Brief Reviews

Plaque Rupture in Humans and Mice

Stephen M. Schwartz, Zorina S. Galis, Michael E. Rosenfeld, Erling Falk
AbstractDespite the many studies of murine atherosclerosis, we do not yet know the relevance of the natural history of
this model to the final events precipitated by plaque disruption of human atherosclerotic lesions. The literature has
become particularly confused because of the common use of terms such as instability, vulnerable, rupture, or even
thrombosis for features of plaques in murine model systems not yet shown to rupture spontaneously and in an animal
surprisingly resistant to formation of thrombi at sites of atherosclerosis. We suggest that use of conclusory terms like
vulnerable and stable should be discouraged. Similarly, terms such as buried fibrous caps that imply preceding
events that are unproven tend to create confusion. We will argue that such terminology may mislead readers by implying
knowledge that does not yet exist. We suggest, instead, a focus on specific processes that various forms of data have
implicated in plaque progression. For example, formation of the fibrous cap, protease activation, and cell death in the
necrotic core can be well described and have all been modeled in well-defined experiments. The relevance of such
well-defined, objective, descriptive observations in the mouse can be tested for relevance against data from human
pathology. (Arterioscler Thromb Vasc Biol. 2007;27:705-713.)
Key Words: plaque rupture murine atherosclerosis fibrous cap vulnerable plaque progression

he term plaque rupture in human pathology is not

controversial. The term has been used for decades to
identify a structural defect in the fibrous cap that separates
a necrotic core of an atherosclerotic plaque from the
lumen, resulting in exposure of the necrotic core to the
blood via the gap in the cap (Figure 1, left panels).15
Often, ruptured human lesions evulse part of the plaque
into the lumen, sometimes resulting in emboli. Exposure of
prothrombotic molecules is presumed to precipitate the
formation of a platelet-rich thrombus.

distinct but related processes within the plaque (hemorrhage),

at the plaque surface (disruption), and over the plaque
(thrombosis), we suggest the use of the terminology described
in the Table in the online supplement (available online at
http://atvb.ahajournals.org). The online version is an expanded version with more thorough discussion of experimental models of possible vulnerable features and a review of
reports of murine lesions that may be representative of human
ruptured plaques which may be too infrequent for use in an
experimental setting.

See pages 697, 714, 969, and 973 and cover

Plaque Rupture Requires a Necrotic Core

Covered by a Fibrous Cap

With the exception of events seen in a small proportion of

atherosclerotic mice,6,7 murine lesions have not as yet progressed to this stage. As a result, the common use of terms
like vulnerable or unstable to describe mouse lesions
implies a conclusion we cannot know is true.8 13 A further
problem is the tendency to overuse the term rupture to
describe murine lesions, including lesions we have described
(Figures 1 and 2). Less severe plaque injuries do occur and,
for clarity, we suggest use of the more general term disruption to refer to any loss of the integrity of the plaque surface,
ranging from a simple loss of endothelial cells to minor
fissures that penetrate into the plaque without exposing the
necrotic core, to frank breakdown of the fibrous cap over a
necrotic core with hemorrhage into the plaque, as is seen in
the murine part of Figure 1. To avoid confusion and enhance
our understanding of the complex interaction between the

It may seem paradoxical that fatty streak lesions (supplemental Figure II), without a fibrous cap and covered only by
endothelium, largely remain intact. Even though the endothelium overlying fatty streaks appears very delicate, any disruption is limited to the presence of apoptotic endothelial
cells and, possibly, the focal adhesion of platelets.14 18 Any
effort to create an animal model of plaque rupture must
presuppose the existence of a fibrous cap overlying a necrotic
core; this combination is required for plaque rupture in
human.

Necrotic Core
Contrary to general expectations, it is not clear that increasing
the rate of cell death in the necrotic core increases the
probability of disruption. Recent efforts to increase the extent

Original received May 18, 2006; final version accepted February 2, 2007.
From the Department of Pathology (S.M.S., M.E.R.), University of Washington, Seattle; the Indiana University and Lilly Research Laboratories
(Z.S.G.), Indianapolis; and the Department of Cardiology (E.F.), University of Aarhus, Denmark.
Correspondence to Stephen M. Schwartz, Department of Pathology, 815 Mercer Street, Room 421, University of Washington, Seattle, WA 98109-4714.
E-mail steves@u.washington.edu
2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org

DOI: 10.1161/01.ATV.0000261709.34878.20

705
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Figure 1. Plaque disruption in humans and mice. Left panel, Photomicrograph and schematic drawing of a ruptured human lesion in a
coronary artery. Characteristic features include the extensive disruption of the thick (compared with mice) fibrous cap, expulsion of
fragments of the lesion into the lumen, and access of blood to the necrotic core. The resulting overlying thrombus, although characteristic of this sort of disruption, is not part of our definition of plaque rupture. The inset shows the full circumference of the vessel,
including the occlusive thrombus. Trichrome stain; collagen blue, and thrombus and hemorrhage red. Right panel, Photomicrograph and schematic drawing of a fissured murine lesion in the innominate/brachiocephalic artery of a 42-week-old male apoE/
mouse fed a chow diet. Characteristic features include the presence of a superficial xanthoma, including xanthoma overlying the lateral
edge of the plaque (lateral xanthoma) which penetrates the thin fibrous cap typical of murine lesions. Plaque disruption occurred in this
lesion likely because of the death of cells in the lateral xanthoma. Movat pentachrome stain; collagen yellow, proteoglycans light
blue, and blood components red.

of the necrotic core have been based on the reasonable

assumption that the necrotic core results from macrophage
death and that death of the macrophage is driven by some
form of apoptosis. Increases in the extent of atherosclerosis
have been reported in response to knockout of the proapoptotic protein p53 in apoE*3-Leiden transgenic mice or
apolipoprotein E deficient (apoE/) mice.19 21 The lesions in
these mice showed an increase in the extent of the necrotic
core. Similarly, transplantation of bone marrow from apoE/
ACAT-1/ mice into apoE/ mice increased cell death
within the lesions, but led to an increase in lesion area.22
Thus, ongoing apoptosis may limit macrophage accumulation
in the lesion, but not affect the rate of necrotic core formation.
Conversely, a reduction in cell death attributable to transplant
of BAX/ cells also led to an increase in lesion area in
fat-fed LDLR/ mice.23 None of these experiments has, as of
yet, resulted in plaques that become disrupted spontaneously.
Studies attempting to model the endogenous mechanism of
formation of the necrotic core have also failed to induce
rupture. Fowler proposed that macrophage death might be the
result of irreversible damage to lysosomes by lipid accumulation.24 Two decades later, Fazio, Tabas, et al separately
showed that inhibition of cholesterol esterification or block-

ing of cholesterol transport from the endoplasmic reticulum

leads to lipid accumulation in plaque macrophages and an
increase in formation of a necrotic core. Consistent with the
paradoxical response to p53 or BAX knockout, these manipulations produced unexpected increases or failure to decrease
plaque mass but not plaque rupture.25 We need to consider
that two or more mechanisms of cell death in the lesion may
produce distinctive results in terms of the size of the necrotic
core. One pathway, primarily apoptotic and dependent on p53
or BCL2-like proteins, may determine rates of foam cell
accumulation without accumulation of necrotic cell debris. A
different pathway, perhaps oxLDL-induced death, the formation of cytotoxic lipids, or simply bulk accumulation may be
required to disrupt the overlying fibrous cap.

Fibrous Cap
Application of terms like vulnerable to the murine fibrous
cap is especially confusing (supplemental materials; Figure
III). The human cap may be hundreds of microns in thickness
and highly cellular or, in other places, may resemble a tendon
with few, RNA-poor fibrocyte-like cells imbedded in a dense
connective tissue matrix.26,27 Murine fibrous caps are less
impressive, perhaps reflecting limitations of lesions growing

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Figure 2. Serial sections of disrupted mouse lesion. This figure contains a series of micrographs showing extensive plaque hemorrhage
that has originated along the margin of aggregated foam cells in an advanced lesion in the innominate/brachiocephalic artery of a
60-week-old chow-fed male apoE/ mouse. Movat pentachrome stain, upper left panel 100 final magnification, upper right panel
200 final magnification, lower left panel 1000 final magnification, lower right panel 1000 final magnification.

in vessels that are so much smaller than their human equivalents. In any case, the murine fibrous cap does not appear
to progress to form dense connective tissue and, instead, is
usually comprised of minimal numbers of thin lamellae of
loosely organized, elastin-rich connective tissue.
Surprisingly, almost nothing is known about the mechanisms controlling formation of the fibrous cap. Although
there have been arguments for a circulating cell origin of the
plaque smooth muscle, a recent article28 provides support for
the traditional view that the fibrous tissue of intima originates
from medial smooth muscle cells responding to cytokines

generated by the xanthomatous macrophages.29 32 Support

for a role for one cytokine in formation of the murine cap
grows from two studies where ablation of PDGF decreased
the number of intimal cells covering the fatty lesion.33,34
Interestingly, under these conditions there appears to be a
decrease in necrotic core formation, suggesting some unknown link between the cap and cell death in the underlying
macrophages.
Experimental manipulations may permit a test of the
importance of fibrous cap thickness. For example, even
though von der Thusen et al were able to produce a decrease

Figure 3. Drawings based on published images summarize the features of 2 lesions described as showing rupture or disruption in the
brachiocephalic artery of apoE/ mice. Left, Plaque hemorrhage penetrating deeply into a necrotic core, originating from the lumen via
a disruption (fissure) through a xanthoma at the edge of the fibrous cap in an old chow-fed apoE/ mouse. Right, Few displaced
erythrocytes located next to foam cells beneath an interrupted endothelium with superimposed mural thrombus in a relatively young,
fat-fed, severely hypercholesterolemic apoE/ mouse. The figures were drawn using painting tools in Photoshop and do not represent
individual published images.

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in cap thickness when they used a p53 adenovirus in apoE/

mice,35 only 3 of 16 mice showed morphological evidence of
cap breaks and only 1 of these showed thrombosis and
hemorrhage. The incidence of disruption, however, was
increased by infusion of phenylephrine, a vasoconstrictor, for
15 minutes. At 24 hours, plaque hemorrhage was seen in 7 of
20 animals, 1 of which showed thrombosis. The adenovirus
approach targets different cell types. In contrast, a novel
induction of apoptosis by targeting smooth muscle cells with
a diphtheria toxin receptor expressed by the SM22- promoter, induced marked thinning of the fibrous cap of atherosclerotic apoE/ mice, loss of collagen and matrix, accumulation of cell debris, and intense intimal inflammation, but did
not induce rupture.18 It would be fascinating to know whether
the latter lesions might have ruptured if exposed to phenylephrine, or if rupture might require death in cells other than
smooth muscle cells.

