Bone morphogenetic protein 6 (BMP6) antagonises experimental proliferative vitreoretinopathy established by TGF-ß2 stimulation in retinal pigment epithelial cells through modulation of the p38 and JNK MAPK pathways.

The formation of the epiretinal fibrotic membrane by retinal pigment epithelial (RPE) cells is a primary pathological change for proliferative vitreoretinopathy (PVR). Bone morphogenetic protein 6 (BMP6) is an antifibrogenic factor in various cells. To date, it is still unknown whether BMP6 can interfere with the fibrogenesis of RPE cells during the progression of PVR. This work aimed to address the relationship between BMP6 and transforming growth factor-ß2 (TGF-ß2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-ß2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-ß2. The treatment of RPE cells with TGF-ß2 resulted in significant increases in proliferation, migration, epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling. These effects were found to be inhibited by the overexpression of BMP6 or exacerbated by the knockdown of BMP6. BMP6 overexpression reduced the phosphorylation of p38 and JNK in TGF-ß2-stimulated RPE cells, while BMP6 knockdown showed the opposite effects. The inhibition of p38 or JNK partially reversed the BMP6-silencing-induced promoting effects on TGF-ß2-elicited fibrogenesis in RPE cells. Taken together, BMP6 demonstrates the ability to counteract the proliferation, migration, EMT, and ECM remodelling of RPE cells induced by TGF-ß2. This is achieved through the regulation of the p38 and JNK MAPK pathways. These findings imply a potential connection between BMP6 and PVR, and highlight the potential application of BMP6 in therapeutic interventions for PVR.

The Effect of Anti-Autotaxin Aptamers on the Development of Proliferative Vitreoretinopathy.

This study investigated the effect of anti-autotaxin (ATX) aptamers on the development of proliferative vitreoretinopathy (PVR) in both in vivo and in vitro PVR swine models. For the in vitro study, primary retinal pigment epithelial (RPE) cells were obtained from porcine eyes and cultured for cell proliferation and migration assays. For the in vivo study, a swine PVR model was established by inducing retinal detachment and injecting cultured RPE cells (2.0 × 106). Concurrently, 1 week after RPE cell injection, the anti-ATX aptamer, RBM-006 (10 mg/mL, 0.1 mL), was injected twice into the vitreous cavity. Post-injection effects of the anti-ATX aptamer on PVR development in the in vivo swine PVR model were investigated. For the in vitro evaluation, the cultured RPE cell proliferation and migration were significantly reduced at anti-ATX aptamer concentrations of 0.5-0.05 mg and at only 0.5 mg, respectively. Intravitreal administration of the anti-ATX aptamer also prevented tractional retinal detachment caused by PVR in the in vivo PVR model. We observed that the anti-ATX aptamer, RBM-006, inhibited PVR-related RPE cell proliferation and migration in vitro and inhibited the progression of PVR in the in vivo model, suggesting that the anti-ATX aptamer may be effective in preventing PVR.

Targeting non-muscle myosin II inhibits proliferative vitreoretinopathy through regulating epithelial-mesenchymal transition.

Proliferative vitreoretinopathy (PVR) is a common complication of rhegmatogenous retinal detachment, eventually leading to vision loss. To date, there are no effective drugs for the treatment of this disease. In this study, we investigated the effect of blebbistatin, a non-muscle myosin II inhibitor, on the ARPE-19 cell line and in a rabbit model of proliferative vitreoretinopathy. In vitro, we found that blebbistatin inhibited the epithelial-mesenchymal transition of retinal pigment epithelial (RPE) cells and inhibited the ability of RPE cells to migrate, proliferate, generate extracellular matrix, and affect contractility. In vivo the PVR model showed that blebbistatin significantly delayed PVR progression. It also partially prevents the loss of retinal function caused by PVR. Our results suggest that blebbistatin is a potential drug with clinical applications for the treatment of PVR.

A randomised controlled trial of adjunctive triamcinolone acetonide in eyes undergoing vitreoretinal surgery for open globe trauma - the ASCOT study.

