© 1998 American Heart Association, Inc.
Original Contributions |
Direct Assessment of Lipoprotein Outflow From In Vivo–Labeled Arterial Tissue as Determined in an In Vitro Perfusion System
From the Wallenberg Laboratory for Cardiovascular Research, University of Göteborg, Göteborg, Sweden.
Correspondence to Dr Tom Björnheden, The Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, S 413 45 Gothenburg, Sweden. E-mail tom.bjornheden{at}wlab.wall.gu.se
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Abstract |
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Abstract—The rate of cholesterol deposition during the atherosclerotic process is determined by the balance between the inflow and outflow of plasma lipoproteins in the arterial wall. Whereas the rate of inflow may be measured directly, the rate of outflow has most often been calculated indirectly from lipoprotein uptake by using the 2-compartment model. One objection against such calculations is that lipoprotein binding is not being considered. In the present study 2 different protocols were used to obtain a direct measure of the outflow of lipoproteins from atherosclerotic rabbit aortas. Thus, 3 rabbits with experimental atherosclerosis were given 125I-LDL intravenously and 3 were given [14C]cholesterol perorally. Twenty-four hours later the aortas were removed and the outflow of label was monitored during in vitro perfusion. Despite the different protocols, our results were consistent and indicated that fractional loss relative to whole tissue was
0.01 pool/h, which is 1 order of magnitude lower
than current estimates based on the 2-compartment model (0.04 to 0.4
pool/h). Furthermore, whereas as much as 2/3 to 3/4 of
the tracer that had entered the arterial wall was
effectively trapped, the remainder equilibrated at a faster rate (0.06
pool/h). In conclusion, it seems that tissue binding constitutes a
prominent and possibly underrated mechanism of lipoprotein deposition,
at least in the atherosclerotic rabbit aorta. Furthermore, this means
that current estimates of lipoprotein exchange parameters
based on the 2-compartment model (eg, fractional loss) may rest on
invalid assumptions and should be regarded with caution.
Key Words: arterial wall • atherosclerosis • lipoprotein outflow
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Introduction |
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Cholesterol deposition is 1 of the prominent features of the developing atherosclerotic lesion, and it seems that the major part of the cholesterol originates from LDL in the blood. The dynamics of the uptake of lipoproteins by the arterial wall has been subjected to a number of studies in animals and humans, both in vivo and in vitro, and it is generally agreed that the uptake is characterized by a bulk-phase transport, whereby the rate of accumulation is inversely related to the size of the lipoprotein. In most cases the rate of entry has been estimated from measurements of the accumulation of labeled lipoproteins in the arterial tissue. However, it is obvious that the net deposition also depends on the outflow of lipoproteins. Estimates of the rate of exit have been made under the assumption that plasma and arterial tissue may be regarded as a 2-compartment system in dynamic equilibrium. Most likely, however, this approach is too simplified, because it is well known that lipoproteins may bind to the matrix of the arterial wall and thus become withdrawn from the freely equilibrating pool to a variable extent.
To address this issue more directly, we used an in vitro perfusion system using the rabbit aorta with or without experimental atherosclerosis. In this system we have studied the outflow of lipoproteins from tissue that had accumulated radioactive lipoproteins in vivo.
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Methods |
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Animals
Male New Zealand White rabbits were used throughout the study. At 2 to 3 months of age, experimental atherosclerosis was induced through a combination of a cholesterol-enriched diet (1% cholesterol, wt/wt, in standard rabbit chow fed ad libitum) and mechanical injury by use of an embolectomy catheter. The method to achieve the mechanical injury was originally described by Baumgartner in 1963,1 and the process of endothelial injury and healing in the rabbit aorta take place during several weeks and months.2 Careful control of the technique makes it possible to obtain a mild injury.3 At the end of the induction period (13±2.4 weeks) a combination of fatty foam cell–rich lesions and fibrous plaque–like lesions were obtained to a varying extent: 0.52±0.18 of the surface area of the perfused aortas was occupied by lesions, corresponding to 0.27±0.12 on a weight per weight basis.
At the time the rabbits were killed (pentobarbital sodium [50
mg/kg], plus bleeding to death), serum cholesterol
and triglyceride levels in these animals were 50.3±19.5
and 3.57±2.58 mmol/L, respectively. In rabbits in which125I-LDL was given in vivo, cholesterol feeding
was interrupted 3 weeks before termination to achieve a high specific
activity in plasma. Their cholesterol and
triglyceride levels were lower at the time of injection:
23.4±22.6 and 2.86±3.72 mmol/L, respectively.
