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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:930-936

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:930-936.)
© 1995 American Heart Association, Inc.

Articles

Effects of Injury on ApoB Kinetics and Concentration in Rabbit Aorta

Gun Olsson; Olov Wiklund; Göran Bondjers

From The Wallenberg Laboratory for Cardiovascular Research, Faculty of Medicine, University of Göteborg, Sweden.

Correspondence to Olov Wiklund, MD, Wallenberg Laboratory, Sahlgren's Hospital, S-413 45 Göteborg, Sweden.


*    Abstract
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 *Abstract
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Abstract Endothelial injury or dysfunction and deposition of lipoproteins and cholesterol are key events during the development of atherosclerosis. We have studied the lipoprotein kinetics in arterial tissue in relation to endothelial injury and re-endothelialization. Endothelial injury was induced in rabbits by use of a balloon catheter. With a specific immunoradiometric assay, apoB levels in arterial tissue were measured at different time points for up to 10 weeks after injury. Forty-five minutes before being killed, the rabbits were injected with 125I-LDL, and influx of LDL was calculated from the accumulation of radioactivity in the arterial tissue. The concentration of apoB in the injured arterial tissue was four times higher than that in control arterial tissue (P<.0001). Within the lesion the concentration was as high in nonendothelialized as in re-endothelialized regions. The tissue pool of apoB was divided into a loosely bound fraction and a tightly bound fraction. The increase of apoB in the injured areas was primarily due to an increase in the tightly bound fraction. The influx of apoB was severalfold higher in nonendothelialized tissue than in re-endothelialized tissue or control areas (P<.005). When retention time was calculated, this was found to dramatically increase (by seven times) the tightly bound pool of apoB in the re-endothelialized areas. In addition to the large increase of a tightly bound apoB pool in injured areas, we found a prolonged retention time of apoB in the lesions, but only in the re-endothelialized areas. This might be due to different composition of the interstitial matrix as well as different diffusion characteristics in the re-endothelialized areas. A prolonged retention time may be important for the local modification of LDL and for the generation of a more atherogenic lipoprotein.


Key Words: apo B • lipoproteins • endothelial injury • atherosclerosis • rabbits


*    Introduction
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 *Introduction
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Atherosclerotic lesions are found at typical predilection sites in the vascular tree. These sites, characterized by decreased endothelial integrity, also occur in nonatherosclerotic arteries.1 2 The fact that endothelial injury and an increased tendency toward atherosclerotic changes are localized in the same regions is an important basis for the response-to-injury hypothesis for atherosclerosis.3 4 5 In line with this hypothesis, various methods have been developed to induce atherosclerosis-like lesions by causing mechanical injury to the intima.6 7 8 9 10

Cholesterol deposition is one of the hallmarks of the atherosclerotic lesion. Consequently, the relationship between endothelial integrity and cholesterol deposition has attracted considerable interest. In the neointima formed after injury, cholesterol deposition is seen, even in normocholesterolemic animals.11 12 13 14 In several studies the relationship between cholesterol accumulation and endothelial integrity has been analyzed. Published data are to some extent contradictory. We have found the highest cholesterol concentration in areas devoid of endothelium,12 while others suggest that re-endothelialized intima has the highest content of cholesterol.13 14 15 16 17 In several studies attempts have been made to study cholesterol kinetics in the intima.18 A more rapid turnover of cholesterol in the nonendothelialized areas has been suggested.12 15 19 20 21 Similar observations have been obtained by use of in vivo labeled LDL. These studies indicated a rapid influx into de-endothelialized intima, balanced by a rapid turnover. In re-endothelialized areas the increased lipid content could be explained by a decreased turnover.19 Also, when in vitro labeled LDL has been used, increased transfer of LDL into de-endothelialized tissue has been observed.22

One attempt to evaluate the actual lipoprotein concentration in the intima was made by Schwenke and Carew.23 24 An interesting conclusion from their study was that retention rather than increased permeability may be the most significant mechanism for lipoprotein deposition in the intima. This is well in line with results from a study by Chang et al,25 who investigated the time course of LDL accumulation in the healing, balloon–de-endothelialized rabbit aorta. Injected labeled LDL was preferentially found at the edge of the regenerating endothelium and remained elevated at this location for at least 40 hours, while in the de-endothelialized areas the labeled LDL disappeared.

