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(Circulation. 2007;116:2053-2061.)
© 2007 American Heart Association, Inc.

Molecular Cardiology

Smooth Muscle Cells Healing Atherosclerotic Plaque Disruptions Are of Local, Not Blood, Origin in Apolipoprotein E Knockout Mice

From the Department of Cardiology, Aarhus University Hospital (Skejby), and Institute of Clinical Medicine, University of Aarhus (J.F.B., E.F.), Aarhus; Department of Molecular Biology, University of Aarhus, Aarhus (C.S.S.); and Department of Endocrinology, Odense University Hospital, Odense (M.K.), Denmark.

Correspondence to Jacob Fog Bentzon, MD, PhD, Department of Cardiology, Research Unit, Aarhus University Hospital, Brendstrupgaardsvej, 8200 Aarhus N, Denmark. E-mail jben{at}ki.au.dk

Received June 18, 2007; accepted August 28, 2007.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Signs of preceding episodes of plaque rupture and smooth muscle cell (SMC)–mediated healing are common in atherosclerotic plaques, but the source of the healing SMCs is unknown. Recent studies suggest that activated platelets adhering to sites of injury recruit neointimal SMCs from circulating bone marrow–derived progenitor cells. Here, we analyzed the contribution of this mechanism to plaque healing after spontaneous and mechanical plaque disruption in apolipoprotein E knockout (apoE^–/–) mice.

Methods and Results— To determine the origin of SMCs after spontaneous plaque disruption, irradiated 18-month-old apoE^–/– mice were reconstituted with bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic apoE^–/– mice and examined when they died up to 9 months later. Plaque hemorrhage, indicating previous plaque disruption, was widely present, but no bone marrow–derived eGFP⁺ SMCs were detected. To examine the origin of healing SMCs in a model that recapitulates more features of human plaque rupture and healing, we developed a mechanical technique that produced consistent plaque disruption, superimposed thrombosis, and SMC-mediated plaque healing in apoE^–/– mice. Mechanical plaque disruption was produced in irradiated apoE^–/– mice reconstituted with eGFP⁺apoE^–/– bone marrow cells and in carotid bifurcations cross-grafted between apoE^–/– and eGFP⁺apoE^–/– mice. Apart from few non–graft-derived SMCs near the anastomosis site in 1 transplanted carotid bifurcation, no SMCs originating from outside the local arterial segment were detected in healed plaques.

Conclusions— Healing SMCs after atherosclerotic plaque disruption are derived entirely from the local arterial wall and not circulating progenitor cells in apoE^–/– mice.

Key Words: atherosclerosis • blood cells • muscle, smooth • thrombosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Plaque rupture with superimposed thrombosis is the major cause of heart attack and death worldwide, but the great majority of plaque ruptures are not lethal, and most are clinically silent.¹ In nonlethal cases, plaques are healed by smooth muscle cells (SMCs) that accumulate at the rupture site, where they secrete extracellular matrix rich in glycosaminoglycans and type III collagen.^2,3 This process restores the integrity of the plaque surface but may cause rapid plaque progression and constrictive remodeling of the arterial wall.^3–5 Serial angiography has shown that many coronary artery stenoses develop in a phasic rather than a linear manner,⁶ and the frequent finding of healed rupture sites in plaques causing stenosis indicates that SMC-mediated plaque healing may be the underlying mechanism.^3,5

Clinical Perspective p 2061

Despite their clinical relevance, the mechanisms governing plaque healing, including the source of the healing SMCs, have been little explored. A longstanding but sparsely documented theory has considered healing to be carried out by local plaque SMCs that are activated by growth factors (eg, platelet-derived growth factor-BB and transforming growth factor-β) and mitogens (eg, thrombin) released or generated during superimposed thrombosis.⁷ Curiously, however, the proliferative response of local plaque SMCs in ruptured human atherosclerotic plaques has been found to be modest.^5,8 Moreover, SMCs in advanced plaques show numerous features of cellular senescence both in culture and in vivo.⁹

