Project description:Metabolic Dysfunction Associated Steatohepatitis (MASH) is characterized by excess circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to the liver sinusoidal endothelial cells (LSEC) and transendothelial migration (TEM) are crucial in the inflammatory process. LSEC under lipotoxic stress develops a pro-inflammatory phenotype known as endotheliopathy. However, the mediators of endotheliopathy remain obscure. Primary mouse LSEC isolated from C57BL/6J mice on chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH, due to a choline-deficient high-fat diet (CDHFD) or FFC diet, were treated with two structurally distinct GSK3 inhibitors [LY2090314, elraglusib (9-ING-41)]. Integrated pathway analysis of mouse LSEC proteome and transcriptome indicates that leukocyte TEM and focal adhesion are major pathways altered in MASH. Kinome profiling of the LSEC phospho-proteome identified GSK3β as the major kinase hub in MASH. GSK3β activating phosphorylation was increased in primary human LSEC treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependant mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC and CDHFD-fed mice, respectively. Immunophenotyping of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib using cytometry by the time of flight showed reduced proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells infiltration.GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and may serve as a potential therapeutic strategy in human MASH.

Project description:Angiocrine signaling by liver sinusoidal endothelial cells (LSEC) regulates liver functions such as liver growth, metabolic maturation, and regeneration. Recently, we identified GATA4 as the master regulator of LSEC specification during development. Here, we studied endothelial GATA4 in the adult liver and in hepatic disease pathogenesis. We generated adult Clec4g-icretg/0xGata4fl/fl (Gata4LSEC KO) mice with deficiency of Gata4 in LSEC. Livers were analyzed by histology, electron microscopy, immunohistochemistry/immunofluorescence, in-situ hybridization, and by expression profiling and ATAC-sequencing of isolated LSEC. For liver regeneration, partial hepatectomy was performed. As models of liver fibrosis, CDAA diet and chronic CCl4 exposure were applied. Human single cell RNAseq data sets were analyzed for endothelial alterations in healthy and cirrhotic livers. Genetic Gata4 deficiency in LSEC in adult mice caused perisinusoidal liver fibrosis, hepatopathy and impaired liver regeneration. Sinusoidal capillarization and LSEC-to-continuous endothelial transdifferentiation were accompanied by a profibrotic angiocrine switch including de novo endothelial expression of hepatic stellate cell-activating cytokine PDGFB. Increased chromatin accessibility and amplification by activated Myc mediated angiocrine PDGFB expression. In CDAA diet-induced perisinusoidal liver fibrosis, LSEC showed repression of GATA4, activation of MYC and the profibrotic angiocrine switch already detected in Gata4LSEC KO mice. Comparison of CDAA-fed Gata4LSEC KO and control mice demonstrated that endothelial Gata4 indeed protects from dietary-induced perisinusoidal liver fibrosis. In human cirrhotic livers, Gata4-positive LSEC and endothelial Gata4 target genes were reduced, while non-LSEC endothelial cells and Myc target genes including PDGFB were enriched. Endothelial GATA4 protects from perisinusoidal liver fibrosis by repressing MYC activation and profibrotic angiocrine signaling on the chromatin level. Therapies targeting the GATA4/MYC/PDGFB/PDGFRβ axis offer a promising strategy for the prevention and treatment of liver fibrosis.

Project description:Liver sinusoidal endothelial cells (LSEC) are highly specialized within the hepatic vascular niche, controlling liver function and disease pathogenesis by angiocrine signaling. Recently, we identified GATA4 as a major transcription factor controlling LSEC development and protecting against liver fibrosis. As the transcription factor c-Maf was strongly downregulated in Gata4-deficient LSEC, we hypothesized that c-Maf might be an important downstream effector of GATA4 in LSEC differentiation and liver fibrogenesis. Clec4g-iCre/Maf fl/fl mice (Maf-LSEC-KO) mice with LSEC-specific Maf deficiency were generated and liver tissue was analyzed by histology, immunofluorescence, and in situ-hybridization at the age of three months. LSECs were isolated for RNA-, ATAC-seq, and single-cell RNA-seq (scRNA-seq) analysis. The expression of MAF and its targets were analyzed in published human scRNA-seq data. Endothelial Maf deficiency resulted in perisinusoidal liver fibrosis without affecting metabolic liver zonation, accompanied by a switch from sinusoidal to continuous endothelial differentiation. Furthermore, endothelial Maf deficiency caused hepatic endothelial proliferation and expression of profibrotic angiocrine factors such as Pdgfb, Igfbp5, Sparcl-1, and Flrt2. scRNA-seq revealed replacement of zonated LSEC subpopulations by capillarized, proliferative, sprouting and secretory endothelial cell subset promoting liver fibrogenesis and angiogenesis. This fundamental dysregulation of LSEC gene expression and differentiation was caused by changes in chromatin accessibility and transcription factor network alterations at promoter and enhancer regions following loss of Maf. Notably, endothelial MAF expression was also significantly reduced in human liver cirrhosis patients. Hepatic endothelial c-Maf protects against metabolic dysfunction-associated steatohepatitis-like liver fibrosis and regulates endothelial differentiation and zonation by controlling chromatin opening. Notably, our findings may open up new avenues to develop angiotargeted strategies for hepatic disease prevention and liver repair.

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Single-cell RNA sequencing reveals endothelial STING-dependent immune remodeling in murine metabolic dysfunction-associated steatohepatitis

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