Ebselen by modulating oxidative stress improves hypoxia-induced macroglial Müller cell and vascular injury in the retina
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.
References (59)
- et al.
Role of oxygen and nitrogen species in experimental uveitis: anti-inflammatory activity of the synthetic antioxidant ebselen
Free Rad. Biol. Med.
(2002) - et al.
Endothelial dysfunction as a modifier of angiogenic response in Zucker diabetic fat rat: amelioration with Ebselen
Kidney Int.
(2004) - et al.
Interleukin-6 receptor-mediated activation of signal transducer and activator of transcription-3 (STAT3) promotes choroidal neovascularization
Am. J. Pathol.
(2007) - et al.
Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression
J. Biol. Chem.
(2003) - et al.
Ebselen, a glutathione peroxidase mimetic seleno-organic compound, as a multifunctional Antioxidant: Implication for inflammation-associated carcinogenesis
J. Biol. Chem.
(2002) - et al.
The Muller cell: a functional element of the retina
Trends Neurosci.
(1996) - et al.
The early research and development of ebselen
Biochem.. Pharmacol.
(2013) - et al.
Ebselen increases cytosolic free Ca2+ concentration, stimulates glutamate release and increases GFAP content in rat hippocampal astrocytes
Toxicology
(2008) Molecular actions of ebselen–an antiinflammatory antioxidant
Gen. Pharmacol.
(1995)Ebselen, a selenoorganic compound as glutathione peroxidase mimic
Free Rad. Biol. Med.
(1993)
Ebselen augments its peroxidase activity by inducing Nrf-2-dependent transcription
Arch. Biochem. Biophys.
Role of Nrf2 in retinal vascular development and the vaso-obliterative phase of oxygen-induced retinopathy
Exp. Eye Res.
Free radicals and antioxidants in normal physiological functions and human disease
Int. J. Biochem. Cell Biol.
Inflammatory cytokines in vitreous fluid and serum of patients with diabetic vitreoretinopathy
J. Diabetes Complicat.
Heme oxygenase-1 induced in muller cells plays a protective role in retinal ischemia-reperfusion injury in rats
Invest. Ophthalmol. Vis. Sci.
Retinopathy of prematurity
Angiogenesis
Antiatherosclerotic and renoprotective effects of ebselen in the diabetic apolipoprotein E/GPx1-double knockout mouse
Diabetes
Site-specific antiatherogenic effect of the antioxidant ebselen in the diabetic apolipoprotein E–deficient mouse
Arterioscler. Thromb. Vasc. Biol.
Retinal vasculopathy is reduced by dietary salt restriction: involvement of glia, ENaCalpha, and the renin-angiotensin-aldosterone system
Arterioscler. Thromb. Vasc. Biol.
Neovascularization is attenuated with aldosterone synthase inhibition in rats with retinopathy
Hypertension
Glutathione peroxidase 1 activity dictates the sensitivity of glioblastoma cells to oxidative stress
Glia
Microvascular modifications in diabetic retinopathy
Curr. Diab. Rep.
Evidence for retinal pathology following interruption of neural regulation of choroidal blood flow: muller cells express GFAP following lesions of the nucleus of Edinger-Westphal in pigeons
Curr. Eye Res.
The significance of neuronal and glial cell changes in the rat retina during oxygen-induced retinopathy
Doc. Ophthalmol.
Neuronal and glial cell abnormality as predictors of progression of diabetic retinopathy
Curr. Pharm. Des.
Expression of acute-phase response proteins in retinal Muller cells in diabetes
Invest. Ophthalmol. Vis. Sci.
Ebselen reduces inflammation and microvascular perfusion failure after blunt skeletal muscle injury of the rat
J. Trauma
Mechanisms and management of retinopathy of prematurity
New. Engl. J. Med.
Muller glia cells regulate Notch signaling and retinal angiogenesis via the generation of 19,20-dihydroxydocosapentaenoic acid
J. Exp. Med.
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- 1
S.M. Tan and D. Deliyanti share equal first authorship.
- 2
J.L. Wilkinson-Berka and J.B. de Haan are joint senior authors.