Detection and Quantification of Lipoprotein (a) in the Arterial Wall of
107 Coronary Bypass Patients
Matthias Rath, Axel Niendorf, Tjark Reblin, Manfred Dietel, Hans Joachim
Krebber, and Ulrike Beisiegel
Journal of Orthomolecular Medicine, August 1992
Lipoprotein (a) [Lp(a)] is a lipoprotein similar to low density lipoprotein
(LDL) in its lipid composition and the presence of apoprotein (apo) B-100.
In contrast to LDL, Lp(a) contains an additional glycoprotein, designated
(a), which is linked to apo B by disulfide bridges. The diameter of the
particle is 250 Å, and it floats in a density range of 1.05 to
1.12 g/ml. The Lp(a) particle contains a high amount of neuraminic acid
due to the highly glycosylated (a),1-44 and only a few areas of helical
structure could be demonstrated in the glycoprotein (a). 5 In spite of
this low lipid-binding capacity, glycoprotein (a) is part of a lipoprotein
and, therefore, it was generally agreed that it should be called apo(a).
Apo(a) is a high molecular weight protein with an apparent molecular
weight of more than 500 kD in sodium dodecyl sulfate-polyacrylamide gradient
gel electrophoresis (SDS-PAGE). 4 A genetically determined heterogeneity
in the form of several bands in SDS-PAGE has been describe. 6,7 The apparent
molecular weight difference cannot be explained by the sialic acid moiety.
6 In fresh human serum, 95% of apo (a) is lipoprotein associated. 8
Recently, a striking homology between the human apo(a) and plasminogen
was demonstrated in both amino acid9, 10 and DNA sequences. 11 Serum
and liver samples from various species were analyzed for the presence
of Lp(a) but only humans, primates, 12 and hedgehogs13 were found to
express apo(a).
Lp(a) was first demonstrated in human plasma by Blumberg et al.14 and
later by Berg and his associates. 15 These and alter studies provided
evidence that Lp(a) is a qualitative and quantitative genetic trait.
16,17,18 Around 70% of the normal population have serum Lp(a) levels
below 25 mg/dl. 19 Utermann et al.6 have postulated that there is a
highly significant association between Lp(a) concentration in serum
and the different apo(a) pheno-types in SDS-PAGE.
In a series of epidemiological studies, a positive correlation of high
serum Lp(a) levels with coronary heart disease (CHD) has been demonstrated.20-23
In imunohistochemical studies, Walton et al.24 detected apo(a) in the
arterial wall; however, Lp(a) was not considered to participate in atherogenesis.
There are several other studies, the first as early as 1958,25 which
extensively analyzed human arterial wall tissue for its lipoprotein content.
None of these studies included Lp(a), and they mainly concentrated on
LDL.26-32 Smith and Slater26 observed a relationship between serum lipid
levels and LDL in the aortic intima of 21 post-mortem samples, and "mobile
and immobilized LDL" were described in atherosclerotic lesions by
Smith et al. 27 To relate the apolipoprotein accumulation in the arterial
wall to the development of arteriosclerosis, Hoff et al. 28 examined
normal intima and plaques and quantified apo B in human arterial fatty
streaks. The most recent work, published by a Finnish group, 33 demonstrated
apo B- and apo E- containing lipoproteins in lesion-free human aortic
intima.
In studies on the possible interaction between LDL and arterial wall,
it has been demonstrated that the binding of Lp(a) to glycosaminoglycans
is stronger than the binding of LDL.34 Similar to modified LDL, dextran
sulfate- modified Lp(a) caused an increase of cholesterol ester accumulation
in amcrophages.35
The aim of this study was to investigate a possible accumulation of
apo(a) in the arterial wall depending on serum Lp(a) concentrations and
to compare these data to the relation between serum and arterial wall
apo B. We did that by quantifying apo(a), apo B, and lipids in fresh
arterial wall tissue. With these experiments, we wanted to determine
whether Lp(a) is an independent risk factor for CHD.
Methods
Patients, Serum and Tissues Samples
Preoperative fasting serum was collected from 306 patients (250 men.
