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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:498-504.)
© 1997 American Heart Association, Inc.

Articles

Uptake by J774 Macrophages of Very-Low-Density Lipoproteins Isolated From ApoE-Deficient Mice Is Mediated by a Distinct Receptor and Stimulated by Lipoprotein Lipase

Wendy L. Hendriks; Femke van der Sman-de Beer; Bart J.M. van Vlijmen; Leonie C. van Vark; Marten H. Hofker; ; Louis M. Havekes

From the TNO–Prevention and Health, Gaubius Laboratory (W.L.H., B.J.M. van V., F. van der S.-de B., L.C. van V., L.M.H), and the MGC–Department of Human Genetics, Leiden University (M.H.H.), Leiden, The Netherlands.

Correspondence to Dr L.M. Havekes, TNO–Prevention and Health, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, PO Box 2215, 2301 CE Leiden, The Netherlands. E-mail lm.havekes{at}pg.tno.nl.

	Abstract

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Abstract Apolipoprotein (apo) E–deficient mice display marked accumulation in the plasma of VLDL deficient in both apoE and apoB100 but containing apoB48, apoA-I, apoCs, and apoA-IV. Since apoE-deficient mice develop severe atherosclerotic lesions with lipid-laden macrophages, we reasoned that the uptake of lipoproteins by intimal macrophages can take place in the absence of both apoE and apoB100. To get more insight into the mechanism of foam cell formation in apoE-deficient mice, we measured the interaction of VLDL from apoE-deficient mice (apoE^null VLDL) with the murine macrophage cell line J774. Scatchard analysis revealed that apoE^null VLDL is bound to J774 cells with a K_d value comparable to that of control VLDL (8.1 versus 4.7 µg/mL) and with a B_max value about half that of control VLDL (40 versus 70 ng/mg cell protein, respectively). ApoE^null VLDL is also taken up and degraded by J774 macrophages via a high-affinity process less efficiently than control mouse VLDL (6-fold and 50-fold less efficiently, respectively). In line with this observation, incubation of J774 cells with 50 µg/mL apoE^null VLDL for 24 hours resulted in an increase in intracellular cholesteryl ester (CE) content, although 5-fold less pronounced than after incubation with 50 µg/mL control mouse VLDL. Under the conditions applied, simultaneous addition of 5 µg/mL lipoprotein lipase (LPL) stimulated the cellular uptake and degradation of apoE^null VLDL about 10-fold and resulted in a 5-fold stimulation of the intracellular CE accumulation, from 9±2 to 46±5 µg CE per milligram cell protein. In contrast to control mouse VLDL, apoE^null VLDL could not compete with ¹²⁵I-labeled LDL for binding to the LDL receptor of J774 cells. Furthermore, neither LDL nor acetylated LDL could compete with ¹²⁵I-labeled apoE^null VLDL for binding to these cells, whereas control mouse VLDL, VLDL from a hypertriglyceridemic patient, and apoE^null VLDL itself were efficient competitors. Thus, VLDL from apoE-deficient mice is taken up by J774 macrophages through recognition by a distinct receptor, which could be the triglyceride-rich lipoprotein receptor. We conclude that in apoE-deficient mice, foam cell formation occurs via a receptor-mediated uptake of apoE^null VLDL, which can be stimulated by the presence of LPL.

Key Words: apoE-deficient mice • macrophages • foam cells • receptor-mediated uptake • lipoprotein lipase

	Introduction

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Apolipoprotein E is a major protein constituent of chylomicrons, VLDL, chylomicron and VLDL remnants, and HDL. One of the major functions of apoE in lipoprotein metabolism is that it serves as a ligand for hepatic receptors, mediating the removal of apoE-containing lipoproteins from the circulation.

