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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2995-3005

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2995-3005.)
© 1997 American Heart Association, Inc.

Articles

Plasma LDL Oxidation Leads to Its Aggregation in the Atherosclerotic Apolipoprotein E-Deficient Mice

Irit Maor; Tony Hayek; Raymond Coleman; ; Michael Aviram

From the Lipid Research Laboratory and the Division of Morphologic Sciences (R.C.), Technion Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel.

Correspondence to Michael Aviram DSc, Lipid Research Laboratory, Rambam Medical Center, Haifa, 31096, Israel. E-mail aviram{at}tx.technion.ac.il


*    Abstract
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 *Abstract
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 down arrowResults
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Abstract Two major modifications of low density lipoprotein (LDL) that can lead to macrophage cholesterol accumulation and foam cell formation include its oxidation and aggregation. To find out whether these modifications can already occur in vivo in plasma and whether they are related to each other, the oxidation and aggregation states of plasma LDL were analyzed in the apolipoprotein E-deficient (E°) transgenic mice during their aging (and the development of atherosclerosis), in comparison to plasma LDL from control mice. Plasma LDL from the E° mice was already minimally oxidized at 1 month of age in comparison to control mice LDL, and it further oxidized with age in the E° mice but not in the control mice. At 6 months of age, the contents of the E° mice LDL-associated cholesteryl ester hydroperoxides, thiobarbituric acid reactive substances, and conjugated dienes were higher by two, three, and twofold, respectively, in comparison to LDL from the young, 1-month-old E° mice. We also investigated the LDL aggregation state in E° mice. In the young E° mice, LDL oxidation was shown in comparison to control mice, but in both groups of young mice their LDL was not aggregated. In the E° mice, however, the LDL aggregation state substantially increased with age, by as much as 125% at 6 months of age compared to the 1-month-old mice, whereas no significant aggregation could be detected in plasma LDL from control mice at the same age. To question the possible effect of LDL oxidation on its subsequent aggregation, LDL oxidation was induced by either copper ions, or by the free radical generator 2,2-azobis-2-amidinopropane hydrochloride, or by hypochlorite. All these oxidative systems led to LDL oxidation (to different degrees) and resulted in a similar, substantial LDL aggregation. These oxidation systems also enhanced the susceptibility of LDL to aggregation (induced by vortexing) by 23%, 28%, or 40%, respectively. To further analyze the relationships between the lipoprotein oxidation and its aggregation, LDL (0.1 mg of protein/mL) was incubated with 5 µmol/L CuSO4 at 37°C in the absence or presence of the antioxidant, vitamin E (25 µmol/L). In the absence of vitamin E, a time-dependent increment in LDL oxidation was noted, which reached a plateau after 2 hours of incubation. LDL aggregation, however, only started at this time point and reached a plateau after only 5 hours of incubation. In the presence of vitamin E, both LDL oxidation and its aggregation were reduced at all time points studied. We extended the vitamin E study to the in vivo situation, and the effect of vitamin E supplementation to the E° mice (50 mg·kg-1·d-1 for a 3-month period) on their plasma LDL oxidation and aggregation states was studied. Vitamin E supplementation to these mice resulted in a 35% reduction in the LDL oxidation state and in parallel, the LDL aggregation state was also reduced by 23%. These reductions in LDL oxidation and aggregation states were accompanied by a 33% reduction in the aortic lesion area, in comparison to nontreated E° mice. We conclude that in E° mice, LDL oxidation, which already took place in the plasma, can lead to the lipoprotein aggregation. These modified forms of LDL were shown to be taken up by macrophages at an enhanced rate, leading to foam cell formation. Thus, the use of an appropriate antioxidant can inhibit the formation of both atherogenic forms of LDL.


Key Words: lipoproteins • apolipoprotein E • oxidized LDL • aggregated LDL • antioxidants


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
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Modifications of LDL, such as its oxidation and aggregation, were shown to be associated with the enhanced uptake of these lipoproteins by macrophages, leading to cellular cholesterol accumulation and foam cell formation, the hallmark of early atherosclerotic lesion.1–4 Evidence for the in vivo occurrence of lipoprotein oxidation in the arterial wall include the presence in the atherosclerotic lesions of oxidized LDL5,6 and of epitopes, which are formed during LDL oxidation.7 Lesion LDL also demonstrates increased electrophoretic mobility and atherogenic biological properties.8 Furthermore, LDL can be oxidized in vitro by arterial wall cells such as endothelial cells,9 smooth muscle cells,10 and macrophages.11-13 Oxidation of LDL was thought to occur primarily in the intimal space, where the LDL may be sequestered from antioxidants which are present in abundance in plasma.14 Recently, an oxidized and cytotoxic electronegative LDL subfraction was isolated from plasma,15 suggesting the possibility that in vivo, under appropriate conditions, formation of oxidized LDL may already occur in plasma. There are several lines of evidence implying that, in addition to oxidation, LDL aggregation also occurs in the arterial wall,16 but little is known about the mechanism responsible for this modification.

In vitro studies demonstrate self-aggregation of LDL that was subjected to brief vortexing17 or to a treatment with phospholipase C18,19 or sphingomyelinase.20 LDL aggregation has also been reported to occur in vitro during extensive oxidation of the lipoproteins,21-23 or following incubation of oxidized LDL with cultured human blood monocytes.24 However, the relationship between LDL oxidation and aggregation in vivo has not yet been explored. Recently, a mouse model for atherosclerosis was created by a gene targeting technique, which produced apolipoprotein E knockout transgenic mice.25 Apo E-deficient (E°) mice are characterized by spontaneous hypercholesterolemia,26 accelerated atherosclerosis, which is substantially increased by high cholesterol diet,27 and lesions similar to those found in humans.28 Several studies suggest the involvement of enhanced lipoprotein oxidation in the accelerated atherosclerosis found in the E° mice,6,29-32 but the interrelationships of plasma LDL oxidation and its aggregation along the development of atherosclerosis have not yet been explored. The results of the present study demonstrate that LDL oxidation occurs in plasma and that this process can lead to lipoprotein aggregation.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
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Mice
Apo E-deficient mice and their control littermates were kindly provided by Dr Jan Breslow, of Rockfeller University. These mice, which were hybrids with a C57BL/6JxCBA/J background, were fed a regular mouse chow diet (Purina chow containing 4% fat). Apo E-deficient mice and control mice were used at 1, 4, and 6 months of age and were matched for sex and body weight.

