3D bioprinted endothelial cell-microglia coculture for diabetic retinopathy modeling.

Diabetic retinopathy (DR) is a common diabetes complication leading to vision impairment or blindness due to retinal vasculature alterations. Hyperglycemia induces structural alterations, inflammation, and angiogenic factor upregulation. Current treatments targeting vascular endothelial growth factor are insufficient for approximately 20% of DR patients, necessitating alternative approaches. Microglia (MG), essential for retinal homeostasis, remains underexplored in DR. This study used digital light processing bioprinting to construct a 3D coculture model of endothelial cells (ECs) and MG under varying glucose conditions, with a hydrogel stiffness of 4.6-7.1 kPa to mimic the extracellular matrix property of retina plexiform. Our results showed that high glucose levels influenced both EC and microglial phenotypes, gene expression, and angiogenic potential. Increasing glucose from 5 mM to 25 mM reduces drug efficacy by 17% for Aflibercept in EC monoculture, and 25% and 30% for Aflibercept and Conbercept in EC-MG coculture, respectively, suggesting that diabetic condition and MG presence could interfere with drug responses. In conclusion, our findings emphasize the importance of cellular interactions and microenvironmental factors in DR therapy, aiming to identify novel strategies and improve understanding of MG's role in disease pathogenesis.

[Design and Development of Cloud Platform of Emergency COVID-19 Nucleic Acid Detection].

According to the characteristics of short time and large amount of samples for out of hospital emergency nucleic acid detection, this study introduces an out of hospital emergency nucleic acid detection cloud platform system, which realizes the functions of rapid identification of the detected person and one-to-one correspondence with the samples, and real-time upload of the detection results to Zhejiang Government service network for quick viewing and statistics, so as to complete the task of national nucleic acid screening efficiently and accurately that we must provide information support.

Synthesis of ponatinib analogues as novel inhibitors of leukemia stem cells.

Aim: To synthesize new analogues of ponatinib and evaluate anti-leukemia cells and cytotoxicity. Methodology & results: The inhibitory activity of compounds 13a and 13c against K562 and HL60 cells was comparable to that of ponatinib (IC50 = 0.74, 0.88 vs 0.64 nM and 0.59, 0.77 vs 0.39 nM, respectively). Compounds 13a and 40b were 34- and 77-fold more potent than ponatinib against KG1a cells (IC50 = 0.091 and 0040 vs 3.6 µM, respectively). Compounds 13a, 13c and 40b also decreased the Abl protein level in the K562 cells, inhibited colony formation in MCF-7 cells and inhibited cell migration in B16BL6 cells. Compound 13a showed low cytotoxicity in 293 cells. Conclusion: Compound 13a was the best lead compound.

Progression Patterns of Myopic Traction Maculopathy in the Fellow Eye After Pars Plana Vitrectomy of the Primary Eye.

Purpose: This retrospective study investigated the patterns and risk factors of progression of myopic traction maculopathy (MTM) of fellow eyes after pars plana vitrectomy (PPV) of primary eyes. Methods: The study population comprised 153 patients with MTM in both myopic eyes who sequentially underwent PPV (2006-2021). Observation periods were from PPV of the primary eye (baseline) to PPV of the fellow (end). MTM was graded based on optical coherence tomography (OCT) images and the ATN (atrophy [A], traction [T], and neovascularization [N]) system. An increase in T grade was considered MTM progression. Results: MTM progressed in 43.8% of fellow eyes during 34.57 ± 34.08 months. The progression of fellow eyes correlated with T grade of primary eyes (P < 0.001). Risk factors for the progression of MTM in fellow eyes were primary eyes in T4-T5, age at baseline <60 years, and fellow eyes with partial posterior vitreous detachment (PVD; P < 0.001, P = 0.042, and P = 0.002, respectively). Fellow eyes in T1/T2 at baseline progressed faster compared with those in T0 (P < 0.001); the annual rate of progression to T3-T5 of the T0 (T1-T2) groups was 9.98% (24.59%). Conclusions: Risk factors for the progression of MTM in fellow eyes included PPV when relatively young, primary eye at high T grade, and partial PVD of the fellow eye. Personalized follow-up for fellow eyes should be based on the severity of MTM of both eyes.

