Identification of a Novel EBF1-Expressing Disease-Associated Arterial Subpopulation in Pulmonary Arterial Hypertension

BACKGROUND: Pulmonary arterial hypertension (PAH) is a debilitating disease with a poor prognosis. However, only a limited number of palliative interventions are currently available. To address this unmet need, it is essential to understand the molecular causes of PAH by identifying unique disease-associated populations and their key molecular regulators during disease progression. METHODS: We constructed a single-cell multiome data set at 4 distinct time points during the pathogenesis of pulmonary hypertension (PH) using endothelial cells isolated from the Sugen5416/hypoxia rat model. We assessed the conservation of novel disease-associated endothelial cells by reanalyzing previously reported single-cell RNA sequencing data from human patients with PAH. We validated the functional importance of EBF1 (early B-cell factor 1) in PAH by performing in silico simulations and an in vivo knockdown mediated by a pulmonary endothelium-targeting associated virus. RESULTS: We found that PH progression gradually altered the transcriptional and epigenetic landscape of the pulmonary endothelium in a stage-specific manner, leading to the emergence of a novel disease-associated arterial endothelial population expressing EBF1. A similar endothelial subset was present in human patients with PAH. Immunofluorescence revealed that EBF1 expression was elevated in both human and rat lungs during PH progression. In silico simulations suggested that EBF1 is required for the pathogenic state, and the associated virus-mediated in vivo knockdown of Ebf1 ameliorated the pathological hallmarks of PH, including right ventricular systolic pressure and right ventricular hypertrophy, when delivered either before the onset of PH or after its establishment. CONCLUSIONS: We identified EBF1 as a key transcriptional regulator of a novel disease-associated population in both humans and rats. We further demonstrated that inhibiting EBF1 within the pulmonary endothelium ameliorates the pathological features of PH, suggesting that EBF1 and its transcriptional targets represent a novel and promising therapeutic avenue for PAH.