Materials
Carrier-free Na125 [I] was obtained from Du Pont, New England Nuclear (Boston, Mass), 2,2-azobis(2-amidinopropane) hydrochloride (AAPH) and 2,2'-azobis(2,4-dimethylvaleronitile) were obtained from Nako Chemical Industries, Ltd., Osaka, Japan. Dulbecco's modified Eagle's medium, fetal cell serum, and phosphate-buffered saline (PBS) were purchased from Biological Industries (Beth Haemek, Israel). Vitamin E was obtained from Sigma Chemical Company.

Lipoproteins
Blood was collected from the apo E-deficient mice and from control mice into tubes containing 1 mmol/L EDTA and 10 µmol/L BHT (butylated hydroxytoluene) in order to prevent oxidation and aggregation of the LDL during the separation process. Plasma cholesterol concentrations were 425±35, 675±40, and 725±45 mg cholesterol/dl of plasma in mice at the ages of 1, 4, and 6 months.

LDL (d=1.019 to 1063 g/mL) was isolated from 4 mL of pooled plasma by sequential density gradient ultracentrifugation as has been described previously.33

To avoid LDL aggregation during ultracentrifugation, plasma derived from the hypercholesterolemic E° mice at the age of 4 or 6 months was diluted with 150 mmol/L NaCl, 1 mmol/L EDTA (pH 7.4), so that the plasma cholesterol concentration in these mice was similar to the plasma cholesterol levels in the young mice. Furthermore, human LDL that was used at the same concentration as the E° mice LDL, and also LDL from control mice, was separated at the same time by the same procedure. The isolated LDL was dialyzed against 150 mmol/L NaCl, 1 mmol/L EDTA (pH7.4) and kept at 4°C for a maximal period of 5 days. Prior to the oxidation studies, LDL was dialyzed against PBS, EDTA-free solution (pH 7.4), at 4°C. LDL was radiolabeled with Na125 I (150 to 250 cpm/ng of protein) by the method of McFarlane as modified for lipoproteins.34 Prior to iodination, butylated hydroxytoluene (BHT, 5 µmol/L) was added to LDL in order to prevent oxidation.

To rule out the possibility that LDL oxidation occurs during its iodination, the amount of malondialdehyde (MDA) equivalents and conjugated dienes in LDL were determined. LDL oxidation measured before and after its iodination was found to be identical (5.4±0.5 nmol/MDA mg of LDL protein and 287±15 nmol of conjugated dienes/mg of LDL protein were measured in LDL from E° mice at age of 4 months before and after iodination).

Characterization of the Isolated Lipoproteins
The protein content of the lipoprotein was measured by the method of Lowry et al.35 Lipoprotein cholesterol,36 triglycerides,37 and phospholipids38 were determined as previously described. The LDL content of vitamin E was determined in the LDL lipid extract by high performance liquid chromatography using RP-8 column (4x25 cm, 5-µm particle size, Merck, Inc.) and methanol/water (94:6, vol/vol) as the mobile phase. The eluate was analyzed spectrophotometrically in terms of absorbance at 292 nm, using {alpha}-tocopherol as a standard.39 Quantification of the various cholesteryl esters in LDL was also determined by high performance liquid chromatography using RP-8 columns and methanol/water (97:3, vol/vol) was used for elution.

Lipoprotein electrophoresis was performed using Hedragel Lipo+Lp(a) kit (Sebia Co. France) and lipoprotein bands were visualized by staining with Sudan black.

LDL Oxidation
Lipoprotein oxidation was performed by incubation of LDL (0.1 mg of protein/mL in EDTA-free PBS) with either 5 µmol/L CuSO4 or with 2 mmol/L of the free radical generator AAPH at 37°C for up to 24 hours. Oxidation of LDL was also carried out on ice by the addition of 1 volume of reagent NaOCl, freshly diluted in phosphate buffer (50 mmol/L) to 4 volumes of LDL solution (0.1mg/mL, final concentration) for 1 hour.40

The susceptibility of LDL to oxidation was assessed by monitoring the changes in absorbance at 234 nm, every 10 minutes for a period of 3 hours, after the addition of 5 µmol/L CuSO4 to freshly isolated LDL (0.1 mg of protein/mL of PBS).

The oxidation state of LDL was assayed by the determination of thiobarbituric acid-reactive substances (TBARS) and was quantified in terms of LDL MDA equivalents.41 The LDL oxidation state was also assessed by the lipid peroxidation test,42 as well as by analysis of the conjugated dienes content of the lipoprotein.43 The LDL cholesterol ester hydroperoxide content was measured in the lipid extract of the lipoprotein (dissolved in 50 µL acetone) using RP-8 column and methanol/water (97:3, vol/vol) for elution.

The absorbance at 234 nm was measured using a standard of cholesteryl linoleate hydroperoxide. The concentration of the standard was determined spectrophotometrically using a molar extinction coefficient of {epsilon}234nm = 2.95x104 mol/L-1.44

Free lysine amino groups in LDL were estimated with trinitrobenzene sulfonic acid. LDL (0.1 mg of protein) was mixed with 1 mL of 4% NaHCO3, pH 8.4, and 50 µL of 0.1% trinitrobenzene sulfonic acid and heated for 1 hour at 37°C. Then the absorbance at 340 nm was measured.45

LDL Aggregation
LDL aggregates were prepared by the vortexing of LDL (0.1 mg of protein/mL) for 120 seconds. Aggregates were also prepared by LDL incubation with phospholipase C (1 U/mL) at 37°C for 1 hour. The susceptibility of LDL to aggregation was determined by LDL vortexing (0.1 mg of protein/mL) for up to 400 seconds and monitoring the changes in absorbance at 680 nm every 10 seconds.

