Pharmacological inhibition of the PLD pathway prevents oxidative stress in retinal pigment epithelium cells expose to high glucose levels

Abstract

Introduction Together with inflammation, oxidative stress (OS) is involved in the pathogenesis of several retinal diseases. Canonical phos- pholipases D (PLD1 and PLD2) hydrolyze phosphatidylcholine (PC) to release choline and phosphatidic acid (PA), which can be dephosphorylated by lipid phosphate phosphatases (LPPs) to diacylglycerol (DAG). We previously demonstrated that PLD1 and 2 mediate the inflammatory response of retinal pigment epithelium (RPE) cells induced by high glucose (HG) levels. Furthermore, a significant increase in reactive oxygen species (ROS) was observed in RPE cells exposed to HG. Objectives This study explores the modulation of OS mediated by PLD inhibition in RPE cells exposed to HG. Methods RPE cells (ARPE-19 and D407) were exposed to HG (33 mM) or control conditions (NG, 5.5 mM). In order to mimic PLD/LPPs activation induced by HG, cells were treated with 100 μM dilauroyl PA (DLPA), 100 μM dioctanoyl glycerol (DOG) or with 50 μM DLPA + 50 μM DOG. PLD1, PLD2, cyclooxygenase-2 (COX-2) and NADPH oxidases (NOX), were inhibited using VU0359595 (PLD1i, 0.5 μM), VU0285655-1 (PLD2i, 0.5 μM), celecoxib (10 μM) or DPI (5 μM), respectively. Immunocytochemistry, fluorescent proves and western blots assays were performed to evaluate reactive oxygen species (ROS), mitochondrial membrane potential (MMP), glutathione (GSH) levels and Nrf-2 pathway Results HG-exposure significantly increased ROS levels and reduced MMP in both RPE lines, with respect to NG. PLD inhibitors prevented both effects in an Nrf2 and COX-2-independent manner. Also, exogenously added DLPA and DOG increased OS and reduced MMP. The NOX inhibitor DPI was able to prevent OS induced in RPE cells exposed to HG and also in cells exposed to DLPA+ DOG. Conclusion Our previous findings together with results presented herein, demonstrate that PLD1 and 2 inhibition not only prevents the in- flammatory response of RPE cells, but also decreases OS generated in RPE cells exposed to HG, possibly through a reduced NOX activity but in an Nrf-2 and COX-2 independent manner. The PLD/LPP pathway constitutes a novel pharmacological target to prevent, at the same time, OS and the inflammatory response, two hallmarks of several retinal diseases.