Single-cell transcriptomics reveals NDRG1/TGF‑β1 associated vascular smooth muscle cell phenotypic switching in chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by a unique combination of mechanical obstruction resulting from organized thrombi and a variable degree of secondary small-vessel remodeling, both of which contribute to increased pulmonary vascular resistance. In this process, pulmonary artery smooth muscle cell (SMC) phenotypic switching plays a critical role in the progression of microvascular disease. However, the underlying molecular mechanisms remain incompletely elucidated. Single-cell RNA sequencing (scRNA-seq) data from CTEPH patients and normal pulmonary artery tissues (GSE224143, GSE228644) were integrated. After data preprocessing, unsupervised clustering, and cell type annotation using Seurat, we focused on SMC subtype analysis and constructed functional landscapes via pseudobulk DESeq2, Gene Ontology (GO) enrichment analysis, Monocle3 pseudotime trajectory analysis, and high-dimensional weighted gene co-expression network analysis (hdWGCNA). Core findings were validated using bulk RNA-seq datasets (GSE84538) and experimental approaches, including Western blotting, immunofluorescence, EdU proliferation assay, and Transwell/scratch wound healing migration assay. scRNA-seq analysis identified 18 distinct cell clusters in pulmonary vascular tissues. Compared with controls, CTEPH tissues showed reduced fibroblasts and increased immune cells (T cells, monocytes/macrophages) and SMCs. Four SMC phenotypes were identified, with a marked expansion of fibroblast-like SMCs in CTEPH and a dynamic transition from contractile to fibroblast-like phenotype confirmed by pseudotime analysis. Upregulated genes in CTEPH SMCs were enriched in extracellular matrix organization and TGF-β signaling pathways (significantly activated). Cross-validation via hdWGCNA, pseudotime trajectory, and bulk RNA-seq data identified NDRG1 (a hypoxia-inducible gene) as a core gene. NDRG1 was significantly overexpressed in CTEPH SMCs and tissues, positively correlating with pulmonary vascular resistance (PVR). Functional experiments showed that NDRG1 knockdown inhibited hypoxia-induced migration/proliferation of human pulmonary artery smooth muscle cells (HPASMCs), suppressed TGF-β/SMAD pathway activation, and reduced fibroblast-like phenotype marker (Vimentin, COL1A1) expression. NDRG1 is associated with SMC phenotypic switching toward the fibroblast-like phenotype and promotes pulmonary vascular remodeling in CTEPH via the TGF-β/SMAD pathway. NDRG1 and related key gene axes may serve as potential therapeutic targets for CTEPH.