Characterization of S100A4-induced inflammatory SMCs in vitro and role of S100A4-expressing medial SMCs during early stages of atherosclerosis in ApoE-/- mice

Abstract

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Swiss National Science Foundation Ksenia Kapitanova, Marie-Luce Bochaton-Piallat Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland During atherosclerotic plaque formation, smooth muscle cells (SMCs) accumulate in the intima and undergo a transition from contractile to synthetic phenotype. We previously isolated contractile/differentiated spindle-shaped (S) and synthetic/dedifferentiated rhomboid (R) SMCs from porcine coronary artery. We showed that S100A4 is a marker of R-SMCs in vitro and intimal SMCs, in pig, human and mouse. In vitro, S100A4-treated SMCs acquire a pro-inflammatory phenotype through toll-like receptor 4-dependent NFkB activation. The neutralization of extracellular S100A4 in ApoE-/- mice induces atherosclerotic plaque stabilization. Using porcine and mouse SMC in vitro models, we aimed at characterizing the profile of S100A4-induced inflammatory SMC. We have shown that porcine and mouse SMCs treated with S100A4 for 2 hours expressed granulocyte macrophage colony stimulating factor (GMCSF) at the mRNA level (as shown by qPCR) in the presence of fetal calf serum or, to a lesser extent, in serum-free medium (Figure 1). After 24 hours of treatment with S100A4, GMCSF was also detected at the protein level by means of Western blotting in porcine SMCs. By means of ELISA, we identified several other inflammatory molecules such as interleukin (IL)-6, IL-33, and IL-1a, released by mouse SMCs after S100A4 stimulation for 6 hours. We will study the impact of SMC-derived GMCSF and other inflammatory molecules on the phenotype of monocytes by FACS using conditioned medium collected from S100A4-treated SMCs. In vivo, we have detected rare S100A4-positive SMCs in the aortic media during early stages of atherosclerosis ApoE-/- mice after 2 weeks of high fat diet (Figure 2). These rare S100A4-positive SMCs could be prone to migrate and accumulate in the intima. We performed single cell RNA sequencing to decipher the phenotype of medial SMCs expressing a-smooth muscle actin (a-SMA aka ACTA2), smooth muscle myosin heavy chains (SMMHCs aka MYH11), and S100A4 compared with SMCs expressing only ACTA2 and MYH11. We primarily identified several upregulated genes expressed in the rare S100A4-positive SMCs, such as decorin, lumican and cartilage oligomeric matrix protein. To further investigate the role of S100A4-expressing medial SMCs during early stages of atherosclerosis, we plan to influence the medial S100A4-positive SMC population by depleting blood-derived monocytes using clodronate-liposomes and/or systemically neutralizing extracellular S100A4 by injecting S100A4 antibodies in ApoE-/- mice under high fat diet for 2 weeks. Our results will be instrumental in deciphering the SMC-monocyte crosstalk and in understanding the role of early activation of S100A4-expressing medial SMCs in atherosclerotic plaque development.