Immunomagnetic exclusion of PECAM-1-positive endothelial cells in fetal mouse liver cell cultures causes impaired growth and gene expression of hepatoblasts and stellate cells

Abstract

Previous studies using mice having defective VEGF signaling have demonstrated that vascular development is indispensable for early hepatic organogenesis. However, not only whether its action lasts during later hepatic development, but also what molecules are involved in that action remains to be determined. The present study was undertaken to examine the effects of primitive sinusoidal endothelial cells on hepatic growth and maturation in primary culture of fetal mouse liver cells, and to determine their molecular mechanisms. When endothelial cells were excluded from E12.5 liver cell cultures by using PECAM-1-antibody-coated magnetic beads, the growth of hepatoblasts and stellate cells was conspicuously reduced and hepatic maturation was also suppressed. Conditioned medium prepared from fetal liver cell cultures containing almost all hepatic cell types stimulated the growth and gene expression of hepatoblasts and stellate cells similarly to the cultures in the presence of endothelial cells. HGF mRNA expression was downregulated in endothelial cellfree cultures of fetal liver cells, and the addition of HGF to the culture medium rescued the cells from the effects of endothelial cell depletion. These data suggest that humoral factors, including HGF, which are produced by endothelial cells or stellate cells, are involved in fetal hepatocyte growth and maturation.Liver primordium develops as an endodermal diverticulum in the anterior intestinal portal region in mammalian embryos, from which hepatic cords invade the subjacent septum transversum mesenchyme, and also extend into the wall of the omphalomesenteric veins and posterior cardinal veins as we thrust our fingers into an inflated balloon (2, 21, 24), leading to the formation of an immature liver having primitive sinusoid structures and abundant hemopoietic cells (22). The primitive sinusoid structures are constructed by endothelial cells and stellate cells, which resemble sinusoids of the adult liver in their cellular configuration and extracellular matrix deposition (22). The immature liver further grows and matures to build functional hepatic lobules in periand postnatal development through successive cellular interactions. The inactivation of the gene for Flk-1, one of the VEGF receptors, resulted in agenesis of vascular development and also various anomalies of organogenesis, including liver primordium formation (13). When fetal hepatoblasts are separated from other hepatic cells (nonparenchymal cells) and cultured alone, their survival rate and growth are conspicuously reduced (17). Although these data show that various cellular interactions are indispensable for normal liver development, it remains to be determined what molecular mechanisms operate between hepatoblast/hepatocyte-endothelial cell and stellate cell-endothelial cell interactions. Several growth factors and cytokines, including HGF, TGFβ, FGFs,