Lipoprotein aggregation state was analyzed by two methods: (1) The turbidity generated by LDL aggregates was measured spectrophotometerically in terms of absorbance at 680 nm.17 (2) The aggregation state of 125I-LDL was analyzed by measuring the precipitated radioactivity obtained after high speed centrifugation (10 000xg for 20 minutes), and expressed as a percentage of the total LDL radioactivity (precipitate+supernatant).

Metabolism of LDL by Macrophages
J-774 A.1 macrophage-like cell line was purchased from American Type Cell Culture (Rockville, Md). Cellular degradation of LDL (derived from the E° mice or from control mice) was measured following cell incubation with 25 µg of protein/mL of 125I-labeled LDL in serum-free Dulbecco's modified Eagle's medium, containing 0.2% bovine serum albumin.

At the end of incubation the medium was removed and subjected to 10% trichloroacetic acid precipitation. The extent of lipoprotein degradation was calculated from the trichloroacetic acid-soluble noniodide radioactivity.46

LDL cholesterol uptake by cells was estimated by measurement of the stimulation of [3H]oleate incorporation into cellular cholesteryl ester.47 The cells were incubated for 18 hours with the lipoprotein, followed by medium removal and a further incubation of the cells with radiolabeled oleate (0.2 mmol/L, 10 µCi/mL [3H]oleate in the presence of 0.07 mmol/L fatty acid-free albumin) for 2 hours at 37°C. The cells were then washed with PBS and cellular lipids were extracted by hexane isopropyl alcohol (3:2, vol/vol) solution. The labeled cholesteryl ester spot was isolated by thin layer chromatography (TLC) on silica gel plates using hexane:diethyl ether: acetic acid solution (130:40:1.5, vol/vol).

Histomorphometry of Aortic Atherosclerotic Lesion Areas
At the end of the experimental period the mice were sacrificed. The heart and entire aorta were rapidly dissected out and immersion fixed in 3% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer with 0.01% calcium at room temperature. The aortic arch was dissected free from the surrounding fatty tissue and the first 4 mm of the ascending aorta (beginning with the aortic valves) was removed and cut transversely with razor blades into four blocks of approximately 1 mm each. The samples were kept in the fixative at room temperature overnight. The samples were then rinsed and stored in 0.1 mol/L sodium cacodylate buffer containing 7.5% wt/vol sucrose prior to treatment with an unbuffered 1% aqueous solution of osmium tetroxide for 4 hours. This was followed by cacodylate rinse and dehydration in ascending ethanols, prior to propylene oxide and embedding in epoxy resin ("Eponate 12", Pelco Int., Redding, Calif). Transverse sections (1 µm) were cut for light microscopy. The prolonged osmium treatment stains the intramural and intracellular lipid a dense black color. The lesion areas were determined using a computerized quantitative image-analysis system (Olympus Cue-2, Lake Success, NY) with morphometric software.

Statistical Analyses
The Student's t test was used in order to analyze the significance of the results. Results are given as mean±SD.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Characterization of Plasma LDL Modifications During the Development of Atherosclerosis in E° Mice
Lipid composition analysis of plasma LDL from E° mice was performed at 3 different ages (1, 4, and 6 months), representing 3 stages in the development of atherosclerosis and compared to that of plasma LDL from aged-matched control mice (Table 1Down). The ratios of LDL cholesterol to protein, cholesterol to phospholipids, and cholesterol to triglycerides were higher in LDL derived from E° mice compared to control mice and increased with the age of E° mice, as demonstrated by 70%, 30%, and 26% increments, respectively, in E° mice at 6 months of age in comparison to young, 1-month-old E° mice (Table 1Down).


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  		Table 1. Plasma LDL Lipid Compositions During Atherogenesis in the Apolipoprotein E-Deficient (E°) Mice

Plasma LDL vitamin E content expressed per mg of LDL cholesterol was reduced in E° mice in comparison to control mice. This ratio was reduced in E° mice with age reaching levels that were as low as 50% of those found in control mice (*P < .01) (Table 1Up). The relative content of LDL cholesteryl linolate and cholesteryl arachidonate (the polyunsaturated fatty acids), out of the total LDL cholesteryl ester, increased with the mice age, and were about 60% higher than their values obtained in LDLs from control mice (Table 1Up). The density of LDL from E° mice at age of 4 months was analyzed by fluorescence-activated cell sorter scanner, using an excitation wavelength of 480 nm and emission wavelength of 610 nm,48 and was found to be about 30% higher in comparison to LDL from age-matched control mice (258 versus 198 density arbitrary units, respectively). Biological activities of the LDL from E° mice in comparison to control LDL were studied by analyses of the macrophage lipoprotein uptake. Incubation of J-774 A.1 macrophages with 125I-LDL (25 µg of protein/mL) from E° or control mice at the age of 4 months, resulted in a 45% higher LDL degradation when using E°-derived LDL in comparison to control LDL (Fig 1ADown). Furthermore, incubation of J-774 A.1 cells with LDL from E° mice resulted in a 142% elevation in the rate of cellular cholesterol esterification in comparison to cellular cholesterol esterification induced by control mice LDL (Fig 1BDown). Two major modifications of LDL that can lead to macrophage cholesterol accumulation during early atherogenesis, include its oxidation and aggregation. To find out whether such modifications occur already in plasma, the oxidation and aggregation state of plasma LDL from E° mice was determined at 1, 4, and 6 months of age. Plasma LDL was already minimally oxidized at 1 month of age in the E° mice and its oxidation state further increased with age from 1 to 6 months (Fig 2Down). At 6 months of age, the content of LDL-associated cholesteryl ester hydroperoxides (Fig 2ADown), TBARS (Fig 2BDown) and conjugated dienes (Fig 2CDown) were higher by 97%, 185%, and 91%, respectively, in comparison to the values obtained in LDL from the young 1-month-old, E° mice (Fig 2Down). Unlike LDL from E° mice, LDL obtained from control mice did not show any age-dependent oxidation (Fig 2Down). Even when expressing LDL oxidation per the lipoprotein cholesterol content rather than the protein content, an age-dependent increment in LDL oxidation by as much as 32% (P<.01) could be demonstrated when comparing lipoprotein from 6 versus 1 months of age mice (Fig 2Down). The LDL free amino groups content (on lysine residues of the LDL apolipoprotein B-100), as measured by the trinitrobenzene sulfonic acid assay was reduced by 30% and 47% in LDL derived from E° mice at 1 and 6 months of age, respectively, in comparison to plasma LDL obtained from control mice at the same age (data not shown). These results demonstrate an increment in LDL oxidation that is associated with the age of the atherosclerotic E° mice.



