Corneal Neovascularization Suppressed by TIMP2 Released from Human Amniotic Membranes

摘要

Purpose: To investigate the effects of culture medium of human amniotic membrane (AM) on corneal neovascularization (CNV) induced by basic fibroblast growth factor (bFGF) in mice. Methods: Culture medium of amniotic membrane was prepared by cultivating AM (with epithelium side up) in EGM basic medium for 3 days, and was collected separately to three groups, e.g. control (EGM only), AM with epithelium (AM) and AM without epithelium (De-AM). Corneal neovascularization was induced in mice by using micropocket assay with Hydron polymer pellets containing 100 ng bFGF. Migration and proliferation of human umbilical cord vein endothelial cells (HUVEC) were performed in Boyden chambers and by using the CyQUANT fluorescence binding assay respectively. The levels of tissue inhibitors of metalloproteinase 1 and 2 (TIMP1, TIMP2) in culture medium were determined by ELISA assay. Results: CNV induced by bFGF was significantly suppressed by culture medium of amniotic membrane. When the medium was applied as an eyedrop 4 times a day for 7 days, the area of CNV was (2.48±0.76) mm~2, (0.64±0.52) mm~2 and (1.96±0.65) mm~2 in control, AM and De-AM group respectively. The migration and proliferation of HUVEC were strongly inhibited by culture medium of AM with epithelium, while the De-AM had no effect on the migration of HUVEC cells. The high level of TIMP2 was found in AM group, but not in De-AM group, while there was no difference in the amount of TIMP1 in medium among three groups. Conclusion: Culture medium of amniotic membrane significantly suppresses the corneal nevovascularization induced by bFGF. The mechanism of which at least in part is that high level of TIMP2 protein secreted or released into the culture medium of AM and inhibition of migration and growth of vascular endothelial cells.