Abstract

Our lab has recently shown that several coronary artery disease (CAD)-protective genes inhibit the transition of smooth muscle cells from plaque-stabilising fibromyocytes (FMCs) to chondrocyte-like chondromyocytes (CMCs) that localise to areas associated with plaque vulnerability, namely acellular and calcified regions. To further investigate the molecular mechanisms underlying the transition from FMCs to CMCs, and to determine the CMC impact on lesional stability, we induced SMC-specific deletion of endochondral master regulator Sox9 and performed single-cell genomics and histological assays in a murine model of atherosclerosis. Whilst deletion of Sox9 resulted in only modest gene expression differences in the contractile SMC and FMC clusters, near total CMC ablation was observed, accompanied by marked downregulation of CMC marker genes, including Col2a1 and Ibsp . Importantly, a substantial decrease in neointimal calcium deposition was also observed. Although Sox9 deletion resulted in closure of chromatin at many CMC genes, overall, a net opening was observed in CMC cluster cells, particularly at genes implicated in FMC-associated processes including inflammation and cytoskeletal dynamics. Peaks of de novo open chromatin were enriched for the motifs of athero-protective, anti-chondrogenic transcription factors, including SMAD3 and AHR, implicating SOX9 as an epigenetic antagonist of anti-chondrogenic programs. In addition, Sox9 ablation substantially reduced total plaque burden in non-aortic root arteries, which was not due to altered serum lipid levels, but which in vitro evidence suggested may in part be due to altered SMC proliferation. Taken together, these data implicate SOX9 as a major regulator of CMC formation and suggest pathological roles for SOX9 beyond intimal calcification. The elucidation of genes and pathways implicated in the FMC to CMC transition could identify novel targets to bias SMC towards plaque-stabilising and away from plaque-destabilising fates.