Study Of ADAR1 Implicates RNA Editing In Vascular Smooth Muscle Cells As A Mechanism Of Coronary Artery Disease Risk

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 double stranded RNA (dsRNA) molecules to prevent activation of the dsRNA sensor MDA5 ( IFIH1 ) that stimulates 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). We hypothesize that smooth muscle cell (SMC) specific RNA editing is required to prevent vascular calcification and inflammation. Here, we demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC). ADAR1 KD in HCASMC causes an ISG response characterized by massive upregulation of key ISG genes (e.g. ISG15, IFI6, IFIH1 ). Importantly, ADAR1 KD stimulates the upregulation of genes with known roles in SMC phenotypic transition and CAD risk, including KLF4 , TCF21 , ATF3 , and ZEB2 . This effect is dependent on activation of MDA5. Additionally, we demonstrate ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro where ADAR1 KD inhibits SMC maturation and dysregulates the calcification gene program in an MDA5 dependent manner. Using scRNAseq data collected from human vascular tissue we show that immunogenic dsRNA are disproportionately expressed in human SMC in atherosclerosis. Similarly, 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 — consistent with MDA5 activation. To further evaluate the role for SMC specific RNA editing in vivo, we generated a conditional SMC specific Adar1 KD mouse ( Adar1 flox/flox , Myh11 CreERT2 , ROSA tdT/+ ). At 2 weeks following SMC- Adar1 KD, 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 ). 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.