title: Knockout of αA-crystallin inhibits ocular neovascularization.
abstract: OBJECTIVE
  The present study aimed to investigate the effects of αA-crystallin on pathologic ocular neovascularization.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study, and αA-crystallin-knockout (CRYAA[-/-]) mice were used for the in vivo study. αA-crystallin was knocked down in HUVECs by using a specific small interfering RNA (siRNA), and the effects of αA-crystallin knockdown on proliferation, migration, tube formation, and apoptosis were evaluated. Enzyme-linked immunosorbent assays were performed to investigate extracellular concentrations of vascular endothelial growth factor (VEGF). In vivo mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) were generated by using CRYAA(-/-) mice. The nonperfused area in the OIR model was measured in flat-mounted retinas, and angiogenesis resulting in CNV was evaluated in retinal sections. Western blot analysis was performed to investigate the phosphorylation status of vascular endothelial growth factor receptor 2 (VEGFR2, high affinity receptor for VEGF), AKT, PLCγ1, FAK, Src, p42/p44MAPK, p38MAPK, caspase-3, and caspase-9 in cultured HUVECs, as well as in the OIR and CNV models.
  
  
  RESULTS
  The CRYAA siRNA not only induced HUVEC apoptosis but also inhibited exogenous and endogenous VEGF-induced cell activities, including proliferation, migration, and tube formation. Pathologic neovascularization was attenuated in the CRYAA(-/-) murine OIR and CNV models. Both in vitro and in vivo, the inhibition of angiogenesis was mediated by the suppression of VEGF secretion and the inhibition of the VEGFR2 signaling pathway; VEGFR2, AKT, PLCγ1, FAK, Src, p42/p44 MAPK, and p38 MAPK all showed reduced phosphorylation levels. In addition, CRYAA knockout led to increased levels of cleaved caspase-9 and caspase-3.
  
  
  CONCLUSIONS
  Knockout of αA-crystallin inhibited pathologic neovascularization through the VEGF and VEGFR2 signaling pathways both in vitro and in vivo. These results suggest that αA-crystallin could be a novel pharmaceutical target for the prevention of ocular neovascularization.
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, China.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Xu
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, 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, China.
authors.forename: Lvzhen
authors.initials: L
authors.lastname: Huang
authors.affiliation: Parkway Health, Inc., Shanghai, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhou
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, 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, China.
authors.forename: Mingwei
authors.initials: M
authors.lastname: Zhao
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D018507:Gene Expression Regulation, Developmental; D061307:Human Umbilical Vein Endothelial Cells / Q000473:pathology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D011247:Pregnancy; D011270:Pregnancy, Animal; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015861:Retinal Neovascularization / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D038202:alpha-Crystallin A Chain / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D038202:alpha-Crystallin A Chain; D012313:RNA
keywords: choroidal neovascularization; human umbilical vein endothelial cell; neovascularization; oxygen-induced retinopathy; vascular endothelial growth factor; αA-crystallin