The role of exogenous/endogenous basic fibroblast growth factor (FGF2) and transforming growth factor beta (TGF beta-1) on human corneal endothelial cells proliferation in vitro

Adult human corneal endothelial cells (HCEC) have extremely low turnover rates but undergo rapid division in vitro when stimulated with soluble growth factors. We have investigated the role played by FGF2 and TGF beta-1 in the regulation of HCEC growth stimulation. HCEC from donors who were over 30 years old were cultured and experiments performed on cultures between the 2nd and the 6th passage in the presence of 5% NCS. Cell counts revealed a maximal stimulation of 2.1x for FGF2 and 1.9x for TGF beta-1 compared to control cultures. When both factors were added, a synergistic effect was noticed with a maximal stimulation of the proliferation rate of 4.5x over controls. In addition, endogenous FGF2 produced by HCEC was quantitated in a sensitive EIA assay. After 5 days in culture, 10(6) cells contained 150 ng FGF2 and 35 ng was extracted from trypsin-digested ECM. Two molar NaCl washes of ECM released 15.6 ng FGF2, which induced a slight mitogenic activity (1.5x over control) in HCEC cultures, which was partially inhibited by an anti-FGF2 antibody. Northern blot analysis of HCEC extracts revealed the presence of FGF receptors R1 and R2 mRNA. The bioactive FGFRs were demonstrated by the toxic effect of a mitotoxin FGF2-SAP. These results suggest that FGF2 could participate in the autocrine regulation of HCEC proliferation and survival. The synergy between exogenously added FGF2 and TGF beta demonstrates that a combination of different growth factors may be important to stimulate proliferation of these cells in vivo.