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

Authors Yu Y, Zou J, Han Y, Quyang L, He H, Hu P, Shao Y, Tu P

Received 1 August 2015

Accepted for publication 4 November 2015

Published 7 December 2015 Volume 2015:9 Pages 6363—6377

DOI https://doi.org/10.2147/DDDT.S93166

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 4

Editor who approved publication: Prof. Dr. Wei Duan


Yao Yu,1,2,* Jing Zou,3,* Yun Han,4 Luowa Quyang,4 Hui He,4 Peihong Hu,2 Yi Shao,2 Ping Tu1

1Nanchang Key Laboratory of Diabetes, Department of Endocrinology and Metabolism, The Third Hospital of Nanchang, Jiangxi, People’s Republic of China; 2Department of Ophthalmology, The First Affiliated Hospital of Nanchang University, Jiangxi Province Clinical Ophthalmology Institute, Jiangxi, People’s Republic of China; 3Department of Ophthalmology, Xiangya Hospital, Central South University, Hunan, People’s Republic of China; 4Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, Fujian, People’s Republic of China

*These authors have contributed equally to this work

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.

Keywords: netrin-1,HUVEC, DR, intravitreal injection, retinal neovas­cularization

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