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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:168.)
© 2005 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Low-Density Lipoprotein From Apolipoprotein E-Deficient Mice Induces Macrophage Lipid Accumulation in a CD36 and Scavenger Receptor Class A-Dependent Manner

Zhenze Zhao; Maria C. de Beer; Lei Cai; Reto Asmis; Frederick C. de Beer; Willem J.S. de Villiers; Deneys R. van der Westhuyzen

From the Department of Internal Medicine and Graduate Center for Nutritional Sciences (Z.Z., M.C.d.B., L.C., R.A., F.C.d.B., W.J.S.d.V., D.R.v.d.W.), University of Kentucky Medical Center, Lexington; and Department of Veterans Affairs Medical Center (Z.Z., M.C.d.B., L.C., F.C.d.B., W.J.S.d.V., D.R.v.d.W.), Lexington, Ky.

Correspondence to D.R. van der Westhuyzen, PhD, Department of Internal Medicine and Graduate Center for Nutritional Sciences, Wethington Health Sciences Building 541, 900 S Limestone St, Lexington, KY 40536. E-mail dvwest1{at}uky.edu

	Abstract

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Objective— To investigate the potential of circulating low-density lipoprotein (LDL), isolated from apolipoprotein E (apoE)-deficient mice (E–/–LDL) and from LDL receptor-deficient mice (Lr–/–LDL), to induce foam cell formation.

Methods and Results— Binding studies using COS-7 cells overexpressing CD36, J774 cells, and mouse peritoneal macrophages (MPMs) unexpectedly showed for the first time that E–/–LDL, which is enriched in cholesterol, is a high-affinity ligand for CD36 and exhibited greater macrophage uptake than Lr–/–LDL or normal LDL. Minimal copper-mediated oxidization of Lr–/–LDL or C57LDL in vitro resulted in increased ligand internalization, although cell uptake of these oxidized LDLs was lower than that of E–/–LDL, even at oxidation levels similar to that found in E–/–LDL. Treatment of MPMs with E–/–LDL and Lr–/–LDL (to a 2- to 3-fold lesser extent), but not normal LDL, resulted in significant cellular cholesteryl ester accumulation and foam cell formation. Experiments using MPMs lacking CD36, scavenger receptor class A (SR-A), or both, indicated a major contribution of CD36 (50%), and to a lesser extent, SR-A (24% to 30%), to E–/–LDL uptake.

Conclusions— Because of its increased state of oxidation and high cholesterol content, LDL in apoE-deficient mice acts in a proatherogenic manner, without requiring further modification in the vascular wall, to induce foam cell formation through its uptake by scavenger receptors.

We investigated the atherogenic capability of circulating LDL from apoE-deficient mice and found that it functions in a proatherogenic manner, even without any further modification in vascular wall, through its uptake by scavenger receptors CD36 and SR-A.

Key Words: apolipoprotein E • macrophages • scavenger receptor • CD36 • SR-A

	Introduction

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On a chow diet, apolipoprotein E (apoE)-deficient (apoE–/–) mice have highly elevated plasma cholesterol levels attributable mainly to the accumulation of very-low-density lipoprotein (VLDL) remnants and develop severe atherosclerotic lesions spontaneously.^1,2 LDL receptor-deficient (LDLr–/–) mice, which have mildly increased plasma cholesterol levels from the accumulation of LDL on a chow diet, are also prone to atherosclerosis, but advanced lesions develop only on a high-fat, high-cholesterol diet.^3–5 Mechanisms by which these lipoprotein remnants induce macrophage foam cell formation and subsequent atherogenesis are uncertain.

