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Cardiovascular Research 2003 58(3):712-720; doi:10.1016/S0008-6363(03)00257-8
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Copyright © 2003, European Society of Cardiology

Formation of apolipoprotein AI–phosphatidylcholine core aldehyde Schiff base adducts promotes uptake by THP-1 macrophages

Zakaria Ahmeda, Amir Ravandib, Graham F Maguirec, Arnis Kuksisb and Philip W Connellyc,d,e,f,*

aDepartment of Pathology and Laboratory Medicine, University of Louisville Medical Center, Louisville, KY 40202, USA
bBanting and Best Department of Medical Research, University of Toronto, Toronto, Canada
cJ. Alick Little Lipid Research Laboratory, St. Michael's Hospital, 38 Shuter Street, Room 1004 WA, Toronto, Ontario M5B 1A6, Canada
dDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
eDepartment of Biochemistry, University of Toronto, Toronto, Canada
fDepartment of Medicine, University of Toronto, Toronto, Canada

connellyp{at}smh.toronto.on.ca

* Corresponding author. Tel.: +1-416-864-6023; fax: +1-416-864-5870.

Received 29 August 2002; accepted 14 January 2003


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: High-density lipoprotein (HDL) is believed to protect against development of atherosclerosis by inhibiting the accumulation of oxidized lipids in low-density lipoprotein (LDL). Paradoxically, HDL lipid is more susceptible to oxidation than LDL lipid. In the present study, we examined the effect of oxidized phospholipids on the uptake of HDL by macrophages. Methods and results: Oxidation of HDL increased formation of phosphatidylcholine core aldehydes that was paralleled by increased covalent binding of phospholipids to HDL protein from 0.96±0.44 to 8.5±1.76 phosphorus/HDL protein (mol/mol). Incubation of apolipoprotein AI with synthetically prepared phosphatidylcholine core aldehydes, 1-palmitoyl-2-[5-oxo]valeroyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-[9-oxo] nonanoyl-sn-glycero-3-phosphocholine, significantly increased the phosphorus:apolipoprotein AI ratio from 1.1±0.5 to 7.2±2.0 and from 0.9±0.6 to 8.5±0.8, respectively. The binding and uptake of phosphatidylcholine core aldehyde–apolipoprotein AI proteoliposomes, by THP-1 macrophages, was similar to that observed for oxidized HDL and oxidized LDL. Conclusion: We conclude that oxidation of HDL increased formation of phosphatidylcholine core aldehyde–apolipoprotein AI Schiff base adducts and enhanced uptake of oxidized HDL by THP-1 macrophages.

KEYWORDS ApoAI, apolipoprotein AI; DiI, 1,1'-dioctadecyl-3,3,3'3'tetramethyl-indocarbocyanine perchlorate; DiO, 3,3'dihexadecyloxacarbocyanine perchlorate; DMPC, dimyristoyl phosphatidylcholine; HDL, high-density lipoproteins; LDL, low-density lipoproteins; PC, phosphatidylcholine; POVPC, 1-palmitoyl-2-[5-oxo]valeroyl-sn-glycero-3-phosphocholine; LC–ESI-MS, liquid chromatography–electrospray ionization-mass spectrometry; PONPC, 1-palmitoyl-2-(9-oxo)nonanoyl-sn-glycero-3-phosphocholine; SDS– PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Protein-bound aldehydes have been proposed as a useful biomarker in degenerative diseases associated with oxidative stress, such as cardiovascular disease and diabetes [1]. Modification of apolipoprotein B by aldehydic fatty acid degradation products plays a crucial role in foam cell formation and in the pathogenesis of atherosclerosis [2]. Macrophage-derived foam cells in atheroma are characterized by co-localization with protein–aldehyde adducts, formed by reactive aldehydes generated from oxidized fragmented fatty acids [3,4] or fragmented fatty acids attached to the glycerol backbone of oxidized phospholipids [5]. Phosphatidylcholine core aldehydes are oxidation products of phosphatidylcholine (PC), generated during oxidation of low-density lipoprotein (LDL) [6,7]. High- or low-molecular-weight aldehydes are chemically-reactive compounds capable of forming stable adducts with amino acids, peptides, and apolipoprotein B [1,8–10]. Modification of apolipoprotein B-containing lipoprotein(s), and modified phospholipids are the main reason for the formation of lipid-laden macrophages in atherosclerotic lesions [3,5]. It has been reported that lipoproteins other than LDL are present in atherosclerotic lesions [11], and that oxidation of these lipoproteins [12,13] could contribute to the initiation of atherosclerosis [14], possibly by modification of the associated proteins. We have recently shown that high-density lipoprotein (HDL) is readily oxidized by the peroxynitrite donor, 3-morpholinosydnonimine, with increased formation of PC core aldehydes [15]. The effect of these products on apolipoprotein AI (apoAI), the major apolipoprotein of HDL, is not known. HDL is sensitive to oxidation [16], as demonstrated by changes in physical characteristics and immunological properties of apoAI and apolipoprotein AII, by modification with aldehydes, 4-hydroxynonenal and malondialdehyde [17].

