Effect of ADAR1 and MDA5 knock down by siRNA on human SMC phenotypic modulation and calcification in vitro
Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression1. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing2. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 (IFIH1)3,4. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing to be an important mechanism of coronary artery disease (CAD) risk2,5. Here, we provide a crucial link between observations in human genetics and the mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque, we implicate the vascular smooth muscle cell (SMC) to have a unique requirement for RNA editing, and that MDA5 activation occurs in SMC phenotypic modulation — demonstrating a cell type and context specific role of RNA editing. We further show Adar1 controls SMC phenotypic state, is required to maintain vascular integrity, and controls progression of vascular calcification. Through this work, we describe a fundamental mechanism of CAD, where cell type and context specific RNA editing and sensing of dsRNA mediates disease progression, bridging our understanding of human genetics and disease causality. Overall design: To investigate the role of ADAR1 and IFIH1 (MDA5) in regulating SMC phenotype in vitro, first we performed an in vitro assay using primary human coronary artery smooth muscle cells (HCASMC) and performed a phenotypic modulation assay with siRNA KD of ADAR1 or ADAR1 and IFIH1 and performed bulk RNA sequencing in both mature SMC phenotype (serum starved) and modulated SMC phenotype (serum stimulated). Second, we performed an in vitro assay of SMC calcification by culturing immortalized HCASMC cells in control or pro-calcification media for 7 days following siRNA KD of ADAR1, ADAR1 and IFIH1, or IFIH1 alone and performed bulk RNA sequencing.