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From the Department of Pathology,*
Institute of Basic
Medical Sciences, University of Tsukuba, Tsukuba, Japan; the Northwest
Lipid Research Laboratory,
University of
Washington, Seattle, Washington; the Research Institute of Yamanouchi
Pharmaceutical Company,
Tsukuba, Japan; and
the Saga Medical School,
Saga, Japan
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Abstract |
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On the basis of a partial amino acid sequence, followed by a complete analysis of the nucleotide sequence of cloned apo(a) complementary DNA (cDNA), extensive homology was revealed between apo(a) and plasminogen.11,12 This has led investigators to suggest that the proatherogenic association of high Lp(a) concentrations may result from its atherogenic potential as a cholesterol-rich lipoprotein particle after receptor-mediated uptake and/or the sequence mimicry between apo(a) and plasminogen, leading to an inhibition of plasminogen’s anti-thrombotic effects13 and anti-proliferative effects on smooth muscle cells (SMCs) through the inhibition of conversion of the active form of transforming growth factor-ß (TGF-ß).14-16 One possible mechanism is that Lp(a) may exert anti-fibrinolytic effects by competitively inhibiting the binding of plasminogen to cells and fibrin.13 Thus far, in vitro data support these hypotheses, but whether or not these mechanisms are operating in vivo remains to be determined. The development of transgenic mice expressing human apo(a) provided an alternative means to study apo(a) functions.17-20 However, unlike in humans, in which nearly all plasma apo(a) is associated with apoB-100, the human apo(a) in the transgenic mice circulates in a free form in the plasma rather than in association with the murine LDL.20 Despite this, expression of human apo(a) resulted in increased aortic fatty streak formation when these apo(a) transgenic mice were fed a high-fat diet,17,21,22 although other studies failed to reproduce the atherogenic effect of the apo(a) in transgenic mice expressing either human apo(a) alone or both human apo(a) and apoB.23,24 These results may suggest that a certain strain of mouse with a different genetic background may be resistant to the atherogenic effects of apo(a).
To define the metabolic and pathological consequences of Lp(a), our laboratory generated transgenic rabbits expressing human apo(a) and showed that human apo(a) is efficiently assembled into Lp(a) in the plasma.25 Recently, we reported that Lp(a) substantially increases the development of atherosclerosis (surface area involvement defined by Sudan IV staining) in transgenic rabbits fed a cholesterol-rich diet.26 To explore the mechanism(s) underlying Lp(a) atherogenicity, the present study was designed with three goals in mind: 1) to determine the influence of Lp(a) on SMC proliferation and dedifferentiation, namely, phenotype changes, during atherosclerosis in transgenic rabbits; 2) to disclose whether the TGF-ß pathway may be involved in the lesion formation; and 3) to determine whether Lp(a) inhibits the fibrinolytic process in transgenic rabbits. Our results revealed that Lp(a) may enhance the lesion development by stimulating SMC proliferation and dedifferentiation possibly through impaired fibrinolytic activity.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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A total of 11 (5 male and 6 female) transgenic and 18 (10 male and 8 female) nontransgenic littermates at the age of 4 to 5 months were fed a diet containing 0.3% cholesterol and 3% soybean oil by weight for 16 weeks as described previously.26 All rabbits were housed in a single cage in a pathogen-free animal facility on a 12-hour dark/12-hour light cycle and had free access to food and water. All animal experiments were performed with the approval of the Animal Research Committee of the University of Tsukuba.
Analysis of Plasma Lipids and Lipoproteins
Plasma total cholesterol and triglycerides were measured using enzymatic assays (Wako Chemicals, Osaka, Japan). Plasma lipoproteins and plasma levels of human apo(a) were determined by an enzyme-linked immunosorbent assay using human recombinant apo(a) as a standard.27,28
Quantitative Analysis for Histology and Immunohistochemistry
Rabbits were euthanized with an overdose injection of sodium
pentobarbital solution (Abbott Laboratories, North Chicago, IL). A
thoracotomy and laparotomy were performed to expose the heart and the
entire arterial tree, which included both common carotid arteries, the
whole aorta, and both common iliac arteries (Figure 1)
. In this study, we attempted to make
histologically quantitative and qualitative analyses on each part of
the arterial wall by measuring the intimal lesion area and quantifying
the cellular components (SMC versus macrophages). For these
purposes, the representative lesion-prone segments of the aorta,
carotid artery, and iliac artery were cut in cross-sections as shown in
Figure 1
, embedded in paraffin, and stained with hematoxylin and eosin
and Elastica-van Gieson.
