Interaction of oxidized low density lipoproteins with both apo B,E and scavenger receptors. A model for its production in vitro

Clinica Chimica Acta. 210 (1992) 93-108 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009-898 l/92/$05.00

93

CCA 05367

Interaction of oxidized low density lipoproteins with both apo B,E and scavenger receptors. A model for its production in vitro Elisabet Vilella, Jorge Joven, Teresa Bargall6, Peter R. Turner and Lluis Masana Unitat de Recerca de Lipids; Facultat de Medicina, Universitat de Barcelona, Hospital de Sam Joan, Reus (Spain)

(Received 21 October 1991; revision received 25 February 1992; accepted 16 June, 1992)

Key words: Minimally oxidized LDL; Lymphocyte; Macrophage; Scavenger receptor

Degradation; Apo B,E receptor;

Oxidation of low density lipoprotein (LDL) has been demonstrated in vivo and directly implicated in the process of foam cell formation. Consequently, a considerable research effort has been devoted to the assessment of the metabolic behaviour of oxidized LDL. We have developed a simple and reproducible model to obtain oxidized LDL consisting of the dialysis of LDL (4 g/l) contained in a cellulose bag against 5 litres of 0.15 M NaCl, 5 PM C&O,+, 0.6 mM FeC13, pH 7.4, 37°C with constant oxygen bubbling. While the resulting particles have a number of physicochemical properties suggesting lipid oxidation, neither apo B fragmentation nor modification in the size and shape were observed. This oxidized LDL showed internalization into cells through both the apo B,E and the scavenger receptors and the rate of removal from the plasma in injected rats was faster than that observed for normal LDL. We suggest that these particles may represent an equivalent to the circulating oxidized LDL postulated in humans.

Intruduction Low density lipoprotein (LDL) is the main transport medium for the delivery of Correspondence to: Ll. Masana, Facultat de Medicina, CfSant Llorenq 21, 43201 Reus, Spain.

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cholesterol to tissues in which incorporation into individual cells is via a specific receptor (apo B/E) mechanism [ 11. A further receptor known as scavenger receptor, described primarily in cultured macrophages [2,3], has been shown to bind altered LDL, but it does not lead to feed back regulation by intracellular cholesterol content [4,5]. Such LDL alterations include a number of chemical modifications (acetylation, methylation, malondialdehyde conjugation, oxidation) which result in an increase in net negative charge of the LDL particle and an increased rate of its incorporation by macrophages. However, among these possible alterations, only oxidized LDL has been found in human plasma [6] and atheromatous plaques [7,8]; this oxidation has been linked to the process of foam cell formation and thus to the development of atherosclerosis [9, lo]; the proposed mechanism for the modification in vivo is lipid oxidation [1 11. Recently we have demonstrated in hypercholesterolemic patients that oxidation of LDL can be reduced by administration of an antioxidative drug, suggesting that oxidation is actually present in circulating plasma lipoproteins [ 121. The oxidation of LDL in vitro can be induced by several methods [ 13- 1.51yielding particles with a considerable apo B degradation which is associated with modified binding by the lipoprotein receptor and cell internalization. As a consequence, it is of considerable interest to study minimally oxidized LDL which may induce in endothelial cells the expression of granulocyte and macrophage colony stimulating factors [16J. We report here a simple and reproducible method to obtain oxidized LDL without apo B fragmentation and its interaction with apo B,E and scavenger receptors. Materials and Methods

Plasma was obtained after an overnight fast from healthy laboratory personnel using 0.37 mM ethylenediaminetetraacetic acid (EDTA) as anticoagulant. The plasma was immediately subjected to preparative ultra-centrifugation (105 000 x g, 24 h, 4’C) according to the recommendations of Schumaker and Puppione [17] in a Kontron TFT 45.6 rotor (Kontron Instruments, Switzerland) to isolate LDL (d = 1.019-l .063 g/ml), which was then recentrifuged under the same conditions and background density so as to concentrate (up to 4-6 g/l of protein) and to wash the LDL free of contaminating lipoproteins and albumin. The samples were then dialyzed against 5,000 volumes of 0.15 M NaCI, 4”C, pH 7.4. Lipoprotein deficient serum (LPDS) was obtained as described by Goldstein et al. [18]. For LDL kinetic and cell degradation studies, native (untreated control) and modified LDL were radiolabelled with 12sI(Amersham International, UK) combining the iodine monochloride method of McFarlane [19] and the modifications of Langer et al. [20] as previously reported [21]. LDL was acetylated with repeated additions of acetic anhydride at 4°C as described by Fraenkel-Conrat [22], Oxidation of LDL was performed under the following ex~rimental conditions: l-2 ml of LDL (4 g/l protein) contained in a dialysis bag of approx. 4-5 cm* surface area, the geometry of which can be roughly adjusted to a bicone with a base

