The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells
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Published on: August 20, 2016
Akane Hongo1, Naoki Okumura1, Makiko Nakahara1
1Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Purpose:
We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs).
Methods:
HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-β-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA.
Results:
Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-β-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580.
Conclusions:
Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.
Insights
A p38 MAPK inhibitor prevented cellular senescence in cultivated human corneal endothelial cells (HCECs). This finding supports using p38 MAPK inhibitors to generate sufficient HCECs for corneal tissue engineering therapies.
Area of Science:
- Ophthalmology
- Cell Biology
- Tissue Engineering
Background:
- Corneal endothelial dysfunction is a significant cause of vision impairment.
- Cell-based therapies offer a promising alternative for treating corneal diseases.
- Cultivating sufficient human corneal endothelial cells (HCECs) for therapy remains a challenge.
Purpose of the Study:
- To investigate the efficacy of a p38 MAPK inhibitor in preventing cellular senescence in HCECs.
- To determine if inhibiting p38 MAPK signaling can improve HCEC yield for tissue engineering.
Main Methods:
- Human corneal endothelial cells (HCECs) were cultured with or without SB203580, a p38 MAPK inhibitor.
- Cell density, morphology, and expression of key proteins (ZO-1, N-cadherin, Na+/K+-ATPase) were assessed.
- Cellular senescence was evaluated using SA-β-gal staining and Western blotting for p16 and p21.
Main Results:
- HCECs cultured with SB203580 exhibited significantly higher cell density and maintained hexagonal morphology.
- SB203580-treated HCECs preserved normal expression patterns of ZO-1, N-cadherin, and Na+/K+-ATPase.
- Inhibition of p38 MAPK reduced SA-β-gal staining and suppressed the expression of senescence markers p16 and p21.
Conclusions:
- p38 MAPK signaling activation contributes to culture-induced senescence in HCECs.
- p38 MAPK inhibitors can counteract HCEC senescence, promoting cell proliferation.
- This strategy holds potential for generating adequate HCECs for corneal tissue engineering therapies.
