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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:144-151

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:144.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Oxidized VLDL Induces Less Triglyceride Accumulation in J774 Macrophages Than Native VLDL Due to an Impaired Extracellular Lipolysis

Miek C. Jong; Wendy L. Hendriks; Leonie C. van Vark; Vivian E. H. Dahlmans; Johanna E. M. Groener; Louis M. Havekes

From TNO-Prevention and Health, Gaubius Laboratory (M.C.J., W.L.H., L.C.v.V., V.E.H.D., L.M.H.), and the Departments of Pediatrics (J.E.M.G.) and of Cardiology and Internal Medicine (L.M.H.), Leiden University Medical Center, Leiden, Netherlands.

Correspondence to Dr M.C. Jong, TNO-Prevention and Health, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, PO Box 2215, 2301 CE Leiden, Netherlands. E-mail mc.jong{at}pg.tno.nl


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Abstract—The present study examined the relative contributions of the different pathways by which oxidatively modified VLDL (oxVLDL) promotes the uptake and intracellular accumulation of lipids in J774 macrophages. VLDL was oxidized for a maximum of 4 hours, resulting in an increase in thiobarbituric acid–reactive substances and an increased electrophoretic mobility on agarose gel. The lipid composition of the relatively moderately oxidized VLDL samples did not differ significantly from that of nonoxidized VLDL samples. The uptake of 125I-labeled VLDL by the J774 cells increased with oxidation time and was completely blocked on coincubation with polyinosinic acid (PolyI), indicating that oxVLDL is taken up by the cells via the scavenger receptor only. Despite the 2-fold increased uptake of oxVLDL protein, the cell association of triglyceride (TG)-derived fatty acids by the J774 macrophages after incubation with oxVLDL was only 50% of that with native VLDL. In line with these observations, the induction of de novo synthesis of TG by J774 cells was {approx}3-fold less efficient after incubation with oxVLDL than after incubation with native VLDL. The induction of de novo synthesis of TG with oxVLDL was even further decreased on simultaneous incubation with PolyI, whereas PolyI did not affect the native VLDL-induced TG synthesis. These results indicate that oxVLDL induces endogenous TG synthesis predominantly through particle uptake via the scavenger receptor and much less via the extracellular lipoprotein lipase (LPL)–mediated hydrolysis of TG, as is the case for native VLDL. In line with these observations, we showed that the suitability of VLDL as a substrate for LPL decreases with oxidation time. Addition of oxVLDL to the LPL assay did not interfere with the lipolysis of native VLDL. However, enrichment of the oxidized lipoprotein particle with native apoC2 was able to fully restore the impaired lipolysis. Thus, from these studies it can be concluded that on oxidation, VLDL becomes less efficient in inducing TG accumulation in J774 cells as a consequence of a defect in apoC2 as an activator for the LPL-mediated extracellular lipolysis.


Key Words: oxidized VLDL • lipolysis • lipid accumulation • macrophages


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The role of triglyceride (TG)-rich lipoproteins in foam cell formation is currently under investigation. Several groups of investigators have shown that human VLDL is capable of inducing cholesteryl ester and TG accumulation in different macrophage culture systems.1 2 3 4 It has been postulated that the mechanisms by which VLDL stimulates cellular lipid accumulation involve at least 2 different pathways2 4 : (1) the receptor-mediated uptake of intact VLDL particles and (2) the direct uptake of free fatty acids (FFAs) as generated by the extracellular lipoprotein lipase (LPL)–mediated hydrolysis of VLDL-TG, followed by intracellular reesterification into lipids. The resulting cholesterol-enriched remnant particles are thereafter taken up via a receptor-mediated process.

In analogy with LDL,5 6 it has been shown that in vitro exposure of ß-VLDL to endothelial cells causes oxidation of this lipoprotein.7 8 This results in a 2- to 3-fold increased degradation by mouse peritoneal macrophages7 and rabbit smooth muscle cells8 compared with unoxidized ß-VLDL and in a 2- to 3-fold increased intracellular cholesterol esterification rate. Isolated human VLDL, like human LDL, was shown to be effectively oxidized in vitro on incubation with free radicals.9 The degradation of oxidized human VLDL (oxVLDL) by mouse peritoneal macrophages was also increased 2-fold compared with native VLDL.10 More recently, it was shown that oxVLDL causes greater accumulation of cholesteryl ester in J774 macrophages than oxLDL.11 Altogether, these data suggest that oxidation of VLDL augments its atherogenic potential and may contribute to foam cell formation in humans.

So far, little is known with respect to the pathways by which oxVLDL promotes uptake and intracellular accumulation of lipids in macrophages. Recent studies by Whitman et al12 showed a decrease in cellular TG levels on incubation with oxidized hypertriglyceridemic VLDL, strongly pointing to a different routing for the uptake of oxidized lipids compared with their nonoxidized counterparts. In the present study, we examined more closely the processing of oxidatively modified forms of human VLDL in the murine macrophage cell line J774. Despite the enhanced cellular (protein) uptake, we found that normolipidemic human oxVLDL induces less TG accumulation in these macrophages than native VLDL. The difference between oxVLDL and normal VLDL in this respect appeared to be due to an impaired LPL-mediated extracellular lipolysis of oxVLDL-TG as a consequence of defective apoC2.