Perfusion of Rabbit Aorta In Vitro
The dissection procedure and the in vitro perfusion system that
was used have been developed to ensure optimal tissue
integrity.4 In brief, under general
anesthesia (premedication: ketamine 7.5 mg/kg and
xylazine 3 mg/kg im; anesthesia: ketamine (50 mg/mL)/xylazine (20
mg/mL), 1:1, 0.35 mL/kg iv initially, plus 0.17 mL/kg per 15 minutes
thereafter) the descending thoracic aorta was removed and dissected,
and the obtained intima-media preparation was fitted into Teflon
cylinders of appropriate size. The cylinders were inserted into 1 of
the legs of a U-shaped glass tube, which served as the perfusion
chamber. The flow of the perfusion medium was adjusted to a steady rate
of 1.5 to 2.0 mL/h. In this system there is no convective flow across
the arterial wall.
In perfusions where 125I-LDL had been given in vivo, the dissection procedure was simplified. Thus, meticulous removal of adventitial fat and blood vessels was performed, but the dissection was not carried down into the media. In this way the interval between the death of the animal and the start of the perfusion could be reduced to <1 hour.
Throughout the study, Eagle's minimum essential medium with Earle's salts and 10 mmol/L NaHCO3 was used, supplemented with 1% nonessential amino acids, 100 µg/mL streptomycin, 100 IU/mL penicillin, 60 mg/mL BSA (Serva Feinbiochimica), and 10% heat-inactivated rabbit lipoprotein–deficient serum (prepared by ultracentrifugation at d>1.21 g/mL). All animal procedures were in accordance with institutional guidelines.
Lipoproteins
Isolation of LDL
LDL (1.019 to 1.063 g/mL) was isolated by sequential
centrifugation from fresh, fasting human plasma in the
presence of 10 mmol/L EDTA. The size of LDL particles was
determined by gradient polyacrylamide gel
electrophoresis,5 giving a peak diameter of 27.4
to 27.8 nm. Ferritin, thyroglobulin, and a pooled LDL standard were
used to calibrate the gel.
Labeling of LDL With 125I
LDL was iodinated with 125I
using the ICl technique as described by
McFarlane6 and modified by Shepherd et
al.7 Specific activities of the lipoproteins were
340 to 770 counts per minute (cpm)/ng apoB protein. ApoB protein was
determined by a quantitative immunoassay.8
Trichloroacetic acid (TCA) precipitability of the preparations was
between 0.97 and 0.99. The LDL preparation was diluted with
nonradioactive lipoprotein from the same donor to obtain the desired
concentration. The final preparation was given to rabbits
intravenously (5.7 to 12.5 mL; 3.2 to 7.0 mg apoB per mL;
7.5 to 12.6 cpm/ng apoB) in the left marginal ear vein.
In Vivo Labeling With
[14C]Cholesterol
In selected rabbits the plasma lipoproteins were labeled with
[14C]cholesterol in vivo. Thus,
0.13 to 0.25 mCi (2.3 to 4.6 µmol)
[4-14C]cholesterol in 0.2 mL
ethanol was added to 0.1 g sodium taurocholate in 10 mL saline and
given via a gastric tube to sedated (ketamine/xylazine) rabbits
24 to 37 hours before the rabbits were killed. By this method, the
major part of the label in plasma is said to be recovered as
cholesterol ester.9 10 In our study
this proportion was 0.84 (0.80 to 0.89), mean and (range). Much of the
label (0.78; 0.66 to 0.86; mean and range) was recovered in the
fraction floating at d=1.019 g/mL.
Experimental Protocol
Three rabbits were given
[14C]cholesterol via gastric tube
and plasma samples were collected at intervals until the aorta was
removed after 25, 36, or 37 hours. In 3 other rabbits125I-LDL was injected into the left marginal ear vein, and
plasma samples were collected after 15 minutes and at intervals until
the aorta was removed after 24 hours.
After dissection, the most proximal 4- to 5-mm portion of the aorta was set aside for analysis, whereas the major part of the aorta was perfused for 24 hours with medium. The whole volume of the perfusate was collected and divided into hourly fractions. At the end of the experiment the tissue was dissected and analyzed.
Analyses
Aortic Tissue
Overt atherosclerotic lesions were cut out and dissected free
from the underlying media under a dissecting microscope. Plaques,
tissue underlying plaques, and noninvolved areas were analyzed
separately. When the adventitia was included in the perfusions, this
layer was also dissected free and analyzed separately. Wet
weight (wet wt) and surface area were measured.
Analysis of samples in experiments with125I -LDL is illustrated in Figure 1
. Total radioactivity was measured on
the nonprocessed tissue. After homogenization in
200 µL saline, samples were centrifuged, and
TCA-precipitable, nonlipid-soluble radioactivity was determined on the
supernatant (mobile pool) and the pellet (bound pool) separately. The
overall recovery of the analysis was 0.81±0.04.
