Shikun He - Academia.edu (original) (raw)

Papers by Shikun He

Investigative Opthalmology & Visual Science, 2015

The purpose of this study was to evaluate expression of methyl-CpG-binding protein 2 (MeCP2) in e... more The purpose of this study was to evaluate expression of methyl-CpG-binding protein 2 (MeCP2) in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR) and to investigate effects of inhibition of MeCP2 and DNA methylation on transforming growth factor (TGF)-β-induced retinal pigment epithelial (RPE) cell transdifferentiation. Expression of MeCP2 and its colocalization with cytokeratin and α-smooth muscle actin (α-SMA) in surgically excised PVR membranes was studied using immunohistochemistry. The effects of 5-AZA-2&amp;amp;amp;amp;amp;amp;amp;#39;-deoxycytidine (5-AZA-dC) on human RPE cell migration and viability were evaluated using a modified Boyden chamber assay and the colorimetric 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Expression of RASAL1 mRNA and its promoter region methylation were evaluated by real-time PCR and methylation-specific PCR. Effects of 5-AZA-dC on expression of α-SMA, fibronectin (FN), and TGF-β receptor 2 (TGF-β R2) and Smad2/3 phosphorylation were analyzed by Western blotting. Effect of short interfering RNA (siRNA) knock-down of MeCP2 on expression of α-SMA and FN induced by TGFβ was determined. MeCP2 was abundantly expressed in cells within PVR membranes where it was double labeled with cells positive for cytokeratin and α-SMA. 5-AZA-dC inhibited expression of MeCP2 and suppressed RASAL1 gene methylation while increasing expression of the RASAL1 gene. Treatment with 5-AZA-dC significantly suppressed the expression of α-SMA, FN, TGF-β R2 and phosphorylation of Smad2/3 and inhibited RPE cell migration. TGF-β induced expression of α-SMA, and FN was suppressed by knock-down of MeCP2. MeCP2 and DNA methylation regulate RPE transdifferentiation and may be involved in the pathogenesis of PVR.

Cytokine, Jan 11, 2015

SIRT1, a NAD(+) -dependent histone deacetylase, has been shown to act as a key regulator of angio... more SIRT1, a NAD(+) -dependent histone deacetylase, has been shown to act as a key regulator of angiogenesis. The purpose of this study was to determine the effects of resveratrol (RSV, a SIRT1 activator) on the vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway and to establish its relevance to choroidal neovascularization (CNV), a blinding complication of age-related macular degeneration. Western blot and ELISA assay showed that RSV inhibited hypoxia-inducible factor (HIF)-1α accumulation and VEGF secretion induced by cobalt chloride (CoCl2) through SIRT1 in human retinal pigment epithelial (hRPE) cells. Furthermore, RSV down-regulated VEGFR2 phosphorylation and activation induced by VEGF in endothelial cells via SIRT1. Thus, the inhibitory effect of RSV on the HIF-1α/VEGF/VEGFR2 signaling axis is mediated, at least in part, through SIRT1. The results suggest that targeting SIRT1 could have therapeutic potential for the treatment of CNV.

PloS one, 2015

Choroidal neovascularization (CNV) is a blinding complication of age-related macular degeneration... more Choroidal neovascularization (CNV) is a blinding complication of age-related macular degeneration that manifests as the growth of immature choroidal blood vessels through Bruch's membrane, where they can leak fluid or hemorrhage under the retina. Here, we demonstrate that the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) can down-regulate the pro-angiogenic hypoxia-inducible factor-1α and vascular endothelial growth factor (VEGF), and up-regulate the anti-angiogenic and neuro-protective pigment epithelium derived factor in human retinal pigment epithelial (RPE) cells. Most strikingly, TSA markedly down-regulates the expression of VEGF receptor-2 in human vascular endothelial cells and, thus, can knock down pro-angiogenic cell signaling. Additionally, TSA suppresses CNV-associated wound healing response and RPE epithelial-mesenchymal transdifferentiation. In the laser-induced model of CNV using C57Bl/6 mice, systemic administration of TSA significantly reduces fluore...