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Antibodies (Abs) used for this study were listed in Table 1
. In this study, we were specifically
interested in SMC phenotypes in the lesions with use of a panel of Abs
against smooth muscle myosin heavy chains (SM1, SM2, SMemb) and basic
transcriptional element binding protein-2 (BTEB2), which mediates the
transcriptional regulation of the SMemb gene during
phenotypic modulation of SMCs.30-32
In addition, we also
attempted to evaluate the relative changes in the contents of
plasminogen and plasminogen activator inhibitor 1 (PAI-1), TGF-ß
(both latent and active forms), and TGF-ß receptors I and II in the
lesions. Evaluations of immunohistochemical staining were made by two
independent trained observers blindly.
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Plasma levels of total amount of PAI-1 in cholesterol-fed rabbits were measured by latex photometric immunoassay (SRL Inc., Tokyo, Japan). To reveal whether transgenic rabbit Lp(a) would inhibit the fibrinolytic process, rabbit plasma in the presence of sodium citrate was immediately frozen and delivered in dry ice to Dr. Maurhofer Olivier (Symphar Co., Geneva, Switzerland) for analysis. Plasma Lp(a) was isolated from Lp(a)-rich fractions of cholesterol-fed transgenic rabbits by ultracentrifugation at a density of 1.12 g/ml33 and analyzed by the euglobulin clot lysis assay as described by others.34 Recombinant human apo(a) prepared from transfected mammalian cells was used as a positive control.
Statistic Analysis
Plasma lipids were compared in the groups of rabbits by using the Student’s t-test, and the lesion analyses were compared using the Mann-Whitney U test for nonparametric analysis. In all cases, statistical significance was set at P < 0.05.
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Results |
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The average human apo(a) levels in transgenic rabbits were
8.1 ± 3.9 mg/dL in males and 8.8 ± 3.3 mg/dL in females;
total cholesterol levels in plasma and body weight were identical
between transgenic and control rabbits (Table 2)
. Agarose gel electrophoresis analysis
revealed that expression of human apo(a) did not lead to marked changes
in lipoprotein profiles in transgenic rabbits compared to nontransgenic
rabbits (Figure 2)
. The lipoprotein
profiles and Lp(a) distribution of cholesterol-fed rabbits were further
studied by sequential density
ultracentrifugation.35
As shown in Figure 3A
, in general, transgenic and
nontransgenic rabbits exhibited a similar pattern of lipoprotein
profiles; there was a marked increase in apoB-containing lipoproteins
including lipoproteins with d < 1.006 (mainly ß-very
low-density lipoprotein) and also those with d = 1.006 to 1.02
(intermediate-density lipoproteins); and those with d = 1.02 to
1.04 (LDL) in both transgenic and nontransgenic rabbits.
Basically, apoB contents in each density fraction of transgenic rabbits
were almost identical to those of nontransgenic rabbits (Figure 3B)
.
Human apo(a) was concomitantly distributed in a range of
apoB-containing lipoproteins: d < 1.006, d = 1.006 to 1.02,
and d = 1.02 to 1.04 g/ml. This indicates that human apo(a) formed
Lp(a) complexes with rabbit apoB (Figure 3C)
even though their
association was not completely covalent because of the difference in
the cysteine residue site between rabbit and human apoB.26
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Table 2
summarizes the results of cross-sectional lesion area in
transgenic and nontransgenic rabbits. In all parts of the aorta (except
abdominal aorta in females) and iliac and carotid arteries, transgenic
rabbits had significantly greater lesions than those of nontransgenic
rabbits although female rabbits, both transgenic and nontransgenic,
developed relatively fewer lesions than their male counterparts in each
group. The reason for less effect of Lp(a) on the lesion formation in
females than that in males may be in part, because of protective
functions of estrogen.36
It is worth noting that lesions were extensive in iliac and carotid
arteries, resulting in a 10- and 18-fold increase in male transgenics
compared to controls. In female control rabbits, no lesions were found
in the selected sections (Table 2)
.
Coronary artery atherosclerosis was evaluated by measuring the intimal
thickness and lesion area of the left main trunk. As shown in Table 3
, male and female transgenic rabbits
showed increased lesion size as compared to control rabbits, although
the enhanced extent of lesions in female transgenic rabbits did not
reach statistical significance. As in the case of the aortas and
carotid and iliac arteries, female rabbits developed less coronary
atherosclerosis than the male animals.
