Critical Role of VEGF as a Direct Regulator of Photoreceptor Function.

Vascular endothelial growth factor (VEGF or VEGF-A), a major pathogenic factor for diabetic and hypoxic blood-retina barrier (BRB) diseases, has been shown to act as a direct functional regulator for neurons in the peripheral and central nerve systems. To determine if VEGF plays a direct role in regulating retinal neuronal function, we established specific experimental procedures and examined the effect of recombinant VEGF (rVEGF) on photoreceptor function with electroretinography (ERG) in mice. In our case, rVEGF caused a significant reduction of scotopic ERG a-wave and b-wave amplitudes and photopic ERG b-wave amplitudes in a dose-dependent manner in dark-adapted wild-type (WT) mice, shortly after the intravitreal delivery of rVEGF in dark. However, the effect of rVEGF on photoreceptor function was nullified in adult Akita diabetic mice. Our data strongly suggest that VEGF is a direct regulator of photoreceptor function and VEGF upregulation contributes significantly to the diabetes-induced reduction of photoreceptor function. In this chapter, we will discuss the relevant background, key experimental procedures and results, and clinical significance of our work.

LONG-TERM EVOLUTION OF MYOPIC RETINOSCHISIS WITH A DOME-SHAPED MACULA AND PREDICTORS OF PROGRESSION AND VISUAL PROGNOSIS.

PURPOSE: To study the long-term natural course of myopic retinoschisis (MRS) with a dome-shaped macula (DSM) and to identify the factors affecting its development and visual prognosis. METHODS: In this retrospective case series study, we followed 25 MRS eyes with a DSM and 68 MRS eyes without a DSM for at least two years and observed changes in optical coherence tomography morphologic features and best-corrected visual acuity. RESULTS: During the mean follow-up of 48.3 ± 13.24 months, the difference in the rate of MRS progression between the DSM and non-DSM groups was not significant ( P = 0.7462). In the DSM group, the patients whose MRS progressed were older and had a higher refractive error than those whose MRS was stable or improved ( P = 0.0301 and 0.0166, respectively). The patients whose DSM was located in the central fovea had a significantly higher progression rate than those whose DSM was located in the parafovea ( P = 0.0421). For all DSM eyes, BCVA did not decrease significantly in eyes with extrafoveal retinoschisis ( P = 0.2500), patients whose best-corrected visual acuity decreased more than two lines had a greater central foveal thickness initially than those whose best-corrected visual acuity decreased less than two lines during the follow-up period ( P = 0.0478). CONCLUSION: A DSM did not delay the progression of MRS. The development of MRS in DSM eyes was associated with age, myopic degree, and DSM location. A higher schisis cavity predicted visual deterioration, and a DSM protected visual function in extrafoveal MRS eyes during the follow-up period.

VEGF as a Direct Functional Regulator of Photoreceptors and Contributing Factor to Diabetes-Induced Alteration of Photoreceptor Function.

Vascular endothelial growth factor (VEGF) is a major therapeutic target for blood-retina barrier (BRB) breakdown in diabetic retinopathy (DR), age-related macular degeneration (AMD), and other hypoxic retinal vascular disorders. To determine whether VEGF is a direct regulator of retinal neuronal function and its potential role in altering vision during the progression of DR, we examined the immediate impact of recombinant VEGF (rVEGF) on photoreceptor function with electroretinography in C57BL6 background wild-type (WT) and Akita spontaneous diabetic mice. Shortly after intravitreal injections, rVEGF caused a significant reduction of scotopic ERG a-wave and b-wave amplitudes and photopic ERG b-wave amplitudes in a dose-dependent manner in dark-adapted 1.5-mo-old WT mice. Compared with WT controls, 5-mo-old Akita spontaneous diabetic mice demonstrated a significant reduction in scotopic ERG a-wave and b-wave amplitudes and photopic ERG b-wave amplitudes. However, the effect of rVEGF altered photoreceptor function in WT controls was diminished in 5-mo-old Akita spontaneous diabetic mice. In conclusion, our results suggest that VEGF is a direct functional regulator of photoreceptors and VEGF up-regulation in DR is a contributing factor to diabetes-induced alteration of photoreceptor function. This information is critical to the understanding of the therapeutic effect and to the care of anti-VEGF drug-treated patients for BRB breakdown in DR, AMD, and other hypoxic retinal vascular disorders.

