J Korean Ophthalmol Soc.  2020 Sep;61(9):999-1009. 10.3341/jkos.2020.61.9.999.

Effect of Cyclosporine A-induced Senescence on Cultured Human Corneal Endothelial Cells

• Jang YK
• Chung TY
• Shin YJ

• KMID: 2506473
• DOI: http://doi.org/10.3341/jkos.2020.61.9.999

Abstract

Purpose
To investigate the in vitro effect of cyclosporine A (CsA)-induced senescence on human corneal endothelial cells (HCECs).
Methods
HCECs were cultured and incubated with 0-100 µM CsA. Senescence-associated β–galactosidase (SA-β-gal) staining was performed. Mitochondrial dehydrogenase activity was assessed using a WST-8 assay kit and mitochondrial membrane potential (∆Ψ m ) was measured using JC-1 dye. Intracellular and mitochondrial formation of reactive oxygen species (ROS) was measured with 2’,7’-dichlorodihydrofluorescein diacetate and MitoSOX probes. Intracellular and mitochondrial calcium levels were measured using Fluo-4 and Rhod-2, respectively. Protein expression was evaluated by Western blotting.
Results
CsA increased the percentage of SA-β-gal-positive cells (p = 0.003) and decreased mitochondrial dehydrogenase activity and ∆Ψ m in a dose-dependent manner (p = 0.029, pp = 0.004). Intracellular and mitochondrial ROS levels increased during incubation with CsA (p = 0.005). CsA at 100 µM increased mitochondrial calcium levels (p = 0.001), whereas intracellular calcium levels decreased at 100 µM CsA (p = 0.029). CsA activated GSK3β and ERK1/2 and reduced ZO-1 expression.
Conclusions
CsA induces senescence in HCECs through oxidative stress and via mitochondria-, GSK3β-, and ERK1/2-dependent pathways. Thus, concentrations of CsA should be monitored.

Keyword

Cyclosporine A; Human corneal endothelial cells; Mitochondrial membrane potential; Mitochondrial oxidative stress; Senescence