This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 26/12/2696    most recent 01.ATV.0000247243.48542.9dv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bentzon, J. F. Articles by Falk, E. Search for Related Content PubMed PubMed Citation Articles by Bentzon, J. F. Articles by Falk, E. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH Medline Plus Health Information Stem Cells Related Collections Mechanism of atherosclerosis/growth factors Animal models of human disease Pathophysiology Genetically altered mice Smooth muscle proliferation and differentiation Related Article

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2696.)
© 2006 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

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

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{at}ki.au.dk

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
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.

Recent studies have concluded that SMCs in atherosclerosis can originate from circulating progenitor cells. In contrast to this hypothesis, we showed by a series of bone marrow and vessel transplantations that SMCs in atherosclerotic plaques are derived entirely from the local vessel wall in apoE knockout mice

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

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
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. 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^+/+ 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

Top Abstract Introduction Methods Results Discussion References

 
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 (n=28) were lethally irradiated and rescued with sex-matched bone marrow from eGFP⁺apoE^–/– mice as previously described (Figure 1a).¹² Age-matched apoE^–/– (n=8) and eGFP⁺apoE^–/– mice (n=8) were included as controls. One mouse died shortly after BM transplantation. Four randomly-chosen BM-transplanted 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.

View larger version (16K): [in this window] [in a new window]   Figure 1. Overview of experimental design and results. a, Analysis of sex-mismatched eGFP+apoE–/– BM 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.

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 apoE^–/– transplanted mice, n=12, and apoE^–/– CCA eGFP⁺apoE^–/– transplanted mice, n=12, 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

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 (n=10 from each group) (Figure 1b).¹⁴ Four mice (eGFP⁺apoE^–/– CCA apoE^–/– transplanted mice, n=2, and apoE^–/– CCA eGFP⁺apoE^–/– transplanted mice, n=2) 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 development¹⁵ 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⁺ cell-containing 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 µm 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 technique, 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 µm [minor axis] x 5 µm [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: P<3.0/number of observations). Sections were taken at least 50 µm apart to ensure that the same SMC was not analyzed twice.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Sex-Mismatched BM Transplantation
The degree of hematopoietic chimerism obtained in eGFP⁺apoE^–/– BM 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 93.4±2.8% (mean±SD, n=23) and 93.7±1.4% (n=12), respectively, which was similar to that of eGFP⁺apoE^–/– positive control mice (92.4±2.0% [n=4] and 92.2±0.8% [n=4], 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.

View larger version (36K): [in this window] [in a new window]   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 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 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 bar=100 µm.

Vascular Pathology in BM Chimeras
In animals euthanized 4 weeks after BM transplantation (n=4), only foam cell lesions were present at the arterial sites selected for analysis, excluding the possibility that SMCs had 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 (n=4, 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

View larger version (85K): [in this window] [in a new window]   Figure 3. Hematopoietic stem cells do not contribute to plaque SMCs. a, Overview of aortic root plaque from eGFP+apoE–/– BM apoE–/– transplanted mouse (32 weeks of age) exhibiting donor-derived 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 bar=200 µm. b, Higher magnification of the area demarcated in a. Fluorescence microscopy combined with DIC imaging (gray scale) to reveal tissue structure. Scale bar=50 µm. 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 bar=25 µm.

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).

View larger version (53K): [in this window] [in a new window]   Figure 4. Sequential immunostaining and FISH technique revealed no male SMA+ cells in plaques in male eGFP+apoE–/– BM 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 bar=50 µm. 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 bar=25 µm. F indicates foam cells; C, Fibrous cap; L, lumen.

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 (n=2+2, 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).

View larger version (89K): [in this window] [in a new window]   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 end-to-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 bar=1 mm.

Of 20 mice in which we performed CCA segment transplantations and collar placements, complete or near-complete 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 (n=4), not a single eGFP⁺SMA⁺ cell 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 (n=5), 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.

View larger version (76K): [in this window] [in a new window]   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 bar=25 µm. 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 bar=25 µm.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 collar-induced 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 BM-derived 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 sex-mismatched 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. 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.

Disclosures

None.