56 women; mean age, 57 years) who were undergoing aortocoronary bypass
surgery in the department of Cardiovascular Surgery. Hamburg University
Clinic. The blood was drawn upon admission to the hospital 48 hours before
the operation. Of the patients, 20% were taking lipid-lowering medicines,
but only 3% had reached normal lipid values with treatment. The control
group was 72 factory workers from a local pharmaceutical company, who
were fasting and were matched for sex and age.
Tissue samples were obtained from 107 of the coronary bypass patients
(mean age 59 years): We used the biopsies routinely taken during an aortocoronary
bypass operation where the vein graft is attached to the ascending aorta.
By histological screening, the biopsies showed different grades of intimal
thickening compared with control tissue of newborns. No biopsies of severe
plaque areas or complicated lesions were examined. Venous samples were
taken from the vena saphena magna, which served as the bypass graft.
The project was approved by the Physicians’ Ethical Commission
of Hamburg.
Post-morten Blood and Tissue
Post-mortem tissue was obtained from autopsy cases within 24 to 28
hours after death. Samples from 11 different individuals were taken
from the ascending aorta and the main stem of the left coronary artery
and exhibited different degrees of atherosclerotic lesions. For immunohistochemistry,
samples were taken from the left descending coronary artery (LAD).
To gain a representative picture of the arterial wall, areas with or
without plaques were used. Other samples were taken as indicated in
the text below. No post-mortem serum was systematically evaluated because
of questionable values due to hemolysis and dilution with other body
fluids. For the lipoprotein particle study, we used the patients’ pre-mortem
blood obtained from the Department of Clinical Chemistry. These samples
were stored as plasma for not more than 24 hours at room temperature
before testing. Lipoproteins, Lipidis, and Apoprotein Determination
Cholesterol was estimated by the use of "Monotest" (CHOD-PAP
method) from Boehringer Mannheim. For triglyceride determination. "Peridichrom" (GPO-PAP)
from Boehringer Mannheim was used. High density lipoprotein (HDL) cholesterol
was quantitated after precipitation of apo B-containing lipoproteins
by phosphotungstic acid/Mg (Boehringer Mannheim, Mannheim, FRG).
The density gradient centrifugation of serum was carried out according
to the method of Redgrave et al. 36 The stepwise gradients were layered
as follows; 3ml of serum adjusted to density d=1.21 g/ml with KBr; 3
ml of 0.9% NaCl, pH7, adjusted to d=1.063 g/ml with KBr; 3ml of the same
solution adjusted to d=1.019 g/ml; and 1 ml of H2O. The spin was carried
out in a TH-641 Sorvall Dupont (Wilmington, DE) rotor from for 21 hours,
200 000 g (40 000 rpm) at 4°C. After the spin, 0.5-ml fractions were
taken from the bottom of the tube (Beckmann fractionation-setup).
The Lp(a) in the 306 bypass patients (Table 1) was measured using radial
immunodiffusion (Immuno, Heidelberg, FRG). The Lp(a) standard from Immuno
Heidelberg was used for this assay and was adjusted to determine the
protein was measured in the isolated Lp(a) and then diluted in lipoprotein-free
serum. The data in this paper, therefore, describe the apo(a) and apo
B [apo B-(a)-complex] in the samples. The tissue apo(a) was calculated
on the assumption that around 45% of the protein in the Lp(a) was apo
B (Ewald Molinari, Immuno GmbH, Vienna, Austria, personal communication).
The 107 patients with tissue samples, apo B and apo(a) in plasma and
tissue homogenates were quantified with enzyme-linked immunosorbent assay
(ELISA) (see below). In plasma, both parameters were also determined by
radioimmunoassy (RIA) from Pharmacia (Uppsala, Sweden). A standard serum
was supplied by Pharmacia to determine the total protein content of the
particle, apo(a) and apo B. This additional assay was used to confirm
the ELISA values by another commercially available method. The correlations
between RIA and ELISA were r=0.9 for apo(a) and r=0.6 for apo B (p<0.001).
The internal standards in our laboratory were in a 10% range. All data
given in this paper for serum and tissue of the 107 coronary bypass patients
were based on the ELISA system.
