YAP is essential for TGF-ß-induced retinal fibrosis in diabetic rats via promoting the fibrogenic activity of Müller cells.

The purpose of this study was to investigate whether Yes-associated protein (YAP) activation and proliferation of retinal Müller cells play a role in the development of TGF-ß-induced retinal fibrosis. We studied the effects of YAP activation on retinal fibrosis in diabetic rats and human retinal Müller cells (hMCs) in vitro. The retinal expression of YAP and fibrogenic molecules in rats was detected using Western blotting and immunohistochemistry. After treatment with transforming growth factor-ß1 (TGF-ß1), the levels of fibrogenic molecules, and the activation of YAP and PI3K/Akt signalling pathway in hMCs were detected with Western blotting. The effect of YAP on retinal fibrotic changes was evaluated using YAP knockdown experiments and YAP inhibitors. Results showed that YAP expression was increased in the retina of diabetic rats along with increased retinal fibrosis. In cultured hMCs, YAP inhibition suppressed TGF-ß1-stimulated hMC differentiation to myofibroblasts and extracellular matrix (ECM) production, while YAP activation promoted hMC differentiation and ECM production independent of TGF-ß1. Furthermore, hMCs cultured on a gel with greater stiffness differentiated into myofibroblasts in a YAP-dependent manner. In diabetic rats, treatment with the YAP inhibitor verteporfin suppressed retinal fibrogenesis. In addition, the TGF-ß1-induced PI3K/Akt signalling pathway mediated YAP activation as well as expression of fibrogenic molecules. The interaction between ECM stiffness and YAP forms a feed-forward process leading to retinal fibrosis. Our work highlights YAP as an essential regulator of pro-fibrotic responses in TGF-ß-induced retinal fibrosis.

Application of pediatric ocular trauma score in pediatric open globe injuries.

AIM: To evaluate the predictive value of pediatric penetrating ocular trauma score (POTS) on the visual outcome in children with open globe injury. METHODS: A retrospective study in 90 children (60 males and 30 females) aged 1-15y (average, 7.48±2.86y) with penetrating ocular trauma was performed. Each patient's POTS was calculated. The effects of POTS on final visual acuity (FVA) were examined. Correlation between factors affecting POTS and the FVA was established. RESULTS: All patients presented with single-eye trauma. The follow-up time was 3-21mo (average, 10.23±3.54mo). Among the 90 cases of penetrating wounds, 71 eyes (78.89%) were injured in Zone I (wound involvement limited to the cornea, including the corneoscleral limbus), 17 eyes (18.89%) were injured in Zone II (wound involving the sclera and no more posterior than 5 mm from the corneoscleral limbus), and 2 eyes (2.22%) were injured in Zone III (wound involvement posterior to the anterior 5 mm of the sclera). Analysis of POTS and FVA showed important correlation between them (r=0.414, P=0.000). Initial visual acuity (P=0.00), age (P=0.02), injury location (P=0.002), traumatic cataract (P=0.00), vitreous hemorrhage (P=0.027), retinal detachment (P=0.003), and endophthalmitis (P=0.03) were found to be statistically significant factors for the FVA outcome. CONCLUSION: Ocular trauma presents serious consequences and poor prognosis in children. The POTS may be a reliable prognostic tool in children with open globe injury.

Exosomes derived from platelet-rich plasma activate YAP and promote the fibrogenic activity of Müller cells via the PI3K/Akt pathway.

The purpose of this study was to investigate the role of exosomes derived from platelet-rich plasma (PRP-Exos) in the regulation of the fibrogenic activity of Müller cells and the underlying mechanism. We studied the effects of PRP-Exos on the fibrogenic activity of human retinal Müller cells (hMCs) in vitro. PRP-Exos were isolated from the plasma of diabetic rats (DM-PRP-Exos) and normal control rats (Nor-PRP-Exos) and then observed by transmission electron microscopy. After treatment with DM-PRP-Exos or Nor-PRP-Exos, the proliferation and migration of hMCs were measured in vitro. Western blotting was conducted to assess the levels of fibrogenic molecules and activation of Yes-associated protein (YAP) and the PI3K-Akt signalling pathway. In cultured hMCs, DM-PRP-Exos but not Nor-PRP-Exos effectively increased the proliferative and migratory activities and improved connective tissue growth factor (CTGF) and fibronectin expression. Genetic and pharmacological suppression of YAP could reduce the proliferative and migratory activities of hMCs induced by DM-PRP-Exo. Additionally, YAP knockdown inhibited the DM-PRP-Exo-induced up-regulation of CTGF and fibronectin. Furthermore, DM-PRP-Exo-induced PI3K-Akt signalling mediated YAP activation and the expression of CTGF and fibronectin. In summary, DM-PRP-Exos, through YAP activation, enhance both the proliferation and fibrogenic activity of Müller cells via the PI3K/Akt pathway.

