Functional Role of HIF-PHDs in ARDS

项目摘要 本申请旨在研究HIF-1 α对缺氧诱导因子HIF-1A的调节。 急性呼吸窘迫综合征（ARDS）期间的脯氨酰羟醛酶（PHDs）。ARDS是一种极端的 由损伤或感染引起的肺部炎症反应。在外科手术患者中， 大手术，并导致发病率和死亡率急剧增加。此外，ARDS深刻地影响了 感染或败血症患者，包括最近的COVID-19大流行。然而，只有一小部分 的病人接受大手术或肺部感染后会发展成急性呼吸窘迫综合征。所以我们 假设内源性适应性反应存在，以保护肺部不发生严重的 炎症导致ARDS。 我们实验室以前的研究表明，肺泡上皮细胞表达的HIF 1A是内源性的， 控制ARDS期间过度肺泡炎症的反馈信号。目前的应用集中在 研究博士生对HIF 1A的“上游”调控。在缺氧或ARDS期间，PHD被抑制， 从而促进HIF的稳定。已知三种PHD亚型（PHD 1 -3）。我们的初步研究 表明PHD 1是肺中最丰富的PHD。使用机械通气诱导 ARDS显示选择性抑制Phd 1。此外，Phd 1或基因缺失的药理学抑制 （Phd 1-/-小鼠）与减弱的肺损伤有关。此外，肺泡Phd 1缺失的小鼠 （Phd 1 loxp/loxp SPC CreER+）在“常规”ARDS或SARS-CoV-2病毒感染期间受到保护。 对介导PHD 1抑制的miRNA的筛选将我们指向了miR-15 a/16。事实上，miR-15 a/16是 转录诱导的HIF 1A和有效地抑制PHD 1在ARDS。此外，过度表达 miR-16的表达与PHD 1抑制和增强的HIF 1A稳定性一起提供肺保护。 因此，我们假设miR-15 a/16依赖的PHD 1抑制和伴随的增强作用可能与miR-15 a/16的表达有关。 HIF 1A稳定功能的内源性前馈回路的关键衰减肺泡 急性呼吸窘迫综合征期间的炎症。我们设计了四个目标来解决这个假设，包括Aim 1， 重点关注miR 15 a/16和PHD 1的相互作用，包括在ARDS患者中的原理验证研究。 在目标2中，我们将在传统的ARDS模型中进行体内研究。在目标3中，我们正在扩大我们的研究 使用我们在UTHealth的BSL-3实验室进行SARS-CoV-2相关ARDS的体内模型。在目标4中， 我们将探索靶向miR-15 a/16-PHD 1通路用于ARDS治疗的治疗策略。 这些研究的成功完成将为我们推进临床试验提供科学基础 靶向个体PHD或miR-15 a/B以预防或治疗手术患者中的ARDS，或 发生病原体相关ARDS的患者，例如在COVID-19期间。

PROJECT SUMMARY This application aims to investigate the regulation of hypoxia-inducible factor HIF1A by HIF- prolylhydroxyalses (PHDs) during acute respiratory distress syndrome (ARDS). ARDS is an extreme inflammatory response of the lungs triggered by injury or infection. In surgical patients, ARDS can occur after major surgery and causes dramatic increases in morbidity and mortality. Moreover, ARDS profoundly impacts patients with infections or sepsis, including the recent COVID-19 pandemic. However, only a minor percentage of patients who undergo major surgery or who have lung infections go on to develop ARDS. Therefore, we hypothesized that endogenous adaptive responses exist to protect the lungs from developing severe inflammation causing ARDS. Previous studies from our laboratory identified alveolar-epithelial expressed HIF1A as an endogenous feedback signal controlling excessive alveolar inflammation during ARDS. The current application is focused on examining the "upstream" regulation of HIF1A by PHDs. During hypoxia or ARDS, PHDs are inhibited, thereby promoting the stabilization of HIFs. Three PHD iso-forms are known (PHD1-3). Our preliminary studies indicate that PHD1 is the most abundant PHD in the lungs. Studies using mechanical ventilation to induce ARDS revealed selective repression of Phd1. In addition, pharmacologic inhibition of Phd1 or genetic deletion (Phd1-/- mice) is associated with attenuated lung injury. Moreover, mice with alveolar deletion of Phd1 (Phd1loxp/loxp SPC CreER+) are protected during "conventional" ARDS or infection with the SARS-CoV-2 virus. A screen for miRNAs that mediate PHD1-repression pointed us towards miR-15a/16. Indeed, miR-15a/16 is transcriptionally induced by HIF1A and effectively represses PHD1 during ARDS. In addition, overexpression of miR-16 provides lung protection in conjunction with PHD1 repression and enhanced HIF1A stabilization. Thus, we hypothesize that miR-15a/16-dependent repression of PHD1 and concomitant enhancement of HIF1A stabilization functions in an endogenous feedforward loop critical for attenuating alveolar inflammation during ARDS. We designed four aims to address this hypothesis, including Aim1, which is focused on the interaction of miR15a/16 and PHD1, including proof-of-principle studies in patients with ARDS. In Aim 2, we will pursue in vivo studies in conventional ARDS models. In Aim 3, we are extending our studies towards in vivo models of SARS-CoV-2-associated ARDS using our BSL-3 laboratory at UTHealth. In Aim 4, we will explore therapeutic strategies targeting the miR-15a/16-PHD1 pathway for ARDS treatments. Successful completion of these studies will give us the scientific foundation to move forward with clinical trials targeting individual PHDs or miR-15a/b towards the prevention or treatment of ARDS in surgical patients or patients experiencing pathogen-associated ARDS, such as during COVID-19.