Potential Mechanism of Platelet GPIIb/IIIa and Fibrinogen on Retinal Vein Occlusion.

PURPOSE: To explore the role of coagulation and fibrinolytic factors, and the potential mechanism of platelet aggregation in the pathogenesis of retinal vein occlusion. METHODS: Coagulation and fibrinolytic parameters in patients with retinal vein occlusion were determined using hemagglutinin and HISCL-5000. Relationships between these elevated parameters and factors representing typical clinical manifestations of retinal vein occlusion were examined, and these parameters were analyzed using a STRING database to indicate the potential role of platelet aggregation. Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) levels were evaluated by flow cytometry after antiplatelet treatment in patients and mouse models. Furthermore, the GPIIb/IIIa ligand fibrinogen in peripheral blood and retina of mouse models was assessed by the turbidimetric method and real-time PCR, respectively. RESULTS: In patients, significant increases in peripheral blood fibrinogen and GPIIb/IIIa levels were observed (p = 0.0040, p < 0.0001, respectively). A positive correlation was observed between macular thickness (MT) and both fibrinogen and GPIIb/IIIa (r = 0.4528, p = 0.0063; r = 0.3789, p = 0.0427, respectively). After intravitreal injections of anti-vascular endothelial growth factor drugs, a significant reduction in fibrinogen levels was observed (p = 0.0072). In addition, the use of antiplatelet drugs resulted in a significant decrease in GPIIb/IIIa (p < 0.0001). In a mouse model, antiplatelet therapy significantly reduced both peripheral blood and retina fibrinogen levels and the overall rate of vein occlusion 3 days after occlusion (p < 0.0005). In addition, the reduction in GPIIb/IIIa levels after antiplatelet therapy was remarkable. CONCLUSION: Fibrinogen and GPIIb/IIIa may be involved in retinal vein occlusion and blocking platelet aggregation may be a new therapeutic approach for retinal vein occlusion.

Trimethylamine N-oxide aggravates vascular permeability and endothelial cell dysfunction under diabetic condition: in vitro and in vivo study.

AIM: To provide the direct evidence for the crucial role of trimethylamine N-oxide (TMAO) in vascular permeability and endothelial cell dysfunction under diabetic condition. METHODS: The role of TMAO on the in vitro biological effect of human retinal microvascular endothelial cells (HRMEC) under high glucose conditions was tested by a cell counting kit, wound healing, a transwell and a tube formation assay. The inflammation-related gene expression affected by TMAO was tested by real-time polymerase chain reaction (RT-PCR). The expression of the cell junction was measured by Western blotting (WB) and immunofluorescence staining. In addition, two groups of rat models, diabetic and non-diabetic, were fed with normal or 0.1% TMAO for 16wk, and their plasma levels of TMAO, vascular endothelial growth factor (VEGF), interleukin (IL)-6 and tumor necrosis factor (TNF)-α were tested. The vascular permeability of rat retinas was measured using FITC-Dextran, and the expression of zonula occludens (ZO)-1 and claudin-5 in rat retinas was detected by WB or immunofluorescence staining. RESULTS: TMAO administration significantly increased the cell proliferation, migration, and tube formation of primary HRMEC either in normal or high-glucose conditions. RT-PCR showed elevated inflammation-related gene expression of HRMEC under TMAO stimulation, while WB or immunofluorescence staining indicated decreased cell junction ZO-1 and occludin expression after high-glucose and TMAO treatment. Diabetic rats showed higher plasma levels of TMAO as well as retinal vascular leakage, which were even higher in TMAO-feeding diabetic rats. Furthermore, TMAO administration increased the rat plasma levels of VEGF, IL-6 and TNF-α while decreasing the retinal expression levels of ZO-1 and claudin-5. CONCLUSION: TMAO enhances the proliferation, migration, and tube formation of HRMEC, as well as destroys their vascular integrity and tight connection. It also regulates the expression of VEGF, IL-6, and TNF-α.

