© 1996 American Heart Association, Inc.
Articles |
Specific Accumulation of Lipoprotein(a) in Balloon-Injured Rabbit Aorta In Vivo
From the Department of Clinical Biochemistry, Rigshospitalet (L.B.N., K.K.), the Department of Clinical Chemistry, Køge Hospital (S.S.), and the Department of Clinical Biochemistry, Herlev Hospital (B.G.N.), University of Copenhagen, Denmark.
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Abstract |
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Abstract To explore whether lipoprotein(a), Lp(a), may accumulate preferentially to LDL in the arterial wall at sites of injury, cholesterol-fed rabbits were injected intravenously with radiolabeled Lp(a) and/or LDL 3.1±0.1 days (mean±SEM, n=30) after a balloon injury of the thoracic aorta. After 5 to 10 minutes' exposure to labeled lipoproteins, more labeled LDL than labeled Lp(a) was recovered in the intima–inner media of the balloon-injured segment (n=9; paired t test, P<.0001); however, the amount of tightly bound labeled lipoprotein was similar for the two lipoprotein fractions. In the second set of experiments, 131I-Lp(a) (or 131I-LDL) was injected 26 hours before and 125I-Lp(a) (or 125I-LDL) 3 hours before the aorta was removed. Permeability and fractional loss of labeled Lp(a) (n=8) versus LDL (n=7) in the balloon-injured aortic intima–inner media were: permeability, 0.46±0.10 µL/cm2 per hour versus 1.41±0.32 µL/cm2 per hour (nonpaired t test, P<.0001); and fractional loss, 0.12±0.02 h-1 versus 0.44±0.05 h-1 (nonpaired t test, P=.0001), respectively. Finally, after 23 hours' exposure to labeled lipoproteins, the total accumulation and the amount of tightly bound labeled Lp(a) in the balloon-injured intima–inner media were, respectively, 174% (n=6; ANOVA, P=.03) and 256% (ANOVA, P=.005) of the values for labeled LDL. For labeled Lp(a) in the balloon-injured compared with the normal aortic intima–inner media, the recovery after 5 to 10 minutes, the permeability, and the accumulation after 23 hours were all increased, whereas the fractional loss was unchanged. These data suggest that the accumulation of Lp(a) is much larger in injured vessels than in normal vessels. Moreover, the data support the idea of a specific accumulation of Lp(a) compared with LDL in injured vessels.
Key Words: angioplasty • atherosclerosis • lipoprotein(a) • low-density lipoprotein • restenosis
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion Appendix References |
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High plasma levels of Lp(a) have been associated with atherosclerotic disease,1 2 as well as with restenosis in coronary bypass vein grafts3 and after percutaneous transluminal coronary angioplasty4 ; however, the role of Lp(a) and other lipoproteins in restenosis remains controversial.5 Transgenic mice expressing human apo(a) are more susceptible to developing atherosclerotic lesions than are control mice,6 which also supports the conclusion that Lp(a) may play an important role in the development of atherosclerosis. The mechanism whereby this may happen remains to be established but could be related to the unique structure of Lp(a).
Lp(a), like LDL, contains apoB and additionally has a glycoprotein, apo(a), attached to apoB by a disulfide bridge.1 2 Apo(a) resembles the fibrinolytic proenzyme plasminogen.7 In vitro, Lp(a) is capable of stimulating foam cell formation,8 inducing growth of smooth muscle cells,9 and inhibiting fibrinolysis.10 Furthermore, Lp(a) binds to fibrin,10 as well as to arterial wall glycosaminoglycans,11 with a higher affinity than LDL. These observations have stimulated the idea that Lp(a) compared with LDL may accumulate selectively in the arterial wall and thereby contribute to the progression of atherosclerosis and possibly also to restenosis after angioplasty.
The present paper explored in vivo the hypothesis that Lp(a) compared with LDL may accumulate selectively in the arterial wall at sites of injury. Permeability, fractional loss, and accumulation of total and tightly bound radiolabeled Lp(a) and LDL in aortic intima–inner media were compared in rabbits 1 to 5 days after a balloon injury of the endothelial layer of the thoracic aorta.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion Appendix References |
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Animals
Thirty white male rabbits (Statens Seruminstitut, Copenhagen, Denmark) weighing 2.7 to 3.8 kg were housed under controlled environmental conditions. Each rabbit received a 1% cholesterol– (USP cholesterol, Sigma) and 5% corn oil– (Oleum Maides BP80, Mecobenzon) enriched chow for 5 to 9 days before the in vivo experiments. The experimental protocols were approved by the Danish government body supervising animal experiments, the "Animal Experiment Inspectorate."
Balloon Injury
The thoracic aorta was balloon injured
3.1±0.1 (range 1 to 5)
days before experiments in vivo as follows. Each rabbit was
anesthetized with pentobarbital (30 to 50 mg/kg IV) and a 4F
embolectomy catheter (Baxter Healthcare Corporation) was introduced
through a femoral artery into the thoracic aorta to about the level of
the first intercostal arteries. The balloon was inflated with 0.75 mL
saline and drawn 2 to 4 cm caudally before being deflated and
withdrawn.
Isolation of Lp(a) and LDL
Six batches of isolated Lp(a) were
used in the present
studies. For each isolation, plasma Lp(a) concentrations were
determined in at least 10 healthy subjects, and Lp(a) was subsequently
isolated from plasma from the 1 or 2 subjects with the highest Lp(a)
concentrations (0.25 to 0.55 mg/mL); plasmas from the 2 subjects were
pooled before lipoprotein isolation. Lp(a) concentrations in plasma and
isolated Lp(a) preparations were measured using a commercially
available turbidimetric assay or rocket immunoelectrophoresis with
rabbit polyclonal antibodies to Lp(a) (DAKO A/S). Antibodies to Lp(a)
have been shown to not cross-react with plasminogen or
apoB. Lp(a) calibrators (DAKO A/S) gave Lp(a) concentrations similar to
those obtained using calibrators from Immuno; presented values
represent total Lp(a) lipoprotein mass.12 Apo(a)
isoforms were determined essentially as described by Utermann et
al.13
Plasma for lipoprotein isolation had a lipoprotein
preservation
cocktail added: Na2 EDTA (final plasma concentration, 1.2
mg/mL), chloramphenicol (80 µg/mL), gentamycin sulfate (80 µg/mL),
benzamidine (10 µg/mL), and aprotinin (10 kallikrein units/mL) (all
from Sigma). Plasma for LDL isolation additionally had
-amino-n-caproic acid (2.6 mg/mL; Sigma) added.