Background: Eyes sustaining open globe trauma are at high risk of severe visual impairment. Proliferative vitreoretinopathy is the most common cause of retinal detachment and visual loss in eyes with open globe trauma. There is evidence from experimental studies and pilot clinical trials that the use of adjunctive steroid medication triamcinolone acetonide can reduce the incidence of proliferative vitreoretinopathy and improve outcomes of surgery for open globe trauma. Objective: The Adjunctive Steroid Combination in Ocular Trauma or ASCOT study aimed to investigate the clinical effectiveness of adjunctive triamcinolone acetonide given at the time of vitreoretinal surgery for open globe trauma. Design: A phase 3 multicentre double-masked randomised controlled trial randomising patients undergoing vitrectomy following open globe trauma to either adjunctive triamcinolone acetonide or standard care. Setting: Hospital vitreoretinal surgical services dealing with open globe trauma. Participants: Patients undergoing vitrectomy surgery who had sustained open globe trauma. Interventions: Triamcinolone acetonide 4 mg/0.1 ml into the vitreous cavity and 40 mg/1 ml sub-Tenon's or standard vitreoretinal surgery and postoperative care. Main outcome measures: The primary outcome was the proportion of patients with at least 10 letters of improvement in corrected visual acuity at six months. Secondary outcomes included retinal detachment secondary to proliferative vitreoretinopathy, retinal reattachment, macula reattachment, tractional retinal detachment, number of operations, hypotony, elevated intraocular pressure and quality of life. Health-related quality of life was assessed using the EuroQol Five Domain and Visual Function Questionnaire 25 questionnaires. Results: A total of 280 patients were randomised; 129 were analysed from the control group and 130 from the treatment group. The treatment group appeared, by chance, to have more severe pathology on presentation. The primary outcome (improvement in visual acuity) and principal secondary outcome (change in visual acuity) did not demonstrate any treatment benefit for triamcinolone acetonide. The proportion of patients with improvement in visual acuity was 47% for triamcinolone acetonide and 43% for standard care (odds ratio 1.03, 95% confidence interval 0.61 to 1.75, p = 0.908); the baseline adjusted mean difference in the six-month change in visual acuity was -2.65 (95% confidence interval -9.22 to 3.92, p = 0.430) for triamcinolone acetonide relative to control. Similarly, the secondary outcome measures failed to show any treatment benefit. For two of the secondary outcome measures, stable complete retinal reattachment and stable macular retinal reattachment, outcomes for the treatment group were significantly worse for triamcinolone acetonide at the 5% level (respectively, odds ratio 0.59, 95% confidence interval 0.36 to 0.99, p = 0.044 and odds ratio 0.59, 95% confidence interval 0.35 to 0.98, p = 0.041) compared with control in favour of control. The cost of the intervention was £132 per patient. Health economics outcome measures (Early Treatment Diabetic Retinopathy Study, Visual Function Questionnaire 25 and EuroQol Five Dimensions) did not demonstrate any significant difference in quality-adjusted life-years. Conclusions: The use of combined intraocular and sub-Tenon's capsule triamcinolone acetonide is not recommended as an adjunct to vitrectomy surgery for intraocular trauma. Secondary outcome measures are suggestive of a negative effect of the adjunct, although the treatment group appeared to have more severe pathology on presentation. Future work: The use of alternative adjunctive medications in cases undergoing surgery for open globe trauma should be investigated. Refinement of clinical grading and case selection will enable better trail design for future studies. Trial registration: This trial is registered as ISRCTN 30012492, EudraCT number 2014-002193-37, REC 14/LNO/1428, IRAS 156358, Local R&D registration CHAD 1031. Funding: This project was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (12/35/64) and will be published in full in Health Technology Assessment; Vol. 27, No. 12. See the NIHR Journals Library website for further project information.

Despite advances in surgical techniques, eye trauma remains a leading cause of blindness and visual impairment. The main cause of trauma is a scarring process within the eye ­ proliferative vitreoretinopathy. There is good evidence from laboratory work and small-scale clinical studies that the addition of a steroid medication, triamcinolone acetonide, given in and around the eye at the time of surgery for eye trauma, can reduce the incidence of proliferative vitreoretinopathy scarring and improve the outcomes of surgery. The Adjunctive Steroid Combination in Ocular Trauma or ASCOT study was a multicentre clinical trial designed to test the use of triamcinolone acetonide as an addition to surgery to improve outcomes in eyes with 'open globe' penetrating injuries. A total of 280 patients were recruited and randomised to receive standard surgery or surgery with the additional steroid (triamcinolone acetonide). No benefit was found from the addition of the steroid medication. The addition of steroid medication was not good value for money. Secondary outcome measures suggested that triamcinolone acetonide may have had a negative effect on outcomes, although this may have been due to the presence of more severe cases amongst the patients allocated to receive the additional steroid (triamcinolone acetonide). The use of adjunctive triamcinolone acetonide in eye trauma cases undergoing surgery is therefore not recommended. Future studies with different additional medications and/or more targeted case selection are indicated to improve outcomes for eyes experiencing penetrating trauma.