A weakness of our present knowledge about lipoprotein deposition after injury has been the absence of quantitative data on the lipoprotein concentrations in tissues. This limits the possibility of making quantitative evaluations of lipoprotein kinetics in the arterial tissue. In an attempt to investigate this, we have developed a method for quantitative evaluation of lipoprotein concentrations in human arterial tissue, based on the primary binding reaction of lipoproteins to antibodies.26 We have also isolated and characterized the major apolipoproteins in rabbit plasma.27 In the present study, we combined these methods to allow studies on rabbit arteries, aiming at an evaluation of the relationship between LDL influx and concentrations in the neointima formed after balloon injury. The significance of the endothelium for both lipoprotein transfer into the tissue and lipoprotein deposition was also evaluated.


*    Methods
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 *Methods
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Animals
A total of 42 male rabbits (New Zealand White, HB Lidköpings Kaninfarm) weighing 1.8 to 2.1 kg at the start of the study were used. The rabbits were fed a standard rabbit diet (Ewos Maintenance food, Ewos AB). The animals were acclimatized to the animal quarters for at least 2 weeks before the experiments. The experiments had been approved by the local experimental animal ethics committee.

De-endothelialization
Essentially the method developed by von Baumgartner and Studer6 was used to induce the formation of atherosclerosis-like lesions. After anesthesia of the animals with xylazine (10 mg/kg body weight) and ketamine (25 mg/kg body weight), the femoral artery of each animal was exposed and incised under aseptic conditions. An embolectomy catheter (EMB No 3F, Shirley Scandinavia AB) was introduced into the aorta. The balloon was then inflated with 0.25 mL saline and the catheter was withdrawn for 3 cm. This procedure was repeated twice. After the balloon was emptied, the catheter was withdrawn from the animal. With this procedure, an intimal thickening could be induced in the upper half of the thoracic aorta and the lower half could serve as an uninjured control. After the catheter was removed, the femoral artery was ligated and the skin lesion was sutured. No complications were encountered during the procedure and no infections in the area of the incision were observed after the operations.

Iodination of Lipoproteins and Antibodies
A narrow density cut of LDL (d=1.030 to 1.055 g/mL) was isolated from normal rabbit plasma as described previously.28 We were not able to demonstrate any contamination by other plasma proteins or lipoproteins using electrophoresis and immunodouble diffusion with antibodies to rabbit apoA-I, apoB, apoC-III, and apoE. LDL was iodinated with the iodine monochloride method as modified by Shepherd et al,29 using 125I with a specific activity of 15 Ci/µg. Most (94% to 98%) of the radioactivity in the labeled lipoproteins could be precipitated in 15% trichloroacetic acid (TCA). After being labeled the lipoproteins migrated as ß-lipoproteins. An antiserum to apoB was prepared by hyperimmunization of sheep with a narrow density cut of rabbit LDL (see above). The antibodies were purified from the serum by immunoadsorption chromatography.30 Human LDL was coupled to CNBr-activated Sepharose 4B (Pharmacia LKB Biotechnology Sverige AB), and the apoB antiserum was passed through the column. The retained antibodies were eluted with 3 mol/L sodium thiocyanate. The retained fraction was then adsorbed onto a Protein G Sepharose 4 Fast Flow (Pharmacia LKB Biotechnology Sverige AB) column for further purification. The isolated antibodies were defined with SDS–polyacrylamide gel electrophoresis; immunodouble diffusion against rabbit LDL, HDL, and VLDL; and Western blot. They appeared to be specific and homogeneous IgG antibodies directed against apoB. The isolated antibodies were iodinated with immobilized preparations of lactoperoxidase (Enzymo-beads, Bio-Rad) by use of 125I, as described by the manufacturer. After being labeled, 95% to 100% of the radioactivity in the antibodies could be precipitated with 15% TCA. Electrophoresis of the labeled antibodies resulted in a single radioactive band.