95

radius of 1 cm and height of 1.3 cm, was incubated at 37°C in 5 litres of 0.15 M NaCl dialysis solution containing 0.6 mM FeCls and 5 FM CuS04 with a constant oxygen bubbling rate of 1 Vmin. For detailed characterization, similar portions of the LDL solution were subjected to this procedure and the extent of oxidation was varied by altering the incubation time. The oxidation process was stopped by dialysis against 0.15 M NaCl, 0.24 mM EDTA, 4°C and pH 7.4. Characterization studies Electrophoretic mobility on cellulose acetate was assessed using 40 mM barbital buffer (pH 8.6) and staining for protein with amido black. Radiolabelled native (unmodified) and modified LDL were also subjected to electrophoresis in preformed 8-25% gradient polyacrylamide gels containing 0.5% sodium dodecylsulphate (SDSPAGE) in a PhastsystemR (Pharmacia) and in gradient 2-16% gels containing 0.1% SDS in a Protean II slab vertical system (Bio-Rad, USA). Normal and delipidated [23] samples were heated for 3 min in a boiling water bath with or without pmercaptoethanol. No differences were found in the electrophoretic mobility of apo B in reduced and non-reduced samples. Bands were stained for protein with silver nitrate and the gels were then exposed to X-ray film (Agfa, Spain) for 20-60 min at room temperature in cassettes equipped with intensifier screens. As a measure of the degree of oxidation, thiobarbituric acid reactive substances (TBARS) were measured in dialysed samples following Nishigaki et al. [24] using malondialdehyde (MDA) as standard. Fatty acid composition was assessed using thin layer chromatography (TLC) and gas chromatography (GC). Lipids were extracted from native and oxidized LDL as described by Folch [25]. The samples were chromatographed on silica gel plates using different eluents for each lipid compound [26] and the bands were visualized with molybdophosphoric acid. Lipid extracts were also converted to fatty acid methyl esters using borotri~uoride-methanol (14% v/v) and were then separated by GC using a 25-m fused silica capillary column (RSL 150 BD) in a DAN1 3800 HR (DANI, Italy) chromatograph. Helium was used as the carrier gas. Injector and detector temperatures were maintained at 25O’C. The column temperature increase was programmed from 120’C to 250°C at a rate of 2”/min. These chromatographic conditions separated all major positional and geometric isomers of fatty acids of 12-24 carbon chain lengths. The major fatty acid methyl ester peaks were identified by reference to the retention times of mixtures of fatty acid methyl ester standards. Esteritied and free cholesterol, phospholipids and triglycerides were determined by enzymatic procedures (Boehringer Mannheim, Germany) in a Monarch 2000 IL (Monarch, Italy) centrifugal analyzer. Proteins in LDL and cells were quantified by the method of Lowry et al. [27] using bovine serum albumin as standard. Apolipoprotein B was determined by nepheIometry (Behring, Ge~any) using goat polyclonal antiserum raised against human apo B. Transmission electron microscopy LDL samples were diluted to 100-250 mg protein/l with 10 mM phosphate buffer,