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In experiments with [14C]cholesterol-labeled lipoproteins, lipids were extracted in chloroform/methanol, 2:1 vol/vol/, and washed.11 Total cholesterol mass (Boehringer Mannheim CHOD-PAP12 ) and total radioactivity were determined on the eluates. Lipids were separated by thin-layer chromatography,13 and the proportion of radioactivity in cholesterol and cholesterol ester was determined from videorecorded images of autoradiograms by using KS400 (Carl Zeiss) image analysis software.
Rabbit Plasma
In experiments with 125I-LDL, apoB mass
and total and TCA-precipitable radioactivity were determined in plasma
samples collected at intervals during the 24 hours preceding the
perfusion. Furthermore, the proportion of the radioactivity recovered
in the d>1.019 g/mL fraction was assessed after ultra
centrifugation.
In experiments with [14C]cholesterol, total cholesterol mass and total and lipid-soluble radioactivity were determined on the plasma samples obtained during the 24 to 37 hours before perfusion. Total and lipid-soluble radioactivity at d<1.019 and d>1.019 g/mL was determined after ultracentrifugation. Lipids were separated by thin-layer chromatography, and the ratio between the amount of radioactivity in cholesterol and that in cholesterol ester was determined through image analysis of videorecorded autoradiograms by using KS400 software (Carl Zeiss).
Perfusates
Total radioactivity was measured in all collected fractions of
the perfusate. In experiments where the aorta had been labeled
with 125I-LDL, lipoprotein-associated radioactivity
was also assessed after separation by exclusion
chromatography on a PD10 column (Pharmacia).
Measurement of Radioactivity
125I radioactivity was measured on a 1282
Compugamma gamma counter (LKB) and 14C
radioactivity on a 1214 Rackbeta liquid scintillation counter (LKB)
with Readygel (Beckman Instruments Inc) as the scintillation cocktail.
Samples were counted to achieve a counting error better than 3% (total
counts >4000), which in some cases meant counting periods of up to 1
hour.
Statistics
The content of radioactivity in the tissue at a given time point
during the perfusion could be extrapolated by adding the sum of the
outflowing activity after this time point to the activity remaining in
the tissue at the end of the experiment. The steadily falling level of
tissue activity that was extrapolated in this way was fitted to
y=A(1+Be-Ct),
where A+B denotes the activity before perfusion,
A the remaining tissue activity at infinity, and
C the fractional loss. Consequently,
A/(A+B) denotes the proportion of the
tracer that was not available for equilibration that is described
by this approximation.
Because the impression was that tissue activity was best described as a 2-phase process, successive curve fits were made to approximate the time course of the second phase in the best possible manner. In this procedure all data points were initially included. Then data points were removed 1 at a time from early time points and onward, the best fit being finally accepted when the SEs of the included parameters were minimal. This fit included the time period from 3 to 5 hours and onward of the obtained data. Marquardt's algorithm as applied in SigmaPlot (Jandel Scientific) was used for the calculations. If not otherwise specified, mean and SD were used to describe the data.
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Results |
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Rabbit Plasma
[14C]Cholesterol
The mean total radioactivity in plasma 25 to 37 hours after an oral dose of 0.13 to 0.25 mCi [14C]cholesterol was 0.23 to 1.0 cpm/nL with a specific activity of 3.4 to 15 cpm/nmol cholesterol. In Figure 2
the
radioactivity is expressed as the proportion of the given dose per
milliliter of plasma. If one assumes a distribution volume of 120
mL,10 this means that 0.30 of the administered
radioactivity was recovered in plasma at 24 hours. After 11 to 37 hours
an average 0.86±0.03 of plasma radioactivity was recovered as
cholesterol ester; 0.77±0.07 of the radioactivity was
associated with lipoproteins floating at d=1.019 g/mL.
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125I-LDL
After the administration of a bolus of125I-LDL intravenously (5.7 to 12.5 mL; 3.2 to
7.0 mg apoB per mL; 7.5 to 12.6 cpm/ng apoB), the TCA-precipitable
radioactivity in plasma rose instantly and then decreased during the
following 24 hours, with a mean of 0.97 to 1.28 cpm/nL. In Figure 2
the
radioactivity is expressed as a proportion of the given dose per
milliliter of plasma. The average apoB concentration in plasma was 0.20
to 0.33 mg/mL and the specific activity 2.82 to 4.49 cpm/ng apoB, which
was 0.40±0.13 of the specific activity in the given preparation. The
TCA-precipitable portion was >0.97 in the injected preparation and
0.93±0.09 in plasma; 0.94±0.05 of the plasma activity was recovered
at d>1.019 g/mL.
