Smooth muscle cell-specific deletion of S100A4 modifies murine atherosclerotic plaques composition

Abstract S100A4, a calcium-binding protein, is crucial in smooth muscle cell (SMC) phenotypic switch, yet its role in atherosclerotic plaque development and SMC plasticity remains unclear. Our previous experiments using an S100A4 neutralizing antibody approach in ApoE-/- atherosclerotic-prone mice showed reduced atherosclerotic burden but did not establish a direct link between the various S100A4-expressing cells and atherosclerosis. By using a carotid artery ligation mouse model, we show that neointimal hyperplasia development is not altered in S100A4 KO mice compared to wild-type mice. In this low-inflammatory context, the intimal thickening and the expression of a-smooth muscle actin (a-SMA) and smooth muscle myosin were similar between both groups. We then used a lineage tracing ApoE-/- mouse model, in which we induced an SMC-specific deletion of S100A4 (SMC-S100A4Δ/Δ). After 12 weeks of a high cholesterol diet, SMC-S100A4Δ/Δ and control (SMC-S100A4wt/wt) aortas were processed for en face Oil Red O staining, immunohistochemistry, and single-cell RNA sequencing (scRNA-seq). SMC-specific deletion of S100A4 reduced the necrotic core area (from 29.75% of total plaque area in SMC-S100A4wt/wt to 18.23% in SMC-S100A4Δ/Δ mice) without affecting total atherosclerotic plaque size, suggesting a more potent impact on plaque composition. Although ScRNA-seq analysis did not reveal major differences in cell population content, Immunofluorescence staining on aortic root plaques showed an increase in YFP+ (from 11.02% to 16.18%) and YFP+Acta2+ SMCs (from 3.25% to 5.1%) in the plaques of SMC-S100A4 Δ/Δ mice accompanied by a relocalization of YFP+ cells towards the fibrous cap, suggesting an alteration in phenotype. Furthermore, the Gene Set Enrichment Analysis revealed that, in SMC-S100A4Δ/Δ mice, mitochondrial metabolism and oxidative respiration genes were downregulated in all SMC clusters. We investigated SMC mitochondrial fitness in vitro and showed that S100A4-/- SMCs have an increased basal oxygen consumption rate and an increased proton leak (fold change of 1.37 and 1.96, respectively), suggestive of differences in cellular metabolic adaptability. Transmission electron microscopy analysis showed alterations in mitochondrial numbers and ultrastructure in S100A4-/- SMCs. These cells also had a reduced migratory activity upon stimulation in comparison to control cells, as seen in wound healing and proliferation assays. We further explored these alterations using metabolic cages and showed that SMC-S100A4Δ/Δ mice presented a higher basal metabolism, energy expenditure, and fatty acid oxidation, resulting in a decrease of adipose tissue and total body weight in comparison to littermate controls. Our research emphasizes the significant role of S100A4 in plaque development, demonstrating how SMC-derived S100A4 knockdown affects plaque evolution, reducing inflammation, and promoting stability likely through influencing SMC phenotypic switch through metabolic pathways.