Microglia enhanced the angiogenesis, migration and proliferation of co-cultured RMECs.

BACKGROUND: Attention is increasingly being given to microglia-related inflammation in neovascular diseases, such as diabetic retinopathy and age-related macular disease. Evidence shows that activated microglia contribute to disruption of the blood-retinal barrier, however, the mechanism is unclear. In this study, we aimed to clarify whether and how microglia affect the function of retinal microvascular endothelial cells (RMECs). METHODS: We activated microglia by Lipopolysaccharides (LPS) stimulation. After co-culturing static or activated microglia with RMECs using the Transwell system, we evaluated the function of RMECs. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) levels in the supernatant from the lower chamber were evaluated by ELISA. Angiogenesis, migration, and proliferation of RMECs were assessed by tube formation, wound healing, and WST-1 assays. The expression levels of tight junction proteins (ZO-1 and occludin) and endothelial markers (CD31 and CD34) were examined by Western blot analysis. RESULTS: We successfully established an LPS-activated microglia model and co-culture system of static or activated microglia with RMECs. In the co-culture system, we showed that microglia, especially activated microglia stimulated VEGF-A and PDGF-BB expression, enhanced angiogenesis, migration, proliferation, and permeability, and altered the phenotype of co-cultured RMECs. CONCLUSIONS: Microglia, especially activated microglia, play important roles in angiogenesis and maintenance of vascular function hemostasis in the retinal microvasculature. The mechanism needs further investigation and clarification.

Development and validation of a Health Policy Model of Type 2 diabetes in Chinese setting.

Aim: Due to the difference in epidemiology and outcomes between eastern and western populations with Type 2 diabetes mellitus (T2DM), an important challenge is determining how useful the outcomes from diabetes models based on western populations are for eastern patients. Consequently, the principal aim of this study was to develop and validate a Health Policy Model (Chinese Outcomes Model for T2DM [COMT]) for supporting Chinese medical and health economic studies. Methods: The model is created to simulate a series of important complications of T2DM diabetes based on the latest Risk Equations for Complications of Type 2 Diabetes, which was adjusted by adding the adjustment regulator to the linear predictor within the risk equation. The validity of the model was conducted by using a total of 171 validation outcomes from seven studies in eastern populations and ten studies in western populations. The simulation cohorts in the COMT model were generated by copying each validation study's baseline characteristics. Concordance was tested by assessing the difference between the identity (45°) line and the best-fitting regression of the scatterplots for the predicted versus observed outcomes. Results: The slope coefficients of the best-fitting regression line between the predicted and corresponding observed actual outcomes was 0.9631 and the R2 was 0.8701. There were major differences between western and eastern populations. The slope and R2 of predictions were 0.9473 and 0.9272 in the eastern population and 1.0566 and 0.8863 in the western population, which showed more perfect agreement with the observed values in the eastern population than the western populations. The subset of macro-vascular and micro-vascular outcomes in the eastern population showed an identical tendency (the slope coefficient was close to 1), and mortality outcomes showed a slight tendency toward overestimation (the slope coefficient was close to 0.9208). Some degree of underprediction of macro-vascular and micro-vascular end points and overprediction of mortality end point was found in the western population. Conclusion: The COMT diabetes model simulated the long-term patient outcomes observed in eastern Asian T2DM patients with prediction accuracy. This study supports the COMT as a credible tool for Chinese healthcare decision makers. Further work is necessary to incorporate new local data to improve model validity and credibility.

Predictive multi-imaging biomarkers relevant for visual acuity in idiopathic macular telangiectasis type 1.