Exosomes derived from platelet-rich plasma mediate hyperglycemia-induced retinal endothelial injury via targeting the TLR4 signaling pathway.

In this study, we aimed to investigate whether exosomes derived from platelet-rich plasma (PRP-Exos) can regulate hyperglycemia-induced retinal injury via targeting the TLR4 signaling pathway. We studied the effects of PRP-Exos on retinal endothelial injury in diabetic rats and human retinal endothelial cells (HRECs) in vitro. Isolated PRP-Exos were observed by transmission electron microscopy and flow cytometry. Samples were obtained from the retinas of rats and cultured HRECs after treatment to analyze reactive oxygen species levels. Immunofluorescence and Western blotting were conducted to assess the levels of adhesion molecules and the TLR4 signaling pathway. The content of CXCL10 in PRP-Exos was analyzed by Western blot. The plasma level of PRP-Exos was greatly increased in diabetic rats. In cultured HRECs, PRP-Exos induced the production of malonyldialdehyde(MDA) and reactive oxygen species(ROS) and inhibited the activity of superoxide dismutase(SOD). Further analysis showed that the activation of the TLR4 pathway by PRP-Exos played a pivotal role in regulating inflammation. The inhibition of the TLR4 pathway by TAK-242 had a robust protective effect on PRP-Exo-induced retinal endothelial injury in vitro and vivo. In addition, PRP-Exo-derived CXCL10 led to retinal endothelial injury, and antagonizing CXCL10 with a CXCL10-neutralizing antibody dramatically attenuated such injury. In summary, PRP-Exos mediate hyperglycemia-induced retinal endothelial injury by upregulating the TLR4 signaling pathway.

Exosomes Derived From Mesenchymal Stem Cells Modulate miR-126 to Ameliorate Hyperglycemia-Induced Retinal Inflammation Via Targeting HMGB1.

Purpose: In this study, we aim to investigate whether mesenchymal stem cell (MSC)-derived exosomes (MSC-Exos) could regulate hyperglycemia-induced retinal inflammation by transferring microRNA-126 (miR-126). Methods: MSC-Exos were isolated from the media of human umbilical cord-derived mesenchymal stem cells (hUCMSCs), and this isolation was followed by the transfer of miR-126. MSC-Exos or MSC-Exos overexpressing miR-126 were intravitreally injected into diabetic rats in vivo and were cocultured with high glucose-affected human retinal endothelial cells (HRECs) in vitro. Plasma samples were obtained from the vitreous of rats and from HREC cells after treatment for ELISA assay. Retinal sections were examined using immunohistochemistry. RT-PCR and Western blotting were conducted to assess the levels of high-mobility group box 1 (HMGB1), NLRP3 inflammasome, and NF-κB/P65 in retinas and HRECs. Results: Our results showed that hyperglycemia greatly increased inflammation in diabetic rats or HRECs exposed to high glucose, increasing the levels of caspase-1, interleukin-1ß (IL-1ß) and IL-18. The administration of MSC-Exos could effectively reverse this reaction. Compared to control MSC-Exos, MSC-Exos overexpressing miR-126 more successfully suppressed the HMGB1 signaling pathway and suppressed inflammation in diabetic rats. The administration of miR-126-expressing MSC-Exos significantly reduced high glucose-induced HMGB1 expression and the activity of the NLRP3 inflammasome in HRECs. Conclusions: miR-126 expression in MSC-Exos reduces hyperglycemia-induced retinal inflammation by downregulating the HMGB1 signaling pathway.

MicroRNA-218-5p inhibit the migration and proliferation of pterygium epithelial cells by targeting EGFR via PI3K/Akt/mTOR signaling pathway.

