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Plaque Rupture in Humans and Mice
Stephen M. Schwartz, Zorina S. Galis, Michael E. Rosenfeld, Erling Falk
Abstract—Despite 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
The term “plaque rupture” in human pathology is notcontroversial. The term has been used for decades toidentify 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).1–5
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.
See pages 697, 714, 969, and 973 and cover
With the exception of events seen in a small proportion of
atherosclerotic mice,6,7 murine lesions have not as yet pro-
gressed 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 “disrup-
tion” to refer to any loss of the integrity of the plaque surface,
ranging from a simple loss of endothelial cells to minorfissures 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
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 ex-
panded version with more thorough discussion of experimen-
tal 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.
Plaque Rupture Requires a Necrotic CoreCovered by a Fibrous Cap
It may seem paradoxical that fatty streak lesions (supplemen-
tal Figure II), without a fibrous cap and covered only by
endothelium, largely remain intact. Even though the endothe-
lium overlying fatty streaks appears very delicate, any dis-
ruption 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 CoreContrary 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
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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 proapo-
ptotic 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 accumu-
lation.24
Two decades later, Fazio, Tabas, et al separatelyshowed 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 manip-
ulations 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 forma-
tion of cytotoxic lipids, or simply bulk accumulation may be
required to disrupt the overlying fibrous cap.
Fibrous CapApplication 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 lessimpressive, perhaps reflecting limitations of lesions growing
Figure 1. Plaque disruption in humans and mice. Left panel, Photomicrograph and schematic drawing of a ruptured human lesion in acoronary artery. Characteristic features include the extensive disruption of the thick (compared with mice) fibrous cap, expulsion offragments of the lesion into the lumen, and access of blood to the necrotic core. The resulting overlying thrombus, although character-istic 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, Photomicro-graph 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 lateraledge of the plaque (lateral xanthoma) which penetrates the thin fibrous cap typical of murine lesions. Plaque disruption occurred in thislesion likely because of the death of cells in the lateral xanthoma. Movat pentachrome stain; collagen yellow, proteoglycans lightblue, and blood components red.
706 Arterioscler Thromb Vasc Biol. April 2007
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in vessels that are so much smaller than their human equiv-
alents. 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 mecha-
nisms 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 un-
known 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 Thüsen et al were able to produce a decrease
Figure 2. Serial sections of disrupted mouse lesion. This figure contains a series of micrographs showing extensive plaque hemorrhagethat has originated along the margin of aggregated foam cells in an advanced lesion in the innominate/brachiocephalic artery of a60-week-old chow-fed male apoE / mouse. Movat pentachrome stain, upper left panel 100 final magnification, upper right panel200 final magnification, lower left panel 1000 final magnification, lower right panel 1000 final magnification.
Figure 3. Drawings based on published images summarize the features of 2 lesions described as showing rupture or disruption in thebrachiocephalic artery of apoE / mice. Left, Plaque hemorrhage penetrating deeply into a necrotic core, originating from the lumen viaa disruption (fissure) through a xanthoma at the edge of the fibrous cap in an old chow-fed apoE / mouse. Right, Few displacederythrocytes 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 representindividual published images.
