Cyclosporine A-induced Senescence in Cultured Human Corneal Endothelial Cells

Abstract

Purpose : To investigate the effect of cyclosporine A (CsA) on senescence in human corneal endothelial cells (HCECs).

Methods : HCECs were cultured and treated with a various concentration of CsA (0-100µM). Senescence-associated β–galactosidase (SA-β-gal staining) staining was performed. Mitochondrial dehydrogenase activity was assessed using a WST-8 assay kit and mitochondrial membrane potential (ΔΨ_m) was measured using JC-1. Intracellular and mitochondrial reactive oxygen species formation was measured by DCF-DA and MitoSOX probe. Intracellular calcium levels were measured using Fluo-4, and mitochondrial calcium levels were measured using Rhod-2. Protein expression of glycogen synthase kinase 3 beta (GSK3β), pGSK3β, ERK 1/2 (extracellular-signal-regulated kinases 1/2) and pERK 1/2 was evaluated by western blotting.

Results : CsA increased Percentage of senescence associated (SA)-gal positive cells increased in a dose-dependent manner (p<0.05). CsA decreased mitochondrial dehydrogenase activity and ΔΨ_m in a dose-dependent manner (p<0.05). Intracellular and mitochondrial reactive oxygen species levels increased in treatment with CsA (p<0.05). CsA increased mitochondrial calcium levels at 100 µM (p<0.05) whereas intracellular calcium levels decreased at 100 µM. CsA attenuated the phosphorylation of GSK3β and ERK1/2.

Conclusions : CsA induced senescence in HCECs through induction of oxidative stress and via mitochodnrial/GSK3β/ERK1/2 dependent pathways.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.