Incidence of Metamorphopsia After Successful Rhegmatogenous Retinal Detachment Surgery: A Systematic Review and Meta-Analysis.

PURPOSES: To investigate the incidence and factors influencing the occurrence of metamorphopsia in patients with simple rhegmatogenous retinal detachment (RRD) after surgery. METHODS: Relevant studies of metamorphopsia were identified by searching in PubMed, Embase, and Cochrane until August 2022. Meta-analysis of the incidence of metamorphopsia after rhegmatogenous retinal detachment surgery was performed using Review Manager 5.4 statistical software. RESULTS: A total of 12 studies reported 1133 participants with 469 patients with postoperative metamorphopsia. The meta-analysis showed a higher incidence of metamorphopsia in macular-off cases compared with macular-on RRD (RR = 2.88, 95% CI: 2.35 to 3.52). The use of perfluorocarbon liquid (PFCL) during pars plana vitrectomy (PPV) reduced the incidence of metamorphopsia (RR = 0.61, 95% CI: 0.41 to 0.92). There was no evidence of any important difference in metamorphopsia between participants in the PPV group and the scleral buckling (SB) group (RR = 1.04, 95% CI: 0.82 to 1.33). There was little or no difference in metamorphopsia between gas and silicon oil (SO) in the PPV group (RR = 0.89, 95% CI: 0.69 to 1.13). CONCLUSION: The incidence of postoperative metamorphopsia is higher in macular-off RRD, and PFCL should be a preferred choice to prevent postoperative metamorphopsia in macula-off RRD cases.

Global, regional, and national burdens of intraocular foreign bodies in children and adolescents from 1990 to 2019: a trend analysis.

BACKGROUND: This study aimed to evaluate trends in global, regional, and national burdens of intraocular foreign bodies among children and adolescents (aged 0 - 19 years) between 1990 and 2019 according to age, sex, and socio-demographic index. METHODS: This study obtained data from the Global Burden of Disease Study 2019 and evaluated the number of cases, rates per 100,000 persons, and average annual percentage changes among children and adolescents. The annual percentage changes in the incidence and years lived with disability rates across various age groups were investigated using joinpoint software. RESULTS: For intraocular foreign bodies in children and adolescents, the incidence and year lived with disability rates decreased in all age groups between 1990 and 2019. However, the number of incident cases and years lived with disability increased from 1091.94 [95% uncertainty interval (UI), 610.91-1839.52] and 89,245 (95% UI, 6.65-18.67) in 1990 to 1134.85 (95% UI, 665.01-1867.50) and 92,108 (95% UI, 32,052-192,153) in 2019, respectively. Age was positively correlated with the number of cases, incidence, and years lived with disability rates. However, there were significant decreases in both the incidence and years lived with disability rates among children and adolescents, especially in the 15-18 years age group, males, and most high-income regions. Notably, the incidence and years lived with disability rates were significantly decreased in middle and high-middle socio-demographic index regions but were increased in low and low-middle socio-demographic index regions. CONCLUSIONS: Despite the remarkable progress between 1990 and 2019 in reducing the global burden of intraocular foreign bodies, there has been an increase in the number of cases, with substantial disparity across age groups, sexes, regions, and countries. Our results could inform more effective strategies for reducing the burden among children and adolescents.

Changing Trends in the Global Burden of Cataract Over the Past 30 Years: Retrospective Data Analysis of the Global Burden of Disease Study 2019.

