Progression Of Proliferative Vitreoretinopathy Research Articles

Role of semaphorin7A in epithelial-mesenchymal transition and proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a multifactorial ocular condition characterized by the development of fibrotic membranes inside the vitreous cavity and on the detached retina, which can result in severe blindness. Semaphorin7A (Sema7a) is involved in axon growth, inflammatory responses, and immune regulation; however, its role in PVR and regulatory mechanisms in retinal pigment epithelium (RPE) cells remains unclear. This study aimed to examine Sema7a in PVR and the underlying mechanisms. Transcriptome sequencing was used to investigate the changes in mRNA expression profiles. Western blotting, immunofluorescence, and real-time polymerase chain reaction (RT-PCR) were utilized to investigate the potential mechanism of Sema7a on epithelial-mesenchymal transition (EMT) in RPE cells. Stimulating RPE cells with transforming growth factor beta-1 (TGF-β1) decreased the levels of epithelial markers but increased those of mesenchymal markers. Based on transcriptome sequencing, many molecules associated with PVR progression were regulated. PVR vitreous fluid proteomics data analysis showed that Sema7a significantly changed at different levels. Silencing Sema7a in RPE cells attenuated TGF-β1-induced EMT and their ability to induce experimental PVR; in contrast, recombinant Sema7a (rSema7a) directly triggered EMT in RPE cells. TGF-β1 induction mechanically activated the PI3k-AKT and MAPK pathways, while Sema7a knockdown by short interfering RNA lowered the phosphorylation of the PI3k-AKT/MAPK signaling pathway. Therefore, Sema7a may be a viable therapeutic target for PVR due to its crucial role in the TGF-β1-induced EMT of RPE cells.

THE METHOD OF DOUBLE CONTRAST IN THE INTRAOPERATIVE DIAGNOSIS OF REGMATOGENIC RETINAL DETACHMENT

Usually, the recurrence rate of regmatogenic retinal detachment (ROS) after surgery ranges from 3% to 30%. In cases where the disease recurs after successful surgery, in 60% of cases the main factor is the progression of proliferative vitreoretinopathy (PVR), which makes it one of the most severe complications that can occur in the postoperative period. In addition, after removal or absorption of a tampon agent (in particular, endotamponade with silicone oil), recurrent retinal detachment may occur due to the inability to detect retinal rupture during surgery.Objective: this study is aimed at evaluating the effectiveness of diagnostic methods used during operations for regmatogenic retinal detachment. After completing the standard stages of central vitrectomy, the group of patients was divided into two equal groups: the first group included patients who received the double contrast method, and the second group (control) – patients who underwent the traditional surgical method.Results and discussion: in patients of the control group, there was a significant increase in the number of relapses of ROS. In addition, it was found that an increase in the number of retinal tears correlated with a greater likelihood of macular involvement; Statistical evaluation has shown that the absence of AST and tight adhesion of the posterior hyaloid membrane to the retina and small retinal tears increase the risk of recurrence of ROS due to the progression of PVR.

Tenascin-C induces transdifferentiation of retinal pigment epithelial cells in proliferative vitreoretinopathy

Proliferation and transdifferentiation of the retinal pigment epithelium (RPE) are hallmarks of proliferative vitreoretinopathy (PVR); however, the critical regulators of this process remain to be elucidated. Here, we investigated the role of tenascin-C in PVR development. In vitro, exposure of human ARPE-19 (hRPE) cells to TGF-β2 increased tenascin-C expression. Tenascin-C was shown to be involved in TGF-β2-induced transdifferentiation of hRPE cells, which was inhibited by pretreatment with tenascin-C siRNA. In PVR mouse models, a marked increase in the expression of tenascin-C mRNA and protein was observed. Additionally, immunofluorescence analysis demonstrated a dramatic increase in the colocalization of tenascin-C with RPE65 or α-smooth muscle actin(α-SMA) in the epiretinal membranes of patients with PVR. There was also abundant expression of integrin αV and β-catenin in the PVR membranes. ICG-001, a β-catenin inhibitor, efficiently attenuated PVR progression in a PVR animal model. These findings suggest that tenascin-C is secreted by transdifferentiated RPE cells and promotes the development of PVR via the integrin αV and β-catenin pathways. Therefore, tenascin-C could be a potential therapeutic target for the inhibition of epiretinal membrane development associated with PVR.

