© 1997 American Heart Association, Inc.
Articles |
Residence Time of Low-Density Lipoprotein in the Normal and Atherosclerotic Rabbit Aorta
the Specialized Center of Research on Arteriosclerosis, Department of Medicine, University of California, San Diego, La Jolla, Calif.
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Previous results from this laboratory found that the arterial low-density lipoprotein (LDL) residence time in lesion-prone aortic sites was longer in hyperlipidemic rabbits before lesion formation than in the corresponding sites in normolipidemic rabbits. The calculation of residence time in the previous study assumed that the arterial wall was homogeneous; the present study reexamines the issue using a method that does not require such an assumption. The concentration of radiolabeled arterial LDL was measured in New Zealand White rabbits killed at several different times (0.5 to 72 hours) after injection of labeled LDL. Using a stochastic analysis, arterial LDL residence time was calculated from the pooled labeled arterial LDL measurements from these rabbits. In these studies, the arterial LDL residence times in normolipidemic and hyperlipidemic rabbits before lesion formation were similar in both the lesion-prone and -resistant sites. However, immediately upon development of early fatty streak lesions, the arterial LDL residence time increased dramatically. After only 16 days of cholesterol feeding, the residence time was 10 times longer in the lesioned aortic arch compared with similar tissue from normolipidemic rabbits (4 to 45 hours). After 21 days of cholesterol feeding, the residence time of LDL in the lesioned aortic arch increased to >25-fold that of normolipidemic tissue. Similar results were observed in the lesioned tissue of the abdominal branchings. This early retention of LDL suggests that significant changes are taking place within the arterial wall during this critical stage of early lesion development.
Key Words: atherosclerosis • residence time • low-density lipoprotein
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Introduction |
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Evidence from animal models and humans suggest that the fatty streak lesion is a precursor of more advanced lesions and fibrous plaques found in clinically significant atherosclerosis.1 2 3 4 5 The fatty streak is characterized by an accumulation of cholesterol ester within foam cells, mostly derived from monocytes/macrophages. The cholesterol found within these cells is derived from plasma lipoproteins, including LDL.6 7 8 Although the link between elevated plasma LDL levels and accelerated atherosclerosis is well established,9 10 11 12 13 the mechanisms by which these increased plasma LDL levels contribute to the atherosclerotic process is uncertain.
An increase in intact undegraded LDL is observed in early atherosclerotic lesions.14 15 16 The intact arterial LDL concentration is determined by the LDL influx into the arterial wall and the LDL efflux from the artery (either by leaving the arterial wall or by degradation within the wall). To what extent the increased LDL concentration in early lesions reflects an increased influx of LDL into the artery or a decreased efflux of LDL from the artery is not clear. Several reports have studied the relative importance of the influx and efflux of LDL in its accumulation in normal and atherosclerotic arteries.17 18 19 20 21 22 23 These studies, performed in several different species and under differing experimental conditions, have found no consensus as to which process contributes most to the increased LDL content in atherosclerotic lesions. In a relevant study from this laboratory, Schwenke and Carew23 reported a greater retention of arterial LDL (ie, a slower efflux) in arterial sites susceptible to atherosclerosis compared with other sites relatively resistant to lesion formation. These studies were done in hypercholesterolemic rabbits before the development of visible fatty streaks. Importantly, this retention of LDL occurred specifically in arterial sites prone to atherosclerosis, suggesting a possible mechanism for the initiation of lesion formation. For example, as LDL remains in the arterial wall, it would presumably be separated from the antioxidants within the blood. Without these protective antioxidants, one could propose that a longer retention of LDL in the artery might increase the chances that oxidative modification could occur to the point of eliciting proatherogenic biological responses.24 25
The calculation of arterial LDL residence time by Schwenke and Carew23 was based on a kinetic model that assumed that the LDL in the arterial wall was present in a single homogeneous compartment exchanging with plasma LDL. However, it is possible that a retention of LDL in specific sites of the arterial wall may result from a "trapping" of LDL (eg, by proteoglycans), thereby generating a second kinetically distinct arterial compartment of LDL. In this scenario, a model of a single homogeneous arterial compartment might be inappropriate, as was mentioned by the authors themselves. Since Schwenke and Carew's findings have important implications about the role of LDL in early lesion formation, we reexamined this issue using a model that does not necessarily assume homogeneity of the LDL pool in the arterial wall. In addition, we extended their studies by measuring LDL residence time in aortic sites immediately after the earliest macroscopically visible fatty streak was formed.
In the present study, the concentration of radiolabeled arterial LDL was determined in a series of animals killed at different times after injection of labeled LDL. By pooling the data from these animals, we derived a curve defining the concentration of labeled arterial LDL over time. With these data, we calculated the arterial residence time using a stochastic method that did not require any assumptions about the number of compartments within the arterial wall.
In contrast to the work of Schwenke and Carew,23 the present study failed to find any prolongation of arterial LDL residence time in the lesion-prone sites of cholesterol-fed rabbits before the appearance of identifiable lesions. However, upon formation of even the earliest fatty streak, a dramatic increase in LDL residence time was observed.
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Materials and Methods |
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LDL Preparation and Labeling
LDL was prepared from the plasma of normal New Zealand White rabbits as described previously.26 Briefly, rabbits were exsanguinated, and whole blood was collected into tubes containing disodium EDTA (4 mmol/L). Plasma was separated from whole blood in a Beckman centrifuge at 2000g for 10 to 15 minutes. LDL was isolated using sequential ultracentrifugation, collecting the density fraction of 1.020 to 1.057 g/mL as described previously.26 The final LDL preparation was dialyzed exhaustively against a buffer containing 0.15 mol/L NaCl, 2 mmol/L EDTA, and 20 mmol/L phosphate, pH 7.4, for 24 to 48 hours (buffer A). LDL protein concentration was assayed by the Lowry method using bovine serum albumin as a standard.27
Each of seven LDL preparations was divided in half; one portion was labeled with 125I and the other with 131I, using 1,3,4,6-tetrachloro-3
,6-diphenylglycouril (Iodogen, Pierce Chemical Co) as previously described.28 The labeled LDL was dialyzed against buffer A for 48 hours to remove unbound radioiodide. The specific activity of these preparations ranged from 84 to 292 cpm/ng (n=7) for 125I-LDL and 131 to 215 cpm/ng (n=7) for 131I-LDL. Approximately 10 to 20 mg of bovine serum albumin was added to these preparations after dialysis to limit potential radiolytic and oxidative damage to the LDL. Two of the seven preparations were characterized by SDS-PAGE. More than 95% of both the 125I and 131I radioactivities was associated with proteins that migrated similarly to apoprotein B, with <5% associated with proteins of an apparent molecular size similar to that of apoprotein E, as has been reported previously in this laboratory using similar protocols.26 Although not done for these preparations, similar radiolabeling protocols in this laboratory indicate that 
5% of the label bound to LDL would be extractable in 2:1 chloroform/methanol.26 29 The LDL preparations were sterilely filtered through a 0.45-µm filter (precoated with a 10 mg/mL albumin solution) and injected into the rabbits 2 to 4 days after labeling and 1 week after initial exsanguination of the donor rabbits. Each rabbit received 9.6±3.9x107 cpm/kg (mean±SD, n=60) of 125I-LDL and 6.7±1.5x107 cpm/kg of 131I-LDL (n=60). The total amount of tracer LDL injected into each rabbit was 0.30±0.08 mg/kg (mean±SD, n=84) of 125I-LDL and 0.35±0.07 mg/kg of 131I-LDL (n=84).
