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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3505-3512.)
© 1997 American Heart Association, Inc.

Articles

Copper-Catalyzed Oxidation Mediates PAF Formation in Human LDL Subspecies

Protective Role of PAF:Acetylhydrolase in Dense LDL

Demokritos C. Tsoukatos; Muriel Arborati; Theodoros Liapikos; Keith L. Clay; Robert C. Murphy; M. John Chapman; ; Ewa Ninio

From the Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece (D.C.T., T.L.); Institut National de la Santé et de la Recherche Medicale (INSERM), Unité de Recherches sur les Lipoprotéines et l'Athérogénèse, U-321, Pavillon Benjamin Delessert, Hôpital de la Pitié, Paris, France (M.A., M.J.C., E.N.); and National Jewish Medical and Research Center, Denver, CO (K.L.C., R.C.M.).

Correspondence to Ewa Ninio, INSERM U-321, Pavillon Benjamin Delessert, Hôpital de la Pitié, Bd de l' Hôpital, 75651 Paris, Cedex 13, France. E-mail: eninio{at}infobiogen.fr

	Abstract

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Abstract Free radical-mediated oxidation of cholesterol-rich LDL plays a key role in atherogenesis and involves the formation of oxidized phospholipids with proinflammatory biological activity. We evaluated the production of platelet-activating factor (PAF), a potent inflammatory mediator, in human LDL subspecies on copper-initiated oxidation (4 µmol/L CuCl₂, 80 µg/mL for 3 hours at 37°C). PAF formation was determined by biological assay of HPLC-purified lipid extracts of copper-oxidized lipoproteins; chemical identity was confirmed by gas chromatographic and mass spectrometric analyses. PAF, characterized as the C16:0 molecular species, was preferentially produced in intermediate LDL (d=1.029 to 1.039 g/mL) (8.6±5.7 pmol PAF/3 h per mg LDL protein) and light LDL (d=1.019 to 1.029 g/mL), but was absent from dense LDL particles (d=1.050 to 1.063 g/mL). As PAF:acetylhydrolase inactivates PAF and oxidized forms of phosphatidylcholine, we evaluated the relationship of lipoprotein-associated PAF:acetylhydrolase to PAF formation. We confirmed that PAF:acetylhydrolase activity was elevated in native, dense LDL (41.5±9.5 nmol/min per mg protein) but low in LDL subspecies of light and intermediate density (d 1.020 to 1.039 g/mL) (3.5±1.6 nmol/min per mg protein) [Tselepis et al, Arterioscler Thromb Vasc Biol. 1995;15:1764–1773]. On copper-mediated oxidation for 3 hours at 37°C, dense LDL particles conserved 20±14% of their initial enzymatic activity; in contrast, PAF:acetylhydrolase activity was abolished in light and intermediate LDL subspecies. Clearly, the elevated PAF: acetylhydrolase activity of dense LDL efficiently diminishes the potential inflammatory role of endogenously formed PAF; nonetheless, formation of proatherogenic lysophospholipids results. In contrast, LDL particles of the light and intermediate subclasses can accumulate PAF on oxidative modification.

Key Words: inflammation • density gradient ultracentrifugation • lipoprotein particle subspecies

	Introduction

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Free radical-mediated oxidation of cholesterol-rich LDL plays a key role in the development of fatty streaks and subsequent formation of lipid-rich, atheromatous plaques.¹ The preferential retention of LDL in the intima on interaction with extracellular matrix components, such as proteoglycans, exposes these particles to oxidative stress, involving the action of reactive oxygen species and of transition metals.² Numerous biologically active compounds are produced on oxidation of LDL lipids, including aldehydes (reviewed in³), oxysterols,⁴ lysophosphatidylcholine,⁵ and oxidized species of phosphatidylcholine.⁶ Oxidized species of phosphatidylcholine have been shown to act in vitro as potent activators of both vascular and circulating cells.⁶ A part of their activity may be due to their structural analogy with PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF-acether),⁷ a potent lipid mediator involved in inflammatory and allergic reactions⁸ that may equally play a crucial role in atherogenesis (reviewed in Reference 9⁹ ). Indeed, PAF receptors may bind by such PAF analogues and transduce signals to internal cell effectors.⁶