Here, we report that oxidation of HDL results in the increase in the amount of tightly-bound phosphorus, consistent with formation of Schiff base adducts of apoAI with PC core aldehydes. Formation of Schiff bases was associated with significant uptake by THP-1 macrophages, of oxidized HDL and apoAI proteoliposomes enriched with PC core aldehydes.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine and dimyristoylglycerophosphocholine (DMPC) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). 1,1'-Dioctadecyl-3,3,3'3' tetramethylindocarbocyanine perchlorate (DiI), and 3,3'dihexadecyloxacarbocyanine perchlorate (DiO), were obtained from Molecular Probes (Eugene, OR, USA). 3-Morpholinosydnonimine, dimethylsulphoxide, lipoprotein deficient serum, diethylenetriamine pentacetic acid and paraformaldehyde were purchased from Sigma (St. Louis, MO, USA). ApoAI was purified and apoAI antibodies were prepared as previously described [15]. 1-Palmitoyl-2 (5-oxo)valeroyl-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-(9-oxo) nonanoyl-sn-glycero-3-phosphocholine (PONPC) were prepared in the laboratory by reductive ozonization [18]. Prior to use, each aldehyde was purified by reversed-phase HPLC to separate it from the carboxylate oxidation product. Choline phospholipids were measured using the phospholipid kit from Boehringer Mannheim. All solvents used in liquid chromatography–mass spectrometry were HPLC grade. Other solvents and chemicals were of reagent grade, provided by local suppliers.

2.2 Isolation and oxidation of lipoproteins
LDL and HDL were isolated from serum taken from subjects fasted for 12–14 h, by ultracentrifugation between densities 1.019–1.063 and 1.063–1.21 g/ml, respectively [19], and dialyzed in 0.1 M phosphate-buffered saline, pH 7.4. HDL (1 mg choline phospholipid/ml) was exposed to 1 mM 3-morpholinosydnonimine for up to 6 h at 37°C in the presence of 100 µM diethylenetriamine pentacetic acid, a metal ion chelator. HDL protein concentration was measured by a modified Lowry method [20]. Oxidation was stopped by the addition of 100 µM butylated hydroxytoluene, and lipid-soluble oxidation products were extracted with chloroform–methanol (2:1, v/v) as described [21].

2.3 Phosphorus assay
Native and oxidized HDL, in phosphate-buffered saline, pH 7.4, were incubated overnight at 4°C in the presence of NaCNBH3 at a final concentration of 30 mM, in order to stabilize the Schiff base adducts of aldehydes and protein amino groups [22]. HDL lipids were extracted using ethanol–ether (3:1, v/v) [23]. Solvent was evaporated under N2 and protein-bound phosphorus was determined immediately, or on samples stored at –20°C. In separate experiments, purified apoAI was incubated at 37°C for 4 h with synthetically-prepared PONPC or POVPC. The mixture was treated with NaCNBH3 and lipids were extracted with ethanol–ether (3:1, v/v). Delipidated protein was dialyzed against water and protein concentration was measured [20]. The amount of phosphorus bound to HDL proteins or apoAI was determined using KH2PO4 as a standard [24]. Briefly, samples were digested overnight with perchlorate at 180°C and, after cooling, phosphorus was measured at 800 nm after addition of ammonium molybdate solution.