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  		Figure 1. Increased macrophage uptake of LDL from E° and from control mice. A, J-774 A.1 macrophages were incubated for 5 hours at 37°C with 25 µg of 125I-labeled LDL protein/mL prior to the determination of LDL degradation. The LDLs were derived from E° mice or from control mice. B, Cells were also incubated with 25 µg of protein/mL of LDL derived from E° or from control mice for 18 hours at 37°C, prior to analysis of the cellular cholesterol esterification rate. Results represent mean±SD of three experiments. *P<.01 (E° LDL versus control LDL).



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  		Figure 2. Age-related increment in LDL oxidation state in E° mice. Plasma LDLs were separated from E° mice or control mice at three different ages (1, 4, and 6 months). The extent of LDL oxidation was determined by analysis of cholesteryl ester hydroperoxides (A), TBARS, measured as MDA equivalents (B), and conjugated dienes (C). Results represent mean±SD of 10 different determinations.

The extent of LDL aggregation was analyzed by measuring LDL absorbance at 680 nm, as well as by measuring the precipitated radioactivity obtained after high speed centrifugation of 125I-labeled LDL. Fig 3Down demonstrates only minimal aggregation in LDL isolated from young E° mice (1-month-old). However, the LDL aggregation state increased with age and the lipoprotein isolated from aged E° mice (6 months old) was 2.4- fold and 2.1-fold more aggregated than LDL from the young E° mice as determined by the two assays of LDL aggregation, respectively.



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  		Figure 3. Age-related increment in LDL aggregation state in E°-mice. Plasma LDLs were separated from E° mice or control mice at three different ages (1, 4, and 6 months). The extent of LDL aggregation was determined by measuring its absorbance at 680 nm (A) or by measuring the precipitated radioactivity obtained after high speed centrifugation (10 000g, 20 minutes) of 125I-labeled LDL. The extent of LDL aggregation is expressed as percentage of total LDL radioactivity (precipitate+supernatant). Results represent mean±SD of 10 different determinations.

Analysis of the lipoprotein aggregation state by measuring the amount of the precipitated 125I-LDL revealed that 8.5% of total 125I-LDL in the 6-month-old E° mice was aggregated (Fig 3BUp). LDL from control mice demonstrated no aggregation at all at any of the studied ages (Fig 3Up). Human LDL, which was separated by the same procedure (in order to rule out the possibility that the isolation procedure caused any particle aggregation), was found to be not aggregated at all.

To better characterize the extent of LDL aggregation, we have separated aggregated and nonaggregated fractions of 125I-LDL derived from E° mice at 6 months of age. Briefly, 5.5 mL of 125 I -labeled LDL (1 mg/mL) from E° mice was mixed with KBr to adjust the solution density to 1.100 g/mL and this layer was overlaid with 5 mL of KBr solution of the same density, followed by centrifugation at 41,000 rpm in a SW 41 rotor for 60 minutes. The top (aggregated LDL) fraction was removed and the remaining (nonaggregated LDL) fraction was floated by a further centrifugation at 41,000 rpm for 18 hours. Aggregated LDL that was made by vortexing served as a control. Analysis of the labeled lipoprotein in each fraction revealed that 9.5±1.0% of the total 125I-LDL was aggregated as it was recovered in the top of the tube. 125I-LDL from control mice at the same age demonstrated no aggregation (0.8±0.1% of the total 125I-LDL was recovered at the top of the tube). The aggregation of LDL in E° mice was further demonstrated by agarose gel electrophoresis. Aggregated particles were visualized at the origin of the gel when analyzing LDL from E° mice at age of 4 or 6 months (Fig 4Down, lanes 2 and 4, respectively). In contrast, no such aggregated particles could be found at the origin of the gel when LDL from control mice at the same ages were used (Fig. 4Down, lanes 1 and 3, respectively). The major band of E° mice LDL migrated faster than the responding LDL control mice, probably because of its increased oxidation state.



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  		Figure 4. Agarose gel electrophoresis of LDLs from E° and control mice. Sudan black-stained gel showing: control LDL at 4 months of age (lane 1), E° mice LDL at 4 months of age (lane 2), control mice at 6 months of age (lane 3), and E° mice at 6 months of age (lane 4). The arrow indicates the origin of the run.

Susceptibility of LDL From E° Mice to Aggregation and Oxidation During the Development of Atherosclerosis
The susceptibility of LDL derived from E° mice at different ages (1 and 6 months) to undergo aggregation was studied by vortexing of the LDL (0.25 mg of cholesterol/mL) for up to 400 seconds and monitoring the changes in the absorbance at 680 nm every 10 seconds (Fig 5ADown). LDL derived from E° mice at 6 months of age (which was oxidized and aggregated) was more susceptible to aggregation than LDL from the young mice which was minimally oxidized and not aggregated, as evident by a threefold increment in the absorbance at 680nm after 400 seconds of vortexing (Fig 5ADown). These results indicate that the extent of LDL oxidation might affect its susceptibility to aggregation by vortexing.