By using gene targeting in murine embryonic stem cells, several groups have generated mice lacking apoE.^{1 2 3} These mice exhibit extreme hypercholesterolemia that is much more severe than that observed in apoE-deficient humans.^{4 5 6} The reason for this difference might be the fact that in mice, particles containing both apoB100 and apoB48 are produced by the liver, whereas in humans, the liver produces only particles containing apoB100.^{7 8} In contrast to apoB100, apoB48 does not function as a ligand for hepatic lipoprotein receptors, and therefore, apoE-deficient mice display a marked accumulation of chylomicrons and VLDL that are deficient in both apoE and apoB100 but that contain apoB48.¹ Since apoE-deficient mice develop extensive atherosclerotic lesions with lipid-laden macrophages, even on a regular chow diet,⁹ we suggest that uptake of lipoprotein particles into macrophages present in the intima can occur in the absence of both apoB100 and apoE. Hence, additional uptake mechanisms may play a role in the process of foam cell formation in apoE-deficient mice.

To provide more insight into the formation of foam cells in mice lacking apoE, we studied the processing of apoE^null VLDL by J774 macrophages, a murine macrophage cell line that is commonly used as a model for foam cell formation and that does not produce apoE.¹⁰

We also investigated the role of LPL in binding and uptake of apoE^null VLDL by J774 cells, since LPL is synthesized by macrophages present in the intima¹¹ and is known to enhance the binding and uptake of LDL and VLDL by different cell types, including macrophages.^{12 13 14 15 16 17}

Our results show that VLDL deficient in both apoE and apoB100 is taken up by J774 macrophages via a receptor-mediated pathway, leading to the accumulation of CE in these cells. In addition, like normal VLDL, this apoE^null VLDL can also be taken up via an LPL-mediated process.

	Methods

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Cells
Murine macrophage-like J774 cells were cultured in 75-cm² flasks in DMEM supplemented with 10% (vol/vol) FCS, 0.85 g/L NaHCO₃, 4.76 g/L HEPES, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine. The cells were incubated at 37°C in an atmosphere containing 5% CO₂ in air. For each experiment, cells were plated in 12- or 24-well plates. The cells were fed every 3 days and used for experiments within 7 days of plating.

Animals and Lipoproteins
Mice were allowed access to food and water ad libitum. ApoE-deficient mice were generated as described¹ and fed a regular chow diet (SRM-A). The control mice were of a genetic background similar to that of the apoE-deficient mice (129 SvxC57BL/6J). To obtain lipoprotein samples from control mice with a lipid composition comparable to that of apoE-deficient mice, control mice were fed a semisynthetic severe high-fat/cholesterol (HFC 0.5%) diet¹⁸ for a period of 4 weeks, composed essentially according to Nishina et al¹⁹ and purchased from Hope Farms, Woerden, The Netherlands. This diet contains 15% cocoa butter, 1% cholesterol, 0.5% cholate, 40.5% sucrose, 10% cornstarch, 1% corn oil, and 4.7% cellulose (all percentages are by weight) and all the required nutrients, minerals, and vitamins. After a 4-hour fasting period, mice were anesthetized with diethyl ether. Blood was collected from apoE-deficient (n=11) and control (n=62) mice by orbital puncture. The d<1.006 g/mL lipoproteins (VLDL) were isolated from the mouse serum by density gradient ultracentrifugation. Human serum was prepared from freshly collected blood either from healthy volunteers or from a type IV hyperlipidemic patient. Human LDL and HTG-VLDL were isolated from the respective sera by density gradient ultracentrifugation as described by Redgrave et al.²⁰ A portion of the LDL sample was acetylated by repeated addition of acetic anhydride as described by Basu et al.²¹ The conversion of LDL into AcLDL was confirmed by agarose gel electrophoresis (100 V, 30 minutes, Paragon lipoprotein electrophoresis kit, Beckman Instruments Inc). The protein content of the lipoprotein samples was determined according to Lowry et al.²²

Total and free cholesterol, triglyceride (without free glycerol), and phospholipid contents of the lipoproteins were measured enzymatically, using commercially available kits (Nos. 236691 and 310328 from Boehringer Mannheim GmbH, No. 337-B from Sigma Chemical Company, and No. 990-54009 from Wako Chemicals GmbH, respectively).

To determine the relative apolipoprotein composition of the lipoproteins, 12.5 µg of lipoprotein protein was applied on SDS–polyacrylamide gradient gels (4% to 20%). After electrophoresis, the protein was stained with Coomassie brilliant blue, followed by densitometric scanning of the bands with a Hewlett Packard ScanJet Plus.