BACKGROUND: Cataracts now account for the largest proportion of the global burden of blindness and vision loss. Understanding the changing trends in the global burden of cataracts over the past 30 years and the next 15 years is of clear significance for the prevention and control of cataracts in key populations. As far as we know, research on the future burden of cataracts is lacking. OBJECTIVE: This study aims to assess the global burden of cataracts over the past 30 years by using age-period-cohort modeling and to estimate trends in the next 15 years. METHODS: Data were obtained from the Global Burden of Disease Study 2019, the United Nations Development Programme, and the WHO (World Health Organization) Global Health Observatory data repository. The assessment of trends and disparities in the number and rate of disability-adjusted life years (DALYs) for cataracts from 1990 to 2019 was conducted. The association between the age-standardized DALY rate (ASDR) and the socio-demographic index (SDI), human development index (HDI), national levels of particulate matter <2.5 µm in diameter (PM2.5), and ambient ultraviolet radiation (UVR) was determined using linear regression analysis. Additionally, we used the Nordpred (Harald Fekjær and Bjørn Møller) age-period-cohort model to predict the cataract burden from 2020 to 2034. RESULTS: Globally, the number of DALYs due to cataract increased from 3,492,604 (95% uncertainty interval [UI] 2,481,846-4,719,629) in 1990 to 6,676,281 (95% UI 4,761,210-9,006,193) in 2019. The ASDRs due to cataract decreased from 93.17 (95% UI 66.14-125.32) in 1990 to 82.94 (95% UI 59.06-111.75) in 2019, with an average annual percentage change of -0.37 (95% CI -0.44 to -0.3; P<.001). Age, female sex, air pollution, smoking, high fasting plasma glucose levels, and a high body mass index were risk factors for the burden of cataracts. SDI and HDI were negatively correlated with ASDRs of cataracts, while PM2.5 and UVR were positively associated with them. Higher DALY rates were also associated with lower SDI (R2=0.1939; P<.001), lower HDI (R2=0.2828; P<.001), national PM2.5 concentration (R2=0.1874; P<.001), and ambient UVR levels (R2=0.2354; P<.001). The prediction model suggested that the number of DALYs due to cataract will continue to rise globally, while the cataract DALY rate will continue to decrease. CONCLUSIONS: While the ASDR of cataracts has decreased, there has been a notable increase in the number of DALYs over the past 30 years. Projections suggest that the global burden of cataracts will continue to rise over the next 15 years. To address this challenge, appropriate prevention and treatment policies must be implemented.

Topical Administration of Bevacizumab to Facilitate the Functional Filtering Bleb After Trabeculectomy in the Rabbit.

Purpose: To evaluate the effects of bevacizumab in 3 different application methods, subconjunctival injection (SCI), hyaluronic acid retardant (HAR), and eye drop (ED), on attenuating scar formation in the filtering bleb. Methods: Trabeculectomy (TRAB) was performed on New Zealand rabbits. TRAB rabbits were intervened with bevacizumab SCI, HAR, ED, or mitomycin C, respectively. Intraocular pressure (IOP) of 1, 7, 14, and 28 days after TRAB was recorded, and the bleb survival rate was analyzed. Bleb height, area, and vascularization were evaluated using anterior segment optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) at 7, 14 and 28 days after surgery. A histopathology examination of the bleb tissue was performed. The expression levels of vascular endothelial growth factor (VEGF)-A, interleukin (IL)-1α, tumor necrosis factor-alpha (TNF-α), transforming growth factor-ß1 (TGF-ß1), and α-smooth muscle actin (α-SMA) were measured by Western blot. Results: Bevacizumab significantly reduced postoperative IOP and increased the survival of the filtering bleb, especially in the ED group. Less vascularization was shown in the SCI, HAR, and ED groups. Histopathological results showed the fewest levels of scarring and fibrosis in the ED group. The local VEGF-A, IL-1α, and TNF-α expression levels after bevacizumab ED were decreased, combined with suppression of TGF-ß1 and α-SMA. Conclusions: Postoperative use of bevacizumab EDs was an effective application method for improving surgical outcomes after TRAB in rabbits. It might be effective in preventing scarring of the filtering bleb by antivascularization and anti-inflammation.

Burden of blindness and vision loss in China over the past 30 years: findings and predictions based on the Global Burden of Disease Study 2019.