Lp(a)
was isolated at 10°C. Plasma was adjusted to a solvent density
of 1.12 g/mL and ultracentrifuged for at least
6.0x108g ·min in a Beckman 55.2 Ti rotor.
After dialysis of the <1.12-g/mL-density fraction against a 0.1-mol/L
phosphate buffer, Lp(a) was adsorbed onto a lysine-Sepharose 4B column
(Pharmacia). The column was washed with the 0.1-mol/L phosphate buffer
and the Lp(a) eluted with 10 mmol/L
-amino-n-caproic acid and 0.5 mol/L NaCl in the
same phosphate buffer. The Lp(a) preparations, with concentrations of
1.3 to 3.2 mg/mL, were passed through 0.22-µm filters (Millex GS
Millipore S.A.) before iodination.
Six batches of LDL (1.019 to 1.063 g/mL) were isolated by sequential ultracentrifugation as previously described.14 In experiments in which LDL and Lp(a) were studied simultaneously, LDL was isolated from the same plasma as Lp(a). In the 3- versus 26-hour experiments, LDL was isolated from a donor with low plasma Lp(a) concentration; this isolated LDL contained less than 0.1% Lp(a) (LDL total lipoprotein mass versus Lp(a) total lipoprotein mass). The concentration of LDL protein was estimated from the absorbance at 220 nm.15
Characterization of Unlabeled Lp(a)
In crossed
immunoelectrophoresis, isolated Lp(a) appeared as a
single peak in a gel containing anti-human serum (DAKO A/S).
Isolated Lp(a) and Lp(a) in whole plasma had a similar electrophoretic
mobility in a pure agarose gel, suggesting that Lp(a) had not been
significantly oxidized during the isolation procedures. Isolated Lp(a)
also migrated as a single band on a nondenaturing 2.5% to 16%
polyacrylamide gradient gel (Isolab Inc).
Lipoprotein Labeling
Preparations of Lp(a) [2 mL, 2.5 to
6.4 mg Lp(a)] and LDL (0.3
to 0.9 mL, 5 mg protein) were mixed with 185 to 370 MBq
125I or 131I (Amersham) and
iodinated using ICl,16 17 as previously
described.14 Unbound iodine was removed with PD-10 columns
before 100 mg of rabbit albumin (Sigma) was added. The
iodination efficiency averaged 60% and was similar for the two
lipoproteins. The specific activities were 0.7 to 2.6x109
cpm/mg Lp(a) total mass and 0.2 to 0.9x109 cpm/mg LDL
protein (0.4 to 2.0x108 cpm/mg LDL total
mass18 ). In labeled Lp(a) and LDL, 95±0.3% and
97±0.4%
of the radioactivity was precipitable with TCA. An average 1.8% and
2.3% of the TCA-precipitable radioactivity in Lp(a) and LDL was
associated with lipids, determined by chloroform/methanol (1:1,
vol/vol) extraction. On nondenaturing 2.5% to 16%
polyacrylamide gradient gels, the radioactivity in labeled
Lp(a) and LDL comigrated with their respective nonlabeled lipoproteins.
Lipoproteins were used for injections within 24 hours of labeling.
Protocol
Three separate experimental protocols were used
(Table 1
).
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To measure total and tightly bound labeled Lp(a) and LDL in the intima–inner media, mixed preparations of 125I-Lp(a) and 131I-LDL (six rabbits used for 5- to 10-minute experiments and six rabbits used for 23-hour experiments) or 131I-Lp(a) and 125I-LDL (three rabbits used for 5- to 10-minute experiments) were injected intravenously into rabbits 5 to 10 minutes or 23 hours before the aorta was removed. The mass of human Lp(a) was increased in two rabbits used in the 5- to 10-minute experiments and in three rabbits used in the 23-hour experiments by an intravenous injection of human <1.12-g/mL-density lipoproteins containing 25 to 33 mg Lp(a) (administered immediately before injection of labeled lipoproteins). The <1.12-g/mL-density lipoprotein fractions were prepared from the same batch of human plasma as was used for isolation of Lp(a) and LDL for labeling.
To measure permeability and fractional loss of labeled Lp(a) from the aortic intima–inner media in eight rabbits, 131I-Lp(a) and 125I-Lp(a) were injected intravenously 3.0±0.1 and 26±0.3 hours before the aorta was removed. Likewise, in seven other rabbits, labeled LDL was injected 3.0±0.1 and 26±0.5 hours before the aorta was removed.
In each rabbit, 6 to 10 blood
samples of 
1 mL were drawn from an ear
vein throughout the 23- to 26-hour period.
Characterization of Normal and Balloon-Injured Aorta
Each
rabbit was intravenously injected with 25 mg of
Evans blue (Merck) dissolved in 5 mL saline, immediately followed by
pentobarbital (50 to 100 mg/kg IV) and removal of the
aorta.14 19 The balloon-injured aortic segment was
clearly blue. A 2- to 3-mm-wide ring was taken from the middle of
the injured segment, fixed immediately in buffered formalin (3.7%),
and examined after staining with hematoxylin-eosin and
Masson-Goldner-elastica. The aorta was divided into four segments:
the normal aortic arch (from the heart to the first intercostal
branches or to the balloon-injured segment; the mean distance from
the heart to the distal edge of this segment was 30±1 mm), the
balloon-injured segment (segment of aorta stained with Evans blue),
the thoracic aorta (from the balloon-injured segment to the celiac
axis), and the abdominal aorta (to the iliac bifurcation). The
intima–inner media in each of these parts was stripped from the
outer media. In the thoracic and abdominal aorta there were
occasionally smaller areas of blue staining, presumably due to damage
during introduction or withdrawal of the embolectomy catheter. Only
data from the aortic arch were used, therefore, to describe conditions
for normal aortic tissue. However, when values from the normal thoracic
and normal abdominal aorta were compared with values from the
balloon-injured aorta, the results and conclusions were similar to
those based on comparison with data from the aortic arch. The mean area
of the normal aortic arch and balloon-injured segment was 4.0±0.2
cm2 and 3.6±0.2 cm2, respectively. The
estimated thickness19 of the intima–inner media of
the normal aortic arch and the balloon-injured aorta was 516±16
µm and 423±16 µm, respectively.
Tightly Bound Lipoproteins
The intima–inner media was
minced with scissors in 2 mL
cold PBS-EDTA. After gentle turning for 2 minutes and
centrifugation at 4°C, 1.8 mL of the supernatant was
transferred to another vial. This procedure was repeated three times.