Murine Plaque Disruption

We (M.E.R., S.M.S.) were the first to report a murine model
with a reproducible frequency of disruption with plaque
hemorrhage.36 Between 30 and 40 weeks of age, about 80%
of lesions in the brachiocephalic arteries of C57BL/6 apoE/
mice showed plaque hemorrhage. Serial sections (Figure 2;
supplemental Figure VI) show that the hemorrhage arises at
the shoulder region where the fibrous cap was either absent or
minimal. Similar lesions were later reported by Renard et al
in the LDLR/ mouse with atherosclerosis accelerated by
diabetes37 and at a lower frequency in apoE/ mice used as
a control.38
The use of serial sections is important, because (intra)plaque hemorrhage might also occur via breakdown of
small intraplaque vessels as have been described in murine
lesions of the aortic arch,39 and a recent study by micro CT40
found a strong correlation between plaque hemorrhage and
the extent of plaque vasa vasorum in atherosclerotic mice.
The CT data did not show neovessels seen in the intima and
provided no data on the brachiocephalic arteries. Intraplaque
vessels have been described in mouse atherosclerotic plaques
of the aorta, but not in brachiocephalic lesions.39 41 We have
not seen intraplaque vessels in the brachiocephalic lesions,
even when we attempted to highlight the vessels by staining
with VE-cadherin antibodies or by perfusion with the vascular tracer, horseradish peroxidase (S.M.S. and M.E.R., unpublished results, 2006). It is therefore unlikely that breakdown of intraplaque vessels accounts for plaque hemorrhage
in lesions of the brachiocephalic artery.
About the same time as our report of plaque hemorrhage,
Jackson and colleagues reported acute plaque rupture with
luminal thrombosis in the brachiocephalic artery of apoE/
mice without convincing evidence of hemorrhage into the
plaque.42,43 They refer to this change as acute plaque
rupture, although as illustrated in our drawing based on their
work (Figure 3, right side), the extent of disruption may be
very small.44 Interpretation of their initial reports was complicated because an unexplained high number of mice died
suddenly and were found decomposed. Reasons for the
frequency of deaths in this model, approximately 25% in 2
months of the diet, have remained unexplained. The absence

of similar data in other studies may relate to strain background, a mixed C57BL/6-129 versus the usual C57BL/6
used as a background in most studies of apoE/, or toxicity
of severe hypercholesterolemia induced by their diet.
In any case, the model described by the Jackson group is
unique in resulting in thrombotic occlusion and possibly
death. That said, the definition of rupture used by this group
bears little resemblance to plaque rupture, as defined in
humans. A more appropriate term for such minimal disruption with thrombosis, if real and not postmortem clots, might
be erosion. Farb et al, as well as others, have used erosion
to describe thrombotic occlusion of human coronary arteries
at autopsy in the absence of breakdown of a fibrous cap and
exposure of a necrotic core.1,45 This lesion characteristically
includes endothelial denudation, though we do not know
whether the endothelial loss is the cause or a result of the
thrombus. Like the lesions reported by Jackson et al, erosion
does not expose a necrotic core, or even require the presence
of a necrotic core, because many of these fatal human lesions
are fibrous lesions without necrotic cores.45
In contrast to our work and the work from Jackson, lesions
approaching the extent of disruption seen in human lesions
(Figure 1) have been seen, as reported by Calara et al6 and
others7 in a few older atherosclerotic mice. Unfortunately, the
incidence, perhaps reflecting real stochastic variables, is too
low to be useful in experimental studies.

Fissures in the Lateral Xanthoma of Mice

Versus Ruptures in Human Plaques
We propose to use fissuring to describe less extensive
breaks in plaques that, if a necrotic core is present, may
extend down to the core, but with no or only minimal loss of
plaque material (supplemental Table). The murine hemorrhagic lesions described above by our group (S.M.S., M.E.R.)
meet this definition better than they meet the criteria for
rupture. Serial sections show that these fissures appear in
xanthomatous areas near the lesion shoulders, rather than
through the fibrous cap itself (Figure 1; supplemental Figure
VI). This sort of disruption through a macrophage-rich cell
mass, to our knowledge, has not been described in human
lesions. Importantly, unlike human plaque rupture, as discussed below, the murine hemorrhagic lesions do not precipitate thrombosis, in contrast to what Jackson et al described
for the much smaller defects in the same artery.43 Thus, we
use the term fissure to describe the degree of surface
disruption required for plaque hemorrhage in mice, but retain
a distinction from human plaque rupture.

Potential Artifacts
Interpretation of small breaks in the endothelium like the
acute plaque rupture described by Johnson et al, eg, their
Figure 1 and 244 are sufficiently small that it may be difficult
to rule out artifacts. Although endothelial death and increased
turnover does happen over atherosclerotic lesions, especially
in shoulder regions of the plaque, regeneration is rapid
enough that denuded areas are rarely seen in well-fixed
tissue.14 18,46 Because atherosclerotic lesions in mice, even
after fixation, are fragile and breaks can occur during handling, it is very valuable, as is the case for the plaque

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Schwartz et al
hemorrhage shown in Figure 1 (see also supplemental Figure
VI), to have certain evidence of some event that could only
occur if the disruption had been in the living animal. Jackson
et al suggest that luminal thrombus may be such a change. In
a recent study where their animals were intentionally euthanized and perfusion fixed to avoid concerns with postmortem
artifacts, thrombotic material was only seen in association
with discontinuities of the plaque surface, implying that the
thrombi were the result of disruption of the luminal surface in
vivo.43 Unfortunately, the article does not provide much
detail on the composition of the thrombus and only a few
displaced erythrocytes beneath an interrupted endothelium
(called intraplaque hemorrhage) were considered enough to
prove that the plaque surface was disrupted before death.44
Moreover, although the mice were reported to have thrombotic material in the lumen,43 no reports have been given on
the pathology of the brains. It would obviously be very
important to find out if this is a model for a thromboembolic
stroke originating in an atherosclerotic artery supplying the
brain.
Identification of extravasation of erythrocytes is obviously
critical to this discussion. In most cases, the distinctive
morphology of the red cells, as seen in conventional stains or
in a Movat stain, is sufficient to identify plaque hemorrhage
in a perfusion-fixed animal. However, caution should be used
when identifying the products of hemolyzed red cells as
hemorrhage. Tinctorial properties alone can be misleading, so
it is useful to identify red cells by electron microscopy or use
specific antibodies to identify red cell proteins.47 The presence of fibrin in lesions would provide independent evidence
for injury, but not proof of disruption, because intramural
coagulation might occur, even without disruption.48 Unfortunately, currently available antibodies are not useful because
of problems with distinguishing fibrinogen from fibrin. Although there have been claims to stain for fibrin in murine
lesions using antibodies,38,44,49 the antibodies used are either
known to be unable to distinguish murine fibrinogen from
fibrin,48 or lack published data demonstrating the needed
specificity.44 The best evidence that fibrin has formed is
electron microscopy showing the characteristic electrondense fibrillar structure with 215 angstrom cross striations.
To date, fibrin has not been seen in spontaneous plaque
hemorrhage by electron microcopy (S.M.S., unpublished
data, 2003). However, a recent study by Gough et al of
lesions disrupted by activated matrix metalloproteinase
(MMP)-9 did demonstrate that large amounts of fibrinogen
(or perhaps fibrin) were present at sites of plaque disruption,
and others have claimed to see luminal fibrin, based on
staining with other antibodies not yet shown to be specific for
fibrin.13,38,50
Another way of supporting a claim that an injury occurs in
vivo is to show that the injury is effected by in vivo actions
of a drug. Jacksons group has reported that their disruptions
were decreased by treatment with pravastatin.44 This confirms
that, as observed by one of the current authors (M.E.R.),51
statin treatment may change the composition of atherosclerotic plaques. However, such changes might also change
fragility, so the experiment does not prove that the observed
disruptions occurred in vivo.44 An in vivo test of endothelial

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709

integrity, such as evidence of hemorrhage through a defect or

use of horseradish peroxidase would be helpful to detect even
minor disruptions, such as occur when endothelial cells die,
or round up during mitosis.52,53
Finally, caution needs to be expressed about identification
of both acute and organized thrombi in arteries. It would be
desirable in reports of thrombi to have more detail about the
thrombus itself. Arterial thrombi formed under rapid flow
conditions are characterized by aggregated platelets and
sheets of fibrin, which are not seen when stagnant blood clots
postmortem, or when blood clots or is crosslinked during an
imperfect perfusion fixation. In older thrombi, cells from the
vessel wall migrate into the thrombus which, of course, is not
seen with postmortem clots, and the thrombus becomes
organized with time. Finally, as discussed above, it is difficult
to distinguish fibrin from fibrinogen, and care needs to be
exercised using special stains or poorly defined antibodies.

