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Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Krüppel-Like Factor 4)

Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Krüppel-Like Factor 4) Arteriosclerosis, Thrombosis, and Vascular Biology BRIEF REVIEW Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Krüppel-Like Factor 4) Carmen Yap ,* Arnout Mieremet ,* Carlie J.M. de Vries, Dimitra Micha , Vivian de Waard ABSTRACT: Multiple layers of vascular smooth muscle cells (vSMCs) are present in blood vessels forming the media of the vessel wall. vSMCs provide a vessel wall structure, enabling it to contract and relax, thus modulating blood flow. They also play a crucial role in the development of vascular diseases, such as atherosclerosis and aortic aneurysm formation. vSMCs display a remarkable high degree of plasticity. At present, the number of different vSMC phenotypes has only partially been characterized. By mapping vSMC phenotypes in detail and identifying triggers for phenotype switching, the relevance of the different phenotypes in vascular disease may be identified. Up until recently, vSMCs were classified as either contractile or dedifferentiated (ie, synthetic). However, single-cell RNA sequencing studies revealed such dedifferentiated arterial vSMCs to be highly diverse. Currently, no consensus exist about the number of vSMC phenotypes. Therefore, we reviewed the data from relevant single-cell RNA sequencing studies, and classified a total of 6 vSMC phenotypes. The central dedifferentiated vSMC type that we classified is the mesenchymal-like phenotype. Mesenchymal-like vSMCs subsequently seem to differentiate into fibroblast-like, macrophage-like, osteogenic-like, and adipocyte-like vSMCs, which contribute differentially to vascular disease. This phenotype switching between vSMCs requires the transcription factor KLF4 (Krüppel-like factor 4). Here, we performed an integrated analysis of the data about the recently identified vSMC phenotypes, their associated gene expression profiles, and previous vSMC knowledge to better understand the role of vSMC phenotype transitions in vascular pathology. GRAPHIC ABSTRACT: A graphic abstract is available for this article. Key Words: atherosclerosis ◼ cardiovascular disease ◼ myocytes ◼ phenotype ◼ smooth muscle ardiovascular diseases are the leading cause of levels of the contractile vSMC markers, capable of phe- death worldwide. Many of these pathologies are notypic switching. There is a growing body of evidence Ccharacterized by progressive cellular modulations that multiple vSMC phenotypes exist in healthy vessels. derived from genetic predisposition, aging, or lifestyle. Single-cell RNA sequencing (scRNA-seq) studies identi- The 3 main cellular layers forming the vessel wall are the fied the presence of specific vSMC-derived cell popula- 5–9 adventitia, media, and the intima, surrounding the lumen. tions. However, there is still limited understanding of In the media, the middle layer, vascular smooth muscle the role of these vSMC phenotypes in physiological and cells (vSMCs) are the major cellular component. These pathological conditions. vSMCs contribute to the integrity of the vessels and are For decades, vSMC activation and dedifferentiation able to adequately respond to stimuli of vasoconstric- has been regarded as the adoption of a single syn- 2,3 tion and vasodilation. In healthy vessels, vSMCs are thetic, proliferative phenotype. However, as revealed regarded as quiescent, differentiated cells, which display by recent scRNA-seq analyses, the diversity of vSMC 4 10 remarkable plasticity. The contractile vSMCs are subject phenotypes is far more sophisticated. The power of to context-dependent changes and studies have shown scRNA-seq is that it provides detailed, unbiased infor- that a subset of vSMCs in healthy tissue express reduced mation on distinct cell populations within healthy or Correspondence to: Vivian de Waard, PhD, Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, University of Amsterdam, Amsterdam UMC, location Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands. Email v.dewaard@amsterdamumc.nl *C. Yap and A. Mieremet contributed equally. For Sources of Funding and Disclosures, see page 2704. © 2021 The Authors. Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited. Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2693 Yap et al Six Shades of vSMCs Illuminated by KLF4 Nonstandard Abbreviations and Acronyms Highlights AHR aryl hydrocarbon receptor • Single-cell RNA sequencing and lineage tracing analyses revealed that vascular smooth muscle cells ATF4 activating transcription factor 4 can be identified beyond the synthetic and contrac- BMP2 bone morphogenetic protein 2 tile profile. Cbfa1 core-binding factor α-1 • We discuss the phenotypic switching of vascular C/EBPβ CCAAT-enhancer-binding protein beta smooth muscle cells from a contractile state to a CVD cardiovascular disease mesenchymal-like, fibroblast-like, macrophage-like, osteogenic-like, or adipocyte-like phenotype. DKK3 dickkopf 3 • The transcription factor KLF4 (Krüppel-like factor 4) DOCK2 dedicator of cytokinesis 2 plays a pivotal role in regulation of vascular smooth ECM extracellular matrix muscle cells phenotype switching and emerges as ER endoplasmic reticulum potential drug target. HDAC9 histone deacetylase 9 • The current challenge is to pinpoint the diverging HDL high-density lipoprotein roles of vascular smooth muscle cell phenotypes and their impact on the broad range of cardiovas- JNK c-Jun N-terminal kinase cular diseases. LDL low-density lipoprotein MEF2C myocyte enhancer factor 2C MGP matrix gla protein mouse models, the fate of vSMCs can be accurately MSX2 Msh Homeobox 2 tracked and provides detailed information of their phe- MYOCD myocardin notypic modulation and contribution to diseased tis- 11,12 sue. In this review, we highlighted the information NKx2-5 NK2 transcription factor related locus 5 obtained about vSMCs and used the recent scRNA- OCT4 octamer-binding transcription factor 4 seq literature to classify 6 different vSMC phenotypes. Olfm2 olfactomedin 2 Besides a contractile phenotype, we distinguish the PDGF-BB platelet-derived growth factor BB mesenchymal-like, fibroblast-like, macrophage-like, PKB protein kinase B osteogenic-like, and adipocyte-like phenotypes. In PLK1 polo-like kinase-1 addition, we address the current insights about stimu- PRKG1 cyclic GMP–dependent protein kinase 1 lating and inhibitory cues mediating vSMC phenotype PRMT5 protein arginine methyltransferase 5 switching. From that analysis, we inferred that tran- RUNX2 runt-related transcription factor 2 scription factor KLF4 (Krüppel-like factor 4) plays a SCA1 stem cell antigen-1 pivotal role in the initial dedifferentiation of vSMCs to scRNA-seq single-cell RNA sequencing the mesenchymal-like phenotype enabling further cel- SOX2 SRY-BOX transcription factor 2 lular changes toward the other 4 vSMC phenotypes. Sp1 stimulating protein-1 SRF serum response factor CONTRACTILE VSMCS TCF21 transcription factor 21 TET2 ten-eleven translocation-2 In a healthy state, vSMCs in the media of the arterial TGF-β transforming growth factor beta vessel wall actively synthesize, secrete, modulate, and maintain the extracellular matrix (ECM) to provide elas- UPR unfolded protein response ticity and strength to the blood vessel. In the vasculature, vSMC vascular smooth muscle cell the vSMCs work in close collaboration with other cells Wnt wingless-related integration site to counterbalance the strong mechanical forces that the ZFP148 zinc finger protein 148 13,14 vessel wall experiences. A single layer of endothelial cells at the luminal side of the vessel forms the tight bar- diseased tissue. Distinct cell populations were identi- rier between vascular lumen and the vessel wall, providing fied by scRNA-seq in the vessel wall, with the major- cues for vSMC relaxation and contraction. Endothelial cell ity concerning immune cells, such as T cells, B cells, function is determined by shear stress, stretching of the myeloid cells, and mast cells. Nonimmune cells in vessel due to heart pulse, and by circulating factors. In atherosclerotic plaques and aortic aneurysm tissue the adventitia, fibroblasts produce and maintain collagen included endothelial cells and vSMCs. The combina- fibers, thereby forming a peripheral structure to preserve tion of scRNA-seq and lineage tracing is extremely vascular integrity at high pressures. In the adventitia of useful as it allows in-depth vSMC phenotypic charac- larger blood vessels, a microvasculature bed is present, terization. With the development of lineage tracing in termed the vasa vasorum. This structure protrudes into 2694 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 the outer layers of the media to provide sufficient oxygen on the different phenotypes of vSMCs as deduced from and nutrients to the multi-layered vSMCs in the media. the scRNA-seq reports with the available knowledge on Contractile vSMCs are regarded as differentiated and specific cues and signal transduction pathways involved quiescent cells under physiological conditions, express- in vSMC phenotype modulation. ing a panel of typical contractile proteins that is crucial to maintain vascular tension. Although the embryonic MESENCHYMAL-LIKE VSMCS origin of vSMCs is diverse, the contractile vSMC phe- notype is considered universal throughout the arte- Extensive lineage tracing studies revealed that con- rial vasculature. Contractile vSMCs are embedded in tractile vSMCs can switch from a contractile status to 29–31 an intricate structure of ECM composed of elastin and a mesenchymal-like phenotype. A mesenchymal-like 13,14,17 collagens as key fibers. The ECM is produced by vSMC is characterized by the ability to proliferate and vSMCs themselves and sequesters growth factors, such self-renew and is marked by reduced expression of con- as those belonging to the TGF-β (transforming growth tractile proteins. This phenotype overlaps with that of factor beta) family. Upon damage of the vessel wall, mesenchymal stem cells, which are defined as stromal the sequestered growth factors are released to induce a cells that have the capacity to differentiate into multiple 19 29,32 local repair response. lineages. Recently, a significant number of scRNA- Contractile vSMCs exhibit an elongated, spindle- seq studies confirmed that among the various vSMC shaped morphology and express a well-characterized phenotypes identified, mesenchymal-like vSMCs are set of contractile markers including smooth muscle actin also present (Table). The selected scRNA-seq studies (ACTA2), smooth muscle myosin heavy chain (MYH11), were performed on human and murine vascular tissue smooth muscle protein 22-alpha (SM22α/TAGLN), of atherosclerotic plaques or aortic aneurysms. Distinct 20–22 smoothelin (SMTN), and calponin (CNN1). Expres- vSMC clusters were specified, and based on their gene sion of these proteins is controlled by the transcription expression profile and available knowledge of upstream factors MYOCD (myocardin) and SRF (serum response modulators, we aimed to identify the main drivers of phe- factor), both of which are involved in the regulation of notypic switches. In this review, we classified stem cell differentiation to contractile vSMCs. These transcrip- marker positive vSMCs as belonging to the mesenchy- tion factors also induce expression of microRNA cluster mal-like phenotype. This included the pioneer cell phe- (miRNA)-143/145, involved in activation of the vSMC notype of Alencar et al and vSMC-derived intermediate 24 8 contractile phenotype. In addition, external stimuli, cells (SEM) by Pan et al. such as TGF-β and heparin, play a pivotal role in pro- Dedifferentiation of vSMCs from a contractile to a moting and maintaining the vSMCs contractile pheno- mesenchymal-like phenotype is driven by external stimuli type. Exposure to these factors leads to upregulation that induce repair and/or proliferation. A key initiating of structural ECM genes and inhibition of vSMC prolif- factor for this phenotypic switch is transcription factor 23,25 29,34–38 eration and migration. The phosphatase and tensin KLF4, which regulates cellular proliferation and homolog (PTEN) also helps to maintain the contractile dedifferentiation. KLF4 also has a crucial function in vSMC phenotype, as in the nucleus PTEN interacts with the induction of cellular pluripotency. In fact, genera- SRF to enhance SRF binding to essential promoter ele- tion of induced pluripotent stem cells from a wide range ments in vSMC contractile genes. MEF2C (myocyte of somatic cells requires KLF4 and 3 other, so-called enhancer factor 2C) was also shown to be essential for Yamanaka, factors: OCT4 (octamer transcription factor contractile vSMC differentiation. A detailed overview 4), SOX2 (SRY-BOX transcription factor 2) and cMYC of the contractile vSMC phenotype in the vasculature is (MYC proto-oncogene, bHLH transcription factor). 