AIMS: To investigate the burden of blindness and vision loss (BVL) in China over the past 30 years according to year, age and sex, and to estimate future predictions. METHODS: We analysed the years lived with disability (YLDs), number of cases, age-standardised YLD rates (ASYRs) and age-standardised prevalence rates (ASPRs) of BVL in China from 1990 to 2019. We focused on changes over time using estimated annual percentage changes (EAPCs). Additionally, we used the Bayesian age-period-cohort model to predict the BVL burden from 2020 to 2030. RESULTS: The number of YLDs and prevalent cases due to BVL increased from 2.57 (95% uncertainty interval (UI) 1.74 to 3.72) and 90.76 million (95% UI 72.21 to 111.92) in 1990 to 5.42 (95% UI 3.61 to 8.02) and 211.67 million (95% UI 168.21 to 259.66) in 2019, respectively. The BVL ASYRs and ASPRs showed a decreasing trend, with EAPCs of -0.13 (95% CI -0.28 to 0.02) and -0.11 (95% CI -0.19 to -0.04), respectively. The elderly and female populations had a higher BVL burden. The numbers of YLDs and cases due to BVL are projected to continue rising to 7.74 and 279.49 million in 2030, respectively. The ASYRs and ASPRs also showed increasing trends. CONCLUSION: While rates of BVL in China have decreased, there has been a notable increase in the number of YLDs and new cases over the past 30 years. Projections suggest that the burden of BVL will continue to rise over the next 11 years. To address this challenge, appropriate policies must be implemented.

SDF­1α/CXCR4 signaling promotes capillary tube formation of human retinal vascular endothelial cells by activating ERK1/2 and PI3K pathways in vitro.

The purpose of this study is to address the effect and mechanism of stromal cell­derived factor­1 (SDF­1)α/chemokine (C­X­C motif) receptor 4 (CXCR4) signaling on capillary tube formation of human retinal vascular endothelial cells (HRECs). The expression of CXCR4 in HRECs was quantified by reverse transcription (RT­PCR) and western blotting. The effects of SDF­1α/CXCR4 signaling in capillary tube formation and migration of HRECs was examined using three­dimensional Matrigel assay and wound scratching assay respectively in vitro. Cell proliferation of HRECs was examined using cell counting kit (CCK)­8 assay in the presence of different concentrations of SDF­1α protein. The effect of SDF­1α/CXCR4 signaling in HREC expression of VEGF, basic fibroblast growth factor (bFGF), IL­8 and intercellular cell adhesion molecule (ICAM)­1 was examined using RT­PCR and western blotting. RT­PCR and western blot analysis revealed CXCR4 was expressed in HRECs. The number of intact capillary tubes formed by HRECs in the presence of SDF­1α was markedly more compared with a PBS treated control group. However, it was reduced with treatment with an CXCR4 antagonist. Wound scratching assay showed a significant increase in the number of migrated HRECs under SDF­1α stimulation and the number was reduced with treatment with an CXCR4 antagonist. RT­PCR and western blotting showed that SDF­1α significantly promoted VEGF, bFGF, IL­8 and ICAM­1 expression in HRECs. The proliferation of HRECs in the presence of SDF­1α was promoted in a dosage­dependent manner. SDF­1α/CXCR4 signaling can increase HREC capillary tube formation through promoting HREC migration, proliferation and expression of VEGF, bFGF, IL­8 and ICAM­1.

Association between Dietary Choline Intake and Diabetic Retinopathy: National Health and Nutrition Examination Survey 2005-2008.

PURPOSE: To explore whether there is an association between dietary choline intake and odds of diabetic retinopathy (DR) in the US diabetic population. METHODS: A cross-sectional study was conducted using the combined data of the National Health and Nutrition Examination Survey (NHANES) 2005-2008 of a complex, multistage, and probability-sampling design. Energy-adjusted choline intake was calculated separately for men and women using the residual method. Binary logistic regression adjusting for covariates was used to identify the variables associated with DR. RESULTS: We included 644 male and 628 female diabetic subjects, which were equivalent to a weighted survey sample of 9,339,124 for males and 10,109,553 for females respectively. Female DR patients consumed more choline than non-DR patients (268.6 mg/d vs 250.9 mg/d; p = .046). The estimated prevalence of DR was 17.4%, 21.9%, and 29.7% across three levels of dietary choline intake in females, respectively. In multivariable logistic-regression models, the odds ratio (OR) of DR for female patients in the highest choline intake group was 2.14 (95% confidence interval [CI], 1.38-3.31; p = .001) compared with those in the lowest intake group. This association was positive but not statistically significant in males. CONCLUSION: Higher intake of dietary choline is associated with increased odds of DR in females, but not in males. Further studies are warranted to investigate the direct role of choline in DR development and determine the recommended daily intake of choline for diabetic patients weighing the pros and cons of dietary choline consumption.

