Ebselen by modulating oxidative stress improves hypoxia-induced macroglial Müller cell and vascular injury in the retina

https://doi.org/10.1016/j.exer.2015.04.015Get rights and content

Highlights

  • Ebselen protects against hypoxia-induced injury of retinal Müller cells.

  • Ebselen reduces oxidative stress, vascular damaging factors and inflammation.

  • Ebselen reduces Müller cell gliosis in oxygen-induced retinopathy (OIR).

  • Ebselen reduces neovascularization and vaso-obliteration in OIR.

Abstract

Oxidative stress is an important contributor to glial and vascular cell damage in ischemic retinopathies. We hypothesized that ebselen via its ability to reduce reactive oxygen species (ROS) and augment nuclear factor-like 2 (Nrf2) anti-oxidants would attenuate hypoxia-induced damage to macroglial Müller cells and also lessen retinal vasculopathy. Primary cultures of rat Müller cells were exposed to normoxia (21% O2), hypoxia (0.5% O2) and ebselen (2.5 μM) for up to 72 h. Oxygen-induced retinopathy (OIR) was induced in C57BL/6J mice while control mice were housed in room air. Mice received vehicle (saline, 5% dimethyl sulfoxide) or ebselen (10 mg/kg) each day between postnatal days 6–18. In cultured Müller cells, flow cytometry for dihydroethidium revealed that ebselen reduced the hypoxia-induced increase in ROS levels, whilst increasing the expression of Nrf2-regulated anti-oxidant genes, heme oxygenase 1, glutathione peroxidase-1, NAD(P)H dehydrogenase quinone oxidoreductase 1 and glutamate-cysteine ligase. Moreover, in Müller cells, ebselen reduced the hypoxia-induced increase in protein levels of pro-angiogenic and pro-inflammatory factors including vascular endothelial growth factor, interleukin-6, monocyte chemoattractant-protein 1 and intercellular adhesion molecule-1, and the mRNA levels of glial fibrillary acidic protein (GFAP), a marker of Müller cell injury. Ebselen improved OIR by attenuating capillary vaso-obliteration and neovascularization and a concomitant reduction in Müller cell gliosis and GFAP. We conclude that ebselen protects against hypoxia-induced injury of retinal Müller cells and the microvasculature, which is linked to its ability to reduce oxidative stress, vascular damaging factors and inflammation. Agents such as ebselen may be potential treatments for retinopathies that feature oxidative stress-mediated damage to glia and the microvasculature.

Introduction

Oxidative stress occurs when there is an imbalance between the excess production of reactive oxygen species (ROS) and the ability to scavenge these ROS by endogenous anti-oxidative factors (Valko et al., 2007). Oxidative stress is a major casual factor in the development of ischemic retinopathies such as retinopathy of prematurity and diabetic retinopathy, which feature ROS-induced up-regulation of pro-angiogenic and pro-inflammatory pathways which damage glia, vascular cells and neurons (Li et al., 2012, Wilkinson-Berka et al., 2014, Wang et al., 2013). Retinopathy of prematurity and diabetic retinopathy are major causes of vision impairment and are escalating in prevalence globally (Chen and Smith, 2007, Hartnett and Penn, 2012, Durham and Herman, 2011). However, treatments that effectively prevent their progression are lacking. Recently, attention has been given to developing treatment strategies that protect against oxidative stress by reducing damaging ROS and enhancing cytoprotective anti-oxidants (Li et al., 2012, Wilkinson-Berka et al., 2014, Wang et al., 2013, Wilkinson-Berka et al., 2013). In this respect, ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a synthetic lipid soluble seleno-organic and low molecular weight compound, is of interest (Schewe, 1995). We previously reported that ebselen reduced ROS levels and organ damage in animal models of diabetic complications (Tan et al., 2013a, Chew et al., 2010, Chew et al., 2009). Furthermore, ebselen is able to bolster endogenous anti-oxidant defenses by activating nuclear factor erythroid-2 related factor 2 (Nrf2)-regulated genes such as heme oxygenase-1 (HO-1), glutathione peroxidase-1 (GPx1), NAD(P)H quinone oxidoreductase 1 (NQO1) and glutamate-cysteine ligase (GCLC) (Tamasi et al., 2004, Lee and Johnson, 2004), which protects against cellular oxidative stress in tissues including the retina (Uno et al., 2010, Xu et al., 2014, Nakamura et al., 2002). Of relevance to ischemic retinopathies is that Nrf2 is predominately found in glia, and notably macroglial Müller cells of both humans and mice (Xu et al., 2014).