20,22,28 given elsewhere. Induction of KLF4 in vSMCs results in a phenotypic Various scRNA-seq analyses confirmed that con- switch from contractile to mesenchymal-like and initiates tractile vSMCs are the most prominent cell type in the the expression of mesenchymal markers such as stem 40,41 healthy vessel wall with the transcriptomic profile just cell antigen-1 (SCA1)/LY6A, CD34, and CD44. Dur- 5,6,8 described. Yet, in combination with lineage tracing, ing this transition, while gaining expression of mesen- additional vSMC populations have been identified with chymal markers, the contractile vSMCs lose expression 5,6,8 12,36,42 distinct gene expression profiles. Once pathological of their contractile markers. Under physiological processes in the vessel wall are initiated, vSMCs respond conditions, a subpopulation of mesenchymal-like vSMCs by changing their phenotype and function. A plethora of resides in the medial and adventitial layer of the arterial 36,43,44 pathological cues induce these changes: factors from wall. Upon injury, mesenchymal-like vSMCs prolif- the circulation, compounds and proteins produced by erate and migrate into the media and intima, to support activated endothelial cells, fibroblasts, perivascular adi- tissue repair, possibly leading to neointimal thicken- 43,45–47 pocytes or inflammatory cells, (lack of) mechanical stress, ing. Expression of the mesenchymal-like vSMC damaged ECM protein fragments, or ECM-derived growth marker SCA1/LY6A increases in vSMCs cultured in vitro, factors. In the next sections, we combine the information after carotid artery ligation and in a subset of vSMCs in Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2695 Yap et al Six Shades of vSMCs Illuminated by KLF4 Table. Overview of scRNA-Seq Studies That Identified vSMC Phenotypes Number of vSMC vSMC phenotype Driver of Marker of Diseased tissue Species phenotypes* classification* switch switch References −/− 31 Ascending aorta atherosclerotic Mouse (ApoE ; 6 Contractile, mesenchymal, KLF4 Chen et al plaque and aneurysm Myh11CreERT2;mT/ fibroblast, macrophage, 2× mGf/f;Tgfbr2f/f) osteogenic, adipocyte Carotid atherosclerotic plaque Human 3 Contractile, mesenchymal, KLF4 Depuydt et al macrophage Brachiocephalic atherosclerotic Mouse (Myh11- 5 Contractile, mesenchymal, KLF4 Alencar et al plaque CreERT2;Rosa- fibroblast, macrophage, 2× −/− eYFP;ApoE ) osteogenic Carotid atherosclerotic plaque Human 5 Contractile, mesenchymal, KLF4 Alencar et al fibroblast, macrophage, 2× osteogenic Marfan syndrome aortic aneurysm Mouse (Fbn1-C1041G/+) 2 Contractile, fibroblast KLF4 Pedroza et al Marfan syndrome aortic aneurysm Human (Fbn1c.7988G>A) 2 Contractile, fibroblast KLF4 Pedroza et al Carotid artery atherosclerotic plaque Human 1 Contractile KLF4 Hartman et al Brachiocephalic artery plaque Mouse (Myh11- 8 Unspecified KLF4 Hartman et al CreERT2;eYFP; −/− Δ/Δ ApoE ;Klf4 ) Infrarenal abdominal aortic aneurysm Mouse (C57BL/6J) 3 2× contractile, mesenchy- KLF4 Zhao et al mal, macrophage Aorta atherosclerotic plaque Mouse (ROSA26Zs- 4 Contractile, mesenchymal, RAR Pan et al −/− Green1/+; Ldlr ; Myh11- fibroblast, macrophage CreERT2) Carotid and coronary artery Human 4 Contractile, mesenchymal, RAR Pan et al atherosclerotic plaque fibroblast, macrophage Aorta atherosclerotic plaque Mouse (Myh11-CreERt2/ 4 Contractile, mesenchymal, SCA1 Dobnikar et al −/− Confetti, ApoE ) osteogenic, macrophage Aortic root and ascending aorta Mouse (TgMyh11-CreERT2; 3 Contractile, fibroblast, TCF21, Kim et al −/− atherosclerotic plaque ROSAtdT/tdT; ApoE ) osteogenic AHR Aortic root and ascending aorta Mouse (TgMyh11-CreERT2; 2 2× contractile, fibroblast TCF21 Wirka et al −/− atherosclerotic plaque ROSAtdT/tdT; ApoE ) Coronary artery atherosclerotic plaque Human 2 Contractile, fibroblast TCF21 Wirka et al Ascending aortic aneurysm Human 3 2× contractile, 2× mesen- ERG↓ Li et al chymal, fibroblast 2× indicates 2 different subtypes described; ERG, erythroblast transformation-specific related gene; KLF4, Krüppel-like factor 4; RAR, retinoic acid receptor; SCA1, stem cell antigen-1; scRNA-seq, single-cell RNA sequencing; TCF21, transcription factor 21; and vSMC, vascular smooth muscle cell. *Number and classification of vSMC phenotypes are indicated according to the categorization used in this review. atherosclerotic plaques. Interestingly, a recent genetic of non-vSMC-derived adventitial SCA1/LY6A–positive fate mapping study reported only minimal involvement cells. Consistent with these in vivo findings, KLF4 over- of SCA1/LY6A-positive vSMCs in atherosclerotic neo- expression in cultured vSMCs promotes the formation of intima formation, suggesting an injury- and/or context- a progenitor cell phenotype with loss of vSMC differen- 48 36 dependent role of the SCA1/LY6A vSMC population. tiation markers. Moreover, KLF4 overexpression inhib- To further investigate the plasticity of human vSMCs its expression of vSMC contractile markers, possibly by switching to mesenchymal-like vSMCs as a source of repressing the expression of MYOCD. reparative vSMCs, it is important to identify the human KLF4 is regulated by various signaling complexes 57,58 SCA1/LY6A orthologue. This will establish whether the at transcriptional and posttranslational levels. After mesenchymal-like vSMC population is also a prerequisite exposure to PDGF-BB (platelet-derived growth factor for tissue regeneration in human vascular disease. BB), a stimulus of vSMC proliferation and phenotype 21,54,59,60 In several mouse vascular disease models, KLF4 sig- switching, elevated levels of KLF4 were identified. naling has been shown to induce vSMC differentiation PDGF-BB induces KLF4 expression via its receptor toward a mesenchymal-like vSMC type in a context- and subsequent transcription factor Sp1 (stimulating 36,50–55 55 dependent manner. In the adventitia, a population protein-1) activation. Interestingly, Sp1 also enhances of vSMC-derived SCA1/LY6A-positive cells is formed ACTA2 expression via TGF-β1 signaling. TGF-β1 is upon induction of KLF4. Targeted deletion of KLF4 known to induce vSMC contractile proteins via activa- in vSMCs resulted in the elective loss of these vSMC- tion of specific SMAD transcription factor family mem- 61 62 derived adventitial SCA1/LY6A-positive cells, but not bers, promoting the vSMC contractile phenotype. 2696 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 Indeed, deficiency of SMAD3 disrupts TGF-β signaling of the aortic wall and a reduced number of contractile and decreases gene expression of the contractile vSMC vSMCs, indicating that miR-143/145 are important 63 51,72 phenotype markers. Thus, there seems to be a dual role in maintaining the contractile vSMC phenotype. for Sp1 in vSMC phenotype modulation. PDGF-BB also In addition, miR-1 repressed KLF4 to regulate vSMC induced DOCK2 (dedicator of cytokinesis 2) and Olfm2 differentiation. Specifically, miR1 expression was 64,65 (olfactomedin 2). DOCK2 inhibits MYOCD-induced increased during differentiation of pluripotent embry- vSMC marker promoter activity. In addition, DOCK2 and onic stem cells toward vSMCs, and inhibition of miR1 64 51 KLF4 cooperatively inhibit MYOCD-SRF interaction. repressed vSMC differentiation. Olfm2 promotes the interaction of SRF with RUNX2 To prevent or reverse the phenotypic transition to (runt-related transcription factor 2), leading eventually to mesenchymal-like vSMCs, the expression of KLF4 can reduction of vSMC marker gene transcription and conse- be suppressed by TGF-β or miR-143/145 to maintain 65 24,73,74 quently vSMC phenotypic modulation. the contractile vSMC phenotype. Indeed, treatment KLF4 activity is modulated by retinoic acid as of mesenchymal-like vSMCs with TGF-β for 3 days 8,50,66 well. Retinoic acid and PDGF-BB have opposite increased ACTA2 expression, indicating an adoption of effects on vSMC proliferation and differentiation by a more contractile phenotype. Also, the transcription changing the phosphorylation and acetylation state of factor erythroblast transformation-specific related gene, KLF4 in different ways, causing it to preferentially bind which is involved in handling reactive oxygen species and 50,66–68 to different regions within the TAGLN promoter. endoplasmic reticulum (ER) signaling, was decreased in Retinoic acid receptor activation leads to the phosphor- mesenchymal-like vSMCs. Increasing the expression of ylation of KLF4, thereby facilitates its acetylation and erythroblast transformation-specific related gene, which subsequent translocation to different transcriptional plays an important role in maintaining normal aortic wall activation domains, alleviating the repression of con- function, promotes the contractile vSMC phenotype. tractile gene expression. Alternatively, mesenchymal-like vSMC may undergo fur- Furthermore, it has been reported in human vSMCs, ther changes into other vSMC phenotypes. human tissue, and mouse models that the DNA-mod- ifying enzyme TET2 (ten-eleven translocation-2) is FIBROBLAST-LIKE VSMCS upregulated in contractile vSMCs and reduced in dedif- ferentiation vSMCs. Knockdown of TET2 inhibited Fibroblasts are cells, which are primarily responsible for expression of MYOCD and SRF with transcriptional the production and modification of ECM proteins, such 76,77 upregulation of KLF4, thus preventing vSMC differen- as collagens and fibronectin throughout tissues. In tiation to contractile vSMCs. Another member of the response to injury, fibroblasts can transition into myo- Krüppel family, namely ZFP148 (zinc finger protein 148) fibroblasts, which generate collagen-rich scar tissue also modulates vSMC phenotype transition by inhibiting to repair a wound. Subsequently, extensive remodeling 76,78–80 vSMC contractile marker expression in a neurofibromin will take place to resolve the scar. Most scRNA- 27,70 1-dependent manner. seq studies have reported fibroblasts-like vSMCs as a In line with the observation that vSMC phenotype phenotype (Table). Generally, the fibroblast-like vSMC switching is context-dependent, sex differences have phenotype is observed following vascular injury, such as also been detected in a recent scRNA-seq study compar- during atherosclerotic progression or aortic aneurysm ing atherosclerotic plaque tissue of males and females. formation. This phenotype is also referred to as myofi- 7,81 Different gene expression profiles were observed with broblasts-like vSMCs or fibromyocytes. a more pronounced KLF4-driven pattern in females Fibroblast-like vSMCs shift toward, but cluster sepa- compared with males. In a vSMC-specific KLF4 knock- rately from, authentic fibroblasts in scRNA-seq analy- out mouse model, expression of several female-biased ses performed in atherosclerotic lesions of murine and genes was observed (FN1 was downregulated; MFAP4, human arteries. Fibroblast-like vSMCs express ACTA2, CNN1, NDRG2, GAS6, and OSR1 were upregulated). SCA1/LY6A, and the fibroblast markers lumican (LUM), Such sex-specific differences in vSMC phenotype are biglycan (BGN), and decorin (DCN). The cells show sub- undervalued and require more attention in future studies. stantially reduced levels of contractile markers (TAGLN, 7,82 The role of miRNAs in the (de)differentiation of CNN1) as compared with contractile vSMCs. Pheno- 51,52 vSMCs has been highlighted in dedicated reviews. type switching to a fibroblast-like vSMC was observed MiR-143 and miR-145 are crucial for the fate of in the thoracic aortic aneurysm of a Marfan syndrome vSMCs, facilitating vSMC differentiation and inhibit- mouse model. Profiling of gene expression in that clus- 51,52 ing proliferation. MiRNA-143/145 are controlled ter revealed enhanced expression of genes involved in by transcription factors NKx2-5 (NK2 transcription adhesion, ECM organization, cellular proliferation, and factor related locus 5) and SRF with its coactivator deposition of collagen. The latter is considered a hall- MYOCD and target KLF4, KLF5, and Sp1. Defi- mark for aortic aneurysm formation, which involves aortic ciency of miRNA143/145 in mice resulted in thinning fibrosis and stiffness. Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2697 Yap et al Six Shades of vSMCs Illuminated by KLF4 A number of factors have been identified that induce be concluded that the fibroblast-like vSMCs are part of this fibroblast-like vSMC phenotype, summarized below. mesenchymal-like vSMCs or an entirely separate cell It has been shown that adventitial SCA1/LY6A–positive type without additional research. However, as the gene fibroblast-like vSMCs arise in a KLF4-dependent man- expression patterns still differ, currently, a distinction ner. The adventitial SCA1/LY6A-derived fibroblast-like between the 2 cell states has been made. vSMCs may even migrate into the intima where they Reverse differentiation from a fibroblast-like pheno- promote a fibrotic response, which stiffens the vessel type back to the contractile vSMCs phenotype has been wall. Interestingly, cholesterol or oxidized phospholipid described for DKK3 (dickkopf 3), which is a Wingless- exposure can also induce a phenotypic switch of vSMCs related integration site (Wnt) inhibitor. DKK3 was shown as shown by induced expression of fibroblast and macro- to be involved in the differentiation of stem and progeni- 40 −/− 88 phage markers. Fibroblast markers upregulated under tor cells to contractile vSMCs in ApoE mice. DKK3 these conditions were fibronectin 1 (FN1), Ecrg4 augurin induces differentiation of SCA1/LY6A-positive mes- precursor (ECRG4), proteoglycan 4 (PRG4), secreted enchymal-like and fibroblasts-like vSMCs to contrac- phosphoprotein 1 (SPP1), lipocalin-2 (LP2), metallopro- tile vSMCs via activation of TGF-β in a Wnt-dependent teinase inhibitor 1 (TIMP1), BGN, and DCN. manner. While TCF21 seems to reduce atherosclerosis Intriguingly, the ER unfolded protein response (UPR) severity by promoting the fibroblast-like vSMC, DKK3 can promote phenotypic switching in vSMCs toward the promotes protective atherosclerotic plaque stabilization fibroblast-like phenotype as well. The ER normally has by increasing the number of contractile vSMCs. There- a low cholesterol content, while accumulation of free fore, it is not yet clear which vSMC phenotype is actually cholesterol in the ER induces membrane dysfunction beneficial in the context of atherosclerosis. 