Role of Hepatocyte Growth Factor in Promoting the Growth of Human Corneal Endothelial Cells Stimulated byl-Ascorbic Acid 2-Phosphate

Abstract

To investigate the mechanisms by which L-ascorbic acid 2-phosphate (Asc-2P) increases the proliferation of human corneal endothelial cells (HCECs). Growth of cultured HCECs was examined in the presence of various antioxidants, including Asc-2P, retinyl acetate (vitamin A), reduced glutathione, oxidized glutathione, carnosine, and sodium alpha-tocopherol phosphate (a water-soluble vitamin E derivative). Synthesis of type I, III, and IV collagen by HCECs cultured with or without Asc-2P was evaluated by measuring cell lysates and conditioned medium with Western blotting, immunocytochemistry, or enzyme-linked immunosorbent assay (ELISA). The gene expression profiles of HCECs cultured with or without Asc-2P were compared by microarray analysis to determine critical proliferative factors, and the proliferative response of these cells to selected factors was tested. Among the antioxidants tested, only Asc-2P promoted the growth of HCECs. Asc-2P did not promote deposition of type I, III, or IV collagen. Microarray analysis revealed that several cytokines were potently upregulated by Asc-2P, but among them, only hepatocyte growth factor (HGF) stimulated HCEC growth. ELISA revealed the upregulation of HGF protein production by Asc-2P, while the stimulatory effect of Asc-2P was abolished by an anti-HGF neutralizing antibody or PHA-665752 (a specific inhibitor of the HGF receptor, c-Met). Asc-2P increases the proliferation of cultured HCECs through upregulation of HGF production via an HGF/c-Met autocrine loop.