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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1707-1715

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1707-1715.)
© 1997 American Heart Association, Inc.

Articles

Uptake of Type III Hypertriglyceridemic VLDL by Macrophages Is Enhanced by Oxidation, Especially After Remnant Formation

Stewart C. Whitman; David B. Miller; Bernard M. Wolfe; Robert A. Hegele; ; Murray W. Huff

From the Departments of Medicine and Biochemistry and the Robarts Research Institute at University of Western Ontario, London, and the Department of Medicine, St Michael's Hospital, University of Toronto (Ont), Canada.

Correspondence to Murray W. Huff, PhD, Robarts Research Institute, 416, University of Western Ontario, London, Ontario, Canada, N6A 5K8. E-mail mhuff{at}julian.uwo.ca


*    Abstract
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Abstract We previously showed that hypertriglyceridemic VLDL (HTG-VLDL, Sf 60 to 400) from subjects with type III (E2/E2) hyperlipoproteinemia do not induce appreciable cholesteryl ester (CE) accumulation in cultured macrophages (J774A.1). In the present study, we examined whether oxidation of type III HTG-VLDL would enhance their uptake by J774A.1 cells. Type III HTG-VLDL were oxidized as measured by both conjugated-diene formation and increased electrophoretic mobility on agarose gels. Both LDL and type III HTG-VLDL undergo oxidation, albeit under different kinetic parameters. From the conjugated-diene curve, type III HTG-VLDL, compared with LDL, were found to have a 6-fold longer lag time, to take 6-fold longer to reach maximal diene production, and to produce a 2-fold greater amount of dienes but at half the rate (all P<.005). Incubation of macrophages with either native type III HTG-VLDL or LDL (50 µg lipoprotein cholesterol/mL media for 16 hours) caused small increases (4-fold and 2.7-fold, respectively) in cellular CE levels relative to control cells (both P=.0001). After 24 hours of CuSO4 exposure, we found that oxidized type III HTG-VLDL and LDL caused a 9.4-fold and 10.5-fold increase, respectively, in cellular CE levels (P=.0001). We next examined whether extending the exposure period for type III HTG-VLDL to CuSO4 beyond 24 hours would further enhance its ability to induce macrophage CE accumulation. After 48 hours of CuSO4 exposure, type III HTG-VLDL and LDL caused 21.3-fold and 11.6-fold increases, respectively, in cellular CE levels (P=.0001). The cellular CE loading achieved with 48 hour–oxidized type III HTG-VLDL was significantly higher than either 24 hour–oxidized type III HTG-VLDL (2.3-fold, P=.003) or 48 hour–oxidized LDL (1.8-fold, P=.012). There was no significant difference between the CE loading achieved by incubation of cells with either 24 hour–oxidized type III HTG-VLDL, 24 hour–oxidized LDL, or 48 hour–oxidized LDL (P>=.518). In this study, we also examined whether partial lipolysis (19% to 50% triglyceride hydrolysis) of type III HTG-VLDL to produce remnants would increase the susceptibility of the lipoprotein to oxidative modification and subsequent cellular CE loading. Forty-eight hour–oxidized type III VLDL-remnants stimulated CE accumulation 30.4-fold over baseline (P=.0001). In contrast, nonoxidized type III VLDL-remnants caused the same very low level of CE loading as did native type III HTG-VLDL (P=.680). The increase in cellular CE levels achieved with 48 hour–oxidized type III VLDL-remnants was significantly higher than that achieved with 48 hour–oxidized type III HTG-VLDL (P=.047). In conclusion, we have shown that oxidized type III HTG-VLDL will induce macrophage CE accumulation well above levels achieved with oxidized LDL. In addition, we also showed that by forming a VLDL-remnant before oxidative modification, we can further enhance macrophage CE accumulation. These results provide a potential mechanism for the atherogenicity of type III HTG-VLDL and their remnants.


Key Words: type III HLP • VLDL • remnants • oxidation • foam cells


*    Introduction
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 *Introduction
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Cholesterol-loaded macrophages, morphologically recognized as foam cells, are characteristic of developing atherosclerotic plaques.1 2 It has been well established that incubation of cultured macrophages with native LDL does not result in either significant cholesterol deposition or foam cell formation.3 However, oxidation of LDL generates a modified form of these lipoproteins whose uptake by macrophages is markedly enhanced, leading to unregulated CE accumulation by the macrophage and foam cell formation.4 5 6 Human VLDL are capable of inducing foam cell formation, without the requirement for oxidation.7 8 9 10 11 Apo E is important in modulating the uptake of VLDL by macrophages,7 8 12 13 14 and previous studies from our laboratory have shown that uptake of VLDL from hypertriglyceridemic subjects (HTG-VLDL) by J774A.1 macrophages requires receptor-competent apo E.7 8

