Cardiovascular Research Advance Access originally published online on November 11, 2008
Cardiovascular Research 2009 81(1):7-8; doi:10.1093/cvr/cvn305
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The origin of intimal smooth muscle cells: are we on a steady road back to the past?
Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, Herestraat 49, Leuven 3000, Belgium
* Corresponding author. Tel: +32 16 33 0578; fax: +32 16 34 5990. E-mail address: bart.degeest{at}med.kuleuven.be
This editorial refers to ‘The origin of post-injury neointimal cells in the rat balloon injury model’ by Rodriguez-Menocal et al.,1 pp. 46–53, this issue.
The origin of intimal smooth muscle cells in native atherosclerosis, post-injury neointima formation, allograft vasculopathy, and vein graft atherosclerosis has been the subject of a confusing literature in the past decade. Rodriguez-Menocal et al.1 show in an elegant series of experiments that neointimal cells in a rat balloon injury model originate from pre-existing vascular cells and only rarely come from circulating progenitor cells derived from the bone marrow or from other sources. Two different experimental approaches in an inbred strain of rats lead to essentially the same conclusions. First, the authors performed balloon angioplasty experiments in the right iliac artery of chimeric rats rescued following lethal irradiation with bone marrow from syngeneic transgenic rats expressing green fluorescent protein (GFP) under the control of a ubiquitously active promoter. Second, injured arteries were transplanted between syngeneic wild-type and GFP transgenic rats. This latter approach may identify smooth muscle cells derived from circulating progenitor cells irrespective whether these originate from the bone marrow or from other tissues. In light of the existing controversy on the contribution of circulating progenitor cells to vascular lesion formation,2–7 it is indicated to look at methodological issues that may explain the apparent contradictory results in the literature.
Before addressing these methodological issues, an important secondary observation of the study should be highlighted. The authors clearly show that both bone-marrow transplantation and syngeneic artery transplantation affect the neointima/media ratio post-balloon injury. Using the neointima/media ratio in non-irradiated and non-transplanted arteries as a reference, this ratio is roughly 1.8-fold lower in the injured arteries of rats with prior irradiation and bone-marrow transplantation, and 1.4-fold higher in transplanted injured arteries. This procedural bias is independent of the GFP genotype of the transplanted bone marrow or of the GFP genotype of the donors or recipients of artery transplantation. Nevertheless, it indicates that a process different from the normal pathophysiology of post-balloon angioplasty intima formation is being investigated. However, it is reassuring that both experimental approaches reach a similar conclusion on the contribution of circulating progenitor cells. An alternative to study the origin of neointimal smooth muscle cells is performing balloon angioplasty in the setting of a parabiosis experiment. This avoids bias secondary to radiation-induced vascular injury or to syngeneic artery transplantation-induced injury.
The discrepant results in the literature on the contribution of circulating progenitor cells to smooth muscle cell accumulation in the neointima are likely due mainly to methodological limitations in tracing the origin of these cells. The authors point out that direct observation of GFP-positive cells in unfixed tissues does not provide adequate resolution and may also induce diffusion of the tracer molecule from sectioned cells. However, immune fluorescence microscopy may also lead to false-positive identification of GFP-positive smooth muscle cells. Inflammatory cells in close contact with smooth muscle cells may lead to erroneous conclusions on the origin of smooth muscle cells if the three-dimensional structure of the tissue is not considered. In other words, overlapping cells may produce artefacts that appear as co-localization of a smooth muscle cell marker and a marker reflecting bone-marrow origin within a single cell in two-dimensional analysis, although the markers are expressed in a smooth muscle cell and an inflammatory cell, respectively. The occurrence of such artefacts will be dependent on the number of inflammatory cells and on the thickness of the section. High-resolution multi-channel sequential confocal scanning microscopy is capable of visualizing three-dimensional vascular structures. Using this technology, single cells can be identified, and co-localization of a smooth muscle cell marker and, for example, GFP in a single cell is more accurately demonstrated. Even so, a double-positive cell could be the product of cell fusion, which may be detected by various techniques.8 This point may be relevant considering the presence of tetraploid smooth muscle cells in arteries.9
Another consideration is that an unequivocal definition and specific and sensitive identification of smooth muscle cells in histological sections is challenging, since they may exhibit a range of phenotypes. This smooth muscle cell phenotype heterogeneity may or may not reflect diverse origins of these cells. The phenotype lies on a continuum between a quiescent contractile cell expressing high levels of smooth muscle-specific isoforms of contractile proteins to a highly proliferative cell that secretes large amounts of extracellular matrix and expresses only low levels of characteristic isoforms of contractile proteins. In addition, myofibroblasts, having a phenotype between a fibroblast and a fully differentiated adult smooth muscle cell, express smooth muscle 
-actin. Thus, positivity for this marker does not necessarily imply the identification of a smooth muscle cell.
