Effect of conditional SMC specific deletion of ADAR1 on mouse aortic single cell transcriptomic profile and effect on atherosclerosis
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 SMC specific ADAR1 on vascular function and progression of atherosclerosis, we used the Adar1 flox mouse crossed onto an Myh11 CreERT2 background, that has additional hyperlipidemia with ApoE-/- and SMC lineage tracing with ROSA tdTomato alleles. First, we performed homozygous deletion of Adar1 by treating 8 week old male mice with tamoxifen (Adar1 fl/fl, Myh11CreERT2, ApoE-/-,  tdTomato+/+). These mice develop severe disease and single cell RNAseq was performed of the ascending aorta and aortic root at 2 weeks post tamoxifen. Control mice were wild type for the Adar1 fl/fl allele ( Myh11CreERT2, ApoE-/-,  tdTomato+/+) and were similarly treated with tamoxifen at 8 weeks of age. To then evaluate the effect of SMC Adar1 haploinsufficiency in atherosclerosis, we treated mice that were heterozygous for the Adar1 flox allele (Adar1 fl/WT, Myh11CreERT2, ApoE-/-,  tdTomato+/+) with tamoxifen at 8 weeks of age, and then put on a high fat diet for 16 weeks to induce atherosclerosis. We then performed single cell RNAseq of the ascending aorta/aortic root for these Adar1 het mice along with controls to evaluate how Adar1 haploinsufficiency modifies atherosclerosis