Differential effects of transforming growth factor-β2 on corneal endothelial cell proliferation - A role of serum factors

Ko Hua Chen, Wen-Ming Hsu, Shui Mei Lee

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Purpose: To determine the influence of serum on transforming growth factor (TGF)-β2 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 [3H]thymidine bioassay. Subconfluent cells were synchronized in the GO (quiescent) phase of the cell cycle by serum starvation for 24 hr. Serum and [3H]thymidine were then added to the cells in the presence or absence of a physiological concentration (5 ng ml-1) of exogenous active TGF-β2. 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-β2 with neutralizing antibody was used to test the cytokine specificity. Results: Without TGF-β2, a linear increase in [3H]thymidine incorporation, indicating S-phase, began approximately 16 hr after serum addition, then plateaued at approximately 24 hr. Serum promoted DNA synthesis of CE in a dose-dependent manner at concentrations of 0.5-10%. In cultures with 10% serum, TGF-β2 (0.5, 1, 5, and 20 ng ml-1) suppressed CE growth dose-dependently. The growth amplitude decreased and the time before S-phase entry, G1 phase, was prolonged to 24 hr. In culture with 1% serum, TGF-β2 (5 ng ml-1) suppressed the CE proliferation by delaying S-phase entry without suppressing growth amplitude. In cultures without serum, TGF-β2 promoted CE growth to a level similar to that of cultures supplemented with 0.5% serum. Conclusions: Responses of cultured CE to exogenous TGF-β2 depended on the concentration of serum in the medium. This result implies a possibility that in vivo serum influx through a compromised bloodocular barrier could influence the CE growth by changing the responses of these cells to TGF-β2 in aqueous humor.

Original languageEnglish
Pages (from-to)61-67
Number of pages7
JournalExperimental Eye Research
Volume75
Issue number1
DOIs
Publication statusPublished - Jan 1 2002
Externally publishedYes

Fingerprint

Transforming Growth Factors
Endothelial Cells
Cell Proliferation
Serum
S Phase
Thymidine
Growth
Starvation
Aqueous Humor
DNA
G1 Phase
Neutralizing Antibodies
Biological Assay
Radioactivity
Cultured Cells
Cell Cycle
Cell Culture Techniques

Keywords

  • Antiproliferative effects
  • Aqueous humor
  • Blood-ocular barrier
  • Cell culture
  • Corneal endothelial cells
  • Cytokine
  • Growth cycle
  • Serum effect
  • Transforming growth factor

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Differential effects of transforming growth factor-β2 on corneal endothelial cell proliferation - A role of serum factors. / Chen, Ko Hua; Hsu, Wen-Ming; Lee, Shui Mei.

In: Experimental Eye Research, Vol. 75, No. 1, 01.01.2002, p. 61-67.

Research output: Contribution to journalArticle

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abstract = "Purpose: To determine the influence of serum on transforming growth factor (TGF)-β2 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 [3H]thymidine bioassay. Subconfluent cells were synchronized in the GO (quiescent) phase of the cell cycle by serum starvation for 24 hr. Serum and [3H]thymidine were then added to the cells in the presence or absence of a physiological concentration (5 ng ml-1) of exogenous active TGF-β2. 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-β2 with neutralizing antibody was used to test the cytokine specificity. Results: Without TGF-β2, a linear increase in [3H]thymidine incorporation, indicating S-phase, began approximately 16 hr after serum addition, then plateaued at approximately 24 hr. Serum promoted DNA synthesis of CE in a dose-dependent manner at concentrations of 0.5-10{\%}. In cultures with 10{\%} serum, TGF-β2 (0.5, 1, 5, and 20 ng ml-1) suppressed CE growth dose-dependently. The growth amplitude decreased and the time before S-phase entry, G1 phase, was prolonged to 24 hr. In culture with 1{\%} serum, TGF-β2 (5 ng ml-1) suppressed the CE proliferation by delaying S-phase entry without suppressing growth amplitude. In cultures without serum, TGF-β2 promoted CE growth to a level similar to that of cultures supplemented with 0.5{\%} serum. Conclusions: Responses of cultured CE to exogenous TGF-β2 depended on the concentration of serum in the medium. This result implies a possibility that in vivo serum influx through a compromised bloodocular barrier could influence the CE growth by changing the responses of these cells to TGF-β2 in aqueous humor.",
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N2 - Purpose: To determine the influence of serum on transforming growth factor (TGF)-β2 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 [3H]thymidine bioassay. Subconfluent cells were synchronized in the GO (quiescent) phase of the cell cycle by serum starvation for 24 hr. Serum and [3H]thymidine were then added to the cells in the presence or absence of a physiological concentration (5 ng ml-1) of exogenous active TGF-β2. 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-β2 with neutralizing antibody was used to test the cytokine specificity. Results: Without TGF-β2, a linear increase in [3H]thymidine incorporation, indicating S-phase, began approximately 16 hr after serum addition, then plateaued at approximately 24 hr. Serum promoted DNA synthesis of CE in a dose-dependent manner at concentrations of 0.5-10%. In cultures with 10% serum, TGF-β2 (0.5, 1, 5, and 20 ng ml-1) suppressed CE growth dose-dependently. The growth amplitude decreased and the time before S-phase entry, G1 phase, was prolonged to 24 hr. In culture with 1% serum, TGF-β2 (5 ng ml-1) suppressed the CE proliferation by delaying S-phase entry without suppressing growth amplitude. In cultures without serum, TGF-β2 promoted CE growth to a level similar to that of cultures supplemented with 0.5% serum. Conclusions: Responses of cultured CE to exogenous TGF-β2 depended on the concentration of serum in the medium. This result implies a possibility that in vivo serum influx through a compromised bloodocular barrier could influence the CE growth by changing the responses of these cells to TGF-β2 in aqueous humor.

AB - Purpose: To determine the influence of serum on transforming growth factor (TGF)-β2 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 [3H]thymidine bioassay. Subconfluent cells were synchronized in the GO (quiescent) phase of the cell cycle by serum starvation for 24 hr. Serum and [3H]thymidine were then added to the cells in the presence or absence of a physiological concentration (5 ng ml-1) of exogenous active TGF-β2. 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-β2 with neutralizing antibody was used to test the cytokine specificity. Results: Without TGF-β2, a linear increase in [3H]thymidine incorporation, indicating S-phase, began approximately 16 hr after serum addition, then plateaued at approximately 24 hr. Serum promoted DNA synthesis of CE in a dose-dependent manner at concentrations of 0.5-10%. In cultures with 10% serum, TGF-β2 (0.5, 1, 5, and 20 ng ml-1) suppressed CE growth dose-dependently. The growth amplitude decreased and the time before S-phase entry, G1 phase, was prolonged to 24 hr. In culture with 1% serum, TGF-β2 (5 ng ml-1) suppressed the CE proliferation by delaying S-phase entry without suppressing growth amplitude. In cultures without serum, TGF-β2 promoted CE growth to a level similar to that of cultures supplemented with 0.5% serum. Conclusions: Responses of cultured CE to exogenous TGF-β2 depended on the concentration of serum in the medium. This result implies a possibility that in vivo serum influx through a compromised bloodocular barrier could influence the CE growth by changing the responses of these cells to TGF-β2 in aqueous humor.

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KW - Cell culture

KW - Corneal endothelial cells

KW - Cytokine

KW - Growth cycle

KW - Serum effect

KW - Transforming growth factor

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