title: Effects of intravitreal injection of netrin-1 in retinal neovascularization of streptozotocin-induced diabetic rats.
abstract: 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.
  
  
  CONCLUSIONS
  Netrin-1 could play a significant role in retinal neovascularization by dual-direction regulating angiogenesis dependent on dosage.
authors.affiliation: Nanchang Key Laboratory of Diabetes, Department of Endocrinology and Metabolism, The Third Hospital of Nanchang, Jiangxi, People's Republic of China ; Department of Ophthalmology, The First Affiliated Hospital of Nanchang University, Jiangxi Province Clinical Ophthalmology Institute, Jiangxi, People's Republic of China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Ophthalmology, Xiangya Hospital, Central South University, Hunan, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zou
authors.affiliation: Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, Fujian, People's Republic of China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Han
authors.affiliation: Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, Fujian, People's Republic of China.
authors.forename: Luowa
authors.initials: L
authors.lastname: Quyang
authors.affiliation: Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, Fujian, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: He
authors.affiliation: Department of Ophthalmology, The First Affiliated Hospital of Nanchang University, Jiangxi Province Clinical Ophthalmology Institute, Jiangxi, People's Republic of China.
authors.forename: Peihong
authors.initials: P
authors.lastname: Hu
authors.affiliation: Department of Ophthalmology, The First Affiliated Hospital of Nanchang University, Jiangxi Province Clinical Ophthalmology Institute, Jiangxi, People's Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Shao
authors.affiliation: Nanchang Key Laboratory of Diabetes, Department of Endocrinology and Metabolism, The Third Hospital of Nanchang, Jiangxi, People's Republic of China.
authors.forename: Ping
authors.initials: P
authors.lastname: Tu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007274:Injections, Intraperitoneal; D058449:Intravitreal Injections; D008297:Male; D009414:Nerve Growth Factors / Q000008:administration & dosage* / Q000378:metabolism* / Q000494:pharmacology; D000075388:Netrin-1; D051381:Rats; D017207:Rats, Sprague-Dawley; D015861:Retinal Neovascularization / Q000378:metabolism*; D013311:Streptozocin / Q000008:administration & dosage; D025521:Tumor Suppressor Proteins / Q000008:administration & dosage* / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; C000617354:Ntn1 protein, rat; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; D013311:Streptozocin
keywords: DR; HUVEC; intravitreal injection; netrin-1; retinal neovascularization