NAD-dependent Signaling and Pulmonary Vascular Remodeling in PAH - PROJECT SUMMARY RATIONALE: Pulmonary vascular remodeling is a pathological feature of Pulmonary Arterial Hypertension (PAH) that ultimately leads to death. Pulmonary Arterial Endothelial Cells (PAECs) are critical regulators of pulmonary artery homeostasis. During PAH, PAECs undergo metabolic changes (Warburg effect) and PAECs dysfunction that leads to a pro-proliferative and apoptosis-resistant phenotype that causes Pulmonary Vascular Remodeling. Current therapies targeted at the vasomotor tone and inflammatory regulators have improved prognosis, however, do not remedy vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting protein in NAD+ synthesis. NAMPT overexpression upregulates glycolysis, promotes pulmonary vascular remodeling, and contributes to PAH pathobiology. The Loss or pharmacological inhibition of NAMPT attenuates PH in animal models, indicating its therapeutic potential. However, there is a gap in our understanding of the cellular mechanisms by which NAMPT promotes PAECs dysfunction and pulmonary vascular remodeling. Sirtuin3 (Sirt 3), an NAD+ dependent deacetylase enzyme involved in mitochondrial homeostasis, has been implicated in PAH, as Sirt 3 KO mice were shown to develop experimental PH. Based on these observations, we hypothesize that in the glycolytic environment characteristic of PAH, Nampt in human PAECs (hPAECs) impairs Sirt 3 function to cause a metabolic reprogramming mediated PAEC dysfunction, and vascular remodeling. METHODS: To investigate the hypothesis, NAMPT, an appropriate control, and a NAMPT construct lacking enzymatic activity will be overexpressed in hPAECs using lentiviral particles at 20 MOI for 24hrs. This model is advantageous as it ensures stable gene expression and further experimentation. Experiments proposed in AIM 1 will permit the evaluation of NAMPT in regulating the function of SIRT3/SOD2 (Manganese-dependent Superoxide Dismutase) axis to promote hPAECs dysfunction. The evaluation will be performed using seahorse metabolic assay, mitochondrial membrane potential assay, oxidative stress assays, SIRT 3 activity assay, and SOD2 activity assay. AIM 2 will address how NAMPT leads to the metabolic reprogramming of PAECs in vivo to promote PAH. Conditional NAMPT EC (Endothelial Cell) overexpressing (OE) mice and conditional NAMPT EC deficient mice (KO) models will be utilized. Hemodynamic studies assessing pulmonary artery remodeling, right ventricular hypertrophy, and right ventricular systolic pressures will be performed, and harvested PAECs subjected to metabolic and SIRT3 activity assays. CONCLUSIONS: These studies when completed will help show that NAMPT overexpression can promote endothelial dysfunction via Sirt3 activity inhibition, resulting in an increase in SOD2 acetylation which could lead to increased reactive oxygen species (ROS) production, promoting vascular remodeling and PH pathogenesis.