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Histological observation revealed that atherosclerotic lesions in
control rabbits were mainly composed of macrophage-derived foam cells
and a relatively small number of SMCs (Figure 4A)
. In apo(a) transgenic rabbits,
however, the atherosclerotic lesions varied from foam cell-rich
lesions to spindle-shaped, cell-rich lesions (Figure 4
; B, C, and D),
presumably SMCs that were either positive (from strong to weak staining
intensity) or negative for smooth muscle 
-actin mAb (see below).
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. The ratios of SMCs to
macrophages defined by each staining area in the lesions were 0.25
(control) versus 1 (transgenic) in the aortic arch and 0.8
(control) versus 1.5 (transgenic) in the coronary artery.
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; B, C, and D), and that were presumably immature SMCs (see
below).
In the pattern 1 and 2 lesions of transgenic rabbits, apo(a) was either
focally present or uniformly located beneath the foam cells (Figure 4, B and C)
. The immunoreactive apo(a) was associated with apoB,
suggesting that the apo(a) was directly from plasma Lp(a). Our previous
study also excluded the possibility that apo(a) was produced de
novo by arterial wall.26
In the pattern 3 lesions,
however, apo(a) was diffusely distributed around extracellular matrix
and only partially co-localized with apoB (Figure 4D)
. Regardless of
the apo(a) deposition patterns, all intimal cells in apo(a)-containing
areas were, almost invariably, not stained by mAb against macrophages.
They were either negative or only weakly positive for anti-smooth
muscle -actin staining. We therefore, initially postulated that
these intimal cells in apo(a)-containing areas may be those of immature
or dedifferentiated SMCs.
To further examine the phenotype changes of these intimal SMCs,
we performed immunohistochemical staining using a panel of mAbs for
myosin heavy chains on a spindle cell-rich lesion. A representative
lesion of aortic arch from a transgenic rabbit is shown in Figure 6
. In this lesion, apo(a) deposition was
evident along the bottom of the lesion (Figure 6A)
. Intimal cells at
the center could not be stained by smooth muscle -actin mAb but SMCs
located in the superficial area and in the media were positive for
smooth muscle -actin mAb (Figure 6B)
. SM1 antibody could detect both
medial and almost all of the intimal SMCs, but SM2 antibody
stained only mature SMCs from the media, suggesting that these intimal
SMCs were immature (Figure 6, C and D)
. To verify this contention, we
further stained the lesions using SMemb mAb, a marker for activated and
immature SMCs, and found that these intimal SMCs were positive for
SMemb mAb (Figure 6E)
. In parallel, we stained the lesions with mAb
against BTEB2, a transcription factor for SMemb. As shown in Figure 6F
,
all intimal SMCs (note the cellular nuclei) but not medial SMCs were
stained by BTEB2 mAb.
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Previous studies using transgenic mice and cultured cells
proposed that Lp(a) may compete with plasminogen to bind to fibrin or
cell receptor, thereby impairing the plasmin generation and
accordingly, delay the formation of the active form of
TGF-ß.14-16
To investigate whether this mechanism was
operating in human apo(a) transgenic rabbits, we performed
immunohistochemical staining using three kinds of specific mAbs either
against both plasminogen and plasmin or plasminogen only (Table 1)
. As
shown in Figure 7
, there was very weak
staining for plasminogen in both transgenic and control rabbits, which
did not enable us to make a direct comparison of these lesions between
transgenic and control rabbits. Weak staining for plasminogen in the
lesions was consistently confirmed by using all three kinds of
antibodies listed in Table 1
. However, the tendency was that the
immunoreactivity of the latent form of TGF-ß1 was increased but the
active form of TGF-ß was reduced in type 3, SMC-rich lesions in
transgenic rabbits compared to foam cell-rich lesions in both
transgenic and control rabbits (Figure 7)
. In such lesions, TGF-ß
receptor II predominated over TGF-ß receptor I (Figure 8)
.
