Smooth Muscle Cells in Atherosclerosis Originate - Arteriosclerosis ...

Smooth Muscle Cells in Atherosclerosis Originate From the Local Vessel Wall and Not 

Circulating Progenitor Cells in ApoE Knockout Mice 

Jacob F. Bentzon, Charlotte Weile, Claus S. Sondergaard, Johnny Hindkjaer, Moustapha 

Kassem and Erling Falk 

Arterioscler Thromb Vasc Biol. 2006;26:2696-2702; originally published online September 28, 

2006; 

doi: 10.1161/01.ATV.0000247243.48542.9d 

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Smooth Muscle Cells in Atherosclerosis Originate From the 

Local Vessel Wall and Not Circulating Progenitor Cells in 

ApoE Knockout Mice 

Jacob F. Bentzon, Charlotte Weile, Claus S. Sondergaard, Johnny Hindkjaer, 

Moustapha Kassem, Erling Falk 

Objective—Recent studies of bone marrow (BM)-transplanted apoE knockout (apoE / ) mice have concluded that a 

substantial fraction of smooth muscle cells (SMCs) in atherosclerosis arise from circulating progenitor cells of 

hematopoietic origin. This pathway, however, remains controversial. In the present study, we reexamined the origin of 

plaque SMCs in apoE / mice by a series of BM transplantations and in a novel model of atherosclerosis induced in 

surgically transferred arterial segments. 

Methods and Results—We analyzed plaques in lethally irradiated apoE / mice reconstituted with sex-mismatched BM 

cells from eGFP apoE / mice, which ubiquitously express enhanced green fluorescent protein (eGFP), but did not find 

a single SMC of donor BM origin among 10 000 SMC profiles analyzed. We then transplanted arterial segments 

between eGFP apoE / and apoE / mice (isotransplantation except for the eGFP transgene) and induced atherosclerosis 

focally within the graft by a recently invented collar technique. No eGFP SMCs were found in plaques that 

developed in apoE / artery segments grafted into eGFP apoE / mice. Concordantly, 96% of SMCs were eGFP in 

plaques induced in eGFP apoE / artery segments grafted into apoE / mice. 

Conclusions—These experiments show that SMCs in atherosclerotic plaques are exclusively derived from the local vessel 

wall in apoE / mice. (Arterioscler Thromb Vasc Biol. 2006;26:2696-2702.) 

Key Words: atherosclerosis smooth muscle cells adult stem cells pathology apoE knockout mice 

Recruitment of smooth muscle cells (SMCs) is a key 

mechanism in the development of atherosclerosis and its 

clinical manifestations. SMCs contribute to plaque volume 

through matrix synthesis, and the accretion of SMCs plays a 

decisive role in the pathogenesis of coronary stenosis. 1 

Conversely, paucity of SMCs in the fibrous cap of plaques 

increases the risk of plaque rupture and life-threatening 

arterial thrombosis. 2 Understanding the origin of plaque 

SMCs may provide new opportunities for controlling their 

ambiguous nature. 

See page 2579 and cover 

Until recently, the only source of SMCs in atherosclerotic 

lesions was considered to be the local vessel wall. According 

to this hypothesis, local SMCs in the arterial media and 

intima modulate into a synthetic and migratory phenotype 

(aka, phenotypic modulation3 ) and form the fibrous component 

of the plaque. This theory was essentially inferred from 

a line of suggestive observations in arterial injury models in 

the 70s and 80s. 4,5 More to the point, Cre/lox fate mapping in 

the apoE knockout (apoE / ) mouse model of atherosclerosis 

recently confirmed that preexisting SMCs, presumably from 

the local media, contribute to plaque SMCs during atherogenesis, 

but the existence of other sources could not be 

excluded by this technique. 6 

In 2002, an alternative and major source of SMCs in 

atherosclerosis was reported, 7 and this new paradigm has 

great impact on current research in this area. 8,9 Based on 

observations in bone marrow (BM)-transplanted apoE / 

mice, it was concluded that a substantial fraction of plaque 

SMCs arise from circulating progenitor cells of hematopoietic 

origin. 7 This pathway holds promise for the development 

of novel therapeutic means of controlling the recruitment and 

accumulation of SMCs in plaques. However, it remains 

questionable whether the quality of the histological documentation 

in that study can support a definitive conclusion, 

especially because the seeming use of unfixed tissue for 

detection of enhanced green fluorescent protein (eGFP) can 

lead to diffusion of the tracer marker from sectioned cells. 10 

Furthermore, reconstitution of apoE / mice with apoE / 

Original received June 13, 2006; final version accepted September 13, 2006. 