*    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 U/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L glutamine. The cells were incubated at 37°C in an atmosphere containing 5% CO2 and 95% air. For each experiment, cells were plated in 6- or 24-well plates. The cells were fed every 3 days and were used for experiments within 7 days after plating. 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) of lipoprotein-deficient human serum (d>1.21 g/mL) instead of FCS. The experiments were subsequently conducted in DMEM containing 1% (wt/vol) BSA.

Lipoproteins
Human VLDL, LDL, and HDL were isolated according to Redgrave et al13 by density-gradient ultracentrifugation of pooled plasma obtained from healthy normolipidemic volunteers after an overnight fast. Immediately after isolation, the lipoprotein fractions were extensively dialyzed against PBS (pH 7.4) containing 10 µmol/L EDTA at 4°C. The protein contents of the VLDL and LDL samples were determined by the method of Lowry et al.14

After isolation, part of the VLDL sample was labeled with glycerol tri[1-14C]oleate (Amersham; specific activity, 61 mCi/mmol) as described by Groener et al.15 In brief, 5 µCi glycerol tri[1-14C]oleate was added to 12.8 µL phosphatidylcholine (100 mg/mL) and 10 µL butylated hydroxytoluene in chloroform (1 mmol/L). After the evaporation of the chloroform under a stream of nitrogen, 1 mL of 50 mmol/L Tris/HCl, pH 7.5, containing 0.27 mmol/L EDTA was added. The suspension was sonicated twice for 5 minutes under nitrogen with a Labsonic 1510 sonicator. The sonicated lipids were added to a mixture of 6.24 mL human lipoprotein-deficient serum, 0.22 mL 0.13-mol/L EDTA, and 0.94 mL 10-mmol/L DTNB. Subsequently, 5 mg of VLDL-TG was added, and the mixture was incubated for 40 hours at 37°C. The VLDL was reisolated by ultracentrifugation as described above. The specific activity ranged from 1500 to 2500 dpm/µg TG.

Oxidation of labeled and unlabeled VLDL (d<1.006 g/mL, 0.5 mg/mL) was performed at 37°C by the addition of a 160-mmol/L aqueous solution of the thermolabile peroxyl radical generator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) (TNO) to a final concentration of 39 mmol AAPH/mg VLDL protein. At various time intervals, 2-mL aliquots of the reaction solution were withdrawn, and the reaction was terminated by addition of cold PBS containing 2 mmol/L ascorbic acid to a final concentration of 2 mol ascorbic acid/mol AAPH. Oxidized lipoproteins were carefully dialyzed overnight at 4°C against 0.1 mol/L Tris/HCl containing 10 µmol/L EDTA, pH 8.5, for use in lipolysis experiments or dialyzed against PBS containing 10 µmol/L EDTA, pH 7.4, for use in binding experiments. Lipid peroxidation was determined by measuring thiobarbituric acid–reactive substances (TBARS)16 with thiobarbituric acid (in 20% trichloroacetic acid) with fresh malonaldehyde-tetramethylacetal as standard. Furthermore, the degree of oxidation was determined by agarose gel electrophoresis (100 V, 30 minutes, Paragon Lipoprotein Electrophoresis kit, Beckman Instruments), and the electrophoretic mobility relative to native VLDL of the different oxVLDL fractions was calculated.

The LDL fraction was acetylated by repeated additions of acetic anhydride as described by Basu et al.17 The conversion of LDL into acetylated LDL (acLDL) was confirmed by agarose gel electrophoresis as described above.

The total and free cholesterol contents of the VLDL samples were measured with commercially available kits (236691 and 310328, Boehringer Mannheim GmbH; 337-B) with cholesterol esterase and cholesterol oxidase used to measure cholesteryl esters and cholesterol, respectively. The VLDL TG content was measured with an enzymatic assay from Sigma (337-B: Sigma Diagnostics), and the phospholipid content was quantified by use of phospholipase D, which hydrolyzes phospholipids to free choline (kit No. 990-54009, Wako Chemicals GmbH). The FFA contents of the lipoprotein samples were measured with the Nefa-C kit (Wako Chemicals GmbH).

The apolipoprotein composition of the VLDL fractions was analyzed by SDS (0.1%)–polyacrylamide gel electrophoresis (SDS-PAGE) using 4% to 20% gradient gels (Gradipore Ltd) or 4% to 25% gradient gels. Proteins were either stained with Serva Blue R or transferred to nitrocellulose membranes (Schleicher and Schuell), followed by incubation with polyclonal rabbit antiserum against human apoC2. Donkey anti-rabbit 125I-labeled IgG (Amersham) was used as a secondary antibody, and detection was performed by scanning of the blots with a Phosphor Imager (Molecular Dynamics).