Animal Studies
Eighty-four female New Zealand White rabbits (2.53±0.32 kg, mean±SD) were used in seven studies of 12 animals each. Six of these studies were conducted with all 12 rabbits fed the same diet, either normal chow or 2% cholesterol–supplemented chow. In the seventh study, the 12 rabbits were divided in half, with 6 rabbits fed normal chow and 6 rabbits fed cholesterol-supplemented chow. The supplemented chow was made by dissolving cholesterol in ether, mixing the solution into normal rabbit chow and allowing the ether to evaporate overnight in a fume hood.
In order to study rabbits both before development of early fatty streaks and after initial fatty streak formation, cholesterol-fed rabbits were divided into three groups based on the duration of cholesterol feeding: 13 to 14 (13/14) days, 15 to 16 (15/16) days, and 21 days of cholesterol feeding. Rabbits were killed at six different time points after the injection of labeled LDL: 0.5, 2, 5, 24, 48, and 72 hours. Because the two shorter cholesterol feeding experiments coincided with the appearance of early foam cells, it was felt that changes in LDL metabolism could be occurring during the time frame of the experiment. Thus, an extra day was added to these experiments in order to average the differences between the duration of cholesterol feeding at the time of injection and at the time of death for the animals injected for long times. For example, rabbits injected 72 and 48 hours before death were injected on days 11 and 12, respectively, and killed on day 14 of the 13/14-day cholesterol experiments. Rabbits injected 24 hours before death were injected on day 12 and killed on day 13. A similar schedule was used for the 15/16-day experiments. After 21 days, the fatty streaks had probably been present for at least 6 days (Fig 1
); thus, the extra day was deemed unnecessary. All animal procedures were in accordance with institutional guidelines.
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To reduce the effect of biological variability and increase the number of data points per time interval, each rabbit was injected intravenously with both 125I-LDL and 131I-LDL at two separate times. In this way, each rabbit provided two independent measurements of the radiolabeled arterial LDL concentration. The 12 rabbits in each experiment were divided into three subgroups, with each subgroup receiving one of the following pairings of injections: 0.5 and 2 hours, 5 and 24 hours, or 48 and 72 hours before death. These pairings allowed for the shorter-term animals (0.5 to 24 hours) to be killed on the desired day of cholesterol feeding (day 13 or 15). The 48- and 72-hour pairing allowed for the averaging of the difference between the duration of cholesterol feeding at the time of injection and the time of death, as described in the preceding paragraph.
Rabbits received 3 mg of NaI intravenously before the injection of radiolabeled LDL to reduce uptake of radioiodide by the thyroid. The disappearance of radiolabeled LDL from the plasma was monitored by taking serial blood samples from each rabbit starting 6 minutes after injection and continuing at increasingly longer intervals (eg, 12, 25, 120, 240 minutes) until the time of death. Immediately before death by sodium pentobarbital (60 mg/kg) administration, the rabbits were anticoagulated with a heparin injection (1000 IU). The systemic circulation was perfused with 1 L of saline in situ at physiological pressure. Immediately thereafter, the animal was perfused with a modified Karnovsky fixative for 10 to 15 minutes.23 Aortas were removed from the aortic valve to the iliac bifurcation. The aorta was cleaned of its loose adventitial tissue and stored in fixative for 24 hours and saline thereafter.
Tissue Sampling
Each aorta was divided into lesion-susceptible sites (ie, aortic arch and major branching points of the abdominal aorta) and lesion-resistant sites (ie, descending thoracic aorta and the abdominal aorta without its major branch points) as described previously.26 The aortic arch was separated from the descending thoracic aorta at the level of the ductus arteriosus. The four major branching points of the abdominal aorta were removed from the rest of the abdominal aorta in triangular sections by cutting 1 mm beneath the flow divider in a "V" shape and then across the top 1 mm above the end of the flow divider and through the lumen of the branching artery. For the rabbits fed cholesterol, the aorta was stained with Sudan IV to visualize lesions.30 With the exception of the abdominal branching points, these arterial samples were further subdivided into either exclusively lesioned or nonlesioned tissue and analyzed independently. Care was taken to avoid "contamination" of nonlesioned tissue in the lesioned tissue samples and vice versa. Because the sites of abdominal branching were quite small, in most cases, the lesioned branches were not subdivided and were thus reported as "mixed" tissue (containing both lesioned and nonlesioned tissue). The exception was in the 21-day cholesterol feeding experiment, where the amount of lesioned tissue in the branching sites of the abdominal aorta was very large. In this experiment, the tissue was further subdivided into solely lesioned tissue; however, the residence time was calculated for both the whole mixed tissue and the lesioned-only tissue.
Artery samples were gently blotted dry on tissue paper and weighed (reported as wet weight tissue).
Radioassay
Plasma and arterial samples were counted in an LKB gamma counter using a double-channel program to correct for overlap in the activity spectra of 125I and 131I. Each plasma sample was typically counted for 2 to 5 minutes. The counting error for the labeled LDL in the plasma was typically <1% for both 125I and 131I samples, with maximal counting errors of 3.6% and 5.6% for 125I and 131I, respectively, in normal rabbits and 1.5% for both 125I and 131I in rabbits fed a cholesterol diet. Because of the much lower level of radioactivity found in the arterial wall, arterial samples were counted for 20 to 30 minutes. The counting error for arterial samples was typically 1% to 2% for both 125I and 131I, with maximal counting errors of 9% for 125I and <8% for 131I in small arterial samples.
Arterial Concentration of Intact LDL
It has been previously demonstrated that fixation with modified Karnovsky's fixative results in retention of protein-bound 125I and 131I in the arterial wall and effectively leaches out non–protein-bound 125I and 131I degradation products such as iodotyrosine and free-labeled iodide.29 31 Thus, the arterial radioactivity is a measure of the radiolabeled concentration of intact LDL in the arterial wall. It is calculated from the radioactivity in the artery (cpm/g) normalized to the radioactivity in the plasma at time zero (cpm/µL). The radioactivity at time zero was calculated by extrapolation of a monoexponential equation fit to data from plasma samples collected 6, 12, and 25 minutes after injection of LDL. When the radiolabeled LDL has equilibrated between the artery and plasma, the radiolabeled arterial LDL can be used to estimate the mass of intact arterial LDL.
