Bidirectional Eph/ephrin signaling in the crosstalk at the tumor-vessel interface
Zusammenfassung der Projektergebnisse
The formation and guidance of specialized endothelial tip cells is essential for both developmental and pathological angiogenesis. Notch1 signalling regulates the generation of tip cells, which respond to gradients of vascular endothelial growth factor (VEGF-A). The molecular cues and signalling pathways that control the guidance of tip cells are poorly understood. Bidirectional signalling by Eph receptors and ephrin ligands represents one of the most important guidance cues involved in axon path finding. In this project, we have shown that ephrinB2 reverse signalling involving PDZ-interactions regulates endothelial tip cell guidance to control angiogenic sprouting and branching in physiological and pathological angiogenesis. In vivo, ephrinB2 PDZ-signalling deficient mice (ephrinB2ΔV) exhibit a reduced number of tip cells with less filopodia extensions at the vascular front in the mouse retina. In pathological settings impaired PDZ-signalling decreases tumour vascularisation and growth. Mechanistically, we have shown that ephrinB2 controls VEGF receptor (VEGFR) 2 internalization and signalling. Importantly, internalization of VEGFR2 is necessary for activation and downstream signalling of the receptor and required for VEGF-induced tip cell filopodia extension. To study angiogenesis ex vivo we have developed a novel technique that consists on the culture of neonatal mouse retina and that allows assessment and quantification of acute responses of developing blood vessels to pharmacological manipulation. The technique has proven to be useful tool to elucidate the molecular mechanisms that underlie the guidance of tip cells in the complex scenario of the angiogenic sprouting process. This culture setting allows the acute stimulation or inhibition of cellular functions of endothelial cells in their physiological environment ex vivo without the necessity to perform retina injections in the animals. The responses of tip endothelial cell sprouting activity and filopodial extension to different angiogenic and angioinhibitory factors can be evaluated within only 4h. The whole process for the retinal explant cultures and stimulation can be completed in 10h. Together, our results suggest that ephrinB2 at the tip cell filopodia regulates the proper spatial activation of VEGFR2 endocytosis and signalling to direct filopodia extension. Blocking ephrinB2 reverse signalling may be used as an attractive alternative or combinatorial antiangiogenic therapy strategy to disrupt VEGFR2 function in tumor angiogenesis.
