Evaluation of meibomian gland dysfunction in type 2 diabetes with dry eye disease: a non-randomized controlled trial.

BACKGROUND: The purpose of this investigation was to evaluate the morphology and physiological function of the meibomian glands between type 2 diabetics with dry eye disease (DED) and control subjects. Doing so will help to better reveal the pathologic mechanisms of meibomian gland dysfunction (MGD) and DED in type 2 diabetes mellitus (T2DM). METHODS: Ninety subjects were divided into the following four groups: DM-DED group: T2DM patients with DED (n = 30); DM control group: DM patients without DED (n = 18); DED group: DED patients without DM (n = 26); and normal control group: normal subjects (n = 16). All participants administered the ocular surface disease index (OSDI) questionnaire, tear meniscus height (TMH), noninvasive Keratograph tear film break-up time (NIKBUT), Schirmer I test (SIT), corneal fluorescein staining (CFS), eyelid margin abnormality examinations, meibum quality and meibomian gland (MG) dropout evaluations. RESULTS: The percentage of MG dropout in the upper and lower lids was significantly higher in the DM-DED group than the DED group (P < 0.05 or P < 0.01). However, there was no significant difference in other MG parameters between these two groups. Oppositely, Significant difference was observed in all of MG parameters except MG dropout in the lower lids comparing DM group with normal controls (P < 0.05 or P < 0.01). While the SIT values decreased in the DM-DED group compared to the DED group (P < 0.05), no significant differences were found in the values of other tear parameters. CONCLUSIONS: The higher prevalence and increased severity of MGD was found in patients with both T2DM and DED compared to those only with DED. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR1800019939, date of registration December 9, 2018, prospectively registered.

Resveratrol inhibits TGF-ß1-induced fibrotic effects in human pterygium fibroblasts.

BACKGROUND: Resveratrol is a polyphenolic phytoalexin which has the properties of anti-oxidant, anti-inflammatory and anti-fibrotic effects. The aim of this study was to investigate the anti-fibrotic effects of resveratrol in primary human pterygium fibroblasts (HPFs) and elucidate the underlying mechanisms. METHOD: Profibrotic activation was induced by transforming growth factor-beta1 (TGF-ß1). The expression of profibrotic markers, including type 1 collagen (COL1), α-smooth muscle actin (α-SMA), and fibronectin, were detected by western blot and quantitative real-time-PCR after treatment with various concentrations of resveratrol in HPFs to investigate the anti-fibrotic effects. Relative signaling pathways downstream of TGF-ß1 were detected by Western blot to assess the underlying mechanism. Cell viability and apoptosis were assessed using CCK-8 assay and flow cytometry to evaluate proliferation and drug-induced cytotoxicity. Cell migration and contractile phenotype were detected through wound healing assay and collagen gel contraction assay. RESULTS: The expression of α-SMA, FN and COL1 induced by TGF-ß1 were suppressed by treatment with resveratrol in dose-dependent manner. The Smad3, mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol-3-kinase (PI3K) / protein kinase B (AKT) pathways were activated by TGF-ß1, while resveratrol attenuated those pathways. Resveratrol also inhibited cellular proliferation, migration and contractile phenotype, and induced apoptosis in HPFs. CONCLUSIONS: Resveratrol inhibit TGF-ß1-induced myofibroblast activation and extra cellular matrix synthesis in HPFs, at least partly, by regulating the TGF-ß/Smad3, p38 MAPK and PI3K/AKT pathways.

Culture and identification of multipotent stem cells in guinea pig sclera.

BACKGROUND: To investigate whether the sclera of guinea pig contains stem cells with multiple differentiation potentials. METHODS: Scleral tissue from guinea pig was separated from the retina and choroid and digested to release single cells. The cells cultured was identified as stem cells by flow cytometric analysis, semiquantitative RT-PCR. Abilities for multipotent differentiation were analyzed by histochemical staining technique (oil-red-O staining, alcian blue staining and alizarin red staining). Scleral fibroblast cell was treated as control group. RESULTS: The cultured scleral stem cells were positive for CD44 and CD105 (mesenchymal stem cell surface markers) by flow cytometry. The cells cultured expressed stem cell markers ABCG2, Notch1, Six2, and Pax6, and the most important component of sclera type I collagen. The positive staining informed that the cells cultured were able to differentiate to adipogenic, chondrogenic, and osteogenic lineages. Scleral fibroblast cell was stained negative by oil-red-O staining and alizarin red staining. Expression of Sox9 in the cells cultured after chondrogenic differentiation significantly increased compared with scleral fibroblast cell. CONCLUSION: The guinea pig sclera contained stem cells with multiple differentiation potentials. The cells were also related to scleral collagen and cartilage related proteins. The finding may provide a new tool to help clarify mechanisms of sclera related disease in further studies.