Permeability of the Artery to LDL
It has been shown that during the first hour after injection, the accumulation of LDL into the arterial wall is largely linear and dominated by the influx process.32 Thus, the animals killed 30 minutes after injection allowed a measure of the entry rate of LDL into the arterial wall. This entry rate of LDL, ie, the permeability coefficient of the arterial wall to LDL, was calculated from the arterial radioactivity (cpm/g) divided by the area under the plasma radioactivity curve (cpm·h/µL). The area under this curve was calculated by integrating a monoexponential equation fit to data measuring the removal of radiolabeled LDL from plasma beginning 6 minutes after injection until the animal's death at 30 minutes. Expressed in this way, the permeability coefficient (µL/g·h) is a clearance rate, measuring the equivalent volume of plasma "cleared" of its LDL by the artery per unit time. This permeability coefficient, although not the classical permeability parameter with units of length/time, allows comparison of the entry of LDL into aortic sites of different animals in a manner independent of plasma LDL levels. This index of permeability has been used similarly by several authors to describe the initial flux or entry of LDL into the arterial wall.19 20 23 33 34 35 The total LDL influx was estimated by multiplying the permeability coefficient by the plasma LDL cholesterol concentration.
Mean Residence Time of Arterial LDL
By use of a stochastic analysis in which the internal compartments of a system are undefined, the mean residence time of a tracer in a system can be determined by the following general relationship (taken from Shipley and Clark,36 page 118, Equation 13):
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The inverse of the integral of xp is the whole-body FCR and can be represented by the following equation:
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Cholesterol Determinations
Plasma cholesterol and triglyceride content were determined in each rabbit before cholesterol feeding and at several intervals (approximately every 4 to 5 days) during feeding up until immediately before injection of radiolabeled LDL. Total plasma cholesterol concentrations (mg/dL) were assayed using an enzymatic technique (high-performance cholesterol reagent, No. 236691, Boehringer Mannheim Diagnostics).26 The concentration of LDL cholesterol was estimated from data in Table 1 of Reference 26. This table gives the percentages of LDL cholesterol as a fraction of total plasma cholesterol at various levels of plasma cholesterol concentrations. The plasma LDL cholesterol levels were estimated by multiplying the total plasma concentration by the appropriate fraction that the LDL cholesterol represented for the level of total plasma cholesterol.
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Plasma Cholesterol Concentrations
The rabbits fed normal chow had an average plasma cholesterol of 74.4±28.2 mg/dL (mean±SD, n=30). Rabbits fed a hypercholesterolemic diet for 13 to 16 days had an average cholesterol level of 1111±379 mg/dL (n=42), an
15-fold increase over normal levels. After 21 days of cholesterol feeding, the average plasma cholesterol level was 1715±361 mg/dL (n=12), 23-fold that of normal-fed rabbits. Within each experiment, the plasma cholesterol values did not differ significantly between any of the injection subgroupings (ie, 0.5/2, 5/24, or 48/72 hours; P<.31 to .97). The whole-body FCR was 0.099±0.022 pools per hour (mean±SD, n=10) in normal rabbits and 0.022±0.004 pools per hour (n=18) in rabbits fed cholesterol for 2 to 3 weeks. The slower FCR in cholesterol-fed rabbits is consistent with the downregulation of LDL receptors due to hypercholesterolemia.38 There was no significant difference in the FCRs between the 125I-LDL and 131I-LDL in either the normal rabbits (P<.68) or the cholesterol-fed rabbits (P<.27). Plasma volumes were calculated by isotope dilution for five of the seven experiments using both the I125- and I131-LDL preparations. There was good agreement between the calculated volumes within each rabbit for the two isotopes. In two of the normal-fed rabbit experiments, the plasma volumes were 80.4±8.93 mL/kg (mean±SD, n=12) and 108±10.78 mL/kg (n=6). In the cholesterol-fed group, the plasma volumes were 26.5±3.1 mL/kg (15/16 day, n=12), 42.34±4.76 mL/kg (13/14 day, n=12), 75.7±16.62 mL/kg (21 day, n=12), and 110±9.5 mL/kg (15/16 day, n=6).
Lesion Development in Lesion-Prone Sites of the Aorta
Very small grossly visible lesions developed in the branch points of the abdominal aorta of rabbits fed cholesterol for 13/14 days. In one of the 13/14-day cholesterol experiments, all 12 of the rabbits exhibited these very early fatty streaks (<27%, by weight, of the branch points contained lesions). In the other 13/14-day experiment, 4 of the 12 rabbits exhibited minuscule lesions, which were in such small proportion of the total tissue that they were removed and the sites were termed "prelesional." After 15/16 days of cholesterol feeding, lesions were also present in the aortic arch (37%, by weight, of tissue ) and throughout the sites of abdominal branching (percent lesioned tissue not determined). Upon histological examination, these lesions appeared to be in very early stages of foam cell formation. The lesion-prone sites had adhering monocytes and macrophages/foam cells one to two layers deep (Fig 1A
). After 21 days of feeding, the fatty streak lesions were further advanced, although still consistent with early stages of lesion development. (Fig 1B). Approximately 53% of the aortic arch and 80% of the branch points of the abdominal aorta, by weight, were lesioned. The intima was significantly thickened, with foam cells several layers thick. Some extracellular lipid accumulation could also be observed.
Arterial Permeability to LDL
The arterial permeability to LDL did not increase after short-term cholesterol feeding before lesion formation (Table 1). In fact, compared with rabbits on a normal diet, the arterial permeability was lower in the aortic arch (by 47% to 71%, P<.002) and the nonbranch region of the abdominal aorta (by 41% to 60%, P<.0001) of rabbits after all days of cholesterol feeding. In the descending thoracic aorta, the permeability was lower (by 32%, P<.003) after 15/16 and 21 days of cholesterol feeding. Multiple comparisons between different experimental conditions at each aortic site were controlled by using a Bonferroni correction factor (significance at P<.015).
The total LDL influx into the artery is determined by the product of the permeability coefficient and the plasma LDL concentration. The plasma LDL concentrations were estimated from the total plasma cholesterol concentration, as described in "Materials and Methods." The plasma LDL concentration increased 7.5- and 11-fold (P<.0001) after 15 and 21 days, respectively. Thus, although the arterial permeability to LDL did not increase after short-term cholesterol feeding before lesion formation, the total LDL influx into the artery increased at all aortic sites. This increased delivery of LDL to the aorta was entirely due to the hypercholesterolemia, not to an increase in permeability.
In the aortic arch, the permeability in the lesioned tissue was significantly greater (P<.05) than that in adjacent nonlesioned tissues by 1.5- to 2-fold. However, this greater permeability in the lesioned tissue was not significantly different than that found in the aortic arch of rabbits fed a normal diet (P<.79 and P<.33 after 15/16 days and 21 days, respectively, on a cholesterol diet). Permeabilities determined for the lesioned branch points of the abdominal aorta were a combination of lesioned and nonlesioned tissue and reported as "mixed" tissue (see "Materials and Methods"). The arterial permeability to LDL was not different between the nonlesioned and mixed tissue for any day of cholesterol feeding (Table 1).