BACKGROUND: To evaluate the structural changes associated with visual acuity (VA) in patients with idiopathic macular telangiectasia (MT) type 1 using multimodal imaging modalities. METHODS: A retrospective study of 14 patients with MT type 1 and of 10 eyes from 10 healthy individuals as age-matched controls was conducted. The medical records of patients who had undergone colour fundus photography, spectral domain optical coherence tomography (OCT), fluorescein angiography and OCT angiography were reviewed. Central macular thickness (CMT), the areas of macular oedema and ellipsoid zone (EZ) disruption, EZ length, disorganization of the retinal inner layers (DRIL) and external limiting membrane (ELM) disruption, as measured by spectral domain OCT; and vascular density and the foveal avascular zones (FAZ) of the superficial capillary plexus (SCP) and deep capillary plexus (DCP), as measured by OCT angiography, were assessed in MT type 1 eyes and correlated with VA. RESULTS: The mean baseline best-corrected VA of MT type 1 eyes was 0.45 ± 0.28. The mean CMT was 385.19 ± 75.21 µm in MT type 1 eyes and 252.43 ± 15.77 µm in contralateral eyes (Z = - 4.113, p < 0.001). The mean vessel density of the DCP was lower in MT type 1 eyes (47.25 ± 4.69%) than in contralateral eyes (53.93 ± 2.94%) and normal eyes (59.37 ± 2.50%) (Z = - 3.492, - 4.099; p < 0.001, < 0.001). The baseline logMAR VA was correlated with CMT (r = 0.682, p = 0.007), SCP density (r = - 0.652, p = 0.012), DCP density (r = - 0.700, p = 0.005), total area of EZ disruption (r = 0.649, p = 0.012); and total lengths of EZ (r = 0.681, p = 0.007), ELM (r = 0.699, p = 0.005) and DRIL (r = 0.707, p = 0.005) disruption in the 1-mm-diameter foveal region in MT type 1 eyes. CONCLUSIONS: Decreased DCP density and the presence of DRIL may be predictive biomarkers of VA in MT type 1. CMT, SCP density, total area of EZ disruption, and lengths of EZ and ELM disruption within the 1-mm-diameter central region were strongly associated with VA.

Activated microglia induce the production of reactive oxygen species and promote apoptosis of co-cultured retinal microvascular pericytes.

PURPOSE: Pericyte apoptosis is a predominant feature of early diabetic retinopathy. In diabetic retinopathy, activated microglia migrate and release proinflammatory cytokines that contribute to disruption of the blood-retinal barrier, neuronal loss, and enhanced ROS production. Reactive oxygen species (ROS) are implicated in pericyte death; however, the mechanism by which activated microglia affect retinal microvascular pericytes is unclear. We hypothesized that activated microglia may promote pericyte apoptosis by enhancing ROS production. METHODS: Lipopolysaccharide (LPS)-activated microglia and pericytes were co-cultured in a cell culture system. Pericyte ROS production and the mitochondrial membrane potential (ΔΨm) were determined by flow cytometry. The pericyte protein expression levels of NADPH oxidase subunits, uncoupling protein 2, nuclear NF-κB-p65, and caspase-3 were determined by western blotting. One-way ANOVAs were used for statistical analysis. RESULTS: LPS successfully activated the microglia, as demonstrated by their morphological and phenotype changes and the significant increase in tumor necrosis factor secretion (P < 0.01). Co-culture with activated microglia significantly up-regulated NADPH oxidase subunits (NOX4, NOX2, and NCF1; P < 0.01) and down-regulated uncoupling protein 2 expression (P < 0.01) in pericytes. Pericyte ROS production increased by 20% in the activated microglia co-cultured group, and was inhibited by pretreatment with diphenyleneiodonium, coenzyme Q10, and N-acetylcysteine. The proapoptotic pericyte changes induced by co-culture with activated microglia included a 9.50% decrease in pericyte ΔΨm and significant increases in NF-κB-p65 nuclear translocation (P < 0.01) and activated caspase-3 (P < 0.01). These proapoptotic effects of activated microglia were inhibited by diphenyleneiodonium. CONCLUSIONS: Our results are consistent with our hypothesis that activated microglia may promote pericyte apoptosis by enhancing ROS production. Further studies are needed to examine retinal microglia activation and the corresponding changes in pericytes in a rat model of diabetes mellitus.

Different Strategies for the Treatment of Age-Related Macular Degeneration in China: An Economic Evaluation.