The human apo E gene is a polymorphic locus having three main alleles ({epsilon}2, {epsilon}3, and {epsilon}4), which in turn give rise to three protein isoforms (E2, E3, and E4, respectively).15 16 Apo E3 and apo E4 have been shown to be the main ligands for removal by the liver of both VLDL and VLDL-remnants.16 Unlike apo E3 and apo E4, the apo E2 (Arg158->131->Cys) isoform has been shown to be defective in its ability to bind to the LDL receptor,17 18 and in subjects homozygous for this isoform, it creates susceptibility to type III HLP.19 Subjects with type III HLP have elevated levels of CE-enriched VLDL as a result of the inability of the hepatic receptors to clear these lipoproteins from the circulation.19 20 Although type III subjects have elevated levels of VLDL and VLDL-remnant particles, they have normal or reduced levels of LDL.19 20 While individuals with type III HLP are at increased risk for atherosclerosis,19 20 the basis for this increased risk is not fully understood. HTG-VLDL isolated from subjects with type IV HLP does contain receptor-competent apo E and will induce CE accumulation in cultured macrophages.7 8 In contrast, coincubation of these same macrophages with type III HTG-VLDL failed to induce an appreciable increase in cellular CE levels.7 8

Like individuals with type III HLP, EKO mice display defective remnant clearance.21 These mice develop atherosclerosis spontaneously,21 22 23 24 25 emphasizing the atherogenic nature of remnants and the importance of functional apo E in the metabolism of TG-rich lipoproteins. In EKO mice, the remnant lipoproteins must therefore cause macrophage lipid accumulation by an apo E–independent mechanism. EKO mice have foam cell lesions that contain epitopes of oxidized lipoproteins and plasma that contains autoantibodies that recognize epitopes on oxidized lipoproteins.24 26 Therefore, oxidative modification of VLDL and VLDL-remnants may be responsible for lesion development in this animal model of atherosclerosis.

In the present study, we tested the hypothesis that oxidative modification of HTG-VLDL (Sf 60 to 400) isolated from subjects with type III HLP leads to foam cell formation in vitro by a mechanism analogous to that which occurs with oxidized LDL. To test this hypothesis, we examined whether type III HTG-VLDL could be oxidized in vitro and whether this modified form of type III HTG-VLDL could induce CE loading of cultured macrophages.


*    Methods
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 *Methods
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Subjects
Subjects were recruited from the Outpatient Endocrinology Lipid Clinics at the London Health Sciences Centre, London, Ontario, and the Lipid Clinic at St Michael's Hospital, Toronto, Ontario. Lipoprotein phenotypes of the HLP subjects were classified according to the criteria of Schaefer and Levy.27 All type III HLP subjects (n=8) were homozygous for the apo E2 isoform, as determined by isoelectric focusing gel electrophoresis28 29 and/or restriction isotyping.30 All type III HLP subjects had elevated plasma cholesterol (8.96±1.39 mmol/L, mean±SEM) and TG (6.94±1.68 mmol/L) levels due to elevated VLDL cholesterol (5.74±1.17 mmol/L) and TG (6.31±1.64 mmol/L) concentrations. LDL preparations used in these experiments were derived from 10 subjects: 1 normolipidemic subject, 4 type III HLP subjects, 3 type IV HLP subjects, and 2 type IIa HLP subjects. These studies were approved by the University of Western Ontario Health Sciences Standing Committee on Human Research, and all subjects gave informed consent before blood sampling.

Lipoprotein Isolation
Sixty to 180 mL of blood was collected from each fasted (12 to 14 hours) subject and placed in tubes containing EDTA-Na2 at a final concentration of 0.15%. Plasma was isolated by centrifugation (Sorvall IEC Centra-8R centrifuge) at 2500 rpm (1000g) for 25 minutes at 4°C. The large VLDL (Sf 60 to 400) subfraction was collected and washed, as described previously,31 by ultracentrifugation in a Beckman L8 ultracentrifuge. The VLDL was first isolated with a Beckman 55.2 Ti rotor (1.75 hours, 40 000 rpm, 12°C) and subsequently washed with a Beckman 70.1 Ti rotor (16 hours, 40 000 rpm, 12°C). After isolation of the VLDL and IDL fractions (d<1.019 g/mL), the LDL (d=1.019 to 1.063 g/mL) fraction was isolated from the infranatant by ultracentrifugation (16 hours, 50 000 rpm, 12°C) in a 55.2 Ti rotor and washed with a 70.1 Ti rotor (16 hours, 50 000 rpm, 12°C). LPDS was prepared as outlined previously.8 The LPDS was assayed for apo C-II and apo E32 and found to be free of these apolipoproteins. Both the VLDL (Sf 60 to 400) and LDL preparations were extensively dialyzed, in the dark and at 4°C, against a 200-fold excess volume of PBS (in mmol/L: NaCl 154, Na2H·7H2O 8, KH2PO4 1.5, and KCl 2.7) containing 10 µmol/L EDTA-Na2. After dialysis, the lipoprotein samples were sterilized with 0.45-µm filters and stored at 4°C.