Multi-omics profiling of retinal pigment epithelium reveals enhancer-driven activation of RANK-NFATc1 signaling in traumatic proliferative vitreoretinopathy

During the progression of proliferative vitreoretinopathy (PVR) following ocular trauma, previously quiescent retinal pigment epithelial (RPE) cells transition into a state of rapid proliferation, migration, and secretion. The elusive molecular mechanisms behind these changes have hindered the development of effective pharmacological treatments, presenting a pressing clinical challenge. In this study, by monitoring the dynamic changes in chromatin accessibility and various histone modifications, we chart the comprehensive epigenetic landscape of RPE cells in male mice subjected to traumatic PVR. Coupled with transcriptomic analysis, we reveal a robust correlation between enhancer activation and the upregulation of the PVR-associated gene programs. Furthermore, by constructing transcription factor regulatory networks, we identify the aberrant activation of enhancer-driven RANK-NFATc1 pathway as PVR advanced. Importantly, we demonstrate that intraocular interventions, including nanomedicines inhibiting enhancer activity, gene therapies targeting NFATc1 and antibody therapeutics against RANK pathway, effectively mitigate PVR progression. Together, our findings elucidate the epigenetic basis underlying the activation of PVR-associated genes during RPE cell fate transitions and offer promising therapeutic avenues targeting epigenetic modulation and the RANK-NFATc1 axis for PVR management.

Internal limiting membrane peeling in vitreoretinal surgery for rhegmatogenous retinal detachment: clinical results, surgical options, and future prospects

Rhegmatogenous retinal detachment (RRD) is one of the leading causes of primary vision disability, while the majority of RRD patients with RRD are people of active working age. Over the past 50 years, retinal detachment surgery has been progressing rapidly, the rate of success increasing up to 90% or higher. However, the reduction of reoperation risks of reoperations, improvement of functional outcomes and increase of postoperative visual acuity remain topical issues. The main reason for repeated surgical interventions is the proliferative vitreoretinopathy progression, which can lead to epiretinal fibrosis, macular oedema, or retinal detachment recurrence. The review is focused on current literature studies that report the results of internal limiting membrane peeling in cases of RRD surgeries and other optional techniques aimed at reducing the risk of the above mentioned postoperative complications.

Bone morphogenetic protein 6 (BMP6) antagonises experimental proliferative vitreoretinopathy established by TGF-β2 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-β2 (TGF-β2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-β2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-β2. The treatment of RPE cells with TGF-β2 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-β2-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-β2-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-β2. 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.

Comparison of vitreous/retinal pigment epithelium relative intensity in proliferative vitreoretinopathy and uveitis

Background: Quantitative measurements of vitreous inflammation using vitreous/retinal pigment epithelium-relative intensity (VIT/RPE-Relative Intensity) have been described recently. In proliferative vitreoretinopathy (PVR), inflammation plays a central role in the pathogenesis, inducing retinal fibrosis and contraction. However, no attempts have yet to be made to analyze the severity of inflammation in PVR progression. Methods: A cross-sectional study was conducted by reviewing OCT image sets obtained from patients divided into four groups: (1) proliferative vitreoretinopathy, (2) intermediate and posterior uveitis, (3) panuveitis, (4) normal healthy eyes in Cipto Mangunkusumo Kirana Eye Hospital between April 2021 – December 2021. OCT images were then analyzed in the ImageJ software for VIT/RPE-relative intensity. Results: A total of 19 PVR eyes, 12 intermediate-posterior uveitis eyes, 16 panuveitis eyes, and 28 normal healthy eyes were recruited for this study. The VIT/RPE-Relative Intensity was significantly higher in PVR eyes (0.415±0.178) than in intermediate-posterior uveitis (0.236±0.043, p=0.002) and panuveitis eyes (0.30±0.07, p=0.023). Compared to the normal eyes, PVR and both uveitis groups have significantly higher VIT/RPE relative intensity (p = <0.001 in each group). Conclusions: VIT/RPE-relative intensity may offer quantitative measurements of vitreous inflammation in the role of the pathogenesis of PVR. Comparison with cellular inflammation in the vitreous is required to validate this finding.