Silibinin Prevents TGFß-Induced EMT of RPE in Proliferative Vitreoretinopathy by Inhibiting Stat3 and Smad3 Phosphorylation.

Purpose: The purpose of this study was to investigate the effects of silibinin on epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) and proliferative vitreoretinopathy (PVR) formation, as well as its underlying molecular mechanism. Methods: Cellular morphological change and EMT molecular markers were evaluated by using phase contrast imaging, qPCR, and Western blot (WB) to investigate the impact of silibinin on the EMT of ARPE-19 cells. Scratch assay and transwell assay were used to study the effect of silibinin on cell migration. An intravitreally injected RPE-induced rat PVR model was used to assess the effect of silibinin on PVR in vivo. RNA-seq was applied to study the molecular mechanism of silibinin-mediated PVR prevention. Results: Silibinin inhibited TGFß1-induced EMT and migration of RPE in a dose-dependent manner in vitro. Moreover, silibinin prevented proliferative membrane formation in an intravitreal injected RPE-induced rat PVR model. In line with these findings, RNA-seq revealed a global suppression of TGFß1-induced EMT and migration-related genes by silibinin in RPEs. Mechanistically, silibinin reduced TGFß1-induced phosphorylation levels of Smad3 and Stat3, and Smad3 nuclear translocation in RPE. Conclusions: Silibinin inhibits the EMT of RPE cells in vitro and prevents the formation of PVR membranes in vivo. Mechanistically, silibinin inhibits Smad3 phosphorylation and suppresses Smad3 nuclear translocation through the inhibition of Stat3 phosphorylation. These findings suggest that silibinin may serve as a potential treatment for PVR.

Transoperative intravitreal dexamethasone implantation in patients with proliferative vitreoretinopathy.

OBJECTIVE: To compare the anatomical results in patients with rhegmatogenous retinal detachment, at least grade B of proliferative vitreoretinopathy, and with a trans-surgical dexamethasone implant vs the control group. We also assessed the diminution of proliferative vitreoretinopathy and the final visual acuity (VA). METHOD: The patients were evaluated clinically and with optical coherence tomography for 10 months. Logistic regression analyses were performed to evaluate the effect of the dexamethasone implant on retinal detachment. Correlational analyses were explored depending on the variables' distribution, and an independent samples t-test was used to compare the VA in both groups. RESULTS: The study included 38 eyes of patients with proliferative vitreoretinopathy: 18 with the implant and 20 for the control group. The evaluation of the main objective showed significant differences (p < 0.05) in the anatomical success between the two groups (61.1% vs. 20%, treatment vs. control); odds ratio of 6.29; 95% confidence interval: 1.5- 26.8; p = 0.013; Nagelkerke's R2 = 0.225. The t-test showed a significant difference in the final VA of the patients (t = 2.047; df = 36; p = 0.048; Cohen's d = 0.66). CONCLUSIONS: Retinal redetachment was less frequent, and better VA was observed, in patients with the dexamethasone implant in comparison with the control group.

OBJETIVO: Comparar los resultados anatómicos en pacientes con desprendimiento de retina regmatógeno, vitreorretinopatía proliferativa a partir de grado B y aplicación de implante de dexametasona transquirúrgico frente a un grupo control. También se valoraron la disminución de la vitreorretinopatía proliferativa y la agudeza visual (AV) final. MÉTODO: Los pacientes se evaluaron clínicamente y con tomografía de coherencia óptica por 10 meses. Se realizaron análisis de regresión logística para evaluar el efecto del implante en el redesprendimiento de retina. Se exploraron análisis correlacionales dependiendo de la distribución de variables y se aplicó la prueba t de muestras independientes para comparar la AV en ambos grupos. RESULTADOS: Se incluyeron 38 ojos de pacientes con vitreorretinopatía proliferativa: 18 con el implante y 20 del grupo control. La evaluación del objetivo principal mostró diferencias significativas (p < 0.05) en el éxito anatómico entre ambos grupos (61.1% en los ojos con tratamiento frente a 20% en el grupo control); razón de momios de 6.29; intervalo de confianza del 95%: 1.5- 26.8; p = 0.013; R2 de Nagelkerke = 0.225. La prueba t mostró una diferencia significativa entre la AV final de los pacientes (t = 2.047; gl = 36; p = 0.048; d de Cohen = 0.66). CONCLUSIONES: Se observó menor redesprendimiento, así como mejor AV, en los pacientes con el implante de dexametasona en comparación con el grupo control.

