The Role And Possible Mechanism Of Angiopoietin In The Formation Of Choroidal Neovascularization

Backgroud Choroidal neovascularization(CNV) disease mainly concludes age-relatedmacular degeneration(AMD), pathologic myopia, and angioid streaks in the fundus and soon, which is one of the important cause of visual impairment. The mechanism of CNV isvery complex.It is regulated by the multiple cytokines and factors.The main effects ofstimulating the CNV formation is hypoxia and Bruch’s membrane damage.The integritydamage of retinal pigment epithelial(RPE)-Bruch membrane-choriocapillaris complexleads to imbalance of angiogenic factors and inhibiting factors.RPE cells secrete vascularendothelial growth factor(VEGF) and various kinds of cytokines activated by Bruch’smembrane rupture and choroidal ischemia, leading to CNV formation.Angiopoietins(Ang) play an important role in the maturity and stability of bloodvessels and regulation of vascular interity. Ang-1and Ang-2have the most close relationship with angiogenesis. Ang-1and Ang-2with Tie2receptor activated byPI3-K/Akt signaling pathways play a biological effect. Ang expression is closely related tothe degree of hypoxia in studies, which is significant for the process of CNV formation,but the specific mechanism is still unclear. Academics have confirmed that Ang andhypoxia inducible factor-1(HIF-1) have common signaling pathways, suggesting that itshould be regulated by the transcription of HIF-1.As a new material inhibiting angiogenesis,3（-5′-hydroxymethyl-2′-furyl）-1-benzylindazole (YC-1) inhibits the transcription of HIF-1.Objective To investigate the effect of the expression of angiopoietin-1(Ang-1) andangiopoietin-2(Ang-2) in CNV formation and in human RPE cells under hypoxiacondition.Methods (1) A total of52male black C57BL/6J mice were induced by the532nm laserphotocoagulation to establish a model of CNV.The mice were evaluated (n=2) in7days,14days and28days later by fundus fluorescein angiography. The mice wererandomly sacrificed at various time points, and then the CNV formation was examined byhistopathological slices. The expressions of Ang-1, Ang-2and HIF-1in the mice wereanalysed by immunofluorescence.(2) Human RPE cells were incubated in a hypoxiachamber. The expressions of Ang-1, Ang-2and hypoxia inducible factor-1(HIF-1) inRPE cells were analysed respectively after hypoxia for0,1,4,12and24hours. Accordingto treatment protocols: the hypoxia and HIF-1inhibitor3-（5′-hydroxymethyl-2′-furyl）-1-benzyl indazole (YC-1) groups after4hours of hypoxia, the expression of Ang-1,Ang-2and HIF-1in RPE cells treated by HIF-1inhibitor YC-1were investigated. Theconcentrations of YC-1were1,10and100μmol·L-1. The expression of Ang-1, Ang-2andHIF-1were analyzed by immunofluorescence and Western blot. All the results weredetermined by one-way analysis of variance.Results (1) The incidence of burns which showed fluorescein leakage was58.3%(42/72),62.5%(30/48) and46%(11/24) in7days,14days and28days later respectively.Histologic studies demonstrated that CNVwas formed in the laser lesions and the RPEcells proliferated in different extent during the study period,and could completely engulf the newly formed vessels in later period.The expressions of Ang-1, Ang-2and HIF-1exsited in normal mice by immunofluorescence. In4days after photocoagulation, Ang-2and HIF-1a immunostaining colocalized in photocoagulation, new blood vessels,peripheral retina and choroid injury area. In7days after photocoagulation, Ang-1andHIF-1a immunostaining colocalized in photocoagulation, new blood vessels, peripheralretina and choroid injury area.(2) Ang-2was mainly found in RPE cells under normoxiccondition and Ang-1and HIF-1were not absent. Ang-1immunostaining colocalizedwith Ang-2in the cytoplasm of HIF-1-positive RPE cells under hypoxia. The expressionof Ang-1and HIF-1both ascended at early stage of hypoxia,the peak-expression time ofHIF-1was at4hours after hypoxia(0.451±0.022,t=5.401, P0.05) and then dropped.The peak-expression of Ang-1protein occurred at24hours（1.342±0.059,t=20.100, P0.01）. However, the expression of Ang-2remained nearly unchangeable（1.542±0.129、1.621±0.131、1.574±0.146、1.624±0.027,t=0.044、0.673、0.244、0.700, all P0.05）.The expression of Ang-1protein（t=9.492、15.487、17.942, all P0.05）was inhibitedby YC-1at concentration of1μmol·L-1,10μmol·L-1or100μmol·L-1groups,following theinhibition of HIF-1（t=21.583、27.154、27.431, all P0.01）whereas the expression ofAng-2（t=0.859、0.178、1.565, all P0.05）almost stayed unchanged.Conclusions The murine CNV model could be induced by532nm laserphotocoagulation, and the incidence of CNV formation reached its peak in14days afterphotocoagulation. Ang-2and HIF-1a immunostaining colocalized in early stage of the CNVformation. Ang-1and HIF-1a immunostaining colocalized in CNV, mainly playing an importantrole in late stage of the CNV formation. On the basis, results in vitro suggest that HIF-1upregulates the expression of Ang-1in a time-and dose-dependent manner, and Ang-2remainsstable in hypoxic RPE cells. The expression of Ang-1is decreased by inhibiting the transcriptionof HIF-1. This may suggest that HIF-1selectively modulate Ang-1expression duringCNV.The expression of Ang-1and Ang-2may affect the formation of HIF-1related CNV,whichmay be partially derived from RPE cells.