MicroRNA-4516 suppresses proliferative vitreoretinopathy development via negatively regulating OTX1.

Proliferative vitreoretinopathy (PVR) progression is associated with TGF-ß2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. In cancer cells, miR-4516 downregulates orthodenticle homeobox 1 (OTX1)-mediated cell invasion. Moreover, OTX1 is shown to be involved in invasion and EMT. The purpose of this study was to assess whether microRNA (miR-4516) suppresses EMT in RPE cells. EMT features were assessed using Western blotting, immunocytochemical staining, scratch-wound healing, modified Boyden chamber assay, and collagen gel contraction assay. For in vivo testing, a rabbit model was used, which involved induction of PVR by injection of transfected spontaneously arising RPE (ARPE) cells into the vitreous chamber. The putative target of miR-4516 was identified by luciferase reporter assay. Results showed that TGF-ß2-induced transdifferentiation and migration of RPE cells was inhibited by miR-4516 delivery. Overexpression of miR-4516 led to upregulation of zonula occludens-1, downregulation of α-smooth muscle actin and vimentin, and cell contractility-all EMT features-in the TGF-ß2-treated ARPE-19 cells. MiR-4516 regulated OTX1 expression negatively by binding to its 3'-UTR. TGF-ß2-induced phosphorylated ERK was inhibited in miR-4516-overexpressing ARPE-19 cells. MiR-4516 suppressed experimental PVR in vitro and in vivo. In conclusion, the overexpression of miR-4516 suppresses TGF-ß2-induced EMT in a PVR model, and its role in PVR depends on OTX1/ERK. Further research is needed to develop a feasible treatment method to prevent and treat PVR.

Repression of Smad4 by MicroRNA-1285 moderates TGF-ß-induced epithelial-mesenchymal transition in proliferative vitreoretinopathy.

The purpose of this study was to assess whether microRNA (miR)-1285 can suppress the epithelial-mesenchymal transition (EMT) in retinal pigment epithelial cells. Expression of miR-1285 was evaluated using quantitative real-time polymerase chain reaction (RT-qPCR). The features of EMT were assessed using Western blotting, immunocytochemical staining, scratch wound healing tests, modified Boyden chamber assay, and collagen gel contraction assay. A rabbit model of proliferative vitreoretinopathy (PVR) was used for in vivo testing, which involved the induction of PVR by injection of transfected ARPE cells into the vitreous chamber. Luciferase reporter assay was performed to identify the putative target of miR-1285. The expression of miR-1285 was downregulated in ARPE-19 cells treated with transforming growth factor (TGF)-ß. Overexpression of miR-1285 led to upregulation of zonula occludens-1, downregulation of α-smooth muscle actin and vimentin, cell migration and cell contractility-all EMT features-in the TGF-ß2-treated ARPE-19 cells. The reporter assay indicated that the 3' untranslated region of Smad4 was the direct target of miR1285. PVR progression was alleviated in the miR-1285 transfected rabbits. In conclusion, overexpression of miR-1285 attenuates TGF-ß2-induced EMT in a rabbit model of PVR, and the effect of miR-1285 in PVR is dependent on Smad4. Further research is warranted to develop a feasible therapeutic approach for the prevention and treatment of PVR.

AR12286 Alleviates TGF-ß-Related Myofibroblast Transdifferentiation and Reduces Fibrosis after Glaucoma Filtration Surgery.

