Phenotypic Switching of Vascular Smooth Muscle Cells in Atherosclerosis

Abstract

The medial layer of the arterial wall is composed mainly of vascular smooth muscle cells (VSMCs). Under physiological conditions, VSMCs assume a contractile phenotype, and their primary function is to regulate vascular tone. In contrast with terminally differentiated cells, VSMCs possess phenotypic plasticity, capable of transitioning into other cellular phenotypes in response to changes in the vascular environment. Recent research has shown that VSMC phenotypic switching participates in the pathogenesis of atherosclerosis, where the various types of dedifferentiated VSMCs accumulate in the atherosclerotic lesion and participate in the associated vascular remodeling by secreting extracellular matrix proteins and proteases. This review article discusses the 9 VSMC phenotypes that have been reported in atherosclerotic lesions and classifies them into differentiated VSMCs, intermediately dedifferentiated VSMCs, and dedifferentiated VSMCs. It also provides an overview of several methodologies that have been developed for studying VSMC phenotypic switching and discusses their respective advantages and limitations.

Keywords: atherosclerosis; cell lineage; dedifferentiation; phenotypes; vascular smooth muscle cells.

Figure 1. VSMC phenotypic switching in atherosclerosis.

The DNA‐modifying enzyme Tet methylcytosine doxygenase 2 (TET2, also known as ten‐eleven translocation‐2) binds in CArG‐rich regions of the MYOCD and SRF genes, and thereby upregulates the expression of these and downstream VSMC contractile genes. TET2 and its product 5‐hydroxymethylcytosine are enriched in contractile VSMCs, while they are reduced in dedifferentiated VSMCs, where the TET2 reduction causes a decrease in the expression of VSMC contractile genes, with concomitant transcriptional upregulation of KLF4. During atherogenesis, VSMCs may transition to intermediately dedifferentiated VSMCs (mesenchymal‐like VSMCs, SEM‐like cells, or Lgals3⁺ VSMCs) and then further to dedifferentiated VSMCs (macrophage‐like VSMCs, or osteoblast‐like VSMCs) or transform directly to dedifferentiated VSMCs (fibroblast‐like VSMCs, myofibroblast‐like VSMCs, macrophage‐like VSMCs, osteoblast‐like VSMCs, or adipocyte‐like VSMCs). Markers typical or potential for the contractile VSMCs are SM22a, SM‐MHC, α‐SMA, and calponin; for the fibroblast‐like phenotype FN1, Lum, Dcn, Bgn, and OPN; for the adipocyte‐like phenotype UCP‐1 and Adipsin, ; for the macrophage‐like phenotype CD68 alone or CD68 and Lgals3, ; for the myofibroblast‐like phenotype PDGFRβ, and S100A4, , ; for the mesenchymal‐like phenotype Sca‐1 and Eng; for the osteoblast‐like phenotype Cbfa1, Msx2, Runx2, and Sox9; for the Lgals3⁺ cells phenotype Lgals3; for the SEM‐like cells phenotype Ly6a, Vcam1 and Ly6c1; for the Mox macrophage subtype HO‐1 and Tsp‐1; for the foam cells phenotype ABCA1, ; for the macrophage‐like phenotype Mac3 and CD68; for the fibrochondrocyte‐like phenotype FN1, Col1a1, Col1a2. ↑ represents VSMC phenotype transformation, represents exacerbation, and T represents inhibition of atherosclerosis. The blue font represents the marker of each phenotype. 5hmC indicates 5‐hydroxymethylcytosine; α‐SMA, α‐smooth muscle actin; ABCA1, ATP‐binding cassette transporter 1; Bgn, biglycan; CArG, CC(A/T‐rich)6GG cis‐regulating element; cbfa1, core‐binding factor α1; CD68, cluster of differentiation 68; CNN, calponin; Col1a1, collagen type I alpha 1 chain; Col1a2, collagen type I alpha 2 chain; Dcn, decorin; ECM, extracellular matrix; ELK; ETS transcription factor ELK1; Eng, endoglin; Fn1, fibronectin 1; GC, G/C repressor element; H3K4me2, histone H3 lysine 4 di‐methylation; HO‐1, heme oxygenase 1; KLF4, Krüppel‐like factor 4; Lgals3, galectin 3; Ly6a, lymphocyte antigen 6 family member A; Ly6c1, lymphocyte antigen 6 family member C1; Lum, lumican; Mac3, lysosomal‐associated membrane protein 2; MCP1, monocyte chemoattractant protein 1; Msx2, Msh homeobox 2; Myocd, myocardin; PDGFRβ, platelet‐derived growth factor receptor beta; OPN, osteophosphorin; Runx2, Runt‐related transcription factor 2; S100A4, S100 calcium binding protein A4; Sca‐1, stem cell antigen‐1; SEM‐like VSMC, stem cell/endothelial cell/monocyte cell‐like VSMC;SM22α, transgelin; SM‐MHC, myosin heavy chain 11; SOX9, SRY‐box transcription factor 9; SRF, serum response factor; TCE, transforming growth factor‐beta control elements; TET2, Tet methylcytosine dioxygenase 2; Tsp‐1, thrombospondin‐1; UCP‐1, uncoupling protein 1; Vcam1, vascular cell adhesion molecule 1; and VSMC, vascular smooth muscle cell.