Molecular vision, 2013

Epigenetics has become an increasingly important area of biomedical research. Increasing evidence... more Epigenetics has become an increasingly important area of biomedical research. Increasing evidence shows that epigenetic alterations influence common pathologic responses including inflammation, ischemia, neoplasia, aging, and neurodegeneration. Importantly, epigenetic mechanisms may have a pathogenic role in many complex eye diseases such as corneal dystrophy, cataract, glaucoma, diabetic retinopathy, ocular neoplasia, uveitis, and age-related macular degeneration. The emerging emphasis on epigenetic mechanisms in studies of eye disease may provide new insights into the pathogenesis of complex eye diseases and aid in the development of novel treatments for these diseases.

PURPOSE. To determine the sensitivity of retinal pigment epithelial (RPE) cells to a vascular end... more PURPOSE. To determine the sensitivity of retinal pigment epithelial (RPE) cells to a vascular endothelial growth factor (VEGF) chimeric toxin. METHODS. A targeted toxin was developed using recombinant methods to fuse VEGF165 to the diphtheria toxin (DT) translocation and enzymatic domain (DT390-VEGF165). Human RPE cells, choroidal endothelial cells (CECs), and scleral fibroblasts were isolated, and a dose-response for DT390-VEGF165 was

PURPOSE. To study the mechanism of the protective effect of hepatocyte growth factor (HGF) in oxi... more PURPOSE. To study the mechanism of the protective effect of hepatocyte growth factor (HGF) in oxidative injury to RPE cells induced by glutathione (GSH) depletion. METHODS. RPE cells were treated with HGF for 24 hours (20 ng/mL) and then were treated with DL-buthionine-(S,R)-sul- foximine (BSO) for an additional 24 hours. Cell death, apopto- sis, and GSH levels were measured. Levels

Investigative ophthalmology & visual science, 1999

To characterize the phagocytosis of extracellular matrix components by retinal pigment epithelial... more To characterize the phagocytosis of extracellular matrix components by retinal pigment epithelial cells and to determine which receptors and signal transduction pathways are involved. Fluorescent latex beads were coated with fibronectin (FN), collagen type I or IV, or thrombospondin and incubated with human retinal pigment epithelial cells for 3 hours. Phagocytosis was quantified by flow cytometry. The effects of adhesion blocking antibodies to cell surface receptors (alpha1, alpha3, alpha5, beta1, alpha5beta1, alphavbeta3, alphavbeta5 integrins and CD36) and inhibitors of specific intracellular signaling pathways (tyrosine kinase phosphatidylinositol 3-kinase [PI3-kinase], protein kinase C [PKC], and mitogen-activated protein kinase) were determined using FN-coated beads. Phagocytosis of FN-coated beads was greater than phagocytosis of beads coated with collagen type I, collagen type IV, or thrombospondin or uncoated controls (P < 0.0005). Anti-alpha5, -beta1, and -alpha5beta1 a...

Yan ke xue bao = Eye science / "Yan ke xue bao" bian ji bu, 2006

To investigate the expression of thrombospondin 1 (TSP-1) in retinal pigment epithelium (RPE) and... more To investigate the expression of thrombospondin 1 (TSP-1) in retinal pigment epithelium (RPE) and choroidal neovascular membranes (CNVMs) from patients with age-related macular degeneration (AMD). Tissue sections from normal human fetal and adult eyes and surgically removed CNVMs were immunostained for TSP-1 localization. Polymerase chain reaction and Western blotting were used to analyze TSP-1 mRNA and protein from human RPE cells, respectively. TSP-1 in the supernatant of cultured RPE cells and eye explants were measured using enzyme-linked immunosorbent assay. MTT assay was used to evaluate the RPE survival after TSP-1 treatment. The strongest immunostaining for TSP-1 was observed in the RPE monolayer around drusen in early AMD. The intensity of TSP-1 staining in normal eye sections was much weaker than that of early AMD and CNVM. TSP-1 mRNA was positive in cultured fetal and adult RPE cells. There was increasing secretion of TSP-1 into the supernatant of cultured RPE and eye exp...