Of the total amount of labeled Lp(a) that was extracted from
balloon-injured aortic intima–inner media, 86±2%, 9±2%,
and 6±1% were extracted in the first, second, and third wash,
respectively. Almost identical results were obtained for normal
intima–inner media and for labeled LDL. Finally, in most cases,
the intimas–inner medias were additionally washed once with 2 mL
of the 10-mmol/L -amino-n-caproic acid buffer used to
elute Lp(a) from the lysine column.
TCA-Precipitable Radioactivity
Proteins in minced aortic
intimas–inner medias, in
supernatants after washing, in aliqouts of plasma, and in labeled
preparations were precipitated with TCA (final concentration 15%,
wt/vol) at 4°C after addition of albumin (100 mg) (human
albumin, fraction V, Sigma). Samples were counted in a
double-channel gamma counter for 42 to 60 minutes (LKB compugamma
1282, Wallac). Typical standard errors of counting rates of
TCA-precipitable 125I and 131I in aortic
tissues and the first washes were <1%.
Determination of Cholesterol
Lipids in TCA precipitates of
aortic intimas–inner medias
were extracted with chloroform/methanol (2:1, vol/vol) and washed twice
with chloroform/methanol (1:1, vol/vol) before the extract was washed
by the procedure of Folch et al.20 Cholesterol
was determined by the Liebermann-Burchard method after
saponification.21 Plasma cholesterol was
determined with the CHOD-PAP enzymatic method (Boehringer
Mannheim).
Two-Tier Rocket Immunoelectrophoresis
To assess
cross-contamination between labeled Lp(a) and LDL,
as well as the amount of labeled free apo(a) in labeled Lp(a),
two-tier rocket immunoelectrophoresis was used. Three separate
bands of 1.25% agarose were applied onto a GelBond film (FMC
Bioproducts). The lower band of the gel was pure agarose, the
middle band contained anti-Lp(a), and the upper band contained
anti-apoB (DAKO A/S). Alternatively, the middle band contained
anti-apoB and the upper band anti-Lp(a). The application spot and
rockets were cut out and gamma counted. The recovery of radioactivity
on the gels was 98.7±1.6% (n=74).
Calculations
TCA-precipitable radioactivity in tissues,
washes, plasma, and
doses was used in the calculations. Total and tightly bound labeled
lipoproteins in the aortic intima–inner media were expressed as
plasma equivalents in nL/cm2: aortic radioactivity
(cpm/cm2) was divided by the initial (5- to 10-minute
experiments) or the time-averaged (23-hour experiments)
radioactivity concentration in plasma (cpm/nL).
Aortic permeability and fractional loss of Lp(a) and LDL from intima–inner media were calculated using a one-pool-compartment model.22 23 24 Plasma radioactivity curves were fitted to double-exponential functions, and fractional loss and permeability were calculated as described previously.24 Crude fractional loss of labeled lipoproteins from the aortic intima–inner media was calculated as recently described.22 The crude fractional loss is a minimal estimate of the "true" fractional loss.
Statistical Analysis
Arterial wall lipoprotein parameters
were evaluated using a two-way layout25 ANOVA with
random effects.26 In this model, for a given
parameter, each rabbit had its own level: the random
effects described variation between rabbits. The total and tightly
bound amounts of labeled lipoproteins after 23 hours' exposure,
lipoprotein permeability, and intimal clearance were all transformed
logarithmically, and crude fractional loss was square root transformed
to approximate normal distributions.
In the initial ANOVA, there was a significant effect of type of tissue (normal or balloon-injured) for all parameters tested, and there was a significant interaction between type of lipoprotein [Lp(a) or LDL] and type of tissue for total amount of labeled lipoproteins in intima–inner media after 5 to 10 minutes' exposure, total and tightly bound amounts of lipoproteins after 23 hours' exposure, permeability, intimal clearance, fractional loss, and crude fractional loss. In those cases, the ANOVA model was reduced to analyze separately the effect of type of lipoprotein for each type of tissue and the effect of injury for each lipoprotein. Paired or nonpaired Student's t tests were used. These calculations were all performed using the proc mixed procedure in the SAS statistical program. Other differences between mean values were analyzed using paired or nonpaired Student's t tests. Linear relations between two parameters were expressed as parametric correlation coefficients. Differences between mean values were considered statistically significant when two-tailed probability values were less than .05; probability values were not corrected for multiple comparisons. All values are expressed as mean±SEM.
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Experimental design and basic characteristics of the 30 rabbits used are shown in Table 1
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Balloon Injury
The balloon-injured aortic segment was easily
identified after
intravenous injection of Evans blue immediately before
removal of aorta: on visual inspection and grading, 91±2% of the
balloon-injured segments was stained blue. On
histological examination of hematoxylin-eosin– and
Masson-Goldner-elastica–stained sections of
balloon-injured aortas, segments from all rabbits showed signs of
injury. No endothelial cells were observed in the
intima. In the media, elastic membranes appeared intact, whereas
occasional necrosis of smooth muscle cells was observed, and in a few
rabbits, infiltration with granulocytes was seen.
The cholesterol content of aortic intima–inner media 3.1±0.1 days after balloon injury was increased compared with the cholesterol content of normal aortic intima–inner media (5.3±1.1 versus 2.7±0.1 nmol/mg wet weight; n=30; paired t test, P<.001).
Labeled Lipoproteins in Plasma
The volume of distribution was
41.4±1.4 mL/kg for labeled LDL
(n=29 injections in 22 rabbits) and 46.1±2.2 mL/kg for labeled
Lp(a)
(n=31 injections in 23 rabbits). In rabbits in which labeled Lp(a) and
labeled LDL were injected simultaneously, the average
volume of distribution was 38.5 mL/kg for both lipoproteins, and linear
regression analysis showed a close positive association between
volumes of distribution for the two lipoproteins (r=.93;
n=15; P<.001); this finding suggests that Lp(a) was not
damaged in these experiments. In contrast, it cannot be excluded that
the larger volume of distribution of labeled Lp(a) used in the 3-
versus 26-hour experiments reflects damage to the Lp(a) particle.
However, in vitro characterization of labeled Lp(a) used in the 3-
versus 26-hour experiments did not reveal any damage of labeled Lp(a)
(see below). Moreover, it seems unlikely that exactly those
preparations and not the ones used for simultaneous studies
on Lp(a) and LDL were altered. It is therefore most likely that the
larger volume of distribution of labeled Lp(a) in rabbits used for the
3- versus 26-hour experiments compared with those used for the 5- to
10-minute and 23-hour experiments simply reflects differences between
rabbits; eg, plasma cholesterol levels and body weights of
the rabbits used for studying Lp(a) in the 3- versus 26-hour
experiments were different from those of the rabbits used in the 5- to
10-minute and 23-hour experiments (Table 1).
