The interaction between autophagy and the epithelial-mesenchymal transition mediated by NICD/ULK1 is involved in the formation of diabetic cataracts.

BACKGROUND: Cataracts are the leading cause of blindness and a common ocular complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) and altered autophagic activity occur during the development of diabetic cataracts. The disturbed interaction of autophagy with EMT in LECs stimulated by high glucose levels may participate in cataract formation. METHODS: A rat diabetic cataract model induced by streptozotocin (STZ) and human lens epithelial cells (HLE-B3) stimulated with a high glucose concentration were employed in the study. These models were treated with rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)), and N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT, an inhibitor of γ-secretase) alone or in combination. Lens opacity was observed and photographed under a slit-lamp microscope. Histological changes in paraffin sections of lenses were detected under a light microscope after hematoxylin and eosin staining. Alterations of autophagosomes in LECs were counted and evaluated under a transmission electron microscope. The expression levels of proteins involved in the EMT, autophagy, and the signaling pathways in LECs were measured using Western blotting and immunofluorescence staining. Cell migration was determined by performing transwell and scratch wound assays. Coimmunoprecipitation (Co-IP) was performed to verify protein-protein interactions. Proteins were overexpressed in transfected cells to confirm their roles in the signaling pathways of interest. RESULTS: In LECs, a high glucose concentration induces the EMT by activating Jagged1/Notch1/Notch intracellular domain (NICD)/Snail signaling and inhibits autophagy through the AKT/mTOR/unc 51-like kinase 1 (ULK1) signaling pathway in vivo and in vitro, resulting in diabetic cataracts. Enhanced autophagic activity induced by rapamycin suppressed the EMT by inducing Notch1 degradation by SQSTM1/p62 and microtubule-associated protein light chain 3 (LC3) in LECs, while inhibition of the Notch signaling pathway with DAPT not only prevented the EMT but also activated autophagy by decreasing the levels of NICD, which bound to ULK1, phosphorylated it, and then inhibited the initiation of autophagy. CONCLUSIONS: We describe a new interaction of autophagy and the EMT involving NICD/ULK1 signaling, which mediates crosstalk between these two important events in the formation of diabetic cataracts. Activating autophagy and suppressing the EMT mutually promote each other, revealing a potential target and strategy for the prevention of diabetic cataracts.

Correlation between neutrophil-to-lymphocyte ratio and clinical manifestations and complications of retinitis pigmentosa.

PURPOSE: The role of inflammation in retinitis pigmentosa (RP) has been receiving additional attention. However, the association between inflammation and the clinical manifestations and complications of RP is still unclear. This study aimed to evaluate the neutrophil-to-lymphocyte ratio (NLR) of RP complicated with cataract and explore the correlations between the NLR and specific clinical features of RP. METHODS: This retrospective study included 79 RP patients complicated with cataract (125 eyes) and 63 age- and sex-matched patients (63 eyes) with age-related cataract (ARC). Patients' ocular examination results were collected and complete blood count results were used to calculate NLRs. The correlations between the NLR of RP patients and the parameters of ocular examinations were analysed. RESULTS: The NLRs of RP patients with cataracts were significantly higher than those of ARC (1.93 ± 0.83 versus 1.65 ± 0.59, p = 0.029). The NLRs increased with the severity of posterior subcapsular cataract (PSC), zonular deficiency, poor preoperative best-corrected visual acuity (LogMAR>1), and visual field defects. Analysis of receiver operating characteristic curves suggested that NLR > 1.36 could predict higher degrees (PSC area >3%, >P1) of PSC (p = 0.002, 95% CI, 0.672-0.934), and that NLR > 2.12 could predict zonular weakness (p = 0.002, 95% CI, 0.665-0.928) in RP. CONCLUSION: The NLRs in RP patients with cataract are not only higher but also associated with several clinical manifestations of RP. The NLR can be a predictive biomarker of higher degrees of PSC (>P1) and zonular weakness in RP before cataract surgery. These results suggest that systemic inflammation may play a role in the pathogenesis of RP.