CCAAT/Enhancer-Binding Protein ß Mediates Oxygen-Induced Retinal Neovascularization via Retinal Vascular Damage and Vascular Endothelial Growth Factor.

OBJECTIVE: To evaluate the role of CCAAT/enhancer-binding protein ß (C/EBP ß (C/EBP. METHODS: Rats with OIR were exposed to alternating hypoxic and hyperopic conditions for 14 days. Then, the rats with OIR were assigned randomly to groups that received intravitreal injections of either shRNA lentiviral particles targeting C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP. RESULTS: In OIR rats, the expression levels of C/EBP ß (C/EBP P < 0.01). The p-C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP P < 0.01). The p-C/EBP ß (C/EBP ß (C/EBP ß (C/EBP P < 0.01). The p-C/EBP. CONCLUSIONS: C/EBP ß shRNA inhibits RNV in OIR. A potential mechanism may be that the activity of C/EBP ß increases with its overexpression, which in turn aggravates the amount of the retinal vascular damage and promotes transcription of VEGF. C/EBP ß might be a new therapeutic target for preventing RNV.ß (C/EBP ß (C/EBP ß (C/EBP.

Correction to: P66Shc expression in diabetic rat retina.

Following publication of the original article.

P66Shc expression in diabetic rat retina.

BACKGROUND: P66Shc is partially localised within the mitochondrial fraction. It is primarily related to the generation of mitochondrial reactive oxygen species and apoptosis. Based on previous studies, we hypothesize that in the retina, p66Shc may exist and affect the development of diabetic retinopathy. The purpose of this study was to investigate p66Shc expression in retinal in streptozotocin-induced diabetic (SD) rats, which may provide a pathway to study the pathogenesis of diabetic retinopathy. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to detect retinal p66Shc mRNA and protein expression in SD rats, respectively. Immunohistochemical staining was applied to detect the location of rat retinal p66Shc expression. TUNEL assay was applied to detect the number of apoptotic cells. RESULTS: P66Shc expression was found in the retina of normal and diabetic rats, and the level of mRNA and protein expression increased with the progression of diabetes mellitus (DM). P66Shc expression was mainly located in the retinal ganglion cell layer and inner nuclear layer. Compared with the normal group, retinal cell tissue apoptosis rate in the D12w group was significantly increased. CONCLUSION: Rat retinal p66Shc expression was mainly in the ganglion cell layer and inner nuclear layer. As the degree of DM progressed, p66Shc expression gradually increased, and the number of apoptotic cells also increased.

G protein-coupled receptor 91 signaling in diabetic retinopathy and hypoxic retinal diseases.

G protein-coupled receptor 91 (GPR91) is a succinate-specific receptor and activation of GPR91 could initiate a complex signal transduction cascade and upregulate inflammatory and pro-angiogenic cytokines. In the retina, GPR91 is predominately expressed in ganglion cells, a major cellular entity involved in the pathogenesis of diabetic retinopathy (DR) and other hypoxic retinal diseases. During the development of DR and retinopathy of prematurity (ROP), chronic hypoxia causes an increase in the levels of local succinate. Succinate-mediated GPR91 activation upregulates vascular endothelial growth factor (VEGF) through ERK1/2-C/EBP ß (c-Fos) and/or ERK1/2-COX-2/PGE2 signaling pathways, which in turn, leads to the breakdown of blood-retina barriers in these disorders. In this review, we will have a brief introduction of GPR91 and its biological functions and a more detailed discussion about the role and mechanisms of GPR91 in DR and ROP. A better understanding of GPR91 regulation may be of great significance in identifying new biomarkers and drug targets for the prediction and treatment of DR, ROP, and hypoxic retinal diseases.

