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(Circulation. 1996;94:1698-1704.)
© 1996 American Heart Association, Inc.

Articles

Oxidation of Plasma Low-Density Lipoprotein Accelerates Its Accumulation and Degradation in the Arterial Wall In Vivo

Klaus Juul, MD*; Lars B. Nielsen, MD*; Klaus Munkholm, MD; Steen Stender, MD, DMSc; Børge G. Nordestgaard, MD, DMSc

the Department of Clinical Biochemistry (K.J., L.B.N., K.M.), Rigshospitalet, and Department of Clinical Biochemistry (B.G.N.), Herlev Hospital, University of Copenhagen, Denmark; and Clinical Institute (S.S.), University of Odense, Denmark.

Correspondence to Dr Børge G. Nordestgaard, Department of Clinical Biochemistry, Herlev Hospital, DK-2730 Herlev, Denmark.

	Abstract

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Background The aim of the present study was to investigate whether oxidized LDL (ox-LDL) in the arterial intima could be derived from LDL already oxidized in plasma.

Methods and Results Rabbits received an intravenous injection of ¹²⁵I-labeled normal LDL (N-LDL) mixed with ¹³¹I-labeled LDL that had been mildly oxidized through exposure to Cu²⁺. The aortic accumulation of undegraded labeled LDL was expressed as plasma equivalents and calculated as radioactivity in the intima/inner media (cpm/cm²) divided by the time-averaged concentration of radioactivity in plasma (cpm/nL): for the thoracic aorta, the accumulation of undegraded ox-LDL in the intima/inner media exceeded that of undegraded N-LDL by 286% (n=6, P<.04), 863% (n=7, P<.02), and 364% (n=8, P<.01) after 1, 3, and 24 hours of exposure, respectively. There was a strong positive association between the extent of oxidation and the excess accumulation of undegraded ox-LDL compared with N-LDL (thoracic aorta; 3 hours of exposure: r=.97, n=14, P<.00001). To measure degradation of N-LDL and ox-LDL, ¹²⁵I-LDL labeled with ¹³¹I-tyramine cellobiose was injected intravenously 24 hours before the aortic intima/inner media was removed: for the thoracic aorta, the accumulation of degradation products from ox-LDL (n=6) exceeded that from N-LDL (n=6) by 301% (P<.04).

Conclusions The present data suggest a novel mechanism: mildly oxidized LDL may circulate in plasma for a period sufficiently long to enter, accumulate, and be degraded in the arterial intima in preference to N-LDL.

Key Words: atherosclerosis • lipoproteins

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Elevated plasma levels of LDL cause accelerated atherogenesis.¹ The mechanism behind this effect is not fully understood, although several lines of evidence support the idea that oxidation of LDL increases its atherogenicity.^{2 3 4} Oxidation of LDL in vitro accelerates its uptake by macrophages and stimulates formation of foam cells,⁵ a key characteristic of the early atherosclerotic lesion. In addition, ox-LDL is cytotoxic⁶ and may also serve as a chemoattractant for monocytes⁷ ; both effects may promote the development of atherosclerosis.

Epitopes characteristic of ox-LDL are detectable in atherosclerotic lesions,^{7 8 9} and particles with properties characteristic of ox-LDL can be extracted from such lesions.^{7 10} It is generally assumed that ox-LDL present in the arterial intima is derived from LDL particles that have been modified within the arterial wall.² This concept that oxidation primarily occurs extravascularly is supported by the finding that addition of plasma inhibits cell-induced oxidation of LDL,² an effect presumably due to the presence of antioxidants in plasma. Furthermore, extensively ox-LDL is removed rapidly from plasma immediately after intravenous injection.^{11 12} Other studies suggest, however, that as much as 10% of LDL in plasma may be oxidized.^{13 14} Such ox-LDL may be produced at sites of inflammation: cells isolated from interstitial inflammatory fluid induce oxidation characteristic changes in LDL.^{15 16} It is equally possible that release of lipoperoxides from endothelial cells or macrophages or that secretion of reactive oxygen species by smooth muscle cells produces ox-LDL in the plasma compartment (for a discussion, see Reference 17).

In the present study, we investigated the hypothesis that mildly oxidized LDL may circulate in plasma for a period sufficiently long to enter, accumulate, and be degraded in the arterial intima to a significant extent. Labeled human LDL that had been mildly oxidized by exposure to Cu²⁺ and labeled human N-LDL were mixed and injected intravenously into normal rabbits; plasma decay and accumulation of undegraded LDL and its degradation products in the arterial intima/inner media of these two LDL species were compared.

	Methods

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The experimental protocols were approved by the Danish government body supervising animal experiments.

LDL Preparation
Blood was drawn from a normocholesterolemic donor into a sterile syringe containing Na₂·EDTA as an antioxidant and anticoagulant (final plasma concentration, 1.2 mg/mL), chloramphenicol (80 µg/mL), gentamicin sulfate (80 µg/mL), benzamidine (10 µg/mL), aprotinin (10 kallikrein units/mL), and -amino-n-caproic acid (2.6 mg/mL) (all from Sigma Chemical Co). LDL (1.019 to 1.063 g/mL) was isolated as previously described with the use of salt solutions containing 0.1 mg/mL Na₂·EDTA.¹⁸ Lipoprotein(a) contamination of the LDL preparations was <3% of the total lipoprotein mass.