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Antibodies
Production fo Ployclonal Anti-apo B
Apo B was prepared from human serum LDL. After density gradient, the
LDL fraction was reduced with 10% (vol/vol) mercaptoethanol; then solid
SDS was added up to 5% and the sample was applied to a Sepharose CL
2B column (94x2.6cm for apo B purification. Antibodies were raised
in rabbits against this purified apo B. The antiserum was purified
by LDL affinity column where apo(a)-free LDL was coupled to CNBr-activated
Sepharose. The purified antibodies were conjugated to peroxidase37
and were used in the ELISA.
Polyclonal anti-apo B from Immuno was used for the apo B ELISA. This
antibody was checked by immonoblotting for its specificity; it recognized
only apo B-100, apo B-48, and the other known fragments of apo B-100.
Monoclonal antibodies against apo B were kindly provided by Yves Marcel
(Montreal, Canada). They were used only to control the validity of the
results obtained with our polyclonal antibodies.
Antibodies against Apo(a)
To establish the ELISA system, monoclonal antibodies, KO 7 and KO 9 (peroxidase
conjugated) against apo (a), were kindly provided by Jean-Charles Fruchard
(Lille, France). 38 For this antibody, we showed by Western blotting
and ELISA that the cross-reactivity with plasminogen was less than
5% Polyclonal anti-apo(a) was produced in our laboratory against the
purified apo(a) (see below) in rabbits.
n parallel, we produced monoclonal antibodies against the isolated apo(a).
The purification was performed by recentrifugation of the density fraction
d=1.08 to 1.15g/ml in a density gradient after reduction with dithiothreitol.39
Two apo(a) isoforms, a3 and a5, were used for immunization (see below).
The antigen was injected into Balb C mice.
The mouse lymphocytes were fused with NSO-1 myeloma cells by using PEG
1500 (Boehringer) and the protocol of Köhler and Milstein.40 Modifications
will be described elsewhere (Beisiegel et al., unpublished observation).
The hybridoma supernatants were screened on 96-well plates coated with
apo(a) or with plasminogen. Ninety percent of the apo(a) positive supernatants
showed cross-reactivity with plasminogen. The hybrids that were specific
for apo(a) were subcloned. In this paper, we used the subclone, 8D3.
All monoclonal and polyclonal antibodies were tested on immunoblots,
and all of them recognized the same banding patterns in 20 patients.
The banding patterns included seven bands: one faster than apo B, one
in the apo B position, and five above the apo B band. We designated
them a1 to a7, from low to high molecular weight.
Homogenization of Tissue
The freshly taken biopsies were rinsed in physiological NaCl several
times. The adventitia was dissected and discarded. The biopsies were
blotted dry and stored in liquid nitrogen until used. The biopsies were
cut into small pieces and homogenized in a Potter glass homogenizer for
3 minutes with 25 m l buffer (10 mM Tris/HCl, pH 8.0, containing 154
mM NaCl, 1mM EDTA, and 1% Tween 20) per milligram wet weight (WW). The
homogenate was then centrifuged at 56 000 rpm for 10 minutes in a Beckmann
TL 100 ultracentrifuge with a TLA-100.2 rotor. The pellet was discarded,
and the supernatant was analyzed for total Tween-soluble cell protein,
total cholesterol, triglycerides, and the Tween-soluble apo(a) and apo
B content. The protein was determined according to the method of Lowry
et al.41 by using bovine serum albumin as the standard. Apo B and apo(a)
were measured with ELISA as described below. The post-mortem tissue samples
were prepared in the same way. For the study of lipoprotein particles,
a slightly different extraction method was applied to the post-mortem
arterial wall. The tissue was cut into small pieces and was gently shaken
overnight at 4°C in 2 ml/g WW of the buffer described above, but
without detergent. After centrifugation, the supernatant was assayed
as describe above.
