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

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 the understanding of the epigenetic pathogenesis of the disease. METHODS: We generated the first single-base resolution pseudouridine (Ψ) landscape in lung tissues of patients with PH using bisulfite-induced deletion sequencing. PUS7 expression was analyzed in hypoxic pulmonary artery endothelial cells, the lung tissues of patients with PH, and SU5416-hypoxia rodent model. The functional roles of PUS7 were investigated through genetic manipulation (PUS7-deficiency cells, adeno-associated virus serotype-mediated overexpression, endothelial cell-specific knockdown, and heterozygous knockout mice) and pharmacological inhibition with NSC107512. RESULTS: Bisulfite-induced deletion sequencing revealed global Ψ dysregulation in the lung tissues of patients with PH. Among PUS family members, PUS7 was the most markedly upregulated in these tissues and in the hypoxic pulmonary artery endothelial cells. Both gene knockdown and pharmacological inhibition with NSC107512 ameliorated PH, whereas adeno-associated virus serotype-mediated PUS7 overexpression exacerbated disease progression. RNA immunoprecipitation sequencing and mutagenesis studies demonstrated that PUS7 bound to and catalyzed Ψ at position 688 of TGFBI (transforming growth factor β-induced protein) mRNA, thereby stabilizing TGFBI and activating phosphatidylinositol 3-kinase-protein kinase B signaling pathway. Furthermore, hypoxia-inducible factor 2α bound directly to the PUS7 promoter, establishing a hypoxia-inducible factor 2α/PUS7/TGFBI/phosphatidylinositol 3-kinase-protein kinase B positive feedback loop that drives PH pathogenesis. CONCLUSIONS: PUS7-mediated pseudouridylation serves as a novel epigenetic driver of PH through the hypoxia-inducible factor 2α/PUS7/TGFBI/phosphatidylinositol 3-kinase-protein kinase B axis, positioning PUS7 as a promising therapeutic target for this devastating disease.