Morphological detection and quantification of lipoprotein (a) deposition
in atheromatous lesions of human aorta and coronary arteries
Axel Niendorf, Matthias Rath, Katrin Wolf, Susanne Peters,
Hartmut Arps, Ulrike Beisiegel, and Manfred Dietel
Virchows Archives of Pathological Anatomy, 1990, Number 417, Pages
Lipoprotein(a) [Lp(a)] is a lipoprotein that closely resembles low-density
lipoprotein (LDL). Both lipoproteins have a similar lipid composition
and both contain apoprotein B (apoB). In addition, Lp(a) consists of
a protein called apoprotein (a) [apo(a)], which is attached to the apoB
by disulphide bonds (Utermann et al. 1969, 1987; Simons et al. 1970;
Ehnholm et al. 1972). Lp(a) was first described by Blumberg et al. (1962)
and Berg (1963). The molecular weight of apo(a) is higher than 500kDa
(Utermann et al. 1987). Lp(a) floats in a density range of 1.05-1.12
g/ml. It binds to the LDL receptor with lower affinity than LDL (Krempler
et al. 1984; Maartmann and Berg 1981; Armstrong et al. 1985). Its clearance
from the plasma has not yet been fully evaluated. Lp(a) is synthesized
in the liver and is probably secreted by hepatocytes, associated with
The pathophysiological relevance of Lp(a) is evident in the positive
correlation between elevated serum levels of Lp(a) and coronary heart
disease shown in a number of epidemiological studies (Wottawa et al.
1984; Schriewer et al. 1984; Dahlen et al. 1986; Armstrong et al. 1986).
Approximately 30% of a normal population express atherogenic serum levels
of Lp(a) that are above 25 mg/dl; 40% of patients suffering from coronary
heart disease express elevated Lp(a) serum levels.
In previous morphological studies apoB has been detected in the arterial
wall by means of immunohistochemistry (Hoff et al. 1977, 1978; Hoff and
Bond 1983; Yomantas et al. 1984; Yamauchi and Hoff 1984; Carter et al.
1987). From these investigations it has been deduced that either LDL
and/ or very low-density lipoprotein (VLDL) are present. Only one study
(Walton et al. 1974) has described apo(a) in the arterial wall. Interestingly,
in that report it was concluded that Lp(a) does not participate in atherogenesis.
Against a background of increasing epidemiological evidence about the
atherogenic properties of Lp(a) and with the availability of an apo(a)-specific
monoclonal antibody, the present study investigates whether or not there
exists a correlation between immunoreactivity of apo(a) and apoB in the
arterial wall. A positive finding indicates that the localization of
apoB has to be related, at least in part, to the presence of Lp(a). Autopsy
tissue from the thoracic aorta and the left coronary artery has been
examined by means of immunohistochemistry (Hsu et al. 1981; Niendorf
et al. 1987) in combination with morphometric analysis in order to determine
whether Lp(a) can be detected preferentially in atheromatous lesions
of the arterial wall.
Materials and methods
Autopsy tissue (n = 74) from patients who died at the age of 0-98 years
(39 females, 35 males, approximately 10 samples representing each decade)
was obtained from the Institutes of Pathology and Forensic Medicine at
the University of Hamburg and was taken between 24 and 47 h post mortem.
The slices were cut from the thoracic aorta in the region of the first
intercostal arteries and from the first 2 cm of the left coronary artery.
From atherosclerotic vessels two samples were taken: one from an atheromatous
area and a second, if possible, from an adjacent area which was grossly
normal. The tissue was formalin-fixed [3.7% buffered in phosphate-buffered
saline (PBS)] and paraffin-embedded by standard procedures. Haematoxylin
and eosin and elastic-van-Gieson staining were performed by routine methods.
Polyclonal goat anti-apoB (Immuno, Vienna) was applied to the tissue
sections in a 1 : 1000 dilution. Monoclonal mouse-anti(a) has been produced
in the laboratory of one of the authors (U. Beisiegel) and will be described
and characterised elsewhere (paper in preparation). This antibody has
been checked for a possible cross-reactivity with plasminogen, which
For immunohistochemistry deparaffinised 2-µm sections were mounted
on Zementit-coated slides. They were allowed to dry at 60¡C overnight.