Observations of Zernecke et al¹⁰ recently suggested an explanation for this paradox. They found that activated platelets adhering to sites of arterial injury facilitated the recruitment of neointimal SMCs from circulating bone marrow (BM)–derived progenitor cells via the expression of P-selectin and stromal cell–derived factor 1. The importance of activated platelets for homing of progenitor cells also has been supported by other experimental studies,^11–13 and the idea is consistent with signs of BM-derived SMCs in human coronary atherosclerosis, in which subclinical plaque ruptures occur.¹⁴

Recently, we and others have shown that plaque SMCs in uncomplicated plaques in young apolipoprotein E knockout (apoE^–/–) mice are recruited entirely from the local arterial wall and that the majority, if not all, descend from SMCs in the arterial media.^15–17 In the present study, we tested the hypothesis that plaque disruption with superimposed thrombosis is the critical event that facilitates smooth muscle progenitor cell recruitment to atherosclerotic plaques. First, we studied whether BM-derived SMCs are present in plaques with spontaneous plaque hemorrhage (indicating recent plaque disruption) in old apoE^–/– mice. Second, we developed a mechanical model of plaque disruption, superimposed thrombosis, and SMC-mediated healing in apoE^–/– mice and determined the origin of SMCs in healed plaques.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
For an expanded Methods section, see the online-only Data Supplement.

Animals
All procedures were approved by the Danish Animal Experiments Inspectorate. ApoE^–/– mice (B6.129P2-Apoe^tm1Unc, Taconic M&B, Ry, Denmark), backcrossed >10 times to C57BL/6 mice, and enhanced green fluorescent protein (eGFP) transgenic C57BL/6 mice [C57BL/6-Tg(ACTB-EGFP)10sb/J, Jackson Laboratories, Bar Harbor, Maine] were crossed to obtain eGFP⁺apoE^–/– mice (hemizygous for the eGFP transgene). All mice were fed normal chow.

BM Transplantation
ApoE^–/– mice (male; age, 18 months; n=34) were lethally irradiated and rescued by tail vein injection of 10⁷ unfractionated BM cells from eGFP⁺apoE^–/– mice (male; age, 2 to 3 months; n=10).^15,18 Hematopoietic chimerism was assessed after 4 weeks by flow cytometry of a peripheral blood sample as previously described and found to be >90% in all cases (see online-only Data Supplement Figure I).¹⁵ Nine mice died before flow cytometry was performed.

Long-Term Hematopoietic Chimeras
A subgroup of the eGFP⁺apoE^–/– BMapoE^–/– transplanted mice (n=10) and age-matched apoE^–/– mice (n=5) were housed until spontaneous death (Figure 1a). Within 24 hours after death, the animals were fixed in immersion in 4% phosphate-buffered formaldehyde for 48 to 72 hours. Cross sections of the carotid bifurcations and longitudinal sections of the aortic arch, including the brachiocephalic trunk and the proximal parts of the left common carotid and subclavian arteries, were prepared for histology (see the detailed description online). Sections were taken at least 50 µm apart.

View larger version (20K): [in this window] [in a new window]   Figure 1. Overview of experiments. a, Long-term hematopoietic chimeras. BM transplants were performed in 18-month-old mice, and mice were housed until spontaneous death. Times of death are indicated by crosses. b, Mechanical disruption of plaques in the CB was produced in BM-transplanted mice (top) and after CB transplantation between apoE–/– and eGFP+apoE–/– mice (bottom). Age of donor and recipient mice at the time of BM or CB transplantation is indicated in parentheses. BMT indicates BM transplantation; PD, mechanical plaque disruption; and CBT, CB transplantation.

Mechanical Plaque Disruption
The other eGFP⁺apoE^–/– BMapoE^–/– transplanted mice (n=15) as well as age-matched apoE^–/– (n=4) and eGFP⁺apoE^–/– (n=2) control mice were subjected to mechanical plaque disruption of carotid bifurcation (CB) plaques (Figure 1b, top). Briefly, mice were anesthetized, and the carotid bifurcation plaque most suitable for the procedure was identified and isolated temporarily between loose 6-0 silk ligatures. Then, a 9-0 spatula needle was inserted into the common carotid artery 0.5 mm proximal to the targeted plaque, advanced distally through the lumen, and inserted repeatedly into the plaque from the luminal side to produce plaque disruption (see the detailed description and the online-only Data Supplement Movie). Immediately after the procedure, mice were randomly allocated to 1 of 3 groups: killed at 30 minutes, 1 week, or 4 weeks after plaque disruption. The control mice were all killed after 4 weeks. Serial sections taken at 100-µm intervals throughout the CB plaques were prepared for histology.

