Tranilast protects against endoplasmic reticulum stress-induced cell death in retinal pigment epithelial cells: Implications for age-related retinal diseases

Abstract

Purpose : Retinal pigment epithelium (RPE) plays an important role in maintaining outer blood-retinal barrier integrity. RPE function declines with age due to endoplasmic reticulum (ER) stress, which results in age-related macular degeneration (AMD). Tanilast, a specific NLRP3 inflammasome inhibitor, has been shown to possess anti-inflammatory potential that so far untested in RPE dysfunction under ER stress. Therefore, the objective of this study is to test the efficacy of tranilast on ER stress-induced RPE dysfunction and cell death.

Methods : ER stress was induced in a RPE cell line ARPE-19 in culture by adding tunicamycin (TM, 1µg/mL) in the presence or absence of 10 µM tranilast for 48 h. Mitochondrial function and membrane potential (Δψm) were measured by cellular ATP level and JC1 assay, respectively. Cell death was determined by measuring the release of lactate dehydrogenase (LDH) into media and by MTT assay. RPE barrier integrity was evaluated by transepithelial electrical resistance (TER) using an electric cell-substrate impedance sensing (ECIS) system and immunostaining of ZO-1, a tight junction protein. 2D gel protein electrophoresis was performed, then stained with SyproRuby^TM dye to determine differential protein profiles. Statistical analysis was performed using One-way ANOVA followed by Bonferroni post-hoc test. p<0.05 was considered statistically significant.

Results : TM treatment of ARPE-19 causes mitochondrial dysfunction (Δψm↓ and ATP↓) and cell death (LDH leakage and reduced MTT activity). These deleterious effects of TM on ARPE-19 are alleviated by treatment with tranilast. Furthermore, we show that TM-induced mitochondrial dysfunction and cell death are associated with a significant reduction in TER suggesting ARPE-19 barrier breakdown, which is prevented by tranilast. In addition, TM-treatment reduces ZO-1 immunostaining accompanied by changes in proteomic profile compared to controls without TM.

Conclusions : Our results show that ER-stress causes mitochondrial damage and RPE barrier dysregulation. The beneficial effects of tranilast on RPE function and cell viability under stress may represent a new interventional therapy to ameliorate early pathological pathways that occur in age-related retinal diseases and vision loss.

This is a 2021 ARVO Annual Meeting abstract.