Endogenous peroxidase was inhibited by a 10-min treatment with methanol-H2O2
(174 ml methanol + 6 ml 30% H2O2). Thereafter tissue sections were washed
for 10 min in PBS and pre-incubated with normal horse serum [apo(a)]
or normal-rabbit serum (apoB) for 30 min at room temperature. The serum
was decanted and the specific first antibody [monoclonal anti-apo(a)
or polyclonalanti-apoB] was applied for 24 h at 6¡ C. Incubation
with the primary antibody was followed by three washes in PBS for 10
min each. After that the sections were incubated with biotinylated secondary
antibodies [horse anti-mouse for Lp(a) detection and rabbit anti-goat
for apoB] was applied for 24 h at 6¡ C. Incubation with the primary
antibody was followed by three washes in PBS for 10 min each. After that
the sections were incubated with biotinylated secondary antibodies [horse
anti-mouse for Lp(a) detection and rabbit anti-goat for apoB] which were
both diluted 1 : 200 in PBS. This was again followed by three washes
of 10 min each. Thereafter the sections were incubated for 30 min with
the avidin-biotin reagents (Vector Laboratories, Burlingame, Calif.)
and washed again three times for 10 min. Sections were then incubated
with diaminobenzidine as a chromogenic substrate for 6 min. They were
rinsed for 5 min in plain water and counter-stained with haemalaum for
5 min, dehydrated in alcohol and mounted in Eukit.
In control incubations non-immune serum of the same species in which
the specific first antibody had been raised (normal mouse serum or normal
goat) was applied instead of the first anybody.
Sections of the arterial wall were classified as "normal" (no
lesion and intimal thickening), "fatty streak" (accumulation
of foam cells), "fibrous plaque" (smooth muscle cell and collagen
accumulation) and "complicated lesion" (any fibrous lesion
including in addition necrosis, haemorrhage, thrombosis or calcification).
Immunoreactivity within the arterial wall was estimated by semiquantitative
methods and a staining score was deduced from the following formula:
SC = (%SALA/100) x (ISA) x (%LATSA/100), (where SC = staining score;
SALA=stained area with regard to lesional area; ISA = intensity of stained
area; LATSA =lesion area with regard to total section area).
This included the area and intensity of staining in a given tissue section.
A value ranging from 0 to 12 thus resulted from the product of intensity
(0-12) and the percentage of a stained area (divided by 100) after consideration
of the percentage of lesional area within the given total section through
the arterial wall. Thus the highest possible staining score was 12. The
morphometric analysis was performed by two independent investigators.
The distribution of staining for apo(a) and apoB was found to be located
almost exclusively in the intima of the arterial wall in both thoracic
aorta and left coronary artery. The media expressed staining for either
antigen in general. The media expressed staining for either antigen in
general. In 5% of the total sample number a restricted immunoreactivity
at the cellular level was observed in smooth muscle cells of the media.
The lumina of adventitial vessles (vasa vasora) showed staining for both
antigens. The adventitia itself expressed a low level of diffuse staining
in some cases.
The deposition pattern of both antigens in the normal intima is summarized
schematically in Fig. 1 A-G. In non-lesional areas there was either no
staining at all (Fig. 2), or fine spot-like or a fine striped staining.
Both patterns were found in the near luminal part and the near media
part (Fig. 3) or were distributed over the whole area of the intima.
In no case was a marked aggregation of staining observed in non-lesional
areas. In contrast, in fibrous plaques and complicated lesions dense
bundle-like staining pattern was seen, for both antigens. Predominant
localization of both antigens within fibrous caps was seen this either
reached to the luminal border of the intima (Fig. 1; CL and FP type A)
or covered the necrotic core of a complicated lesion or was found at
the edges of necrotic cores (Fig. 5). The distribution patterns of apo(a)
and apoB in lesional areas are summarized in Fig. 1 H - L.
At the cellular level predominant localization of apo(a) and apoB in
the extracellular matrix has been observed.
Exceptionally, some foam cells within fatty streaks or complicated lesions
(Fig. 4) and a few smooth muscle cells of the media contain certain amounts
of both antigens. In summary apo(a) and apoB are located extracellularly
Comparison of the distribution pattern of both antigens with regard
to localization and intensity exhibits a high degree of congruency for
apo (a)- and apoB staining in all lesion areas. Localization of apo(a)
and apoB is shown to be present in atheromatous lesions of the aorta
as well as in the coronary artery as shown in Figs. 2 Ð 5 [apo(a)
localization]. The co-localization of both proteins is demonstrated in
Figs. 6 and 7.
Quantification of immunoreactivity shows a significantly more intensive
staining for both antigens in regions of the intima that show either
fatty streaks, fibrous plaques or complicated lesions than in areas of
normal intima. Plaque area as a total has a staining score (mean value
for both lipoproteins in aorta and coronary artery) of 6.5 compared with
2.5 in non-lesional areas. The most pronounced aggregation of both proteins
is observed in the coronary artery exhibiting a staining score of 5.1
for apo(a) and 6.7 for apoB in lesional areas compared with 1.2 [apo
(a)] and 1.8 (apoB) in non-lesional areas. All values for the immunoreactivity
are listed in Table 1.
Increasing numbers of epidemiological studies (Wottawa et al. 1984;
Schriewer et al. 1984; Dahlen et al. 1986; Armstrong et al. 1986) have
indicated the value of investigation the distribution of apo(a) and apoB
in a larger number of cases. The intriguing immunological problem that
results from a close similarity between plasminogen and apo(a) (Havekes
et al. 1981; Floren et al. 1981) was resolved by the availability of
a monoclonal antibody that detects the apo(a) and does not cross-react.