[14C]Cholesterol Loading and
Washout
Twenty-five, 37, and 36 hours before the animals were killed, an
oral dose of [14C]cholesterol was
given to 3 rabbits, resulting in a steady increase in plasma
radioactivity, with a time-averaged mean of 0.63±0.39 cpm/nL (Figure 2). The aorta was taken out and perfusion ensued. Normalized to the
average total radioactivity in plasma, the uptake in the whole aortic
tissue of the sample before perfusion was 67.9±21.7 nL/mg wet wt,
whereas the activity was 41.7±21.3 nL/mg wet wt in the incubated
tissue at the end of the perfusion after 24 hours. In dissected plaques
the uptake was 198±29 and 92.3±27.5 nL/mg wet wt before and after
perfusion, respectively. The activity in noninvolved tissue was very
low, and reliable values were obtained in only 2 aortas before
perfusion, ie, 12.6 and 12.6 nL/mg wet wt and in 1 aorta after
perfusion, 9.6 nL/mg wet wt. Paired analysis indicated that
0.47±0.14 of the radioactivity remained in the plaques after the
washout phase. In noninvolved tissue the proportion was 3/4.
In whole tissue the activities before and after perfusion are not
directly comparable, because the proportion of plaque to noninvolved
area is not necessarily the same in the sample obtained before
perfusion and in the incubated aorta. However, when this difference was
taken into account, the uptake in the whole perfused aorta before
washout could be extrapolated to 64.6±28.3 nL/mg wet wt, indicating
that the proportion of the activity that remained after perfusion was
0.66±0.05 (paired analysis).
The content of radioactivity in the tissue at a given time point during
the perfusion could be extrapolated by adding the sum of the outflowing
activity after this time point to the activity remaining in the tissue
at the end of the experiment (Figure 3).
With this procedure the normalized uptake of label before perfusion
could be estimated at 55.2±28.3 nL/mg wet wt, which is 0.82±0.11
(paired ratio) of the content estimated from sample analysis.
Nonlinear regression indicated that there was a proportion of the
label, 0.65±0.08, that was not equilibrating with the perfusion medium
at the time scale that was used. The fractional loss from the
proportion that seemed to equilibrate was 0.051±0.016 pool/h. Based on
the total content of label in the tissue, however, the estimate was
lower, ie, 0.011±0.003 pool/h, averaged over the 24 hours of
perfusion. Almost 90% (0.86±0.03) of the radioactivity in plasma was
recovered as cholesterol ester. In the tissue, the
proportion was somewhat lower both before perfusion (0.71±0.07) and
after (0.74±0.02).
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The average specific activity of total cholesterol in the tissue (0.56±0.39 cpm/nmol) was 0.059±0.026 of that in plasma (9.4±5.8 cpm/nmol). The specific activity in the tissue did not change during the perfusion, and the specific activity was similar in plaque and in nonplaque areas.
125I-LDL Loading and Washout
Three rabbits with experimental atherosclerosis
were given 100 µCi 125I-LDL
intravenously 24 hours before they were killed. After an
initial peak the TCA-precipitable radioactivity in plasma fell
gradually (fractional catabolic rate 0.06) with a time-averaged mean
of 1.11±0.17 cpm/nL at a specific activity of 3.86±0.74 cpm/ng apoB
during the 24 hours (Figure 2).
In samples collected during the initial dissection, the normalized uptake of TCA-precipitable radioactivity before perfusion was 35.3±11.5 nL/mg wet wt in whole tissue. After the 24 hours of cold perfusion, the remaining uptake was 22.3±8.2 nL/mg wet wt, constituting 0.64±0.10 of the activity at the beginning of the washout phase (paired analysis). In dissected plaques the uptake was 84.7±12.4 and 70.7±37.2 nL/mg wet wt and in noninvolved tissue, 10.3±4.1 and 5.33±1.78 nL/mg wet wt at the respective time points. These figures indicated that 0.64±0.10, 0.80±0.32, and 0.53±0.07 of the radioactivity remained after the washout phase in whole tissue, plaques, and noninvolved tissue, respectively. During these experiments the difference in the degree of atherosclerosis was minimal between the preperfusion sample and the incubated aorta and required no compensatory corrections during the calculations.
The outflow of radioactivity from the incubated tissue was used to
extrapolate the tissue activity during perfusion as described before
(Figure 3). With this procedure the normalized uptake of label before
perfusion could be estimated at 31.9±11.6 nL/mg wet wt, which is
0.91±0.13 of the uptake measured on the initial samples. The data
indicated a 2-phase pattern of outflow, with a more rapid release
during the first few hours and a slower decline during the major part
of the washout period (Figure 3). Nonlinear regression during the
latter phase indicated that the tissue concentration of LDL did not
approach zero with time, suggesting that a proportion of the label,
0.76±0.06, belonged to a nonequilibrating pool. The fractional loss of
the label from the equilibrating pool was 0.067±0.020 pool/h.
