Ephrin-B2 Controls VEGF Receptor Endocytosis & Angiogenic Sprouting

Angiogenesis specifically describes the sprouting of new blood vessels from the existing vasculature. This process requires the formation of endothelial tip cells, highly motile cells that invade the surrounding tissue.1 Endothelial tip cells guide the direction of new blood vessel growth by extending sensory filopodia that react to chemical signals in the surrounding environment. Endothelial cell proliferation and migration are primarily regulated by members of the vascular endothelial growth factor (VEGF) family. VEGF is known to regulate the generation of tip cells through the Notch-1 signaling pathway.2 However, the underlying mechanisms that direct tip cell migration and vessel guidance remain enigmatic.

Emerging research suggests the cues that govern capillary attraction and repulsion are dependent on the same molecules that orchestrate axon migration. For example, the expression of Ephrin-B2, an established axon guidance molecule, was shown to be essential for early angiogenic remodeling.3, 4 Two recent papers tested the hypothesis that the VEGF and Ephrin signaling pathways cooperate to regulate angiogenic growth.5, 6 Wang and colleagues reported that selective inhibition of the Ephrin-B2 gene in endothelial cells caused a significant decrease in sprouting angiogenesis.5 In contrast, microinjection of an Ephrin-B2 expression construct into individual cells in an endothelial monolayer induced migration, the dynamic extension of cellular protrusions, and invasive behavior.

VEGF and Ephrin Signaling Pathways Cooperate During Vessel Guidance.
View Larger Image 		VEGF and Ephrin Signaling Pathways Cooperate During Vessel Guidance. In the developing retina, endothelial sprouts migrate across an astrocyte scaffold.6 Endothelial tip cell filopodia direct vessel growth by reacting to guidance cues in the surrounding environment. Following stimulation by EphB4, Ephrin-B2 reverse signaling promotes the endocytosis of phosphorylated VEGF R dimers, in a PDZ domain dependent manner.5, 6 Internalized VEGF receptors subsequently exert pro-angiogenic effects via the PKC, Akt, and ERK signaling pathways. VEGF signaling is attenuated by dephosphorylation of VEGF R at the plasma membrane by phosphatases, such as CD148.

A unique feature of Ephrin/Eph signaling is that Ephrin ligands can also act as receptors and Eph receptors can function as ligands. To clarify this bidirectional relationship, an Ephrin ligand binding to an Eph receptor induces forward signaling that is dependent on a catalytically active intracellular Eph kinase domain. In addition, Ephrin ligands can also reverse signal into their host cell.7 For transmembrane Ephrin-B ligands, reverse signaling is dependent on tyrosine phosphorylation sites and a PDZ binding motif in the cytoplasmic domain.8

To investigate the importance of reverse signaling during angiogenesis, Sawamiphak and colleagues created Ephrin-B2 delta V mice, in which the cytoplasmic PDZ binding motif of Ephrin-B2 was impaired by targeted deletion of a single valine residue.6 The authors reported decreased angiogenic vessel sprouting and reduced filopodia density in the retinas of Ephrin-B2 delta V mice compared to wild-type control littermates. These data support the hypothesis that Ephrin-B2 reverse signaling controls vessel sprouting by promoting cell tip filopodia extension during angiogenesis.

The effects of VEGF are dependent on the internalization of VEGF receptors, which subsequently promotes signaling in the endosomal compartment.2 Both studies highlighted here examined the role of Ephrin-B2 in VEGF-induced angiogenesis. Sawamiphak et al. showed that the Ephrin-B2 PDZ domain was required for VEGF-A-induced internalization and activation of VEGF Receptor 2 (VEGF R2) in endothelial cells.6 In parallel, Wang et al. showed that knockout of the Ephrin-B2 gene severely impaired VEGF-C-induced VEGF R3 endocytosis and pathway activation, suggesting Ephrin-B2 and VEGF are functionally linked.5 Interestingly, in the absence of VEGF, recombinant Ephrin-B2 or EphB4 triggered internalization of VEGF R3 but did not activate markers of VEGF signaling. This supports the theory that Ephrin and VEGF pathways cooperate to promote angiogenesis.5

Further studies by Sawamiphak and colleagues tested the hypothesis that Ephrin-B2 regulates VEGF signaling during pathological angio­genesis. In an orthotopic glioma model, tumor size in Ephrin-B2 delta V mice was severely reduced compared to tumors in wild-type control littermates.6 This effect was associated with a substantial decrease in tumor vascularization, indicating that a common mechanism of angiogenesis occurs in physiological and pathological settings. Because blockade of VEGF/VEGF R signaling is routinely used as a treatment for cancer patients, inhibition of Ephrin-B2 may represent an alternative or combinatorial anti-angiogenic therapy.9 This strategy is supported by a recent study that used a specific EphB4 kinase inhibitor to suppress VEGF-driven angiogenesis in vivo.10 In addition, inhibition of Ephrin-B2/EphB4 signaling reduced tumor size in a transgenic mouse model of pancreatic cancer (RIP1-Tag2), an effect that was enhanced by combinatorial blockade of Notch signaling.11

References

  1. Jakobsson, L. et al. (2010) Nat. Cell Biol. 12:943.
  2. Phng, L.K. et al. (2009) Dev. Cell 16:70.
  3. Adams, R. et al. (1999) Genes & Dev. 13:295.
  4. Adams, R. et al. (2001) Cell 104:57.
  5. Wang, Y. et al. (2010) Nature 465:483.
  6. Sawamiphak, S. et al. (2010) Nature 465:487.
  7. Xu, N.J. & M. Henkemeyer (2009) Nat. Neurosci. 12:268.
  8. Makinen, T. et al. (2005) Genes & Dev. 19:397.
  9. Scehnet, J.S. et al. (2009) Blood 113:245.
  10. Martiny-Baron, G. et al. (2010) Angiogenesis 13:259.
  11. Djokovic, D. et al. (2010) BMC Cancer 10:641.

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