CB Transplantation
CBs containing advanced atherosclerotic plaques were transplanted from old apoE^–/– mice into young eGFP⁺apoE^–/– mice (n=12) and from old eGFP⁺apoE^–/– mice into young apoE^–/– mice (n=4) (Figure 1b, bottom). A subset of these transplantations were sex mismatched (female apoE^–/– CBmale eGFP⁺apoE^–/– mice, n=4). The others were sex matched (both male and female).

The microsurgical technique was essentially as previously described (see the detailed description online).¹⁵ The proximal anastomosis was prepared between recipient and donor common carotid arteries. The distal anastomosis was made between the recipient common carotid artery and the donor internal or external carotid artery, depending on plaque localization. The carotid branch not used for anastomosis was ligated. Mechanical plaque disruption of transplanted plaques was performed immediately after reestablishment of blood flow. Mice were killed after 30 minutes (n=2) or 4 weeks (n=14). Serial sections were collected at 100-µm steps throughout the disrupted CB plaques.

Immunohistochemistry and Fluorescence In Situ Hybridization
Antibodies against smooth muscle -actin (SMA) and Mac2 were used to identify SMCs and macrophages, respectively. SMA is considered the most sensitive, although not specific, marker for plaque SMCs.¹⁶ Validity was confirmed by observing the expected intracellular distribution of SMA (subplasmalemmal) and Mac2 (cytoplasmic) and by lack of staining when the specific antibody was replaced by an irrelevant antibody of the same isotype and concentration.

Perls’ Prussian blue (iron) staining was performed to detect plaque hemorrhage in sections from the old hematopoietic chimeric mice and nontransplanted controls. When indicated, anti-eGFP antibody was used to detect eGFP and Sudan Black B to stain and quench the autofluorescence of lipofuscin in these mice.¹⁹ Sequential SMA immunostaining and fluorescence in situ hybridization for the Y chromosome was performed on CB plaque sections from sex-mismatched CB-transplanted mice and sex-matched controls as previously described.¹⁵

Fluorescence Microscopy
Sections were examined in an Olympus (Ballerup, Denmark) Cell-R fluorescence microscope equipped with differential interference contrast (DIC) optics and motorized focus. SMA⁺ cell profiles in the range of small SMC nuclear profiles or larger (from 3 µm [minor axis] x5 µm [major axis]) were analyzed for colocalization of eGFP and SMA by direct inspection at x400 magnification. Deconvolution of wide-field z-axis image stacks (0.2-µm optical section thickness) was performed on all plaque areas suspicious of previous needle injury using a blind 3-dimensional deconvolution algorithm (Autoquant Deblur 9.3, Media Cybernetics, Bethesda, Md). Nucleated SMC profiles, defined as cells in which the nucleus was circumscribed by SMA⁺ staining, were analyzed for the presence of the Y chromosome.

Statistical Analysis
The log-rank test was used for survival analysis after BM transplantation. One-tailed 95% confidence Clopper-Pearson limits (CLs) were calculated for the occurrence of disruption and thrombosis after needle injury. CLs for the number of double-positive eGFP⁺SMA⁺ cells were calculated by the approximation described by Hanley et al²⁰ (95% CL, <3.0/number of observations).

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Widespread Plaque Hemorrhage in Old ApoE^–/– Mice
Plaque hemorrhage, assumed to be a sign of recent spontaneous plaque disruption, has been reported in the brachiocephalic trunk of apoE^–/– mice from 30 weeks of age and in the aortic root and descending aorta from 34 weeks of age.^21,22

To investigate the potential recruitment of BM-derived SMCs to hemorrhagic plaques, we studied a cohort of old eGFP⁺apoE^–/– BMapoE^–/– transplanted mice (n=10) at the time of spontaneous death (Figure 1a). Median survival time after BM transplantation (at 18 months of age) was 6 months compared with 11 months in nonirradiated age-matched apoE^–/– controls (n=5) (P=0.02, log-rank test), indicating late detrimental effects of irradiation. Times of death in BM-transplanted mice are indicated in Figure 1a. Causes of death were not determined.

