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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2579.)
© 2006 American Heart Association, Inc.

Editorials

Origin of Neointimal Smooth Muscle

We’ve Come Full Circle

Mark H. Hoofnagle; James A. Thomas; Brian R. Wamhoff; Gary K. Owens

From the Department of Molecular Physiology and Biological Physics (M.H.H., J.A.T., G.K.O.) and the Department of Medicine, Cardiovascular Division (B.R.W.), University of Virginia, Charlottesville.

Correspondence to Dr Gary K. Owens, Department of Molecular Physiology and Biological Physics, University of Virginia, MR5 Room 1220, 415 Lane Road, PO Box 801394, Charlottesville, VA 22908. E-mail gko{at}virginia.edu

The origin of smooth muscle cells (SMCs) in atherosclerotic lesions has been a hotly debated topic for more than four decades. Until recently, it was widely accepted that the majority of smooth muscle cells in atherosclerotic lesions originate from the medial layer of the vessel wall, with local SMC proliferation and migration leading to remodeling of the vessel wall and vessel lumen.^1,2 Recent reports in the literature, however, claimed that SMCs in atherosclerotic lesions originate primarily from circulating bone marrow progenitor cells.^3,4 Where these cells originate from is not merely academic, because the cells that give rise to SMCs within atherosclerotic lesions would likely serve as targets for current and future therapeutic interventions. The bone marrow–derived origin suggested by these articles would indicate that attempts to control SMC phenotype in in-stent restenosis, transplant atherosclerosis, or fibrous cap rupture should be targeted toward these cells from the marrow. However, we cautioned in 2004 that if anything, the bone marrow–derived intimal SMC paradigm was "Lost in Transdifferentiation",⁵ and further studies were needed to clearly resolve this critically important issue.

See page 2696

In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Bentzon and colleagues provide excellent evidence that bone marrow–derived cells do not give rise to vascular SMCs in hyperlipidemia-induced atherosclerotic plaques in the apoE knockout mouse.⁶ These investigators studied plaques in apoE^–/– mice that had their bone marrow reconstituted from sex-mismatched apoE^–/–/eGFP⁺ animals, and then performed meticulous high-resolution z-axis sectioning of atherosclerotic lesions induced by Western diet to try to identify transdifferentiated cells staining for both the eGFP/Y chromosome lineage tags and the SMC marker smooth muscle -actin (SMA). Of critical importance, and in contrast to many previous studies in this area where investigators examined relatively small numbers of intimal SMCs using low-resolution immunomicroscopy, Bentzon et al examined nearly 4000 lineage-traced cells in atherosclerotic lesions derived from these apoE^–/– mice and based on extensive high-resolution z-axis sectioning found no colocalization of eGFP or sex-mismatched bone marrow–lineage markers and SMC markers, indicating no transdifferentiation of bone marrow derived cells into intimal SMCs. Moreover, using a sophisticated model of collar-induced lesion formation (that the authors assert more closely mimics human lesions) in transplanted syngeneic carotid arteries, they observed that SMCs within atherosclerotic lesions originated from the transplanted vessel. These results are completely consistent with a series of recent studies that have used high-resolution confocal analyses and shown little or no contribution of bone marrow–derived cells to SMC lineages in a variety of models including systemic hypoxia-induced arteriogenesis,⁷ hindlimb ischemia-induced angiogenesis/collateralization,⁸ regenerative lung growth,⁹ and tumor angiogenesis.^8,10 Indeed, each of these articles showed that anything less than use of high-resolution confocal microscopy led to counting of false-positive transdifferentiation events. Results are also consistent with a more general study of this topic by Wagers et al¹¹ indicating limited transdifferentiation of hematopoietic stem cells in any tissue using a parabiotic mouse model that lacks the confounding variables of irradiation and transplant. Finally, as described in detail our 2004 commentary on this topic,⁵ a fundamental flaw in virtually all previous studies claiming transdifferentiation of bone marrow–derived cells into SMCs was an over-reliance on use of the marker SMA, which we know can be expressed by many lineages other than SMCs.¹² Whereas the absence of SMA as demonstrated by Bentzon et al provides compelling evidence ruling out SMC differentiation, the presence of this marker alone is not sufficient to establish SMC identity. Taken together, results provide what we believe is compelling evidence that so called transdifferentiation of bone marrow–derived cells into SMC lineages either does not occur at all or is a relatively rare event, at least in the models that have been rigorously studied to date.

In hindsight, the results of Bentzon et al should not have been too surprising because of the abundant literature from the 1970s and 1980s on proliferative and migratory responses of SMCs in various models of injury and hyperlipidemia. In particular, interested readers should seek out some of the seminal articles in the field that precisely demonstrated the nature of SMC proliferation and migration from the media in various animal models.^13–19 Of these studies, two classic articles by Wilbur Thomas and colleagues in particular are difficult to reconcile with the bone marrow derivation of SMCs hypothesis in hyperlipidemic models of vascular disease. The first, an EM ultra-structural study of mitotic SMCs in atherosclerotic lesions, indicated mature SMCs dividing in the media were the source of proliferative SMCs.¹⁹ The second, a thymidine-lineage tracing study, demonstrated that medial SMC investing atherosclerotic lesions have a polyclonal origin.¹⁷ These findings seem to be inconsistent with a small or clonal stem-cell population dividing and differentiating within a lesion to form SMCs, and more congruous with proliferation and expansion of phenotypically modulated SMCs in the media of the vessel wall.

