Abstract Body (Do not enter title and authors here): Background: The cellular heterogeneity and molecular mechanisms underlying human pulmonary arterial hypertension (PAH) remain incompletely characterized. We aimed to construct a comprehensive single-cell lung atlas of human PAH to elucidate shifts in cellular composition and disease-specific gene expression.

Methods:Flash-frozen lung tissues were collected from 10 healthy donors and 30 PAH patients, including idiopathic PAH (IPAH), drug/toxin-induced PAH, and PAH secondary to systemic-to-pulmonary shunting, representing diverse sexes, ethnicities, and ages. Fixed lung tissues were processed for single-cell RNA sequencing and single-cell resolution spatial transcriptomics using 10X Genomics Xenium 5K gene panel. We analyzed changes in cell proportions, gene expression, and pathways across disease subtypes, sexes, and age groups.

Results: We identified four major cell lineages and 34 distinct clusters. We discovered a novel subtype: COL15A1⁺ systemic arterial endothelial cells (SAECs), characterized by co-expression of arterial EC markers and COL15A1. Spatial transcriptomics analysis and immunostaining confirmed that SAECs localized within plexiform and occlusive vascular lesions in PAH lungs. SAECs were enriched for genes involved in migration, shear stress response, and coagulation. PAH SAECs exhibited strong upregulation of genes linked to angiogenesis, epithelial-mesenchymal transition (EMT), apical surface remodeling, and Wnt/β-catenin signaling. Transcriptional factor prediction showed that SAECs identity was maintained by SOX18, E2F6 and SOX6. Ligand-receptor interaction demonstrated that SAECs produce the most significantly autocrine and paracrine signaling in PAH lungs. Cellular lineage pseudotime analysis revealed SAECs might come from arterial ECs or systemic venous ECs in PAH. Global cell proportion analysis showed consistent PAH-associated increases in SAECs, systemic venous ECs, secondary crest myofibroblasts (SCMF), and alveolar fibroblast 2. Notably, female PAH patients exhibited reduced mast cells, CD8⁺ T cells, and vascular smooth muscle cells, but enrichment of SAECs, SCMFs, and platelets. EMT emerged as a core upregulated pathway across all major cell types in PAH.

Conclusion:Our integrative single-cell atlas of human PAH lungs identifies a previously unrecognized SAECs and provides deep insight into the molecular and cellular landscape of PAH across disease subtypes and sexes.