Introduction: Adenosine-to-inosine RNA editing is an epitranscriptional regulator of RNA metabolism, catalyzed by the adenosine deaminases acting on RNA-1/-2 (ADAR1/2). 

Hypothesis: Whether the RNA editor, ADAR2, controls ischemia-triggered myocardial inflammation remains unknown. 

Methods: Immune cell recruitment to ischemic tissues was analysed in three mouse genetic models (global and inducible vascular endothelial cell (iEC)-restricted ADAR2 and IL6ST knockout) using three experimental ischemic disease models (hindlimb, heart, and cremaster muscle ischemia). Pharmacological studies using a chimeric protein of the natural inhibitor of IL-6-trans-signaling, soluble gp130 further confirmed the involvement of IL-6-trans-signaling in the ADAR2 mediation effect on ischemia-induced inflammation. The translational value was confirmed in biopsies from patients with ischemic heart disease. Molecular biology and transcriptome-wide studies involved RNA editing assays, site-specific RNA disruption studies, RNA immunoprecipitation, mutagenesis, stability, RNAi and gain- and loss of function mutant protein assays in primary ECs. 

Results: ADAR2 (Adarb1) is induced by >2-fold in endothelium of murine and human ischemic heart disease biopsies. Genetic ablation of global or EC ADAR2 diminishes ischemia-induced heart and skeletal muscle inflammation in mice. ADAR2 is required for IL-6-signal transducer (IL6ST) expression, the co-receptor of IL-6, and thus, IL-6 trans-signaling, evidenced by STAT3-phosphorylation and downstream adhesion molecule expression. Genetic and pharmacological studies underlined the causative involvement of IL-6/IL6ST in ischemia-induced leukocyte trafficking. Intravital imaging of IL-6-inflamed/ ischemic venules showed that rolling and adhesion of leukocyte subsets to vascular wall were impaired by 2-fold in Adarb1^-/-/tg or iEC-Adarb1^-/-. Similarly, leukocyte trafficking was defective during IL-6 sterile peritonitis. ADAR2-IL6ST axis was nearly 2-fold increased in murine and human ischemic heart disease and strongly associated with leukocyte tissue infiltration. ADAR2-deficient transcriptome revealed a selective upregulation of a conserved group of miRNAs targeting the IL6ST mRNA. At single-nucleotide level, site-selective RNA editing mapping and disruption studies as well as Drosha assays revealed that RNA editing of the primary miRNAs stem loops is a determinant for suppressing Drosha recruitment thus inhibiting the maturation of miRs targeting IL6ST mRNA. Multilayered rescue studies including miR-inhibitors restored IL6ST levels after ADAR2 deficiency. 

Conclusions: ADAR2-induced RNA editing controls leukocyte trafficking to ischemic injury, thus regulating myocardial inflammation in patients with ischemic heart disease.