ELISA Techniques
Tissue Determinations
For quantification of apo B and apo(a) in the arterial wall homogenates
, we used a sandwich ELISA technique, a modification of the ELISA system
described by Vu-Dac et al.38 For the Apo(a) ELISA, 96-well plates were
coated with monoclonal anti-apo(a) (KO7) in a concentration of 25m
g/ml overnight at room temperature. Washing, blocking, and dilutions
were performed in 0.1 M phosphate-buffered saline (PBS), pH 7.4, containing
1% bovine serum albumin (Sigma, fraction V) and 1% Tween 20. After
washing and blocking, the homogenized samples were applied in quadruplicate
or duplicate. At the end of the incubation (2hours at 37°C), the
homogenates from parallel wells (duplicates and quadruples) were taken
out and pooled, and 50m l of the pool was transferred onto a second
plate coated with anit-apo B (see below). The original plate coated
with anti-apo(a) was washed, and then half of each set of quadruples
or duplicates was treated with monoclonal anti-apo(a) [KO 9 (peroxidase
conjugate) diluted 1:4000]. The other half was treated with our plolyclonal
anti-apo B (peroxidase conjugated, diluted 1:100) to determine the
amount of apo B associated with the apo(a) [[apo B-(a) complex). For
the quantification of the apo B, we used the apo B standard on an anti-apo
B coat as described for the apo B ELISA. The incubations with the detection
antibodies were performed at 37°C for 2 hours. After washing, the
KO 9 peroxidase antibody was visualized with 100 m l of o-phenyldiamine
in 0.1 M citrate buffer (pH 5.0). The reaction was stopped after 10
minutes by the addition of 100 m l 1 N HCL. The anti-apo B peroxidase
was treated as described below.
To evaluate our assay systems, we used post-mortem tissue, Performing
triplicates, the intra- and interassay varianaces were below 10% for
both apoproteins. This was particularly necessary, since in 45 cases,
we had only extremely small sample volumes (less than 500m l, from which
all measurements had to be made), and we were not able to do all ELISA
measurements in quadruplicate on the first plate. Therefore, we did not
have duplicates for all final apo(a) and apo B measurements of all tissue
samples.
The maximum capacity in the apo(a) ELISA was 35 ng/well, and if we stayed
in this limit (by using the adequate dilutions), less than 5% of apo(a)
could be measured in the transferred homogenates in a subsequent ELISA.
The same kinds of transfer assay were performed with serum and lipoprotein
fractions and were thereby proven to be dependable. This control was
necessary to ensure that only apo B-containing particles were transferred
and not residual Lp(a), which might not have been bound in the first
incubation.
The apo B ELISA was used to determine the total apo B in fresh homogenates
as well as to measure apo B particles that did not contain any apo(a)
in the homogenates transferred from the apo(a) ELISA. The plates were
coated with polyclonal anti-apo B antibodies (Immuno) in a 1:1000 dilution.
The incubations were done under the same conditions as described for
the apo(a) ELISA. The apo B was detected by using our peroxidase-conjugated
polyclonal anti-apo B antibody. As substrate, 1,2-phenylenediaminedihydrochloride
(Boehringer Ingellheim, Garching, FRG) was used, and, after 10 minutes,
8 N H2SO4 was added to stop the reaction.
With the combination of these two ELISAs, we could differentiate between
the apo B linked to the apo(a) [apo B-(a) complex] and apo B not associated
with apo (a). In 32 cases, the sum of these twoapo B-containing subfractions
was comparable to the total apo B as determined by the same ELISA (Table
3).
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Plasma Determinations
For the quantification of apo(a) in the plasma samples, the same ELISAs
were used; however, no Tween was added to the buffers. Apo B quantification
in plasma included 0.05% Tween 20 in all buffers.
Standards
As a principle, all standards were treated exactly the same way as the
samples. For the determination of apo (a) in the tissue samples, we
therefore added 1% Tween 20 into the standard, since that was the Tween
concentration we used in the homogenates and in all ELISA buffers.
In the apo(a) ELISA, the Lp(a) standard serum supplied by Immuno was
used. We compared the Immuno standard with Lp(a) and apo(a) isolated
from a serum pool. The isolated samples were examined for their protein
content with the method of Lowry et al.41 The Lp(a) was found to be in
good correspondence with the standard with a deviation of less than 10%
and the apo (a) was overestimated by around 50% as expected from using
the "apo B-(a)" standard. Thus, for the calculation of the
arterial wall apo (a), we might have overestimated the apo (a) content,
depending on the ratio of free apo (a) in the tissue.
