Neuronal Guidance Cue Slit2 And Robo1,Robo4 Receptors Regulate Corneal Neovascularization

Background and Objective:Corneal diseases, which include infections, chemical and mechanical injuries, immunologic diseases and degenerations, are a major cause of blindness worldwide. Corneal neovascularization (NV) is involved in many corneal diseases as an important pathological process. Although a number of regulatory factors have been identified to play a role, the molecular mechanisms of corneal NV are still not fully understood and there is no ideal therapy for corneal NV currently as a result. Therefore, further investigation of pathological mechanisms that control corneal NV is needed to improve our understanding of this complex process. To elucidate the molecular mechanisms of corneal NV from a whole new angle, we focus our attention on the nervous system that possesses similar structure to the vascular system. Nerves and blood vessels are in close proximity and in connection with each other, sharing anatomical similarities and functional parallels. Given the behavioral similarities between nervous and vascular system, there may be molecular cross talk and common cues. Four families of classical neuronal guidance cues have so far been identified as regulators of vascular development:the Ephrins, Netrins, Semaphorins and Slits. We focus on the Slits family that regulates monodirectionally in nervous system. Our objective was to investigate whether neuronal guidance cue Slit2 and Roundabout (Robo) receptors are involved in corneal NV. Methods:Corneal NV model in rats was induced by implantation of agarose-coated gelfoam pellets containing basic fibroblast growth factor (bFGF) into corneal stroma. Differential expression of Slit2 and Robol-4 between normal and neovascularized cornea was detected by real-time RT-PCR and visualized by immunohistochemistry and in situ hybridization. Primary human umbilical vein endothelial cells (HUVECs) were harvested and their expression of Robol-4 was detected by RT-PCR. Recombinant human Slit2 protein was prepared and the effect of it on the migration of vascular endothelial cells was examined using cell migration assay. Results:Agarose-coated gelfoam pellets were able to induce well-localized and reproducible corneal NV model. A significant down-regulation of Slit2 and a strong up-regulation of Robol and Robo4 were seen in neovascularized cornea when compared with normal cornea (P<0.05). Slit2, Robol and Robo4 were throughout the epithelium in normal cornea and markedly weak or absent in epithelium in neovascularized cornea, with Robol and Robo4 being prominent in vascular endothelial cells invading the stroma. Primary HUVECs were confirmed to express both Robol and Robo4 receptors and their migration was inhibited by Slit2 (P<0.05). Conclusion:This is the first study to assess the association between Slit2 and corneal NV. Our findings suggest that the interaction of Slit2 with Robol and Robo4 receptors plays an essential role in inhibiting pathological neovascular processes of the cornea and may represent a new therapeutic target for corneal NV.