LncRNA MALAT1 Regulates miR-144-3p to Facilitate Epithelial-Mesenchymal Transition of Lens Epithelial Cells via the ROS/NRF2/Notch1/Snail Pathway.

Diabetic cataract is a common complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a key event in the development of diabetic cataracts. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been reported to be highly expressed in different tissues of diabetic patients. This study is aimed at investigating the function and mechanism of MALAT1 in the regulation of EMT in human LECs under high glucose conditions. MALAT1, α-smooth muscle actin (α-SMA), fibronectin (FN), and nuclear factor erythroid-derived 2-like 2 (NRF2) were highly expressed in the LECs of diabetic cataract patients and in the human LECs under high glucose conditions; meanwhile, the decreased expressions of E-cadherin and zonula occludens 1 (ZO-1) were detected. Knockdown of MALAT1 could significantly reduce ROS, prevent EMT, arrest S phase cell cycle, and suppress the expression of total NRF2 and its nucleus translocation in LECs. Furthermore, after NRF2 was knocked down, total NRF2, α-SMA, and FN in cells, and NRF2, Notch intracellular domain (NICD), and Snail were decreased in the nucleus. Using bioinformatics methods, we predicted that MALAT1 and NRF2 shared the same microRNA-144-3p (miR-144-3p) combining site. Luciferase reporter coupled with qRT-PCR assays revealed that miR-144-3p was a target of MALAT1, which was confirmed to downregulate miR-144-3p in the LECs. In addition, after transfection of miR-144-3p mimics or inhibitor, western blot assay demonstrated that miR-144-3p negatively regulated the expression of total NRF2, α-SMA, and FN in cells, and NRF2, NICD, and Snail in the nucleus without affecting Kelch-like ECH-associated protein 1 (KEAP1). Finally, we confirmed that transfection of shMALAT1 inhibited NRF2 expression, and its mediated EMT could be rescued by miR-144-3p inhibitor; transfection of pcDNA3.1-MALAT1 promoted NRF2 expression, and its mediated EMT could be reversed by miR-144-3p inhibitor. In summary, we demonstrate that MALAT1 regulates miR-144-3p to facilitate EMT of LECs via the ROS/NRF2/Notch1/Snail pathway.

Activation of autophagy inhibits epithelial to mesenchymal transition process of human lens epithelial cells induced by high glucose conditions.

Subcapsular cataracts are common phenotype of diabetic cataracts, and abnormal lens epithelial cells (LECs) under the lens capsules have been considered to involve in the pathogenesis. Our previous studies have shown that the epithelial to mesenchymal transition (EMT), which is responsible for the LECs to lose their original polarity and tight junctions, occurs in a diabetic cataract mouse model. Autophagy is known to function in the EMT process in multiple tissues. However, the relationship between autophagy and EMT process in LECs has not yet been fully demonstrated. We found that high glucose retreatment reducing expression level of E-cadherin, an epithelial marker, but increasing that of α-smooth muscle actin (α-SMA), a mesenchymal marker, by Western blot and immunoflurence staining assays, and increased the cell migration by Transwell assay in human lens epithelial cell line HLE-B3. High glucose retreatment also led to impairment of autophagy, representing by downregulation of Beclin, LC3II/LC3I, and reducing the number of autophagosomes. Activation of autophagy by rapamycin could prevent high glucose-induced EMT. In addition, the levels of p62 and Snail were increased in high glucose-treated HLE-B3 cells, and their interactions were demonstrated by co-immunoprecipitation and immunoflurence staining, but all these changes were attenuated by application of rapamycin. These findings delineated a novel autophagy-mediated mechanism, p62 might mediate Snail underlying high glucose-induced EMT in LECs, suggesting a potential therapeutic approach for diabetic cataract by regulating autophagy.

Effects of bradykinin on TGF­ß1­induced epithelial­mesenchymal transition in ARPE­19 cells.

