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

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, [2000-

Blood-Retinal Barrier/*metabolism ; Diabetic Retinopathy/*metabolism ; Epithelial Cells/*metabolism ; Macular Degeneration/*metabolism ; Mitochondria/*metabolism ; Oxidative Phosphorylation/*drug effects ; Retinal Pigment Epithelium/*metabolism; Blood-Retinal Barrier/drug effects ; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacokinetics ; Cell Line ; Cell Survival/drug effects ; Electric Impedance ; Electron Transport/drug effects ; Enzyme Inhibitors/pharmacokinetics ; Humans ; Mitochondria/drug effects ; Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors ; Oligomycins/pharmacokinetics ; Retinal Pigment Epithelium/drug effects ; Rotenone/pharmacokinetics

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 (R b ), cell-matrix interactions (α), and the cell membrane capacitance (C m ). 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 C m ) 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 R b in a dose-dependent manner compared to basolateral resistance α or C m . 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.

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P30 EY004068 United States EY NEI NIH HHS; R01 EY023992 United States EY NEI NIH HHS; American Heart Association Grant 18CDA34080403 American Heart Association

Keywords: AMD; ARPE-19; DR; ECIS; RPE; mitochondria; oxidative phosphorylation; uncouplers

0 (Enzyme Inhibitors)
0 (Oligomycins)
03L9OT429T (Rotenone)
370-86-5 (Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone)
EC 3.6.3.- (Mitochondrial Proton-Translocating ATPases)

Date Created: 20210807 Date Completed: 20210914 Latest Revision: 20240403

20240403

PMC8348500

10.3390/ijms22158130

34360894