Proteolysis and Murine Lesion Disruption

Although there is widely held belief that proteases play a
critical role in disruption and rupture of the human lesion,
studies of protease expression in advanced lesions in experimental animals have produced confusing results.38,54,55 It is
important to realize that a protease, which might disrupt a
fibrous cap in a thick human lesion, may have very different
effects in the thinner vessel wall and more macrophage-rich
lesions seen in most experimental animals. For example, the
induction of aneurysm, but not rupture, by proteases induced
by angiotensin in atherosclerotic mice could be a result of the
difference in vessel wall structure in the murine model.56
Falkenberg and colleagues expressed urokinase in the
endothelium overlying atherosclerotic lesions in fat-fed rabbits, rather than mice, to take advantage of the greater
accessibility of the endothelium for viral gene transfer. The
urokinase plasminogen activator (uPA)-transduced arteries
had 70% larger intimas than control-transduced arteries,
smaller lumens, and evidence for degradation of elastic
laminae. Along with genetic data on elastin mutations from
others,57 these data suggest that elastin may serve to keep the
artery open, and that loss of elastin as a result of endothelialtargeted overexpression may allow inward pathological remodeling as is found in some advanced atherosclerotic
disease.
The most extensively studied molecular candidates for
rupture-producing proteases are the MMPs. Until recently,
most of these studies produced evidence only for changes the
authors considered as important for stability of lesions without objective evidence of disruption. For example, using the
apoE/ mouse, Johnson and colleagues studied double
knockouts for MMPs 3, 7, 9, and 12.13 Knockouts of 3 and 9
produced larger lesions with more buried fibrous caps, a
feature we will discuss below. In contrast, MMP-12 and
MMP-7 knockouts showed increased smooth muscle cell
content. The authors interpreted these data as evidence that
the normal function of MMP-3 and 9 are protective; MMP-7
is neutral; whereas MMP-12 is destabilizing. Overexpression
studies give a very different and more complex set of
conclusions, dependent on when MMPs are expressed and
activated. MMP-1 is an interstitial collagenase and would be

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expected to promote plaque rupture. Lemaitre et al54 expressed human MMP-1 under a macrophage-specific promoter in apoE/ mice. To their surprise, overexpression of
MMP-1 resulted in decreased experimental lesion size with
no evidence of plaque rupture. MMP-9 has received the most
attention. Increased MMP-9 activity and expression are
detected in the shoulders of advanced human lesions58 correlated with degradation of collagen, suggesting that MMP-9
would be destabilizing.9 However, MMP-9 also promotes in
interstitial collagen assembly59 by smooth muscle cells,
which might lead one to expect MMP-9 to contribute to the
mechanical strength of the plaque. The actual effect turns out
to be even more complex, depending on how the enzyme is
delivered and how it is activated. Increased transient expression of MMP-9 via intraluminal adenoviral delivery, largely
confined to the vessel lining,60 did not produce any form of
fibrous cap disruption. Instead, there was intralesional hemorrhage attributed to neoangiogenesis, as well as increased
outward (expansive) remodeling without increasing macrophage infiltration. The latter is in good agreement with the
previously reported effect of MMP-9. MMP-9 deficiency in
the MMP-9/ apoE/ mouse impaired the compensatory
enlargement of the carotid artery characteristic of lesion
development seen in the apoE/ mouse,61 as well as in
human atherosclerotic lesions.62 Interestingly, outward arterial remodeling is also a characteristic associated with human
plaque rupture, pointing out that we simply know too little to
predict how an enzyme may act in the complex plaque
milieu.63,64 Evidence for the importance of knowing where a
protease is activated comes from Gough et al. Transplanted
macrophages expressing an auto-activating form of MMP-9
induced plaque disruption in 9 of 10 mice when overexpressed in vivo in advanced atherosclerotic lesions of apoE/
mice, as compared with frequencies of about 1 of 9 in the
controls.38 Thus, MMP-9, expressed in the right place and
time, can rupture the plaque.
In summary, lesion disruption, in 1 case approaching the
severity of human plaque rupture,38 has been caused experimentally by interventions with proapoptotic stimuli and with
targeted delivery of MMP-9.

Consequences of Plaque Disruption in Mice

Surprisingly, hemorrhagic lesions in murine plaques do not
develop luminal thrombus, even though the hemorrhage
infiltrates the necrotic core. Fibrin is absent in the hemorrhage itself, even when studied by electron microscopy
(S.M.S. and M.E.R., unpublished data, 2003). Although the
failure to form fibrin in the lesion or to develop a thrombus is
disappointing, it is not entirely surprising. Fibrin is not seen
when normal rat arteries undergo injury with an inflated
balloon catheter.65 This, however, reflects the lack of tissue
factor in nonatherosclerotic vessels.66 The claim by Jackson
et al to see spontaneous luminal thrombosis is important, but
remains to be confirmed by others.
The other consequence of previous plaque rupture in man
is the presence of layered scars containing organized thrombotic debris.1,67 69 By analogy, Jackson and his colleagues
propose that previous episodes of rupture in mice may be
represented by buried fibrous caps.43 In 2005, buried

Figure 4. Layered plaque in murine brachiocephalic artery. Layered lesion with multiple fibrous caps (arrows) in the innominate/
brachiocephalic artery of a 40-week-old chow-fed female
apoE/ mouse. Movat pentachrome stain, 100 final
magnification.

fibrous caps (smooth muscle cellrich layers, invested with

elastin and usually overlain with foam cells) were described
within plaques, associated with positive staining for fibrin.44
In our opinion, the published pictures (Figures 1C, 4A, and
B44) appear quite dissimilar to healed plaque rupture in
humans (supplemental Figure IV), where the Sirius red
collagen stain and polarized light has been used to detect a
discrete defect in the old and dense collagen of the cap (type
I, yellow), filled in by newer and more loosely arranged
collagen (type III, green) containing an increased density of
smooth muscle cells.69 Moreover, mural thrombi have not as
yet been described by our groups or by other investigators,
even though layered lesions are often seen in more advanced
murine lesions in our own studies. The more obvious hypothesis, in our opinion, is that buried caps represent episodic
plaque growth with formation of superficial fatty streaks, ie,
xanthomas, over older lesions resulting in a layered plaque
phenotype, as shown in Figure 4 (and supplemental Figure
V). This interpretation is consistent with the morphology
showing intermediate stages of cap formation associated with
superficial xanthomas and with recent cell kinetic studies
showing that fresh macrophages are deposited on the surface
of later lesions, rather than appearing within the lesions.38,70
The answer, ultimately, will require either better evidence for
mural thrombus formation or, perhaps, an experimental test
of the buried cap phenomenon, possibly using the p53 model,
the diphtheria toxin model, or the MMP-9 model to study the
response to intentionally induced plaque disruptions.

Opportunities
If we leave behind the need to define terms carefully, there
are several experimental opportunities to discuss.
It may be important to remember that almost all of the
work in mice described here was done in a single genetic
background, ie, the C57BL/6 strain. In 1985, Paigen and her
colleagues71 screened strains of mice for their ability to form
fatty streaks and identified C57BL/6 as especially susceptible

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independently of lipid levels. She suggested that the gene for
this trait be called Ath1. The nature of Ath1 could be very
important to our discussion of advanced lesions. Recently, the
gene for OX40 ligand, an inflammatory mediator in the tumor
necrosis factor (TNF)/Fas death receptor ligand family, has
been identified as a major part of the C57BL/6 atherosclerosis-susceptible phenotype.72 Recent evidence from Pei et al
shows that the susceptibility of C57BL/6 is intrinsic to the
vessel wall.73 Identification of the specific atherosclerosis
sensitivity genes, combined with new methods for accelerating analysis of murine genetic crosses,74 may make it possible
to cross such regions into other strains and look for loci that
contribute to plaque progression and rupture.
One example of such a genetic approach may come from
the obvious fact that the advanced atherosclerotic plaque is a
lesion of age. Oxidation is a major topic of research in
atherosclerosis and in aging. Most of this is beyond the
purview of this review, other than to note that most of the
experimental studies have focused, once again, on the effect
of antioxidants on fatty streak formation, rather than on
features of the advanced plaque.29,75 Moreover, oxygen and
other free radical products are not the only issues in relation
to aging. For example, humans with a splicing defect in lamin
A, develop fatal arteriosclerotic vascular disease in their
teens, despite an absence of lipid disorders, hypertension, or
diabetes.76 At least to date, mice with similar mutations have
not been reported to develop accelerated atherosclerotic
disease. However, a recent study suggests that the lamin
mutation is associated with loss of medial smooth muscle, a
late feature in most human atherosclerosis and one that
appears to be exacerbated in humans with progeria.77,78
Finally, autopsy studies in humans show that many plaque
ruptures occur without forming an occlusive thrombus. It is
not possible to overestimate the importance of understanding
why some plaque disruptions, even the mild disruption seen
in erosions, lead to occlusive thrombus, whereas more extensive disruption, ie, plaque rupture, can occur with little
consequence. Here, the contrast in the 2 models of disease in
the murine brachiocephalic artery is quite dramatic. In the
model we have studied (M.E.R., S.M.S.), spontaneous, obviously extensive plaque injury does not result in thrombosis. In
the other model discussed above from Jackson and his
colleagues, the same site, but with strain differences and a
different diet, shows a subtle, but apparently thrombogenic
plaque injury severe enough, perhaps, to lead to the animals
deaths. Regardless of the semantics of plaque rupture, this
difference needs to be studied and clarified.

Disclosures
None.

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2. Falk E. Plaque rupture with severe pre-existing stenosis precipitating
coronary thrombosis. Characteristics of coronary atherosclerotic plaques
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Plaque Rupture in Humans and Mice

Stephen M. Schwartz1,5, Zorina S. Galis2, Michael E. Rosenfeld4, Erling Falk3
1

Department of Pathology, University of Washington, Seattle, WA 98109

Department of Surgery, Indiana University, and Lilly Research Laboratories, Indianapolis, IN
46285
3
Department of Cardiology (Research), Aarhus University Hospital (Skejby), Aarhus, Denmark
8200
4
Department of Pathobiology, University of Washington, Seattle, WA 98109
5
Corresponding author:
Stephen M. Schwartz
Department of Pathology
University of Washington
815 Mercer Street, Room 421
Seattle, WA 98109-4714
Phone: 206-543-0258
Fax: 206-897-1540
e-mail: steves@u.washington.edu
2

Please Note: The print version is an abbreviated version of this full length text. The full text
includes much more complete discussions, especially about murine experimental models that
test processes and features posited to contribute to plaque vulnerability, about models combining
prothrombotic with atherosclerotic phenotypes, and about the literature reporting infrequent occurence
of murine lesions that may, as compared with the common lesions seen in mice,
come closer to ruptured human plaques but are difficult to use because of a low incidence.
All figures in the online version are reproduced here, however to simplify cross reference between the
versions, we have numbered the online references using Roman numerals. The equivalent figure
numbers, for figures present in both versions, are identified in the online figure legends.
Bookmarks to the Roman numbered figures are included to assist the reader.
Figure legends are linked via dashed red boxes to the first citation in the text.
.

Page 1 of 60

Abstract
Recent efforts to use murine models of atherosclerosis to model the advanced human
plaque have become confused by use of terms such as unstable and vulnerable that imply
conclusions beyond the available evidence. Even the term rupture has been used in confusing
ways that may have little to do with the events described in humans. In this review we will
describe existing models of murine plaque rupture, place these in the context of what we know
about the development of lesions in the two species, and introduce a more precise terminology,
designed to be applicable to both men and mice. Finally we will suggest possible new
experimental directions for future studies of the advanced murine lesion.