In the apo B ELISA, the apo B standard from Immuno was used when plasma
samples were measured. For the tissue homogenates, we prepared our
own pure apo B standard in 1% Tween 20. Apo B was isolated with column
chromatography and was delipidated and solubilized in SDS. For the
ELISA, apo B was dialyzed extensively against 1% Tween 20 to minimize
the residual SDS content and to reach the same Tween content as the
tissue samples. The protein content was determined by the method of
Lowry et al.41
SDS-
PAGE and Immunoblotting
Serum and tissue homogenates were delipidated in acetone/ethanol 1:1
/vol/vol). Samples were prepared in 0.02 M ethylmorpholine containing
5% SDS, were reduced with 10% mercaptoethanol at 100°C, and were
applied to an 8% PAGE with 0.1% cross-linker according to the method
of Neville.42 Subsequent and immunoblotting was performed as previously
describe. 43 The legends to the figures indicate which antibodies were
for incubation of the blots.
Morphologic Methods
For immunohistochemistry, the tissue was rinsed in PBS and was fixed
immediately in 3.7% PBS-buffered formalin. Following standard procedures,
the tissue was paraffin-embedded, cut, and mounted on coated glass sides.
Sections of autopsy material were histologically stained with hematoxylin-eosin
and elastica-van Gieson. Lesions were classified into fatty streak, fibrous
plaque, of complicated lesions according to common histological criteria.
Immunohistochemical localization of apo B and apo (a) was performed by
means of the avidin-biotin-peroxidase (ABC) method.44,45 The above-mentioned
polyclonal rabbit ant-apo B and rabbit anti-apo(a), which were produced
in our laboratory, as well as monoclonal ant-apo(a) antibodies (8D3)
were applied to the sections. Controls consisted of replacement of the
first antibody with a nonimmune serum of the same species.
Statistical Methods
For statistical evaluation, all parameters with a skewed distribution,
such as serum Lp(a), tissue apo(a), and apo B, were transformed logarithmically
for correlations. The statistical significance for these values was calculated
with the Mann-Whitney U test.
Table 1.
Results
The serum lipid parameters from 306 patients who underwent aortocoronary
bypass surgery and had angiographically determined CHD were measured
and compared with an age-matched control group of normal factory workers
(Table 1). The comparison of these groups showed significant differences
in serum triglyceride (p<0.0001) and HDL cholesterol (p<0.0001).
Total serum cholesterol was higher in the CHD group, even though it was
not highly significant (p<0.01). In addition to the common lipoprotein
parameters, serum Lp(a) was measured. In Table 1, the percentage of subjects
with serum Lp(a) levels greater that 25 mg/dl is shown (16% in controls
and 40% in the CHD group, p<0.0001). Lp(a9 values were expressed as
mg/dl apo B-(a) complex measured with immunological techniques. Mean
values are also given in Table 1, but note that Lp(a) is not normally
distributed, as shown in Figure 1. The difference between the groups
is clearly confirmed by the mean values: 14 mg/dl in the controls and
25 mg/dl in the bypass patients.
From 107 of the 306 bypass patients, we obtained arterial wall tissue
samples from the ascending aorta at the time of surgery. The tissue samples
were used to demonstrate a possible correlation between lipoprotein levels
in serum and arterial wall tissue. No biopsies of severe plaque areas
were used, although the degree of intimal thickening varied.
Figure 1.
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Our main interest was to study apo B- and apo (a)- containing particles
in the tissue. A special ELISA technique (described under Methods) allowed
us to determine the concentration of apo(a), apo(a)-linked apo B, and
apo B not linked to apo(a) in the same sample. The lipoprotein parameters
analyzed in the tissue samples are given in Table 2. Note that the standard
deviation in Table 2 is very high, reflecting the fact that the lipid
and protein content in the biopsy samples varied considerably, due to
the degree of intimal thickening.
In patients with high serum Lp(a) levels, the apo(a) content in aortic
biopsies were, as well as the apo(a)- linked apo B, significantly higher
than in patients with low serum Lp(a). This led to an increase of total
apo B in the arterial wall of patients with Lp(a) greater than 25mg/dl.
All other parameters in Table 2 were not significantly influence by
the Lp(a) serum level.
Table 2.