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  		Figure 5. Age-related increment in the susceptibility of LDL from E° mice to aggregation and oxidation. LDLs (0.25 mg of cholesterol/mL) derived from E° mice (at 1 and 6 months of age) were aggregated by vortexing for up to 400 seconds. The extent of LDL aggregation was spectrophotometrically monitored every 10 seconds at 680 nm (A). LDL oxidation was induced by its incubation with 5 µmol/L CuSO4 for 3 hours at 37°C. The extent of LDL oxidation was monitored every 10 minutes at 234 nm (B). Result are given for 1 representative experiment out of 3\E experiments performed.

LDL susceptibility to oxidation was assessed by continuously monitoring the changes in absorbance at 234 nm (an index for the formation of conjugated dienes), following LDL incubation with 5 µmol/L CuSO4 (Fig 5BUp). After 200 minutes of incubation, LDL oxidation increased by twofold in the aged mice, in comparison to the young mice as shown by the maximal increment in conjugated dienes formation (Fig 5BUp), demonstrating an increased propensity to oxidation of LDL from the highly atherosclerotic E° mice in comparison to the young nonatherosclerotic mice.

In the present study LDL was isolated from plasma of E° and control mice in order to estimate its aggregation state and also its susceptibility to aggregation. To find whether our finding in the isolated particles really reflects the state of particle aggregation while they are circulating in plasma, we performed the following study. LDL (0.5 mg of cholesterol/mL) isolated from E° mice or from control mice at 6 months of age was incubated for 18 hours at 37°C in the absence or presence of the plasma fraction d>1.063 g/mL (obtained either from the E° mice or control mice at the same age). At the end of the incubation, we determined the LDL aggregation state and its susceptibility to aggregation by vortexing of the LDL for up to 180 seconds. The lipoprotein was reisolated by ultracentrifugation at d<1.063 g/mL and its absorbance was monitored at 680 nm. Fig 6Down demonstrated that LDL from E° mice that was incubated in PBS was threefold more aggregated than LDL from control mice, and its susceptibility to aggregation by vortexing was also found to be increased by 4.5-fold, in comparison to LDL from control mice (Fig 6Down). Incubation of LDL from E° mice with the plasma fraction of d>1.063 g/mL derived from the same mice, did not change its aggregation state but resulted in a 51% decrement in its susceptibility to aggregation while incubation of E° mice LDL with the plasma fraction of d>1.063 g/mL from control mice did not change its aggregation state but led to a 75% decrement in its susceptibility to aggregation (Fig 6ADown). Similar results were obtained with control LDL from control mice. Incubation of control LDL with d>1.063 g/mL plasma fraction from the same mice or from E° mice did not change its aggregation state, but led to a 63% and 35% reduction in its susceptibility to aggregation, respectively (Fig 6BDown). These results thus suggest that the aggregation state of the isolated LDL particles reflect their situation in plasma. Furthermore, plasma from control mice showed inhibitory effect on LDL aggregation in comparison to plasma derived from E° mice.



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  		Figure 6. Effect of plasma components on the susceptibility of LDL to aggregation. LDLs (0.5 mg of cholesterol/mL) derived from E° mice or from control mice at 6 months of age were incubated for 18 hours at 37°C with the plasma fraction of d>1.063 g/mL from either E° or control mice. At the end of the incubations, LDL was aggregated by vortexing for up to 180 seconds, followed by reisolation of the LDL (at d<1.063 g/mL) and monitoring of the absorbance at 680 nm.

The Effect of LDL Aggregation on Its Oxidation
Since LDL derived from E° mice at 6 months of age was more aggregated and more oxidized than LDL from the young mice, we questioned the possibility that LDL aggregation can lead to its oxidation and/or increase its susceptibility to oxidation. For these studies we used human LDL obtained from healthy subjects (after ensuring that these LDL preparations were not oxidized, nor aggregated). LDL aggregates were prepared by vortexing for up to 120 seconds, and also by LDL (0.1 mg of protein/mL) incubation with phospholipase C (PLase C, 1 U/mL) for up to 60 minutes. As shown in Table 2Down, on both increasing the vortexing time of LDL (Table 2Down, A) or on increasing the incubation time of LDL with PLase C (Table 2Down, B), a dose-dependent increment in the formation of LDL aggregates was shown (Table 2Down). These increments in LDL aggregation, however, did not lead to enhanced LDL oxidation (Table 2Down). Analysis of the effect of LDL aggregation on its susceptibility to oxidation revealed that the increase in LDL aggregation state by increasing the time of vortexing (Fig 7ADown), and also by increasing the incubation time of LDL with PLase C (1 U/mL) (Fig 7BDown) was accompanied by a striking decrease (by up to 44% and 33%, respectively) in the susceptibility of the LDL to oxidation induced by 5 µmol/L CuSO4. We also found that the increase in LDL aggregation state, either by vortexing the LDL or by incubation with PLase C, led to reduced susceptibility of LDL to oxidation by using the aqueous soluble radical generator AAPH (2 mmol/L) by up to 28% and 20%, respectively (values of 25, 18, and 20 nmol of MDA/mL of nonaggregted LDL, vortexed LDL, and PLase C-LDL, respectively, were obtained after 3 hours of lipoprotein incubation with AAPH). Similar results showing reduced susceptibility of the aggregated LDL to oxidation were obtained using the lipid-soluble radical generator 2,2'-azobis(2,4-dimethylvaleronitrile) (2 mmol/L) values of 22, 14, and 15 nmol of MDA/mL of nonaggregated LDL, vortexed LDL, and PLase C LDL, respectively, were obtained after 3 hours of oxidation with 2,2'-azobis(2,4-dimethylvaleronitrile). Based on these results which demonstrated that LDL aggregation could not lead to enhanced LDL oxidation, we conclude that the increased oxidation state and susceptibility of LDL derived from the aged E° mice to oxidation could not be attributed to the higher aggregation state and increased susceptibility to aggregation in comparison to young mice.