96

150 mM NaCl (pH 7.4) and then dialysed against 125 mM ammonium acetate, 2.6 mM ammonium carbonate, 0.126 mM EDTA, 4°C and pH 7.4. The samples were mixed with sodium phosphotungstate (1% w/v, pH 7.4) and applied to Formvar carbon-coated grids and immediately examined under a Zeiss 10 C/CR (Zeiss, Germany) electron microscope at 80 kV [28]. The mean particle size was calculated by measuring across two diameters of 100 particles in each sample. l&and-receptor interaction studies To assess the affmity of oxidized LDL for the apo B/E receptor, LDL degradation studies were performed with cultured lymphocytes, essentially as described by Bilheimer et al. [29]. Human lymphocytes were obtained from venous blood collected into sodium heparinate (10 U/ml), isolated on Ficoll-Paque (Pharmacia) by the method of Biiyum [30] and maintained in RPM1 1640 medium supplemented with glutamine (2 mM), penicillin (100 U/ml) streptomycin (100 &ml) (Gibco, UK) and LPDS (10% v/v) for 72 h in order to achieve maximal receptor expression. Cell suspension (2 ml) was dispensed into 60-mm Petri dishes at a concentration of 2 x lo6 cells/ml with increasing concentrations (10, 20, 30 hg/ml) of ‘*‘I-native LDL and incubated at 37°C in 5% CO, humidified atmosphere for 6 h. The effect of lo-fold excess unlabelled LDL, native or oxidized for 8, 12 or 40 h, was examined. In addition, ~om~tition assays were performed using 1251-native LDL (20 pg/ml) incubated with increasing concentrations (20,40,80, 160 &ml) of unlabelled native or 40-h oxidized LDL. Oxidized LDL ligand interactions with the scavenger receptor were assessed using rat peritoneal macrophages. Resident peritoneal macrophages from unstimulated rats were harvested [31] and cultured in RPM1 1640 containing 10% fetal calf serum, glutamine and penicillin-streptomycin. The cells (2 x lo5 cell/ml) were dispensed in 24 multi-well plates (16-mm diameter wells) and allowed to adhere overnight. Just prior to the addition of radioiodinated LDL, the original medium containing the non-adherent cells was removed and replaced with fresh RPM1 1640 containing 10% LPDS in place of the fetal calf serum. The macrophages were incubated with increasing concentrations of ‘251-oxidized LDL (10, 20 and 30 mg/l) and the effect of the addition of lo-fold excess native LDL was observed. Assays were performed using LDL that had been oxidized for 8, 12 and 40 h. Ligand displacement competitive assays were performed with 20 mg/l of ‘251-40-hoxidized LDL as the ligand and increasing concentrations (20, 40, 80 and 160 mg/l) of native, acetylated or oxidized LDL as competitors. For validation purposes, acetylated [ 12’I]LDL competitive binding assays were also performed using 20 mg/l of 1251-acetylated LDL competing with 20, 40, 80 and 160 mg/l native or acetylated unlabelled LDL. Analyses were performed in triplicate, all assays including cell-free blanks which were processed similarly. After incubation for 6 h, the ceils were centrifuged (800 x g, 10 min) from the media, washed with 150 mM NaCI, solubilized in 0.5 M NaOH and assayed for protein content by the method of Lowry. The separated culture medium was treated with trichloroacetic acid (0.6 M final cont.) and the acid-soluble fraction extracted with chloroform to remove unreacted 1251.After gamma counting in an LKB Wallac 1275 gamma counter (Pharmacia-LKB, Sweden)

Fig. 1. Electrophoretic mobiiity of native and oxidized low density Iipoprotein (LDL). Samples were run in cellulose acetate electrophoresis with barbital buffer. (A) Protein bands stained with amido black (P = plasma, numbers indicate the incubation time in hours and A = acetylated LDL). (B) Correlation between the distance from the anode and the incubation time.

98

and the subtraction of the blanks. the receptor [ lz51]LDL degraded/mg cell protein/6 h.

activity was expressed as ng

LDL kinetic studied For experiments in vivo, LDL from human and rat plasma was isolated and portions oxidized (40 h) and radiolabeled as described above, but at a lower specific activity. Four sets of male Sprague-Dawley rats (3 animals in each set) weighing 190-230 g were anesthetized with ketamine (15 mgikg) and injected via a tail vein with approximately 5 &Zi of radiola~lled native rat LDL (nrLDL), oxidized rat LDL (orLDL), native human LDL (nhLDL) or oxidized human LDL (ohLDL) in 0.5 ml saline. Blood samples (50 ~1 each) were taken from the retro-orbital space at approximately 0.08,0.3, 0.8, 2,4, 7, 2, 24,28 and 48 h, microcentrifuged to separate the blood cells, and portions of the plasma counted using a gamma counter (LKB Wallac 1275). The plasma radioactivity/time decay curve was plotted as a fraction of the first post-injection plasma sample and the fractional catabolic rate (FCR) of

4

8

12

16

20

24

26

32

38

40

44

TCME(hours) Fig. 2. Thiobarbitu~c acid reactive su~tances (TBARS) measured in LDL after different incubation time. Open symbols represent aliquots of LDL exposed to the same conditions in the absence of 5 pM CuSO.+, 0.6 mM FeCI, and oxygen bubbling.