Labeled
Lp(a) was removed from plasma at a faster rate than labeled LDL
(Fig 1); 23 hours after injection 19±3% (n=14,
data
from 3- versus 26-hour and 23-hour experiments combined) of labeled
Lp(a) and 54±2% (n=13) of labeled LDL remained in plasma
(nonpaired
t test, P<.001). The corresponding values in the
three rabbits used in the 23-hour-accumulation experiments with a
mean plasma Lp(a) concentration of 0.16±0.03 mg/mL after mass
injection of Lp(a) were 25±6% and 60±3% for Lp(a) and LDL,
respectively (paired t test, P<.01).
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, 
) and 131I- (
,
) labeled LDL and Lp(a). Values are expressed as percent of plasma
radioactivity after 5 to 10 minutes. Data were from two rabbits used in
the 3- versus 26-hour experiments.
Two-tier
rocket immunoelectrophoresis was used to estimate the
relative amount of labeled free apo(a) in plasma (Table 2). The
fraction of radioactivity in labeled Lp(a) that
migrated through anti-apoB–containing gel and precipitated in
anti-Lp(a)–containing gel, ie, labeled free apo(a), increased only
slightly, from 1% to 3% during 23 hours. However, the fraction of
labeled Lp(a) in plasma that migrated through anti-Lp(a)–containing
gel and precipitated in anti-apoB–containing gel, ie, labeled
"Lp(a)-," increased from 7% to 19% during 23 hours of
circulation in vivo. The values for labeled Lp(a) and labeled LDL in
preparations used for injections were similar to the values in plasma
at 5 to 10 minutes (data not shown).
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Comparing gel filtration profiles
of labeled lipoproteins used for
injection and labeled lipoproteins in plasma 23 hours after injection,
no small disintegration products of labeled Lp(a) and LDL were
observed (Fig 2). The recovery of labeled Lp(a) and LDL
were 87% to 98% (see legend for Fig 2). It cannot be excluded
that
loss of labeled Lp(a) during gel filtration
chromatography partly represents aggregation of
labeled Lp(a) or labeled free apo(a) on the top of the column. However,
the idea that a large fraction of the radioactivity in plasma was
present in free apo(a) after injection of labeled Lp(a) cannot be
supported by the results of two-tier rocket immunoelectrophoresis
(see above).
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Total and Tightly Bound Lipoproteins After 5 to 10 Minutes'
Exposure
After 5 to 10 minutes' exposure to labeled
lipoproteins, the
total amount of labeled LDL in the balloon-injured aortic
intima–inner media was larger than that of Lp(a) (paired
t test, P<.0001; Fig 3). However,
the tightly bound amount of labeled LDL was similar to that of labeled
Lp(a). The two rabbits that received a supplementary mass injection of
human Lp(a) before injection of labeled lipoproteins had plasma Lp(a)
concentrations of 0.10 and 0.15 mg/mL; the results obtained in these
two rabbits were similar to those obtained in the rabbits with only
trace amounts of Lp(a) in plasma (data not shown).
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The total and
tightly bound amounts of labeled LDL and labeled Lp(a) in
the normal aortic intima–inner media were similar after 5 to
10 minutes' exposure (Fig 3).
The percentage of
the total amount of labeled lipoproteins in the
intima–inner media that was extractable with 10 mmol/L
-amino-n-caproic acid after three preceding washes with
phosphate buffer was less than 3% in the balloon-injured aorta and
about 1% in the normal aorta for both Lp(a) and LDL.
Comparison of normal and balloon-injured aortic intima–inner media showed statistically significant differences between the two tissues (ANOVA, P<.0001): both the total and tightly bound amounts of labeled LDL and labeled Lp(a) were significantly higher in the balloon-injured than in the normal aortic intima–inner media (paired t tests, P<.001).
Permeability and Fractional Loss
In the balloon-injured
aorta, both permeability and fractional
loss in the intima–inner media were larger for labeled LDL than
for labeled Lp(a) (nonpaired t tests, P<.0001;
Fig 4). Also, crude fractional loss from
balloon-injured aortic intima–inner media was larger for
labeled LDL (89±1% per 26 hours; n=7) than for labeled Lp(a)
(70±8%
per 26 hours; n=8) (nonpaired t test, P=.001).
In
accordance with a larger loss of labeled LDL compared with labeled
Lp(a) from balloon-injured aortic intima–inner media during 3
hours, the intimal clearance of labeled LDL during this time
underestimated the aortic permeability relatively more than did the
intimal clearance of labeled Lp(a) (Fig 4). However, these
results
could be influenced by differences in the balloon injury between
rabbits used to study labeled LDL and Lp(a) (Table 1).
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.0001 (nonpaired
t tests) compared with LDL in balloon-injured aorta.
In normal aorta, permeabilities and fractional losses from the
intima–inner media of labeled LDL and labeled Lp(a) were similar
(Fig 4). Crude fractional loss from normal aortic
intima–inner
media was also similar for labeled LDL and labeled Lp(a): 68±6% per
26 hours (n=7) and 74±4% per 26 hours (n=8),
respectively.
Comparison of normal and balloon-injured aorta showed
that
permeabilities to both LDL and Lp(a) were markedly increased in
balloon-injured compared with normal aorta (paired t
tests, P<.0004; Fig 4). The fractional loss of
labeled LDL
was higher in the balloon-injured than in the normal aortic
intima–inner media (paired t test,
P<.0001), whereas the fractional loss of labeled Lp(a) was
similar in normal and balloon-injured aortic intima–inner
media (Fig 4).
Total and Tightly Bound Lipoproteins After 23 Hours'
Exposure
In balloon-injured aortic intima–inner media, the total
accumulation and amount of tightly bound labeled Lp(a) were 174%
(paired t test, P=.03) and 256% (paired
t test, P=.005), respectively, of the amounts of
labeled LDL after 23 hours' exposure to labeled lipoproteins (Fig
5). In the three rabbits that received a supplementary
mass injection of human Lp(a) before injection of labeled lipoproteins,
the total accumulation during 23 hours of labeled Lp(a) and LDL was
1.30±0.86 µL/cm2 and 0.73±0.26
µL/cm2, respectively.
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In normal aortic
intima–inner media, the total accumulation of
labeled LDL was significantly larger than that of Lp(a) (paired
t test, P=.004; Fig 5). However, there
was no
significant difference in the tightly bound amounts of labeled LDL and
Lp(a) in the normal aortic intima–inner media (paired t
test, P=.19).