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We further examined the effect of isolated Lp(a) from
transgenic rabbit plasma on euglobulin clot lysis time. As shown in
Figure 9
, Lp(a) from transgenic rabbits
apparently delayed the clot lysis at the dose of 0.8 
6.4 mg/dL
compared to control rabbit plasma. Furthermore, transgenic rabbits had
higher levels of total plasma PAI-1 than did nontransgenic rabbits
(Figure 10)
. Immunohistochemical
staining showed that in the lesions, PAI-1 was produced almost by all
kinds of vascular cells including endothelial cells, SMCs, and
macrophages (Figure 10)
.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The results of this study provide further evidence that Lp(a) increases the susceptibility to atherosclerosis in transgenic rabbits on a cholesterol-rich diet. Transgenic rabbits showed uniform enhancement of the lesion size in all parts of the aortic tree. Compared to nontransgenic rabbits, in which the lesions were mainly macrophage-derived foam cells, the lesions of transgenic rabbits were predominantly enriched in SMCs, suggesting that Lp(a) promotes the proliferation of SMCs during the lesion development of atherosclerosis. All these features were not documented in the previous studies using transgenic mice in which human apo(a) was unable to bind to murine apoB to form Lp(a) in plasma.20 On a high-fat diet, human apo(a) transgenic mice developed more fatty streaks (rather than other types of lesions) in the aortic sinus.17 Expression of human apo(a) in transgenic rabbits, however, aggravated atherosclerosis in all arterial beds examined, and furthermore, the lesions were characterized by a marked proliferation of SMCs. Especially noteworthy in this study was the significant increase of lesions in muscular arteries, carotid, iliac, and coronary arteries, in transgenic rabbits compared to control rabbits. This diffuse spatial distribution may mimic the situation in humans in that high levels of Lp(a) lead to the increased incidence of coronary, peripheral vascular, and cerebrovascular diseases in patients.2
Apo(a) Deposition and SMC Proliferation in Atherosclerosis
We investigated the relationship between apo(a) deposition and
lesion quality. In the lesions of transgenic rabbits, human apo(a) was
mainly deposited in extracellular matrix but not apparently associated
with foam cells. Consistent with our previous observation, Lp(a) may
not, if at all, contribute to the formation of foam cells. Of
particular note, apo(a)-deposited areas contained numerous immature
SMC-like cells based on the finding that the lesions were negative or
only weakly positive for smooth muscle -actin staining. This notion
has been further strengthened by the fact that these SMCs are negative
for SM2 but positive for SM1 and SMemb and a transcript factor for
SMemb, BTEB2 mAbs. SM1 is constitutively expressed at all developmental
stages of SMCs, whereas SM2 is a marker of mature SMCs and appears only
after birth.30,31
On the other hand, SMemb is an embryonic
isoform of myosin heavy chain, the expression of which is prominent in
fetal aorta and normally down-regulated during
development.30
SMemb expression is up-regulated in
immature or activated SMCs such as neointimal SMCs resulting from
vascular injury.32
Taken together, these results indicate
that intimal SMCs in transgenic rabbits are either immature or in the
state of dedifferentiation and resemble those of neointimal
synthetic-type SMCs from balloon injury or restenosis after
percutaneous transluminal coronary angioplasty.32
We thus
suggest that Lp(a) may not only stimulate the migration and
proliferation of medial SMCs toward the intima, but also mediate the
phenotypic alteration of SMCs toward an immature state.
Lp(a) and TGF-ß Pathway
Several previous studies have demonstrated that human Lp(a) and
apo(a) stimulate the proliferation of SMCs in vitro through
the inhibition of plasminogen activation and consequent reduction of
plasmin.14
In this context, reduced generation of plasmin
leads to inhibition of latent TGF-ß activation, which is an inhibitor
of SMC growth. Grainger et al14
also showed that in
transgenic mice expressing human apo(a) at the levels of 3.8 mg/dL,
there was approximately threefold less active plasmin in the vessel
wall than that in normal mice and the active form of TGF-ß was
significantly reduced.15
In the current study, we also
attempted to examine this hypothesis in transgenic rabbits using
immunohistochemical staining. Unfortunately, we found that the total
contents (as observed by immunostaining intensity) of plasminogen in
the lesions were extremely low and seemingly uncomparable between
transgenic and control rabbits. Immunohistochemical staining revealed
that latent form of TGF-ß tended to be comparatively increased and
accompanied by concomitant reduction of active form of TGF-ß in
SMC-rich lesions in transgenic rabbits but not in other lesions of
transgenic and nontransgenic rabbits, suggesting that the presence of
apo(a) deposition in the arterial wall might, if at all, lead to
inhibition of TGF-ß activation. However, one should be cautious in
interpreting this observation in terms of
Lp(a)-plasminogen-TGF-ß1-SMC growth scenario derived from
immunohistochemical staining because 1) the cellular components in such
apo(a)-containing areas (SMC-rich) of transgenic rabbits were
substantially different from those of other types of lesions
(macrophage-derived foam cell-rich) in transgenic and control rabbits;
and 2) the net amounts of autocrine/paracrine production of TGF-ß1
from different types of cells (SMC versus macrophages) in
the lesions may not be directly compared. Moreover, increased type
II/type I TGF-ß receptor ratio as shown in Figure 8
is also not in
favor of the Lp(a)-plasminogen-TGF-ß1-SMC hypothesis. It is possible
that Lp(a) may stimulate growth of SMCs via the independent mechanisms
from the inhibition of TGF-ß activation.37
Therefore, it
is still uncertain whether Lp(a) has a direct effect on SMC
proliferation and dedifferentiation either dependent on or independent
of the inhibition of TGF-ß activation or both.