Page 2 of 60

Review
The object of this review is to address the question, How do we best use murine models
of atherosclerosis to model the processes that lead to rupture and thrombotic occlusion in human
atherosclerosis? Despite the many successes of the murine model in understanding the early
lesions of atherosclerosis, as we will see, it is much less clear that the murine lesion, even the
dramatically disrupted lesion shown in Figure I, is a model for the ruptured, thrombogenic
lesions seen in the coronaries, carotid arteries, or other portions of the human arterial tree
afflicted by atherothrombosis.
The problem has, in our opinion, become particularly severe because of the common use
of terms such as instability, vulnerable, rupture, or even thrombosis for features of
plaques in murine model systems not yet shown to rupture spontaneously and in animals
surprisingly resistant to formation of thrombi at sites of atherosclerosis. Similarly, terms such as
buried fibrous caps that infer preceding events that are unproven tend to create confusion. We
will argue that such terminology may mislead readers by implying knowledge that does not yet
exist.

Not all Disruption is Rupture

The human lesion shown in Figures I and IV are typical of lesions described as ruptured.
The fibrous caps have broken down, fragments of the lesion have been evulsed into the lumen, the
overlying endothelium is no longer intact, blood has gained access to the necrotic core via the
break in the fibrous cap, and the lumen is filled with thrombus. Before going any further, it is
obvious that this typical example of a ruptured human plaque shows a level of disruption that is
very different from less extensive disruptions, including the massive hemorrhage into a murine
Page 3 of 60

plaque shown in the right panel of Figure I. We will use the general term disruption to refer to
any loss of the integrity of the plaque surface, ranging from a simple loss of endothelial cells to
minor fissures that penetrate into the plaque without exposing the necrotic core, to frank
breakdown of the fibrous cap over a necrotic core with plaque hemorrhage into the plaque, as is
seen in the murine part of Figure I, to the frank plaque rupture as seen in humans with acute
coronary artery occlusion due to plaque disruption. The review will also attempt to offer a
consistent set of terms that can be applied to different extents of disruption both in experimental
lesions in animals and in lesions occurring spontaneously in humans (Table 1).

Fatty Streaks do not Rupture

In order to compare potentially rupture-prone murine versus human lesions, it is
important to begin with a lesion we all agree does not rupturethe fatty streak. Figure II
compares the histology of human and murine fatty streaks. Fatty streaks, sometimes called early
lesions, are comprised of fat-filled macrophages that have accumulated in the intima. In formal
pathological terminology, these are xanthomas, i.e. focal masses of fat-filled macrophages.
Similar xanthomas are seen in extravascular connective tissue sites in people with severe
hyperlipidemia. Equation of the term early lesion with the term fatty streak may be
misleading, since xanthomatous accumulations of cells in the intima can be seen in older people,
as well as in the vessels of younger people, and recent studies in mice show that monocytes can
continue contributing to intimal masses even in older lesions in mice.1-3 Even though the fatty
streaks seem very fragile with only a thin layer of endothelium separating the foam cells from the
lumen, in mice and human fatty streaks do not rupture, so it is misleading to describe features
such as accumulation of proteases, accumulation of connective tissue, incidence of apoptosis or
appearance of smooth muscle as making a fatty streak lesion more or less likely to rupture.
Page 4 of 60

Moreover, as we will note below, at least in mice, new intimal xanthomas can appear on
top of existing lesions, especially in the shoulder regions of the plaques where they are called
lateral xanthomas. As we will discuss, these new xanthomas appear to be a frequent site of
extensive plaque disruption with hemorrhage into the plaque, as seen in Figure I.

Formation of the Fibrous Cap in Mice and Humans

While the fatty streak is usually described as the early lesion in mice and in humans, the
identity of the early human lesion is less clear. Humans do show fatty streaks, i.e. masses of
xanthomatous macrophages, especially in areas of flow separation downstream of flow dividers
in the thoracic aorta. The more clinically significant human lesions, however, are lesions that
arise in areas of spontaneous intimal hyperplasia. Such areas arise spontaneously during the first
year of life at branch sites in the coronary arteries and carotid arteries. In adulthood, these sites
will be primary locations of complex atherosclerotic lesions.4 As described in the American
Heart Association classification, these sites show lipid deposition occurring deep within the preformed intimal thickening.5 The location of these early, deep lesions correlates well with that
of adult advanced lesions.6 Moreover, the fibrous cap, i.e. connective tissue covering over adult
human atherosclerotic lesions is monoclonal,7 suggesting that the cells of the intimal thickening
over deep lesions seen in children may expand over time to become the fibrous cap of the
characteristic adult lesion (below).6 Thus, the tissue that forms the fibrous cap overlying the
necrotic core of advanced lesions may precede the formation of lesions in humans. Since no
similar spontaneous intimal hyperplasia is seen in mice, it is possible that mechanisms of
disruption of the fibrous cap are also different in the two species.
Surprisingly, almost nothing is known about the mechanisms controlling formation of the
fibrous cap in the atherosclerotic mouse. Review articles have, until recently, assumed that the
Page 5 of 60

fibrous tissue of intima originates from medial smooth muscle cells responding to cytokines
generated by the xanthomatous macrophages, perhaps in response to oxidized lipids.6, 8-10 This
model grew out of studies of the response of the vessel to balloon angioplasty, which was
interpreted as evidence that intima is derived by migration of medial cells in response to PDGF,
TGF-, FGF, inter alia. Support for a PDGF hypothesis grows from two studies where ablation
of PDGF decreased the number of intimal cells covering the fatty lesion.11, 12
Controversial studies have made the origins of the smooth muscle cells that make up any
intimal lesion, including the atherosclerotic plaque, confusing. These studies using cell labeling
techniques and bone marrow transplant in multiple models of intimal formation in mice and in
humans suggested that at least some of the smooth muscle-like cells in intimal lesions, especially
in transplant vasculopathy, are derived from sources exogenous to both the media and the intima,
i.e. from the circulation or the adventitia.13-16 While the transplant studies are clear, 14 the studies
of atherosclerosis are less convincing, especially in terms of the frequency of this exogenous
origin, and more recent studies in atherosclerotic mice have not been able to confirm that smooth
muscle cells in atherosclerotic plaques of mice originate in the bone marrow and reach the
plaque via the blood.17-19 Again, it may be important to remember that we do not know that all
forms of intima are formed in the same way.
Murine atherosclerotic lesions and, presumably, human fatty streak lesions do, with time,
develop a layer of fibrous tissue over the xanthomatous fatty streak lesions. The fibrous cap
overlying a necrotic core in human lesions contains collagen, elastin, and proteoglycans. This
layer may be hundreds of microns in thickness and highly cellular (Figure III). In some places the
human atherosclerotic fibrous cap resembles a tendon with few, RNA-poor fibrocyte-like cells
imbedded in a dense matrix of collagen and elastin.20 In other places, the fibrous cap is more
Page 6 of 60

highly cellular with many cytoplasm-rich intimal cells, presumably of the smooth muscle
lineage, although that has become a controversial topic due to a lack of the usual markers for this
cell type.13, 20 In the coronary arteries, ruptured caps are usually less than 100 microns thick and
highly inflamed.21 The murine fibrous caps are less impressive, perhaps reflecting limitations of
lesions growing in vessels that are so much smaller than their human equivalents. In any case,
the murine fibrous cap does not appear to progress to form dense connective tissue and,
instead, is usually comprised of minimal numbers of lamellae of loosely organized, elastin-rich
connective tissue. The fibrocellular structure overlying a necrotic core is often interdigitated
with smooth muscle-like cells and macrophages. The most superficial of these layers may be
only a few microns thick. Later sections of this review will discuss the relevance of breaks in
this very thin structure to the usual disruptions defined as plaque rupture in human lesions.

Formation of the Necrotic Core

Since any effort to create an animal model of plaque rupture must presuppose the existence of a
necrotic core,1 the simplest approach to an experimental model of atherosclerosis may be to
increase the extent of the necrotic core. Recently, efforts to do this have been based on the
reasonable assumption that the necrotic core results from macrophage death and that death of the
macrophage is driven by some form of apoptosis.
The experimental literature on the effects of anti-apoptotic genes on formation of the
necrotic core in mice is confusing. Increases in the extent of atherosclerosis have been reported
in response to knockout of the pro-apoptotic protein p53 in apoE*3-Leiden transgenic mice or
apolipoprotein E deficient (apoE-/-) mice.22-24 The lesions in these mice showed an increase in
the extent of the necrotic core. Similarly, transplantation of bone marrow from apoE-/- x ACAT1-/- mice into apoE-/- mice increased cell death within the lesions, but led to an increase in lesion
Page 7 of 60

area.25 Thus, ongoing apoptosis may limit macrophage accumulation in the lesion, but not affect
the rate of necrotic core formation. Conversely, a reduction in cell death due to transplant of
BAX-/- cells also led to an increase in lesion area in fat-fed LDLR-/- mice.26
An explanation for the paradox may come from the presence of two or more different
mechanisms for macrophage death proposed for the plaque. The first specific mechanism was
proposed over twenty years ago. Fowler et al proposed that macrophage death might be the
result of irreversible damage to lysosomes by lipid accumulation.27 Two decades later, Fazio,
Tabas, and their colleagues have separately shown that inhibition of cholesterol esterification or
blocking of cholesterol transport from the endoplasmic reticulum leads to lipid accumulation in
plaque macrophages and an increase in formation of a necrotic core. Consistent with the
paradoxical response to p53 or BAX knockout, these manipulations produced unexpected either
increases to, or failure to decrease, plaque mass.28 Tabas has identified the mechanism of cell
death due to cholesterol storage as a form of caspase-dependent apoptosis induced by a stress
response to lipid accumulation in the endoplasmic reticulum.28 A very different caspaseindependent form of death occurs, at least in vitro, to oxidized LDL.29 Although not
demonstrated in vivo, oxLDL-dependent death of plaque macrophages is consistent with a large
amount of speculation that oxidized lipids lead to plaque progression.30 In this regard, it is
important to remember the complex mixture of lipids and lipid products in a plaque. It is
reasonable to consider that many oxidized lipids are detergents, and detergents may cause cell
death by disrupting cell membranes.29, 31-34
Finally, little attention has been given to cytotoxic products of the inflammatory cells in
plaques.35 Perhaps we need to consider that two or more mechanisms of cell death in the lesion
may produce distinctive results in terms of the size of the necrotic core. One pathway, primarily
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apoptotic and dependent on p53 or BCL2-like proteins, may determine rates of foam cell
accumulation without accumulation of necrotic cell debris. The other pathway, oxLDL-induced
death independent of apoptosis, may be required for accumulation of necrotic material. In
summary, as of 2006, we do not know the pathway or pathways leading to formation of the
necrotic core. Without that core, plaque rupture would be a moot issue.

