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

解析复杂疾病的基因组架构，此前一直基于这样的核心认知：遗传变异通过调控基因表达来影响疾病风险¹。然而，最新研究揭示，常见自身炎症性疾病与冠状动脉疾病（coronary artery disease, CAD）的疾病遗传力中，有显著组分是通过遗传变异调控RNA的转录后修饰——即腺苷至肌苷（A-to-I）RNA编辑²所介导。这种常见RNA修饰由ADAR酶（ADAR enzymes）催化，其中ADAR1可编辑特定的免疫原性双链RNA（double stranded RNA, dsRNA），以阻断双链RNA（double stranded RNA, dsRNA）感受器MDA5（IFIH1）的激活³,⁴。多项人类遗传学研究数据表明，RNA编辑受损与dsRNA感知增强是冠状动脉疾病（CAD）风险升高的重要致病机制²,⁵。本研究搭建了人类遗传学观测结果与驱动CAD进展的细胞生物学机制之间的关键关联。通过分析人类动脉粥样硬化斑块，我们证实血管平滑肌细胞（vascular smooth muscle cell, SMC）对RNA编辑具有独特的需求，且MDA5激活发生在SMC表型转化过程中——这证明了RNA编辑的细胞类型与背景特异性作用。我们进一步证实，Adar1可调控SMC的表型状态，是维持血管完整性所必需，并可调控血管钙化的进展。本研究阐明了CAD的一项核心致病机制：细胞类型与背景特异性的RNA编辑及dsRNA感知介导疾病进展，打通了人类遗传学与疾病因果关系的认知鸿沟。实验设计：为探究SMC特异性ADAR1对血管功能及动脉粥样硬化进展的作用，我们将Adar1条件性敲除（flox）小鼠与Myh11 CreERT2背景小鼠杂交，同时引入载脂蛋白E敲除（ApoE-/-）以构建高脂血症模型，并搭配ROSA tdTomato等位基因进行SMC谱系示踪。首先，我们对8周龄雄性小鼠经他莫昔芬处理，实现Adar1的纯合敲除（基因型为Adar1 fl/fl, Myh11CreERT2, ApoE-/-, tdTomato+/+）。该组小鼠会出现严重病变，并在他莫昔芬处理2周后，对升主动脉与主动脉根部进行单细胞RNA测序（single cell RNAseq, scRNA-seq）。对照组小鼠的Adar1 fl/fl等位基因为野生型（基因型为Myh11CreERT2, ApoE-/-, tdTomato+/+），并于8周龄时同样接受他莫昔芬处理。为评估SMC Adar1单倍体不足（haploinsufficiency）对动脉粥样硬化的影响，我们对Adar1 flox等位基因为杂合型的小鼠（基因型为Adar1 fl/WT, Myh11CreERT2, ApoE-/-, tdTomato+/+）于8周龄时经他莫昔芬处理，随后给予16周高脂饮食以诱导动脉粥样硬化。之后，我们对该Adar1杂合小鼠及其对照组小鼠的升主动脉/主动脉根部进行单细胞RNA测序，以探究Adar1单倍体不足如何调控动脉粥样硬化的进展。