2.4 Formation of apoAI-PC core aldehyde Schiff base adducts
ApoAI proteoliposomes were prepared according to Sorci-Thomas et al. [26] using apoAI–POVPC (PONPC)–DMPC–cholesterol in a molar ratio 1:18:82:5. Phosphatidylcholine core aldehyde proteoliposomes were incubated for 6 h at 37°C, to allow Schiff base adduct formation. The complexes were then reduced with 30 mM NaCNBH3 for 1 h at 4°C, to convert the Schiff base adducts to stable amide structures and dialyzed overnight. Modification of apoAI by phosphatidylcholine core aldehyde was determined by phosphorus assay, as described above. ApoAI was also analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as described [15].

2.5 Cell culture
THP-1 cells, a human monocyte cell line, were obtained from American Type Culture Collection (TIB 202) and propagated in RPMI 1640, 10% fetal calf serum, containing penicillin (100 units/ml) and streptomycin (100 µg/ml), at 37°C in 5% CO2. Cells were plated at a density of 1x106 cells/ml in medium containing 10% fetal calf serum. The cells were then washed extensively with serum-free RPMI medium, diluted to the appropriate density and used as described [25].

2.6 Preparation of DiI–HDL and DiI–PC core aldehyde proteoliposomes
Native HDL and LDL, oxidized HDL and oxidized LDL and apoAI proteoliposomes, containing POVPC or PONPC, reduced with NaCNBH3 as described above, were labeled with either DiI or DiO as described [27]. Briefly, 300 µl of DiI solution in dimethyl sulfoxide (3 mg/ml) was added slowly to 5 ml HDL (2 mg protein/ml), predialyzed in 0.1 M phosphate-buffered saline, pH 7.4. The mixture was agitated gently and incubated for 8 h at 37°C under nitrogen in the dark. DiI-labeled HDL and oxidized HDL were resuspended in lipoprotein-deficient serum, reisolated by ultracentrifugation (d1.063–1.21 g/ml) and assessed by agarose gel electrophoresis. DiI-labeled lipoproteins or phosphatidylcholine core aldehyde proteoliposomes were also purified from excess label, using Sephadex G-25 (medium grade) column chromatography (Amersham Biosciences, Piscataway, NJ, USA).

2.7 Interaction of oxidized HDL with THP-1 cells
THP-1 cells were seeded on culture slides placed in 24-well culture plates at a density of 1x104 cells/ml in RPMI-1640 medium with 10% fetal calf serum. Transformation of monocyte to macrophage was performed by adding 164 nM phorbol myristate acetate for 36 h, prior to incubation with labeled lipoproteins. The cells were then washed with serum-free RPMI-1640 medium, followed by incubation with DiI-labeled oxidized HDL, DiI–oxidized HDL, DiO-labeled native LDL, DiO-labeled oxidized LDL or DiI–phosphatidylcholine core aldehyde proteoliposomes (POVPC or PONPC). Cell viability exceeded 90%, as determined by trypan blue exclusion. THP-1 macrophages were incubated for 2 h at 37°C with DiI-labeled lipoproteins or proteoliposomes on slides in culture plates. Plates were washed extensively with formalin–phosphate-buffered saline, pH 7.4 (4:96, v/v) as described [27]. Slides were removed from culture plates and examined by confocal microscopy. Fluorescent confocal microscopy was performed using an argon laser with excitation at 488 nm and fluorescence emission at 501 nm for detection of DiO-labeled complexes and using a helium neon laser with excitation at 553 nm and fluorescence emission at 570 nm for detection of DiI-labeled complexes.

