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

此 A1 应用重点关注细胞外烟酰胺磷酸核糖基转移酶的关键作用 (eNAMPT) 驱动肺血管炎症和多器官内皮细胞 (EC) 通透性，这些事件 对于增加 ARDS 死亡率和 COVID-19-ARDS 血管内型至关重要。我们初步确定 eNAMPT 作为一种利用基因组强化的新型 ARDS 和呼吸机诱发性肺损伤 (VILI) 治疗靶点 过度机械应力/VILI 的方法以及细胞和临床前研究。我们证明 eNAMPT 是 新型 ARDS 生物标志物，其血浆 eNAMPT 水平随着病毒/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 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 评估临床前 ARDS 炎性肺损伤及其严重程度。我们已经证明 eNAMPT 会产生这些伤害 作为损伤相关分子模式蛋白 (DAMP) 和主要调节因子发挥严重作用 通过 Toll 样受体 4 (TLR4) 的新型连接，进化上保守的炎症级联反应。我们的前任 引用大鼠和猪 ARDS/VILI 模型的临床前数据验证了 eNAMPT 的功效 中和人源化单克隆抗体 (ALT-100) 可减少 eNAMPT 和 LPS 诱导的 TLR4 激活和 NFκB- 驱动细胞因子的产生、肺通透性和炎症性肺损伤。进一步询问和验证 eNAMPT 作为 ARDS 治疗靶点，具体目标 #1 将扩展先前显示 ROS 生成的研究 ARDS 刺激（缺氧、高氧、机械应力、细胞因子）诱导 NAMPT 表达和 NAMPT 亲 运动 SNP 显着增加 eNAMPT 血浆水平和 ARDS 死亡率风险（减少无呼吸机 天，ARDS 死亡率增加）。 SA #1 将描述三个关键转录因子（缺氧- 诱导因子 HIF1/2、NRF2)、NAMPT 和 TLR4 启动子 SNP，以及遗传/ep- 中的 DNA 甲基化位点 NAMPT 和 TLR4 启动子活性的基因调控。由于 eNAMPT 分泌是炎症启动的关键 SA #2 将机械性地探索 TLR4 和机械应力的新调节 通过细胞外囊泡形成、炎性体激活和 ABC 转运刺激 eNAMPT 分泌 呃。随着 eNAMPT 中和 ALT-100 mAb 的治疗逆转了 Akt1 硝化的急剧增加， MAP 激酶效应器激活，并减少 Akt/mTOR 去泛素化，SA #3 将剖析结构/功能 参与 eNAMPT-TLR4 结合并刺激 EC 通透性增加的机制 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 活性的影响。 最后，SA #4 将优化 eNAMPT ALT-100 mAb 剂量和给药时间，作为治疗策略 临床前大鼠和猪 ARDS/VILI 模型。 EC 分泌 eNAMPT 和 eNAMPT/TLR4 的剖析 参与 ARDS/VILI 病理学将加速 ALT-100 mAb 的开发，这是一项可行的战略 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.