Earlier studies have shown that VLDL, VLDL/intermediate-density lipoprotein (IDL), and the non-high-density lipoprotein (HDL) fraction from apoE–/– mice induce modest lipid accumulation in cultured macrophages and that modification of these lipoproteins stimulates further lipid uptake, evidently by receptors other than scavenger receptor class A (SR-A) or CD36.^6–9 However, a role for SR-A as well as CD36 in macrophage lipid accumulation has been demonstrated in vitro and in vivo. In vitro studies showed that SR-A and CD36 expression in macrophages accounts for 75% to 90% of degradation of LDL modified by acetylation or oxidation.¹⁰ Studies with SR-A- or CD36-deficient mice showed that disruption of either receptor in mice lacking apoE partially inhibits uptake of acetylated LDL or oxidized LDL (oxLDL) in macrophages and retards atherosclerotic progression.^11,12

To date, no studies have specifically investigated the capability of naturally occurring LDL from hyperlipidemic models (eg, mice lacking apoE) to induce foam cell formation. The study of LDL is potentially important because LDL from apoE–/– mice shows evidence of being oxidized to some extent in vivo.^13,14 Furthermore, compared with VLDL/IDL particles,^6–9 smaller LDL particles more readily enter the arterial wall and accumulate in the subendothelial space, where they may become modified and subsequently taken up by macrophages in an unregulated manner by scavenger receptors.^15,16 In this study, we investigated the abilities of LDL from apoE–/– mice, LDLr–/– mice, and C57BL/6 mice to induce foam cell formation. Our results surprisingly show that circulating E–/–LDL, and to a lesser extent, Lr–/–LDL, are actual ligands for scavenger receptors, particularly CD36, and induce significant macrophage cholesteryl ester (CE) accumulation.

	Methods

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Mice
ApoE–/–, LDLr–/–, and C57BL/6 mice were obtained from Jackson Laboratories. CD36–/– mice were kindly provided by R.L. Silverstein (Weill Medical College of Cornell University, New York, NY). SR-A–/– mice were kindly provided by M.F. Linton (Vanderbilt University Medical Center, Nashville, Tenn). CD36/SR-A double knockout mice were cross-bred in our laboratory. All of the mice were on a C57BL/6 background, aged 6- to 8-weeks old, and maintained in a pathogen-free facility under equal light/dark cycles with free access to water and food. All procedures were approved by the Veterans Administration Medical Center institutional animal use and care committee.

Cell Culture
COS-7 cells and J774 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin (GIBCO/BRL). Mouse peritoneal macrophages (MPMs) were harvested 4 days after intraperitoneal injection of 1 mL 2% bio-gel beads by peritoneal lavage using ice-cold PBS. Cells were washed, counted, and seeded at 2.5x10⁶ cells/12-well plate in DMEM complete medium containing 10% heat-inactivated FCS and 1000 IU/mL recombinant human M-colony-stimulating factor (CSF). Adherent cells were washed after overnight incubation and used.

LDL Particle Isolation and Radiolabeling
ApoE–/–, LDLr–/–, and C57BL/6 mice (20 to 30 each) were fasted 6 hours before blood collection. To minimize oxidation, collected blood was immediately mixed with EDTA (final concentration 2 mmol/L) and cooled on ice. LDL (d=1.019 to 1.063 g/mL) was isolated rapidly by sequential ultracentrifugation using a VTi 90 rotor (Beckman Coulter) as described previously.¹⁷ The isolated LDLs were stored under N₂ gas at 4°C and were used immediately after preparation. Minimally oxidized mouse LDLs were prepared by dialysis against 150 mmol/L NaCl, pH 7.4, containing 5 µmol/L CuSO₄ at 4°C for 2, 4, or 8 hours. Oxidation was terminated by the addition of 10 µmol/L EDTA. Lipid peroxide (LPO) content of LDL formed during oxidation was measured by a modified iodometric method.¹⁸

Lipoproteins were iodinated by the iodine monochloride method.¹⁹ The range of specific activity of iodinated lipoproteins was 50 to 200 cpm/ng protein, and lipid iodination was <0.1% of iodination on protein. The integrity of all labeled lipoproteins was verified by SDS-PAGE and gradient gel electrophoresis. The electrophoretic mobility in nondenaturing agarose gels of E–/–LDL was similar to that of Lr–/–LDL. Lipid compositions were determined enzymatically (WAKO Chemicals). Protein was quantified by Lowry method.²⁰

