Immunological evidence for the accumulation of lipoprotein(a) in the atherosclerotic
lesion of the hypoascorbemic guinea pig
Matthias Rath M.D. and Linus Pauling Ph.D.
Lp(a) is a low density lipoprotein particle with an additional glycoprotein,
named apoprotein(a) [apo(a)]. The c-DNA sequence of apo(a) shows a striking
homology of apo(a) and plasminogen with multiple repeats of kringle,
one kringle , and a protease domain. Lp(a) competitively inhibits the
binding of plasminogen to immobilized fibrin, fibrinogen, and the plasminogen
receptor on endothelial cells and it attenuates clot lysis induced by
tissue-type plasminogen activator. Therefore Lp(a) was assumed to be
the missing link between atherosclerosis and thrombosis. Lp(a) has been
shown in various epidemiological studies to be positively associated
with coronary heart disease and other forms of atherosclerosis. 5,6Furthermore,
a significant positive correlation between Lp(a) concentrations in human
plasma and arterial wall has been established SUP7and the development
of atherosclerotic lesions correlates with the degree of Lp(a) deposition
in the arterial wall. We observed that Lp(a) had primarily been found
in the plasma of species that have lost the ability to synthesize ascorbate
and we have consequently formulated the hypothesis that Lp(a) is a surrogate
for ascorbate. 9 According to this hypothesis ascorbate and Lp(a) share
common properties such as the promotion of cell repair and would be able
to replace one another under physiological and pathophysiological conditions.
Beside man and other primates, the guinea pig is known to have lost the
ability to synthesize ascorbate. It was therefore of interest to look
for Lp(a) in the plasma of the guinea pig. Earlier studies had shown
that ascorbate deficiency induces atherosclerosis in the guinea pig.10,11
This is in contrast to most other species, where atherosclerosis must
be induced by a high-fat diet or other atherogenic stimuli. It was therefore
of particular interest whether Lp(a) would also be found in the atherosclerotic
lesion of the hypoascorbemic guinea pig.
Material and Methods
In a pilot experiment 3 female Hartley guinea pigs with an average weight
of 800 g and an approximate age of 1 year were studied. All animals were
treated in accordance with the National Institutes of Health Principles12and
the Animal Welfare Act Regulations13for the utilization and care of vertebrate
animals. The animals received ascorbate free guinea pig chow. In addition
one animal received an extreme hypoascorbic diet with 2 mg ascorbate/kg
body weight/d. Another animal received 4 mg/kg BW/d. The 3rd animal served
as a control, receiving 40 mg ascorbate/kg BW/d. Blood was drawn from
the anesthetized animals by heart puncture and collected into EDTA-containing
tubes at the beginning, after 10 days, and after 3 weeks, when the animals
were sacrificed. Plasma was stored at -80°C until analyzed. At necropsy
the animals were anesthetized with metophane and were exsanguinated.
Heart, aorta, and other organs were taken for further biochemical and
histological analysis. The aorta was excised, the adventitia was carefully
removed, and the vessel was opened longitudinally. Subsequently the aorta
was placed on a dark metric paper and color slides were taken. The picture
of the proximal part of the aorta including the aorta ascendens and the
aortic arch was projected and thereby magnified by an approximate linear
factor 10. The circumference and the lesion areas were marked and measured
with a digitalized planimetry system (Sigma Scan, Jandell Scientific,
Sausalito, California). The degree of atherosclerosis was expressed by
the ratio of plaque area and compared to total area of the proximal aorta.
To confirm the data from the pilot study we conducted a comprehensive
guinea pig study which will be reported in detail separately. For the
purpose outlined here 22 male animals with a mean weight of 550 g and
an approximate age of 5 months were included. One group of 8 animals
served as a control and received 40 mg ascorbate/kg BW/d (group A). To
induce hypoascorbemia 8 animals were fed 2 mg ascorbate/kg/d (group B).
The animals were sacrificed after 5 weeks as described above.
Determination of Lp(a) was performed by SDS-polyacrylamide gels according
to Neville14 followed by Westernblotting.15 40 ml of guinea pig plasma
and 20 mg (ww) of arterial wall homogenate were applied in delipidated
form per lane of the gel. The immunodetection of apo(a) was performed
using a polyclonal anti-human apo(a) antibody (Immuno, Vienna, Austria)
followed by a rabbit anti-sheep antibody (Sigma, St. Louis) and then
gold-conjugated goat anti-rabbit antibody with subsequent silver enhancement
(Bio Rad, Richmond, California). In the same way a polyclonal anti-plasminogen
antibody (Sigma, St. Louis) was used.
On the hypothesis that apo(a) is a surrogate for ascorbate, the blood
of the guinea pigs was analyzed for its content of this protein. With
use of SDS-PAGE and subsequent immunoblotting a distinct immunoreactivity
for apo(a) was detected in the plasma of all animals. Figure 1 shows
an immunoblot with an anti-apo(a) antibody. All guinea pig plasma samples
showed an immunoreactivity with a commercially available antibody against
apo(a). To exclude any cross-reactivity of the polyclonal anti-apo(a)
antibody ,e.g. with apoB-100, the immunoblots of guinea pig plasma
were also incubated with a polyclonal antibody against plasminogen.