Table 3 compares the lipoprotein content of aortic biopsies to venous
samples (n=32). The mean values for total apo B and apo(a) in the veins
were significantly lower, 55% and 27% respectively, of the aortic biopsy
values. Total cholesterol and triglyceride levels were also much lower
in veins (33% and 73% of the aortic values). Table 3 also includes the
measurements for apo B linked to apo (a) and not linked to apo(a) (see
Methods) for the aortic biopsies. The calculated sum of these two values
(42.9m g/mg WW). In this subgroup of patients, 83% of the apo B was apo(a)
linked. It seems, however, that the amount of apo B-(a) complex varied
considerably (see Table 2).
he tissue lipoprotein parameters were correlated to the serum lipoprotein
values in Table 4. The results shown in this table are confirmed in Figure
2A, where the correlation (r=0.556, p<0.001) between serum and arterial
wall apo (a) is shown in more detail. In contrast, no significant correlation
could be found between serum apo B and arterial wall apo B, as shown
in Figure 2B (r00.0999, p=NS).
Table 3.
The next question we raised was whether apo(a) could be detected in
the arterial wall as an intact protein or whether it might be already
partially degrade. In 8% SDS-PAGE and Western blotting, intact apo(a)
with its t normal high molecular weight was seen (Figure 3). In addition,
the majority of immunodetectable apo B was found to be still intact as
a 500-kD protein band (data not shown). We demonstrated that the apo(a)
isoform pattern in the arterial wall corresponded to the serum pattern
(Figures 3,4, and 5). Moreover, in 10 arterial wall samples, we separated
the three main layers of the thoroughly washed arterial wall and showed
the following distribution: most of apo(a) was present in the intima,
there were traces in the media, and none was detected in the well-washed
adventitia. These data were confirmed in 100 immunohistochemical preparations,
were apo(a) and apo B were mainly detected in the intima.
Post-mortem arterial wall samples with different areas of intimal surface
covered with atherosclerotic lesions were analyzed to determine the amount
of lipids, apo(a), and apo B in relation to the percentage of plaque
area (Table 5). We divided the samples into two groups according to the
macroscopically visible plaque area (<50% or >50%). Included were
21 arterial wall tissue samples of the aorta and the left coronary artery
from 11 patients. While triglycerides and protein did not differ between
the two groups, cholesterol, apo(a), and apo B were greater in the group
with >50% plaque area.
Table 4.
As in the bypass samples, we wanted to determine whether apo(a) and
apo B were still intact as high molecular weight proteins in the post-mortem
tissue. Figure 4 shows that most of the apo(a) was intact in its high
molecular weight position on 8% SDS-PAGE. Figure 4 also shows that the
apo(a) pattern was comparable between serum, aorta, and coronary artery.
Moreover, in the post-mortem tissue, we studied the apo(a) content of
the ascending aorta at the typical location where the biopsies were taken
during bypass surgery, and we compared this to the content of the stem
region of the right ascending (RA) and left ascending (LA) coronary artery,
as well as to the branching region of the LAD coronary artery and the
circumflex coronary artery (CX). The different arterial wall sections
contained comparable amounts of apo(a) and expressed the same isoform
pattern (Figure 5). Only the branching regions of the coronary arteries
(fatty streak) contained slightly more apo(a). This figure shows again
the absence of apo(a) in the media (compare Figure 3).
Immunohistochemistry of the post-mortem arterial wall was performed
parallel to the biochemical analysis. Three controls showed a complete
lack of staining (Figure 6A) while the polyclonal and monoclonal apo(a)
antibodies revealed comparable results (data not shown). Apo B and apo(a)
were found to be exclusively located in the intima. Atheromatous lesions
were significantly more severely affected than regular segments (Figure
6B).
Figure2.
Comparing the distribution of apo B and apo(a), a tight association
of both was observed (Figures 6C and 6D). Patterns of apo B and apo(a)
deposits were either almost congruent with a slightly stronger staining
for apo B, or matched at least in major parts. Apo B and apo(a) were
found to be primarily associated with extracellular structures in a bundle-like
staining pattern (Figure 6C and 6D). However, in fatty streaks, some
foam cells could be identified as bearing the apo B and/or apo(a) intracellularly
( Niendorf et al., unpublished observations).
Figure3.
