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 endothelium (LSEC) is a prime example for organ-specific microvascular differentiation and functions. Disease-associated capillarization of LSEC in vivo and dedifferentiation of LSEC in vitro indicate the importance of the hepatic microenvironment. To identify the LSEC-specific molecular differentiation program in the rat, we used a two-sided gene expression profiling approach comparing LSEC freshly isolated ex vivo with both lung microvascular endothelial cells (LMEC) and with LSEC cultured for 42h. The LSEC signature consisted of 48 genes both down-regulated in LMEC and in LSEC upon culture (FC>7 in at least one comparison); qRT-PCR confirmation of these genes included numerous family members and signalling pathway-associated molecules. The LSEC differentiation program comprised distinct sets of growth (wnt2, Fzd4, 5, 9, wls, VEGFR1, 2, 3, Nrp2) and transcription factors (Gata4, Lmo3, Tcfec, Maf) as well as endocytosis-related (Stabilin-1/2, Lyve1 and Ehd3) and cytoskeleton-associated molecules (Rnd3/RhoE). Specific gene induction in cultured LSEC versus freshly isolated LSEC as well as LMEC (Esm-1, Aatf) and up-regulation of gene expression to LMEC levels (CXCR4, Apelin) confirmed true transdifferentiation of LSEC in vitro. In addition, our analysis identified a novel 26 kDa single-pass transmembrane protein, liver endothelial differentiation-associated protein (Leda)-1, that was selectively expressed in all liver endothelial cells and preferentially localized to the abluminal cell surface. Upon forced over-expression in MDCK cells, Leda-1 was sorted baso-laterally to E-cadherin-positive adherens junctions suggesting functional involvement in cell adhesion and polarity. Conclusion: Comparative microvascular analysis in rat identified a hepatic microenvironment-dependent LSEC-specific differentiation program including the novel junctional molecule Leda-1. Highly pure liver sinusoidal endothelial cells (LSEC) and lung microvascular endothelial cells (LMEC) were isolated by enzymatic digestion, density gradient centrifugation and MACS sorting and subjected to RNA extraction and affymetrix hybridization without any culturing step (LSEC_0h, LMEC_0h). LSEC were also plated on collagen coated dishes and RNA extraction and Affymetrix hybridization was carried out after 2h and 42h in culture (LSEC_2h, LSEC_42h).

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Mus musculus

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