Mouse VLDL and human LDL were radioiodinated by using the ¹²⁵I-monochloride method described by Bilheimer et al.²³ The specific activity ranged from 90 to 400 cpm/ng protein. After iodination, the lipoprotein samples were dialyzed extensively against PBS (pH 7.4), stabilized with 1% (wt/vol) HSA, stored at 4°C, and used within 2 weeks.

Whenever unlabeled lipoproteins were used, immediately after isolation, dialysis against PBS containing 10 µmol/L EDTA and subsequently DMEM was performed at 4°C.

LPL
LPL was partly purified from fresh bovine milk by using heparin-Sepharose chromatography as described previously.²⁴

Binding and Uptake of Lipoproteins by J774 Macrophages
The J774 cells were cultured in 24-well plates as described above. Twenty-four hours before each experiment, the cells were washed with DMEM containing 1% (wt/vol) BSA and further incubated with DMEM containing 5% (vol/vol) lipoprotein-deficient serum (d<1.21 g/mL) instead of FCS. The binding and uptake studies were subsequently conducted in medium containing no serum components other than the respective lipoproteins and 1% (wt/vol) BSA.

The binding of ¹²⁵I-labeled lipoproteins to the cells in the absence or presence of 5 µg/mL LPL was determined after a 3-hour incubation at 4°C with different concentrations of ¹²⁵I-labeled lipoprotein, either in the presence or absence of a 20-fold excess of the respective unlabeled lipoprotein. The concentrations of the labeled lipoproteins in the respective incubations are described in the text and figure legends. The receptor-mediated (specific) cell binding was calculated by subtracting the amount of labeled lipoproteins that was cell bound after incubation in the presence of the excess of unlabeled lipoprotein (nonspecific) from the amount of labeled lipoprotein that was cell bound after incubation in the absence of unlabeled lipoprotein (total cell binding). After removal of the medium, the cells were washed four times with ice-cold PBS containing 0.1% (wt/vol) BSA and subsequently once with PBS without BSA. Cells were then dissolved in 1 mL of 0.2 mol/L NaOH. Protein content was measured by the method of Lowry et al.²² In an aliquot, the radioactivity represented the amount of cell-bound lipoprotein.

The association and degradation of ¹²⁵I-labeled lipoproteins in the absence or presence of 5 µg/mL LPL was determined after a 3-hour incubation at 37°C with 10 µg/mL ¹²⁵I-labeled lipoprotein either in the absence or presence of 200 µg/mL of the respective unlabeled lipoprotein. At the end of the incubation period, a fraction of the medium was removed to determine the amount of lipoprotein degraded, as described previously.^{25 26} After removing the remaining portion of the medium, the cells were washed four times with ice-cold PBS containing 0.1% (wt/vol) BSA and subsequently with PBS without BSA. To measure the cell-associated (bound plus internalized) lipoprotein fraction, the washed cells were dissolved in 1 mL 0.2 mol/L NaOH and an aliquot of the cell lysate was counted for radioactivity.²⁵ Another aliquot was used for protein determination according to Lowry et al.²²

To identify the nature of the receptor that mediates the binding of apoE^null VLDL to J774 macrophages, competition experiments were performed in which J774 cells were incubated for 3 hours at 4°C with either 10 µg/mL ¹²⁵I-labeled human LDL, 10 µg/mL ¹²⁵I-labeled apoE^null VLDL, or 10 µg/mL ¹²⁵I-labeled HTG-VLDL in the presence of different concentrations of the indicated unlabeled lipoprotein samples. Thereafter, cells were washed and binding at 4°C was measured as described above.

