Chronic photo-oxidative stress and subsequent MCP-1 activation as causative factors for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries. Although pathogenic factors, such as oxidative stress, inflammation and genetics are thought to contribute to the development of AMD, little is known about the relationships and priorities between these factors. Here, we show that chronic photo-oxidative stress is an environmental factor involved in AMD pathogenesis. We first demonstrated that exposure to light induced phospholipid oxidation in the mouse retina, which was more prominent in aged animals. The induced oxidized phospholipids led to an increase in the expression of monocyte chemoattractant protein-1, which then resulted in macrophage accumulation, an inflammatory process. Antioxidant treatment prevented light-induced phospholipid oxidation and the subsequent increase of monocyte chemoattractant protein-1 (also known as C-C motif chemokine 2; CCL2), which are the beginnings of the light-induced changes. Subretinal application of oxidized phospholipids induced choroidal neovascularization, a characteristic feature of wet-type AMD, which was inhibited by blocking monocyte chemoattractant protein-1. These findings strongly suggest that a sequential cascade from photic stress to inflammatory processes through phospholipid oxidation has an important role in AMD pathogenesis. Finally, we succeeded in mimicking human AMD in mice with low-level, long-term photic stress, in which characteristic pathological changes, including choroidal neovascularization formation, were observed. Therefore, we propose a consecutive pathogenic pathway involving photic stress, oxidation of phospholipids and chronic inflammation, leading to angiogenesis. These findings add to the current understanding of AMD pathology and suggest protection from oxidative stress or suppression of the subsequent inflammation as new potential therapeutic targets for AMD.

Bioinformatics and in vitro study reveal the roles of microRNA-346 in high glucose-induced human retinal pigment epithelial cell damage.

AIM: To study microRNAs (miRNAs) and their potential effects in high glucose-induced human retinal pigment epithelial cell damage. METHODS: We screened the GSE52233 miRNA expression dataset for differentially expressed miRNAs (DEMs). The target genes of the top 10 DEMs were predicted using miRWalk 2.0 database, followed by function enrichment and protein-protein interaction analysis. miRNA expression was determined in the human retinal pigment epithelial cell line ARPE-19 treated with high glucose (HG) by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Cell proliferation was determined using cell counting kit (CCK)-8 assay. Cell cycle, apoptosis, and reactive oxygen species (ROS) levels were determined by flow cytometry. The direct interaction between miRNA and targets was validated using dual-luciferase reporter assay. RESULTS: Thirty-nine DEMs were screened, and we predicted 125 miRNA-mRNA pairs for the top 10 DEMs, including 119 target genes of seven DEMs such as miR-346, which was upregulated in diabetic retinopathy (DR). miR-346 target genes were substantially enriched in the regulation of intracellular transport and retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathway. Expression of three upregulated and downregulated miRNAs were verified by qRT-PCR in HG-treated ARPE-19 cells. Expression of miR-346 was elevated in HG treated ARPE-19 cells in a dose-dependent manner. HG inhibited cell proliferation and induced apoptosis, which were partly reversed by transfecting an miR-346 inhibitor, which even decreased the ROS levels elevated due to HG. Argonaute 2 (AGO2) was a target of miR-346. CONCLUSION: miR-346 is a key miRNA and plays an important role in HG-induced damage in human retinal pigment epithelial cells.

Clinical research of EX-PRESS drainage device and modified trabeculectomy combined with intravitreal conbercept treatment for neovascular glaucoma.

AIM: To evaluate the efficacy and safety of modified trabeculectomy (experimental group) and implantation of EX-PRESS drainage device (control group), combined with intravitreal conbercept injection for neovascular glaucoma (NVG). METHODS: Totally 30 patients with NVG were selected from June 2014 to June 2017, and randomly divided into experimental group and control group. All patients were underwent intravitreal conbercept (0.5 mg/0.05 mL) treatment before surgery. Modified trabeculectomy was performed in MT group, while EX-PRESS drainage device implantation was performed in EX group. The success rates, best corrected visual acuity (BCVA), intraocular pressure (IOP), filtering bleb and complications were observed and compared. RESULTS: The differences of success rate, BCVA and filtering bleb were not statistically significant 12mo after the surgery (P>0.05), however, the difference of IOP at 1d, 1wk, 1, 3, and 6mo after surgery was statistically significant (F time=390.64, P time<0.0001) between two groups. The interactions between two groups in the given time showed no significant difference (F intergroup×time=0.181, P intergroup×time=0.57), and also there was no significant difference in IOP between the two groups (F=3.16, P=0.09). The results of pairwise comparison at each time point showed no significant difference in IOP between 1d and 1wk, 3 and 6, 3mo and 12mo after surgery (P>0.05), while the results at other time point indicate statistical differences (P<0.05). CONCLUSION: The modified trabeculectomy and the implantation of EX-PRESS drainage device have clinical application value in reducing IOP and postoperative complications of refractory NVG.

