Stable Expression of Thioredoxins in RPE Cells Increases Cell Viabilities During Oxidative Stress

Abstract

Purpose:: Thioredoxins (TRXs) is an endogenous redox-active multifunctional protein with antioxidant. To investigate the crucial role of TRXs against oxidative stress, we transferred TRXs (TRX-1 or TRX-2) gene into RPE cells using an adeno-associated virus (AAV) vector and monitored cell viabilities during oxidative stress.

Methods:: The viral vector constructs (AAV-Trx1, or -2) were made by subcloning Trx-1 or -2 fused to a fluorescent protein into an adeno-associated virus vector cassette including a CAG promoter. The AAV-Trx vector particles were produced in 293T cells based on the helper free system. ARPE-19 cells infected with the AAV-TRX1 or -TRX2 were maintained with DMEM containing 10 % of fetal bovine serum (FBS). ARPE-Trxs cells plated onto 96 well culture dishes for the cell viability test. For the induction of oxidative stress, C2-ceramide or cysteine free medium was used. Cell viabilities were assessed using the cell Titer 96 aqueous non-radioactive cell proliferation assay. The mitochondrial membrane potentials were analyzed using the membrane-potential-sensitive dye, JC-1.

Results:: Cell viabilities of ARPE-19 cells were about 40 % and 70 % of survival induced by 40 µM of C2-ceramide and cysteine free (cys-free) medium, respectively. Mitochondrial depolarization was observed in each treatment. Increased cell viability was observed in ARPE-Trx1 cells exposed to oxidative stress by cys-free medium but not C2-ceramide. The membrane depolarization in ARPE cells was observed in each oxidative stress. The rate of membrane depolarization in ARPE-Trx1 cells exposed to cys-free medium was decreased. But those in C2-ceramide treatment were not changed.

Conclusions:: Trx-1 protects RPE cells from oxidative stress by inhibiting the loss of mitochondrial membrane potential.

Keywords: oxidation/oxidative or free radical damage • retinal pigment epithelium • antioxidants