All lipoprotein samples were analyzed for protein content by a modification of the Lowry method33 for free fatty acids with a diagnostic kit from Wako (NEFAC kit 990-75401) and for TG, free cholesterol, total cholesterol, and phospholipid with diagnostic kits from Boehringer Mannheim GmbH Diagnostica (kits 450032 without free glycerol, 310328, 1442350, and 691844, respectively).

Bovine Milk LPL Isolation
Bovine skim milk LPL was partially purified by a modification of the method of Socorro and Jackson34 as described previously.35 The LPL activity in each eluted fraction was determined by measuring the amount of free fatty acids released from a predetermined amount of a commercially obtained TG emulsion (Intralipid, Pharmacia Inc). The LPL activity assay was conducted in 16x100-mm borosilicate glass tubes (Fisher Scientific) by adding, in order, the following: 300 µL LPL buffer (0.15 mol/L NaCl, 0.2 mol/L Tris, pH 8.2), 5% (wt/vol) FAF-BSA (fraction V, Sigma Chemical Co), and 12% (vol/vol) normolipidemic human plasma (as a source of apo C-II), 10 µL deionized H2O, 10 µL lipase solution, and 100 µL Intralipid containing 1.82 µmol TG. After incubation for 30 minutes at 37°C in a shaking water bath, the reaction was stopped by addition of 2 mL isopropyl alcohol–3N H2SO4 40:1 (vol/vol), 1 mL H2O, and 2.5 mL hexane, with vigorous vortexing for 1 minute. The phases were allowed to separate by standing for 20 minutes at room temperature. The hexane layer was removed into new 16x100-mm glass test tubes, and the hexane extraction step was then repeated once. The pooled hexane fractions were evaporated under N2. Chloroform (1 mL) was added to each tube, and aliquots were taken for subsequent fatty acid determination. To these aliquots, 750 µL of a 1% (vol/vol) solution of Triton X-100 in chloroform was added, the solutions were mixed and dried under N2, and the amount of free fatty acids was determined with a spectrophotometry-based free-fatty-acid assay kit (see above). One unit of LPL activity is defined as 1 µmol free fatty acid released · mL enzyme solution-1 · h-1.

VLDL-Remnant Preparation
Remnant-like particles of type III HTG-VLDL (Sf 60 to 400) were formed in vitro under sterile conditions by incubation of the type III HTG-VLDL with LPL (0.0638 U/50 µg total lipoprotein cholesterol) in the presence of a 5% (wt/vol final concentration) solution of FAF-BSA made up in PBS. TG hydrolysis was allowed to proceed at 37°C in a tissue culture incubator for 8 hours. The VLDL-remnants were reisolated by adjustment of the reaction buffer to a final density of 1.019 g/mL followed by ultracentrifugation in a Beckman 70.1 Ti rotor (16 hours, 40 000 rpm, 12°C). The VLDL-remnant preparations were dialyzed and sterilized by filtration as stated above. Percent TG hydrolysis was calculated from the following formula: % hydrolysis=100-[(TG/CE of VLDL-remnantx100)/(TG/CE of native VLDL)]. In all remnant-forming assays, the percentage of TGs hydrolyzed ranged from 19.0% to 50.0%.

Oxidized Lipoprotein Preparation
The dialyzed and sterile type III HTG-VLDL, type III VLDL-remnant, and LDL preparations were oxidized in vitro according to a modification of the protocol described by Steinbrecher et al.36 Briefly, reactions were performed under sterile conditions by incubation of the lipoprotein preparation (200 µg protein/mL) with CuSO4 (5.0 µmol/L) in EDTA-free PBS for either 24 or 48 hours at 37°C and in the absence of light. A control reaction for each sample was run by substitution of EDTA (200 µmol/L) for CuSO4 (sham-oxidized lipoproteins). The reactions were stopped by immediate placement of the samples on ice, followed by the addition of EDTA (200 µmol/L) and BHT (40 µmol/L). The sham-oxidized and oxidized lipoprotein preparations were then reisolated by adjustment of the reaction buffer to a final density of either 1.063 g/mL (sham-oxidized/oxidized type III HTG-VLDL and VLDL-remnants) or 1.10 g/mL (sham-oxidized/oxidized LDL) with buffer A at d=1.34 g/mL, followed by ultracentrifugation with a Beckman 70.1 Ti rotor spun for 16 hours at 50 000 rpm and 12°C. The sham-oxidized and oxidized HTG-VLDL, VLDL-remnant, and LDL preparations were dialyzed and sterilized by filtration as stated above. Filtration of the oxidized lipoprotein preparations also served to remove any aggregated lipoproteins.