PAF is synthesized by various activated proinflammatory cells, including endothelial cells, platelets, monocytes, and macrophages (reviewed in reference⁸), all of which are known to contribute to the development of atherosclerotic plaques (reviewed in reference¹⁰). Oxidized phosphatidylcholine and PAF are hydrolyzed by PAF: acetylhydrolase (acetylhydrolase, EC 3.11.48), the Ca²⁺-independent enzyme that inactivates PAF by hydrolyzing its sn-2 acetate group and thus converting it to lyso PAF.^11–14 Acetylhydrolase in plasma is associated mainly with LDL and HDL.¹³ Recently, we have shown that the association of acetylhydrolase activity with LDL particles is heterogenous.^15,16 Furthermore, we demonstrated that acetylhydrolase is associated primarily with small, dense LDL of d=1.050 g/mL to 1.063 g/mL of elevated atherogenicity, but also with the apo A-I-containing VDHL subclass (VHDL-1) of d=1.156 g/mL to 1.179 g/mL.¹⁶ Acetylhydrolase is present in several cells and tissues, including monocytes and macrophages,^17,18 platelets,¹⁹ erythrocytes,²⁰ spleen and liver cells.²¹ The acetylhydrolase that has been recently cloned from human monocyte-derived macrophages²² may represent a major acetylhydrolase activity associated with LDL. In this context, our earlier studies suggested that distinct forms of acetylhydrolase are associated with different LDL subspecies,¹⁶ although their cellular sources have not as yet been established.

The oxidative modification of LDL involves the hydrolytic transformation of its content of oxidized phosphatidylcholine to lysophosphatidylcholine.²³ Such hydrolysis has been reported to be mediated by the LDL-associated acetylhydrolase.^24,25 On oxidative modification of LDL, however, acetylhydrolase activity dramatically decreases and thus oxidized LDL is devoid of its anti-inflammatory property.^24,25 Indeed we showed recently that PAF is produced on copper-mediated oxidation of LDL in which acetylhydrolase was irreversibly inhibited with PMSF.²⁵ In this respect, it is relevant that an earlier report described the recovery of PAF from native LDL preparations isolated either by sequential ultracentrifugation or by affinity column chromatography, although its chemical structure was not validated by mass spectrometry.²⁶

The aim of the present study was to evaluate the potential relationship between the level of acetylhydrolase activity in distinct LDL subspecies to formation of PAF in each particle subpopulation. We show that PAF is produced in the intermediate LDL subclass on copper-mediated oxidation; in contrast, PAF formation was not detectable in dense LDL unless its elevated acetylhydrolase activity had been fully inactivated with Pefabloc. Clearly, the elevated acetylhydrolase activity in dense LDL efficiently diminishes the potential inflammatory role of endogenously formed PAF in these atherogenic particles.

	Methods

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Materials
PAF C16:0 was obtained as a powder from Novabiochem. The solution of PAF C16:0 was dissolved at a final concentration of 5 mmol/L in ethanol (80% vol/vol), mixed with 1-O-hexadecyl-2-[³H-acetyl]-sn-glycero-3-phosphocholine (10 Ci/mmol; DuPont-New England Nuclear), dried under a stream of nitrogen and redissolved in a solution containing fatty acid-free bovine serum albumin (BSA)/saline (0.25%), to obtain a [³H-acetyl]PAF solution of 25 mmol/L. [1-octadecyl-³H] PAF (80–180 Ci/mmol) was from Amersham International (Amersham, UK). [1-octadecenyl-³H] PAF was prepared as described in.²⁷ Pefabloc SC (4-[2-aminoethyl benzenesulfonyl fluoride, Pefabloc) and BCA protein reagent were from Pierce Ltd. Liquid scintillation fluid (Optiphase Hi-Safe 3) was supplied by E.G.G. Berthold. Lipase from Rhizopus arrhizus was supplied by Boehringer Mannheim, Germany. Purified sphingomyelin from bovine brain, phosphatidylcholine from egg yolk, and lysophosphatidylcholine were from Sigma Chemical Co.

Fractionation of Plasma Lipoproteins
Subjects were healthy, normolipidemic volunteers (all females) who had fasted overnight and whose plasma Lp(a) levels were less than 0.1 mg/mL. Venous blood was collected into glass tubes containing EDTA (3 µmol/L), from which plasma was rapidly separated by low-speed centrifugation (1000g, 20 min) at 4°C. Immediately after collection of plasma, gentamycin (50 µg/mL) and EDTA (0.3 mmol/L) were added. Lipoproteins were fractionated by isopycnic density gradient ultracentrifugation using a Beckman SW41 Ti rotor at 40 000 rpm for 44 hours in a Beckman XL 70 centrifuge at 15°C, as described previously.²⁸ All fractions were analyzed for their protein content by the BCA method and their purity was evaluated by SDS-polyacrylamide gel electrophoresis. Subsequently, equal volumes of certain gradient fractions were pooled to constitute the lipoprotein subfractions, as follows: fractions 1 and 2 corresponding to VLDL+IDL (d<1.019 g/mL) were discarded; 3 and 4 (LDL-1; d=1.019 g/mL to 1.023 g/mL); 5 and 6 (LDL-2; d=1.023 to 1.029 g/mL) were denominated as the light LDL; 7 and 8 (LDL-3; d=1.029 g/mL to 1.039 g/mL) as the intermediate LDL; 9 and 10 (LDL-4; d=1.039 g/mL to 1.050 g/mL); 11 and 12 (LDL-5; d=1.050 g/mL to 1.063 g/mL) were designated as the dense LDL.

