oxPC hydroxyalkenals are converted to oxPC-furans there is no evidence of competition with the remaining precursor, and CD36 binding goes down. Finally, because CD36 expression in transfected cells theoretically might not faithfully represent the binding activity of naturally occurring CD36, additional studies were performed using mouse peritoneal macrophages. SUV containing the oxPCCD36, but not their down-stream oxPC-furans, bound to the peritoneal macrophages (Fig. 5E; data for HOHA-PC and oxPC-furan(2) shown).

oxPC-furans Are Generated in Vivo—To demonstrate that formation of oxPC-furans occurs in vivo, studies were performed using an animal model of stroke, a pathophysiological process associated with marked increases in oxidant stress within a tissue rich in phospholipids containing polyunsaturated fatty acids. One day following ligation of a middle cerebral artery, control and infarcted brain tissues were examined for evidence of oxPC-furan generation in vivo. Briefly, brain hemispheres from mice were excised, rinsed of free blood, and individually submerged in argon-sparged antioxidant mixture containing butylated hydroxytoluene and the metal chelator diethylenetriaminepentaacetic acid and sealed in gas-tight vials overlaid with argon, and then the vials were snap frozen in liquid nitrogen. For analyses, frozen tissue/buffer was pulverized in mortar and pestle submerged within liquid nitrogen, and then the lipids were rapidly extracted from the fine tissue powder and examined by LC/MS/MS as described under "Methods." Fig. 6 illustrates typical LC/MS/MS chromatograms of tissue extracts. Each of the oxPC-furans anticipated as products of PL-PC, PA-PC, and DHA-PC oxidation were detected by their characteristic parent -> daughter ion transitions and retention times.


Figure 6
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  		FIGURE 6.
oxPC-furan species are present in mice cerebral ischemic tissues. One day following induction of stroke by chronic ligation of an internal carotid artery, infarcted and contralateral control hemispheres were harvested, and phospholipids were analyzed by LC/MS/MS as described under "Methods." Authentic synthetic oxPC-furans (shown in lower channels in A–C, respectively) were applied to confirm the endogenous oxPC-furan peak in ischemic brain tissues (multiple reaction monitoring tracings shown in the upper channels in A–C).

 
To further confirm the formation of oxPC-furans in vivo, additional studies were performed. Following injection of lipid extract from ischemic brain tissues onto the reverse phase HPLC, post-column eluent was split with one portion diverted into the mass detector and the remainder into a fraction collector. HPLC fractions corresponding to those where specific oxPC-furans eluted were then treated with phospholipase A2, dried under nitrogen, and then reanalyzed by LC/ESI/MS/MS to test for the presence of multiple parent -> daughter ion transitions at the appropriate retention time characteristic of the nonderivatized fatty acid furans. Each of the fatty acid furans was observed. For illustrative purposes, the data for fatty acid furan(2) are shown in Fig. 7A. Note the presence of analytes with multiple characteristic parent -> daughter ion transitions and retention times identical to that of the synthetic fatty acid furan(2) in nonderivatized form (Fig. 7A). Independent conformation of the presence of each fatty acid furan was further achieved by analyzing the phospholipase A2-treated extracts following derivatization to either their corresponding TBDMS or pentafluorobenzyl esters. For illustrative purposes, the data for fatty acid furan(2) are again shown in Fig. 7 (B and C). Note the presence of analytes with multiple characteristic parent -> daughter ion transitions and retention times identical to that of the synthetic fatty acid furan(2) in its corresponding TBDMS or pentafluorobenzyl ester forms. Finally, using LC/ESI/MS/MS based quantification methods (as in Fig. 2), we monitored each oxPC-furan in ischemic versus control brain tissues. A marked enrichment in each of the oxPC-furans monitored was noted within ischemic brain hemisphere 24 h following stroke compared with that observed in noninfarcted control tissues (Fig. 8). These data collectively provide unequivocal evidence to demonstrate the presence of the structurally defined oxPC-furan molecular species in vivo, as well as their formation at sites of inflammation, such as following cerebral ischemia.


