title: Antiangiogenesis effects of endostatin in retinal neovascularization.
abstract: OBJECTIVE
  Pathological retinal angiogenesis is a major cause of vision loss. Endostatin is a natural antiangiogenesis antitumor protein that is widely used in cancer studies. In this study, we investigated the efficacy and potential mechanisms of endostatin for the prevention of retinal neovascularization both in vitro and in vivo.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) were used for the in vitro studies. HUVECs were incubated with endostatin or the vascular endothelial growth factor (VEGF) and endostatin for different time points. Cell proliferation, migration, cell cycling, and tube formation studies were carried out using a Cell Counting Kit-8 assay, a Transwell assay, flow cytometry, and a Matrigel assay, respectively. Enzyme-Linked Immunosorbent Assay (ELISA) was used to study VEGF and pigment epithelial-derived factor (PEDF) protein secretion from the HUVECs at different time points. A murine oxygen-induced retinopathy (OIR) model was used for the in vivo studies. Seven-day-old C57BL/6J pups (p7) were exposed to 75% oxygen for 5 days. On p12, the animals were returned to a normal atmosphere and were immediately injected intravitreously with 1.5 μL of a 5 mg/mL endostatin solution. At p18, the mice were perfused with fluorescein-dextran-FITC, and their retinas were flat mounted to measure the nonperfused area. Retinal VEGF and PEDF levels were also measured by ELISA Kits in the OIR mice at p18.
  
  
  RESULTS
  In vitro, endostatin inhibited HUVEC proliferation in a dose-dependent manner and also inhibited HUVEC proliferation in a VEGF-containing medium. Additionally, endostatin can inhibit migration, tube formation, and VEGF secretion in HUVECs, while also inducing apoptosis in HUVECs at several time points. These effects were statistically significant when compared to the control group (P<0.05). In vivo, a single intravitreous injection of endostatin reduced the retinal nonperfused area from 30% in the control group to 23% in the treatment group (P<0.0001). Intravitrous injection of endostatin reduced VEGF levels in retinas, while it increased PEDF levels.
  
  
  CONCLUSIONS
  Endostatin showed convincing inhibitory effects on angiogenesis both in vitro and in vivo. The inhibitory effects may be, at least partly, resulted from the restoration of the PEDF/VEGF ratio. These data suggest that endostatin could offer an innovative pharmaceutical strategy for the prevention of retinal neovascularization.
authors.affiliation: Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital , Beijing, PR China .
authors.forename: Yu-jing
authors.initials: YJ
authors.lastname: Bai
authors.forename: Lv-zhen
authors.initials: LZ
authors.lastname: Huang
authors.forename: Ai-yi
authors.initials: AY
authors.lastname: Zhou
authors.forename: Min
authors.initials: M
authors.lastname: Zhao
authors.forename: Wen-zhen
authors.initials: WZ
authors.lastname: Yu
authors.forename: Xiao-xin
authors.initials: XX
authors.lastname: Li
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D003911:Dextrans; D004195:Disease Models, Animal; D043169:Endostatins / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005452:Fluoresceins; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009414:Nerve Growth Factors / Q000378:metabolism; D010100:Oxygen / Q000633:toxicity*; D015861:Retinal Neovascularization / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D012178:Retinopathy of Prematurity / Q000188:drug therapy* / Q000209:etiology / Q000473:pathology; D015843:Serpins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D020533:Angiogenesis Inhibitors; D003911:Dextrans; D043169:Endostatins; D005136:Eye Proteins; D005452:Fluoresceins; D009414:Nerve Growth Factors; D010100:Oxygen; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor; C052526:fluorescein-dextran