Radioiodination of VLDL and oxVLDL samples was performed by the 125I-monochloride method described by Bilheimer et al.18 The specific activity ranged from 100 to 300 cpm/ng protein, whereas the degree of lipid labeling in VLDL after iodination varied between 9% and 15%. After iodination, the lipoprotein samples were extensively dialyzed against PBS (pH 7.4), stored at 4°C, and used within 2 weeks.

Interaction of VLDL and oxVLDL With J774 Macrophages
Lipoprotein Uptake
The J774 cells were cultured in 24-well plates as described above. The association and degradation of 125I-labeled VLDL and oxVLDL was determined after a 4-hour incubation at 37°C with 10 µg/mL 125I-labeled lipoprotein in a final incubation volume of 0.5 mL, either in the absence or in the presence of a 20-fold excess of the respective unlabeled lipoprotein. The receptor-mediated (specific) cell association and degradation was calculated by subtracting the amount of labeled lipoproteins that was associated or degraded after incubation in the presence of an excess of unlabeled lipoprotein (nonspecific) from the amount of labeled lipoprotein that was cell-bound after incubation in the absence of unlabeled lipoprotein (total). At the end of the incubation period, a fraction of the medium was removed to determine the amount of lipoprotein degraded as described previously.19 After the remaining portion of the medium had been removed, the cells were washed 4 times with ice-cold PBS containing 0.1% (wt/vol) BSA and subsequently with PBS without BSA. To measure the cell-associated lipoprotein fraction, the washed cells were dissolved in 1 mL 0.2-mmol/L NaOH, and an aliquot of the cell lysate was counted for radioactivity.19 Another aliquot was used for protein determination according to Lowry et al.14 In the respective figures, lipoprotein uptake is expressed as the sum of cell-associated and degraded lipoproteins.

The effect of acLDL and polyinosinic acid (PolyI) on the receptor-mediated uptake of 125I-labeled oxVLDL was determined in competition experiments. Therefore, J774 cells were incubated for 4 hours at 37°C with 10 µg/mL of 125I-labeled oxVLDL in the presence of different concentrations of unlabeled oxVLDL, acLDL, or PolyI, as described in the text and the figure legends. Thereafter, the association and degradation of 125I-oxVLDL were determined exactly as described above.

Effect on Cellular and Medium Lipids
J774 cells were cultured in 6-well plates, and VLDL was labeled in the TG with glycerol tri[1-14C]oleate and subsequently oxidized as described above. The effect of 14C-TG-VLDL and 14C-TG-oxVLDL (dissolved in DMEM containing 1% [wt/vol] FFA-free BSA) on the lipid content of the cells and the medium was determined after incubation of the J774 cells at 37°C with 50 µg/mL of VLDL-TG for different time periods. Incubation volumes were 1 mL/well. After the incubation period, culture dishes were placed on ice, and 800 µL of the medium was used for lipid extraction. Thereafter, the rest of the medium was removed, and the cells were washed 4 times with PBS containing 0.1% (wt/vol) BSA and subsequently once with PBS without BSA. The cells were harvested in 1 mL PBS with a rubber policeman and resuspended by 3 successive slow passages through a syringe needle (25 gauge). Samples (100 µL) were taken for protein determination and lipid extraction (800 µL), and 50 µL of the cell lysate was counted for determination of total uptake of [14C]oleate. Cellular and medium lipids were extracted with methanol/chloroform (2:1, vol/vol) as described by Bligh and Dyer20 after the addition of glycerol tri[1-3H]oleate as an internal standard for cellular TG and [3H]palmitic acid as an internal standard for FFA in the medium. The lipids were separated by thin-layer chromatography with hexane/diethylether/acetic acid (83:16:1) as developing solvent. Bands were visualized by phosphoimaging, and those representing the FFA, TG, phospholipids, and cholesteryl ester fraction were scraped into vials and counted for radioactivity.

De Novo TG Synthesis
The J774 cells were cultured in 6-well plates as described above. The de novo synthesis of TG was measured after a 4-hour incubation of the cells at 37°C with 1 mL of DMEM containing 1% (wt/vol) of FFA-free BSA in the presence or in the absence of VLDL or oxVLDL (100 µg TG/mL), with or without the addition of PolyI (100 µg/mL), as described in the text and figure legends. During the incubation, [1(3)-3H]glycerol (4.4 µCi/mL, 25 µmol/L at final concentration) was present in the medium in each well. After the incubation period, culture dishes were placed on ice, the medium was removed, and the cells were washed 4 times with PBS containing 0.1% (wt/vol) BSA and subsequently once with PBS without BSA. The cells were suspended in 1 mL PBS as described above, and samples (100 µL) were taken for the measurement of protein. Subsequently, lipids were extracted from the cell suspension (800 µL) as described above, except that glycerol tri[1-14C]oleate was added as an internal standard. After iodine staining, the spots containing the TG fraction were scraped into vials and assayed for radioactivity by scintillation counting.

