Objective: A novel metalloproteinase, Pregnancy Associated Plasma Protein-A (PAPP-A), has been implicated in the development
of atherosclerosis, and is under consideration as a novel biomarker for acute coronary syndrome and unstable plaque.
The aim of this study was to determine if PAPP-A overexpression directly contributes to plaque vulnerability
Methods: Apolipoprotein E knock-out (ApoE KO) mice, a model of atherosclerotic development, and ApoE KO mice overexpressing
PAPP-A transgene in arterial smooth muscle (ApoE KO/Tg) were fed a high fat diet for 20 or 40 weeks starting at
seven weeks of age. At harvest, the brachiocephalic artery (BCA) was fixed, embedded in paraffin, sectioned and stained for
Results: After 20 weeks on high fat diet, significantly more ApoE KO/Tg mice had BCA lesions with a necrotic core and fibrous
cap than did ApoE KO mice. After 40 weeks on high fat diet all mice had BCA plaques with necrotic cores, but plaque
progression with healed ruptures (i.e., buried fibrous caps), and plaque inflammation were greater in BCA of ApoE KO/Tg
mice than in ApoE KO mice.
Conclusion: Overexpression of PAPP-A in arterial smooth muscle of ApoE KO mice is associated with accelerated plaque
progression and development of vulnerable and ruptured plaque.
Pregnancy associated plasma protein-A; Brachiocephalic artery; Plaque stability; Vulnerable plaque; Apolipoprotein
E knock-out mice
Atherosclerosis, one of the major diseases of industrialized
nations, represents a complex response to chronic vascular
injury that involves several cell types and associated cytokines,
growth factors and enzyme systems [1-3]. Among the
spectrum of events, injurious agents promote the infiltration
of monocytes from the circulation, and these in turn become
lipid-laden macrophages (foam cells) forming fatty streaks
in the intima of the vessel. Transition from relatively simple
fatty streaks to more advanced lesions is associated with accumulation
of smooth muscle cells in the luminal space that
proliferate, take up modified lipoproteins and synthesize extracellular
matrix. Complex lesions are characterized by a
necrotic lipid-rich core covered by a fibrous cap of smooth
muscle cells or fibroblasts.
In humans, lipid-laden plaques with thin or uneven fibrous
cap are the most prone to rupture ('vulnerable' plaque), especially
at the shoulder region of eccentric plaques [4,5]. Unfortunately,
identification of culprit and vulnerable plaques
usually comes after the fact, (i.e., at autopsy or coronary
atherectomy), since imaging systems generally evaluate luminal
narrowing but not plaque composition. Thus, the quest
for treatment options to prevent plaque progression would
benefit from better understanding of the pathobiology of atherosclerosis
and the use of animal models that produce vulnerable
plaques "at risk" for rupture .
Pregnancy associated plasma protein-A (PAPP-A),
a newly recognized metalloproteinase in the Insulin-like
Growth Factor (IGF) system, has been implicated in vascular
repair processes in vitro and in vivo , and as a circulating
biomarker for acute coronary syndrome in humans [8,9]. Furthermore,
there is strong PAPP-A immunostaining in human
autopsy samples of vulnerable atherosclerotic plaque that is
associated with activated macrophages and smooth muscle cells, especially in the structurally-weakened shoulder region
of an eccentric culprit plaque .
To determine if PAPP-A contributes directly to atherosclerotic
plaque development, we crossed PAPP-A knock-out
(KO) mice with apolipoprotein E (ApoE) KO mice, the latter
being an established murine model of atherosclerosis .
Compared to ApoE KO mice, the ApoE/PAPP-A double KO
mice had significantly reduced aortic plaque burden and delayed
progression from fatty streaks to complex lesions .
Conversely, ApoE KO mice overexpressing PAPP-A in arterial
smooth muscle had significantly increased aortic lesion area
. However, the effect of PAPP-A overexpression on plaque
vulnerability has not been evaluated. Thus, this study was designed
to test the hypothesis that targeted overexpression of
PAPP-A in arterial smooth muscle of ApoE KO mice accelerates
the development of atherosclerotic lesions with morphometric
characteristics of vulnerable plaque and plaque rupture.