Lipid and Lipoprotein Analysis
The lipid contents of lipoprotein subfractions were analyzed by enzymatic methods using BioMerieux kits (Marcy l'Etoile) for total cholesterol, free cholesterol, phospholipids, and triglycerides.²⁹ Cholesteryl ester mass was calculated as 1.67x(free cholesterol mass).²⁸

Oxidation of Lipoprotein Subfractions by Copper Ions
Isolated LDL subfractions were submitted to oxidation in PBS of pH 7.4 in a final volume of 2 mL containing 80 µg/mL LDL protein and 4 µmol/L CuCl₂. Incubations were carried out at 37°C for 3 hours and terminated by the addition of EDTA (0.01%, final concentration). Aliquots were withdrawn before and after oxidation in order to assay the activity of acetylhydrolase and to measure the content of MDA by the TBARS assay. The content of lipid peroxides was assessed by commercial kit.³⁰ The remaining samples were submitted to extraction with chloroform: methanol: water (1:1:0.9, v/v/v) as described by Bligh and Dyer³¹ and brought to dryness under a nitrogen stream. Samples containing lipids and PAF were kept at -20°C for further purification and analysis.

Purification of PAF
Samples containing crude lipid extracts were submitted to thin layer chromatography on silicagel G plates and developed in a mixture of chloroform:methanol:water (65:35:6, v/v/v) as mobile phase. The bands corresponding to the Rf of synthetic standard PAF were scraped off, extracted, dried (as described above), and assayed for PAF content. After oxidation of the different LDL subspecies, characterization of the TLC-purified aggregating activity as PAF was performed by studying its resistance on treatment with lipase from Rhizopus arrhizus. Incubation with lipase 1000 U/mL in 0.1 mol/L borate buffer at pH 6.5 was performed in polypropylene tube for 18 hours at 30°C.³²

In the next step, the dry residues containing PAF were suspended in 25 µL of HPLC mobile phase (ammonium acetate [10 mmol/L])/acetonitrile/methanol (120: 140: 40, v/v/v) before separation of the molecular species of PAF on a reversed-phase Spherisorb C6 column (Touzart et Matignon). The retention times of PAF molecular species were determined using [³H] labeled PAF C16:0, C18:0, and C18:1 standards as described earlier.²⁷ Fractions were collected, extracted with chloroform, dried, and assayed for PAF content. The yield of platelet aggregating activity of PAF on separation by reversed-phase HPLC varied between 70% and 80%.

PAF Analysis by GC/MS
The biologically active material that was recovered from reversed-phase HPLC with the retention time of PAF C16:0 was further analyzed by GC/MS as described.³³ In brief, the samples from reversed-phase HPLC were added to tubes that contained 2 ng of the stable, isotopically labeled variant of PAF, 1-O-hexadecyl-2(D3)-acetyl-glycero-3-phosphocholine. The samples were redissolved in ethanol and applied to silica solid phase extractor tubes (Varian). The tubes were washed with 4 mL ethanol and then eluted with 4 mL of methanol:water(4:1). The samples were then dried and subjected to phospholipase C cleavage. The diglycerides thus produced were extracted into methylene chloride, dried, and then derivatized with pentafluorobenzoyl chloride. The pentafluorobenzoyl derivatives were subsequently analyzed by negative ion chemical ionization GC/MS with a Finnigan Mat SSQ70 mass spectrometer, as described.³³

PAF Bioassay
Dry, purified samples containing PAF were redissolved in a small volume of ethanol (60%, v/v) for quantitation of PAF by the thromboxane A2- and ADP-independent aggregation of washed rabbit platelets, as previously described.³⁴ The aggregating activity of the samples was measured over the linear portion of the calibration curve established with 5 pg to 50 pg synthetic PAF C16:0. The results are expressed as equivalent pmol of PAF per mg/LDL protein. The specific PAF antagonist WEB 2086 (4 µmol/L, a gift from Boehringer Ingelheim, Germany)³⁵ totally inhibited platelet aggregation induced by all samples. Various samples were treated with 1000 U/mL of lipase A1 from Rhizopus arrhizus³² and platelet aggregation was again measured in order to estimate the percentage of platelet aggregation induced by PAF and by the sn1 ester analog of PAF. Lipase A1 from Rhizopus arrhizus exclusively hydrolyzes the sn1 ester bond of the ester analog of PAF, but not the sn1 ether bond of PAF.