From the Departments of Cardiology (J.F.B., E.F.), Aarhus University Hospital, and Institute of Clinical Medicine, Aarhus University; the Department 

of Plastic Surgery (C.W.), Aarhus University Hospital; the Department of Molecular Biology and Institute of Clinical Medicine (C.S.S.), Aarhus 

University; the Department of Gynecology and Obstetrics (J.H.), Aarhus University Hospital; and the Department of Endocrinology (M.K.), Odense 

University Hospital, Denmark. 

Correspondence to Jacob Bentzon, MD, Department of Cardiology, Research Unit, Aarhus University Hospital, Brendstrupgaardsvej, 8200 Aarhus N, 

Denmark. E-mail jben@ki.au.dk 

© 2006 American Heart Association, Inc. 

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000247243.48542.9d 


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BM as performed in that experiment provides powerful 

protection against the development of atherosclerosis. 11 

To address these concerns, we repeated the BM transplantation 

experiment in apoE / mice with a number of methodological 

modifications but could not replicate the results. To 

confirm and extend these findings, we then studied atherosclerosis 

induced in surgically transferred arterial segments. 

Here, we show that atherosclerotic plaque SMCs are derived 

from the local vessel wall and not circulating smooth muscle 

progenitor cells in apoE / mice. 

Methods 

For a detailed Methods section, please see the online data supplement, 

available online at http://atvb.ahajournals.org. 

Transgenic Animals 

The Danish Animal Experiments Inspectorate approved all procedures. 

ApoE / mice (B6.129P2-Apoe tm1Unc ; Taconic M&B, Ry, 

Denmark), backcrossed more than 10 times to C57BL/6 mice, and 

eGFP C57BL/6 mice (C57BL/6-Tg[ACTB-EGFP]1Osb/J; Jackson 

Laboratories, Bar Harbor, Me), were intercrossed to obtain 

eGFP apoE / mice (hemizygous for the eGFP transgene). 

Bone Marrow Transplants 

ApoE / mice (n28) were lethally irradiated and rescued with 

sex-matched bone marrow from eGFP apoE / mice as previously 

described (Figure 1a). 12 Age-matched apoE / (n8) and 

eGFP apoE / mice (n8) were included as controls. One mouse 

died shortly after BM transplantation. Four randomly-chosen BMtransplanted 

mice were killed after 4 weeks. The other mice were 

changed from chow to high fat diet (21% saturated fat, 1.5% 

cholesterol, Hope Farms, Woerden, Netherlands) to accelerate 

atherogenesis and then killed at 20 or 32 weeks of age. 

Atherosclerosis Induced in Transplanted 

Arterial Segments 

We performed orthotopic transplantations of common carotid artery 

(CCA) segments between eGFP apoE / and apoE / mice 

(eGFP apoE / CCA 3 apoE / transplanted mice, n12, and 

apoE / CCA 3 eGFP apoE / transplanted mice, n12, excluding 

perioperative deaths). Transplantations were isogenic apart from the 

eGFP transgene and immunogenicity of eGFP has been reported to 

be minimal in the background C57BL/6 strain. 13 

Bentzon et al Origin of SMCs in Atherosclerosis in ApoE / Mice 2697 

Figure 1. Overview of experimental design and results. a, Analysis of sex-mismatched eGFP apoE / BM 3 apoE / transplanted mice 

using eGFP or Y chromosome as tracers. *Per total number of SMA profiles analyzed. † Per total number of nucleated SMA profiles 

analyzed. BMT indicates bone marrow transplantation; HF, high-fat diet; AR, aortic root; AA, aortic arch; BT, brachiocephalic trunk; 

AbA, Abdominal aorta; ThA, descending thoracic aorta. b, Analysis of atherosclerotic plaques induced in transplanted CCA segments. 

CCAT indicates common carotid artery segment transplantation. See text for description of additional control experiments. 


http://atvb.ahajournals.org/ by guest on June 4, 2013 

All grafted vessels were patent after 6 weeks. Then, localized 

atherosclerosis was induced by placement of a constrictive perivascular 

collar around the distal part of the graft using a technique 

slightly modified from von der Thüsen et al (n10 from each group) 

(Figure 1b). 14 Four mice (eGFP apoE / CCA 3 apoE / transplanted 

mice, n2, and apoE / CCA 3 eGFP apoE / transplanted 

mice, n2) were treated identically with the exception that no collars 

were inserted. 

Immunohistochemistry 

SMCs were identified by staining for smooth muscle -actin 

(SMA). This abundant protein is considered the most sensitive, 

though not specific, SMC marker. SMA is expressed early in 

embryonic development 15 and lost late in phenotypic modulation, 3 

and it is the marker on which previous conclusions on BM origin of 

plaque SMCs have been based. 7,16 Specificity of SMA stainings 

was confirmed by observing the expected subplasmalemmal distribution 

of SMA in plaque SMCs and by negative isotype control 

stainings. The Mac2 epitope was used as a marker for 

plaque macrophages. 

