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

内皮 eNAMPT 分泌和 TLR4 信号传导在 ARDS 血管内型中的作用

• 批准号: 10440855
• 负责人: Joe G. N. Garcia
• 金额: $ 23.27万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-20 至 2022-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10440855
• 关键词: 2019-nCoV; ATP-Binding Cassette Transporters; Acute Respiratory Distress Syndrome; Address; Attenuated; Automobile Driving; Bacterial Infections; Binding; Biological Markers; Blood Circulation; Blood Vessels; COVID-19 pandemic; COVID-19/ARDS; Cell secretion; DNA Methylation; Data; Deubiquitination; Development; Dimerization; Dissection; Dose; Endothelial Cells; Endothelium; Event; Exposure to; FDA approved; FRAP1 gene; Family suidae; Functional disorder; Genetic; Genomics; Hyperoxia; Hypoxia; Hypoxia Inducible Factor; Inflammasome; Inflammation; Inflammatory; Knockout Mice; Ligation; Lung; Mechanical Stress; Mechanical ventilation; Mitogen-Activated Protein Kinases; Modeling; Molecular; Monoclonal Antibodies; Mus; Pattern; Permeability; Pharmacotherapy; Plasma; Pre-Clinical Model; Predisposition; Production; Proteins; Rattus; Regulation; Risk; Role; SARS-CoV-2 infection; Severities; Signal Transduction; Site; Small Interfering RNA; Stimulus; Structure; TLR4 gene; Therapeutic; Time; UCHL1 gene; Ubiquitination; Vascular Permeabilities; Ventilator; Ventilator-induced lung injury; Viral; cytokine; druggable target; efficacy validation; epigenetic regulation; extracellular; extracellular vesicles; hypoxia inducible factor 1; lung injury; lung vascular inflammation; mortality; new therapeutic target; nicotinamide phosphoribosyltransferase; nitration; novel; pre-clinical; preclinical study; promoter; response; therapeutic target; transcription factor; vascular inflammation

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.

该A1应用集中在细胞外烟酰胺磷酸贝糖基转移酶的关键作用上 （ENAMPT）驱动肺血管注射和多器官内皮细胞（EC）渗透性，事件 对于增加ARDS死亡率和COVID-19-ads血管内型的核心。我们最初确定了 使用基因组密集型的陶器作为一种新型ARDS和呼吸机诱导的肺损伤（VILI）治疗靶标 过度机械应力/VILI的方法以及细胞和临床前研究。我们表明enampt是一个 具有血浆enampt水平的新型ARDS生物标志物，响应病毒/细菌感染和博览 一定要进行机械通风。重要的是，使用有条件的EC特异性NAMPT KO小鼠，我们最近有 表明，EC对ARDS病理生物学的贡献包括向循环中分泌的ENAMPT分泌。 临床前炎性肺损伤和严重程度。我们已经表明，ENAMPT会产生这些不变 通过充当损伤相关的分子模式蛋白（潮湿）和主调节剂的丰富影响 通过Toll-like受体4（TLR4）的新结扎结扎，进化保存的炎症级联反应。我们的前 引用大鼠和猪ARDS/VILI模型中的临床前数据验证了Enampt-的效率 中和人源化的mAb（ALT-100）在减少ENAMPT和LPS诱导的TLR4激活和NFκB-中中和 驱动的细胞因子产生，肺部渗透性和炎症性肺损伤。进一步审问和验证 ENAMPT作为ARDS治疗靶点，具体目标＃1将扩展先前的研究，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 Moter SNP可显着提高ENAMPT等离子体水平和ARDS死亡率的风险（无呼吸机降低） 天数增加了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节。因为ENAMPT分泌是启动fordram-的关键 机械级联激活，SA＃2将机械探索TLR4和机械应力的新调节 通过细胞外场地形成，炎症体激活和ABC转运刺激的ENAMPT分泌 - ers。随着用ENAMPT中和化的Alt-100 mAb处理，Akt1硝化的急剧增加， MAP激酶效应子激活并减少AKT/MTOR去泛素化，SA＃3将剖析结构/功能 ENAMPT-TLR4结合和刺激的EC渗透性的涉及的机制，具有特定的焦点 在EC细胞骨架驱动的屏障功能障碍中，在MAP激酶效应子P90RSK上，Akt1硝化和UCHL1活性。 最后，SA＃4将优化Enampt Alt-100 mAb剂量和交付时间作为治疗策略 临床前大鼠和猪ARDS/VILI模型。 EC分泌ENAMPT和ENAMPT/TLR4的解剖 参与ARDS/VILI病理生物学将加速ALT-100 MAB开发，这是一种可行的策略 减弱炎症性肺损伤，EC渗透性和ARDS/VILI死亡率。