Purpose. To assess the cost-effectiveness of bevacizumab compared to ranibizumab, verteporfin photodynamic therapy (PDT), and usual care for the treatment of age-related macular degeneration (AMD) in China. Methods. A Markov model was developed according to patient visual acuity (VA) in the better-seeing eye (Snellen scale). Four cohorts of patients were treated with one of the following therapies: bevacizumab, ranibizumab, PDT, or usual care. Clinical data related to treatments were obtained from published randomized clinical trials. Direct medical costs and resource utilization in the Chinese health care setting were taken into account. Health and economic outcomes were evaluated over a lifetime horizon. Sensitivity analyses were performed. Results. Treatment with ranibizumab provided the greatest gains in quality-adjusted life-years (QALYs). The cost per marginal QALY gained with bevacizumab over usual care was $1,258, $3,803, and $2,066 for the predominantly classic, minimally classic, and occult lesions, respectively. One-way sensitivity analysis showed considerably influential factors, such as utility values and effectiveness data. Probabilistic sensitivity analysis indicated that, compared to usual care, PDT and ranibizumab most cases would be cost-effective in the bevacizumab arm at a threshold of $7,480/QALY. Conclusion. Bevacizumab can be a cost-effective option for the treatment of AMD in the Chinese setting.

Pediatric posttraumatic endophthalmitis.

BACKGROUND: To identify the etiology, pars plana vitrectomy results, culture results, and visual outcome of endophthalmitis in the pediatric age group. METHODS: Fifteen eyes of 15 consecutive pediatric patients who were treated and followed up for endophthalmitis between July 2011 and December 2012 were included in this study. Combined pars plana vitrectomy and intravitreal norvancomycin plus ceftazidime injection was performed on all 15 eyes. The vitreous samples were obtained by a standard pars plana vitrectomy. The specimens were sent to microbiology facility for gram staining, culturing, and sensitivity testing. RESULTS: The mean age of the patients was 5.3 ± 2.8 years (3-10 years). The mean follow-up time was 9.7 ± 1.5 months (7-12 months). Preoperatively, the visual acuities of the patients were as follows: light perception (n = 7), hand movements (n = 4), and counting fingers from 30 cm (n = 1), and these values were not available in three patients (n = 3). Postoperatively, the final best corrected visual acuity was 20/200 or better in six (40 %), counting of fingers in four (26.7 %), light perception to hand movements in two (13.3 %), and no light perception in one (6.7 %), with results being unavailable for two patients. Positive culture results were obtained from ten eyes (66.7 %). A single species was isolated in eight eyes, and multiple organisms were isolated in two eyes. CONCLUSIONS: Penetrating ocular trauma was the most common cause of pediatric endophthalmitis in this consecutive series of endophthalmitis. Staphylococci and Streptococcus species were the most common isolated organisms. Patients with multiple organisms had poor visual recovery. Visual outcomes were poor in this series in spite of vitrectomy being performed immediately on all patients.

Strategies to Screen for Diabetic Retinopathy in Chinese Patients with Newly Diagnosed Type 2 Diabetes: A Cost-Effectiveness Analysis.

To investigate the cost-effectiveness of different screening intervals for diabetic retinopathy (DR) in Chinese patients with newly diagnosed type 2 diabetes mellitus (T2DM). Chinese healthcare system.Chinese general clinical setting. A cost-effectiveness model was developed to simulate the disease course of Chinese population with newly diagnosed with diabetes. Different DR screening programs were modeled to project economic outcomes. To develop the economic model, we calibrated the progression rates of DR that fit Chinese epidemiologic data derived from the published literature. Costs were estimated from the perspective of the Chinese healthcare system, and the analysis was run over a lifetime horizon. One-way and probabilistic sensitivity analyses were performed. Total costs, vision outcomes, costs per quality-adjusted life year (QALY), the incremental cost-effectiveness ratio (ICER) of screening strategies compared to no screening. DR screening is effective in Chinese patients with newly diagnosed T2DM, and screen strategies with ≥4-year intervals were cost-effective (ICER <$7,485 per QALY) compared to no screening. Screening every 4 years produced the greatest increase in QALYs (11.066) among the cost-effective strategies. The screening intervals could be varied dramatically by age at T2DM diagnosis. Probabilistic sensitivity analyses demonstrated the consistency and robustness of the cost-effectiveness of the 4-year interval screening strategy. The findings suggest that a 4-year interval screening strategy is likely to be more cost-effective than screening every 1 to 3 years in comparison with no screening in the Chinese setting. The screening intervals might be tailored according to the age at T2DM diagnosis.

