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

摘要： 全球COVID-19大流行突出了肺血管炎症的关键作用， 多器官内皮细胞（EC）通透性在ARDS死亡率和前所未有的COVID-19-ARDS vas中的作用 眼内型（1）。该A1项目#2应用程序仍然专注于eNAMPT（细胞外）的关键作用 烟酰胺磷酸核糖基转移酶）在驱动肺血管炎症和多器官内皮细胞 细胞（EC）通透性，是增加ARDS死亡率的核心事件。我们最初将eNAMPT确定为 利用基因组密集方法的新型ARDS和呼吸机诱导的肺损伤（VILI）治疗靶点 以及过度机械应力/VILI的细胞和临床前研究。我们发现eNAMPT是一种新型的ARDS 血浆eNAMPT水平响应于病毒/细菌感染和暴露于MEN而增加的生物标志物， 机械通气重要的是，利用条件性EC特异性Nampt KO小鼠，我们最近表明， EC通过eNAMPT分泌到循环中对ARDS病理生物学的贡献，从而驱动前 临床上ARDS的炎性肺损伤及严重程度。eNAMPT通过充当 损伤相关分子模式蛋白（DAMP）和进化保守的炎症的主要调节因子， 通过Toll样受体4（TLR 4）的新型连接的炎性级联。我们在小鼠、大鼠和猪中获得的令人兴奋的数据 ARDS/VILI模型已经验证了eNAMPT中和人源化mAb（ALT-100）在再灌注中的功效。 诱导eNAMPT和LPS诱导的TLR 4活化、NFκ B驱动的细胞因子产生、肺通透性和 炎性肺损伤为了进一步询问eNAMPT作为ARDS治疗靶点，SA #1将在 研究表明，ROS产生的ARDS刺激（缺氧，高氧，机械应激，细胞因子）， 诱导NAMPT表达并表征三个关键转录因子（缺氧诱导因子）的作用 HIF 1a/2a，NRF 2），NAMPT/TLR 4启动子SNP和DNA甲基化在遗传/表观遗传调控中的作用 NAMPT/TLR 4启动子活性。SA #2将机械地探索TLR 4和机械的新调节。 应激刺激的eNAMPT分泌是启动炎症级联反应的关键事件，通过细胞外 较大囊泡形成、炎性小体活化和ABC转运蛋白。利用Core B的CRISPR/Cas9基因- SA #3将剖析参与eNAMPT-TLR 4结合的结构/功能机制， EC渗透性增加，特别关注MAP激酶效应子p90 rsk、Akt 1硝化和UCHL 1 EC细胞膜驱动的屏障功能障碍的活性。最后，利用核心C专业知识，SA #4将优化eN- AMPT ALT-100 mAb给药，定义ALT-100递送的治疗窗，并定义潜在的ALT-100 利用临床前大鼠和猪ARDS/VILI模型的mAb与其他PPG治疗方式的协同作用。这 项目#2与每个PPG项目的高度整合将阐明eNAMPT和eNAMPT/TLR 4的EC分泌 参与ARDS/VILI病理学，并将加速ALT-100 mAb作为一种可行的策略，以减轻 炎性EC渗透性和降低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.