The percentage of the total amount of
labeled lipoproteins in the
intima–inner media that was extractable with 10 mmol/L
-amino-n-caproic acid after three preceding washes with
phosphate buffer was 1.9±0.5% in the balloon-injured aorta for
labeled Lp(a) and 2.2±0.1% for labeled LDL. In the normal aorta, the
corresponding values were 4.0±0.5% for Lp(a) and 2.3±0.3% for
LDL
(paired t test, P<.005).
Comparison of normal and
balloon-injured aortic intima–inner
media showed that the total accumulation as well as the tightly bound
amounts of both labeled LDL and Lp(a) were markedly increased in the
balloon-injured aortic intima–inner media (paired t
tests, P<.01; Fig 5).
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion Appendix References |
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Effect of Balloon Injury
The present study compared the kinetics of LDL and Lp(a) in the rabbit arterial wall 1 to 5 days after a balloon injury. At this stage, the total cholesterol content of balloon-injured aortic intima–inner media was increased compared with the normal aortic intima–inner media. However, the formation of neoendothelium was minimal: no endothelial cells were observed on histological evaluation, although an average of 9% of the balloon-injured segment was not stained by Evans blue. This variation in staining intensity of the balloon-injured segment may be due to inhomogeneous damage by the balloon catheter or possibly local reendothelialization, which were not detected in the biopsies used for histological examination. A lack of endothelial cells in the histological sections is in accordance with data from a previous study,27 although another study28 found evidence for reendothelialization 1 week after a balloon injury of the abdominal aorta in rabbits.
Schwenke and Zilversmit have previously investigated the lipoprotein permeability of balloon-injured aorta within the first days after injury. In one study,29 the lipoprotein permeability was markedly increased, whereas in another study,24 using a milder balloon injury, albumin but not lipoprotein permeability was increased in the balloon-injured aorta. In the present study, the aortic permeability to both LDL and Lp(a) was markedly increased in the balloon-injured aorta.
In the two previous papers mentioned,24 29 fractional loss of esterified cholesterol and albumin was increased in balloon-injured compared with normal aorta. In accordance with this previous data, fractional loss of labeled LDL was markedly increased in balloon-injured compared with the normal aortic intima–inner media in the present study, whereas there was no difference in fractional loss of labeled Lp(a) between normal and balloon-injured aorta. Fractional loss of labeled LDL and Lp(a) from the normal aortic arch was 0.13±0.03 h-1 and 0.11±0.01 h-1, respectively, in the present study. These data compare favorably with a previous paper,30 in which the mean fractional loss of intact LDL in the aortic arch of five rabbits that were cholesterol fed for 8 days was 0.11 h-1. Thus, the present data are in accordance with the notion that a balloon injury induces a marked increase in permeability of the arterial wall to LDL and to Lp(a), which leads to an increase in aortic cholesterol content, despite an accompanying increased fractional loss of LDL from the injured intima–inner media.
Labeled Lp(a) and Labeled LDL in Balloon-Injured Aortic
Intima–Inner Media
The present data suggest that in normal
vessels the
accumulation of Lp(a) and LDL is much lower than in injured vessels and
that this difference between normal and injured vessels reflects an
increased permeability of the injured vessel. Moreover, the data
support the idea of a specific accumulation of Lp(a) compared with LDL
in injured vessels, despite a possible larger permeability of LDL than
of Lp(a) at sites of injury (see below).
The average fractional loss of labeled Lp(a) was 27% of that of labeled LDL in the balloon-injured aorta. Some of this difference may be ascribed to differences between rabbits used for Lp(a) studies and rabbits used for LDL studies, ie, the relative number of severely injured aortas, the average number of days from injury to removal of aorta, and the average cholesterol content of the injured aorta were larger for the LDL rabbits than for the Lp(a) rabbits. However, there was a large overlap between the two groups of rabbits in number of days between injury and removal of aorta (LDL range, 3 to 5 days; Lp(a) range, 2 to 4 days) and some overlap in aortic cholesterol content (LDL range, 2.1 to 36.5 nmol/mg wet weight; Lp(a) range, 1.8 to 4.7 nmol/mg wet weight). Despite these similarities, there was no overlap between fractional loss values for Lp(a) (range, 0.07 h-1 to 0.18 h-1) and LDL (range, 0.23 h-1 to 0.65 h-1). Furthermore, a subgroup of the rabbits used for studying fractional loss of LDL had a mean cholesterol content of the injured aorta (3.1±0.5 nmol/mg wet weight; n=3) similar to that in rabbits used for studying fractional loss of Lp(a) (3.7±1.4 nmol/mg wet weight). The mean fractional loss of LDL in this subgroup was 0.55±0.08 h-1, which is significantly larger than that of Lp(a) (0.12±0.02 h-1) (nonpaired t test, P<.0001). Thus, the present difference in fractional loss of Lp(a) and LDL is believed primarily to reflect differences between the two lipoprotein species rather than differences between rabbits used for Lp(a) and LDL studies.
It may also be speculated that the higher permeability to LDL than to Lp(a) in the balloon-injured aorta could be related to a longer period from injury to removal of aorta and/or more severe damage to aortas of rabbits used for LDL and than for Lp(a) studies. This idea is supported by the previous finding that severe injury increases the aortic influx of lipoproteins 2 to 5 days after injury,29 whereas mild injury did not.24 Also, in contrast to fractional loss values, there was a large overlap in permeability to LDL (range, 167 to 2549 nL/cm2 per hour) and to Lp(a) (range, 107 to 1040 nL/cm2 per hour) between the two groups.
In the normal aorta, the amount of labeled lipoprotein
in the
intima–inner media after 5 to 10 minutes' exposure primarily
reflects plasma contamination rather than labeled lipoproteins that
have entered the arterial wall.14 In the
balloon-injured aorta, on the other hand, the lipoprotein
permeability was on average 9 to 25 times larger than in the normal
aorta in the present study (Fig 4). Moreover, in a previous
study,
the blood contamination of normal and balloon-injured rabbit aorta
was similar when labeled red blood cells were injected 4 to 10 minutes
before the aorta was removed.31 Thus, influx of labeled
lipoprotein presumably contributed significantly to the total amount of
labeled lipoproteins in balloon-injured aortic intima–inner
media even after 5 to 10 minutes. This may suggest that the larger
amount of labeled LDL than of Lp(a) after 5 to 10 minutes in the
injured intima–inner media reflects a higher permeability to LDL
than to Lp(a), which is in accordance with data from the 3- versus
26-hour experiments.
