Novel Involvement of eNAMPT/TLR4 Signaling in PAH Vascular Remodeling and Right Ventricular Dysfunction

University Of Florida, Gainesville FL

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Abstract

ABSTRACT: This extensively revised R01 resubmission remains committed to addressing the unmet need for curative thera- pies in pulmonary arterial hypertension (PAH) via a therapeutic focus that targets the eNAMPT/Toll-like recep- tor-4 (TLR4) signaling cascade. We speculate that eNAMPT/TLR4 signaling is a key contributor to accelerated cellular phenotypic switching involving endothelial cells (ECs), smooth muscle cells (SMCs) and monocyte/mac- rophages (Mo/Macs) in the perivascular niche, events that culminate in persistent “sterile inflammation”, pulmo- nary vascular remodeling and right ventricular (RV) fibrosis/dysfunction. With the growing appreciation of the role of lung mechano-immunity elicited by cyclic stretch and shear stress, it is important to recognize that eN- AMPT (extracellular nicotinamide phosphoribosyltransferase) binds TLR4 and is a novel and highly mechano- sensitive DAMP (damage-associated molecular pattern protein) and a highly druggable PAH target. We have shown eNAMPT/TLR4 signaling is a key innate immunity inflammatory cascade involved in the severity of sev- eral inflammatory, fibrotic and neoplastic disorders including PAH. NAMPT expression is robustly increased in PBMCs and in remodeled lung vessels from PAH subjects and elevated plasma eNAMPT levels are associated with RV dysfunction. GWAS studies showed NAMPT promoter SNPs in PAH subjects are linked to right heart catheterization-verified abnormalities in PVR and RV function. Importantly, the eNAMPT-neutralizing ALT-100 mAb, currently in Phase 2A ARDS clinical trials, effectively reverses established preclinical PH caused by hy- poxia/Sugen (Hy/Su) or monocrotaline exposures. The heavily revised Specific Aims (SAs) in this A1 R01 renewal application are designed to confirm eNAMPT’s role in PAH pathobiology and to translate ALT-100 mAb into a PAH therapeutic strategy. SA #1 will mechanistically detail eNAMPT secretion following mechanical stress/PAH agonist-induced inflammasome activation in lung ECs and pulmonary artery (PA) SMCs derived from PAH patients, focusing on novel involvement of NAMPT ubiquitination and autophagy in NAMPT secretion. SA #2 will utilize biophysical and structural imaging modalities (CryoEM, HDX, SPR, Mass Photometry) to define eNAMPT/TLR4 structure/function interactions and the influence of eNAMPT cysteine oxidation. Newly-proposed SA #3 will utilize scRNA sequencing and spatial transcriptomics to examine the influence of eNAMPT/TLR4 autocrine/paracrine signaling in driving EC mesenchymal transition (EndMT), SMC contractile-to-proliferative phenotypic transition (SmcCPPT), and monocyte/macrophage phenotypic transition (MoMacPT) leading to peri- vascular inflammation, vascular remodeling and RV dysfunction. SA #4 will assess potential synergy of ALT-100 mAb with sotatercept, an FDA-approved PAH therapy, and will generate a precision medicine NAMPT genotyp- ing risk score to stratify ‘at risk’ PAH subjects for clinical trials targeting the eNAMPT/TLR4 pathway. Thus, these A1 R01 renewal studies will define eNAMPT/TLR4 involvement in PAH/PH pathobiology and accelerate trans- lation of the ALT-100 mAb as a PAH therapeutic strategy that addresses several unmet needs in PAH.