The aim of the present study was to investigate the effects of bradykinin (BK) on an epithelial-mesenchymal transition (EMT) model in retinal pigment epithelium (RPE) cells through exposure to transforming growth factor­ß1 (TGF­ß1). The aim was to improve the effect of BK on proliferative vitreoretinopathy (PVR) progression, and to find a novel method of clinical prevention and treatment for PVR. The morphology of ARPE­19 cells was observed using an inverted phase­contrast microscope. A Cell Counting Kit­8 was used to assess the effects of TGF­ß1 on the proliferation of ARPE­19 cells. Western blotting and immunofluorescence were used to detect the expression levels of the epithelial marker E­cadherin, mesenchymal markers α­smooth muscle actin (SMA) and vimentin, and phosphorylated (p) mothers against decapentaplegic homolog (Smad)3 and Smad7 of the TGF/Smad signaling pathway. Wound healing tests and Transwell assays were performed to detect cell migration ability. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis was performed to detect the expression levels of pSmad3 and Smad7 in the TGF/Smad signaling pathway. The results revealed that the addition of 10 ng/ml TGF­ß1 resulted in the expression of factors associated with EMT in ARPE­19 cells. BK decreased the expression levels of the mesenchymal markers α­SMA and vimentin, and increased the expression of the epithelial marker E­cadherin. BK decreased cell migration in TGF­ß1­induced EMT. These effects were reversed by HOE­140, a specific BK 2 receptor antagonist. BK significantly downregulated the expression of pSmad3 and upregulated the expression of Smad7 in TGF­ß1­treated ARPE­19 cells, and the protective alterations produced by BK were inhibited by HOE­140. In conclusion, 10 ng/ml TGF­ß1 resulted in EMT in ARPE­19 cells and BK served a negative role in TGF­ß1­induced EMT. BK had effects in TGF­ß1­induced EMT by upregulating the expression of Smad7 and downregulating the expression of pSmad3 in TGF­ß/Smad signaling pathway, indicating that BK may be a novel and effective therapy for PVR.

The Effect of CM082, an Oral Tyrosine Kinase Inhibitor, on Experimental Choroidal Neovascularization in Rats.

The aims of this study were to evaluate the effects of CM082 on the development of choroidal neovascularization (CNV) in a laser-induced CNV rat model and to determine the drug concentration in the ocular tissues. After the laser-induced CNV model was established in rats, CM082 was orally administered. The effects of CM082 on the CNV lesions were assessed using fundus fluorescein angiography (FFA), CNV histology, and retinal pigment epithelium- (RPE-) choroid-sclera eyecup analysis. The concentrations of CM082 in the plasma and eye tissues were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results of FFA, histology, and RPE-choroid-sclera eyecup analysis demonstrated that the CM082-treated (10 mg/kg/d or 30 mg/kg/d) rats exhibited significantly less neovascularization than did the control group. The total concentration of CM082 in the eyes (172.86 ± 57.11 ng/g) was similar to that in the plasma (196.87 ± 73.13 ng/ml). Within the eye, the concentrations of CM082 and its metabolites were highest in the retina-sclera. The orally administered CM082 thus effectively passed through the blood-retina barrier (BRB) to reach the retina in the Brown Norway rats. Therefore, at both 10 mg/kg/d and 30 mg/kg/d, CM082 was able to reduce CNV lesions in the laser-induced CNV rat model.

Dry eye and corneal sensitivity after small incision lenticule extraction and femtosecond laser-assisted in situ keratomileusis: a Meta-analysis.

AIM: To assess the corneal sensitivity and the incidences of dry eye after small incision lenticule extraction (SMILE) and femtosecond laser-assisted in situ keratomileusis (FS-LASIK). METHODS: The Meta-analysis was performed using RevMan 5.3. We searched on PubMed from inception to March 2016. Summary weighted mean difference (WMD) and 95% confidence intervals (CIs) were used to analyze the datum. Random-effects or fixed-effects models were chosen up to between-study heterogeneity. The main outcomes were composed of the Ocular Surface Disease Index (OSDI) scores, tear film break-up time (TBUT), Schirmer Test and corneal sensitivity. RESULTS: Eight eligible studies including 772 eyes (386 in SMILE group and 386 in FS-LASIK group) were identified. The parameters have no significiant difference heterogeneity between SMILE and FS-LASIK group preoperatively. There were significant differences between the two groups in OSDI scores at one and three months postoperatively, in TBUT at one and three months postoperatively, in corneal sensitivity at one week, about one month and three months postoperatively. However, there was no significant difference observed in Schirmer Test at the follow-up periods. CONCLUSION: Compare to FS-LASIK, dry eye and the corneal sensitivity recover better in the SMILE group, in first three months after the surgery.