Acetylhydrolase Assay
Acetylhydrolase activity was measured in the gradient subfractions, as previously described,¹⁶ using 4 µg of protein from the dialyzed lipoprotein samples and 10 µL of [³H-acetyl]PAF (final concentration, 25 µmol/L; specific activity, 16,890 dpm/nmol). After preincubation at 37°C, the reaction was initiated and performed for 10 minutes at 37°C. The acetylhydrolase activity in lipoprotein subfractions was stable at 4°C for at least two weeks; such activity corresponded closely to that described earlier in native LDL before Cu²⁺-oxidation.³⁶

Quantification of Ether-Containing Phosphatidylcholine and Lyso PAF
Total lipids contained in the lipoprotein subspecies before and after 3 hours of oxidation were extracted as described by Bligh and Dyer.³¹ The crude extracts were subjected to TLC as described above. After brief exposure to iodine vapor, the bands corresponding to the Rf of standard phosphatidylcholine and lysophosphatidylcholine were scraped off the plate and extracted. Aliquots of purified phospholipids were submitted to phosphorous assay^37,38 and the remaining samples were subjected to acid hydrolysis in order to eliminate plasmalogens.³⁹ In short, the samples containing phospholipids were dissolved in 1.5 mL diethyl ether and treated for 2 minutes with 1 mL concentrated HCl. After several washes with water to remove traces of acidity, the organic phase was evaporated to dryness under a stream of nitrogen and the remaining phospholipids were submitted to mild alkaline hydrolysis;⁴⁰ they were then dissolved in 1 mL CHCl₃: CH₃OH (1:4, v/v) and 100 µL of 1.2N NaOH in CH₃OH: H₂O (1:1, v/v) were added. After 20 minutes incubation at 60°C, the remaining phospholipids were extracted with 2 mL CHCl₃: CH₃OH (1:1, v/v) to which 1 mL isobutanol and 2 mL of water were added. The upper phase was discarded, and after several washes with water to remove any alkali, the chloroform phase was evaporated to dryness under a stream of nitrogen. The alkyl-ether-linked lysophosphatidylcholine (lyso PAF) was dissolved in 200 µL pyridine and chemically acetylated to PAF with 200 µL acetic anhydride.⁴¹ The alkyl-ether-linked phosphatidylcholine and lyso PAF were quantified as PAF equivalents using the bioassay, as described above.

Statistical Analysis
Results are expressed as mean±SD. Mean values were compared by the Mann-Whitney nonparametric test, with significance defined at a value of P=.05.

	Results

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Characterization of LDL Gradient Subfractions
We first evaluated PAF formation among plasma LDL subfractions during Cu²⁺-initiated oxidation. After fractionation of plasma lipoprotein species by isopycnic density gradient ultracentrifugation,²⁸ gradient fractions were pooled to constitute the LDL subspecies described in "Methods." The correspondence of density fractions to LDL subspecies was assessed on the basis of chemical, physical, and immunological analysis and corresponded well to previous findings in normolipidemic subjects.^28,42,43 The density profiles of the concentrations of protein and cholesteryl esters in LDL subspecies from normolipidemic female subjects are summarized in Fig 1. The peak concentration of both lipids and protein was typically found in LDL of the intermediate subclass (LDL-3, d=1.029 g/mL to 1.039 g/mL).

View larger version (30K): [in this window] [in a new window]   Figure 1. Graphs show the mass distribution of cholesteryl esters and protein as a function of density on fractionation by isopycnic density gradient ultracentrifugation (n=4). After centrifugation, fractions of 0.4 mL were collected from each gradient. The LDL subfractions were reconstituted by mixing equal volumes of the individual fractions, as described in "Methods." The significance of the results was determined with the Mann-Whitney nonparametric test: *P<.05 compared with LDL-1, LDL-2, and LDL-5, **P<.05 compared with LDL-1 and LDL-2.

