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

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 (Kruppel-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.

Keywords: atherosclerosis; cardiovascular disease; myocytes; phenotype; smooth muscle.

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 CNN1, SMTN, ACTA2, TAGLN, MYH11, and MYOCD,^,,, for the mesenchymal phenotype KLF4, CD34, CD44, and SCA1/LY6a,^,,, for the fibroblast phenotype FN1, BGN, DCN, and COL1A1, for the macrophage phenotype CD45, CD68, ITGAM, 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.

Figure 2.

KLF4 (Krüppel-like factor 4) facilitates phenotypic switching of vascular smooth muscle cells (vSMCs). Schematic overview of the role of KLF4 in vSMC phenotype switching. In contractile vSMCs (1), the transcription factor complex MYOCD (myocardin)-SRF (serum response factor) induces the expression of contractile genes. The transcriptional activity of KLF4 is balanced by protein ubiquitination and subsequent degradation. Acetylated KLF4, induced by all-trans retinoic acid stimulation, alleviates the KLF4-induced repression of contractile gene expression.^, In the mesenchymal vSMC phenotype (2), KLF4 expression is increased and stabilized to repress the expression of contractile genes^,, by reduction of formation of the MYOCD-SRF complex. In the fibroblast-like vSMC (3), the unfolded protein response (UPR) induces KLF4 expression, which affects the vSMC phenotypic switch by an unknown mechanism.^, The switching toward a macrophage-like vSMC (4) also requires UPR and/or oxidized LDL (oxLDL) uptake to increase KLF4, which by an unknown mechanism induced the switch toward foam cell formation. The transition to an osteogenic-like vSMC (5) is facilitated by a KLF4-mediated activation of RUNX2 (runt-related transcription factor 2) or SOX9-induced transcriptional programmes, which allows calcification by the release of extracellular vesicles (EVs).^,,,, The phenotype switch to an adipocyte-like vSMC (6) could hypothetically require activation of KLF4, leading to a KLF4-mediated increase in C/EBP and subsequent PPARy (peroxisome proliferator-activated receptor gamma) transcription factor activation to induce adipogenesis and uptake of triglycerides (TGs). Green arrow indicates expression of contractile proteins, and red arrow indicates expression of phenotype specific genes. Ac indicates acetylation; C, cytoplasm; N, nucleus; and Ub, ubiquitination.