Fluorescence In Situ Hybridization 

The Y chromosome was visualized in a subset of SMA stained 

aortic root sections. First, z-axis image stacks of SMA cellcontaining 

areas were acquired and stored. Then, cover slips were 

removed and the immunostained sections were pretreated in 

10 mmol/L sodium citrate, pH 6.0 (2 hours, 80°C) and 0.025% 

pepsin solution (10 minutes, 37°C; Sigma), which totally extinguished 

eGFP fluorescence and deteriorated the SMA staining 

signal. Y chromosomes were then detected by a fluorescein isothiocyanate 

(FITC)-conjugated paint probe using the protocol recommended 

by the manufacturer (Cambio), and the phenotype of cells 

with Y nuclei was identified in the stored images. This sequential 

technique allows for a robust analysis, because the cell phenotype is 

analyzed before the harsh pretreatment necessary for fluorescence in 

situ hybridization (FISH) distorts morphology. 

Microscopic Analysis 

Sections were examined in an Olympus Cell-R epifluorescence 

microscope system equipped with differential interference contrast 

(DIC) optics and motorized focus. Deconvolution analysis on widefield 

z-axis image stacks (0.3 m optical thickness) was performed 

in a subset of sections (aortic root sections for sequential FISH 

analysis and CCA plaques) by using a blind 3D deconvolution 

algorithm (Autoquant Deblur 9.3; Autoquant Imaging). This tech-

2698 Arterioscler Thromb Vasc Biol. December 2006 

nique, like confocal microscopy, yields high signal-to-noise images 

of thin optical sections. 

SMA cell profiles in the range of small SMC nuclei or larger 

(from 3 m [minor axis] 5 m [major axis]) were analyzed for 

colocalization. Because all SMCs contain a nucleus of relatively 

similar size, this strategy is an accurate analysis of the presence of 

eGFP SMA cells and avoids many interpretational problems 

presented by smaller autofluorescent structures and overlapping parts 

of closely opposed eGFP and SMA cells. The number of 

analyzed SMA profiles was counted to estimate statistical power. 

In BM transplanted mice, we estimated the total number by counting 

a representative subset (34%) of the sections. Nucleated SMA 

profiles, defined as cells where the nucleus were circumscribed by 

SMA stain, were counted and analyzed for combined Y chromosome 

and SMA signal. 

Statistical Analysis 

Rare binomially distributed events approach the Poisson distribution, 

and a 95% confidence limit for double-positive cells were calculated 

by this approximation (95% confidence limit: P3.0/number of 

observations). Sections were taken at least 50 m apart to ensure that 

the same SMC was not analyzed twice. 

Results 

Sex-Mismatched BM Transplantation 

The degree of hematopoietic chimerism obtained in 

eGFPapoE/ BM 3 apoE/ transplanted mice was assessed 

by flow cytometry of peripheral blood leukocytes. The 

fraction of fluorescent cells in BM transplanted mice 4 and 24 

weeks after transplantation was 93.42.8% (meanSD, 

n23) and 93.71.4% (n12), respectively, which was 

similar to that of eGFPapoE/ positive control mice 

(92.42.0% [n4] and 92.20.8% [n4], respectively; 

Figure 2a). The sustained presence of eGFP leukocytes 

documents the replacement of hematopoietic stem cells, 

which are the only long-term self-renewing cells in the 

hematopoietic system. 17 Plasma lipid values are described in 

supplemental Table I. 

Vascular Pathology in BM Chimeras 

In animals euthanized 4 weeks after BM transplantation 

(n4), only foam cell lesions were present at the arterial sites 

selected for analysis, excluding the possibility that SMCs had 



Figure 2. The hematopoietic system in irradiated 

apoE / mice was reconstituted with 

eGFP apoE / cells after BM transplantation. 

a, Flow cytometry of peripheral blood 

leukocytes. Left panel, Cells within a defined 

forward-side scatter gate that encompassed 

the major leukocyte populations were analyzed. 

Right panel, Green fluorescence of 

leukocytes obtained from an apoE / mouse 

(top), an eGFP apoE / mouse (middle), and 

an eGFP apoE / BM 3 apoE / transplanted 

mouse (bottom). b, Macrophage 

foam cells detected by the murine macrophage 

marker Mac2 were eGFP in BM 

transplanted mice as expected. Aortic root 

plaque from eGFP apoE / BM 3 apoE / 

transplanted mouse (32 weeks of age). 