Müller cells play a crucial role in maintaining retinal function as well as the integrity of the vasculature (Fletcher et al., 2010, Reichenbach and Bringmann, 2010). In ischemic retinopathies, Müller cells experience oxidative-induced damage and in response exhibit a reactive phenotype demonstrated by up-regulated expression of the intermediate filament, glial fibrillary acidic protein (GFAP) (Prentice et al., 2011, Fletcher et al., 2010, Fitzgerald et al., 1990, Kim et al., 1998) and increased production of vascular damaging factors and inflammatory mediators including cytokines, chemokines and adhesion molecules (Gerhardinger et al., 2005, Reichenbach and Bringmann, 2010). The anatomical association of Müller cell processes with the retinal microvasculature means that damage to Müller cells can promote severe injury to blood vessels including capillary degeneration and neovascularization (Newman and Reichenbach, 1996, Hu et al., 2014). Whether ebselen can confer protection against hypoxia-induced oxidative stress in retinal Müller cells and also improve vascular injury in ischemic retinopathies has not been explored.

We hypothesized that ebselen would improve the oxidative status, gliotic and pro-angiogenic and pro-inflammatory phenotype of Müller cells exposed to hypoxia, with a concomitant reduction in retinal microvascular injury. These studies were performed in primary cultures of rat Müller cells as well as in a robust mouse model of retinopathy of prematurity known as oxygen-induced retinopathy (OIR).

Section snippets

Animals

Animals were obtained from the Alfred Medical Research and Education Precinct (AMREP) Animal Services, Melbourne, Victoria, Australia. Experimental procedures adhered to guidelines of the National Health and Medical Research Council (NHMRC) of Australia's Code for the Care and Use of Animals for Scientific Purposes and were approved by the AMREP Animal Ethics Committee.

Primary cultures of rat Müller cells

Primary cultures of rat Müller cells were studied as previously described (Deliyanti et al., 2012, Deliyanti et al., 2014).

Ebselen reduced the levels of ROS in cultured Müller cells

Flow cytometry for DHE showed that hypoxia significantly increased the levels of ROS in Müller cells compared to normoxia controls (Fig. 1A, B). Ebselen had no effect on ROS levels in Müller cells cultured in normoxia, but decreased the hypoxia-induced increase in ROS (Fig. 1A, B). Exposure of Müller cells to hypoxia and ebselen did not influence cell survival (Fig. 1C).

Ebselen up-regulated anti-oxidant genes and reduced pro-angiogenic and pro-inflammatory factors as well as GFAP in cultured Müller cells

The exposure of Müller cells to hypoxia did not alter the mRNA and protein levels of the anti-oxidant genes, HO-1 (Fig. 2A, B)

Discussion

The effect of ebselen on hypoxia-induced Müller cell injury and OIR has not been described. The major findings of this study are that ebselen has the ability to improve the hypoxia-induced reactive phenotype of retinal Müller cells by modulating oxidative stress with a reduction in ROS and an increase in anti-oxidant genes, as well as reducing gliosis and angiogenic and inflammatory mediators. Importantly, these protective effects of ebselen on the health of Müller cells in vitro, extended to a

Acknowledgments

SMT was supported by Juvenile Diabetes Research Foundation International Postdoctoral Fellowship. JW-B is a Senior Research Fellow of the National Health and Medical Research Council of Australia. Supported in part by the Victorian Government′s OIS Program.

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