We chose to use LDL obtained from the same normocholesterolemic human donor in all experiments in an attempt to minimize variation in oxidation between batches of ox-LDL. In pilot studies, ¹²⁵I-labeled rabbit LDL and ¹³¹I-labeled human LDL were injected intravenously into two rabbits 24 hours before removal of the aorta. The plasma decay of the two labeled LDL species as well as their accumulation in the aorta were similar. This supports the idea that human LDL and rabbit LDL interact similarly with the arterial wall in the rabbit. Therefore, the present use of human LDL instead of rabbit LDL probably does not affect the present data.

Labeling and Oxidation of LDL
Immediately before iodination, purified LDL (4.7 to 6.6 mg protein/mL)¹⁹ was equilibrated with PBS (40 mmol/L Na₂HPO₄, 7 mmol/L NaH₂PO₄, 90 mmol/L NaCl, pH 7,4) with the use of PD-10 columns (Pharmacia).

LDL (5 mg of protein) was labeled with 185 to 370 MBq of ¹²⁵I or ¹³¹I (Amersham) within 72 hours of isolation using iodine monochloride.^{18 20 21} Unbound iodine was removed with a PD-10 column equilibrated with PBS. The labeling efficiency ranged from 7% to 74%; specific activities were 0.8 to 17x10⁹ cpm ¹²⁵I/mg of LDL protein and 0.4 to 3.9x10⁹ cpm ¹³¹I/mg of LDL protein.

TC (50 nmol) was labeled with 370 MBq of ¹³¹I using Iodogen (Pierce Chemical Co).^{22 131}I-TC was transferred to a vial containing 10 µL of NaHSO₃ (0.1 mol/L) and 5 µL of NaI (0.1 mol/L) and activated by the addition of 20 µL of cyanuric chloride (2.5 mmol/L in acetone) followed by 5 µL of NaOH (0.02 mol/L) and 10 µL of acetic acid (0.015 mol/L). ¹²⁵I-LDL (5 mg of protein), which had been adjusted to pH 9 to 10 by the addition of borate buffer (0.3 mol/L), was immediately added to the activated ligand. After 20 minutes, unbound ¹³¹I-TC was removed using a PD-10 column equilibrated with PBS containing NH₄HCO₃ (0.1 mol/L).²² The efficiency by which ¹³¹I-TC was bound to ¹²⁵I-LDL was 5% and 7%; the specific activity was 2.8 to 4.7x10⁸ cpm ¹³¹I/mg of LDL protein.

Rabbit albumin (20 mg/mg LDL protein), ascorbic acid (final concentration, 50 µmol/L), Na₂EDTA (200 µmol/L), and butylated hydroxytoluene (40 µmol/L) were added to ¹²⁵I-LDL and aliquots of ¹³¹I-TC/¹²⁵I-LDL immediately after labeling. This addition of albumin and antioxidants completely prevented an otherwise increased formation of conjugated dienes²³ on incubation of LDL (200 µg/mL) with Cu²⁺ (5 µmol/L) at room temperature for up to 24 hours. This LDL was designated N-LDL.

¹³¹I-LDL and aliquots of ¹³¹I-TC/¹²⁵I-LDL were diluted to a concentration of 200 µg protein/mL, and CuSO₄ (final concentration, 5 µmol/L) was added. After 2, 4, or 8 hours of incubation at room temperature, albumin and antioxidants were added as described above; this LDL was designated ox_2h-LDL, ox_4h-LDL, and ox_8h-LDL, respectively.

N-LDL and ox-LDL were mixed and maintained at +4°C for a maximum of 16 hours before injection. In addition, TC-labeled LDL was dialyzed against PBS with antioxidants for 12 hours at +4°C before injection. Preparations of labeled LDL were filtered through 0.22-µm filters (Millex GS Millipore SA) before injection.

On precipitation with 15% TCA at +4°C, 95% of the radioactivity in N-LDL and ox-LDL was protein associated; of this, <2% was lipid soluble, ie, extractable with chloroform/methanol (1:1 vol/vol). For ¹³¹I-TC–labeled LDL, 92±2% (n=4) of the ¹³¹I was protein associated and 1.4±2% was lipid soluble.

Characterization of Labeled LDL
Fixed density ultracentrifugation of aliquots of preparations used for injections and of plasma samples was performed at +4°C for 2.7x10⁸ gxmin with a 50.3 Ti Beckman rotor. Densities were adjusted to 1.019, 1.063, 1.12, and 1.21 g/mL using NaBr solutions containing Na₂EDTA (0.27 µmol/mL).

Gel filtration chromatography was performed on a Sephacryl S-500 HR gel (Pharmacia). The column dimensions were 2.6x100 cm, and the flow rate was 30 mL/h. Lipoproteins were eluted with PBS. Void and total volume were determined with Lipofundin (B Braun Melsungen AG) and ²²Na (Amersham), respectively.