Assessing the protective effects of cryptotanshinone on CoCl2­induced hypoxia in RPE cells.

The development of several retinal diseases is closely related to hypoxia. As a component of the Traditional Chinese medicine Salvia miltiorrhiza, the effects of cryptotanshinone (CT) on retinal cells under hypoxic conditions are not well understood. The aim of the present study was to explore how CT exerted its protective effects on retinal pigment epithelium (RPE) cells under hypoxic conditions induced by cobalt chloride (CoCl2). The effects of CT were investigated using a Cell Counting Kit­8 assay, Annexin V­FITC/PI staining, reverse transcription­quantitative PCR and western blotting in ARPE­19 cells. CT (10 and 20 µM) reduced the CoCl2­induced increase in vascular endothelial growth factor expression and hypoxia­inducible transcription factor­1α expression in ARPE­19 cells. Additionally, CT alleviated hypoxia­induced apoptosis by regulating Bcl­2 and Bax protein expression. CT treatment also reduced the increase in the mRNA levels of IL­6, IL­1ß and TNF­α induced by CoCl2. In summary, CT may protect RPE cells against apoptosis and inflammation in CoCl2­induced hypoxia, and these results warrant further in vivo study into its value as a drug for treating hypoxic eye diseases.

Inhibition of PDGF-BB reduces alkali-induced corneal neovascularization in mice.

The aim of the present study was to investigate the role of platelet­derived growth factor (PDGF)­BB/PDGF receptor (R)­ß signaling in an experimental murine corneal neovascularization (CrNV) model. Experimental CrNV was induced by alkali injury. The intra­corneal expression of PDGF­BB was examined using immunohistochemistry. The effect of PDGF­BB on CrNV was evaluated using immunofluorescence staining. The expression levels of PDGFR­ß in human retinal endothelial cells (HRECs) under normal conditions or following cobalt chloride treatment, which induced hypoxic conditions, was assessed using reverse transcription­quantitative PCR. The effect of exogenous treatment of PDGF­BB on the proliferation, migration and tube formation of HRECs under normoxic or hypoxic conditions was evaluated in vitro using Cell Counting Kit­8, wound healing and 3D Matrigel capillary tube formation assays, respectively. The results indicated that the intra­corneal expression levels of the proteins of PDGF­BB and PDGFR­ß were detectable on days 2 and 7 following alkali injury. The treatment with neutralizing anti­PDGF­BB antibody resulted in significant inhibition of CrNV. The intra­corneal expression levels of vascular endothelial growth factor A, matrix metallopeptidase (MMP)­2 and MMP­9 proteins were downregulated, while the expression levels of thrombospondin (TSP)­1, TSP­2, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)­1 and ADAMTS­2 were upregulated significantly in mice treated with anti­PDGF­BB antibody. The expression levels of PDGFR­ß were upregulated in HRECs under hypoxic conditions compared with those noted under normoxic conditions. Recombinant human PDGF­BB promoted the proliferation, migration and tube formation of HRECs under hypoxic conditions. The data indicated that PDGF­BB/PDGFR­ß signaling was involved in CrNV and that it promoted endothelial cell proliferation, migration and tube formation. The pro­angiogenic effects of this pathway may be mediated via the induction of pro­angiogenic cytokine secretion and the suppression of anti­angiogenic cytokine secretion.

Elevated plasma trimethylamine-N-oxide levels are associated with diabetic retinopathy.