Calculations based on total tissue activity would give a considerably
lower fractional loss, ie, 0.010±0.006 pool/h, averaged over the 24
hours of perfusion. Exclusion chromatography of the
outflowing perfusate indicated that 0.62±0.18 of the activity
was associated with lipoprotein particles and this proportion did not
seem to change with time during the perfusion. Under the assumption
that the outflow of degradation products is much faster than that
of lipoprotein particles, this means that the fractional loss of the
lipoprotein particles per se might be even lower, possibly 0.04
pool/h, from the equilibrating pool and 0.006 pool/h relative to the
total tissue activity.
The samples were homogenized in saline to differentiate easily equilibrating LDL (the mobile pool, ie, the TCA-precipitable radioactivity in the supernatant) from the slowly equilibrating LDL (the bound pool, ie, the TCA-precipitable activity in the pellet).
Before perfusion 0.11±0.03 of the LDL belonged to the mobile pool. After perfusion the proportion was somewhat less, 0.078±0.030, or 0.73±0.11 of the initial value. Although the LDL uptake in plaques was 1 range of order higher than that in the adjacent media, the proportion of mobile LDL was similar.
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Discussion |
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In most cases calculations of the outflow of lipoproteins from the arterial wall have been based on lipoprotein uptake and the use of a simple 2-compartment model.3 14 15 16 In such a model, lipoprotein binding is not considered, and this introduces a systematic error that is negligible in short-term experiments but may become considerable after 24 hours, an end point that is commonly used.3 14 15 16 Another source of error is, of course, the crucial assumption that the arterial wall may be regarded as 1 homogeneous, readily equilibrating compartment. This error was effectively circumvented by Tozer and Carew in a recent study,17 in which they used a stochastic approach insensitive to the characteristics of the dynamic exchange process. However, other approximations were introduced, and determinations of outflow still rested on indirect observations.
The main idea with the present study was to develop a method for direct measurement of the outflow of lipoproteins from the arterial wall. To this end, aortas of rabbits with experimental atherosclerosis were loaded with radioactive lipoproteins in vivo, and the outflow of radioactivity was studied in a well-defined in vitro perfusion system.
Two different techniques were used to load the aortic tissue:125I-LDL injected intravenously (I)
and 14C-labeled lipoprotein after an oral dose of
[14C]cholesterol (C). In
I, the injected LDL was continuously degraded and the
arterial lumen was exposed to gradually falling levels of
lipoprotein (Figure 2). Much (0.93±0.09) of the radioactivity in
plasma was TCA-precipitable, and 0.94±0.05 was recovered at
d>1.019 g/mL, indicating that a major part of the
radioactive label belonged to the LDL fraction. In C, the
administered [14C]cholesterol
appeared in plasma at steadily rising levels of radioactivity during
the loading phase (Figure 2). A major part of the radioactivity
appeared as cholesterol ester (0.86±0.03). As much as
0.77±0.07 of the label most likely belonged to the ß-VLDL fraction
floating at d=1.019 g/mL. Thus, whereas in I the
aorta was exposed to human LDL particles labeled in the protein moiety,
the major part of the label in C resided in the
cholesterol ester portion of homologous lipoprotein
particles, most likely ß-VLDL.
The normalized uptake in whole tissue at the beginning of the washout phase was 35.3±11.5 and 55.2±28.3 nL/mg wet wt in I and C respectively, which is similar to published data for atherosclerotic monkeys14 18 and rabbits with16 or without15 19 atherosclerosis. In I the uptake reflected the entry of LDL particles into the arterial wall, whereas in C the uptake represented a combination of lipoprotein uptake and physical exchange of unesterified cholesterol. If cholesterol ester is used as a label of lipoprotein particles,11 the normalized uptake would be similar (57.2±16.5 nL/mg wet wt), indicating that a major part of the tissue radioactivity at the beginning of the washout phase represented lipoproteins that had been taken up. The uptake in C was considerably higher than in I, which may partly reflect the longer loading period in C (25, 37, and 36 hours) than in I (24 hours). Another explanation might be that whereas the final degradation product in I (free iodine) readily left the tissue, this was not the case in C. In fact, 0.37 of the outflow in I was of low molecular weight, indicating that the lipoprotein uptake in I could have been as high as 56 nL/mg wet wt [35.3/(1-0.37)] if the degradation products had equilibrated at the same rate as the LDL particles.
