Slit2 signaling through Robo1 and Robo2 is required for retinal neovascularization

Abstract

Ocular neovascular diseases are a leading cause of blindness. Vascular endothelial growth factor (VEGF) blockade improves vision, but not all individuals respond to anti-VEGF treatment, making additional means to prevent neovascularization necessary. Slit-family proteins (Slits) are ligands of Roundabout (Robo) receptors that repel developing axons in the nervous system. Robo1 expression is altered in ocular neovascular diseases, and previous in vitro studies have reported both pro- and anti-angiogenic effects of Slits. However, genetic evidence supporting a role for Slits in ocular neovascularization is lacking. Here we generated conditional knockout mice deficient in various Slit and Robo proteins and found that Slit2 potently and selectively promoted angiogenesis via Robo1 and Robo2 in mouse postnatal retina and in a model of ocular neovascular disease. Mechanistically, Slit2 acting through Robo1 and Robo2 promoted the migration of endothelial cells. These receptors are required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation. Thus, Slit2 blockade could potentially be used therapeutically to inhibit angiogenesis in individuals with ocular neovascular disease.

Slit-Robo signaling

“…This has indeed been shown in human endothelial cells in vitro, where a ROBO1/ROBO4 heterodimer promotes cell migration (Sheldon et al, 2009). In blood vessels, Slit-Robo cooperates with vascular endothelial growth factor A (Vegfa) to induce neovascularization, and this involves the scaffolding proteins Nck1/2 and Rac1 (Dubrac et al, 2015;Rama et al, 2015). Interestingly, a splice isoform of Slit2 (generated by splicing at exon 15) has a stronger effect on vessel tube formation and angiogenesis than its canonical counterpart, possibly in a Robo-independent manner (Yang et al, 2015).…”

“…Vertebrate Robo4 is selectively expressed by endothelial cells and has been reported to control angiogenesis and blood vessel permeability (Bedell et al, 2005;Huminiecki et al, 2002;Jones et al, 2009). Many studies have linked Robo4 function to Slit (Enomoto et al, 2016;Fritz et al, 2015;Jones et al, 2009;Yu et al, 2014), although it was subsequently proposed that Robo4 does not bind Slits (Koch et al, 2011), with genetic evidence in knockout mice also failing to support a direct link between Robo4 and Slits (Rama et al, 2015). Endothelial cells also express Robo1/2 receptors and Slits, in particular Slit2, which favors angiogenesis by promoting endothelial cell motility and polarity (Dubrac et al, 2016;Rama et al, 2015).…”

“…Many studies have linked Robo4 function to Slit (Enomoto et al, 2016;Fritz et al, 2015;Jones et al, 2009;Yu et al, 2014), although it was subsequently proposed that Robo4 does not bind Slits (Koch et al, 2011), with genetic evidence in knockout mice also failing to support a direct link between Robo4 and Slits (Rama et al, 2015). Endothelial cells also express Robo1/2 receptors and Slits, in particular Slit2, which favors angiogenesis by promoting endothelial cell motility and polarity (Dubrac et al, 2016;Rama et al, 2015). Given the overlapping expression pattern of Robo1/2 and Robo4, it is possible that Slit functions through receptor heterodimers without direct binding to Robo4.…”

Truncated netrin-1 contributes to pathological vascular permeability in diabetic retinopathy

“…Potential effector molecules with the propensity to mediate neurovascular communication in retinal health and disease are classical neuronal guidance cues and include netrins (20,21), semaphorins (17,18,(22)(23)(24), ephrins (25,26), and slits (27)(28)(29). While initially identified as mediators of neuronal circuit formation during embryogenesis, these cues play equally prominent roles in vascular (30) and immune function (18,31,32).…”

Making Connections: Guidance Cues and Receptors at Nonneural Cell–Cell Junctions

“…Robo1 and Robo2 have pro-angiogenic effects (Wang et al 2003;Rama et al 2015). On the other hand, Robo4 stabilizes the vasculature and decreases transendothelial permeability, and the molecular mechanisms underlying these effects have been well described ( Fig.…”