Animal Studies
White male rabbits of the Danish Country Strain (Statens Seruminstitut) weighing 2.7 to 4.1 kg received normal rabbit chow (Altromin) before injection of labeled LDL into the left ear vein, with an average of 1.7±0.1x10⁸ cpm ¹²⁵I/kg body wt and 0.6±0.06x10⁸ cpm ¹³¹I/kg body wt.

To compare the accumulation of undegraded N-LDL and ox-LDL in the arterial intima/inner media in the same rabbit, N-LDL and ox_8h-LDL were mixed and injected intravenously into conscious rabbits 1 hour (n=6), 3 hours (n=7), or 24 hours (n=8) before the aorta was removed; other rabbits received an intravenous injection of N-LDL mixed with either ox_2h-LDL (n=3) or ox_4h-LDL (n=4) 3 hours before the aorta was removed. Degradation and accumulation of undegraded LDL in the arterial intima/inner media and selected tissues were determined using intravenous injections of normal (n=6) or oxidized (n=6) ¹³¹I-TC/¹²⁵I-LDL 24 hours before the aorta was removed. Plasma contamination of the intima/inner media was determined by the use of an intravenous injection of N-LDL and/or ox_8h-LDL (n=11) 5 to 10 minutes before the aorta was removed. Blood samples were taken from the right ear vein into tubes containing Na₂EDTA.

The rabbits were killed with an intravenous injection of pentobarbital (50 to 100 mg/kg) before the circulation was perfused through a cannula placed in the left ventricle of the heart. In rabbits used for measuring accumulation of undegraded LDL in the aorta, the circulation was perfused with 1000 mL of cold saline before the aorta was removed. After removal of adventitial tissue, the aorta was fixed with pins on a corkboard, and the area was outlined on graph paper. The aorta was then divided into the aortic arch, the thoracic aorta, and the abdominal aorta.¹⁸ The intima/inner media was immediately separated from the outer media with a pair of forceps and minced with scissors in 0.9 mL of PBS, and 100 mg of bovine albumin (fraction V, Sigma) was added before proteins were precipitated at +4°C with 15% TCA.

In rabbits used for measuring degradation and accumulation of undegraded LDL in the aorta and selected tissues, the circulation was perfused with 500 mL of cold saline followed by perfusion with 500 mL of half-strength Karnofsky's fixative. After removal of adventitial tissue, the aorta was opened longitudinally and fixed for an additional 20 to 24 hours in half-strength Karnofsky's fixative (which results in retention in the tissue of protein bound ¹²⁵I and of ¹³¹I-TC²⁴ ) before the intima/inner media was separated from the outer media. Selected tissues were also removed and fixed for 20 to 24 hours in half-strength Karnofsky's fixative.

A 2-mm-wide specimen from the most distal part of the aortic arch was taken from 20 aortas, and the intima/inner media and outer media were separated; tissues were fixed in formalin and stained with Orcein. The intima/inner media contained intima and 15±1 elastic membranes from the media, whereas the outer media contained 10±1 elastic membranes from the media as well as occasional small amounts of adventitial tissue.

The mean luminal areas for saline-perfused and fixed aortas were similar in the aortic arch, thoracic aorta, and abdominal aorta. Furthermore, there was no difference in the number of elastic membranes in the intima/inner media of saline- and fixative-perfused aortas. However, the average wet weight of fixed intima/inner media was an average of 76% of the average wet weight of saline-perfused intima/inner media.

Determination of Radioactivity
Aliquots (0.1 mL) of plasma and doses diluted with cold human plasma were added to 0.9 mL of PBS before precipitation with TCA (15%). Precipitates and supernatants were counted in a double-channel counter (LKB Compugamma 1282, Wallac); SE values of total count were usually <1%.

Calculations
Calculations were based on protein bound radioactivity (ie, TCA-precipitable radioactivity or radioactivity after fixation in half-strength Karnofsky's fixative).

The accumulation (A; nL/cm²) of undegraded labeled LDL and undegraded labeled LDL plus its degradation products combined in the intima/inner media after 1, 3, or 24 hours of exposure to labeled lipoproteins was corrected for plasma contamination and calculated as the following: where T is the total amount of radioactivity in the intima/inner media (cpm/cm² luminal surface area), C is the plasma contamination (nL/cm² luminal surface area), c_end is the plasma radioactivity concentration at the end of the experiment (cpm/nL), and c_avg is the time-averaged concentration of radioactivity in plasma during the experiment (cpm/nL); plasma decay curves were fitted to monoexponential (1-hour experiments) or double-exponential (3- or 24-hour experiments) functions, and the time-averaged radioactivity concentration was calculated through the use of integration.