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To learn more about the form in which apo B and apo(a) is associated
in the arterial wall, we analyzed post-mortem tissue extracted in NaCl
without detergent in a KBr density gradient. We measured the total and
free cholesterol, triglycerides, phospholipids, apo B, and apo(a) in
the extract and in the different density fractions. Figure 7 shows the
distribution of cholesterol, apo B, and apo(a) in the density gradient
of aortic extracts from two different samples (see legend). While no
apo B was detected in the bottom fraction, 30% to 40% of apo(a) was found
in this lipid-free form. There was apo B and apo(a) in the density range
of Lp(a) and LDL, where most of the cholesterol was measured. In other
experiments, we also demonstrated that the distribution of triglycerides
and phospholipids was mainly associated with the LDL and Lp(a) regions
(data not shown).
Figure 4.
Figure 5.
Table 5.
Figure 6.
Figure 7.
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Discussion
As background for our study, we analyzed lipoproteins in 306 CHD patients
who underwent coronary bypass surgery. In this cohort, we confirmed
earlier studies on lipoproteins and CHD. The importance of hyperlipidemia
as a risk factor for CHD has recently been confirmed in a study of
40 bypass patients who underwent a second operation.46 In addition
to higher serum and LDL cholesterol levels, this study determined the
CHD patients have significantly higher triglyceride values 47,48,49
in most of the cases associated with lower high density lipoprotein
cholesterol.50
The most striking difference between normal controls and bypass patients
in our study was the serum Lp(a) level (p<0.0001). Forty percent
of the patients had Lp(a) levels greater than 25 mg/dl, while only
16% of the controls surpassed this level. This supports the epidemiological
evidence from others and our own group that Lp(a) has to be considered
as an atherosclerotic particle.51 However, to date, the pathophysiological
background for the atherogenicity of Lp(a) has not been studied extensively.
While Walton et al.24 detected apo(a) in the arterial wall, they did
not think that it participates in atherogenesis.
The level of apo(a) in the arterial wall tissue samples of 107 bypass
patients compared with their serum Lp(a) showed a strong correlation.
No significant correlation could be found between serum and tissue
cholesterol of serum and tissue apo B. The apo(a)-linked apo B showed
a correlation with Lp(a) as a risk factor for the development of arteriosclerosis
independent from LDL.
The described results differ from earlier studies, 26 which found a relationship
between high serum lipid levels and LDL in aortic intima. Based on
our data, we speculate that the lack of correlation between serum and
tissue apo B is due to intracellular degradation of LDL in the arterial
wall.
If tissue lipoprotein values are analyzed (Table 2) according to the
serum Lp(a) of the patients (under or above 25 mg/dl), two striking
facts are seen: there is a high correlation between high serum Lp(a)
and the concentration of apo(a) in the arterial wall, and there is
a considerable amount of apo B linked to apo(a) (40% to 83%) in the
aortic biopsies. Thus, high serum Lp(a) levels can contribute significantly
to the deposition of apo B in the arterial all. These data were confirmed
by immunohistochemistry; in post-mortem arterial wall tissue, most
of the apo B was co-localized with apo(a). The fact that apo(a), as
well as apo B, are immunologically detectable suggests that major parts
of Lp(a) accumulate extracellularly instead of being digested in the
cells.
To prove the suggestion from the immunohistochemistry that apo B and
apo(a) might still be intact proteins, we performed SDS-PAGE. Apo B
was found at 513 kD and apo(a), in the molecular weight range around
and above apo B; these weights correspond to their normal total molecular
weight. Moreover, the isoforms described for apo(a) in serum can be
demonstrated in the comparable pattern in the arterial wall. Whether
this is a general observation needs to be evaluated. Intact apo(a)
was found in the biopsies as well as in the post-mortem samples, a
particularly surprising fact.
For the quantification experiments, we compared different detergents
for extraction of the tissue samples and found that in 1% Tween 20,
the measurable amounts of apo B were comparable to those in 3% Triton
(TX100), which was used by Hoff et al.28 Both detergents left some
SDS-soluble apo B in the pellet, but Tween buffer was slightly superior
to Triton for the detection of apo(a).
Apo B has been measured in several studies with rather different results.
