Smooth muscle cells in atherosclerosis: clones but not carbon copies

Abstract

Our knowledge of the contribution of vascular smooth muscle cells (SMCs) to atherosclerosis has greatly advanced in the previous decade with the development of techniques allowing for the unambiguous identification and phenotypic characterization of SMC populations within the diseased vascular wall. By performing fate mapping or single-cell transcriptomics studies, or a combination of both, the field has made key observations: SMCs populate atherosclerotic lesions by the selective expansion and investment of a limited number of medial SMCs, which undergo profound and diverse modifications of their original phenotype and function. Thus, if SMCs residing within atherosclerotic lesions and contributing to the disease are clones, they are not carbon copies and can play atheroprotective or atheropromoting roles, depending on the nature of their phenotypic transitions. Tremendous progress has been made in identifying the transcriptional mechanisms biasing SMC fate. In the present review, we have summarized the recent advances in characterizing SMC investment and phenotypic diversity and the molecular mechanisms controlling SMC fate in atherosclerotic lesions. We have also discussed some of the remaining questions associated with these breakthrough observations. These questions include the underlying mechanisms regulating the phenomenon of SMC oligoclonal expansion; whether single-cell transcriptomics is reliable and sufficient to ascertain SMC functions and contributions during atherosclerosis development and progression; and how SMC clonality and phenotypic plasticity affects translational research and the therapeutic approaches developed to prevent atherosclerosis complications. Finally, we have discussed the complementary approaches the field should lean toward by combining single-cell phenotypic categorization and functional studies to understand further the complex SMC behavior and contribution in atherosclerosis.

Keywords: Cell differentiation; Cell plasticity; Coronary artery disease; Transcriptomics; Vascular cell; Vascular disease.

Fig 1

In vivo smooth muscle cell (SMC) tracking systems for investigation of SMC phenotypic transitions and clonality. A, SMC fate mapping with a combination of highly specific and tamoxifen-inducible Myh11-Cre-ER^T2 (tamoxifen-inducible estrogen receptor Cre recombinase) transgene with a floxed-STOP reporter allowing for rigorous tracking of Myh11⁺ SMCs during atherosclerotic plaque formation and progression. Tamoxifen treatment induced definitive expression of the reporter in Myh11⁺ medial SMCs and their progeny and tracking of SMC investment and downstream phenotypic transitions. B, SMC clonality tracking. Random tamoxifen-induced recombination of multicolor reporter systems (eg, rainbow, confetti) in Myh11⁺ cells permitted determination of clonal pattern of SMCs investing the lesion. Oligoclonal expansion was characterized by the predominant presence of a few monocolor patches of SMCs within the lesion. In contrast, an intermixing of SMCs and equal representation of the different reporters were associated with polyclonal expansion. C, Dual SMC-lineage tracing (example of dual lineage tracing used by Alencar et al¹⁰). Dual lineage tracing systems are useful to study the transitions between phenotypic states and to characterize intermediary states. The system developed by Alencar et al allows for the precise tracking of all Myh11⁺ SMCs (red) and the identification of the subset of these cells activating the gene Lgals3 at any time during atherosclerosis progression (green). This dual lineage tracking requires the combination of Dre ER^T2-and Cre-mediated excision of Rox and LoxP sites, respectively, and a multicolor reporter.

Fig 2

Proposed experimental pipeline for studying smooth muscle cell (SMC) phenotypes and function in atherosclerosis. This experimental pipeline is based on SMC lineage tracing animal models and the integration of multiple technical platforms, including single cell RNA sequencing, spatial and temporal studies by high-resolution microscopy and slide-sequencing, and in vitro and in vivo studies investigating the functional relevance of putative phenotypic regulators in SMCs.

Fig 3

TCF21 and KLF4 mediate vascular smooth muscle cell (SMC) phenotypic transitions in atherosclerosis. The transcription factors TCF21 and KLF4 play central and opposite roles in regulating SMC phenotypic modulation in atherosclerosis. TCF21 mediates SMC transition to fibromyocytes and population of the fibrous cap by ACTA2⁺ SMCs. In contrast, KLF4 controls transitions to foam and chondrogenic cells. It has been postulated that the expression of LGALS3 and/or SCA1 marks an early and intermediate state mediating SMC investment within the lesion and further transitions. The exact interdependence between these states and transitions requires further investigation.