The lipoprotein oxidation reactions were monitored at both the early and late stages of modification by assaying for conjugated-diene formation and changes in lipoprotein electrophoretic mobility, respectively. The conjugated-diene assays were conducted in parallel with the oxidation reactions according to the protocol of Kleinveld et al.37 The kinetics of conjugated-diene production at 37°C were determined by continuous monitoring of the changes in absorbance at 234 nm over a period of 20 hours. A 5-µg sample of each lipoprotein preparation (native, sham-oxidized, or oxidized) was subjected to agarose gel electrophoresis36 to determine changes in lipoprotein electrophoretic mobility. The mobility of each lipoprotein sample relative to BSA (5 µg/lane) was used as a measure of the degree of oxidative modification. The lipoprotein and BSA in each lane were visualized with Coomassie brilliant blue R250 (Bio-Rad).

Cell Culture
J774A.1 cells, a murine macrophage–like cell line that secretes LPL but not apo E,38 39 were used in this study. Because the J774A.1 cells do not secrete apo E, the potential for confounding effects introduced by the presence of functional cell-secreted apo E was eliminated. The J774A.1 cells were obtained from the American Type Culture Collection and were maintained in culture as outlined previously.8 For all experiments in the present report, J774A.1 cells were plated in six-well (35-mm) culture plates (Linbro, Flow Laboratories Inc) in 2.0 mL of DMEM (low glucose) (Gibco) containing 10% FCS (Flow Laboratories Inc) and grown for 2 to 3 days. When the monolayers had become 70% to 80% confluent, the medium was replaced with DMEM containing 5% LPDS. The final albumin concentration in the medium was 0.13%. For each lipoprotein preparation, 50 µg total lipoprotein cholesterol/mL medium was added to duplicate or in some instances triplicate wells of cells and was incubated for 16 hours at 37°C.

Quantitative Analysis of Cellular Lipids
The cell-lipoprotein incubations were terminated by two washes with buffer B (0.15 mol/L NaCl, 50 mmol/L Tris, 0.2% [wt/vol] FAF-BSA, pH 7.4) and two additional washes with buffer B without FAF-BSA. The lipids were extracted in situ with two 30-minute incubations with 1.0 mL of hexane-isopropanol 3:2 (vol/vol), with the solvents from each extraction being pooled. To each dish, 1 mL of 0.1N NaOH was added and incubated overnight at room temperature to digest the cells. Cell protein was determined as stated above. Cellular CE mass was determined by gas-liquid chromatography and cellular TG mass by the method of Neri and Frings40 as outlined previously.8

Statistical Analysis
In each experiment, duplicate and in some instances triplicate cell culture wells were used for each specific lipoprotein preparation, with the resulting values combined to give a mean value for that particular experiment. Mean values from separate experiments were then used to calculate a group mean±SEM for each condition. Statistical significance between control and experimental group mean values was assessed with a Student's unpaired t test. A value of P<.05 was considered statistically significant.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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Type III HTG-VLDL, like LDL, was susceptible to oxidative modification after exposure to CuSO4 as assessed by the formation of conjugated dienes (Fig 1Down, Table 1Down). From the conjugated-diene curve, type III HTG-VLDL (compared with LDL) was found to have a 6-fold longer lag time, took 6-fold longer to reach maximal diene production, and produced a 2-fold greater amount of dienes but at half the rate (Table 1Down, P=.005).



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  		Figure 1. Line graph shows representative curves of conjugated-diene formation generated from oxidation of LDL ({bigtriangleup}), type III HTG-VLDL ({circ}), and type III VLDL-remnants ({bullet}). Conjugated-diene curves were produced by incubating (37°C) lipoprotein preparations (50 µg protein/mL) with CuSO4 (5.0 µmol/L) in EDTA-free PBS and continuously monitoring changes in absorbance at 234 nm over a period of 20 hours. Sham oxidations ({blacktriangleup}) were conducted by replacing CuSO4 with EDTA (200 µmol/L).


