To identify robust pulmonary arterial hypertension (PAH)-associated genes through multi-cohort transcriptomic integration and single-cell resolution analyses, and to elucidate their cellular origins and potential links to immune microenvironment remodeling.

Differential expression analysis of PAH and control was conducted on three independent transcriptome cohorts (GSE217438, GSE117261, GSE33463) respectively, and the intersection was taken to obtain consistent candidate genes across cohorts. Build a protein-protein interaction (PPI) network based on STRING to locate key nodes; The co-expression network was constructed using WGCNA and module-trait association and functional enrichment were carried out. Further analyze the single-cell transcriptome data of lung tissue to clarify the cellular origin of RGS1 and conduct quasi-temporal analysis in the mononuclear macrophage lineage. At the same time, compare the differences in cell communication between the control and PAH and screen the key ligand-receptor axes. Finally, a rat PAH model induced by MCT (60 mg/kg) was constructed. The expression changes of RGS1 were verified by RT-qPCR of alveolar macrophages, Western blot and immunohistochemistry of lung tissue.

The intersection of differentially expressed genes across three cohorts indicates candidate genes exhibit strong cross-cohort stability, with the immune-regulatory gene RGS1 further identified. In all three independent cohorts, RGS1 was significantly upregulated in the PAH group (P<0.05). Protein interaction network analysis revealed RGS1 occupies a central position within the network and interacts with multiple G protein signaling and immune-related molecules, suggesting its potential involvement in GPCR-mediated immune pathways. WGCNA analysis revealed that the dark gray module containing RGS1 was highly correlated with the PAH phenotype (cor=0.43, P=5e-5). Genes in this module were enriched in pathways including adaptive immune response, immunoglobulin-mediated response, and lymphocyte-mediated immunity. Single-cell analysis revealed that RGS1 is predominantly highly expressed in immune cells. The proportion of macrophages significantly increased in the PAH group, accompanied by enrichment of inflammation-related pathways (P<0.05). The results of pseudotime series showed that monocyte-macrophage migration occurred in a continuous state along the trajectory, and the expression of RGS1 changed dynamically with pseudotime. Cell communication analysis showed that the number and intensity of overall communication were increased in PAH group, and the probability of ligand-receptor pair PPIA-BSG communication was significantly increased in PAH group, suggesting that the signal network was remodled. The expressions of RGS1 mRNA and protein in alveolar macrophages in the experimental group were significantly up-regulated in the MCT animal model (P<0.01), and the positive signal of RGS1 in lung tissue was enhanced by IHC.

RGS1 may be involved in the remodeling of the immune microenvironment in PAH and serve as an immune-related biomarker and potential therapeutic target.