**3. Existence of immature SMCs in vascular disease**

SMCs exhibit the diversity of phenotypes in the vascular lesions. A panel of antibodies for SMC differentiation markers enables to identify a wide spectrum of their phenotypes in experimental and human vascular diseases. In the arterial lesions after acute mechanical injury and in chronic atherosclerotic plaques of experimental animals, intimal SMC often express lower levels of SM-MHC isoforms SM1 and SM2, whereas the medial SMC usually stain positively for SM-MHC and α-actin. While SM1 expression appears at the late stage of vascular development, SM2 expression increases later, particularly after birth [2, 35-37]. SM2 thus serves as a sensitive marker of mature SMC. Aikawa et al. demonstrated that intimal SMC of the rabbit aorta 4 months after mechanical injury and the initiation of a high-cholesterol diet showed an immature phenotype as gauged by decreased SM2 immunoreactivity (Figure 1, top panels) [38]. Interestingly, dietary cholesterol lowering for 8 or 16 months induced the increased expression of SM2 in intimal SMC to the levels similar to those of medial SMC (Figure 1, bottom panels). Such SMC plasticity may depend on microenvironmental cues. Alterna‐ tively, different microenvironment may affect the balance of SMC subpopulations. Thus, the recovery of SM2 expression by lipid lowering may result from either "redifferentiation" of the same group of intimal SMC, the expansion of a subset of SMC with a mature phenotype, or the repopulation of immature SMC by mature SMC.

In human coronary arteries, SMC begin to lose their SM2 expression even in the physiological intima of young adults with no obvious signs of atherosclerotic changes such as macrophage accumulation [28]. In more advanced plaques, immature SMCs often exist in areas where macrophages accumulate. In Figure 2, SM2 was undetectable in SMC identified by SMα-actin and SM1. Co-existence of intraplaque microvessels and macrophages may indicate active recruitment of circulating monocytes into this area. A pro-inflammatory microenvironment produced by activated macrophages via the release of IL-1β, PDGF-BB, and other factors and production of oxidative stress may have promoted phenotypic modulation of SMC in this region. As we discuss later, some of these immature SMC may have originated from a subset of monocytes. Alternatively, some SMCs may have transdifferentiated into macrophage-like cells. Understanding of such crosstalk and potential interchangeability between SMCs and macrophages may provide important insight into the identification of new therapeutic targets. Of note, "redifferentiation" or "repopulation" of intimal SMC after mechanical injury also occurs in human coronary arteries. The differentiation state of SMC reduces a first few months after percutaneous coronary intervention while it increases over time [27]. The key question is where those intimal SMCs originate from.
