Cellular Models of PAH-Associated Molecular Defects as a Tool for Identifying New Therapeutic Targets

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Loss-of-function mutations in bone morphogenetic protein type II receptor (BMPR2) are the most common genetic cause of PAH accounting for 70% of hereditary cases and 10-20% of idiopathic PAH. PAH patients with BMPR2 mutations are younger at diagnosis, demonstrate more severe hemodynamic compromise, and have an increased risk of death or lung transplantation compared to patients without these mutations. However, even in patients genotypically normal at the BMPR2 locus, expression is markedly reduced in end-stage PAH lungs, suggesting that BMPR2 might have a more general role in disease pathogenesis. We have previously shown that BMPR2 siRNA gene silencing in human pulmonary artery endothelial cells (PAECs) produced phenotypic, transcriptomic and functionally significant signaling changes that closely recapitulated many of the abnormalities and pathogenic mechanisms associated with advanced PAH (Awad KS and Elinoff JM et al. Am J Physiol Lung Cell Mol Physiol. 2016). Sub-Project 1: Application of a Rare Genetic Defect in Glucose Metabolism as a Model for Investigating Mechanisms Underlying Vascular Remodeling in PAH Aim 1: Determine the phenotypic consequences of glucose-6-phosphatase catalytic subunit 3 (G6PC3)-silencing in human pulmonary artery and human pulmonary microvascular endothelial cells (ECs). Aim 2a: Investigate the impact of G6pc3 deficiency on pulmonary vascular function in vivo using knockout mice under both normoxic and chronic hypoxic (10% FiO2 for 3 weeks) conditions. Aim 2b: Characterize the cellular phenotype of lung endothelial cells isolated from G6pc3 knockout (G6pc3-/-) mice and wild-type (WT) controls. Aim 3: Develop and characterize patient-specific in vitro models of endothelial dysfunction using induced pluripotent stem cell (iPSC)-derived endothelial cells from patients with pathogenic G6PC3 mutations. Metabolic reprogramming and abnormal glucose homeostasis have emerged as prominent features contributing to proliferative vascular remodeling in PAH. Likewise, metabolic disorders such as type 1a glycogen storage disease due to glucose-6-phosphatase catalytic subunit 1 (G6PC1) deficiency can lead to pulmonary vascular disease that is histologically indistinguishable from idiopathic PAH. We hypothesize that investigations of rare but well characterized genetic causes of disrupted cellular energy homeostasis will provide valuable insight into how metabolic reprogramming contributes to PAH pathobiology. In contrast to G6PC1, which is exclusively expressed in gluconeogenic organs, G6PC3 is a ubiquitously expressed enzyme that maintains intracellular glucose homeostasis by catalyzing the hydrolysis of glucose-6-phosphate to glucose in the endoplasmic reticulum. Loss-of-function mutations in G6PC3 lead to an autosomal recessive, multi-system syndrome of severe congenital neutropenia with a broad phenotypic spectrum that includes a high incidence of congenital heart defects. A subset of affected patients exhibits Dursun syndrome, a triad of congenital neutropenia, atrial septal defect and PAH. Notably, G6PC3 protein expression is decreased in lung tissue from PAH patients. Furthermore, in vitro studies of BMPR2 mutant pulmonary endothelial cells revealed accumulation of intracellular glucose-6-phosphate, the proximal substrate of G6PC3. While the effect of G6PC3 deficiency on neutrophil function has been thoroughly studied, little is known about its impact on the vasculature. Sub-Project 2: Impact of PAH-Associated Molecular Defects on Immune Effector Cell Function Contribution to Inflammatory Vascular Remodeling. Study Aim: Characterize the effects of BMPR2 loss-of-function on primary human elutriated monocytes and monocyte-derived macrophages in vitro. Despite clinical associations of PAH with autoimmunity and infection, evidence of local and systemic immune activation in patients, and animal models suggesting that inflammation can serve as a second hit, the consequences of PAH-associated genetic defects on immune effector cell function and activation is largely unknown. Recruitment of circulating cells of a monocyte/macrophage lineage appear necessary for the development of hypoxia-induced PH and recent studies have implicated a causal role for circulating myeloid-derived cells in the development of PAH-associated vascular remodeling. Furthermore, we recently completed a meta-analysis of genome-wide blood expression profiling in PAH and identified several inflammatory signaling pathways as significantly overrepresented including a prominent interferon gene signature (Elinoff JM et al. Am J Physiol Lung Cell Mol Physiol. 2020). Building upon our findings in human pulmonary artery endothelium, we are now examining the impact of PAH-associated genetic defects in human monocytes and monocyte-derived macrophages in order to better understand the impact of dysregulated BMPR2 signaling on immune effector cells. Sub-Project 3: Translating Promising Therapeutic Targets Identified In Vitro Study Aim: Determine whether RB-50-LV29 (abbreviated RB), a PI3K/AKT pathway inhibitor, can halt the progression of pulmonary vascular remodeling in a rat PAH model that closely mimics human disease. In collaboration with Dr. Robert Danner, our program has developed six distinct in vitro models of PAH-associated molecular defects in order to identify promising new treatments. Importantly, activation of the PI3K/AKT pathway is a prominent, shared feature across all six of our model systems. The PI3K/AKT signaling pathway, known to be activated in many cancers, leads to apoptosis resistance and increased cell proliferation, features demonstrated in each of our in vitro models and in cells from PAH patients. Leniolisib is a PI3K inhibitor developed by Novartis that is completing a Phase 3 trial for activated PI3Kdelta syndrome (APDS). Notably, leniolisib has been very well tolerated over long periods of time in children with this disorder (Rao et al., Blood 2017) and reversed the hyperproliferative, apoptosis resistant cellular phenotype seen in our in vitro models of PAH. In collaboration with Novaris, we have obtained RB-50-LV29, the tool compound for leniolisib, in order to begin pre-clinical testing in a rat PAH model.