July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
The phospholipase D pathway modulates the inflammatory response of retinal pigment epithelium cells exposed to high glucose concentrations
Author Affiliations & Notes
  • Melina Valeria Mateos
    Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB)-CONICET, Bahía Blanca, Buenos Aires, Argentina
    Depto. de Biología, Bioquímica y Farmacia (DBByF), Universidad Nacional del Sur (UNS), Bahía Blanca, Buenos Aires, Argentina
  • Paula Estefanía Tenconi
    Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB)-CONICET, Bahía Blanca, Buenos Aires, Argentina
    Depto. de Biología, Bioquímica y Farmacia (DBByF), Universidad Nacional del Sur (UNS), Bahía Blanca, Buenos Aires, Argentina
  • Vicente Bermúdez
    Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB)-CONICET, Bahía Blanca, Buenos Aires, Argentina
  • Gerardo Martín Oresti
    Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB)-CONICET, Bahía Blanca, Buenos Aires, Argentina
    Depto. de Biología, Bioquímica y Farmacia (DBByF), Universidad Nacional del Sur (UNS), Bahía Blanca, Buenos Aires, Argentina
  • Gabriela Alejandra Salvador
    Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB)-CONICET, Bahía Blanca, Buenos Aires, Argentina
    Depto. de Biología, Bioquímica y Farmacia (DBByF), Universidad Nacional del Sur (UNS), Bahía Blanca, Buenos Aires, Argentina
  • Norma María Giusto
    Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB)-CONICET, Bahía Blanca, Buenos Aires, Argentina
    Depto. de Biología, Bioquímica y Farmacia (DBByF), Universidad Nacional del Sur (UNS), Bahía Blanca, Buenos Aires, Argentina
  • Footnotes
    Commercial Relationships   Melina Mateos, None; Paula Tenconi, None; Vicente Bermúdez, None; Gerardo Oresti, None; Gabriela Salvador, None; Norma Giusto, None
  • Footnotes
    Support  Fundación Florencio Fiorini (Subsidio para Investigación en Ciencias Biomédicas 2016), Comisión de Investigaciones Científicas de la Prov. de Buenos Aires (CIC), Universidad Nacional del Sur (UNS, PGI 24/B226 8388), Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT; PICTs 2013-0987, 2013-2317 and 2014-3352).
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 5357. doi:
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    • Get Citation

      Melina Valeria Mateos, Paula Estefanía Tenconi, Vicente Bermúdez, Gerardo Martín Oresti, Gabriela Alejandra Salvador, Norma María Giusto; The phospholipase D pathway modulates the inflammatory response of retinal pigment epithelium cells exposed to high glucose concentrations. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5357.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Diabetic retinopathy (DR) is one of the main causes of visual dysfunction and blindness in working-age adults in which chronic hyperglycemia, oxidative stress and inflammation are key pathogenic players. The aim of this work was to study the role of the phospholipase D (PLD) pathway in retinal pigment epithelium (RPE) cells exposed to an in vitro DR model.

Methods : Human RPE cell lines (ARPE-19 and D407) were exposed to high glucose (HG) concentrations (16.5 or 33 mM) or to normal glucose concentration (NG, 5.5 mM) for 4, 24 or 72 h. Osmotic controls were performed with mannitol (Man). After experimental treatment western blot (WB), immunocytochemistry and qPCR assays were performed. Cell viability was evaluated using the MTT reduction assay and PLD activity was measured as [3H]-phosphatidylethanol ([3H]-PEth) generation from [3H]-phosphatidylcholine in the presence of 0.4% ethanol.

Results : Exposure to HG increased reactive oxygen species levels and caspase-3 cleavage and reduced cell viability after 72 h of incubation. In addition, short term HG exposure (4 h) induced the activation of early events, that involve PLD and ERK1/2 signaling, nuclear factor kappa B (NF-κB) nuclear translocation and IκB phosphorylation. An increment in pro-inflammatory interleukins (IL-6 and IL-8) and cyclooxygenase-2 (COX-2) mRNA levels was observed after 24 h of HG exposure. The effect of selective pharmacological PLD1 (VU0359595) and PLD2 (VU0285655-1) inhibitors demonstrated that ERK1/2 and NF-κB activation were downstream events of both PLD isoforms. 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. Furthermore, the inhibition of PLD1, PLD2, the MEK/ERK pathway (with U0126) and COX-2 (with celecoxib) prevented the loss of cell viability induced by HG.

Conclusions : Our results demonstrate that HG exposure induces PLD activation in RPE cells, leading to ERK1/2 activation, IκB degradation, NF-κB nuclear translocation and expression of pro-inflammatory ILs and COX-2 and reduced cell viability. Our findings are the first evidence that classical PLDs participate in the inflammatory response of RPE cells exposed to HG and leads us to postulate these signaling enzymes as potential therapeutic targets for DR treatment.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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