ABSTRACT
Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.
Subject(s)
Blood Vessels/embryology , Embryo, Mammalian/blood supply , Endothelial Cells/metabolism , Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Blood Vessels/cytology , Calcium-Binding Proteins , Cell Adhesion , Cell Count , EGF Family of Proteins , Embryo, Mammalian/abnormalities , Embryo, Mammalian/cytology , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/blood supply , Embryo, Nonmammalian/cytology , Endothelial Cells/cytology , In Situ Hybridization , Mice , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Zebrafish/abnormalities , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
A key feature of recovery from liver fibrosis is hepatic stellate cell (HSC) apoptosis, which serves the dual function of removing the major source of neomatrix and tissue inhibitors of metalloproteinases thereby facilitating matrix degradation. The mechanisms regulating HSC apoptosis remain undefined but may include the interaction of nerve growth factor (NGF) with its receptor, p75, on HSC. In this study, by TaqMan polymerase chain reaction in situ hybridization and immunohistochemistry, we demonstrate that NGF is expressed by hepatocytes during fibrotic injury. Peak hepatocyte expression of NGF (48 hours after CCl(4) injection) coincides with maximal rate of apoptosis of HSC by terminal dUTP nick-end labeling staining. Addition of recombinant NGF to HSC in tissue culture causes a dose-dependent increase in apoptosis. NGF regulates nuclear factor (NF)-kappaB activity, reducing p50/p65 binding detected by electromobility shift assay and reduced NF-kappaB CAT reporter activities from both basal unstimulated levels and after NF-kappaB induction by tumor necrosis factor. In each case, a relative reduction in NF-kappaB binding was associated with a significant increase in caspase 3 activity. These data provide evidence that NGF is expressed during fibrotic liver injury and may regulate number of activated HSCs via induction of apoptosis.