**6. Monocytes and macrophages in vascular disease**

In atherosclerosis research, monocytes and macrophages have called particular attention. Cardiovascular risk factors, such as dyslipidemia, hypertension, smoking, and diabetes mellitus, promote sustained activation of monocytes and macrophages. Activated monocytes and macrophages critically contribute to multiple processes of atherogenesis from the initiation to the acute onset of devastating complications [52-54]. Dyslipidemia provokes monocytosis by expanding the pro-inflammatory subset of circulating monocytes [55]. These inflammatory monocytes attach to vascular EC and invade the subendothelial space. Mono‐ cytes then differentiate into macrophages, although how recruited monocytes are retained and differentiate into macrophages remains incompletely understood [55, 56]. Monocytes recruit‐ ment to the subendothelial space mediated by chemokines, such as MCP-1 [57, 58] and C-C chemokine-5 (CCL5) [59], and adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on activated ECs, initiates the early processes of atherogenesis [60, 61]. The secretion of collagenolytic MMPs by atheroscler‐ otic plaque macrophages may weaken the protective fibrous cap, causing plaque disruption and thrombosis [62-65].

### **7. Monocytes, subsets, and functions in atherosclerosis**

Several subsets may exist in circulating monocyte in mice and humans. At least two distinct subsets of mouse monocytes are identified via differential expression levels of Ly-6C, CX3CR1, and CCR2 [66]. One subset exhibiting Ly-6ChighCX3CR1lowCCR2+ is designated as pro-inflam‐ matory monocytes, and the other subset with Ly-6ClowCX-3CR1highCCR2<sup>−</sup> as resident or patrolling monocytes [67, 68]. Human monocytes are often classified into three subsets based on the expression levels of CD14 and CD16 [69]. The classical subset CD14++CD16<sup>−</sup> CCR2high may correspond to mouse Ly-6Chigh pro-inflammatory monocytes, whereas the nonclassical subset CD14+ CD16++CCR2low may be equivalent to mouse Ly-6Clow patrolling monocytes [70-72]. The intermediate subset is identified as CD14++CD16+ cells [73]. CD14++CD16<sup>−</sup> CCR2high cells appear to preferentially enter sites of atherosclerosis [74, 75]. In mice, Ly-6Chigh monocytes in peripheral circulation were induced by hypercholesterolemia [76]. Similarly, in human, elevated CD14++CD16<sup>−</sup> monocyte levels are associated with an increased risk of cardiovascular events [77]. Human monocyte subpopulations are also associated with the status of athero‐ sclerosis [78, 79].

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‐

In atherosclerosis research, monocytes and macrophages have called particular attention. Cardiovascular risk factors, such as dyslipidemia, hypertension, smoking, and diabetes mellitus, promote sustained activation of monocytes and macrophages. Activated monocytes and macrophages critically contribute to multiple processes of atherogenesis from the initiation to the acute onset of devastating complications [52-54]. Dyslipidemia provokes monocytosis by expanding the pro-inflammatory subset of circulating monocytes [55]. These inflammatory monocytes attach to vascular EC and invade the subendothelial space. Mono‐ cytes then differentiate into macrophages, although how recruited monocytes are retained and differentiate into macrophages remains incompletely understood [55, 56]. Monocytes recruit‐ ment to the subendothelial space mediated by chemokines, such as MCP-1 [57, 58] and C-C chemokine-5 (CCL5) [59], and adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on activated ECs, initiates the early processes of atherogenesis [60, 61]. The secretion of collagenolytic MMPs by atheroscler‐ otic plaque macrophages may weaken the protective fibrous cap, causing plaque disruption

Several subsets may exist in circulating monocyte in mice and humans. At least two distinct subsets of mouse monocytes are identified via differential expression levels of Ly-6C, CX3CR1,

matory monocytes, and the other subset with Ly-6ClowCX-3CR1highCCR2<sup>−</sup> as resident or patrolling monocytes [67, 68]. Human monocytes are often classified into three subsets based

may correspond to mouse Ly-6Chigh pro-inflammatory monocytes, whereas the nonclassical

on the expression levels of CD14 and CD16 [69]. The classical subset CD14++CD16<sup>−</sup>

is designated as pro-inflam‐

CCR2high

ing SMα-actin and SM22α [50, 51].

240 Muscle Cell and Tissue

and thrombosis [62-65].

**6. Monocytes and macrophages in vascular disease**

**7. Monocytes, subsets, and functions in atherosclerosis**

and CCR2 [66]. One subset exhibiting Ly-6ChighCX3CR1lowCCR2+

In addition to the influence of phenotypes of infiltrating monocytes, as macrophage precursors, microenvironmental cues in the vascular wall may determine macrophage phenotype. In vitro studies have established the paradigm of macrophage heterogeneity. IFNγ induces a proinflammatory phenotype of macrophages (M1). IL-4 promote non/anti-inflammatory macro‐ phages (M2) [74, 80]. The in vivo significance of such macrophage polarization has driven many investigations in the contexts of cardiovascular and metabolic disorders [81-86]. More specific terms, M(IFNγ) and M(IL-4), rather than M1 and M2, have been recently proposed [87].

Hyperlipidemia causes the generation of monocytes in bone marrow through medullary hematopoiesis and thus induces monocytosis. Hematopoietic stem and progenitor cells, however, may relocate to the splenic red pulp and differentiate into Ly-6Chigh monocytes [88]. The spleen as a monocyte pool may participate in further accumulation of monocytes/ macrophages in peripheral organs such as atherosclerotic plaques. The fate and function of Ly-6Clow monocytes also remain incompletely understood. These cells may patrol the endo‐ thelium for injury and infection and also promote wound healing [89].