Mean Residence Time of LDL in Aorta
The mean residence times were similar in normal-fed and cholesterol-fed rabbits before lesion formation at all aortic sites (Table 2). Fig 2 shows representative plasma and arterial radiolabeled LDL concentration curves from a normal rabbit experiment and from a cholesterol-fed rabbit experiment before lesion formation. Note that the terminal decay of radiolabeled LDL from both the plasma and the artery was considerably slower in the hypercholesterolemic rabbits. However, despite the apparent differences between these two graphs, the calculated residence times of LDL in nonlesioned areas of the abdominal aorta were not different (8.86 versus 8.96 hours). The slower terminal decay of radiolabeled LDL from the artery in the hypercholesterolemic rabbits appeared to be entirely accounted for by the slower removal rate of LDL from the plasma.
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With the appearance of small Sudan-stainable lesions, the arterial LDL residence time increased markedly (Table 3). In fact, with increasing duration of cholesterol feeding, the residence time of LDL was progressively prolonged. In the branch points of the abdominal aorta after 13001/days of cholesterol feeding, with less than 27% (by weight) of the tissue lesioned, the arterial LDL residence time was two times longer than the residence time in corresponding sites of cholesterol-fed rabbits before lesion formation (18 versus 9.28 hours, respectively). After 15/16 days of cholesterol feeding, the LDL residence time in the abdominal branching points was longer than that in nonlesioned tissue by 5-fold (45 hours). After 21 days of feeding, the LDL residence time was >10 times that in cholesterol-fed rabbits before fatty streak formation (99 hours).
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In the aortic arch, where the lesioned tissue did not contain significant nonlesioned portions, the results were even more dramatic. After 15/16 days of cholesterol, the arterial LDL residence time was 45 hours, 10 times that of cholesterol-fed rabbits before lesion development. After 21 days, the arterial LDL residence time was 119 hours, an increase of >25-fold. Fig 3 shows the greater radiolabeled LDL concentration in the arterial wall and slower terminal decay of arterial LDL found in the lesioned tissue compared with adjacent nonlesioned tissue.
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Discussion |
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The present study introduces a stochastic method for measuring the average residence time of arterial LDL that does not depend on the number of LDL compartments (pools) within the arterial wall. Although several investigators have used similar experimental procedures, analysis of their data either was based on a kinetic model with a single arterial compartment or did not include a determination of the arterial LDL residence time.20 39 40 By use of this stochastic method, the major findings are that the arterial residence time of LDL is similar in the nonlesioned atherosclerosis-prone sites of hypercholesterolemic and normolipidemic rabbits. However, immediately on detection of the earliest fatty streak, the arterial LDL residence time increases markedly. This increase varies directly with the duration of cholesterol feeding.
It is necessary to point out that the present method is not without some assumptions. The calculation of arterial residence time from a composite arterial LDL concentration curve assumes that it is possible to pool arterial measurements from different rabbits. As the calculated residence times were remarkably consistent between experiments, it appears that this is a reasonable assumption. This calculation also assumes that one can adequately extrapolate the arterial LDL concentration to infinity using the slope derived from the last three time points. In the normal rabbits and the cholesterol-fed rabbits before lesion formation, the terminal slope was well defined, and the area under the extrapolated region represented a small fraction of the total LDL concentration. These extrapolated areas were typically between 20% to 40% of the total calculated areas (occasionally higher), with the higher percentages usually from the cholesterol-fed rabbits. Thus, errors due to the extrapolation should be small. After lesion formation, however, the terminal decay of LDL from the artery was much slower, and the extrapolated area contributed a larger fraction to the total labeled LDL concentration. The extrapolated area represented 40% to 60% of the total LDL concentration in the 13/14- and 15/16-day cholesterol feeding experiments and 87% (aortic arch) and 90% (abdominal branching points) in the 21-day experiment. Thus, some degree of error (especially in the 21-day experiment) was involved in the extrapolation of the arterial LDL curve to infinity in the lesioned tissue. However, it should be remembered that although the values for the LDL residence time in the 21-day experiment may not be exact, the observed kinetics indicate that the residence time of LDL in these lesions is much longer than that observed in either the earlier lesions or in the nonlesioned tissue. Although a balance must be made between the level of desirable radioactivity for such experiments and the duration of the study, it would be preferable for future studies measuring LDL residence time in lesioned tissue to be extended to include later time points. Nonetheless, multiple experiments (ie, 15/16 days of cholesterol feeding) provided very similar results.
As with all tracer experiments, the present method assumes that the injected LDL is an adequate tracer of endogenous LDL. As LDL isolated from normolipidemic rabbits was used in both the normal and hypercholesterolemic rabbits, one might question whether this LDL appropriately traces the endogenous LDL in hypercholesterolemic rabbits. Changes in lipid and apoprotein composition that occur upon cholesterol feeding might produce important alterations in cellular uptake and degradation. However, studies done in this laboratory have shown that LDL isolated from normal and hypercholesterolemic rabbits is catabolized at similar rates when injected into hypercholesterolemic rabbits.26 Still, one cannot rule out the possibility that differences in the metabolism of normal and hypercholesterolemic LDL exist at the tissue level. Since the focus of this present work was on investigating potential alterations that occur within the arterial wall, we chose to control the potential variation in particle behavior by using LDL derived from normal animals for both diet conditions.
In cholesterol-fed rabbits with plasma cholesterol levels >1000 mg/dL (after 2 to 3 weeks of 2% cholesterol), the proportion of cholesterol that is in the form of LDL is fairly small, 15%.26 The majority of the cholesterol in these rabbits is found within ßVLDL and, to a lesser extent, IDL. Thus, it is necessary to consider that ßVLDL and IDL might also play a significant role in fatty streak formation in these rabbits.41 42 43 However, LDL was chosen for these studies to allow comparison with human atherosclerosis, in which LDL potentially plays a more dominant role. In addition, it is technically much more difficult to perform tracer studies on VLDL because of its rapid rate of metabolism into other lipoproteins.
