[Suppression of corneal neovascularization by conditioned culture medium of human amniotic membrane]

To investigate the effects of the conditioned culture medium (CCM) of human amniotic membrane (AM) on corneal neovascularization (CNV) induced by basic fibroblast growth factor (bFGF) in mice.

AM (with epithelium side up) was cultured in EGM basic medium for 3 days, and the CCM was collected. The CCM consisted of three groups: 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 measured in Boyden chambers and by CyQUANT fluorescence binding assay, respectively. The levels of tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1, TIMP-2) in the CCM were determined by ELISA assay.

CNV induced by bFGF was significantly suppressed by the CCM of amniotic membrane. When CCM 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 groups, respectively. The migration and proliferation of HUVEC were strongly inhibited by the CCM of AM with epithelium, while the De-AM had no effect on the migration of HUVEC cells. A high level of TIMP-2 was found in AM group, but not in De-AM group. There were no differences in the amount of TIMP-1 in medium among these three groups.

CCM of amniotic membrane significantly suppresses the CNV induced by bFGF. One of the mechanisms of CCM-mediated suppression is that a high level of TIMP-2 protein is secreted or released into the CCM by AM, which can inhibit the migration and growth of vascular endothelial cells.