Scar formation can cause the failure of glaucoma filtration surgery. We investigated the effect of AR12286, a selective Rho-associated kinase inhibitor, on myofibroblast transdifferentiation and intraocular pressure assessment in rabbit glaucoma filtration surgery models. Cell migration and collagen contraction were used to demonstrate the functionality of AR12286-modulated human conjunctival fibroblasts (HConFs). Polymerase chain reaction quantitative analysis was used to determine the effect of AR12286 on the production of collagen Type 1A1 and fibronectin 1. Cell migration and collagen contraction in HConFs were activated by TGF-ß1. However, compared with the control group, rabbit models treated with AR12286 exhibited higher reduction in intraocular pressure after filtration surgery, and decreased collagen levels at the wound site in vivo. Therefore, increased α-SMA expression in HConFs induced by TGF-ß1 could be inhibited by AR12286, and the production of Type 1A1 collagen and fibronectin 1 in TGF-ß1-treated HConFs was inhibited by AR12286. Overall, the stimulation of HConFs by TGF-ß1 was alleviated by AR12286, and this effect was mediated by the downregulation of TGF-ß receptor-related SMAD signaling pathways. In vivo results indicated that AR12286 thus improves the outcome of filtration surgery as a result of its antifibrotic action in the bleb tissue because AR12286 inhibited the TGF-ß receptor-related signaling pathway, suppressing several downstream reactions in myofibroblast transdifferentiation.

Antifibrotic role of low-dose mitomycin-c-induced cellular senescence in trabeculectomy models.

PURPOSE: We assessed whether mitomycin-C (MMC) has different antifibrotic mechanisms in trabeculectomy wound healing. METHODS: We identified 2 concentrations of MMC as "low-dose" by using WST-1 assay, Lactic dehydrogenase assay, and fluorescence-activated cell sorting flow cytometry. Senescence-associated ß-galactosidase (SA-ß-gal) and fibrotic gene expression was examined through immunocytochemistry, flow cytometry, real-time quantitative reverse transcription polymerase chain reaction, Western blotting, zymography, and modified scratch assay in vitro. In vivo, 0.1 mL of MMC or normal saline was injected to Tenon's capsule before trabeculectomy in a rabbit model. SA-ß-gal expression, apoptotic cell death, and collagen deposition in sites treated and not treated with MMC were evaluated using terminal dUTP nick end labeling assay and histochemical staining. Bleb function and intraocular pressure (IOP) levels were examined 3, 7, 14, 21, 28, and 35 days after trabeculectomy. RESULTS: In vitro, human Tenon's fibroblast (HTF) senescence was confirmed by observing cell morphologic change, SA-ß-gal accumulation, formation of senescence-associated heterochromatin, increased p16INK4a and p21CIP1/WAF1 expression, lower percentage of Ki-67-positive cells, and decreased COL1A1 release. Increased expression of α-SMA, COL1A1, and Smad2 signaling in TGF-ß1-induced stress fibers were passivated in senescent HTFs. In addition, cellular migration enhanced by TGF-ß1was inactivated. In vivo, histological examination indicated increased SA-ß-gal accumulation, lower apoptosis ratios, and looser collagen deposition in sites treated with 0.2 µM MMC. Low-dose MMC-induced cellular senescence prolonged trabeculectomy bleb survival and reduced IOP levels in a rabbit model. CONCLUSION: Low-dose MMC-induced cellular senescence is involved in the antifibrotic mechanism of trabeculectomy wound healing.

Protective effects of hypercapnic acidosis on Ischemia-reperfusion-induced retinal injury.

Ischemia-reperfusion (I/R) injury is associated with numerous retinal diseases, such as diabetic retinopathy, acute glaucoma, and other vascular retinopathies. Hypercapnic acidosis (HCA) has a protective effect on lung, myocardial, and central nervous system ischemic injury models. However, no study has evaluated its protective effects in an experimental retinal I/R injury model. In this study, retinal I/R injury was induced in Sprague Dawley rats by elevating the intraocular pressure to 110 mmHg for 60 minutes. HCA was induced before and after the injury. After 24 hours, the terminal dUTP nick end labeling assay was performed. Moreover, the ratios of cleaved caspase-3/total caspase-3, phosphorylated IκB/IκB, and phosphorylated p38 were measured through Western blotting. After 7 days, the rats' aqueous humor was analyzed. In addition, electroretinography and retinal thickness measurement were performed in the rats. Moreover, the retinal neural cell line RGC-5 was exposed to 500 µM H2O2 for 24 hours to induce a sustained oxidative stress in vitro. The effects of HCA were evaluated by comparing oxidative stress, MAPK signals, NF-κB signals, survival rates, and apoptosis rates in the RGC-5 cells before and after H2O2 exposure. We further investigated whether the potent I/R-protective heat shock protein (HSP) 32 contribute to protective effects of HCA. Our results indicated that HCA has protective effects against retinal I/R injury both in vivo and in vitro, at multiple levels, including antiapoptotic, anti-inflammatory, antioxidative, and functional retinal cell protection. Further research clarifying the role of HCA in retinal I/R injury prevention and treatment is warranted.