Conditions for PAF Formation in LDL Subspecies on Oxidation
In preliminary experiments, we established optimal conditions for PAF formation: 80 µg protein of LDL subfraction/mL in PBS, 4 µmol/L Cu²⁺ and incubation for 3 hours at 37°C. Under such conditions, the formation of conjugated dienes in all LDL subspecies reached a maximum (data not shown) in accordance with previous studies from our own and other groups.³ Next we evaluated the level of lipid peroxides in the oxidized subspecies, which attained values ranging from 220±96 nmol/mg LDL protein in LDL-5 (d=1.050 g/mL to 1.063 g/mL) to 371±144 nmol/mg LDL protein (n=3) in LDL-1 (d=1.019 g/mL to 1.029 g/mL. In native LDL subfractions, the lipoperoxide level typically did not exceed 25±3 nmol/mg LDL protein (n=3). We also evaluated the production of MDA on oxidation; the formation of this aldehyde, as assessed by the TBARS assay, ranged from 56±24 nmol/mg protein in dense LDL-5 to 115±58 nmol/mg protein (n=3) in light LDL. In native subfractions, the MDA level was 17±24 nmol/mg LDL protein. However, no significant difference could be detected between LDL subfractions in the accumulation of lipid peroxides and MDA during copper-mediated oxidation (Mann-Whitney nonparametric test). Nonetheless, and as previously reported by ourselves and others,^42,44 small dense LDL are distinct in displaying reduced oxidative resistance (measured as the lag phase for conjugated diene formation on copper-mediated oxidation) relative to LDL particles of the light and of the intermediate subclasses.

Formation of PAF Among LDL Subspecies on Oxidation
On oxidation induced by incubation of lipoprotein species with copper ions, significant amounts of PAF were produced in LDL-2 and LDL-3, attaining 4.6±3.4 pmol/mg and 8.6±5.7 pmol/mg protein respectively (n=4) (Fig 2A). Among dense LDL particles, PAF was produced only in LDL-4 (3.3±3.8 pmol/mg protein LDL subfraction). However, no PAF was formed in LDL-5 particles, with the exception of a trace amount in one of four subjects. Only traces of PAF were formed in LDL-1 particles (2.4±0.9 pmol/mg protein LDL subfraction). In agreement with our previous results,¹⁶ the activity of acetylhydrolase was preferentially associated with small, dense particles of LDL-5 (Fig 2B). Oxidation of lipoprotein subspecies for 3 hours decreased the activity of acetylhydrolase by at least 86±7% (n=4) in the majority of subspecies; 20±14% of the initial activity still remained however in LDL-5 particles, representing 8±4 nmol/min per mg prot. (n=4) (Fig 2). Such residual activity was more elevated than the initial acetylhydrolase level found in all other lipoprotein subfractions, except for LDL-4. In two separate experiments, using different subjects, we studied the time course of PAF formation in LDL-3 on Cu²⁺-induced oxidation in parallel with acetylhydrolase inhibition. Indeed, PAF began to be measurable as soon as the activity of acetylhydrolase decreased to less than 0.8 nmol/min per mg protein in both preparations and its formation was linear up to 6 hours (data not shown). We next inhibited the activity of acetylhydrolase in LDL-3 and LDL-5 subfractions with Pefabloc⁴⁵ before oxidation with copper ions. As expected, the level of PAF production in LDL-3 was not modified as compared to untreated control fractions (8.4 pmol/mg versus 7.4 pmol/mg LDL protein). In contrast, the production of PAF increased from 0.6 pmol/mg to 3.9 pmol/mg LDL protein) in LDL-5 after treatment with Pefabloc. These experiments suggested that acetylhydrolase protects small, dense LDL from the accumulation of PAF on oxidation.

View larger version (19K): [in this window] [in a new window]   Figure 2. Graphs show (A) formation of PAF and (B) activity of acetylhydrolase in LDL subfractions from normolipidemic human plasmas obtained as described in Fig 1 and submitted to oxidation for 3 hours in the presence of 4 µmol/L CuCl2 and 80 µg/mL protein. (A) PAF was extracted and purified by TLC before bioassay on washed rabbit platelets as described in "Methods." Less than 10 fmol PAF/mg protein was formed in control, nonoxidized lipoprotein fractions. Results are means of 4 different donors and the significance of the results was determined with the Mann-Whitney nonparametric test: #P<.05 compared with LDL-1 through LDL-3; §P<.05 compared with LDL-1 and LDL-5. (B) Acetylhydrolase activity was determined with 25 µmol/L of 13H-acetyl PAF as the substrate. Graph shows acetylhydrolase activity in control, nonoxidized (hatched), and oxidized lipoprotein fractions (solid). Bar graph shows mean±SD of acetylhydrolase activity (duplicate determinations; coefficient of variation <10%) in LDL subfractions from plasma of 4 different subjects. The significance of the results was determined with the Mann-Whitney nonparametric test. The results before and after oxidation were significantly different in all LDL subfractions with P<.05, #P<.005, *P<.05 compared with LDL-1 through LDL-5.