Nintedanib inhibits normal human vitreous-induced epithelial-mesenchymal transition in human retinal pigment epithelial cells.

PURPOSE: In this study, we aim to investigate the potential of nintedanib as a therapeutic approach to proliferative vitreoretinopathy (PVR), which is the leading cause of failure in retinal detachment repair. PVR is characterized by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, and understanding the effects of nintedanib on EMT in the normal human vitreous (HV)-induced RPE cells is crucial. METHODS: Our research focuses on assessing the impact of nintedanib on HV-induced EMT in human retinal pigment epithelial (ARPE-19) cells in vitro. We employed various techniques, including quantitative real-time PCR (qPCR), western blot analysis, and immunofluorescence staining, to evaluate the mRNA and protein expression of EMT biomarkers in HV-induced ARPE-19 cells. Additionally, we measured the proliferation of RPE cells using cell counting, CCK-8, and Ki-67 assays. Migration was assessed through wound healing and transwell migration assays, while contraction was determined using a collagen gel contraction assay. Morphological changes were examined using phase-contrast microscopy. RESULTS: Our results demonstrate that nintedanib selectively attenuates the upregulation of mesenchymal markers in HV-induced ARPE-19 cells, at both the mRNA and protein levels. Furthermore, nintedanib effectively suppresses the HV-induced proliferation, migration, and contraction of ARPE-19 cells, while maintaining the cells' basal activity. These findings strongly suggest that nintedanib exhibits protective effects against EMT in ARPE-19 cells and could be a promising therapeutic option for PVR. CONCLUSIONS: By elucidating the anti-EMT effects of nintedanib in HV-induced RPE cells, our study highlights the potential of this oral triple tyrosine kinase inhibitor in the treatment of PVR. These findings contribute to the growing body of research aimed at developing novel strategies to prevent and manage PVR, ultimately improving the success rates of retinal detachment repair.

Using Advanced Bioinformatics Tools to Identify Novel Therapeutic Candidates for Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) is the dreaded cause of failure following retinal detachment repair; however, no cures or preventative therapies exist to date. The purpose of this study was to use bioinformatics tools to identify drugs or compounds that interact with biomarkers and pathways involved in PVR pathogenesis that could be eligible for further testing for the prevention and treatment of PVR. Methods: We queried PubMed to compile a comprehensive list of genes described in PVR to date from human studies, animal models, and genomic studies found in the National Center for Biotechnology Information database. Gene enrichment analysis was performed using ToppGene on PVR-related genes against drug-gene interaction databases to construct a pharmacome and estimate the statistical significance of overrepresented compounds. Compounds with no clinical indications were filtered out from the resulting drug lists. Results: Our query identified 34 unique genes associated with PVR. Out of 77,146 candidate drugs or compounds in the drug databases, our analysis revealed multiple drugs and compounds that have significant interactions with genes involved in PVR, including antiproliferatives, corticosteroids, cardiovascular agents, antioxidants, statins, and micronutrients. Top compounds, including curcumin, statins, and cardiovascular agents such as carvedilol and enalapril, have well-established safety profiles and potentially could be readily repurposed for PVR. Other significant compounds such as prednisone and methotrexate have shown promising results in ongoing clinical trials for PVR. Conclusions: This bioinformatics approach of studying drug-gene interactions can identify drugs that may affect genes and pathways implicated in PVR. Predicted bioinformatics studies require further validation by preclinical or clinical studies; however, this unbiased approach could identify potential candidates among existing drugs and compounds that could be repurposed for PVR and guide future investigations. Translational Relevance: Novel repurposable drug therapies for PVR can be found using advanced bioinformatics models.

Human Wharton's Jelly Mesenchymal Stem Cell Secretome Modifies the Processes of Neuroprotection and Epithelial-Mesenchymal Transition in Retinal Pigment Epithelium at Transcriptional Level.