Plaques containing extravasated erythrocytes were present at the postmortem examination in 7 of 10 BM-transplanted mice (1 to 2 plaques per mouse) and in 2 of 5 age-matched nonirradiated apoE^–/– controls (1 plaque per mouse) (Figure 2). Intracellular iron-containing hemosiderin pigment, a sign of erythrophagocytosis and thus preceding plaque hemorrhage,²³ was identified by Perls’ Prussian blue staining in plaques from 8 of 10 BM-transplanted mice (1 to 3 plaques per animal) and in 3 of 5 nonirradiated apoE^–/– controls (1 plaque per animal) (Figure 2). Hemosiderin-positive cells most frequently coincided with intraplaque erythrocytes but were found in other locations as well. Prussian blue staining also was notable in extracellular structures (see online-only Data Supplement Figure II). There appeared to be no predilection site for plaque hemorrhages among the sites examined (lesser aortic arch curvature, brachiocephalic trunk, branch points of left common carotid and subclavian arteries, and CBs). Superimposed thrombosis was not seen.

View larger version (164K): [in this window] [in a new window]   Figure 2. Erythrocytes (E) and hemosiderin pigment (blue) present in a plaque near the branch point of the left subclavian artery in a BM-transplanted mouse. Longitudinal section. Perls’ Prussian blue, Kernechtrot counterstain, and DIC. P indicates plaque; L, lumen; and M, tunica media. Scale bar=50 µm.

SMCs in Hemorrhagic Plaques Are Not of Hematopoietic Origin
Plaque foam cells in the old BM-transplanted mice were double positive for a macrophage marker (Mac2) and the tracking marker eGFP (Figure 3a), confirming the ability of the applied techniques to trace the progeny of hematopoietic stem cells. However, no plaque SMCs, identified by positive staining for SMA, were found to be eGFP⁺ whether detected by the natural fluorescence of eGFP (7650 SMA⁺ profiles analyzed; 95% CL, <0.04%; Figure 3b and 3c) or by an anti-eGFP antibody (4820 SMA⁺ cell profiles analyzed; 95% CL, <0.06%; Figure 3d).

View larger version (48K): [in this window] [in a new window]   Figure 3. a, Foam cells (Fo) in the shoulder region of a fibrofatty plaque in the lesser curvature of the aortic arch. As expected, they are Mac2+ (red) and eGFP+ (green), yielding a yellow overlay in the combined image. Scale bar=50 µm. See also online-only Data Supplement Figure III. b, Plaque from the lesser curvature of the aortic arch. Genetic tracing using eGFP fluorescence (green) reveals no BM-derived SMA+ (red) cells. Scale bar=200 µm. c, Magnification of b. Scale bar=50 µm. d, Tracing of BM-derived cells by immunohistochemical detection of eGFP (green). No eGFP+SMA+ cells are seen. Scale bar 50 µm. e and f, Autofluorescence of intracellular pigment quenched by Sudan Black B (black) in an SMA- and eGFP-immunostained section adjacent to d. e, DIC microscopy; f, fluorescence microscopy. No eGFP+SMA+ cells are unmasked by the quenching of autofluorescence. Scale bars=50 µm. Blue indicates DAPI; gray scale, DIC; M, tunica media; F, fibrous cap; and L, lumen.

Cells exhibiting strong autofluorescence in the red and green color spectrum (yielding yellow in the combined image) often were present in plaques (pronounced examples are shown in Figure 3c and 3d). Sudan black B stained these cells and quenched the autofluorescence, indicating that the fluorescent pigment was lipofuscin (Figure 3e and 3f). Importantly, the autofluorescent cells could not be mistaken for eGFP⁺SMA⁺ cells (false positives) in which the red fluorescent SMA staining is confined to the subplasmalemmal space. Neither did quenching of lipofuscin autofluorescence reveal otherwise masked eGFP⁺SMA⁺ cells (false negatives) whether by tracing of natural eGFP fluorescence (2430 SMA⁺ profiles analyzed; 95% CL, <0.1%) or by immunofluorescent detection of eGFP (4690 SMA⁺ profiles analyzed; 95% CL, <0.06%; Figure 3f).