Preparation of the Aorta
The rabbits were killed 1 day to 10 weeks after operation. Five unmanipulated rabbits were also used in the study. To distinguish re-endothelialized and unmanipulated areas from de-endothelialized areas by gross examination, all rabbits were injected intravenously with 4 mL 0.45% Evans blue, dissolved in saline, 40 minutes before being killed. Two to six rabbits, at 0, 2, 5, and 10 weeks after catheterization, were injected intravenously with 125I-LDL 5 minutes before Evans blue was injected. The short time of exposure to labeled LDL was chosen to be within the linear phase of LDL accumulation in the arterial wall.31 Blood samples were drawn from the marginal ear vein before Evans blue and 125I-LDL were injected. Blood samples for radioactivity determinations were collected at the time the rabbits were killed. In a separate group of four rabbits, the 125I radioactivity was followed by blood samples every 5 minutes from the injection until the rabbits were killed 45 minutes later. From the radioactivity decay curve, the mean exposure of the rabbits to the 125I-LDL could be calculated and related to the specific activity in the blood samples at the time of death. Separate rabbits were used for this purpose, because we have previously shown drastic effects on endothelial integrity even after a short time of stress32 such as repeated blood sampling.

Before the injection of 125I-LDL and Evans blue, the animals were lightly anesthetized by an intramuscular injection with ketamine (25 mg/kg body weight, four consecutive injections). They were killed with an overdose of pentobarbital sodium (25 mg/kg body weight).

After the injection of pentobarbital, a midline incision was made in each rabbit and the aorta was removed. The aorta was rinsed with saline to remove contaminating blood and opened longitudinally to expose the luminal surface. After removal of surface fluid, the intima-media was dissected from the adventitia under a dissecting microscope. During dissection the tissue was kept moist, but without a surplus of fluid to avoid elution of buffer-extractable apoB from the samples. Nonendothelialized areas were separated from re-endothelialized areas. The intima-media from the unmanipulated thoracic aorta was taken as the control. The different tissue samples were photographed to allow the determination of surface areas. The tissue samples were then cut into smaller pieces and put into preweighed sealed Eppendorf tubes and weighed on a microbalance. The wet weights of the samples were 13 mg to 110 mg. The tissue samples from the rabbits injected with 125I-LDL were counted in a gamma counter (1282 CompuGamma, LKB Wallac).

After being weighed, the tissue samples were incubated four times for 30 minutes at room temperature in 0.2 to 0.5 mL 0.05 mol/L Tris-HCl, 10 mmol/L CaCl2, pH 7.4, in a shaker at 200 rpm. After each incubation, the samples were centrifuged at 20°C for 5 minutes at 15 000g and the supernatants were recovered and analyzed for apoB content (see below) and radioactivity. Lipoproteins released by this procedure will be referred to as "loosely bound apoB" or "buffer-extractable apoB." After these extractions, the tissue samples were incubated in 800 U/mL collagenase (Sigma type I, 450 U/mg protein) in the same buffer solution as above for 5 hours at 37°C. After digestion, the suspension of tissue debris was centrifuged at 15 000g for 15 minutes at 20°C and the supernatants were recovered. The lipoproteins in this fraction will be referred to as "tightly bound apoB." Supernatants from both buffer and collagenase incubations were frozen at -80°C for subsequent determination of apoB. The level of immunoreactive apoB as well as the radioactivity was so low that we had to refrain from doing TCA precipitations in those tissue samples in which tissue apoB was to be determined. In separate experiments, we found that almost no TCA-soluble radioactivity (<3%) was found in the tightly bound apoB fraction. In the loosely bound fraction, the TCA-soluble radioactivity was far higher: 74.5% in normal arterial tissue, 42.3% in de-endothelialized injured tissue, and 67.4% in re-endothelialized injured tissue. When mean retention times and transfer rates were calculated for total LDL and loosely bound LDL in different tissue fractions, the data were corrected for TCA-soluble radioactivity.