BACKGROUND: Retinal pigment epithelium (RPE) cells are potential targets for treating retinal detachment (RD) and proliferative vitreoretinopathy (PVR), considering the importance of neuroprotection and epithelial-mesenchymal transition (EMT) of RPE in these conditions. This study investigated the effect of human Wharton's jelly mesenchymal stem cell secretome (WJMSC-S) on the expression of genes involved in both neuroprotection and EMT in RPE cells in vitro (TRKB, MAPK, PI3K, BDNF, and NGF). METHODS: RPE cells from passages 5-7 were treated with WJMSC-S (or the vehicle culture medium as control) for 24 h at 37◦C and subsequently subjected to RNA extraction and cDNA synthesis. Gene expression level was evaluated using real-time PCR in the treated versus control cells. RESULTS: The results of our study showed that WJMSC-S led to a significant downregulation in three out of five studied gene expression (MAPK, TRKB, and NGF), and simultaneously, remarkably upregulated the expression of the BDNF gene. CONCLUSIONS: According to the present data, WJMSC-S can affect the EMT and neuroprotection processes at the mRNA level by suppressing EMT and promoting neuroprotection in RPE cells. This finding may have positive clinical implications in the context of RD and PVR.

In vitro laboratory models of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR), the most common cause of recurrent retinal detachment, is characterized by the formation and contraction of fibrotic membranes on the surface of the retina. There are no Food and Drug Administration (FDA)-approved drugs to prevent or treat PVR. Therefore, it is necessary to develop accurate in vitro models of the disease that will enable researchers to screen drug candidates and prioritize the most promising candidates for clinical studies. We provide a summary of recent in vitro PVR models, as well as avenues for model improvement. Several in vitro PVR models were identified, including various types of cell cultures. Additionally, novel techniques that have not been used to model PVR were identified, including organoids, hydrogels, and organ-on-a-chip models. Novel ideas for improving in vitro PVR models are highlighted. Researchers may consult this review to help design in vitro models of PVR, which will aid in the development of therapies to treat the disease.

Low-Dose Intravitreal Methotrexate for Proliferative Vitreoretinopathy.

BACKGROUND AND OBJECTIVE: Proliferative vitreoretinopathy (PVR) has been mitigated by intravitreal methotrexate (MTX) 400 µg/0.1 mL in several studies. Here, we evaluate the results from a lower dose of MTX, 200 µg/0.05 mL. MATERIALS AND METHODS: We identified and reviewed records of patients with grade ≥C1 PVR who were treated with 200 µg/0.05 mL MTX injections: during PVR surgery and every 2 weeks thereafter. RESULTS: Twenty-four eyes met inclusion criteria with a mean of 5.6 injections and follow-up ranging 6 to 56 months. The retina was reattached in 19 of 24 eyes (79%) after a single surgery and in 5 of 24 eyes (21%) after one additional PVR surgery. Visual acuity improved from baseline logMAR 1.63 to 0.97 at 12 months (P < .001), with 5 of 20 achieving 20/60 or better and 16 of 20 achieving 20/200 or better. One eye developed a transient corneal abrasion that resolved within 1 week. CONCLUSION: Low-dose MTX (200 µg/0.05 mL) during and after PVR surgery resulted in good rates of retinal reattachment and visual acuity recovery. [Ophthalmic Surg Lasers Imaging Retina 2023;54(3):139-146.].

Pharmacological Therapy of Proliferative Vitreoretinopathy: Systematic In Vitro Comparison of 36 Pharmacological Agents.

Purpose: Proliferative vitreoretinopathy (PVR) is currently treated surgically. Reliable pharmaceutical options would be desirable, and numerous drugs have been proposed. This in vitro study is intended to systematically compare and determine the most promising candidates for the treatment of PVR. Methods: A structured literature review was conducted in the "PubMed" database to identify previously published agents proposed for medical treatment of PVR -36 substances that met the inclusion criteria. Toxicity and antiproliferative effects were evaluated on primary human retinal pigment epithelial (hRPE) using colorimetric viability assays. The seven substances with the widest therapeutic range between toxicity and no longer detectable antiproliferative effect were then validated with a bromodeoxyuridine assay and a scratch wound healing assay using primary cells derived from surgically excised human PVR membranes (hPVR). Results: Among 36 substances, 12 showed no effect on hRPE at all. Seventeen substances had a significant (P < 0.05) toxic effect of which nine did not have an antiproliferative effect. Fifteen substances significantly reduced hRPE proliferation (P < 0.05). The seven most promising drugs with the highest difference between toxicity and antiproliferative effects on hRPE were dasatinib, methotrexate, resveratrol, retinoic acid, simvastatin, tacrolimus, and tranilast. Whereof resveratrol, simvastatin, and tranilast additionally showed antiproliferative and dasatinib, resveratrol, and tranilast antimigratory effects on hPVR (P < 0.05). Conclusion: This study presents a systematic comparison of drugs that have been proposed for PVR treatment in a human disease model. Dasatinib, resveratrol, simvastatin, and tranilast seem to be promising and are well-characterized in human use.

