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

National Heart, Lung, And Blood Institute

Investigators

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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 dysregulation in pulmonary arterial hypertension (PAH) has emerged as a significant area of research. Understanding how intracellular glucose homeostasis contributes to vascular remodeling may provide insights into underlying mechanisms of this chronic disease. 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. This work may also improve our understanding of other chronic diseases characterized by endothelial cell dysfunction and metabolic dysregulation such as obesity, diabetes and atherosclerosis. Based on our observation that G6pc3-/- mice demonstrated an exaggerated response to chronic hypoxia, in vitro experiments were continued in FY25 to further investigate the cellular phenotype of G6PC3-deficient, primary human pulmonary artery endothelial cells (PAECs). Similar to the “prototypic model” of PAH-associated endothelial dysfunction observed in BMPR2-deficient cells, G6PC3-deficient PAECs exhibited a hyperproliferative and migratory phenotype. Oxidative phosphorylation reserve was reduced and hexokinase 2 (HK2) and phosphoinositide-dependent kinase 1 (PDK1) expression was increased in G6PC3-silenced PAECs, consistent with a shift toward glycolytic metabolism. Basal endothelin-1 mRNA expression was increased in G6PC3-silenced PAECs and suppression of EDN1 transcription mediated by the laminar flow mimetic Yoda1, was blunted. Yoda1-induced NOS3 mRNA expression and NOS3 activation (phosphorylation at Ser 1177) was also compromised in G6PC3-silenced PAECs. Consistent with blunted induction of NOS3, histone H3 lysine 27 acetylation, an epigenetic modification at enhancer and promoter regions associated with activation of transcription, was reduced in G6PC3-silenced PAECs. Collectively, these findings suggest important cross-talk between altered cellular metabolism and endothelial dysfunction that is directly relevant to PAH targeted therapy. Sub-Project 2: Mechanisms Leading to Interferon Activation in Caveolin-1 (CAV1)-Deficient Pulmonary Artery Endothelial Cells (PAECs) 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 (Gairhe et al. PNAS 2021). In FY25, we continued investigations into the mechanisms underlying STAT1 activation following CAV1 loss in human PAECs. Using two different small molecule inhibitors, we previously demonstrated that blocking soluble adenylyl cyclase (ADCY10) attenuated ROS production, STAT1 and NOS3 phosphorylation and cell proliferation in CAV1-silenced PAECs. Subsequent experiments have been completed to determine whether ADCY10 inhibition alters IFN target gene expression following IFN stimulation and/or CAV1-silencing. ADCY10 expression was examined in explanted lung tissue from PAH patients as well as Cav1 knockout mice. Finally global RNA sequencing and proteomic profiling of blood samples from PAH patients with disease associated CAV1 mutations is underway to determine whether chronic IFN-associated inflammation is present and/or associated with clinical severity. Sub-Project 3: Efficacy of PI3K/AKT Pathway Inhibition on Pulmonary Vascular Remodeling in Rat Models of Pulmonary Arterial Hypertension 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 in children with activated PI3K-delta syndrome and reversed the hyperproliferative, apoptosis-resistant cellular phenotype in our in vitro PAH cellular models (Awad et al. Int J Mol Sci 2024; Wang et al. AJP Lung 2024). In collaboration with Novaris/Pharming, we first tested RB-50-LV29 (abbreviated RB), a tool compound for leniolisib, in our rat SU5416-hypoxia PAH model. More recently we have obtained leniolisib for additional in vivo studies (CCM 19-03, CCM 19-07 and CCM 23-01). In FY25, we continued a comprehensive, multipart study investigating the efficacy of PI3K/AKT pathway inhibition on pulmonary vascular remodeling in pre-clinical PAH models. Sub-Project 4: Determine the Contribution of CD44 on the Proliferative, Hypermigratory Phenotype of BMPR2-Deficient Pulmonary Artery Endotheial Cells (PAECs) Genome-wide expression profiling of BMPR2-silenced PAECs uncovered CD44, a non-kinase transmembrane glycoprotein, among the top 20 upregulated transcripts (Awad and Elinoff et al. AJP Lung 2016). Therapeutic strategies that block CD44 or reduce its expression are currently in various stages of development for cancer. Therefore a better mechanistic understanding of the potential contribution of CD44 to vascular remodeling in PAH is directly aligned with our program’s overall goal. In FY25, we continued in vitro work exploring the effect of CD44 gene silencing on the cellular phenotype and genome-wide transcriptome of BMPR2-PAECs. In our PAH cellular model we are examining the effects of a well-tolerated, peptide inhibitor of CD44 that has been investigated in clinical trials of patients with various advanced cancers. Lastly we are continuing to develop an endothelial targeted-Cd44 knockout murine model using the Cre/lox recombination system and plan to determine whether these mice are protected from the development of chronic hypoxia- and/or SU5416/hypoxia-induced pulmonary hypertension.