The FCR can be used as an indirect index of the condition of the injected LDL preparation. For example, if the LDL were significantly oxidized during handling, one might see a large increase in the rate at which the LDL leaves the plasma. In these studies, the FCR for the cholesterol-fed rabbits was similar to previously published results from this laboratory26 and slightly faster than previous results (eg, 0.066 h-1 and 0.078 h-1)26 29 in the normal-fed rabbits. It is unlikely that the slightly faster FCR in normal rabbits is due to significant oxidation of LDL, because one would expect the FCR of oxidized LDL to be extremely rapid.44 45 46 47 It is difficult to know exactly why the present FCR is faster, but it could be due to biological variation between the rabbits used in the studies, differences in the isolated LDL preparation itself, or differences in the radiolabeled preparation. It is important to point out, however, that these rates are similar to or slower than other published FCRs for LDL in normal-fed rabbits.48 49 50
Alternatively, it is possible that if only a portion of the LDL preparation was unphysiologically modified, it might not be detectable from the FCR measurement. In this case, a measure of the plasma volume by isotope dilution could indicate whether a portion of the LDL preparation was rapidly removed from the plasma. Two of the calculated plasma volumes were similar to those recorded in the literature22 26 : 26.5±3.1 and 42.34±4.76 mL/kg. The remaining estimated plasma volumes were 2- to 3-fold higher than the reported values. These larger plasma volumes suggest that a portion of the LDL preparation was potentially modified and rapidly removed from the plasma. However, the modified LDL was probably removed so quickly from the plasma (most likely by the liver)44 that it would not be a factor in the residence time calculations. Furthermore, the normalization to the experimentally determined apparent dose in both the numerator and the denominator of the residence time equation would cancel out any miscalculation. There is a possibility that arterial foam cells could also be rapidly taking up the LDL from the plasma and, in this way, could affect the residence time calculations. This is unlikely because the residence times in the two 15/16-day cholesterol experiments were similar despite two differing plasma volumes: 26.5 and 110.4 mL/kg. In addition, large plasma volumes (80.4 and 108.3 mL/kg) occurred in the normal-fed rabbits, where there presumably were no arterial foam cells.
One of the LDL preparations characterized by SDS-PAGE did not show a significant breakdown of apoprotein B but did have a larger than normal plasma volume, 80 mL/kg. In this case, it is difficult to determine the cause of the larger calculated plasma volume. It is possible that the LDL has been modified in a manner not discernible under the experimental conditions used during SDS-PAGE.
The present finding of similar arterial LDL residence times in normal-fed and cholesterol-fed rabbits is in agreement with a study in pigeons that also found no change in LDL efflux (the inverse of the residence time) in nonlesioned arterial sites of normocholesterolemic and hypercholesterolemic animals.18 However, this conclusion is in contrast to that of Schwenke and Carew,23 who found that the LDL residence time was prolonged 2- to 3-fold in lesion-prone sites of cholesterol-fed rabbits before the appearance of fatty streaks. Schwenke and Carew estimated arterial LDL residence times using a model that assumed that the arterial LDL was a single homogeneous pool that exchanged with plasma LDL. To understand the disparity between these two studies, we used our more comprehensive data set in an attempt to evaluate the validity of this model in defining the characteristics of LDL metabolism in the arterial wall. Using their model, the parameters of influx (ie, the permeability coefficient) and efflux (ie, the inverse of the residence time) were constrained to the experimental values determined in this study. Using the computer program SAAM II,51 model simulations of the arterial LDL concentration were compared with the actual arterial LDL observations. In normal rabbits, these simulations were found to exhibit faster terminal decays of LDL from the artery than those observed experimentally (Fig 4A). This discrepancy suggests the possible existence of an additional compartment within the arterial wall, as was discussed in the previous work.23 For example, this compartment could be a pool of LDL bound to the extracellular matrix that exchanges with unbound LDL at a rate that is slow compared with that of the terminal decay from plasma.
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In contrast to the results in normal rabbits, the model adequately predicted the kinetics of the arterial LDL concentration in cholesterol-fed rabbits before lesion formation (Fig 4B). It is possible that the exchange rate of the bound LDL in the additional arterial compartment postulated in normal rabbits is similar to the LDL plasma decay rate in cholesterol-fed rabbits. The slower decay of LDL from the plasma in cholesterol-fed animals could mask the presence of the exchange pool or additional compartment, and the model would thus appear to provide a good fit of the data.
However, it is also possible (given a lower permeability coefficient and a lower efflux than experimentally observed) that the model could predict the arterial LDL concentration very well. Thus, in another test of the validity of the Schwenke and Carew model,23 all of the model parameters were estimated, and a best fit was determined for the observed measurements. Similar to the test with the constrained model parameters, the solution did not fit the experimental data in normal rabbits but performed well in the cholesterol-fed rabbits before lesion formation. In normal rabbits, the model again predicted a faster terminal decay of LDL from the artery than what was observed experimentally. Additionally, the arterial LDL residence times predicted from the model were shorter in normal rabbits than those determined using the present stochastic method. These results further support the conclusion that in normal rabbits, a model of the arterial wall as a single homogeneous compartment does not adequately describe the arterial system. It suggests that in a kinetic analysis, the arterial wall might be better described with two or more compartments. One could postulate that the prolonged LDL residence time observed in the previous study23 was due to an underestimation of the residence time in normal rabbits.
However, one cannot conclusively say that the residence time of LDL does not increase before fatty streak formation. It is important to remember that changes in arterial LDL residence time could be a very localized phenomenon. The work done by Stemerman et al52 suggests that very focal differences in transport properties exist within the arterial wall. They found increases in LDL permeability of up to 47-fold in localized spots (<1 millimeter in diameter) that had been stained with horseradish peroxidase when compared with areas unstained by horseradish peroxidase.52 It is possible that similarly localized changes in LDL residence time occurred in our studies but were not observed because of the analysis of much larger tissue sections.
It should also be noted that the development of atherosclerosis is not a sudden process but one of gradual formation. Since our method detects lesions only as they become visible through Sudan IV staining, it is possible that the sensitivity of this method does not allow a measure of small or localized changes in either lesion formation or LDL residence time.
Nonetheless, our results did show that the arterial LDL residence time increased dramatically with the presence of even the smallest Sudan-stainable lesion. The increase in arterial retention of LDL over a short period of cholesterol feeding varied directly with the duration of cholesterol feeding. These data suggest that the character of the lesion and/or the arterial wall changes rapidly in the rabbit, within 2 to 3 weeks of cholesterol feeding. This increased residence time in lesioned tissue is in agreement with previous preliminary results from this laboratory, which showed an increase in LDL residence time (up to 14-fold) in lesioned tissue after 9 weeks of cholesterol feeding.53
After 21 days of cholesterol feeding, the abdominal branch was extensively lesioned, and the remaining small nonlesioned areas were separated from the rest of the tissue. For comparison with other abdominal branch sites, the entire tissue was analyzed. However, it was interesting to note that with the nonlesioned areas removed, the LDL residence time in the lesioned tissue was 128 hours, an increase from 99 hours in the mixed tissue. Thus, the nonlesioned tissue appeared to dilute the longer residence time found in the lesioned tissue. This result provides additional evidence that an increase in LDL residence time generally occurs in the areas with lesions but not in those without lesions.
