The inflammatory response of retinal pigment epithelium cells exposed to high glucose concentrations: the role of classical phospholipases D

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

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Diabetic retinopathy (DR) is one of the main causes of visual dysfunction and blindness in working-age adults. Chronic hyperglycemia, oxidative stress and inflammation are key players in the pathogenesis of DR. The aim of the present work is to study the role of classical phospholipases D (PLD1 and PLD2) in retinal pigment epithelium (RPE) cells exposed to an in vitro DR model induced by high glucose (HG) concentrations.ARPE-19 and D407 human RPE cells were exposed to normal glucose concentration (Control condition or NG, 5.5 mM) or to HG concentrations (16.5 or 33 mM) for 4, 24 or 72 h. To study the role of classical PLDs and the ERK pathway cells were pre-incubated for 1 h with EVJ (VU0359595, 0.15 μM) to inhibit PLD1 activity, with APV (VU0285655-1, 0.5 μM) to inhibit PLD2 or U0126 (10 µM) to inhibit the MEK/ERK pathway, prior to cell exposure to HG. Statistical analysis was performed using ANOVA followed by Bonferroni´s test and p-values ≤ 0.05 were considered statistically significant.Our results demonstrate that 72 h exposure to HG reduced cell viability and increased caspase-3 cleavage. In addition, HG exposure for 4 h induced early and concatenated events, as PLD, ERK and nuclear factor kappa B (NFκB) activation. NFκB activation induced by HG correlated with the increment in pro-inflammatory interleukins (IL-6 and IL-8) and cyclooxygenase-2 (COX-2) mRNA levels (p ≤ 0.0001). The effect of pharmacological inhibitors demonstrated that ERK and NFκB activation were dependent on both PLD isoforms. Furthermore, the increment in IL-6 and COX-2 mRNA levels induced by HG was reduced to control levels in cells pre-incubated with both PLD inhibitors. However, the rise in IL-8 mRNA levels was only reduced in cells incubated with the PLD1 inhibitor, EVJ.In conclusion, our findings demonstrate that PLD1 and PLD2 mediate the inflammatory response of RPE cells exposed to HG, pointing to the potential use of classical PLDs as a therapeutic target for DR treatment.