De-differentiation of synthetic vascular smooth muscle in human coronary artery plaque fibrous cap to macrophage-like vascular smooth muscle promotes plaque instability

Background Myocardial infarction is the leading cause of death globally, primarily due to coronary artery atherosclerosis (CAA). CAA is characterized by plaque development from lipid accumulation, immune cell infiltration and smooth muscle cell phenotype changes within the vessel wall. Up to 70% of myocardial infarctions occur when plaques rupture due to fibrous cap instability. The mechanism by which plaque instability develops in advanced atherosclerosis is poorly understood. Purpose Our aim was to identify key cell types involved in fibrous cap formation and degradation in human coronary arteries. We hypothesized that synthetic vascular smooth muscle cells (VSMCs) promote fibrous cap formation in CAA, and that plaque instability is a result of their de-differentiation into a lipid-scavenging, macrophage-like phenotype in the later stages of plaque progression. Methods Eight cryopreserved human left anterior descending (LAD) coronary artery samples with varying degrees of CAA progression were analysed. Advanced single-cell RNA sequencing (scRNAseq) and spatial transcriptomics were performed on fixed samples using probe-based transcript targeting and subsequent sequencing, followed by analysis with the Seurat package in R Studio. A detailed analysis was conducted to assess cellular heterogeneity, localisation and interactions within each CAA sample. Results ScRNAseq analysis of 32,481 cells identified nine major cell clusters, including endothelial cells, VSMCs, fibroblasts, macrophages, B cells, T cells, leukocytes, adipocytes, and neuronal cells. Four VSMC subtypes were identified: contractile, synthetic, proinflammatory, and macrophage-like. Gene ontology enrichment analysis revealed distinct biological processes in each VSMC subtype (Table 1). Spatial analysis showed varying prevalence of VSMC subtypes across different stages of plaque progression Gene signatures for synthetic VSMCs were lowly expressed in healthy samples, with enriched expression in the fibrous cap of plaques in early stages of disease, but not in the later stages. Further, Macrophage-like VSMC gene signatures were absent in healthy arteries, expressed within the plaque in early-stage plaques and highly enriched within the fibrous cap of more advanced plaques. Conclusion This study provides new insights into mechanism of VSMC heterogeneity in human CAA and highlights an important potential role of fibrous cap synthetic VSMCs in promoting plaque instability, by converting to a macrophage-like phenotype within the fibrous cap. Future work should focus on therapeutic strategies targeting these cellular transitions to prevent plaque rupture.