Adenoviral Vectors Infection
CD36 adenovirus (AdCD36) was prepared as described previously.²¹ AdNull (provided by Dr D.J. Rader, University of Pennsylvania, Philadelphia) is a recombinant virus with analogous adenoviral sequences containing no transgene. COS-7 cells were seeded in 12-well plates 48 hours before assays (2.5x10⁵ cells per well). Overexpression was performed by addition of AdNull and AdCD36 at a viral dose of 1000 particles per cell 24 hours before assay. At this dose, 95% of cells expressed mCD36 as shown by indirect immunofluorescence. Western blotting for CD36 was performed as described previously.²¹

Ligand Binding and Uptake Assays
Cell association and degradation assays, described previously,²² were performed at 37°C in DMEM supplemented with 0.5% essentially fatty acid-free BSA, 1% penicillin and streptomycin, and radiolabeled lipoprotein. All values are shown in terms of LDL protein, which is associated or degraded by the cells. K_d and B_max values were determined by nonlinear regression analysis of receptor-specific values using Prism software (GraphPad Software).

Lipid Accumulation in MPM Cells Exposed to Lipoproteins
MPM cells were collected, seeded, and incubated for 1 day as described above and then incubated with different lipoproteins (150 µg/mL) in DMEM complete medium at 37°C for 2 days. To visualize lipid droplets by oil red O staining, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 minutes, rinsed once quickly in 60% isopropyl alcohol, stained in 0.3% oil red O solution for 10 minutes, washed briefly in 60% isopropyl alcohol, and then mounted using 50% glycerol. To determine CE accumulation, lipids were extracted by incubation for 30 minutes with 2 mL hexane/isopropanol (3:2 by volume), dried under nitrogen gas, dissolved in chloroform with 0.5% Triton X-100, dried again under nitrogen gas, and then resuspended in 0.225 mL water by vortex and incubation at 37°C for 15 minutes. Aliquots of lipid extract were used for determination of total cholesterol and unesterified cholesterol by enzymatic kits (WAKO Chemicals).

Statistics
Data were expressed as mean±SD. Results were analyzed by Student t test. A value of P<0.05 was considered significant.

	Results

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Distinct Lipid and Apolipoprotein Composition of E–/–LDL, Lr–/–LDL, and C57LDL
The lipid composition of LDL particles from 3 types of mice, namely apoE–/–, LDLr–/–, and C57BL/6 mice, differed significantly in composition, the greatest difference being between E–/–LDL and C57LDL (Table). Protein and triglyceride mass percentage values increased in the order of E–/–LDL to Lr–/–LDL to C57LDL, whereas cholesterol (free and esterified cholesterol) and phospholipids decreased in the same order. E–/–LDL is therefore relatively enriched in total and free cholesterol. LDLs also differ in their apolipoprotein composition (Figure I, available online at http://atvb.ahajournals.org). E–/–LDL contains apoB-48 as the major apolipoprotein, as well as significant amounts of apoB-100, apoA-IV, and apoA-I. Lr–/–LDL and C57LDL each contain apoB-100 as the major apoB species, together with apoB-48, apoE, and apoA-I in Lr–/–LDL and apoE and apoA-I in C57LDL. Small amounts of apoA-II and C peptides are found in each of the LDLs.

View this table: [in this window] [in a new window]   Lipid Composition of Mouse LDL Particles

CD36 Binds and Internalizes E–/–LDL
COS-7 cells expressed CD36 through adenovirus-mediated gene transfer at a comparable level to that found in the macrophage cell line, J774 (Figure 1A). E–/–LDL and Lr–/–LDL exhibited saturable high-affinity association to CD36, and association of Lr–/–LDL was 4-fold lower than E–/–LDL (Figure 1B). Significant degradation of E–/–LDL was observed, whereas degradation of Lr–/–LDLwas markedly lower (10- to 15-fold) than that of E–/–LDL (Figure 1C). These data indicate that E–/–LDL is a high-affinity ligand for CD36 and is efficiently internalized by the receptor.

