Background: Endothelial cell (EC) pathobiology drives pulmonary hypertension (PH), but confusion over the evolution of EC phenotypes in this disease has persisted for decades. EC senescence, a state of stable cell cycle arrest, has been reported in PH, but the regulatory features are unknown. Led by our prior work showing deficiency of iron-sulfur (Fe-S) clusters in PH, we found that a Fe-S biogenesis protein, frataxin (FXN), controls senescence in pulmonary ECs. This may occur in Friedreich’s ataxia (FRDA), a disease marked by genetic FXN deficiency, cardiomyopathy, and often PH. Here, we offer a new model of EC biology in PH, where FXN loss promotes genotoxic stress and senescence in a pulmonary EC subset with low enough FXN. Senescent ECs then promote inflammation and drive many PH subtypes, including PH of FRDA and left heart disease. Hypothesis: We propose FXN deficiency, driven by genetic or acquired means, orchestrates Fe-S-dependent genotoxic stress, enforcing EC senescence and multiple PH subtypes. Aim 1. Determine if FXN deficiency drives DNA damage to enforce EC senescence. By study of human pulmonary artery and microvascular ECs and via genome editing of FXN mutations in inducible pluripotent stem cell (iPSC)-derived ECs from FRDA patients, we will study the role of FXN in genomic stress and EC senescence. Via study of circulating factors, histology, and single cell RNA sequencing, we will assess EC senescence in plasma and rare lung specimens from PAH patients and FRDA and HCM patients with pulmonary vascular disease. We expect to see a dose- dependent orchestration of FXN activities converging on EC senescence and PH. Aim 2. Determine if EC FXN deficiency depends upon senescence and myeloid inflammation to drive PH. In mice models of Groups 1 PAH and Group 2 PH due to FRDA, by using EC FXN-/- (KO) technology and adeno-associated virus delivery of FXN and its binding partner ISCU to pulmonary ECs in vivo, we will assess EC genomic stress, senescence, inflammation, and PH. EC-specific p16 KO mice and CX3CR1 KO mice will be used to define if EC FXN depends upon senescence and downstream myeloid inflammation to control PH. Thus, we aim to prove a new causative model of EC biology in PH – one that deconvolutes the confusion over EC heterogeneity that has plagued this field for decades. Aim 3. Determine if a novel GSTP1 inhibitor increases FXN and reverses multiple PH subtypes. We found that a GSTP1 inhibitor increases FXN and ISCU and improves PH. We will define this drug’s efficacy for ameliorating Groups 1-2 PH models and if FXN and GSTP1 are crucial for its function (via FXN and GSTP1/2 KO mice). If so, we could define an entirely new Fe-S-specific therapy for PH and FRDA. Significance: Via unique human and rodent discovery platforms, we will investigate an EC Fe-S cluster- senescence axis controlling multiple PH subtypes. Our work could explain the evolution of EC biology in PH and shift molecular paradigms, particularly for FRDA and...