Cellular Accumulation of Lipid
J774 cells were cultured in 12-well plates as described above. Twenty-four hours before the start of the experiment, DMEM supplemented with 5% (vol/vol) lipoprotein-deficient serum instead of FCS was added to the cells. At the start of the experiment, fresh DMEM containing 1% HSA (wt/vol) and 50 µg/mL lipoprotein protein either in the presence or absence of 5 µg/mL LPL was added in triplicate dishes of cells and incubated for 24 hours at 37°C. Control incubations were performed with DMEM/1% HSA without any further additions or with DMEM/1% HSA with 5 µg/mL LPL. At the end of the incubation period, the cells were washed four times with 1 mL of PBS containing 0.1% (wt/vol) BSA followed by one wash with PBS alone. Intracellular lipid content was determined as described by Havekes et al.²⁷ Briefly, the cells were harvested by scraping with a rubber policeman and resuspended by three successive slow passages through a syringe needle (G25). Samples (100 µL) were taken for measurement of protein. Lipids were extracted from the cell suspension using methanol/chloroform (2:1, vol/vol) as described by Bligh and Dyer²⁸ after addition of cholesteryl acetate (2 µg) as an internal standard. The lipids were separated using high-performance thin layer chromatography. Subsequently the lipid bands were quantified densitometrically on a Shimadzu CS910 chromatograph scanner at 380 nm, and areas under the curve were integrated by using a data processor.

	Results

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Lipid and Apolipoprotein Composition of ApoE^null VLDL and Control VLDL
As expected, the cholesterol, triglyceride, and phospholipid levels are similar in VLDL isolated from control mice fed a high-fat, high-cholesterol diet (HFC 0.5%) and in VLDL from apoE-deficient mice fed a regular chow diet (Table 1).

View this table: [in this window] [in a new window]   Table 1. Lipid Composition of Mouse VLDL

In apoE^null VLDL, apoB48, apoA-IV, and apoCs are the major protein constituents, whereas small amounts of apoA-I are also found (Fig 1). No apoB100 or apoE could be detected in this lipoprotein sample. In control mouse VLDL, apoE is the major apolipoprotein, whereas equal amounts of apoB100 and apoB48 are found, in addition to apoA-IV and apoCs.

View larger version (80K): [in this window] [in a new window]   Figure 1. SDS–polyacrylamide gel electrophoresis of apoEnull VLDL and control VLDL. About 12.5 µg of lipoprotein protein was applied on 4% to 20% SDS–polyacrylamide gradient gels. After electrophoresis and staining with Coomassie brilliant blue, the relative apolipoprotein composition of the lipoproteins was determined by densitometric scanning. Lane 1, marker; lane 2, apoEnull VLDL; and lane 3, control VLDL.

Binding and Uptake of Lipoproteins by J774 Macrophages
J774 macrophages were incubated with increasing concentrations of ¹²⁵I-labeled apoE^null VLDL or control VLDL in the absence or presence of a 20-fold excess of unlabeled lipoprotein for 3 hours at 4°C to measure the total and nonspecific binding, respectively. In Fig 2A and 2B, the upper curve represents total binding (specific plus nonspecific) of apoE^null VLDL and control VLDL, respectively, whereas the lower curve represents nonspecific binding, which occurs in the presence of excess unlabeled lipoprotein. The calculated difference between the two curves represents specific binding (middle curve). J774 cells express a saturable binding site for both apoE^null VLDL and control VLDL. Scatchard plot analysis, shown in the insets of the graphs, reveals that the dissociation constants (K_d) are 8.1 and 4.7 µg/mL for apoE^null VLDL and control VLDL, respectively; the B_max of apoE^null VLDL and control VLDL is 40 and 70 ng/mg cell protein, respectively.

View larger version (19K): [in this window] [in a new window]   Figure 2. Binding curves of 125I-labeled apoEnull VLDL and control VLDL to J774 macrophages. The J774 macrophages were incubated for 3 hours at 4°C with increasing concentrations (from 2.5 to 50 µg/mL) of 125I-labeled apoEnull VLDL (A) or control VLDL (B) in the absence or presence of a 20-fold excess of unlabeled lipoprotein to measure total (upper curve) and nonspecific (lower curve) binding, respectively, as described in "Methods." The specific binding (middle curve) was calculated by subtracting the nonspecific binding from the total binding. Values represent the mean±SD of four measurements. Insets represent the respective Scatchard analysis.

