PMID- 34360894 OWN - NLM STAT- MEDLINE DCOM- 20210914 LR - 20240403 IS - 1422-0067 (Electronic) IS - 1422-0067 (Linking) VI - 22 IP - 15 DP - 2021 Jul 29 TI - Relative Contribution of Different Mitochondrial Oxidative Phosphorylation Components to the Retinal Pigment Epithelium Barrier Function: Implications for RPE-Related Retinal Diseases. LID - 10.3390/ijms22158130 [doi] LID - 8130 AB - 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-Ztheta software to investigate in depth the effects of these inhibitors on three separate barrier parameters: cell-cell interactions (R(b)), cell-matrix interactions (alpha), 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, alpha, 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 alpha 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 alpha 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. FAU - Guerra, Michael H AU - Guerra MH AD - Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 East Canfield, Detroit, MI 48201, USA. FAU - Yumnamcha, Thangal AU - Yumnamcha T AD - Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 East Canfield, Detroit, MI 48201, USA. FAU - Singh, Lalit P AU - Singh LP AD - Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 East Canfield, Detroit, MI 48201, USA. FAU - Ibrahim, Ahmed S AU - Ibrahim AS AUID- ORCID: 0000-0001-8480-6252 AD - Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 East Canfield, Detroit, MI 48201, USA. AD - Department of Pharmacology, School of Medicine, Wayne State University, 540 East Canfield, Detroit, MI 48201, USA. AD - Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt. LA - eng GR - P30 EY004068/EY/NEI NIH HHS/United States GR - R01 EY023992/EY/NEI NIH HHS/United States GR - American Heart Association Grant 18CDA34080403/American Heart Association/ PT - Journal Article DEP - 20210729 PL - Switzerland TA - Int J Mol Sci JT - International journal of molecular sciences JID - 101092791 RN - 0 (Enzyme Inhibitors) RN - 0 (Oligomycins) RN - 03L9OT429T (Rotenone) RN - 370-86-5 (Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone) RN - EC 3.6.3.- (Mitochondrial Proton-Translocating ATPases) SB - IM MH - Blood-Retinal Barrier/drug effects/*metabolism MH - Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacokinetics MH - Cell Line MH - Cell Survival/drug effects MH - Diabetic Retinopathy/*metabolism MH - Electric Impedance MH - Electron Transport/drug effects MH - Enzyme Inhibitors/pharmacokinetics MH - Epithelial Cells/*metabolism MH - Humans MH - Macular Degeneration/*metabolism MH - Mitochondria/drug effects/*metabolism MH - Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors MH - Oligomycins/pharmacokinetics MH - Oxidative Phosphorylation/*drug effects MH - Retinal Pigment Epithelium/drug effects/*metabolism MH - Rotenone/pharmacokinetics PMC - PMC8348500 OTO - NOTNLM OT - AMD OT - ARPE-19 OT - DR OT - ECIS OT - RPE OT - mitochondria OT - oxidative phosphorylation OT - uncouplers COIS- The authors declare no conflict of interest. EDAT- 2021/08/08 06:00 MHDA- 2021/09/15 06:00 PMCR- 2021/07/29 CRDT- 2021/08/07 01:08 PHST- 2021/06/23 00:00 [received] PHST- 2021/07/19 00:00 [revised] PHST- 2021/07/23 00:00 [accepted] PHST- 2021/08/07 01:08 [entrez] PHST- 2021/08/08 06:00 [pubmed] PHST- 2021/09/15 06:00 [medline] PHST- 2021/07/29 00:00 [pmc-release] AID - ijms22158130 [pii] AID - ijms-22-08130 [pii] AID - 10.3390/ijms22158130 [doi] PST - epublish SO - Int J Mol Sci. 2021 Jul 29;22(15):8130. doi: 10.3390/ijms22158130.