84,85 and subsequent ER stress, causing UPR. Moreover, the UPR promotes a macrophage-like vSMC phenotype, MACROPHAGE-LIKE VSMCS which may explain the appearance of both fibroblast- like and macrophage-like vSMCs in atherosclerotic Macrophages play a central role in all stages of inflam- plaques. Even without cholesterol exposure, chemically mation and healing and recruit other immune cells to induced UPR is sufficient to cause phenotype switching initiate an appropriate immune response to clear debris 40 89 of vSMCs to fibroblast-like or macrophage-like vSMCs. and combat pathogens. In atherosclerosis, macro- The underlying mechanism of this phenotypic switch is phages are known to clear the vascular wall of oxidized linked to the UPR effector ATF4 (activating transcrip- LDL (low-density lipoprotein), in the process becom- tion factor 4). ATF4 prevents proteasomal degradation ing foam cells. As stated earlier, vSMCs can acquire of KLF4, and this enhanced KLF4 expression promotes a macrophage-like phenotype, even with phagocytic 40 91 atherosclerotic plaque formation. properties, as reported in 6 out of eleven selected The vSMC-specific knockout of TCF21 (transcription scRNA-seq studies (Table). Phenotype switching from factor 21) in hyperlipidemic apolipoprotein E deficient a contractile vSMC, via the mesenchymal-like to a mac- −/− (ApoE ) mice led to fewer fibroblast-like vSMCs in the rophage-like vSMC, is associated with development of 7 29,91,92 protective fibrous cap of the atherosclerotic lesions. atherosclerosis. Moreover, high TCF21 expression is associated with Macrophage-like vSMCs are typically indicated by decreased coronary artery disease risk in humans, pos- expression of LGALS3 and classical macrophage mark- sibly due to a more stable and fibroblast-like vSMC-rich ers such as CD11b, CD45, CD68, CD116, and F4/80 plaque. In addition, TCF21 is activated early on in coro- (for murine macrophages). Phenotype switching to nary artery disease and directly inhibits SMAD3-medi- macrophage-like vSMCs also involves KLF4 signal- 29,91,92 ated gene expression, thereby reducing expression of ing. A conditional KLF4 knockout in an athero- the contractile markers. Together, these studies point sclerotic mouse model results in reduced vSMC-derived to TCF21 as a regulator of vSMC phenotype switching mesenchymal-like cells and macrophage-like cells, plus toward a protective fibroblast-like vSMC population in a marked reduction in lesion size and increased plaque 29,33 atherosclerosis. stability. Similar macrophage-like vSMCs have been 6,8,33,71 The vSMC phenotypes distinct from the contractile identified in human atherosclerotic plaques. A low 7,8 type are often described as modulated vSMCs. Gene number of cells within the vSMC group was KLF4 posi- expression profiles of these modulated vSMCs comprise tive, indicating that vSMCs probably only have transient markers of mesenchymal-like and fibroblast-like but also KLF4 expression. One of these vSMC clusters was char- 7,8,40 of macrophage-like or osteogenic-like cells. Many acterized by ACTA2, LGALS3, and CD68 expression, fibroblast-like vSMCs originate from the mesenchymal- typical for the macrophage-like phenotype. However, like pool of vSMCs, although it is challenging to distin- identification of macrophage-like cells is often based on guish between these phenotypes. It is also not clear if different macrophage or foam cell markers between the all fibroblast-like vSMCs first, and entirely, transition via scRNA-seq studies, with LGALS3 and CD68 being the the mesenchymal-like vSMC state. Therefore, it cannot most common markers. 2698 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 KLF4 gene expression in vSMCs facilitates foam cell from cells and transport to the liver to remove cholesterol formation by enhancing the uptake of cholesterol-rich from the periphery. Exposure to HDL reduces the mac- lipoproteins. While classically lipid-laden foam cells rophage-like vSMC phenotype by increasing MYOCD were considered to be solely derived from monocytes/ and miR-143/145 expression in vSMCs in vitro. The macrophages, it has become evident that vSMCs with question remains whether macrophage-like vSMCs have a macrophage-like phenotype are abundantly present similar functions as monocyte-derived macrophage sub- 94 8 in the atherosclerotic plaque. Upon high cholesterol sets in atherosclerosis. exposure, expression of KLF4 was induced. This trans- forms contractile vSMCs into macrophage-like vSMCs, OSTEOGENIC-LIKE VSMCS a cell population contributing to disease progression. Switching of vSMCs to a macrophage-like phenotype The natural function of chondrocytes is to form and coincides with loss of the contractile vSMC factor maintain cartilage, while that of osteoblasts is to gen- 91,96,97 102,103 MYOCD. Conversely, gain of MYOCD expression erate bone tissue. During ossification, hypertrophic inhibits macrophage-like vSMC accumulation in athero- chondrocytes produce a unique ECM that mineralizes, sclerotic lesions in vivo. One of the Yamanaka factors, enabling cells to differentiate into osteoblasts. Both OCT4, also plays a role in regulating vSMC phenotypic chondrocytes and osteoblasts are of mesenchymal ori- transition, but interestingly in a contrasting way com- gin and share a common precursor. Switching from a pared with KLF4. Deficiency of KLF4 or OCT4 resulted mesenchymal-like vSMC phenotype to a chondrocyte- in opposite patterns of gene expression in vSMCs. like or osteoblast-like vSMC has been identified in 4 out During atherosclerosis, deficiency of KLF4 showed of the 11 scRNA-seq studies (Table). In this review, we reduced lesion size, while OCT4 deficiency showed an classified chondrocyte-like and osteoblast-like vSMC increase in lesion size. Decreased lesion size was in phenotypes together as osteogenic-like vSMCs. 33,98 turn consistent with increased plaque stability. Deposition of calcium phosphate can drive vSMC phe- Various scRNA-seq analyses have been performed on notype switching to contribute to various cardiovascular 4,7,8,31,33,99 −/− 105,106 atherosclerotic plaques of mice. In ApoE mice diseases. Calcification of the intimal layer is associ- fed a western diet, vSMCs expressing Lgals3 compose up ated with arterial obstruction and atherosclerotic plaque to two thirds of all vSMCs in the atherosclerotic lesion. rupture, while calcification of the medial layer is associ- This macrophage-like phenotype was similarly found by ated with vessel stiffening leading to heart failure. In −/− 29,31,33,90,93 others in ApoE mice, as well as in LDL recep- addition, vascular calcification has been associated with −/− 8 tor knockout (LDLR ) mice. Through fate mapping, it hypertension, osteoporosis, rheumatoid arthritis, chronic appears that these macrophage-like vSMCs were derived kidney disease, diabetes type II, and aortic aneurysm for- 8 106–108 from a multipotent vSMC-derived intermediate cell state, mation. Interestingly, overexpression of the Twist which bear similarity to the mesenchymal-like vSMCs. family BHLH transcription factor 1 (TWIST1), a coronary Recent evidence even suggests that a considerable sub- artery disease risk gene, in rat aortic vSMCs increases set of the plaque may originate from dedifferentiated cell proliferation and decreases calcification, whereas 100 109 vSMCs, which proliferate in a clonal fashion. TWIST1 knockdown has the opposite effect. Of note, Other extracellular stimuli playing a role in phenotypic TWIST1 was also found to be differentially expressed in switching to macrophage-like vSMCs are nitric oxide and several vSMC clusters in ascending aorta aneurysm tis- natriuretic peptides. In response to these compounds, sue analyzed by scRNA-seq. vSMCs generate cyclic GMP, which induces vasodilation, The osteogenic-like vSMC phenotype is charac- enhancing blood flow. Interestingly, PRKG1 (cyclic terized by a loss of contractile markers (SM22α and GMP–dependent protein kinase 1) activation also con- ACTA2) and an increase in calcification markers, such tributes to the formation of macrophage-like vSMCs as osteogenic transcription factors MSX2 (Msh Homeo- that reside within the atherosclerotic plaque. Under box 2), Cbfa1 (core-binding factor α-1, also known as atherogenic conditions, vSMCs migrate to the athero- RUNX2), and Sp7/Osterix, as well as the chondrogenic 4,106,107,110 sclerotic plaque (intima), which in Prkg1-deficient mice transcription factor SOX9. Other markers such remain in the medial layer. Using cell-fate mapping, it was as osteopontin, osteocalcin, alkaline phosphatase, col- shown that Prkg1 is involved in phenotype switching of lagen II, and collagen X were reported as markers of 106,111 vSMCs to macrophage-like vSMCs in the plaques. In line this osteogenic-like vSMC phenotype as well. The with this, postnatal ablation of Prkg1 in murine vSMCs expression of RUNX2 and SOX9 is a main determinant resulted in smaller lesions. This study also demon- of the osteogenic-like vSMC phenotype, since these 2 strates that macrophage-like vSMCs are derived from transcription factors drive the osteoblast or chondro- 101 106,112 mature vSMCs that migrated into the plaque. cyte phenotype, respectively. Reverse differentiation of the macrophage-like vSMC Deficiency of RUNX2 in vSMCs prevents differen- phenotype is accomplished by HDL (high-density lipo- tiation into the osteogenic-like vSMC phenotype, shown 107,113,114 protein). HDL is responsible for the efflux of cholesterol both in vitro and in vivo. In vitro, RUNX2, Osterix, Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2699 Yap et al Six Shades of vSMCs Illuminated by KLF4 and alkaline phosphatase expression is required to drive ADIPOCYTE-LIKE VSMCS the calcification process. This process also involves An adipocyte-like vSMC phenotype has only been the PKB (protein kinase B) or AKT and c-JNK (Jun described in a single scRNA-seq study (Table). Using an N-terminal kinase) signaling pathways. Human vSMCs in vivo fate mapping approach, with either a constitutive or exposed to a calcifying medium show decreased AKT an inducible Myh11-driven Cre mouse model, it has been phosphorylation and a transient increase in JNK activ- demonstrated that vSMCs are able to adapt toward this ity. This is in line with the observation that elevated adipocyte-like phenotype. These adipocyte-like vSMC levels of AKT and JNK protect from vascular calcifica- were classified as beige adipocytes, which are also known tion. Another pathway involves Wnt signaling, which as inducible brown adipocytes. Brown/beige adipocytes promotes vSMC differentiation to osteogenic-like cells regulate thermogenesis by producing heat when burning primarily through RUNX2. Activation of the Wnt cas- fatty acids in the presence of an UCP-1 (uncoupling pro- cade in osteogenic-like vSMCs increases BMP2 (bone tein-1), while white adipocytes store triglycerides to save morphogenetic protein 2) signaling, which plays a 122,123 energy. Sustained thermogenic activation leads to significant role in vascular calcification. BMPs are part the browning of white adipose tissue, in which a popula- of the TGF-β superfamily, which signal via the SMAD tion of adipocytes differentiate into beige adipocytes. transcription factors -1 and -5, regulating RUNX2. Given that only 1 scRNA-seq study reports adipocyte-like Interestingly, in disease models associated with vascu- vSMCs, this may indicate that a higher barrier exists for lar calcification, KLF4 was demonstrated to decrease differentiation towards an adipocyte-like vSMC type or expression of vSMC differentiation makers and induced that cues to induce this phenotype are not widespread in osteogenic genes. KLF4 was also demonstrated atherosclerotic and aneurysm tissue. to regulate RUNX2 transcription, with knockdown of Adipocyte-like vSMCs express the crucial beige adi- KLF4 inhibiting upregulation of RUNX2 and vSMC pocyte marker UCP1, as mentioned earlier, as well as calcification. In addition, KLF4 enhanced chondro- PPARγ (peroxisome proliferator-activated receptor cyte differentiation as characterized by upregulation of gamma) coactivator 1 alpha (PPARGC1A), transmem- SOX9 and downregulation of the chondrocyte dediffer- brane protein 26 (TMEM26), PR-domain containing 16 entiation marker Col1α1. In the context of scRNA- (PRDM16), and the temperature-sensitive ion channel seq, loss of KLF4 in vSMCs coincided with reduction transient receptor potential cation channel subfamily V of an osteogenic phenotype. However, the exact link member 1 (TRPV1). Upon 7-day cold exposure, these between KLF4 and osteogenic-like cells is not fully cells are able to mature further to brown adipocyte-like clear and requires further research. vSMC expressing UCP1, adiponectin (ADIPOQ), cell An in vivo study recapitulated vSMC differentiation death inducing DFFA like effector A (CIDEA), and iodo- into an osteogenic-like phenotype, by demonstrating thyronine deiodinase 2 (DIO2). Furthermore, KLF4 is that MGP (matrix gla protein)-deficient mice develop suggested to regulate early adipogenesis to induce C/ severe calcification of vSMCs in arterial blood ves- EBPβ (CCAAT-enhancer-binding protein beta). C/EBPβ sels. Interestingly, MGP-deficient mice also lacking stimulates expression of PPARγ, which is required for HDAC9 (histone deacetylase 9) had a 40% reduction adipocyte differentiation. However, if this pathway is in aortic calcification with improved survival. Thus also activated in vSMC by KLF4 has yet to be determined. the presence of HDAC9 causes progression toward Conversion of contractile vSMCs to adipocyte-like the osteogenic-like vSMC phenotype induced by the vSMCs within the vessel wall is presumably not without absence of MGP. consequences, but as this phenotype is least studied, this Another pathway involved in the suppression of cal- will require more research to understand its relevance. cification is based on aryl hydrocarbon receptor (AHR)- mediated gene expression. This transcription factor is involved in stem cell maintenance and cellular differen- EXPANDING THE VIEW ON VSMC tiation and is a downstream target of TCF21. TCF21 PHENOTYPES promotes vSMC phenotype switching to fibroblast-like cells and knockdown of AHR resulted in switching of Recent scRNA-seq analyses of human and mouse vas- fibroblast-like vSMCs to the osteogenic-like vSMCs. This cular tissues disclose a previously not well-defined diver- is in line with the ability of AHR to suppress SOX9 and sity of arterial vSMC phenotypes. We reasoned that the RUNX2 expression. Interestingly, TCF21 knockdown classification of vSMC phenotypes should be expanded prevented both the fibroblast-like and osteogenic-like beyond the traditional exclusive division of contractile vSMC differentiation, indicating that the fibroblast-like and synthetic vSMCs. In this review, we summarized the phenotype may first be required before transition towards current evidence for the presence of 6 distinct vSMC the osteoblast-like phenotype can occur. Taken phenotypes in (diseased) vascular tissue: contractile, together, TCF21 can be considered a driver and AHR an mesenchymal-like, fibroblast-like, macrophage-like, inhibitor of these vSMC phenotypes. osteogenic-like, and adipocyte-like vSMCs (Figure 1A). 