2.8 Flow cytometry assay
THP-1 cells were prepared and treated with phorbol myristate acetate, as described above. In separate wells, DiI–HDL, DiI–oxidized HDL, DiO–LDL, DiO–oxidized LDL, in addition to DiI–POVPC or DiI–PONPC-containing proteoliposomes, were added at 25, 50 and 75 µg/ml in serum-free RPMI-1640 medium. Cells were incubated in culture plates for 2 h at 37°C. Cells were washed extensively with 0.5% paraformaldehyde in phosphate-buffered saline, pH 7.4 and prepared for flow cytometry analysis as described [28]. Cells were detached with a rubber policeman and resuspended in 0.5% paraformaldehyde fixative solution prepared in phosphate-buffered saline, pH 7.4 and evaluated by flow cytometry. Flow cytometry experiments measured 10 000 individual cells gated for macrophages, based on their forward and side light-scatter profiles. The mean fluorescence intensity was measured using Coulter FACSORT and CELL QUEST software (Coulter Becton Dickinson, San Jose, CA, USA).

2.9 Liquid chromatography–electrospray ionization-mass spectrometry
Lipid-soluble HDL oxidation products were extracted and the molecular species of the oxidation products were identified, based on the molecular mass provided by ESI-MS, the knowledge of the fatty acid composition of the phospholipid classes and the relative HPLC retention time of the PC standards as described [8].

2.10 Statistical analysis
Means, standard deviation and graphical curve fitting were done using GRAPHPAD–Prism software (San Diego, CA, USA). All results are expressed as mean±standard deviation.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Formation of phosphatidylcholine core aldehydes and modification of apoAI
We have previously shown that oxidation of HDL by 3-morpholinosydnonimine results in the accumulation of 1-palmitoyl (stearoyl)-2-[9-oxo]nonanoyl-sn-glycero-phosphocholine and 1-palmitoyl (stearoyl)-2-[5-oxo]valeroyl-sn-glycero-3-phosphocholine. Aldehydes can form Schiff base adducts with the {epsilon}-amino groups of lysine residues of apolipoproteins. Schiff bases are chemically unstable and reversible. Thus, in order to study these complexes, we first reduced the Schiff base form to a chemically stable adduct. Following the protocol of reduction and exhaustive solvent extraction, native HDL contained 0.9±0.4 mol phosphorus/mol of HDL protein. After 20 h of oxidation, HDL contained 8.5±1.75 mol of phosphorus/mol of HDL protein (Fig. 1A).


Figure 1
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  		Fig. 1 (A) Binding of oxidized phospholipids, as determined by phosphorus:protein molar ratio of delipidated HDL proteins recovered from oxidized HDL (close square) and native HDL (open square). The results represent the mean±S.D. of four experiments. (B) Phosphorus tightly bound to native apoAI (striped bar) and apoAI following incubation with POVPC (solid bar) or PONPC (open bar). The results are the mean±S.D. of three experiments.

 
We hypothesized that adduct formation would occur in the absence of oxidation when phosphatidylcholine core aldehydes were incubated with apoAI. To test this, apoAI proteoliposomes were made using either PONPC or POVPC. The apoAI–PONPC and apoAI–POVPC proteoliposomes were reduced with NaCNBH3. The phosphorus:protein molar ratio of apoAI increased, following a 6-h incubation with either POVPC or PONPC from 1.1±0.5 to 7.2±2.0, and from 0.9±0.6 to 8.5±0.8, respectively (Fig. 1B).

SDS–PAGE of oxidized HDL showed that a portion of apoAI was modified to form apoAI dimer, apoAI:AII heterodimer, and apoAI trimer (Fig. 2, left panel). The identity of these proteins was confirmed by Western blotting using anti-apoAI and anti-apoAII antibodies (data not shown). Modification of apoAI by phosphatidylcholine core aldehydes was characterized by SDS–PAGE (Fig. 2, right panel). Native apoAI was monomeric with a molecular weight of 28 kDa (Fig. 2A, right panel). Following incubation of apoAI with phosphatidylcholine core aldehydes, apoAI appeared as one broad band with a higher apparent molecular weight. PONPC caused a greater shift (Fig. 2C) than POVPC (Fig. 2B). Notably, there was no evidence for crosslinking of apoAI.