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  		Table 1. CuSO4-Induced Formation of Conjugated Dienes

On the basis of the considerably longer lag phase to oxidation exhibited by type III HTG-VLDL, we extended the duration of VLDL exposure to CuSO4 from 24 to 48 hours. This increase in incubation time was carried out to ensure that the VLDL preparations were becoming sufficiently modified by oxidation to achieve maximal cellular uptake. Oxidative modification (24 and 48 hours) of both type III HTG-VLDL and LDL caused a significant increase (P<.001) in the REM of these modified lipoproteins on agarose gels compared with their native counterparts (Table 2Down). No significant difference existed between the REM of 24 hour–oxidized type III HTG-VLDL versus 24 hour–oxidized LDL (P=.12) or 48 hour–oxidized LDL (P=.42) (Table 2Down). Comparison of the REM for 24 hour–oxidized LDL versus 48 hour–oxidized LDL also showed no significant difference (P=.24; Table 2Down). In contrast, 48 hour–oxidized type III HTG-VLDL had a significantly greater REM than both 24 hour–oxidized type III HTG-VLDL and 24 hour–oxidized LDL (P<.0003; Table 2Down).


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  		Table 2. REM of Lipoproteins on Agarose Gels

The compositions of the lipoproteins used in the cell studies are summarized in Table 3Down. Oxidative modification of type III HTG-VLDL for 24 hours or type III HTG-VLDL and type III VLDL-remnants for 48 hours caused no significant change in any of the compositional characteristics measured (Table 3Down). In addition to a 30.3% reduction (P=.035) in the ratio of TG to CE, the process of in vitro remnant formation also caused a 2-fold (P=.01) increase in the ratio of free fatty acid to protein of the newly formed remnants (Table 3Down). In contrast to oxidation of VLDL and VLDL-remnants, oxidation of LDL for either 24 or 48 hours caused a significant decrease in the ratio of LDL total cholesterol to protein (P<.01; Table 3Down). The observed decrease in total cholesterol once LDL had been oxidized has been reported previously by other laboratories.36 41


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  		Table 3. Characteristics of LDL, VLDL, and VLDL-Remnants Used in the Cell Studies1

Incubation of J774A.1 cells with either native type III HTG-VLDL or native LDL resulted in a 4-fold and a 2.7-fold increase, respectively, in cellular CE levels relative to control cells incubated in lipoprotein-deficient media (both P=.0001; Fig 2ADown). The increases in cellular CE levels induced by incubating cells with either native type III HTG-VLDL or native LDL were not significantly different from each other (P=.18; Fig 2ADown). With 24 hours of oxidation used as a starting point for lipoprotein modification, oxidized type III HTG-VLDL and oxidized LDL caused a 9.4-fold and 10.5-fold increase, respectively, in cellular CE levels relative to control cells (both P=.0001; Fig 2ADown). The extents of cellular CE loading achieved with 24 hour–oxidized type III HTG-VLDL versus 24 hour–oxidized LDL were not significantly different from each other (P=.75; Fig 2ADown). After 48 hours of exposure to CuSO4, oxidized type III HTG-VLDL and oxidized LDL caused a 21.3-fold and 11.6-fold increase, respectively, in cellular CE levels compared with control cells (both P=.0001; Fig 2ADown). The extent of cellular CE loading achieved with 48 hour–oxidized type III HTG-VLDL was significantly higher than that observed with either 24 hour–oxidized type III HTG-VLDL (P=.003) or 48 hour–oxidized LDL (P=.01; Fig 2ADown). There was no significant difference in the amount of cellular CE loading achieved by incubating cells with LDL oxidized for either 24 or 48 hours (P=.74; Fig 2ADown). Even though the LDL preparations used in these experiments were derived from 10 subjects with different blood lipid phenotypes, no significant difference in cellular uptake between each preparation was found.



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  		Figure 2. Comparison of type III HTG-VLDL and LDL to cause cellular cholesteryl ester (A) and TG (B) accumulation in J774A.1 macrophages after either 24 or 48 hours of oxidative modification. Lipoprotein cholesterol (50 µg/mL medium) of various lipoprotein preparations: type III HTG-VLDL, native (n=10), oxidized for 24 hours (n=5), or oxidized for 48 hours (n=5) and LDL native (n=11), oxidized for 24 hours (n=5), or oxidized for 48 hours (n=6) was incubated with J774A.1 macrophages for 16 hours. Values are mean±SEM for number of experiments indicated for each incubation condition. a, P=.0001 relative to control cells. b, P<.006 relative to native counterparts. c, P=.003 relative to 24 hour–oxidized type III HTG-VLDL and P=.01 relative to 48 hour–oxidized LDL. d, P<.03 relative to 24 and 48 hour–oxidized LDL. Cellular CE and TG levels were determined as described in "Methods."

Incubation of J774A.1 cells with native type III HTG-VLDL resulted in a 12.2-fold increase in cellular TG levels compared with control cells (Fig 2BUp, P=.0001). This finding is consistent with observations reported previously by this laboratory.8 In contrast to native type III HTG-VLDL, incubation of cells with either 24 or 48 hour–oxidized type III HTG-VLDL resulted in only 3.2-fold and 4.9-fold increases, respectively, in cellular TG levels compared with control cells (both P=.0001; Fig 2BUp). This difference between native and oxidized type III HTG-VLDL was statistically significant (P=.006). No differences with respect to the incubation conditions (based on lipoprotein TG concentrations) could be found to explain the observed reduction in cellular TG levels achieved with oxidized (24 and 48 hours) type III HTG-VLDL versus native type III HTG-VLDL (Table 3Up).