In Vitro Lipolysis of VLDL and oxVLDL
In vitro lipolysis experiments were performed as previously described.21 Briefly, VLDL samples were incubated at 37°C in 0.1 mol/L Tris/HCl, pH 8.5, in the presence of 2% (wt/vol) albumin (essentially FFA-free) and 0.2 U of commercially available bovine LPL (Sigma Chemical Co). After 10 minutes, the reaction was stopped by the addition of 50 mmol/L KH2PO4, 0.1% Triton-X100, pH 6.9 (Merck). The assay was performed with 4 different concentrations of VLDL-TG in the range of 0.1 to 0.5 mmol/L with duplicate FFA measurements. FFAs were quantified as described above.

In a different set of experiments, polyI (100 µg/mL) was added to native VLDL before incubation with LPL at 37°C. In another set of experiments, nonoxidized and oxidized VLDL fractions were incubated with human HDL (1 mg VLDL protein:6 mg HDL protein) for 1 hour at 37°C to allow exchange of apolipoproteins. Thereafter, apolipoprotein-enriched VLDL fractions were reisolated by ultracentrifugation to remove HDL particles, and lipolysis experiments were performed as described above.

In Vitro Lipolysis of VLDL With HSPG-Bound LPL to Assay LPL Activity
An in vitro assay to study the lipolysis of VLDL by heparan sulfate proteoglycan (HSPG)–bound LPL was developed previously.22 Briefly, wells of a 96-well microtiter plate were incubated with 0.1 µg HSPG (Sigma) in 75 µL PBS for 18 hours at 4°C. Aspecific binding sites were blocked by incubation with 1% (wt/vol) BSA in 100 µL PBS for 1 hour at 37°C. Subsequently, wells were incubated with 1 U LPL (Sigma) in 75 µL Tris-glycerol buffer (0.1 mol/L Tris, 20% [vol/vol] glycerol, pH 8.5) for 1 hour at 4°C. Unbound LPL was removed by washing the plates 3 times with 0.1 mol/L Tris. To examine the effect of oxidized lipoproteins on LPL activity, HSPG-LPL–coated plates were first incubated for 1 hour at 37°C with either native VLDL or oxVLDL4 (see below) (0.3 mmol/L TG). Thereafter, plates were washed 3 times with 0.1 mol/L Tris to remove all VLDL. Subsequently, the catalytic activity of the remaining HSPG-bound LPL was assayed upon the addition of nonoxidized VLDL-TG samples (ranging from 0.1 to 0.5 mmol/L TG) to the wells and incubation of the plate for 20 minutes at 37°C. The reaction was terminated by the addition of 1% (vol/vol) Triton X-100 in 0.1 mmol/L Tris and cooling on ice. The FFA release by LPL bound to HSPG was measured enzymatically as described above and was linear for the 20 minutes assayed.

The ability of oxVLDL to inhibit LPL activity was further examined on the addition of a constant amount of oxVLDL4 (0.2 mmol/L TG) to a range of native VLDL samples (0.1 to 0.5 mmol/L TG) in the HSPG-LPL assay solution. The rate of FFA release was measured as described above.

Statistical Analysis
The statistical significance between native VLDL and oxVLDL mean values was assessed with Student’s unpaired t test. A value of P<0.05 was considered statistically significant.


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Characterization of VLDL Samples
Human VLDL was incubated with the peroxyl radical generator AAPH for different time periods (0, 1, 2, 3, and 4 hours of incubation: VLDL, oxVLDL1, oxVLDL2, oxVLDL3, and oxVLDL4, respectively). The oxidative properties of VLDL were analyzed by TBARS assay and electrophoretic mobility on agarose gel. As shown in the TableDown, the oxidative changes in VLDL were accompanied by a gradual increase in TBARS/mg VLDL protein and an increase in relative electrophoretic mobility on agarose gel compared with that of native VLDL. The VLDL cholesterol, TG, phospholipid, and FFA composition did not change significantly during 4 hours of incubation with AAPH (TableDown). Furthermore, SDS-PAGE analysis of oxVLDL showed a significant depletion in apoB, apoE, and apoC with increasing oxidation time (Figure 1Down). VLDL that was oxidized for 4 hours was used in further experiments.


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  		Table 1. Chemical Composition and Relative Electrophoretic Mobility of VLDL Samples After Oxidation



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  		Figure 1. SDS-PAGE analysis of VLDL and oxVLDL. VLDL fractions (10 µg of protein) were subjected to SDS (0.1%)-gel electrophoresis (4% to 20% gradient gel), and proteins were stained with Serva Blue R. Lane 1, molecular weight markers; lane 2, native VLDL; lane 3, 1 hour oxVLDL; lane 4, 2 hours oxVLDL; lane 5, 3 hours oxVLDL; lane 6, 4 hours oxVLDL.