Degradation of labeled LDL in the intima/inner media was calculated as the accumulation of degradation products of labeled LDL in the intima/inner media, ie, the difference between the accumulation of ¹³¹I-TC and of ¹²⁵I (in nL/cm²) in the same rabbit; ¹²⁵I represents undegraded LDL. Degradation of labeled LDL in the intima/inner media and in selected tissues was also expressed as the fraction of the total plasma pool irreversibly degraded as previously described^{24 25} ; the fractional catabolic rate was calculated as described previously.²⁶ The fraction of the dose irreversibly degraded by the entire body during 24 hours calculated from a two-compartment model^{24 25} was an average of 78% for N-LDL but >100% for ox_8h-LDL. These data suggest that the two-compartment model originally proposed²⁵ is not appropriate for ox_8h-LDL or, possibly, that a large fraction of ox_8h-LDL had been removed from plasma during the 5 to 10 minutes between injection of ox_8h-LDL and the first blood sampling. Because 98% of labeled ox_8h-LDL was observed to have disappeared from plasma after 24 hours, it was therefore estimated that 90% of the total amount of ox_8h-LDL had been irreversibly degraded 24 hours after injection; however, the use of estimates of a minimum of 78% or a maximum of 100% did not change the results or conclusions regarding degradation of ox_8h-LDL (expressed as a fraction of plasma pool) in comparison with N-LDL.

Plasma contamination values calculated as radioactivity in the intima/inner media after a 5- to 10-minute exposure (cpm/cm²) divided by the plasma radioactivity concentration at termination (cpm/nL) were similar for N-LDL and ox_8h-LDL: an average of 35.3±7.0 nL/cm² (n=11) in the aortic arch, 28.9±6.5 nL/cm² in the thoracic aorta, and 39.0±5.1 nL/cm² in the abdominal aorta. Importantly, regardless of whether corrected for plasma contamination, the overall results and conclusions of the present study remain the same.

By multiplying the mean values per cm² of aortic arch, thoracic aorta, and abdominal aorta by 27, 31, and 33, respectively, the values can be converted to mean values per gram of wet weight of intima/inner media.

The volume of distribution was determined as the injected dose (cpm) divided by the initial plasma radioactivity concentration (cpm/nL) and by the body weight of the rabbit (kg).

Statistical Analysis
The aim of the present study was to compare accumulation and degradation of N-LDL and ox-LDL. Differences between N-LDL and ox-LDL were analyzed with the paired Wilcoxon test when N-LDL and ox-LDL were studied simultaneously and with the unpaired Wilcoxon test when N-LDL and ox-LDL were studied in separate animals (Instat program, version 1.14; 1990, GraphPAD Software). The association between the extent of oxidation and the excess aortic accumulation of undegraded ox-LDL compared with N-LDL was evaluated with the use of Spearman's rank correlation coefficient. Probability values are two tailed. Values are given as mean±SEM.

	Results

Top Abstract Introduction Methods Results Discussion References

 
The ox-LDL and N-LDL were characterized by agarose gel electrophoresis, fixed density ultracentrifugation, gel filtration chromatography, and rate of removal from plasma in vivo. Compared with N-LDL, ox-LDL had an increased density (Fig 1) and electrophoretic mobility on agarose gel (data not shown). These differences between ox-LDL and N-LDL increased with increasing exposure time of labeled LDL to Cu²⁺ (from 2 to 8 hours of incubation with Cu²⁺) (data not shown).

View larger version (32K): [in this window] [in a new window]   Figure 1. Density distribution of N-LDL and ox8h-LDL in injected doses (n=6) and plasma after 1 hour (n=6), 3 hours (n=5), and 24 hours (n=7), respectively. Values are mean±SEM.

Gel filtration chromatography profiles of ox_8h-LDL and N-LDL were similar except that ox-LDL consistently tended to elute before N-LDL (Fig 2). On gel filtration of 8-hour oxidized ¹³¹I-TC/¹²⁵I-LDL, ¹³¹I and ¹²⁵I coeluted (data not shown). No evidence of fragmentation of ox-LDL was detected with ultracentrifugation and agarose gel electrophoresis followed by autoradiography or on gel filtration chromatography.

View larger version (23K): [in this window] [in a new window]   Figure 2. Gel filtration profiles of N-LDL and ox8h-LDL. Recoveries of radioactivity were 97% and 96% for N-LDL and ox8h-LDL, respectively. Vt and Vo indicate the total and void volume, respectively.

Labeled LDL in Plasma
On intravenous injection, the volume of distribution was 37±1 mL/kg for N-LDL and 43±1 mL/kg for ox-LDL (n=45, P<.001). Fractional catabolic rate was 9.7±1.2 d^-1 (n=14) for ox_8h-LDL and 1.7±0.2 d^-1 (n=14) for N-LDL (P<.001). The difference in plasma decay between N-LDL and ox-LDL increased with increasing exposure time to Cu²⁺ (Fig 3). Plasma decay curves for ¹²⁵I and ¹³¹I were similar after intravenous injection of both normal and oxidized ¹³¹I-TC/¹²⁵I-LDL (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 3. Line plot showing plasma decay curves of N-LDL, ox2h-LDL, ox4h-LDL, and ox8h-LDL. Values are mean±SEM and are expressed as percentage of total plasma radioactivity concentration 5 to 10 minutes after intravenous injection.