Figure 2
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  		Fig. 2 (Left panel) Protein stain of SDS–PAGE of HDL exposed to 3-morpholinosydnonimine for 0 to 20 h. (Right panel) Protein stain of SDS–PAGE of apoAI proteoliposomes containing apoAI–dimyristoylphosphatidylcholine (DMPC) (A), apoAI–DMPC–POVPC (B) or apoAI–DMPC–PONPC (C). The result is representative of three experiments. M, molecular weight markers.

 
3.2 Binding and uptake of oxidized HDL with THP-1 macrophages
The binding and uptake of DiI-labeled oxidized HDL or phosphatidylcholine core aldehyde apoAI proteoliposomes by THP-1 macrophages was studied by confocal microscopy. Oxidized LDL was used as a positive control. Fig. 3 shows confocal microscopy images of binding and uptake of fluorescently-labeled native and oxidized lipoproteins. DiI-labelled oxidized HDL was co-incubated with DiO-labeled oxidized LDL (25 µg protein/ml) to assess co-localization. The binding and uptake of DiI-labeled native HDL (Fig. 3A) or DiO-labeled native LDL (Fig. 3B) showed a diffuse pattern of fluorescence of low intensity. In contrast to native HDL, DiI-labeled oxidized HDL, when incubated alone with THP-1 cells, resulted in significant binding and uptake (results not shown). Co-incubation of DiI-labeled oxidized HDL (Fig. 3C and E) and DiO-labeled oxidized LDL (Fig. 3D and E) with THP-1 macrophages showed an overlapping distribution of oxidized HDL and oxidized LDL (Fig. 3E. The yellow color results from the co-localization of green and red).


Figure 3
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  		Fig. 3 Confocal microscopy showing binding and uptake of fluorescently labeled lipoproteins by THP-1 macrophages. DiI-labeled HDL (A); DiI-labelled LDL (B); DiI-labeled oxidized HDL (C) in the presence of DiO-labeled oxidized LDL (D). Superimposed images of uptake of DiI-labeled oxidized HDL and DiO-labeled oxidized LDL (E). Preparation of cells, incubation conditions and analysis by confocal microscopy were as detailed under experimental procedures. The images are representative of three separate experiments. Images were taken at 60x magnification and are representative of three separate experiments.

 
Next, we compared the binding and uptake of DiI-labeled proteoliposomes containing either POVPC or PONPC with the binding and uptake of DiO-labeled oxidized LDL (Fig. 4). Confocal microscopy of DiI-labeled POVPC-containing proteoliposomes (Fig. 4B) in the presence of DiO-labeled oxidized LDL (Fig. 4C and D) showed predominantly a morphological pattern of overlapping fluorescence of DiI-labeled POVPC proteoliposomes and DiO-labeled oxidized LDL. There were instances of a cell binding DiI-labeled POVPC proteoliposomes but not DiO-labeled oxidized LDL. The frequency and reproducibility of this phenomena was not investigated further. Confocal microscopy of DiI-labeled PONPC-containing proteoliposomes (Fig. 4E) in the presence of DiO-labeled oxidized LDL (Fig. 4F and G) showed a morphological pattern of overlapping fluorescence of DiI-labeled PONPC proteoliposomes and DiO-labeled oxidized LDL. There was little uptake of DiI-labeled DMPC proteoliposomes (Fig. 4A). Similar images were seen for the uptake of DiI-labeled POVPC or PONPC proteoliposomes in the absence of DiO-labelled oxidized LDL (results not shown). In order to quantitate and demonstrate a dose–response curve for the binding and uptake of oxidized HDL or phosphatidylcholine core aldehyde proteoliposomes by THP-1 macrophages, we used flow cytometry to measure the mean cell-associated fluorescence intensity. The flow cytometry light scattering profile of THP-1 macrophages provided a unimodal distribution, indicating that the cells were homogeneous (Fig. 5A). Incubation of DiI-labeled native HDL at 50 µg protein/ml or 75 µg protein/ml with THP-1 macrophages for 2 h at 37°C, showed little shift in the flow cytometry trace (Fig. 5B and C). In contrast, DiI-labeled oxidized HDL showed a dose-dependent increase in cell-bound fluorescence intensity, when incubated at 25, 50 or 75 µg/ml (Fig. 5D–F). The fluorescence intensity increased about 15-fold during incubation of DiI-labeled oxidized HDL, compared to 2-fold with DiI-labeled HDL (Fig. 5, inset).


