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Abstract

Endothelial cells (EC) display heterogeneity in morphology and function to meet the specific requirements of the respective organ. EC are no passive cell layer, but rather control development, homeostasis, and regeneration by interacting with their microenvironment via angiocrine factors. A prime example of angiodiversity are the discontinuous and fenestrated capillaries of the livers, referred to as liver sinusoidal EC (LSEC). LSEC are characterized by fenestrations without diaphragm, lack of a basement membrane and expression of various scavenger receptors. They regulate functions such as liver growth, zonation, and regeneration by secreting specific angiokines including WNT2 and HGF. Recently, transcription factor GATA4 was identified as a master regulator of hepatic endothelial differentiation during development. The early embryonic Gata4 deletion in Stab2 positive LSEC causes capillarization of the endothelium, hypoplasia and fibrosis resulting in fetal anemia and late embryonic lethality. In order to investigate the function of Gata4 in vivo in the adult liver, Clec4g Cre deleter mice were used to generate adult Gata4LSEC-KO mice since analyses of Clec4gCre;Rosa26-YFP reporter mouse showed late embryonic (E17.5) Cre activity in CD31+ EC. Gata4LSEC-KO mice were born at the expected mendelian ratio and were viable. However, Gata4LSEC-KO livers showed macroscopic irregularities, were significantly smaller as well as serum values indicated liver damage and metabolic alterations. Adult endothelial Gata4 deletion led to perisinusoidal liver fibrosis and increased collagen deposition. Gene expression profiling revealed 403 genes significantly dysregulated in LSEC of Gata4LSEC-KO mice and a LSEC-to-continuous endothelial transdifferentiation. Furthermore, capillarization of liver sinusoids was accompanied by the formation of a continuous basement membrane. LSEC restricted Gata4 deficiency caused impaired angiocrine signaling resulting in impaired metabolic zonation of the liver. In addition, profibrotic angiocrine factors were upregulated due to Gata4 deletion including stellate cell-activating mitogenic factor Pdgfb. For the first time LSEC were identified as main source of Pdgfb in Gata4LSEC-KO livers by duplex ISH and Pdgfb expression was amplified by activated MYC. Moreover, ATAC-Sequencing of Gata4LSEC-KO LSEC revealed that GATA4 mediated its repressive function by regulating chromatin accessibility. Analyses of regeneration after partial hepatectomy (PHx) revealed an impaired capability of regenerative hypertrophy caused by missing Wnt2 induction in the early phase of regeneration in Gata4LSEC-KO livers. In addition, liver regeneration following a single CCl4 injection was also impaired in Gata4LSEC-KO mice due to a delayed uptake and/or metabolization of CCl4. These data indicate that GATA4 acts as a master regulator of sinusoidal endothelial differentiation in the adult liver, as endothelial deletion of Gata4 results in LSEC capillarization, profibrotic angiocrine signaling responsible for development of perisinusoidal liver fibrosis, and deficiency in the regenerative capacity of the liver.