Abstract Rationale Hepatopulmonary syndrome (HPS) and portopulmonary hypertension (POPH) are serious vascular complications of chronic liver disease, the former featuring intrapulmonary vascular dilatation and hypoxemia, the latter characterized by elevated pulmonary vascular resistance. Despite overlapping hepatic backgrounds, the intrahepatic cellular and molecular mechanisms driving these divergent pulmonary outcomes remain poorly defined. We applied single-cell RNA sequencing (scRNA-seq) to human liver specimens from HPS, POPH, cirrhotic non-HPS, and healthy control subjects to delineate disease-specific hepatic remodeling, identify unique non-parenchymal and cholangiocyte signatures, and uncover pathways distinguishing HPS and POPH. Methods Liver tissue samples were obtained from controls (n = 4), HPS (n = 6), POPH (n = 4), and cirrhotic non-HPS (n = 6). Single-cell libraries were generated using the 10x Genomics Chromium platform. Data integration and batch correction were performed with Seurat and Harmony. Comparative analyses included differential gene expression and Hallmark gene-set enrichment (GSEA) across epithelial, endothelial, immune, and stromal compartments. Results Compared with controls, all cirrhotic groups showed broad hepatic remodeling with expansion of non-parenchymal immune/stromal populations, including Kupffer cells, hepatic stellate cells (HSCs), T cells, and plasma cells, and increased cholangiocytes. Relative to non-HPS cirrhosis, HPS livers displayed a distinct immune-stromal landscape marked by higher resting HSCs and Kupffer cells but fewer activated HSCs, fibroblasts, and cholangiocytes, suggesting a quiescent yet pro-angiogenic microenvironment. POPH livers shared a similar pattern but showed pronounced plasma cell expansion, indicating divergent immune activation. To define molecular programs underlying these shifts, Hallmark GSEA was performed across hepatic cell types. In HPS versus non-HPS, cholangiocytes showed enrichment of apical surface and reactive oxygen species (ROS) pathways; hepatocytes upregulated xenobiotic metabolism and G2/M checkpoint signaling; Kupffer cells activated mesenchymal remodeling, complement, and ROS pathways with suppressed TNF-α signaling; and resting HSCs displayed angiogenesis enrichment. Direct comparison of HPS and POPH revealed upregulated apical junction signaling in activated HSCs, enhanced unfolded protein response and fatty acid metabolism in fibroblasts, and increased coagulation signaling in hepatocytes of HPS, together with reduced interferon-γ and TNF-α activity in proliferating T/NK cells. These data highlight disease-specific hepatic remodeling that may underline divergent pulmonary vascular phenotypes. Conclusions Human liver scRNA-seq reveals cellular and pathway signatures distinguishing HPS and POPH: HPS shows Kupffer cell activation, cholangiocyte oxidative stress programs, and angiogenic signaling in resting HSCs, whereas POPH features plasma cell expansion and immune dysregulation. These hepatic signatures may contribute to divergent pulmonary vascular phenotypes in cirrhosis and suggest disease-specific therapeutic targets. This abstract is funded by: NIH
Zheng et al. (Fri,) studied this question.