Sub-Project 1: Rare Genetic Defect in Glucose Metabolism as a Model for Investigating Mechanisms Underlying Vascular Remodeling in PAH Glucose-6-phosphatase catalytic subunit 3 (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. While the effect of G6PC3 deficiency on neutrophil function has been thoroughly studied, little is known about its impact on the vasculature. We hypothesize that investigation of a rare but well-characterized genetic cause of disrupted cellular energy homeostasis will provide valuable insight into how metabolic reprogramming contributes to PAH pathobiology. Aim 1: Determine the phenotypic consequences of G6PC3-silencing in human pulmonary artery and human pulmonary microvascular endothelial cells (ECs). In FY21, G6PC3 loss in primary human pulmonary vascular ECs produced a proliferative, apoptosis-resistant, hypermigratory phenotype. Consistent with mitochondrial dysfunction, spare respiratory capacity was reduced in primary human PAECs following in G6PC3 knockdown . Aim 2: Investigate the impact of G6pc3 deficiency on pulmonary vascular function in vivo using knockout mice under both normoxic and chronic hypoxic conditions. In FY21, longitudinal cardiac assessments in G6pc3 knockout (KO) mice revealed gradual biventricular dilation over time without obvious development of pulmonary hypertension (PH) under normoxic conditions. In FY22, cryorecovery, initial breeding and colony expansion were performed for G6pc3 global knockout mice. Future studies are planned using both chronic hypoxia and the combination of SU5416 and chronic hypoxia to induce PH. Furthermore, in collaboration with the NHLBI Transgenic Core, we began the initial steps necessary for generating an endothelial-specific G6pc3 knockout strain for further study under conditions that induce PH. Zygotes were injected with G6pc3 CRISPR reagents in order to insert an upstream and downstream loxP site. Tails were collected from the resultant mice and genotyped to check for proper insertion. These murine studies are done under Animal Study Proposal (ASP)# CCM 20-03. Aim 3: Develop and characterize patient-specific in vitro models of endothelial dysfunction using induced pluripotent stem cell (iPSC)-derived endothelial cells. 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 PAECs produced phenotypic, transcriptomic and functionally significant signaling changes that closely recapitulated many of the abnormalities and pathogenic mechanisms associated with advanced PAH (Awad and Elinoff et al. AJP Lung 2016). Recently, comprehensive in vitro characterization of CAV1 deficiency in human lung endothelium revealed a proliferative, interferon (IFN)-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 et al. PNAS 2021). In FY21, we continued investigations into the mechanisms underlying the activation of IFN signaling following CAV1 loss in 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 NOS3 and/or NADPH oxidase to ROS production following CAV1 loss. In FY22, additional experiments confirmed that ROS, as determined by the CellRox assay, are increased in PAECs following CAV1 silencing. Peroxynitrite production, assessed by immunoblotting for 3-nitrotyrosylated, was also elevated in CAV1-deficient cells. MnTBAP, a cell-permeable superoxide dismutase (SOD) mimetic, effectively scavenged ROS but did not reduce STAT1 phosphorylation in CAV1-deficient PAECs. In contrast NOS3 co-silencing not only blocks STAT1 phosphorylation (Gairhe et al. PNAS 2021), but also decreases cell proliferation, and in preliminary experiments, diminishes ROS generation in CAV1-deficient PAECs. NOS3 phosphorylation (Ser1177), a post-translational modification that can activate either nitric oxide and/or superoxide production depending on the cellular context, is also increased in CAV1-deficient PAECs. In addition to AKT, protein kinase A (PKA) phosphorylates NOS3 at S1177. Using small molecule inhibitors, we examined the role of adenylyl cyclase (AC) and PKA on phosphorylation of both STAT1 and NOS3 in PAECs following CAV1 silencing. Interestingly, while inhibiting transmembrane AC did not alter levels of STAT1 and NOS3 phosphorylation, two different soluble AC inhibitors (KH7 and LRE1) potently reduced phosphorylation of both targets and reduced cell proliferation. Similarly, STAT1 and NOS3 phosphorylation were also reduced in CAV1-deficient PAECs following treatment with H89, an inhibitor of PKA. Sub-Project 3: Translating Promising Therapeutic Targets Identified In Vitro Importantly, activation of the PI3K/AKT pathway is a prominent, shared feature across our models of PAH-associated molecular defects. Leniolisib is a PI3K-delta inhibitor that has been very well tolerated over long periods of time in children with activated PI3K-delta syndrome 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 and oral gavage was ultimately selected as the preferred route. In vivo testing in our rat PAH model is underway. In FY22, mRNA and protein expression levels of different PI3K isoforms were examined in PAECs. Notably, PI3K beta was the most abundantly expressed isoform, followed closely by alpha, then delta, and PI3K gamma expression was the lowest. Future work using canonical activators of PI3K/AKT relevent to PAH pathobiology (e.g. sphingosine-1 phosphate, PDGF, VEGF) will be used to investigate which isoform(s) are necessary for PI3K/AKT activation in PAECs. The relative expression of the different PI3K isoforms and the degree of cross-reactivity between the various small molecule inhibitors of PI3K will be important for understanding how to best target PI3K/AKT activation in the pulmonary vasculature.