Experimental Models to Study Epithelial-Mesenchymal Transition in Proliferative Vitreoretinopathy.

Proliferative vitreoretinal diseases (PVDs) encompass proliferative vitreoretinopathy (PVR), epiretinal membranes, and proliferative diabetic retinopathy. These vision-threatening diseases are characterized by the development of proliferative membranes above, within and/or below the retina following epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) and/or endothelial-mesenchymal transition of endothelial cells. As surgical peeling of PVD membranes remains the sole therapeutic option for patients, development of in vitro and in vivo models has become essential to better understand PVD pathogenesis and identify potential therapeutic targets. The in vitro models range from immortalized cell lines to human pluripotent stem-cell-derived RPE and primary cells subjected to various treatments to induce EMT and mimic PVD. In vivo PVR animal models using rabbit, mouse, rat, and swine have mainly been obtained through surgical means to mimic ocular trauma and retinal detachment, and through intravitreal injection of cells or enzymes to induce EMT and investigate cell proliferation and invasion. This review offers a comprehensive overview of the usefulness, advantages, and limitations of the current models available to investigate EMT in PVD.

p21CIP/WAF1 saRNA inhibits proliferative vitreoretinopathy in a rabbit model.

PURPOSE: Proliferative vitreoretinopathy (PVR) is a disease process resulting from proliferation of retinal pigment epithelial (RPE) cells in the vitreous and periretinal area, leading to periretinal membrane formation and traction and eventually to postoperative failure after vitreo-retinal surgery for primary rhegmatogenous retinal detachment (RRD). The present study was designed to test the therapeutic potential of a p21CIP/WAF1 (p21) inducing saRNA for PVR. METHODS: A chemically modified p21 saRNA (RAG1-40-53) was tested in cultured human RPE cells for p21 induction and for the inhibition of cell proliferation, migration and cell cycle progression. RAG1-40-53 was further conjugated to a cholesterol moiety and tested for pharmacokinetics and pharmacodynamics in rabbit eyes and for therapeutic effects after intravitreal administration in a rabbit PVR model established by injecting human RPE cells. RESULTS: RAG1-40-53 (0.3 mg, 1 mg) significantly induced p21 expression in RPE cells and inhibited cell proliferation, the progression of cell cycle at the G0/G1 phase and TGF-ß1 induced migration. After a single intravitreal injection into rabbit eyes, cholesterol-conjugated RAG1-40-53 exhibited sustained concentration in the vitreal humor beyond at least 8 days and prevented the progression of established PVR. CONCLUSION: p21 saRNA could represent a novel therapeutics for PVR by exerting a antiproliferation and antimigration effect on RPE cells.

DAPL1 prevents epithelial-mesenchymal transition in the retinal pigment epithelium and experimental proliferative vitreoretinopathy.

Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a hallmark of the pathogenesis of proliferative vitreoretinopathy (PVR) that can lead to severe vision loss. Nevertheless, the precise regulatory mechanisms underlying the pathogenesis of PVR remain largely unknown. Here, we show that the expression of death-associated protein-like 1 (DAPL1) is downregulated in PVR membranes and that DAPL1 deficiency promotes EMT in RPE cells in mice. In fact, adeno-associated virus (AAV)-mediated DAPL1 overexpression in RPE cells of Dapl1-deficient mice inhibited EMT in physiological and retinal-detachment states. In a rabbit model of PVR, ARPE-19 cells overexpressing DAPL1 showed reduced ability to induce experimental PVR, and AAV-mediated DAPL1 delivery attenuated the severity of experimental PVR. Furthermore, a mechanistic study revealed that DAPL1 promotes P21 phosphorylation and its stabilization partially through NFκB (RelA) in RPE cells, whereas the knockdown of P21 led to neutralizing effects on DAPL1-dependent EMT inhibition and enhanced the severity of experimental PVR. These results suggest that DAPL1 acts as a novel suppressor of RPE-EMT and has an important role in antagonizing the pathogenesis of experimental PVR. Hence, this finding has implications for understanding the mechanism of and potential therapeutic applications for PVR.