Interaction of VLDL and oxVLDL With J774 Cells
To investigate the effects of oxidation on the cellular processing of TG-rich lipoprotein particles by macrophages, both native and oxVLDL-induced protein uptake and lipid accumulation by J774 cells was determined after a 4-hour incubation at 37°C. As shown in Figure 2ADown, the uptake (expressed as the sum of the cell-associated and degraded lipoprotein) of 125I-labeled oxVLDL4 by J774 cells was 2.5-fold higher than that of 125I-labeled native VLDL. To examine the effect of oxidation on the accumulation of TG-derived lipids in J774 cells, VLDL was used that was labeled in the TG moiety with glycerol tri[1-14C]oleate and subsequently oxidized (14C-TG-VLDL and 14C-TG-oxVLDL4). As shown in Figure 2BDown, after incubation with oxVLDL4, the total uptake of 14C-oleate by J774 cells is significantly decreased compared with that after incubation with native VLDL (14.5±0.6% versus 27.2±2.1% of total lipid added to the cells, respectively). Lipid extraction of the cell lysate and subsequent analysis by TLC showed that the decrease in 14C-oleate uptake on incubation with oxVLDL4 was reflected by a strong decrease in intracellular TG accumulation (Figure 2BDown). Thus, in contrast to the protein uptake, the TG-derived fatty acid uptake by J774 cells is reduced after incubation with oxVLDL compared with native VLDL.



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  		Figure 2. Effect of oxidation on VLDL protein uptake and lipid accumulation by J774 cells. A, The J774 cells were incubated for 4 hours at 37°C with 10 µg/mL 125I-VLDL (open bars) and 125I-oxVLDL4 (solid bars). The uptake (expressed as cell-associated plus degraded lipoprotein) was determined as described in Methods. The values represent the mean±SD of 4 different experiments. B, Glycerol tri[1-14C]oleate-labeled VLDL (open bars) and oxVLDL4 (solid bars, 50 µg TG/mL medium) were incubated with J774 cells for 4 hours at 37°C. The uptake of total lipid and TG (depicted as the percentage of the total amount of lipid [activity] added to the cell) were determined as described in Methods, and values represent the mean±SD of 4 experiments. *P<0.05, indicating the difference between native VLDL and oxVLDL4 by unpaired Student’s t test.

To investigate the mechanisms underlying the decreased cellular uptake of TG-derived oleate on incubation with oxVLDL4, the relative contributions of the different pathways through which lipid accumulation is thought to occur were assessed for oxVLDL4. First, we show that acLDL is able to compete efficiently with the uptake (association and degradation) of 125I-labeled oxVLDL4 (Figure 3ADown). These results suggest that oxVLDL4 is taken up via the macrophage scavenger receptor and not via LDL or VLDL receptors. Furthermore, we show that the uptake (association and degradation) of 125I-labeled oxVLDL4 was completely inhibited by 100 µg/mL of PolyI (Figure 3BDown). Thus, through a complete inhibition of the scavenger receptor–mediated uptake of oxVLDL4 via PolyI, the role of the second pathway involved in the intracellular lipid accumulation, ie, the extracellular VLDL-TG lipolysis with subsequent cellular FFA uptake, can be determined.



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  		Figure 3. Competition of 125I-labeled oxVLDL with acLDL and PolyI. J774 cells were incubated for 4 hours at 37°C with 10 µg/mL of 125I-oxVLDL4 in the presence or absence of different concentrations of oxVLDL4 ({circ}) and acLDL (•, A), or PolyI (B). The uptake (expressed as cell-associated plus degraded lipoprotein) was determined as described in Methods. Values are given as a percentage of the uptake in the absence of any competitor and represent the mean±SD of 3 experiments.

Therefore, the oxVLDL-induced intracellular lipid accumulation was investigated by measuring the de novo synthesis of TG by J774 macrophages after incubation with VLDL and oxVLDL4, either in the absence or in the presence of PolyI. In this experimental approach, newly formed FFAs from either extracellular lipolysis or intracellular lysosomal hydrolysis of VLDL-TG are reesterified with [3H]glycerol into [3H]TG. In accordance with the decreased TG accumulation after incubation with oxVLDL4 (Figure 2BUp), Figure 4Down shows that the induction in the de novo synthesis of TG by J774 macrophages after incubation with oxVLDL4 is {approx}7-fold compared with control incubations without VLDL, whereas incubation with native VLDL induced the de novo TG synthesis by {approx}20-fold. This lower induction in TG synthesis upon incubation with oxVLDL4 compared with incubation with native VLDL cannot be explained by differences in TG content of the respective VLDL preparation (TableUp). Nearly complete inhibition of the receptor-mediated uptake of oxVLDL4 by PolyI caused a further lowering in the induction of the de novo synthesis of TG by {approx}2-fold (Figure 4Down). PolyI, at a concentration of 100 µg/mL, had little effect on TG synthesis upon incubation with native VLDL (Figure 4Down) and did not interfere with the LPL-mediated hydrolysis of VLDL-TG (72±3 versus 67±2 µmol FFA · L-1 · min-1 at a concentration of 0.2 mmol/L VLDL-TG in the presence or absence of PolyI, respectively). Thus, these results indicate that the extracellular lipolysis, which has been shown to mediate intracellular TG accumulation through the release and subsequent uptake of FFA, may be impaired on oxidation of TG-rich lipoproteins.