LDL is possibly retained in the lesioned tissue by one or more of the following methods: (1) formation of lipoprotein aggregates,54 55 (2) sequestration of LDL within the macrophage,56 57 and (3) binding of LDL to proteoglycans in the extracellular matrix.58 59 60
Regarding the formation of lipoprotein aggregates, one could speculate that the rabbit aorta has a certain capacity to process LDL. Above a threshold value, the "excess" LDL may form aggregates that, because of their greater size, limit the efflux of LDL from the artery. Several reports have indicated that the influx of macromolecules into the arterial wall is inversely proportional to the molecule's diameter.33 61 62 Recently, it was observed that the efflux of macromolecules from the artery follows similar principles.22 Thus, these larger aggregated particles might be relatively restricted from leaving the arterial wall. In the rabbits fed cholesterol for 15/16 and 21 days, although the permeability of the artery to LDL was only 2-fold greater in the lesioned tissue, the estimated plasma LDL pool increased to 11-fold. Thus, the total influx of LDL into the lesioned artery was elevated by 22-fold, possibly reaching the aorta's "processing" capacity. This idea of a limited arterial processing capacity has some validity, because it has been shown that after 2 hours of a bolus injection of LDL, focal regions of normal rabbit aortic intimas show clustering of LDL-sized particles.54 Some of these particles appear to fuse into larger lipidlike structures. These larger structures are similar to those observed in 10-day cholesterol-fed rabbits,63 suggesting that aggregation of LDL may be occurring, in vivo, after excessive LDL uptake.
It is also possible that the retention of LDL observed in the present study results from a sequestration of LDL within the macrophage. Recent studies have shown that oxidized LDL, although it is taken up more rapidly by macrophages, is degraded within the cell at a rate significantly slower than that for native LDL.56 57 It is possible that upon initiation of lesion formation in these cholesterol-fed rabbits, the arterial LDL becomes oxidized and subsequently is taken up by macrophages. Because of its slower metabolism, some of the oxidized LDL might not be degraded and thus is retained in the artery within the time course of the experiment. This could contribute to the higher measurements of radiolabeled arterial LDL concentration and residence time.
Finally, it has long been recognized that LDL can bind to arterial proteoglycans and, more specifically, with their glycosaminoglycan side chains. Several lines of evidence suggest that interactions between LDL and arterial proteoglycans could be a mechanism of retention of LDL in the atherosclerotic lesion. Immunohistological studies have shown a very similar distribution between apoprotein B and glycosaminoglycans in atherosclerotic lesions.58 64 65 LDL-glycosaminoglycan complexes have been isolated from atherosclerotic lesions in humans and experimental animals,59 66 67 and LDL and glycosaminoglycans form soluble and insoluble complexes in vitro.60 68 In addition, increases in specific glycosaminoglycans (eg, chondroitin sulfate) have been observed in atherosclerotic lesions of experimental animals and in regenerating endothelium.69 70 Positive correlations have been observed between increasing apoprotein B content and chondroitin/dermatan sulfates with age and with early lesion formation in humans.71 72 73 Thus, as Schwenke and Carew23 previously speculated, the hypercholesterolemic stimulus could induce an alteration in proteoglycan metabolism such that the binding of LDL to the extracellular matrix is enhanced. Concurrent with the recruitment of monocytes and formation of foam cells, an increased binding of LDL to proteoglycans could augment the lesion-forming process.
All of these potential mechanisms of retention of LDL within the arterial wall could be atherogenic or are suggestive of atherogenic processes. One such process is that aggregated LDL can be recognized by macrophage receptors and internalized, thus contributing to foam cell formation.74 75 It has also been shown, in vitro, that the reversible interaction of LDL with arterial chondroitin sulfate proteoglycans increases both the susceptibility of LDL to oxidation and its uptake by human macrophages.76 77 In addition, there is some evidence to suggest that the binding of LDL to matrix components could promote LDL aggregation.63 Furthermore, the increased retention of LDL could prolong the exposure of LDL to oxidizing stimuli and thus increase the likelihood of proatherogenic modifications. The dramatic increases in residence time observed in the present study over a short period of time suggest that all these mechanisms may be occurring simultaneously.
Although the residence time of LDL did not increase with cholesterol feeding before lesion formation, the prolongation of LDL residence time was certainly concurrent with the presence of the earliest fatty streak. These findings indicate that significant changes take place within the arterial wall during this critical stage of early lesion development. Future studies are necessary to understand the mechanisms by which LDL is being retained in early lesions.
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Acknowledgments |
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This article is dedicated to the memory of Dr Thomas E. Carew. His loss is felt greatly. Dr Tozer thanks Paula Sicurello, Simone Green, and Mercedes Silvestre for their excellent technical assistance and is also greatly indebted to Dr William F. Beltz for his helpful advice and insights on kinetic modeling. This manuscript was submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences at the University of California, San Diego. Thomas Carew served as Dr Tozer's thesis advisor until his death, at which time Drs Daniel Steinberg and Shu Chien took over as advisors. Their help and advice were essential in the completion of this manuscript and many thanks are extended to them.
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Reprint requests to Eileen Collins Tozer, The Scripps Research Institute, VB2, 10550 N Torrey Pines Rd, La Jolla, CA 92037. E-mail ecollins@scripps.edu
Received September 16, 1996; accepted November 19, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Strong JP, McGill HC. The natural history of coronary atherosclerosis. Am J Pathol. 1961;40:37-49.
2. Stary HL. Evolution and progression of atherosclerosis in the coronary arteries of children and adults. In: Bates SR, Giangloff EC, eds. Atherogenesis and Aging. New York, NY: Springer-Verlag; 1987.
3.
Small DM. Progression and regression of atherosclerotic lesions: insights from lipid physical biochemistry. Arteriosclerosis. 1988;8:103-129.
4. Poole JCF, Florey HW. Changes in the endothelium of the aorta and the behaviour of macrophages in experimental atheroma of rabbits. J Pathol Bacteriol. 1958;75:245-251.[Medline] [Order article via Infotrieve]
5. Stary HC, Malinow MR. Ultrastructure of experimental coronary artery atherosclerosis in cynomolgus macaques: a comparison with lesions of other primates. Atherosclerosis. 1982;43:151-175.[Medline] [Order article via Infotrieve]
6.
Newman HA, Zilversmit DB. Quantitative aspects of cholesterol flux in rabbit atheromatous lesions. J Biol Chem. 1962;237:2078-2084.
7. Christensen S. Transfer with labeled cholesterol across the aortic intimal surface of normal and cholesterol-fed cockerels. J Atheroscler Res. 1964;4:151-160.
8. Zilversmit DB. Cholesterol flux in the atherosclerotic plaque. Ann N Y Acad Sci. 1968;149:710-724.[Medline] [Order article via Infotrieve]
9. Lipid Research Clinics Program. The Lipid Research Clinics coronary primary prevention trial results, II: the relationship of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA. 1984;257:3233-3240.
10.
Solberg LA, Strong JP. Risk factors and atherosclerotic lesions: a review of autopsy studies. Arteriosclerosis. 1983;3:187-198.
11. Tyroler HA. Lowering plasma cholesterol levels decreases risk of coronary heart disease: an overview of clinical trials. In: Steinberg D, Olefsky JM, eds. Hypercholesterolemia and Atherosclerosis. New York, NY: Churchill Livingstone; 1987:99-116.
12. Goldstein JL, Brown MS. The low density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897-930.[Medline] [Order article via Infotrieve]
13.
