ScRNA-seq of Lineage-tracing Mice Identifies Smooth Muscle Cell Phenotypic Alterations in Aortic Aneurysms and Dissections

ScRNA-seq analysis revealed that aortic stress induced the transition of SMCs from a primary contractile phenotype to proliferative, extracellular matrix (ECM)-producing, inflammatory, and dying phenotypes. Lineage tracing showed the complete transformation of SMCs to fibroblasts and macrophages. ScATAC-seq analysis indicated that these phenotypic alterations were controlled by chromatin remodeling marked by the reduced chromatin accessibility of contractile genes and the induced chromatin accessibility of genes involved in ECM, inflammation, and cell death. IRF3, a pro-inflammatory transcription factor activated by cytosolic DNA, was identified as a key driver for the transition of aortic SMCs from a contractile phenotype to an inflammatory phenotype. In cultured SMCs, cytosolic DNA signaled through its sensor STING-TBK1 to activate IRF3, which bound and recruited EZH2 to contractile genes to induce repressive H3K27me3 modification and contractile gene suppression. In contrast, dsDNA-STING-IRF3 signaling induced inflammatory gene expression in SMCs. In Sting-/- mice, the aortic stress–induced transition of SMCs into an inflammatory phenotype was prevented, and SMC populations were preserved. Finally, profound SMC phenotypic alterations in diverse directions were detected in human ATAAD tissues. To understand aortic SMC dynamics during AAD development, we performed the single-cell transcriptomic analysis of aortic tissues collected from Myh11-CreERT2+ mT/mG lineage-tracing mice that were infused with Saline or angiotensin II (AngII; 1000 ng/kg/min) for 7 days. SMCs became green in color upon Cre induction, whereas non-SMCs remained red in color. For mice infused with AngII, aortas were separated as intramural hematoma/dissection (IMH/D) and non-IMH/D according to the condition of aorta. For each group, single-cell suspensions from the ascending thoracic aorta of 3 mice were pooled. SMCs (GFP+ cells) and non-SMCs (RFP+ cells) from the ascending aorta of AngII-infused mice were separated by using flow cytometry and individually processed for scRNA-seq. In total, 6 samples were submitted for scRNA-seq analysis.