Human Plaque Rupture vs. Murine Plaque Disruption

The term plaque rupture has been used by pathologists and cardiologists for decades to
identify a structural defect (a disruption) in the fibrous cap that separates a necrotic core of an
atherosclerotic plaque from the lumen, resulting in exposure of the necrotic core to the blood via
the gap in the cap (Figure I, left panel, and IV).1, 36-39 Often, ruptured human lesions evulse part
of the plaque into the lumen, sometimes resulting in emboli. Exposure of prothrombotic proteins
is presumed to precipitate the formation of a platelet-rich thrombus.
To our knowledge, murine lesions approaching this extent of disruption have only been
seen anecdotally by Calara et al40 and others41 in a few older atherosclerotic mice. A model
reported as having a reproducible frequency of disruption with plaque hemorrhage was first
described in the brachiocephalic artery of the apoE-/- mutation in the C57BL/6 background. The
brachiocephalic artery, sometimes called the innominate artery, was chosen for careful study
because analysis of lesions in the complete arterial tree of chow-fed apoE-/- mice showed that the
most consistent site for development of complicated atherosclerotic lesions in these mice is the
brachiocephalic artery.42 Between 30 and 40 weeks of age, about 80% of these lesions showed
plaque hemorrhage (Figures I and VI). Serial sections of these lesions showed that the hemorrhage
arose at the shoulder region where the fibrous cap was either absent or minimal. Similar lesions
were later reported by Renard et al in the LDLR-/- mouse with atherosclerosis accelerated by
Page 9 of 60

diabetes{Renard, 2004 11461 /id} and at a lower frequency in apoE-/- mice used as a
control.{Gough, 2006 12315 /id}
It is important to point out that interpretation of this model depends on the frequency of
plaque hemorrhage. As pointed out by Michael Davies, however, identification of plaque
hemorrhage with disruption of the lumen surface is complicated, because hemorrhage within a
plaque can also originate from microvessels arising from the adventitia.45 In a seminal paper in
1951, Geiringer attributed hemorrhage into the plaque to a breakdown of a subset of intraplaque
vessels characterized by thin walls.46 The endothelium of these intraplaque vessels in humans
shows a very high level of cell replication and apoptosis, accounting for a large part of the
replication and apoptosis in advanced lesions and suggesting that these vessels may be very
fragile.47 Such vasa vasorum-derived microvessels are nearly universal in advanced human
lesions48-52 and can account for blood flows comparable to renal clearance.53, 54 The distinction
between the two types of plaque hemorrhage is important, because intraplaque hemorrhage
originating from labile microvessels within the plaque does not expose the necrotic core to the
lumen and probably cannot serve as the basis for a luminal thrombus. Returning to the murine
lesions, small intraplaque vessels have been described by Molton et al in murine lesions of the
aortic arch.55 Intriguingly, Langheinrich et al56 have recently used micro CT to examine the
relationship of adventitial vascularity to lesion progression. The possibility that the intraplaque
hemorrhages arise from vasa vasorum was supported by a strong correlation between intraplaque
hemorrhage and the extent of plaque vasa. Unfortunately, they did not correlate the CT data with
the extent of neovessels seen in the intima itself and did not provide data on the brachiocephalic
arteries where most hemorrhage has been seen.56 Despite these possibilities, it is unlikely that
breakdown of these vessels accounts for the data in the brachiocephalic artery. First, as seen in

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Figure VI, serial sections of the hemorrhages in the brachiocephalic artery routinely show
continuity with the lumen via disruption of xanthomas located at the edge of the murine fibrous
cap that can also be seen in intact form in lesions without hemorrhage into the plague, Figure V.
Second, intraplaque vessels have been described in mouse atherosclerotic plaques of the aorta,
but not in brachiocephalic lesions.55, 57 We have not seen such vessels in the brachiocephalic
lesions, even when we attempted to highlight the vessels by staining with VE-cadherin
antibodies or by perfusion with the vascular tracer, horseradish peroxidase (SMS and MER,
unpublished results).
About the same time as the report of plaque hemorrhage, Jackson and colleagues claimed
to describe acute plaque rupture with luminal thrombosis in the brachiocephalic artery of apoE/-

mice with unconvincing evidence of hemorrhage into the plaque.58, 59 They refer to this change

as acute plaque rupture, although as illustrated in the drawing based on their work, Figure VII, the
extent of disruption may be very small.60 Interpretation of their initial reports was complicated
because an unexplained high number of mice died suddenly and were found decomposed.
Reasons for the frequency of deaths in this model, approximately 25% in two months of the diet,
remain unexplained, but might relate to strain background, a mixed C57BL/6-129 versus the
usual C57BL/6 used as a background in most studies of apoE-/-, or toxicity of severe
hypercholesterolemia induced by their diet. Although all the dead mice were reported to have
thrombotic material in the lumen,59 no reports have been given on the pathology of the brains. It
would obviously be very important to find out if this is a model for a thromboembolic stroke
originating in an atherosclerotic artery supplying the brain.
Our major concern with these papers is the equation of a very small disruption of the
luminal surface, perhaps only the loss of a few endothelial cells, with plaque rupture with the
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characteristic extensive disruption of the plaque structure and penetration into the necrotic core
(See Figure I.). The observation of small areas of endothelial injury in atherosclerosis is not
entirely new. In the 1980s there was an extensive debate about whether endothelial denudation
or endothelial death was an early event in atherosclerotic lesion development. The consensus,
over time, was that denudation was a rare and not an initiating event in formation of lesions,61, 62
although until now no one has suggested that such small injuries were related to plaque rupture.
Although endothelial death and increased turnover does happen, especially in shoulder regions of
the plaque, regeneration is rapid enough that denuded areas are rarely seen.61, 63-67 Some of the
issues in these older studies, including quality of fixation and criteria for determining that an
endothelial cell is missing or dead remain valid today. Atherosclerotic lesions in mice, even
after fixation, are fragile and breaks can occur during handling. It is, therefore, very valuable, as
is the case for the plaque hemorrhage shown in Figure I, to have certain evidence of some event
that could only occur if the disruption had been in the living animal.
The most impressive part of the observations from Jackson and colleagues may be the
claim to see luminal thrombus. In a recent study where their animals were intentionally
sacrificed and perfusion fixed to avoid concerns with post mortem artifacts, thrombotic material
was only seen in association with disruption, and no thrombi were seen in the absence of
discontinuities of the plaque surface, implying that the thrombi were the result of disruption of
the luminal surface in vivo.59 Unfortunately, the paper does not provide much detail on the
composition of the thrombus and only a few displaced erythrocytes beneath an interrupted
endothelium (called intraplaque hemorrhage) were considered enough to prove that the plaque
surface was ruptured before death.60 Furthermore, the presence of a necrotic core was not
required. Finally, at least as shown in the figures, e.g. Figures 1 and 2 in Johnson et al,60 the

Page 12 of 60

defects are sufficiently small that it may be difficult to eliminate possible postmortem or
handling artifacts.
Although this may be a form of disruption, the definitions used by this group and their
illustrations bear little resemblance to plaque rupture, as defined in humans. If the thrombi are
real and not postmortem clots, a more appropriate term for such minimal disruption may be
erosion (Table 1). Erosion, as used in the analysis of human lesions, implies endothelial
denudation, even though the critical feature of the lesion is thrombus. Farb et al, as well as
others, have described thrombotic occlusion of human coronary arteries at autopsy in the absence
of breakdown of a fibrous cap and exposure of a necrotic core.1, 49 This lesion is called erosion1,
49

and characteristically includes endothelial denudation, even though we do not know whether

the endothelial loss was the cause of the thrombus or a result of the thrombus. Like the lesions
reported by Jackson et al, erosion does not expose a necrotic core, or even require the presence
of a necrotic core, since many of these fatal human lesions are fibrous lesions without necrotic
cores.49

Fissures in the Lateral Xanthoma of Mice vs. Ruptures in Human Plaques

The final term in our effort to create an inter-species lexicon is fissure. Davies used
fissure as a synonym for rupture,45 but others have used it less stringently. The term plaque
rupture is usually used, as we will use it here and in Table 1, for a structural defect (a gap) in
the fibrous cap exposing the necrotic core and often with loss of plaque material into the lumen.
Rupture often, but far from always, leads to a flow-limiting thrombosis and an acute ischemic
event.68-70 We propose to use fissuring to describe less extensive breaks in plaques that, if a
necrotic core is present, may extend down to the core, but without exposing it because there is no
or only minimal loss of plaque material. As already noted, the relationship of the extent of
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disruption to the extent of thrombosis, i.e. erosion vs. fissure vs. rupture is not totally clear.
Occlusive thrombosis can also occur with erosion or fissure and ruptures may occur without
occlusive thrombosis.71
The murine hemorrhagic lesions described above by our group (SMS, MER) meet this
definition of fissures better than they meet the criteria for rupture. Moreover, the disruption
appears to be in xanthomatous areas rather than through the fibrous cap itself (Figure I). As
already discussed, the shoulder regions in these mice are overlaid by xanthomas very much like
the early lesions seen in younger animals (Figure V). Plaque fissures may be a result of cell
death in these xanthomas, although studies of cell death have not as yet shown localization to the
disrupted sites. It may be relevant that cell death occurring at such a superficial region of the
plaque is consistent with studies discussed below where pro-apoptotic treatments delivered
locally at the lesion surface promote rupture, while more diffuse expression of pro-apoptotic
genes produced only changes in lesion mass or size of the necrotic core72.
This sort of disruption through a macrophage-rich cell mass, to our knowledge, has not
been described in human lesions. Disruptions of the murine lesions also differ from plaque
rupture due to the absence of extensive lesion breakdown with loss of material from the plaque.
Finally, as discussed below, the murine hemorrhagic lesions do not precipitate thrombosis, in
contrast to what Jackson et al described for the much smaller defects in the same artery.59 Thus,
we would use the term fissure to describe the degree of surface disruption required for plaque
hemorrhage in the mouse, but retain a distinction from the far more extreme breakdown of
fibrous cap usually described as human plaque rupture.