Steinberg D. Lipoproteins and atherosclerosis: a look back and a look ahead. Arteriosclerosis. 1983;3:283-301.
14. Hoff HF, Bond MG. Accumulation of lipoproteins containing apo B in the aorta of cholesterol-fed cynomolgus monkeys. Atherosclerosis. 1982;43:329-339.[Medline] [Order article via Infotrieve]
15. Hollander W, Paddock J, Colombo M. Lipoproteins in human atherosclerotic vessels, I: biochemical properties of arterial low density lipoproteins, very low density lipoproteins, and high density lipoproteins. Exp Mol Pathol. 1979;30:144-171.[Medline] [Order article via Infotrieve]
16. Smith EB. Transport, interactions and retention of plasma proteins in the intima: the barrier function of the internal elastic lamina. Eur Heart J. 1990;11:72-81.
17. Ghosh S, Armstrong ML, Megan MB, Cheng FH. Arterial uptake indices of low density lipoproteins after fatty streak formation in Cynomolgus monkeys. Cardiovasc Res. 1987;21:14-20.[Medline] [Order article via Infotrieve]
18.
Schwenke DC, St Clair RW. Influx, efflux, and accumulation of LDL in normal arterial areas and atherosclerotic lesions of Whilte Carneau pigeons with naturally occurring and cholesterol-aggravated aortic atherosclerosis. Arterioscler Thromb. 1993;13:1368-1381.
19. Ghosh S, Finkelstein JN, Moss DB, Schweppe JS. Evaluation of the permeability parameters (influx, efflux and volume of distribution) of arterial wall for LDL and other proteins. Adv Exp Med Biol. 1975;67:191-204.
20.
Alavi M, Moore S. Kinetics of low density lipoprotein interactions with rabbit aortic wall following balloon catheter deendothelialization. Arteriosclerosis. 1984;4:395-402.
21. Wootton R, Baskerville P, Turner P, Insell M, Shaikh M, LaVille A, Quiney J, Browse NL, Lewis B. A method for quantifying lipoprotein flux rates between plasma and arterial intima in vivo. Clin Phys Physiol Meas. 1987;8:65-74.[Medline] [Order article via Infotrieve]
22.
Nordestgaard BG, Wootton R, Lewis B. Selective retention of VLDL, IDL, and LDL in the arterial intima of genetically hyperlipidemic rabbits in vivo: molecular size as a determinant of fractional loss from the intima-inner media. Arterioscler Thromb Vasc Biol. 1995;15:534-542.
23.
Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits, II: selective retention of LDL vs. selective increases in LDL permeability in susceptible sites of arteries. Arteriosclerosis. 1989;9:908-918.
24. 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]
25. Parthasarathy S, Steinberg D, Witztum JL. The role of oxidized low-density lipoproteins in the pathogenesis of atherosclerosis. Annu Rev Med. 1992;43:219-225.[Medline] [Order article via Infotrieve]
26.
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.
27.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
28.
Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3,6-diphenylglycoluril. Biochem Biophys Res Commun. 1978;80:849-857.[Medline]
[Order article via Infotrieve]
29.
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.
30. Holman RL, McGill HC, Strong JP, Geer JC. The natural history of atherosclerosis: the early aortic lesions as seen in New Orleans in the middle of the 20th century. Am J Pathol. 1957;226:497-509.
31.
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.
32.
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.
33.
Stender S, Zilversmit DB. Transfer of plasma lipoprotein components and of plasma proteins into aortas of cholesterol-fed rabbits: molecular size as a determinant of plasma lipoprotein influx. Arteriosclerosis. 1981;1:38-49.
34.
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.
35. Bell FP, Gallus AS, Schwartz CJ. Focal and regional patterns of uptake and the transmural distribution of 131I-fibrinogen in the pig aorta in vivo. Exp Mol Pathol. 1974;20:281-292.[Medline] [Order article via Infotrieve]
36. Shipley RA, Clark RE. Tracer Methods for In Vivo Kinetics, Theory and Applications. New York, NY: Academic Press Inc; 1972.
37. Olsson DM. A sequential simplex program for solving minimization problems. J Qual Technol. 1974;6:53-57.
38.
Brown MS, Goldstein JL. Receptor-mediated endocytosis: insights from the lipoprotein receptor system. Proc Natl Acad Sci U S A. 1979;76:3330-3337.
39.
Duncan LE, Cornfield J, Buck K. Circulation of iodinated albumin through aortic and other connective tissues of the rabbit. Circ Res. 1958;6:244-255.
40. Finkelstein JN, Ghosh S, Schweppe JS. Kinetic analysis of in vivo lipoprotein flux in the normal rabbit aorta. Artery. 1976;2:161-172.
41. Nordestgaard BG, Lewis B. Intermediate density lipoprotein levels are strong predictors of the extent of aortic atherosclerosis in the St. Thomas's Hospital rabbit strain. Atherosclerosis. 1991;87:39-46.[Medline] [Order article via Infotrieve]
42.
Blankenhorn DH, Alaupovic P, Wickham E, Chin HP, Azen SP. Prediction of angiographic change in native human coronary arteries and aortocoronary bypass grafts: lipid and nonlipid factors. Circulation. 1990;81:470-476.
43.
Tatami R, Mabuchi H, Ueda K, Ueda R, Hara T, Kametani T, Ito S, Koizumi J, Ohta M, Miyamoto S, Nakayama A, Kanaya H, Oiwake H, Genda A, Takeda R. Intermediate-density lipoprotein and cholesterol-rich very low density lipoprotein in angiographically determined coronary artery disease. Circulation. 1981;64:1174-1184.
44.
Nagelkerke JF, Havekes L, van Hinsbergh VWM, van Berkel TJC. In vivo catabolism of biologically modified LDL. Arteriosclerosis. 1984;4:256-264.
45. Khouw AS, Parthasarathy S, Witztum JL. Radioiodination of low density lipoprotein initiates lipid peroxidation: protection by use of antioxidants. J Lipid Res. 1993;34:1483-1496.[Abstract]
46. Wunder A, Stehle G, Sinn H, Schrenk HH, Neufeld B, Dempfle CE, Dresel HA, Harenberg FE, Maier-Borst W, Heene DL. The injection of heparin prolongs the plasma clearance of oxidized low density lipoprotein in the rat. Thromb Res. 1995;78:139-149.[Medline] [Order article via Infotrieve]
47. Deigner HP, Friedrich E, Sinn H, Dresel HA. Scavenging of lipid peroxidation products from oxidizing LDL by albumin alters the plasma half-life of a fraction of oxidized LDL particles. Free Radic Res Commun. 1992;16:239-246.[Medline] [Order article via Infotrieve]
48. Portman OW, Illingworth DR, Alexander M. The effects of hyperlipidemia on lipoprotein metabolism in squirrel monkeys and rabbits. Biochim Biophys Acta. 1975;398:55-71.[Medline] [Order article via Infotrieve]
49.