AIMS: To determine the relationship between plasma levels of trimethylamine-N-oxide (TMAO) and odds of diabetic retinopathy (DR). METHODS: A cross-sectional study was conducted. Blood samples were obtained from 122 type 2 diabetes mellitus (T2DM) patients with or without DR. Multivariable logistic regression analyses were performed to identify the association between plasma TMAO and DR. The diagnostic value of plasma TMAO was assessed by the area under the receiver operating characteristic curve (AUROC) and integrated discrimination improvement (IDI). RESULTS: In the T2DM patients, plasma levels of TMAO were significantly higher in patients with DR compared with those without DR (P = 0.001). As logarithmic (ln) transformation of TMAO increased per standard deviation (SD), there was higher probability to have DR [odds ratio (OR) = 2.31; P = 0.005]. As ln-transformed TMAO increased per SD, the severity of DR was more likely to get worse (OR = 2.05; P = 0.004). In the diagnostic model, the addition of TMAO contributed to the improvement in AUROC from 0.646 to 0.734 (P = 0.043), and the IDI was 10.7% (P < 0.001). CONCLUSION: Elevated levels of plasma TMAO were associated with higher odds and worse severity of DR in T2DM patients, and further investigation is required for the causality of this association.

Proteomic analysis of anti-angiogenic effects by conbercept in the mice with oxygen induced retinopathy.

AIM: To analyze the retinal proteomes with and without conbercept treatments in mice with oxygen-induced retinopathy (OIR) and identify proteins involved in the molecular mechanisms mediated by conbercept. METHODS: OIR was induced in fifty-six C57BL/6J mouse pups and randomly divided into four groups. Group 1: Normal17 (n=7), mice without OIR and treated with normal air. Group 2: OIR12/EXP1 (n=14), mice received 75% oxygen from postnatal day (P) 7 to 12. Group 3: OIR17/Control (n=14), mice received 75% oxygen from P7 to P12 and then normal air to P17. Group 4: Lang17/EXP2 (n=21), mice received 75% oxygen from P7 to P12 with intravitreal injection of 1 µL conbercept at the concentration of 10 mg/mL at P12, and then normal air from P12 to P17. Liquid Chromatography-Mass Spectrometry (LC-MS)/MS data were reviewed to find proteins that were up-regulated after the conbercept treatment. Gene ontology (GO) analysis was performed of conbercept-mediated changes in proteins involved in single-organism processes, biological regulation, cellular processes, immune responses, metabolic processes, locomotion and multiple-organism processes. RESULTS: Conbercept induced a reversal of hypoxia-inducible factor 1 signaling pathway as revealed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and also induced down-regulation of proteins involved in blood coagulation and fibrin clot formation as demonstrated by the Database for Annotation, Visualization and Integrated Discovery (DAVID) and the stimulation of interferon genes studies. These appear to be risk factors of retinal fibrosis. Additional conbercept-specific fibrosis risk factors were also identified and may serve as therapeutic targets for fibrosis. CONCLUSION: Our studies reveal that many novel proteins are differentially regulated by conbercept. The new insights may warrant a valuable resource for conbercept treatment.

Extraction, Purification, Characterization, and Antiangiogenic Activity of Acidic Polysaccharide from Buddleja officinalis.

Firstly, optimal parameters of crude polysaccharide from Buddleja officinalis were obtained as follows: ratio of water to raw material of 26 : 1, ultrasonic power of 240 W, ultrasonic time of 45 min, and ultrasonic temperature of 62°C. Secondly, acidic polysaccharide (APBOM) from Buddleja officinalis was successfully acquired with the yield of 9.57% by using DEAE-52 cellulose and Sephadex G-100 gel column chromatography. Then, we found that total polysaccharide content of APBOM was 94.37% with a sulfuric acid group of 1.68%, uronic acid content of 17.41%, and average molecular weight of 165.4 kDa. Finally, APBOM was confirmed to have significant antiangiogenic effects.