Microarray profiling of long non-coding RNA (lncRNA) associated with hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an inherited disorder with around 1400 known mutations; however the molecular pathways leading from genotype to phenotype are not fully understood. LncRNAs, which account for approximately 98 % of human genome, are becoming increasingly interesting with regard to various diseases. However, changes in the expression of regulatory lncRNAs in HCM have not yet been reported. To identify myocardial lncRNAs involved in HCM and characterize their roles in HCM pathogenesis. METHODS: Myocardial tissues were obtained from 7 HCM patients and 5 healthy individuals, and lncRNA and mRNA expression profiles were analyzed using the Arraystar human lncRNA microarray. Real-time PCR was conducted to validate the expression pattern of lncRNA and mRNA. Gene ontology (GO) enrichment and KEGG analysis of mRNAs was conducted to identify the related biological modules and pathologic pathways. RESULTS: Approximately 1426 lncRNAs (965 up-regulated and 461 down-regulated) and 1715 mRNAs (896 up-regulated and 819 down-regulated) were aberrantly expressed in HCM patients with fold change > 2.0. GO analysis indicated that these lncRNAs-coexpressed mRNAs were targeted to translational process. Pathway analysis indicated that lncRNAs-coexpressed mRNAs were mostly enriched in ribosome and oxidative phosphorylation. CONCLUSION: LncRNAs are involved in the pathogenesis of HCM through the modulation of multiple pathogenetic pathways.

Ranibizumab versus bevacizumab for ophthalmic diseases related to neovascularisation: a meta-analysis of randomised controlled trials.

BACKGROUND: Bevacizumab is believed to be as effective and safe as ranibizumab for ophthalmic diseases; however, its magnitude of effectiveness and safety profile remain controversial. Thus, a meta-analysis and systematic review appears necessary. METHODS: PubMed and EMBASE were systematically searched with no restrictions. All relevant citations comparing ranibizumab and bevacizumab were considered for inclusion. Pooled effect estimates were obtained using a fixed- and random-effects meta-analysis. RESULTS: Nine independent randomised-controlled clinical trials (RCTs) involving 2,289 participants were identified. Compared with bevacizumab, the overall combined weighted mean difference (WMD) of the mean change in visual acuity for ranibizumab was 0.52 letters (95% CI -0.11-1.14). The odds ratios (ORs) of gaining ≥15, gaining 5-14, losing 5-14 and losing ≤15 letters were 1.10 (95% CI 0.90-1.33), 0.93 (95% CI 0.77-1.11), 0.89 (95% CI 0.65-1.22) and 0.95 (95% CI 0.73-1.25), respectively. The risk of serious systemic events increased by 17% (95% CI 6%-27%, p = 0.0042) for bevacizumab treatment in comparison with ranibizumab. No statistically significant differences between the two treatments were found for the nonfatal arterial thrombotic events, ocular serious adverse, death from vascular and all causes events. CONCLUSIONS: Bevacizumab is not inferior to ranibizumab as a treatment for achieving visual acuity. The use of bevacizumab was associated with an increased risk of developing serious systemic events. Weighing the costs and health outcomes is necessary when selecting between bevacizumab and ranibizumab for ophthalmic diseases. Due to the limitations of the available data, further research is needed.

Functional analysis of a survivin-like gene in Bombyx mori.

