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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 [email protected]

© 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.

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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: [email protected]

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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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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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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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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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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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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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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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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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

ruptura de la placa en

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