RNA editing controls pulmonary endothelial pathophenotypes in pulmonary hypertension

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ABSTRACT The primary objective of this proposal is to generate a scientific discovery program in pulmonary vascular disease to ensure a robust pathway for Dr. Woodcock to develop into an independent research scientist. This objective will be accomplished through a combination of active mentorship, didactic training, enrichment activities, and research. The core of the research focuses on the role of RNA editing, a post-transcriptional regulation mechanism, in pulmonary hypertension. Pulmonary hypertension (PH) is a lethal disease without a cure. Early apoptosis in pulmonary artery endothelial cells (PAECs) is a key trigger for the development of PH. However, there is a critical knowledge gap regarding the mechanisms or key factors controlling PAEC pathophenotypes that drive this deadly disease, and no effective therapeutics exist for PH that target this key cell type. Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA editing enzyme that converts adenosine to inosine (A-to-I) in genome-encoded RNA transcripts and plays a vital role in gene regulation and cardiac function. However, the roles of ADAR1-mediated RNA editing in the regulation of pulmonary vascular function are unknown. Our preliminary data indicate that ADAR1 plays a crucial role in the regulation of PAEC survival and the cytosolic innate immunity response. To identify enriched genes in PAECs that are modified and regulated by ADAR1 RNA editing activity, we recently performed in vitro RNA sequencing in ADAR1 knockdown PAECs. From this analysis, we found that the circadian gene nocturnin (NOCT) contains two active ADAR1 RNA editing sites. Non-edited NOCT transcript is stable and accumulates within PAECs lacking ADAR1 RNA editing. Forced expression of NOCT upregulated key interferon transcriptional factor IRF7 and promoted apoptosis in PAECs. NOCT knockout (KO) mice mitigated PH induced by hypoxia and IL6 exposure. Lastly, silenced NOCT restored PAEC apoptosis and immunity response triggered by ADAR1 deficiency. In our pilot studies, we explored key elements of this concept by showing that ADAR1 RNA editing can control NOCT expression to regulate PAEC functions and PH. Given these findings, we hypothesize that ADAR1 RNA editing deficiency promotes PH development due to induction of pulmonary artery endothelial cell apoptosis via aberrant NOCT- driven innate immunity activation. This hypothesis will be addressed in the experiments of the following Specific Aims: (1) to determine the role of NOCT in the innate immunity-dependent PAEC survival response; (2) to determine the effect of ADAR1-specific RNA editing on NOCT expression and function in PAECs; and (3) to define the impact of endothelial ADAR1 deficiency on NOCT-IFN signaling in PH in vivo. This novel mechanism may have important implications for how RNA editing regulates PA endothelial functions related to PH pathogenesis, and its elucidation will advance our understanding of PH development and ultimately lead to novel strategies for developing effective therapeutics to treat PH. This project will provide me with advanced expertise in RNA biology and pulmonary physiology that will strengthen my development into an independent investigator.