Transcription factors regulate GPR91-mediated expression of VEGF in hypoxia-induced retinopathy.

Hypoxia is the most important factor in the pathogenesis of diabetic retinopathy (DR). Our previous studies demonstrated that G protein-coupled receptor 91(GPR91) participated in the regulation of vascular endothelial growth factor (VEGF) secretion in DR. The present study induced OIR model in newborn rats using exposure to alternating 24-hour episodes of 50% and 12% oxygen for 14 days. Treatment with GPR91 shRNA attenuated the retinal avascular area, abnormal neovascularization and pericyte loss. Western blot and qRT-PCR demonstrated that CoCl2 exposure promoted VEGF expression and secretion, activated the ERK1/2 signaling pathways and upregulated C/EBP and AP-1. Knockdown of GPR91 inhibited ERK1/2 activity. GPR91 siRNA transduction and the ERK1/2 inhibitor U0126 inhibited the increases in C/EBP ß, C/EBP δ, c-Fos and HIF-1α. Luciferase reporter assays and a chromatin immunoprecipitation (ChIP) assay demonstrated that C/EBP ß and c-Fos bound the functional transcriptional factor binding site in the region of the VEGF promoter, but not C/EBP δ. Knockdown of C/EBP ß and c-Fos using RNAi reduced VEGF expression. Our data suggest that activation of the GPR91-ERK1/2-C/EBP ß (c-Fos, HIF-1α) signaling pathway plays a tonic role in regulating VEGF transcription in rat retinal ganglion cells.

Population-based survey of prevalence, causes, and risk factors for blindness and visual impairment in an aging Chinese metropolitan population.

AIM: To assess the prevalence, causes, and risk factors for blindness and visual impairment among elderly (≥60 years of age) Chinese people in a metropolitan area of Shanghai, China. METHODS: Random cluster sampling was conducted to identify participants among residents ≥60 years of age living in the Xietu Block, Xuhui District, Shanghai, China. Presenting visual acuity (PVA) and best-corrected visual acuity (BCVA) were checked by the Early Treatment Diabetic Retinopathy Study (ETDRS) visual chart. All eligible participants underwent a comprehensive eye examination. Blindness and visual impairment were defined according to World Health Organization (WHO) criteria. RESULTS: A total of 4190 persons (1688 men and 2502 women) participated in the study, and the response rate was 91.1%. Based on PVA, the prevalence of blindness was 1.1% and that of visual impairment was 7.6%. Based on BCVA, the prevalence of blindness and visual impairment decreased to 0.9% and 3.9%, respectively. Older (≥80 years of age) women, with low educational levels and smoking habits, exhibited a significantly greater chance for blindness and visual impairment than did those with high educational levels and no smoking habits (P<0.05). Based on PVA and BCVA, the main causes of blindness were cataract, myopic maculopathy, and age-related macular degeneration (AMD). CONCLUSION: Our findings help to identify the population in need of intervention, to highlight the need for additional eye healthcare services in urban China.

Baclofen Protects Primary Rat Retinal Ganglion Cells from Chemical Hypoxia-Induced Apoptosis Through the Akt and PERK Pathways.

