Lipoprotein (a) in the arterial wall
U. Beisiegel, A. Niendorf, K. Wolf, T. Reblin and M. Rath
EUROPEAN HEART JOURNAL (1990) 11 (SUPPL. E), 174-183
Lp(a) was first demonstrated by Berg and his associates (1). They described
Lp(a) as a human lipoprotein polymorphism. Characterization of Lp(a)
revealed that it is very similar to LDL in lipid composition and presence
of apo B-100. In contrast to LDL it contains an additional glycoprotein,
designated apo (a) (2,3). Apo (a) is linked to apo B by disulfide bridges.
Lp(a) is found in all human beings, but in variable amounts, which are
genetically determined. Size isoforms have been described for apo (a)
(4,5) which were categorized by Utermann et al.(6) into five homozygous
phenotypes and the respective double-band heterozygous forms. A highly
significant association of the phenotypes with the serum level of Lp(a)
could be demonstrated (7,8). Recently, striking homology between human
apo (a) and plasminogen was demonstrated in both amino acid (9,10) and
c-DNA (11) sequences. This unexpected homology between an apolipoprotein
and an important protein of the coagulation system led Goldstein and
Brown to speculate that "these dramatic findings may provide the
long-sought link between lipoproteins and the clotting system" (12).
In a series of epidemiological studies, a positive correlation of high
serum Lp(a) levels with CHD has been demonstrated (13-15). The pathomechanism
by which Lp(a) contributes to the development of atherosclerotic plaques
has not yet been studied extensively. As early as 1958, human arterial
wall was analyzed for its lipoprotein content (16). This and alter studies
mainly concentrated on LDL in the vessel tissue. LDL was measured in
arterial wall and compared with serum lipid levels (17) and apo B was
quantitated in normal intima compared with fibrous plaques (18). Moreover
LDL and lipoprotein-like particles were extracted from human aorta and
analyzed by physical, chemical and immunological means (19,20).
The aim of the present study was to analyze human arterial wall tissue
for the presence of Lp(a). In biochemical studies we measured Lp(a) in
arterial wall and extracted lipoproteins from the tissue (21). With immunohistochemistry
we tried to localize apo (a) in different areas of arterial wall tissue
(22) and compared the pattern with the deposition apo B and fibrin in
consecutive tissue sections (22,23).
Materials and method
Fasting blood samples were taken from the patients after admission to
the hospital one or two days before the bypass operation. It was not
always possible to obtain fasting samples from blood donors, thus the
difference in triglycerides and VLDL might be even more distinct in this
Cholesterol and triglycerides, as well as HDL cholesterol was determined
with enzymatic test kits from Boehringer Mannheim. VLDL cholesterol concentration
was calculated from the triglyceride levels measured (triglycerides:
5) and LDL cholesterol was calculated by the Friedewald formula (LDL
cholesterol = total cholesterol Ð VDL cholesterol Ð HDL cholesterol).
Apo B was determined by turbidometric method from Behring Diagnostika
(Marburg, F.R.G.) using standards and control sera from the same company.
Lp(a) was measured by radioimmunoassay from Pharmacia/LKB (Freiburg,
F.R.G.) using standards and control sera from Pharmacia/LKB.
We used as tissue samples the biopsies routinely taken during the aortocoronary
bypass operation where the vein graft is attached to the ascending aorta.
The biopsies were around 10-30 mg and by histological screening they
showed different grades of intimal thickening than control tissue. No
severe plaque areas or complicated lesions were examined. Venous samples
were taken from the vena sephena magna, which served as the bypass graft.
Biochemical analysis of the tissue samples has been described elsewhere.
Apo(a) and apo B were determined by a special ELISA system, which allowed
differentiation of apo B linked to apo (a) and free apo B. Standard immunohistochemical
procedures were followed (22).
Anti-apo(a) monoclonal antibody was prepared in our laboratory (21)
and polyclonal anti-apo B antiserum was raised in a rabbit. For detection,
the ABC-method (avidin-biotin-complex) was used.
For morphometric quantitation, performed by two independent investigators,
a staining score was deduced from the formula : [staining score = (percentage
of stained area with regard to the lesion area/100) x (intensity of stained
area) x (percentage of lesion with regard to total section area/100)]
This gave a value between 0 and 12.
