A smooth muscle cell gene-regulatory network critical for the development of advanced-stage and symptomatic atherosclerosis [human]

While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remain poorly understood. Single cell RNA sequencing (scRNAseq) data generated with SmartSeq2 (~8000 genes/cell) in nearly 19,000 single cells isolated during atherosclerosis progression in mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease (CAD) patients in the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) study.Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by three smooth-muscle cells (SMC), and three macrophage (MP) subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type pro-inflammatory/Trem2-high lipid-associated (MP) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of three arterial wall GRNs: GRN33 (MP), GRN39 (SMC) and GRN122(MP) with major contributions to CAD heritability and strong associations with clinical scores of coronary atherosclerosis severity (SYNTAX/Duke scores). The presence and pathophysiological relevance of GRN39 was verified in five independent RNAseq datasets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM, in cultured human vascular SMCs.By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a CAD framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced-stage, symptomatic atherosclerosis.