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  		Table 2. The Effect of LDL Aggregation on Its Oxidation by Copper Ions



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  		Figure 7. Effect of LDL aggregation state on its susceptibility to oxidation. LDL aggregates were prepared by LDL (0.1 mg of protein/mL) vortexing for up to 120 seconds (A) and also by LDL incubation with PLase C (1 U/mL) at 37°C for up to 60 minutes (B). At the end of incubation, the extent of LDL aggregation and oxidation was determined. The susceptibility of these aggregated LDLs to oxidation was studied by lipoprotein incubation with 5 µmol/L CuSO4 for 3 hours at 37°C followed by determination of LDL associated TBARS. Results represent mean±SD of 3 different determinations.

The Effect of LDL Oxidation on Its Aggregation
Based on our findings, which demonstrated that in E° mice LDL oxidation precedes its aggregation, we hypothesized that LDL oxidation in plasma can lead to its aggregation. In order to explore the effect of LDL oxidation on its aggregation, we oxidized LDL by different mechanisms and assessed its aggregation state. For this purpose, human LDL (0.1 mg of protein/mL) was oxidized by incubation with CuSO4 (5 µmol/L) or AAPH (2 mmol/L) or by the reagent hypochlorite.

At the end of the incubation period, LDL oxidation and aggregation states were analyzed. LDL oxidation by all the above mechanisms was associated with its enhanced aggregation state (Table 3Down). The extent of LDL aggregation (as evident by measuring the absorbance at 680 nm) was similar in all three preparations of the oxidized lipoprotein even though there were considerable differences in the content of LDL-associated TBARS (Table 3Down). We next studied the effect of LDL oxidation by the three methods (Table 3Down), on its susceptibility to aggregation by vortexing, in comparison to control nonoxidized LDL (Fig 8Down). Noteworthy was the observation that the susceptibility of LDL which was oxidized by hypochlorite, to aggregation by vortexing was the highest in comparison to the two other preparations of LDLs that were oxidized with CuSO4 or with AAPH and contained higher content of MDA equivalents (Fig 8Down). These results may suggest that the amount of LDL-associated TBARS is not the major determinant of LDL aggregation, but different oxidation products that are formed in the different oxidation systems might differently affect the susceptibility of the oxidized LDL to undergo self-aggregation.


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  		Table 3. The Effect of LDL Oxidation, by Different Mechanisms, on its Aggregation



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  		Figure 8. Effect of LDL oxidation by different mechanisms on its susceptibility to aggregation. LDL (0.1 mg of protein/mL) was oxidized by three different mechanisms (CuSO4, AAPH, and NaOCl). The susceptibility of these oxidized LDLs to aggregation was studied by vortexing of the lipoprotein and monitoring the extent of LDL aggregation at 680 nm.

To study the relationships between the extent of LDL oxidation and its aggregation, human LDL (0.1 mg of protein/mL) was incubated with 5 µmol/L CuSO4 for up to 24 hours and the extent of LDL oxidation and aggregation at several time points was studied. In these experiments the effect of the antioxidant vitamin E on these two processes was also studied. As shown in Fig 9ADown, when LDL was oxidized in the absence of vitamin E (Control LDL), a time-dependent increment in lipoprotein-associated TBARS, which reached a plateau after 2 hours of incubation was demonstrated (Fig 9ADown). LDL aggregation, however, only started at this time point and reached a plateau only after 5 hours of incubation (Fig 9BDown). Vitamin E (25 µmol/L) inhibited both LDL oxidation and aggregation processes at all studied time points (Fig 9Down). After 3 hours of incubation, for example, LDL oxidation and aggregation states were inhibited by 85% and 33%, respectively (Fig 9Down). At longer time points, the inhibitory effect of vitamin E on LDL oxidation was lower (secondary to vitamin E consumption under the oxidative conditions) and after 24 hours of incubation LDL oxidation was inhibited by 25% (Fig 9ADown) and its aggregation, by 29% (Fig 9BDown).



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  		Figure 9. The in vitro effect of vitamin E on LDL oxidation and aggregation. One representative experiment is shown out of 4 similar studies. Human LDL (0.1 mg of protein/mL) was incubated at 37°C with 5 µmol/L CuSO4 in the absence (control) or presence of 25 µmol/L vitamin E for up to 24 hours. The extent of LDL oxidation (A) and aggregation (B) was determined along the time of incubation. Representative experiment (out of 3 different experiments) is shown.

Effect of Vitamin E on LDL Oxidation and Aggregation and on the Development of Atherosclerosis in E° Mice
To further analyze the relationship between LDL oxidation and aggregation in vivo, the effect of vitamin E supplementation to the E° mice on their plasma LDL oxidation and aggregation states was studied. Two groups of 15 apo E-deficient mice each, aged 2 months were fed a regular chow diet for a period of 3 months.

