Abstract Body: Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vascular remodeling, increased vascular resistance, and eventual right heart failure. Elucidating the cellular and molecular mechanisms underlying PAH is critical to developing novel therapies.
Hypothesis: Integrating single-cell RNA sequencing (scRNAseq) with high-resolution spatial transcriptomics (ST) will provide a deeper understanding of the cellular and molecular changes driving PAH onset and progression.
Methods: Lung tissues from 10 healthy donors and 30 PAH patients, including idiopathic, drug/toxin-induced, and systemic/pulmonary shunt–associated PAH, were obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI). Specimens underwent fixed scRNAseq and high-resolution ST on 10X Genomics Xenium platform. Data from both methods were integrated to characterize cellular and molecular alterations in PAH.
Results: Integrated scRNAseq and ST analysis identified 34 distinct cell types, including endothelial cells (ECs), stromal cells, immune cells, and epithelial cells. Cell proportion analysis demonstrated an increase of alveolar fibroblast 2, secondary crest myofibroblasts, alveolar type 0 cells, and dendritic cells and a decrease in aerocytes, vascular smooth muscle cells, neutrophils and patrolling monocytes in PAH. GSEA analysis showed that upregulated pathways related to angiogenesis, androgen response, coagulation, and epithelial-mesenchymal transition (EMT), while downregulated pathways enriched in IL2-STAT5 signaling, MYC targets V2 signaling, and P53 pathway in PAH lungs. EMT is the most significant upregulated pathway. Within EC subclusters, we identified a novel arterial EC subtype (SAEC) in PAH lungs, characterized by expression of COL15A1, CXCL12, JAG2 and LTBP4. Notably, cell proportion analysis showed an increase in systemic venous ECs (SVECs), venous ECs, and SAECs along with a reduction in aerocytes in PAH lungs. ST mapping revealed SAECs and SVECs enriched within plexiform lesions, further confirmed by immunostaining for COL15A1. Ligand-receptor interaction analysis indicated SAECs and SVECs as key mediators of cellular communication in PAH lungs.
Conclusions: This integrative analysis of scRNAseq and ST offers a detailed cellular and molecular landscape of PAH lungs, highlighting unique cell populations and molecular markers. These insights pave the way for novel therapeutic strategies targeting cellular crosstalk in PAH.