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  		Figure 4. Effect of inhibition of receptor-mediated uptake of oxVLDL on de novo TG synthesis in J774 cells. J774 cells were incubated for 4 hours at 37°C with DMEM/1% BSA containing [1(3)-3H]glycerol (4.4 µCi/mL, 25 µmol/L), either in the absence of lipoproteins (control) or in the presence of VLDL or oxVLDL4 (100 µg TG/mL medium), with (solid bars) or without (open bars) the addition of PolyI (100 µg/mL). After incubation, the J774 cells were washed and resuspended in 1 mL PBS. Subsequently, lipids were extracted from the cell suspension, and TG was determined as described in Methods. The values represent the mean±SD of 4 different experiments. *P<0.05, native VLDL vs oxVLDL4; **P<0.05, incubation with PolyI vs without PolyI by unpaired Student’s t test.

Lipolysis of oxVLDL
To investigate whether the extracellular lipolysis of oxVLDL4 is hampered, the amount of FFA released into the medium was measured during a 4-hour incubation of the J774 cells with 14C-TG-VLDL and 14C-TG-oxVLDL4 (labeled in oleate). Significantly lower amounts of 14C-oleate appeared in the medium after incubation with oxVLDL4 than with native VLDL (8.2±0.5 versus 28.2±1.8 µg/mL), indeed suggesting an impaired extracellular lipolysis of oxVLDL-TG. However, 14C-oleate present in medium can be derived either from extracellular hydrolysis of VLDL-TG or intracellular hydrolysis of lipid pools. Thus, to directly investigate the effect of oxidation on the LPL-mediated hydrolysis of VLDL-TG, different VLDL samples with various degrees of oxidation were incubated with purified bovine LPL. Figure 5Down shows that the amount of FFA released after the addition of LPL decreases with oxidation time. Thus, oxidation decreases the suitability of VLDL as substrate for LPL, leading to a defective TG hydrolysis.



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  		Figure 5. In vitro lipolysis of VLDL and oxVLDL. VLDL was oxidized for 0, 1, 2, 3, or 4 hours as described in Methods and dialyzed against 0.1 mol/L Tris/HCl, pH 8.5. VLDL-TG samples, ranging from 0.1 to 0.5 mmol/L, were incubated at 37°C in the presence of 2% (wt/vol) FFA-free albumin and 0.2 U of LPL. The FFA release is expressed per unit LPL and represents the mean±SD of at least 4 experiments. The results for 0.3 mmol/L TG are shown; similar results were obtained with other TG concentrations. *P<0.05, native VLDL vs oxVLDL samples by unpaired Student’s t test.

To investigate whether oxidation of VLDL has a direct inhibitory effect on the enzyme activity of LPL, we performed 2 different in vitro lipolysis assays with the system in which LPL was bound to HSPG.22 In the first assay, we tested whether oxidized lipoproteins would irreversibly damage LPL activity by measuring the FFA release of native VLDL after preincubation of the HSPG-LPL–coated wells with oxidized lipoproteins. As shown in Figure 6Down, preincubation with oxVLDL4 did not significantly alter the HSPG-LPL–mediated FFA release of VLDL-TG compared with preincubation with native VLDL, indicating that LPL is still catalytically active after withdrawal of oxidized lipoproteins. In the second in vitro lipolysis assay, we carried out a substrate curve for native VLDL in the presence or absence of oxVLDL4 (0.2 mmol/L TG). The LPL-mediated FFA release of native VLDL-TG in the absence of oxVLDL4 was not significantly different from that in the presence of the oxidized lipoprotein fraction (Figure 6Down), providing further evidence that oxidative modification of the lipoprotein particle does not directly inhibit LPL.



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  		Figure 6. Effect of oxVLDL on the enzyme activity of LPL. LPL was bound to HSPGs in a 96-well microtiter plate and preincubated with native VLDL (left) or oxVLDL4 (middle). Thereafter, VLDL samples were removed, and the remaining activity of the HSPG-bound LPL was assayed as described in Methods. The lipolysis of native VLDL was also assayed in the presence of oxVLDL4 (0.2 mmol/L) (right). Depicted values represent the mean±SD of 3 experiments with FFA release at a TG concentration of 0.3 mmol/L. Similar results were obtained with VLDL-TG concentrations at 0.2 and 0.5 mmol/L.

