Endothelial ADAR1 Deficit Induces the NOCT-IRF7 Axis in Pulmonary Hypertension.

Abstract

BACKGROUND: Early apoptosis of pulmonary artery endothelial cells (PAECs) is a driver of vascular remodeling and pulmonary hypertension (PH), but its regulation is poorly defined. Adenosine deaminase acting on RNA 1 (ADAR1, gene name) is an RNA editing enzyme that converts adenosine to inosine (A-to-I) in RNA transcripts and participates in RNA metabolism. While deficiency in ADAR1-mediated RNA editing stimulates cellular innate immunity signaling and can promote apoptosis, the exact ADAR1 RNA editing targets and downstream mechanisms regulating PAEC survival are unknown. We sought to define the functions and targets of ADAR1-dependent RNA editing that control pulmonary endothelial pathophenotypes in PH. METHODS: ADAR1 or Nocturnin (NOCT) expression and A-to-I RNA editing levels were evaluated in human PAH lungs by immunofluorescent staining and single cell RNA sequencing, respectively. Mice carrying a human missensemutation and genetic deletion ofwith interleukin-6 (il6) transgene were studied in chronic hypoxia-induced PH in vivo models. RESULTS: ADAR1 expression was downregulated in the pulmonary vascular endothelium and in lung tissue of human and mouse PH. Global A-to-I RNA editing was decreased in lungs from PAH patients and hypoxic PH mice. In vitro, hypoxia, a PH trigger, downregulated ADAR1 in PAECs. Circadian genewas identified as a direct ADAR1 target which carries two active A-to-I RNA editing sites in the 3'UTR. In human PAH lungs, NOCT editing levels were reduced, while NOCT protein level increased. Correspondingly, in vitro,silencing increasedmRNA levels, thus inducing dsRNA-MDA5 sensing interferon signaling and PAEC apoptosis. Importantly, silencing ofreversed these changes. Forced NOCT expression phenocopied the effect of ADAR1 knockdown, upregulating interferon signaling molecules and increasing apoptosis. Chronically hypoxic PH mice carrying humanmutation displayed worsened PH. Forced adeno-associated virus (AAV) expression ofimproved monocrotaline-induced PH in rats. Genetic deletion ofmitigated PH in hypoxic il6-expressing transgenic PH mice, emphasizing the crucial role of NOCT in PH pathogenesis. CONCLUSIONS: Hypoxia-induced ADAR1 deficiency upregulatesexpression to induce PAEC interferon signaling activation, PAEC apoptosis, and PH. This study provides impetus to target the ADAR1-NOCT axis for more effective diagnostics and therapeutics for PH.