Defect in lysosomal enzyme trafficking and sorting is associated with irreversibility of pulmonary arterial hypertension.

Background: Patients with congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH) have a reversible stage, during which shunt closure reverses PAH. However, PAH is irreversible beyond a certain time point, and the molecular mechanisms underlying the switch from reversible to irreversible PAH remain poorly understood. Methods: Three transcriptomic datasets were obtained from the Gene Expression Omnibus database. Weighted gene co-expression network analysis (WGCNA) was performed to identify gene modules significantly associated with PAH irreversibility. Serum samples were collected from 40 patients with CHD-PAH, 20 patients with congenital heart disease (CHD), and 20 healthy controls. Protein levels in serum were quantified using enzyme-linked immunosorbent assay. Results: WGCNA generated 16 gene modules, and one of the modules showing the highest positive correlation with PAH irreversibility was recognized as the key module. GO and KEGG pathway analyses of key module revealed that PAH irreversibility was associated with lysosome. Pathview analysis indicated dysfunctional lysosome in irreversible PAH, including dysregulation of lysosomal enzymes and impaired lysosomal transport and acidification. Comprehensive characterization of lysosome identified a set of downregulated genes, including Gnptab, M6pr, and Arf1, whose expression patterns shifted significantly during the transition from reversible to irreversible PAH. Functional enrichment analysis linked these genes to lysosomal enzyme trafficking and sorting. The downregulation of M6PR, a receptor involved in lysosomal trafficking and sorting, was validated using independent datasets and clinical samples. Additionally, eight lysosomal hub genes were identified, including Ctsd, Ctsk, Ctsb, Ctsa, Pld3, Tpsab1, Lgals3 and Smpdl3a; among them, serum protein levels of CTSD, CTSB, and LGALS3 were significantly elevated in CHD patients with irreversible PAH compared to those with reversible PAH. Conclusion: This study identified dysfunctional lysosome in irreversible PAH, and the loss of compensation for lysosomal trafficking and sorting may be associated with a switch from reversible to irreversible PAH, thus providing novel insights into the molecular mechanisms underlying irreversible PAH.