Mechanical Plaque Disruption
In murine plaques containing hemorrhage, the presence and extent of plaque disruption usually remain concealed, and superimposed thrombosis or clear signs of plaque healing were not observed in the material presented above. To circumvent this limitation of atherosclerosis in apoE^–/– mice, we developed a needle injury technique that allowed us to control the spatial and temporal occurrence of plaque disruption, superimposed thrombosis, and SMC-mediated healing (Figure 4a).

View larger version (77K): [in this window] [in a new window]   Figure 4. a, Schematic of mechanical plaque disruption in a CB plaque (left). Same procedure after transplantation of an apoE–/– CB into an eGFP+apoE–/– mouse (right). b, A transplanted CB 4 weeks after mechanical disruption. DA indicates distal anastomosis; PA, proximal anastomosis. *The site of needle insertion. Scale bar=1 mm. c through e, Plaque (P) disruption with a superimposed platelet-rich thrombus (T) 30 minutes after the procedure. A rupture site (R) in the fibrous cap is visible, allowing access of blood from the lumen (L) into a necrotic core. Scale bars=100 µm.

SMCs Healing Mechanical Disruptions Are Not of Hematopoietic Origin
To characterize the type of plaque injury inflicted by the needle and to examine whether cells of hematopoietic origin contribute to plaque healing, we produced mechanical disruptions in carotid plaques in eGFP⁺apoE^–/– BMapoE^–/– transplanted mice.

Mice were killed at various time points as indicated in Figure 1b. Acute injury to the plaque surface and thrombosis were seen in all of the 5 mice killed after 30 minutes. One example is shown in Figure 4c through 4e; the rest are shown in online-only Data Supplement Figure IV. Because this sample was drawn at random, it can be used to estimate the confidence interval of injured plaques in the BM-transplanted mice that were left to survive until 1 or 4 weeks after mechanical disruption (95% CL, >55%). After 1 week, no residual luminal thrombi were observed. After 4 weeks, distinct wedge-shaped areas of SMC accumulation, indicative of healed disruption sites, were identified in 4 of 6 BM-transplanted mice (Figure 5a through 5c). This pattern was not observed in plaques removed from the contralateral uninjured CBs (4 plaques analyzed) or in age-matched controls (4 mice, 6 plaques analyzed) (data not shown).

View larger version (87K): [in this window] [in a new window]   Figure 5. Healing SMCs after mechanical plaque disruption are derived from local sources. a through c, SMA staining of a CB plaque 4 weeks after mechanical plaque disruption in an eGFP+apoE–/–apoE–/– BM-transplanted mouse. The magnified area shows abnormal plaque architecture indicative of healed injury. No eGFP+SMA+ double-positive cells are seen. d through f, Healed disruption site in an apoE–/– CB plaque transplanted into an eGFP+apoE–/– mouse recipient. A small intraplaque lumen is seen, indicating past injury to the plaque surface. Abundant extracellular eGFP is present in the adventitia (please see note in the online-only Data Supplement). No eGFP+SMA+ cells were identified. g through i, Similar plaque in an eGFP+apoE–/– CBapoE–/– transplanted mouse. SMA+ cells are eGFP+. Wide-field fluorescence (a, d, g) and deconvoluted thin optical sections combined with DIC (b and c, e and f, h and i). Red indicates SMA; green, eGFP; blue, DAPI; L, lumen; M, media; and A, adventitia. Scale bars=50 µm. See individual color channels in online-only Data Supplement Figure VI.

Importantly, not a single eGFP⁺ SMA⁺ cell among 3560 analyzed SMA⁺ cells was identified in serial sections of the healed plaques in BM-transplanted mice (Figure 5a through 5c). By comparison, SMA⁺ cells were almost uniformly eGFP⁺ in healed plaques in positive control eGFP⁺apoE^–/– mice (n=2; 469 of 478 [98%] analyzed SMA⁺ cell profiles; see online-only Data Supplement Figure V).