Figure 2. Extracellular stimuli and intracellular mediators of VSMC phenotypic transitions.

The colored lines indicate the respective pathways from the different extracellular stimuli (PDGF,, TGF‐β, cholesterol,, , , OxLDL,, , FGF23, ATRA, POVPC,, and adipogenic differentiation medium ²⁷ ) via the highlighted intracellular mediators to induce the transition of contractile VSMCs to various dedifferentiated VSMCs (fibroblast‐like VSMCs, adipocyte‐like VSMCs, macrophage‐like VSMCs, myofibroblast‐like VSMCs, mesenchymal‐like VSMCs, osteoblast‐like VSMCs, and Lgals3⁺ VSMCs). ↑ indicates activation, and T represents inhibition. AKT indicates AKT serine/threonine kinase 1; Atf‐6, activating transcription factor 6; ATRA, all‐trans‐retinoic acid; BCLAF1, BCL2‐associated transcription factor 1; Bgn, biglycan; CRABP2, cellular retinoic acid‐binding protein 2; CD68, cluster of differentiation 68; Dcn, decorin; Eng, endoglin; FGF23, fibroblast growth factor 23; Hif‐1α, hypoxia inducible factor 1 subunit alpha; FN1, fibronectin 1; KLF4, Krüppel‐like factor 4; Ire‐1, serine/threonine‐protein kinase 1; Lgals3, galectin 3; Lum, lumican; Ly6a, lymphocyte antigen 6 family member A; Ly6c1, lymphocyte antigen 6 family member C1; LXR, liver X receptor; Msx2, Msh homeobox 2; Nrf‐2, nuclear factor‐erythroid 2‐related factor 2; OxLDL, oxidized low‐density lipoprotein; PDGF, platelet‐derived growth factor; mTOR, mechanistic target of rapamycin kinase; Oct‐4, POU class 5 homeobox 1; OPN, osteophosphorin; Myocd, myocardin; OPN, osteophosphorin; PDGFRβ, platelet‐derived growth factor receptor beta; Pert, PKR‐like ER kinase; PI3K, phosphatidylinositol 3‐kinase; pELK1/2, phospho‐ETS transcription factor 1 and 2; RA, retinoic acid; Runx2, runt‐related transcription factor 2; S100A4, S100 calcium‐binding protein A4; Sca‐1, stem cell antigen‐1; SEM‐like VSMC, stem cell/endothelial cell/monocyte cell‐like VSMC; Sox9, sex‐determining region Y‐box 9; SRF, serum response factor; Sirt1, sirtuin 1; Sirt3, sirtuin 3; SREBP1, sterol regulatory element‐binding protein 1; TGF‐β, transforming growth factor‐β; POVPC, 1‐palmitoyl‐2‐(5′‐oxo‐valeroyl)‐sn‐glycero‐3‐phosphocholine; UCP‐1, uncoupling protein 1; Vcam1, vascular cell adhesion molecule 1; and VSMC, vascular smooth muscle cell.

Figure 3. Inducers of VSMC phenotypic transitions in vitro.

Induction conditions for transformation of VSMC phenotypes (mesenchymal stem cell‐like, Lgals3⁺, macrophage‐like,, , myofibroblast‐like, fibroblast‐like, osteoblast‐like, and adipocyte‐like ²⁷ ). *The calcification medium consists of Dulbecco's Modified Eagle Medium containing sodium pyruvate, 15% fetal bovine serum, 10 mmol/L β‐glycerophosphate, 100 μmol/L insulin, and 50 μg/mL ascorbic acid., , **The adipogenic differentiation medium consists of Dulbecco's modified Eagle's medium/Ham's F‐12 medium (1:1, by volume) or Medium 199, containing 15 mmol/L 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid, 33 μmol/L biotin, 17 μmol/L pantothenate, 1.2 μmol/L human insulin, 100 nmol/L dexamethasone, 1 nmol/L triiodothyronine, 0.25 mmol/L 3‐isobutyl‐1‐methylxanthine, and antibiotics. α‐SMA indicates α‐smooth muscle actin; Lgals3, galectin 3; Bgn, biglycan; Cbfa1, runt‐related transcription factor 2; CD68, cluster of differentiation 68; C/EBP, CCAAT/enhancer binding protein; Dcn, decorin; FN1, fibronectin 1; IL‐1β, interleukin‐1β; Itgb3, integrin beta 3; MBD‐Cholesterol, methyl‐beta‐cyclodextrin cholesterol; PDGF‐BB, platelet‐derived growth factor‐BB; SM22α, transgelin; TGF‐β, transforming growth factor‐β; Sca‐1, stem cell antigen‐1; SM‐MHC, myosin heavy chain 11; and VSMC, vascular smooth muscle cell.