Green indicates eGFP; Red, Mac2; Yellow, 

overlay of eGFP and Mac2; Blue, DAPI; Gray 

scale, DIC. Color channels are shown separately 

to facilitate interpretation. L indicates 

lumen; F, foam cells; M, tunica media. Scale 

bar100 m. 

entered the intima already before full reconstitution of the 

hematopoietic system with eGFP cells was confirmed. At 20 

weeks of age, all mice had developed fibrofatty plaques in the 

aortic root, and fibrofatty plaques were also present in the 

aortic arch, brachiocephalic trunk, abdominal aorta, and to a 

variable extent in the descending thoracic aorta at 32 weeks of 

age. 

Atherosclerotic Plaque SMCs Are Not Derived 

From Hematopoietic Stem Cells 

As an internal validation of the experiment, the hematopoietic 

origin of plaque macrophages was verified by the demonstration 

of cells double-positive for eGFP and the murine 

macrophage marker Mac2 in plaques in BM transplanted 

mice (Figure 2b). 

SMA cells were predominantly located in the fibrous 

cap separating the core of the plaque from the arterial lumen. 

As expected, SMA cells were almost uniformly eGFP in 

eGFP apoE / positive control mice (n4, 32 weeks of age, 

580 of 598 SMA cell profiles analyzed). In agreement with 

observations reported by others, SMA expression was lost 

in major parts of the media underlying atherosclerotic plaques 

(Figure 3). 7 

We did not identify a single eGFP SMA cell among 

10 000 SMA cell profiles analyzed in 154 sections from 

multiple sites of the arterial tree in 23 BM-transplanted mice 

(95% confidence limit 0.03%) (Figures 1a and Figure 3; 

supplemental Figure I). The identical result was reached 

using blind 3D deconvolution performed on a subset of the 

same sections from the aortic root (Figure 4). 

As an independent tracing method, we detected Y chromosomes 

in SMA immunostained sections from the aortic root 

(Figure 4). Not once did we convincingly detect a Y chromosome 

in 367 analyzed nucleated SMA profiles in 

plaques from male-to-female BM-transplanted mice. In one 

inconclusive case, a SMA profile circumscribed two nuclear 

profiles, one of which was Y chromosome . A positive 

Y chromosome signal was detected in 114 of 209 analyzed 

nucleated SMA cell profiles (54%) in female-to-male 

BM-transplanted mice, which was similar to that detected in

plaques from the control apoE / and eGFP apoE / male 

mice (n22, 32 weeks of age, 48 of 94 analyzed nucleated 

SMA cells) and in the range predicted from the thickness 

of the section and the nuclear size. 

SMCs Are Derived From the Local Vessel Wall in 

Cross-Grafted Arterial Segments 

Our observations in BM-transplanted mice showed that differentiation 

of hematopoietic stem cells to plaque SMCs is 

exceedingly rare if it occurs at all. To evaluate whether 

circulating smooth muscle progenitor cells of nonhematopoietic 

origin can contribute to atherosclerotic plaque SMCs, we 

then analyzed SMC origin in atherosclerotic lesions that were 

induced proximal to a constrictive collar in cross-grafted 

CCA segments (Figures 1b and 5a). 

Of 20 mice in which we performed CCA segment transplantations 

and collar placements, complete or near-complete 




Figure 3. Hematopoietic stem cells do not contribute 

to plaque SMCs. a, Overview of aortic root 

plaque from eGFP apoE / BM 3 apoE / transplanted 

mouse (32 weeks of age) exhibiting donorderived 

eGFP foam cells (green) and the formation 

of a fibrous cap of recipient-derived SMA 

cells (red only). L indicates lumen; C, fibrous cap; 

F, foam cells; M, tunica media; A, tunica adventitia. 

Scale bar200 m. b, Higher magnification of the 

area demarcated in a. Fluorescence microscopy 

combined with DIC imaging (gray scale) to reveal 

tissue structure. Scale bar50 m. c-e, Further 

analysis of the area demarcated in b. With the 

superimposed DIC image, it is often possible to 

visualize cell boundaries that would otherwise 

escape detection by fluorescence microscopy. 

Arrows indicate two separate cellular structures 

appearing as depressions in the relief-like DIC 

image (c), one of which stains positive for SMA 

(d, red channel). The neighboring cell is eGFP (e, 

red and green channels). No eGFP SMA 

double-positive cells are present. Scale 

bar25 m. 

graft occlusions were present in 5 mice at time of sacrifice. In 

4 mice, no significant lesions were found, and in 2 mice, 

extensive lesion formation extending into the graft from the 

proximal anastomosis was present. These were all excluded 

from the analysis. Most likely, the cause of these technical 

failures was inconsistency in tightening of the ligature around 

the collar to yield appropriate constriction. None of the 

CCA-transplanted mice in which no collar was placed developed 

lesions in the grafted artery apart from a small mural 

thrombus in one. 

