PUS7-Mediated Pseudouridylation of TGFBI Drives Vascular Remodeling in Pulmonary Hypertension II

Background: Pulmonary hypertension (PH) is a life-threatening cardiovascular disorder characterized by irreversible pulmonary vascular remodeling and poor prognosis. RNA pseudouridylation, the most evolutionarily conserved RNA epigenetic modification, and its catalytic enzyme pseudouridine synthase 7 (PUS7) remained uncharacterized in PH, representing a major gap in understanding the epigenetic pathogenesis of the disease. Methods: We generated the first single-base resolution pseudouridine (Ψ) landscape in lung tissues of PH patients using bisulfite-induced deletion sequencing (BID-seq). PUS7 expression was analyzed in hypoxic pulmonary artery endothelial cells (PAECs), the lung tissues of PH patients, and Su5416-hypoxia rodent model. The functional roles of PUS7 were investigated through genetic manipulation (PUS7-deficiency cells, AAV-mediated overexpression, endothelial cell-specific knockdown, and heterozygous knockout mice) and pharmacological inhibition using NSC107512. Results: BID-seq revealed global Ψ dysregulation in the PH patient lung tissues. Among PUS family members, PUS7 was the most markedly upregulated in these tissues and in the hypoxic PAECs. Both gene knockdown and pharmacological inhibition with NSC107512 ameliorated PH, whereas AAV-mediated PUS7 overexpression exacerbated disease progression. RNA immunoprecipitation sequencing and mutagenesis studies demonstrated that PUS7 bound to and catalyzed Ψ at position 688 of Transforming Growth Factor Beta Induced Protein (TGFBI) mRNA, thereby stabilizing TGFBI and activating Phosphatidylinositol 3 Kinase (PI3K)-Protein Kinase B (AKT) signaling pathway. Furthermore, hypoxia-inducible factor 2α (HIF-2α) directly bound the PUS7 promoter, estabilishing a HIF-2α/PUS7/TGFBI/PI3K-AKT positive feedback loop that drives PH pathogenesis. Conclusions: PUS7-mediated pseudouridylation serves as a novel epigenetic driver of PH via the HIF-2α/PUS7/TGFBI/PI3K-AKT axis, positioning PUS7 as a promising therapeutic target for this devastating disease.