We next examined whether partial in vitro hydrolysis would render the VLDL lipoprotein more susceptible to oxidative modification and in turn allow the lipoprotein to induce an even greater amount of cellular CE loading. Type III VLDL-remnants, like native type III HTG-VLDL, were susceptible to oxidative modification after exposure to CuSO4 as assessed by the formation of conjugated dienes (Fig 1Up, Table 1Up). From the conjugated-diene curve, type III VLDL-remnants (compared with native type III HTG-VLDL) were found to have a similar lag time, to take slightly less time to reach maximal diene production, and to produce a similar amount of conjugated dienes but at almost twice the rate (Table 1Up).

No differences in REMs were seen between type III VLDL-remnants, sham-oxidized type III VLDL-remnants, and oxidized type III VLDL-remnants (Table 2Up). The REM of the VLDL-remnant preparations was found to equal that of the oxidized type III HTG-VLDL preparations and to be greater than that of the oxidized LDL preparations (P<.001; Table 2Up).

Type III VLDL-remnants increased cellular CE levels 4.6-fold compared with control cells (P=.0001; Fig 3ADown). This level of cellular CE accumulation was found not to be significantly different from the cellular CE levels induced by native type III HTG-VLDL (P=.68; Fig 3ADown). In contrast, type III VLDL-remnants oxidized for 48 hours caused a 30.4-fold increase in cellular CE levels compared with control cells (P=.0001; Fig 3ADown). The increase in cellular CE levels achieved with 48 hour–oxidized type III VLDL-remnants was significantly higher than the 21.3-fold increase achieved by incubating cells with 48 hour–oxidized type III HTG-VLDL (P=.047; Fig 3ADown).



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  		Figure 3. Comparison of 48 hour–oxidized type III HTG-VLDL, 48 hour–oxidized type III VLDL-remnants, and their nonoxidized counterparts to cause cellular cholesteryl ester (A) and TG (B) accumulation in J774A.1 macrophages. Lipoprotein cholesterol (50 µg/mL of medium) of various lipoprotein preparations: type III VLDL, native (n=10) or oxidized for 48 hours (n=5) and type III VLDL-remnants, nonoxidized (n=5) or oxidized for 48 hours (n=5) was incubated with J774A.1 macrophages for 16 hours. Values are mean±SEM for number of experiments indicated for each incubation condition. a, P<.006 relative to nonoxidized counterpart. b, P=.047 relative to 24 hour–oxidized type III HTG-VLDL. Cellular CE and TG levels were determined as described in "Methods."

Unmodified type III VLDL-remnants were found to induce a 10.4-fold increase in cellular TG levels compared with control cells (P=.0001, Fig 3BUp). However, oxidized type III VLDL-remnants were able to bring about only a 4.9-fold increase in cellular TG levels compared with control cells (P=.0001, Fig 3BUp). Once again, this difference could not be explained by differences in the incubation conditions based on TG concentrations between the native and oxidized type III VLDL-remnants (Table 3Up).


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Our findings present the first evidence that oxidized type III HTG-VLDL is more effective than oxidized LDL at inducing CE loading of cultured macrophages. This study is also the first to directly compare the susceptibility of human VLDL versus LDL to oxidation in vitro. In addition, these experiments showed that lipolysis of type III HTG-VLDL, followed by oxidative modification, further enhanced macrophage CE loading. These findings are consistent with the results of Palinski et al24 that atherogenesis in EKO mice is related to enhanced oxidation of remnant lipoproteins.

Copper-induced oxidation of LDL involves the peroxidation of polyunsaturated fatty acids found within the LDL phospholipid monolayer, which transforms them to reactive aldehyde-derivatized fragments36 42 such as malondialdehyde and 4-hydroxynonenal.43 Once created, these reactive aldehydes undergo Schiff-base reactions with the {epsilon}-amino groups of lysine residues on the LDL apo B-100 molecule.36 42 This type of modification neutralizes the net positive charge of apo B-100,3 36 44 which reduces its affinity for the LDL receptor and increases its affinity for the acetyl-LDL (scavenger) receptor. Because the scavenger receptor, unlike the LDL receptor, is not downregulated by increases in cellular CE levels,44 45 the uptake of oxidized LDL by this pathway results in unregulated accumulation of cellular CE and subsequent foam cell formation. During the early stages of the oxidative process, the extent of lipoprotein modification can be monitored by assay for the formation of conjugated dienes, which are transiently formed during the conversion of polyunsaturated fatty acids to reactive aldehydes.37 The end stage of modification can be assessed by monitoring changes in the electrophoretic mobility of the oxidized lipoproteins on agarose gels. This study has shown that although both LDL and VLDL can undergo oxidation, the two lipoproteins do so under different kinetic parameters. LDL undergoes oxidation much sooner and at a greater rate than HTG-VLDL. However, once HTG-VLDL begins to be oxidized, a greater number of conjugated dienes (and hence reactive aldehydes) can be generated than with LDL.