To examine whether oxidation of VLDL modifies apoC2 in a manner that prevents it from acting as a cofactor for the LPL-mediated hydrolysis of TG, oxVLDL4 fractions were incubated with HDL to allow supplementation with native apoC2. As shown by Western blot analysis, the amount of human apoC2 on native VLDL and oxVLDL4 fractions remained unaltered upon incubation with HDL (see inset in Figure 7ADown). In line with these results, preincubation with HDL did not affect the lipolysis rate of native VLDL, nor did it restore the impaired LPL-mediated FFA release of oxVLDL4 (Figure 7ADown). From these data, it can thus be hypothesized that either (1) native apoC2 is unable to bind to the oxVLDL particle or (2) oxidative modification of VLDL lipid decreases its suitability as substrate for LPL independent of the presence of apoC2. To discriminate between these 2 possibilities, apoC2-deficient (apoC2-/-) VLDL was isolated from a hypertriglyceridemic patient, oxidized, and incubated with HDL before the in vitro LPL-mediated lipolysis assay. As shown in the inset of Figure 7BDown, both native and oxidized apoC2-/- VLDLs were enriched with human apoC2 on incubation with HDL. Furthermore, incubation of apoC2-/- VLDL and oxidized apoC2-/- VLDL with HDL effectively restored the LPL-mediated release of FFA to control levels (native VLDL) (Figure 7BDown), indicating that native human apoC2 binds to the oxVLDL particle and is able to overcome the inhibitory action of oxidation on VLDL lipolysis.



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  		Figure 7. Effect of HDL incubation on the lipolysis of VLDL. VLDL and oxVLDL4 (A) and apoC2-deficient (C2-/-) VLDL and oxC2-/-VLDL4 (B) were assayed for their suitability as substrate for LPL without (open bars) or with (solid bars) previous HDL incubation (to allow exchange of apolipoproteins). Values are the mean±SD of 3 experiments with FFA release at a TG concentration of 0.3 mmol/L. Similar results were obtained with VLDL-TG concentrations at 0.1 and 0.2 mmol/L. Oxidized apoC2-deficient VLDL displayed a relative electrophoretic mobility of 1.80 vs nonoxidized VLDL after 4 hours of oxidation. The insets represent Western blot analysis of the respective VLDL fractions (16 µg of VLDL protein) for human apoC2. Note the more intensive staining of human apoC2 on oxidation, which is most likely caused by the oxidative modification of apoC2 (diffuse band on protein staining, see TableUp), rather than reflecting greater amounts of apoC2 associated with the lipoprotein particle. *P<0.05, VLDL fractions with vs without previous HDL incubation by unpaired Student’s t test.


*    Discussion
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 *Discussion
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In the present study, we examined the effect of oxidative modification on the processing of human VLDL-TG in the murine macrophage cell line J774. Previous studies have shown that J774 cells secrete LPL,23 but not apoE,24 into the culture medium. We showed that the uptake of oxVLDL protein by J774 macrophages was significantly increased compared with native VLDL (Figure 2AUp). The uptake of oxVLDL was competitively inhibited by an excess of unlabeled acLDL and completely blocked by coincubation with PolyI. Since PolyI is known to efficiently block ligand binding to the scavenger receptor class A,25 we conclude that the uptake of oxVLDL is mediated primarily via the scavenger receptor. Similar data on the enhanced uptake of oxVLDL have previously been published by other groups.7 10 However, despite this increased protein (ie, particle number) uptake, we showed that incubation of J774 cells with oxVLDL resulted in a 2- to 3-fold less efficient accumulation of TG compared with native VLDL (Figures 2BUp and 4Up). This apparent paradox can be explained by earlier observations that the uptake of VLDL-TG components by macrophages, in contrast to VLDL-protein uptake, involves 2 different pathways2 4 23 26 : (1) receptor-mediated uptake of the intact VLDL particle and (2) uptake of FFAs generated by the extracellular LPL-mediated lipolysis of VLDL-TG, followed by intracellular reesterification into TG. Thus, in addition to its so-called "bridge function," through which LPL can enhance the cellular uptake of lipoproteins,27 28 29 30 31 32 LPL also plays an important role in the cellular lipid accumulation by mediating the extracellular lipolysis of TG-rich lipoproteins.4

We present evidence that oxidation of VLDL results in a less efficient intracellular TG accumulation because of a defect in the second pathway, ie, a decreased suitability of oxVLDL as a substrate for LPL. Although the LPL-mediated lipolysis of VLDL-TG gradually decreased with oxidation time (to 25% of control levels), some residual lipolysis activity could still be observed for the maximally oxidized VLDL fraction (oxVLDL4) (Figure 5Up). In agreement with these observations, simultaneous incubation of oxVLDL4 with PolyI was not able to completely eliminate the de novo synthesis of TG to control values (Figure 4Up). As discussed earlier, Whitman et al12 showed that oxidation of apoE2/E2 ß-VLDL isolated from type III hyperlipidemic patients resulted in a decreased accumulation of TG in J774 cells compared with native type III VLDL. Because oxidized apoE2/E2 VLDL is taken up at an enhanced rate via the scavenger receptor, whereas the receptor-mediated uptake of native apoE2/2 VLDL is severely hampered by a defect of apoE2 in binding to the LDL receptor, their results support our present data and further sustain the important role of extracellular LPL–mediated lipolysis in the intracellular accumulation of TG.

