Abstract

Background: Hepatic stellate cells (HSCs) are the main drivers of liver fibrosis. During The Liver Meeting 2022, we demonstrated that glycolysis deficiency selectively in HSCs attenuated liver fibrosis in vivo. The aim of the present study is to understand how HSC-specific glycolysis promotes liver fibrosis by altering signaling pathways and HSC interactions with the surrounding cell types in a spatial manner.

Methods: Liver fibrosis was induced by carbon tetrachloride (CCl₄) administration in glycolysis-deficient mice, where hexokinase 2 (HK2) was deleted selectively in PDGFRβ⁺ HSCs (HK2^∆HSC), and their HK2^fl/fl littermate controls. 10X Genomics Visium platform was employed to examine spatial gene expression. Data were normalized using SCTransform and integrated using Harmony algorithm. Unsupervised clustering was performed using Louvain algorithm. In-house and public single cell RNA-seq data (GSE175939, GSE108097) were integrated for cell type deconvolution using RCTD algorithm. Differential co-localization analysis between cell types was performed using Fisher’s z test.

Results: RNA was collected and sequenced from each of the 55-µm spatial spots, each spot including several cells. Based on conserved gene expression, the spots were classified into 4 clusters (0-3) across the conditions. Clusters 0 and 3 expressed the peri-portal marker Cyp2f2, while clusters 1 and 2 expressed the peri-central marker Cyp2e1 as well as collagen 1α1 and 3α1 (adj. p<0.05). Gene ontology analysis (Panther 2023) of differential gene expression showed that HSC-specific glycolysis regulated distinct pathways during liver fibrosis, such as amino acid metabolism in the peri-portal clusters and extracellular matrix, vitamine A and glucosaminoglycan-related pathways in the peri-central clusters (FDR<0.02). These results suggest that glucosaminoglycan metabolism closely correlates to HSC activation and matrix deposition in the pericentral areas. In addition, a neighborhood analysis was performed to study how HSC-specific glycolysis regulates cell-to-cell interactions. CCl₄-mediated fibrosis was accompanied by an increased colocalization between HSCs and macrophages, T cells and dendritic cells (p<0.05), which were significantly reduced when HK2 was selectively deleted in HSCs (p<0.05).

Conclusion: Our results suggest that HSC-specific glycolysis promotes liver fibrosis by regulating distinct metabolic pathways in peri-central versus peri-portal zones and facilitating the interaction between HSCs and immune cells in vivo.