As presented in Fig 3A, at 10 µg labeled lipoprotein per milliliter, the high-affinity (receptor-mediated) binding of ¹²⁵I-labeled apoE^null VLDL by J774 macrophages is low compared with ¹²⁵I-labeled control VLDL (13±6 versus 57±2 ng/mg cell protein, respectively). Addition of 5 µg/mL LPL resulted in a 20-fold increase of the binding of control VLDL to J774 macrophages, whereas the binding of apoE^null VLDL was stimulated about 100-fold on addition of LPL (compare Fig 3A with 3B and note the difference in scale).

View larger version (40K): [in this window] [in a new window]   Figure 3. Binding, association, and degradation of apoEnull VLDL and control VLDL in the absence or presence of LPL. The binding (A and B), association (C and D), and degradation (E and F) of apoEnull VLDL and control VLDL were measured on incubation of the cells with 10 µg/mL labeled lipoprotein at 4°C and 37°C for a period of 3 hours in the absence (A, C, and E) or presence (B, D, and F) of 5 µg/mL LPL. Binding, association, and degradation are expressed as nanograms labeled lipoprotein per milligram cell protein and were determined as described in "Methods." Values represent the mean±SD of three measurements.

ApoE^null VLDL was also associated (bound plus internalized) and degraded by J774 cells via a high-affinity process but to a much lesser extent than control VLDL (55±5 and 15±10 versus 315±19 and 642±22 ng/mg of cell protein, respectively; Fig 3C and 3E). The association and degradation of control VLDL by J774 cells were stimulated 2-fold and 1.5-fold, respectively, on addition of 5 µg/mL LPL. The association and degradation of apoE^null VLDL were enhanced by a factor of 8 and 16, respectively, in the presence of 5 µg/mL LPL (compare Fig 3C with 3D and 3E with 3F, respectively).

Our data indicate that ¹²⁵I-labeled apoE^null VLDL is bound and internalized by J774 cells via a specific saturable receptor, since its binding, association, and degradation are reduced on addition of an excess amount of unlabeled apoE^null VLDL. To identify the nature of the receptor involved in the binding and uptake of apoE^null VLDL, competition experiments were performed. On addition of 20 µg/mL unlabeled LDL, the binding of ¹²⁵I-labeled human LDL by J774 cells was reduced to about 60% of the control binding (Fig 4). Unlabeled control VLDL was much more efficient in competing with ¹²⁵I-labeled LDL (reduction up to 30% of the control binding). This high efficiency of control VLDL in competing for the binding of LDL can be explained by the fact that control mouse VLDL is relatively rich in apoE, whereas human LDL consists of apoB100 only. ApoE^null VLDL was not able to compete with ¹²⁵I-labeled LDL for binding to the J774 cells. As shown in Fig 5, the binding of ¹²⁵I-labeled apoE^null VLDL by J774 cells was inhibited to a similar extent (60% of control value) on addition of either unlabeled apoE^null VLDL, control VLDL, or human HTG-VLDL. On the contrary, addition of unlabeled LDL or AcLDL did not significantly reduce the binding of ¹²⁵I-apoE^null VLDL. The binding of ¹²⁵I-labeled HTG-VLDL to the cells was inhibited to 20% of control value by both unlabeled HTG-VLDL and control VLDL and to 55% of control value by LDL. ApoE^null VLDL and AcLDL were not able to compete with ¹²⁵I-labeled HTG-VLDL for binding to J774 macrophages. From these experiments we conclude that the binding of apoE^null VLDL does not occur via the LDL receptor or the scavenger receptor but that a receptor also recognizing control mouse VLDL and HTG-VLDL is involved in the binding of apoE^null VLDL to J774 macrophages.

View larger version (20K): [in this window] [in a new window]   Figure 4. Competition for binding of 125I-labeled human LDL to J774 macrophages by various unlabeled lipoprotein samples. Competition studies were performed by incubating J774 cells with 10 µg/mL 125I-labeled LDL for 3 hours at 4°C in the presence of the indicated amounts of unlabeled LDL (), apoEnull VLDL (), and control VLDL (). The binding is expressed as a percentage of the value in the absence of competitor and is determined as described in "Methods." Values represent the mean±SD of four measurements.

