RNA editing controls pulmonary endothelial pathophenotypes in pulmonary hypertension

摘要 该提案的主要目的是在肺血管疾病方面产生一个科学发现计划 以确保伍德考克博士发展成为一名独立研究科学家的强大途径。这一目标 将通过积极的指导，教学培训，丰富活动， research.该研究的核心集中在RNA编辑的作用上，这是一种转录后调控 机制，在肺动脉高压。肺动脉高压（PH）是一种无法治愈的致命疾病。早期 肺动脉内皮细胞（PAECs）的凋亡是PH发展的关键触发因素。然而， 在控制PAEC病理表型的机制或关键因素方面存在严重的知识缺口 导致这种致命疾病，目前还没有针对这种关键细胞类型的有效治疗方法。 腺苷脱氨酶作用于RNA 1（ADAR 1）是一种双链RNA编辑酶， 在基因组编码的RNA转录物中，腺苷转化为肌苷（A-to-I），在基因调控中起着至关重要的作用， 心脏功能然而，ADAR 1介导的RNA编辑在肺血管调节中的作用， 功能未知。我们的初步数据表明，ADAR 1在PAEC的调节中起着至关重要的作用 存活和胞质固有免疫应答。为了鉴定PAEC中富集的基因， 受ADAR 1 RNA编辑活性的调控，我们最近在ADAR 1敲低中进行了体外RNA测序， PAEC。从这个分析中，我们发现昼夜节律基因NOCT含有两个活性ADAR 1 RNA， 编辑网站。未编辑的NOCT转录物是稳定的，并在缺乏ADAR 1 RNA编辑的PAEC内积累。 NOCT的强制表达上调了关键干扰素转录因子IRF 7，并促进了细胞凋亡。 PAEC。NOCT敲除（KO）小鼠减轻由缺氧和IL 6暴露诱导的PH。最后，沉默的NOCT 恢复由ADAR 1缺陷引发的PAEC凋亡和免疫应答。在试点研究中，我们 通过显示ADAR 1 RNA编辑可以控制NOCT表达， 根据这些发现，我们假设ADAR 1 RNA编辑缺陷 通过异常NOCT诱导肺动脉内皮细胞凋亡促进PH的发展- 驱动先天免疫激活。这一假设将在以下具体的实验中得到解决。 目的：（1）确定NOCT在先天免疫依赖性PAEC存活反应中的作用;（2） 确定ADAR 1特异性RNA编辑对PAEC中NOCT表达和功能的影响;以及（3） 定义内皮ADAR 1缺陷对PH中NOCT-IFN信号传导的影响。这种新颖的机制 可能对RNA编辑如何调节与PH相关的PA内皮功能具有重要意义 发病机制，它的阐明将促进我们对PH发展的理解，并最终导致新的 发展有效的治疗策略来治疗PH。这个项目将为我提供先进的专业知识 在RNA生物学和肺生理学方面，这将加强我成为一名独立研究者的发展。

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.