Bulk and single-cell transcriptomics for identification of potential diagnostic biomarkers associated with pulmonary arterial hypertension and integrated stress response and experimental validation

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal vascular disorder characterized by irreversible remodeling of the pulmonary arteries, but the systematic role of the integrated stress response (ISR)-related genes (ISR-RGs) in its pathogenesis remains unexplored. OBJECTIVE: This study aimed to identify and validate ISR-associated potential biomarkers for PAH using integrative bioinformatics, single-cell transcriptomics, and experimental approaches. METHODS: We analyzed bulk RNA-seq datasets (GSE38267, GSE131793) and a single-cell RNA sequencing (scRNA-seq) dataset (GSE210248) from PAH patients and controls. A total of 529 ISR-RGs were integrated. Differential expression analysis, machine learning, and functional enrichment analyses were employed to identify potential biomarkers. A diagnostic nomogram was constructed and validated. Immune infiltration, regulatory networks, drug-gene interactions, and molecular docking were further investigated. Key cellular mechanisms were elucidated via scRNA-seq, and biomarker expression was validated using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and Enzyme-linked immunosorbent assay (ELISA) in clinical whole blood samples. RESULTS: = 0.432). Gene set enrichment analysis (GSEA) revealed their involvement in translation, neutrophil degranulation, and glucocorticoid pathways. The scRNA-seq highlighted their cell-type-specific expression in T cells and monocytes. Molecular docking identified CAMONSERTIB as a potent ATM-targeting drug (binding energy: -10.2 kcal/mol), and its therapeutic effects on pulmonary arterial hypertension were validated through both animal and cellular models. RT-qPCR and Elisa confirmed upregulation of ABCC4 and downregulation of ATM, SERPINH1 and ZBTB40 in PAH patients. CONCLUSIONS: This study identified four novel ISR-related biomarkers for PAH and elucidated their roles in immune infiltration and cellular remodeling. The findings provided new insights into PAH pathogenesis and offered potential tools for diagnosis and targeted therapy.