The magnitude of the lag phase of a typical conjugated-diene curve has been shown to be directly proportional to the amount of antioxidants present within the target lipoprotein.46 47 48 Of the various antioxidants in a given lipoprotein subclass, {alpha}-tocopherol has been shown to be the most potent.46 47 48 The difference between the lengths of the lag portion of the VLDL and LDL conjugated-diene curves probably arises because the VLDL particles contain, on average, many more antioxidant molecules (such as {alpha}-tocopherol) per lipoprotein than does LDL47 49 and would therefore be protected to a greater extent against the initiation of oxidation. Because VLDL particles are larger than LDL, they contain more polyunsaturated fatty acid substrate for oxidation. As our data show, this would produce more conjugated dienes from VLDL after oxidation commences. Conjugated dienes are formed at twice the rate in LDL versus type III HTG-VLDL because the surface phospholipid monolayer of the smaller, denser LDL particles may have an enhanced vulnerability to oxidation by external agents such as CuSO4.42 This notion is supported by the fact that the rate of conjugated-diene formation of type III VLDL-remnants was higher than that achieved with native type III HTG-VLDL.

Most studies involving oxidized lipoproteins and their ability to induce foam cell formation have focused on oxidized LDL.50 In the past decade, however, several groups have examined the atherogenic potential of oxidized VLDL. Most of these studies have used human VLDL isolated from normolipidemic49 51 52 53 54 or hypercholesterolemic (type IIa) subjects52 or ß-VLDL isolated from hypercholesterolemic rabbits.55 56 57 Of the human VLDL studies, only one directly examined the ability of oxidized VLDL to be taken up by cultured macrophages.53 The oxidation of VLDL from normolipidemic subjects after 24 hours of exposure to CuSO4 resulted in 4-fold more degradation of the iodinated lipoprotein in mouse peritoneal macrophages compared with native VLDL.53 Four other studies examined the oxidation of human VLDL as assessed by increases in REMs,51 the formation of thiobarbituric acid–reactive substances,51 52 54 and/or the formation of hydroperoxides.49 51 These studies showed that human VLDL was susceptible to oxidation; however, only the study by Jurgens et al51 examined both VLDL and LDL under similar oxidative stresses. In that study, which used CuCl2 as an oxidant for 24 hours, oxidized VLDL and oxidized LDL from normolipidemic subjects were found to have very similar REMs and levels of thiobarbituric acid–reactive substances.

Oxidation studies examining ß-VLDL from cholesterol-fed rabbits focused on the ability of radioiodinated oxidized ß-VLDL to be degraded by SMCs56 or macrophages55 57 and the ability of oxidized ß-VLDL to induce an increase in macrophage cholesterol esterification.55 Two of these rabbit studies compared oxidized ß-VLDL (d<1.006 g/mL)55 or oxidized IDL (d 1.006 to 1.019 g/mL)58 with oxidized LDL. In the study by Parthasarathy et al,55 oxidized ß-VLDL (24 hours with CuSO4) was found to be degraded by macrophages at half the rate of oxidized LDL. In contrast, Haratz et al58 found that SMC-induced oxidative modification of IDL particles, which were used as a model for VLDL-remnants, enhanced their rate of degradation (1.3- to 2.4-fold higher) by macrophages compared with SMC-modified LDL. Results from studies of rabbit ß-VLDL are difficult to extrapolate to humans because humans have no lipoprotein counterpart to rabbit ß-VLDL. Although type III HTG-VLDL contains ß-migrating lipoproteins, in contrast to rabbit ß-VLDL, in their native form they are taken up poorly by J774A.1 macrophages.8 Our present experiments have shown that human type III HTG-VLDL can be oxidized in vitro and that oxidized type III HTG-VLDL significantly enhances CE loading of cultured macrophages compared with native type III HTG-VLDL and with oxidized LDL. Our results differ from those of Parthasarathy et al55 because we found that 48 hour–oxidized type III HTG-VLDL is actually more atherogenic than 48 hour–oxidized LDL. The reason for this discrepancy is not clear; however, it is possible that the oxidation period of 24 hours used for rabbit ß-VLDL was not sufficient to modify the ß-VLDL to the same extent as LDL.