Thus, 2 quite different aortic preparations were used to study the
washout of radioactivity (Figure 4): In
C the major part of the label probably resided in the
cholesterol ester portion in the core of homologous
ß-VLDL particles, whereas the remainder most likely was distributed
between intracellular cholesterol ester from degraded
lipoprotein particles and free cholesterol, reflecting
inflow through physical exchange and hydrolysis of
cholesterol ester. In I the radioactivity
resided in the protein moiety of human LDL particles and in their
degradation products.
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During the 24-hour washout phase the rate of outflow seemed somewhat
faster during the first few hours than during the rest of the perfusion
(Figure 3). During the latter phase (from 3 to 5 hours and onward)
outflow data fitted well to a negative exponential function,
y=A(1+Be-Ct).
In neither I nor C was the outflow of
radioactivity constant, but it decreased slowly with time at a rate
that indicated that only a proportion of the radioactivity that had
been taken up during the loading phase was freely equilibrating with
the incubation medium (Figure 3). It seemed that as much as 0.76±0.06
and 0.65±0.08 of the activity in I and C,
respectively, belonged to a tissue pool that did not equilibrate at the
time scale used. This implies that a major portion of the lipoprotein
that had entered the arterial wall at the beginning of the
washout phase had become effectively trapped. Our interpretation of the
data is illustrated in Figure 5, in which
this bound pool is represented by the dotted line and the
experimental data by the heavy line. The thin line depicts the washout
curve that would have been expected if all the tracer had equilibrated
freely. The size of the bound pool is compatible with early data by
Srinavasan et al,20 who estimated that 0.47 of
LDL was tissue bound after 4 hours. Similarly, approximately half of
LDL was tissue bound in early, short-term experiments at our
laboratory.21 Also in a recent study, 0.50 to
0.60 of LDL was defined as tightly bound after 23
hours.15 As an alternative measure, the bound LDL
pool was also estimated as the saline-nonextractable radioactivity of
the tissue homogenates. By this method the bound pool
seemed larger (0.90±0.03 and 0.92±0.03 before and after the washout
phase, respectively), which might reflect that some of this
nonextractable activity was released during perfusion. Furthermore,
only 1 extraction in saline was performed, whereas others have used
protracted20 or repeated21
extractions, even including EDTA.15 In
I the proportion of the outflowing radioactivity that was
associated with LDL particles was 0.62±0.18 as assessed by exclusion
chromatography. The remaining 0.37
represented low-molecular-weight degradation products,
indicating a degradation rate similar to values that we have calculated
earlier by other methods.16 This proportion did
not seem to change with time, suggesting a fairly constant degradation
rate of the mobile LDL pool during the 24-hour washout phase.
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The fractional loss in I and C calculated from
the outflow data (0.067±0.020 and 0.051±0.017 pool/h, respectively)
was similar to the rate deduced from uptake data only in animals (0.08
to 0.09,14 0.04 to 0.10,3
and 0.11 to 0.42 pool/h15 ) and in humans (0.11 to
0.13 pool/h22 ). However, our data cannot be
compared directly with these figures, because our results refer to the
fractional loss from only a part of the tissue lipoprotein pool, ie,
the mobile pool (cf Figure 5), which only constituted 0.24±0.06 and
0.35±0.08 of the total tissue content in I and
C, respectively. For comparison, our data were recalculated
relative to the total tissue content, as was the case in the quoted
studies. This recalculation gave an apparent fractional loss that was
considerably lower, ie, 0.010±0.006 and 0.011±0.003 pool/h,
respectively, for I and C. Roughly the same low
value of the fractional loss was also obtained when calculations were
made from our data on tissue content only before and after perfusion,
ie, 0.017 pool/h. These rates are 1 order of magnitude lower than the
quoted determinations based on the accumulating radioactivity in the
2-compartment model, in which lipoprotein binding was disregarded.
Ghosh et al14 deliberately ignored lipoprotein
retention, stating that it amounted to <1/20 the inflow, but
our data and those of others15 20 do not
vindicate this statement. However, it is disturbing that an increased
accumulation due to lipoprotein binding would lead to a flattening of
the uptake-versus-time curve, which would give too low rather than too
high an estimate of the fractional loss. One way to reconcile these
observations is to introduce a "very fast equilibrating pool" or to
adopt the concept of plasma contamination23
(Figure 5). This would lead to high uptake at early (3 hours) time
points, thus restoring the shape of the curve, whereby the 2 types of
errors might actually tend to cancel each other. Our observation that
the rate of outflow of label from the tissue was higher during the very
first hours of the washout phase (Figure 3) supports this view.