Schwartz et al Plaque Rupture in Humans and Mice 707
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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- pro-
moter, induced marked thinning of the fibrous cap of athero-
sclerotic apoE / mice, loss of collagen and matrix, accumu-
lation 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 phenyl-
ephrine, 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 modelwith 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 (in-
tra)plaque hemorrhage might also occur via breakdown of
small intraplaque vessels as have been described in murinelesions 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 vascu-
lar tracer, horseradish peroxidase (S.M.S. and M.E.R., un-
published results, 2006). It is therefore unlikely that break-
down 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 com-
plicated 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 2months of the diet, have remained unexplained. The absence
of similar data in other studies may relate to strain back-
ground, 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 disrup-
tion 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 MiceVersus 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 hemor-rhagic 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 dis-
cussed below, the murine hemorrhagic lesions do not precip-
itate 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 ArtifactsInterpretation 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 han-dling, it is very valuable, as is the case for the plaque
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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 eutha-
nized 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 throm-
botic 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 pres-
ence of fibrin in lesions would provide independent evidence
for injury, but not proof of disruption, because intramural
coagulation might occur, even without disruption.48 Unfortu-
nately, currently available antibodies are not useful becauseof problems with distinguishing fibrinogen from fibrin. Al-
though 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 electron-
dense 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. Jackson’s 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 atheroscle-
rotic plaques. However, such changes might also change
fragility, so the experiment does not prove that the observeddisruptions occurred in vivo.44 An in vivo test of endothelial
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 acritical role in disruption and rupture of the human lesion,
studies of protease expression in advanced lesions in exper-
imental 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 rab-
bits, rather than mice, to take advantage of the greateraccessibility 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 endothelial-
targeted overexpression may allow inward pathological re-
modeling 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 with-
out 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 andactivated. MMP-1 is an interstitial collagenase and would be
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expected to promote plaque rupture. Lemaitre et al54 ex-
pressed human MMP-1 under a macrophage-specific pro-
moter 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 corre-
lated 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 expres-
sion 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 hem-
orrhage attributed to neoangiogenesis, as well as increased
outward (expansive) remodeling without increasing macro-
phage infiltration. The latter is in good agreement with thepreviously 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 arte-
rial 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 overex-
pressed 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 experi-
mentally by interventions with proapoptotic stimuli and with
targeted delivery of MMP-9.
Consequences of Plaque Disruption in MiceSurprisingly, hemorrhagic lesions in murine plaques do not
develop luminal thrombus, even though the hemorrhage
infiltrates the necrotic core. Fibrin is absent in the hemor-
rhage 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 throm-
botic debris.1,67–69 By analogy, Jackson and his colleagues
propose that previous episodes of rupture in mice may berepresented by “buried fibrous caps”.43 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.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 asyet 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 hypoth-
esis, 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.
OpportunitiesIf 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 formfatty streaks and identified C57BL/6 as especially susceptible
Figure 4. Layered plaque in murine brachiocephalic artery. Lay-ered 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
finalmagnification.
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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 atherosclero-
sis-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 accelerat-
ing 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 onfeatures 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 plaqueruptures 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 exten-
sive 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, obvi-
ously 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.
DisclosuresNone.
References1. Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from
sudden coronary death: a comprehensive morphological classification
scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2000;
20:1262–1275.
2. Falk E. Plaque rupture with severe pre-existing stenosis precipitating
coronary thrombosis. Characteristics of coronary atherosclerotic plaquesunderlying fatal occlusive thrombi. Br Heart J . 1983;50:127–134.
3. Arbustini E, Dal BB, Morbini P, Burke AP, Bocciarelli M, Specchia G,
Virmani R. Plaque erosion is a major substrate for coronary thrombosis in
acute myocardial infarction. Heart . 1999;82:269–272.
4. Davies MJ. The pathophysiology of acute coronary syndromes. Heart .
2000;83:361–366.
5. Virmani R, Kolodgie FD, Burke AP, Finn AV, Gold HK, Tulenko TN,
Wrenn SP, Narula J. Atherosclerotic plaque progression and vulnerability
to rupture: angiogenesis as a source of intraplaque hemorrhage. Arte-
rioscler Thromb Vasc Biol. 2005;25:2054 –2061.
6. Calara F, Silvestre M, Casanada F, Yuan N, Napoli C, Palinski W.
Spontaneous plaque rupture and secondary thrombosis in apolipoprotein
E-deficient and LDL receptor-deficient mice. J Pathol. 2001;195:
257–263.
7. Zhou J, Moller J, Danielsen CC, Bentzon J, Ravn HB, Austin RC, Falk E.
Dietary supplementation with methionine and homocysteine promotes
early atherosclerosis but not plaque rupture in ApoE-deficient mice.
Arterioscler Thromb Vasc Biol. 2001;21:1470 –1476.
8. Galis ZS. Vulnerable plaque: the devil is in the details. Circulation.
2004;110:244–246.
9. Shah PK, Galis ZS. Matrix metalloproteinase hypothesis of plaque
rupture: players keep piling up but questions remain. Circulation. 2001;
104:1878–1880.