View larger version (21K): [in this window] [in a new window]   Figure 5. Competition for binding of 125I-labeled apoEnull VLDL to J774 macrophages by various unlabeled lipoprotein samples. Competition studies were performed by incubating J774 cells with 10 µg/mL 125I-labeled apoEnull VLDL for 3 hours at 4°C in the presence of the indicated amounts of unlabeled apoEnull VLDL (), control VLDL (), LDL (), AcLDL (), or HTG-VLDL (). The binding is expressed as a percentage of the value in the absence of competitor and is determined as described in "Methods." Values represent the mean±SD of four measurements.

Accumulation of CE in J774 Macrophages After Incubation With Different Lipoproteins
As apoE-deficient mice develop atherosclerotic plaques containing lipid-laden macrophages (foam cells), we wondered whether apoE^null VLDL, which is deficient in both apoE and apoB100, can cause accumulation of CE in J774 macrophages. The data presented in Table 2 demonstrate that incubation of J774 cells with apoE^null VLDL resulted in a threefold increase in cellular CE content. Simultaneous incubation with LPL stimulated the cellular CE content by an additional factor of 5, reaching similar levels as obtained after incubation with control VLDL. However, simultaneous addition of LPL did not stimulate the cellular CE level in J774 cells after incubation with control VLDL.

View this table: [in this window] [in a new window]   Table 2. CE Content in J774 Cells After Incubation With ApoEnull VLDL and Control VLDL

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
ApoE-deficient mice have been shown to display a marked accumulation in the plasma of chylomicron and VLDL remnants, which are deficient in both apoE and apoB100. ApoE and apoB100 are also commonly assumed to be responsible for the receptor-mediated uptake of lipoproteins by macrophages. Thus, since apoE-deficient mice develop extensive atherosclerotic lesions with lipid-laden macrophages, uptake of lipoproteins by macrophages can take place in the absence of both apoB100 and apoE. In the present paper we studied the mechanisms by which apoE^null VLDL can be taken up by macrophages by using the murine macrophage cell line J774, which does not produce apoE itself.¹⁰

We found that apoE^null VLDL is bound by J774 macrophages via a high-affinity, saturable receptor with a dissociation constant (K_d) of the same order of magnitude as that of control VLDL (Fig 2). Furthermore, uptake of apoE^null VLDL resulted in the accumulation of CE in these cells. The LDL receptor was not expected to be a candidate for the uptake of apoE^null VLDL, since apoE and apoB100, which are both absent on the surface of this VLDL, are known to be the only ligands for this receptor. We found that the scavenger receptor was also not involved in the uptake by J774 macrophages of apoE^null VLDL. However, since the scavenger receptor is involved in the uptake of oxidized lipoproteins by macrophages²⁹ and lipoprotein oxidation occurs in the intima of apoE-deficient mice,^{30 31 32} it is plausible that in vivo, the scavenger receptor does indeed contribute to foam cell formation and subsequent development of atherosclerosis in apoE-deficient mice.

Gianturco et al^{33 34} and Ramprasad et al^{35 36} described a distinct receptor that is able to mediate the uptake of HTG-VLDL and that has been denominated as the TGRLP receptor. They reported that this receptor plays a major role in the uptake of HTG-VLDL by human monocyte-macrophages^{33 35 36} and murine macrophages.³⁴ Recently, Gianturco et al³⁷ reported that neither apoB100 nor apoE is necessary to mediate the binding of HTG-VLDL to this receptor and that apoB48 is sufficient for this purpose. The fact that apoB48 and not apoE or apoB100 is present on the surface of apoE^null VLDL supports the hypothesis that the binding and uptake of apoE^null VLDL by macrophages occurs via this TGRLP receptor. This hypothesis is strongly sustained by our observation that HTG-VLDL does indeed compete with VLDL from apoE-deficient mice for binding to the J774 macrophages (Fig 5). The observation that an excess concentration of apoE^null VLDL does not compete for the binding of ¹²⁵I-labeled HTG-VLDL, is not in disaccord with our hypothesis, since HTG-VLDL also binds to the cells via apoB100 and apoE, as shown by the finding that both control mouse VLDL and LDL can compete for the binding of ¹²⁵I-labeled HTG-VLDL (Fig 6). Obviously, this part of the HTG-VLDL binding cannot be competed with by apoE^null VLDL.