Circulating miRNAs as Potential Biomarkers of Age-Related Macular Degeneration.

Backgroud: Age-related macular degeneration (AMD) is one of the leading causes of irreversible blindness of the elder people. This research was intended to demonstrate the different expression of microRNAs (miRNA) in AMD patients and whether they can be used as biomarkers for AMD. METHODS: MiRNAs expression was measured by microarray of 6 AMD cases and 6 gender matched controls. In a larger-sample case-control study with 126 AMD cases and 140 controls, whole blood samples were detected for the differences of miRNA expression. RESULTS: A total of 216 differentially expressed miRNAs (111 increased and 105 decreased miRNAs) were detected from circulating miRNA microarray. Expanded case-control study results showed that the expression of miR-27a-3p, miR-29b-3p and miR-195-5p was increased significantly. Moreover, the level of miR-27a is higher in patients with wet AMD compared to patients with dry AMD. All 3 miRNAs showed a potential diagnostic value for AMD. CONCLUSION: Circulating miRNA levels were significantly varied in AMD patients. Three miRNAs, miR-27a-3p, miR-29b-3p and the miR-195-5p, might be potential diagnostic biomarkers for AMD.

Effect of bradykinin on TGF-ß1-induced retinal pigment epithelial cell proliferation and extracellular matrix secretion.

BACKGROUND: To evaluate the effect of bradykinin (BK) on TGF-ß1-induced retinal pigment epithelial (RPE) cell proliferation and extracellular matrix secretion and to elucidate the relationship between BK and the Erk/Akt signaling pathway. METHODS: The effects of BK on TGF-ß1-induced RPE cell proliferation were examined via CCK-8 assay. Cell culture supernatant collagen I concentrations were measured via ELISA. Fibronectin (Fn), matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA and protein expression levels were measured via q-PCR and Western blotting, respectively. Changes in Akt/Erk phosphorylation induced by BK and HOE-140 were evaluated via Western blotting. RESULTS: TGF-ß1 stimulated ARPE-19 cell proliferation, which was inhibited by BK, whose effects were inhibited by HOE-140. BK inhibited TGF-ß1-induced collagen I, Fn and MMP-2 secretion in RPE cells, and these effects were inhibited by HOE-140. BK also inhibited TGF-ß1-induced Akt phosphorylation in RPE cells, and these effects were blocked by HOE-140. BK had no significant effect on Erk-mediated signaling. CONCLUSIONS: The findings from this study indicate that BK could be novel therapeutic targets for the treatment of PVR.

Effects of orthokeratology on the progression of low to moderate myopia in Chinese children.

BACKGROUND: To investigate the effectiveness of orthokeratology (ortho-k) in reducing the development of myopia in Chinese children with low to moderate myopia. METHODS: This was a retrospective study. In the ortho-k group, there were141 subjects, and the average age was (9.43 ± 1.10) years. The average spherical equivalent refractive error (SER) was (-2.74 ± 1.15)D, with examinations performed 1, 7, 30, and 90 days and 12 months after the patients started wearing ortho-k lenses. In the control group, there were 130 subjects, and the average age was (9.37 ± 1.00) years. The average SER was (-2.88 ± 1.39)D, with examinations performed every 6 months. Axial elongation, which is an important parameter reflecting the progression of myopia, was measured at baseline from the same IOLMaster each time by the same masked examiner and was compared between the groups after 1 year. The subjects were divided into two sub-groups according to age to further study the development of myopia at different ages. An unpaired t-test, paired t-test, Chi-square test and Spearman test were performed to analyze the data. RESULTS: After 1 year, the average axial elongation was (0.27 ± 0.17) mm in the ortho-k lens group and (0.38 ± 0.13) mm in the control group, with a significant difference between the groups (P < 0.001). Axial elongation was not correlated with SER but had a negative correlation with initial age (ortho-k group: r s = -0.309, p < 0.01; control group: r s = -0.472, p < 0.01). The percentages of individuals with fast myopic progression (axial elongation > 0.36 mm per year) were 38.0 % among younger children (7.00 to 9.40 years) and 24.3 % among older children (9.40 to 12.00 years), whereas the respective percentages were 76.5 and 12.9 % in the control group. When SER ranged from -5.0D to -6.0D, the axial elongation in the ortho-k group was 57.1 % slower than that in the control group. CONCLUSIONS: Ortho-k lenses are effective in controlling myopic progression in Chinese children, particularly in younger children and in children with higher myopia.