One group was treated with vitamin E (50 mg/kg/d) and the other group served as a control. Comparison of plasma lipids revealed that mice treatment with vitamin E for a 3-month period did not affect the concentrations of plasma and LDL cholesterol in comparison to the values obtained in nontreated E° mice (685±35 and 697±42 mg of cholesterol/dL plasma, and 2.7±0.3 and 2.8±0.3 mg of LDL cholesterol/mg protein respectively). LDL derived from vitamin E-treated mice, however, contained twofold higher concentration of vitamin E in comparison to control nontreated mice (14.5±1.8 and 7.1±0.8 µg of vitamin E/mg LDL protein, respectively). Vitamin E supplementation to the E° mice resulted in a 35% reduction in the LDL oxidation state (7.8±0.95 and 5.7±0.2 nmol of MDA equivalents/mg LDL protein were associated with LDL derived from the control and the vitamin E-treated mice, respectively) (Fig 10ADown). Vitamin E also inhibited the susceptibility of the LDL to oxidation that was induced by CuSO4 (5 µmol/L), as evident by a significant (P<.01, n=3), 21% reduction in the TBARS content formed after 3 hours of LDL oxidation (38±3 and 30±2 nmol of MDA equivalents/mg LDL protein were associated with LDL derived from nontreated placebo, and from vitamin E-treated mice, respectively). Vitamin E supplementation to the E° mice also resulted in a reduction (by 23%) in LDL aggregation state. These results were obtained by reading the absorbance of the lipoproteins at 680 nm, as shown in Fig 10BDown, and also after radiolabeling the lipoproteins with 125I, and measuring the precipitated radioactivity as described under Methods (data not shown). These results further demonstrate the possible association between LDL oxidation and its aggregation. Since both modifications (oxidation and aggregation) of LDL were shown to be associated with the development of atherosclerosis in E° mice, we also studied the effect of vitamin E supplementation to the E° mice on the extent of the aortic lesion. Quantitation of the lesion area in aorta that was separated from the placebo and from vitamin E-treated mice demonstrated that 3-month treatment with vitamin E reduced the lesion area by 33% in comparison to the nontreated placebo mice (Fig 10CDown).



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  		Figure 10. Effect of vitamin E supplementation to E° mice on the states of LDL oxidation, LDL aggregation, and on the size of their lesion. Vitamin E (50 mg·kg-1·d-1) was supplemented to E° mice (2 months old) for a 3-month period. At the end of the study, plasma LDLs were separated and their oxidation (A) and aggregation (B) states were determined and compared to those of control, nontreated mice. The size of the aortic surface covered by atherosclerotic lesions (C) was measured as described in "Methods." Results represents mean±SD of 15 different determinations. *P<.01 (LDL derived from vitamin E-treated mice versus LDL from control mice).


*    Discussion
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The present study demonstrated, that in the apolipoprotein E-deficient (E°) atherosclerotic mice LDL oxidation, which already occurs in the plasma, could lead to lipoprotein aggregation. These two modifications are associated with macrophage cholesterol accumulation, foam cell formation, and enhanced atherogenicity. LDL oxidation and aggregation are known to increase lipoprotein atherogenicity and it is generally thought that these modifications, especially oxidation, occur primarily in the intimal space, where LDL may be sequestered from antioxidants which are present in abundance in plasma.14 To date no direct evidence has been provided for the occurrence of LDL oxidation in plasma prior to the appearance of aortic lesions. Furthermore, the changes in LDL oxidation during the development of atherosclerosis have not yet been characterized. Based on our previous studies,6,32 that demonstrated that plasma LDL from E° mice is minimally oxidized in comparison to LDL from control mice, we further analyzed the oxidation state of LDL from E° mice at different ages, which represent different stages in the development of the atherosclerotic lesions. We demonstrated that plasma LDL from E° mice, but not that from control mice, was already oxidized at 1-month of age, a stage where no lesion was found in the mice aorta. LDL oxidation further increased with age, paralleled by the development of extensive atherosclerosis at 6 months of age. These results are consistent with recent immunocytochemical studies which demonstrated that atherosclerotic lesions from the E° mice contains epitopes of oxidized LDL29,30 and that serum from these mice contained circulating autoantibodies against such oxidation-specific epitopes. Additional evidence for the in vivo occurrence of oxidative modification of LDL in mice was shown in E° mice that were supplemented with the antioxidant N,N'-diphenyl-1,4-phenylenediamine.31 Lipoproteins (d<1.019g/mL) from N,N'-diphenyl-1,4-phenylenediamine-treated animals showed a greater resistance to in vitro oxidation than lipoproteins from control mice, and the extent of atherosclerosis was significantly reduced in the N,N'-diphenyl-1,4-phenylenediamine-treated mice compared to nontreated mice.31 Our results, which demonstrated that the formation of oxidized LDL may occur already in plasma, are also supported by the findings of minimally oxidized electronegative LDL subfraction in humans, as well as in plasma of animals.15,49-51 However, it has been suggested that circulating oxidized LDL particles may originate from the extravascular space, an hypothesis that remains to be established.51

The mechanism responsible for LDL oxidation in plasma has not yet been explored. Injury to the endothelial cells lining the vessel wall by any mechanism has the potential to stimulate free radical reactions which can induce LDL oxidation.1,2,10 Myeloperoxidase, which is found in abundance in neutrophils and monocytes, may also play a key role in the initiation of lipid peroxidation in plasma LDL.52 Oxidation of plasma LDL in E° may thus result from the oxidative stress which exists in these hypercholesterolemic atherosclerotic mice and was found to depend on intrinsic as well as on extrinsic factors.43 We have previously demonstrated in hypercholesterolemic patients,53 as well as in the E° mice,31 that high plasma LDL concentrations are associated with the increased susceptibility of the lipoprotein to oxidation. Hypocholesterolemic therapy, which removes aged LDL that is more prone to oxidation from the plasma, leads to the inhibition of LDL oxidizability.54,55 The LDL antioxidants vitamin E and ß-carotene were shown to protect the lipoprotein from lipid peroxidation, whereas the low content of these antioxidants in the lipoprotein contributes to its increased propensity for oxidation.1,14,43 The present study demonstrated that plasma LDL from the atherogenic E° mice showed increased content of cholesterol as well as, decreased content of vitamin E in comparison to the control mice LDL. These modifications in the E° mice LDL increased with their age and with the development of atherosclerosis, and thus they can all contribute to the enhanced oxidation state of LDL.

