Role of hepatocyte growth factor in promoting the growth of human corneal endothelial cells stimulated by L-ascorbic acid 2-phosphate

• Published in: Investigative ophthalmology & visual science Vol. 53; no. 12; pp. 7583 - 7589
• Main Authors: Kimoto, Miwa, Shima, Nobuyuki, Yamaguchi, Masahiro, Amano, Shiro, Yamagami, Satoru
• Format: Journal Article
• Language: English
• Published: United States 09.11.2012
• Subjects: Antineoplastic Agents - pharmacology; Ascorbic Acid - analogs & derivatives; Ascorbic Acid - pharmacology; Blotting, Western; Cell Proliferation - drug effects; Cells, Cultured; Endothelium, Corneal - drug effects; Endothelium, Corneal - growth & development; Endothelium, Corneal - metabolism; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Developmental - drug effects; Hepatocyte Growth Factor - biosynthesis; Hepatocyte Growth Factor - genetics; Humans; RNA - genetics; Tissue Array Analysis
• ISSN: 1552-5783; 1552-5783
• DOI: 10.1167/iovs.12-10146

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.