Effect of Cyclosporine A-induced Senescence on Cultured Human Corneal Endothelial Cells
10.3341/jkos.2020.61.9.999
- Author:
Yoon Kyung JANG
1
;
Tae-Young CHUNG
;
Young Joo SHIN
Author Information
1. Department of Ophthalmology, Hallym University College of Medicine, Seoul, Korea
- Publication Type:Original Article
- From:Journal of the Korean Ophthalmological Society
2020;61(9):999-1009
- CountryRepublic of Korea
-
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.
