Pharmacological inhibition of the phospholipase D 1 and 2 prevents oxidative stress in retinal pigment epithelium cells exposed to high glucose levels

ECHEVARRIA M. S.; TENCONI P. E.; GIUSTO N. M.; MATEOS M. V.

Buenos Aires

Simposio; Frontiers in Bioscience; 2022

Instituto Max Planck

Objective: Oxidative stress (OE) and inflammation are involved in the pathogenesis of severalretinal diseases. We previously demonstrated that classical phospholipase D isoforms (PLD1and 2) mediate the inflammatory response of retinal pigment epithelium (RPE) cells induced byhigh glucose (HG) levels. The aim of the present work was to study the relationship betweenOE and PLD activation observed in RPE cells exposed to HG.Methods: RPE cells (ARPE-19 and D407) were exposed to HG (33 mM) or to normal glucoselevels (NG, 5.5 mM) for 24 h. To inhibit PLD1, PLD2 and cyclooxygenase-2 (COX-2) VU0359595(PLD1i, 0.5 μM), VU0285655-1 (PLD2i, 0.5 μM) or celecoxib (COX-2i, 10 μM) were used,respectively. ROS production was assessed using the probe DCDCDHF. Mitochondrialmembrane potential was evaluated by labeling the cells with the probe Mitotracker.Immunocytochemistry assays (ICC) and western blots were performed to evaluate Nuclearfactor erythroid 2–related factor2 (Nrf-2) pathway.Results: HG-exposure increased significantly ROS levels and reduced mitochondrial membranepotential in both RPE lines, with respect to NG conditions. PLD inhibitors (PLDi) prevented botheffects. COX-2i was not able to prevent OE induced by HG. ICC showed Nrf-2 nucleartranslocation in cells exposed to HG, what is not observed when cells were pre-treated withPLDi. Nrf-2 activation correlated with an increased heme oxygenase-1 (42%) and superoxidedismutase-1 (43%) expression in HG-exposed cells, no differences were observed in cellstreated with PLDi with respect to NG condition.Conclusions: Our results demonstrate that PLD1 and PLD2 inhibition not only prevents theinflammatory response of RPE cells, but also decreases OE generated in RPE cells exposed toHG in a Nrf-2 and COX-2 independent manner. Further experiments are needed to fullyelucidate the mechanisms by which the PLD pathway mediates OE in RPE cells exposed toinflammatory injury.