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      Unfolded Protein Response Pathways Correlatively Modulate Endoplasmic Reticulum Stress Responses in Rat Retinal Müller Cells

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          Abstract

          Background

          Endoplasmic reticulum stress (ERS) in the retinal Müller cells is a key factor contributing to the retinal inflammation and vascular leakage in diabetic retinopathy (DR). This study was to investigate the underlying mechanisms through which the 3 main unfolded protein response (UPR) pathways regulate ERS and to examine the expression levels of vascular endothelial growth factor (VEGF) in Müller cells in vitro.

          Methods

          Rat Müller cell lines were stimulated with high glucose to mimic a diabetic environment in vitro. PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) were downregulated or upregulated with shRNA or overexpression plasmids. The transfected Müller cells were cultivated in high glucose medium for 48 hours. Expression of glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP1), ATF6, and VEGF was examined with immunofluorescence and western blot.

          Results

          Our data indicated that ERS was found in both high glucose and osmotic control groups. Overexpression or downregulation of UPR pathways effectively increased or reduced the production of GRP78, ATF4, XBP1, ATF6, and VEGF, respectively. These 3 signaling pathways had similar regulatory effects on VEGF.

          Conclusion

          The 3 UPR-mediated inflammatory pathways were dependent on each other. Inhibition any of these signaling pathways in UPR might be a potential therapeutic target for DR.

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          Most cited references23

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          New functions of Müller cells.

          Müller cells, the major type of glial cells in the retina, are responsible for the homeostatic and metabolic support of retinal neurons. By mediating transcellular ion, water, and bicarbonate transport, Müller cells control the composition of the extracellular space fluid. Müller cells provide trophic and anti-oxidative support of photoreceptors and neurons and regulate the tightness of the blood-retinal barrier. By the uptake of glutamate, Müller cells are more directly involved in the regulation of the synaptic activity in the inner retina. This review gives a survey of recently discoved new functions of Müller cells. Müller cells are living optical fibers that guide light through the inner retinal tissue. Thereby they enhance the signal/noise ratio by minimizing intraretinal light scattering and conserve the spatial distribution of light patterns in the propagating image. Müller cells act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as soft substrate required for neurite growth and neuronal plasticity. Müller cells release neuroactive signaling molecules which modulate neuronal activity, are implicated in the mediation of neurovascular coupling, and mediate the homeostasis of the extracellular space volume under hypoosmotic conditions which are a characteristic of intense neuronal activity. Under pathological conditions, a subset of Müller cells may differentiate to neural progenitor/stem cells which regenerate lost photoreceptors and neurons. Increasing knowledge of Müller cell function and responses in the normal and diseased retina will have great impact for the development of new therapeutic approaches for retinal diseases. Copyright © 2013 Wiley Periodicals, Inc.
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            Müller Cell-Derived VEGF Is Essential for Diabetes-Induced Retinal Inflammation and Vascular Leakage

            OBJECTIVE Vascular endothelial growth factor (VEGF-A or VEGF) is a major pathogenic factor and therapeutic target for diabetic retinopathy (DR). Since VEGF has been proposed as a survival factor for retinal neurons, defining the cellular origin of pathogenic VEGF is necessary for the effectiveness and safety of long-term anti-VEGF therapies for DR. To determine the significance of Müller cell-derived VEGF in DR, we disrupted VEGF in Müller cells with an inducible Cre/lox system and examined diabetes-induced retinal inflammation and vascular leakage in these conditional VEGF knockout (KO) mice. RESEARCH DESIGN AND METHODS Leukostasis was determined by counting the number of fluorescently labeled leukocytes inside retinal vasculature. Expression of biomarkers for retinal inflammation was assessed by immunoblotting of TNF-α, ICAM-1, and NF-κB. Vascular leakage was measured by immunoblotting of retinal albumin and fluorescent microscopic analysis of extravascular albumin. Diabetes-induced vascular alterations were examined by immunoblotting and immunohistochemistry for tight junctions, and by trypsin digestion assays for acellular capillaries. Retinal integrity was analyzed with morphologic and morphometric analyses. RESULTS Diabetic conditional VEGF KO mice exhibited significantly reduced leukostasis, expression of inflammatory biomarkers, depletion of tight junction proteins, numbers of acellular capillaries, and vascular leakage compared to diabetic control mice. CONCLUSIONS Müller cell-derived VEGF plays an essential and causative role in retinal inflammation, vascular lesions, and vascular leakage in DR. Therefore, Müller cells are a primary cellular target for proinflammatory signals that mediates retinal inflammation and vascular leakage in DR.
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              Glial reactivity, an early feature of diabetic retinopathy.

              To characterize early structural gliotic reactions in retinal Müller cells, astrocytes, and microglia in experimentally induced diabetes. Rats were rendered diabetic by streptozotocin injection and killed after 2, 4, 12, or 20 weeks. Cell densities were determined in flatmounted retinas or transverse semithin sections. Expression of glial fibrillary acidic protein (GFAP) was localized on frozen sections or flatmounts by immunofluorescence and confocal microscopy, and GFAP content was evaluated by Western blot analysis. Microglial cells were visualized by binding of isolectin B4 or staining with antibodies to phosphotyrosine residues. The integrity of the blood-retinal barrier was assessed by intravenous injection of Evans blue. The density of Müller cells and microglia was significantly increased at 4 weeks of diabetes compared with nondiabetic controls. GFAP expression in Müller cells was not detected at 4 weeks but was prominent at 12 weeks. The number of astrocytes was significantly reduced at 4 weeks in the peripapillary and far peripheral retina. Shape changes of microglial cells indicated functional activation. Leakage of the blood-retinal barrier was observed at 2 weeks of hyperglycemia, the earliest time point investigated. The leakage of the blood-retinal barrier before glial reactivity suggests that glia are early targets of vascular hyperpermeability. The individual glial cell types react differentially to the diabetic state. Müller cells undergo hyperplasia preceding GFAP expression, and microglial cells are activated, whereas astrocytes regress. This glial behavior may contribute decisively to the onset and development of neuropathy in the diabetic retina.
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                Author and article information

                Contributors
                Journal
                J Ophthalmol
                J Ophthalmol
                JOPH
                Journal of Ophthalmology
                Hindawi
                2090-004X
                2090-0058
                2019
                24 February 2019
                : 2019
                : 9028483
                Affiliations
                Department of Ophthalmology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China
                Author notes

                Academic Editor: Carsten H. Meyer

                Author information
                http://orcid.org/0000-0003-2174-4669
                Article
                10.1155/2019/9028483
                6409019
                0e2e397e-d75d-4b3a-b223-de1176a79d55
                Copyright © 2019 Shengyu Wu et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 15 October 2018
                : 23 December 2018
                : 23 January 2019
                Funding
                Funded by: National Natural Science Foundation of China
                Award ID: 81271024
                Categories
                Research Article

                Ophthalmology & Optometry
                Ophthalmology & Optometry

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