**12. SMC transdifferentiation into macrophages**

cytes and fibroblasts. Considerable overlaps exist in the gene expression profiles among human monocytes, macrophages, fibrocytes, and fibroblasts [121]. Human fibrocytes also may differentiate into cells with characteristics of adipocytes, chondrocytes, and osteoblasts [122, 123]. In human peripheral blood, 0.1% to 0.5% of nucleated cells are circulating fibrocytes that express type 1 and 2 collagens, vimentin, and SMα-actin [124]. Because no single marker can unequivocally identify fibrocytes, the combined use of collagen and other surface markers, including CD34, CD45, and CD68, is a common approach. More recent studies have used a

bar=50 µm. i Indicates intima; m, media; and a, adventitia (Modified from Ref 14 by Iwata et al.).

**Figure 4. Bone marrow CD11b+Ly-6C+ cells migrate to the arterial wall and express SM α-actin.** CD11b+Ly-6C+ cells within the bone marrow of SM α-actin-EGFP mice were sorted and adoptively transferred into wild-type mice with femoral arteries that had been subjected to wire-induced arterial injury. Four weeks after the injury, EGFP+ cells corre‐ sponding to CD11b+Ly-6C+-derived SM α-actin+ cells (arrows) were found in the walls of the injured vessel. Scale

The fibrous cap of human atherosclerotic lesions contains fibrocytes expressing procollagen I and CD34 [125]. Subendothelial SMα-actin–positive myofibroblasts expressing the monocyte marker CD68 have been found in lipid-rich areas of the atherosclerotic intima in human aorta [126]. The overexpression of TGF-β1 resulted in the increased accumulation of fibrocytes in atherosclerotic plaques of Apoe-/- mice [127]. The pro-inflammatory monocyte subset CD14<sup>+</sup>

CCR2high may be precursors of fibrocytes. The expression profile of marker genes

indicates considerable overlaps between fibrocytes, SMPC, smooth muscle–like cells, and monocytes/macrophages, suggesting the importance to clarify the relationship in lineages and functions between these cell types to unfold intertwined mechanisms for atherosclerosis and

combination of CD45RO, 25F9, and S100A8/A9 or CD49.

provide insight into the development of new classes of therapeutics.

+ CD16−

244 Muscle Cell and Tissue

In addition to the potential existence of intimal SMC or SMC-like cells of monocytes origin, accumulating evidence suggests transdifferentiation of SMC into macrophage lineage. Rong et al. demonstrated that cholesterol or oxidized LDL loading of SMCs suppresses SMC marker expression, but induces macrophage markers (e.g., CD68, Mac-2, and ABCA1) and phagocytic activity in cultured SMCs, raising the possibility that some macrophages within lesions may originate from SMC [20]. Bentzon et al. presented evidence that a significant fraction of lesional cells of Apoe-/- mice immunopositive for the macrophage marker Mac2 are not of the bone marrow origin [21]. Medial SMCs may undergo clonal expansion, loose classical SMC marker expression, and convert to macrophage-like cells in mouse atherosclerotic plaques [22]. Immunohistological analysis of human coronary atherosclerotic lesions demonstrated that a subpopulation (>40%) of macrophage foam cells (CD68 and oil-red O positive) co-express SMα-actin [23]. Caplice et al. showed that more than 90% of SM α-actin-expressing cells within lesions are not of the hematopoietic cell lineage [24]. More recently, Vengrenyuk et al. demonstrated that cholesterol loading converts vascular SMCs into cells similar to macrophage foam cells via the mechanisms dependent on micro RNA-143/145, and the transcription factor myocardin, and its co-activator serum response factor, responsible for SMC differentiation [25]. These studies indicate more dynamic crosstalk between SMC and monocytes/macro‐ phage lineages than traditionally thought (Figure 5).

**Figure 5. Possible transdifferentiation processes between SMC and macrophage lineages in the atherosclerotic pla‐ ques.** The evidence suggests possible transdifferentiation between cells in the SMC and monocyte/macrophage lineag‐ es within the plaque. Various studies have demonstrated this concept in two directions: from SMC to macrophage foam cells (Ref. 20-25) and from monocytes to smooth muscle–like cells (Ref. 13, 14, 18, 19, 111,) and addressed its po‐ tential contribution to the pathogenesis of vascular disease.

### **13. Conclusions and future perspective**

The evidence has used cutting-edge technologies, particularly in mouse models, to propose the substantial heterogeneity of SMCs and monocytes/macrophages. Due to technical diffi‐ culties in identifying SMC and monocyte/macrophage lineages in lesions, addressing the origin and the functionality of each cell type remains challenging. Lineage tracing of lesional cells in humans particularly requires highly sophisticated technologies. Gomez et al. recently reported a rigorous method with detection of histone modification at specific gene loci of SM-MHC gene [133]. Such specific cell lineage tracing methods will serve as powerful tools to provide insights into the crosstalk between SMCs and macrophages in human atherosclerosis. In addition, the use of multidisciplinary strategies, involving in vitro models, animal experi‐ ments, human samples, and more systemic approaches such as network analysis may help to unfold complex mechanisms for human atherogenesis. In addition, such strategies may identify new classes of therapeutic targets for atherosclerosis and its devastating complications such as myocardial infarction and stroke, and may help to evaluate the effects of new thera‐ peutics.
