Differential effects of transforming growth factor-beta2 on corneal endothelial cell proliferation-A role of serum...

To determine the influence of serum on transforming growth factor (TGF)-beta2 mediated effects on the proliferation of corneal endothelial cells (CE).

Rat CE were grown in explant culture and the proliferation of CE was measured by [(3)H]thymidine bioassay. Subconfluent cells were synchronized in the G0 (quiescent) phase of the cell cycle by serum starvation for 24hr. Serum and [(3)H]thymidine were then added to the cells in the presence or absence of a physiological concentration (5ngml(-1)) of exogenous active TGF-beta2. Radioactivity was measured at various time points to detect DNA synthesis. These experiments were repeated without adding serum after serum starvation. Preincubation of exogenous TGF-beta2 with neutralizing antibody was used to test the cytokine specificity.

Without TGF-beta2, a linear increase in [(3)H]thymidine incorporation, indicating S-phase, began approximately 16hr after serum addition, then plateaued at approximately 24hr. Serum promoted DNA synthesis of CE in a dose-dependent manner at concentrations of 0.5-10%. In cultures with 10% serum, TGF-beta2 (0.5, 1, 5, and 20ngml(-1)) suppressed CE growth dose-dependently. The growth amplitude decreased and the time before S-phase entry, G1 phase, was prolonged to 24hr. In culture with 1% serum, TGF-beta2 (5ngml(-1)) suppressed the CE proliferation by delaying S-phase entry without suppressing growth amplitude. In cultures without serum, TGF-beta2 promoted CE growth to a level similar to that of cultures supplemented with 0.5% serum.

Responses of cultured CE to exogenous TGF-beta2 depended on the concentration of serum in the medium. This result implies a possibility that in vivo serum influx through a compromised blood-ocular barrier could influence the CE growth by changing the responses of these cells to TGF-beta2 in aqueous humor.