2700 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 Figure 1. Schematic overview of vascular smooth muscle cell (vSMC) phenotypes. A, Overview of vSMC phenotypes and associated gene expression markers. The contractile phenotype is associated with the expression of contractile genes. Mesenchymal-like vSMCs lose expression of these contractile markers and adopt expression of a number of specific markers including the key player of phenotype switching: KLF4 (Krüppel-like factor 4). From this vSMC phenotype, differentiation to fibroblast-, macrophage-, osteogenic-, or adipocyte-like phenotypes seems to occur. Genes typical for the contractile phenotype are 5,7,141,142 CNN1, SMTN, ACTA2, TAGLN, MYH11, and MYOCD, for the mesenchymal phenotype KLF4, CD34, CD44, and SCA1/ 36,37,143,144 7 LY6a, for the fibroblast phenotype FN1, BGN, DCN, and COL1A1, for the macrophage phenotype CD45, CD68, ITGAM, 7,93,145 106,107,120 LGALS3, ITGAM, and ADGRE1, for the osteogenic phenotype SP-7, SOX9, MSX2, and RUNX2, and for the adipocyte phenotype UCP1, ELOVL3, ADIPOQ, PRDM16, and PPARGC1A. B, Activating and inhibitory stimuli of phenotype switching in vSMCs. Stimuli that mediate specific phenotype switching are indicated. Blue arrows and names indicate a transition to the mesenchymal- like vSMC, red arrows and names indicate a transition to the contractile-like vSMC, and black arrow and names indicate a transition to the other phenotypes in A and B. AHR indicates aryl hydrocarbon receptor; BMP2, bone morphogenetic protein 2; DKK3, dickkopf 3; HDAC9, histone deacetylase 9; HDL, high-density lipoprotein; MGP, matrix gla protein; PDGF-BB, platelet-derived growth factor BB; RA, retinoic acid; and TGF-β, transforming growth factor beta. Combining the scRNA-seq data on vSMC subtypes with as none of the phenotypes seem to be a final state of knowledge on vSMC function, one may speculate that cellular differentiation. Rather, vSMCs can go back and there is an initial transition of the contractile vSMC dedif- forth between different phenotypes, triggered by specific ferentiating into mesenchymal-like vSMCs, followed by stimuli (Figure 1B). The exact phenotypic composition of differentiation towards the other 4 vSMC phenotypes. vSMCs in the vessel wall may determine vascular pathol- A remarkable characteristic of vSMCs is their plasticity, ogy in various cardiovascular diseases, and at present Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2701 Yap et al Six Shades of vSMCs Illuminated by KLF4 the consequence of the relative prevalence of these 6 shades of vSMC is not fully understood. Moreover, pre- sumably there are even more vSMC flavors as indicated by 2 distinct contractile, mesenchymal- and osteogenic- 7,31,33,41,75 like vSMC populations in different studies, and 8 undefined vSMC populations in the study from Hartman et al. (Table). KLF4 AND ITS CRUCIAL ROLE IN VSMC PHENOTYPE SWITCHING The transcription factors KLF4, retinoic acid receptor, TCF21, AHR, and erythroblast transformation-specific related gene are all to some extent involved in vSMC phenotype switching in cardiovascular diseases, with a key role for KLF4. Therefore, we summarize the cur- rent knowledge on modulation of KLF4 to interfere with vSMC phenotype switching. KLF4 is known to regulate gene expression via different mechanisms, not merely as Figure 2. KLF4 (Krüppel-like factor 4) facilitates phenotypic DNA-binding transcription factor, which may in part switching of vascular smooth muscle cells (vSMCs). explain its involvement in the different vSMC phenotypes. Schematic overview of the role of KLF4 in vSMC phenotype The sections above substantiate the importance of switching. In contractile vSMCs (1), the transcription factor complex KLF4 in relation to vSMC phenotype switching. The dis- MYOCD (myocardin)-SRF (serum response factor) induces the tinct role of KLF4 per vSMC phenotype with respect to expression of contractile genes. The transcriptional activity of KLF4 is balanced by protein ubiquitination and subsequent degradation. gene regulation and transcription factor cooperation is Acetylated KLF4, induced by all-trans retinoic acid stimulation, context-dependent and affected by additional stimuli, alleviates the KLF4-induced repression of contractile gene as summarized in Figure 2. In the contractile vSMC 50,68 expression. In the mesenchymal vSMC phenotype (2), KLF4 phenotype, the activated transcription factor complex expression is increased and stabilized to repress the expression of 50,55,66 MYOCD-SRF induces the expression of contractile pro- contractile genes by reduction of formation of the MYOCD- SRF complex. In the fibroblast-like vSMC (3), the unfolded protein teins. In this situation, KLF4 expression is balanced response (UPR) induces KLF4 expression, which affects the vSMC by protein ubiquitination and subsequent degrada- 40,99 phenotypic switch by an unknown mechanism. The switching tion. Posttranslational modification through acetylation toward a macrophage-like vSMC (4) also requires UPR and/or of KLF4 changes its binding to preferential DNA sites oxidized LDL (oxLDL) uptake to increase KLF4, which by an unknown to alleviate the repression of contractile gene expres- mechanism induced the switch toward foam cell formation. The transition to an osteogenic-like vSMC (5) is facilitated by a KLF4- sion. In the mesenchymal-like vSMC phenotype, KLF4 mediated activation of RUNX2 (runt-related transcription factor expression is upregulated, which reduces MYOCD-SRF 2) or SOX9-induced transcriptional programmes, which allows complex formation and thereby inhibits expression of 4,106,107,110,127 calcification by the release of extracellular vesicles (EVs). 50,54,55,66 contractile proteins. KLF4 then increases mes- The phenotype switch to an adipocyte-like vSMC (6) could enchymal marker expression: SCA1, CD34, and CD44. hypothetically require activation of KLF4, leading to a KLF4-mediated increase in C/EBP and subsequent PPARy (peroxisome proliferator- When KLF4 signaling is enhanced in the fibroblast-like activated receptor gamma) transcription factor activation to induce vSMC phenotype, this is linked to the UPR and its effec- adipogenesis and uptake of triglycerides (TGs). Green arrow tor factor ATF4. KLF4 has also been associated with indicates expression of contractile proteins, and red arrow indicates macrophage foam cell formation by enhancing uptake of expression of phenotype specific genes. Ac indicates acetylation; C, cholesterol-rich lipoproteins. Switching of vSMCs toward cytoplasm; N, nucleus; and Ub, ubiquitination. a macrophage-like phenotype may be induced by KLF4 through enhanced UPR or lipid uptake, which can lead to with a vSMC-specific conditional deficiency of KLF4. 40,94 foam cell formation. The transition toward an osteo- These mice show an increase in the number of contrac- genic-like phenotype requires the induction of RUNX2 tile vSMCs, and a substantial reduction of macrophage- or SOX9 to induce vascular calcification by releasing like and osteogenic-like vSMCs. Moreover, Chen et al 4,106,107,110,127 extracellular vesicles. Activation of KLF4 observed a decrease in overall atherosclerotic burden may be required for switching toward an adipocyte-like and a reduction in the development of aortic aneurysms phenotype, which could lead to adipogenesis through C/ (regardless of cholesterol levels), in vSMC-specific KLF4 EBPβ and PPARγ signaling. knockout mice. A KLF4 signature was also identified C1041G/+ In atherosclerosis, Alencar et al identified a reduc- in aneurysmal aortic tissue of fibrillin-1 (Fbn1) tion in lesion size and increased plaque stability in mice Marfan syndrome mice. Furthermore, using an in vitro 2702 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 induced pluripotent stem cell–derived vSMC model with tissues collected at a single point in time, especially in distinct FBN1 mutations from Marfan syndrome patients, human end-stage disease. To understand the dynam- knockdown of KLF4 restored Fbn1 fiber deposition, ics of vSMC phenotype plasticity between the 6 vSMC vSMC proliferation defects, and contractile function. phenotypes, phenotype switching over time needs to In contrast, a marked reduction in KLF4 expression was be explored in detail. To date, only Pan et al performed observed in the aorta of very young Fbn1 hypomorphic scRNA-seq and lineage tracing in mice over various time (mgR/mgR model) Marfan mice, which was concomi- points and suggested that this method provided both tant with an increase in contractile vSMCs. Together, sensitive and specificity for tracking vSMC behaviors these data may indicate that the contractile population of during atherosclerosis. vSMCs is unable to undergo phenotype switching during In addition, we argued that the data point in the direc- 5,130 early stages of aneurysm formation. However, vSMC- tion of the mesenchymal-like cells giving rise to the specific KLF4-deficient Marfan mice are yet to be gener- other vSMC phenotypes; however, further experimental ated to precisely determine the effect of KLF4 in aortic validation is needed to fully support this hypothesis. Fur- aneurysm formation. thermore, it remains challenging to recognize the vSMC These findings emphasize KLF4 as an interesting origin of a cell population, especially in nonlineage traced potential drug target to diminish vascular disease. As human tissue samples. However, even conventional lin- there are no highly selective KLF4 antagonists, KLF4 eage tracing studies have their limitations; they can only expression can be targeted by indirect inhibitors, of mark the fate of the contractile vSMC but not the fate which several are in preclinical development. One of of once contractile vSMCs that underwent phenotype these inhibitors is designed to interfere with the methyla- switching with the loss of their contractile markers. tion of KLF4 by PRMT5 (protein arginine methyltransfer- Limitations and potential biases should also be ase 5). Since KLF4 has a short half-life, governed by considered when analyzing and interpreting different VHL-VBC ubiquitin-protein ligase, methylation of amino scRNA-seq datasets. As often, the data rely on a sin- acids R374, R376, and R377 prolong protein stability. gle-cell suspension representative of all cell populations The small molecule antagonist WX2–43 was designed present within tissue samples exposed to tissue disrup- to interfere with the PRMT5-KLF4 interaction, prevent- tion. scRNA-seq also lacks spatial information about the ing the methylation, thus promoting degradation. Another distribution of the different subpopulations within tissues, indirect inhibitor of KLF4 targets its interaction with and different processes can cause variations in transcript PLK1 (polo-like kinase-1). This kinase phosphorylates levels that might not reflect differences in protein lev- KLF4 at Ser234, promoting protein stability, previously els or cellular functions. Moreover, the lack of depth of linked to hyperplastic intima of injured vessels. The scRNA-seq also makes it difficult to investigate low- experimental drug BI6727, originally developed as an expression genes, which are often transcription factors. anticancer drug, is a highly selective, potent PLK1 inhibi- Although the transcriptomic signature in defining tor. Exposure to BI6727 results in reduced expression of vSMC states and transitions offers a wealth of infor- KLF4 and increased protein turnover due to reduced sta- mation, it is yet to be determined what this means on bility. Given that the translation of miRNAs into clinical a protein level and whether these vSMC-derived cells medicine will become more common in the future, it is of have similar functionality to their classical counterparts importance that several miRNAs have been reported to (eg, macrophage-like vSMC compared with a bone interact with KLF4 mRNA, and may serve as blueprint marrow-derived macrophage). The vSMC-derived cells for therapeutic oligonucleotides. The miRNAs miR- seem to be heavily influenced by their environment to 1, miR-25, miR-29, miR-143, miR-145, and miR-375 trigger the transition and may not always provide posi- induce RNAi-mediated silencing of KLF4 protein expres- tive consequences. 92,135–138 sion. Further research will establish the potential As for the function of the different vSMCs pheno- of targeting KLF4 in the context of the different car- types, their diverging roles in the broad range of CVDs diovascular diseases, where KLF4 may be beneficial in remain to be discovered. It will eventually pinpoint which one, but detrimental in another vascular disease type. In type is beneficial or harmful in specific vascular disor- addition, potential side effects of repressed KLF4 should ders. This knowledge is crucial for the development of not be overlooked, as KLF4 inhibition has been shown to innovative disease intervention strategies. 139,140 delay wound healing and elevate insulin resistance. ARTICLE INFORMATION Received June 3, 2021; accepted August 20, 2021. DISCUSSION AND PERSPECTIVES Affiliations The scRNA-seq technique, together with lineage trac- Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, ing, opens up a whole new world of possibilities to study University of Amsterdam, Amsterdam UMC, Location Academic Medical vSMCs in the normal and diseased vessel wall. Until Center, The Netherlands (C.Y., A.M., C.J.M.d.V., V.d.W.). Department of Clinical now, the scRNA-seq has been performed on (diseased) Genetics, Amsterdam Cardiovascular Sciences, Vrije Universiteit Amsterdam, Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2703 Yap et al Six Shades of vSMCs Illuminated by KLF4 Amsterdam UMC, Location VU University Medical Center, Amsterdam, The thoracic aortic aneurysms and dissections. Arterioscler Thromb Vasc Biol. Netherlands (D.M.). 2017;37:26–34. doi: 10.1161/ATVBAHA.116.303229 16. Majesky MW. Developmental basis of vascular smooth muscle diver- Acknowledgment sity. Arterioscler Thromb Vasc Biol. 2007;27:1248–1258. doi: 10.1161/ We thank Dr Dave Speijer for critical reading of the article. ATVBAHA.107.141069 17. Pfaltzgraff ER, Bader DM. Heterogeneity in vascular smooth muscle cell Sources of Funding embryonic origin in relation to adult structure, physiology, and disease. Dev Dyn. 2015;244:410–416. doi: 10.1002/dvdy.24247 This study was supported by Amsterdam Cardiovascular Sciences PhD grant 18. Intengan HD, Schiffrin EL. Structure and mechanical properties of resis- 2019 and Zeldzame Ziekten Fonds via AMC foundation (C. Yap, D. Micha, V. de tance arteries in hypertension: role of adhesion molecules and extracel- Waard) and by Health Holland TKI-Public Private Partnership grant 22532 (A. lular matrix determinants. Hypertension. 2000;36:312–318. doi: 10.1161/ Mieremet, V. de Waard). 01.hyp.36.3.312 Disclosures 19. Robertson IB, Rifkin DB. 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DOI: 10.1161/ATVBAHA.121.316600 November 2021 2707 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Arteriosclerosis Thrombosis and Vascular Biology Wolters Kluwer Health

Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Krüppel-Like Factor 4)

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Abstract

Arteriosclerosis, Thrombosis, and Vascular Biology BRIEF REVIEW Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Krüppel-Like Factor 4) Carmen Yap ,* Arnout Mieremet ,* Carlie J.M. de Vries, Dimitra Micha , Vivian de Waard ABSTRACT: Multiple layers of vascular smooth muscle cells (vSMCs) are present in blood vessels forming the media of the vessel wall. vSMCs provide a vessel wall structure, enabling it to contract and relax, thus modulating blood flow. They also play a crucial role in the development of vascular diseases, such as atherosclerosis and aortic aneurysm formation. vSMCs display a remarkable high degree of plasticity. At present, the number of different vSMC phenotypes has only partially been characterized. By mapping vSMC phenotypes in detail and identifying triggers for phenotype switching, the relevance of the different phenotypes in vascular disease may be identified. Up until recently, vSMCs were classified as either contractile or dedifferentiated (ie, synthetic). However, single-cell RNA sequencing studies revealed such dedifferentiated arterial vSMCs to be highly diverse. Currently, no consensus exist about the number of vSMC phenotypes. Therefore, we reviewed the data from relevant single-cell RNA sequencing studies, and classified a total of 6 vSMC phenotypes. The central dedifferentiated vSMC type that we classified is the mesenchymal-like phenotype. Mesenchymal-like vSMCs subsequently seem to differentiate into fibroblast-like, macrophage-like, osteogenic-like, and adipocyte-like vSMCs, which contribute differentially to vascular disease. This phenotype switching between vSMCs requires the transcription factor KLF4 (Krüppel-like factor 4). Here, we performed an integrated analysis of the data about the recently identified vSMC phenotypes, their associated gene expression profiles, and previous vSMC knowledge to better understand the role of vSMC phenotype transitions in vascular pathology. GRAPHIC ABSTRACT: A graphic abstract is available for this article. Key Words: atherosclerosis ◼ cardiovascular disease ◼ myocytes ◼ phenotype ◼ smooth muscle ardiovascular diseases are the leading cause of levels of the contractile vSMC markers, capable of phe- death worldwide. Many of these pathologies are notypic switching. There is a growing body of evidence Ccharacterized by progressive cellular modulations that multiple vSMC phenotypes exist in healthy vessels. derived from genetic predisposition, aging, or lifestyle. Single-cell RNA sequencing (scRNA-seq) studies identi- The 3 main cellular layers forming the vessel wall are the fied the presence of specific vSMC-derived cell popula- 5–9 adventitia, media, and the intima, surrounding the lumen. tions. However, there is still limited understanding of In the media, the middle layer, vascular smooth muscle the role of these vSMC phenotypes in physiological and cells (vSMCs) are the major cellular component. These pathological conditions. vSMCs contribute to the integrity of the vessels and are For decades, vSMC activation and dedifferentiation able to adequately respond to stimuli of vasoconstric- has been regarded as the adoption of a single syn- 2,3 tion and vasodilation. In healthy vessels, vSMCs are thetic, proliferative phenotype. However, as revealed regarded as quiescent, differentiated cells, which display by recent scRNA-seq analyses, the diversity of vSMC 4 10 remarkable plasticity. The contractile vSMCs are subject phenotypes is far more sophisticated. The power of to context-dependent changes and studies have shown scRNA-seq is that it provides detailed, unbiased infor- that a subset of vSMCs in healthy tissue express reduced mation on distinct cell populations within healthy or Correspondence to: Vivian de Waard, PhD, Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, University of Amsterdam, Amsterdam UMC, location Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands. Email v.dewaard@amsterdamumc.nl *C. Yap and A. Mieremet contributed equally. For Sources of Funding and Disclosures, see page 2704. © 2021 The Authors. Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited. Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2693 Yap et al Six Shades of vSMCs Illuminated by KLF4 Nonstandard Abbreviations and Acronyms Highlights AHR aryl hydrocarbon receptor • Single-cell RNA sequencing and lineage tracing analyses revealed that vascular smooth muscle cells ATF4 activating transcription factor 4 can be identified beyond the synthetic and contrac- BMP2 bone morphogenetic protein 2 tile profile. Cbfa1 core-binding factor α-1 • We discuss the phenotypic switching of vascular C/EBPβ CCAAT-enhancer-binding protein beta smooth muscle cells from a contractile state to a CVD cardiovascular disease mesenchymal-like, fibroblast-like, macrophage-like, osteogenic-like, or adipocyte-like phenotype. DKK3 dickkopf 3 • The transcription factor KLF4 (Krüppel-like factor 4) DOCK2 dedicator of cytokinesis 2 plays a pivotal role in regulation of vascular smooth ECM extracellular matrix muscle cells phenotype switching and emerges as ER endoplasmic reticulum potential drug target. HDAC9 histone deacetylase 9 • The current challenge is to pinpoint the diverging HDL high-density lipoprotein roles of vascular smooth muscle cell phenotypes and their impact on the broad range of cardiovas- JNK c-Jun N-terminal kinase cular diseases. LDL low-density lipoprotein MEF2C myocyte enhancer factor 2C MGP matrix gla protein mouse models, the fate of vSMCs can be accurately MSX2 Msh Homeobox 2 tracked and provides detailed information of their phe- MYOCD myocardin notypic modulation and contribution to diseased tis- 11,12 sue. In this review, we highlighted the information NKx2-5 NK2 transcription factor related locus 5 obtained about vSMCs and used the recent scRNA- OCT4 octamer-binding transcription factor 4 seq literature to classify 6 different vSMC phenotypes. Olfm2 olfactomedin 2 Besides a contractile phenotype, we distinguish the PDGF-BB platelet-derived growth factor BB mesenchymal-like, fibroblast-like, macrophage-like, PKB protein kinase B osteogenic-like, and adipocyte-like phenotypes. In PLK1 polo-like kinase-1 addition, we address the current insights about stimu- PRKG1 cyclic GMP–dependent protein kinase 1 lating and inhibitory cues mediating vSMC phenotype PRMT5 protein arginine methyltransferase 5 switching. From that analysis, we inferred that tran- RUNX2 runt-related transcription factor 2 scription factor KLF4 (Krüppel-like factor 4) plays a SCA1 stem cell antigen-1 pivotal role in the initial dedifferentiation of vSMCs to scRNA-seq single-cell RNA sequencing the mesenchymal-like phenotype enabling further cel- SOX2 SRY-BOX transcription factor 2 lular changes toward the other 4 vSMC phenotypes. Sp1 stimulating protein-1 SRF serum response factor CONTRACTILE VSMCS TCF21 transcription factor 21 TET2 ten-eleven translocation-2 In a healthy state, vSMCs in the media of the arterial TGF-β transforming growth factor beta vessel wall actively synthesize, secrete, modulate, and maintain the extracellular matrix (ECM) to provide elas- UPR unfolded protein response ticity and strength to the blood vessel. In the vasculature, vSMC vascular smooth muscle cell the vSMCs work in close collaboration with other cells Wnt wingless-related integration site to counterbalance the strong mechanical forces that the ZFP148 zinc finger protein 148 13,14 vessel wall experiences. A single layer of endothelial cells at the luminal side of the vessel forms the tight bar- diseased tissue. Distinct cell populations were identi- rier between vascular lumen and the vessel wall, providing fied by scRNA-seq in the vessel wall, with the major- cues for vSMC relaxation and contraction. Endothelial cell ity concerning immune cells, such as T cells, B cells, function is determined by shear stress, stretching of the myeloid cells, and mast cells. Nonimmune cells in vessel due to heart pulse, and by circulating factors. In atherosclerotic plaques and aortic aneurysm tissue the adventitia, fibroblasts produce and maintain collagen included endothelial cells and vSMCs. The combina- fibers, thereby forming a peripheral structure to preserve tion of scRNA-seq and lineage tracing is extremely vascular integrity at high pressures. In the adventitia of useful as it allows in-depth vSMC phenotypic charac- larger blood vessels, a microvasculature bed is present, terization. With the development of lineage tracing in termed the vasa vasorum. This structure protrudes into 2694 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 the outer layers of the media to provide sufficient oxygen on the different phenotypes of vSMCs as deduced from and nutrients to the multi-layered vSMCs in the media. the scRNA-seq reports with the available knowledge on Contractile vSMCs are regarded as differentiated and specific cues and signal transduction pathways involved quiescent cells under physiological conditions, express- in vSMC phenotype modulation. ing a panel of typical contractile proteins that is crucial to maintain vascular tension. Although the embryonic MESENCHYMAL-LIKE VSMCS origin of vSMCs is diverse, the contractile vSMC phe- notype is considered universal throughout the arte- Extensive lineage tracing studies revealed that con- rial vasculature. Contractile vSMCs are embedded in tractile vSMCs can switch from a contractile status to 29–31 an intricate structure of ECM composed of elastin and a mesenchymal-like phenotype. A mesenchymal-like 13,14,17 collagens as key fibers. The ECM is produced by vSMC is characterized by the ability to proliferate and vSMCs themselves and sequesters growth factors, such self-renew and is marked by reduced expression of con- as those belonging to the TGF-β (transforming growth tractile proteins. This phenotype overlaps with that of factor beta) family. Upon damage of the vessel wall, mesenchymal stem cells, which are defined as stromal the sequestered growth factors are released to induce a cells that have the capacity to differentiate into multiple 19 29,32 local repair response. lineages. Recently, a significant number of scRNA- Contractile vSMCs exhibit an elongated, spindle- seq studies confirmed that among the various vSMC shaped morphology and express a well-characterized phenotypes identified, mesenchymal-like vSMCs are set of contractile markers including smooth muscle actin also present (Table). The selected scRNA-seq studies (ACTA2), smooth muscle myosin heavy chain (MYH11), were performed on human and murine vascular tissue smooth muscle protein 22-alpha (SM22α/TAGLN), of atherosclerotic plaques or aortic aneurysms. Distinct 20–22 smoothelin (SMTN), and calponin (CNN1). Expres- vSMC clusters were specified, and based on their gene sion of these proteins is controlled by the transcription expression profile and available knowledge of upstream factors MYOCD (myocardin) and SRF (serum response modulators, we aimed to identify the main drivers of phe- factor), both of which are involved in the regulation of notypic switches. In this review, we classified stem cell differentiation to contractile vSMCs. These transcrip- marker positive vSMCs as belonging to the mesenchy- tion factors also induce expression of microRNA cluster mal-like phenotype. This included the pioneer cell phe- (miRNA)-143/145, involved in activation of the vSMC notype of Alencar et al and vSMC-derived intermediate 24 8 contractile phenotype. In addition, external stimuli, cells (SEM) by Pan et al. such as TGF-β and heparin, play a pivotal role in pro- Dedifferentiation of vSMCs from a contractile to a moting and maintaining the vSMCs contractile pheno- mesenchymal-like phenotype is driven by external stimuli type. Exposure to these factors leads to upregulation that induce repair and/or proliferation. A key initiating of structural ECM genes and inhibition of vSMC prolif- factor for this phenotypic switch is transcription factor 23,25 29,34–38 eration and migration. The phosphatase and tensin KLF4, which regulates cellular proliferation and homolog (PTEN) also helps to maintain the contractile dedifferentiation. KLF4 also has a crucial function in vSMC phenotype, as in the nucleus PTEN interacts with the induction of cellular pluripotency. In fact, genera- SRF to enhance SRF binding to essential promoter ele- tion of induced pluripotent stem cells from a wide range ments in vSMC contractile genes. MEF2C (myocyte of somatic cells requires KLF4 and 3 other, so-called enhancer factor 2C) was also shown to be essential for Yamanaka, factors: OCT4 (octamer transcription factor contractile vSMC differentiation. A detailed overview 4), SOX2 (SRY-BOX transcription factor 2) and cMYC of the contractile vSMC phenotype in the vasculature is (MYC proto-oncogene, bHLH transcription factor). 