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Heba Mohamed Saleh, Khaled Hussein, Ahmed S Ibrahim, Khaled Elmasry, Nehal M El-Sherbiny, David Fulton, Mohamed Al-Sayed Al-Shabrawey; NOX4 Stabilizes Barrier Function of Human Retinal Endothelial Cells and Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3216. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
NADPH oxidase 4 (NOX4) is one of the 7 isoforms of the NADPH oxidase family. NOX4 is regulated at the gene level, does not require cytosolic subunits for activation and generates H2O2 in preference to O2−. These facts make it different than other NOX isoforms. Integrity of blood retinal barrier (BRB) is essential for proper retinal health. BRB consists of inner and outer barriers where the retinal endothelial cells and retinal pigment epithelial cells constitute the major cells that maintain both barriers, respectively. Hence, in the current study we are investigating the impact of modulating NOX4 expression on barrier function of human retinal endothelial cells (HRECs) and human retinal pigment epithelial cells (ARPE19).
HRECs were cultured in EBM2 medium with 5% FBS. ARPE19 were cultured in DMEM medium with 10% FBS. Electrical cell-substrate impedance sensing (ECIS) was used to measure transcellular electrical resistance (TER) in HRECs and ARPE19. Cells were serum starved after reaching confluence within the ECIS machine. Inhibition of basal levels NOX4 was done using 20 µM of NOX4 inhibitor (GKT137831) and compared to vehicle treated. Overexpression of NOX4 was performed using NOX4 Adenovirus (AdV) and compared to control AdV. TER was normalized to the start of treatment and was measured over 48-72 hours.
Inhibition of the basal levels of NOX4 using 20 µM of GKT137831 significantly attenuates TER in both cell types. In HRECs, significant decrease in normalized TER (pValue < 0.05) started after 24 hours of treatment and reached almost 15% (pValue <0.001) at 60 hours of treatment. Concerning ARPE19 cells, inhibition of NOX4 induced initial increase in TER for the first 24 hours of treatment followed by significant decrease in TER by 5% at 72 hours (pValue<0.05). Moreover, over expression of NOX4 significantly improved TER in HRECs by 5% (pValue<0.05) within 48 hours of adding virus. In addition, NOX4 AdV significantly enhanced TER in ARPE19 cells starting at 24 hours of adding virus (pValue<0.05) and reached more than 15% at 72 hours (pValue<0.001).
NOX4 might be important in stabilizing both outer and inner BRB. This adds another evidence to the protective effect of NOX4 in its damaging/protective paradox, which highlights the need for further studies before considering the therapeutic potentials of NOX4 inhibitors.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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