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

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Abstract

Sub-Project 1: Rare Genetic Defect in Glucose Metabolism as a Model for Investigating Mechanisms Underlying Vascular Remodeling in PAH 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. 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). In FY21, ongoing experiments revealed that G6PC3 loss in primary human pulmonary vascular ECs produced a proliferative, apoptosis-resistant, hypermigratory phenotype. Furthermore, spare respiratory capacity was reduced in primary human PAECs following in G6PC3 knockdown consistent with mitochondrial dysfunction. Endothelial and mesenchymal cell markers were assessed by quantitative real-time PCR and western blot following G6PC3-silencing in PAECs. Preliminary data suggest evidence of endothelial-to-mesenchymal transition, consistent with the proliferative and migratory phenotype of these cells. Additionally, samples from in G6PC3-silenced PAECs were collected for global metabolomic and genome-wide transcriptomic profiling. Aim 2: 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. In FY21, longitudinal cardiac assessments in G6pc3 knockout (KO) mice were completed. Preliminary analysis revealed gradual biventricular dilation over time without obvious development of pulmonary hypertension (PH) under normoxic conditions. These murine studies were done under Animal Study Proposal (ASP)# CCM 20-03. Future studies are planned using both chronic hypoxia and the combination of SU5416 and chronic hypoxia to induce PH. In collaboration with the NHLBI Transgenic Core Facility, we also plan to create an endothelial-specific G6pc3 knockout strain for further study under conditions that induce PH. Aim 3: Develop and characterize patient-specific in vitro models of endothelial dysfunction using induced pluripotent stem cell (iPSC)-derived endothelial cells. Induced pluripotent stem cell (iPSC) lines have been obtained from two different patients harboring known pathogenic G6PC3 mutations. Refinement of the protocol to differentiate these iPSCs into endothelial cell lines as well as the assays necessary for confirmation of cellular phenotype is underway. Sub-Project 2: The Contribution of Reactive Oxygen Species to Activation of Interferon Signaling in Cellular Models of PAH 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). Recently, comprehensive in vitro characterization of CAV1 deficiency in human lung endothelium revealed a proliferative, interferon-biased inflammatory phenotype driven by constitutively activated STAT and AKT signaling. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 mutations. Finally, inhibiting JAK/STAT and/or PI3K/AKT reversed this aberrant cell phenotype and may ameliorate vascular remodeling in PAH (Gairhe S et al. Proc Natl Acad Sci USA 2021). In FY21, we continued in vitro investigations into the mechanisms underlying the activation of interferon (IFN) signaling following CAV1 loss in primary human PAECs. In preliminary experiments, higher levels of cytosolic reactive oxygen species (ROS) were detected in CAV1-silenced PAECs compared to control cells transfected with a non-targeting siRNA. Catalase, superoxide dismutase and a cell permeable superoxide dismutase mimetic are being used to determine whether inactivating ROS in CAV1-silenced PAECs will ameliorate STAT1 activation, a surrogate for IFN signaling. Small molecule inhibitors and siRNA gene silencing are being utilized to determine the contribution of eNOS and/or NADPH oxidase to ROS production following CAV1 loss. Lastly, RNA isolated from peripheral blood mononuclear cells of PAH patients enrolled in our Natural History Study (13-CC-0012) will be used to assess whether IFN signaling is similarly activated in vivo based on a standardized, 28-gene IFN response score. Sub-Project 3: Translating Promising Therapeutic Targets Identified In Vitro In collaboration with Dr. Robert Danner, our programs combined have 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-delta inhibitor that is being investigated in patients with activated PI3K-delta 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 PAH cellular models. In collaboration with Novaris/Pharming, we have obtained RB-50-LV29 (abbreviated RB), a tool compound for leniolisib, for testing in our rat SU5416-hypoxia PAH model. The pre-clinical studies associated with this project are Animal Study Proposal (ASP) # CCM 19-03 and CCM 19-07. In FY21, we completed pharmacokinetic testing in rodents comparing three different routes of RB administration. Oral gavage was ultimately selected as the preferred route and in vivo testing in our rat PAH model is underway. The primary outcome of the study is to determine whether RB, a PI3K/AKT pathway inhibitor, can reduce pulmonary pressure and halt the progression of pulmonary vascular remod