SPARC knockdown attenuated TGF-ß1-induced fibrotic effects through Smad2/3 pathways in human pterygium fibroblasts.

PURPOSE: Secreted protein acidic and rich in cysteine (SPARC), a matricellular glycoprotein, has been found to regulate processes involved in fibrotic diseases. The aim of this study was to investigate the anti-fibrotic effects of SPARC in primary human pterygium fibroblasts (HPFs) and elucidate the underlying mechanisms. METHODS: The expression of SPARC in HPFs was knocked down by RNA interference-based approach. Subsequently, we examined the expression of profibrotic markers induced by transforming growth factor-ß1 (TGF-ß1), including type 1 collagen (COL1), α-smooth muscle actin (α-SMA), and fibronectin (FN). The changes in signaling pathways and matrix metalloproteinases (MMPs) were also detected by western blotting. The cellular migration ability, proliferation ability, apoptosis, and contractile phenotype were detected using the wound healing assay, Cell Counting Kit-8 assay, flow cytometry, and collagen gel contraction assay, respectively. The interaction between SPARC and TGF-ß RII was detected by Co-IP RESULTS: Silencing of SPARC inhibited the basal and TGF-ß1-induced expression of COL1, α-SMA, and FN in HPFs, and suppressed the expression of p-Smad2, p-Smad3, Smad4 and MMP2, MMP9. The downregulation of SPARC also attenuated the cell migration and contractile phenotype of HPFs. SPARC could bind to TGF-ßRII under TGF-ß1 treatment. However, knockdown of SPARC did not affect the proliferation and apoptosis of HPFs. CONCLUSION: SPARC knockdown attenuated the fibrotic effect induced by TGF-ß1 at least in part by inactivating the Smad2/3 pathways in HPFs. Therefore, SPARC may be a promising therapeutic target for the treatment of pterygium.

Meibum lipid composition in type 2 diabetics with dry eye.

PURPOSE: The purpose of this investigation was to analyze and compare the composition of meibum between type 2 diabetics with dry eye disease (DED) and control subjects to better reveal the pathologic mechanisms of the meibomian gland degeneration (MGD) and DED in type 2 diabetes mellitus (T2DM). METHODS: 90 subjects were divided into the following 4 groups: DM-DED group: T2DM patients with DED (n = 30); DM control group: DM patients without DED (n = 18); DED group: DED patients without DM (n = 26); naive control group: normal subjects (n = 16). The lipid composition of meibum samples collected from these subjects was analyzed by high-pressure liquid chromatography-mass spectrometry (HPLC-MS) system. The content of lipid features from 12 major lipid classes was compared among the 4 groups. RESULTS: A significantly lower level of triacylglycerols (TG) and wax esters (WE) was found between DM-DED patients and normal controls (P < 0.01), whereas the level of Cholesteryl Ester (CE) in DM-DED patients increased compared with DED patients (P < 0.05). The level of (O-acyl)-omega-hydroxy fatty acids (OAHFA) in DM-DED patients was significantly lower than that in normal controls (P < 0.01). An opposite higher level of phospholipids (PLs) was observed in DM-DED patients than that in normal controls (P < 0.01). CONCLUSIONS: T2DM could influence the expression of meibum lipids to further aggravate DED and MGD. Lower expression of TG,WE and OAHFA, higher expression of CE and PLs were discovered in meibum lipids of T2DM-DED.

Plasma exosomal proteomic studies of corneal epithelial injury in diabetic and non-diabetic group.