View larger version (21K): [in this window] [in a new window]   Figure 1. CD36-expressing COS-7 cell association and degradation of E–/–LDL and Lr–/–LDL. COS-7 cells were treated with AdCD36 or control AdNull virus for 24 hours as described in Methods. A, Immunoblot for CD36 (10-µg cell protein per lane). Lanes 1 and 2, AdCD36-treated cells; lanes 3 and 4, AdNull-treated cells; lanes 5 and 6, J774 cells. Concentration-dependent cell association (B) or degradation (C) of 125I-E–/–LDL and 125I-Lr–/–LDL at 37°C for 2 hours was quantified as described in Methods. Shown are CD36-specific values, which were calculated as the difference between AdCD36-treated cells and AdNull-treated cells. Apparent association Kd values for E–/–LDL and Lr–/–LDL were 16±2 and 23±5 µg/mL; and Bmax values were 1667±60 and 447±45 ng ligand/mg cell protein, respectively.

Macrophages Bind and Take Up E–/–LDL and Lr–/–LDL in a CD36 and SR-A-Dependent Manner
E–/–LDL and Lr–/–LDL associated with J774 cells at 37°C in a high-affinity saturable manner with an 2-fold greater association of E–/–LDL than Lr–/–LDL (Figure 2A). A more marked difference between 2 LDLs was seen in ligand degradation that was 5-fold higher for E–/–LDL than Lr–/–LDL (Figure 2B). The greater difference in degradation of the 2 LDLs, compared with the difference in their cell association, suggests a lower efficiency of internalization of Lr–/–LDL compared with E–/–LDL, or that Lr–/–LDL is internalized but is more resistant to degradation, or that some Lr–/–LDL is bound to surface sites that do not mediate internalization.

View larger version (22K): [in this window] [in a new window]   Figure 2. J774 cell association and degradation of E–/–LDL and Lr–/–LDL. J774 cells were incubated with 125I-E–/–LDL and 125I-Lr–/–LDL at 37°C for 2 hours at the indicated concentration. Cell association (A) and degradation (B) were quantified as described in Methods. Apparent association Kd values for E–/–LDL were 4.4±0.7 and 6.4±1.0 µg/mL, respectively; and Bmax values were 1121±50 and 653±32 ng protein/mg cell protein, respectively.

As observed in J774 cells, E–/–LDL exhibited significantly greater degradation (2.5-fold) than Lr–/–LDL in MPMs from normal mice (Figure 3A). Degradation was significantly reduced (50%) for E–/–LDL, to a lesser extent (24% reduction) for Lr–/–LDL, in MPMs lacking CD36. Decreased ligand degradation was also observed in SR-A–/– cells (Figure 3B). The SR-A dependent contribution was similar for E–/–LDL (30%) and Lr–/–LDL (25%). In MPMs lacking CD36 and SR-A, degradation of E–/–LDL or Lr–/–LDL was further decreased, resulting in similar degradation values for 2 ligands. Cell association values showed similar trends as those seen with ligand degradation (data not shown). These results indicate that CD36 and SR-A are responsible for the difference in macrophage uptake of the 2 ligands, and that CD36 contributes more to the uptake of E–/–LDL than SR-A.

View larger version (21K): [in this window] [in a new window]   Figure 3. Cellular degradation of E–/–LDL and Lr–/–LDL in MPM cells. MPMs were collected and seeded as described in Methods and then incubated for 5 hours at 37°C with 125I-E–/–LDL or 125I-Lr–/–LDL, each at the concentration 10 µg/mL. Cell degradation in C57 and CD36–/– MPMs (A), and in C57, SR-A–/–, and CD36/SR-A double knockout MPMs (B) were determined as described in Methods. +P<0.05 vs C57; *P<0.05 vs E–/–LDL.