Essential Contribution of Macrophage Tie2 Signalling in a Murine Model of Laser-Induced Choroidal Neovascularization.

Wet age-related macular degeneration (AMD), which can cause progressive blindness, is characterised by choroid neovascularization (CNV) in the macular area. Although close attention has been paid to AMD, and anti-vascular endothelial growth factor (VEGF) drugs are available, its complex pathogenesis is still elusive. Tie2-expressing macrophages (TEMs) have been found to promote angiogenesis in remodel tissues and tumours. This study aimed to elucidate the role of macrophage Tie2 signalling in laser-induced CNV (LCNV). We observed that TEMs were responsible for the severity of CNV. Mechanistically, TEM deletion resulted in impaired LCNV due to the suppression of inflammatory angiogenesis and the promotion of apoptosis. We also observed that TEMs prevented apoptosis of b.End3 cells, but promoted their migration, proliferation and tube formation via VEGF, extracellular signal-regulated kinase (ERK) and v-akt murine thymoma viral oncogene (AKT)-dependent signalling pathways. The flow cytometry results comparing dry AMD patients and healthy controls with wet AMD patients showed that the percentage of Tie2+CD14+ cells was higher in the wet AMD patients' peripheral blood. This study demonstrates that Tie2 expression by macrophages intensifies CNV in LCNV murine models, thereby proposing an additional intervention option to inhibit CNV.

Essential contribution of macrophage Tie2 signal mediated autophagy in laser-induced choroidal neovascularization.

Autophagy plays critical roles in various ocular diseases, including age-related macular degeneration (AMD). Tie2-expressing macrophages (TEMs) play crucial roles in angiogenesis. To investigate the role of TEMs and autophagy in the development of AMD, we employed macrophage-specific Tie2 knockout mice and used a laser-induced choroidal neovascularization (CNV). The results showed that TEMs can promote CNV formation by up-regulating the level of autophagy. These results were further verified by in vitro cell experiments that peritoneal macrophages from Tie2 knockout mice can inhibit the expression of autophagy-related factors and inhibit the expression of angiogenic factor of VEGF by activating AMPK signaling pathway. Our results suggest that TEMs and macrophage Tie2 signal mediated-autophagy play critical role in experimental CNV, and they may be novel preventive targets for AMD treatment.

Blockade of insulin receptor substrate-1 inhibits biological behavior of choroidal endothelial cells.

AIM: To investigate the effects of blockade of insulin receptor substrate-1 (IRS-1) on the bio-function of tube formation of human choroidal endothelial cells (HCECs). METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to determine the expression level of IRS-1 and phospho-IRS-1 in HCECs. Tube formation of HCECs was analyzed using three dimensional in vitro Matrigel assay with or without IRS-1 blockage via IRS-1 inhibitor (GS-101) and vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. In addition, cell counting kit (CCK)-8 and Transwell migration assay were exerted to analyze the effects of blockade of IRS-1 on the bio-function of proliferation and migration of HCECs, respectively. The apoptosis of HCECs was examined using flow cytometry (FCM). RESULTS: RT-PCR and Western blot revealed that IRS-1 phospho-IRS-1 were expressed in HCECs and the expression level was enhanced by stimulation of VEGF-A. The number of tube formation was decreased significantly in GS-101 treated groups compared to phosphate buffered saline (PBS) treated control groups. Furthermore, both cell proliferation and migration of HCECs were decreased in the presence of GS-101. FCM analysis showed that the apoptosis of HCECs was enhanced when the cells were treated with GS-101. Western blot also showed that the expression level of cleaved-caspase 3 in GS-101 treated group was higher than that in control group. CONCLUSION: Blockade of IRS-1 can inhibit tube formation of HCECs through reducing cell proliferation and migration and promoting cell apoptosis.

High glucose: activating autophagy and affecting the biological behavior of human lens epithelial cells.