In all other mice, advanced plaques had developed focally 

immediately proximal to the constrictive collar with an area 

of unaffected vessel wall separating the lesion from the 

proximal anastomosis site (Figure 5b). In plaques that had 

formed in apoE / artery segments grafted into 

eGFP apoE / mice (n4), not a single eGFP SMA cell 

Figure 4. Sequential immunostaining and FISH 

technique revealed no male SMA cells in 

plaques in male eGFP apoE / BM 3 female 

apoE / transplanted mice. The figure shows analysis 

of an aortic root plaque from a mouse 20 

weeks of age. a, SMA immunostained sections 

were analyzed and the digitized images were 

stored. Deconvoluted optical section is shown. 

Arrows indicate nuclei that proved to contain a Y 

chromosome with subsequent FISH. Red indicates 

SMA; Green, eGFP; Blue, DAPI. Scale 

bar50 m. b, FISH for Y chromosome. Fluorescence 

of eGFP is completely extinguished by the 

FISH procedure, and only the green fluorescence 

resulting from the FITC-conjugated Y chromosome 

paint probe is visible. Deteriorated SMA staining 

(red channel) is not shown. The phenotype of cells 

with Y nuclei is identified by comparing with the 

stored image (a). c-e, Larger magnifications of a 

and b. Scale bar25 m. F indicates foam cells; 

C, Fibrous cap; L, lumen.


Figure 5. Schematic of the model of constrictive collar-induced 

atherosclerosis in surgically transferred CCA segments. First, 

the graft was interpositioned in the recipient CCA by two endto-end 

anastomoses. Then after 6 weeks, a slightly constrictive 

piece of silicone tubing was positioned around the distal part of 

the graft, and this induced the formation of an atherosclerotic 

plaque in the grafted CCA immediately proximal to the collar in 

the course of 10 weeks. b, Atherosclerotic plaque in the transplanted 

CCA 10 weeks after collar insertion. PA indicates proximal 

anastomosis; C, Collar; DA, distal anastomosis; P, plaque. 

Scale bar1 mm. 

was detected in 54 serial sections (10 to 16 per plaque), 

encompassing 684 SMC profiles (95% confidence limit, 

0.4%; Figure 6a through 6c). Concordantly, in plaques 

induced in eGFP apoE / artery segments grafted into 

apoE / recipients (n5), nearly all SMA cells were 

eGFP (Figure 6d through 6f). We analyzed 34 sections from 

this type of lesion (6 to 9 per plaque) and found that 469 of 

487 (96%) SMC profiles were clearly eGFP , which was 

similar to that seen in plaques from positive control 

eGFP apoE / mice. 

Discussion 

In this study, we traced the origin of plaque SMCs in different 

models of atherosclerosis in the apoE / hyperlipidemic 

mouse. First, we attempted and failed to reproduce the 

previous finding that hematopoietic stem cells can contribute 

to plaque SMCs in BM chimeric apoE / mice. 7 Second, we 

directly evaluated and confirmed that plaque SMCs are 

derived from cells of the local vessel wall in a novel model of 

collar-induced atherosclerosis in surgically transferred artery 

segments. 

Induction of Atherosclerosis in Surgically 

Transferred CCA Segments 

An important premise for basing our conclusions on the 

collar-induced atherosclerosis model is that the pathogenesis 

is equivalent to that of spontaneously developing atherosclerosis. 

An extensive number of observations support this 

assumption. Lesions in the constrictive collar model develop 

immediately proximal to the collar, presumably elicited by 

low wall shear stress in this region, 18 and are strictly 

dependent on the presence of hypercholesterolemia. 14 Thus, 



lesion development in this model appears to depend on the 

two key etiologic factors known for spontaneous atherosclerosis. 

These features distinguish the constrictive collar model 

fundamentally from any other mechanical means of inducing 

intimal lesions, including the classic loose cuff model of 

neointima formation. 19 Furthermore, constrictive collarinduced 

lesions are pathoanatomically reminiscent of spontaneously 

developing atherosclerosis with respect to the 

presence of macrophage foam cells (supplemental Figure II), 

cholesterol crystals, fibrous caps, and necrotic cores (Figure 

6d). It can never be proved that constrictive collar-induced 

and spontaneous atherosclerosis are identical pathological 

entities, but it seems unlikely that disease processes that 

resemble each other to such detail in etiology and morphology 

would differ in a key mechanism such as SMC 

recruitment. 

Origin of SMCs in Different Vascular 

Disease Models 

The present study is only one of several recent studies to have 

(re)investigated the origin of lesional SMCs in arterial disease, 

and not the first to have reached a conclusion that 

conflicts with others. Surprisingly few groups have examined 

the most prevalent arterial disease, atherosclerosis, 7,16 but 

analogous investigations have been carried out in a number of 

other vascular disease models. 8,9 

The major discussion stemming from these studies has 

been over the question whether lesional SMCs can originate 

from BM cells. Besides the report of Sata et al on atherosclerosis 

in apoE / mice, 7 this has been described to occur in 

human atherosclerosis, 16 and in rodent models of wire injury, 

7,20 ferric chloride injury, 21 and allotransplantation arteriopathy. 

