Activation of the VEGF-A/ERK/PLA2 Axis Mediates Early Retinal Endothelial Cell Damage Induced by High Glucose: New Insight from an In Vitro Model of Diabetic Retinopathy.

Giovanni Giurdanella,Giuliana Mannino,Debora Lo Furno,Federica Conti,Gabriella Lupo,Florinda Gennuso,Salvatore Salomone,Carmelina Daniela Anfuso,Filippo Drago,Claudio Bucolo

doi:10.3390/ijms21207528

Giovanni Giurdanella, Giuliana Mannino + Show 8 more

Open Access

https://doi.org/10.3390/ijms21207528

Copy DOI

Abstract

Early blood retinal barrier (BRB) dysfunction induced by hyperglycemia was related to increased pro-inflammatory activity of phospholipase A2 (PLA2) and the upregulation of vascular endothelial growth factor A (VEGF-A). Here, we tested the role of VEGF-A in high glucose (HG)-induced damage of human retinal endothelial cells (HRECs) mediated by Ca++-dependent (cPLA2) and Ca++-independent (iPLA2) PLA2s. HRECs were treated with normal glucose (5 mM, NG) or high glucose (25 mM, HG) for 48 h with or without the VEGF-trap Aflibercept (Afl, 40 µg/mL), the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3; 15 µM), the iPLA2 inhibitor bromoenol lactone (BEL; 5 µM), or VEGF-A (80 ng/mL). Both Afl and AACOCF3 prevented HG-induced damage (MTT and LDH release), impairment of angiogenic potential (tube-formation), and expression of VEGF-A mRNA. Furthermore, Afl counteracted HG-induced increase of phospho-ERK and phospho-cPLA2 (immunoblot). VEGF-A in HG-medium increased glucose toxicity, through upregulation of phospho-ERK, phospho-cPLA2, and iPLA2 (about 55%, 45%, and 50%, respectively); immunocytochemistry confirmed the activation of these proteins. cPLA2 knockdown by siRNA entirely prevented cell damage induced by HG or by HG plus VEGF-A, while iPLA2 knockdown produced a milder protective effect. These data indicate that VEGF-A mediates the early glucose-induced damage in retinal endothelium through the involvement of ERK1/2/PLA2 axis activation.

Highlights

Blood retinal barrier (BRB) dysfunction represents an early pathological event in diabetic retinopathy (DR) [1,2]
Effects of Aflibercept and phospholipase A2 (PLA2) Inhibitors on Retinal Endothelial Cell Damage Induced by HG. It has been previously reported (i) that high glucose-induced toxicity in human retinal endothelial cells (HRECs) depends on PLA2 activity [19,29], and (ii) that the induction of cPLA2 is mediated by vascular endothelial growth factor A (VEGF-A) signaling (II) [35,37]
We further investigated the role of VEGF-A/PLA2 axis blockade by testing the tube formation capability ofWHe RfuErCthserchinavlelestniggaeteddbtyheHroGle, ionf VprEeGsFe-nAc/ePLoAf 2AaflxiibsebrlcoecpkatdoerbPyLtAes2tininghthibeittuobres.foTrumbaetifoonrmation capability of HRECs challenged by HG, in presence of Aflibercept or PLA2 inhibitors

Summary

Introduction

Blood retinal barrier (BRB) dysfunction represents an early pathological event in diabetic retinopathy (DR) [1,2]. Following pro-inflammatory stimuli, cPLA2 mediates arachidonic acid (AA) release through a Ca2+-dependent mechanism of translocation from cytosol to perinuclear membranes [21]. We tested the hypothesis that VEGF-A impairs human retinal endothelial cells (HRECs) subjected to high glucose through Ca++-dependent (cPLA2) and Ca++-independent (iPLA2) phospholipase A2. In this respect, HRECs were treated with a high concentration of glucose, to mimic the early stage of DR; thereafter, the effects of the VEGF-trap Aflibercept, PLA2 blockade by chemical agents or knock down by siRNA, and exogenous VEGF-A were evaluated

Methods

Results

Conclusion