Oxidation of Plasma Low-Density Lipoprotein Accelerates Its Accumulation and Degradation in the Arterial Wall In Vivo
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.
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.
The experimental protocols were approved by the Danish government body supervising animal experiments.
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 ε-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 17×109 cpm 125I/mg of LDL protein and 0.4 to 3.9×109 cpm 131I/mg of LDL protein.
TC (50 nmol) was labeled with 370 MBq of 131I using Iodogen (Pierce Chemical Co).22 131I-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 4.7×108 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 ox2h-LDL, ox4h-LDL, and ox8h-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 ≥2.7×108 g×min 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 2.6×100 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.
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±0.1×108 cpm 125I/kg body wt and 0.6±0.06×108 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 ox8h-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 ox2h-LDL (n=3) or ox4h-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 ox8h-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 γ counter (LKB Compugamma 1282, Wallac); SE values of total count were usually <1%.
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), cend is the plasma radioactivity concentration at the end of the experiment (cpm/nL), and cavg 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 described24 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 model24 25 was an average of 78% for N-LDL but >100% for ox8h-LDL. These data suggest that the two-compartment model originally proposed25 is not appropriate for ox8h-LDL or, possibly, that a large fraction of ox8h-LDL had been removed from plasma during the 5 to 10 minutes between injection of ox8h-LDL and the first blood sampling. Because ≈98% of labeled ox8h-LDL was observed to have disappeared from plasma after 24 hours, it was therefore estimated that ≈90% of the total amount of ox8h-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 ox8h-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 ox8h-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).
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.
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).
Gel filtration chromatography profiles of ox8h-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.
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 ox8h-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).
The distribution of radioactivity between the various density fractions in plasma remained different for N-LDL and ox8h-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 ox8h-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 ox8h-LDL and N-LDL did not reach statistical significance in the aortic arch (Fig 4⇓). However, accumulation of undegraded ox8h-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 ox8h-LDL than undegraded N-LDL had accumulated in the intima/inner media (Table 1⇓). The difference between undegraded N-LDL and ox8h-LDL after 1 and 3 hours was most pronounced in the thoracic and abdominal aortas.
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 ox2h-LDL, N-LDL and ox4h-LDL, or N-LDL and ox8h-LDL 3 hours before the aorta was removed.
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 ox8h-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 ox8h-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
In aortic outer media, liver, spleen, kidney, heart, lung, and adipose tissue, the degradation rate of ox8h-LDL was also greater than that of N-LDL. In contrast, there was no difference between degradation rates of ox8h-LDL and N-LDL in the adrenals or in the small intestine (Table 2⇑).
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 ox8h-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 ox8h-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 ox8h-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
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.
*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.
- Copyright © 1996 by American Heart Association
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