20,22,28 given elsewhere. Induction of KLF4 in vSMCs results in a phenotypic Various scRNA-seq analyses confirmed that con- switch from contractile to mesenchymal-like and initiates tractile vSMCs are the most prominent cell type in the the expression of mesenchymal markers such as stem 40,41 healthy vessel wall with the transcriptomic profile just cell antigen-1 (SCA1)/LY6A, CD34, and CD44. Dur- 5,6,8 described. Yet, in combination with lineage tracing, ing this transition, while gaining expression of mesen- additional vSMC populations have been identified with chymal markers, the contractile vSMCs lose expression 5,6,8 12,36,42 distinct gene expression profiles. Once pathological of their contractile markers. Under physiological processes in the vessel wall are initiated, vSMCs respond conditions, a subpopulation of mesenchymal-like vSMCs by changing their phenotype and function. A plethora of resides in the medial and adventitial layer of the arterial 36,43,44 pathological cues induce these changes: factors from wall. Upon injury, mesenchymal-like vSMCs prolif- the circulation, compounds and proteins produced by erate and migrate into the media and intima, to support activated endothelial cells, fibroblasts, perivascular adi- tissue repair, possibly leading to neointimal thicken- 43,45–47 pocytes or inflammatory cells, (lack of) mechanical stress, ing. Expression of the mesenchymal-like vSMC damaged ECM protein fragments, or ECM-derived growth marker SCA1/LY6A increases in vSMCs cultured in vitro, factors. In the next sections, we combine the information after carotid artery ligation and in a subset of vSMCs in Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2695 Yap et al Six Shades of vSMCs Illuminated by KLF4 Table. Overview of scRNA-Seq Studies That Identified vSMC Phenotypes Number of vSMC vSMC phenotype Driver of Marker of Diseased tissue Species phenotypes* classification* switch switch References −/− 31 Ascending aorta atherosclerotic Mouse (ApoE ; 6 Contractile, mesenchymal, KLF4 Chen et al plaque and aneurysm Myh11CreERT2;mT/ fibroblast, macrophage, 2× mGf/f;Tgfbr2f/f) osteogenic, adipocyte Carotid atherosclerotic plaque Human 3 Contractile, mesenchymal, KLF4 Depuydt et al macrophage Brachiocephalic atherosclerotic Mouse (Myh11- 5 Contractile, mesenchymal, KLF4 Alencar et al plaque CreERT2;Rosa- fibroblast, macrophage, 2× −/− eYFP;ApoE ) osteogenic Carotid atherosclerotic plaque Human 5 Contractile, mesenchymal, KLF4 Alencar et al fibroblast, macrophage, 2× osteogenic Marfan syndrome aortic aneurysm Mouse (Fbn1-C1041G/+) 2 Contractile, fibroblast KLF4 Pedroza et al Marfan syndrome aortic aneurysm Human (Fbn1c.7988G>A) 2 Contractile, fibroblast KLF4 Pedroza et al Carotid artery atherosclerotic plaque Human 1 Contractile KLF4 Hartman et al Brachiocephalic artery plaque Mouse (Myh11- 8 Unspecified KLF4 Hartman et al CreERT2;eYFP; −/− Δ/Δ ApoE ;Klf4 ) Infrarenal abdominal aortic aneurysm Mouse (C57BL/6J) 3 2× contractile, mesenchy- KLF4 Zhao et al mal, macrophage Aorta atherosclerotic plaque Mouse (ROSA26Zs- 4 Contractile, mesenchymal, RAR Pan et al −/− Green1/+; Ldlr ; Myh11- fibroblast, macrophage CreERT2) Carotid and coronary artery Human 4 Contractile, mesenchymal, RAR Pan et al atherosclerotic plaque fibroblast, macrophage Aorta atherosclerotic plaque Mouse (Myh11-CreERt2/ 4 Contractile, mesenchymal, SCA1 Dobnikar et al −/− Confetti, ApoE ) osteogenic, macrophage Aortic root and ascending aorta Mouse (TgMyh11-CreERT2; 3 Contractile, fibroblast, TCF21, Kim et al −/− atherosclerotic plaque ROSAtdT/tdT; ApoE ) osteogenic AHR Aortic root and ascending aorta Mouse (TgMyh11-CreERT2; 2 2× contractile, fibroblast TCF21 Wirka et al −/− atherosclerotic plaque ROSAtdT/tdT; ApoE ) Coronary artery atherosclerotic plaque Human 2 Contractile, fibroblast TCF21 Wirka et al Ascending aortic aneurysm Human 3 2× contractile, 2× mesen- ERG↓ Li et al chymal, fibroblast 2× indicates 2 different subtypes described; ERG, erythroblast transformation-specific related gene; KLF4, Krüppel-like factor 4; RAR, retinoic acid receptor; SCA1, stem cell antigen-1; scRNA-seq, single-cell RNA sequencing; TCF21, transcription factor 21; and vSMC, vascular smooth muscle cell. *Number and classification of vSMC phenotypes are indicated according to the categorization used in this review. atherosclerotic plaques. Interestingly, a recent genetic of non-vSMC-derived adventitial SCA1/LY6A–positive fate mapping study reported only minimal involvement cells. Consistent with these in vivo findings, KLF4 over- of SCA1/LY6A-positive vSMCs in atherosclerotic neo- expression in cultured vSMCs promotes the formation of intima formation, suggesting an injury- and/or context- a progenitor cell phenotype with loss of vSMC differen- 48 36 dependent role of the SCA1/LY6A vSMC population. tiation markers. Moreover, KLF4 overexpression inhib- To further investigate the plasticity of human vSMCs its expression of vSMC contractile markers, possibly by switching to mesenchymal-like vSMCs as a source of repressing the expression of MYOCD. reparative vSMCs, it is important to identify the human KLF4 is regulated by various signaling complexes 57,58 SCA1/LY6A orthologue. This will establish whether the at transcriptional and posttranslational levels. After mesenchymal-like vSMC population is also a prerequisite exposure to PDGF-BB (platelet-derived growth factor for tissue regeneration in human vascular disease. BB), a stimulus of vSMC proliferation and phenotype 21,54,59,60 In several mouse vascular disease models, KLF4 sig- switching, elevated levels of KLF4 were identified. naling has been shown to induce vSMC differentiation PDGF-BB induces KLF4 expression via its receptor toward a mesenchymal-like vSMC type in a context- and subsequent transcription factor Sp1 (stimulating 36,50–55 55 dependent manner. In the adventitia, a population protein-1) activation. Interestingly, Sp1 also enhances of vSMC-derived SCA1/LY6A-positive cells is formed ACTA2 expression via TGF-β1 signaling. TGF-β1 is upon induction of KLF4. Targeted deletion of KLF4 known to induce vSMC contractile proteins via activa- in vSMCs resulted in the elective loss of these vSMC- tion of specific SMAD transcription factor family mem- 61 62 derived adventitial SCA1/LY6A-positive cells, but not bers, promoting the vSMC contractile phenotype. 2696 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 Indeed, deficiency of SMAD3 disrupts TGF-β signaling of the aortic wall and a reduced number of contractile and decreases gene expression of the contractile vSMC vSMCs, indicating that miR-143/145 are important 63 51,72 phenotype markers. Thus, there seems to be a dual role in maintaining the contractile vSMC phenotype. for Sp1 in vSMC phenotype modulation. PDGF-BB also In addition, miR-1 repressed KLF4 to regulate vSMC induced DOCK2 (dedicator of cytokinesis 2) and Olfm2 differentiation. Specifically, miR1 expression was 64,65 (olfactomedin 2). DOCK2 inhibits MYOCD-induced increased during differentiation of pluripotent embry- vSMC marker promoter activity. In addition, DOCK2 and onic stem cells toward vSMCs, and inhibition of miR1 64 51 KLF4 cooperatively inhibit MYOCD-SRF interaction. repressed vSMC differentiation. Olfm2 promotes the interaction of SRF with RUNX2 To prevent or reverse the phenotypic transition to (runt-related transcription factor 2), leading eventually to mesenchymal-like vSMCs, the expression of KLF4 can reduction of vSMC marker gene transcription and conse- be suppressed by TGF-β or miR-143/145 to maintain 65 24,73,74 quently vSMC phenotypic modulation. the contractile vSMC phenotype. Indeed, treatment KLF4 activity is modulated by retinoic acid as of mesenchymal-like vSMCs with TGF-β for 3 days 8,50,66 well. Retinoic acid and PDGF-BB have opposite increased ACTA2 expression, indicating an adoption of effects on vSMC proliferation and differentiation by a more contractile phenotype. Also, the transcription changing the phosphorylation and acetylation state of factor erythroblast transformation-specific related gene, KLF4 in different ways, causing it to preferentially bind which is involved in handling reactive oxygen species and 50,66–68 to different regions within the TAGLN promoter. endoplasmic reticulum (ER) signaling, was decreased in Retinoic acid receptor activation leads to the phosphor- mesenchymal-like vSMCs. Increasing the expression of ylation of KLF4, thereby facilitates its acetylation and erythroblast transformation-specific related gene, which subsequent translocation to different transcriptional plays an important role in maintaining normal aortic wall activation domains, alleviating the repression of con- function, promotes the contractile vSMC phenotype. tractile gene expression. Alternatively, mesenchymal-like vSMC may undergo fur- Furthermore, it has been reported in human vSMCs, ther changes into other vSMC phenotypes. human tissue, and mouse models that the DNA-mod- ifying enzyme TET2 (ten-eleven translocation-2) is FIBROBLAST-LIKE VSMCS upregulated in contractile vSMCs and reduced in dedif- ferentiation vSMCs. Knockdown of TET2 inhibited Fibroblasts are cells, which are primarily responsible for expression of MYOCD and SRF with transcriptional the production and modification of ECM proteins, such 76,77 upregulation of KLF4, thus preventing vSMC differen- as collagens and fibronectin throughout tissues. In tiation to contractile vSMCs. Another member of the response to injury, fibroblasts can transition into myo- Krüppel family, namely ZFP148 (zinc finger protein 148) fibroblasts, which generate collagen-rich scar tissue also modulates vSMC phenotype transition by inhibiting to repair a wound. Subsequently, extensive remodeling 76,78–80 vSMC contractile marker expression in a neurofibromin will take place to resolve the scar. Most scRNA- 27,70 1-dependent manner. seq studies have reported fibroblasts-like vSMCs as a In line with the observation that vSMC phenotype phenotype (Table). Generally, the fibroblast-like vSMC switching is context-dependent, sex differences have phenotype is observed following vascular injury, such as also been detected in a recent scRNA-seq study compar- during atherosclerotic progression or aortic aneurysm ing atherosclerotic plaque tissue of males and females. formation. This phenotype is also referred to as myofi- 7,81 Different gene expression profiles were observed with broblasts-like vSMCs or fibromyocytes. a more pronounced KLF4-driven pattern in females Fibroblast-like vSMCs shift toward, but cluster sepa- compared with males. In a vSMC-specific KLF4 knock- rately from, authentic fibroblasts in scRNA-seq analy- out mouse model, expression of several female-biased ses performed in atherosclerotic lesions of murine and genes was observed (FN1 was downregulated; MFAP4, human arteries. Fibroblast-like vSMCs express ACTA2, CNN1, NDRG2, GAS6, and OSR1 were upregulated). SCA1/LY6A, and the fibroblast markers lumican (LUM), Such sex-specific differences in vSMC phenotype are biglycan (BGN), and decorin (DCN). The cells show sub- undervalued and require more attention in future studies. stantially reduced levels of contractile markers (TAGLN, 7,82 The role of miRNAs in the (de)differentiation of CNN1) as compared with contractile vSMCs. Pheno- 51,52 vSMCs has been highlighted in dedicated reviews. type switching to a fibroblast-like vSMC was observed MiR-143 and miR-145 are crucial for the fate of in the thoracic aortic aneurysm of a Marfan syndrome vSMCs, facilitating vSMC differentiation and inhibit- mouse model. Profiling of gene expression in that clus- 51,52 ing proliferation. MiRNA-143/145 are controlled ter revealed enhanced expression of genes involved in by transcription factors NKx2-5 (NK2 transcription adhesion, ECM organization, cellular proliferation, and factor related locus 5) and SRF with its coactivator deposition of collagen. The latter is considered a hall- MYOCD and target KLF4, KLF5, and Sp1. Defi- mark for aortic aneurysm formation, which involves aortic ciency of miRNA143/145 in mice resulted in thinning fibrosis and stiffness. Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2697 Yap et al Six Shades of vSMCs Illuminated by KLF4 A number of factors have been identified that induce be concluded that the fibroblast-like vSMCs are part of this fibroblast-like vSMC phenotype, summarized below. mesenchymal-like vSMCs or an entirely separate cell It has been shown that adventitial SCA1/LY6A–positive type without additional research. However, as the gene fibroblast-like vSMCs arise in a KLF4-dependent man- expression patterns still differ, currently, a distinction ner. The adventitial SCA1/LY6A-derived fibroblast-like between the 2 cell states has been made. vSMCs may even migrate into the intima where they Reverse differentiation from a fibroblast-like pheno- promote a fibrotic response, which stiffens the vessel type back to the contractile vSMCs phenotype has been wall. Interestingly, cholesterol or oxidized phospholipid described for DKK3 (dickkopf 3), which is a Wingless- exposure can also induce a phenotypic switch of vSMCs related integration site (Wnt) inhibitor. DKK3 was shown as shown by induced expression of fibroblast and macro- to be involved in the differentiation of stem and progeni- 40 −/− 88 phage markers. Fibroblast markers upregulated under tor cells to contractile vSMCs in ApoE mice. DKK3 these conditions were fibronectin 1 (FN1), Ecrg4 augurin induces differentiation of SCA1/LY6A-positive mes- precursor (ECRG4), proteoglycan 4 (PRG4), secreted enchymal-like and fibroblasts-like vSMCs to contrac- phosphoprotein 1 (SPP1), lipocalin-2 (LP2), metallopro- tile vSMCs via activation of TGF-β in a Wnt-dependent teinase inhibitor 1 (TIMP1), BGN, and DCN. manner. While TCF21 seems to reduce atherosclerosis Intriguingly, the ER unfolded protein response (UPR) severity by promoting the fibroblast-like vSMC, DKK3 can promote phenotypic switching in vSMCs toward the promotes protective atherosclerotic plaque stabilization fibroblast-like phenotype as well. The ER normally has by increasing the number of contractile vSMCs. There- a low cholesterol content, while accumulation of free fore, it is not yet clear which vSMC phenotype is actually cholesterol in the ER induces membrane dysfunction beneficial in the context of atherosclerosis. 