ABSTRACT Pulmonary arterial hypertension (PH) is a progressive disease leading to pulmonary vascular remodeling, increased pulmonary arterial pressures, declining right ventricular (RV) function, right heart failure, and death. No curative therapies are currently available. In this F31 application, the PI will train in the acclaimed translational laboratory of Joe GN Garcia, MD and seek to address the unmet need for novel effective PAH therapeutic strategies by focusing on eNAMPT (extracellular nicotinamide phosphoribosyltransferase). eNAMPT is a novel damage-associated molecular pattern protein (DAMP) and PAH target identified by the Garcia lab utilizing genomic–intensive approaches. Intracellular NAMPT mainly serves as a rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) synthesis. However, secreted extracellular eNAMPT (from leukocytes, lymphocytes, endothelium, epithelium) ligates the Toll-like receptor 4 (TLR4) to potently activate this inflammatory signaling cascade. eNAMPT/TLR4 participation in innate immunity inflammatory responses is key to the severity of several serious inflammatory disorders (ARDS, lung fibrosis) including PH with elevated plasma eNAMPT levels in PH subjects correlating with RV dysfunction. Increased eNAMPT secretion is influenced by NAMPT promoter SNPs and NAMPT transcriptional regulation by anti-oxidant response elements and hypoxia response elements induced by transcription factors such as HIF-2α. We have demonstrated that the eNAMPT/TLR4 pathway is highly druggable as a humanized eNAMPT-neutralizing mAb effectively reduced the severity of preclinical PH. Several key gaps remain, however, in fundamentally understanding the role of eNAMPT in PH pathobiology. For example, it remains unclear as to whether endothelial cells (ECs) or smooth muscle cells (SMCs) are the primary target cell for eNAMPT involvement in PH. In Specific Aim #1 (SA), the PI will examine EC- and SMC-specific NAMPT promoter responses to PAH stimuli that increase NAMPT transcriptional activities and translation. SA #2 will investigate the EC- and SMC-specific intracellular mechanisms contributing to eNAMPT secretion into the circulation by PAH stimuli focusing on the role of NAMPT dimerization, inflammasome and ABC transporter activation and generation of extracellular vesicles. Finally, utilizing human and rodent tissues, SA #3 will define EC- and SMC-specific eNAMPT-induced responses by examining cytosolic Ca2+ signaling, cell proliferation/activation, cell survival, EMT activity and angiogenic activity as readouts. Together, these studies will provide key novel mechanistic insights into eNAMPT’s influence on vascular remodeling and PH pathobiology.