ApoB Determination
An immunoradiometric assay (IRMA), originally developed for the determination of human apoB in arterial tissue,26 was modified for use in rabbit tissue. Fifteen micrograms of sheep anti–rabbit-LDL in 200 µL PBS (10 mmol/L sodium phosphate buffer, pH 7.4, with 150 mmol/L NaCl and 0.05% NaN3 [wt/vol]) was adsorbed on polystyrene balls 6.4 mm in diameter (Precision Plastic Balls) by incubation of the balls for 20 hours at 4°C. After incubation, the balls were postcoated overnight at 4°C with 200 µL 10% dry milk (wt/vol) in PBS to block residual protein binding sites. BSA was used instead of dry milk as the blocking agent in earlier phases of the experiments. Dry milk, however, was easier to rinse away and also blocked the residual protein-binding sites more effectively than BSA without affecting the immunological reactivity. Before each determination, the balls were washed four times with 400 µL PBS. The samples were then added in appropriate dilutions made in PBS with 5% dry milk (wt/vol) and 0.1 mol/L EDTA. After the polystyrene balls were rinsed four times with 400 µL PBS with 5% dry milk (wt/vol) and 0.1 mol/L EDTA, the labeled antibody was added. A total of 1250 ng (60000 cpm) IgG in 200 µL PBS with 5% dry milk (wt/vol) and 0.1 mol/L EDTA was added to each ball. After being incubated for 20 hours at 4°C, the balls were rinsed four times with 400 µL PBS containing 0.1% Tween 20. Radioactivity bound to the balls was determined in a gamma counter. A reference serum from rabbits was used for standardization of the assay. This serum had been characterized earlier with regard to apolipoprotein and lipid content.31 The reference serum was incubated under the same conditions as the tissue samples. Previous experiments had established that the concentrations of both the primary and the secondary antibodies were in excess of the antigen. The apoB concentrations in the tissue samples were calculated from a standard curve obtained from the serum samples. Each standard and tissue sample was run in triplicate. Values giving a coefficient of variation greater than 15% were excluded. To study the effects that incubation in Tris buffer and collagenase might have on the immunological reactivity of apoB, plasma LDL was incubated in Tris buffer and collagenase for various periods of times at 37°C. When purified LDL was incubated with collagenase or Tris buffer for 5 hours, we observed a decrease in immunoreactivity of up to 60%. However, if whole serum was treated the same way, the decrease was always less than 20%, with a very small variation between samples and between days. A decrease in immunoreactivity of 20% was also seen when purified LDL was incubated in the presence of arterial tissue. Thus, we conclude that the incubations as such most likely had only a small effect on the observed tissue apoB levels.

Estimation of Serum Apolipoprotein and Lipid Concentrations
The concentrations of serum cholesterol and triglycerides were determined in a Gilford System 3500 autoanalyzer with enzymatic colorimetric methods (respectively, Monotest Cholesterol and Test-Combination Triglycerides GPO-PAP, Boehringer Mannheim GmbH). ApoB and apoA-I were determined with rocket immunoelectrophoresis.27

Calculations and Statistical Methods
Clearance of LDL (microliters per gram wet weight, divided by hours) was calculated by dividing the radioactivity in tissue (cpm [counts per minute] per gram wet weight) by the area under the curve for serum radioactivity (cpm per microliter, times hours). The area under the curve was calculated by integrating a monoexponential equation fit to data for the declining radioactivity in serum from injection until the time the rabbits were killed. Mean residence time for LDL was calculated as follows: tissue apoB content (nanograms per milligram wet weight) divided by flux rate (nanograms per milligram wet weight, divided by hours). For loosely bound LDL, TCA-soluble radioactivity, calculated as described above, was subtracted. Values are given as medians and ranges. Statistical analyses of differences between groups were analyzed with the Mann-Whitney U test or, in the case of paired data, with Wilcoxon's signed rank test. The effects of time and groups were analyzed with multifactor ANOVA. All statistical calculations were made with the STATGRAPHICS program for the personal computer (Statistical Graphics Corp).