Retinal ganglion cells (RGCs) consume large quantities of energy to convert light information into a neuronal signal, which makes them highly susceptible to hypoxic injury. This study aimed to investigate the potential protection by baclofen, a GABAB receptor agonist of RGCs against hypoxia-induced apoptosis. Cobalt chloride (CoCl2) was applied to mimic hypoxia. Primary rat RGCs were subjected to CoCl2 with or without baclofen treatment, and RNA interference techniques were used to knock down the GABAB2 gene in the primary RGCs. The viability and apoptosis of RGCs were assessed using cell viability and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, Hoechst staining, and flow cytometry. The expression of cleaved caspase-3, bcl-2, bax, Akt, phospho-Akt, protein kinase RNA (PKR)-like ER kinase (PERK), phospho-PERK, eIF2α, phospho-eIF2α, ATF-4 and CCAAT/enhancer-binding protein homologous protein (CHOP) were measured using western blotting. GABAB2 mRNA expression was determined using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Our study revealed that CoCl2 significantly induced RGC apoptosis and that baclofen reversed these effects. CoCl2-induced reduction of Akt activity was also reversed by baclofen. Baclofen prevented the activation of the PERK pathway and the increase in CHOP expression induced by CoCl2. Knockdown of GABAB2 and the inactivation of the Akt pathway by inhibitors reduced the protective effect of baclofen on CoCl2-treated RGCs. Taken together, these results demonstrate that baclofen protects RGCs from CoCl2-induced apoptosis by increasing Akt activity and by suppressing the PERK pathway and CHOP activation.

Comparative analysis of three purification protocols for retinal ganglion cells from rat.

PURPOSE: To make comparative analyses of the common three purification protocols for retinal ganglion cells (RGCs), providing a solid practical basis for selecting the method for purifying RGCs for use in subsequent experiments. METHODS: Rat RGCs were isolated and purified using three methods, including two-step immunopanning (TIP) separation, two-step immunopanning-magnetic (TIPM) separation, and flow cytometric (FC) separation. Immunocytochemical staining, quantitative real-time PCR, flow cytometry, electrophysiology, and Cell Counting Kit-8 (CCK-8) analyses were performed to compare the purity, yield, and viability of the RGCs. RESULTS: The RGC yields from the TIP, TIPM, and FC methods were 24.60±15.98 × 10(4), 5.28±4.42 × 10(4), and 5.4±2.7 × 10(3) per retina, respectively. We easily controlled the relative purity of the RGCs with the FC method and even reached 100% of the maximum expected purity. However, the RGC purity was only 80.97±5.45% and 95.41±3.23% using the TIP and TIPM methods, respectively. The contaminant cells were mainly large, star-shaped, glial fibrillary acidic protein (GFAP)-positive astrocytes and small, round, syntaxin 1-positive amacrine cells with multiple short neurites. The RGCs purified with FC could not be cultured successively in our study; however, the TIP-RGCs survived more than 20 days with good viability, while the TIPM-RGCs survived less than 9 days. CONCLUSIONS: The three protocols for purifying the RGCs each had its own pros and cons. The RGCs isolated by the TIP method exhibited the highest viability and yield but had low purity. The purity of the RGCs isolated with the FC method could reach approximately 100% but had a low yield and cell viability. The TIPM method was reliable and produced RGCs with considerable purity, yield, and viability. This study provides a solid practical basis for selecting the method for purifying RGCs for use in subsequent experiments.

The MAPK signaling pathway mediates the GPR91-dependent release of VEGF from RGC-5 cells.

Vascular endothelial growth factor (VEGF) is one of the major regulatory molecules in diabetic retinopathy (DR). In our previous study, we demonstrated that succinate levels were elevated in the retinas of diabetic rats and that the knockdown of the succinate receptor, G-protein-coupled receptor 91 (GPR91), inhibited the release of VEGF and attenuated retinal vascular disorder in the early stages of DR. In the present study, we examined the signaling pathways involved in the GPR91-dependent release of VEGF in the retinal ganglion cell line, RGC-5. The cells were infected with a lentiviral small hairpin RNA (shRNA) expression vector targeting GPR91 (LV.shGPR91). Immunofluorescence staining revealed that GPR91 was predominantly localized in the cell bodies of the RGC-5 cells. RT-qPCR, western blot analysis and ELISA indicated that succinate exposure upregulated VEGF expression, activated the extracellular signal-regulated protein kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways and led to the release of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). The knockdown of GPR91 inhibited ERK1/2 and JNK activity, but did not inhibit the activation of the p38 MAPK pathway. The increase in COX-2 expression and the release of PGE2 were inhibited by transduction with LV.shGPR91 and ERK1/2, JNK and COX-2 inhibitors. The expression and release of VEGF showed similar results. Cell Counting Kit-8 (CCK-8) assays revealed that the shRNA-mediated knockdown of GPR91 decreased the proliferation of RF/6A cells cultured in succinate-conditioned medium. Our data suggest that GPR91 modulates the succinate-induced release of VEGF through the MAPK/COX-2/PGE2 signaling pathway.