Materials and Methods
Transgenic PAPP-A overexpression and atherosclerosis.
PAPP-A Tg mice (FVB genetic background) were crossed with
ApoE KO mice (C57BL/6 and 129 genetic background), as
previously described . It is of note that transgene expression
is driven by the minimal SM22a promoter that had been
modified by deletion of the repressor elements, which are triggered
by vessel injury . The highest PAPP-A transgene expresser,
Tg6' , was used for this study. Offspring from this
mating, heterozygous for the ApoE gene and positive for high
level PAPP-A transgene expression, were then intercrossed to
produce ApoE KO mice and ApoE KO mice expressing the
PAPP-A transgene, (ApoE KO/Tg). These littermates, males
and females housed separately up to five per cage, were fed a
high-fat, Western-style diet [21% by weight (42% of calories)
fat and 0.15% by weight cholesterol (Harland Tekland, South
Eaton, MA)] for 20 and 40 week starting at 7 weeks of age.
This protocol was reviewed and approved by the Institutional
Animal Care and Use Committee of Mayo Clinic.
At harvest, the Brachiocephalic Artery (BCA) was fixed in situ
by perfusion with Phosphate Buffered Saline (PBS)-formalin
at physiological pressure. Individual arteries were removed,
placed in PBS-buffered formalin, and fixed for 24 hours before
paraffin embedding. Cross-sections (5.0 Ám thick) were
collected over the length of the BCA. Each end and middle
sections were stained with hematoxylin and eosin. Adjacent
sections were stained with Verhoeff-Van Gieson (Accustain;
Sigma-Aldrich, St. Louis, MO). Microscopic analysis was
performed by an expert cardiovascular pathologist (WDE)
, blinded to genotype, and according to criteria used in
anatomic pathology and modified for mice (Table 1). Fisher's
exact test was used for statistical comparisons between ApoE
KO and ApoE KO/Tg mice.
Immunohistochemistry. De-paraffinized sections of
BCA were stained for macrophages using F4/80 as primary
antibody, as described previously .
Body weights of ApoE KO and ApoE KO/Tg mice are shown in Table 2.There were no differences between the two strains
of mice prior to or 20 and 40 weeks after high fat diet. Summarized
data for the histopathology review of BCA plaque
morphology are presented in Table 3 for mice 20 weeks on
high fat diet and in Table 4 for mice 40 weeks on high fat diet.
There was no evidence of luminal thrombus, surface erosion,
or plaque hemorrhage in any of the sections, and all plaques
After 20 weeks on high fat diet, significantly (P = 0.002)
more ApoE KO mice that were overexpressing PAPP-A had a
necrotic core in BCA plaques than did ApoE KO mice negative
for the transgene (Table 3). There was no significant difference
between the two groups of mice in terms of plaque grade and
internal elastic membrane (IEM) disruption, and there was no
calcification or inflammation in either group. Figure 1presents a grade 2 (of 4) plaque (based on 25% increments of luminal
narrowing in cross-sectional area) having a necrotic core with
cholesterol clefts. There are foam cells without a fibrous cap
as well as within and beneath the fibrous cap. There is also an
example of disruption of the IEM with extension of the plaque
into the media. Interestingly, chondrocyte-like cells were only
seen in the BCA plaque of ApoE KO, and plaque progression
was only seen in the BCA of ApoE KO/Tg mice. However,
numbers were small.
After 40 weeks on a high fat diet, the BCA plaques of
all mice had necrotic cores, and there were no differences between
the ApoE KO and ApoE KO/Tg mice in terms of IEM
disruption, presence of chondrocyte-like cells (Figure 2A), or
calcification (Figure 2B). However, plaque progression and
inflammation were substantially greater in BCA of the ApoE
KO/Tg mice than in ApoE KO mice (Table 4). This difference did not reach statistical significance (P = 0.07) likely due
to the small group sizes. Plaque progression indicated healed
ruptures (i.e., multiple layers of necrotic cores interspersed by
fibrous tissue). Examples of these buried plaques and associated
macrophage staining are presented in Figure 3.