10. de Nooijer R, von der Thusen JH, Verkleij CJ, Kuiper J, Jukema JW, van
der Wall EE, van Berkel JC, Biessen EA. Overexpression of IL-18
decreases intimal collagen content and promotes a vulnerable plaque
phenotype in apolipoprotein-E-deficient mice. Arterioscler Thromb Vasc
Biol. 2004;24:2313–2319.
11. Libby P, Galis ZS. Cytokines regulate genes involved in atherogenesis.
Ann N Y Acad Sci. 1995;748:158 –168.
12. Johnson JL, Baker AH, Oka K, Chan L, Newby AC, Jackson CL, George
SJ. Suppression of atherosclerotic plaque progression and instability by
tissue inhibitor of metalloproteinase-2: involvement of macrophage
migration and apoptosis. Circulation. 2006;113:2435–2444.
13. Johnson JL, George SJ, Newby AC, Jackson CL. Divergent effects of
matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque sta-
bility in mouse brachiocephalic arteries. Proc Natl Acad Sci U S A.
2005;102:15575–15580.
14. Hansson GK, Schwartz SM. Evidence for cell death in the vascular
endothelium in vivo and in vitro. Am J Pathol. 1983;112:278 –286.
15. Bylock A, Bondjers G, Jansson I, Hansson HA. Surface ultrastructure of
human arteries with special reference to the effects of smoking. Acta
Pathol Microbiol Scand [ A]. 1979;87A:201–209.16. Hansson GK, Chao S, Schwartz SM, Reidy MA. Aortic endothelial cell
death and replication in normal and lipopolysaccharide-treated rats. Am J
Pathol. 1985;121:123–127.
17. Lin SJ, Jan KM, Chien S. Role of dying endothelial cells in transendo-
thelial macromolecular transport. Arteriosclerosis. 1990;10:703–709.
18. Weinbaum S, Tzeghai G, Ganatos P, Pfeffer R, Chien S. Effect of cell
turnover and leaky junctions on arterial macromolecular transport. Am J
Physiol. 1985;248:H945–H960.
19. van Vlijmen BJ, Gerritsen G, Franken AL, Boesten LS, Kockx MM,
Gijbels MJ, Vierboom MP, van Eck M, van De WB, van Berkel TJ,
Havekes LM. Macrophage p53 deficiency leads to enhanced atheroscle-
rosis in APOE*3-Leiden transgenic mice. Circ Res. 2001;88:780–786.
20. Guevara NV, Kim HS, Antonova EI, Chan L. The absence of p53
accelerates atherosclerosis by increasing cell proliferation in vivo. Nat
Med . 1999;5:335–339.
21. Mercer J, Figg N, Stoneman V, Braganza D, Bennett MR. Endogenousp53 protects vascular smooth muscle cells from apoptosis and reduces
atherosclerosis in ApoE knockout mice. Circ Res. 2005;96:667–674.
22. Su YR, Dove DE, Major AS, Hasty AH, Boone B, Linton MF, Fazio S.
Reduced ABCA1-mediated cholesterol efflux and accelerated atheroscle-
rosis in apolipoprotein E-deficient mice lacking macrophage-derived
ACAT1. Circulation. 2005;111:2373–2381.
23. Liu J, Thewke DP, Su YR, Linton MF, Fazio S, Sinensky MS. Reduced
macrophage apoptosis is associated with accelerated atherosclerosis in
low-density lipoprotein receptor-null mice. Arterioscler Thromb Vasc
Biol. 2005;25:174–179.
24. Shio H, Haley NJ, Fowler S. Characterization of lipid-laden aortic cells
from cholesterol-fed rabbits. III. Intracellular localization of cholesterol
and cholesteryl ester. Lab Invest . 1979;41:160–167.
25. Feng B, Zhang D, Kuriakose G, Devlin CM, Kockx M, Tabas I.
Niemann-Pick C heterozygosity confers resistance to lesional necrosis
and macrophage apoptosis in murine atherosclerosis. Proc Natl Acad SciU S A. 2003;100:10423–10428.