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
The arterial injury induced by the balloon catheter method resulted in the formation of an intimal thickening. By 2 weeks after the operation a neointima had formed and was observed both in areas still devoid of endothelium and in areas that had been re-endothelialized. This was obvious also from the increased weight-to-area ratio found in these regions (P<.05, data not shown). During the course of the experiment, the relationship between weight and area was constant in the lesions. The concentration of total apoB-reactive material in the lesions is presented in Fig 1Down. An increase was observed during the first few weeks, and a maximum concentration of four times higher than that in control tissue was reached after 35 days (P<.02 compared with normal tissue). During the first few days, there was also an increased concentration of apoB in normal, unmanipulated tissue (Fig 1Down). This probably reflected acute effects from operative stress. No difference between areas with intimal thickening devoid of endothelium and areas that had been re-endothelialized could be demonstrated.



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  		Figure 1. Bar graph shows total apoB content in intima-media of rabbit aortas at various time points after de-endothelialization. Values are medians; bars indicate ranges. Number of rabbits is shown above each bar. NEA indicates uninjured tissue; DEA, de-endothelialized area; REA, re-endothelialized area; and w.w., wet weight.

The extraction procedures allowed us to differentiate between loosely bound and tightly bound apoB. In Fig 2Down, top panel, the concentrations for loosely bound apoB are presented. Two to 10 weeks after injury, the concentrations of apoB in this tissue pool were about four times higher in the lesion than in the surrounding normal arterial tissue (P<.001). There was no consistent difference between de-endothelialized and re-endothelialized tissue. The concentration of apoB in the tightly bound fraction (Fig 2Down, bottom panel) was about 10 times higher in the lesion than in the control tissue (P<.0001). At all time points more tightly bound apoB was found in the de-endothelialized than in the re-endothelialized tissue (P<.005), reflecting a higher percentage of the total apoB in the tightly bound pool in the de-endothelialized tissue (P<.0005).



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  		Figure 2. Bar graphs show loosely bound (top) and tightly bound (bottom) apoB content in intima-media of rabbit aortas at various periods after de-endothelialization. Values are medians; bars indicate ranges. Number of rabbits is shown above each bar. NEA indicates uninjured tissue; DEA, de-endothelialized area; REA, re-endothelialized area; and w.w., wet weight.

No statistically significant correlations were observed between tissue apoB on the one hand (in either of the tissue fractions) and serum triglycerides, apoA-I, or apoB on the other. However, positive correlations between tissue apoB and serum cholesterol were observed in both the loosely bound and the tightly bound pools in the normal tissue (r=.41, P<.02 and r=.46, P<.01, respectively) and the re-endothelialized tissue (r=.60, P<.01 and r=.77, P<.0001, respectively).

The uptake of labeled LDL in the lesion and in control arterial tissue is presented in Fig 3Down. The clearance of LDL from plasma was higher in areas of the lesion devoid of endothelium than in surrounding normal arterial tissue (P<.0005) or re-endothelialized areas (P<.005) of the lesion. No consistent differences between re-endothelialized areas of the lesion and control areas were observed. When the radioactivity in different pools of apoB was analyzed, the uptake of labeled LDL in the loosely bound fraction was found to be higher in de-endothelialized areas than in control tissue (P<.02) or in re-endothelialized tissue (P<.05) (Fig 4Down, top panel). However, the difference in the uptake of labeled LDL into the tightly bound fraction of apoB was even larger when areas of the lesions devoid of endothelium were compared with either control tissue (P<.0001) or re-endothelialized tissue (P<.001) (Fig 4Down, bottom panel). This difference was reflected in a higher percentage of labeled LDL being found in the tightly bound fraction in de-endothelialized areas than in re-endothelialized areas or normal arterial tissue (P<.0001).



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  		Figure 3. Bar graph shows total influx of 125I-LDL into intima-media of rabbit aortas at various periods after de-endothelialization. 125I-LDL was injected 45 minutes before the rabbits were killed. Data are calculated using wet weights and are corrected for trichloroacetic acid–soluble radioactivity. Values are medians; bars indicate ranges. The number of rabbits is shown above each bar. NEA indicates uninjured tissue; DEA, de-endothelialized area; and REA, re-endothelialized area.



