Abstract
Purpose :
Disruption of retinal pigment epithelial (RPE) barrier integrity is a hallmark feature of age-related macular degeneration (AMD), 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. This study aimed to investigate the role of mitochondrial bioenergetics in maintaining RPE barrier functionality RPE.
Methods :
We used ECIS system to monitor in real time the barrier integrity of RPE cell line (ARPE-19) after treatment with one of four mitochondrial electron transport chain (ETC) inhibitors (Rotenone for complex 1, Oligomycin for Complex IV, trifluoromethoxy carbonylcyanide phenylhydrazone FCCP for uncoupling ATP synthesis from ETC, and cobalt chloride (CoCl2) as hypoxia-mimetic agent). We investigated how the resistance across ARPE-19 cells changes in three separate parameters: Rb (paracellular resistance between cells), α (basolateral resistance between RPE and its substrate), and Cs (cell membrane capacitance). Statistical analysis was performed using ANOVA test followed by Tukey post hoc test with p<0.05 considered significant.
Results :
Treatment with CoCl2 and FCCP induced a significant reduction in the total resistance and thus the barrier function of ARPE-19 in a dose dependent manner. Treatment with rotenone decreased barrier function in a non-dose dependent fashion, whereas only treatment with a high dose oligomycin (10 µM vs 1 µM) disrupted barrier function. Specifically, the ECIS program’s modelling of paracellular resistance, basolateral resistance, and capacitance showed that both CoCl2-induced hypoxia and FCCP- induced uncoupling decreased paracellular resistance and basolateral resistance, but they both led to increases in membrane capacitance, suggestive of cellular swelling. Sufficient inhibition of ATP-synthase (high dose oligomycin), disrupted both paracellular and basolateral resistances; and finally, complex 1 inhibition only disrupted the basolateral resistance.
Conclusions :
Our findings suggest that the ECIS is a powerful tool for measuring in real time how the barrier function of RPE cells changes following mitochondrial dysfunction, and recommend its further use in investigation of RPE dysfunction as it relates to the multifactorial pathogenesis of dry AMD.
This is a 2021 ARVO Annual Meeting abstract.