Investigation On The Effect Of AOC2 On Choroidal Neovasularizaton In Vitro And In Vivo

Part ? the effect of AOC2 in angiogenesis by overexpression of AOC2 in HREC Objective To investigate the role of amine oxidase-copper containing 2,?AOC2?in angiogenesis by over expressing AOC2 in human retinal endothelial cell lines?HRECs?.Methods The AOC2 mRNA level of HRECs were determined by quantitative PCR?qPCR?in normal,hypoxia and inflammatic conditons mimicked by cobalt chloride?CoCl₂?and lipopolysaccharide?LPS?respectively.The over expression of AOC2 in HRECs was established by infection with lenti-virus loaded human AOC2 cDNA?OE group?and the lenti-virus contain EGFP was infected to HRECs as negative control?NC group?,uninfected HRECs as control group.The proliferation of each groups were assessed by cell counting kit-8?CCK-8?assay.The migration of each group was detected by scrathing wound healing assay and modified Boyden Chamber migration assay?Transwell?.Tube formation was evaluated by HRECs from NC and OE group cultured in three dimension?3D?Matrigel.The mRNA level of angio-related regulator moleiculars such as vascular endothelial growth factor?VEGF?-A,PDGF-B,MMP-2,MMP-9,TSP-1 and TIMP-1 were detected by using qPCR.The level of phosphorylated FAK and ERK1/2 were evaluated by western boltting?WB?.Finally,the level of F-actin polymization were observed by staining with Alexa Fluor 568 phalloidin.Results The expression of AOC2 within control HRECs was detectable but in a low abundance,expression of AOC2 in control HRECs was upregulated when treated with CoCl₂ and LPS for 12 hours?h?.The fold change of AOC2 is 4.19?P<0.01?when treated with 300mM CoCl₂,treated with 2.5mg/mL LPS,the chang was 4.02 fold?P<0.01?.The proliferation of OE group was increased when compared with NC group after cultured for 48 h.OD_OE,1.90±0.19 vs OD_NC,1.27±0.08?P<0.01?.Both of migration as well as tube formation of HRECs were enhanced in OE group.Migrated distance of OE is 517.50±29.90mm,while the distance of NC is 453.30±15.25mm?P<0.05?,the numbers of migrated cells in Transwell were 84.50±7.94 within OE group and 62.67±4.95 with NC group?P<0.05?.The tube length of OE group at 6 h is 27.46±2.56 mm,while length of NC group is 23.46±0.94mm?P<0.05?,the tube branch points were 137±10.12 in OE group and 112±4.33 in NC group?P<0.05?.The overexpression of AOC2 could upregulated VEGF-A,MMP-2 and MMP-9 mRNA level while downregulated expression of TSP-1 and TIMP-1.The fold chang of VEGF-A in OE group compaired with NC group is+3.24?P<0.05?,fold change of MMP-2 is+3.13?P<0.05?,chang of MMP-9 is+1.99?P<0.05?,change of TSP-1 is-5.75?P<0.01?and fold of TIMP-1 is-3.25?P<0.01?.In western blotting,the level of phosphorylated FAK was up-scaled in OE group,the level of pFAK is upregulated with+1.88 fold compared with NC group?P<0.01?.Finally,the level of F-actin polymerization was enhanced in OE group.Conclution AOC2 promote angiogenesis in vitro by enhanced the proliferation,migration and tube formation of HRECs,the up-regulation of pro-angiogenic cytokines and inhibition of anti-angiogenesis factors may invove in these positive effects,the activated FAK pathway and F-actin polymization may result in enhanced mobility of HRECs which AOC2 overexpressed.Part ? The role of AOC2 in experimental choroidal neovascularizationObjective To investigate the role of AOC2 in the development of experimental choroidal neovascularization?CNV?with AOC2 knock out?KO?mice.Methods Wild type C57BL/6 mice?WT?were prepared for laser-induced CNV.The expression of AOC2 was assessed in the early stage of CNV at day 2,day 4 and day 7.AOC2-deficient?AOC2-/-?mice were generated from C57BL/6 mice.The genotype of AOC2-/-mice were confirmed by a T7 EI mutation detection kit.The exudation of CNV was asscessed by FFA at day 7,leakage of CNV was rated from grade 0 to grade 3.After mice perfused with FITC-dextran at day 14,the CNV area was detected by using choroidal flat-mounts,the area of CNV was recorded under confocal microscope.The exudative grade and area of new vessels were compared between KO and WT mice.Total RNA was extracted from retinal pigment epithelium-choroid-sclera compex.The expression of angio-related cytokines was investigated incluing VEGF-A,PDGF-B,TSP-1 and ADAMTS-1 via using quantitative PCR.Results The CNV induced with laser was detectable from day 7 to day 28,the development of CNV was reach maxium level at day 14.The expression of AOC2 was detectable at the early stage of CNV?from day 2 to day 7?,the m RNA level of AOC2 was increased at day 4 and declined at day 7,compare with day 2 the expression of AOC2 is upscaled 4.8 fold?P<0.001?.Both of KO and WT mice were formed CNV after laser induction.As to leakage grade and area of CNV,there was no significant difference between KO and WT groups,although KO mice showed a slight decrease.The m RNA level of VEGF-A was downregulated?VEGF-A downscaled 1.42,P<0.05?while anti-angiogenic cytokine TSP-1 was upregulated?TSP-1 upscaled 1.31 fold P<0.05?.Conclusion AOC2 was detected in the formation of CNV in murine model,the level of AOC2 was upregulated at day 4 in experimental CNV.Whith AOC2 knocked out,the expression of VEGF-A was downregulated and TSP-1 was upregulated,but the CNV in KO mice was not inhibited effectively.