Schwartz et al Plaque Rupture in Humans and Mice 711
by guest on January 20, 2015http://atvb.ahajournals.org/ Downloaded from
http://atvb.ahajournals.org/http://atvb.ahajournals.org/http://atvb.ahajournals.org/http://atvb.ahajournals.org/
-
8/9/2019 Ruptura de La Placa en
8/70
26. Kolodgie FD, Virmani R, Burke AP, Farb A, Weber DK, Kutys R, Finn
AV, Gold HK. Pathologic assessment of the vulnerable human coronary
plaque. Heart . 2004;90:1385–1391.
27. Adams LD, Geary RL, Li J, Rossini A, Schwartz SM. Expression pro-
filing identifies smooth muscle cell diversity within human intima and
atherosclerotic plaque fibrous cap: loss of RGS5 distinguishes the cap
SMC. Arterioscler Thromb Vasc Biol. 2006;26:319–325.
28. Bentzon JF, Weile C, Sondergaard CS, Hindkjaer J, Kassem M, Falk E.
Smooth muscle cells in atherosclerosis originate from the local vesselwall and not circulating progenitor cells in ApoE knockout mice. Arte-
rioscler Thromb Vasc Biol. 2006;26:2696–2702. .
29. Glass CK, Witztum JL. Atherosclerosis. The road ahead. Cell. 2001;104:
503–516.
30. Schwartz SM. The intima: A new soil. Circ Res. 1999;85:877–879.
31. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease.
N Engl J Med . 2005;352:1685–1695.
32. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med . 1999;
340:115–126.
33. Sano H, Sudo T, Yokode M, Murayama T, Kataoka H, Takakura N,
Nishikawa S, Nishikawa SI, Kita T. Functional blockade of platelet-
derived growth factor receptor-beta but not of receptor-alpha prevents
vascular smooth muscle cell accumulation in fibrous cap lesions in
apolipoprotein E-deficient mice. Circulation. 2001;103:2955–2960.
34. Kozaki K, Kaminski WE, Tang J, Hollenbach S, Lindahl P, Sullivan C,
Yu JC, Abe K, Martin PJ, Ross R, Betsholtz C, Giese NA, Raines EW.Blockade of platelet-derived growth factor or its receptors transiently
delays but does not prevent fibrous cap formation in ApoE null mice.
Am J Pathol. 2002;161:1395–1407.
35. von der Thusen JH, van Vlijmen BJ, Hoeben RC, Kockx MM, Havekes
LM, van Berkel TJ, Biessen EA. Induction of atherosclerotic plaque
rupture in apolipoprotein E / mice after adenovirus-mediated transfer
of p53. Circulation. 2002;105:2064–2070.
36. Seo HS, Lombardi DM, Polinsky P, Powell-Braxton L, Bunting S,
Schwartz SM, Rosenfeld ME. Peripheral vascular stenosis in apoli-
poprotein E-deficient mice. Potential roles of lipid deposition, medial
atrophy, and adventitial inflammation. Arterioscler Thromb Vasc Biol.
1997;17:3593–3601.
37. Renard CB, Kramer F, Johansson F, Lamharzi N, Tannock LR, von
Herrath MG, Chait A, Bornfeldt KE. Diabetes and diabetes-associated
lipid abnormalities have distinct effects on initiation and progression of
atherosclerotic lesions. J Clin Invest . 2004;114:659–668.
38. Gough PJ, Gomez IG, Wille PT, Raines EW. Macrophage expression of
active MMP-9 induces acute plaque disruption in apoE-deficient mice.
J Clin Invest . 2006;116:59–69.
39. Moulton KS, Vakili K, Zurakowski D, Soliman M, Butterfield C, Sylvin
E, Lo KM, Gillies S, Javaherian K, Folkman J. Inhibition of plaque
neovascularization reduces macrophage accumulation and progression of
advanced atherosclerosis. P r oc Natl A cad Sci U S A. 2003;100:
4736–4741.
40. Langheinrich AC, Michniewicz A, Sedding DG, Walker G, Beighley PE,
Rau WS, Bohle RM, Ritman EL. Correlation of vasa vasorum neovas-
cularization and plaque progression in aortas of apolipoprotein E( / )/
low-density lipoprotein( / ). double knockout mice. Arterioscler
Thromb Vasc Biol. 2006;26:347–352.