Lp(a) and Fibrinolysis
The inhibition of plasminogen activation by Lp(a) has also been suggested to contribute to the development of vascular lesions by inhibiting fibrinolysis. It is of interest that we found that transgenic rabbits had higher plasma levels of PAI-1 than did nontransgenic rabbits and Lp(a) isolated from transgenic plasma inhibited the fibrinolytic process, suggesting that Lp(a) does possess prothrombogenic properties. Furthermore, Lp(a) may enhance the production of PAI-1 from the arterial wall thereby exacerbating this process.38 Although the biological significance and relevance of this anti-fibrolytic effect of Lp(a) to atherosclerosis remains to be defined, these results suggest that apo(a) transgenic rabbits may be potentially susceptible to thrombosis and may be specifically useful for studying pathophysiology of thrombosis.
Transgenic rabbits had similarly high levels of total cholesterol as
did control rabbits on a cholesterol-rich diet (Table 2)
. However,
atherogenic lipoproteins, ß-very low-density lipoprotein, and
intermediate-density lipoprotein from transgenic rabbits were markedly
enriched in human apo(a) (Figure 3)
, and, therefore, it is likely that
the increased amount of human apo(a) in these atherogenic particles was
the principal factor underlying the significant enhancement in the
atherosclerosis and impaired fibrinolytic activity in these animals.
Enrichment of apo(a) might increase the binding of these lipoproteins
to proteoglycans with the arterial wall intima,22
leading
to the retention of atherogenic lipoproteins in the intima, thus
accelerating atherogenesis. We recognize that the major atherogenic
lipoproteins in cholesterol-fed rabbits are those of hepatic remnant
lipoproteins, ß-very low-density lipoprotein, whereas in humans, LDLs
are the main atherogenic lipoproteins. It is necessary to have an
appropriate animal (human-like) model in which both Lp(a) and LDL
levels are elevated. Currently, we have generated WHHL transgenic
rabbits that express higher levels of Lp(a) than normal transgenic
rabbits and have elevated LDL levels,28
as in human
familial hypercholesterolemia. In humans, the risk of elevated Lp(a)
concentrations is significantly increased in patients who also have
high levels of LDL cholesterol.39,40
Therefore, it may be
particularly interesting to use WHHL transgenic rabbits to address
several issues related to pathophysiological functions of Lp(a) in both
atherosclerosis and thrombosis and also to test pharmacological
intervention to lower Lp(a) to inhibit the progression of
atherosclerosis.
In summary, we have shown that human apo(a) transgenic rabbits develop more extensive atherosclerosis than normal rabbits in the aorta, iliac artery, carotid artery, and coronary artery in response to a cholesterol-rich diet. Increased atherosclerosis in transgenic rabbits is associated with SMC proliferation and dedifferentiation, possibly related to impaired fibrinolytic activity. Although the molecular mechanism underlying this effect remains to be elucidated, the current results favor the notion that Lp(a) enhances the lesion progression of atherosclerosis by modifying the SMC phenotype.
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Acknowledgements |
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Footnotes |
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Supported by Grants-in-Aid for Scientific Research (10470046, 11470048, 11557016) from the Ministry of Education, Science, and Culture of Japan and the Japan Society for the Promotion of Sciences (JSPS-RFTF96I00202).
Accepted for publication October 8, 2001.
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), and smooth muscle
