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

摘要

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 μg/mL or 5 μg/mL. We intravitreally injected 0.1 μg/mL or 5 μg/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 μg/mL ( P <0.05), while 5 μg/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 μg/mL?netrin-1 =17.67±3.39 μm, 5?μg/mL?netrin-1 =28.50±1.31 μm, phosphate-buffered saline [PBS]-treated =17.67±3.39?μm; b-wave: 0.1 μg/mL?netrin-1?=44.67±4.80 μm, 5 μg/mL?netrin-1 =97.17±9.63 μm, PBS-treated?=44.67±4.80 μm) 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 μg/mL?netrin-1 treated rats, 21.37±3.64 pg/mL; 5 μg/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 μg/mL ( P <0.05), while 0.1 μg/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 angiogenesis dependent on dosage.