Western blot analyses of the tissue samples were performed with 6% SDS-polyacrylamide
gel electrophoresis. From the aorta both the intima and media were dissected
and solubilized together (without the adventitia). Following electrophoresis,
the proteins were transferred to nitrocellulose. Apo (a) was detected
with monoclonal antibodies on the nitrocellulose and the bands stained
with a second antibody conjugated to horseradish peroxidase. [sitemap]
To confirm the described evidence that Lp(a) serum levels above 25 mg
dl-1 are more frequent in patients with CHD than in normal controls we
compared 300 healthy blood donors with 235 patients who underwent coronary
bypass operations (Rath et al., manuscript in preparation). For both
groups, the various lipoprotein parameters are shown in Table 1. All
lipid parameters except HDL (P<0.0045) were highly significantly different
in the two groups (P<0.0001. Lp(a) is not normally distributed, but
shows a highly skewed distribution. Therefore, in addition to the mean,
the percentage of persons with Lp(a) >25 mg dl-1 is given for both
groups. The means differ with P<0.0004 and in the CHD group 1.6 times
more persons have Lp(a) > 25 mg dl-1 (45% vs 27% in the controls).
From 107 of the 235 bypass patients, we were able to obtain an aortic
biopsy taken routinely during operation. In these tissue samples, we
measured lipids, total protein and apoproteins as described by Rath et
al. (21). Table 2 shows the comparison of tissue values for patients
with serum Lp(a) levels>and< 25mg dl-1. A clear difference was
seen in apo(a) and apo(a)-linked apo B according to serum Lp(a) level,
while free apo B, cholesterol, triglycerides and total protein showed
no significant difference.
In a smaller group of patients (n=32) we obtained venous samples in
addition to aortic biopsies. Comparing the two different types of vessels
the following results were obtained : in the venous tissue lower values
for cholesterol (1.4 compared with 4.3 mg.g-1WW) and apo (a) (2.4 compared
with 11.1m g.g-1WW) were found. These values are comparable with the
data on normal saphenous veins from Cushing et al. (24). They describe
apo (a) in the venous tissue with <2 ng.mg-1WW and apo B with 3.3
ng.mg-1WW and for apo B 70 ng.mg-1WW, demonstrating a net accumulation
of these apoproteins in the veins from the time of their grafting in
the arterial bed (24).
Comparing areas of the arterial wall with no visible plaque with various
stages of atherosclerotic plaques, we observed a more than twofold increase
using semquantitative morphological measurements (Fig 10) (22). Biochemical
data from autopsy tissue confirm this accumulation of Lp(a) in plaque
areas. In a small number of cases, we analyzed pot-mortem arterial wall
with plaque areas of <50% or >50%. The means differed by 14% (38m
g.g-1WW versus 44m g.g-1WW).
We tried to determine, by immunohistochemical methods, where in the arterial
wall the Lp(a) accumulates. Slices from post-mortem aortic and coronary
tissue from 74 patients (aged 1-98 years) were stained with monoclonal
anti-apo(a) (22). Fig. 2a demonstrates apo (a) in the thickened intima
of an early plaque formation. In Fig. 2b, a fibrous plaque is shown
with a massive accumulation of apo (a). In both examples the apo(a)
is located extracellularly in the intima, which is more clearly demonstrated
in Fig. 3, where differential interference contrast microscopy was
used. The higher magnification shows that apo(a) is extracellularly
associated with fibrous structures; no intracellular staining can be
seen. In few cases, however, we could demonstrate apo(a) intracellularly
in foam cells. It can also be shown by biochemical means that apo (a)
accumulates in the intima rather than in other layers of the vessel
wall (Fig.4). In several aortic tissue samples the intima and media
were dissected and separately analyzed. Two apo(a) bands are only detectable
in the intima. They represent the two apo(a) isoforms which we had
also determined in the patientsÕ serum. It is important to note
that we found apo (a) as intact protein even in autopsy tissue; no
significant proteolytic degradation seemed to affect this high seemed
to affect this high molecular weight protein.
From autopsy tissue, adjacent slices of the aorta were stained with
monoclonal anti-apo(a) or polyclonal anti-apo B to investigate a possible
co-localization of the two protein components of Lp(a). In all cases
we found a strict co-localization of apo(a) and apo B (22). A representative
example is given in Fig. 5. We found only very few areas where apo B
alone was stained.