Safety threshold of intravitreal activated protein-C.

PURPOSE: Although activated protein C (APC) is effective in preventing the death of retinal neurons in ischemic retinopathy, it is not known at what concentrations it becomes retinotoxic. The purpose of this study was to determine the concentrations of intravitreal APC that are safe and those that are toxic for the retina, using rabbit eyes. METHODS: The left eyes of 12 rabbits received an intravitreal injection of 1.5 to 150 µg of APC in 0.1 ml of saline. The fellow eyes were treated with an intravitreal injection of the same amount of saline. Slit-lamp examination, fundus examination, fluorescein angiography (FA), and electroretinograms (ERGs) were performed before and at different times after the injection. The eyes were enucleated at 6 months after the injection and examined histologically. RESULTS: The clinical and histological differences between the control eyes and the eyes that had APC injections up to 15 µg were not significant. Localized retinal edema was observed in two of three eyes with 150 µg of APC immediately after the injection. In these two eyes, chorioretinal atrophy was observed in the area of the retinal edema at 6 months, which corresponded with a hyperfluorescent area in the FA images and focal retinal degeneration histologically. No significant changes were detected in the full-field ERGs in the eyes treated even with 150 µg of APC throughout the observation period. CONCLUSION: Our results show that an intravitreal injection of APC at a dose ≤15 µg is safe, but 150 µg of APC can be toxic to the retina.

Integrin ß1 subunit signaling is involved in the directed migration of human retinal pigment epithelial cells following electric field stimulation.

AIMS: Direct current electric fields (EFs) can induce directed cell migration in a wide variety of cells, and this has been proven to be of importance in wound healing. Here we observed the effects of EFs on cultured human retinal pigment epithelial (RPE) cells and explored the possible involvement of integrin ß1 subunit signaling in the process. METHODS: Cultured human RPE cells were exposed to an EF at 5 V/cm for 3 h. The rate and directionality of cell migration were quantified. The distribution of integrin ß1 subunit was measured by immunohistochemistry and the expression of integrin ß1 subunit and phosphorylated focal adhesion kinase (FAK) was determined by PCR and Western blotting. Experiments were performed in the presence or absence of anti-integrin ß1 subunit antibody. RESULTS: During exposure to an EF at 5 V/cm for 3 h, the separated human RPE cells and wounded RPE monolayer demonstrated a cathodal-directed migration. The distribution of integrin ß1 subunit in the cells was also polarized to the cathode, and the expression in mRNA and its protein level were obviously increased. Furthermore, exposure to EFs of 5 V/cm triggered the phosphorylation of FAK in human RPE cells. In contrast, blocking of integrin ß1 subunit suppressed the directed migration of RPE cells and reduced the activation of FAK in EFs. CONCLUSIONS: These findings indicate that EF exposure results in directed migration of the separated RPE cells and RPE monolayer. These effects may partially act through the activation of integrin ß1 subunit signaling.

Magnetic nanoparticles conjugated with "RPE cell -MCP-1 antibody -VEGF antibody" compounds for the targeted therapy of age-related macular degeneration: a hypothesis.

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly throughout the world. Treatment of AMD utilizing retinal pigment epithelium (RPE) transplantation represents a promising therapy. However, simplex RPE transplantation can only replace the diseased RPE cells, but has no abilities to stop the development of AMD. It has been indicated that oxidization triggers the development of AMD by inducing the dysfunction and degeneration of RPE cells, which results in the upregulation of local monocyte chemotactic protein-1 (MCP-1) expression. MCP-1 induces macrophage recruiment which triggers local inflammation. As a result, the expression of vascular endothelial growth factor (VEGF) is upregulated by MCP-1 mediated inflammation and results in the formation of choroidal neovascularization (CNV). We accordingly propose a targeted therapy of AMD by subretinal transplanting the compound of RPE cell, MCP-1 antibody, and VEGF antibody and using a magnetic system to guide RPE cell compounds conjugated with superparamagnetic iron oxide nanoparticles (SPIONs). Furthermore, SPION-labelled RPE cells can be tracked and detected in vivo by non-invasive magnetic resonance imaging (MRI). This novel RPE cell transplantation methodology seems very promising to provide a new therapeutic approach for the treatment of AMD.

