Slit2 signaling through Robo1 and Robo2 is required for retinal neovascularization

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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.

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Figure 1: Slit2 promotes retinal angiogenesis.
Figure 2: Abnormal retinal angiogenesis in Robo1- and Robo2-knockout mice.
Figure 3: Slit2 promotes sprouting angiogenesis through Robo1 and Robo2.
Figure 4: ROBO1 and ROBO2 regulate Slit2- and VEGF-A–induced RAC1 activation.
Figure 5: Targeting Slit2–Robo signaling blocks angiogenesis in OIR.

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Acknowledgements

This project was supported by grants from the Agence Nationale de la Recherche (ANR; ANR-11BSV102502 to A.C. and A.E.), the Fondation pour la Recherche Médicale (“Programme Équipe FRM” to A.C.; DEQ20120323700), Sanofi-Fovea (to A.C.), Fondation Leducq (Artemis Transatlantic Network of Excellence, to A.E.) and the Thome Foundation for Age-Related Macular Degeneration (to A.E.). It was performed in the frame of the LABEX LIFESENSES (reference ANR-10-LABX-65), supported by French state funds managed by the ANR within the Investissements d'Avenir program under reference ANR-11-IDEX-0004-02. We thank F. Sennlaub for help with OIR experiments; J. Sahel and T. Debeir for helpful suggestions; Y. Zagar, M. Belle and C. Dominici for technical help; and K. Lyer for help with the initial phenotypic analysis. We also thank M. Tessier-Lavigne for the Slit1- and Slit2-knockout mice, J. Livet for the CAG:CreERTM mouse line and R. Adams for the Cdh5:CreERT2 mouse line. VEGF-A–AP constructs were provided by C. Ruiz de Almodovar (Vesalius Research Center, Leuven, Belgium). pGEX-PAK-CRIB was a kind gift from M. Schwartz (Yale University, New Haven, Connecticut, USA). The Slit2(lox) mutant mouse line was established at the Mouse Clinical Institute (Institut Clinique de la Souris, Strasbourg, France) in the Targeted Mutagenesis and Transgenesis Department.

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A.C. and A.E. supervised the project. A.C., A.E., N.R. and A.D. designed the experiments and wrote the manuscript. N.R., A.D., T.M., R.-A.N.C., G.G., B.C. and L.P.-F. performed the experiments and analyzed the data. L.M. generated the Robo1; Robo2lox mice.

Corresponding authors

Correspondence to Anne Eichmann or Alain Chédotal.

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Competing interests

This project was supported in part by a grant from Sanofi-Fovea to A.C.

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Rama, N., Dubrac, A., Mathivet, T. et al. Slit2 signaling through Robo1 and Robo2 is required for retinal neovascularization. Nat Med 21, 483–491 (2015). https://doi.org/10.1038/nm.3849

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