The role of SLIT-ROBO signaling in proliferative diabetic retinopathy and retinal pigment epithelial cells

PURPOSE: SLIT-ROBO signaling acts as a cue in neuronal guidance and plays a role in vasculogenesis and angiogenesis. The aim of this study is to explore the effects of robo1 and slit2 on the formation of fibrovascular membranes (FVMs) in samples from patients with proliferative diabetic retinopathy. The effects of advanced glycation end products (AGEs) on robo1 and slit2 expression in human retinal pigment epithelium (RPE) cells and the role of recombinant N-SLIT2 protein in human RPE cell regulation were investigated. METHODS: Immunohistochemistry was performed to determine the presence and distribution of robo1 and slit2 in FVMs, and to confirm the effects of SLIT-ROBO signaling on FVM formation. The expression levels of robo1 and slit2 in RPE cells under basal and differential concentrations of AGEs were measured using real-time reverse transcription-polymerase chain reaction (RT–PCR), immunoblotting, or enzyme-linked immunosorbent assay. LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), was used to help determine the AGE signaling mechanism. Recombinant N-SLIT2 protein was used to study the effects of slit2 on RPE cells in vitro. Cell proliferation, migration, and cell cycling were assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay assay (MTT) assay, a Boyden chamber assay, and flow cytometry. Real-time RT–PCR and enzyme-linked immunosorbent assay were used to study vascular endothelial growth factor (VEGF) mRNA expression in and VEGF protein secretion from RPE cells. RESULTS: Robo1 and Slit2 were expressed in FVMs in RPE cells coimmunostained for pancytokeratin. AGEs resulted in an increase in robo1 and slit2 levels in RPE cells, and inhibition of PI3K-blocked robo1 and slit2 expression. Recombinant N-SLIT2 protein increased proliferation, attachment, and migration of the RPE cells, and these cells demonstrated significant accumulation in the S phase compared to control cells. Furthermore, RPE cells treated with exogenous N-SLIT2 protein had higher levels of VEGF mRNA expression and VEGF protein secretion (p<0.05). CONCLUSIONS: Robo1 and slit2 may play a role in the formation of FVMs. The presence of AGEs increased levels of robo1 and slit2 in human RPE cells via signaling through the PI3K/Akt pathway. Recombinant N-SLIT2 protein increased the biologic activity of RPE cells, as well as the expression of VEGF. From these results, we may conclude that SLIT-ROBO signaling potentially contributes to the development of diabetic retinopathy.