Direct extracellular NAMPT involvement in pulmonary hypertension and vascular remodeling transcriptional regulation by SOX and HIF-2a

We previously demonstrated involvement of NAMPT (nicotinamide phosphoribosyltransferase) in pulmonary arterialhypertension(PAH) and now examine NAMPT regulation and extracellular NAMPT's (eNAMPT's) role in PAHvascular remodeling. NAMPT transcription and protein expression in human lung endothelial cells were assessed in response to PAH-relevant stimuli (PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor], TGF-b1 [transforming growth factor-b1], and hypoxia). Endothelial-tomesenchymal transition was detected by SNAI1 (snail family transcriptional repressor 1) and PECAM1 (platelet endothelial cell adhesion molecule 1) immunofluorescence. An eNAMPTneutralizing polyclonal antibody was tested in a PAH model of monocrotaline challenge in rats. Plasma eNAMPT concentrations, significantly increased in patients with idiopathic pulmonary arterial hypertension, were highly correlated with indices of PAH severity. eNAMPT increased endothelial-to-mesenchymal transition, and each PAH stimulus significantly increased endothelial cell NAMPT promoter activity involving transcription factors STAT5 (signal transducer and activator of transcription 5), SOX18 (SRY-box transcription factor 18), and SOX17 (SRY-box transcription factor 17), a PAH candidate gene newly defined by genome-wide association study. The hypoxia-induced transcription factor HIF-2a (hypoxiainducible factor-2a) also potently regulated NAMPT promoter activity, and HIF-2a binding sites were identified between 2628 bp and 2328 bp. The PHD2 (prolyl hydroxylase domain-containing protein 2) inhibitor FG-4592 significantly increased NAMPT promoter activity and protein expression in an HIF-2a-dependent manner. Finally, the eNAMPT-neutralizing polyclonal antibody significantly reduced monocrotaline-induced vascular remodeling, PAH hemodynamic alterations, and NF-kB activation. eNAMPT is a novel and attractive therapeutic target essential to PAH vascular remodeling.