OBJECTIVE: Diabetic Keratopathy (DK) is one of the significant complications of type II diabetes (T2DM) with pathogenesis not yet clarified. Since hyperglycemia is able to change the protein components contained in plasma exosomes, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as feasible to analyze the expression of plasma exosomal proteins in patients with T2DM and non-diabetic patients respectively, find critical biological markers, and explore the mechanism of DK as well as potential therapeutic targets. METHOD: Blood and clinical information of corneal epithelial injury in a diabetic group (the study group) and a non-diabetic group (the control group), who were patients admitted to the Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine from July 2020 to November 2020, were collected. The qEV size exclusion method was adopted to separate exosomes from plasma. The exosomes were then identified through transmission electron microscopy (TEM), nanoparticle tracking analyzer (NTA), and Western blot. The plasma exosomes of the study group and the control group were quantitatively analyzed by proteomics. A bioinformatics method is utilized to screen differential proteins and the expression of the differential proteins was verified by Western blot. RESULT: TEM indicated that the exosomes had a double-concave disc-like appearance, with a size of about 100 nm, and Western blot expressed as CD63 and TSG101. The plasma exosomes of the study group and the control group were analyzed by quantitative proteomics with a total number of 952 proteins detected of which 245 proteins existed in the ExoCarta exosomal protein database. Through adoption of P-value to screen credible differential proteins, the heat map displayed 28 differential proteins, 7 upregulated proteins, and 21 downregulated proteins; the volcano map displayed 7 upregulated proteins and 22 downregulated proteins; the PPI interaction map displayed 12 upregulated proteins and 18 downregulated proteins. Through GO enrichment analysis, it was identified that the differential protein participated in the main biological processes and was involved in regulating the cell's stimulation response to insulin, the insulin receptor signaling pathway, and the activity of glycosylphosphatidylinositol phospholipase D as well as anti-oxidation. The enriched cell components include main components such as exosomes, blood particles, and cytoplasm. KEGG enrichment analysis indicated that the target protein FLOT2 was mainly concentrated in insulin-related signaling pathways. Western blot indicated that the expression of FLOT2 in the study group was lower compared with the control group while the expression of Exo70 was higher. CONCLUSION: Proteomic analysis of the study group and the control group displayed a variety of proteins in plasma exosomes. The downregulated protein FLOT2 in the study group was closely related to the occurrence, development, and complication of DK in T2DM patients. The expression status of plasma FLOT2 protein in T2DM patients is expected to be a biomarker for diagnosing and monitoring of DK.

Effects of asymmetric dimethylarginine on bovine retinal capillary endothelial cell proliferation, reactive oxygen species production, permeability, intercellular adhesion molecule-1, and occludin expression.

PURPOSE: Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase, is associated with impaired endothelial dysfunction, such as chronic heart failure, hypertension, diabetes, and pulmonary hypertension. The effects of ADMA on cell proliferation, reactive oxygen species (ROS) production, cell permeability, intercellular adhesion molecule-1 (ICAM-1), and tight-junction protein occludin levels in bovine retinal capillary endothelial cells (BRCECs) were investigated. METHODS: A cell proliferation assay was performed using the novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and an electron coupling reagent. Intracellular ROS levels were determined using the fluorescent probe CM-H(2)DCFDA. Horseradish peroxidase was used for a permeability assay. ICAM-1 and tight-junction protein occludin were assessed by western blotting and quantitative real-time PCR. RESULTS: Cell proliferation was significantly inhibited by ADMA. ADMA increased intracellular ROS generation in BRCECs. The increased ROS production induced by ADMA was markedly inhibited by the angiotensin II receptor-blocker telmisartan, the angiotensin-converting enzyme inhibitor benazepril, the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyliodonium (DPI), or the antioxidant and free-radical scavenger N-acetyl-L-cysteine (NAC). ADMA significantly increased horseradish peroxidase (HRP) permeability in BRCECs. Benazepril, telmisartan, DPI, and NAC downregulated cell permeability. ADMA markedly upregulated ICAM-1 expression in BRCECs, which were downregulated by telmisartan, DPI, and NAC. ADMA significantly downregulated occludin expression in BRCECs. Benazepril and telmisartan upregulated occludin expression in BRCECs exposed to ADMA. CONCLUSIONS: Our results provide the first reported evidence that ADMA has potent adverse effects on cell proliferation, intracellular ROS generation, cell permeability, levels of ICAM-1, and the tight-junction protein occludin. Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and antioxidants are effective inhibitors of the adverse effects of ADMA.

Acid-sensing ion channel 1a is involved in retinal ganglion cell death induced by hypoxia.