The survivin (svv) gene is a newly discovered member of the inhibitors of apoptosis gene family. In recent years, svv has been confirmed to have an anti-apoptosis function and to play a critical role in cell division. We identified a survivin-like gene in the silkworm, Bombyx mori (Bm-svv). In this study, to gain insight into its function, a baculovirus expression system was used to express the Bm-svv gene in insect cell lines. The recombinant viruses were then used as a vector to transform insect cells, and cell activity was determined using the Cell Counting Kit-8 (CCK-8), which is usually employed for detecting mammalian cell number. The results indicated that the Bm-svv gene plays a role in the cell growth arrest or apoptosis induced by viruses. Furthermore, the CCK-8 kit is effective in determining the activity of insect cells.

Supplementation with antioxidants attenuates transient worsening of retinopathy in diabetes caused by acute intensive insulin therapy.

BACKGROUND: To investigate whether insulin can increase the production of reactive oxygen species (ROS) in bovine retinal microvascular endothelial cells (BRECs), the role of antioxidants in the insulin-induced exacerbation of diabetic retinopathy and the related mechanisms. METHODS: BRECs were cultured in either 5 or 30 mM glucose for 3 days before stimulation with 100 nM insulin for 24 h or incubated with 1 mM apocynin, 100 µM LY294002, 50 µM U0126, 2 µM GF109203X, 250 U/ml catalase, 100 µg/ml ascorbic acid, 100 µM α-lipoic acid and 50 µM α-tocopherol before stimulation with 100 nM insulin. H(2)O(2) (200 µM) was added to cells to measure the VEGF protein expression. Intracellular ROS was measured by immunofluorescence and flow cytometry, superoxide anion measurement was done by cytochrome c reduction, and VEGF protein was measured by ELISA analysis. RESULTS: Insulin or (and) high glucose significantly increased intracellular ROS production in BRECs, and pretreatment of the cells with apocynin and LY294002 decreased insulin-induced superoxide anion production. Neither pretreatment with GF109203X nor U0126 showed an effect on the superoxide anion production. Ascorbic acid, α-lipoic acid, and α-tocopherol also decreased superoxide anion production. Furthermore, H(2)O(2) increased VEGF protein expression in BRECs and catalase suppressed insulin-induced VEGF protein expression. CONCLUSIONS: Insulin can increase ROS production through an NAD(P)H, phosphatidylinositol 3´-kinase-dependent mechanism in bovine retinal microvascular endothelial cells ex vivo. ROS can regulate insulin-induced VEGF expression. Supplementation with antioxidants may help to attenuate the transient worsening of retinopathy in diabetes caused by acute intensive insulin therapy.

[Epidemiologic analysis on severe fever with thrombocytopenia syndrome in Hubei province, 2010].

OBJECTIVE: To identify the epidemic characteristics and risk factors of an emerging infectious disease-severe fever with thrombocytopenia syndrome (SFTS) in Hubei province. METHODS: Active surveillance program on SFTS was set up in monitoring sites-hospitals, at the township level or above, in Suizhou, Huanggang and Wuhan from January to December, 2010. Specific surveillance program on SFTS was launched across the province in hospitals above the county level. Cases that matched the definition of surveillance case were identified and reported to Centers for Disease Control and Prevention (CDCs). Cases were interviewed and their blood samples collected and detected using PCR and virus isolation. We also conducted serum antibody surveys among healthy population and livestock and surveillance on vector ticks in those high-epidemic areas. RESULTS: 188 cases that matched the definition of surveillance case and 21 deaths were reported in 11 cities, 32 countries and 100 towns in 2010, with an incidence rate of 0.33/10(6). The fatality rate was 11.2%. Data showed that the patients were from hilly areas at the altitude elevated between 28-940 meters. The epidemic period was between April and December with the peak from May to September. The youngest case was an 11-year old, while the eldest was 81 with median age as 56-year old. 95.3% of the patients were farmers. All Patients did not have the history of traveling, two weeks before the onset of SFTS. 93.6% of the patients engaged in different kind of work which was associated with agriculture. 52.8% of the patients had been exposed to ticks. 22.0% of the patients had been bitten by ticks. Skin injury was found in 64.2% of the patients. Samples from 129 cases (68.6%) were collected and detected, with 67.4% of them (87 cases) showed positive by Real time-PCR for SFTS virus. An elevation in antibody titer by a factor of four or evidence of sero-conversion was observed in 11 patients; SFTS virus was isolated from 2 patients. The total antibody positive rates were 3.8%, 55.0% (6/11), 36.7% (2/3) and 80.0% (4/5) respectively in healthy population, dogs, sheep and cows. Ticks from grass, cattle and sheep were detected positive by Real time-PCR. CONCLUSION: Most cases of SFTS in Hubei were infected by SFTS virus, and cases of livestock were infected by SFTS virus. Ticks might serve as an important vector. Skin injury, exposure to tick bites seemed to be the risk factors.