7,22 Others, however, have not been able to detect 

BM-derived SMCs in allotransplantation arteriopathy, 12 and 

it has been reported not to be involved in the pathogenesis of 

vein graft atherosclerosis, 23 ligation-induced neointima, 19 and 

neointima formed within a loose cuff. 19 

It is conceivable that part of the disparity in these findings, 

including the conflict between our results and those of Sata et 

al, 7 is attributable to methodological or interpretational differences. 

For instance, it is strikingly difficult, despite a 

considerable literature, to find compelling images of BMderived 

SMA cells with the expected SMC morphology. 

But it is also reasonable to think that biological differences 

between models are important. Even though intimal SMC 

accumulation is the common hallmark of many types of 

vascular disease, the etiology and pathogenesis of lesion 

development differ. Interestingly, recruitment of circulating 

smooth muscle progenitor cells has predominantly been 

reported in models with significant endothelial disruption and 

platelet deposition, and experimental evidence indicates that 

SDF expressed by adhering platelets facilitates homing of 

circulating progenitor cells. 24 This hypothesis offers a possible 

explanation for the discrepancy between our results and 

those reported for coronary atherosclerosis in sexmismatched 

BM transplanted patients. 16 It is possible that 

asymptomatic plaque rupture with mural thrombus in humans, 

which is not seen in the mouse model, is critical in 

mediating homing of circulating progenitor cells.

The finding of intimal SMCs that do not originate from the 

graft or from hematopoietic stem cells in rodent models of 

vein graft atherosclerosis and allotransplantation arteriopathy 

has led to the theory that circulating smooth muscle progenitor 

cells of nonhematopoietic origin exist and participate in 

vascular lesion formation. 25 It is, however, important to 

realize that migration of SMCs from the contiguous vasculature 

into the graft in those types of studies has not been 

conclusively excluded. For instance, Hu et al found that 40% 

of SMCs in atherosclerotic lesions of vein grafts were not 

derived from the local vessel wall or from hematopoietic stem 

cells, but the experimental design did not allow to distinguish 

between SMCs migrating through the anastomosis sites and 

homing and differentiation of SMCs from nonhematopoietic 

circulating progenitor cells. 23 If migrating medial SMCs were 

the source of these cells, then the fact that our lesions 

developed isolated from the recipient vasculature by a stretch 

of unaffected donor vessel (Figure 6a), whereas lesions in the 

vein graft model did not, may explain the differences in the 

result obtained. Another explanation could be the inherent 

differences between SMCs of arterial and venous origin. 26 

Local Source of Plaque SMCs 

Our experiments were not designed to discriminate between 

candidate sources for plaque SMCs within the vascular wall. 




Figure 6. Analysis of atherosclerotic 

plaques induced in surgically transferred 

CCA segments demonstrated that 

plaque SMC are derived from the local 

arterial wall. a-c, All SMA cells were 

eGFP - in plaques developing in apoE / 

CCA segments grafted into 

eGFP apoE / mice. The eGFP signal 

visible in the top left corner of a is an 

artifact caused by diffusion into the outer 

medial layer of perivascular extracellular 

eGFP, which was abundantly present 

around transplanted vessels in 

eGFP apoE / mice. Red indicates 

SMA; green, eGFP; blue, DAPI; grayscale, 

DIC; L, lumen; C, fibrous cap; F, 

foam cells; M, tunica media. Scale 

bar25 m. d-e, Conversely, in plaques 

induced in eGFP apoE / CCA segments 

grafted into apoE / mice, virtually 

all SMA cells were eGFP . L indicates 

lumen; C, fibrous cap; NC, necrotic core; 

F, foam cells; M, tunica media. Scale 

bar25 m. 

Several alternatives to vascular SMCs have been proposed, 

including endothelial-to-SMC differentiation and invasion of 

fibroblasts or stem cell progeny from the tunica adventitia. 

27,28 However, the identification of the local arterial wall as 

the origin of plaque SMCs established in this study combined 

with the observation of Feil et al that a major fraction of 

plaque SMCs originate from SM22 cells, 6 pinpoint that in 

mice—that have no resident intimal SMCs—tunica media is 

the major contributor to SMCs in atherosclerotic plaques. 

Conclusions 

Our experiments demonstrate that atherosclerotic plaque 

SMCs are derived exclusively from the local vessel wall in 

apoE / mice. This observation strongly supports the original 

hypothesis that these cells originate from local vascular 

SMCs and disagrees with the proposed role of circulating 

smooth muscle progenitor cells in atherogenesis. These findings 

have implications for future research into the mechanisms 

by which the fibrous component of atherosclerosis 

develops. 

