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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.

The blood–retinal barrier (BRB) is strongly compromised in diabetic retinopathy (DR) due to the detachment of pericytes (PCs) from retinal microvessels, resulting in increased permeability and impairment of the BRB. Western blots, immunofluorescence and ELISA were performed on adipose mesenchymal stem cells (ASCs) and pericyte-like (P)-ASCs by co-cultured human retinal endothelial cells (HRECs) under hyperglycemic conditions (HG), as a model of DR. Our results demonstrated that: (a) platelet-derived growth factor receptor (PDGFR) and its activated form were more highly expressed in monocultured P-ASCs than in ASCs, and this expression increased when co-cultured with HRECs under high glucose conditions (HG); (b) the transcription factor Nrf2 was more expressed in the cytoplasmic fraction of ASCs and in the P-ASC nuclear fraction, under normal glucose and, even more, under HG conditions; (c) cytosolic phospholipase A2 activity and prostaglandin E2 release, stimulated by HG, were significantly reduced in P-ASCs co-cultured with HRECs; (d) HO-1 protein content was significantly higher in HG-P-ASCs/HRECs than P-ASCs/HRECs; and (e) VEGF-A levels in media from HG-co-cultures were reduced in P-ASCs/HRECs with respect to ASCs/HRECs. The data obtained highlighted the potential of autologous differentiated ASCs in future clinical applications based on cell therapy to counteract the damage induced by DR. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is characterized by morphologic and metabolic alterations in endothelial cells (ECs) and pericytes (PCs) of the blood–retinal barrier (BRB). The loss of interendothelial junctions, increased vascular permeability, microaneurysms, and finally, EC detachment are the main features of DR. In this scenario, a pivotal role is played by the extensive loss of PCs. Based on previous results, the aim of this study was to assess possible beneficial effects exerted by adipose mesenchymal stem cells (ASCs) and their pericyte-like differentiated phenotype (P-ASCs) on human retinal endothelial cells (HRECs) in high glucose conditions (25 mM glucose, HG). P-ASCs were more able to preserve BRB integrity than ASCs in terms of (a) increased transendothelial electrical resistance (TEER); (b) increased expression of adherens junction and tight junction proteins (VE-cadherin and ZO-1); (c) reduction in mRNA levels of inflammatory cytokines TNF-α, IL-1β, and MMP-9; (d) reduction in the angiogenic factor VEGF and in fibrotic TGF-β1. Moreover, P-ASCs counteracted the HG-induced activation of the pro-inflammatory phospho-ERK1/2/phospho-cPLA2/COX-2 pathway. Finally, crosstalk between HRECs and ASCs or P-ASCs based on the PDGF-B/PDGFR-β axis at the mRNA level is described herein. Thus, P-ASCs might be considered valuable candidates for therapeutic approaches aimed at countering BRB disruption in DR. [ABSTRACT FROM AUTHOR]

A pericyte-like differentiation of human adipose-derived mesenchymal stem cells (ASCs) was tested in in vitro experiments for possible therapeutic applications in cases of diabetic retinopathy (DR) to replace irreversibly lost pericytes. For this purpose, pericyte-like ASCs were obtained after their growth in a specific pericyte medium. They were then cultured in high glucose conditions to mimic the altered microenvironment of a diabetic eye. Several parameters were monitored, especially those particularly affected by disease progression: cell proliferation, viability and migration ability; reactive oxygen species (ROS) production; inflammation-related cytokines and angiogenic factors. Overall, encouraging results were obtained. In fact, even after glucose addition, ASCs pre-cultured in the pericyte medium (pmASCs) showed high proliferation rate, viability and migration ability. A considerable increase in mRNA expression levels of the anti-inflammatory cytokines transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) was observed, associated with reduction in ROS production, and mRNA expression of pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and angiogenic factors. Finally, a pmASC-induced better organization of tube-like formation by retinal endothelial cells was observed in three-dimensional co-culture. The pericyte-like ASCs obtained in these experiments represent a valuable tool for the treatment of retinal damages occurring in diabetic patients. [ABSTRACT FROM AUTHOR]