Abstract 4137829: Comprehensive Single-Cell Atlas of the Human Lung in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a complex cardiopulmonary disease characterized by progressive vascular remodeling. Although various immune and vascular cell populations have been implicated in the pathobiology of PAH, a comprehensive assessment of the various cell types in human PAH lungs is still lacking. Methods: Single-nucleus RNA sequencing was performed on lung samples from the Pulmonary Hypertension Breakthrough Initiative using the 10x Genomics Chromium Fixed RNA Profiling workflow. Rigorous quality control measures were implemented, including the detection and removal of ambient RNA using CellBender and doublets using scDblFinder. Data normalization and dimensionality reduction were performed using Seurat, and alignment by sample and sequencing batch was conducted using Harmony. Cells were annotated by mapping onto the Human Lung Cell Atlas using Azimuth. Differential gene expression analysis was conducted using a robust pseudobulk approach with edgeR to limit false positive findings. Gene set enrichment analysis was performed using the Hallmark pathways. Results: Lung samples from a total of 67 individuals were profiled, including 42 PAH and 25 age- and sex-matched control lungs. After quality control filtering, over 800,000 cells were captured, with a median of 12,000 cells per sample. Clustering uncovered over 30 cell types, representing endothelial, stromal, immune, and epithelial lineages of the lung. A median of 2,500 transcripts and 1,500 genes were detected per cell. Differentially expressed genes between PAH and control were detected in all major cell populations (FDR < 0.05). Comparative analysis across all cell types revealed interferon response in monocyte subpopulations and endothelial-to-mesenchymal transition in endothelial subpopulations as top upregulated pathways. Conclusion: Comprehensive single-nucleus profiling of human lungs in PAH revealed extensive gene and pathway dysregulation across diverse cell types. This rich dataset provides a valuable resource for the research community, offering opportunities to advance and refine our understanding of the cellular pathobiology of PAH.