Efficacy and safety of different doses of a slow-release corticosteroid implant for macular edema: meta-analysis of randomized controlled trials.

BACKGROUND: The purpose of this meta-analysis was to assess the efficacy and safety of intravitreal corticosteroid implants for macular edema. METHODS: A total of 3,586 patients from previously reported randomized controlled trials were included. The meta-analysis was performed using RevMan 5.2. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were calculated, employing random-effects or fixed-effects models according to between-study heterogeneity. The main outcome measures were the ORs for effects and safety of intravitreal corticosteroid implants. RESULTS: Four eligible studies were included. Compared with the sham group, the ORs for ≥15 letter improvement of visual acuity in the high-dose and low-dose groups were 1.89 (95% CI 1.33-2.69, P=0.0004) and 1.62 (95% CI 1.10-2.41, P=0.02), respectively. The weight mean differences in central retinal thickness increases were -75.46 (95% CI -90.29, -60.63, P<0.0001) and -46.47 (95% CI -92.08, -0.86, P=0.05), respectively. However, the ORs for increased intraocular pressure in both intervention groups were higher than in the sham group, and were 11.50 (95% CI 7.24-18.28, P<0.00001) and 10.30 (95% CI 6.49-16.36, P<0.00001), respectively. The incidence of cataract was 7.25 (95% CI 5.68-9.25, P<0.00001) and 3.56 (95% CI 1.28-9.96, P=0.02) in the two intervention groups, respectively. There was no significant difference between the intervention groups except for the incidence of cataract in which the OR was 1.59 (95% CI 1.28-1.97, P<0.001). CONCLUSION: Intravitreal corticosteroid implants are effective in treating macular edema. However, the efficacy is not related to corticosteroid dose.

Steroids as an adjunct for reducing the incidence of proliferative vitreoretinopathy after rhegmatogenous retinal detachment surgery: a systematic review and meta-analysis.

BACKGROUND: This meta-analysis was performed to determine the effectiveness of steroids as an adjunct following rhegmatogenous retinal detachment (RRD) surgery. METHODS: RRD patients with or without proliferative vitreoretinopathy (PVR) were included. The treatment group included patients in whom steroids were used as an adjunct and a control group in which placebo was used. Only randomized controlled trials were included. We searched the main electronic databases and included studies published until July 2014. PVR odds ratio, visual acuity, retinal reattachment rate, and complications were evaluated in three trials. RESULTS: Three randomized controlled trials were included in the meta-analysis. There was no significant difference in the incidence of postoperative PVR between groups (heterogeneity I (2)=48%, P=0.14). However, the incidence of postoperative PVR was lower in the treatment group (I (2)=0%, P<0.0001) than in the control group when a PVR grade C study was excluded. There was no statistically significant difference in postoperative visual acuity between the treatment and control groups (odds ratio -0.18; 95% confidence interval -0.38, 0.02; P=0.08). The two groups had similar results for primary/final retinal reattachment and reoperation rate. There was no significant difference in postoperative intraocular pressure. CONCLUSION: This systematic review demonstrates that steroids may significantly reduce the incidence of postoperative PVR grade B or lower following RRD surgery.