Abstract 12901: ADAR1 RNA Editing in Vascular Smooth Muscle Maintains Vascular Integrity, Regulates Cell State Transition, and is a Novel Mechanism of Coronary Artery Disease Risk

Abstract

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes. ADAR1 serves a crucial function to edit specific immunogenic double stranded RNA (dsRNA) molecules to prevent the dsRNA sensor, MDA5 ( IFIH1 ), activating an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism in coronary artery disease (CAD). Here, we reveal in human atherosclerosis, smooth muscle cells (SMCs) express immunogenic dsRNA at markedly higher levels compared to all other cell types. We demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC). Loss of RNA editing through ADAR1 KD in HCASMC causes an ISG response characterized by upregulation of ISG genes (i.e. ISG15, IFI6, IFIH1 ) in addition to genes with known roles in SMC phenotypic transition and CAD risk (i.e. KLF4 , TCF21 , and ZEB2 ). ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro and in atherosclerosis prone mice. scRNAseq analysis reveals SMC to have increasing ISG expression as cells transition from mature SMC to a more calcified chondromyocyte phenotype. To further evaluate the role for SMC RNA editing in vivo , we generated a conditional SMC specific Adar1 KD mouse ( Adar1 flox/flox , Myh11 CreERT2 , ROSA tdT/+ , ApoE -/- ). At 2 weeks following SMC- Adar1 KD, we observe 50% mortality, where mice demonstrate a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. This effect was not seen with an endothelial cell specific deletion model ( Adar1 flox/flox , Cx40 CreERT2 ). scRNAseq of aortic tissue shows that SMC- Adar1 KD causes ISG activation throughout the vessel wall, SMCs show loss of mature SMC markers, and there is robust macrophage and T cell infiltration. Through evaluation of ligand-receptor interaction, we show that Ccl5:Ccr5 is the principal regulator of cell infiltration following SMC- Adar1 KD. Here we provide key evidence to support the observations from human genetics that the ADAR1-dsRNA-MDA5 axis is a causal mechanism in vascular disease and warrants further investigation.