Decorin Prevents Retinal Pigment Epithelial Barrier Breakdown Under Diabetic Conditions by Suppressing p38 MAPK Activation.

PURPOSE: The purpose of this study was to determine the effect of decorin on the barrier function of human retinal pigment epithelial (RPE) cells under high-glucose (HG) plus hypoxia conditions. METHODS: Human RPE (ARPE-19) cells were cultured for 18 days in normal glucose (5.5 mM) or HG (25 mM) medium. In addition, to mimic the hypoxic impact which occurs in diabetic retinopathy, cells were treated with 100 µM CoCl2 during the last 2 days of the experiment. Decorin, 100 nM, was applied 1 hour before CoCl2 was added. Retinal pigment epithelial barrier function was evaluated by measuring transepithelial electrical resistance (TER) and apical-basolateral permeability of fluorescein isothiocyanate (FITC)-dextran. The content and distribution of tight junction proteins (claudin-1, occludin, and zonula occludens-1 [ZO-1]) were examined by Western blotting and immunofluorescence. p38 mitogen-activated protein kinase (MAPK) phosphorylation was evaluated by Western blotting, and small interfering RNA transfection to p38 MAPK was also performed in ARPE-19 monolayers. RESULTS: High-glucose plus hypoxia significantly increased FITC-dextran permeability, paralleled by decreased TER. Decorin reversed both of these effects. High-glucose plus hypoxia-induced reduction and disorganization of occludin and ZO-1 were also reversed by decorin. Decorin prevented the activation of p38 MAPK induced by hypoxia. Silence of p38 MAPK by RNA interference also inhibited the breakdown of ARPE-19 cell monolayer induced by HG plus hypoxia. CONCLUSIONS: Retinal pigment epithelial barrier disruption induced by HG plus hypoxia was prevented by decorin through suppression of p38 MAPK activation, which could present a new therapeutic strategy for inhibition of diabetic macular edema.

ERK1/2/COX-2/PGE2 signaling pathway mediates GPR91-dependent VEGF release in streptozotocin-induced diabetes.

PURPOSE: Retinal vascular dysfunction caused by vascular endothelial growth factor (VEGF) is the major pathological change that occurs in diabetic retinopathy (DR). It has recently been demonstrated that G protein-coupled receptor 91 (GPR91) plays a major role in both vasculature development and retinal angiogenesis. In this study, we examined the signaling pathways involved in GPR91-dependent VEGF release during the early stages of retinal vascular change in streptozotocin-induced diabetes. METHODS: Diabetic rats were assigned randomly to receive intravitreal injections of shRNA lentiviral particles targeting GPR91 (LV.shGPR91) or control particles (LV.shScrambled). Accumulation of succinate was assessed by gas chromatography-mass spectrometry (GC-MS). At 14 weeks, the ultrastructure and function of the retinal vessels of diabetic retinas with or without shRNA treatment were assessed using hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and Evans blue dye permeability. The expression of GPR91, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and cyclooxygenase-2 (COX-2) were measured using immunofluorescence and western blotting. COX-2 and VEGF mRNA were determined by quantitative RT-PCR. Prostaglandin E2 (PGE2) and VEGF secretion were detected using an enzyme-linked immunosorbent assay. RESULTS: Succinate exhibited abundant accumulation in diabetic rat retinas. The retinal telangiectatic vessels, basement membrane thickness, and Evans blue dye permeability were attenuated by treatment with GPR91 shRNA. In diabetic rats, knockdown of GPR91 inhibited the activities of ERK1/2 and COX-2 as well as the expression of PGE2 and VEGF. Meanwhile, COX-2, PGE2, and VEGF expression was inhibited by ERK1/2 inhibitor U0126 and COX-2 inhibitor NS-398. CONCLUSIONS: Our data suggest that hyperglycemia causes succinate accumulation and GPR91 activity in retinal ganglion cells, which mediate VEGF-induced retinal vascular change via the ERK1/2/COX-2/PGE2 pathway. This study highlights the signaling pathway as a potential target for intervention in DR.