99

LD IL was calculated from the resulting bi-exponential curves by the curve {Teeling teclmique of Mathews [32]. Sta tistical analysis 1The unpaired t statistic for small numbers was used to compare mean diffe:rences bet ween study groups. All P values < 0.05 were considered statistically signi &ant.

Fig. 3. Electron microscope photographs representing native LDL and 40-h oxidized LDL. Bar 50 nm, original magnification 50,000 x

too Results Physicochemical

changes observed in LDL during the oxidation

procedure

A consistent feature of the oxidation process was the gradual net increase in electronegative charge (Fig. 1A); the incubation time correlating directly with the distance from the anode (r = 0.99, P < 0.001, Fig. 1B). The increased electrophoretic mobility was less marked than that observed for acetylated LDL, which was approximately 1.9-fold that of LDL incubated for 20 h. Progressive oxidation also produced a net increase of MDA-equivalent TBARS (Fig. 2), reaching a plateau between 12 and 24 h incubation. After approximately 12 h incubation, oxidized LDL showed a progressive loss of colour and enhanced fluorescence when exposed to an UV (360 nm) transilluminator. The particles that had been oxidized for up to 40 h were similar in size and shape to native LDL as seen under the transmission electron microscope. An increase in

Fig. 4. Autoradiogram of ‘251-native LDL and ‘Z5I-24-h oxidized LDL. Lane 1, delipidated native LDL (4 pg protein); lane 2, delipidated 24-h oxidized LDL (1.3 tip); lane 3, non-delipidated native HDL (12 Pg, used as a control); lane 4, non-deiipidated native LDL (2.5 pg); lane 5. non-delipidated 24-h oxidized LDL (1.2 pg).

-

I.

0.1 0.6 3.2 4.9 61.6 21.3 17.1

22:5n-3 22:6n-3 24:0 24: In-9

72.5 6.8 20.7

-

3.8 14.2 16.9 6.8 -

~ 35.7 13.2 51.1

2.3 5.1 48.5 10.5 0.5 0.4 0.1 0.3 0.2 0.2 0.2 0.8 0.3 -

2.2 28.3

III

18.3 47.7 34.0

0.3

0.6 5.2 0.2

2.0 I.2 32.0 40.4 0.7 0.3

I.1 16.0

IV

3.2 7.0 62.4 15.8 21.7

4.0 -

I.1 1.8

0.3

0.2 1.4

0.8 38.2 0.5 14.8 14.2 12.4

I

41.0 10.5 48.5

-

3.2 33.5 2.5 4.3 46.0 10.5 -

II

12-h oxidized

36.6 5.1 58.3

-

-

-

0.4

2.6 29.4 2.6 4.6 55.7 4.7 -

III

LDL

LDL

I = phospholipids, II = free fatty acids, III = triglycerides, IV = esterilied acid mass in each lipid fraction for a representative sample. Dispersion

Saturated Polyun Monoun

20:5n-3 22:o 22: In-9 22:4n-6

20:2n-6 20:3n-9 20:3n-6 20:4n-6

18:3n-6 18:3n-3 20:o 20:ln-9

0.4 14.9 II.6 14.0 0.1 1.3 0.1 0.3 0.1 1.6 4.2 0.1 3.9 -

16:ln-7 18:O 18:ln-9+7 18:2n-6

15.8 42.5

II

I

0.6 37.7

LDL

of fatty acids in native and oxidized

Native

composition

14:o 16:O

Percentage

TABLE

3.9 5.9 76.6 2.0 21.5

4.7

0.3 1.6 0.1

I.1 47.8 0.7 17.5 14.8 1.6

I

61.0 0.0 39.0

4. I 52.4 2.2 4.5 36.8

II

57.5 0.1 42.4

-

-

4.4 48.0 2.3 5.1 40. I 0.1‘

III

LDL

53.1 0.2 46.7

5.3 45.7 3.4 2.1 43.3 0.2

IV

4.0 5.2 80.0 I.7 18.3

4.7

-

1.6

1.3 50. I 0.6 18.3 12.5 1.7

I

72.6 0.0 27.4

-

1.0 8.4 26.4

7.3 56.9

II

40-h oxidized

-

cholesterol, - = not detected. These are values that represent between samples was negligible (< 0.8%).

