A p-Tyr42 RhoA Inhibitor Promotes the Regeneration of Human Corneal Endothelial Cells by Ameliorating Cellular
Hyeon Jung Kim1,2, Jin Sun Hwang1,2, Kyung Bo Noh1,2
1Department of Ophthalmology, Hallym University Medical Center, Hallym University College of Medicine, Seoul 07442, Republic of Korea.
Abstract:
The development of treatment strategies for human corneal endothelial cells (hCECs) disease is necessary because hCECs do not regenerate in vivo due to the properties that are similar to senescence. This study is performed to investigate the role of a p-Tyr42 RhoA inhibitor (MH4, ELMED Inc., Chuncheon) in transforming growth factor-beta (TGF-β)- or H2O2-induced cellular senescence of hCECs. Cultured hCECs were treated with MH4. The cell shape, proliferation rate, and cell cycle phases were analyzed. Moreover, cell adhesion assays and immunofluorescence staining for F-actin, Ki-67, and E-cadherin were performed. Additionally, the cells were treated with TGF-β or H2O2 to induce senescence, and mitochondrial oxidative reactive oxygen species (ROS) levels, mitochondrial membrane potential, and NF-κB translocation were evaluated. LC3II/LC3I levels were determined using Western blotting to analyze autophagy. MH4 promotes hCEC proliferation, shifts the cell cycle, attenuates actin distribution, and increases E-cadherin expression. TGF-β and H2O2 induce senescence by increasing mitochondrial ROS levels and NF-κB translocation into the nucleus; however, this effect is attenuated by MH4. Moreover, TGF-β and H2O2 decrease the mitochondrial membrane potential and induce autophagy, while MH4 reverses these effects. In conclusion, MH4, a p-Tyr42 RhoA inhibitor, promotes the regeneration of hCECs and protects hCECs against TGF-β- and H2O2-induced senescence via the ROS/NF-κB/mitochondrial pathway.
Insights
A novel p-Tyr42 RhoA inhibitor, MH4, promotes human corneal endothelial cell (hCEC) regeneration. MH4 protects hCECs from senescence by targeting the ROS/NF-κB/mitochondrial pathway.
Area of Science:
- Ophthalmology
- Cell Biology
- Regenerative Medicine
Background:
- Human corneal endothelial cells (hCECs) lack in vivo regeneration capacity due to senescence-like properties.
- Developing effective treatments for hCEC diseases is crucial for maintaining corneal health.
Purpose of the Study:
- To investigate the therapeutic potential of a p-Tyr42 RhoA inhibitor, MH4, in preventing cellular senescence in hCECs.
- To elucidate the mechanisms by which MH4 affects hCEC proliferation, cell cycle, and response to senescence-inducing agents.
Main Methods:
- Treatment of cultured hCECs with MH4, transforming growth factor-beta (TGF-β), or hydrogen peroxide (H₂O₂).
- Analysis of cell proliferation, cell cycle, cell adhesion, and E-cadherin expression.
- Evaluation of mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential, NF-κB translocation, and autophagy markers (LC3II/LC3I).
Main Results:
- MH4 enhanced hCEC proliferation, altered cell cycle distribution, and improved cell adhesion and E-cadherin expression.
- MH4 treatment attenuated TGF-β- and H₂O₂-induced increases in mitochondrial ROS and NF-κB nuclear translocation.
- MH4 reversed the decrease in mitochondrial membrane potential and suppressed autophagy induced by TGF-β and H₂O₂.
Conclusions:
- MH4, a p-Tyr42 RhoA inhibitor, promotes hCEC regeneration and protects against senescence.
- The protective effects of MH4 involve modulating the ROS/NF-κB/mitochondrial pathway and autophagy.
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