Altered retinal microRNA expression profiles in early diabetic retinopathy: an in silico analysis.

PURPOSE: MicroRNAs (miRNAs) - as negative regulators of target genes - are associated with various human diseases, but their precise role(s) in diabetic retinopathy (DR) remains to be elucidated. The aim of this study was to elucidate the involvement of miRNAs in early DR using in silico analysis to explore their gene expression patterns. METHODS: We used the streptozotocin (STZ)-induced diabetic rat to investigate the roles of miRNAs in early DR. Retinal miRNA expression profiles from diabetic versus healthy control rats were examined by miRNA array analysis. Based on several bioinformatic systems, specifically, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified signatures of the potential pathological processes, gene functions, and signaling pathways that are influenced by dysregulated miRNAs. We used quantitative real-time polymerase chain reaction (qRT-PCR) to validate six (i.e. those with significant changes in expression levels) of the 17 miRNAs that were detected in the miRNA array. We also describe the significant role of the miRNA-gene network, which is based on the interactions between miRNAs and target genes. RESULTS: GO analysis of the 17 miRNAs detected in the miRNA array analysis revealed the most prevalent miRNAs to be those related to biological processes, olfactory bulb development and axonogenesis. These miRNAs also exert significant influence on additional pathways, including the mitogen-activated protein and calcium signaling pathways. Six of the seventeen miRNAs were chosen for qRT-PCR validation. With the exception of a slight difference in miRNA-350, our results are in close agreement with the differential expressions detected by array analysis. CONCLUSIONS: This study, which describes miRNA expression during the early developmental phases of DR, revealed extensive miRNA interactions. Based on both their target genes and signaling pathways, we suggest that miRNAs perform critical regulatory functions during the early stages of DR evolution.

RNA Interference Targeting Snail Inhibits the Transforming Growth Factor ß 2-Induced Epithelial-Mesenchymal Transition in Human Lens Epithelial Cells.

Epithelial-msenchymal transition (EMT) contributes to posterior capsule opacification (PCO) type of cataract. Transcription factors Snail is a key trigger of EMT activated by transforming growth factor ß (TGF ß ). This study was done to investigate the effect of Snail targeting siRNA on TGF ß 2-induced EMT in human lens epithelial cells. TGF ß 2 treatment of cultured human epithelial cell line (HLEB3) upregulated the expression of Snail and the EMT relevant molecules such as vimentin and α -SMA but downregulated the expression of keratin and E-cadherin. After the stimulation of TGF ß 2, the HLEB3 cells became fibroblast-like in morphology, and the junctions of cell-cell disappeared. TGF ß 2 treatment also enhanced migration ability of HLEB3 cells. TGF ß 2-induced Snail expression and EMT were significantly inhibited by Snail siRNA. By analyzing the response characteristics of HLEB3 in TGF ß 2-induced EMT model with/without Snail-specific siRNA, we concluded that Snail is an element in the EMT of HLEB3 cells induced by TGF ß 2. Snail siRNA targeting can block the induced EMT and therefore has the potential to suppress the development of PCO.