Ras-related C3 botulinum toxin substrate 1 activation is involved in the pathogenesis of diabetic retinopathy.

This study used a streptozotocin (STZ)-induced rat model of diabetes to investigate whether Ras-related C3 botulinum toxin substrate 1 (Rac1) was involved in the pathogenesis of diabetic retinopathy. The effects of Rac1 inhibition on vascular endothelial (VE)-cadherin and ß-catenin expression in high glucose-induced rat retinal endothelial cells (RRECs) were additionally examined. Rac1 activation in the retinas from STZ-induced diabetic rats and in high glucose-induced RRECs was measured by reverse transcription-quantitative polymerase chain reaction analysis, immunohistochemistry and western blot analysis. The expression levels of VE-cadherin and ß-catenin were also examined with or without Rac1 inhibition through small interfering (si)RNA transfection. STZ-induced diabetes was associated with an increase in the vascular permeability of the retina. Furthermore, Rac1 activation was increased in the retina of STZ-induced diabetic rats and in high glucose-induced RRECs compared with that in the controls. Immunohistochemistry showed that immunostaining of Rac1 was localized in the outer plexiform, inner nuclear, inner plexiform and ganglion cell layers and in the retinal microvasculature of rats. The expression of ß-catenin was increased in the retinas of the diabetic rats at four, eight and 12 weeks after the induction of diabetes compared with that in the controls. Additionally, Rac1 activation was required for the high glucose-induced VE-cadherin expression decrease and for ß-catenin expression in high glucose-induced RRECs. Rac1 inhibition by Rac1-siRNA transfection effectively prevented hyperpermeability, ß-catenin expression and the VE-cadherin expression decrease in high glucose-induced RRECs. In conclusion, diabetes affects the expression of Rac1 in the retina. Rac1 may be involved in the diabetes-induced damage and/or alterations to the blood-retinal barrier through changes in VE-cadherin and ß-catenin expression.

Activated protein C rescues the retina from ischemia-induced cell death.

PURPOSE: Ischemia causes severe and persistent visual loss in many eye diseases, including central retinal vein occlusion (CRVO) and diabetic retinopathy. Activated protein C (APC) has been demonstrated to reduce the cell death associated with ischemia in the brain and kidney. This study was performed to examine the ability of APC to rescue hypoxia-induced retinal cell death in vitro and in vivo. METHODS: Retinal pigment epithelium (RPE) and photoreceptor cells were placed in either a normoxic or a hypoxic chamber. Immediately before they were subjected to ischemia, the cultures were treated with APC (3-240 µg/mL). Incubation was followed by an MTT assay to determine the number of viable cells. The activity of caspase-3, -8, and -9 in RPE cells was also analyzed. Various concentrations of APC were intravitreally injected in a rat CRVO model, followed by TUNEL staining to detect the in vivo effects of APC. RESULTS: Lower concentrations of APC (0.3-30 µg/mL) showed a cell-protective effect against hypoxia in vitro, whereas higher concentrations (≥120 µg/mL) demonstrated cytotoxicity in both RPE and photoreceptor cells. Caspase-3, -8, and -9 were activated when the cells were exposed to hypoxia, but this activation was significantly inhibited by APC. Experimental CRVO-induced retinal cell apoptosis was reduced dramatically by intravitreal injection of APC. CONCLUSIONS: APC can reduce ischemia-induced cytotoxicity both in vitro and in vivo via blocking the activation of caspase-3, -8, and -9. APC may be a promising candidate for protecting the retina from ischemia.

Coordinated expression of Ets-1, pERK1/2, and VEGF in retina of streptozotocin-induced diabetic rats.