PURPOSE: Loss of retinal ganglion cells (RGCs) during retinal ischemia is the potentially blinding mechanism that underlies several sight-threatening disorders. Fluctuations in extracellular pH are associated with such pathological conditions. It has been demonstrated that the retina is a functionally distinct region of central neurons that are known to contain acid-sensing ion channels (ASICs), which are depolarizing conductance channels directly activated by protons. This study was conducted to determine whether ASIC1a channels in RGCs are essential for ischemia-induced cell death. METHODS: Expression of ASIC1a channels was detected in primary cultures of rat RGCs and in retinal sections. The patch-clamp technique in the conventional whole-cell configuration was used to examine the currents evoked by acid in the cultured RGCs. Intracellular Ca(2+) ([Ca(2+)]i) elevation was detected by Ca(2+) imaging. Furthermore, hypoxia-induced cell death in RGC cultures was measured by methyl thiazolyl tetrazolium assay. RESULTS: RGCs expressed a high density of ASIC1a channels. The expression and function of ASIC1a channels were upregulated after hypoxia in cultured RGCs. Ratiometric Ca(2+) imaging showed that RGCs responding to a drop in pH presented an increase in the concentration of (Ca(2+))i. Acute blockade of ASIC1a channels with the specific inhibitor amiloride or psalmotoxin 1 reduced RGC death in vitro. CONCLUSIONS: Based on these novel findings, we conclude that ASIC1a plays a role in RGC death induced by hypoxia. Therefore, neuroprotective strategies in glaucoma could include tools to improve the ability of these neurons to survive the cytotoxic consequences of ASIC1a activation.

Association between Inflammatory Factors in the Aqueous Humor and Hyperreflective Foci in Patients with Intractable Macular Edema Treated with Antivascular Endothelial Growth Factor.

BACKGROUND: To evaluate the correlations between the inflammatory factors in the aqueous humor and hyperreflective foci (HRF) in patients with intractable macular edema treated with antivascular endothelial growth factor (anti-VEGF). METHODS: This study included 17 patients with intractable macular edema (ME) treated with anti-VEGF agents. Inflammatory factors in the aqueous humor were measured by the Cytometric Beads Array before injection, and the numbers of HRF pre- and post-anti-VEGF treatment were counted from four different directions (90 degrees, 45 degrees, 180 degrees, and 135 degrees) in the SD-OCT images, respectively, before treatment and one month after treatment. The correlations between inflammatory factors and the numbers of HRF were assessed. RESULTS: The numbers of HRF were reduced significantly after anti-VEGF treatment. The change in the HRFs at the 90-degree location was significantly positively correlated with IL-8 and VCAM-1. The change of all HRFs was significantly positively correlated with IL-8. The HRFs before the treatment also had a positive correlation with IL-8 and VCAM-1. CONCLUSION: After anti-VEGF treatment, the numbers of HRF in intractable ME declined greatly. The higher the levels of IL-8 and VCAM-1 before treatment, the more significant the reduction of HRF after anti-VEGF treatment, which indicated that HRF could be an effective noninvasive imaging indicator for evaluating the effect of anti-VEGF on intractable macular edema. The OCT images at the 90-degree location could better show the inflammatory reaction of patients and also had better clinical significance for the prognosis evaluation of ME associated with inflammation.

Identification of Novel Choroidal Neovascularization-Related Genes Using Laplacian Heat Diffusion Algorithm.

Choroidal neovascularization (CNV) is a type of eye disease that can cause vision loss. In recent years, many studies have attempted to investigate the major pathological processes and molecular pathogenic mechanisms of CNV. Because many diseases are related to genes, the genes associated with CNV need to be identified. In this study, we proposed a network-based approach for identifying novel CNV-associated genes. To execute such method, we first employed a protein-protein interaction network reported in STRING. Then, we applied a network diffusion algorithm, Laplacian heat diffusion, on this network by selecting validated CNV-related genes as the seed nodes. As a result, some novel genes that had unknown but strong relationships with validated genes were identified. Furthermore, we used a screening procedure to extract the most essential genes. Eleven latent CNV-related genes were finally obtained. Extensive analyses were performed to confirm that these genes are novel CNV-related genes.

A Random Walk-Based Method to Identify Candidate Genes Associated With Lymphoma.

