The expression of the Slit-Robo signal in the retina of diabetic rats and the vitreous or fibrovascular retinal membranes of patients with proliferative diabetic retinopathy

Abstract

Purpose: The Slit-Robo signal has an important role in vasculogenesis and angiogenesis. Our study examined the expression of Slit2 and its receptor, Robo1, in a rat model of streptozotocin-induced diabetes and in patients with proliferative diabetic retinopathy.

Methods: Diabetes was induced in male Sprague-Dawley rats via a single, intraperitoneal injection of streptozotocin. The rats were sacrificed 1, 3 or 6 months after the injection. The expression of Slit2 and Robo1 in retinal tissue was measured by real-time reverse transcription polymerase chain reaction (RT-PCR), and protein levels were measured by western blotting and immunohistochemistry. Recombinant N-Slit2 protein was used to study the effects of Slit2 on the expression of VEGF in vivo. The concentration of Slit2 protein in human eyes was measured by enzyme-linked immunosorbent assay in 27 eyes with proliferative diabetic retinopathy and 28 eyes in control group. The expression of Slit2, Robo1 and VEGF in the excised human fibrovascular membranes was examined by fluorescence immunostaining and semi-quantitative RT-PCR.

Results: The expression of Slit2 and Robo1 in the retina was altered after STZ injection. Recombinant N-Slit2 protein did not increase the retinal VEGF expression. Vitreous concentrations of Slit2 were significantly higher in the study group than in the control group. In the human fibrovascular membranes of the study group, the co-localization of VEGF with the markers for Slit2 and Robo1was observed. The expression of Slit2 mRNA, Robo1 mRNA, and VEGF mRNA was significantly higher in human fibrovascular proliferative diabetic retinopathy membranes than in the control membranes.

Conclusions: The alteration of Slit2 and Robo1 expression in the retinas of diabetic rats and patients with proliferative diabetic retinopathy suggests a role for the Slit-Robo signal in the various stages diabetic retinopathy. Further studies should address the possible involvement of the Slit-Robo signal in the pathophysiological progress of diabetic retinopathy.

Fig 1. The expression of Slit2 and Robo1 mRNA in the STZ diabetic rat retinas relative to the control rat retinas as measured by real-time PCR.

A: The expression of Slit2 mRNA was increased after 1, 3 and 6 months of diabetes than age matched control group. (P<0.05). B: The expression of Robo1 mRNA was significantly increased after 1, 3 and 6 months compared to controls. Values are the means ± SD of at least three independent experiments. Asterisks denote values significantly different between the diabetic and control groups (P<0.05). The relative expression level of the control group cell was set to 1.

Fig 2. The expression of Slit2 and Robo1 protein in the retinas of STZ diabetic rats relative to the control rat retinas as measured by immunoblotting and normalized to β-actin expression.

A: A representative photograph of the western-blot analysis of Slit2 expression. B: A representative photograph of the western-blot analysis of Robo1 expression. C: The relative Slit2 protein levels in the age matched control group and diabetic rats after 1, 3 and 6 months. D: The relative Robo1 protein levels in the age matched control group and diabetic rats after 1, 3 and 6 months. Values are the means ±SD of at least three independent experiments. Asterisks denote values significantly different from the diabetic rat group compared to the control group (p<0.05). Slit2 expression after 1 and 3 months was significantly increased compared to controls (p<0.05); however, slit2 did not vary significantly at 6months compared to controls (p>0.05). Robo1 expression after 1, 3 and 6 months was significantly increased compared to controls (p<0.05); Western blot image was analyzed by a tool called “Image J”. The pixel intensity of the control group was set to 100%. C1, C3, C6: control group1, 3, 6 months; D1, D3, D6:diabetic rat group1, 3, 6 months.

Fig 3. A–D: Slit2 antibody staining of rat retina sections.

A: control group; B: 1 month after diabetes induction; C: 3 months after diabetes induction; D: 6 months after diabetes induction. Scale bar 100 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Fig 4. A–D: Robo1 antibody staining of rat retina sections.

A: control group; B: 1 month after diabetes induction; C: 3 months after diabetes induction; D: 6 months after diabetes induction. Scale bar 100 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Fig 5. The retinal levels of VEGF were quantified by using the ELISA technique.

Compared with the retina of control group, the retina of diabetic animals demonstrate a 2.71-fold increase in VEGF levels (P<0.05). Diabetic animals of treatment with recombinant N-Slit2 protein does not significantly increase the retinal VEGF levels (P>0.05).

Fig 6. The vitreous concentration of Slit2 in patients with PDR and in patients with idiopathic preretinal membrane or macular holes.

Box plots showing the vitreous concentration of Slit2 in patients with PDR and in patients with idiopathic preretinal membrane or macular holes, with a statistically significant difference between the groups (P<0.05).

Fig 7. The vitreous concentration of Slit2 in different patients.

Box plots showing the vitreous concentration of Slit2 in patients with PDR (23 patients with vitreous hemorrhage and 4 patients without vitreous hemorrhage) and in 28 patients with idiopathic preretinal membrane or macular holes, with a statistically significant difference between PDR with vitreous hemorrhage and patients with idiopathic preretinal membrane or macular holes, (P<0.05),However, there is no difference between proliferative diabetic retinopathy with vitreous hemorrhage or not.(P>0.05)

Fig 8. Expression of Slit2 and VEGF in the FVMs of eyes with PDR and the preretinal membranes of control patients without DR.

(A-D) Immunostaining for Slit2 (A), VEGF (B), and DAPI (C) in FVMs from eyes with PDR. Staining intensities of Slit2 were strong (A) and co-localized with VEGF (D). (E-H) Staining of Slit2 (E), VEGF (F), and DAPI (G) in the preretinal membranes of control patients without DR. Weak staining of Slit2 (E) and insignificant staining of VEGF (F) were observed. Scale bar 50 μm.

Fig 9. Expression of Robo1 and VEGF in the FVMs of eyes with PDR and the preretinal membranes of control patients without DR.

(A-D) Immunostaining for Robo1 (A), VEGF (B), and DAPI (C) inFVMs from eyes with PDR. Staining intensities of Robo1 were strong (A) and co-localized with VEGF (D). (E-H) Staining of Robo1 (E), VEGF (F), and DAPI (G) in the preretinal membranes of control patients without DR. Weak staining of Robo1 (E) and insignificant staining of VEGF (F) were observed. Scale bar 50 μm.

Fig 10. RT-PCR analysis showed the mRNA expression of Slit2, Robo1, VEGF, and ß-actin in the FVMs of different patients.

A: RT-PCR amplification of Slit2, Robo1, VEGF, and ß-actin in the FVMs of six patients with PDR (panels 1–6) and in the membranes of six patients with idiopathic epiretinal membranes (panels 7–12).B: A representative photograph of the mRNA analysis of Slit2 expression. C: A representative photograph of the mRNA analysis analysis of Robo1 expression. D: A representative photograph of the mRNA analysis analysis of VEGF expression. Asterisks denote values significantly different to the control group (P<0.05).