The distribution of radioactivity between the various density fractions in plasma remained different for N-LDL and ox_8h-LDL throughout the experimental period (Fig 1).

Accumulation of Undegraded LDL in Aortic Intima/Inner Media
Twenty-four hours after a simultaneous intravenous injection, the accumulation of undegraded ox_8h-LDL was significantly greater than that of undegraded N-LDL in the aortic intima/inner media of the thoracic and abdominal aorta, whereas this difference between ox_8h-LDL and N-LDL did not reach statistical significance in the aortic arch (Fig 4). However, accumulation of undegraded ox_8h-LDL was significantly greater than that of undegraded N-LDL after 24 hours' exposure to ¹³¹I-TC/¹²⁵I-LDL in all three aortic segments (Fig 5). Also, 1 and 3 hours after intravenous injection, more undegraded ox_8h-LDL than undegraded N-LDL had accumulated in the intima/inner media (Table 1). The difference between undegraded N-LDL and ox_8h-LDL after 1 and 3 hours was most pronounced in the thoracic and abdominal aortas.

View larger version (26K): [in this window] [in a new window]   Figure 4. Accumulation in aortic intima/inner media of undegraded N-LDL and ox8h-LDL after 24 hours of exposure to labeled lipoproteins. Accumulation of undegraded labeled LDL was calculated as radioactivity in intima/inner media (cpm/cm2) divided by the time-averaged plasma radioactivity concentration (cpm/nL). Values obtained in the same tissue are connected by a line.

View larger version (31K): [in this window] [in a new window]   Figure 5. Accumulation of undegraded LDL and its degradation products in aortic intima/inner media after 24 hours of exposure to normal or 8-hour oxidized 131I-TC/125I-LDL. Accumulation of undegraded LDL and its degradation products combined was calculated as 131I in intima/inner media (cpm/cm2) divided by the time-averaged plasma concentration of 131I (cpm/nL). Accumulation of undegraded LDL only was calculated as 125I in intima/inner media (cpm/cm2) divided by the time-averaged plasma concentration of 125I (cpm/nL). Values are mean±SEM. *P<.005 and **P<.04 compared with N-LDL.

View this table: [in this window] [in a new window]   Table 1. Accumulation of Undegraded ox8h-LDL and N-LDL in Aortic Intima/Inner Media After 1- and 3-Hour Exposure

There was a strong positive association in all three aortic segments between the excess accumulation of undegraded ox-LDL compared with undegraded N-LDL and the extent of oxidation of ox-LDL determined in vivo (Fig 6); rabbits used for this analysis were injected intravenously with mixed preparations of N-LDL and ox_2h-LDL, N-LDL and ox_4h-LDL, or N-LDL and ox_8h-LDL 3 hours before the aorta was removed.

View larger version (21K): [in this window] [in a new window]   Figure 6. Excess accumulation of undegraded ox-LDL compared with undegraded N-LDL in aortic intima/inner media after 3 hours of exposure plotted as a function of the extent of oxidation of ox-LDL. The excess accumulation is expressed as the accumulation of ox-LDL minus that of N-LDL; 20 has been added to this value for statistical reasons. The extent of oxidation is expressed as the time-averaged plasma radioactivity concentration of N-LDL (in percentage of plasma radioactivity concentration 5 to 10 minutes after intravenous injection) minus that of ox-LDL. Depicted r values represent Spearman's correlation coefficient.

Degradation of LDL in Aortic Intima/Inner Media and Other Tissues
The accumulation of degradation products of labeled LDL in the intima/inner media was significantly larger for ox_8h-LDL than for N-LDL in all three aortic segments (Fig 5).

Also, the degradation rate expressed as fraction of the plasma pool irreversibly degraded was significantly greater for ox_8h-LDL than for N-LDL in the intima/inner media of the aortic arch, thoracic aorta, and abdominal aorta (Table 2); the present values for N-LDL are similar to previously published degradation rates for normal LDL in rabbit aorta.^{24 25 27}

View this table: [in this window] [in a new window]   Table 2. Degradation Rates for ox8h-LDL and N-LDL in Selected Rabbit Tissues After 24-Hour Exposure to 131I-TC/125I-LDL

In aortic outer media, liver, spleen, kidney, heart, lung, and adipose tissue, the degradation rate of ox_8h-LDL was also greater than that of N-LDL. In contrast, there was no difference between degradation rates of ox_8h-LDL and N-LDL in the adrenals or in the small intestine (Table 2).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Results of the present study suggest that mildly ox-LDL as a minor fraction of the total plasma LDL pool can circulate in plasma for a period long enough to enter the arterial wall to a significant extent: the density of ox-LDL in plasma remained increased compared with that of N-LDL at 1, 3, and 24 hours after intravenous injection.