84,85 and subsequent ER stress, causing UPR. Moreover, the UPR promotes a macrophage-like vSMC phenotype, MACROPHAGE-LIKE VSMCS which may explain the appearance of both fibroblast- like and macrophage-like vSMCs in atherosclerotic Macrophages play a central role in all stages of inflam- plaques. Even without cholesterol exposure, chemically mation and healing and recruit other immune cells to induced UPR is sufficient to cause phenotype switching initiate an appropriate immune response to clear debris 40 89 of vSMCs to fibroblast-like or macrophage-like vSMCs. and combat pathogens. In atherosclerosis, macro- The underlying mechanism of this phenotypic switch is phages are known to clear the vascular wall of oxidized linked to the UPR effector ATF4 (activating transcrip- LDL (low-density lipoprotein), in the process becom- tion factor 4). ATF4 prevents proteasomal degradation ing foam cells. As stated earlier, vSMCs can acquire of KLF4, and this enhanced KLF4 expression promotes a macrophage-like phenotype, even with phagocytic 40 91 atherosclerotic plaque formation. properties, as reported in 6 out of eleven selected The vSMC-specific knockout of TCF21 (transcription scRNA-seq studies (Table). Phenotype switching from factor 21) in hyperlipidemic apolipoprotein E deficient a contractile vSMC, via the mesenchymal-like to a mac- −/− (ApoE ) mice led to fewer fibroblast-like vSMCs in the rophage-like vSMC, is associated with development of 7 29,91,92 protective fibrous cap of the atherosclerotic lesions. atherosclerosis. Moreover, high TCF21 expression is associated with Macrophage-like vSMCs are typically indicated by decreased coronary artery disease risk in humans, pos- expression of LGALS3 and classical macrophage mark- sibly due to a more stable and fibroblast-like vSMC-rich ers such as CD11b, CD45, CD68, CD116, and F4/80 plaque. In addition, TCF21 is activated early on in coro- (for murine macrophages). Phenotype switching to nary artery disease and directly inhibits SMAD3-medi- macrophage-like vSMCs also involves KLF4 signal- 29,91,92 ated gene expression, thereby reducing expression of ing. A conditional KLF4 knockout in an athero- the contractile markers. Together, these studies point sclerotic mouse model results in reduced vSMC-derived to TCF21 as a regulator of vSMC phenotype switching mesenchymal-like cells and macrophage-like cells, plus toward a protective fibroblast-like vSMC population in a marked reduction in lesion size and increased plaque 29,33 atherosclerosis. stability. Similar macrophage-like vSMCs have been 6,8,33,71 The vSMC phenotypes distinct from the contractile identified in human atherosclerotic plaques. A low 7,8 type are often described as modulated vSMCs. Gene number of cells within the vSMC group was KLF4 posi- expression profiles of these modulated vSMCs comprise tive, indicating that vSMCs probably only have transient markers of mesenchymal-like and fibroblast-like but also KLF4 expression. One of these vSMC clusters was char- 7,8,40 of macrophage-like or osteogenic-like cells. Many acterized by ACTA2, LGALS3, and CD68 expression, fibroblast-like vSMCs originate from the mesenchymal- typical for the macrophage-like phenotype. However, like pool of vSMCs, although it is challenging to distin- identification of macrophage-like cells is often based on guish between these phenotypes. It is also not clear if different macrophage or foam cell markers between the all fibroblast-like vSMCs first, and entirely, transition via scRNA-seq studies, with LGALS3 and CD68 being the the mesenchymal-like vSMC state. Therefore, it cannot most common markers. 2698 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 KLF4 gene expression in vSMCs facilitates foam cell from cells and transport to the liver to remove cholesterol formation by enhancing the uptake of cholesterol-rich from the periphery. Exposure to HDL reduces the mac- lipoproteins. While classically lipid-laden foam cells rophage-like vSMC phenotype by increasing MYOCD were considered to be solely derived from monocytes/ and miR-143/145 expression in vSMCs in vitro. The macrophages, it has become evident that vSMCs with question remains whether macrophage-like vSMCs have a macrophage-like phenotype are abundantly present similar functions as monocyte-derived macrophage sub- 94 8 in the atherosclerotic plaque. Upon high cholesterol sets in atherosclerosis. exposure, expression of KLF4 was induced. This trans- forms contractile vSMCs into macrophage-like vSMCs, OSTEOGENIC-LIKE VSMCS a cell population contributing to disease progression. Switching of vSMCs to a macrophage-like phenotype The natural function of chondrocytes is to form and coincides with loss of the contractile vSMC factor maintain cartilage, while that of osteoblasts is to gen- 91,96,97 102,103 MYOCD. Conversely, gain of MYOCD expression erate bone tissue. During ossification, hypertrophic inhibits macrophage-like vSMC accumulation in athero- chondrocytes produce a unique ECM that mineralizes, sclerotic lesions in vivo. One of the Yamanaka factors, enabling cells to differentiate into osteoblasts. Both OCT4, also plays a role in regulating vSMC phenotypic chondrocytes and osteoblasts are of mesenchymal ori- transition, but interestingly in a contrasting way com- gin and share a common precursor. Switching from a pared with KLF4. Deficiency of KLF4 or OCT4 resulted mesenchymal-like vSMC phenotype to a chondrocyte- in opposite patterns of gene expression in vSMCs. like or osteoblast-like vSMC has been identified in 4 out During atherosclerosis, deficiency of KLF4 showed of the 11 scRNA-seq studies (Table). In this review, we reduced lesion size, while OCT4 deficiency showed an classified chondrocyte-like and osteoblast-like vSMC increase in lesion size. Decreased lesion size was in phenotypes together as osteogenic-like vSMCs. 33,98 turn consistent with increased plaque stability. Deposition of calcium phosphate can drive vSMC phe- Various scRNA-seq analyses have been performed on notype switching to contribute to various cardiovascular 4,7,8,31,33,99 −/− 105,106 atherosclerotic plaques of mice. In ApoE mice diseases. Calcification of the intimal layer is associ- fed a western diet, vSMCs expressing Lgals3 compose up ated with arterial obstruction and atherosclerotic plaque to two thirds of all vSMCs in the atherosclerotic lesion. rupture, while calcification of the medial layer is associ- This macrophage-like phenotype was similarly found by ated with vessel stiffening leading to heart failure. In −/− 29,31,33,90,93 others in ApoE mice, as well as in LDL recep- addition, vascular calcification has been associated with −/− 8 tor knockout (LDLR ) mice. Through fate mapping, it hypertension, osteoporosis, rheumatoid arthritis, chronic appears that these macrophage-like vSMCs were derived kidney disease, diabetes type II, and aortic aneurysm for- 8 106–108 from a multipotent vSMC-derived intermediate cell state, mation. Interestingly, overexpression of the Twist which bear similarity to the mesenchymal-like vSMCs. family BHLH transcription factor 1 (TWIST1), a coronary Recent evidence even suggests that a considerable sub- artery disease risk gene, in rat aortic vSMCs increases set of the plaque may originate from dedifferentiated cell proliferation and decreases calcification, whereas 100 109 vSMCs, which proliferate in a clonal fashion. TWIST1 knockdown has the opposite effect. Of note, Other extracellular stimuli playing a role in phenotypic TWIST1 was also found to be differentially expressed in switching to macrophage-like vSMCs are nitric oxide and several vSMC clusters in ascending aorta aneurysm tis- natriuretic peptides. In response to these compounds, sue analyzed by scRNA-seq. vSMCs generate cyclic GMP, which induces vasodilation, The osteogenic-like vSMC phenotype is charac- enhancing blood flow. Interestingly, PRKG1 (cyclic terized by a loss of contractile markers (SM22α and GMP–dependent protein kinase 1) activation also con- ACTA2) and an increase in calcification markers, such tributes to the formation of macrophage-like vSMCs as osteogenic transcription factors MSX2 (Msh Homeo- that reside within the atherosclerotic plaque. Under box 2), Cbfa1 (core-binding factor α-1, also known as atherogenic conditions, vSMCs migrate to the athero- RUNX2), and Sp7/Osterix, as well as the chondrogenic 4,106,107,110 sclerotic plaque (intima), which in Prkg1-deficient mice transcription factor SOX9. Other markers such remain in the medial layer. Using cell-fate mapping, it was as osteopontin, osteocalcin, alkaline phosphatase, col- shown that Prkg1 is involved in phenotype switching of lagen II, and collagen X were reported as markers of 106,111 vSMCs to macrophage-like vSMCs in the plaques. In line this osteogenic-like vSMC phenotype as well. The with this, postnatal ablation of Prkg1 in murine vSMCs expression of RUNX2 and SOX9 is a main determinant resulted in smaller lesions. This study also demon- of the osteogenic-like vSMC phenotype, since these 2 strates that macrophage-like vSMCs are derived from transcription factors drive the osteoblast or chondro- 101 106,112 mature vSMCs that migrated into the plaque. cyte phenotype, respectively. Reverse differentiation of the macrophage-like vSMC Deficiency of RUNX2 in vSMCs prevents differen- phenotype is accomplished by HDL (high-density lipo- tiation into the osteogenic-like vSMC phenotype, shown 107,113,114 protein). HDL is responsible for the efflux of cholesterol both in vitro and in vivo. In vitro, RUNX2, Osterix, Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2699 Yap et al Six Shades of vSMCs Illuminated by KLF4 and alkaline phosphatase expression is required to drive ADIPOCYTE-LIKE VSMCS the calcification process. This process also involves An adipocyte-like vSMC phenotype has only been the PKB (protein kinase B) or AKT and c-JNK (Jun described in a single scRNA-seq study (Table). Using an N-terminal kinase) signaling pathways. Human vSMCs in vivo fate mapping approach, with either a constitutive or exposed to a calcifying medium show decreased AKT an inducible Myh11-driven Cre mouse model, it has been phosphorylation and a transient increase in JNK activ- demonstrated that vSMCs are able to adapt toward this ity. This is in line with the observation that elevated adipocyte-like phenotype. These adipocyte-like vSMC levels of AKT and JNK protect from vascular calcifica- were classified as beige adipocytes, which are also known tion. Another pathway involves Wnt signaling, which as inducible brown adipocytes. Brown/beige adipocytes promotes vSMC differentiation to osteogenic-like cells regulate thermogenesis by producing heat when burning primarily through RUNX2. Activation of the Wnt cas- fatty acids in the presence of an UCP-1 (uncoupling pro- cade in osteogenic-like vSMCs increases BMP2 (bone tein-1), while white adipocytes store triglycerides to save morphogenetic protein 2) signaling, which plays a 122,123 energy. Sustained thermogenic activation leads to significant role in vascular calcification. BMPs are part the browning of white adipose tissue, in which a popula- of the TGF-β superfamily, which signal via the SMAD tion of adipocytes differentiate into beige adipocytes. transcription factors -1 and -5, regulating RUNX2. Given that only 1 scRNA-seq study reports adipocyte-like Interestingly, in disease models associated with vascu- vSMCs, this may indicate that a higher barrier exists for lar calcification, KLF4 was demonstrated to decrease differentiation towards an adipocyte-like vSMC type or expression of vSMC differentiation makers and induced that cues to induce this phenotype are not widespread in osteogenic genes. KLF4 was also demonstrated atherosclerotic and aneurysm tissue. to regulate RUNX2 transcription, with knockdown of Adipocyte-like vSMCs express the crucial beige adi- KLF4 inhibiting upregulation of RUNX2 and vSMC pocyte marker UCP1, as mentioned earlier, as well as calcification. In addition, KLF4 enhanced chondro- PPARγ (peroxisome proliferator-activated receptor cyte differentiation as characterized by upregulation of gamma) coactivator 1 alpha (PPARGC1A), transmem- SOX9 and downregulation of the chondrocyte dediffer- brane protein 26 (TMEM26), PR-domain containing 16 entiation marker Col1α1. In the context of scRNA- (PRDM16), and the temperature-sensitive ion channel seq, loss of KLF4 in vSMCs coincided with reduction transient receptor potential cation channel subfamily V of an osteogenic phenotype. However, the exact link member 1 (TRPV1). Upon 7-day cold exposure, these between KLF4 and osteogenic-like cells is not fully cells are able to mature further to brown adipocyte-like clear and requires further research. vSMC expressing UCP1, adiponectin (ADIPOQ), cell An in vivo study recapitulated vSMC differentiation death inducing DFFA like effector A (CIDEA), and iodo- into an osteogenic-like phenotype, by demonstrating thyronine deiodinase 2 (DIO2). Furthermore, KLF4 is that MGP (matrix gla protein)-deficient mice develop suggested to regulate early adipogenesis to induce C/ severe calcification of vSMCs in arterial blood ves- EBPβ (CCAAT-enhancer-binding protein beta). C/EBPβ sels. Interestingly, MGP-deficient mice also lacking stimulates expression of PPARγ, which is required for HDAC9 (histone deacetylase 9) had a 40% reduction adipocyte differentiation. However, if this pathway is in aortic calcification with improved survival. Thus also activated in vSMC by KLF4 has yet to be determined. the presence of HDAC9 causes progression toward Conversion of contractile vSMCs to adipocyte-like the osteogenic-like vSMC phenotype induced by the vSMCs within the vessel wall is presumably not without absence of MGP. consequences, but as this phenotype is least studied, this Another pathway involved in the suppression of cal- will require more research to understand its relevance. cification is based on aryl hydrocarbon receptor (AHR)- mediated gene expression. This transcription factor is involved in stem cell maintenance and cellular differen- EXPANDING THE VIEW ON VSMC tiation and is a downstream target of TCF21. TCF21 PHENOTYPES promotes vSMC phenotype switching to fibroblast-like cells and knockdown of AHR resulted in switching of Recent scRNA-seq analyses of human and mouse vas- fibroblast-like vSMCs to the osteogenic-like vSMCs. This cular tissues disclose a previously not well-defined diver- is in line with the ability of AHR to suppress SOX9 and sity of arterial vSMC phenotypes. We reasoned that the RUNX2 expression. Interestingly, TCF21 knockdown classification of vSMC phenotypes should be expanded prevented both the fibroblast-like and osteogenic-like beyond the traditional exclusive division of contractile vSMC differentiation, indicating that the fibroblast-like and synthetic vSMCs. In this review, we summarized the phenotype may first be required before transition towards current evidence for the presence of 6 distinct vSMC the osteoblast-like phenotype can occur. Taken phenotypes in (diseased) vascular tissue: contractile, together, TCF21 can be considered a driver and AHR an mesenchymal-like, fibroblast-like, macrophage-like, inhibitor of these vSMC phenotypes. osteogenic-like, and adipocyte-like vSMCs (Figure 1A). 