Postoperative use of bevacizumab as an antifibrotic agent in glaucoma filtration surgery in the rabbit.

PURPOSE: To evaluate the efficacy of bevacizumab as an antifibrotic agent after trabeculectomy in rabbits. METHODS: Forty-two rabbits underwent trabeculectomy and were randomly assigned to receive a postoperative course of seven subconjunctival injections of bevacizumab (1.25 mg, 25 mg/mL), 5-fluorouracil (5-FU; 5 mg, 50 mg/mL), or balanced salt solution (BSS; 0.1 mL, control). Bleb survival and characteristics were evaluated over a 30-day period. The animals were killed on postoperative day (PD)10, PD20, and PD30. Histology and immunohistochemistry of the surgical eyes was performed to evaluate and grade the amount of scarring and fibrosis in each group. RESULTS: Bevacizumab significantly improved the outcome of filtration surgery in this model. Bevacizumab prolonged bleb survival compared with the 5-FU and control groups (16.0 +/- 1.3 days vs. 6.9 +/- 0.6 and 7.4 +/- 0.85 days, respectively; P < 0.001). Bevacizumab-treated eyes had significantly larger and higher blebs than the control and 5-FU-treated groups (P < 0.05). Histologic analysis revealed that eyes treated with bevacizumab had significantly less postoperative scarring at the microscopic level at PD10 and PD20 (P = 0.009). CONCLUSIONS: Postoperative subconjunctival injection of bevacizumab is associated with improved trabeculectomy bleb survival in the rabbit model. Bevacizumab may be a useful agent for improving success and limiting scar tissue formation after trabeculectomy.

Comparison of clear corneal phacoemulsification combined with 25-gauge transconjunctival sutureless vitrectomy and standard 20-gauge vitrectomy for patients with cataract and vitreoretinal diseases.

PURPOSE: To compare effectiveness and outcomes of clear corneal phacoemulsification combined with 25-gauge transconjunctival sutureless vitrectomy and standard 20-gauge vitrectomy system for patients with clinically significant cataract and vitreoretinal diseases. SETTING: Department of Ophthalmology, Tri-Service General Hospital, Taipei, Taiwan, Republic of China. METHODS: Clear corneal phacoemulsification combined with 25-gauge transconjunctival sutureless vitrectomy was performed in the study group (15 eyes). Patients in the control group (15 eyes) had combined surgery with clear corneal phacoemulsification and the existing 20-gauge vitrectomy system, providing a comparison between 2 groups with regard to preoperative and postoperative best corrected visual acuity (BCVA), intraocular pressure, and intraoperative and postoperative complications. The additional outcome measurements in both groups were duration of the surgical procedures, time to return to preoperative corneal clarity, time to achieve stable vision, and subjective ocular irritation postoperatively. RESULTS: Postoperatively, BCVA improved in 12 eyes (80%) in the study group and in 11 eyes (73.3%) in the control group. Mean operative time for opening and closing the sclerotomy was significantly greater in the control group than in the study group. Time to return to preoperative corneal clarity and time to achieve stable vision showed no significant difference between the groups. Compared with the control group, postoperative ocular irritation in patients in the study group was considerably reduced. CONCLUSIONS: Combined sutureless surgery needed shorter setup time for sclerotomy and caused less postoperative ocular irritation than combined surgery with 20-gauge vitrectomy. Therefore, this type of procedure would be a good option for selected cases with cataract and vitreoretinal diseases.