SMCs in Healed Plaques Are Derived From the Local Artery Segment
To evaluate whether any cell type in the circulating blood contributes to SMCs in healed plaques, we produced plaque disruptions in CBs that were transplanted from apoE^–/– mice into the common carotid artery of eGFP⁺apoE^–/– mice (Figures 1b, 4a, and 4b).

Plaque disruption with superimposed thrombosis was confirmed in both mice (n=2) killed after 30 minutes (data not shown). Four weeks after plaque disruption, focal and distinct accumulations of SMCs indicative of healed plaque disruption sites were recognized in 8 of 10 mice (Figure 5d through 5f). In one of these animals, the presence of multiple lumina indicated that a thrombus had been recanalized. Obliteration of the transplanted CB was found in 1 mouse.

Among 7980 SMA⁺ profiles analyzed in serial sections of the healed plaques from apoE^–/– CBeGFP⁺apoE^–/– transplanted mice, only 4 were eGFP⁺ (non–graft derived). They were all located in the section taken closest to the distal anastomosis in 1 mouse, strongly indicating that these few cells had migrated from the contiguous recipient vasculature.

Consistently, in the reverse experiment in which eGFP⁺apoE^–/– CBs were grafted into apoE^–/– mice (n=4), 2896 of 2982 SMA⁺ cellular profiles in healed plaques were eGFP⁺ (97%) (Figure 5g through 5i). This fraction was similar to that found in eGFP⁺apoE^–/– positive control mice (see above).

As an independent tracking marker for blood-derived SMA⁺ cells, we stained Y chromosomes in sections from the subset of mice in which CB transplantations were performed sex mismatched (female to male) and from sex-matched positive controls (male to male). No Y chromosomes were detected among 432 analyzed SMA⁺ cell nuclei in healed female apoE^–/– CB plaques transplanted into male eGFP⁺apoE^–/– mice (n=4) (Figure 6). In comparison, the Y chromosome was detectable in 45 of 98 nucleated SMA⁺ cell profiles in male apoE^–/– CB plaques transplanted into male eGFP⁺apoE^–/– mice.

View larger version (35K): [in this window] [in a new window]   Figure 6. a, CB plaque 4 weeks after mechanical plaque disruption in a female apoE–/– CBmale eGFP+apoE–/– transplanted mouse. Multiple lumina are present, indicating recanalization of a luminal thrombus. Scale bar=200 µm. b, Larger magnification of a. Gray scale indicates DIC. Scale bar=50 µm. c, Detection of Y chromosomes in the SMA-stained section using a FITC-conjugated paint probe (green). Maximum projection of deconvoluted image stack. eGFP fluorescence is totally extinguished by the fluorescence in situ hybridization procedure, and the deteriorated SMA+ staining (red channel) is not shown. The phenotype of cells with Y+ nuclei (marked by arrows) can be identified in the stored image of the SMA immunostaining (b). No Y+SMA+ cells are seen. Red indicates SMA; green, eGFP; blue, DAPI; L, lumen; M, tunica media; P, plaque; and A, adventitia.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The healing process at plaque rupture sites has been described in humans through the study of autopsy or atherectomy specimens.^2,3,5 First, the superimposed thrombus is partially lysed by the fibrinolytic system. Then, synthetic-type SMCs accumulate at the rupture site and secrete a fibrous extracellular matrix that seals the plaque defect and organizes the residual thrombus. Finally, the plaque is re-endothelialized. Possibly, scar-like contraction of healing SMCs pulling on fibrin fibrils lead to concomitant constrictive remodeling of the arterial segment.^4,24

Research into the cellular and molecular mechanisms that control this sequence of events, however, has been limited, at least partly by the lack of a feasible and widely accepted animal model. In the present study, we describe a novel mechanical model of murine plaque disruption, superimposed thrombosis, and plaque healing and show that healing SMCs are of local arterial wall origin and not from circulating progenitor cells.