RESCUE INTRAVITREAL METHOTREXATE TREATMENT FOLLOWING EARLY RECOGNITION OF PROLIFERATIVE VITREORETINOPATHY.

PURPOSE: To report a case of proliferative vitreoretinopathy (PVR) in a man with recurrent retinal detachment successfully managed without surgical intervention following the initiation of intravitreal methotrexate injections to arrest progression of PVR. METHODS: Report of a case. RESULTS: A 60-year-old man presented to the retina clinic 4 weeks after undergoing vitrectomy for rhegmatogenous retinal detachment and was found to have an inferior recurrent retinal detachment. He underwent repeat vitrectomy and scleral buckling with successful reattachment of the retina in the immediate postoperative period. At postoperative Week 2, preretinal membranes were noted inferiorly with stretching of the causative retinal break and localized subretinal fluid, consistent with early PVR. The patient underwent immediate laser barricade, and a course of intravitreal methotrexate injections was started. At the final follow-up 7 months later, the retina was fully attached without progression of PVR. CONCLUSION: Intravitreal methotrexate may play a role in arresting progression of early postoperative PVR and obviating the need for surgical intervention.

Inhibition of proliferative vitreoretinopathy by a newly developed methotrexate loaded drug carrier in vitro.

PURPOSE: Repeated intravitreal injections of methotrexate for proliferative vitreoretinopathy, a rare ocular condition that can cause vision loss, have shown beneficial effects in recent clinical studies. The purpose of this study was to develop a slow-release, long-term drug carrier composed of the polymer polylactide-co-glycolide and methotrexate that can be injected intravitreally. METHODS: The required composition of the drug carrier was modeled using pharmacokinetic parameters based on current literature. Release kinetics were determined using an ocular pharmacokinetic model. Epiretinal PVR-membranes were harvested during pars plana vitrectomy and subsequently transferred to cell culture. The effect of the drug carrier on cell migration was investigated using time-lapse microscopy and a scratch-induced migration assay. The colorimetric WST-1-assay and a live-dead-assay were performed to determine viability, and the BrdU-assay was applied for proliferation. RESULTS: The release profile showed an initial and a final burst of methotrexate with an intervening steady state that lasted 9-11 weeks. It showed inhibitory effects on pathobiological processes in human PVR-cells in vitro. Cell velocity in the time-lapse assay, migration in the scratch assay (p = 0.001), and proliferation in the BrdU assay (p = 0.027) were reduced after addition of the drug carrier. These effects occurred without causing a reduction in viability in the WST-1 assay (p > 0.99) and the live-dead assay. CONCLUSION: The methotrexate-loaded drug carrier can maintain a stable concentration for 9-11 weeks and influence the pathobiological process of PVR cells in vitro. Therefore, it represents a potential therapeutic orphan drug for PVR.

TSPAN4-positive migrasome derived from retinal pigmented epithelium cells contributes to the development of proliferative vitreoretinopathy.

BACKGROUND: Proliferative vitreoretinopathy (PVR) is a blind-causing disease initiated by the activation of retinal pigmented epithelium (RPE) primarily induced by TGF-ß families. Migrasome is a recently discovered type of extracellular vesicle related to cell migration. RESULTS: Here, we used ex vivo, in vitro, and in vivo models, to investigate the characteristics and functions of migrasomes in RPE activation and PVR development. Results indicated that the migrasome marker tetraspanin-4 (TSPAN4) was abundantly expressed in human PVR-associated clinical samples. The ex vivo model PVR microenvironment is simulated by incubating brown Norway rat RPE eyecups with TGF-ß1. Electron microscope images showed the formation of migrasome-like vesicles during the activation of RPE. Further studies indicated TGF-ß1 increased the expression of TSPAN4 which results in migrasome production. Migrasomes can be internalized by RPE and increase the migration and proliferation ability of RPE. Moreover, TSPAN4-inhibited RPE cells are with reduced ability of initiating experimental PVR. Mechanically, TSPAN4 expression and migrasome production are induced through TGF-ß1/Smad2/3 signaling pathway. CONCLUSION: In conclusion, migrasomes can be produced by RPE under PVR microenvironment. Migrasomes play a pivotal role in RPE activation and PVR progression. Thus, targeting TSPAN4 or blocking migrasome formation might be a new therapeutic method against PVR.