Lymphoma is a serious type of cancer, especially for adolescents and elder adults, although this malignancy is quite rare compared with other types of cancer. The cause of this malignancy remains ambiguous. Genetic factor is deemed to be highly associated with the initiation and progression of lymphoma, and several genes have been related to this disease. Determining the pathogeny of lymphoma by identifying the related genes is important. In this study, we presented a random walk-based method to infer the novel lymphoma-associated genes. From the reported 1,458 lymphoma-associated genes and protein-protein interaction network, raw candidate genes were mined by using the random walk with restart algorithm. The determined raw genes were further filtered by using three screening tests (i.e., permutation, linkage, and enrichment tests). These tests could control false-positive genes and screen out essential candidate genes with strong linkages to validate the lymphoma-associated genes. A total of 108 inferred genes were obtained. Analytical results indicated that some inferred genes, such as RAC3, TEC, IRAK2/3/4, PRKCE, SMAD3, BLK, TXK, PRKCQ, were associated with the initiation and progression of lymphoma.

Sirt1 attenuates diabetic keratopathy by regulating the endoplasmic reticulum stress pathway.

AIMS: The objectives of this study were to explore physiological and pathological changes in the corneas of diabetic rats by intervening in the expression of silent information regulator 1 (Sirt1) and to investigate whether Sirt1 can regulate the activation of endoplasmic reticulum stress (ERS) while influencing corneal epithelial cell apoptosis under high glucose conditions. MATERIALS AND METHODS: Using 8-week old Sprague-Dawley rats, we established a model of type 1 diabetes, with or without Sirt1 intervention. Clinical evaluation was performed once per week. Primary rat corneal epithelial cells (RCECs) were cultured by combining Sirt1 intervention under high glucose conditions. Generation of reactive oxygen species (ROS), apoptosis, and the expression of Sirt1 and ERS-related proteins were evaluated in rat corneal tissues and RCECs. KEY FINDINGS: During the intervention, clinical evaluation of the ocular surface, ROS generation, apoptosis, and protein expression of ERS-related proteins in corneal tissue and cultured RCECs were altered with Sirt1expression levels. SIGNIFICANCE: Sirt1 expression influences the pathological progression of diabetic keratopathy, plays an important role in regulating the ERS pathway, and decreases corneal epithelial cell apoptosis.

PRMT-1 and DDAHs-induced ADMA upregulation is involved in ROS- and RAS-mediated diabetic retinopathy.

Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase, is generated in presence of type 1 protein arginine N-methyltransferase (PRMT-1) and is metabolized by dimethylarginine dimethylaminohydrolases (DDAHs). Reportedly ADMA is associated with endothelial dysfunction. The aim of this study is to investigate whether PRMT-1- and DDAHs-induced ADMA increase in diabetic rat retina and high glucose-treated bovine retinal capillary endothelial cells (BRCECs) is involved in reactive oxygen species (ROS)- and renin-angiotensin system (RAS)-mediated diabetic retinopathy. Rats were divided into four groups: sham-injected group, streptozotocin (STZ)-induced diabetic model group, STZ-induced diabetic model plus 12-week ACEI benazepril treatment group, and STZ-induced diabetic model plus 12-week ARB telmisartan treatment group. BRCECs were exposed to 5mM glucose, 30mM glucose, and 30mM glucose plus benazepril, telmisartan, diphenyliodonium (NADPH oxidase inhibitor, DPI), or N-Acetyl-l-cysteine (antioxidant and free radical scavenger, NAC) until passage four. We found that the concentrations of ADMA were significantly elevated in the plasma of diabetic rat models, and were significantly reduced by benazepril or telmisartan. DDAHs expression was decreased and PRMT-1 expression was increased in diabetic rat retina, which was reversed by benazepril. Telmisartan decreased PRMT-1 expression and increased DDAH II expression, but had no effect on DDAH I expression. In vitro, BRCECs exposed to high glucose had elevated ROS production, decreased cGMP, increased PRMT-1 expression, and decreased DDAH activity and DDAH II expression. Coincubating BRCECs with benazepril, telmisartan, DPI or NAC reversed the effects of high glucose. It can be concluded that PRMT-I and DDAHs-induced upregulation of ADMA levels might be involved in ROS- and RAS-mediated diabetic retinopathy.

[Epidemiologic study of dry eye in populations equal or over 20 years old in Jiangning District of Shanghai].