Surprisingly, the accumulation in the intima/inner media of undegraded ox-LDL, expressed as plasma equivalents, exceeded that of N-LDL at 1, 3, and 24 hours after intravenous injection. The positive association between the excess accumulation of undegraded ox-LDL compared with undegraded N-LDL and the extent of oxidation supports the idea that oxidation causes specific changes in the LDL particle, which facilitate its accumulation in the arterial intima. The extent of oxidation was estimated in vivo from the disappearance of ox-LDL from plasma compared with that of N-LDL in the same rabbit, simply because a previous study suggested that accelerated in vivo clearance from plasma is a very sensitive biological marker of oxidation of LDL.¹¹

The accumulation of N-LDL in the intima/inner media was similar to that observed in previous studies from our laboratory.^{18 28} Because N-LDL and ox-LDL were mixed before injection, this suggests that ox-LDL by itself or endotoxins in the preparations of the two labeled LDL species did not affect the barrier function of the arterial wall under the present experimental conditions.

A greater lag time for the release of labeled degradation products of ox-LDL compared with N-LDL²⁹ may have contributed to the observed greater accumulation of undegraded ox-LDL compared with undegraded N-LDL in the arterial wall after 24 hours' exposure. However, accumulation of degradation products of LDL, determined with the use of TC-labeled LDL, was greater for ox-LDL than for N-LDL after 24 hours' exposure; this finding cannot be accounted for by a greater lag time for the degradation of ox-LDL than for N-LDL.

Small LDL particles enter the arterial wall faster than large LDL particles.³⁰ The present greater accumulation of ox-LDL than of N-LDL, however, could not be explained by a difference in size between the two LDL species since oxidation increased the size of LDL slightly (Fig 2); this is in accordance with size evaluations of in vivo ox-LDL with electron microscopy.¹³ Electrostatic forces may also affect the passage of plasma macromolecules across the arterial endothelium.³¹ Because the glycocalyx of the endothelial surface is negatively charged³² and because oxidation increased the negative charge of LDL, one would expect ox-LDL to have entered the intima/inner media slower than N-LDL, not faster, as was observed. It is, however, possible that transfer of ox-LDL across the endothelial barrier occurs at microdomains with a positive charge: such microdomains have been identified and correspond to structures involved in endocytosis and transcytosis of anionic proteins.³² Alternatively, greater unidirectional transfer of ox-LDL than of N-LDL into the intima/inner media may reflect that more ox-LDL than N-LDL is taken up by the endothelial cells as opposed to increased transfer of ox-LDL across the endothelial cells into the subendothelial space.³³ The transfer of LDL into aorta is thought to occur as a nonspecific molecular sieving,^{30 34} whereas uptake of N-LDL and ox-LDL by endothelial cells presumably involves receptors.

The accumulation of undegraded labeled LDL in the arterial intima represents the combination of transfer into and loss from the intima/inner media of labeled LDL as a result of efflux back to plasma and degradation. To estimate the approximate fractional loss of ox_8h-LDL from the intima/inner media, mean values of plasma and aortic radioactivity after 3 and 24 hours' exposure were used³⁵ : the estimated fractional losses of N-LDL and ox_8h-LDL were 0.09 versus 0.05 h^-1, 0.07 versus 0.10 h^-1, and 0.09 versus 0.11 h^-1 in the aortic arch, thoracic aorta, and abdominal aorta, respectively. Thus, a lower loss, as opposed to increased influx, apparently was of minor importance for the greater accumulation of undegraded ox-LDL compared with undegraded N-LDL in the intima/inner media in the present study. This notion is in accordance with a previous study by Chang et al³⁶ suggesting that focal sequestering of LDL in arterial lesions is independent of oxidation of the LDL particle.

The present study suggests that oxidation of plasma LDL increases its degradation in the aortic intima/inner media compared with N-LDL. This observation could reflect selective uptake of ox-LDL directly from plasma by putative receptors on the luminal surface of endothelial cells, by degradation by endothelial or other cells after increased accumulation of undegraded ox-LDL in the intima/inner media, or by a combination of the two. Aortic endothelial cells can degrade acetylated³⁷ and acetoacetylated³⁸ LDL in vitro, and incubation of endothelial cells with ox-LDL induced intracellular accumulation of apolipoprotein B that was apparently resistant to degradation.³⁹ However, smooth muscle cells also apparently degraded ox-LDL in the present study: ox-LDL was degraded faster than N-LDL in the outer media.

The present data suggest that ox-LDL is degraded more rapidly than N-LDL in the liver and spleen but not in the adrenals or the small intestine. This result may reflect that oxidation of LDL abolishes its recognition by the "classic" LDL receptor and that degradation of LDL in the adrenals and small intestine predominantly is LDL receptor mediated, which is not the case in liver and spleen.³⁸ We were concerned that LDL oxidized by Cu²⁺ incubation may interact differently with the arterial wall than does LDL that has been oxidized in vivo. However, the Cu²⁺ ox-LDL used in the present study shares physical and chemical characteristics with LDL oxidized in vivo, ie, an increased electrophoretic mobility on agarose gels, a higher density, and a slightly greater size than N-LDL.^{10 13 14} Furthermore, Cu²⁺/ox-LDL competitively inhibited macrophage degradation of lesion-derived LDL¹⁰ as well as LDL oxidized by cultured endothelial cells.²⁹ These observations support the notion that the metabolism of Cu²⁺/ox-LDL in the present study to a large extent mimics the metabolism of LDL oxidized in vivo; however, the present data cannot conclusively exclude that Cu²⁺/ox-LDL may behave differently in vivo compared with LDL oxidized in vivo.