41. Yang X, Thomas DP, Zhang X, Culver BW, Alexander BM, Murdoch
WJ, Rao MN, Tulis DA, Ren J, Sreejayan N. Curcumin inhibits platelet-derived growth factor-stimulated vascular smooth muscle cell function
and injury-induced neointima formation. Arterioscler Thromb Vasc Biol.
2006;26:85–90.
42. Johnson JL, Jackson CL. Atherosclerotic plaque rupture in the apoli-
poprotein E knockout mouse. Atherosclerosis. 2001;154:399–406.
43. Williams H, Johnson JL, Carson KG, Jackson CL. Characteristics of
intact and ruptured atherosclerotic plaques in brachiocephalic arteries of
apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol. 2002;
22:788–792.
44. Johnson J, Carson K, Williams H, Karanam S, Newby A, Angelini G,
George S, Jackson C. Plaque rupture after short periods of fat feeding in
the apolipoprotein E-knockout mouse: model characterization and effects
of pravastatin treatment. Circulation. 2005;111:1422–1430.
45. Farb A, Burke AP, Tang AL, Liang TY, Mannan P, Smialek J, Virmani
R. Coronary plaque erosion without rupture into a lipid core. A frequent
cause of coronary thrombosis in sudden coronary death. Circulation.1996;93:1354–1363.
46. Davies PF, Reidy MA, Goode TB, Bowyer DE. Scanning electron
microscopy in the evaluation of endothelial integrity of the fatty lesion in
atherosclerosis. Atherosclerosis. 1976;25:125–130.
47. MacDougall ED, Kramer F, Polinsky P, Barnhart S, Askari B, Johansson
F, Varon R, Rosenfeld ME, Oka K, Chan L, Schwartz SM, Bornfeldt KE.
Aggressive very low-density lipoprotein (VLDL). and LDL lowering by
gene transfer of the VLDL receptor combined with a low-fat diet regimen
induces regression and reduces macrophage content in advanced athero-
sclerotic lesions in LDL receptor-deficient mice. Am J Pathol. 2006;168:
2064–2073.
48. Ikari Y, Yee KO, Hatsukami TS, Schwartz SM. Human carotid artery
smooth muscle cells rarely express alpha(v)beta3 integrin at sites of
recent plaque rupture. Thromb Haemost . 2000;84:338–344.
49. Braun A, Trigatti BL, Post MJ, Sato K, Simons M, Edelberg JM,
Rosenberg RD, Schrenzel M, Krieger M. Loss of SR-BI expression leads
to the early onset of occlusive atherosclerotic coronary artery disease,
spontaneous myocardial infarctions, severe cardiac dysfunction, and pre-
mature death in apolipoprotein E-deficient mice. Circ Res. 2002;90:
270–276.
50. Zhang S, Picard MH, Vasile E, Zhu Y, Raffai RL, Weisgraber KH,
Krieger M. Diet-induced occlusive coronary atherosclerosis, myocardial
infarction, cardiac dysfunction, and premature death in scavenger
receptor class B type I-deficient, hypomorphic apolipoprotein ER61 mice.
Circulation. 2005;111:3457–3464.
51. Bea F, Blessing E, Bennett B, Levitz M, Wallace EP, Rosenfeld ME.
Simvastatin promotes atherosclerotic plaque stability in apoE-deficient
mice independently of lipid lowering. Arterioscler Thromb Vasc Biol.
2002;22:1832–1837.
52. Wen GB, Weinbaum S, Ganatos P, Pfeffer R, Chien S. On the time
dependent diffusion of macromolecules through transient open junctions
and their subendothelial spread. 2. Long time model for interaction
between leakage sites. J Theor Biol. 1988;135:219 –253.
53. Weinbaum S, Ganatos P, Pfeffer R, Wen GB, Lee M, Chien S. On the
time-dependent diffusion of macromolecules through transient open
junctions and their subendothelial spread. I. Short-time model for cleft
exit region. J Theor Biol. 1988;135:1–30.
