Lgals3-Transitioned Inflammatory Smooth Muscle Cells: Major Regulators of Atherosclerosis Progression and Inflammatory Cell Recruitment

The contribution of vascular smooth muscle cells (SMCs) in atherosclerosis was largely underestimated until the development of genetic lineage tracing mouse models.^1,2 SMCs can contribute up to 70% of all cells in atherosclerotic plaque, most of which exhibit a dedifferentiated phenotype characterized by low expression of SMC contractile genes and upregulation of ECM (extracellular matrix) and proinflammatory genes.^3,4 Some plaque SMCs exhibit expression of markers overlapping with macrophages, such as Lgals3 (galectin 3) and CD68.^4,5 Although later rectified as a transition SMC state rather than bona fide macrophages,^6,7 the functional significance of Lgals3⁺ cells in atherosclerosis was supported by the correlation between decreased Lgals3⁺ content with decreased plaque size and increased stability.⁴

The plasticity of SMCs extends beyond Lgals3⁺ cells. Lineage tracing studies have confirmed that under specific context, SMCs adopt phenotypes resembling osteochondrogenic cells,⁸ fibroblast-like cells,^6,7 as well as multipotent mesenchymal stem cell–like progenitor cells.^8,9,10 SMC plasticity, while intriguing and leading to important functional consequences of vascular disease, posts a unique challenge when studying their fate and function. The most common and well described SMC lineage tracing system, which takes advantage of an inducible, Myh11-Cre driver and fluorescent reporter, lacks the capability to distinguish various SMC-derived subpopulations. Application of single-cell transcriptomics technology in the field has provided a glimpse of the true scale of plasticity of SMCs, but essential tools to genetically manipulate molecular pathways in specific SMC lineage subpopulations in an accurate fashion are lacking.

In this issue of ATVB, Owsiany et al¹¹ took a step toward overcoming these difficulties by generating an elegant dual recombinase lineage tracing mouse model, which captures the phenotypic modulation of SMCs in real time with the switch between tdTomato and eGFP (enhanced green fluorescent protein) fluorescence (Figure). Using a SMC-specific Klf4 (Kruppel-like 4) KO (knockout) mouse model, the authors demonstrated that SMCs express MCP1 (monocyte chemoattractant protein-1) in vitro and in vivo in a Klf4-dependent manner. MCP1 is a CC family chemokine that functions to recruit monocytes to the site of inflammation. The involvement of MCP1 in atherogenesis is highlighted by studies where genetic or pharmacological inhibition of the MCP1-CCR2 (C-C motif chemokine receptor 2) axis decreased the atherosclerotic plaque burden and altered plaque composition in mouse models.^12–16 Furthermore, circulating and in-plaque MCP1 levels are positively associated with plaque vulnerability and long-term risk of stroke and cardiovascular mortality.^17–19 The feasibility and clinical effectiveness of anti-inflammatory therapeutics for the treatment of atherosclerosis has long been investigated.²⁰ Despite encouraging preclinical results, clinical trials of such therapeutics yielded inconsistent results, frequently accompanied by the increased risk of fatal infections.²¹ In the current study, Owsiany et al¹¹ made the paradoxical discovery that selective haploid loss of MCP1 in all SMCs resulted in larger lesions with reduced fibrous caps compared with WT and homozygous MCP1 KO mice. This was associated with increased accumulation of plaque Ly6C-hi macrophages. Although the exact reason for this phenotype is unknown, this result does highlight the risk of systemic anti-inflammatory therapy. Indeed, when the authors depleted MCP1 specifically in Lgals3-transitioned SMCs using the dual recombinase lineage tracing system, a more stable plaque phenotype was observed. Single-cell RNA sequencing was performed to further examine the transcriptomics changes induced by MCP1 depletion. The sequencing data support the conclusions from the imaging studies. These findings suggest that the development of a localized, cell type–accurate anti-inflammatory agent delivery system, although technically challenging, might be needed to overcome some of the adverse effects observed with systemic anti-inflammatory treatments in atherosclerosis. Alternatively, leveraging epigenetic and pharmacological agents that target key signaling pathways controlling inflammation and SMC phenotype, rather than directly altering cytokines and chemokines, might be a viable approach to maintain the differentiated phenotype of SMCs in atherosclerosis.^22–24

The dual recombinase lineage tracing system demonstrated here by Owsiany et al¹¹ offers a unique tool to dissect gene regulatory networks controlling the function and phenotype of Lgals3-transitioned SMCs. With minor modifications, this model can be utilized in the study of other subpopulations of SMCs with high specificity. Since it has been proposed that Lgals3-positive SMCs represent an early transitional phenotypic state,²⁵ an interesting question is how does this fit into the clonal expansion model²⁶ that SMCs in the plaque are derived from a common ancestor in the setting of atherosclerosis. Does MCP1 play a role in the expansion and migration of these ancestor SMCs? Further, does the embryonic memory of SMCs in different vascular beds impact their MCP1 secreting capability and response? The work from Owsiany et al opens the possibility of future investigations into these questions.