Role of Endothelial eNAMPT Secretion and TLR4 Signaling in the ARDS Vascular Endotype

这个A1的应用重点是细胞外烟酰胺磷酸核糖转移酶的关键作用 (ENAMPT)在推动肺血管炎症和多器官内皮细胞(EC)通透性方面的作用，事件 是增加急性呼吸窘迫综合征死亡率和新冠肺炎-急性呼吸窘迫综合征血管内型的核心。我们最初确定了 ENAMPT作为一种新的ARDS和呼吸机诱导肺损伤(VILI)的基因组密集治疗靶点 过度机械应力的入路、细胞学和临床前研究/VILI。我们展示了eNAMPT是一个 一种新的ARDS生物标志物，其血浆eNAMPT水平随着病毒/细菌感染和博览会的反应而升高 当然是机械通风。重要的是，利用有条件的EC特异性NAMPT KO小鼠，我们最近已经 研究表明，EC在ARDS病理生物学中的作用包括eNAMPT分泌到循环中，从而驱动血管紧张素转换酶的生成。 ING临床前ARDS炎性肺损伤及严重程度。我们已经证明，eNAMPT产生这些注射- 作为损伤相关分子模式蛋白(DAMP)的Rious效应和 通过新的Toll样受体4(TLR4)的连接，进化保守的炎症级联反应。我们的前任- 引用了大鼠和猪ARDS/VILI模型的临床前数据，证实了eNAMPT- 中和人源化单抗(ALT-100)降低eNAMPT和内毒素诱导的TLR4活化和NF-κB- 驱动细胞因子的产生、肺通透性和炎症性肺损伤。进一步审问和验证 ENAMPT作为ARDS的治疗靶点，特殊目标#1将扩展先前的研究，这些研究表明ROS产生 ARDS刺激(低氧、高氧、机械应激、细胞因子)诱导NAMPT表达和NAMPT前体 更多SNPs显著增加eNAMPT血浆水平和ARDS死亡率(减少无呼吸机 天数，ARDS死亡率增加)。SA#1将描述三个关键转录因子的作用(缺氧- 诱导因子HIF1/2、NAMPT和TLR4启动子SNP，以及Genetic/EP中的DNA甲基化位点 NAMPT和TLR4启动子活性的遗传调控。因为eNAMPT的分泌是启动炎症的关键- 人工级联激活，SA#2将从机械上探索TLR4-和机械应力-的新调节- 通过细胞外小泡形成、炎性小体激活和ABC转运刺激eNAMPT的分泌。 艾尔斯。随着eNAMPT中和ALT-100单抗的处理逆转了Akt1硝化的急剧增加， MAP激酶效应器激活，Akt/mTOR去泛素化减少，SA#3将剖析其结构/功能 ENAMPT-TLR4结合和刺激的EC通透性增加的机制 在EC细胞骨架驱动的屏障功能障碍中，MAP激酶效应器p90rsk、Akt1硝化和UCHL1活性。 最后，SA#4将优化eNAMPT ALT-100单抗的剂量和给药时间，作为一种治疗策略 临床前期大鼠和猪ARDS/VILI模型。ENAMPT和eNAMPT/TLR4对EC分泌的剖析 参与ARDS/VILI病理生物学将加速ALT-100单抗的开发，这是一种可行的战略，以 减轻炎症性肺损伤、EC通透性和ARDS/VILI死亡率。

This A1 application is focused on the critical role of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) in driving lung vascular inflammation and multi-organ endothelial cell (EC) permeability, events that are central to increasing ARDS mortality and the COVID-19-ARDS vascular endotype. 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 expo- sure to mechanical ventilation. Importantly, utilizing conditional EC–specific NAMPT KO mice, we have recently shown that EC contributions to ARDS pathobiology includes eNAMPT secretion into the circulation thereby driv- ing preclinical ARDS inflammatory lung injury and severity. We have shown that eNAMPT produces these inju- rious effects by functioning as a damage-associated molecular pattern protein (DAMP) and master regulator of evolutionarily-conserved inflammatory cascades via novel ligation of the Toll–like receptor 4 (TLR4). Our ex- citing preclinical data in both rat and porcine ARDS/VILI models have validated the efficacy of the eNAMPT- neutralizing humanized mAb (ALT-100) in reducing eNAMPT- and LPS-induced TLR4 activation and NFκB- driven cytokine production, lung permeability and inflammatory lung injury. To further interrogate and validate eNAMPT as a ARDS therapeutic target, Specific Aim #1 will extend prior studies which showed ROS-generating ARDS stimuli (hypoxia, hyperoxia, mechanical stress, cytokines) to induce NAMPT expression and NAMPT pro- moter SNPs to significantly increase eNAMPT plasma levels and risk of ARDS mortality (reduced ventilator-free days, increased ARDS mortality). SA #1 will characterize the role of three key transcription factors (hypoxia- inducible factors HIF1/2, NRF2), NAMPT and TLR4 promoter SNPs, and DNA methylation sites in genetic/ep- igenetic regulation of NAMPT and TLR4 promoter activities. As eNAMPT secretion is key to initiation of inflam- matory cascade activation, SA #2 will mechanistically explore novel regulation of TLR4- and mechanical stress- stimulated eNAMPT secretion via extracellular vesicle formation, inflammasome activation, and ABC transport- ers. As treatment with the eNAMPT-neutralizing ALT-100 mAb reversed the dramatic increases in Akt1 nitration, MAP kinase effector activation, and reduced Akt/mTOR deubiquitination, SA #3 will dissect the structure/function mechanisms involved in eNAMPT-TLR4 binding and stimulated increases in EC permeability with specific focus on MAP kinase effector p90rsk, Akt1 nitration, and UCHL1 activity in EC cytoskeletal-driven barrier dysfunction. Finally, SA #4 will optimize eNAMPT ALT-100 mAb dosing and time of delivery as a therapeutic strategy in preclinical rat and porcine ARDS/VILI models. The dissection of EC secretion of eNAMPT and eNAMPT/TLR4 participation in ARDS/VILI pathobiology will accelerate ALT-100 mAb development, an actionable strategy to attenuate inflammatory lung injury, EC permeability and ARDS/VILI mortality.