The current study, showing a low to negligible contribution of bone-marrow-derived cells to smooth muscle cell accumulation in the intima of balloon injured rat arteries, is in agreement with data in a murine model of transplant arteriosclerosis5 and with recent studies on the origin of smooth muscle cells in native atherosclerosis6 and in healing atherosclerotic plaque disruptions7 in apolipoprotein E-deficient mice. In the transplant arteriosclerosis study,5 bone-marrow transplantations were performed with transgenic SM-LacZ mice expressing ß-gal under the control of the smooth muscle-specific protein SM22 promoter. This strategy avoids the specificity problem associated with detection of co-localization of bone-marrow and smooth muscle cell markers. Notwithstanding the lack of contribution of bone-marrow-derived cells, smooth muscle cells in murine models of allograft vasculopathy appear to be predominantly recipient derived. Whether this represents migration and proliferation of medial smooth muscle cells of anastomosed arteries of the recipient into the graft, or the contribution of circulating smooth muscle cell progenitor cells of a non-bone-marrow origin, is unclear.2,5
The results of the current and other recent studies lead us back to the original hypothesis on the origin of intimal smooth muscle cells, namely that they arise from medial smooth muscle cells that start proliferating and migrating in response to injury.10 An addition to this paradigm is that progenitor cells in the adventitia may be another local source of smooth muscle cells.11 In a similar vein, the authors of the current study show that Matrigel-embedded vascular smooth muscle cells from primary culture applied to the adventitial region migrate into the intima in response to injury and contribute to intimal smooth muscle cell accumulation. In contrast, following application of bone-marrow cells, smooth muscle cells derived from seeded cells were only rarely observed in the intima.
The existence of smooth muscle cell progenitor cells12–14 in the blood should not be doubted. However, there is a huge difference between the potential to differentiate to smooth muscle cells and the actual occurrence of such an event in vivo. Following homing to the subendothelium in vivo, the fate and potential differentiation of smooth muscle cell progenitor cells is dependent on micro-environmental cues such as cytokines, growth factors, extracellular matrix proteins, and mechanical forces induced by blood flow. These signals, such as platelet-derived growth factor (PDGF)-BB interacting with PDGF receptor-ßß or collagen type IV interacting with integrin 1ß1, may be adequately provided in a specific and well-controlled in vitro micro-environment but are likely much more difficult to be obtained in a complex in vivo micro-environment. The possibility that certain pathophysiological conditions provide the right micro-environmental conditions for in vivo smooth muscle cell differentiation of progenitor cells remains open. The inherent limitations of animal models in mimicking human pathophysiology should be taken into account. Studies in humans with sex-mismatch bone-marrow transplantation,15 using stringent criteria for the identification of bone-marrow-derived smooth muscle cells, are required to further resolve the existing controversies.
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The Center for Molecular and Vascular Biology is supported by Excellentiefinanciering KU Leuven (EF/05/013).
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The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology.
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