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

此A1申请集中于细胞外烟酰胺磷酸核糖基转移酶的关键作用 （eNAMPT）在驱动肺血管炎症和多器官内皮细胞（EC）渗透性中的作用， 是增加ARDS死亡率和COVID-19-ARDS血管内皮型的核心。我们最初发现 eNAMPT作为一种新的ARDS和呼吸机诱导的肺损伤（VILI）治疗靶点，利用基因组密集型 方法和细胞和临床前研究过度机械应力/VILI。我们展示了eNAMPT是一个 新的ARDS生物标志物，血浆eNAMPT水平响应于病毒/细菌感染和暴露而增加， 一定要机械通气。重要的是，利用条件性EC特异性NAMPT KO小鼠，我们最近 表明EC对ARDS病理学的贡献包括eNAMPT分泌到循环中，从而驱动 临床前ARDS炎症性肺损伤及严重程度。我们已经证明，eNAMPT产生这些损伤- 作为一种损伤相关分子模式蛋白（DAMP）和主调节因子， 通过Toll样受体4（TLR 4）的新型连接，进化保守的炎症级联反应。我们的前- 引用大鼠和猪ARDS/VILI模型的临床前数据已经验证了eNAMPT的功效， 中和人源化mAb（ALT-100）降低eNAMPT和LPS诱导的TLR 4活化和NFκB- 驱动的细胞因子产生、肺渗透性和炎性肺损伤。为了进一步询问和验证 eNAMPT作为ARDS治疗靶点，具体目标#1将扩展先前的研究，这些研究显示ROS生成 ARDS刺激（缺氧、高氧、机械应激、细胞因子）诱导NAMPT表达和NAMPT原- 显著增加eNAMPT血浆水平和ARDS死亡率风险（减少无呼吸机 天，增加ARDS死亡率）。SA #1将表征三个关键转录因子的作用（缺氧- 诱导因子HIF 1 α/2 β，NRF 2），NAMPT和TLR 4启动子SNP，以及遗传/表型中的DNA甲基化位点。 NAMPT和TLR 4启动子活性的遗传调控。由于eNAMPT分泌是引发炎症的关键， 炎症级联激活，SA #2将机械地探索TLR 4和机械应力的新调节， 通过细胞外囊泡形成、炎性小体活化和ABC转运刺激eNAMPT分泌- 呃。随着使用eNAMPT中和ALT-100 mAb治疗逆转了Akt 1硝化的急剧增加， MAP激酶效应子激活和Akt/mTOR去泛素化减少，SA #3将剖析结构/功能 参与eNAMPT-TLR 4结合的机制，并刺激EC通透性增加，具有特异性焦点 MAP激酶效应p90 rsk，Akt 1硝化，和UCHL 1活性在EC细胞驱动的屏障功能障碍。 最后，SA #4将优化eNAMPT ALT-100 mAb给药和给药时间作为治疗策略， 临床前大鼠和猪ARDS/VILI模型。内皮细胞分泌eNAMPT和eNAMPT/TLR 4的研究 参与ARDS/VILI病理学研究将加速ALT-100 mAb的开发，这是一项可行的策略， 减轻炎性肺损伤、EC渗透性和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.