Page 14 of 60

Potential Artifacts
Consideration when comparing models for the advanced, complicated plaque needs to
include a number of possible artifacts.
One class of artifacts may arise simply because of the tiny size of murine arteries. For
example, interpretation of reports of occlusion of murine coronary arteries should consider that
even modest-sized retrovalvular lesions may occlude the orifices of the coronary arteries because
the orifices run through the lesions and have very small lumens. There is no equivalent human
structure with similar geometry. Studies of lesions at these sites need very careful serial section
analysis in order to be taken as evidence for a process comparable to rupture and thrombosis
occurring in a human coronary artery.73-75 Even in the aorta or major arterial branches, the small
lumen size and thin medial wall of murine vessels raises the question of whether vessels of this
caliber might be expected to reproduce a human pathology. Thus, a fibrous cap of the extent
seen in advanced human lesions simply cannot develop in the smaller caliber murine arteries.
On the other hand, because the murine media is also very thin, as we will review, overexpression
of enzymes that are believed to cause plaque rupture in the thick-walled human lesion may
instead cause remodeling or aneurysms in the thin-walled murine lesion.
Extravasation of erythrocytes and fibrin formation are obviously valuable criteria for plaque
disruption. In most cases, the distinctive morphology of the red cells, as seen in conventional
stains or in a Movats stain, is sufficient to identify plaque hemorrhage in a perfusion-fixed
animal. However, caution should be used when identifying the products of hemolyzed red cells
as hemorrhage. Tinctorial properties alone can be misleading, so it is useful to identify red cells
by electron microscopy or use specific antibodies to identify red cell proteins.76 The presence of
fibrin in lesions would provide independent evidence, but not proof of disruption, since
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coagulation might occur even without disruption. Moreover, fibrin antibodies, as discussed
elsewhere in this review, are not useful because of problems with distinguishing fibrinogen from
fibrin. Although there have been claims to stain for fibrin in murine lesions using antibodies,44,
60, 75

the antibodies used are either known to be unable to distinguish murine fibrinogen from

fibrin,77 or lack published data demonstrating the needed specificity.60 The best evidence that
fibrin has formed is electron microscopy showing the characteristic electron-dense fibrillar
structure with 215 angstrom cross striations. To date, fibrin has not been seen in spontaneous
plaque hemorrhage by electron microcopy (SMS, unpublished data). However, a recent study by
Gough et al of lesions disrupted by activated MMP-9 did demonstrate that large amounts of
fibrinogen (or perhaps fibrin) were present at sites of plaque disruption, and others have claimed
to see luminal fibrin, based on staining with other antibodies not yet shown to be specific for
fibrin.44, 74, 78
Autolysis, fixation and handling of fragile lesions is obviously of greater concern in the
models described by Jackson and colleagues, since the changes seen in the luminal interface
were relatively minor. That these disruptions were decreased by treatment with pravastatin60
certainly confirms that, as observed by one of the current authors (MER),79 statin treatment may
change the composition of atherosclerotic plaques. However, such changes might also change
fragility, so it does not prove that the observed disruptions were real and occurred in vivo.60 An
in vivo test of endothelial integrity, such as evidence of hemorrhage through a defect or perfusion
with horseradish peroxidase would be helpful. This tracer is able to detect even minor
disruptions, such as occur when endothelial cells die, or even during the rounding up associated
with mitosis.80, 81

Page 16 of 60

Finally, caution needs to be expressed about identification of both acute and organized
thrombi in arteries. It would be desirable in reports of thrombi to have more detail about the
thrombus itself. Arterial thrombi formed under rapid flow conditions are characterized by
aggregated platelets and sheets of fibrin, which are not seen when stagnant blood clots
postmortem or when blood clots or is crosslinked during an imperfect perfusion fixation. In
older thrombi, cells from the vessel wall migrate into the thrombus which, of course, is not seen
with postmortem clots, and the thrombus becomes organized with time. Finally, as discussed
above, it is difficult to distinguish fibrin from fibrinogen and care needs to be exercised using
special stains or poorly defined antibodies.

Experimental Approaches to Murine Lesion Disruption

The focus of this section will be on experimental efforts that manipulate specific factors
thought to be plausible causes of plaque disruption. It is important to point out that these
experiments test specific hypotheses. It is worth noting, however, that atherosclerotic plaques
can be disrupted in many other ways, including physical damage with a balloon catheter,
application of physical force to the lesion, or application of a known cytotoxic agent. In one
extreme form of such mechanical manipulation, Sasaki et al recently reported on the induction of
plaque rupture in the chow-fed apoE-/- mouse following ligation of the left common carotid
artery and placement of a cuff around the vessel.82 The relevance of this ischemic, occluded
vessel to a spontaneously disrupted lesion occurring in the presence of luminal and adventitial
blood flow is difficult to determine.83
It is also important to note that experimental approaches need to be evaluated relative to
the lesion being disrupted. As discussed above, the fibrous caps identified in some papers60
are, at best, thin tenuous sheets of connective tissue bearing little resemblance to the fibrous caps
Page 17 of 60

seen in advanced human lesions. Paradoxically, it is worthwhile noting that xanthomatous

lesions comprised of macrophages with no fibrous cap did not rupture, even when macrophages
were targeted with pro-apoptotic agents. Lesion mass changed, but these lesions did not rupture,
perhaps because cell death occurred mainly within the foam cell mass rather than at the
surface.22-24 Rupture of these xanthomatous lesions, or murine lesions with thin fibrous caps,
may require cell death at the interface between plaque and lumen. For example, von der Thsen
et al were able to produce a decrease in cap thickness when they used a p53 adenovirus in apoE-/mice.72 Only three of 16 mice showed morphological evidence of cap breaks, and only one of
these showed thrombosis and hemorrhage. The incidence of rupture was increased by infusion
of phenylephrine, a vasoconstrictor, for 15 minutes. At 24 hours, plaque hemorrhage was seen in
7 of 20 animals, one of which showed thrombosis. The adenovirus approach presumably was
non-selective in targeting different cell types. In contrast, a novel induction of apoptosis by
targeting smooth muscle cells with a diphtheria toxin receptor expressed by the SM22-
promoter, induced marked thinning of the fibrous cap of atherosclerotic apoE-/- mice, loss of
collagen and matrix, accumulation of cell debris, and intense intimal inflammation, but did not
induce rupture67. It would be fascinating to know whether the latter lesions might have ruptured
if exposed to phenylephrine, or if rupture might require death in cells other than smooth muscle
cells.
The molecules most studied in attempts to rupture plaque are proteases. Besides the
sought for outcome, studies of protease expression in advanced lesions in experimental animals
have produced unexpected results in regards to remodeling and angiogenesis.44, 84, 85 Falkenberg
and colleagues expressed urokinase in the endothelium overlying atherosclerotic lesions in fatfed rabbits. This was done in rabbits to take advantage of the greater accessibility of the

Page 18 of 60

endothelium for viral gene transfer. The uPA-transduced arteries had 70% larger intimas than
control-transduced arteries, smaller lumens, and evidence for degradation of elastic laminae.
Along with genetic data on elastin mutations from others,86 these data suggest that elastin may
serve to keep the artery open, and that loss of elastin as a result of endothelial-targeted
overexpression may allow inward pathological remodeling as is found in some advanced
atherosclerotic disease. In this case, an effort to rupture the plaque may have led to some
understanding of the expansive remodeling seen in atherosclerotic vessels.
The most extensively studied molecular candidates for rupture-producing proteases are
the MMPs where both knockout and overexpression studies have been performed. Using the
apoE-/- mouse, Johnson and colleagues studied double knockouts for MMPs 3, 7, 9, and 12.78
Knockouts of 3 and 9 produced larger lesions with more buried fibrous caps, a feature we will
discuss below. In contrast, MMP-12 and MMP-7 knockouts showed increased smooth muscle
cell content. The authors interpreted these data as evidence that the normal function of MMP-3
and 9 are protective, MMP-7 is neutral, while MMP-12 is destabilizing. Overexpression studies
give a very different and more complex set of conclusions, dependent on when MMPs are
expressed and activated. MMP-1 is an interstitial collagenase and would be expected to promote
plaque rupture. Lemaitre et al84 expressed human MMP-1 under a macrophage-specific
promoter in ApoE-/- mice. To their surprise, overexpression of MMP-1 resulted in decreased
experimental lesion size with no evidence of plaque rupture. The MMP that has received the
most attention is MMP-9. Increased MMP-9 activity and expression are detected in the
shoulders of advanced human lesions87 correlated with degradation of collagen, suggesting that
MMP-9 would be destabilizing.88 However, MMP-9 also promotes in interstitial collagen
assembly89 by smooth muscle cells, which might lead one to expect MMP-9 to contribute to the

Page 19 of 60

plaques mechanical strength. The actual effect turns out to be even more complex, depending
on how the enzyme is delivered and how it is activated. Increased transient expression of MMP9 via intraluminal adenoviral delivery, largely confined to the vessel lining,90 did not produce
any form of fibrous cap disruption. Instead, there was intra-lesional hemorrhage attributed to
neoangiogenesis, as well as increased outward (expansive) remodeling without increasing
macrophage infiltration. The latter is in good agreement with the previously reported effect of
MMP-9. MMP-9 deficiency in the MMP-9-/- apoE-/- mouse impaired the compensatory
enlargement of the carotid artery characteristic of lesion development seen in the apoE-/mouse,91 as well as in human atherosclerotic lesions.92 Interestingly, expansive or outward
arterial remodeling is also a characteristic associated with human plaque rupture, pointing out
that we simply know too little to predict how an enzyme may act in the complex milieu of the
plaque.93, 94 Perhaps the best evidence for the importance of knowing when and where a protease
will be activated comes from a recent study by Gough et al. These researchers transplanted
macrophages expressing an auto-activating form of MMP-9 inducing plaque disruption in 9/10
mice when overexpressed in vivo in advanced atherosclerotic lesions of apoE-/- mice, as
compared to frequencies of about 1/9 in the controls.44 Thus, MMP-9, expressed in the right
place and time can rupture the plaque. Whether this extent of disruption is comparable to human
plaque rupture is an issue we will discuss in the section below.
In summary, lesion disruption, in one case approaching the severity of human plaque
rupture,44 has been caused experimentally by interventions with pro-apoptotic stimuli and with
targeted delivery of MMP-9. These experiments at the least provide a proof of concept that
murine lesions can rupture.