Acknowledgments 

We thank Sandra H. van Heiningen and Erik Biessen for instructions 

on the constrictive collar model, and Helle Quist, Birgitte Sahl, and 

Merete Dixen for excellent technical assistance.


Sources of Funding 

This study was funded by the Danish Medical Research Council and 

the Danish Heart Foundation. 

None. 

Disclosures 

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Supplementary Methods 

Transgenic mice 

ApoE -/- mice (Taconic M&B, Ry, Denmark) and eGFP + mice (Jackson Laboratories, Bar Harbor, 

ME), both backcrossed more than ten times to C57BL/6 mice, were intercrossed to obtain 

eGFP + apoE -/- mice (hemizygous for the eGFP transgene). 1,2 Genotyping for apoE was performed 

using primers recommended by the Jackson Laboratory (5-GCCTAGCCGAGGGAGAGCCG-3, 5- 

TGTGACTTGGGAGCTCTGCAGC-3, and 5-GCCGCCCCGACTGCATCT-3). Phenotyping for 

eGFP was done using a Woods UV lamp. 

Bone marrow transplantations 

Crude bone marrow was flushed from femurs and tibias from eGFP + apoE -/- mice 8 weeks of age 

(n=7). Cells were washed twice in RPMI-1640 (Invitrogen) and filtered through a 70 μm mesh. 

ApoE -/- mice (n=28) 8 weeks of age were irradiated with 10 Gy from a 137 Cs source and 10 7 

unfractionated bone marrow cells were injected into a tail vein 4 hours later. Mice were housed with 

filter tops and administered oxytetracyclin (2g/l Terramycin vet. 20%) one day prior to and 7 days 

after transplantation. 

Flow cytometry 

Blood (50 μl) was lysed in erythrocyte lysis buffer (NH4Cl 8.02 g/L, NaHCO3 0.84 g/l, Na2EDTA 

0.37 g/l). The fraction of green fluorescent leukocytes in peripheral blood was measured in a 

forward-side scatter gate that was enriched for CD45 + cells (>98%) and excluded only a few CD45 + 

cells (~2%) as established in pilot experiments. 



Plasma lipids analysis 

Plasma total cholesterol and triglycerides were measured on a Vitros 950 analyzer (Ortho-Clinical 

Diagnostics). HDL cholesterol (HDL-C) was measured enzymatically on a Kone 30 analyzer 

(Thermo) using kits from ABX (Triolab, Copenhagen, Denmark). 

Common carotid artery segment transplantation and collar placement 

Donor common carotid arteries (CCA) were flushed with 100 IU/ml heparin saline and kept in 

RPMI-1640 at room temperature. Recipient mice were anesthetized with isoflurane (induction 5%, 

maintenance 1.5%-2%) and the right CCA was accessed through a midline neck incision. Heparin 

was administered (200 IU/kg i.m.) before microvascular clamps were applied. The recipient CCA 

was then divided and the donor CCA segment was interposed by two end-to-end anastomoses of 

four symmetrically placed 11-0 polyamide single sutures (Ethicon, Johnson & Johnson, Birkerød, 

Denmark). Total operation time was 75-90 minutes. Perioperative mortality was 15 per cent. 

Postoperative analgetics (buprenorphine, 0.1 mg/kg s.c. repeated every 12 hours for 3 days) were 

administered. An instructional video of the procedure can be obtained from the corresponding 

author. 

Six weeks after CCA transplantation, mice were reoperated under isoflurane anesthesia and collars 

made from 0.75 mm silicone tubing (inner diameter 0.31 mm, HelixMark, Helix Medical Inc., CA, 

USA) were positioned around the most distal segment of the graft and secured by a single 7-0 

ligature. 

Tissue processing 

Anesthetized mice (pentobarbital 5 mg i.p.) were killed by exsanguination, pressure-fixed with a 

phosphate-buffered (pH 7.2) formaldehyde solution (4%) through the left ventricle for 5 minutes, 



and immersion-fixed for 6 hours at room temperature. The aortic root, aortic arch, right CCA, 

brachiocephalic trunk, abdominal aorta and descending thoracic aortas were removed, cryoprotected 

in a sucrose solution (25 % w/v for 24 h + 50 % w/v for 24 h), embedded in O.C.T TM compound 

(Sakura Finetek, Pro-Hosp, Vaerloese, Denmark), and snap-frozen in liquid nitrogen-chilled 

methanol:acetone (1:1). Specimens were sectioned at 4-5 μm thickness. 