It has been estimated that 5% to 20% of the LDL particles in human and monkey plasma may be oxidized.^{13 14} If it is assumed that 5% of the LDL in plasma is oxidized to the same extent as the ox_8h-LDL that was used in the present study, the mass accumulation of undegraded ox-LDL during 1 hour would contribute 11%, 14%, and 40% to the total LDL accumulation in the aortic arch, thoracic aorta, and abdominal aorta, respectively (calculated from data in Table 1). Furthermore, ox-LDL would contribute 28%, 23%, and 33% to the total degradation of plasma LDL per gram of tissue per day in the aortic arch, thoracic aorta, and abdominal aorta, respectively (calculated from data in Table 2). However, the exact concentration and extent of oxidation of LDL particles in plasma await further exploration.

In summary, the present data support the hypothesis that mildly oxidized LDL in plasma accumulates and is degraded faster than N-LDL in the arterial wall: depending on the concentration of ox-LDL in plasma, this novel pathway may make an important contribution to the accumulation of ox-LDL and its degradation products in the arterial wall during the development of atherosclerosis.

	Selected Abbreviations and Acronyms

 

N-LDL = normal LDL ox-LDL = oxidized LDL TC = tyramine cellobiose TCA = trichloroacetic acid

	Acknowledgments

 
This study was supported by the Danish Heart Foundation. The TC used in the study was a most generous gift from Dr Daniel Steinberg, Department of Medicine, University of California, San Diego.

	Footnotes

 
*Dr Juul and Dr Nielsen contributed equally to this work.

Dr Nielsen's present address: Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, Calif.

Received October 9, 1995; revision received April 3, 1996; accepted April 9, 1996.

	References

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1. Pyorala K, De Backer G, Graham I, Poole-Wilson P, Wood D. Prevention of coronary heart disease in clinical practice: recommendations of the Task Force of the European Society of Cardiology, European Atherosclerosis Society and European Society of Hypertension. Eur Heart J. 1994;15:1300-1331.[Free Full Text]
   
2. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915-924.[Medline] [Order article via Infotrieve]
   
3. Steinberg D. Modified forms of low-density lipoprotein and atherosclerosis. J Intern Med. 1993;233:227-232.[Medline] [Order article via Infotrieve]
   
4. Penn MS, Chisolm GM. Oxidized lipoproteins, altered cell function and atherosclerosis. Atherosclerosis. 1994;108(suppl):S21-S29.
   
5. Henriksen T, Mahoney EM, Steinberg D. Enhanced macrophage degradation of biologically modified low density lipoprotein. Arteriosclerosis. 1983;3:149-159.[Abstract/Free Full Text]
   
6. Chisolm GM, Morel DW. Lipoprotein oxidation and cytotoxicity: effect of probucol on streptozotocin-treated rats. Am J Cardiol. 1988;62:20B-26B.[Medline] [Order article via Infotrieve]
   
7. Quinn MT, Parthasarathy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc Natl Acad Sci U S A. 1987;84:2995-2998.[Abstract/Free Full Text]
   
8. Yla-Herttuala S, Palinski W, Butler SW, Picard S, Steinberg D, Witztum JL. Rabbit and human atherosclerotic lesions contain IgG that recognizes epitopes of oxidized LDL. Arterioscler Thromb. 1994;14:32-40.[Abstract/Free Full Text]
   
9. Rosenfeld ME, Palinski W, Yla-Herttuala S, Butler S, Witztum JL. Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis. 1990;10:336-349.[Abstract/Free Full Text]
   
10. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.
    
11. Steinbrecher UP, Witztum JL, Parthasarathy S, Steinberg D. Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL: correlation with changes in receptor-mediated catabolism. Arteriosclerosis. 1987;7:135-143.[Abstract/Free Full Text]
    
12. Nagelkerke JF, Havekes L, van Hinsbergh VW, van Berkel TJ. In vivo catabolism of biologically modified LDL. Arteriosclerosis. 1984;4:256-264.[Abstract/Free Full Text]
    
13. Avogaro P, Bon GB, Cazzolato G. Presence of a modified low density lipoprotein in humans. Arteriosclerosis. 1988;8:79-87.[Abstract/Free Full Text]
    
14. Hodis HN, Kramsch DM, Avogaro P, Bittolo Bon G, Cazzolato G, Hwang J, Peterson H, Sevanian A. Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL-). J Lipid Res. 1994;35:669-677.[Abstract]
    
15. Raymond TL, Reynolds SA, Swanson JA. In vitro incubation of low-density lipoproteins with inflammatory cells causes enhanced degradation by macrophages in culture. Inflammation. 1987;11:335-344.[Medline] [Order article via Infotrieve]
    
16. Raymond TL, Reynolds SA. Lipoproteins of the extravascular space: alterations in low density lipoproteins of interstitial inflammatory fluid. J Lipid Res. 1983;24:113-119.[Abstract]
    
17. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785-1792.
    