Nature protocols, 2007

We outline current in vitro and in vivo models for experimental proliferative vitreoretinopathy (... more We outline current in vitro and in vivo models for experimental proliferative vitreoretinopathy (PVR) and provide a detailed protocol of our standardized in vivo PVR model. PVR is the leading cause of failed surgical procedures for the correction of rhegmatogenous retinal detachment. The pathogenesis of this multifactorial condition is still not completely understood. Experimental models for PVR help us understand the factors that play a role in the pathogenesis of the disease process in a controlled manner and allow for reproducible preclinical assessment of novel therapeutic interventions. We describe a cell injection model in detail that uses homologous retinal pigment epithelial (RPE) cell cultures to induce PVR over a 2-8 week period.

Investigative ophthalmology & visual science, 2001

To determine whether vascular endothelial growth factor (VEGF) regulates angiopoietin (Ang)-1 and... more To determine whether vascular endothelial growth factor (VEGF) regulates angiopoietin (Ang)-1 and -2 expression in retinal pigment epithelial (RPE) cells. Expression of VEGF, Ang1, and Ang2 in surgically removed human choroidal neovascular membranes (CNVMs) was analyzed by double-label confocal immunofluorescence microscopy. Total RNA was extracted from cultured human RPE cells treated with VEGF for mRNA analysis. Northern blot analysis was performed to examine the time course and dose response of Ang1 and Ang2 mRNA expression. mRNA stability and nuclear run-on analyses were performed. Secreted Ang1 and Ang2 protein levels in conditioned media from RPE cells were examined by Western blot analysis. Ang1 and Ang2 immunostaining colocalized with VEGF-positive stromal cells in human CNVMS: Ang1 and Ang2 mRNAs were expressed by cultured serum-starved RPE cells. VEGF upregulated Ang1 mRNA in a time- and dose-dependent manner without a significant change in Ang2 mRNA. Ang1 and Ang2 mRNAs i...

Investigative ophthalmology & visual science, 2002

To evaluate the effect of hepatocyte growth factor (HGF) on the integrity and function of tight j... more To evaluate the effect of hepatocyte growth factor (HGF) on the integrity and function of tight junctions and adherens junctions in the retinal pigment epithelial (RPE) monolayer. Fresh bovine eyes were dissected to obtain 2- to 3-mm(2) explants of intact RPE with underlying choroid and sclera. Explants were cultured with or without HGF (20 ng/mL) for various periods (20 minutes to 72 hours). Junction integrity was assessed by transmission and scanning electron microscopy; localization, expression, and phosphorylation of junction proteins; and measurement of transepithelial resistance (TER), diffusion of fluorescent labeling in the plasma membrane, and the migration of RPE cells from the monolayer. Untreated explants consisted of polarized cells with apical microvilli and well-developed tight and adherens junctions. After HGF treatment, the explants showed loss of tight and adherens junctions ultrastructurally, diffusion of fluorescent label from apical to lateral membrane domains, ...

PURPOSE. To determine the antiangiogenic effects of peroxisome proliferator-activated receptor (P... more PURPOSE. To determine the antiangiogenic effects of peroxisome proliferator-activated receptor (PPAR)-g agonists on ocular cells involved in the pathogenesis of choroidal neovascularization (CNV) in vitro and on experimental laser photocoagulation-induced CNV in vivo. METHODS. PPAR-g expression in human retinal pigment epithelial (RPE) cells and bovine choroidal endothelial cells (CECs) was determined using an RNase protection assay and Western blot analysis.

Retina, 2006

Matrix metalloproteinases (MMP)-2 and -9 play an important role in the pathogenesis of choroidal ... more Matrix metalloproteinases (MMP)-2 and -9 play an important role in the pathogenesis of choroidal neovascularization (CNV). Retinal pigment epithelial cells (RPE) are an important source of MMPs in the outer retinal environment, however little is known about the local factors that modulate MMP secretion in these cells. The purpose of this study was to determine the effects of CNV involved growth factors and the extracellular matrix molecule fibronectin on MMP-2 and -9 secretion by cultured human RPE. MMP-2 and -9 secretion was studied using gelatin zymography, Western blot, and ELISA assay of RPE culture supernatants. The effects of stimulating the cells for 36 hours with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bGFG), tumor necrosis factor-alpha (TNF-alpha), or fibronectin (FN), all angiogenic factors found in CNV membranes, was determined. Resting RPE cells secreted MMP-2 but not MMP-9. Stimulation with TNF-alpha induced secretion of MMP-9 and increased the secretion of MMP-2. MMP-2 secretion was also increased by stimulation with FN and VEGF, but not bFGF. The results indicated that the angiogenic molecules VEGF, FN, and TNF-alpha stimulate MMP-2 and -9 secretion from RPE and thus further promote CNV.