The amount varied between 2 and 15 mg/g dry tissue29,31 and 5 to 50
m g/g52 or up to 600m g/g dry tissue.53,54 The 2 to 15 mg/g were described
for human material, while the values in micrograms per gram were from
swine54 and monkey studies.52,53 We detected apo B in a mean range
from 40 to 70 m g/g wet tissue in our detergent-extracted tissue samples
with the ELISA technique. Our data actually corresponded much better
to the animal studies and did not reach values in the range of milligrams
per gram as described by Hoff et al.29,31 for human tissue. However,
we consider our results more reasonable since it is unlikely that apo
B content is of the same order of magnitude as total protein in detergent-solubilized
tissue (18mg/g). We believe that 0.2% for aortic material and 0.12%
for venous samples are more realistic for the percentage of apo B from
total protein.
No determination of apo(a) in the arterial wall has been published so
far. Ylä-Hertuala et al.33 failed to detect apo(a) in the arterial
wall by radial immunodiffusion (RID).On the basis of our results, we
conclude that the RID method is not sensitive enough to allow a quantification
of apo(a) in arterial wall tissue.
When the apo(a) and apo B content of the aortic biopsies were compared
with the venous samples, significantly lower values were found in the
venous tissue. Under physiological conditions, thee seemed to be no
comparable accumulation of lipoproteins in the veins. Hoff et al.,51
however, showed that serum Lp(a) levels were significantly associated
with the degree of stenosis in saphenous vein grafts.
In 10 biopsies, as well as in post-mortem tissue samples, we dissected
the different tissue layers and analyzed the intima separate from the
media and adventitia. With biochemical and immunohistochemical methods,
we showed that apo B and apo(a) are mainly localized in the intima;
there were only traces in the media and nothing was seen in the washed
adventitia. Therefore, the lipoproteins must have entered the arterial
wall via the endothelium rather than being contaminations from the
vasa vasorum. With the post-mortem tissues, we had the chance to study
different vessel areas and detected apo(a) with no differences in the
individual isoform pattern. We have not yet quantitatively differentiated
the various areas.
The next question was whether Lp(a) can be extracted from the tissue
as lipoprotein particles. In preliminary studies with density gradient
ultracentrifugation, we demonstrated that 50% to 65% of apo(a) was
lipid-associated in the density range of 1.05 to 1.1 g/ml, and we found
20% of the apo B in this density fraction. We detected 70% to 80% of
apo B in the density rage of 1.02 to 1.05 g/ml. No apo B was found
in the lipid-free bottom. We propose that these LDL-like particles
are at least partly derived from Lp(a) that lost the apo(a) glycoprotein.
The dissociated apo(a) was detected in the bottom fraction of the gradient.
The stripping of apo(a) from the particle may be due to post-mortem
changes in the tissue, because a similar dissociation was observed
in serum samples that had been stored at room temperature for 24 hours.
This hypothesis would also explain the strict co-localization of apo
B and apo(a) in the immunohistochemistry. We were not able to obtain
enough biopsy material to measure the free apo(a) content in fresh
arterial wall tissue; therefore, we cannot fully verify the apo(a)
values, which were calculated on the assumption that most of the apo(a)
was in the apo B-(a) complex.
In addition to the already discussed set of data, we describe in this
paper some preliminary results obtained by comparing different vessel
wall sections in relation to the plaque area. The wall pieces with
more than 50% visible plaque showed considerably higher values for
apo(a), apo B, and cholesterol as compared with the samples with less
than 50% plaque area. The protein content and triglycerides were comparable
in both tissue samples. Hoff et al.28 found also higher amounts of
apo B in normal aorta than plaque using Triton for extraction. The
exact definition of plaque grades will play an important role in the
final answer to this question as well as the methods of extraction
and quantification.
In conclusion, this is the first study showing a positive correlation
of Lp(a) serum levels with apo(a) and apo B accumulation in the arterial
wall. Also, the presence of intact apo (a) and Lp(a)-like particles in
human arterial wall was demonstrated for the first time. We assume that
in earlier studies on LDL-like particles in the arterial wall, the apo(a)
might have been missed and, at least partly, Lp(a)-like particles might
have been isolated. Our studies imply that Lp(a) enters the arterial wall
and accumulates extracellularly, where apo(a) and apo B can be co-localized
with immunohistochemistry. Further studies are necessary to define the
way by which Lp(a) enters the vessel wall.
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