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Consequences of Plaque Disruption in Mice

The hemorrhagic lesions seen spontaneously in the brachiocephalic arteries of mice do
not develop the luminal thrombus seen in human occlusive coronary artery disease. This cannot
be explained simply by the lack of extensive exposure of the necrotic core seen in human lesions
(Figure IV), because there also appears to be a lack of fibrin in the hemorrhage itself. We have
not seen fibrin by electron microscopy in the murine lesions showing plaque hemorrhage (SMS
and MER, unpublished data). Although the failure to form fibrin in the lesion or to develop a
thrombus is disappointing, it is not entirely surprising. Fibrin is not seen when normal arteries
undergo injury with an inflated balloon catheter.95 Thrombi might form transiently and be
dissolved by plasmin. Rapid fibrinolysis could be very important in an animal with small caliber
vessels.
Despite the lack of thrombosis associated with plaque hemorrhage in mice, there is
anecdotal evidence that murine lesions can thrombose spontaneously under some
circumstances,40, 41 including induction of rupture by MMP-944 and development of platelet-rich
arterial thrombosis after physical injuries to the vessel wall.96-98 Genetic manipulation should
make it possible to combine models of disruption with thrombosis. Recently, Eitzman et al
introduced a procoagulant mutation into apoE-/- mice.99 Surprisingly, the mutation in Factor V
led to an increase in total plaque mass. These investigators did not observe plaque hemorrhage
or occlusive vascular disease; however, the study did not include the serial sections of the
brachiocephalic artery needed to demonstrate plaque disruption. They did, however, report
finding fibrin in aortic plaques, and suggested that this implied previous plaque disruption
with hemorrhage and persistent fibrin. Again, the obvious caveat arises of a need for better
antibodies or confirmation by electron microscopy, especially since fibrinogen itself normally
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accumulates in lesions, presumably due to altered permeability or increased proteoglycan content

in lesions without disruption100, 101. Alternatively, Khatri et al have recently described elevated
intra-lesional angiogenesis following carotid artery ligation in a transgenic mouse overexpressing
p22phox.102 Like other angiogenic vessels, these are fragile.47 This mouse offers a potential
approach to intentional induction of intraplaque hemorrhage.

Buried Caps as Stigmata for Past Rupture

In humans, it is widely believed that past episodes of plaque rupture, depending on their
age, can be identified by the presence of a particular pattern of scarring.1, 68-70 These scars, often
containing evidence of hemorrhage or thrombus, offer plausible evidence for past plaque
ruptures (Figure IV). A somewhat different lesion feature has received attention from Jackson and
his colleagues. They propose that previous episodes of rupture may be represented by buried
fibrous caps. The term was introduced but neither defined, nor illustrated by pictures, in a
paper from 2002.59 In 2005, buried fibrous caps (smooth muscle cell-rich layers, invested with
elastin and usually overlain with foam cells) were described within plaques, associated with
positive staining for fibrin.60 Judged from the published pictures (Figures 1C and 4A and B in
reference,60 the buried caps do not bear much resemblance to healed plaque rupture in humans,
where the Sirius red collagen stain and polarized light has been used to detect a discrete defect in
the old and dense collagen of the cap (type I; yellow), filled in by newer and more loosely
arranged collagen (type III, green) containing an increased density of smooth muscle cells.68
If we accept the claim to see mural thrombin in these studies, then perhaps the buried
caps represent organized, old mural thrombi. However, as discussed above, the presence of
mural thrombi has not as yet been described in other models, even though layered lesions are
often seen in more advanced murine lesions. The more obvious hypothesis, in our opinion, is
Page 22 of 60

that buried caps represent episodic plaque growth with formation of superficial xanthomas
over older lesions resulting in a layered plaque phenotype, as shown in Figure VIII. This
interpretation is consistent with the morphology (Figure VIII) showing intermediate stages of cap
formation associated with superficial xanthomas and with recent cell kinetic studies showing that
fresh macrophages are deposited on the surface of later lesions rather than appearing within the
lesions.3, 44 The answer, ultimately, will require either better evidence for mural thrombus
formation or, perhaps, an experimental test of the buried cap phenomenon, perhaps using the p53
model, the diphtheria toxin model, or the MMP-9 model to study the response to intentionally
induced plaque disruptions.

Modeling Murine Plaque Progression

It is reasonable to imagine that the factors controlling formation of fatty streaks/intimal
xanthoma also determine the formation of the advanced lesions. The role of lipid accumulation
or depletion in the late lesions seems clear. In one study, LDLR-/- mice fed a high-fat diet for 16
weeks developed advanced lesions with fibrous caps and lipid-rich necrotic cores in the
brachiocephalic artery.103 A low-fat diet decreased plasma cholesterol levels from 21.0 2.6 to
8.4 0.6 mM/L, but lesions did not regress. However, when levels of VLDL/LDL were further
lowered by using a helper-dependent adenovirus encoding the VLDL receptor, lesion size
regressed, and there was an 89% reduction in macrophages compared to controls. Similarly,
Zhang et al were able to prevent myocardial infarction by dietary restriction in a highly
accelerated model of coronary atherosclerosis combining mutations in scavenger receptor B1 and
apoE.74
As another example of lesion progression behaving as might be expected, it is widely
believed that hyperglycemia and angiotensin accelerates disruption in murine lesions. Renard et
Page 23 of 60

al reported that diabetes in LDLR-/- mice accelerated both xanthoma formation and the
appearance of plaque hemorrhage similar to that shown in Figure I.43 Finally, angiotensin II,
independent of its effect on blood pressure, is able to promote both the extent of initial lesions
and aneurysm formation at a specific site in the abdominal aorta.104 Unlike diabetes, there is no
evidence that angiotensin increases the frequency of plaque hemorrhage in the murine
brachiocephalic carotid lesions. Perhaps proteases activated by angiotensin cause rupture in a
human lesion, but aneurysm in a murine lesion. Implication of distinct mechanisms for the early
and late phases of atherosclerosis, moreover, is suggested by a recent report claiming that
knockout of 5-lipoxygenase, contrary to other reports, did not block lesion initiation, but
protected from aneurysm formation in advanced lesions.105
At a more mechanistic level than lipid accumulation, there has begun to be exploration of
the hypothesis that specific molecules required for formation of the murine fatty streak are also
important in the processes leading to advanced lesions. For example, knockouts of MCP-1 or its
receptor, CCR2, have a dramatic effect in preventing the formation of initial lesions, but bone
marrow transplant studies of macrophages deficient in CCR2 show no effect in alleviating the
advanced lesion.106 This observation suggests that macrophage influx, an obviously critical step
in forming the fatty streak, may play much less of a role once the lesion is established.91, 106
Similarly, a study of estrogen-treated castrated male apoE-/- mice found that the protective effects
of this hormone were limited to blocking the formation of new xanthomatous lesions, but
progression of existing lesions was unaffected by the treatment.107 This difference in early
versus late lesion mechanisms suggests that macrophage subsets differing in CCR2 expression
could be involved in influx into established, as opposed to new, lesions.108 Other potent anti- or

Page 24 of 60

proinflammatory factors, such as the role of T-regulatory cells in formation of intimal

xanthomas, have yet to be explored in advanced lesions.74, 109

Opportunities
After more than a decade of studies on the origins of atherosclerotic lesions in mice, we
are just beginning to see experiments, such as those described here, that explore the most
important question of how plaques rupture. If we leave behind the need to define terms
carefully, there are several experimental opportunities to discuss.
The most obvious approach is the one we have already seen, i.e. proofs of concept that
test the ability of particular manipulation, e.g. induction of MMP-9 to cause acute plaque
disruption. Presumably with time, we will read of the efficacy of additional cytokines or
proteases. The challenge with such studies is going to be to find ways of proving that such
events happen spontaneously, and when inhibited, prevent plaque disruption. The simple
appearance of an enzyme or cytokine in ruptured human lesions is necessary evidence, but as we
discussed in the case of MMP-9, not sufficient proof that the process actually occurs. Other
evidence in man might include evidence for polymorphisms and, finally, therapeutic trials. For
the mouse to contribute to such evidence, however, we probably need to show that the targeted
molecules can contribute to disruption and that inhibition of the pathway also works in the
mouse.
The best opportunity may be in the genetics of the mouse itself. It may be important to
remember that almost all of the work in mice described here was done in a single genetic
background, i.e. the C57BL/6 strain. In 1985, Paigen and her colleagues110 screened strains of
mice for their ability to form fatty streaks and identified C57BL/6 as especially susceptible. She

Page 25 of 60

suggested that the gene for this trait be called Ath1. The nature of Ath1 could be very important
to our discussion of advanced lesions. Recently, the gene for OX40 ligand, an inflammatory
mediator in the TNF/Fas death receptor ligand family, has been identified as a major part of the
C57BL/6 atherosclerosis-susceptible phenotype.111 Progress like this in mapping sites in the
mouse needed to initiate lesion development, combined with new methods for accelerating
analysis of murine genetic crosses,112 may make it possible to cross such regions into other
strains and look for loci that contribute to plaque progression and rupture.
One example of such a genetic approach may come from recent work in humans on the
genetics of aging. The exigencies of grants generally encourage relatively short-term models;
however, aging is obviously a risk factor for advanced atherosclerosis. The aging mechanism
that has received some study in atherosclerosis is the role of oxidation. Most of this is beyond
the purview of this review, other than to note that most of the experimental studies have focused,
once again, on the effect of anti-oxidants on xanthoma formation.8, 113 However, oxygen and
other free radical products are not the only issues in relation to aging. For example, humans with
the Hutchinson-Guilford progeria mutation, a splicing defect in lamin A, develop fatal
arteriosclerotic vascular disease in their teens, despite an absence of lipid disorders,
hypertension, or diabetes.114 At least to date, mice with similar mutations have not been reported
to develop accelerated atherosclerotic disease. However, a recent study suggests that the lamin
mutation is associated with loss of medial smooth muscle, a late feature in most human
atherosclerosis and one that appears to be exacerbated in humans with progeria.115, 116
Finally, as noted above, we have not yet begun to learn about the consequences of murine
plaque disruption. The most obvious and crucial gap, and possibly the most important
therapeutic opportunity, is in our lack of a model combining disruption with thrombosis and
Page 26 of 60

coagulation. As already mentioned, autopsy studies in humans show that many plaque ruptures
occur without forming an occlusive thrombus. It is not possible to overestimate the importance
of understanding why some plaque disruptions, even the mild disruption seen in erosions, lead to
occlusive thrombus, while more extensive disruption, i.e. plaque rupture, can occur with little
consequence. Here the contrast in the two models of disease in the murine brachiocephalic
artery is quite dramatic. In the model we have studied (MER, SMS), spontaneous, obviously
extensive plaque injury does not result in thrombosis. In the other model discussed above from
Jackson and his colleagues, the same site, but with strain differences and a different diet, shows a
subtle but thrombogenic plaque injury severe enough, perhaps, to lead to the animals death.
Regardless of the semantics of plaque rupture, this difference needs to be studied and clarified.

