Placental growth factor-1 and epithelial haemato–retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy

Abstract

Aims/hypothesis Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. Methods ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato-retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. Results In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 into the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasmic translocation of junction proteins. Conclusions/interpretation Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

VEGF modulation of retinal pigment epithelium resistance

“…The ARPE-19 cell line expresses several RPE-specific markers (CRALBP and RPE65) and forms polarized monolayers; however, the TER generated by these cells is normal at 40-70% less than the primary RPE cells in culture (Dunn et al 1996;Geisen et al 2006;Hartnett et al 2003;Jin et al 2002). In the current study, both primary cells and the ARPE-19 cell line produced monolayers that formed continuous tight-junctioned complexes between cells and generated TER that was consistent with previous studies (Dunn et al 1996;Geisen et al 2006;Miyamoto et al 2007). Although the TER values obtained in ARPE-19 cells were lower than those in primary porcine cells, the administration of VEGF to either cell type resulted in a robust (30-50%) drop in the TER within 5 hours of treatment.…”

“…In the vascular endothelium, VEGF increases paracellular permeability by the activation of VEGF-R2 receptors (Autiero et al 2003;Gille et al 2001;Shen et al 1999). However, a recent study by Miyamoto and colleagues concluded that the VEGF-R1 receptor subtype mediated VEGF-induced changes in the barrier properties of ARPE-19 cell monolayers (Miyamoto et al 2007). Like Miyamoto and colleagues, we identified VEGF-A 165 as an effective cytokine in regulating RPE barrier function.…”

“…The utilized cell line and culture conditions were different, as well as the source of the agonist ligands. Also the route of VEGF administration is not clear in their paper (Miyamoto et al 2007). These seemingly minor changes may make a considerable difference, because the activity of VEGF receptors can be complicated by a series of additional factors including agonist-induced crosstalk between VEGF-R1 and VEGF-R2, hetero-dimerization between individual receptor subunits, and hetero-dimerization of the agonists themselves (Autiero et al 2003); moreover, additional receptors may enhance VEGF receptor signaling (Neuropilin-1) (Becker et al 2005).…”

“…In vascular endothelial cells, there is growing evidence that increases in permeability and proliferation are mediated by VEGF-R2 activation, and within the choroid, that these actions can be opposed by VEGF-R1 stimulation (Autiero et al 2003;Gille et al 2001;Nozaki et al 2006;Shen et al 1999). However, a recent study of the RPE concluded that VEGF-R1 receptor activation is responsible for the VEGF-induced increase in epithelial permeability (Miyamoto et al 2007). Our studies were designed to clarify how VEGF modulates RPE barrier properties.…”

Fenofibric acid prevents retinal pigment epithelium disruption induced by interleukin-1β by suppressing AMP-activated protein kinase (AMPK) activation

“…In fact, treatment of RPE cells with either serum, interferon-γ, tumour necrosis factor-α, hepatocyte growth factor (HGF), IL-1β or placental growth factor-1 (PLGF-1) increases permeability and alters the levels or content of tight junction molecules [14][15][16][17][18]. As IL-1β plays an essential role in the development of DR [19][20][21][22], we decided to use this cytokine to provoke the breakdown of the RPE cell monolayer and to test the potential preventive effects of fenofibrate.…”