OBJECTIVE: To investigate the prevalence of dry eye in populations equal or over 20 years old in Jiangning District, Shanghai, China. METHODS: This was a cross-sectional study. From September 2008 to January 2009, 6 small districts including 21,102 people of Jiangning District were randomly selected as survey venues by Department of Ophthalmology in First People's Hospital affiliated to Shanghai Jiaotong University. Then, 1266 people as the selected residents were enrolled, which was figured out through the random cluster sampling procedure. Every participant completed dry eye questionnaire, the ocular surface disease index (OSDI), and a series of examination including slit-lamp microscope, tear-film break-up time (BUT) , Schirmer I test, and fluorescein staining of the cornea (F1). The diagnosis of dry eye was referred to the well-accepted domestic diagnostic criteria The SPSS11. 0 software was used to analyze the database, t . test, chi2 test, one-way-ANOVA and Logistic regression were used for analysis. RESULTS: One thousand and eighty five residents finally took part in this study, and the inclusion ratio was 85.70%. Three hundred and twenty six individuals, including 101 men and 225 women, were diagnosed as dry eye, and the prevalence rate was 30.05%. The prevalence of dry eye in the female (33.78%) was higher than that of the male (24.11%) (chi2 = 11.46, P < 0.01). The prevalence of dry eye in people over 50 years old was higher than that under 50 years (chi2 = 94.50, P < 0.01). The figure of Schirmer I test and BUT decreased in elder people, at the same time the scores of Fl and MGD increased. Meanwhile, the score of OSDI in dry eye patients was significantly higher than that in non-dry eye individuals. The relative risk factors of dry eye were gender, age, wearing contact lens, long-time using of eye solutions, taking anti-allergy drugs. CONCLUSIONS: The prevalence of dry eye in female is higher than that in the male. And the prevalence of dry eye increases following the aging process. Relative risk factors of dry eye are gender, age, wearing contact lens, long-time using of eye solutions, taking anti-allergy drugs.

High-glucose treatment regulates biological functions of human umbilical vein endothelial cells via Sirt1/FOXO3 pathway.

BACKGROUND: Hyperglycaemia-induced angiogenesis plays an important role in diabetic retinopathy (DR). This study aimed to investigate the role of sirtuin1 (Sirt1)/forkhead box O3 (FOXO3) pathway in the effects of high-glucose on human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were divided into normal control group (5 mM glucose), high glucose group (30 mM), 30 mM glucose + shsirt1 group, 30 mM glucose + Sirt1 over-expression group (30 mM + Sirt1), 30 mM glucose + Sirt1 agonist SRT group, 30 mM glucose + SRT + FOXO3 silencing group (30 mM + SRT + siFOXO3). Cell proliferation, migration, invasion and apoptosis were determined. RESULTS: High glucose treatment reduced the expression of Sirt1 and FOXO3 in HUVECs. However, Sirt1 over-expression or SRT attenuated the high-glucose-induced inhibition of HUVEC proliferation and migration as well as reduced their apoptosis. In contrast, Sirt1 silencing deteriorated the high-glucose induced inhibition of HUVEC proliferation and migration and further increased HUVEC apoptosis. FOXO3 expression increased with the increase in Sirt1 expression, which was accompanied by enhanced cellular functions. These were abolished after FOXO3 silencing. In addition, Sirt1/FOXO3 regulated HUVEC activities via peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). CONCLUSIONS: Sirt1/FOXO3 pathway is essential for the survival of endothelial cells under high-glucose and plays an important role in the development of diabetes-induced retinal vascular endothelial injury.

High glucose causes apoptosis of rabbit corneal epithelial cells involving activation of PERK-eIF2α-CHOP-caspase-12 signaling pathway.

