Differential effects of transforming growth factor-beta2 on corneal endothelial cell proliferation-A role of serum factors.
OBJECTIVE
To determine the influence of serum on transforming growth factor (TGF)-beta2 mediated effects on the proliferation of corneal endothelial cells (CE).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Chen
Ko Hua
KH
Department of Ophthalmology, Taipei Veterans General Hospital, National Yang-Ming University, #201, Shih-Pai Road, Section II, Taipei, 11217, Taiwan. khchen@vghtpe.gov.tw
Hsu
Wen Ming
WM
Lee
Shui Mei
SM
D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects / Q000502:physiology*; D003470:Culture Media; D004727:Endothelium / Q000187:drug effects / Q000502:physiology; D007546:Isoquinolines / Q000494:pharmacology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D016196:S Phase / Q000502:physiology; D044005:Tetrahydroisoquinolines; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology*
D003470:Culture Media; D007546:Isoquinolines; D044005:Tetrahydroisoquinolines; D016212:Transforming Growth Factor beta; C058571:soquinolol