Models of Plaque Disruption in ApoE^–/– Mice
Owing to the detailed descriptions of several groups, it is well established that spontaneous plaque hemorrhages occur in aging apoE^–/– mice.^21,22,25 Because the described hemorrhages, at least in some anatomic locations, are found in plaques without intraplaque neovessels, the source must be luminal blood entering the plaque through a defect in the plaque surface (ie, by definition, spontaneous plaque disruption).^26–29

The value of these discrete disruptions for studies of plaque healing in general and of platelet-mediated progenitor cell recruitment in particular, however, is open to questions.^28,29 First, superimposed thrombi are rarely found on spontaneously disrupted plaques in apoE^–/– mice,³⁰ and none were detected in the present study. It is possible, however, that transient thrombi form over spontaneously disrupted plaques in apoE^–/– mice but that they escape detection because of rapid lysis. This is consistent with our observation that thrombi over mechanically disrupted plaques persisted for <1 week. Second, no definitive signs of spontaneous SMC-mediated plaque healing have been described. The presence of layers of fibrous tissue within some murine plaques (buried fibrous caps) has been suggested to reflect healed disruptions, but this remains to be documented.^28,29

To circumvent these unanswered questions, we based most of our analysis on a novel model in which plaque disruption was produced by passing a microsurgical needle repeatedly into advanced carotid plaques from the luminal side. This technique is related to but appears to be more efficient and to produce more specific plaque injury than the model reported previously by Reddick et al³¹ in which atherosclerotic segments of apoE^–/– mouse abdominal aorta were squeezed between forceps. Our needle injury produced plaque disruption and superimposed thrombi in most, if not all, treated mice (95% CL, >55%). Like superimposed thrombi in humans, the thrombi were platelet rich, demonstrating that they had formed under rapid blood flow. Moreover, plaque disruption was followed after 4 weeks by the appearance of focal and distinct accumulations of SMCs that resembled healed rupture sites in humans.³

Healing SMCs Are of Local, Not Blood, Origin
The longstanding paradigm in the field has considered plaque healing to be mediated by local proliferating plaque SMCs.⁷ Recent studies in arterial injury models, however, have suggested an alternative mechanism by which the superimposed thrombotic response on ruptured plaques may mediate recruitment of healing SMCs from a circulating pool of BM-derived smooth muscle progenitor cells.^{10–13,32–34} Using both spontaneous and mechanical plaque disruptions in apoE^–/– mice as model systems, we addressed this novel hypothesis, but we found that the healing SMCs were derived entirely from the local vessel wall with no contribution from circulating progenitor cells.

First, not a single BM-derived SMC was detected in BM-transplanted apoE^–/– mice whether in plaques with spontaneous plaque hemorrhage (median follow-up, 6 months) (Figure 3) or in healed plaques 4 weeks after mechanical plaque disruption (Figure 5a through 5c). Because reconstitution of the irradiated mice with eGFP⁺ hematopoietic stem cells in both of these experiments was confirmed by flow cytometry and by the finding of eGFP⁺ macrophages in plaques (online-only Data Supplement Figure I, Figure 3a, and data not shown), we conclude that hematopoietic stem cells do not contribute to healing SMCs in apoE^–/– mice.

Second, to investigate whether healing SMCs could originate from circulating cells of nonhematopoietic origin (eg, from mesenchymal stem cells in the BM or from non-BM sources), we analyzed mechanically disrupted plaques in apoE^–/– CBs that were surgically inserted into the bloodstream of eGFP⁺apoE^–/– mice. In these mice, all circulating progenitor cells were of eGFP⁺ genotype regardless of their possible site of production. Furthermore, circulating progenitor cells were present at physiological levels, which may not be the case after BM transplantation. These experiments showed that no circulating cell type contributes to healing SMCs 4 weeks after mechanical plaque disruption (Figure 5d through 5f). Although 4 non–graft-derived SMCs were detected in 1 transplanted mouse, this small cluster of cells was found in the outermost serial section, strongly indicating that they had migrated from the contiguous recipient artery.

