**5. The origin of intimal SMC**

As discussed, SMCs participate in the development of atherosclerotic plaques and the onset of acute thrombotic complications. Lesional SMCs show dynamic changes in their phenotypes depending on the disease context and the stage of each disease. Where do they come from? Many studies have led to the traditional theory that intimal SMC originate from the tunica media via proliferation and migration. More recently, several lines of evidence have indicated that intimal SMC or intimal cells that possess phenotypes similar to SMC (often called "SMlike cells") may originate from sources other than the media, including circulating precursors, adventitial cells and local stem cells [15, 32]. The relative contribution of each of these sources, however, remains obscure, and may also depend on the disease context in humans, and models or species in experimental animals [48]. Figure 3 illustrates possible sources of intimal SMC or SM-like cells.

In lipid-laden intimal SMCs, cholesterol accumulation induces cell death. SMC death and subsequent necrosis promote a series of pro-inflammatory events: the release of pro-inflam‐ matory cytokines including monocyte chemo-attractant protein-1 (MCP-1) and IL-1β from the dying and surrounding SMCs [44]; migration and proliferation of adjacent SMCs [45], MCP-1 mediated monocyte infiltration. MCP-1 and IL-1β modulate SMC phenotype, growth, and MMP production [46]. Such a cascade of events accelerates a positive feedback loop of vascular inflammation. To assess the anti-inflammatory and anti-atherosclerotic effects of a monoclonal anti-human IL-1β antibody, a randomized, placebo-controlled trial entitled CANTOS is

**Figure 2. Decreased SMC differentiation in macrophage-rich regions of human coronary arteries.** Immunohisto‐ chemistry of SMC differentiation markers in the left ascending coronary artery (LAD) of a 78-year-old male had devel‐ oped the thickened intima. SM α-actin covers a wide spectrum of SMC differentiation. SMC-specific myosin heavy chains isoforms are definitive markers of differentiated SMC. In particular, SM2 identifies mature SMC. In this athero‐ sclerotic human artery, medial SMC stained positively for all three markers, suggesting an apparently "normal" phe‐ notype. Intima SMC exhibited the diversity of SMC phenotype. Some SM α-actin expressing cells were not immunoreactive for SM1 and SM2 antibodies. Especially, many SMC did not express the detectable level SM2 in the area where many macrophages (CD68) accumulated. They may have undergone phenotypic modulation ("de-differen‐ tiation") due to a pro-inflammatory microenvironment induced by neighboring macrophages. As an alternative possi‐ bility, some of these SM2-negative cells probably co-expressed SM α-actin, SM1, and CD68, an intermediate phenotype between SMC and macrophage lineages. Of note, intraplaque microvessels surrounded by macrophages may have re‐ cruited a subset of activated monocytes as precursor cells for smooth muscle–like cells of the bone marrow origin.

As discussed, SMCs participate in the development of atherosclerotic plaques and the onset of acute thrombotic complications. Lesional SMCs show dynamic changes in their phenotypes depending on the disease context and the stage of each disease. Where do they come from? Many studies have led to the traditional theory that intimal SMC originate from the tunica media via proliferation and migration. More recently, several lines of evidence have indicated

currently ongoing in high-risk cardiovascular patients [47].

**5. The origin of intimal SMC**

238 Muscle Cell and Tissue

**Figure 3. Potential sources of intimal smooth muscle cells (SMCs) 1)** In response to injury or inflammatory stimuli, medial SMC undergo phenotypic modulation, migrate into the subendothelial space, and form the tunica intima. Resi‐ dent progenitor cells in the media may contribute to intima formation. **2)** Circulating progenitor cells such as mesen‐ chymal stem cells and activated monocytes may engraft in the intima and contribute to lesion development. Other organs, including the spleen, fat, and skeletal muscles may also release SMC progenitors. **3)** The adventitia may con‐ tain SMC precursors (resident stem cells), and contribute to intima formation, particularly after mechanical injury. **4)** Potential transdifferentiation of SMCs into macrophage- or macrophage-like cells may also contribute to atherogenesis. (Modified from Ref. 48 by Fukuda et al.)

Recent cell lineage studies have indicated that some bone marrow–derived cells express SMCspecific markers, while SMCs can display proteins associated with macrophages [15, 32]. For instance, a subpopulation of circulating monocytes may become SM-like cells in the intima [14], whereas SMC can transdifferentiate into macrophage-like cells. [25]. Therefore, the origin and fate of SMC in vascular lesions are not so clear as we traditionally thought [49]. We need to overcome several challenges to explore seemingly complex, intertwined mechanisms [32]. The limited availability of lineage-tracing methods that enable to identify the specific origin of SMC cells, particularly in disease contexts where cells tend to reduce the expression of differentiation markers. Therefore, some lesional SMCs may not be identified (false negative). As suggested by many studies, cell types other than SMCs in vascular lesions can express SMα-actin (false positive). According to a study by Caplice et al. up to 10% of cells in advanced atherosclerotic lesions of human coronary arteries expressing SMα-actin are of the myeloid lineage [24]. TGF-β or thrombin may induce macrophage expression of SMC markers includ‐ ing SMα-actin and SM22α [50, 51].