Characterization of PAF
The biologically active compound formed among lipoprotein subfractions on oxidation was characterized as PAF by classical criteria including its Rf on TLC,⁴⁶ its resistance to treatment with lipase A1 from Rhizopus arrhizus,³² and its ability to aggregate washed rabbit platelets in the presence of aspirin and an ADP scavenging system³⁴ (data not shown). In addition, such PAF-like activity generated in the LDL-3 subfraction was further characterized by reversed HPLC and GC/MS analysis. As shown in Fig 3A, PAF was eluted as a single peak with the retention time of synthetic PAF C16:0 on reversed phase HPLC; its typical reconstructed ion chromatogram obtained during GC/MS analysis for the specific ion m/z 552 (endogenous hexadecyl PAF diglyceride) and m/z 555 (from the stable isotopically labeled internal standard diglyceride derivative) is shown in Fig 3B. Quantitation of PAF was achieved by comparison of the ratio of m/z 552 (from native PAF) to m/z 555 (from the stable isotopically labeled, internal standard) with a standard curve constructed by addition of various known amounts of authentic hexadecyl PAF to 2 ng of the internal standard and measurement of the resulting ratios of m/z 552 to m/z 555.

View larger version (18K): [in this window] [in a new window]   Figure 3. Graphs show analysis of PAF extracted and purified by reversed phase HPLC from LDL-3 (d=1.029 g/mL to 1.039 g/mL), which was submitted to oxidation for 3 hours in the presence of 4 µmol/L of CuCl2 and 80 µg/mL LDL protein: (A) reversed phase HPLC profile and (B) reconstructed ion chromatogram obtained during GC/MS analysis for the specific ions m/z 552 (endogenous hexadecyl PAF diglyceride) and m/z 555 (from the stable isotopically labeled internal standard diglyceride derivative). (A) The synthetic radiolabeled C16:0, C18:1 and C18:0 PAF were used as standards. Each analysis has been performed using material pooled from 3 subjects. (B) The D3-PAF was used as internal standard. The ions at m/z 552 and m/z 555 correspond to endogenous PAF and internal standard (2 ng) added to the biological system before extraction and conversion to the pentafluorobenzoyl diglyceride, as previously described. The ratio of the ion abundance area of the elution of the D3-PAF diglyceride to that of unlabeled PAF diglyceride was used to calculate the quantity of 1-hexadecyl-2-acetyl-3-pentafluorobenzoyl-glycerol.

Quantitation of Alkyl-Ether-Linked Phosphatidylcholine and Lyso PAF Content of Lipoprotein Subspecies
Human plasma contains trace amounts of ether-linked phospholipids⁴⁷ that are mainly present in LDL as plasmalogens.³⁹ The 1 to O-alkenyl linkage of plasmalogens is rapidly degraded on oxidation.⁴⁸ Thus, we investigated the distribution of PAF precursors, namely alkyl-ether-linked phosphatidylcholine species and their major hydrolytic product (ie, lyso PAF), in LDL subpopulations in order to evaluate their potential contribution to PAF formation.

The content of alkyl-ether-linked phosphatidylcholine in LDL subfractions was low and ranged from 0.24 to 0.53% of total phosphatidylcholine content (Table I). Such low levels of this precursor pool (range from 1.7±0.4 nmol/mg to 3.9±1.1 nmol/mg protein) were sufficient however to ensure PAF formation, which attained 8.6±5.7 pmol/mg protein (n=4) in LDL-3 (Fig 3A).

View this table: [in this window] [in a new window]   Table 1. Quantitative Distribution of Phosphatidylcholine and Alkyl Ether–Linked Phosphatidylcholine Between Human LDL Subfractions

The content of the PAF metabolite, lyso PAF, in LDL subfractions was low before oxidation (26±5 pmol/mg to 60±15 pmol/mg protein, n=4), but increased several-fold however in all LDL subspecies after incubation for 3 hours with copper ions (Fig 4B). In dense LDL (LDL-4 and LDL-5), a 12-fold and 17-fold elevation of lyso PAF level was observed on oxidation, respectively. In parallel, a marked decrease in alkyl-ether-linked phosphatidylcholine content occurred, thereby supporting the hypothesis that alkyl-ether-linked phosphatidylcholine species are indeed the precursors of lyso PAF as shown in Fig 4A.