	Footnotes

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

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Mann J, Davies MJ. Mechanisms of progression in native coronary artery disease: role of healed plaque disruption. Heart. 1999; 82: 265–268.[Abstract/Free Full Text]
2. Schwartz SM, Virmani R, Rosenfeld ME. The good smooth muscle cells in atherosclerosis. Curr Atheroscler Rep. 2000; 2: 422–429.[Medline] [Order article via Infotrieve]
3. Chamley-Campbell J, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev. 1979; 59: 1–61.[Free Full Text]
4. Stemerman MB, Ross R. Experimental arteriosclerosis. I. Fibrous plaque formation in primates, an electron microscope study. J Exp Med. 1972; 136: 769–789.[Abstract]
5. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest. 1983; 49: 327–333.[Medline] [Order article via Infotrieve]
6. Feil S, Hofmann F, Feil R. SM22alpha modulates vascular smooth muscle cell phenotype during atherogenesis. Circ Res. 2004; 94: 863–865.[Abstract/Free Full Text]
7. 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.[CrossRef][Medline] [Order article via Infotrieve]
8. Xu Q. The impact of progenitor cells in atherosclerosis. Nat Clin Pract Cardiovasc Med. 2006; 3: 94–101.[CrossRef][Medline] [Order article via Infotrieve]
9. Sata M. Role of circulating vascular progenitors in angiogenesis, vascular healing, and pulmonary hypertension: lessons from animal models. Arterioscler Thromb Vasc Biol. 2006; 26: 1008–1014.[Abstract/Free Full Text]
10. Jockusch H, Voigt S, Eberhard D. Localization of GFP in frozen sections from unfixed mouse tissues: immobilization of a highly soluble marker protein by formaldehyde vapor. J Histochem Cytochem. 2003; 51: 401–404.[Abstract/Free Full Text]
11. Linton MF, Atkinson JB, Fazio S. Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation. Science. 1995; 267: 1034–1037.[Abstract/Free Full Text]
12. Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, Dietrich H, Xu Q. Smooth muscle cells in transplant atherosclerotic lesions are originated from recipients, but not bone marrow progenitor cells. Circulation. 2002; 106: 1834–1839.[Abstract/Free Full Text]
13. Tian C, Bagley J, Kaye J, Iacomini J. Induction of T cell tolerance to a protein expressed in the cytoplasm through retroviral-mediated gene transfer. J Gene Med. 2003; 5: 359–365.[CrossRef][Medline] [Order article via Infotrieve]
14. der Thusen JH, van Berkel TJ, Biessen EA. Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Circulation. 2001; 103: 1164–1170.[Abstract/Free Full Text]
15. Hungerford JE, Owens GK, Argraves WS, Little CD. Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers. Dev Biol. 1996; 178: 375–392.[CrossRef][Medline] [Order article via Infotrieve]
16. Caplice NM, Bunch TJ, Stalboerger PG, Wang S, Simper D, Miller DV, Russell SJ, Litzow MR, Edwards WD. Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation. Proc Natl Acad Sci U S A. 2003; 100: 4754–4759.[Abstract/Free Full Text]
17. Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity. 1994; 1: 661–673.[CrossRef][Medline] [Order article via Infotrieve]
18. Cheng C, van HR, de WM, van Damme LC, Tempel D, Hanemaaijer L, van Cappellen GW, Bos J, Slager CJ, Duncker DJ, van der Steen AF, de CR, Krams R. Shear stress affects the intracellular distribution of eNOS: direct demonstration by a novel in vivo technique. Blood. 2005; 106: 3691–3698.[Abstract/Free Full Text]
19. Tanaka K, Sata M, Hirata Y, Nagai R. Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res. 2003; 93: 783–790.[Abstract/Free Full Text]
20. Zernecke A, Schober A, Bot I, von Hundelshausen P, Liehn EA, Mopps B, Mericskay M, Gierschik P, Biessen EA, Weber C. SDF-1{alpha}/CXCR4 Axis Is Instrumental in Murine Neointimal Hyperplasia and Recruitment of Smooth Muscle Progenitor Cells. Circ Res. 2005; 96: 784–791.[Abstract/Free Full Text]
21. Schafer K, Schroeter MR, Dellas C, Puls M, Nitsche M, Weiss E, Hasenfuss G, Konstantinides SV. Plasminogen activator inhibitor-1 from bone marrow-derived cells suppresses neointimal formation after vascular injury in mice. Arterioscler Thromb Vasc Biol. 2006; 26: 1254–1259.[CrossRef][Medline] [Order article via Infotrieve]
22. Shimizu K, Sugiyama S, Aikawa M, Fukumoto Y, Rabkin E, Libby P, Mitchell RN. Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy. Nat Med. 2001; 7: 738–741.[CrossRef][Medline] [Order article via Infotrieve]
23. Hu Y, Mayr M, Metzler B, Erdel M, Davison F, Xu Q. Both donor and recipient origins of smooth muscle cells in vein graft atherosclerotic lesions. Circ Res. 2002; 91: e13–e20.[Abstract/Free Full Text]
24. Massberg S, Konrad I, Schurzinger K, Lorenz M, Schneider S, Zohlnhoefer D, Hoppe K, Schiemann M, Kennerknecht E, Sauer S, Schulz C, Kerstan S, Rudelius M, Seidl S, Sorge F, Langer H, Peluso M, Goyal P, Vestweber D, Emambokus NR, Busch DH, Frampton J, Gawaz M. Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo. J Exp Med. 2006; 203: 1221–1233.[Abstract/Free Full Text]
25. Hillebrands JL, Klatter FA, Rozing J. Origin of vascular smooth muscle cells and the role of circulating stem cells in transplant arteriosclerosis. Arterioscler Thromb Vasc Biol. 2003; 23: 380–387.[Abstract/Free Full Text]
26. Deng DX, Spin JM, Tsalenko A, Vailaya A, Ben-Dor A, Yakhini Z, Tsao P, Bruhn L, Quertermous T. Molecular signatures determining coronary artery and saphenous vein smooth muscle cell phenotypes: distinct responses to stimuli. Arterioscler Thromb Vasc Biol. 2006; 26: 1058–1065.[Abstract/Free Full Text]
27. Hu Y, Zhang Z, Torsney E, Afzal AR, Davison F, Metzler B, Xu Q. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest. 2004; 113: 1258–1265.[CrossRef][Medline] [Order article via Infotrieve]
28. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004; 84: 767–801.[Abstract/Free Full Text]