In this study, we present morphological evidence that overexpression
of PAPP-A in arterial smooth muscle of ApoE KO
mice is associated with accelerated plaque progression and development
of vulnerable and ruptured plaque (Figure 4).
After 20 weeks on high fat diet, there was a significant
increase in the number of ApoE KO/Tg mice bearing BCA
plaque with a necrotic core. After 40 weeks on high fat diet,
these BCA plaques showed numerous buried fibrous caps,
which are indicative of an unstable plaque phenotype and are
a surrogate marker of plaque rupture [16-19]. This layered appearance
in mouse BCA plaques is similarly observed in human
coronary arteries, and it has been suggested that healed
plaque ruptures play a role in plaque progression and sudden
coronary death in humans .
There has been controversy in the literature about
whether plaque rupture occurs in mice. Issues arise from
the definitions of plaque vulnerability and rupture and sudden death in mice versus humans [16,21-24]. Also, several
of the studies in mice have evaluated plaque stability in the
aortic sinus, which is not a typical site affected by atherosclerotic
disease in mice or humans. Lesions in the aortic sinus
are relatively refractory to destabilization . The major site
of predilection for developing vulnerable plaque and plaque
rupture in mice is the BCA/innominate artery connecting the
aortic arch to the right common carotid and right subclavian
arteries . Williams et al.  reported a high frequency
of plaque instability in the BCA and spontaneous death by
myocardial infarction in ApoE KO mice after 40-60 weeks on
a high fat diet. Similarly, the BCA is a site for advanced lesions
in humans .
There were no sudden deaths in ApoE KO/Tg mice
in this study, even after 40 weeks on high fat diet. As nicely
discussed by Jackson et al. , the requirement for luminal
thrombus to define plaque rupture in mice needs to be reconsidered,
since occlusive thrombi are unlikely to be found in mice given their active fibrinolytic system and rapid thrombolysis.
On the other hand, buried fibrous caps are indicators
of repeated episodes of non-fatal rupture and repair in humans
and mice [6,16-20].
In this study, we also observed an interesting pattern
of lipid-laden macrophages above, within, and beneath fibrous
caps, especially in BCA plaque of ApoE KO/Tg mice, and an
increase in plaque inflammation in ApoE KO/Tg mice. Inflammation
is known to play a critical role in the progression
of atherosclerosis [1,26]. Indeed, macrophages in shoulder
regions of plaque are considered to be vulnerable to rupture.
It is of note that pro-inflammatory cytokines associated with
atherosclerotic plaque development are potent stimulators of
PAPP-A expression in human arterial smooth muscle and endothelial
cells [27, 28].
The mechanism(s) by which PAPP-A promotes plaque
progression and instability is unclear. Our working hypothesis
is that activated macrophages in plaque synthesize pro-inflammatory
cytokines that stimulate vascular smooth muscle and
endothelial cells to synthesize and secrete PAPP-A . Thus,
PAPP-A can function in an autocrine/paracrine fashion as an
important amplification point in plaque progression. That a
key function of PAPP-A is to increase local IGF available for
receptor activation  suggests an IGF-dependent mechanism.
However, in contrast to what we found with PAPP-A
overexpression, smooth muscle-specific IGF-I overexpression
in ApoE KO mice was reported to have no effect on plaque
burden, macrophage accumulation, oxidative stress, inflammation,
or smooth muscle cell apoptosis, but it increased features
of plaque stability, i.e., increased smooth muscle cell content
and decreased necrotic core size in the aortic valve . It
remains to be determined whether these different morphologies
reflect IGF-independent effects of PAPP-A or are due to
analyses at different sites.
In a recent study by Zhao et al. , serum PAPP-A
correlated positively with necrotic core area. Patients with unstable
angina and with no-reflow after percutaneous coronary
intervention had higher serum PAPP-A, coronary plaques
with higher percentage necrotic core area, more thin-cap fibroatheromas,
and ruptured plaque as measured by virtual
histology intravascular ultrasound. These findings, in conjunction
with ours, suggest PAPP-A not only as a marker of
vulnerable plaque, but also as a potential therapeutic target to
limit plaque progression.
This work was supported by National Institutes of Health
award HL 074871 to CAC.