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Article

Real-Time Monitoring the Effect of Cytopathic Hypoxia on Retinal Pigment Epithelial Barrier Functionality Using Electric Cell-Substrate Impedance Sensing (ECIS) Biosensor Technology

1
Department of Ophthalmology, Visual, and Anatomical Sciences, School of Medicine, Wayne State University, 540 East Canfield, Gordon Scott Hall (room 7133), Detroit, MI 48201, USA
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Department of Pharmacology, School of Medicine, Wayne State University, 540 East Canfield, Gordon Scott Hall (room 7133), Detroit, MI 48201, USA
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Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
*
Author to whom correspondence should be addressed.
Academic Editor: Silvia Bisti
Int. J. Mol. Sci. 2021, 22(9), 4568; https://doi.org/10.3390/ijms22094568
Received: 19 March 2021 / Revised: 20 April 2021 / Accepted: 23 April 2021 / Published: 27 April 2021
(This article belongs to the Special Issue Retinal Function and Morphology in Health, Aging and Disease)
Disruption of retinal pigment epithelial (RPE barrier integrity is a hallmark feature of various retinal blinding diseases, including diabetic macular edema and age-related macular degeneration, but the underlying causes and pathophysiology are not completely well-defined. One of the most conserved phenomena in biology is the progressive decline in mitochondrial function with aging leading to cytopathic hypoxia, where cells are unable to use oxygen for energy production. Therefore, this study aimed to thoroughly investigate the role of cytopathic hypoxia in compromising the barrier functionality of RPE cells. We used Electric Cell-Substrate Impedance Sensing (ECIS) system to monitor precisely in real time the barrier integrity of RPE cell line (ARPE-19) after treatment with various concentrations of cytopathic hypoxia-inducing agent, Cobalt(II) chloride (CoCl2). We further investigated how the resistance across ARPE-19 cells changes across three separate parameters: Rb (the electrical resistance between ARPE-19 cells), α (the resistance between the ARPE-19 and its substrate), and Cm (the capacitance of the ARPE-19 cell membrane). The viability of the ARPE-19 cells and mitochondrial bioenergetics were quantified with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and seahorse technology, respectively. ECIS measurement showed that CoCl2 reduced the total impedance of ARPE-19 cells in a dose dependent manner across all tested frequencies. Specifically, the ECIS program’s modelling demonstrated that CoCl2 affected Rb as it begins to drastically decrease earlier than α or Cm, although ARPE-19 cells’ viability was not compromised. Using seahorse technology, all three concentrations of CoCl2 significantly impaired basal, maximal, and ATP-linked respirations of ARPE-19 cells but did not affect proton leak and non-mitochondrial bioenergetic. Concordantly, the expression of a major paracellular tight junction protein (ZO-1) was reduced significantly with CoCl2-treatment in a dose-dependent manner. Our data demonstrate that the ARPE-19 cells have distinct dielectric properties in response to cytopathic hypoxia in which disruption of barrier integrity between ARPE-19 cells precedes any changes in cells’ viability, cell-substrate contacts, and cell membrane permeability. Such differences can be used in screening of selective agents that improve the assembly of RPE tight junction without compromising other RPE barrier parameters. View Full-Text
Keywords: age related macular degeneration (AMD); diabetic macular edema (DME); cytopathic hypoxia; retinal pigment epithelial cells (RPE); ARPE-19; CoCl2; seahorse; ECIS modeling; Rb resistance; alpha resistance; impedance; capacitance; barrier integrity age related macular degeneration (AMD); diabetic macular edema (DME); cytopathic hypoxia; retinal pigment epithelial cells (RPE); ARPE-19; CoCl2; seahorse; ECIS modeling; Rb resistance; alpha resistance; impedance; capacitance; barrier integrity
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MDPI and ACS Style

Guerra, M.H.; Yumnamcha, T.; Ebrahim, A.-S.; Berger, E.A.; Singh, L.P.; Ibrahim, A.S. Real-Time Monitoring the Effect of Cytopathic Hypoxia on Retinal Pigment Epithelial Barrier Functionality Using Electric Cell-Substrate Impedance Sensing (ECIS) Biosensor Technology. Int. J. Mol. Sci. 2021, 22, 4568. https://doi.org/10.3390/ijms22094568

AMA Style

Guerra MH, Yumnamcha T, Ebrahim A-S, Berger EA, Singh LP, Ibrahim AS. Real-Time Monitoring the Effect of Cytopathic Hypoxia on Retinal Pigment Epithelial Barrier Functionality Using Electric Cell-Substrate Impedance Sensing (ECIS) Biosensor Technology. International Journal of Molecular Sciences. 2021; 22(9):4568. https://doi.org/10.3390/ijms22094568

Chicago/Turabian Style

Guerra, Michael H., Thangal Yumnamcha, Abdul-Shukkur Ebrahim, Elizabeth A. Berger, Lalit P. Singh, and Ahmed S. Ibrahim. 2021. "Real-Time Monitoring the Effect of Cytopathic Hypoxia on Retinal Pigment Epithelial Barrier Functionality Using Electric Cell-Substrate Impedance Sensing (ECIS) Biosensor Technology" International Journal of Molecular Sciences 22, no. 9: 4568. https://doi.org/10.3390/ijms22094568

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