Considering the results of SDS-PAGE and co-localization studies, we
wanted to determine whether Lp(a)-like lipoproteins could be extracted
from the arterial wall. We had to use post-mortem tissue for this experiment
since we did not obtain enough fresh tissue. Nevertheless, we were able
to isolate particles within the density range of Lp(a) which contained
lipids as well as apo(a) and apo B. In addition, we detected apo B in
the density range of LDL and a comparable amount of apo (a) at the bottom
of the gradient (data not shown) (21). Our conclusion from these data
are that Lp(a) can indeed be extracted from arterial wall. We also assume
that the apo(a) at the bottom of the gradient was split from the particles
which appear at LDL density in post-mortem samples. Further extraction
experiments with fresh tissue should clarify this.
In our latest studies, we used adjacent slices of pot-mortem aortic
tissue to analyze further the areas in which Lp(a) is detected in the
intima. With anti-fibrinogen antibodies (also recognizing fibrin and
other breakdown products of fibrinogen) we looked for a possib le co-localization
of apo(a) and fibrin deposition. Fibrin and apo(a) were found in the
same areas of the intima (Fig.6), and this co-localization indicate that
Lp(a) might be associated with fibrin in the arterial wall (Wolf, K.
et al. manuscript in preparation).
Before the main discussion it should be mentioned that we found, in
contrast to most earlier studies, no highly significant difference in
HDL cholesterol levels between the two groups investigated (blood donors
and bypass patients). This disparity cannot be explained by the sex distribution
in the two groups since they were comparable (percentage females = 21%
in the controls and 24% in the CHD group). The other lipoprotein parameters
we found increased in the CHD group, which is in good agreement with
most former reports.
Studies over the past 30 years on lipoproteins and the pathobiology
of the arterial wall have concentrated on LDL as an important factor
in plaque development. LDL contributes to the foam cell formation mainly
in its oxidized form. Today it is necessary, besides LDL and oxidized
LDL, to consider LP (a) in studies on lipoproteins in arterial wall.
An accumulation of the two protein constituents of Lp(a), apo (a) and
apo B, in the intima of the arterial wall was demonstrated by biochemical
quantification, and Western blotting demonstrated that apo (a) is in
the intima of the arterial wall and is still intact as a high molecular
weight band. Immunohistochemical studies revealed a mainly extracellular
co-localization of both proteins. These experiments indicate an accumulation
of the intact Lp(a) particle in the intima; Lp(a)-like lipoproteins could
also be extracted from arterial wall tissue.
We found very few areas where apo B alone was stained by immunohistochemistry.
This could either mean that there is no accumulation of intact LDL in
the analyzed samples or the LDL accumulates in exactly the same areas
as Lp(a). The measurement of free apo B in the fresh samples in addition
to a comparable amount of apo(a) and apo(a)-linked apo B would indicated
some deposit of intact LDL-apo B. In this connection it has, however,
to be questioned whether in former experiments where lipoprotein were
extracted from arterial wall, the apo(a) might have been overlooked and
Lp(a) had been isolated in addition to or instead of LDL.
It can be concluded from these data that most of the Lp(a) is not taken
up by macrophages and degraded intracellularly, as is LDL. Lp(a) rather
seems to invade the arterial wall very early in plaque development and
becomes bound to extracellular structures, where it accumulates over
the years without being degraded. A possible mechanism for this accumulation
is the interaction of Lp(a) with glycosaminoglycans, as has been described
by Dahlen et al. (25) and Bihari-Varga et al. (26) Salonen et al. (27)
recently described apo(a) binding to fibronectin, a major substance in
the basal membrane of the arterial wall.
Another line of thought on the pathophysiology of Lp(a) was introduced
by the demonstration of homology with plasminogen. Competitively, apo(a)
might bind to fibrin and thereby inhibit the action of plasminogen. Recently,
evidence for such a mechanism was reported (28). If such inhibition occurs
in vivo, fibrinolysis could be affected and the thrombi might remain
on the endothelial surface. Our study supports this hypothesis by the
co-localization of apo(a) and fibrin in the arterial wall. This might
be due to the thrombi remaining on the endothelial surfaces since in
presence of Lp(a) the fibrin and the apo(a) will eventually become part
of the plaque and present the described co-localization pattern.
Following the same path, it was demonstrated (29,30) that Lp(a) can
bind to the plasminogen receptor of endothelial cells. Lp(a) possibly
enters the intima via this pathway. Moreover, apo(a) is reported to have
proteolytic activity, as does plasminogen, first demonstrated versus
These data show that Lp(a) contributes to plaque development by extracellular
accumulation and only a minor amount is taken up in foam cells. This
indicates that the role Lp(a) plays in plaque formation differs from
that of LDL. More careful studies are necessary to evaluate the present
data and finally elucidate the role of Lp(a) in atherosclerosis.
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