An impaired LPL-mediated lipolysis of oxVLDL can be explained by either (1) greater amounts of FFAs associated with oxVLDL; (2) a direct effect of oxidation on the enzyme activity of LPL; (3) inactivation of apoC2, an essential cofactor for LPL activation; or (4) oxidative modification of VLDL lipid, rendering the TG component defective as a substrate for LPL. The first possibility could be excluded, because similar levels of FFAs were associated with native VLDL and oxVLDL (TableUp). Furthermore, we showed that LPL enzyme activity was not directly affected upon incubation with oxVLDL (Figure 6Up). The possible inactivation of apoC2 upon oxidation was supported by SDS-gel electrophoresis, showing significant depletion of apoC as well as other apolipoproteins with increasing oxidation time (Figure 1Up). In addition, enrichment of the oxidized apoC2-deficient VLDL particle with native apoC2 restored the impaired LPL-mediated lipolysis of oxVLDL (Figure 7BUp). Thus, we can conclude from these studies that oxidation decreases the suitability of VLDL as a substrate for LPL because of a defective apoC2, rather than because of modification of VLDL-TG. In contrast to oxidized apoC2–deficient VLDL, we were not able to restore the lipolysis of apoC2 containing oxVLDL on incubation with HDL, most likely because oxVLDL could not be supplemented with native apoC2 (see inset in Figure 7AUp). Although previous studies have shown that apoCs rapidly exchange between VLDL and HDL fractions upon incubation,33 the present findings strongly suggest that oxidatively modified apoC2 is no longer able to transfer between lipoproteins.

In conclusion, the role of LPL in atherogenesis may be dual, depending on where it is localized. In the liver, the role of LPL would be antiatherogenic, because it enhances the hepatic uptake of atherogenic lipoproteins by its bridging function. Conversely, in the intima of the vessel wall, LPL can serve as a proatherogenic factor in several ways: (1) through bridging between the HSPGs and lipoproteins, LPL enhances the uptake of these lipoproteins by smooth muscle cells and macrophages, leading to pronounced foam cell formation; and (2) through its enzymatic function as a lipase, LPL plays an important stimulatory role in the accumulation of VLDL-derived lipid in macrophages. This latter pathway, however, depends strongly on the level of oxidation of the VLDL particles. We observed that oxidative modification of VLDL into particles that are recognized by the scavenger receptor leads to a 2-fold enhanced particle uptake by macrophages, whereas the overall TG accumulation was reduced 2-fold compared with that of native VLDL. Thus, considering the lipid accumulation in macrophages as fundamental to foam cell formation, our results would point to an antiatherogenic effect of VLDL oxidation in the intima. However, 2 questions need to be answered before we can speculate any further on the possible antiatherogenic effect of VLDL oxidation as being relevant for the in vivo situation. (1) Does oxidation of VLDL occur in vivo in the intima to the extent required for rendering these particles defective as a substrate for LPL? and (2) at what time after entering the intima does this VLDL oxidation occur, ie, before or after the LPL-mediated lipolysis?

It has been well established that LDL can infiltrate the arterial intima and that subsequent oxidative modification of this lipoprotein particle enhances its atherogenic potential.34 Whether oxVLDL is also present in vivo remains to be determined. However, considering the facts that particles in the VLDL size range have been isolated from the human aortic intima35 and human aortic atherosclerotic plaques36 and that VLDL undergoes oxidation in vitro similarly to LDL,9 we hypothesize that VLDL oxidation may indeed occur in the intima in vivo. Whether this oxidative modification is severe enough to inhibit the LPL-mediated lipolysis is at present subject to speculation. In addition, because it may be expected that in the intima, the VLDL particle will be lipolyzed immediately after entry, it may be suggested that the oxidative modification becomes manifest only after lipolysis of most of the VLDL-TG, ie, after VLDL particles have been converted into VLDL remnant particles. Recently, it was shown that oxVLDL remnant particles are most effective in the accumulation of cholesteryl ester in macrophages.11 Taking all these considerations into account, we suggest that in the intima, LPL-mediated lipolysis of VLDL TGs and oxidative modification of VLDL (remnant) particles represent 2 proatherogenic steps: (1) on entry into the intima, the action of LPL leads to enhanced TG accumulation in macrophages, and subsequently, (2) oxidative modification of VLDL remnants promotes cholesteryl ester accumulation in macrophages.


*    Acknowledgments
 
This study was financially supported by the Dutch Heart Foundation (projects 92.337 and 97.067). We thank Wim Bax for preparing the 14C-labeled VLDL samples and Hans van der Boom for excellent technical assistance. We are grateful to Dr Ulrike Beisiegel for providing us with plasma from an apoC2-deficient patient.

Received May 5, 1999; accepted September 2, 1999.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
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 up arrowDiscussion
 *References
 

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