Moerlein SM, Daugherty A, Sobel BE, Welch MJ. Metabolic imaging with gallium-68 and indium-111-labeled low density lipoprotein. J Nucl Med. 1991;32:300-307.
50.
Hay RV, Fleming RM, Ryan JW, Williams KA, Stark VJ, Lathrop KA, Harper PV. Nuclear imaging analysis of human low density lipoprotein biodistribution in rabbits and monkeys. J Nucl Med. 1991;32:1239-1245.
51. Berman M, Weiss MF. SAAM Manual. Washington, DC: US Government Printing Office; 1978. US Dept of Health, Education, and Welfare Publication NIH 78-180.
52.
Stemerman MB, Morrel EM, Burke KR, Colton CK, Smith KA, Lees RS. Local variation in arterial wall permeability to low density lipoprotein in normal rabbit aorta. Arteriosclerosis. 1986;6:64-69.
53. Schwenke DC, Carew TE. Determination of LDL degradation rate, content, and residence time in lesioned and nonlesioned aorta. Circulation. 1987;76(suppl IV):IV-313. Abstract.
54.
Nievelstein PFEM, Fogelman AM, Mottino G, Frank JS. Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein: a deep-etch and immunolocalization study of ultrarapidly frozen tissue. Arterioscler Thromb. 1991;11:1795-1805.
55. Simionescu N, Vasile E, Lupu F, Popescu G, Simionescu M. Prelesional events in atherogenesis: accumulation of extracellular cholesterol-rich liposomes in the arterial intima and cardiac valves of the hyperlipidemic rabbit. Am J Pathol. 1986;123:109-125.[Abstract]
56.
Sparrow CP, Parthasarathy S, Steinberg D. A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoproteins. J Biol Chem. 1989;264:2599-2604.
57.
Lougheed M, Zhang HF, Steinbrecher UP. Oxidized low density lipoprotein is resistant to cathepsins and accumulates within macrophages. J Biol Chem. 1991;266:14519-14525.
58. Curran RC, Crane WA. Mucopolysaccharides in the atheromatous aorta. J Pathol Bacteriol. 1962;84:405-412.[Medline] [Order article via Infotrieve]
59. Srinivasan SR, Dolan P, Radhakrishnamurthy B, Pargaonkar PS, Berenson GS. Lipoprotein-acid mucopolysaccharide complex of human atherosclerotic lesions. Biochim Biophys Acta. 1975;388:58-70.[Medline] [Order article via Infotrieve]
60.
Iverius PH. The interaction between human plasma lipoproteins and connective tissue glycosaminoglycans. J Biol Chem. 1972;247:2607-2613.
61.
Nordestgaard BG, Tybjaerg-Hansen A, Lewis B. Influx in vivo of low density, intermediate density, and very low density lipoproteins into aortic intimas of genetically hyperlipidemic rabbits: roles of plasma concentration, extent of aortic lesion, and lipoprotein particle size as determinants. Arterioscler Thromb. 1992;12:6-18.
62. Nordestgaard BG, Zilversmit DB. Large lipoproteins are excluded from the arterial wall in diabetic cholesterol-fed rabbits. J Lipid Res. 1988;29:1491-1500.[Abstract]
63. Frank JS, Fogelman AM. Ultrastructure of the intima in WHHL and cholesterol-fed rabbit aortas prepared by ultra-rapid freezing and freeze-etching. J Lipid Res. 1989;30:967-978.[Abstract]
64. Walton KW, Williamson N. Histological and immunofluorescent studies on the evolution of the human atheromatous plaque. J Atheroscler Res. 1968;8:599.[Medline] [Order article via Infotrieve]
65. Hoff HT, Jackson RL, Mao SJ, Gotto AM. Localization of low density lipoproteins in atherosclerotic lesions from human normolipemics employing a purified fluorescent labeled antibody. Biochim Biophys Acta. 1974;351:407.[Medline] [Order article via Infotrieve]
66. Hollander W. Unified concept on the role of acid mucopolysaccharides and connective tissue proteins in the accumulation of lipids, lipoproteins and calcium on the atheroslcerotic plaque. Exp Mol Pathol. 1976;25:106-120.[Medline] [Order article via Infotrieve]
67. Srinivasan SR, Radhakrishnamurthy B, Dalferes ER, Berenson GS. Collagenase-solubilized lipoprotein-glycosaminoglycan complexes of human aortic fibrous plaque lesions. Atherosclerosis. 1979;34:105-118.[Medline] [Order article via Infotrieve]
68. Camejo G, Lopez A, Lopex F, Quinones J. Interaction of low density lipoproteins with arterial proteoglycans: the role of charge and sialic acid content. Atherosclerosis. 1985;55:93-105.[Medline] [Order article via Infotrieve]
69. Hoff H, Wagner W. Plasma low density lipoprotein accumulation in aortas of hypercholesterolemic swine correlates with modifications in aortic glycosaminoglycan composition. Atherosclerosis. 1986;61:231-236.[Medline] [Order article via Infotrieve]
70. Alavi M, Moore S. Glycosaminoglycan composition and biosynthesis in the endothelium-covered neointima of de-endothelialized rabbit aorta. Exp Mol Pathol. 1985;42:389-400.[Medline] [Order article via Infotrieve]
71. Yla-Herrtuala S, Sumuvuori H, Karkola K, Mottonen M, Nikkari T. Glycosaminoglycans in normal and atherosclerotic human coronary arteries. Lab Invest. 1986;54:402-407.[Medline] [Order article via Infotrieve]
72.
Hollman J. Schmidt A, Barthold van Bassewitz D, Buddecke E. Relationship of sulfated glycosaminoglycans and cholesterol content in normal and arteriosclerotic human aorta. Arteriosclerosis. 1989;9:154-158.
73.
Yla-Herttuala S, Solakivi T, Hervonen J, Laaksonen H, Mottonen M, Pesonen E, Raekallio J, Akerblom H, Nikkari T. Glycosaminoglycans and apolipoproteins B and A1 in human aortas: chemical and immunological analysis of lesion-free aortas from children and adults. Arteriosclerosis. 1987;7:333-340.
74.
Khoo JC, Miller E, McLoughlin P, Steinberg D. Enhanced macrophage uptake of low density lipoprotein after self-aggregation. Arteriosclerosis. 1988;8:348-358.
75.
Suits AG, Heinecke JW, Chait A. Novel mechanism for macrophage uptake of low density lipoprotein after self aggregation. Proc Natl Acad Sci U S A. 1989;86:2713-2717.
76. Hurt E, Bonjers G, Camejo G. Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages. J Lipid Res. 1990;31:443-454.[Abstract]
77.
Hurt-Camejo, E, Camejo G, Rosengren B, Lopez F, Ahlstrom C, Fager G, Bondjers G. Effect of arterial proteoglycans and glycosaminoglycans on low density lipoprotein oxidation and its uptake by human macrophages and arterial smooth muscle cells. Arterioscler Thromb. 1992;12:569-583.
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