Effects of hepatocyte growth factor, transforming growth factor-beta1 and epidermal growth factor on bovine corneal epithelial cells under epithelial-keratocyte interaction in reconstruction culture.
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-beta1 (TGF-beta1) 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-beta1- 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-beta1 induced significantly higher intakes of nuclear BrdU of the epithelial cells than HGF. In lamellar differentiation of the epithelial cells, TGF-beta1- 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-beta1-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-beta1 promotes proliferation and lamellar differentiation of corneal epithelial cells through keratocyte-mediated stimulation.
Nishimura
T
T
Department of Ophthalmology, Saga Medical School, Saga, Japan.
Toda
S
S
Mitsumoto
T
T
Oono
S
S
Sugihara
H
H
D000818:Animals; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000187:drug effects*; D016895:Culture Media, Serum-Free; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D007150:Immunohistochemistry; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D016895:Culture Media, Serum-Free; D016212:Transforming Growth Factor beta; D004815:Epidermal Growth Factor; D017228:Hepatocyte Growth Factor