Characterization of peroxynitrite-oxidized low density lipoprotein binding to human CD36
Introduction
Several lines of evidence indicate a central role for oxidized low density lipoprotein (OxLDL) in the pathogenesis of atherosclerosis. Of particular relevance to atherogenesis has been the demonstration that OxLDL is recognized by scavenger receptors and contributes to the formation of foam cells (reviewed in [1], [2], [3], [4]). Based on in vitro studies, a variety of additional atherogenic properties have been attributed to OxLDL, including: the recruitment, retention, and proliferation of macrophages in the intima due to the release of chemotactic factors; enhanced expression of endothelial cell adhesion molecules; the induction of macrophage signaling pathways that include phosphatidylinositol 3-kinase [5], [6], [7], [8]; and cytotoxicity of endothelial cells and inhibition of NO-mediated vasodilation [9], [10], [11].
The exact mechanisms whereby LDL is oxidized in vivo are unknown. A variety of methods have been used to generate OxLDL in vitro, including endothelial or smooth muscle cell-mediated oxidation [12], [13], [14] and cell-free systems, in which oxidation is induced with: transition metals such as copper or iron [14], [15]; peroxynitrite [16], [17], [18]; myeloperoxidase [19], [20], [21]; or lipoxygenase [22], [23]. Because of its simplicity, generating maximally oxidized LDL by exposure to CuSO4 for 18–24 h, has become a standard method. However, Cu2+ oxidation does not correspond to a physiological event since free copper is not present in vivo. Furthermore, the ligand generated by maximally oxidizing LDL with CuSO4 may not be representative of OxLDL found in vivo, which is of particular importance for studies assessing the structure-function relationships of lipoprotein receptors with their ligands and the biologic effects of OxLDL [3].
A number of more physiologic mechanisms by which LDL may be oxidized in vivo have been identified. These include lipoxygenase enzymes, glycoxidation, peroxynitrite, and myeloperoxidase [16], [19], [20], [21], [22], [23]. Although the relative contribution of each of these to oxidative modification of LDL in vivo is unknown, recent evidence supports a role for peroxynitrite [24], [25]. Endothelial cells, monocytes, macrophages and neutrophils at the lesion site have the potential to produce both nitric oxide (NO) and superoxide (O2−) [18], [25], [26], [27]. NO and O2− rapidly react to generate peroxynitrite (ONOO−) which is a potent oxidizer of LDL in vitro [17], [28], [29]. In vitro, ONOO− reacts with tyrosine to generate 3-nitrotyrosine, which has been shown to be a highly stable and specific marker for oxidative modification of LDL by peroxynitrite [24], [30]. Recent studies, indicating that LDL isolated from atherosclerotic lesions had 90-fold higher levels of 3-nitrotyrosine than plasma from healthy controls, provide strong evidence for the involvement of peroxynitrite oxidation of LDL in vivo [24], [25].
Several receptors have been implicated in the binding and uptake of OxLDL in vitro, including CD36, AcLDL-R, SR-BI, CD68/macrosialin and LOX-1 [31], [32], [33], [34], [35], [36]. The in vivo roles of these receptors in OxLDL uptake have yet to be elucidated, but recent studies support a role for CD36. CD36 is expressed on cells (monocytes/macrophages, endothelial cells and platelets) [37], and is known to participate in atherosclerosis. It has been shown to be a high affinity receptor for OxLDL and cells transfected with CD36 bind and internalize the particle [31], [34], [38]. It has recently been reported that CD36 also binds HDL, VLDL and LDL, but does not internalize these ligands [39], [40]. Evidence for an in vivo role for CD36 comes from studies by Nozaki et al. [41]. Macrophages from CD36-deficient individuals were shown to have a low capacity to bind, internalize and degrade OxLDL and they accumulated significantly less cholesterol ester than control macrophages. It has recently been shown that OxLDL up-regulates expression of CD36 via a PPARγ pathway [42], [43]. This could lead to enhanced uptake of OxLDL and foam cell formation via a positive feedback loop. NF-κB binding activity is enhanced following OxLDL binding to TNF-α activated cells expressing CD36, therefore, CD36 mediates signal transduction events in response to OxLDL [44].
Given the growing evidence supporting a role for both peroxynitrite-mediated oxidation of LDL and CD36 in atherogenesis, the objectives of this study were to compare and contrast Cu2+ and peroxynitrite oxidation of LDL, to characterize the biochemical changes in the particle and to examine the binding of LDL, oxidized by each method, to the scavenger receptor CD36. It was demonstrated that peroxynitrite-OxLDL is a suitable physiological model for studying lipoprotein-receptor and cellular interactions.
Section snippets
Isolation and oxidation of LDL
Human LDL was isolated from pooled serum by ultracentrifugation between densities 1.019 and 1.063g/ml at 45 000 rpm for 18 h at 10°C, using a model 50.3 Ti rotor in an L8-80 centrifuge (Beckman Instruments Inc. Fullerton, CA) [45]. Isolated LDL was dialyzed against 0.15 M NaCl, 0.24 mM Na2EDTA at pH 7.4 and diluted with 1 M glycine/NaOH buffer, pH 10.0, prior to iodination with Na125I (Amersham, IL) [46]. Unbound iodine was removed by chromatography on QAE-Sephadex A-25 (Pharmacia, Baie d'Urfe,
Kinetics of modification of LDL following Cu2+ and SIN-1 treatment
3-Morpholinosydnonomine hydrochloride (SIN-1) was selected as the source of peroxynitrite in these experiments since the reaction of SIN-1 with aqueous solutions immediately results in the simultaneous generation of equivalent amounts of NO and O2− that interact rapidly to form ONOO− [18]. ONOO− generated using SIN-1 oxidizes LDL [16], [53]. An analysis of the electrophoretic migration of LDL, on agarose gels, revealed an increase in the mobility of LDL following oxidative modification by
Discussion
A variety of techniques are used for oxidation of LDL in vitro and the LDL particles generated are complex and have not been fully characterized as to their chemical nature and extent of the damage to the LDL particle. Moreover, not all methods of oxidation are physiologically relevant. The aims of this study were 2-fold: (1) to determine whether a more physiological method of oxidation of LDL using SIN-1 to generate peroxynitrite, produces an oxidized particle with similar chemical and
Acknowledgements
This research was supported by the Medical Research Council of Canada (MT-13721) and the Heart and Stroke Foundation of Ontario (NA-3391). RG is supported by the Natural Sciences and Engineering Research Council of Canada. KCK was supported by a Career Award from the Ontario Ministry of Health.
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