PLD1 and PLD2 mediate retinal pigment epithelium cell damage induced by high glucose concentrations.

SALVADOR, G.A.; TENCONI, P.E.; GIUSTO, N.M.; MATEOS, M.V.

Chamonix

Congreso; 57th International Conference on the Bioscience of Lipids (ICBL); 2016

Diabetic retinopathy (DR) is one of the most serious complication of diabetes and the main cause of blindness among working-age people. Hyperglycemia, oxidative stress and inflammation are key players in the pathogenesis of DR. The retinal pigment epithelium (RPE) is essential for the integrity and function of the retina, thus elucidating the effects of inflammatory processes in these cells could lead to the discovery of new therapeutic targets for the treatment of retinal degenerative diseases. The objective of the present work was to describe the effects and signaling events elicited by high glucose (HG) concentrations in RPE cells. In particular, our aim was to study the role of classical phospholipases D (PLD1 and PLD2) in this model of DR. PLD1 and PLD2 hydrolyze phosphatidylcholine (PC) to generate phosphatidic acid (PA), that can be further dephosphorylated by lipid phosphate phosphatases in order to produce diacylglycerol (DAG). Thus, the PLD pathway acts as a modulator of several PA and DAG-responding signaling cascades.ARPE-19 cells (human RPE cell line) were exposed to HG (16.5 and 33 mM) or to normal glucose (5 mM, NG) concentrations for 4 (33 mM glucose) or 72 h (16.5 mM glucose) in order to mimic a peak of and a sustained hyperglycemia. Osmotic controls were performed with mannitol. During sustained HG, cell viability (evaluated by the MTT reduction assay) was reduced by 30% and reactive oxygen species (ROS) generation was increased 5 times with respect to NG. The sustained exposure to HG reduced peroxiredoxin (PRX) expression but did not affect superoxide dismutase 1 (SOD1) expression. After a short exposure to HG (4 h) we observed activation of the extracellular signal-regulated kinase (ERK1/2) and translocation of the transcription factor nuclear factor κ B (NFκB) to the nucleus. Moreover, PLD activity (measured as the generation of [3H ]-Phosphatidylethanol) was increased by 80% with respect to cells exposed to NG. Pharmacological inhibition of PLD1 and PLD2 activities (wiht EVJ and APV) prevented ERK1/2 activation as well as ERK1/2 dependent HG-induced NFκB nuclear translocation. Furthermore, the HG-induced loss in cell viability was prevented by both PLD inhibitors, U0126 (MEK/ERK1/2 pathway inhibitor) and by the cyclooxygenase-2 (COX-2) inhibitor (Celecoxib).Our results demonstrate that subsequent activation of PLD1 and PLD2, ERK1/2 and NFκB mediates RPE cell damage during HG condition, possibly through COX-2 induction as well as NFκB-regulated inflammatory cytokines expression. Our findings point to the potential use of classical PLDs as therapeutic targets for the treatment of ocular inflammatory diseases, such as DR.