The findings in our study rest on the pattern of outflow of tracer from the incubated tissue, ie, the observation that the outflow seemed to level off with time earlier than would have been expected in a simple 2-compartment model. The calculated parameters (bound pool and fractional loss) were remarkably similar between the 2 incubation setups despite the fact that the outflowing tracees were different and not distinctly defined. One obvious explanation is, of course, that the collected radioactivity in effect represents outflow of lipoprotein particles to a high extent. In I we showed that at least 0.62 of the outflowing activity could be attributed to LDL particles. In C the physical state of the outflowing activity was not studied for technical reasons, but it is likely that a large proportion of the activity represented outflow of lipoprotein particles, because cholesterol ester from degraded lipoproteins is highly insoluble in aqueous media and no vehicle was available to facilitate the outgoing transport of free cholesterol via physical exchange.
The dynamics of the exchange process during the loading phase (Figure 5) is not essential for our calculations, but the size of the bound
pool suggests that the rate of deposition must lie within the same
range as the rate of entry, pointing to a considerable capacity of the
arterial wall to bind lipoproteins. Although it cannot be
ruled out that the size of the bound pool, as defined in our in vitro
system, partly reflected the incubation conditions, it should be
pointed out that binding itself took place in vivo. At the same time it
may seem hard to imagine a rate of deposition within the same range as
the inflow in the long run, since the bound pool also will eventually
reach saturation. Then again, crude calculations do in fact indicate
that several weeks would be required to load the
cholesterol pool of the atherosclerotic rabbit aorta even
if all inflowing lipoprotein cholesterol was deposited.
(Assume a cholesterol clearance rate of 2 to 10 nL/mg wet
wt per hour11 14 16 at a plasma
cholesterol level of 25 mmol/L and a
cholesterol concentration in the tissue of 100 nmol/mg wet
wt [data from the present study].)
One inherent limitation in our approach was that it did not allow the differentiation between lesion and nonlesion areas but gave a composite picture of the outflow. However, the decrease in tracer concentration during the washout phase did not differ substantially between total tissue, plaque, and nonlesion areas, ie, 0.41±0.16, 0.32±0.28, and 0.47±0.07. Furthermore, although the concentration of tracer in lesions was higher than in other areas, the proportion of the total tracer content of the aorta that resided in lesions was not higher than 0.66±0.19. This means that the total outflow pattern was not dominated by either, so it seems that the major conclusions that we have drawn were valid for both.
Lipoprotein deposition in the arterial wall is determined
by lipoprotein inflow and lipoprotein outflow. Whereas measurement of
the rate of inflow is fairly straightforward, the assessment of
lipoprotein outflow is more controversial and most commonly rests on
indirect conclusions from determinations of lipoprotein uptake. In the
present study we used 2 different protocols to obtain a direct
measure of the fractional loss of lipoproteins from atherosclerotic
rabbit aorta. Our results indicated that fractional loss related to
whole tissue was 0.01 pool/h, which is 1 order of magnitude lower
than current estimates based on the 2-compartment model (0.04 to 0.4
pool/h). Furthermore, whereas as much as 2/3 to 3/4 of
the tracer that had entered the arterial wall was
effectively trapped, the remainder equilibrated at a faster rate (0.06
pool/h).
It appears likely that binding to resident proteoglycans constitutes the major mechanism of the observed lipoprotein entrapment.21 24 25 Such entrapment has been suggested to facilitate lipoprotein modification, thereby contributing to the atherogenic potential of the deposited lipoproteins.26 27
In conclusion it seems that tissue binding constitutes a prominent and possibly underrated mechanism of lipoprotein deposition, at least in atherosclerotic rabbit aorta. Furthermore, this implies that current estimates of lipoprotein exchange parameters based on the 2-compartment model (eg, fractional loss) may rest on invalid assumptions and should be regarded with caution.
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Acknowledgments |
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This study was supported by the Swedish Heart Lung Foundation and the Swedish Medical Research Council (project No. 4531) (to Göran Bondjers).
Received October 7, 1998; accepted May 29, 1998.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Baumgartner HR. Eine neue Methode zur Erzeugung von Thromben durch gezielte Überdehnung der Gefässwand. Z Ges Exp Med. 1963;137:227–232.
- Stemerman MB, Spaet TH, Pitlick F, Cintron J, Lejnieks I, Tiell ML. Intimal healing: the pattern of re-endothelialization and intimal thickening. Am J Pathol. 1977;125:87.
- Schwenke DC, Zilversmit DB. The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit, 1: studies during the first two days after mild aortic injury. Atherosclerosis. 1989;77:99–103.
-
Björnheden T, Bylock A, Hansson GK, Bondjers G.
A system for long-term perfusion of rabbit aorta in vitro.
Arteriosclerosis. 1983;3:366–382.
