Effects of plasma kallikrein inhibitors in an in vitro RPE oxidative stress model

Purpose Plasma Kallikrein (PK) Inhibitors (PKI) are a promising treatment for retinal diseases such as diabetic macular edema, where PK activity is implicated. Our aim was to set up an in vitro model of chronic oxidative stress in the retinal pigment epithelium cell line ARPE19 and use it to test the effect of 8 PKI on cell viability and proliferation. Methods ARPE19 cells were grown to a sub-confluence stage, synchronized, and exposed to different dilutions of glucose oxidase (GOx) from Aspergillus niger, culture medium (− control) and benzalkonium chloride (+ control) for 24 hr. Cell viability and apoptosis were analyzed (MTT assay and caspase-3/7 detection, respectively). The oxidative stressed cells were exposed to 8 different PKI developed by KalVista (Salisbury, UK) at 100 nm and 1 μm. Also, ARPE19 cells were exposed to PKI previously and simultaneously to GOx and cell viability and proliferation were assessed (alamarBlue® assays). Results Exposure of ARPE19 to GOx induced dose-dependent decreased cell viability and increased cell apoptosis. A sub-lethal GOx dose reducing viability by 30% and increasing apoptosis by 59% was chosen for PKI exposure experiments. Exposure of GOx stressed cells to PKI for 24 hr did not further reduce cell viability after 5 days. No effect on cell proliferation was observed when GOx stressed cells were either pre-treated or simultaneously exposed to PKI. Conclusions GOx oxidative sub-lethal stress model in ARPE19 cells may be used for screening new drugs with therapeutic potential for retinal diseases. The PKI did not reduce GOx-induced cytotoxic effects in RPE cells suggesting plasma kallikrein has no role in this stress mechanism. Support: FP7-PEOPLE-2013-IAPP (612218/3D-NET) and Regional Junta de Castilla y Leon Scholarship/European Social Fund Program (Va040-13).