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Ko-Hua Chen, Deshea L. Harris, Nancy C. Joyce; TGF-β2 in Aqueous Humor Suppresses S-Phase Entry in Cultured Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 1999;40(11):2513-2519.
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purpose. Corneal endothelium in vivo is arrested in G1, the phase of the cell
cycle that prepares cells for DNA synthesis. In many cell types,
transforming growth factor (TGF)-β inhibits proliferation by inducing
G1-phase arrest. Evidence indicates that corneal endothelial cells
synthesize mRNA for TGF-β1 and are also bathed in aqueous humor that
contains TGF-β2 (mainly in a latent form). As such, this cytokine may
maintain the corneal endothelium in a G1-phase–arrested state in vivo.
The purpose of these studies was to determine the effect of exogenous
TGF-β2 and TGF-β2 in aqueous humor on DNA synthesis in cultured
corneal endothelial cells.
methods. Rat corneal endothelial cells were grown in explant culture and
identified by morphology and reverse transcription–polymerase chain
reaction using primers for specific corneal cell markers. Subconfluent
cells were synchronized in the G0 phase (quiescence) by serum
starvation for 24 hours. Serum was then added to the cells in the
presence or absence of exogenous TGF-β2 or activated rat aqueous
humor. [3H]Thymidine was added, and radioactivity was
measured at various time points to detect DNA synthesis. Preincubation
of exogenous TGF-β2 or activated rat aqueous humor with neutralizing
antibody was used to test for cytokine specificity.
results. A linear increase in [3H]thymidine incorporation began
approximately 16 hours after serum addition, and peak incorporation
occurred at approximately 24 hours. Exposure of cells to serum plus
TGF-β2 suppressed [3H]thymidine incorporation in a
dose-dependent manner at concentrations ranging from 5 pg/ml to 5
ng/ml. [3H]Thymidine incorporation was also suppressed in
cells exposed to serum plus rat aqueous humor diluted 1:10.
Neutralizing antibody reversed the effects of both exogenous TGF-β2
and aqueous humor.
conclusions. Exogenous TGF-β2 and TGF-β2 in aqueous humor suppress S-phase entry
of rat corneal endothelial cells. These results suggest that this
cytokine in aqueous humor could help maintain the corneal endothelium
in a G1-phase–arrested state in vivo.
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