View larger version (25K): [in this window] [in a new window]   Figure 4. Graphs show (A) the content of alkyl-ether-linked phosphatidylcholine species and (B) the content of lyso PAF in LDL subfractions, isolated as described in the to Fig 1., before (hatched) and after oxidation for 3 hours in the presence of 4 µmol/L CuCl2 and 80 µg/mL LDL protein (solid). Bar graph shows mean±SD of 4 independent experiments using samples of different donors. The results (A and B) before and after oxidation in each subfraction were significantly different in the Mann-Whitney nonparametric test with P<.03. Panel A: asterisks show the significant differences between LDL subfractions before oxidation (*P<.05 compared with LDL-5; **P<.03 compared with LDL-1); after oxidation (##P<.03 compared with LDL-1 through LDL-4). Panel B: asterisks show significant differences between LDL subfractions before oxidation (*P<.05 compared with LDL-5); after oxidation (##P<.03 compared with LDL-1 through LDL-5).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We show for the first time that a significant amount of a single molecular species of PAF, bearing a C16:0 alkyl chain at the sn-1 position of glycerol, is formed on copper-initiated oxidation of LDL particles of the intermediate subclass (d=1.029 g/mL to 1.039 g/mL). Equally, the amount of PAF formed on oxidation was closely related to the endogenous PAF-acetylhydrolase activity of individual LDL subclasses. Earlier attempts to demonstrate PAF formation in oxidized LDL were not successful,⁴⁹ except when the activity of PAF-degrading acetylhydrolase had been irreversibly inactivated with serine protease inhibitors.²⁵

As acetylhydrolase is mainly associated with small, dense LDL (1.050 g/mL to 1.063 g/mL),¹⁶ we speculated that PAF would not be formed in such LDL particles, at least when the enzyme is maintained in its active form. Indeed, we now show the lack of PAF formation on in vitro copper-induced oxidation of dense LDL. Elevated levels of the precursor/metabolite of PAF, lyso PAF, were attained during oxidation of dense LDL, representing approximately 50% of the initial level of alkyl-ether-linked phosphatidylcholine. Such lyso PAF accumulation probably arose from the action of the acetylhydrolase,²³ which also degrades oxidized forms of phosphatidylcholine generated on oxidation.¹⁴ Nonetheless, the activity of acetylhydrolase decreased on oxidation by 80%±14 (n=4), but remained sufficiently elevated to ensure hydrolysis of PAF and its analogues. Indeed, the acetylhydrolase activity that remained in dense LDL-5 after oxidation was still superior to that of other LDL subfractions before oxidation. We were also able to show that the irreversible inhibition of acetylhydrolase with Pefabloc⁴⁵ permitted PAF formation and accumulation on oxidation. When the accumulation of lysophospholipids in lipoproteins attains concentrations of 10 µmol/L to 100 µmol/L, then it may be deleterious to cells of the vascular wall.⁵ Thus, we hypothesize that both lyso PAF and lysophosphatidylcholine, which are associated with dense LDL, are potentially accessible to the cellular enzymes that may reacylate them into inactive phospholipids bearing a long fatty acid chain at the sn-2 position of glycerol. However, they may also serve as precursors of PAF, or its sn-1 acyl analogue. Indeed, human neutrophils and macrophages produce PAF when they are supplemented with exogenous lyso PAF.^50,51 We cannot therefore exclude a deleterious role of such acetylhydrolase-generated lysophospholipids in the arterial wall.⁵

Our reversed-phase HPLC and GC/MS analyses revealed that PAF formed in intermediate LDL particles corresponded to the C16:0 species. PAF C16:0 has been identified as the major molecular species of this inflammatory mediator in many cell types, including human neutrophils^52,53 and macrophages;⁵¹ however, other species including PAF C18:1 and C18:0 are also formed in these cells.^27,52,53 It is relevant that PAF C16:0 was demonstrated to be the most potent molecular species of PAF in several biological models,⁵⁴ as well as in platelet aggregation.⁵⁵

Our results do not explain the mechanism(s) of PAF formation in oxidized LDL. Nonetheless, uncontrolled free radical-catalyzed oxidation of polyunsaturated fatty acids attached to the sn-2 position of phospholipids can produce several break-down products that structurally resemble PAF and possess biological activities that may be mediated by PAF receptors.⁶ The presence of an acetate group at the sn-2 position in such lipids was not observed unless the activity of acetylhydrolase had been irreversibly inhibited with serine esterase inhibitors.²⁵

An alternative hypothesis that could explain the formation of PAF in the intermediate LDL subclass concerns the enzymatic reaction catalyzed by LCAT or possibly some other enzyme. Indeed, Liu and Subbaiah⁵⁶ recently reported that transacetylation between lyso PAF and a phospholipid bearing the acetyl group may be catalyzed by LCAT. However this reaction requires a donor of acetate, which could not be identified as yet under our oxidation conditions.