In contrast to the aortic radioactivity values after 5 to 10 minutes' exposure, the accumulation during 23 hours represents the combination of influx and loss of labeled lipoprotein. The larger accumulation of labeled Lp(a) than of labeled LDL during 23 hours (calculated as arterial wall radioactivity divided by the mean plasma radioactivity concentration) supports the idea that the lower fractional loss is more important than the lower permeability for the long-term accumulation of labeled Lp(a) compared with LDL.
Previous studies have divided arterial wall radioactivity by the final plasma radioactivity concentration after 24 hours' exposure to iodinated LDL to estimate the apparent concentration of LDL in the arterial wall.32 33 If this approach was used in the present 23-hour experiments, the apparent content of Lp(a) and LDL in balloon-injured intima–inner media was 3.5±0.9 µL/cm2 and 1.2±0.2 µL/cm2 (paired t test on logarithmically transformed data, P=.003). However, since labeled lipoproteins in plasma and balloon-injured intima–inner media may not have equilibrated 23 hours after the intravenous injection of labeled lipoproteins, arterial wall radioactivity divided by the final plasma radioactivity concentration after 23 hours' exposure may overestimate the arterial wall pool size of Lp(a) by 51% and that of LDL by 9% (details are described in the "Appendix"). When these estimates were used to correct the apparent content of Lp(a) and LDL in the 23-hour experiments, the pool size of Lp(a) and LDL in the arterial wall was on average 2.3±0.6 µL/cm2 for Lp(a) and 1.1±0.2 µL/cm2 for LDL (paired t test on logarithmically transformed data, P<.02). Thus, the overall conclusion of a specific accumulation of Lp(a) relative to LDL in balloon-injured intima–inner media seems valid even when it is taken into account that labeled lipoproteins in plasma and the arterial wall may not have equilibrated after 23 hours' exposure.
Labeled Lp(a) and Labeled LDL in Normal Aortic Intima–Inner
Media
In the normal aortic intima–inner media, the total
accumulation during 23 hours of labeled Lp(a) was lower than that of
labeled LDL. This suggests that specific accumulation of Lp(a) compared
with LDL is limited to injured arterial sites. This
observation contrasts with a previous study in mice, which suggested
that more labeled Lp(a) than LDL accumulates in a normal aorta 24 hours
after intravenous injection of labeled
lipoproteins.34 The divergence between the previous and
present studies may reflect that the previous study used
autoradiography to quantitate aortic radioactivity
accumulation, whereas the present used gamma counting. Also,
species differences may play a role. In the present study, the
fraction of labeled Lp(a) that was extractable from normal aorta with
-amino-n-caproic acid was significantly higher than that
of LDL after 23 hours' exposure. Therefore, albeit the present
experimental designs were not able to demonstrate differences in
fractional loss or permeability of labeled Lp(a) and LDL in the normal
artery, there may be subtle differences in the metabolism
of Lp(a) and LDL in normal arterial tissue, which could
affect the relative accumulation of Lp(a) compared with LDL during
longer time periods than were studied in the present experiments.
The lack of difference in fractional loss and permeability of Lp(a) and
LDL in normal aorta disagrees with the observed lower accumulation of
Lp(a) than of LDL during 23 hours; however, permeability and fractional
loss were determined in different animals for Lp(a) and LDL. This means
that a difference between the two lipoprotein species is more difficult
to demonstrate in the 3- versus 26-hour experiments than in the 23-hour
experiment, in which the two labeled lipoproteins were injected
simultaneously into the same rabbit for direct
comparison.
Potential Limitations of the Present Study
It may be argued
that the differences between Lp(a) and LDL
resulted from the possible absence of endogenous Lp(a) in
contrast to the presence of large amounts of LDL in rabbit plasma.
However, the observed difference between labeled Lp(a) and labeled LDL
in accumulation during 23 hours was also found when the plasma Lp(a)
concentration was increased to human levels by intravenous
injection of the <1.12-g/mL-density lipoprotein fraction of human
plasma.
Metabolism of labeled Lp(a) in plasma may produce labeled
non-Lp(a) particles, which might interact differently than Lp(a) with
the arterial wall. When iodinated Lp(a) was
injected into humans, an increasing fraction of the total amount of
plasma radioactivity appeared in LDL-like particles, whereas no
radioactivity was found in free apo(a) or nonlipoprotein
particles.35 36 In the present study, the fraction of
Lp(a) radioactivity in LDL-like or "Lp(a)-" particles increased
from an initial 7% to 19% during 23 hours. Also, the gel filtration
profile of labeled Lp(a) in plasma gave some indications of formation
of LDL-like particles: on the gel filtration profile of labeled Lp(a)
in plasma 23 hours after injection, a shoulder corresponding to
LDL-sized particles may be observed (see Fig 2). Using two-tier
rocket immunoelectrophoresis, the fraction of Lp(a) radioactivity in
plasma present in free apo(a) was found to increase from an initial
1% to 3% 23 hours after injection of labeled Lp(a).
To estimate the influence of labeled Lp(a)- and labeled free apo(a) in plasma on the observed permeability and fractional loss of labeled Lp(a), the mean arterial wall radioactivity 3 and 26 hours after IV injection of labeled Lp(a) was corrected for the estimated contribution from labeled free apo(a) and labeled Lp(a)- the following way: assuming that the fraction of total radioactivity in plasma that was present in Lp(a)- and free apo(a) increased linearly from 0 hours to 26 hours (from 7% to 19% and from 1% to 3%, respectively), plasma radioactivity decay curves were constructed for Lp(a)-, free apo(a), and intact Lp(a).
The contribution of labeled Lp(a)- to
radioactivity in the
balloon-injured aorta was then estimated (using Equation 3 in
the
"Appendix"). Lp(a)- presumably is similar to LDL; it was
therefore assumed that Lp(a)- interacts with the balloon-injured
segment like LDL. Fractional loss and permeability of LDL were not
determined in the rabbits used for studying Lp(a). Alternatively, mean
values of fractional loss (0.44 h-1) and
permeability (1.4 µL/cm2 per hour) that were obtained in
the 3- versus 26-hour studies on LDL were used; then, 15% and 11% of
the total radioactivity in the injured aortic intima–inner media
would be attributable to Lp(a)- 3 hours and 26 hours after injection of
labeled Lp(a). Permeability and fractional loss of free apo(a) in the
balloon-injured aorta are unknown. However, since the size of
apo(a) is comparable with that of albumin and HDL, it was
assumed as a best guess that in the balloon-injured
intima–inner media, the apo(a) permeability was two times that of
the uncorrected Lp(a) permeability and that the fractional loss of
apo(a) was 0.99 h-1 (in normal rabbits,
the aortic permeability of albumin and HDL was about 1.9 and
2.3 times as high as the permeability of LDL,37 and the
fractional loss of albumin was on average 0.99
h-1 from a balloon-injured rabbit
aorta24 ); then, 0.8% and 0.5% of the total radioactivity
in the balloon-injured intima–inner media was attributable to
labeled free apo(a) after 3 and 26 hours' exposure, respectively. A
worst case was also considered in which the apo(a) permeability was 5
times that of Lp(a) and the fractional loss of labeled apo(a) was zero;
then, 7% and 39% of the total radioactivity in the injured
intima–inner media would be attributable to labeled free
apo(a).