AIM: To investigate the effect of high concentration of glucose (HCG) on double stranded RNA-activated protein kinase-like ER kinase (PERK)-eukaryotic initiation factor-2α (eIF2α)-transcription factor C/EBP homologous protein (CHOP)-cysteine aspartate specific proteinase (caspase-12) signaling pathway activation and apoptosis in rabbit corneal epithelial cells (RCECs). METHODS: RCECs were treated by different concentrations of glucose for 0-48h. The expressions of PERK, p-PERK, eIF2α, p-eIF2α, 78 kDa glucose-regulated protein 78 (GRP78), CHOP, B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-2-associated X protein (Bax) and caspase-12 were determined by Western blot. Apoptosis was detected by TUNEL assay. Meanwhile, the function of PERK-eIF2α-CHOP-caspase-12 signaling pathway activation in high glucose-induced apoptosis was evaluated using PERK inhibitor, GSK2606414. RESULTS: HCG significantly promoted the expression of p-PERK, p-eIF2α, GRP78, CHOP, Bax and cleaved caspase-12 in RCECs (P<0.05), while remarkably decreased the expression of Bcl-2 and caspase-12 (P<0.05), and the alterations caused by glucose were in concentration- and time-dependent manners. Meanwhile, PERK and eIF2α expressions were not affected in all groups (P>0.05). TUNEL assay showed that the apoptosis rate of RCECs in the HCG group increased significantly in contrast with that in the normal concentration of glucose or osmotic pressure control group (P<0.05), and the apoptosis rate increased with the increase of glucose concentration within limits (P<0.05). GSK2606414 down-regulated the expression of p-PERK and p-eIF2α in the HCG group (P<0.05), while still did not affect the expression of PERK and eIF2α among groups (P>0.05). Correspondingly, GSK2606414 also significantly reduced the apoptosis rate induced by high glucose (P<0.05). CONCLUSION: HCG activates PERK-eIF2α-CHOP-caspase-12 signaling pathway and promotes apoptosis of RCECs.

Pediatric penetrating keratoplasty in Shanghai: a retrospective multiple centre study from 2003 to 2007.

BACKGROUND: With advances in pediatric keratoplasty over the last two decades, the success rate of surgery has increased remarkably. However, few epidemiological studies in this field have been performed in China. The present study investigated the indications and characteristics of pediatric penetrating keratoplasty in Shanghai. METHODS: All records of pediatric keratoplasty performed on 156 eyes in 149 children (< 14 years) at four ophthalmic units in Shanghai during a 5-year period (2003 - 2007) were used for this retrospective study. Patients were from the Eye, Ear, Nose & Throat Hospital of Fudan University, Shanghai No.10 People's Hospital, Shanghai No.1 People's Hospital, and Shanghai Peace Ophthalmology Hospital; and included mostly school-age children (97 boys and 59 girls). RESULTS: The median age at surgery was 9 years with an interval quartile range of 6-12 years. Scarring after keratitis (29.5%, 46/156) and traumatic corneal scar (19.2%, 30/156) were the most common indications. Best Corrected Snellen visual acuity (BCSVA) was reported only in 72% (112/156) of cases. Visual acuity outcomes were significantly better for keratoplasty 1 year postoperatively compared to preoperative visual acuity (P = 0.001). Of all patients, 13% (14/112) achieved a BCSVA of 6/18 or better. None of the indications was associated with a higher rate of failure compared to after 1 year follow-up. CONCLUSION: We obtained valuable information on pediatric keratoplasty in Shanghai. There was no significant difference in graft survival rate among the two indications. Vision outcomes after corneal transplantation in Chinese children were significantly better compared with pre-operation status. In conclusion, corneal graft survival can be achieved in childhood.

High Concentration of Glucose Increases Reactive Oxygen Species Generation and Apoptosis Induced by Endoplasmic Reticulum Stress Pathway in Rabbit Corneal Epithelial Cells.

OBJECTIVE: To investigate the effect of high concentration of glucose on reactive oxygen species (ROS) production in rabbit corneal epithelial cells (RECEs) and explore whether the increased ROS initiates the apoptosis process of RECEs through oxidative stress and endoplasmic reticulum (ER) stress pathway. METHODS: RECEs were treated by different concentrations of glucose for a while, and then the production of ROS was detected by flow cytometry. The expressions of PERK, p-PERK, Akt, p-Akt, and CHOP were determined by western blot, and the cell viability was measured by Cell Counting Kit-8 (CCK-8). Flow cytometry was used to detect the early apoptosis rate. Meanwhile, the effects of N-acetyl-L-cysteine (NAC), an active oxygen inhibitor, on the experimental results were observed. RESULTS: Compared with the normal glucose concentration group, the fluorescence intensity of ROS in the high concentration (1 mM glucose) of glucose group was significantly increased (P < 0.05). NAC-inhibited ROS production was induced by high concentration of glucose (P < 0.05).Western blot demonstrated that the expressions of the p-PERK and CHOP increased significantly (P < 0.05), the p-Akt expression decreased (P < 0.05), and the PERK and Akt expressions did not change significantly in the high concentration of glucose group compared to the normal concentration group. CCK-8 results revealed that compared with the normal concentration of glucose group, the cell activity of the high concentration of glucose group decreased. For the cells in the high concentration of glucose group, the cell survival rate of NAC-treated cells was higher than that of untreated (P < 0.05). The flow cytometry results indicated that the early apoptosis rate of the cells in the high concentration of glucose group increased in contrast with that in the normal concentration of glucose group (P < 0.05). Treating the cells in the high concentration of glucose group with NAC could reduce the cell apoptosis resulted from high glucose (P < 0.05). CONCLUSIONS: High concentration of glucose may induce the formation of ROS which leads to oxidative stress and ER stress in RECEs and even leads to cell apoptosis. The reactive oxygen inhibitor, NAC, can play a protective character in the high concentration of glucose environment. These results might provide theoretical basis for the study of the diabetes-related dry eye.

