Forward EphB4 signaling in endothelial cells controls cellular repulsion and segregation from ephrinB2 positive cells.
Contact-dependent interactions between endothelial cells (ECs), as well as between ECs and mural cells, play a key role in the formation of a regular vascular system and the assembly of the vessel wall. Recent studies have identified ephrinB2 and EphB4 as markers and makers of arteriovenous differentiation during vascular development. On the basis of these findings, we hypothesized that Ephephrin interactions in the vascular system mediate distinct propulsive and repulsive effector functions that provide guidance signals for the proper spatial organization of the developing vasculature. Utilizing a set of specialized endothelial differentiation and angiogenesis assays, the present study was aimed at studying vascular morphogenic functions of endothelial EphB4 and ephrinB2 activation. EphrinB2-Fc acts anti-adhesively and induces detachment of ECs, as demonstrated by (1) inhibition of adhesion to ephrinB2-Fc-coated culture dishes, (2) detachment of ECs grown as differentiated 3D spheroids, and (3) endothelial denudation of explanted fragments of umbilical vein. Conversely, soluble ephrinB2-Fc inhibits lateral cell migration, vascular endothelial growth factor (VEGF) gradient-driven chemotaxis, capillary-like network formation and sprouting angiogenesis. In turn, soluble EphB4-Fc is pro-adhesive and stimulates EC migration and sprouting angiogenesis. EphrinB2-mediated repulsive signals are transduced by EphB4, as demonstrated by EphrinB2-Fc inhibition of sprouting angiogenesis of constitutively EphB4-overexpressing ECs. Confrontation experiments of EphB4-overexpressing ECs with ECs overexpressing full-length or truncated ephrinB2 that lacks the cytoplasmic catalytic domain demonstrated that forward EphB4 signaling with EphB4 tyrosine phosphorylation restricts intermingling of cells and supports cellular segregation. Taken together, these data identify distinct propulsive and repulsive effector functions of endothelial ephrinB2 and EphB4 that mediate spatial positional signals during angiogenesis and vessel assembly.
Department of Vascular Biology and Angiogenesis Research, Institute of Molecular Oncology, Tumor Biology Center, Breisacher Strasse 117, 79106 Freiburg, Germany.
Tim
T
Füller
Thomas
T
Korff
Adrienne
A
Kilian
Gudrun
G
Dandekar
Hellmut G
HG
Augustin
D002196:Capillaries / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D020134:Catalytic Domain / Q000187:drug effects / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology; D018920:Coculture Techniques; D003463:Cues; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D036388:Ephrin-B2 / Q000819:agonists / Q000378:metabolism*; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D036224:Receptor, EphB4 / Q000819:agonists / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D018874:Spheroids, Cellular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors
D036388:Ephrin-B2; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D036224:Receptor, EphB4