Oxidized type III HTG-VLDL and oxidized type III VLDL-remnants caused an increase in cellular CE levels but a decrease in cellular TG levels compared with their nonoxidized counterparts. We previously showed that HTG-VLDL–induced CE and TG accumulation in J774A.1 macrophages occurs by different mechanisms.8 We demonstrated that after interaction with cellular LPL, VLDL-TG are hydrolyzed extracellularly, with the resulting free fatty acids being subsequently taken up by the macrophage and reesterified into TG within the cell. Lipolysis proceeds until apo E epitopes are exposed, allowing the TG-depleted remnant, containing all the CE, to be taken up via an apo E–mediated process.8 A possible explanation for the decreased ability of oxidized type III HTG-VLDL to induce TG loading would be oxidative modification and subsequent inactivation of lipoprotein-associated apo C-II. Inactivation of apo C-II, which is the coenzyme for LPL, would effectively eliminate the ability of macrophage-released LPL to act on the oxidized VLDL particles. Although in this study we did not directly examine whether apo C-II was modified, Keidar et al53 previously showed that oxidation of VLDL from normolipidemic subjects did result in the fragmentation of the C apolipoproteins as well as apo B-100 and apo E.

The unexpected increase in the electrophoretic mobility of both the unmodified and sham-oxidized type III VLDL-remnants is most likely related to an increase in the free-fatty-acid content of the lipoproteins (Table 3Up). Studies by Gordon59 and Herbst et al60 have shown that enrichment of LDL with free fatty acids (LDL collected from postheparin plasma) directly caused an increase in the REM of LDL. A similar type of lipoprotein modification may have taken place during the formation of type III VLDL-remnants in vitro. Chung et al61 showed that when rapid and extensive lipolysis of TG-rich lipoproteins occurs in sera of hypertriglyceridemic subjects or in postprandial lipemic sera from normolipidemic subjects, albumin present in the sera binds only a small portion (14% to 35%) of the free fatty acids generated by lipolysis, with the majority becoming partitioned (bound) to lipoproteins, particularly VLDL-remnants. It is therefore conceivable that during remnant formation in vitro, the free fatty acids partitioned to VLDL-remnants rather than to albumin. This would increase the REM of the remnant, as was shown for LDL in the studies by Gordon59 and Herbst et al.60 Despite this increased electrophoretic mobility, which suggested that the VLDL-remnants would be more readily taken up by macrophages, VLDL-remnant preparations induced the same low level of cellular CE accumulation as did native type III HTG-VLDL. In addition, 48 hour–oxidized type III HTG-VLDL and 48 hour–oxidized type III VLDL-remnants have similar REMs, but the latter showed a 2-fold greater enhancement of CE accumulation relative to the former. These findings therefore demonstrate that not all processes that alter the REM of a lipoprotein necessarily enhance the ability of that lipoprotein to be taken up by macrophages.47

In conclusion, the present experiments clearly demonstrate that human type III HTG-VLDL and their remnants can be oxidatively modified in vitro, leading to enhanced macrophage uptake. This provides a potential mechanism to explain foam cell formation in type III hyperlipidemia. The enhanced cellular CE accumulation by oxidized type III VLDL-remnants compared with oxidized type III HTG-VLDL suggests that the former may be the more atherogenic of the two lipoprotein particles in vivo. We hypothesize that in vivo macrophage-secreted LPL may first aid in the trapping of partially catabolized type III HTG-VLDL within the extracellular matrix and to the macrophage cell surface. After further TG hydrolysis by LPL, the anchored type III VLDL-remnants may undergo cell-induced oxidation by one or more of the possible cellular mechanisms that have been proposed for LDL modification.62 Oxidative modification of the VLDL-remnants would allow these modified lipoproteins to be rapidly taken up via the scavenger receptor on macrophages and SMCs, resulting in CE accumulation and foam cell formation.


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
CE = cholesteryl ester
EKO = apo E knockout
FAF = fatty acid–free
HLP = hyperlipoproteinemia
HTG = hypertriglyceridemic
LPDS = lipoprotein-deficient serum
LPL = lipoprotein lipase
REM = relative electrophoretic mobility
Sf = Svedberg flotation unit
SMC = smooth muscle cell
TG = triglyceride


*    Acknowledgments
 
This work was supported by a Medical Research Council of Canada grant (MT8014) to Dr Huff. Dr Whitman is a recipient of a Medical Research Council of Canada Studentship. Dr Miller is a recipient of a Heart and Stroke Foundation of Canada Research Fellowship. Drs Hegele and Huff are Career Investigators of the Heart and Stroke Foundation of Ontario. We are grateful to Sandra Kleinstiver and Cynthia Sawyez for their expert technical assistance.


*    Footnotes
 
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and printed in abstract form (Circulation. 1995;92[suppl I]:I-289).


*    References
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