Decorin inhibits angiogenic potential of choroid-retinal endothelial cells by downregulating hypoxia-induced Met, Rac1, HIF-1α and VEGF expression in cocultured retinal pigment epithelial cells.

Choroidal neovascularization (CNV) is one of the most common causes of severe vision loss. Decorin, a multiple receptor tyrosine kinase inhibitor, has been recently shown to play an important regulatory role in angiogenic response. This study aims to investigate whether the overexpression of decorin in retinal pigment epithelial (RPE) cells under hypoxia alters the in vitro angiogenic ability of cocultured choroid-retinal endothelial cells and to explore the possible mechanisms involved. Human RPE cells (ARPE-19) were subjected to hypoxia with or without decorin pretreatment, and RNA interference technique was used to knock down the Met gene in ARPE-19 cells. Cell viability was determined using the Cell Counting Kit-8 assay. Expression of Met, Rac1 and hypoxia-inducible factor-1 alpha (HIF-1α) was evaluated by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Vascular endothelial growth factor (VEGF) expression was evaluated by enzyme-linked immunosorbent assay (ELISA) and qRT-PCR. We then constructed a recombinant lentiviral vector carrying the decorin gene to transduce ARPE-19 cells. The overexpression of decorin in transduced RPE cells was confirmed by qRT-PCR and western blot. The transduced RPE cells were then cocultured with rhesus macaque choroid-retinal endothelial cells (RF/6A) in a transwell coculture system to observe the effects of decorin overexpression in ARPE-19 cells on the proliferation, migration and tube formation of RF/6A cells. In response to hypoxia, the VEGF concentrations in the culture supernatants increased greatly at 24 and 48 h, and this effect was inhibited significantly and nearly equally in the presence of 50-200 nM decorin. Decorin pretreatment before hypoxia exposure effectively reduced the hypoxia-induced expression of Met, Rac1, HIF-1α and VEGF in ARPE-19 cells. Transfection of small interfering RNA against Met to ARPE-19 cells also resulted in significant downregulation of Rac1, HIF-1α and VEGF under hypoxia, and this effect was similar to that noted with decorin pretreatment alone or with their combination. Results from the coculture system showed that the overexpression of decorin in ARPE-19 cells significantly inhibited the proliferation, migration and tube formation of RF/6A cells. These results indicate that Met pathway activation plays an important role in the upregulation of VEGF in RPE cells under hypoxia. Decorin may interfere with angiogenesis by downregulating hypoxia-induced Met, Rac1, HIF-1α and VEGF expression in RPE cells, which suggests a potential strategy for the inhibition of CNV.

Inhibition of high glucose-induced VEGF release in retinal ganglion cells by RNA interference targeting G protein-coupled receptor 91.