54. Lemaitre V, O’Byrne TK, Borczuk AC, Okada Y, Tall AR, D’Armiento
J. ApoE knockout mice expressing human matrix metalloproteinase-1 in
macrophages have less advanced atherosclerosis. J Clin Invest . 2001;107:
1227–1234.
55. Falkenberg M, Tom C, DeYoung MB, Wen S, Linnemann R, Dichek DA.
Increased expression of urokinase during atherosclerotic lesion devel-opment causes arterial constriction and lumen loss, and accelerates lesion
growth. Proc Natl Acad Sci U S A. 2002;99:10665–10670.
56. Daugherty A, Manning MW, Cassis LA. Angiotensin II promotes ath-
erosclerotic lesions and aneurysms in apolipoprotein E-deficient mice.
J Clin Invest . 2000;105:1605–1612.
57. Brooke BS, Bayes-Genis A, Li DY. New insights into elastin and vascular
disease. Trends Cardiovasc Med . 2003;13:176–181.
58. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of
matrix metalloproteinases and matrix degrading activity in vulnerable
regions of human atherosclerotic plaques. J Clin Invest . 1994;94:
2493–2503.
59. Johnson C, Galis ZS. Matrix metalloproteinase-2 and -9 differentially
regulate smooth muscle cell migration and cell-mediated collagen orga-
nization. Arterioscler Thromb Vasc Biol. 2004;24:54–60.
60. de Nooijer R, Verkleij CJ, von der Thusen JH, Jukema JW, van der Wall
EE, van Berkel TJ, Baker AH, Biessen EA. Lesional overexpression of matrix metalloproteinase-9 promotes intraplaque hemorrhage in advanced
lesions but not at earlier stages of atherogenesis. Arterioscler Thromb
Vasc Biol. 2006;26:340–346.
61. Lessner SM, Martinson DE, Galis ZS. Compensatory vascular
remodeling during atherosclerotic lesion growth depends on matrix
metalloproteinase-9 activity. Arterioscler Thromb Vasc Biol. 2004;24:
2123–2129.
62. Glagov S, Weisenberg E, Zarins CK, Stankunavicius R, Kolettis
GJ. Compensatory enlargement of human atherosclerotic coronary
arteries. N Engl J Med . 1987;316:1371–1375.
63. Schoenhagen P, Vince DG, Ziada KM, Tsutsui H, Jeremias A, Crowe TD,
Nissen SE, Tuzcu EM. Association of arterial expansion (expansive
remodeling) of bifurcation lesions determined by intravascular ultra-
sonography with unstable clinical presentation. Am J Cardiol. 2001;88:
785–787.
64. Schoenhagen P, Ziada KM, Kapadia SR, Crowe TD, Nissen SE, TuzcuEM. Extent and direction of arterial remodeling in stable versus unstable
712 Arterioscler Thromb Vasc Biol. April 2007
by guest on January 20, 2015http://atvb.ahajournals.org/ Downloaded from
http://atvb.ahajournals.org/http://atvb.ahajournals.org/http://atvb.ahajournals.org/http://atvb.ahajournals.org/
-
8/9/2019 Ruptura de La Placa en
9/70
coronary syndromes : an intravascular ultrasound study. Circulation.
2000;101:598–603.
65. Reidy MA, Schwartz SM. Arterial endothelium: Assessment of in vivo
injury. Exp Molec Pathol. 1984;41:419–434.
66. Courtman DW, Schwartz SM, Hart CE. Sequential injury of the rabbit
abdominal aorta induces intramural coagulation and luminal narrowing
independent of intimal mass: extrinsic pathway inhibition eliminates
luminal narrowing. Circ Res. 1998;82:996–1006.
67. Burke AP, Kolodgie FD, Farb A, Weber DK, Malcom GT, Smialek J,
Virmani R. Healed plaque ruptures and sudden coronary death: evidence
that subclinical rupture has a role in plaque progression. Circulation.
2001;103:934–940.
68. Rioufol G, Gilard M, Finet G, Ginon I, Boschat J, ndre-Fouet X. Evo-
lution of spontaneous atherosclerotic plaque rupture with medical
therapy: long-term follow-up with intravascular ultrasound. Circulation.