Neurosurgery, 1994

Hypericin, a polycyclic aromatic dione isolated from plants, is presently being clinically evalua... more Hypericin, a polycyclic aromatic dione isolated from plants, is presently being clinically evaluated as an antiviral agent in the treatment of human immunodeficiency virus (HIV) infection. In addition, it is known to be a potent protein kinase C inhibitor. To evaluate its potential as an inhibitor of glioma growth, an established (U87) and low-passage glioma line (93-492) were treated with hypericin in tissue culture for a period of 48 hours after passage. Hypericin inhibited the glioma growth in a dose-related manner, with a marked inhibition of growth in the low-micromolar concentration range (e.g., in line U87 and low-passage line 93-492, a concentration of hypericin of 10 mumol/L produced 62 and 76% decreases in [3H]thymidine uptake, respectively). Because the reported inhibitory effects of protein kinase C are enhanced by visible light, [3H]thymidine uptake was measured in both the presence and the absence of visible light. In glioma line A172, the presence of light slightly increased the inhibitory effect of hypericin. Moreover, an apoptosis (i.e., programmed cell death) assay was performed to determine whether the treatment of glioma cells with hypericin was cytostatic or cytocidal. Cells were harvested, and purified deoxyribonucleic acid (DNA) was analyzed by agarose gel electrophoresis. DNA from cells treated with hypericin for 48 hours exhibited a classical "ladder" pattern of oligonucleosome-sized fragments characteristic of apoptosis. These data suggest that the proven safe drug hypericin may have potential as an antiglioma agent; we suggest clinical trials.

Journal of Neuroimmunology, 2006

Inflammatory mediators have been proposed to play a critical role in the pathogenesis of choroida... more Inflammatory mediators have been proposed to play a critical role in the pathogenesis of choroidal neovascularization, a blinding complication of age-related macular degeneration. We evaluated the expression of TNF-alpha in human choroidal neovascular membranes and found that it colocalized with cells expressing VEGF, angiopoietin (Ang)-1 and Ang2. In cultured choroidal endothelial cells we found that TNF-alpha increased Ang2 mRNA (increased transcription) and protein levels prior to those of Ang1 and VEGF. The results raise the possibility that during neovascularization, TNF-alpha may modulate endothelial plasticity and survival by sequential inactivation of Tie2 followed by activation of Tie2 and VEGF receptors.

Investigative Ophthalmology & Visual Science, 2010

EphB4 receptor (EphB4) and its ligand (EphrinB2) play an important role in the regulation of cell... more EphB4 receptor (EphB4) and its ligand (EphrinB2) play an important role in the regulation of cell adhesion, growth, and migration. The purpose of this study was to determine the effects of EphB4 blockade by soluble EphB4 (sEphB4) on retinal pigment epithelial (RPE) cell migration and proliferation, induced by platelet-derived growth factor-BB (PDGF), and to establish its relevance to proliferative vitreoretinopathy (PVR). The expression of EphB4 and EphrinB2 in early-passage human RPE cells and in human PVR membranes was evaluated by confocal microscopy. The effect of sEphB4 (0.1-3 microg/mL) on PDGF (20 ng/mL)-induced RPE migration and proliferation was evaluated using a modified Boyden chamber assay and an MTT assay, respectively. Attachment to basement membrane matrix and fibronectin was assayed by MTT. Phosphorylation of FAK and p42/44 mitogen-activated protein kinase (MAPK) in retinal pigment epithelium was determined by Western blot analysis after exposure to sEphB4. The effect of sEphB4 on the phosphorylation of EphB4/EphrinB2 was demonstrated with the use of immunoprecipitation assays. EphrinB2 and EphB4 were expressed on human RPE cells in vitro and in cells within human PVR membranes. sEphB4 blocked EphB4 and EphrinB2 phosphorylation in RPE cells in vitro. sEphB4 reduced RPE migration in response to PDGF stimulation (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01). Similarly, sEphB4 inhibited RPE attachment and proliferation in a dose-dependent manner (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). PDGF-induced phosphorylation of FAK and MAPK was inhibited by sEphB4. EphB4 and EphrinB2 are expressed in RPE cells and PVR membranes. sEphB4 inhibits PDGF-induced RPE cell attachment, proliferation, and migration. This effect may result from the inhibition of FAK and MAPK phosphorylation.