Generation of trans-arachidonic acid under nitrative stress is associated with upregulation of thromponsdin-1 in diabetic rats.

BACKGROUND: Trans-arachidonic acids (TAAs), newly discovered markers of nitrative stress and the major products of nitrogen dioxide (NO2(·))-mediated isomerization of arachidonic acid (AA), represent a new mechanism of NO2(·)-induced toxicity. It has been reported that TAAs were generated in oxygen-induced microvascular degeneration model and TAAs were also generated in a diabetic retinopathy (DR) model. In this study, we examined high glucose-induced nitrative stress damage and TAAs levels and explored the possible mechanisms for DR caused by reactive nitrogen species. METHODS: Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ) at 60 mg/kg. Bovine retinal capillary endothelial cells (BRECs) were selectively cultured and incubated with normal or high glucose. The serum TAAs and AA in diabetic rats were measured by the gas chromatography and mass spectrometry (GC/MS) method. The ratio of peak area of TAAs to AA with selected ion of 79 was estimated by a group t-test. Thrombospondin-1 (TSP-1) in the rat retinas and BRECs extracts were examined by Western blotting. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) protein was examined by Western blotting in BRECs incubated with high glucose. RESULTS: The TAAs to AA ratio (TAAs/AA) was significantly increased in the serum at 8, 12 and 16 weeks after STZ injection (P < 0.05), with no noticeable change found at 2 or 4 weeks (P > 0.05). Expression of TSP-1 in the retina of diabetic rats was progressively elevated according to the duration of diabetes. TSP-1 expression was increased in BRECs incubated with high glucose at 48 hours. Moreover, high glucose also increased ERK1/2 expression, which peaked at 30 minutes and then decreased in the following 48 hours. CONCLUSION: An elevation of TAAs/AA is associated with high glucose-induced nitrative stress, which probably involves upregulation of TSP-1 through activating ERK1/2.

Different effects of low- and high-dose insulin on ROS production and VEGF expression in bovine retinal microvascular endothelial cells in the presence of high glucose.

BACKGROUND: Clinical trials have demonstrated that acute intensive insulin therapy may cause transient worsening of retinopathy in type 1 and type 2 diabetes patients. However, the related mechanism still remains controversial. The purpose of the present study was to investigate the effect of insulin on the mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) production, UCP-2 and VEGF expression in bovine retinal microvascular endothelial cells (BRECs) in the presence of normal or high glucose and the related mechanisms. METHODS: BRECs were isolated as primary cultures and identified by immunostaining. Passage BRECs were initially exposed to normal (5 mM) or high glucose (30 mM) for 3 days, with equimolar L: -glucose supplemented for osmotic equation. Then the cells were treated with 1 nM, 10 nM, or 100 nM insulin for 24 h: △Ψm and ROS production were determined by JC-1 and CM-H2DCFDA, respectively. Expression of UCP-2 and VEGF mRNA was determined by real-time RT-PCR; expression UCP-2 and VEGF protein was determined by Western-blotting analysis. A general ROS scavenger N-acetylcysteine (NAC, 10 mM) and an NADPH oxidase inhibitor apocynin (1 mmol/l) were added 1 h before treatment with 100 nM insulin. RESULTS: Insulin increased △Ψm, ROS production, and expression of UCP-2 and VEGF in BRECs at normal glucose (5 mM) in a dose-dependent manner. Low-dose insulin (1 nM) decreased △Ψm, ROS production, and UCP-2, VEGF expression in BRECs at high glucose (30 mM); and high-dose insulin (10 nM, 100nM) recovered △Ψm, ROS production, and UCP-2, VEGF expression. Pretreatment of cells with NADPH oxidase inhibitor apocynin significantly suppressed 100 nM insulin-induced ROS production (p < 0.01, one-way ANOVA). Pretreatment of cells with ROS scavenger N-acetylcysteine completely blocked insulin-induced UCP-2 expression (p < 0.01, one-way ANOVA) and significantly suppressed VEGF expression (p < 0.01, one-way ANOVA). CONCLUSIONS: High-dose insulin-induced ROS production and VEGF expression in BRECs in the presence of high glucose might be one of the reasons for the transient worsening of diabetic retinopathy during intensive insulin treatment.