Figure 4
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  		Fig. 4 Confocal microscopy showing binding and uptake of fluorescently-labeled lipoproteins and liposomes by THP-1 macrophages. DiI-labeled DMPC proteoliposomes (A); DiI-labeled POVPC proteoliposomes (B) in the presence of DiO-labeled oxidized LDL (C); superimposed images of uptake of DiI-labeled POVPC proteoliposomes and DiO-labeled oxidized LDL (D); DiI-labeled PONPC proteoliposomes (E) in the presence of DiO-labeled oxidized LDL (F); superimposed images of DiI-labeled PONPC proteoliposomes and DiO-labeled oxidized LDL (G). Preparation of cells, concentrations of lipoproteins and liposomes, incubation conditions and analysis by confocal microscopy were as detailed under Methods. Images were taken at 60x magnification and are representative of three separate experiments.

 

Figure 5
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  		Fig. 5 Flow cytometry profiles of mean fluorescence intensity with increasing oxidized HDL binding and uptake by THP-1 macrophages. (A) Untreated THP-cells; (B) 50 µg/ml DiI-labeled HDL; (C) 75 µg/ml DiI-labeled HDL; (D) 25 µg/ml DiI-labeled oxidized HDL; (E) 50 µg/ml DiI-labeled oxidized HDL: (F) 75 µg/ml DiI-labelled oxidized HDL. The inset panel represents the increase of mean fluorescence intensity as determined by flow cytometry for DiI-labelled HDL (open squares) and DiI-labelled oxidized HDL (solid squares). Preparation of THP-1 macrophages, lipoprotein incubation conditions and flow cytometry analysis were as detailed under Methods. The results are representative of three separate experiments.

 
The uptake of DiI-labeled oxidized HDL, compared to DiI-labeled oxidized LDL and DiI-labeled POVPC and DiI-labeled PONPC proteoliposomes, by THP-1 macrophages, is shown in Fig. 6. There were dose-dependent increases in fluorescence intensity, due to increased binding and uptake of oxidized HDL, oxidized LDL, and phosphatidylcholine core aldehyde proteoliposomes (Fig. 6). PONPC proteoliposomes achieved maximal binding and uptake at 25 µg/ml, whereas POVPC proteoliposomes achieved maximal binding and uptake at 50 µg/ml. In contrast, while the binding and uptake of oxidized HDL and oxidized LDL showed overlapping curves, neither achieved maximal signal intensity at 75 µg/ml, the highest concentration tested. Further validation of the proportion of fluorescent label incorporated into each complex would be required to definitively interpret these differences.


Figure 6
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  		Fig. 6 Flow cytometry determination of uptake of DiI-labeled oxidized HDL (open square) and DiI-labeled oxidized LDL (closed square), DiI-labeled PONPC proteoliposomes (closed circle), and DiI-labeled POVPC proteoliposomes (open circle). The increase in the fluorescence intensity was determined as described under Methods. Preparation of THP-1 macrophages, incubation conditions and analysis by flow cytometry were as detailed under Methods. The results are representative of three separate experiments.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study demonstrates that oxidation of HDL increased the phosphorus content of HDL proteins. This was paralleled by increased formation of the major oxidized phospholipids, PONPC and POVPC. Modification of HDL proteins by phosphatidylcholine core aldehydes was replicated by addition of POVPC or PONPC to apoAI proteoliposomes. The chain length of core ester aldehyde had no effect on the phosphorus incorporation into apoAI. However, the change in the electrophoretic migration of modified apoAI correlated with the molecular mass of the aldehyde. Modification of apoAI, following incubation with PONPC or POVPC, was distinguishable from apoAI modification in oxidized HDL, shown by the absence of apoAI dimers, consistent with PONPC and POVPC being monovalent aldehydes.