2700 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 Figure 1. Schematic overview of vascular smooth muscle cell (vSMC) phenotypes. A, Overview of vSMC phenotypes and associated gene expression markers. The contractile phenotype is associated with the expression of contractile genes. Mesenchymal-like vSMCs lose expression of these contractile markers and adopt expression of a number of specific markers including the key player of phenotype switching: KLF4 (Krüppel-like factor 4). From this vSMC phenotype, differentiation to fibroblast-, macrophage-, osteogenic-, or adipocyte-like phenotypes seems to occur. Genes typical for the contractile phenotype are 5,7,141,142 CNN1, SMTN, ACTA2, TAGLN, MYH11, and MYOCD, for the mesenchymal phenotype KLF4, CD34, CD44, and SCA1/ 36,37,143,144 7 LY6a, for the fibroblast phenotype FN1, BGN, DCN, and COL1A1, for the macrophage phenotype CD45, CD68, ITGAM, 7,93,145 106,107,120 LGALS3, ITGAM, and ADGRE1, for the osteogenic phenotype SP-7, SOX9, MSX2, and RUNX2, and for the adipocyte phenotype UCP1, ELOVL3, ADIPOQ, PRDM16, and PPARGC1A. B, Activating and inhibitory stimuli of phenotype switching in vSMCs. Stimuli that mediate specific phenotype switching are indicated. Blue arrows and names indicate a transition to the mesenchymal- like vSMC, red arrows and names indicate a transition to the contractile-like vSMC, and black arrow and names indicate a transition to the other phenotypes in A and B. AHR indicates aryl hydrocarbon receptor; BMP2, bone morphogenetic protein 2; DKK3, dickkopf 3; HDAC9, histone deacetylase 9; HDL, high-density lipoprotein; MGP, matrix gla protein; PDGF-BB, platelet-derived growth factor BB; RA, retinoic acid; and TGF-β, transforming growth factor beta. Combining the scRNA-seq data on vSMC subtypes with as none of the phenotypes seem to be a final state of knowledge on vSMC function, one may speculate that cellular differentiation. Rather, vSMCs can go back and there is an initial transition of the contractile vSMC dedif- forth between different phenotypes, triggered by specific ferentiating into mesenchymal-like vSMCs, followed by stimuli (Figure 1B). The exact phenotypic composition of differentiation towards the other 4 vSMC phenotypes. vSMCs in the vessel wall may determine vascular pathol- A remarkable characteristic of vSMCs is their plasticity, ogy in various cardiovascular diseases, and at present Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2701 Yap et al Six Shades of vSMCs Illuminated by KLF4 the consequence of the relative prevalence of these 6 shades of vSMC is not fully understood. Moreover, pre- sumably there are even more vSMC flavors as indicated by 2 distinct contractile, mesenchymal- and osteogenic- 7,31,33,41,75 like vSMC populations in different studies, and 8 undefined vSMC populations in the study from Hartman et al. (Table). KLF4 AND ITS CRUCIAL ROLE IN VSMC PHENOTYPE SWITCHING The transcription factors KLF4, retinoic acid receptor, TCF21, AHR, and erythroblast transformation-specific related gene are all to some extent involved in vSMC phenotype switching in cardiovascular diseases, with a key role for KLF4. Therefore, we summarize the cur- rent knowledge on modulation of KLF4 to interfere with vSMC phenotype switching. KLF4 is known to regulate gene expression via different mechanisms, not merely as Figure 2. KLF4 (Krüppel-like factor 4) facilitates phenotypic DNA-binding transcription factor, which may in part switching of vascular smooth muscle cells (vSMCs). explain its involvement in the different vSMC phenotypes. Schematic overview of the role of KLF4 in vSMC phenotype The sections above substantiate the importance of switching. In contractile vSMCs (1), the transcription factor complex KLF4 in relation to vSMC phenotype switching. The dis- MYOCD (myocardin)-SRF (serum response factor) induces the tinct role of KLF4 per vSMC phenotype with respect to expression of contractile genes. The transcriptional activity of KLF4 is balanced by protein ubiquitination and subsequent degradation. gene regulation and transcription factor cooperation is Acetylated KLF4, induced by all-trans retinoic acid stimulation, context-dependent and affected by additional stimuli, alleviates the KLF4-induced repression of contractile gene as summarized in Figure 2. In the contractile vSMC 50,68 expression. In the mesenchymal vSMC phenotype (2), KLF4 phenotype, the activated transcription factor complex expression is increased and stabilized to repress the expression of 50,55,66 MYOCD-SRF induces the expression of contractile pro- contractile genes by reduction of formation of the MYOCD- SRF complex. In the fibroblast-like vSMC (3), the unfolded protein teins. In this situation, KLF4 expression is balanced response (UPR) induces KLF4 expression, which affects the vSMC by protein ubiquitination and subsequent degrada- 40,99 phenotypic switch by an unknown mechanism. The switching tion. Posttranslational modification through acetylation toward a macrophage-like vSMC (4) also requires UPR and/or of KLF4 changes its binding to preferential DNA sites oxidized LDL (oxLDL) uptake to increase KLF4, which by an unknown to alleviate the repression of contractile gene expres- mechanism induced the switch toward foam cell formation. The transition to an osteogenic-like vSMC (5) is facilitated by a KLF4- sion. In the mesenchymal-like vSMC phenotype, KLF4 mediated activation of RUNX2 (runt-related transcription factor expression is upregulated, which reduces MYOCD-SRF 2) or SOX9-induced transcriptional programmes, which allows complex formation and thereby inhibits expression of 4,106,107,110,127 calcification by the release of extracellular vesicles (EVs). 50,54,55,66 contractile proteins. KLF4 then increases mes- The phenotype switch to an adipocyte-like vSMC (6) could enchymal marker expression: SCA1, CD34, and CD44. hypothetically require activation of KLF4, leading to a KLF4-mediated increase in C/EBP and subsequent PPARy (peroxisome proliferator- When KLF4 signaling is enhanced in the fibroblast-like activated receptor gamma) transcription factor activation to induce vSMC phenotype, this is linked to the UPR and its effec- adipogenesis and uptake of triglycerides (TGs). Green arrow tor factor ATF4. KLF4 has also been associated with indicates expression of contractile proteins, and red arrow indicates macrophage foam cell formation by enhancing uptake of expression of phenotype specific genes. Ac indicates acetylation; C, cholesterol-rich lipoproteins. Switching of vSMCs toward cytoplasm; N, nucleus; and Ub, ubiquitination. a macrophage-like phenotype may be induced by KLF4 through enhanced UPR or lipid uptake, which can lead to with a vSMC-specific conditional deficiency of KLF4. 40,94 foam cell formation. The transition toward an osteo- These mice show an increase in the number of contrac- genic-like phenotype requires the induction of RUNX2 tile vSMCs, and a substantial reduction of macrophage- or SOX9 to induce vascular calcification by releasing like and osteogenic-like vSMCs. Moreover, Chen et al 4,106,107,110,127 extracellular vesicles. Activation of KLF4 observed a decrease in overall atherosclerotic burden may be required for switching toward an adipocyte-like and a reduction in the development of aortic aneurysms phenotype, which could lead to adipogenesis through C/ (regardless of cholesterol levels), in vSMC-specific KLF4 EBPβ and PPARγ signaling. knockout mice. A KLF4 signature was also identified C1041G/+ In atherosclerosis, Alencar et al identified a reduc- in aneurysmal aortic tissue of fibrillin-1 (Fbn1) tion in lesion size and increased plaque stability in mice Marfan syndrome mice. Furthermore, using an in vitro 2702 November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 BRIEF REVIEW - VB BRIEF REVIEW - VB Yap et al Six Shades of vSMCs Illuminated by KLF4 induced pluripotent stem cell–derived vSMC model with tissues collected at a single point in time, especially in distinct FBN1 mutations from Marfan syndrome patients, human end-stage disease. To understand the dynam- knockdown of KLF4 restored Fbn1 fiber deposition, ics of vSMC phenotype plasticity between the 6 vSMC vSMC proliferation defects, and contractile function. phenotypes, phenotype switching over time needs to In contrast, a marked reduction in KLF4 expression was be explored in detail. To date, only Pan et al performed observed in the aorta of very young Fbn1 hypomorphic scRNA-seq and lineage tracing in mice over various time (mgR/mgR model) Marfan mice, which was concomi- points and suggested that this method provided both tant with an increase in contractile vSMCs. Together, sensitive and specificity for tracking vSMC behaviors these data may indicate that the contractile population of during atherosclerosis. vSMCs is unable to undergo phenotype switching during In addition, we argued that the data point in the direc- 5,130 early stages of aneurysm formation. However, vSMC- tion of the mesenchymal-like cells giving rise to the specific KLF4-deficient Marfan mice are yet to be gener- other vSMC phenotypes; however, further experimental ated to precisely determine the effect of KLF4 in aortic validation is needed to fully support this hypothesis. Fur- aneurysm formation. thermore, it remains challenging to recognize the vSMC These findings emphasize KLF4 as an interesting origin of a cell population, especially in nonlineage traced potential drug target to diminish vascular disease. As human tissue samples. However, even conventional lin- there are no highly selective KLF4 antagonists, KLF4 eage tracing studies have their limitations; they can only expression can be targeted by indirect inhibitors, of mark the fate of the contractile vSMC but not the fate which several are in preclinical development. One of of once contractile vSMCs that underwent phenotype these inhibitors is designed to interfere with the methyla- switching with the loss of their contractile markers. tion of KLF4 by PRMT5 (protein arginine methyltransfer- Limitations and potential biases should also be ase 5). Since KLF4 has a short half-life, governed by considered when analyzing and interpreting different VHL-VBC ubiquitin-protein ligase, methylation of amino scRNA-seq datasets. As often, the data rely on a sin- acids R374, R376, and R377 prolong protein stability. gle-cell suspension representative of all cell populations The small molecule antagonist WX2–43 was designed present within tissue samples exposed to tissue disrup- to interfere with the PRMT5-KLF4 interaction, prevent- tion. scRNA-seq also lacks spatial information about the ing the methylation, thus promoting degradation. Another distribution of the different subpopulations within tissues, indirect inhibitor of KLF4 targets its interaction with and different processes can cause variations in transcript PLK1 (polo-like kinase-1). This kinase phosphorylates levels that might not reflect differences in protein lev- KLF4 at Ser234, promoting protein stability, previously els or cellular functions. Moreover, the lack of depth of linked to hyperplastic intima of injured vessels. The scRNA-seq also makes it difficult to investigate low- experimental drug BI6727, originally developed as an expression genes, which are often transcription factors. anticancer drug, is a highly selective, potent PLK1 inhibi- Although the transcriptomic signature in defining tor. Exposure to BI6727 results in reduced expression of vSMC states and transitions offers a wealth of infor- KLF4 and increased protein turnover due to reduced sta- mation, it is yet to be determined what this means on bility. Given that the translation of miRNAs into clinical a protein level and whether these vSMC-derived cells medicine will become more common in the future, it is of have similar functionality to their classical counterparts importance that several miRNAs have been reported to (eg, macrophage-like vSMC compared with a bone interact with KLF4 mRNA, and may serve as blueprint marrow-derived macrophage). The vSMC-derived cells for therapeutic oligonucleotides. The miRNAs miR- seem to be heavily influenced by their environment to 1, miR-25, miR-29, miR-143, miR-145, and miR-375 trigger the transition and may not always provide posi- induce RNAi-mediated silencing of KLF4 protein expres- tive consequences. 92,135–138 sion. Further research will establish the potential As for the function of the different vSMCs pheno- of targeting KLF4 in the context of the different car- types, their diverging roles in the broad range of CVDs diovascular diseases, where KLF4 may be beneficial in remain to be discovered. It will eventually pinpoint which one, but detrimental in another vascular disease type. In type is beneficial or harmful in specific vascular disor- addition, potential side effects of repressed KLF4 should ders. This knowledge is crucial for the development of not be overlooked, as KLF4 inhibition has been shown to innovative disease intervention strategies. 139,140 delay wound healing and elevate insulin resistance. ARTICLE INFORMATION Received June 3, 2021; accepted August 20, 2021. DISCUSSION AND PERSPECTIVES Affiliations The scRNA-seq technique, together with lineage trac- Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, ing, opens up a whole new world of possibilities to study University of Amsterdam, Amsterdam UMC, Location Academic Medical vSMCs in the normal and diseased vessel wall. Until Center, The Netherlands (C.Y., A.M., C.J.M.d.V., V.d.W.). Department of Clinical now, the scRNA-seq has been performed on (diseased) Genetics, Amsterdam Cardiovascular Sciences, Vrije Universiteit Amsterdam, Arterioscler Thromb Vasc Biol. 2021;41:2693–2707. DOI: 10.1161/ATVBAHA.121.316600 November 2021 2703 Yap et al Six Shades of vSMCs Illuminated by KLF4 Amsterdam UMC, Location VU University Medical Center, Amsterdam, The thoracic aortic aneurysms and dissections. Arterioscler Thromb Vasc Biol. Netherlands (D.M.). 2017;37:26–34. doi: 10.1161/ATVBAHA.116.303229 16. Majesky MW. Developmental basis of vascular smooth muscle diver- Acknowledgment sity. Arterioscler Thromb Vasc Biol. 2007;27:1248–1258. doi: 10.1161/ We thank Dr Dave Speijer for critical reading of the article. ATVBAHA.107.141069 17. Pfaltzgraff ER, Bader DM. Heterogeneity in vascular smooth muscle cell Sources of Funding embryonic origin in relation to adult structure, physiology, and disease. Dev Dyn. 2015;244:410–416. doi: 10.1002/dvdy.24247 This study was supported by Amsterdam Cardiovascular Sciences PhD grant 18. Intengan HD, Schiffrin EL. Structure and mechanical properties of resis- 2019 and Zeldzame Ziekten Fonds via AMC foundation (C. Yap, D. Micha, V. de tance arteries in hypertension: role of adhesion molecules and extracel- Waard) and by Health Holland TKI-Public Private Partnership grant 22532 (A. lular matrix determinants. Hypertension. 2000;36:312–318. doi: 10.1161/ Mieremet, V. de Waard). 01.hyp.36.3.312 Disclosures 19. Robertson IB, Rifkin DB. 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