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  		Figure 4. Bar graphs show influx of 125I-LDL into loosely bound (top) and tightly bound (bottom) pools of apoB in intima-media of rabbit aortas at various periods after de-endothelialization. 125I-LDL was injected 45 minutes before the rabbits were killed. Data are calculated using wet weights and are corrected for trichloroacetic acid–soluble radioactivity. Values are medians; bars indicate ranges. The number of rabbits is shown above each bar. NEA indicates uninjured tissue; DEA, de-endothelialized area; and REA, re-endothelialized area.

The mean residence time for apoB in various pools and tissue segments is presented in Fig 5Down. The mean residence time for apoB in the loosely bound pool (Fig 5Down, top panel) was somewhat longer in the lesion than in the control tissue (P<.05), with a mean residence time of about 5 hours. In the tightly bound fraction the mean residence time was shorter in areas of the lesion devoid of endothelium than in control tissue (P<.05) or re-endothelialized tissue (P<.005) (Fig 5Down, bottom panel). Thus, the de-endothelialized areas are characterized by a larger pool of tightly bound apoB with a fast turnover, whereas in the re-endothelialized areas the pool is smaller and has a slow turnover.



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  		Figure 5. Bar graphs show retention time for loosely bound (top) and tightly bound (bottom) apoB in intima-media of rabbit aortas at various periods after de-endothelialization. 125I-LDL was injected 45 minutes before the rabbits were killed. Data are corrected for trichloroacetic acid–soluble radioactivity. Values are medians; bars indicate ranges. The number of rabbits is shown above each bar. NEA indicates uninjured tissue; DEA, de-endothelialized area; and REA, re-endothelialized area.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study we used the methodology for the determination of apoB in arterial tissue earlier described for human tissue by Lindén et al.26 We have here used the method on rabbit tissue. This method, IRMA, is based on the primary binding reaction between antigen and antibody. Although there are no previous quantitative studies on apoB in arterial tissue in rabbits, there is a series of studies on human arterial tissue in which various immunological methods such as electroimmunoassay33 34 or competitive radioimmunoassays35 were used. The levels of apoB observed in normal rabbit tissue are two orders of magnitude lower than those in normal human tissue.26 34 35 36 Apparently this is not due to methodological differences because different methods, including IRMA, used on human tissue give approximately the same result. The conclusion that the lower levels found in rabbits actually are species related is supported by the fact that reported indirect estimates give a very similar level of apoB in rabbit arterial tissue.37 Thus, although variation in epitope exposure is hard to exclude, the consistency between different studies and methods supports the conclusion that our method gives a reliable estimate of apoB in arterial tissue.

The levels of apoB obtained from the rabbit tissue were very low. Therefore, it was impossible to further characterize the different fractions of apoB in terms of composition and immunoreactivity. We also had to perform studies on TCA precipitability in separate control experiments. This may lend some uncertainty to the corrections of the loosely bound LDL data. However, differences between the individual animals were small, (<10%) and the corrections do not affect the major conclusions of the study. The relatively large fraction of TCA-soluble iodine in the loosely bound fraction of LDL most likely reflects a fast diffusion of free iodine from plasma into the tissue. From earlier studies it can be deduced that significant amounts of apoB are not degraded in the aorta during the first 45 minutes after injection of the tracer.38

In several animals we observed increased levels of apoB in normal tissue as well as during the first week. This may be explained by the stress induced by the operation. Stress has been shown to cause endothelial injury. For example, Pettersson et al32 reported a higher frequency of injured endothelial cells in the aorta even after a short time of stress.