View larger version (22K): [in this window] [in a new window]   Figure 6. Competition for binding of 125I-labeled HTG-VLDL to J774 macrophages by various unlabeled lipoprotein samples. Competition studies were performed by incubating J774 cells with 10 µg/mL 125I-labeled HTG-VLDL for 3 hours at 4°C in the presence of the indicated amounts of unlabeled HTG-VLDL (), apoEnull VLDL (), control VLDL (), LDL (), or AcLDL (). The binding is expressed as a percentage of the value in the absence of competitor and is determined as described in "Methods." Values represent the mean±SD of four measurements.

It has previously been shown by several groups that LPL stimulates the binding and uptake of LDL and VLDL by different cell types, including macrophages, via bridging between the lipoprotein and the heparan sulfate proteoglycans present on the plasma membrane.^{12 13 14 15 16 17} In the present paper we present data that the binding and uptake of apoE^null VLDL by J774 macrophages is also stimulated on addition of LPL. The finding that the binding of VLDL is stimulated by LPL to a greater extent than both the association and the degradation (Fig 3) suggests that a large part of the LPL-mediated VLDL binding at 37°C is reversible. This would mean that not all the VLDL bound via LPL to the J774 cells is taken up and subsequently degraded. Another explanation could be that under the conditions applied (J774 cells incubated 3 hours at 37°C), the LPL-bound VLDL is not internalized and/or degraded as rapidly as VLDL bound to the receptor directly. At present we cannot discriminate between the two possibilities. The increased LPL-mediated uptake of apoE^null VLDL resulted in an increased accumulation of CE in these cells (Table 2). In vivo, LPL is synthesized by macrophages, positively correlated with the amount of intracellular cholesterol.¹¹ Furthermore, in atherosclerotic lesions, high amounts of LPL protein are formed mainly in macrophage-rich intimal regions.³⁸ Thus, in apoE-deficient mice, the LPL-mediated uptake of VLDL by macrophages may play an important role in the development of atherosclerosis.

The role of apoE in the development of atherosclerotic lesions may be dual. On one hand, the apoE-mediated uptake of lipoproteins by cells of the arterial wall will lead to lipid accumulation and foam cell formation. On the other hand, apoE plays a role in the cholesterol efflux from cholesterol-laden macrophages to extracellular cholesterol acceptors.³⁹ Thus, the absence in apoE-deficient mice of apoE-mediated cholesterol efflux may also contribute to the marked atherosclerosis observed in these mice. This is sustained by the finding of Bellosta et al⁴⁰ that macrophage-specific expression of human apoE is able to prevent or delay the development of atherosclerotic lesions in apoE-deficient mice, even in the presence of high levels of atherogenic lipoproteins.

In summary, in addition to the absence of an apoE-mediated cholesterol efflux from macrophages in apoE-deficient mice, we present evidence that there may be at least two additional processes that contribute to the development of atherosclerosis in apoE-deficient mice. First, VLDL is taken up by macrophages via a specific saturable receptor, possibly the TGRLP receptor, which leads to the accumulation of CE in these cells. Second, this accumulation of CE by macrophages in the intima may be enhanced by production of LPL by macrophages in the intima.

	Selected Abbreviations and Acronyms

 

AcLDL = acetylated LDL apo = apolipoprotein apoEnull VLDL = VLDL from apoE-deficient mice CE = cholesteryl ester DMEM = Dulbecco's modified Eagle's medium FCS = fetal calf serum HSA = human serum albumin HTG-VLDL = VLDL from hypertriglyceridemic patients LPL = lipoprotein lipase TGRLP = triglyceride-rich lipoprotein

	Acknowledgments

 
This study is financially supported by the Netherlands Heart Foundation (project No. 92.337). Dr M.H. Hofker is awarded a Dr E. Dekker Established Investigatorship of the Netherlands Heart Foundation (D95.022). We would like to thank Hans van der Boom for excellent technical assistance.

Received February 26, 1996; accepted July 10, 1996.

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