Pterygium is a common ocular surface disease which could result in various ocular surface symptoms. MicroRNAs play an important role in the development of various eye diseases. However, the role of microRNAs in the pathogenesis of pterygium is rarely reported. Our research aims to analyze the relationship between miR-218-5p and Epidermal Growth Factor Receptor (EGFR) in human pterygium tissues and cultured Human Pterygium Epithelial Cells (hPECs). Furthermore, the EGFR/PI3K/Akt/mTOR signaling pathway was firstly verified in pterygium. Pterygium tissues and normal bulbar conjunctival tissues were obtained from surgery, and primary hPECs were cultured in vitro. Cell transfection, Quantitative real-time PCR (qRT-PCR), Western blotting, Luciferase reporter assay and Scratch Wound Healing Assay were performed. Our data demonstrated that miR-218-5p was decreased and EGFR was increased in pterygium tissues than normal conjunctival tissues. In transfected hPECs, our results indicated that upregulated miR-218-5p significantly suppressed the expression level of EGFR via PI3K/Akt/mTOR pathway. In addition, the migration and proliferation of hPECs was promoted by miR-218-5p inhibitor and retarded by miR-218-5p mimics. And knockdown of EGFR significantly inhibit hPECs migration. Taken together, miR-218-5p downregulated the expression of EGFR via PI3K/Akt/mTOR pathway in pterygium tissues and hPECs and inhibited hPECs migration and proliferation. The microRNA-218-5p-EGFR-PI3K/Akt/mTOR axis should be further investigated for the potential treatment of pterygium.

[Retracted] Slit-miR-218-Robo axis regulates retinal neovascularization.

Following the publication of this article, it was brought to our attention that five of the Figures contained in this study were published entirely, or in part, in the following publication, on which several of us were co-authors: Han S, Kong YC, Sun B, Han QH, Chen Y and Wang YC: microRNA-218 inhibits oxygen-induced retinal neovascularization via reducing the expression of roundabout 1. Chin Med J (Engl) 129: 709-715, 2016. While we had intended that these papers offered different research perspectives, we were reminded of the fact that, in submitting the article to International Journal of Molecular Medicine, the work described therein was required to be "original research that has not been published previously, and is not under consideration for publication elsewhere, in whole or in part". Therefore, owing to the redundancy in the data between these publications, the above paper is to be retracted. All the authors have agreed to the retraction. We sincerely apologize for our misunderstanding, and deeply regret any inconvenience this mistake has caused.[the original article was published in the International Journal of Molecular Medicine 37: 1139-1145, 2016; DOI: 10.3892/ijmm.2016.2511].

microRNA-218 Inhibits Oxygen-induced Retinal Neovascularization via Reducing the Expression of Roundabout 1.

BACKGROUND: The mechanisms of pathological retinal neovascularization (RNV) remain unknown. Several microRNAs were reported to be involved in the process of RNV. Oxygen-induced retinopathy (OIR) is a useful model to investigate RNV. Our present work explored the expression and the role of microRNA-128 (miR-218) in oxygen-induced RNV. METHODS: OIR was used to establish RNV model. The expression level of miR-218 in the retina from OIR mice was assessed by quantitative real-time reverse transcriptase polymerase chain reaction. Fluorescein angiography was performed in retinae of OIR mice, and RNV was quantified by hematoxylin and eosin staining to evaluate the effect of pCDH-CMV-miR-218 intravitreal injection on RNV in OIR mice. Roundabout 1 (Robo1) expression was detected by Western blotting in mouse retinal vascular endothelial cells expressing a high or low level of miR-218 and retinal tissues from OIR mice. Cell migration was evaluated by scratch wound assay. RESULTS: In OIR mice, the expression level of miR-218 was significantly down-regulated (P = 0.006). Retinal Robo1 expression was significantly increased at both mRNA and protein levels (P = 0.001, 0.008; respectively). miR-218 intravitreal injection inhibited retinal angiogenesis in OIR mice, and the restoration of miR-218 in retina led to down-regulation of Robo1. CONCLUSIONS: Our experiments showed that restoration of miR-218 inhibited retinal angiogenesis via targeting Robo1. MiR-218 contributed to the inhibition of retinal angiogenesis and miR-218 might be a new therapeutic target for preventing RNV.