[Clinical analysis of keratoplasty for 121 children in Shanghai].

OBJECTIVE: To determinate the clinical characteristics of children underwent corneal transplantation in Shanghai, including the indications, the age and gender distributions, etc.. METHODS: Records of 122 keratoplasties in 122 eyes of 121 children below 17 years old, undertaken from January 1, 2003 to January 1, 2006, were gathered by four ophthalmic units in Shanghai as a multiple centre study and the data were analyzed by the statistic software SPSS 13.0 retrospectively, especially on the indication, the age and gender distributions. RESULTS: One hundred and twenty one patients were enrolled in this study. Traumatic corneal scar (22.3%) and scarring after keratitis (19.8%) were the leading indications for keratoplasty, followed by congenital corneal opacity (11.6%), keratoconus (10.7%), corneal ulceration (9.1%), graft rejection (5.0%), band keratopathy (5.0%), chemical injury (5.0%), thermal burns (4.1%), corneoconjunctival dermatosis (4.1%) and congenital hereditary corneal endothelial dysfunction (3.3%). The median age was 11.0 years with the interval quartile range at 8 - 14 years. The number of male patients (77) was approximately twice as many as the female patients (44). Ninety-five patients underwent penetrating keratoplasty (78.5%), 19 patients underwent lamellar keratoplasty (15.7%) and 7 patients underwent combined penetrating keratoplasty with other surgeries (5.8%). CONCLUSIONS: Traumatic corneal scar and scarring after keratitis were the leading indications. The age of operated cases mainly in the school age. Male is more frequent than the female.

The injection of DisCoVisc into the anterior chamber improved corneal preservation and transplantation for cornea blind patients.

PURPOSE: In this study, we aimed to provide a new method of corneal preservation by injecting DisCoVisc into the anterior chamber of eyeballs and evaluate its efficiency for corneal transplantation. METHODS: Three pairs of eyeballs (n=6) were preserved by DisCoVisc viscoelastic agent, and the corneas were stored for 1 to 6 days. Then, the structure and morphology of cornea were analyzed by hematoxylin-eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and transmission electron microscopy (TEM). The corneas preserved by this method were transplanted into 15 patients with corneal disease and the efficacy was assessed. RESULTS: Epithelial cells and endothelial cells were intact and a normal morphology was preserved in both fresh corneas and corneas stored for 1-6 days by DisCoVisc viscoelastic agent. Corneal endothelial cells did not appear apoptosis in fresh corneas and corneas stored for 1-3 days, whereas a few apoptosis positive cells were shown on the 4th day. The results of TEM showed that all corneas had active corneal endothelial cells with normal nuclei and homogenous nucleoplasm. Desmosomes and hemidesmosomes were closely connected. Mild nuclear pyknosis and autophagic cell death were only found from the 6th day, and mitochondria appeared a little bubble from the 5th day. Visual acuity in 11 of the 15 patients receiving transplantation of the preserved corneas was improved by more than 0.5. Average corneal endothelial cell counts, areas of corneal endothelial cells, and CV% of average area were not affected during the 6-month follow-up. Compared to the values obtained one-month postoperatively, the values of corneal thickness were significantly reduced in the three-month and six-month periods. CONCLUSIONS: Corneal preservation technology with the injection of DisCoVisc viscoelastic agent may effectively extend the preservation time of corneas for five days, which could be used for patients as penetrating keratoplasty surgery.