The notion that LDL in the arterial wall may be present either in a loosely bound or in a more tightly bound or "immobilized" pool has been discussed for quite some time.36 39 Loosely bound LDL can be extracted from the arterial tissue with conventional buffer extractions. Tightly bound lipoproteins may then be released by incubation of the tissue with proteolytic enzymes36 or detergents.39 In our experience, the treatment with collagenase seemed to be the mildest way to extract the tightly bound pool. Elastase had a larger effect on the immunoreactivity of apoB. For human tissue, our data obtained with collagenase also are close to those obtained with detergents.26 The proportion of tightly bound LDL increases with cholesterol deposition in the tissue.40 41 In normal human arterial tissue, most of the lipoproteins are in the loosely bound pool. When such lipoproteins are extracted, they have been found to have several characteristics that suggest that they have been subjected to oxidative modification.42 43 The lipoproteins in the tightly bound pool, on the other hand, appear to be aggregated.40 41

The mechanisms involved in LDL immobilization to the arterial wall have been disputed. Originally, when proteolytic enzymes were used to release this lipoprotein fraction, plasmin was the most efficient of the enzymes tested.36 Therefore, binding of LDL to fibrin or fibrinogen in the arterial wall was considered as a mechanism for lipoprotein immobilization.44 As an alternative to this hypothesis, an association between LDL deposition and proteoglycans has been given considerable attention. Thus, in experimental animals, LDL deposits preferentially in areas of glycosaminoglycan accumulation.45 46 Additionally, soluble complexes of LDL and glycosaminoglycan have been isolated from human aortic lesions,47 and LDL interacts specifically with the glycosaminoglycans of intact arterial proteoglycans.48 Finally, a specific interaction between LDL and elastin has been observed.49 Thus, it is still uncertain which component(s) in the arterial wall might bind LDL, and several possibilities should be considered.

The retention time of LDL in the tightly bound fraction was particularly short in areas of the lesion without endothelium, with a mean residence time of less than 5 hours. In contrast, the retention time of this pool in the re-endothelialized areas was long. Our earlier studies suggest that variation in the retention of LDL may be related to differences in proteoglycan composition in different areas of the plaque. Thus, proliferating smooth muscle cells produce proteoglycans with higher affinities to LDL.50 The affinity of LDL for human chondroitin sulfate–rich proteoglycans is high and is mediated by specific interaction between peptide segments in apoB and chondroitin sulfate chains of the proteoglycans.48 51 In vitro these interactions lead to the selective retention of a fraction of LDL particles with lower molecular weight and larger exposure of the apoB protein. As a consequence of both the selective retention of certain lipoprotein particles and the structural changes during binding to proteoglycans, the sensitivity of the lipoproteins to oxidative modification increases.52 53 After oxidation, the affinity of LDL to proteoglycan decreases, and a shift toward the loosely bound pool of LDL might be anticipated. Taken together, these studies may support the suggestion that the loosely bound LDL pool may be oxidatively modified.41 42

The concentration of apoB was as high in the re-endothelialized region of the lesion as in areas devoid of endothelial lining. However, the retention time of the lipoproteins was much longer in the re-endothelialized regions, especially in the tightly bound fraction. Because the oxidative modification of LDL is a time-dependent process and because the retention of LDL in the tightly bound, "vulnerable" pool is long, the long retention in itself may lead to an increased concentration of oxidatively modified LDL. The possibility that retention of LDL with a decreased turnover of the lipoproteins may be significant in atherogenesis has also been proposed by Schwenke and Carew.23 24 Because oxidatively modified LDL is taken up with high avidity in macrophages, leading to foam cell formation,54 selective retention of LDL, possibly by proteoglycans, followed by oxidative modification may explain the increased frequency of foam cells in re-endothelialized regions of lesions induced by injury.13 14 55

In conclusion, the apoB concentration was increased in lesions compared with normal tissue, but there was no difference between de-endothelialized and re-endothelialized areas. In contrast, there was a dramatic difference in influx rate between areas devoid of endothelium and endothelialized areas. In de-endothelialized areas we observed a large pool of tightly bound apoB with a fast turnover, while in the re-endothelialized areas the tightly bound pool was small with a long residence time. These data emphasize that the kinetics of lipoproteins in the tissue may be of great significance for local lipoprotein modification and subsequent development of foam cells and advanced atherosclerotic lesions.


*    Acknowledgments
 
This study was supported by grants from the Swedish Medical Research Council (project No. 4531), the Swedish Association Against Heart and Chest Diseases, the Sahlgren's Hospital Foundations, and the Medical Faculty of Göteborg.

Received January 4, 1995; accepted March 31, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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