AIM: To clarify the effect of autophagy on human lens epithelial cells (HLECs) under high glucose conditions. METHODS: HLECs were cultured with different concentrations of glucose and 3-methyladenine (3-MA); the expression of autophagy-related protein LC3B was detected by Western blotting and immunofluorescence histochemistry. The migration of HLECs was quantified by scratch wound assay and the expression of transforming growth factor-ß (TGF-ß) was measured by real-time polymerase chain reaction. RESULTS: Compared with 5 mmol/L normal glucose treatment, 40 mmol/L glucose treatment can significantly increase the generation of autophagosome in HLECs, which could be inhibited by 0.375 mmol/L 3-MA treatment. The migration of HLECs and the expression of TGF-ß in HLECs induced by high glucose were significantly suppressed by 0.375 mmol/L 3-MA treatment. CONCLUSION: Autophagy promotes HLECs cell migration and increases the expression of TGF-ß after exposed to high glucose, which may relate to the development of diabetic cataract.

Streptozotocin­induced diabetic mice exhibit reduced experimental choroidal neovascularization but not corneal neovascularization.

The present study aimed to investigate the effects of diabetes mellitus (DM) on the generation of experimental corneal neovascularization (CrNV) and choroidal neovascularization (ChNV). Diabetes was induced in mice by intraperitoneal injection of streptozotocin (STZ). Experimental CrNV and ChNV were induced by alkali injury and laser photocoagulation, respectively. CrNV and ChNV were compared between the STZ­induced diabetic mice and control mice two weeks after injury. Relative expression of angiogenic factors was quantified by reverse transcription­quantitative polymerase chain reaction, and progenitor cell or macrophage accumulation in the early phase following injury was examined by flow cytometric analysis. Compared with the alkali­injured normal mice, the alkali­injured diabetic mice (STZ­induced) exhibited no significant difference in CrNV occurrence, whereas the laser­injured diabetic mice exhibited significantly reduced levels of ChNV compared with those of the laser­injured control animals. The laser­induced intrachoroidal mRNA expression levels of angiogenic factors, including vascular endothelial growth factor, hypoxia­induced factor­1α, chemokine (C­C motif) ligand 3, and stromal cell­derived factor­1α, were reduced in the laser­injured diabetic mice when compared with laser­injured control mice. Furthermore, the laser­induced intrachoroidal infiltration of c­Kit+ progenitor cells was impaired in the laser­injured diabetic mice compared with the laser­injured control mice. Overall, diabetes did not exert a significant effect on the generation of experimental CrNV. However, diabetes reduced laser­induced ChNV through downregulation of intrachoroidal progenitor cell infiltration and angiogenic factor expression.

B-cell leukemia/lymphoma 10 promotes angiogenesis in an experimental corneal neovascularization model.

PURPOSE: Corneal neovascularization (CrNV) arises from many causes including corneal inflammatory, infectious, or traumatic insult, and frequently leads to impaired vision. This study seeks to determine the role of B-cell leukemia/lymphoma 10 (BCL-10) in the development of experimental CrNV. METHODS: Corneas from BCL-10 knockout (KO) mice and wild-type (WT) mice were burned by sodium hydroxide (NaOH) to create the CrNV model and neovascular formation in the corneas was assessed 2 weeks later. Intracorneal macrophage accumulation and the expression of angiogenic factors were quantified by flow cytometric analysis (FCM) and real-time PCR, respectively. RESULTS: The amount of CrNV was determined 2 weeks after alkali burn. Compared to WT mice, the amount of CrNV in BCL-10 KO mice was significantly decreased. FCM revealed that F4/80-positive macrophages were markedly decreased in BCL-10 KO mice compared with WT mice. Reverse transcription PCR showed that the mRNA expression levels of intracorneal vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (bFGF) and monocyte chemotactic protein 1 were reduced in BCL-10 KO mice compared with WT mice. CONCLUSION: BCL-10 KO mice exhibited reduced alkali-induced CrNV by suppressing intracorneal macrophage infiltration, which subsequently led to decreased VEGF-A and bFGF expression, suggesting that BCL-10 may become a potential clinical intervening target of CrNV.