28.3 25.0 46.7

0.3 0.8 0.2

1.9 24.5 2.7 1.9 44.0 23.5 0.1 0.1

IV

24-h oxidized

percentage

63.1 0.0 36.9

36.9

10.3 52.8

III

LDL

of total fatty

95.6 0.0 4.4

4.4 36.0 -

10.5 49. I

IV

Z

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heterogeneity was observed in the former as indicated by the greater standard deviations associated with the particle diameter measurements (21.52 f 0.3 nm vs. 21.52 f 0.6; mean jc SD.) (Fig. 3). Oxidized LDL when radiolabelled and subjected to SDS-PAGE, showed no apolipoprotein degradation (Fig. 4). No time-dependent change in the composition of the oxidized particles in terms of apo-B, total cholesterol, unesterilied cholesterol, triglyceride or phospholipid was observed. With prolonged incubations (>24 h), however, a trend towards a decrease in esterified cholesterol and triglyceride values appeared. TLC analysis showed a gradual disappearance of the bands representing the less polar polyunsaturated fatty acids (PUFA) of the cholesteryl esters moiety, concomitant with a time-dependent increase of bands of the neutral lipids. A progressive loss of phosphatidylcholine together with the appearance of unidenti~ed, more polar, bands in the more oxidized samples were also observed (data not shown). Results obtained by gas chromatographic analysis of native LDL and LDL after a 12, 24 or 40-h oxidation period (Table I) showed that, relative to the native LDL, there was a decrease in PUFAs (particularly linoleic, C18:2; linolenic, C18:3; arachidonic acid, C20:4) together with a net increase in saturated fatty acids (principally myristic, C14:O; palmitic, C16:O; and stearic acid, C18:O) and the appearance of unusual unidentified breakdown-products of the fatty acid chains (data not shown). Lig~~d-receptor i~teruction in lymphocytes and mac~o~hages In lymphocytes, the addition of a IO-fold excess of unlabelled oxidized or native LDL effectively reduced the degradation of labeled oxidized LDL (Fig. 5A); the degree of displacement being greater with the LDL oxidized for 12-h than for 40-h. Similarly, increasing amounts of unlabelled native LDL effectively displaced labeled native and oxidized LDL from binding sites on lymphocytes (Fig. 5B). Results of the ligand-receptor interaction assays using rat resident peritoneal macrophages are presented in Figs. 6A and 6B. The 40-h oxidized LDL was more completely degraded than the 12-h sample (Fig. 6A). When assessed in competition studies (Fig. 6B) the 40-h oxidized LDL was, slightly (5%) displaced by native LDL, but moderately so (60%) by oxidized LDL and completely displaced by acetylated LDL. In vivo catabolism

ofoxidized LDL in rats

Figure 7 shows the radioactivity/time plasma decay curves for native human LDL (nhLDL), oxidized human LDL (ohLDL), native rat LDL (nrLDL) and oxidized rat LDL (orLDL). FCR values (means f S.D.) are shown in Table II. The removal rate of the 40-h oxidized rat LDL (FCR = 0.190 ho.006 pools/h) was significantly higher (P I 0.001) than that observed with native rat LDL (FCR = 0.087 f 0.005 pools/h). Similar changes were observed when native and oxidized human LDL were injected in the rat, but FCR values were 2-fold higher than those observed with homologous LDL.

103

A

e +-

?G

I

1

I

I

10 20 30 LDL CONCENTRATION (pg apoprotein/ml)

io

io

‘io

CONCENTRATION

OF UNLABELLED

lb0 LDL

(pg apoprotein/ml)

Fig. 5. Human lymphocyte degradation of native LDL. (A) Saturation curve of native [ “‘I]LDL (R) and competing with a IO-fold excess of native LDL (n), 40-h oxidized LDL (0) and 12-h oxidized LDL (A). (B) The effect of increasing concentrations of unlabelled native LDL (m or 40-h oxidized LDL (0) on lymphocyte degradation of native “%LDL. Each point represents the mean of triplicate experiment f SD.