Slit-miR-218-Robo axis regulates retinal neovascularization.

miR-218 is an important intronic microRNA (miRNA or miR) which is known to regulate angiogenesis in tumors. The present study aimed to investigate the effects of miR-218, as well as its host genes, Slit2 and Slit3, on oxygen-induced retinal neovascularization (RNV) and to explore the associated mechanisms of action. For this purpose, a mouse model of oxygen-induced retinopathy (OIR) was established. The expression levels of miR-218-1 and miR-218-2, as well as those of their host genes, Slit2 and Slit3, were determined by RT-qPCR. Fluorescein angiography was performed on the retinas of the mice with OIR, and RNV was quantified by H&E staining in order to evaluate the effect of pCDH-CMV-miR-218 intravitreal injection on RNV in the mouse model of OIR. Roundabout, axon guidance receptor, homolog 1 (Robo1) expression was detected in mouse retinal vascular endothelial cells expressing high or low levels of miR-218 and in retinal tissues from mice with OIR by western blot analysis. Cell migration was evaluated by a scratch wound assay. We noted that in the mice with OIR, the expression level of miR-218 was significantly downregulated. We also noted that Robo1 expression was suppressed by miR-218. Furthermore, in the mice with OIR, the expression level of miR-218 was significantly downregulated, and that of miR-218-1 and its host gene, Slit2, was concomitantly downregulated as well. The restoration of miR-218 inhibited retinal angiogenesis by targeting Robo1. Taken together, our findings suggest that the Slit2-miR-218-Robo1 axis contributes to the inhibition of retinal angiogenesis and that miR-218 may be a new therapeutic target for preventing RNV.

Six-year clinical study of firework-related eye injuries in North China.

PURPOSE: To analyse the demographic data, clinical characteristics, management and prognosis of patients with firework-related eye injuries. METHODS: A retrospective review was performed of patients with eye injuries related to fireworks referred to TianJin Eye Hospital in North China from 2008 to 2013. Demographic information, clinical features, management and visual outcome were analysed and prognosis factors were evaluated. RESULTS: Ninety-nine patients (86 men) with 118 eye injuries were enrolled in the study. The average age of the patients was 32.0±20.5 years; 70/99 (70.7%) were aged >20 years. Eighty-one of the patients had been lighting the fireworks while the rest were bystanders. The main ophthalmic manifestations were hyphaema, vitreous haemorrhage, corneal/sclera/corneoscleral open globe injury, eyelid laceration, traumatic cataract, retinal/choroid detachment, endophthalmitis and intraocular foreign body (IOFB). Ninety patients required surgical intervention including repair of open globe injury, vitrectomy, cataract extraction and enucleation. 56/118 eyes (47.5%) received multiple operations. After treatment, final best-corrected visual acuity (BCVA) significantly improved (p=0.015). Some factors were significantly correlated with better final BCVA, including initial BCVA (p=0.036), closed globe injury (p=0.031), absence of endophthalmitis (p=0.014), absence of IOFB (p=0.024) and absence of retinal detachment (p=0.046). CONCLUSIONS: Firework-related eye injuries mainly occur in adult men and result in severe visual damage. The most common clinical manifestations are hyphaema and vitreous haemorrhage. Better initial BCVA and closed globe injury have a better visual result while endophthalmitis, IOFB and retinal detachment have a negative visual outcome. Improved eye protection, along with enhanced public education and legal ban on fireworks, could reduce the incidence of eye injuries.

[Advance studies of Slit-Robo signal pathway and its roles in ocular neovascularisation].

The migration and patterning of axons and blood vessels share similar guidance mechanisms. Slits and their Roundabout (Robo) receptors were initially characterized as repulsive guidance cues for neuronal axons and mediate the migration of neuronal precursor cells during neural development. In recent years, the research of Slit/Robo signal pathway on neovascularization has become one of hot topics. This review will focus on the role of Slit/Robo signal pathway in ocular neovascularization to promote the research of Slit/Robo signaling on ophthalmology.

Small interference RNA targeting vascular endothelial growth factor gene effectively attenuates retinal neovascularization in mice model.