Summary
We suggest that use of conclusory terms like vulnerable, stable, or buried caps, as
has often been done in this field, should be discouraged.78, 88, 117-120
With that caveat, it is clear that murine models have only begun to produce fully
convincing spontaneous models for the advanced lesion. However, it is also true that very few
models have been studied, especially models on genetically diverse backgrounds. There are also,
as already cited, a number of papers where the effects of specific enzymes, apoptotic molecules,
angiotensin, and diabetes have been used successfully to manipulate plaque progression as
determined by specific end points: size of the necrotic core, frequency of breaks in the fibrous
cap, frequency of plaque hemorrhage, and aneurysm formation.

Page 27 of 60

Acknowledgements
This work was supported by the following grants: NIH PO1 HL03174, NIH 5R37
HL26405, NIH RO1 HL58083, and NIH RO1 HL 72411-01 (SMS); partial research funding
through NIH RO1 HL64689 and RO1 HL71061 (ZSG); and NIH RO1 HL076748 (MER). We
wish to thank Soren Dalager-Pedersen for providing the left of Figure II (human fatty streak).

Page 28 of 60

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Table 1. Terminology: Terms describing disruption of

atherosclerotic plaques
Plaque Disruption: We suggest use of this term as a generic term, including any lesion where
continuity of the surface is lost. Previously, plaque disruption has been used as a synonym for
plaque rupture,38, 121 or for all lesions with missing endothelium even if there is no break in the
underlying plaque.5, 122-124
Endothelial Denudation: We prefer endothelial denudation to the term erosion because
erosion has come to be used when endothelial denudation occurs with thrombosis,48, 49 even
though denudation does occur both in humans and animal models without formation of a
thrombus.
Erosion: Erosion has come to be used when endothelial denudation occurs with thrombosis in
the absence of breaks in the fibrous cap and, therefore without access to a necrotic core.48, 49
Erosion may even occur over lesions that lack a necrotic core. The term is confusing since it
conflates denudation and thrombosis, even though we do not know that the two are causally
connected.
Plaque Rupture: Plaque rupture is a term, used consistently by those studying coronary
thrombosis.1, 36-38 It implies a structural defect (a gap) in the fibrous cap that separates a necrotic
core of an atherosclerotic plaque from the lumen, resulting in exposure of the necrotic core to the
blood via the gap in the cap. Plaque material is lost, either to form embolic fragments or tethered
fragments that may protrude as flaps into the blood stream. Endothelial denudation, fissures or
microvessel-derived intraplaque hemorrhage do not expose a necrotic core, and consequently, do
not meet the definition of plaque rupture.
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Plaque Fissure: This term is best used for breaks in plaques that do not expose a necrotic core.
In a fissure there is no or minimal loss of plaque material. Michael Davies reintroduced the
term125 and used it synonymously with plaque rupture,38 however, it is also used to refer to
limited areas of disruption that do not meet the criteria for rupture. Murine lesions arising by
disruption of the lateral xanthoma may be classified as plaque fissure.
Healed Fissure/Rupture: Fissures and ruptures may heal forming scarred lesions that may
include thrombotic debris. In mice, there is speculation that periodic ruptures could produce
multilayered lesions; however, such lesions are more likely to arise simply by episodic plaque
growth, with new tissue (e.g., a xanthoma) forming atop an older lesions.
Necrotic core: A hypocellular plaque cavity devoid of collagen and containing necrotic debris
and cholesterol clefts. It originates, at least in part, from dead lipid-laden foam cells and has
been called the graveyard of dead macrophages.126 Synonyms: lipid core,38 atheromatous
gruel,121 and pultaceous debris.127
Fibrous cap: The fibrocellular structure that separates the necrotic core from the blood. Thus, if
a plaque does not contain a necrotic core, a fibrous cap cannot be defined. Endothelium alone
does not make a fibrous cap.
Plaque hemorrhage: Bleeding (extravasation of erythrocytes) into or within a plaque. It may
originate either from the lumen through a disrupted cap or from breakdown of defective
microvessels within the plaque. When possible, we use the term plaque hemorrhage for the
former and intraplaque hemorrhage for the latter. It is very important to distinguish plaque
hemorrhage originating by disruption of the fibrous cap from intraplaque hemorrhage arising by
breakdown of microvessels within the plaque.

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Vulnerable plaque: Although commonly used to mean a plaque containing a large necrotic core
with an overlying thin and inflamed fibrous cap, this and related terms like unstable and
thrombosis-prone50 are conclusionary rather than anatomically meaningful terms. It would be
better to use anatomically meaningful terms, since we have imperfect data on which plaques are
at high risk of rupture, thrombosis, and/or becoming the culprit for a clinical event (e.g. heart
attack).
Thrombus: A solid mass formed from fibrin and platelets in vivo and within the vascular
lumen.51 In contrast to postmortem clots (and some red thrombi) formed in stagnant blood,
thrombi formed in flowing blood during life contain clumps of platelets and/or fibrin
membranes.128, 129

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Figure I. Plaque disruption in humans and mice

Figure I (print figure 1.) Left panel, Photomicrograph and schematic of

a ruptured human lesion in a coronary artery. Characteristic features include the extensive
disruption of the thick (compared to mice), typically human, fibrous cap, expulsion of fragments
of the lesion into the lumen and access of blood to the necrotic core. The resulting overlying
thrombus, although characteristic of this sort of disruption is not part of our definition of plaque
rupture. The inset shows the full circumference of the vessel, including the occlusive thrombus.
Trichrome stain; collagen = blue, and thrombus and hemorrhage = red. Right panel,
Photomicrograph and schematic of a fissured murine lesion in the innominate/brachiocephalic
artery of a 42-week-old male apoE-/- mouse fed a chow diet. Characteristic features include the
presence of a superficial xanthoma, including xanthoma overlying the lateral edge of the plaque
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(lateral xanthoma) that penetrates the thin fibrous cap typical of murine lesions. Plaque
disruption occurred in this lesion likely because of the death of cells in the lateral xanthoma.
Movats pentachrome stain; collagen = yellow, proteoglycans = light blue, and blood
components = red.

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Figure II. The fatty streak: a xanthoma of the intima

Figure II. (online only) Despite the assumption that thin fibrous caps are "vulnerable," atty streak
lesions, other than the lateral xanthomas, never show disruption. Left, A human xanthoma, traditionally
called a fatty streak, formed within intima of the internal carotid artery from a 53-year-old man.
Inset: Macrophage foam cells within adaptive intimal thickening. iem, internal elastic membrane.
Hematoxylin-eosin stain. Right, A murine xanthoma (aggregate of foam cells) formed beneath
the endothelium without preexisting intimal thickening of the aorta from a 20-week-old apoE-/mouse. Movats pentachrome stain.

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Figure III. Fibrous cap in humans and mice

Figure III. (online only) Left, An advanced atherosclerotic plaque in human

coronary artery illustrating a small necrotic core covered by a thick and collagen-rich fibrous
cap. Well-formed fibrous caps like this one are rarely, if ever, seen in mice. Trichrome stain;
collagen = blue. Right, This figure shows an example of a distinct fibrous cap (black arrows)
covering a partly calcified necrotic core (fibro-fatty nodule) and a thin cap covering a lateral
xanthoma (red arrow) in an advanced lesion in the innominate/brachiocephalic artery of a 60week-old chow-fed male apoE-/- mouse. Movats pentachrome stain.

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Figure IV. Ruptured coronary artery lesion in human plaque,

with healing.

Figure IV. (online only) As noted in the text, human lesions show much thicker,
better-organized fibrous caps than those seen in murine lesions (See Figures I.,
V., VII. and VIII.). In human lesions, multiple layers of connective tissue are believed
to represent multiple episodes of plaque disruption or mural thrombus followed by
wound healing. In this plaque, a gap in the old collagen covered by presumably more loosely
structured collagen is suggestive of a healed plaque rupture. Although no acute
rupture was identified, radiographic contrast (c) material injected postmortem is seen
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mixed with erythrocytes (plaque hemorrhage) in necrotic cores deep in the plaque.
Trichrome stain, rendering collagen blue, blood components red, and contrast grey.

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Figure V. Murine lateral xanthoma

Figure V. (print Figure 4.) This figure shows a xanthoma (aggregate of foam cells)
that has formed along the lateral margin (red arrow) and fibrous caps covering
submerged aggregates of foam cells (core formation) in a lesion within the
innominate/brachiocephalic artery of a 40-week-old chow-fed male apoE-/mouse. Movats pentachrome stain.

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Figure VI. Serial sections of disrupted mouse lesions

Figure VI. (online only) This figure contains a series of micrographs showing extensive
plaque hemorrhage that has originated along the margin of aggregated foam cells in an
advanced lesion in the innominate/brachiocephalic artery of a 60-week-old chow-fed male
apoE-/- mouse. Movats pentachrome stain, upper left panel 100X final magnification,
upper right panel 200X final magnification, lower left panel - 1,000X final magnification,
lower right panel 1,000X final magnification.

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Figure VII. Drawings based on published images summarize of

the features of two lesions described as showing rupture or
disruption in the brachiocephalic artery of apoE-/- mice.

Figure VII. (print Figure 3.) Left, a plaque hemorrhage penetrating deeply into a necrotic core,
originating from the lumen via a disruption (fissure) through a xanthoma at the edge of the fibrous
cap in an old chow-fed apoE-/- mouse. Right, a few displaced erythrocytes located next to foam
cells beneath an interrupted endothelium with superimposed mural thrombus in a relatively young,
fat-fed, severely hypercholesterolemic apoE-/- mouse. The figures were drawn using painting tools
in Photoshop(R) and do not represent individual published images.

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Figure VIII. Layered plaque in murine brachiocephalic artery.

Figure VIII. (print Figure 4.) Layered lesion with multiple fibrous caps (arrows) in the
innominate/brachiocephalic artery of a 40-week-old chow-fed female apoE-/- mouse.
Movats pentachrome stain, 100X final magnification.

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Plaque Rupture in Humans and Mice

Stephen M. Schwartz, Zorina S. Galis, Michael E. Rosenfeld and Erling Falk
Arterioscler Thromb Vasc Biol. 2007;27:705-713; originally published online March 1, 2007;
doi: 10.1161/01.ATV.0000261709.34878.20
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright 2007 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636

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