Relative Contribution of Different Mitochondrial Oxidative Phosphorylation Components to the Retinal Pigment Epithelium Barrier Function: Implications for RPE-Related Retinal Diseases

Abstract

Disruption of retinal pigment epithelial (RPE) barrier integrity is involved in the pathology of several blinding retinal diseases including age-related macular degeneration (AMD) and diabetic retinopathy (DR), but the underlying causes and pathophysiology are not completely well-defined. Mitochondria dysfunction has often been considered as a potential candidate implicated in such a process. In this study, we aimed to dissect the role of different mitochondrial components; specifically, those of oxidative phosphorylation (OxPhos), in maintaining the barrier functionality of RPE. Electric cell-substrate impedance sensing (ECIS) technology was used to collect multi-frequency electrical impedance data to assess in real-time the barrier formation of the RPE cells. For this purpose, the human retinal pigment epithelial cell line—ARPE-19—was used and treated with varying concentrations of specific mitochondrial inhibitors that target different steps in OxPhos: Rotenone for complex I (the largest protein complex in the electron transport chain (ETC)); oligomycin for ATP synthase; and carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) for uncoupling ATP synthesis from the accompanying ETC. Furthermore, data were modeled using the ECIS-Zθ software to investigate in depth the effects of these inhibitors on three separate barrier parameters: cell–cell interactions (Rb), cell–matrix interactions (α), and the cell membrane capacitance (Cm). The viability of ARPE-19 cells was determined by lactate dehydrogenase (LDH) Cytotoxicity Assay. The ECIS program’s modeling demonstrated that FCCP and thus OxPhos uncoupling disrupt the barrier function in the ARPE-19 cells across all three components of the total resistance (Rb, α, and Cm) in a dose-dependent manner. On the other hand, oligomycin and thus ATP synthase inhibition mostly affects the ARPE-19 cells' attachment to their substrate evident by a significant decrease in α resistance in a dose-dependent manner, both at the end and throughout the duration of the experiment. On the contrary, rotenone and complex I inhibition mostly affect the ARPE-19 paracellular resistance Rb in a dose-dependent manner compared to basolateral resistance α or Cm. Our results clearly demonstrate differential roles for different mitochondrial components in maintaining RPE cell functionality in which uncoupling of OxPhos is a major contributing factor to the disruption barrier function. Such differences can be used in investigating gene expression as well as for screening of selective agents that improve the OxPhos coupling efficiency to be used in the therapeutic approach for treating RPE-related retinal diseases.

Relative Importance of Different Elements of Mitochondrial Oxidative Phosphorylation in Maintaining the Barrier Integrity of Retinal Endothelial Cells: Implications for Vascular-Associated Retinal Diseases

“…When comparing the effects of the three mitochondrial inhibitors on the various resistance parameters between RPE cells in our previous study [ 17 ] to HRECs in the present work, there are key similarities and differences that need to be highlighted. In RPE cells, rotenone disrupted R b in a dose-dependent fashion, while in HRECs, we showed that a single concentration of 1 µM rotenone completely abolished R b .…”

“…The effect of different inhibitors targeting different steps in mitochondrial OxPhos on the cellular barrier functionality of HRECs was evaluated by measuring the overall impedance [Z; ohms (Ω)] using ECIS ® Zθ (theta) biosensor technology (Applied Biophysics Inc., Troy, NY, USA) followed by mathematical modeling as described previously [ 16 , 17 ]. Before seeding the cells, the electrode substrate array of the 96-well plate (96W20idf PET, Applied Biophysics, Inc.) was prepared by coating with 50 µL of 100 µM cysteine (Applied Biophysics) for 30 min, followed by aspiration.…”

Endogenous and Exogenous Regulation of Redox Homeostasis in Retinal Pigment Epithelium Cells: An Updated Antioxidant Perspective

“…ROS (superoxide radicals) appear in the RPE during ER stress as a byproduct of oxidative phosphorylation, which occurs largely in the mitochondria. Superoxide radicals are massively generated in dark processes of the mitochondrial respiratory chain, as well as by exposure to visible light [ 94 , 95 ].…”

“…Mitochondrial OS in the RPE leads to metabolic dysfunction in both the RPE and retinal photoreceptors [ 95 , 101 ]. Mitochondrial DNA is more susceptible to oxidative damage in neuropathologies [ 102 ].…”