Immunohistochemistry 

SMCs were identified by staining for smooth muscle α-actin (SMαA) using biotinylated mouse 

monoclonal anti-SMαA (1:50, Clone 1A4, Neomarkers MS-113-B, AH Diagnostics, Aarhus, 

Denmark) followed by Alexa 594-conjugated streptavidin (1:200, Molecular Probes, Invitrogen, 

Taastrup, Denmark). Endogenous biotin was blocked with an avidin-biotin blocking kit (Vector 

Labs, VWR, Albertslund, Denmark) and a biotinylated irrelevant monoclonal antibody (1:50, 

Neomarkers NC-1390-B) of the same isotype was used as negative control. We would expect our 

two-layer immunofluorescence technique to be at least as sensitive as the one-layer method used by 

Sata et al. 3 To further ensure sufficient sensitivity of our staining procedure, we initially compared 

this method to SMαA staining visualized by high-sensitive tyramide signal amplification (TSA TM 

Biotin System, Perkin Elmer, Hvidovre, Denmark), and found that both techniques gave similar 

results in terms of number of SMαA + cells identified (data not shown). 

The Mac2 epitope was stained as a marker for plaque macrophages by using rat anti-mouse Mac2 

antibody (1:500, CL8942AP, Cedarlane Labs, Trichem Aps, Frederikssund, Denmark) followed by 

Texas Red-conjugated goat anti-rat secondary antibody (1:200, Jackson Immunoresearch, 

Cambridgeshire, UK). An irrelevant rat monoclonal antibody (1:50, R2a00, Caltag, Trichem) was 

used as negative control. 



Fluorescence in situ hybridization (FISH) 

The Y chromosome was visualized in sections by a FITC-conjugated chromosome paint probe 

(Cambio, Cambridge, UK). Sections were first immunostained for SMαA, and high-power fields of 

SMαA + cells were photographed. Then, the cover slips were removed and the sections were 

pretreated in 10 mM sodium citrate, pH 6.0 (2h, 80°C) (Bie & Berntsen, Aarhus, Denmark), 

digested in pepsin solution (0.025%, 10 min, 37 °C, Sigma), fixed in 4% paraformaldehyde and 

treated in freshly prepared acetic acid (0.25% acetic anhydride in 0.1 M triethanolamine-HCl, pH 

8.0, 10 min, room temperature (Sigma)) to reduce unspecific binding of the probe. Two μl paint 

probe was applied to dehydrated sections under cover slips, and probe and tissue were denatured at 

60 °C for 10 min followed by overnight hybridization at 37 °C. Slides were washed in 50% 

formamide/1xSSC (15 min, 37°C) and in 2xSSC (15 min, 37°C), before incubation in DAPI 

solution and mounting in Slowfade Light Antifade. 

Supplementary References 

(1) Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N. Generation of mice carrying a 

mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc Natl 

Acad Sci U S A. 1992 May 15;89:4471-4475. 

(2) Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. 'Green mice' as a source of 

ubiquitous green cells. FEBS Lett. 1997 May 5;407:313-319. 

(3) Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, 

Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the 

pathogenesis of atherosclerosis. Nat Med. 2002;8:403-409. 



Supplementary Table I. Plasma lipids in bone marrow transplanted mice at sacrifice 

Total cholesterol HDL cholesterol Triglycerides 

mmol/L 

mmol/L 

mmol/L 

20 weeks of age 

(n=11) * 

20.3±6.6 0.43±0.26 0.81±0.26 

32 weeks of age 

(n=12) † 

*On high fat diet. † On chow diet. 

11.7±1.6 0.31±0.18 0.79±0.31 



Supplementary Figure I 

Supplementary Figure I. Not a single plaque SMC derived from donor bone marrow cells 

administered at bone marrow transplantation was found. Two high power fields of an aortic root 

plaque from an eGFP + apoE -/- BM → apoE -/- transplanted mouse (32 weeks of age) are shown. All 

SMαA + cells are eGFP + . Color channels are shown separately [SMαA in (a,d), eGFP in (b,e) and 

combined (c,f)] to facilitate interpretation. L. Lumen. C. Fibrous cap. F. Foam cells. M. Tunica 

media. Red, SMαA. Green, eGFP. Greyscale, differential interference contrast. Scale bar 50 μm. 



Supplementary Figure II 

Supplementary Figure II. Collar-induced atherosclerosis in transplanted common carotid artery 

(CCA) segments is pathoanatomically reminiscent of spontaneously developed atherosclerosis in 

apoE -/- mice. In this figure the occurrence of foam cells is shown in an eGFP + apoE -/- CCA graft 

anastomosed into an apoE -/- recipient mouse. As expected, foam cells in the plaque are derived from 

the circulation (eGFP - ) and positive for the macrophage marker Mac2. Red, Mac2. Green, eGFP. L, 

lumen, C. fibrous cap. F, foam cells. Scale bar 50 μm.

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