Comparison of vitreous/retinal pigment epithelium relative intensity in proliferative vitreoretinopathy and uveitis

Background: Quantitative measurements of vitreous inflammation using vitreous/retinal pigment epithelium-relative intensity (VIT/RPE-Relative Intensity) have been described recently. In proliferative vitreoretinopathy (PVR), inflammation plays a central role in the pathogenesis, inducing retinal fibrosis and contraction. However, no attempts have yet to be made to analyze the severity of inflammation in PVR progression. Methods: A cross-sectional study was conducted by reviewing OCT image sets obtained from patients divided into four groups: (1) proliferative vitreoretinopathy, (2) intermediate and posterior uveitis, (3) panuveitis, (4) normal healthy eyes in Cipto Mangunkusumo Kirana Eye Hospital between April 2021 – December 2021. OCT images were then analyzed in the ImageJ software for VIT/RPE-relative intensity. Results: A total of 19 PVR eyes, 12 intermediate-posterior uveitis eyes, 16 panuveitis eyes, and 28 normal healthy eyes were recruited for this study. The VIT/RPE-Relative Intensity was significantly higher in PVR eyes (0.415±0.178) than in intermediate-posterior uveitis (0.236±0.043, p=0.002) and panuveitis eyes (0.30±0.07, p=0.023). Compared to the normal eyes, PVR and both uveitis groups have significantly higher VIT/RPE relative intensity (p = <0.001 in each group). Conclusions: VIT/RPE-relative intensity may offer quantitative measurements of vitreous inflammation in the role of the pathogenesis of PVR. Comparison with cellular inflammation in the vitreous is required to validate this finding.

Расширенный подход к первичной микрохирургической обработке обширных проникающих ранений склеры с одномоментной витрэктомией и герметизацией хориоретинального дефекта

Background: the current strategy for managing extensive zone 3 scleral lacerations implies delayed subtotal vitrectomy. A high rate of poor treatment outcomes requires novel approaches to surgical debridement of lacerations given anatomical and pathogenic specifics. Aim: to assess the treatment results of extensive zone 3 scleral laceration surgery by simultaneous vitrectomy and chorioretinal wound debridement. Patients and Methods: 14 patients (14 eyes) diagnosed with extensive penetrating scleral/corneoscleral laceration and polymorphic intraocular lesions associated with grade 4–5 visual impairment were enrolled. All patients underwent primary microsurgical debridement using a technique that involved external debridement and suturing of scleral laceration, single-step subtotal vitrectomy, and internal debridement of the chorioretinal wound. The chorioretinal wound was filled with pure platelet-rich plasma (P-PRP). Endotamponade was performed using silicone oil (n=9) or air-gas mixture (n=5). Results: after 6 months, visual acuity (VA) was estimated to be 0.02–0.5 in 12 patients and light perception in 2 patients. During the follow- up, no phthisis bulbi was reported. The progression of proliferative vitreoretinopathy (formation of intraretinal adhesions or subretinal bands involving healthy retina) was reported in 3 patients (21.4%). Chorioretinal scars were characterized by local nonaggressive proliferation, no prominence in the vitreous cavity, or proliferation of tissue beyond the scars. Conclusion: our approach to severe eye injury management has demonstrated positive outcomes by providing a stable anatomical result, significant improvement in early and late postoperative VA. KEYWORDS: open globe injury, primary microsurgical debridement, proliferative vitreoretinopathy, scleral lacerations, vitrectomy, platelet- rich plasma. FOR CITATION: Shamkin S.S., Subbotina S.N., Stepanyants A.B., Salov D.V. Advanced approach to primary microsurgical debridement of extensive scleral lacerations involving simultaneous vitrectomy and chorioretinal wound closure. Russian Journal of Clinical Ophthalmology. 2023;23(3):124–128 (in Russ.). DOI: 10.32364/2311-7729-2023-23-3-3.

