Role of hepatocyte growth factor in promoting the growth of human corneal endothelial cells stimulated by L-ascorbic acid 2-phosphate.
OBJECTIVE
To investigate the mechanisms by which L-ascorbic acid 2-phosphate (Asc-2P) increases the proliferation of human corneal endothelial cells (HCECs).


METHODS
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.


RESULTS
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).


CONCLUSIONS
Asc-2P increases the proliferation of cultured HCECs through upregulation of HGF production via an HGF/c-Met autocrine loop.
Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation, Kobe, Japan.
Miwa
M
Kimoto
Nobuyuki
N
Shima
Masahiro
M
Yamaguchi
Shiro
S
Amano
Satoru
S
Yamagami
D000970:Antineoplastic Agents / Q000494:pharmacology; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D012313:RNA / Q000235:genetics*; D046888:Tissue Array Analysis
D000970:Antineoplastic Agents; C011669:ascorbate-2-phosphate; D012313:RNA; D017228:Hepatocyte Growth Factor; D001205:Ascorbic Acid