18. Nielsen LB, Stender S, Kjeldsen K, Nordestgaard BG. Effect of angiotensin II and enalapril on transfer of low-density lipoprotein into aortic intima in rabbits. Circ Res. 1994;75:63-69.[Abstract/Free Full Text]
    
19. Zilversmit DB, Shea TM. Quantitation of apoB-48 and apoB-100 by gel scanning or radio-iodination. J Lipid Res. 1989;30:1639-1646.[Abstract]
    
20. McFarlane AS. Efficient trace-labelling of proteins with iodine. Nature. 1958;182:53.[Medline] [Order article via Infotrieve]
    
21. Bilheimer DW, Eisenberg S, Levy RI. The metabolism of very low density lipoprotein proteins, I: preliminary in vitro and in vivo observations. Biochim Biophys Acta. 1972;260:212-221.[Medline] [Order article via Infotrieve]
    
22. Pittman RC, Carew TE, Glass CK, Green SR, Taylor CA, Attie AD. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem J. 1983;212:791-800.[Medline] [Order article via Infotrieve]
    
23. Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun. 1989;6:67-75.[Medline] [Order article via Infotrieve]
    
24. Schwenke DC, Carew TE. Quantification in vivo of increased LDL content and rate of LDL degradation in normal rabbit aorta occurring at sites susceptible to early atherosclerotic lesions. Circ Res. 1988;62:699-710.[Abstract/Free Full Text]
    
25. Carew TE, Pittman RC, Marchand ER, Steinberg D. Measurement in vivo of irreversible degradation of low density lipoprotein in the rabbit aorta: predominance of intimal degradation. Arteriosclerosis. 1984;4:214-224.[Abstract/Free Full Text]
    
26. Matthews CME. The theory of tracer experiments with ¹³¹I-labeled plasma proteins. Phys Med Biol. 1957;2:36-53.[Medline] [Order article via Infotrieve]
    
27. Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits, I: focal increases in arterial LDL concentration precede development of fatty streak lesions. Arteriosclerosis. 1989;9:895-907.[Abstract/Free Full Text]
    
28. Nielsen LB, Nordestgaard BG, Stender S, Kjeldsen K. Aortic permeability to LDL as a predictor of aortic cholesterol accumulation in cholesterol-fed rabbits. Arterioscler Thromb. 1992;12:1402-1409.[Abstract/Free Full Text]
    
29. Sparrow CP, Parthasarathy S, Steinberg D. A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein. J Biol Chem. 1989;264:2599-2604.[Abstract/Free Full Text]
    
30. Nordestgaard BG, Zilversmit DB. Comparison of arterial intimal clearances of LDL from diabetic and nondiabetic cholesterol-fed rabbits: differences in intimal clearance explained by size differences. Arteriosclerosis. 1989;9:176-183.[Abstract/Free Full Text]
    
31. Baldwin AL, Chien S. Endothelial transport of anionized and cationized ferritin in the rabbit thoracic aorta and vasa vasorum. Arteriosclerosis. 1984;4:372-382.[Abstract/Free Full Text]
    
32. Simionescu M, Simionescu N. Functions of the endothelial cell surface. Annu Rev Physiol. 1986;48:279-293.[Medline] [Order article via Infotrieve]
    
33. Kim MJ, Dawes J, Jessup W. Transendothelial transport of modified low-density lipoproteins. Atherosclerosis. 1994;108:5-17.[Medline] [Order article via Infotrieve]
    
34. Wiklund O, Carew TE, Steinberg D. Role of the low density lipoprotein receptor in penetration of low density lipoprotein into rabbit aortic wall. Arteriosclerosis. 1985;5:135-141.[Abstract/Free Full Text]
    
35. Schwenke DC, Zilversmit DB. The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit, 1: studies during the first two days after mild aortic injury. Atherosclerosis. 1989;77:91-103.[Medline] [Order article via Infotrieve]
    
36. Chang MY, Lees AM, Lees RS. Low-density lipoprotein modification and arterial wall accumulation in a rabbit model of atherosclerosis. Biochemistry. 1993;32:8518-8524.[Medline] [Order article via Infotrieve]
    
37. Stein O, Stein Y. Bovine aortic endothelial cells display macrophage-like properties towards acetylated ¹²⁵I-labelled low density lipoprotein. Biochim Biophys Acta. 1980;620:631-635.[Medline] [Order article via Infotrieve]
    
38. Pitas RE, Boyles J, Mahley RW, Montgomery Bissel D. Uptake of chemically modified low density lipoproteins in vivo is mediated by specific endothelial cells. J Cell Biol. 1985;100:103-117.[Abstract/Free Full Text]
    
39. Pittman RC, Carew TE, Attie AD, Witztum JL, Watanabe Y, Steinberg D. Receptor-dependent and receptor-independent degradation of low density lipoprotein in normal rabbits and in receptor-deficient mutant rabbits. J Biol Chem. 1982;257:7994-8000.[Abstract/Free Full Text]
    

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