In these experiments, oxidation of HDL increased apoprotein phosphorus content ~8-fold. Previous reports showed that modification of apolipoprotein B by oxidation with copper increased binding of oxidized phospholipids up to 20-fold. This involved 20–30% of the lysine residues of apolipoprotein B [30].

In the present experiments, the modification of apoAI, whether during oxidation of HDL or during incubation with PONPC or POVPC, significantly increased uptake by THP-1 macrophages in a manner indistinguishable from the uptake of oxidized LDL. The uptake of DMPC proteoliposomes by THP-1 macrophages was much lower compared to the uptake of phosphatidylcholine core aldehyde proteoliposomes. These data are consistent with the requirement for specific modifications of the apolipoproteins for recognition by receptors expressed on macrophages, rather than selectivity for a specific apolipoprotein [31]. These results are consistent with the immunohistochemical identification of apoAI in atherosclerotic lesions [12,13]. The intracellular location of apoAI and apoAII in the intima of early stage human coronary and aortic lesions [12] is consistent with the binding and uptake of oxidized HDL by THP-1 cells.

The presence of oxidized phospholipid–apolipoprotein B adducts in vivo is supported by their detection with monoclonal antibodies specific for PONPC, their elevated plasma levels in acute coronary syndromes [32], and their immunologic detection in atherosclerotic lesions [4,33,34]. Hedrick et al. [35] identified oxidized phospholipid epitopes on mouse apoAI 3 days after introducing a hypercholesterolemic diet, providing in vivo evidence for the presence of oxidized phospholipids in HDL. Our data indicate that this modification is functionally significant and could result in the uptake of oxidized HDL by macrophages.

It has been reported that reactive aldehydes, derived from glucose, also modify HDL, but do not promote uptake by macrophages [36]. Combined with the results presented here, the modification of apoAI, following HDL oxidation, and its uptake by THP-1 macrophages suggests a specificity for oxidized phospholipids. Previously, oxidized HDL has been shown to induce scavenger receptors in macrophages [37]. However, the effect of oxidized HDL on the macrophage scavenger receptor expression, whether alone or in the presence of oxidized LDL, was not specifically studied in these experiments.

CD36 and SR-BI are the most likely candidate receptors relevant to atherosclerosis and macrophage foam cell formation [37]. Gillotte et al. [38], reported that oxidized HDL was by far the most potent competitive inhibitor of oxidized LDL binding to SR-BI, while native HDL, LDL or acetylated LDL were less effective competitors. SR-BI shows a broad ligand specificity, as it recognizes native HDL [29], oxidized LDL, anionic phospholipids, apoptotic cells, HDL apolipoproteins and probably other ligands yet to be identified [37,39–41]. Consistent with these reports, non-apoB-containing lipoproteins [42], may play crucial roles in foam cell formation. The presence of apoAI-containing lipoproteins in macrophage and smooth muscle cells in human atherosclerotic tissues provides further evidence for HDL oxidation in vivo [11–13].

It is concluded that the anti-atherogenic potential of HDL becomes compromised by HDL oxidation, modification of apoAI, and uptake of oxidized HDL into THP-1 macrophages. Both oxidized HDL and oxidized LDL may serve to initiate atherosclerosis.

Time for primary review 28 days.


   Acknowledgements
 
We would like to thank Judy Trogadis at St. Michael's Hospital for confocal microscopy analysis, Cheryl Smith, Department of Immunology, University of Toronto for flow cytometry analysis, and Drs. Ian Crandall and Jianshe Zhang, Institute of Medical Sciences, University of Toronto for their assistance in tissue culture. This work was funded by grant no. T4027 and T4948 from the Heart and Stroke Foundation of Ontario.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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