These control experiments showed the same immuno-reactivity pattern.
It has been known that ascorbate deficiency induces atherosclerosis
in the guinea pig. It was therefore of interest to study the process
of atherogenesis in this animal model and to further analyze the atherosclerotic
plaque. One-year-old animals as well as animals approximately six months
old were used as described in materials and methods. The animals receiving
an adequate amount of ascorbate (group A) were essentially free of atherosclerotic
lesions (Figure 2 A). By contrast, at both ages atherosclerotic lesions
could be induced by feeding a diet low in ascorbate. The older animals
showed a pronounced plaque formation that was most prominent in the aortic
arch and the branching regions of the intercostal and abdominal arteries
(Figure 2 B). In the six-month-old animals only early lesions could be
found after five weeks of ascorbic-acid deficiency. The difference in
plaque area of the proximal aorta between group A and group B was 25%
for the period of 5 weeks.
Since immunological evidence for the presence of Lp(a) in the plasma
of the guinea pig was obtained it was of interest to see whether this
lipoprotein is also a constituent of the atherosclerotic lesion in this
animal. Using SDS-PAGE followed by immunoblotting we were able to detect
distinct immunoreactivity for apo(a) in the homogenate of the atherosclerotic
lesion of the hypoascorbemic guinea pig (Figure 1, lane 11). Only a trace
of immunoreactivity was found in the control animal (lane 12).
We have recently observed that Lp(a) is mainly found in the plasma of
animals that have lost the ability to synthesize ascorbate. Consequently
we postulated the presence of Lp(a) in the plasma of the guinea pig.
A study confirmed our assumption and we were able to detect apo(a)
in the plasma of this species. We therefore conclude that Lp(a) is
a constituent of the lipoprotein pattern of the guinea pig. This observation
substantiates our hypothesis that apo(a) is a surrogate for ascorbate;
it does not, however, exclude the possibility of detection of apo(a)
in some other species, which might endogenously synthesize ascorbate.
The guinea pig was used in this study also for its unique inducibility
of atherosclerosis. We could confirm earlier reports that a significant
reduction of dietary ascorbate is sufficient to cause atherosclerotic
plaques in this animal model without any additional dietary modification
or other stimuli. Since we concluded that Lp(a) is present in the plasma
of the guinea pig it was of particular interest to determine whether
this lipoprotein could be detected in the atherosclerotic plaque of this
The crucial finding of this report is the immunological evidence for
an accumulation of apo(a) in the atherosclerotic plaque of the hypoascorbemic
guinea pig. In analogy to human atherosclerosis,7 we conclude that the
lipoprotein particle Lp(a) accumulates in the atherosclerotic lesion
and that Lp(a) contributes to plaque formation in this animal model.
Because of the analogies between the guinea pig and the human system
with respect to the lack of endogenous ascorbate supply as well as the
role of Lp(a) in atherogenesis, the guinea pig should be an ideal animal
model for future atherosclerosis research.
Our knowledge about the atherogenetic process in recent years has profited
by an increased understanding of the role of cholesterol and lipid metabolism
in general. Less attention has been paid to the extracellular matrix
and particularly to the role of ascorbate in maintaining its integrity.
It is of interest that already toward the end of the last century the
German pathologist Rudolf Virchow described the early stage of atherosclerosis
as a "certain loosening of the connective-tissue ground substance" followed
by lipid infiltration.16 These observations have been supported in the
meantime by electron-microscope studies.17 These observations and the
data reported here may contribute to a more comprehensive understanding
of human atherogenesis.
In this context it will be of particular interest to identify the underlying
mechanism of the accumulation of Lp(a) in the arterial wall leading to
plaque development. It is likely that ascorbate deficiency increases
the infiltration of all lipoproteins into the arterial wall due to the
disintegration of the endothelial cell lining and the impairment of the
extracellular matrix at low ascorbate concentrations .11,18< In accordance
with our hypothesis that apo(a) is a surrogate for ascorbate, Lp(a) accumulation
in the arterial wall would be a consequence of the cellular and extracellular
disintegration caused by a decrease of tissue ascorbate concentrations.
It seems likely therefore that ascorbate depletion leads to an increase
in apo(a) synthesis and our preliminary data support this mechanism.
As discussed earlier low ascorbate levels may enhance the selective accumulation
of Lp(a) in the arterial wall by decreasing the ratio of hydroxylisine
to lysine in different components of the arterial wall which in turn
would enhance the binding of Lp(a). Further studies are also needed to
determine the degree of lipidperoxidation and foam cell formation19,20
at low ascorbate concentrations. Independent of the potential pathomechanisms
involved, the most important therapeutic finding of this study is the
fact that appropriate amounts of ascorbate prevent the development of
atherosclerotic plaques and the deposition of Lp(a) in the arterial wall.
Since the atherogenicity of Lp(a) in humans is established beyond doubt
by epidemiological, biochemical, and histological studies, our findings
have significant implications for the future treatment of cardiovascular
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