Effect of conditional SMC specific deletion of ADAR1 with constitutive deletion of RNA sensor MDA5 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 expression. 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 editing. 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) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing by MDA5 to be an important mechanism of coronary artery disease (CAD) risk. Here, we provide a crucial link between observations in human genetics and 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 ISG induction occurs in SMC phenotypic modulation, implicating MDA5 activation. Through culture of human coronary artery SMCs, generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background with additional constitutive deletion of MDA5 (Ifih1), and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state by regulating Mda5 activation, is required to maintain vascular integrity, and controls progression of atherosclerosis and 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 and RNA sensor MDA5 on vascular function and progression of atherosclerosis, we used the Adar1 flox mouse crossed onto an Myh11 CreERT2 background, with additional constitutive either homozygous or heterozygous deletion of Mda5 (Ifih1), that has additional hyperlipidemia with ApoE-/- and SMC lineage tracing with ROSA tdTomato alleles. In our prior experiments, 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. In the submitted data, SMC Adar1 deletion was performed with additional constitutive homozygous deletion of MDA5 (Adar1 fl/fl, Ifih1-/-, Myh11CreERT2, ApoE-/-,  tdTomato+/+). Control mice were wild type for the Adar1 fl/fl allele and Ifih1 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, in our prior experiments 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. In the submitted dataset, these SMC Adar1 het mice where then crossed onto the Mda5 het background where scRNAseq was performed at the same 16 week timepoint.