[Abstract/Free Full Text] - Gambert P, Bouzerand-Gambert C, Athias A, Farnier M, Lallemant C. Human low density lipoprotein subfractions separated by gradient gel electrophoresis: composition, distribution and alterations induced by cholesteryl ester transfer protein. J Lipid Res. 1990;31:1199–1210.[Abstract]
- McFarlane AS. Efficient trace-labeling of proteins with iodine. Nature. 1958;182:53–57.[Medline] [Order article via Infotrieve]
- Shepherd J, Bedford DK, Morgan HG. Radioiodination of human low density lipoprotein: a comparison of four methods. Clin Chim Acta. 1976;97:97–109.
- Laurell B. Electroimmunoassay. Scand J Clin Lab Invest.. 1972;29:21–37.
- Stender S, Zilversmit DB. Arterial influx of esterified cholesterol from two plasma lipoprotein fractions and its hydrolysis in vivo in hypercholesterolemic rabbits. Atherosclerosis. 1981;39:97–109.[Medline] [Order article via Infotrieve]
-
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.
[Abstract/Free Full Text] - Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;205:920–922.
- Kattermann R, Jaworek D, Moller G, Assmann G, Bjorkhem I, Svensson L, Borner K, Boerma G, Leijnse B, Desager JP, et al. Multicentre study of a new enzymatic method of cholesterol determination. J Clin Chem Clin Biochem. 1984;22:245–251.[Medline] [Order article via Infotrieve]
- Gloster J, Fletcher RF. Quantitative analysis of serum lipids with thin-layer chromatography. Clin Chim Acta. 1966;13:235–240.[Medline] [Order article via Infotrieve]
- 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]
-
Nielsen LB, Stender S, Kjeldsen K, Nordestgaard BG.
Specific accumulation of lipoprotein(a) in balloon-injured rabbit aorta
in vivo. Circ Res. 1996;78:615–626.
[Abstract/Free Full Text] - Björnheden T, Babyi A, Bondjers G, Wiklund O. Accumulation of lipoprotein fractions and subfractions in the arterial wall, determined in an in vitro perfusion system. Atherosclerosis. 1996;123:43–56.[Medline] [Order article via Infotrieve]
-
Tozer EC, Carew TE. Residence time of low density
lipoprotein in the normal and atherosclerotic rabbit aorta. Circ
Res. 1997;80:208–218.
[Abstract/Free Full Text] - Lin DS, Conner WE, Wissler RW, Vesselinovitch D, Hughes R. A comparison of the turnover and metabolism of cholesterol in normal and atherosclerotic monkey aortas. J Lipid Res. 1980;21:192–201.[Abstract]
-
Haarbo J, Nielsen LB, Stender S, Christiansen C. Aortic
permeability to LDL during estrogen therapy. Arterioscler
Thromb. 1994;14:243–247.
[Abstract/Free Full Text] - Srinavasan SR, Vijayagopal P, Dalferes ER, Abbate B, Radhakrishnamurthy B, Berenson GS. Low density lipoprotein retention by aortic tissue: contribution of extracellular matrix. Atherosclerosis. 1986;62:201–208.[Medline] [Order article via Infotrieve]
-
Olsson G, Wiklund O, Bondjers G. Effects of injury on
apoB kinetics and concentration in rabbit aorta. Arterioscler
Thromb Vasc Biol. 1995;15:930–936.
[Abstract/Free Full Text] -
Shaikh M, Wootton R, Nordestgaard BG, Baskerville P,
Lumley JS, La Ville AE, Quiney J, Lewis B. Quantitative studies of
transfer on vivo of low density, Sf 12–60, and Sf 60–400 lipoproteins
between plasma and arterial intima in humans.
Arterioscler Thromb. 1991;11:569–577.
[Abstract/Free Full Text] -
Nielsen LB, Nordestgaard BG, Stender S, Kjeldsen K.
Aortic permeability to LDL as a predictor of aortic
cholesterol accumulation on cholesterol-fed
rabbits. Arterioscler Thromb. 1992;12:1402–1409.
[Abstract/Free Full Text] - Hoff H, Bond G. Apolipoprotein B localization in coronary atherosclerotic plaques from cynomolgus monkeys. Artery. 1983;12:104–116.[Medline] [Order article via Infotrieve]
-
Hurt-Camejo E, Olsson U, Wiklund O, Bondjers G, Camejo
G. Cellular consequences of the association of apoB lipoproteins with
proteoglycans: potential contribution to atherogenesis.
Arterioscler Thromb Vasc Biol. 1997;17:1011–1017.
[Free Full Text] - 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]
-
Williams KJ, Tabas I. The response-to-retention
hypothesis of early atherogenesis. Arterioscler Thromb Vasc
Biol. 1995;15:551–561.
[Free Full Text]
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