Related Article:

Origin of Neointimal Smooth Muscle: We’ve Come Full Circle

Mark H. Hoofnagle, James A. Thomas, Brian R. Wamhoff, and Gary K. Owens
Arterioscler. Thromb. Vasc. Biol. 2006 26: 2579-2581. [Full Text] [PDF]

This article has been cited by other articles:

				H. Iwata and M. Sata Origin of Cells That Contribute to Neointima Growth Circulation, June 17, 2008; 117(24): 3060 - 3061. [Full Text] [PDF]

				Q. Xu Stem Cells and Transplant Arteriosclerosis Circ. Res., May 9, 2008; 102(9): 1011 - 1024. [Abstract] [Full Text] [PDF]

				J. F. Bentzon, E. Falk, C. S. Sondergaard, and M. Kassem Response to Letter Regarding Article, "Smooth Muscle Cells Healing Atherosclerotic Plaque Disruptions Are of Local, Not Blood, Origin in Apolipoprotein E Knockout Mice" Circulation, April 22, 2008; 117(16): e318 - e318. [Full Text] [PDF]

				S. J. House and H. A. Singer CaMKII-{delta} Isoform Regulation of Neointima Formation After Vascular Injury Arterioscler. Thromb. Vasc. Biol., March 1, 2008; 28(3): 441 - 447. [Abstract] [Full Text] [PDF]

				Y. Diao, S. Guthrie, S.-L. Xia, X. Ouyang, L. Zhang, J. Xue, P. Lee, M. Grant, E. Scott, and M. S. Segal Long-Term Engraftment of Bone Marrow-Derived Cells in the Intimal Hyperplasia Lesion of Autologous Vein Grafts Am. J. Pathol., March 1, 2008; 172(3): 839 - 848. [Abstract] [Full Text] [PDF]

				Y. S. Chatzizisis, M. Jonas, A. U. Coskun, R. Beigel, B. V. Stone, C. Maynard, R. G. Gerrity, W. Daley, C. Rogers, E. R. Edelman, et al. Prediction of the Localization of High-Risk Coronary Atherosclerotic Plaques on the Basis of Low Endothelial Shear Stress: An Intravascular Ultrasound and Histopathology Natural History Study Circulation, February 26, 2008; 117(8): 993 - 1002. [Abstract] [Full Text] [PDF]

				K. Tanaka, M. Sata, T. Natori, J.-r. Kim-Kaneyama, K. Nose, M. Shibanuma, Y. Hirata, and R. Nagai Circulating progenitor cells contribute to neointimal formation in nonirradiated chimeric mice FASEB J, February 1, 2008; 22(2): 428 - 436. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 26/12/2696    most recent 01.ATV.0000247243.48542.9dv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bentzon, J. F. Articles by Falk, E. Search for Related Content PubMed PubMed Citation Articles by Bentzon, J. F. Articles by Falk, E. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH Medline Plus Health Information Stem Cells Related Collections Mechanism of atherosclerosis/growth factors Animal models of human disease Pathophysiology Genetically altered mice Smooth muscle proliferation and differentiation Related Article