After corrections of plasma and arterial wall radioactivity for radioactivity in Lp(a)- and free apo(a), as described above, permeability and fractional loss of intact Lp(a) in the balloon-injured intima–inner media were recalculated to be 0.42 µL/cm2 per hour and 0.10 h-1 for the best guess and 0.43 µL/cm2 per hour and 0.16 h-1 for the worst case. The mean permeability was 0.46 µL/cm2 per hour, and the mean fractional loss was 0.12 h-1 for Lp(a) when calculated using total plasma and aortic radioactivity. These calculations suggest that even if the worst-case assumptions are applied, the observed contamination of total Lp(a) radioactivity in plasma with radioactivity in Lp(a)- and free apo(a) does not affect the overall conclusions of the present paper.
Calculation of fractional loss is based on the assumption that the lipoprotein kinetics in aortic intima–inner media can be described by a one-pool-compartment model. This has not been proven for the balloon-injured aorta, but previous studies have found the assumption to be reasonably appropriate for iodinated LDL in normal and atherosclerotic aorta of rabbits22 and monkeys.23 Also, it should be noted that crude fractional loss, in which the calculations are not based on the one-compartment assumption,22 was also larger for labeled LDL than for labeled Lp(a) in the balloon-injured aortic intima–inner media. Furthermore, in contrast to the marked difference between LDL and Lp(a) in balloon-injured aortic intima–inner media, fractional loss, as well as crude fractional loss, was similar for the two lipoproteins in the normal aortic intima–inner media; it should be noted that fractional loss of Lp(a) and LDL was determined in different groups of rabbits but that the comparison between injured and normal aorta was done in the same animal. This consideration further suggests that the observed difference between Lp(a) and LDL in the balloon-injured aortic intima–inner media was not a result of differences in plasma metabolism of labeled Lp(a) and labeled LDL but rather reflects differences in metabolism of the two lipoprotein species within the injured arterial wall.
The present Lp(a) studies were performed with Lp(a) isolated from 10 different persons with different isoforms of apo(a), including the F, S1, S2, S3, S4, and S6 isoforms. In most studies, however, two or three apo(a) isoforms were studied simultaneously. Therefore, it cannot be excluded that the present differences between LDL and Lp(a) are enhanced or reduced for certain apo(a) isoforms.
Mechanisms for Specific Accumulation of Lp(a) Compared With LDL in
Injured Intima–Inner Media
Removal of the arterial endothelium
by
balloon injury reduces the anticoagulant capacity of the
arterial surface,38 favoring deposition of
fibrin. Also, production of
glycosaminoglycans is increased within the first 4
days after injury.39 Specific accumulation of Lp(a)
compared with LDL in the injured arterial wall may thus be
related to the larger capacity of Lp(a) than of LDL to bind to
fibrin40 or to form complexes with arterial
wall glycosaminoglycans and
proteoglycans.11 In the present study, binding of
labeled Lp(a) to lysine residues in balloon-injured aortic
intima–inner media was investigated; after extensive washing of
the aortic intima–inner media with buffer, Lp(a) was extracted in
the presence of a competitor for lysine binding residues, ie,
-amino-n-caproic acid. The amount of labeled Lp(a)
extracted with -amino-n-caproic acid from injured aortic
intima–inner media was quantitatively insignificant compared with
the total amount of labeled Lp(a) present. Furthermore,
-amino-n-caproic acid extracted labeled Lp(a) and labeled
LDL with similar efficiency from the injured tissue. The present
study, therefore, cannot support the proposition that selective
accumulation of Lp(a) compared with LDL in balloon-injured aortic
intima–inner media is mediated by binding of Lp(a) to lysine
residues on the fibrin surface. Nevertheless, binding of Lp(a) to
fibrin may be of importance since non–lysine-mediated binding
of Lp(a) to fibrin has been described.40 Also, it is
possible that labeled Lp(a) was incorporated into fibrin clots in the
injured intima–inner media: Lp(a) can be extracted from human
atherosclerotic lesions after digestion with
plasmin.41
Implications for Atherosclerosis,
Restenosis, and Thrombotic Occlusion After
Angioplasty
In the present study, balloon injury of the aorta was
performed an average of 3 days before studying accumulation of Lp(a)
and LDL in the aorta. This procedure enabled study of the relative
accumulation of Lp(a) and LDL in an in vivo model of fibrin deposition
at the surface of the arterial wall with
simultaneous exposure of subendothelial
components of the arterial wall to plasma lipoproteins.
Cholesterol feeding was commenced 5 to 9 days before
measurement of lipoprotein accumulation in the arterial
wall to minimize loss of LDL from the normal arterial
intima via degradation of labeled LDL via the LDL receptor, which may
account for 40% to 50% of the LDL degradation in normal rabbit
intima42 ; cholesterol feeding of rabbits for 8
or 16 days decreases the fractional degradation rate of LDL in
aorta.30 Cholesterol feeding of rabbits,
however, produces lipoprotein particles unlike those present in
human plasma. Accordingly, it cannot be excluded that such lipoproteins
affected the interaction of human Lp(a) and LDL with the normal or
injured intima–inner media in the present study and thus
affected the present results. Given this reservation, the
present data suggest that balloon injury accelerates the
accumulation of Lp(a) in the arterial wall and,
furthermore, that the accumulation of Lp(a) may exceed that of LDL at
sites of injury, possibly because of a preferential retention of Lp(a)
compared with LDL. As discussed above, the latter notion may reflect
incorporation of Lp(a) into intramural fibrin at sites of injury.
Binding of Lp(a) to intramural fibrin after endothelial
injury may inhibit fibrinolysis10 and may
by this mechanism facilitate thrombotic occlusion at sites of
angioplasty.
