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

Abstract

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.

Figure 1

Robo1 and slit2 expression in fibrovascular membranes from a 66-year-old patient with a 15-year history of diabetes. A, B, E, F: Immunofluorescent staining shows Robo1-positive (A), slit2-positive (E), and cytokeratin-positive (B, F) staining in fibrovascular membranes (FVMs). C, D, G, H: Cell nuclei were stained with 4’, 6’ -diamino-2-phenylindole (DAPI; C, G), and the colocalization of Robo1 and pancytokeratin (D), and slit2 and cytokeratin (H) are also shown. Bar graphs denote 100 μm.

Figure 2

Effects of advanced glycation end-products and LY294002 on Robo1 and slit2 mRNA levels in human retinal pigment epithelium cells as measured by real-time RT–PCR. A: Robo1 expression in human retinal pigment epithelium (RPE) cells was significantly increased at the mRNA (mRNA) level after a 24 h treatment of advanced glycation end products (AGEs) as measured by real-time RT–PCR. LY294002 (10 uM) blocks the expression of robo1 under conditions of AGEs (20 ug/ml). B: Slit2 expression in human RPE cells was also increased at the mRNA level after AGEs. The expression of slit2 in response to AGEs (20 ug/ml) decreased after treating cells with LY294002 (10 uM) versus treating the cells with AGEs (20 ug/ml) alone. Values provided are the mean±SD of three independent experiments. Asterisks denote values significantly different between the treated and control groups (p<0.05). Double asterisks denote values significantly different between the LY294002 (10 uM) group and the AGEs (20 ug/ml) group (p<0.01). The relative expression level of the control group cell was set to 1.

Figure 3

Protein expression of Robo1 in human retinal pigment epithelium cells was measured by immunoblotting with normalization to β-actin expression in retinal pigment epithelium cells. A: A representative photograph of the immunoblot analysis for Robo1 expression in human retinal pigment epithelium (RPE) cells. B: Relative Robo1 protein levels between the control group, advanced glycation end-products (AGEs) group, and LY294002-treated group. Values provided are mean±SD of three independent experiments. Asterisks denote values significantly different between the treated group and control group (p<0.05). Double asterisks denote values significantly different between the LY294002 (10 uM) group and AGEs (20 ug/ml) group (p<0.01).The pixel intensity of the control group was set to 1.

Figure 4

Effects of increasing concentrations of advanced glycation end-products and LY294002 on the secretion of slit2 protein in human retinal pigment epithelium cells. Cells were treated with increasing concentrations of advanced glycation end-products (AGEs) ranging from 10 to 100 ug/ml and LY294002 for 24 h in serum-free media. Slit2 protein levels in human retinal pigment epithelium (RPE) cells were measured using an enzyme-linked immunosorbent assay, and it was demonstrated that AGEs resulted in an increase in slit2 secretion. LY294002 was shown to decrease the expression of slit2 protein under AGE treatment conditions (20 ug/ml). Values are reported as the mean±SD from three independent experiments. Asterisks denote values significantly different between the treated group and control group (p<0.05). Double asterisks denote values significantly different between the LY294002 (10 uM) group and the AGEs treatment (20 ug/ml) group (p<0.01).

Figure 5

Effects of recombinant N-SLIT2 protein on human retinal pigment epithelium cell proliferation. Retinal pigment epithelium (RPE) cell proliferation was measured using an MTT assay at 24 h. Values reported are mean±SD from three independent experiments. Asterisks denote values significantly different from those of cells treated with recombinant N-SLIT2 protein compared to control cells (p<0.05).

Figure 6

Effects of recombinant N-SLIT2 protein on the attachment of human retinal pigment epithelium cells. Cell attachment was assessed after 6 h of incubation and subsequent MTT assay. Values are reported as mean±SD of four independent experiments. Asterisks denote values significantly different from those of cells treated with recombinant N-SLIT2 protein compared to control cells (p<0.01). The absorbance of the control cells was set to 1.

Figure 7

Effects of recombinant N-SLIT2 protein on human retinal pigment epithelium cell migration. The migratory activity of cells was estimated based on the number of cells that had traveled through the filter of the chamber. A: Control human retinal pigment epithelium migration. B: Migration of cells treated with recombinant N-SLIT2 protein (10 ng/ml). C: Migration of cells treated with recombinant N-SLIT2 protein (100 ng/ml). D: Relative migration of cells in the control and recombinant N-SLIT2 protein groups. Values are reported as the mean±SD from three independent experiments. The results demonstrate that the number of migratory cells in the recombinant N-SLIT2 protein group was greater than that in the control group (D, * p<0.01). Migration of cells in the control group was set to 1.

Figure 8

Effects of recombinant N-SLIT2 protein on the cell cycles in human retinal pigment epithelium cells. A: Cell cycle of control retinal pigment epithelium (RPE) cells. B: Cell cycle of RPE cells treated with recombinant N-SLIT2 protein (10 ng/ml). C: Cell cycle of RPE cells treated with recombinant N-SLIT2 protein (100 ng/ml). D: Data from the RPE cell cycle distribution of the control group and the recombinant N-SLIT2 protein group. Flow cytometric analysis demonstrates the effects of recombinant N-SLIT2 protein on the human RPE cell cycle. The x-axis represents fluorescence intensity on a logarithmic scale and the y-axis represents the number of events. The results show that the fraction of cells in the G1 phase has decreased and the proportion of cells in the S phase has increased in the presence of recombinant N-SLIT2 protein. Values are the mean±SD from three independent experiments. The proportion of G0/G1, G2, and S phase cells was demonstrated to be decreased in RPE cells treated with recombinant N-SLIT2 protein compared to control cells (D, *p<0.05).

Figure 9

Effects of recombinant N-SLIT2 protein on vascular endothelial growth factor mRNA levels in human retinal pigment epithelium cells. Vascular endothelial growth factor (VEGF) mRNA expression in human retinal pigment epithelium (RPE) cells was significantly increased with increasing concentrations of recombinant N-SLIT2 protein as measured by real-time RT–PCR 24 h after recombinant N-SLIT2 administration. Values are reported as the mean±SD from three independent experiments. Asterisks denote values significantly different between the treatment group and control group (p<0.05). The relative expression level of the control group cell was set to 1.

Figure 10

Effects of recombinant N-SLIT2 protein on vascular endothelial growth factor protein levels in human retinal pigment epithelium cells. Vascular endothelial growth factor (VEGF) protein levels were measured by enzyme-linked immunosorbent assay, and it was determined that VEGF secretion from human retinal pigment epithelium (RPE) cells increased in a direct manner with increasing administration of recombinant N-SLIT2 protein (0.1, 1, 10, 100 ng/ml). Values are reported as the mean±SD from three independent experiments. Asterisks denote values significantly different between the treatment group and control group (p<0.05).