What is the potential physiological significance of PAF formation in intermediate LDL and of its diminished production in small, dense LDL? Our results indicate that small, dense LDL are efficiently protected, at least against production of PAF during a short period (3 hours) of copper-mediated oxidation, as compared to intermediate LDL particles. As we have shown earlier, small, dense LDL are not only the privileged carriers of acetylhydrolase,¹⁶ but also transport the majority of the tissue factor pathway inhibitor in plasma.⁴³ Indeed, both of these factors may exert antithrombotic properties of key importance in the thrombotic complications of atherosclerosis, ie, during plaque rupture. Moreover, acetylhydrolase may participate in the detoxification of PAF and PAF-like phospholipids, with short chains at the sn-2 position of glycerol, during the initiation of the formation of atheromatous plaques when minimally modified LDL are present⁵⁷ and when activated vascular and circulating cells produce PAF and release it into the intima. In this context, it is relevant that earlier studies have established that activated human endothelial cells, monocytes, and neutrophils synthesize PAF. We also demonstrated recently that stimulated human monocyte-derived macrophages and cholesterol-loaded macrophage foam cells could represent a major source of PAF in the plaque.⁵¹ Furthermore, our present data indicate that PAF is produced on oxidation of intermediate LDL and that it may exert proinflammatory, proaggregatory, and proatherogenic effects in arterial intima. Indeed, the presence of PAF in atheromatous plaques from canine and human coronary arteries has been documented,⁵⁸ and a close link between tumor necrosis factor-induced angiogenesis and the in situ formation of PAF has been described.⁵⁹ Furthermore, PAF contributes to the increased permeability of the endothelial monolayer⁶⁰ and is involved in the release of tissue-type plasminogen activator from vessel walls.⁶¹ Recent studies showed that PAF plays a pivotal role in the lymphocyte-mediated expression of tissue factor by endothelial cells⁶² and thus PAF participates in thrombus formation. Moreover, PAF stimulates transcription of heparin-binding epidermal growth factor in monocytes through an enhanced nuclear factor kB activity, and therefore PAF is equally implicated in smooth muscle cell proliferation.⁶³ Taken together, the potent actions of PAF on platelet aggregation,⁸ superoxide⁶⁴ and elastase⁶⁵ release, and the initiation of eicosanoid synthesis via arachidonate release from phospholipids⁶⁶ are consistent with a key role of this mediator in atherogenesis.

Our present data reveal that the production of PAF in lipoproteins is dependent on a low level of acetylhydrolase activity. We presume that the level of PAF in the arterial intima is controlled by both lipoprotein-associated and macrophage-associated acetylhydrolase. In this context, it is relevant that we have shown recently,^25,36 and now confirm using distinct LDL particle subpopulations, that acetylhydrolase is progressively inactivated on oxidation of LDL, which thus loses its ability to protect against the proinflammatory actions of PAF. However, we have clearly demonstrated that small, dense LDL particles may conserve a part of their initial acetylhydrolase activity on copper-mediated oxidation, thereby protecting them from the formation of PAF and presumably other PAF-like molecules.

In conclusion, we have shown that a distinct, C16:0 molecular species of PAF is produced in intermediate LDL particles (d=1.029 g/mL to 1.039 g/mL) on copper-induced oxidation, suggesting that they may exert multiple proinflammatory, proatherogenic and prothrombogenic effects. In contrast, oxidation of small, dense LDL particles (d=1.050 g/mL to 1.063 g/mL) does not result in formation of significant amounts of PAF, unless their elevated acetylhydrolase activity is decreased to undetectable levels. Nonetheless, substantial amounts of atherogenic lysophospholipids (ether- or ester-linked) are formed that may accumulate at micromolar levels in arterial tissue.

	Selected Abbreviations and Acronyms

 

PAF = platelet-activating factor LDL = low-density lipoproteins HDL = high-density lipoproteins VHDL = very high-density lipoproteins PBS = phosphate-buffered saline MDA = malondialdehyde TBARS = thiobarbituric acid reactive substances GC/MS = gas chromatography/mass spectrometry LCAT = lecithin-cholesterol acyltransferase D3-PAF = deuterium-labeled platelet-activating factor

	Acknowledgments

 
These studies were partially supported by INSERM, by the Franco-Hellenic Program PLATON, by ARCOL (Comité Français de Coordination des Recherches sur l'Athérosclérose et le Cholestérol), and by research grants from the European Community (PL 951115 and 963191) and the National Institutes of Health (HL 34303).

Received February 19, 1997; accepted April 28, 1997.

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