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 o...