Role of S100A4 in the crosstalk between smooth muscle and inflammatory cells in atherosclerosis

Abstract Background During atherosclerotic plaque formation, smooth muscle cells (SMCs) accumulate in the intima and undergo a transition from a contractile to a synthetic phenotype. We previously showed that S100A4 is a marker of the synthetic SMCs in vitro and intimal SMCs in pig, human and mouse. In vitro, S100A4-treated SMCs acquire a pro-inflammatory phenotype. The systemic neutralization of extracellular S100A4 in ApoE-/- mice induces atherosclerotic plaque stabilization. Purpose To characterize the role of S100A4-expressing SMCs in atherosclerosis. Methods and results We established a coculture model between S100A4-stimulated human aortic SMCs and human blood-derived monocytes. Flow cytometry analysis of monocytes exposed to S100A4-treated SMCs for 3 days showed an upregulation of markers typical of inflammatory M1 (HLA-DR) and anti-inflammatory M2 (CD206) macrophages (fig. 1). Macrophages also upregulated mRNA expression of NPC Intracellular cholesterol transporter 1 and 2 by qPCR, suggesting modifications in their cholesterol metabolism. In vivo, in an SMC-lineage-tracing ApoE-/-mouse model, we identified S100A4-expressing SMCs at the onset of atherosclerosis in aortic media of 11-week-old mice fed a high cholesterol diet (HCD) for 3 weeks. Medial SMCs were characterized by means of single-cell RNA sequencing. Interestingly, 10 out of 11 identified clusters expressed S100A4, demonstrating broad and early involvement of S100A4 in shaping medial SMC identity. We assumed that blood-derived monocytes might be a driving force of early S100A4 activation in SMCs. To mitigate the impact of monocytes, 8-week-old ApoE-/- mice were fed an HCD for 9 weeks and injected with either clodronate-encapsulated or empty liposomes during the first 3 weeks. No differences in S100A4 expression in the media were observed by qPCR. Unexpectedly, ApoE-/- mice treated with clodronate demonstrated larger atherosclerotic lesions compared to control animals as measured by OilRedO staining (fig. 2). Studies are in progress to define the plaque composition. Conclusion Our findings shed light on the role of S100A4 in the crosstalk between SMCs and inflammatory cells in atherosclerosis and the fine-tuning of the phenotype of medial SMCs prior to their accumulation in the intima. We suggest that temporary monocyte depletion at the onset of the disease alters the mechanisms underlying plaque formation, worsening the outcome of the disease, while not affecting S100A4 expression in medial SMCs.