2004;110:2875–2880.
69. Mann J, Davies MJ. Mechanisms of progression in native coronary artery
disease: role of healed plaque disruption. Heart . 1999;82:265–268.
70. Gough PJ, Raines EW. Gene therapy of apolipoprotein E-deficient mice
using a novel macrophage-specific retroviral vector. Blood . 2003;101:
485–491.
71. Paigen B, Morrow A, Brandon C, Mitchell D, Holmes P. Variation in
susceptibility to atherosclerosis among inbred strains of mice. Athero-
sclerosis. 1985;57:65–73.
72. Wang X, Ria M, Kelmenson PM, Eriksson P, Higgins DC, Samnegard A,
Petros C, Rollins J, Bennet AM, Wiman B, de Faire U, Wennberg C,
Olsson PG, Ishii N, Sugamura K, Hamsten A, Forsman-Semb K, Lager-
crantz J, Paigen B. Positional identification of TNFSF4, encoding OX40
ligand, as a gene that influences atherosclerosis susceptibility. Nat Genet .
2005;37:365–372.
73. Pei H, Wang Y, Miyoshi T, Zhang Z, Matsumoto AH, Helm GA, Tellides
G, Shi W. Direct evidence for a crucial role of the arterial wall in control
of atherosclerosis susceptibility. Circulation. 2006;114:2382–2389.
74. Drake TA, Schadt EE, Lusis AJ. Integrating genetic and gene expression
data: application to cardiovascular and metabolic traits in mice. Mamm
Genome. 2006;17:466–479.
75. Kris-Etherton PM, Lichtenstein AH, Howard BV, Steinberg D, Witztum
JL. Antioxidant vitamin supplements and cardiovascular disease. Circu-
lation. 2004;110:637–641.
76. Al-Shali KZ, Hegele RA. Laminopathies and atherosclerosis. Arterioscler
Thromb Vasc Biol. 2004;24:1591–1595.
77. Yang SH, Bergo MO, Toth JI, Qiao X, Hu Y, Sandoval S, Meta M,
Bendale P, Gelb MH, Young SG, Fong LG. Blocking protein farnesyl-
transferase improves nuclear blebbing in mouse fibroblasts with a
targeted Hutchinson-Gilford progeria syndrome mutation. Proc Natl Acad
Sci U S A. 2005;102:10291–10296.
78. Varga R, Eriksson M, Erdos MR, Olive M, Harten I, Kolodgie F, Capell
BC, Cheng J, Faddah D, Perkins S, Avallone H, San H, Qu X, Ganesh S,
Gordon LB, Virmani R, Wight TN, Nabel EG, Collins FS. Progressive
vascular smooth muscle cell defects in a mouse model of Hutchinson-
Gilford progeria syndrome. Proc Natl Acad Sci U S A. 2006;103:
3250–3255.
Schwartz et al Plaque Rupture in Humans and Mice 713
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Page 1 of 60
Plaque Rupture in Humans and Mice
Stephen M. Schwartz1,5
, Zorina S. Galis2, Michael E. Rosenfeld
4, Erling Falk
3
1Department of Pathology, University of Washington, Seattle, WA 98109
2Department of Surgery, Indiana University, and Lilly Research Laboratories, Indianapolis, IN
462853 Department of Cardiology (Research), Aarhus University Hospital (Skejby), Aarhus, Denmark
82004Department of Pathobiology, University of Washington, Seattle, WA 98109
5Corresponding author: Stephen M. Schwartz
Department of Pathology
University of Washington815 Mercer Street, Room 421
Seattle, WA 98109-4714
Phone: 206-543-0258
Fax: 206-897-1540
e-mail: steves@u.washington.edu
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 thattest 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 repr oduced here, however to simplify cross reference between the
versions, we have numbered the online references using Roman numerals. The equivalent figurenumbers, 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.
.
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Page 2 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.
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Page 3 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
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Page 4 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 rupture—the 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.
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Page 5 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 pre-
formed 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
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Page 6 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-β, 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
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Page 8 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 caspase-
independent 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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Page 9 of 60
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
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Page 10 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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Page 11 of 60
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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Page 12 of 60
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
top related