The results from this study reveal a staining pattern for apo(a) and
apoB located in the intima of the arterial wall with a preference for
lesions is predominantly in the extracellular space, and is congruent
in adjacent sections.
Determination of staining intensity in lesions and non-lesional areas
clearly demonstrates that apo(a) and apoB are enriched in lesions. This
finding supports the epidemiological observation that Lp(a) is associated
with the appearance of atherosclerosis. Both antigens show a comparable
tissue distribution in the thoracic aorta and the coronary artery. Since
the aorta exhibits fatty streaks that are composed of foam cells more
frequently than the coronary artery, with its rather fibrous or complicated
lesions, it is noteworthy that the number of intracellularly stained
cells is higher in the aorta than in the coronary vessel. Under the premise
that intimal thickening can be considered to be a physiological age-related
process it is important to note that deposition of both proteins occurs
in parts of the intima not affected by morphologically detectable lesions.
However, this is observed to a much lesser extent than in lesions of
the arterial wall. The demonstration of protein deposition in non-lesional
areas might be interpreted as the detection of earliest stages of the
development of atheromatous lesions.
The mechanism of accumulation of lipoproteins in the arterial wall is
still under debate (Kratzin et al. 1972; Smith et al. 1976; Hollander
et al. 1979; Hoff et al. 1979). LDL is suspected to be modified and enriched
in the intima that has undergone a primary injury at the site of the
endothelial barrier. Concerning the Lp(a) particle two hypotheses are
under discussion: Lp(a) as a lipoprotein might diffuse into the intima
and either stick to the intercellular components such as glycosaminoglycans
or be directly taken up by monocyte-derived macrophages or smooth muscle
cells. Since Lp(a) is known to bind to glycosaminoglycans with a higher
affinity than does LDL (Smith et al. 1976; Hollander et al. 1979) this
particle is predisposed to be entrapped in the intercellular matrix.
Subsequently it might be phagocytosed, in the sense of a repair or clearance
mechanism and phagocytosis and receptor-mediated endocytosis lead to
lysosomal degradation of the incorporated ligand. One would not expect
ligands which have been undergone lysosomal degradation to be detectable
by immunological methods and it seems unlikely that we are detecting
apoproteins that have undergone phagocytosis.
We therefore favour a second hypothesis to explain Lp(a) accumulation
in the arterial wall, based on the similarity of this lipoprotein with
plasminogen. The apo(a) molecule is made up predominantly of kringle
structures having a high degree of homology with plasminogen (Eaton et
al. 1987; McLean et al. 1987). Lp(a) might therefore bind to sites where
fibrinogen is polymerized. In this regard Lp(a) might play a fatal role
interfering with fibrinolysis, which has to occur in a regular manner
to prevent fibrotic organization of mural thrombi triggering the formation
of complicated lesions. Preliminary results from our group confirm speculations
about a Lp(a)-fibrin association. Morphologically a striking congruency
of fibrinogen and apo(a) was observed in a limited number of cases (data
not shown). The distribution patterns of both proteins with regard to
the intra- and extracellular localization also favour a mechanism where
Lp(a) either sticks to extracellular matrix components or is entrapped
in thrombi, due to its similarity with plasminogen. Biochemical data
(Rath et al. 1989) reveal a considerable amount of gradient-extracted
apo(a) associated with intact lipoprotein particles. This finding corroborates
the concept of a predominantly extracellular deposition. In this work
we find apo(a) and apoB to be deposited mainly extracellularly, although
weak intracellular staining within foam cells can be observed occasionally.
In previous morphological studies the distribution pattern of apoB has
been determined (Hoff et al. 1977, 1978; Hoff and Bond 1983; Yomantas
et al. 1984; Yamauchi and Hoff 1984; Carter et al. 1987). The results
of these investigators are confirmed with regard to tissue distribution.
The presence of apoB indicates that VLDL or LDL must be present, and
from the findings of this study the presence of LDL cannot be excluded.
The major new conclusions is that immunoreactivity for apoB has to be
related to the presence of Lp(a) at least in part, since apo(a) and apoB
are detected in congruency. One should therefore keep in mind the possibility
that Lp(a) is present when apoB is detected in atheromatous lesions.
Walton (1974) first reported the observation of similar distribution
patterns for apo(a) and apoB. However, he found it to be "rather
unlikely that the finding of apo (a)-immunoreactivity in patients with
clinical evidence of coronary artery disease has any special sinister
significance". On the basis of our data we can clearly contradict
In conclusion, this work describes Lp(a) for the first time as an atherogenic
particle that can be detected immunologically by the demonstration of
apo (a). It has a preferential deposition in atheromatous lesions and
should be considered as a lipoprotein that deserves close attention with
regard to its diagnostic and therapeutic implications.
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