Figure 7
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  		FIGURE 7.
Fatty acid furan generation in vivo following liberation from oxPC-furans by phospholipase A2. A, LC/MS/MS analysis of nonderivatized lipids recovered from oxPC-furan containing fractions (e.g. as in Fig. 6) following phospholipase A2 treatment, as described under "Methods." Also shown are parallel LC/MS/MS analyses of authentic synthetic fatty acid furan(2) (i.e. 3-(2-furyl)propanic acid). B and C, LC/MS/MS analysis of TBDMS and pentafluorobenzyl (PFB) esters of lipids recovered from oxPC-furan containing fractions (e.g. as in Fig. 6) following phospholipase A2 treatment, as described under "Methods." Also shown are parallel LC/MS/MS analyses of authentic synthetic fatty acid furan(2) TBDMS (B) and pentafluorobenzyl (C) esters. Within each panel, the structure of the analyte monitored and the proposed fragmentation pathway for the daughter ions monitored are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Since the seminal observation that transfection of nonphagocytic cells with CD36 confers professional phagocytic activity (1), the scavenger receptor has been shown to perform critical housekeeping functions as part of the innate immune system, including macrophage phagocytosis of oxidized lipoproteins (10, 14, 15, 27), senescent or apoptotic cells (1, 26),4 and effete rod outer segments (16, 28, 29) (Fig. 8). Although the role of CD36 in these phagocytic functions has not been debated, recent studies have questioned the role of CD36 in atherogenesis (30). Regardless, the initial step in carrying out its phagocytic functions involves CD36 receptor recognition of oxidized lipoproteins and cell surface ligands. Choline glycerophospholipids possessing a conserved structural motif, an oxidatively truncated sn-2 acyl group with a terminal {gamma}-hydroxy(or oxo)-{alpha},beta-unsaturated carbonyl (oxPCCD36), were recently shown to serve as endogenous high affinity ligands for CD36 within lipoproteins and cell membranes and to be generated by multiple oxidation pathways through initiation of lipid peroxidation (14–16). The present studies extend our understanding of one potential metabolic fate of a subset of oxPCCD36, phospholipid hydroxyalkenals: cyclization and generation of CD36-inert oxPC-furans.


Figure 8
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  		FIGURE 8.
oxPC-furans are formed in vivo following stroke and are enriched within ischemic cerebral tissue. The tissue content of the indicated oxPC-furans within ischemic cerebral hemisphere (CVA) and sham-operated controls (Ctrl) was determined in tissue harvested 1 day following ligation of the middle cerebral artery. The data plotted are the means ± S.D. from four sham operated (control) and eight middle cerebral artery occluded mice.

 


Figure 9
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  		FIGURE 9.
Model of potential pathways impacting upon formation and decay of oxidized phospholipids that serve as high affinity ligands for the scavenger receptor CD36. CytoC, cytochrome c; RNS, reactive nitrogen species; MPO, myeloperoxidase; NOX, NAD(P)H oxidase; LPO, lipoxygenase; PAF-AH, platelet-activating factor acetylhydrolase; OB-PL, ON-PL, and OV-PL, the 4-oxobutyric acid, 9-oxononanoic acid, and 5-oxovaleric acid ester of 2-lyso-PC; A-PL, G-PL, and S-PL, azeleic acid, glutaric acid, and succinic acid ester of 2-lyso-PC; oxPLCD36, oxidized phospholipids that bind to the scavenger receptor CD36; oxPC-furan, oxidized phospholipids with sn-2 acyl group that incorporates a terminal 2-furyl carbonyl.

 
Fig. 9 represents a schematic illustration of pathways potentially involved in the formation and removal of oxidized phospholipid ligands for the scavenger receptor CD36. Numerous potential oxidative pathways can trigger initiation of lipid peroxidation and formation of oxidized phospholipid species possessing a CD36-binding motif (15, 16). Once formed, the fate of oxPCCD36 will no doubt depend upon the chemical reactivity of the specific oxPCCD36 formed and the environment in which it is produced. For example, prior studies have shown that oxPCCD36 may be further oxidized to generate oxPC possessing shorter sn-2 fatty acyl chains with terminal aldehyde or carboxylic acid groups (14, 16) (Fig. 9). Some are reported to have potent pro-inflammatory activities, such as the 5-oxovaleric acid ester of 2-lyso-PC (31). In addition, oxidized phospholipids like oxPCCD36 may potentially serve as substrates for phospholipases as part of either membrane remodeling or signal transduction pathways. For example, some phospholipases and platelet-activating factor acetylhydrolase may selectively use phospholipids harboring oxidized fatty acids at the sn-2 position as substrates (32,33) and may thus serve as a catabolic pathway for oxPCCD36.

One subclass of oxPCCD36 is of particular interest because of their chemical reactivity, phospholipid hydroxyalkenals. These electrophilic lipid oxidation products are analogous to 4-hydroxy-2-nonenal and may thus react with nucleophilc groups on proteins and amino phospholipids forming covalent adducts (17, 34). The present studies demonstrate an additional metabolic pathway for phospholipid hydroxyalkenals: conversion into stable oxPC-furans. The presumed mechanism (Fig. 1B) likely involves a rate-limiting trans-cis isomerization step that permits intramolecular cyclization to occur forming a cyclic hemiacetal. A subsequent irreversible dehydration step would then form the stable oxPC-furan species. Studies using synthetic phospholipid hydroxyalkenals demonstrated clear precursor -> product relationships between structurally defined phospholipid hydroxyalkenals derived from PA-PC, PL-PC, and DHA-PC (HOOA-PC, HODA-PC, and HOHA-PC, respectively) and novel oxPC species possessing a sn-2 acyl group that incorporates a terminal furyl moiety (oxPC-furan(3), oxPC-furan(7), and oxPC-furan(2), respectively) (Fig. 3).