Effect of Oxidation on LDL Uptake and Degradation
We next assessed the extent to which lipoprotein oxidation might contribute to the enhanced uptake of E–/–LDL. LPO content in freshly isolated E–/–LDL was 2.5-fold higher than that in Lr–/–LDL or C57LDL (Figure 4). Oxidation over short periods in vitro increased LPO content in Lr–/–LDL and C57LDL. CD36-dependent degradation in COS-7 cells of minimally oxidized Lr–/–LDLs or C57LDLs was increased with increasing degree of oxidation. A similar increase in degradation of Lr–/–LDL with increasing levels of ligand oxidation was also observed in MPMs from C57BL/6J mice (data not shown). Despite the increased degradation observed with increased ligand oxidation, degradation of 4-hour and even 8-hour oxidized Lr–/–LDL or C57LDL was lower than that of E–/–LDL, although these ligands had levels of oxidation similar to E–/–LDL. Values for ligand cell association showed similar trends to those observed for degradation (data not shown). These results indicate that an increased level of oxidation of E–/–LDL might account, at least in part, for its greater uptake and degradation.

View larger version (25K): [in this window] [in a new window]   Figure 4. Cellular degradation of oxidized mouse LDLs. LDLs were oxidized for the indicated times as described in Methods. LPO content of LDLs is expressed as nmol/mg protein. CD36-specific degradation of native and oxLDL ligands (E–/–LDL, Lr–/–LDL, and C57LDL) was quantified as in Figure 2. +P<0.05 vs oxidized Lr–/–LDL (4 hours and 8 hours); *P<0.05 vs E–/–LDL; P<0.05 vs oxidized C57LDL (4 hours and 8 hours).

E–/–LDL Induces Foam Cell Formation in a CD36- and SR-A-Dependent Manner
E–/–LDL exposure induced significant lipid droplet formation in macrophages, and Lr–/–LDL induced fewer lipid droplets and C57LDL none (Figure 5A). Lipid droplets after exposure to E–/–LDL were distinctly reduced in CD36–/– MPMs, and further reduced but still seen in CD36/SR-A double knockout macrophages.

View larger version (48K): [in this window] [in a new window]   Figure 5. Macrophage foam cell formation induced by E–/–LDL and Lr–/–LDL. MPMs from C57, CD36–/–, and CD36/SR-A double knockout mice were collected and incubated at 37°C for 2 days in complete DMEM media (with 10% FBS and 1000 IU/mL M-CSF) containing E–/–LDL, Lr–/–LDL, or C57LDL at a concentration of 150 µg/mL. A, Cells stained with oil red O. Higher (x60) magnification images in C57 macrophages are shown in the top row. B, Lipids were extracted and measured as described in Methods. CE mass is expressed relative to that in E–/–LDL-treated C57 cells, which was 48.3±16.6 µg CE/mg cell protein. +P<0.05 vs C57; *P<0.05 vs E–/–LDL.

E–/–LDL treatment caused significant cellular CE accumulation in C57 MPMs to levels 2-fold greater than with Lr–/–LDL, whereas C57LDL led to negligible CE accumulation (Figure 5B). CE accumulation induced by E–/–LDL or Lr–/–LDL was significantly reduced in MPMs lacking CD36 or SR-A, and further reduced in cells lacking the 2 receptors. In CD36–/– cells, the reduction of CE accumulation was greater for E–/–LDL (50%) and less for Lr–/–LDL (20%). These results show that E–/–LDL, and to a lesser extent, Lr–/–LDL, increase more foam cell formation in a CD36- and SR-A-dependent manner.

	Discussion

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The marked difference observed in MPMs between binding and internalization of E–/–LDL and Lr–/–LDL was also shown in macrophage cell line J774 that expresses CD36 and SR-A. These cells do not express apoE,²³ indicating that E–/–LDL internalization occurred by an apoE-independent mechanism. This was confirmed by enhanced uptake of E–/–LDL in MPMs from apoE–/– mice (data not shown). Unlike Lr–/–LDL, E–/–LDL contains apoB-48 as the major apolipoprotein and very small amounts of apoB-100.²⁴ Together, these results demonstrate that uptake of E–/–LDL was not dependent on LDL receptor or a related receptor, such as LRP, that recognizes either apoB-100 or apoE as ligands. The pathway by which CD36 mediates the internalization of lipoprotein ligands such as oxLDL is still poorly defined.²⁵ It was reported recently that CD36-mediated endocytosis of oxLDL uses lipid rafts in a pathway that does not require caveolin-1 or clathrin.²⁶ Our results in J774 cells similarly indicate the uptake of E–/–LDL via a caveolin-1-independent pathway because J774 cells lack caveolin-1.²⁷

