ABSTRACT: The global COVID-19 pandemic has dramatically highlighted the critical role of lung vascular inflammation and multi-organ endothelial cell (EC) permeability in ARDS mortality and the unprecedented COVID-19-ARDS vas- cular endotype (1). This A1 Project #2 application remains focused on the critical role of eNAMPT (extracellular nicotinamide phosphoribosyltransferase) in driving lung vascular inflammation and multi-organ endothelial cell (EC) permeability, events that are central to increasing ARDS mortality. 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 exposure to me- chanical ventilation. Importantly, utilizing conditional EC–specific Nampt KO mice, we have recently shown that EC contributions to ARDS pathobiology via eNAMPT secretion into the circulation, thereby driving pre- clinical ARDS inflammatory lung injury and severity. eNAMPT produces these injurious effects by functioning as a damage-associated molecular pattern protein (DAMP) and master regulator of evolutionarily-conserved inflam- matory cascades via novel ligation of the Toll–like receptor 4 (TLR4). Our exciting data in mouse, rat and porcine ARDS/VILI models have validated the efficacy of the eNAMPT-neutralizing humanized mAb (ALT-100) in re- ducing eNAMPT- and LPS-induced TLR4 activation, NFκB-driven cytokine production, lung permeability and inflammatory lung injury. To further interrogate eNAMPT as an ARDS therapeutic target, SA #1 will extend prior studies which showed ROS-generating ARDS stimuli (hypoxia, hyperoxia, mechanical stress, cytokines) to in- duce NAMPT expression and characterize the role of three key transcription factors (hypoxia-inducible factors HIF1a/2a, NRF2), NAMPT/TLR4 promoter SNPs, and DNA methylation in genetic/epigenetic regulation of NAMPT/TLR4 promoter activities. SA #2 will mechanistically explore novel regulation of TLR4- and mechanical stress-stimulated eNAMPT secretion, a key event to initiation of inflammatory cascade activation, via extracellu- lar vesicle formation, inflammasome activation, and ABC transporters. Utilizing Core B's CRISPR/Cas9- gener- ated EC lines, SA #3 will dissect the structure/function mechanisms involved in eNAMPT-TLR4 binding and increases in EC permeability with specific focus on MAP kinase effector p90rsk, Akt1 nitration, and UCHL1 activity in EC cytoskeletal-driven barrier dysfunction. Finally, utilizing Core C expertise, SA #4 will optimize eN- AMPT ALT-100 mAb dosing, define the therapeutic window for ALT-100 delivery, and define potential ALT-100 mAb synergy with other PPG therapeutic modalities utilizing preclinical rat and porcine ARDS/VILI models. This high integration of Project #2 with each PPG Project will...