The high content of LDL-cholesteryl ester-18:2+cholesteryl ester 20:4 in the E° mice, in comparison to the control mice, may also be responsible for the enhanced susceptibility of their LDL to oxidation. The modest LDL oxidation state in E° mice, in comparison to control mice, may have not affected the levels of LDL-cholesteryl ester polyunsaturated fatty acids concentration, but may first affect surface LDL phospholipid-associated fatty acids. Furthermore, other lipoprotein characteristics such as size and density can also influence the extent of LDL oxidation. Small dense LDL is more susceptible to oxidation than large bayonet LDL.56 In this study we demonstrated that the density of LDL from E° mice was about 30% higher in comparison to LDL from age-matched control mice and this may have also contributed to the enhanced LDL oxidation state in E° mice. Unlike the LDL oxidation characteristics already shown in the young, 1-month-old E° mice, this LDL was not aggregated. However, the LDL aggregation state increased with the age of the E° mice in parallel to the increment in the LDL oxidation state and to the development of atherosclerosis. Several lines of evidence demonstrated that LDL aggregation, which is known to increase LDL atherogenicity, like its oxidation, also occurs in the arterial wall.16,57-59 However, no evidence has been provided for LDL aggregation in plasma. Based on our present findings that in E° mice, plasma LDL oxidation occurred prior to its aggregation, we suggest that LDL oxidation in the plasma might lead to lipoprotein aggregation. To test this hypothesis, we performed several in vitro studies that demonstrated that oxidation of LDL by different mechanisms always led to an increase in its aggregation. Additional support to the causal in vivo effect of LDL oxidation on its aggregation in the E° mice was provided by the findings that plasma LDL from the old mice, which was oxidized and aggregated, was more susceptible to aggregation than LDL from the young mice, which was less oxidized and not yet aggregated. The present study thus demonstrated, that in the E° mice plasma LDL oxidation is related to its aggregation. This is further supported by the observation that the supplementation of the antioxidant vitamin E to these mice for a 3-month period resulted in a significant reduction in both LDL oxidation and aggregation states. These effects of vitamin E could be attributed to the direct interaction of the vitamin with LDL, similar to our demonstration of its inhibitory effect on LDL oxidation (and aggregation) in vitro. The in vivo results demonstrating that LDL oxidation is related to its aggregation in the E° mice are in good agreement with those of Hoff et al,21 who showed that LDL aggregation occurs during an extensive in vitro oxidation of the lipoprotein. Furthermore, recently Steinbrecher et al60 demonstrated that the extent of aggregation of oxidized LDL was significantly affected by the extent of its oxidation. None of these studies, however, examined the relationship between LDL oxidation and its aggregation in vivo, and the possible contribution of these modifications to the development of atherosclerosis. In the present study we demonstrated such relationships in vivo using the E° mice both directly and indirectly, by using antioxidants. The exact mechanism leading to oxidation-induced aggregation of plasma LDL in the E° mice is not yet clear.

This oxidative burden on E° mice LDL leads to the formation of aggregated LDL, probably as a result of the changes that occur on the lipoprotein surface net charge, secondary to the action of LDL-associated phospholipases and the formation of new phospholipid derivatives. Furthermore, oxidation products of the LDL lipid constituents can bind to the lipoprotein surface apo B-100 and phospholipids, causing changes in the lipoprotein physicochemical properties, and finally leading to LDL aggregation. Proteins which have been exposed to oxygen radicals underwent progressive covalent cross-linking, thus exhibiting an altered primary structure.61,62 Aldehydes such as MDA and 4-hydroxynonenal, which are produced by the decomposition of lipid hydroperoxides, were found to interact covalently with apo B-100, thus leading to LDL aggregation.63 In addition, LDL oxidized by myloperoxidase could also undergo aggregation by cross-linking tyrosine residues in separate LDL particles,52 and hypochlorite, which is generated by myeloperoxidase, can induce the aggregation of LDL by cross-linking lysine residues.64 In this study we also proved that LDL aggregation, induced by vortexing, or by its incubation with PLase C, was associated with reduced (not increased) LDL susceptibility to oxidation by different agents (copper ions, AAPH and 2,2'-azobis(2,4-dimethylvaleronitrile)). Thus LDL aggregation cannot be considered as the cause of the enhanced oxidation of LDL in E° mice. Finally, we have also demonstrated that supplementation of the antioxidant vitamin E to E° mice not only inhibited LDL oxidation and its subsequent aggregation, but also inhibited atherogenesis, as evident by a significant reduction in the mice aortic lesion area, in comparison to nontreated atherosclerotic mice thus showing that lipoprotein oxidation and aggregation may indeed contribute to atherogenic lesions in E° mice.

It is concluded from our study that the progression of atherosclerosis in the apo E-deficient mice is related to the lipoprotein modifications in the plasma. However, it should be mentioned that the observed progress of lipoprotein modifications and atherosclerosis in these mice is primarily due to impaired lipoprotein catabolism and to an altered lipoprotein biosynthesis. Apolipoprotein E is a ligand for the receptors that bind and take up chylomicron remnants and very low density lipoproteins. Lack of plasma apo E thus (as is the case in the apo E-deficient mice), results in a massive accumulation of cholesterol-rich VLDL-like remnants and also LDL-like particles.

The prolonged circulation of these atherogenic lipoproteins in the plasma of the E° mice contribute to their enhanced susceptibility to oxidative and to aggregative stress. Furthermore, plasma HDL levels in E° mice are about 50% lower than those found in control mice.26 The low concentration of HDL and of its major apolipoprotein, apo A-I in plasma, might further reduce its protective effect against LDL aggregation. Indeed, in the present study we have demonstrated that incubation of LDL with the plasma fraction of d>1.063 g/mL derived from control mice led to a better protective effect against LDL aggregation in comparison to plasma from E° mice.

Both oxidized LDL and aggregated LDL found in plasma can enter the arterial wall, where further and more extensive oxidation and aggregation processes can take place. The enhanced cellular uptake of these modified forms of LDL by arterial wall macrophages can then lead to foam cell formation and to the development of the complicated atherosclerotic lesion.


*    Selected Abbreviations and Acronyms
 
AAPH = 2,2-azobis-2-amidinopropane hydrochloride
PLase = phospholipase
MDA = malondialdehyde
PBS = phosphate-buffered saline
TBARS = thiobarbituric acid reactive substances

Received November 27, 1996; accepted March 14, 1997.


*    References
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