BACKGROUND: The mechanism of retinal neovascularization is not understood completely. Many growth factors are involved in the process of retinal neovascularization, such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF), which are the representatives of angiogenic and antiangiogenic molecules respectively. Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization. The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF). METHODS: In vitro, cultured EOMA cells were transfected with VEGF-siRNA (psi-HI(TM)/EGFP/VEGF siRNA) and Lipofectamine(TM) 2000 for 24, 48, and 72 hours, respectively. Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting. In vivo, OIR model mice were established, the mice (C57BL/6J) received an intra-vitreal injection of 1 µl of mixture of psi-HI(TM)/EGFP/VEGF siRNA and Lipofectamine 2000. Expressions of retinal VEGF and PEDF protein were measured by Western blotting, retinal neovascularization was observed by fluorescein angiography, and quantified. RESULTS: In vitro psi-HI(TM)/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression. In vivo, with decreased VEGF and VEGF-PEDF ratio, significant attenuation of neovascular tufts, avascular regions, tortuous, and dilated blood vessels were observed in the interfered animals. CONCLUSIONS: VEGF plays an important role in OIR, and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo, accompanied by the downregulation of VEGF-PEDF ratio, and simultaneous attenuation of retinal neovascularization was also observed. These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression, which represents a possible therapeutic strategy.

[Expression of vascular endothelial growth factor and pigment epithelium derived factor in mouse oxygen-induced retinopathy and its significance].

OBJECTIVE: To investigate the expression and significance of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in oxygen-induced mouse retinopathy. METHODS: This experiment was a control experiment study. Newborn C57BL-6 mice were exposed to hyperoxia, and then returned to normoxia to induce retinal neovascularization. Mice were sacrificed at postnatal days 12, 14 and 17 and the retina were processed for RT-PCR, Western Blot and fluorescein angiography. Analysis of variance and Dunnett's t3 was used to compare the mRNA and protein level of VEGF and PEDF between experiment and control groups. Statistical difference was considered significant at a P value less than 0.05. RESULTS: At any of the time points tested, there was significant difference at VEGF protein level (A value) between the experiment (0.47 +/- 0.12, 2.15 +/- 0. 46, 5.49 +/- 0.97) and control (l.81 +/- 0.50, 0.90 +/- 0.05, 0.88 +/- 0.91) groups (P = 0.009, 0.010, 0.000, respectively); the same situation occurred at PEDF protein level (P = 0.002, 0.046, 0.000, respectively). At postnatal day 12, 14 and 17, a significant difference at VEGF protein level was observed among the different experiment groups (P = 0.002, 0.001, 0.000, respectively); the same situation occurred at PEDF protein level (P = 0.009, 0.010, 0.000, respectively). There was significant difference at VEGF mRNA level between the experiment and control groups (P = 0.001, 0.000, 0.001); at postnatal day 12 and 14, the same situation occurred at PEDF protein level (P = 0.001, 0.000, respectively), but there was no significant difference at postnatal day 17 (P = 0.612). At postnatal day 12, 14 and 17, a significant difference at VEGF mRNA level was observed among different experiment groups (P = 0.000, 0.001, 0.000, respectively); the same situation occurred at PEDF mRNA level (P = 0.000, 0.001, 0.000, respectively). The time course of the decrease of PEDF was consistent with the increase of VEGF expression. VEGF/PEDF ratio change was correlated with the development and progression of retinal neovascularization. The time course of PEDF mRNA down-regulation was consistent with the VEGF mRNA up-regulation, similar with the changes of the protein. The change of VEGF and PEDF mRNA was prior to that of the protein. CONCLUSIONS: One of the mechanisms for development of retinal neovascularization is the changes of VEGF\PEDF level in the retina.

[Fluorescein angiography of retinal neovascularization model in mice].

OBJECTIVE: To investigate a high molecular weight fluorescein angiography in the application of retinal neovascularization model in mouse. METHODS: retinal neovascularization model was induced by exposure mouse to an environment containing high concentration of oxygen. High molecular weight fluorescein isothiocyanate dextran were perfused through the left ventricle directly, then the mouse eyes were enucleated and fixed with 4% paraformaldehyde. The retina was separated from the eyecup and flat mounting was performed on a gelatin coated slide. The vasculature was examined under fluorescent microscope. RESULT: The whole retinal vasculature was clearly visualized under fluorescent microscope. By focusing on different layer of the tissue, superficial, deep vascular plexus and connecting vessels also could be distinguished. The neovascular response occurred at the junction between the vascular and the avascular retina. CONCLUSION: High molecular weight fluorescein angiography can be applied for retinal neovascularization evaluation.