Role of endostatin in the in vitro anti–angiogenic activities expressed by human limbal epithelial cells cultivated on amniotic membrane

Abstract

Abstract: : Purpose: Previously, we have shown that the in vitro anti–angiogenic activities of human limbal epithelial cells (HLE) were enhanced when HLE were cultivated on amniotic membrane (AM), and were most prominent when HLE cultivated on denuded AM were previously cocultured with 3T3 fibroblasts (HLE/DAM/3T3). The purpose of this study is therefore to identify factors responsible for such activities. Methods: HLE were grown from limbal explant on cell culture insert, on insert overlaid with intact or denuded AM, and on denuded AM cocultured with 3T3 fibroblasts without air–lifting. The cells were kept with SHEM with 5% FBS until 80 to 90% confluence, then serum–and EGF–free culture supernatant was collected and used for ELISA for tissue inhibitor of metalloproteinase–1 (TIMP–1), thrombospondin–1 (TSP–1), pigment epithelium–derived factor (PEDF), and endostatin (proteolytic fragment of collagen XVIII). Neutralizing antibody was used to block the inhibitory effect on the proliferation and migration of human umbilical vein endothelial cells (EC) by culture supernatant of HLE/DAM/3T3, and EC differentiation (tube formation) by coculture with HLE/DAM/3T3. Results: Quantitation of TIMP–1, TSP–1, PEDF, and endostatin revealed that only the level of endostatin showed a paralleled increased expression by HLE cultivated on AM. The increase was most significant when HLE cultivated on denuded AM were previously cocultured with 3T3 fibroblasts. After correcting the measured values on a per cellular protein basis, there was still a significant increase compared to HLE cultured on insert only (250%, p = 0.007). Endostatin content in the AM kept at 37 °C incubator for 3 weeks were negligible. Neutralizing antibody to endostatin substantially abrogated the inhibitory effect on EC proliferation and migration , but was less effective on EC differentiation. Conclusions: The anti–angiogenic effect of HLE was enhanced when HLE were cultivated on AM and cocultured with 3T3 fibroblasts, and endostatin–related anti–angiogenic factor may play a major role. This finding justifies the clinical application of combined AM and limbal transplantation or the transplantation of cultivated limbal epithelial cells on AM, especially in corneas affected by marked inflammation and neovascularization.

Keywords: cornea: epithelium • neovascularization • cornea: basic science