The controversy over the origin of SMCs within atherosclerotic lesions demonstrates the critical importance of using high-resolution confocal or deconvolution microscopy for any lineage tracing study, as well as rigorous statistical analysis of large numbers of individual cells in evaluating complex cell populations. It is somewhat regrettable, in hindsight, that progress in determining the origin of SMCs within atherosclerotic lesions was hindered by reports demonstrating the bone marrow origin of SMC without appropriate confocal microscopic analysis and/or study design of sufficient power. Indeed, the results of Bentzon and colleagues suggest a potential fundamental methodological flaw of previous studies demonstrating transdifferentiation of circulating stem cells into SMCs (Figure).

View larger version (30K): [in this window] [in a new window]   Potential for false colocalization without confocal/deconvolution microscopy. In the upper panel the importance of higher 3-dimensional resolution for identifying colocalization of signal in tissue sections is demonstrated. The higher resolution of confocal/deconvolution microscopy allows for thinner optical sections to be imaged, reducing the likelihood of false-positive colocalization from cell overlap. In the lower panel we summarize the current state of our understanding of SMC migration and proliferation in atherosclerosis. Bentzon et al demonstrated that bone marrow–derived cells do not costain with SmActin-positive cells in atherosclerotic lesions with superior 3D techniques, thus our understanding of SMC origin in atherosclerotic lesions has come full circle.

It should also be noted that hyperlipidemic models of atherosclerosis may be distinct from transplant atherosclerosis/arteriosclerosis (TA) models as well as injury-induced vascular lesion models. Based on several studies indicating a significant contribution of SMCs from marrow in these models, we believe a bone marrow or local stem cell origin of SMCs is possible in these situations,^20–22 although these studies do suffer from the same lack of high-resolution 3D microscopic methods. Nevertheless, both severe vascular injury and TA are likely to elicit very different vascular responses than ordinary atherosclerosis or responses to systemic or local hypoxia. Indeed, the higher bone marrow contributions in TA may be related to multiple observations in the literature that host-versus-graft disease, a component of TA, results in medial necrosis, which may prime the media for progenitor cell investment and transdifferentiation. Similarly, there is evidence from Han et al showing that significant transdifferentiation of bone marrow cells into intimal SMC-like cells only occurred with severe but not modest injury.²³ As such, the phenomenon of bone marrow transdifferentiation may be limited to models in which all or a significant fraction of medial SMCs have been eliminated either by mechanical damage or immune mechanisms. This being said, results of bone marrow lineage studies must be reconciled with the classic series of studies by Monika and Alexander Clowes, Stephen Schwartz, and colleagues in the 1980s that provided compelling evidence that severe injury of the rat carotid artery can elicit more than 50% of the medial SMC to enter the cell cycle, and that intimal lesion cells are derived from a combination of migration of medial SMC into the intima as well as proliferation of both medial and intimal SMCs.^14,15

Bentzon et al with this report provide a definitive answer concerning the origin of SMCs in lesions within a mouse model of spontaneous atherosclerosis and in a sophisticated constrictive collar model, that will certainly have a large impact on the field. Yet, we must always be cautious in applying conclusions of these animal models to human disease. The quintessential experiment in humans that would prove or disprove the bone marrow origin of SMCs is, of course, not possible, because it would require confocal image analysis of the atherosclerotic lesions of several individuals whose bone marrow has been replaced with an autologous sample ex vivo engineered to express a lineage tracer. Alternatively, a careful analysis to determine whether bone marrow–derived cells contribute SMCs in lesions in humans could be done using pathologic samples from female recipients (X,X) of male donor (X,Y) bone marrow, similar to the excellent studies of Caplice et al,²⁴ except using the higher resolution microscopic techniques and z-sectioning with confocal or deconvolution microscopy, similar to Bentzon et al, as well as more definitive markers for SMC lineage including smooth muscle myosin heavy chain,^25–27 myocardin,²⁸ LPP,²⁹ and others,¹² as well as markers for leukocytes to eliminate possible false-positives. However, even in this scenario, one must recognize that positive results may be attributable to the uncontrolled variables of bone marrow transplant/graft-versus-host disease, irradiation, chemotherapy, and use of immune-suppressant drugs in these patients, which may limit the relevance of such results to other more organic mechanisms of lesion formation in humans.

Herein, Bentzon et al definitively demonstrate that SMCs in lesions within a mouse model of spontaneous atherosclerosis originate entirely from the local blood vessel wall. This article exemplifies excellent, thorough analyses, the use of high-resolution techniques, and the need, when major scientific paradigms are overturned, to demand better explanations for how new theories incorporate previous valid data and amplify our understanding of the older literature. Their study illuminates the methodological deficiencies that suggested bone marrow origins of intimal SMC in vivo; their data are consistent with the well-established literature in the field, and as a result, their study has brought us full circle in our understanding of origins of SMC in vascular pathologies.

	Acknowledgments

 
Disclosures

None.

	References

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