Retina Vessel Clinic Course, Comprehensive Gene-expression Profile In OIR Murine And The Role Of Edn2 In HUVEC

PURPOSE. To observe the clinic course and determine a profile of gene expression in retinas at different postnatal day of wild-type murine and oxygen-induced retinapathy (OIR) murine. To investigate the role of Edn2 in human umbilical vein endothelial cells (HUVEC) proliferation.METHODS. Clinical course of twild-type and OIR (induced according to standard protocols) murine was evaluated on retinal Flat-mounts after fluorescein isothiocyanate-conjugated dextran perfusion at different time point. To identify altered expressive genes by hybridizing to Mouse Whole Genome (Phalanx Mouse Whole Genome OneArrayTM Microarray), containing 29,922 mouse genome probes, in five experimental groups: Group 1—P8 in OIR(the early response to hyperoxia) and wild-type(control group) C57BL/6J; Group 2—P12 in OIR (the late response to hyperoxia) and wild-type(control group) C57BL/6J; Group 3—P13 in OIR(the early response to relative hypoxia) and wild-type(control group) C57BL/6J; Group 4—P13 in OIR C57BL/6J and P9 in wild-type(control group) C57BL/6J(vessel branch spouts from superficial to deeper layer); Group 5—P9 and P30(control group) in wild-type C57BL/6J(immature and mature retina vessel). Regulated expression of selected genes was confirmed by real-time PCR (RT-PCR). To silence Edn2 gene and detect expression of Edn2,Mmp2,Mmp-9,Timp-2, Vegfa, Vtn by Migration Test, Western Blot, Real-time PCR, and Elisa test.RESULTS. In wile-type murine, the vessels entered the inner retina from the optic nerve head about P1. The superficial vessels continued to grow and remodeled in radial fashion, reaching the retinal periphery about P7. Branching spouts from the superficial vascular network grow into the deeper layer of the retina about P9, completing three vessel layers by approximately P16. In OIR murine, after 24 hours of hyperoxia, the central retina exhibited a central vasoconstriction. The degree of both central avascular area and central vasoconstriction were maximal from P8 to P12, Blood-vessel tortuosity was maximal on P15, and extraretinal neovascularisation was maximal from P17 to P19. Many genes associated with inflammation and angiogenesis were upregulated/downregulated on P8 in OIR (than normal) murine when central avascular area appeared, corresponding to the early response to hyperoxia; Many genes associated with inflammation were upregulated/downregulated on P12 in OIR (than normal) murine, corresponding to the the late response to hyperoxia; Many genes associated with inflammation and angiogenesis were upregulated/downregulated on P13 in OIR murine when the retinal angiogenesis and vasoconstriction began to appear (than P13 in normal murine), corresponding to the early response to relative hypoxia; Many genes associated with inflammation and angiogenesis were upregulated/downregulated on P13 in OIR than P9 normal murine when the retinal microvascular were budding from superficial to deep layer; and many genes associated with development were upregulated/downregulated on P9 than P30 normal murine when the retinal vessel were mature. Many of the same genes at different time point have different expressive change respectively in OIR and normal murine. And real-time PCR analyses showed a homology of gene-expression pattern. Edn2 is closely related with Vegfa, Timp2, Vtn.CONCLUSIONS. This work compared altered genes expression between vesseles regression and angiogeneses induced by oxygen, and serves as a comprehensive resource for the study of retinal neovascularization and identification of potential rational targets for antiangiogenic therapic. Edn2 play an important role in angiogenesis.