title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta.
abstract: OBJECTIVE
  Choroidal neovascularization (CNV) is a major cause of vision loss in retinal diseases such as age-related macular degeneration (AMD). Previously, we demonstrated that semaphorin3A (Sema3A), which is a chemorepellent guidance molecule, inhibited the formation of retina neovascularization. In the present study, we investigated the antiangiogenic effects of Sema3A on transforming growth factor beta (TGF-β) in vitro and in vivo.
  
  
  METHODS
  Enzyme-linked immunosorbent assays (ELISAs) were used to measure the TGF-β levels in the vitreous humor of patients with AMD and controls. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study, and a laser-induced CNV mouse model was prepared for the in vivo study. The HUVECs were incubated with TGF-β and Sema3A. The proliferation, migration, apoptosis, and tube formation of the cells were then measured using BrdU, Transwell, flow cytometry, and Matrigel assays, respectively, and the SMAD2/3 signaling pathways were analyzed using western blot analysis. The C57BL/6J mouse retina was exposed to a laser to induce choroidal neovascularization (CNV), and Sema3A was injected intravitreously. After 14 days, fundus fluorescein angiography was performed to evaluate the leakage area of the CNV. The vascular endothelial growth factor (VEGF) and TGF-β concentrations in the retina-choroid complex were measured with ELISA. Components of the p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), and SMAD2/3 signaling pathways in the Sema3A-treated groups were analyzed using western blotting.
  
  
  RESULTS
  In this study, we first verified that the vitreous TGF-β level was higher in patients with neovascular AMD than in the controls. We also showed that Sema3A inhibited TGF-β-induced HUVEC proliferation, migration, and tube formation and inhibited the downstream SMAD2/3 signaling pathway. Sema3A also induced TGF-β-stimulated HUVEC apoptosis and inhibited the response of TGF-β in vitro. In vivo, the TGF-β level was increased in the CNV mouse model. Sema3A not only inhibited laser-induced CNV formation but also inhibited the uptake of VEGF and TGF-β. In the western blot analysis, Sema3A was shown to inhibit the phosphorylation of p38 MAPK, ERK1/2, and JNK and to inhibit the SMAD2/3 signaling pathway after Sema3A treatment in CNV mice.
  
  
  CONCLUSIONS
  Sema3A can be applied as a useful, adjunctive therapeutic strategy for preventing CNV formation.
authors.affiliation: Key Laboratory of Vision Loss and Restoration, Ministry of Education; Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases; Department of Ophthalmology, Peking University People's Hospital, Beijing, 100044, China.
authors.forename: Yujing
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Key Laboratory of Vision Loss and Restoration, Ministry of Education; Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases; Department of Ophthalmology, Peking University People's Hospital, Beijing, 100044, China.
authors.forename: Shuting
authors.initials: S
authors.lastname: Liang
authors.affiliation: Key Laboratory of Vision Loss and Restoration, Ministry of Education; Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases; Department of Ophthalmology, Peking University People's Hospital, Beijing, 100044, China.
authors.forename: Wenzhen
authors.initials: W
authors.lastname: Yu
authors.affiliation: Key Laboratory of Vision Loss and Restoration, Ministry of Education; Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases; Department of Ophthalmology, Peking University People's Hospital, Beijing, 100044, China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Vision Loss and Restoration, Ministry of Education; Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases; Department of Ophthalmology, Peking University People's Hospital, Beijing, 100044, China.
authors.forename: Lvzhen
authors.initials: L
authors.lastname: Huang
authors.affiliation: Key Laboratory of Vision Loss and Restoration, Ministry of Education; Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases; Department of Ophthalmology, Peking University People's Hospital, Beijing, 100044, China.
authors.forename: Mingwei
authors.initials: M
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Vision Loss and Restoration, Ministry of Education; Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases; Department of Ophthalmology, Peking University People's Hospital, Beijing, 100044, China.
authors.forename: Xiaoxin
authors.initials: X
authors.lastname: Li
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016022:Case-Control Studies; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002829:Choroid / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D020256:Choroidal Neovascularization / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D005786:Gene Expression Regulation / Q000187:drug effects*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D058449:Intravitreal Injections; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D040121:Semaphorin-3A / Q000378:metabolism / Q000494:pharmacology*; D015398:Signal Transduction; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D014822:Vitreous Body / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D040121:Semaphorin-3A; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4