104

30 rpoprotoin/ml)

20

40

CONCENTFIATION

80 OF UNUBELLEO

160 LDL (pg apoprotsin/ml)

Fig. 6. Rat peritoneal macrophage degradation of native LDL and oxidized LDL. (A) Saturation curve of 12-h (A) and 40-h 0 oxidized [‘2sI]LDL. (B) Competition assay for degradation of ‘25I-4O-hoxidized LDL (open symbols) or iZ51-acetylated LDL (closed symbols) by IO-fold excess native LDL (circles), 40-h oxidized LDL (triangles) and acetylated LDL (squares). Each point represents the mean of triplicate experiment f SD.

I

I

2

12

I

L

I

24

35

48

TIME (hours) Fig. 7. Plasma decay curves of native human LDL (A), oxidized human (0). native rat LDL (A) and oxidized rat LDL (0) in rats. 40-h oxidized LDL and native LDL were radioiodinated and injected into a tail vein. Sequential plasma samples were obtained from retro-orbital space. The radioactivity at each point is expressed as the percent in the plasma of initial dose. Each point represents the mean of three experiments.

Discussion The oxidized LDL observed in humans probably represent a heterogeneous mixture of particles and the oxidative induction of apo B degradation seems to be the final step in this process. Oxidized LDL isolated from plasma has been shown not

106

TABLE II. Fractional catabolic rate values for native rat LDL (nrLDL), oxidized rat LDL (orLDL) native human LDL (nhLDL) and oxidized human LDL (ohLDL) In rats. Values in third column represent FCR related to FCR of nrLDL and in the later FCR of oxidized LDL respect to native LDL LDL

FCR f S.D.

FCRJnrFCR

nrLDL orLDL nhLDL ohLDL

0.0866 0.1900 0.0250 0.0996

I

1

2.2 0.3 1.1

2.2 1 3.9

f f f f

0.005 0.006 0.005 0.005

FCR/nFCR

to have fragmented apo B [6] while that obtained from atheromatous plaques is a mixture of intact and fragmented apo B particles [33,34]. The degree of LDL oxidation may explain, at least in part, the discrepancies in the biological behaviour of oxidized LDL described in the literature; most authors decribing oxidative cleavage of peptide bonds in apolipoprotein B [12,15,35]. With the rationale that highly oxidized LDL are without physiological significance in humans we have studied the method to obtain oxidized particles without further modi~~ations in the apo B moiety. The actual limiting factors were shown to be the initial LDL concentration, the avoidance of direct contact with the oxidative mixture and the surface area exposed to the oxidative conditions. Oxidation was confirmed by the increased electrophoretic mobility (Fig. 1) and the increased TBA reactivity. Oxidized LDL showed an apparently similar chemical composition when compared with native LDL. However, with increasing incubation times, progressive loss of linoleic, linolenic and arachidonic acid in phospholipids, cholesteryl esters and acylglycerides as well as an increase in fatty acid breakdown products were observed by TLC and gas chromatography. The absence of aggregation was confirmed by transmission electron microscopy and the measurement of particle size, although a mild degree of particle heterogeneity was noted. It was further confirmed by the finding that a more prolonged process of oxidation (> 60 h) in our particular system yields a product with severe structural damage leading to the formation of particle aggregates. The lack of protein fragmentation was also extensively investigated; the silver staining and the autoradiographs obtained (Fig. 4) conning the absence of apo B cleavage, Moreover, our oxidized LDL particles cross-react with antibodies against native human apo B although when injected into mice they raised specific antibodies not reacting with native LDL (data not shown). The integrity of the apo B molecule in the oxidized LDL particle would explain the interaction of this modified LDL with the apo B/E receptor. On the other hand, the oxidized lipids and its products are probably responsible for the particle interaction with the scavenger receptor. Our results show that the oxidized LDL clearly competed with native LDL for the lymphocyte receptor (apo B/E receptor) but was also extensively degraded by macrophages, suggesting a dual interaction with both receptors. Kinetic experiments in rats confirm, in vivo, the in vitro observations. Both human and rat oxidized LDL were rapidly cleared from plasma, indicating the ex-

107

istence of specific mechanisms, to remove modified LDL in vivo, as it was suggested by Nagelkerke et al from equivalent experiments 1361. In conclusion, the LDL particles oxidized under these conditions, without apo B fragmentation, showed a biological behaviour similar to that observed with other modified LDL but the role of apo B/E receptors in the catabolism of these particles cannot be neglected. These particles shared characteristics of oxidized LDL isolated in humans by other authors and as such may be useful in further studies to ascertain the mechanisms of foam cell formation.

References

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9 10

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14 15 16

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