Investigative Ophthalmology & Visual Science, 2008

Purpose-To investigate the role of connective tissue growth factor (CTGF) in the pathogenesis of ... more Purpose-To investigate the role of connective tissue growth factor (CTGF) in the pathogenesis of proliferative vitreoretinopathy (PVR).

Investigative Ophthalmology & Visual Science, 2005

The purpose of this study was to evaluate the effect of a soluble monomeric form of the EphB4 ext... more The purpose of this study was to evaluate the effect of a soluble monomeric form of the EphB4 extracellular domain (sEphB4) on choroidal endothelial cell (CEC) migration and tube formation and on experimental laser-induced choroidal neovascularization (CNV). EphrinB2 and EphB4 expression in CECs was investigated by Western blot analysis and immunohistochemistry. Effects of sEphB4 (0.5-3 microg/mL) on CEC migration were evaluated with a modified Boyden chamber assay. Tube formation was assayed in CEC cultures in collagen gel. CNV was induced in rats by laser photocoagulation. The effects of intravitreal injection of sEphB4 on CNV development were evaluated at day 14 by fluorescein angiography (FA), confocal volumetric analysis of isolectin-B4 labeled flatmounts, and histologic examination of CNV membranes. CEC cells express both EphB4 and EphrinB2, according to Western blot analysis. Immunohistochemical sections of rat eye showed immunoreactivity for both EphB4 and EphrinB2 in the choroidal endothelium. sEphB4 reduced CEC migration in response to vascular endothelial growth factor (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01). Similarly, sEphB4 inhibited CEC tube formation in a dose-dependent manner. EphB4, and to a lesser extent EphrinB2, were detected on vascular channels within laser-induced CNV membranes. Intravitreal injection of sEphB4 inhibited laser-induced CNV formation. CNV membranes showed a reduction in leakage score (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), and membrane volumes were reduced in size (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Histologic analysis revealed that vascularity was reduced in sEphB4-treated membranes. Recombinant soluble monomeric EphB4 exerts an inhibitory effect on choroidal angiogenesis in vitro and in vivo. It should be further evaluated for its potential as a novel therapy for CNV.

Graefe's Archive for Clinical and Experimental Ophthalmology, 2008

Induction of glucose-regulated protein (GRP)-78 in the endoplasmic reticulum (ER) is a protective... more Induction of glucose-regulated protein (GRP)-78 in the endoplasmic reticulum (ER) is a protective mechanism cells use to adapt to ER stress. We evaluated the expression of GRP-78 and its regulation by an oxidant tert-butyl hydroperoxide (tBH) in human retinal pigment epithelium (RPE) cells. We used a carboxy-H2-DCFDA staining method to detect tBH-induced accumulation of reactive oxygen species (ROS) in RPE cells, and analyzed the expression of GRP-78 in normal human fetal and adult retinas and in cultured human RPE cells by immunohistochemistry. The effects of tBH (10-100 microM) on GRP-78 and on growth arrest and DNA damage inducible genes 153 (GADD153) protein and mRNA expression were studied using Western blot and real-time polymerase chain reaction. Sections of fetal retinas were negative for GRP-78. Adult retinas showed moderate cytoplasmic GRP-78 staining in the RPE and choroid. tBH-induced ROS accumulation in RPE cells showed partial colocalization with the ER. GRP-78 and GADD153 mRNA and protein expression in cultured RPE cells were significantly upregulated by treatment with tBH. tBH increases oxidative stress, increases accumulation of ROS in the ER, and upregulates expression of GRP-78 and GADD153. This supports the connection between oxidative stress and ER stress, and suggests that GRP-78 may serve a protective role in the RPE response to oxidative stress.