That oxPC-furans might be formed in vivo as was observed (Figs. 6, 7, 8) might seem surprising, given the abundance of nucleophilic targets in biological matrices capable of reacting with the electrophilic hydroxyalkenal moiety. However, two factors likely contribute to phospholipid hydroxyalkenal conversion into oxPC-furans, even in complex biological matrices. First, when tethered to a phospholipid glycerol backbone and incorporated within a membrane bilayer, the reactive electrophilic group on phospholipid hydroxylakenals may experience a relatively inert hydrophobic environment if buried within the acyl chains of the bilayer, limiting exposure to nucleophilic targets. Second, the irreversible formation of stable oxPC-furan species was likely detectable because many of the covalent adducts formed by hydroxyalkenals are reversible, such as generation of Schiff bases and some Michael adducts (18, 35–38). Indeed, our studies with coincubation of high concentrations of albumin with SUV containing oxPC hydroxyalkenals demonstrated marked increases in the rate of oxPC hydroxyalkenal depletion; however, the overall yields of oxPC-furans was only slightly attenuated (Fig. 3D). These findings suggest that electrophilic lipid species like hydroxyalkenals exist in equilibrium between many reversible covalent adducts with nucleophilic targets and their free forms. The reversibly bound forms of oxPC hydroxyalkenals can thus serve as a reservoir or buffer and are still potentially available to slowly form downstream more stable and irreversible reaction products, such as the oxPC-furans.

Perhaps one of the more remarkable aspects of the present studies is the finding that oxPC possessing furan groups are endogenous oxidation products formed both in model membranes and in vivo. Although furan-containing fatty acids have been identified in multiple organisms, as far as we are aware, the present studies represent the first reported description of furan-containing phospholipids as endogenous products in a mammalian system. Since furan fatty acids were first identified in the seed oil of Exocarpus cupressiformis (Santalaceae) in 1966 (39), numerous furan fatty acid analogues, including within phospholipids and cholesterol esters, have been identified in organisms including algae, plants, bacteria, murine sponges, and fish (23, 40). Fatty acid furans recovered from natural products have been attributed with a variety of biological activities, ranging from functioning as anti-oxidant cytoprotectants, to promoters of cell cytotoxicity (reviewed in Ref. 23). Whether the oxPC-furans identified herein possess similar biological activities remains to be determined. In contrast to their oxPCCD36 precursors, oxPC-furans no longer bind to CD36, both when expressed on transfected cells or mouse peritoneal macrophages (Fig. 5). Transformation of oxPCCD36 into oxPC-furans is thus anticipated to attenuate the physiological eat me signal that triggers CD36-mediated phagocytosis. The unique structure and stability of oxPC-furans may permit these unusual phospholipids to serve as molecular fingerprints for generation of phospholipid hydroxyalkenals in vivo. Further studies on the biological activities of these intriguing oxidized phospholipids are warranted.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants P01 HL076491, P01 HL077107, HL70621, HL61878, GM21249, and HL53315 and supported in part by Cleveland Clinic Foundation General Clinical Research Center Grant M01 RR018390. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported in part by a fellowship from the American Heart Association. Back

2 To whom correspondence should be addressed: Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, 9500 Euclid Ave., NE-10, Cleveland, OH 44195. Tel.: 216-445-9763; Fax: 216-636-0392; E-mail: hazens{at}ccf.org.

3 The abbreviations used are: LDL, low density lipoprotein; DHA-PC, PA-PC, PL-PC, and PO-PC, the docosahexaenoic acid, arachidonic acid, linoleic acid, and oleic acid ester of 2-lyso-PC; Di-I, 1,1'-dioctadecyl-3,3,3',3'-tetramethlyindocarbocyanine perchlorate; DT-PC, 1,2-ditridecanoyl-sn-glycero-3-phosphatidylcholine; ESI, electrospray ionization; GFP, green fluorescent protein; HODA-PC, HOHA-PC, and HOOA-PC, the 9-hydroxy-12-oxododec-10-enoic acid, 4-hydroxy-7-oxohept-5-enoic acid, and 5-hydroxy-8-oxoocta-6-enoic acid ester of 2-lyso-PC; HPLC, high performance liquid chromatography; LC, liquid chromatography; LDL, low density lipoprotein; lyso-PC, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine; MS, mass spectrometry; oxPC, oxidized phosphatidylcholine molecular species; oxPC-furan, oxidized phospholipids with sn-2 acyl group that incorporates a terminal 2-furyl carbonyl; oxPC-furan(2), 1-palmitoyl-2-(3-(2-furyl)propanoyl)-sn-glycero-3-phosphatidylcholine; oxPC-furan(3), 1-palmitoyl-2-(4-(2-furyl)butanoyl)-sn-glycero-3-phosphatidylcholine; oxPC-furan(7), 1-palmitoyl-2-(8-(2-furyl)octanoyl)-sn-glycero-3-phosphatidylcholine; oxPCCD36, oxPC that bind to the scavenger receptor CD36; SUV, small unilamellar vesicle(s); PC, phosphatidylcholine; TBDMS, tert-butyldimethylsilyl. Back

4 M. E. Greenberg, M. Sun, R. Zhang, M. Febbraio, R. Silverstein, and S. L. Hazen, submitted for publication. Back



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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