Short-Term In Vitro ROS Detection and Oxidative Stress Regulators in Epiretinal Membranes and Vitreous from Idiopathic Vitreoretinal Diseases

Abstract

Background. A plethora of inflammatory, angiogenic, and tissue remodeling factors has been reported in idiopathic epiretinal membranes (ERMs). Herein we focused on the expression of a few mediators (oxidative, inflammatory, and angiogenic/vascular factors) by means of short-term vitreal cell cultures and biomolecular analysis. Methods. Thirty-nine (39) ERMs and vitreal samples were collected at the time of vitreoretinal surgery and biomolecular analyses were performed in clear vitreous, vitreal cell pellets, and ERMs. ROS products and iNOS were investigated in adherent vitreal cells and/or ERMs, and iNOS, VEGF, Ang-2, IFNγ, IL18, and IL22 were quantified in vitreous (ELISA/Ella, IF/WB); transcripts specific for iNOS, p65NFkB, KEAP1, NRF2, and NOX1/NOX4 were detected in ERMs (PCR). Biomolecular changes were analyzed and correlated with disease severity. Results. The higher ROS production was observed in vitreal cells at stage 4, and iNOS was found in ERMs and increased in the vitreous as early as at stage 3. Both iNOS and NOX4 were upregulated at all stages, while p65NFkB was increased at stage 3. iNOS and NOX1 were positively and inversely related with p65NFkB. While NOX4 transcripts were always upregulated, NRF2 was upregulated at stage 3 and inverted at stage 4. No significant changes occurred in the release of angiogenic (VEGF, Ang-2) and proinflammatory (IL18, IL22 and IFNγ) mediators between all stages investigated. Conclusions. ROS production was strictly associated with iNOS and NOX4 overexpression and increased depending on ERM stadiation. The higher iNOS expression occurred as early as stage 3, with respect to p65NFkB and NRF2. These last mediators might have potential prognostic values in ERMs as representative of an underneath retinal damage.