effects of intravitreal injection of netrin-1 in retinal neovascularization of streptozotocin-induced diabetic rats

Background: In a previous study, we confirmed that netrin-1 acts as an antiangiogenic factor by inhibiting alkali burn-induced corneal neovascularization in rats. Here, we continue working on the role of netrin-1 in retinal neovascularization. Methods: Using an in vitro angiogenesis assay, we detected the effects of netrin-1 on human umbilical vein endothelial cell tube formation, viability and proliferation, migration, and invasion at concentrations of 0.1 µ g/mL or 5 µ g/mL. We intravitreally injected 0.1 µ g/mL or 5 µ g/mL netrin-1 into streptozotocin-induced rats to assess retinal neovascularization using retinal electrophysiology and electroretinography, enzyme-linked immunosorbent assay, fundus fluoresce in angiography, measurement of inner blood retinal barrier, retinal hematoxylin-eosin staining, and retinal flat-mount fluorescence assays. Results: Human umbilical vein endothelial cell tube formation, viability and proliferation, migration, and invasion were upregulated by netrin-1 at a concentration of 0.1 µ g/mL ( P  0.05), while 5 µ g/mL netrin-1 had an opposite effect ( P  0.05) in our in vitro angiogenesis assay. Retinal electrophysiology testing revealed that intravitreal injection of netrin-1 affected the amplitude of a- and b-waves (a-wave: 0.1 µ g/mL netrin-1 = 17.67 ± 3.39 µ m, 5 µ g/mL netrin-1 = 28.50 ± 1.31 µ m, phosphate-buffered saline [PBS]-treated = 17.67 ± 3.39 µ m; b-wave: 0.1 µ g/mL netrin-1 = 44.67 ± 4.80 µ m, 5 µ g/mL netrin-1 = 97.17 ± 9.63 µ m, PBS-treated = 44.67 ± 4.80 µ m) and the expression of VEGF-A (no-treatment rats, 9.29 ± 0.80 pg/mL; PBS-treated rats, 19.64 ± 3.77 pg/mL; 0.1 µ g/mL netrin-1 treated rats, 21.37 ± 3.64 pg/mL; 5 µ g/mL netrin-1 treated rats, 9.85 ± 0.54 pg/mL, at 6 weeks after induction). By comparing fluoresce in angiography, level of inner blood retinal barrier breakdown (% of control), retinal hematoxylin-eosin staining, and collagen-IV fluorescence assays in the retinas of PBS-treated rats, netrin-1 was found to suppress and reverse retinal neovascularization at a concentration of 5 µ g/mL ( P  0.05), while 0.1 µ g/mL netrin-1 ( P  0.05) led to an increase in the number of new retinal blood vessels, after 6 weeks’ injection. Conclusion: Netrin-1 could play a significant role in retinal neovascularization by dual-direction regulating µ g/ml, 5.0 µ g/ml, 10.0 µ g/ml or 50.0 µ g/ml) for 36, 48, and 72 hours, growth of the hUVecs was assessed by a ccK-8 assay. The highest value of cell viability and proliferative capacity was detected in the group with 0.1 µ g/ml of netrin-1. The inhibitory effects of netrin-1 $ 5.0 µ g/mL on cell viability and proliferative capacity were similar. We performed a flow cytometric analysis of hUVecs treated with different concentrations of netrin-1 ( B ). The hUVecs that were cultured with 1% Bsa, 0.1 µ g/ml or 5.0 µ g/ml netrin-1 were stained with propidium iodide and analyzed by flow cytometry. The percentage of cells in the S phase was significantly higher in the group treated with 0.1 µ C The g2 + s/ G1 ratio was significantly higher in the group treated with 0.1 D