[Clinical analysis of sarcoidosis of ocular adnexa].

OBJECTIVE: To study the diagnosis and treatment of 17 patients with sarcoidosis in ocular adnexa. METHODS: The clinical data of 17 cases with sarcoidosis in ocular adnexa treated during 1993 and 2008 were retrospectively analyzed. All patients underwent surgical treatment, and the diagnosis was proven histopathologically. RESULTS: Among the 17 cases, 4 were male, and 13 were female. The patients' age ranged from 15 to 70 years, with a mean of 46.9 years. The lesions were located at the orbit (8 cases), lacrimal grand (5 cases) and eyelids (4 cases). Fourteen cases complained of the presence of a local mass, 2 cases complained of exophthalmos and 1 swelling of eyelids. Concurrent systemic sarcoidosis was present in 7 cases. Three cases coincided with lung sarcoidosis, 3 cases with uveitis and 1 case with dermatopathy. Angiotensin converting enzyme analysis was performed in 6 cases; 4 of them were elevated. Computer tomography was performed in 12 cases; in 11 cases it presented as moderate density parenchymatous mass, and in the remaining one it presented as hypodensity cystic mass. B scan of 5 cases showed hypoechoic parenchymatous homogeneous mass. None of 14 cases relapsed after 1 to 15 years follow-up. CONCLUSIONS: Ocular adnexal sarcoidosis usually presents as local mass and it should be included in the differential diagnosis of orbital and ocular adnexal lesions. Excision of localized mass alone could achieve satisfactory outcomes for isolated lesions, while for diffuse or systematic lesions, corticosteroid treatment should be prescribed routinely.

Inhibition of pathologic retinal neovascularization by a small peptide derived from human apolipoprotein(a).

PURPOSE: To evaluate the effect of KV11, a novel 11-mer peptide from human apolipoprotein(a), against retinal neovascularization and to study its penetration and the possible toxicity to the retina. METHODS: Wound-healing, a modified Boyden chamber, and MTS assays were used to evaluate the effect of KV11 on the migration and proliferation of bovine retinal capillary endothelial cells (BRCECs) induced by vascular endothelial growth factor (VEGF) in vitro. The antiangiogenic effect of KV11 was also studied with a mouse model of oxygen-induced retinopathy. Then, FITC-labeled KV11 was injected into the vitreous of normal rabbits, the retinal penetration was determined by confocal laser-scanning microscope, and further confirmed by UPLC/MS analysis of KV11 in tissue extracts. Electrophysiological tests and histologic examinations were used to study the possible toxicity of KV11 against rabbit neuroretina after intravitreal administration. RESULTS: KV11 inhibited VEGF-induced BRCEC migration but not proliferation and reduced the pathologic neovascularization in a mouse model, without affecting normal retinal vasculature. FITC-labeled KV11 appeared in the retina within 30 minutes after injection and diffused to all layers 3 hours later. The transfer of KV11 from the vitreous to the retina was confirmed by UPLC/MS data. Electrophysiologic tests and histologic examinations revealed no evident functional or morphologic abnormalities in rabbit neuroretina after KV11 injection. CONCLUSIONS: It is concluded that the novel peptide KV11 is an effective inhibitor of retinal pathologic angiogenesis with a sufficient retinal penetration and a favorable safety profile and may provide a promising alternative for ocular antiangiogenic therapy.