Robo receptors in angiogenesis and ocular vaso-proliferative disease – RoboVision

Ocular vaso-proliferative diseases are the most common causes of vision loss in the industrial world, affecting millions of people worldwide. They are observed in age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), retinopathy of prematurity (the main cause of blindness in young children) and corneal inflammation. All these diseases are characterized by excessive angiogenesis accompanied by endothelial barrier breakdown and vascular leak, which leads to oedema, haemorrhage and retinal detachment compromising vision. To date, the treatment of excessive angiogenesis relies largely on inhibition of a single factor, vascular endothelial growth factor (VEGF), with some therapeutic success. However, other factors in addition to VEGF contribute to angiogenesis and identification of alternative pathways to block excessive angiogenesis and vascular leak is of enormous therapeutic interest.
Partner 1 and others have shown that several receptors for axon guidance molecules including Neuropilins, the Netrin receptor UNC5B and Roundabout 4 (Robo4) can also modulate blood vessel growth in the retina. Among those, Robo4 is an endothelial cell (EC)-specific member of the Robo receptor family. Using a large-scale protein-protein interaction screen with the Robo4 extracellular domain (ECD), partner 1 found that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. They have shown that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of VEGF. Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity.  Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4-/- mice, but not in Unc5b deficient mice, indicating that Robo4 counteracts VEGF by binding and signaling through UNC5B and plays a key role in the stabilization of the retinal vasculature. These data suggest that soluble Robo4 could be useful as a novel anti-angiogenic treatment for ocular diseases.
Robo4 had previously been thought to act by binding to Slit1-3 proteins, the major chemorepellents for growing axons in the developing nervous system. Slit proteins have been shown to inhibit migration and permeability of EC treated with VEGF, and to inhibit angiogenesis during ocular neovascularization in mice, suggesting that Slit proteins could be used as an anti-angiogenic molecule to treat ocular vaso-proliferative disease. However, the molecular mechanisms underlying Slit action in the vasculature remain to be determined. The cellular source of Slits for retina vessels is also unknown. Last, Robo4 has also been show to bind to Robo1 a well-known Slit receptor suggesting that Slits could modulate Robo4 function upon binding to Robo1. However, there is no direct evidence supporting a role for Robo1 or Robo2 receptors in the retina vasculature. Partner 2 has generated or collected various Robo and Slit knockout lines, including conditional alleles, which allow deleting these molecules in the eye to study the consequences on vessel development and stability.