The relevance of gross endothelial denudation in formation of atherosclerosis has been questioned: detailed ultrastructural studies have shown that developing atherosclerotic lesions are covered by an intact endothelium and that the structure of lesions induced by mechanical injury is not exactly identical to that of atherosclerotic lesions (review in References 43 through 45). On the other hand, focal endothelial cell death occurs, and it is conceivable that microscopic loss of endothelium could be involved in initiation of atherosclerotic lesions. Furthermore, balloon injury of the arterial endothelium induces increased matrix formation,39 which is also observed in atherosclerotic lesions, and retention of lipoproteins by matrix components most likely plays a central role in early atherogenesis.46 Although it may be argued that accumulation of Lp(a) at sites of injury may lead to accumulation of lipids in the arterial wall during formation of neointima,41 the morphology of the aorta after the acute injury was clearly different from the morphology of atherosclerotic lesions. Extrapolation of the present results to atherogenesis may therefore not be valid and if attempted, it should be done only with utmost caution.
The tissue events that follow a balloon injury may better mimic events following percutaneous transluminal angioplasty.45 47 Accelerated accumulation of Lp(a) in the intima–inner media after balloon injury may lead to a stimulation of smooth muscle cell growth by the inhibitory effect of Lp(a) on formation of active transforming growth factor ß,9 which in theory may contribute to restenosis.
In summary, the present results suggest that balloon injury of the thoracic aorta of rabbits leads to accelerated accumulation of both LDL and Lp(a) in the intima–inner media. Further, the smaller fractional loss and the larger total accumulation after 23 hours' exposure of labeled Lp(a) compared with LDL in the balloon-injured intima–inner media together suggest a specific accumulation of Lp(a) compared with LDL at sites of endothelial injury, despite a possible larger permeability to LDL. Whether Lp(a) also accumulates to a larger extent than LDL in atherosclerotic lesions remains to be determined.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Danish Heart Foundation, the Danish Medical Research Council, and the Danish foundations "Novo's Fond Komite" and "Overlæge Johan Boserup og Lise Boserups Legat." Thanks are extended to Thomas Schieke, PhD, Department of Biostatistics, University of Copenhagen, for help in the statistical analysis; to Professor A. Niendorf, Department of Pathology, University of Hamburg, for preparing histological sections and evaluating lesion severity in balloon-injured aortas; and to Dr Matti Jauhiainen, National Public Health Institute, Helsinki, Finland, for determining apo(a) isoforms. Karen Rasmussen, Hanne Damm, and Kurt S. Jensen provided skillful technical assistance.
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Footnotes |
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Reprint requests to Dr Børge G. Nordestgaard, Department of Clinical Biochemistry, Herlev Hospital, DK-2730 Herlev, Denmark.
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Appendix |
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TopAbstractIntroductionMaterials and Methods Results Discussion Appendix References |
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Aortic radioactivity divided by the radioactivity concentration in plasma at late time points can be used to estimate the arterial pool size of intact lipoproteins in a system in which the plasma radioactivity concentration is constant, or in a system in which the rate by which labeled lipoproteins are lost from the arterial wall is high compared with the rate by which labeled lipoproteins are removed from the plasma compartment. However, this may not be the case for Lp(a) in the present study, as described in detail below.
In the one-pool-compartment model the kinetics of labeled lipoproteins in the arterial intima can be described by:
 | (1) |
with two unknowns: ki (permeability) and ke (fractional loss). P(t) is the plasma radioactivity concentration, and A(t) is the aortic radioactivity at time t.
To solve this equation, plasma radioactivity decay curves were fitted to double-exponential functions:
 | (2) |
in which H1, H2, g1, and g2 are constants: g1 and H1 are slope and intercept for the initial rapid removal of radioactivity from plasma; g2 and H2 are slope and intercept for the late slow removal of radioactivity. Thus, g1>g2.
Equation 1 can be
solved as described in Reference 2424 :
 | (3) |
 |
The aortic radioactivity divided by the plasma radioactivity concentration as a function of time can then be written as:
 | (4) |
 |
This
equation can be rewritten as:
 | (4A) |
, a and c converge toward 0
at late
time points if ke>g2 (a and c
converge toward 
if ke<g2). Since
g1>g2, b converges toward 0
and d converges toward ki/(ke-g2) at late
time points. %In the present study, ke>g2; therefore:
 | (5) |
If, on the other hand, ke<g2, then:
 | (6) |
Because the predicted pool size of labeled lipoproteins is equal to:
 | (7) |
the
aortic radioactivity divided by the plasma concentration at
late time points is only a reasonable estimate for the pool size if
g2 
ke.
In the present 3- versus 26-hour experiments, the average g2 for LDL was 0.025 h-1, whereas ke was 0.13 h-1 and 0.435 h-1 in the normal and balloon-injured aorta, respectively. Accordingly, at very late time points, A(t)/P(t) would overestimate the pool size of LDL by 24% and 6% in normal and balloon-injured aorta. For Lp(a), g2 was 0.057 h-1 and ke was 0.114 h-1 and 0.116 h-1 in normal and balloon-injured aorta, respectively. At very late time points, therefore, A(t)/P(t) would overestimate the pool size of Lp(a) by 99% and 95% in the normal and balloon-injured aorta, respectively.
To further illustrate
that A(t)/P(t) cannot accurately estimate the
arterial pool size of labeled lipoproteins in the
present studies, A(t)/P(t) versus time curves were constructed for
LDL and Lp(a) in normal and balloon-injured aorta (Fig 6) using
Equation 4; values for average plasma decays,
permeability, and fractional loss of Lp(a) and LDL were entered in the
equation. From Fig 6 it appears that A(t)/P(t) at 23 hours is
497
nL/cm2 and 3436 nL/cm2 for LDL in normal and
balloon-injured aorta, respectively, and 703 nL/cm2 and
6028 nL/cm2 for Lp(a) in normal and balloon-injured
aorta, respectively. Using Equation 6, the pool size of LDL is
57.2/0.13=440 nL/cm2 [88% of A(t)/P(t) after 23
hours]
and 1409/0.435=3154 nL/cm2 [92% of A(t)/P(t) after 23
hours] in normal and balloon-injured aorta, respectively; for
Lp(a) the pool size is 53/0.114=465 nL/cm2 [66% of
A(t)/P(t) after 23 hours] in normal aorta and 463/0.116=3991
nL/cm2 [66% of A(t)/P(t) after 23 hours] in
balloon-injured aorta.
In conclusion, these calculations illustrate that A(t)/P(t) is not necessarily similar to the arterial wall pool size of labeled lipoproteins at late time points.
Received August 29, 1995; accepted December 22, 1995.
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