Notch Signaling in the Hepatic Endothelial Regulome Mediates a Dual Reaction Pattern of Liver Fibrosis and Angiodysplasia

Background & Aims Unbalanced Notch signaling in liver sinusoidal endothelial cells leads either to vascular malformations or metabolic dysfunction–associated steatohepatitis–like liver fibrosis. Here, we investigated this dual reaction pattern for interactions of Notch signaling with the Alk1 and Gata4 pathways, which we hypothesized to contribute to aberrant angiogenesis or to liver fibrosis, respectively. Methods Liver tissue of Clec4g-Cre/Rosa26 N1ICD-IRES-GFP (NICD LSEC-OE ) mice was studied by computed tomography, ultrasound, and (immuno)-histology. Pathway analysis was performed using RNA sequencing, assay for transposase-accessible chromatin with sequencing, or single-cell RNA sequencing data from liver sinusoidal endothelial cells of NICD LSEC-OE , Gata4-deficient (Gata4 LSEC-KO ), Alk1-deficient (Alk1 LSEC-KO ), or choline-deficient, l-amino acid-defined–fed mice. Results NICD LSEC-OE mice showed simultaneous development of angiodysplasia and perisinusoidal liver fibrosis with loss of liver sinusoidal endothelial cell identity and hepatic stellate cell activation. Transcriptome analysis revealed a stronger overlap of NICD LSEC-OE liver sinusoidal endothelial cells with Gata4 LSEC-KO than Alk1 LSEC-KO liver sinusoidal endothelial cells. Similarly, TOBIAS footprint analyses of NICD LSEC-OE liver sinusoidal endothelial cells showed major downregulation of GATA4 DNA binding, while DNA binding of Alk1-associated transcription factors remained unchanged. Correspondingly, angiocrine factors Vwf driving liver fibrosis and Efnb2 supporting arterial angiogenesis were upregulated in NICD LSEC-OE and Gata4 LSEC-KO liver sinusoidal endothelial cells, but not in Alk1 LSEC-KO liver sinusoidal endothelial cells in vivo. Notch activation of liver sinusoidal endothelial cells in vitro induced VWF and EFNB2 expression, but downregulation of ALK1-target gene ID1 . In turn, EFNB2 induced up-regulation of proangiogenic genes VEGFR3 and RGCC in liver sinusoidal endothelial cells. In addition, short-term diet–induced metabolic dysfunction–associated steatohepatitis aggravated liver fibrosis in NICD LSEC-OE mice by largely Notch-independent pathogenic liver sinusoidal endothelial cell programs. Conclusions Notch-overactivated liver sinusoidal endothelial cells drive liver fibrosis via Gata4 loss, whereas they promote angiodysplasia largely independent of Alk1 signaling. Preventing pathogenic angiogenesis and liver fibrosis requires understanding the complex interactions within the liver sinusoidal endothelial cell regulome and the hepatic vascular niche.