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

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. ADAR1 (adenosine deaminase acting on RNA 1, gene name ADAR ) is an RNA editing enzyme that converts adenosine to inosine in RNA transcripts and participates in RNA metabolism. Although 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 AND RESULTS: ADAR1 expression was downregulated in the pulmonary vascular endothelium and in the lung tissue of human and mouse PH. Global adenosine to inosine RNA editing was decreased in lungs from patients with PAH and hypoxic PH mice. In vitro, hypoxia, a PH trigger, downregulated ADAR1 in PAECs. By RNA sequencing of PAECs after ADAR1 knockdown, we identified the circadian gene NOCT (nocturnin) as a direct ADAR1 target. NOCT was found to carry 2 active adenosine-to-inosine RNA editing sites in the 3′UTR. By single-cell RNA sequencing of human PAH lungs, NOCT editing levels were reduced, while NOCT protein and transcript levels increased. Correspondingly, in vitro, ADAR1 silencing increased NOCT mRNA levels, thus inducing double-strand RNA-MDA5 (melanoma differentiation-associated protein 5) sensing interferon signaling and PAEC apoptosis. Importantly, silencing of NOCT reversed these changes. Forced NOCT expression phenocopied the effect of ADAR1 knockdown, upregulating interferon signaling molecules and increasing apoptosis. This ADAR1-NOCT axis was studied across multiple rodent models of disease. Chronically, hypoxic PH mice carrying a human missense mutation in ADAR displayed worsened PH. Forced adeno-associated virus expression of Adar improved monocrotaline-induced PH in rats. Genetic deletion of Noct mitigated PH in hypoxic interleukin 6-expressing transgenic PH mice, emphasizing the crucial role of NOCT in PH pathogenesis. CONCLUSIONS: Hypoxia-induced ADAR1 deficiency upregulates NOCT expression 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.