Finally, the use of the Y chromosome as an alternative tracking marker excluded the theoretical possibility that the few percent of plaque SMCs that were not detectably eGFP⁺ in healed plaques of eGFP⁺apoE^–/– CBapoE^–/– transplanted mice (Figure 5g through 5i) or in eGFP⁺apoE^–/– positive controls (online-only Data Supplement Figure V) were derived from a special subset of circulating progenitor cells with low eGFP expression.

Candidate Sources of Healing SMCs
Our findings support the classic theory that plaque healing is mediated by proliferation and matrix secretion of local plaque or medial SMCs. The experiments, however, were not designed to distinguish between sources of SMCs within the arterial wall; thus, our data do not exclude the possibility that some SMCs may differentiate from local stem cells. Hu et al³⁵ have described a population of Sca-1⁺ cells in the arterial adventitia that have the potential to differentiate to SMCs in vitro and to contribute to vein graft neointimal SMCs in vivo. In their experiments, however, Sca-1⁺ cells gave rise only to significant numbers of neointimal SMCs when proliferation and migration of local vein SMCs were blocked by irradiation of the vein graft. When atherosclerotic plaque disruptions heal, large populations of SMCs, some of which are proliferating,^5,8,31 are present in the plaque and adjacent media; hence, the contribution of adventitial stem cells in this situation would be expected to be insignificant.

Contrasting Observations
Previous reports of BM-derived neointimal SMCs after guidewire injury to nondiseased arteries in apoE^–/– and wild-type mice are not easy to reconcile with the findings of the present study.^10,33 It is possible, as first suggested by Han and colleagues,³⁴ that the varying degree of local SMC injury in different models may be important. Plaque disruption involves limited local SMC injury, but passing a guidewire through a small artery causes massive damage to medial SMCs in murine models.¹³ This may incapacitate local sources for repair and, in turn, by unknown mechanisms facilitate recruitment of circulating progenitor cells.^13,34 However, because Li and colleagues,³⁶ even after total allogenic eradication of local medial SMCs in their vasculopathy model, did not observe BM-derived neointimal SMCs, this hypothesis cannot by itself explain the variable results obtained in the field.³⁶

As recently pointed out by Hoofnagle and colleagues,¹⁶ it is plausible that methodological problems in immunofluorescence analyses can explain many previous observations of BM-derived SMCs. Most important, an absolute prerequisite for valid analysis, which is very often not fulfilled, is clear single-cell resolution of the microscopic data. Also often overlooked is the fact that myofilaments and hence SMA, which is essentially the sole SMC marker used within this field of research, are located predominantly in the periphery of neointimal SMCs, whereas the perinuclear region is occupied by the organelles involved in protein synthesis (Figures 3, 5, and 6).³⁷ If single neointimal SMCs with this characteristic distribution of SMA cannot be discerned in histological sections, it is highly questionable whether tissue preparation, staining specificity, and microscopic resolution are of sufficiently high quality to confirm cellular colocalization of SMCs with a BM tracking marker. Those interested in the possibility of BM-derived SMCs are encouraged to read the literature while bearing this in mind.

Conclusions
Consistent plaque disruption, superimposed thrombosis, and SMC-mediated healing can be induced by endovascular needle injury to advanced carotid plaques in apoE^–/– mice. Using this model, we found that healing SMCs are entirely derived from the local artery with no contribution from circulating progenitor cells. This supports the longstanding theory that plaque healing is mediated by local proliferating SMCs.

	Acknowledgments

 
We thank Helle Quist, Birgitte Sahl, and Merete Dixen for excellent technical assistance.

Sources of Funding

Funding for this work was provided by the Danish Medical Research Council, the Danish Heart Foundation, Fonden til Lægevidenskabens Fremme, and the University of Aarhus Research Foundation.

Disclosures

Dr Falk is on the Scientific Program Board of the High-Risk Plaque Initiative, BG Medicine, Waltham, Mass, and a consultant to Boston Scientific Corporation, Natick, Mass. The other authors resport no conflicts.

	References

Top Abstract Introduction Methods Results Discussion References

 
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	Footnotes

 
The online-only Data Supplement, which contains an expanded Methods section, a movie, and Figures I through VI, is available at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.10.1161/107.722355.

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