PURPOSE: To investigate the role played by E26 transformation-specific-1 (Ets-1), a transcription factor, and extracellular signal-regulated kinase 1/2 (ERK1/2) in the expression of vascular endothelial growth factor (VEGF), and the interaction of Ets-1 and ERK1/2 in the retina of diabetic rats. METHODS: Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). To follow the time course in the expression of Ets-1, phosphorylated ERK1/2 (pERK1/2), and VEGF, rats were killed at 1, 2, 4, and 8 weeks after the injection of STZ, and total proteins were extracted from the isolated retinas. An adenovirus vector encoding dominant-negative Ets-1 and an inhibitor of PD98059 was injected intravitreally to investigate the effects of Ets-1 blockade and ERK1/2 inhibition on the expression of VEGF. Four weeks after the first intravitreal injection, total proteins and total RNA were extracted from the retinas for Western blot and Northern blot analyses. RESULTS: The expression of Ets-1, pERK1/2, and VEGF in the retina increased in a time-dependent manner after STZ injection. The phosphorylation of ERK1/2 and protein level of VEGF were significantly reduced following intravitreal Ets-1. Inhibition of ERK1/2 phosphorylation resulted in a significant reduction in the expression of Ets-1 and the level of VEGF protein. CONCLUSIONS: These results indicate that in the retina of STZ-induced diabetic rats: (1) the alterations of Ets-1, pERK1/2, and VEGF are approximately synchronized; (2) the phosphorylation of ERK1/2 is regulated by the expression of Ets-1; (3) the production of Ets-1 protein is dependent on the ERK1/2 pathway, and (4) the protein level of VEGF is regulated by both Ets-1 expression and ERK1/2 phosphorylation. We propose that VEGF, Ets-1, and ERK1 act synergistically in the development of diabetic retinopathy.

Up-regulation of integrin-linked kinase in the streptozotocin-induced diabetic rat retina.

OBJECTIVES: This study investigated whether integrin-linked kinase (ILK) is involved in the pathogenesis of diabetic retinopathy, by analyzing the expression and activity of ILK in the retina from a streptozotocin (STZ)-induced rat model of diabetes. METHODS: ILK expression in the retina from both control and STZ-induced diabetic rats was measured by reverse transcription polymerase chain reaction, immunohistochemistry and Western blot analysis. The expressions of Akt and FOXO1A, the downstream molecules of ILK, were also examined. RESULTS: The present study showed that the STZ-induced diabetes was associated with the increase in the vascular permeability in the retina. This elevated vascular permeability increased with the progression of diabetic retinopathy. Meanwhile, the results also showed that the expression of ILK increased in protein and mRNA levels in the retina of STZ-induced diabetic rats. Immunohistochemistry showed that immunostaining of ILK was localized in the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), the ganglion cell layer (GCL) and the retinal microvasculature of rats. However, the expression of Akt was reduced in the retinas at 8 and 12 weeks and increased in the retinas at 4 weeks after induction of diabetes. Meanwhile, the expression of the FOXO1A protein increased in the retinas at 8 and 12 weeks and decreased in the retinas at 4 weeks after induction of diabetes. The FOXO1A immunostaining was also observed in the retinal microvasculature and in the OPL, INL, IPL and GCL of rat retinas. CONCLUSION: These results indicate that diabetes affects the expression of ILK in the retina. ILK may be involved in the diabetes-induced damage and/or alterations of neural and microvascular structures.

[The effect of copolymer-1 on the apoptosis and the IL-6R expression of the retinal ganglion cells in chronic elevated intraocular pressure rat model].

AIM: To observe the effect of autoimmune response induced by copolymer-1 (COP-1) on apoptosis of retinal ganglion cells (RGCs) and IL-6R expression on the RGCs in chronic elevated intraocular pressure (EIOP) rat models. METHODS: Thirty SD rats were randomly divided into 3 groups, namely normal control group, mock-immunized EIOP group and COP-1-immunized EIOP group. Cauterization of episcleral vein was used to set up rat's EIOP model. The normal control rats were not immunized, whereas the rats in the other two groups were immunized via i.p injection with normal saline and COP-1 at hindfeet, respectively. The expression of the IL-6R on RGCs was detected by immunohistochemical staining. The apoptosis of the RGCs was examined by TUNEL staining. RESULTS: The number of the apoptotic RGCs in the COP-1- immunized EIOP group was notably lower than that in mock-immunized EIOP group (P<0.05). IL-6R were expressed on RGCs in all 3 groups, and expression level of IL-6R increased in the following order: normal control group, mock-immunized EIOP group and COP-1-immunized EIOP group (P<0.05). CONCLUSION: The autoimmune response induced by COP-1 protects the RGCs from apoptosis under the condition of chronic EIOP and results in increased IL-6R expression on RGCs. These results suggest that increased IL-6R expression on RGCs induced by COP-1 immunization with the protection of neurons resulted from autoimmune response is related.