Role of Endothelial eNAMPT/NAMPT secretion and TLR4 signaling in the ARDS Vascular Endotype

摘要： 全球新冠肺炎大流行戏剧性地突显了肺血管炎症和 多器官内皮细胞通透性在急性呼吸窘迫综合征死亡率和史无前例的新冠肺炎-急性呼吸窘迫综合征血管病变中的作用 圆形内型(1)。此A1项目2应用程序的重点仍然是eNAMPT(细胞外)的关键作用 烟酰胺磷酸核糖转移酶在肺血管炎症和多器官内皮中的作用 细胞(EC)通透性，这是增加ARDS死亡率的核心事件。我们最初将eNAMPT确定为 利用基因组密集型方法治疗ARDS和呼吸机诱导肺损伤(VILI)的新靶点 以及过度机械应力的细胞和临床前研究/VILI。我们展示了eNAMPT是一种新型的ARDS 血浆eNAMPT水平随着病毒/细菌感染和暴露于Me而升高的生物标志物- 机械通风。重要的是，利用条件性EC特异性NAMPT KO小鼠，我们最近已经证明 EC通过eNAMPT分泌进入循环，从而推动Pre-PRE在ARDS病理生物学中的作用 临床ARDS炎症性肺损伤及严重程度。ENAMPT通过发挥以下功能来产生这些有害效果 一种损伤相关的分子模式蛋白(DAMP)，是进化保守的炎症反应的主要调节因子。 交配通过新型的Toll样受体4(TLR4)的连接而发生。我们在小鼠、大鼠和猪身上的令人兴奋的数据 ARDS/VILI模型验证了eNAMPT中和人源化mAb(ALT-100)在再感染中的有效性。 诱导eNAMPT和内毒素诱导的TLR4活化，NFκB驱动的细胞因子的产生，肺通透性和 炎症性肺损伤。为了进一步将eNAMPT作为ARDS的治疗靶点，SA#1将在 研究表明，产生ROS的ARDS刺激(低氧、高氧、机械应激、细胞因子)对In- Duce NAMPT的表达及三个关键转录因子(低氧诱导因子)的作用 HIF1a/2a，NRF2)，NAMPT/TLR4启动子SNPs和DNA甲基化在遗传/表观遗传调控中的作用 NAMPT/TLR4启动子活性。SA#2将机械地探索TLR4-和机械的新调节 应激刺激的eNAMPT分泌，是启动炎症级联激活的关键事件，通过细胞外- 大泡形成、炎性小体激活和ABC转运蛋白。利用Core B的CRISPR/CAS9-GENER- SA#3将剖析eNAMPT-TLR4结合所涉及的结构/功能机制，并 EC通透性增加，特别关注MAP激酶效应蛋白p90rsk、Akt1硝化和UCHL1 EC细胞骨架驱动的屏障功能障碍中的活动。最后，利用核心C专业知识，SA#4将优化EN- AMPT ALT-100单抗剂量，确定ALT-100传递的治疗窗口，并定义潜在的ALT-100 利用临床前大鼠和猪ARDS/VILI模型，MAb与其他PPG治疗方法的协同作用。这 项目2与每个PPG项目的高度集成将阐明EC分泌eNAMPT和eNAMPT/TLR4 参与ARDS/VILI病理生物学，并将加速ALT-100单抗作为一种可行的减毒策略 炎性EC通透性和降低ARDS/VILI死亡率。

ABSTRACT: The global COVID-19 pandemic has dramatically highlighted the critical role of lung vascular inflammation and multi-organ endothelial cell (EC) permeability in ARDS mortality and the unprecedented COVID-19-ARDS vas- cular endotype (1). This A1 Project #2 application remains focused on the critical role of eNAMPT (extracellular nicotinamide phosphoribosyltransferase) in driving lung vascular inflammation and multi-organ endothelial cell (EC) permeability, events that are central to increasing ARDS mortality. We initially identified eNAMPT as a novel ARDS and ventilator-induced lung injury (VILI) therapeutic target utilizing genomic–intensive approaches and cellular and preclinical studies of excessive mechanical stress/VILI. We showed eNAMPT is a novel ARDS biomarker with plasma eNAMPT levels increasing in response to viral/bacterial infection and exposure to me- chanical ventilation. Importantly, utilizing conditional EC–specific Nampt KO mice, we have recently shown that EC contributions to ARDS pathobiology via eNAMPT secretion into the circulation, thereby driving pre- clinical ARDS inflammatory lung injury and severity. eNAMPT produces these injurious effects by functioning as a damage-associated molecular pattern protein (DAMP) and master regulator of evolutionarily-conserved inflam- matory cascades via novel ligation of the Toll–like receptor 4 (TLR4). Our exciting data in mouse, rat and porcine ARDS/VILI models have validated the efficacy of the eNAMPT-neutralizing humanized mAb (ALT-100) in re- ducing eNAMPT- and LPS-induced TLR4 activation, NFκB-driven cytokine production, lung permeability and inflammatory lung injury. To further interrogate eNAMPT as an ARDS therapeutic target, SA #1 will extend prior studies which showed ROS-generating ARDS stimuli (hypoxia, hyperoxia, mechanical stress, cytokines) to in- duce NAMPT expression and characterize the role of three key transcription factors (hypoxia-inducible factors HIF1a/2a, NRF2), NAMPT/TLR4 promoter SNPs, and DNA methylation in genetic/epigenetic regulation of NAMPT/TLR4 promoter activities. SA #2 will mechanistically explore novel regulation of TLR4- and mechanical stress-stimulated eNAMPT secretion, a key event to initiation of inflammatory cascade activation, via extracellu- lar vesicle formation, inflammasome activation, and ABC transporters. Utilizing Core B's CRISPR/Cas9- gener- ated EC lines, SA #3 will dissect the structure/function mechanisms involved in eNAMPT-TLR4 binding and increases in EC permeability with specific focus on MAP kinase effector p90rsk, Akt1 nitration, and UCHL1 activity in EC cytoskeletal-driven barrier dysfunction. Finally, utilizing Core C expertise, SA #4 will optimize eN- AMPT ALT-100 mAb dosing, define the therapeutic window for ALT-100 delivery, and define potential ALT-100 mAb synergy with other PPG therapeutic modalities utilizing preclinical rat and porcine ARDS/VILI models. This high integration of Project #2 with each PPG Project will elucidate EC secretion of eNAMPT and eNAMPT/TLR4 participation in ARDS/VILI pathobiology, and will accelerate ALT-100 mAb as an actionable strategy to attenuate inflammatory EC permeability and reduce ARDS/VILI mortality.