To investigate ferroptosis-cuproptosis crosstalk in pulmonary arterial hypertension (PAH) and its potential association with pulmonary vascular immune microenvironment remodeling.

PAH-related bulk transcriptomic and single-cell RNA sequencing datasets were obtained from the GEO database. Differentially expressed genes between PAH(n=76) and normal lung tissues(n=42) were identified, and ferroptosis, cuproptosis and ferroptosis-cuproptosis crosstalk scores were calculated using ssGSEA/GSVA. Candidate crosstalk genes were screened by intersecting PAH differentially expressed genes, ferroptosis/cuproptosis-related genes and crosstalk score-related genes, followed by expression validation, correlation analysis and GO/KEGG functional enrichment analysis. Single-cell RNA sequencing data were further used for cell annotation, cell type-specific score evaluation and core gene distribution analysis. Cell Chat was applied to assess intercellular communication changes in PAH lung tissues. In addition, a PAH animal model(n=40) was used to validate hemodynamic alterations, key molecular expression by qRT-PCR and Western blot.

Ferroptosis-, cuproptosis- and crosstalk-related transcriptional features were markedly altered in PAH lung tissues: the ferroptosis, cuproptosis and crosstalk scores were all significantly lower in PAH than in normal lungs (P=5.3×10^5, 0.028 and 0.00029, respectively), and ferroptosis score was positively correlated with cuproptosis score (R=0.42, P=0.019). Sixteen candidate genes were obtained by intersection analysis, among which DLAT, SAT1, MTF1, SLC7A11, GCLM and NCOA4 were identified as core candidate genes involved in ferroptosis-cuproptosis crosstalk (DLAT was upregulated whereas the other five genes were downregulated in PAH; P=8.8×10^6, 2.0×10^4, 3.5×10^4, 0.0073, 6.7×10⁵ and 7.3×10^4, respectively). Functional enrichment analysis showed that these genes were mainly enriched in ferroptosis 7 genes, lipoic acid metabolism 4 genes, glutathione metabolism 3 genes, and cysteine and methionine metabolism 2 genes. Single-cell analysis (n=9) identified 16 cell types and revealed cell type-specific alterations of the crosstalk score in pulmonary vascular endothelial cells, smooth muscle cells and macrophages, all of which showed significantly lower crosstalk scores in PAH (P<0.05, P<0.01 and P<0.05, respectively). CellChat analysis showed increased interaction number (2 067 vs. 2 802) and interaction strength (32.759 vs. 58.673) in PAH, with enhanced macrophage communication with endothelial cells, fibroblasts and smooth muscle cells through signaling axes including THBS1-CD47, SELPLG-SELE, PECAM1-PECAM1 and FN1-(ITGA5+ ITGB1). Animal experiments showed significantly increased right ventricular systolic pressure (approximately 23 vs. 49 mmHg, P<0.01) and Fulton index (approximately 0.23 vs. 0.42, P<0.001) in the PAH group. Moreover, DLAT was upregulated at both mRNA and protein levels (P<0.0001 and P<0.001), whereas SAT1, MTF1, SLC7A11, GCLM and NCOA4 were downregulated (P<0.05).

Ferroptosis-cuproptosis crosstalk is dysregulated in PAH lung tissues and mainly involves pulmonary vascular endothelial cells, smooth muscle cells and macrophages. This dysregulation may participate in pulmonary vascular remodeling by regulating glutathione metabolism, lipoic acid metabolism and macrophage-vascular wall cell communication.