In vitro antiangiogenic activity in ex vivo expanded human limbocorneal epithelial cells cultivated on human amniotic membrane

PURPOSE. To compare the in vitro antiangiogenic activities of ex vivo expanded human limbocorneal epithelial (HLE) cells cultivated on preserved human amniotic membrane (AM) and to identify factors responsible for the activities. METHODS. The antiangiogenic effects were compared of culture media conditioned by AM, HLE cells, or HLE cells cultivated on intact AM (HLE/IAM), on denuded AM (HLE/DAM), or on DAM cocultured with 3T3 fibroblasts (HLE/DAM/3T3). A monolayer culture of human umbilical vein endothelial cells (ECs) was used in a proliferation and migration assay. ECs suspended in type I collagen gel were used to assess capillary tube formation. Quantitative analyses of tissue inhibitor of metalloproteinase (TIMP)-1, thrombospondin (TSP)-1, pigment epithelium-derived factor (PEDF), and endostatin (proteolytic fragment of collagen XVIII) were performed by ELISA. Immunoconfocal microscopy was performed to localize the site of endostatin expression in HLE cells and AM. RESULTS. HLE cell- but not AM-conditioned medium (CM) inhibited the proliferation and migration of ECs, and coculture of HLE cells, but not of AM, with ECs inhibited capillary tube formation. Although some data from HLE cells alone are not significantly different from the control, increased inhibitory activity was expressed by HLE/IAM and HLE/DAM and was most significantly expressed by HLE/DAM/3T3. Quantitation of TIMP-1, TSP-1, PEDF, and endostatin revealed that only the level of endostatin showed an increased expression by HLE cells cultivated on AM. Neutralizing antibody to endostatin substantially abrogated the inhibitory effect on EC proliferation and migration, but was less effective on EC differentiation, Endostatin signal was more prominent in the basement membrane zone of HLE cells cultivated on denuded AM than in those cultivated on intact AM. CONCLUSIONS. The antiangiogenic effect of HLE cells was enhanced when they were cultivated on AM and cocultured with 3T3 fibroblasts, and endostatin-related antiangiogenic factor may play a major role. This highlights the significance of cell-matrix and cell-cell interaction in the regulation of antiangiogenic factor secretion by HLE cells.