Effects of Hepatocyte Growth Factor, Transforming Growth Factor-β1 and Epidermal Growth Factor on Bovine Corneal Epithelial Cells under Epithelial–Keratocyte Interaction in Reconstruction Culture

Abstract

In the cornea, corneal epithelial cells are in close contact with keratocytes: the epithelial cells organize thickened lamellar structure on a layer of keratocytes embedded in extracellular matrix (ECM). Thus, growth factors are expected to critically regulate corneal component cells under epithelial–keratocyte interaction. The purpose of this study is to clarify effects of hepatocyte growth factor (HGF), transforming growth factor-β1 (TGF-β1) or epidermal growth factor (EGF) on corneal epithelial cells under epithelial–keratocyte interaction. We examined proliferation and differentiation of the epithelial cells in a simple corneal reconstruction culture composed of an epithelial cell layer on the keratocyte-containing stromal layer, using three-dimensional collagen gel matrix culture. We observed the morphological change by phase contrast microscopy, and conducted histological and immunohistochemical examinations. The epithelial proliferation was examined by nuclear bromodeoxy-uridine (BrdU) uptake. In the reconstructed cornea under epithelial–keratocyte interaction, EGF-, TGF-β1- and HGF-treated cells formed a thickened epithelial layer consisting of 5–6, 5–6 and 3–4 cells, respectively. In fact, both EGF and TGF-β1 induced significantly higher intakes of nuclear BrdU of the epithelial cells than HGF. In lamellar differentiation of the epithelial cells, TGF-β1- or HGF-treated cells formed a triple lamellar structure specific for the in vivo corneal epithelium: basal, middle and superficial layers are composed of cuboidal basal-like cells, spindle wing-like cells and flat superficial-like cells, respectively. TGF-β1-treated cells formed a more markedly thickened epithelial layer than HGF-treated cells. In contrast, EGF formed a single lamellar structure consisting of cuboidal cells. These results suggest that those growth factors regulate proliferation and/or lamellar differentiation of corneal epithelial cells under epithelial–keratocyte interaction. The most interesting result was that TGF-β1 promotes proliferation and lamellar differentiation of corneal epithelial cells through keratocyte-mediated stimulation.