Previous studies of apoB-containing lipoproteins from apoE–/– mice showed induction of foam cell formation, and the identity of the receptor(s) responsible for lipid uptake in those studies is not known.^6–9 The present study shows for the first time that E–/–LDL, unlike Lr–/–LDL, is a circulating lipoprotein fraction that is an efficient ligand for CD36. One explanation for this is a greater degree oxidation in E–/–LDL. Studies have documented previously that E–/–LDL is oxidized in vivo, for example, autoantibodies to oxLDL epitopes are found in apoE–/– mouse plasma,^13,14 and E–/–LDL shows increased lipid peroxidation.^28,29 As shown in this study, freshly isolated E–/–LDL has a 2.5-fold higher LPO content than Lr–/–LDL or C57LDL, probably the result of the different degrees of hypercholesterolemia in these mice. CD36, unlike SR-A, has a preference for less or "minimal" oxLDL.^25,30 CD36 can mediate uptake of modified lipoprotein particles through an interaction with the oxidized lipid moiety,³¹ which can be in the lipid phase or covalently attached to apoB.^31,32 For this reason, CD36, rather than SR-A, may be the preferred receptor for E–/–LDL that has undergone minimal oxidation. This is supported by our experiments using minimally oxidized Lr–/–LDL or C57LDL, which indicate that minimal ligand oxidation might contribute to the greater ligand activity of E–/–LDL.

Another modification that E–/–LDL undergoes in vivo is aggregation,^33,34 a process that is enhanced by E–/–LDL oxidation³⁴ as well as by sphingomyelin hydrolysis.^9,35,36 Uptake of aggregated lipoproteins could occur through the process of patocytosis.^37,38 Although we cannot rule out an effect of ligand aggregation in our experiments, filtering the E–/–LDL ligand using a 0.22-µm filter to remove any larger aggregates immediately before incubation with macrophages had no effect on foam cell formation (data not shown). We conclude that the greater efficiency of E–/–LDL uptake by scavenger receptors, together with the greater content of cholesterol carried in each E–/–LDL particle, account for the greater macrophage lipid accumulation caused by E–/–LDL compared with Lr–/–LDL or control LDL.

A modest level of E–/–LDL uptake and CE accumulation occurs even in MPMs lacking CD36 and SR-A, indicating the possible involvement of another receptor(s). These candidates include other scavenger receptors that interact with modified lipoproteins.³⁹ We showed previously that scavenger receptor BI, an HDL receptor, did not mediate the uptake of apoB-containing lipoproteins from apoE–/– mice, despite high-affinity, high-capacity binding.²⁴ Macrosialin, a type D scavenger receptor, has been shown by ligand blotting to bind oxLDL.^40–42 However, we reported recently that this receptor, which is predominantly an intracellular protein, does not bind oxLDL at macrophage cell surface, and is therefore unlikely to mediate E–/–LDL-induced lipid accumulation.¹⁷ Other candidates include lectin-like oxLDL receptor 1, a macrophage receptor containing a collagenous domain, and endothelial cell scavenger receptor. Such pathways are unlikely to contribute to Lr–/–LDL uptake because there was negligible CE accumulation induced by this ligand in CD36/SR-A double knockout macrophages.

In summary, we show for the first time that a circulating plasma LDL fraction namely, LDL from apoE–/– mice, functions as an efficient ligand for CD36 without requiring further modification and promotes macrophages foam cell formation. Enhanced uptake of E–/–LDL by scavenger receptors, particularly CD36, may contribute to the spontaneous development of atherosclerosis in apoE-deficient mice.

	Acknowledgments

 
This work was supported in part by National Institutes of Health Grants AA-00292 (W.J.S.d.), HL-65730 and HL-63763 (D.R.v.). The authors thank Xin Shi, Wei Shi, Jason Cupp, and Nathan Whitaker for excellent technical assistance, and Nancy R. Webb for critical review of this manuscript.

Received August 4, 2004; accepted October 14, 2004.

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