Recent research using a rat oxygen-induced retinopathy model has demonstrated that the G protein-coupled receptor 91 (GPR91) of retinal ganglion neurons is the principal respondent to succinate and consequently induces the release of angiogenic factor vascular endothelial growth factor (VEGF). The aim of this study was to determine whether GPR91 modulate the release of VEGF from retinal ganglion cells in a high-glucose model in vitro and to dissect the role of GPR91 in the pathogenesis of diabetic retinopathy. We constructed a lentiviral small hairpin RNA (shRNA) expression vector targeting GPR91 (LV.shGPR91) and infected the retinal ganglion cell line RGC-5 to obtain stably transduction system. The knockdown effect of GPR91 was detected by Western blotting. After incubation with succinate and various concentrations of glucose, the expression of VEGF in RGC-5 cells was evaluated by real-time PCR and Western blotting, and the release of VEGF protein was measured using an ELISA assay. Conditioned media were also collected, and the effects of proliferation and migration of RF/6A cells, a vascular endothelial cell line, were evaluated by CCK-8 and Transwell assays. The phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK) in RGC-5 cells after exposure to high glucose were evaluated by Western blotting. Following a single exposure of RGC-5 cells to the encoding lentivirus, more than 80% of infected cells expressed GFP at 72 h, and the level of GPR91 protein was significantly downregulated. GPR91 shRNA inhibited the cell survival rates of RGC-5 cells incubated with high glucose (F = 21.36, P = 0.002). The mRNA and protein expression of VEGF in LV.shGPR91 RGC-5 cells decreased markedly compared to that of LV.shScrambled or untransduced control cells incubated with different concentrations of glucose or succinate (P < 0.01). The VEGF protein level in medium from RGC-5 cells treated with high glucose (F = 57.43, P = 0.000) or succinate (F = 241.91, P = 0.000) was also downregulated when transduced with GPR91 shRNA. The siRNA-mediated knockdown of GPR91 was also found to inhibit the proliferation of RF/6A cells in high glucose-stimulated (t = 8.21, P = 0.001) or succinate-stimulated (t = 3.36, P = 0.028) conditioned media. However, the siRNA-mediated knockdown of GPR91 suppressed the migration of RF/6A cells incubated with moderate levels of glucose (t = 2.97, P = 0.018). The exposure of RGC-5 cells to high glucose activated ERK1/2 and JNK MAPK signaling blocking by GPR91 shRNA (P < 0.01). These results indicate that GPR91 modulates the high glucose-induced VEGF release of RGC-5 cells, possibly by inhibiting ERK1/2 and JNK MAPK signaling.

Polymorphisms of the WRN gene and DNA damage of peripheral lymphocytes in age-related cataract in a Han Chinese population.

Werner syndrome is caused by mutations in the DNA repair Werner helicase (WRN) gene and characterized by accelerated aging including cataracts. Age-related cataract (ARC) cases (N = 504) and controls (N = 244) were recruited from a population-based study to evaluate the association of single-nucleotide polymorphisms (SNPs) of WRN and another DNA repair gene (human 8-oxoguanine DNA N-glycosylase 1) with ARC. Among the five SNPs tested, only WRN rs1346044 was found to be significantly associated between cases and controls before multiple-testing adjustment. The minor C allele of rs1346044 was associated with ARC with an odds ratio (OR) of 0.66, suggesting a protective role of the C allele for developing ARC. The stratification analysis on the subtypes of ARC showed that rs1346044 was significantly associated with cortical cataract, but not with nuclear, posterior subcapsular, and mixed types after multiple-testing adjustment (OR = 0.51, p< 0.01). The genetic model analysis showed that the results fit the dominant model (OR = 0.44, p < 0.001). The comet assay used to assess the extent of DNA damage in peripheral lymphocytes of ARC cases found that the DNA damage in lymphocytes from patients with CC genotype was significantly less than that in patients with TT genotype. We concluded that the C allele of rs1346044, a non-synonymous SNP resulting in the conversion of Cys to Arg at amino acid position 1367 of WRN, alters susceptibility to ARC, especially the cortical type of the disease, in the Han Chinese. The underlying mechanism of its protective role might be related to the improved DNA repair function.

Evaluation of miR-122-regulated suicide gene therapy for hepatocellular carcinoma in an orthotopic mouse model.

OBJECTIVE: Intratumoral administration of adenoviral vector encoding herpes simplex virus (HSV) thymidine kinase (TK) gene (Ad-TK) followed by systemic ganciclovir (GCV) is an effective approach in treating experimental hepatocellular carcinoma (HCC). However, hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy. miR-122 is an abundant, liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines. These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation. METHODS: By inserting miR-122 target sequences (miR-122T) in the 3' untranslated region (UTR) of TK gene, we constructed adenovirus (Ad) vectors expressing miR-122-regulated TK (Ad-TK-122T) and report genes. After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model, we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T. RESULTS: Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment. Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo, resulting in an 11-fold improvement of tumor-specific transgene expression. Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor. The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy. Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose, no liver damage was found in Ad-TK-122T group. CONCLUSIONS: miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.