Mechanisms of Epithelial-Mesenchymal Transition and Prevention of Dispase-Induced PVR by Delivery of an Antioxidant αB Crystallin Peptide.

Proliferative Vitreoretinopathy (PVR) is a refractory retinal disease whose primary pathogenesis involves the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. At present, there is no effective treatment other than surgery for PVR. The purpose of this study was to investigate the effect of αB crystallin peptide (αBC-P) on EMT in PVR. We have previously shown that this peptide is antiapoptotic and regulates RPE redox status. Subconfluent primary human RPE (hRPE) cells were stimulated by TGFβ2 (10 ng/mL) with or without αBC-P (50 or 75 μg/mL) for 48 h and expression of EMT/mesenchymal to epithelial transition (MET) markers was determined. Mitochondrial ROS (mtROS) generation in hRPE cells treated with TGFβ2 was analyzed. The effect of TGFβ2 and αBC-P on oxidative phosphorylation (OXPHOS) and glycolysis in hRPE was studied. RPE cell migration was also assessed. A PVR-like phenotype was induced by intravitreal dispase injection in C57BL/6J mice. PVR progression and potential therapeutic efficiency of αBC-Elastin-like polypeptides (ELP) was studied using fundus photography, OCT imaging, ERG, and histologic analysis of the retina. αSMA, E-cadherin, Vimentin, Fibronectin and, RPE65, and CTGF were analyzed on Day 28. Additionally, the amount of VEGF-A in retinal cell lysates was measured. The EMT-associated αSMA, Vimentin, SNAIL and SLUG showed a significant upregulation with TGFβ2, and their expression was significantly suppressed by cotreatment with αBC-P. The MET-associated markers, E-cadherin and Sirt1, were significantly downregulated by TGFβ2 and were restored by αBC-P. Incubation of hRPE with TGFβ2 for 24 h showed a marked increase in mitochondrial ROS which was noticeably inhibited by αBC-ELP. We also showed that after TGFβ2 treatment, SMAD4 translocated to mitochondria which was blocked by αBC-ELP. Mitochondrial oxygen consumption rate increased with TGFβ2 treatment for 48 h, and αBC-P co-treatment caused a further increase in OCR. Glycolytic functions of RPE were significantly suppressed with αBC-P (75 μg/mL). In addition, αBC-P significantly inhibited the migration from TGFβ2 treatment in hRPE cells. The formation of proliferative membranes was suppressed in the αBC-ELP-treated group, as evidenced by fundus, OCT, and H&E staining in dispase-induced PVR in mice. Furthermore, ERG showed an improvement in c-wave amplitude. In addition, immunostaining showed significant suppression of αSMA and RPE65 expression. It was also observed that αBC-ELP significantly reduced the expression level of vimentin, fibronectin, and CTGF. Our findings suggest that the antioxidant αBC-P may have therapeutic potential in preventing PVR by reversing the phenotype of EMT/MET and improving the mitochondrial function in RPE cells.

Intravitreal Tissue Plasminogen Activator Injection for the Treatment of Proliferative Vitreoretinopathy in a Rabbit Model

Introduction: The purpose of this study was to evaluate the effect of intravitreal injection of tissue plasminogen activator (tPA) on proliferative vitreoretinopathy (PVR). Methods: PVR was induced in a rabbit model by intraocular injection of dispase (0.05 U/0.1 mL). Progression of PVR was followed by indirect ophthalmic examination. Following 6 weeks, 5 animals received intravitreal injection of 25 µg/0.1 mL tPA and four were injected with balanced salt solution (BSS). Animals were euthanized at 48 h following tPA/BSS injection, and eyes were enucleated for histological evaluation and staining with α-smooth muscle actin (αSMA) and Sirius Red. Results: Following tPA injection, one eye had a reduction in PVR from grade 2 to 1 and three eyes remained stable. Following BSS, PVR grade was unchanged in three eyes. In one eye in each group, the severity of PVR could not be assessed due to limited view. Staining with αSMA showed reduced presence of fibroblasts in eyes injected with tPA compared with those injected with BSS. Collagen type I and III, demonstrated by Sirius Red staining, was reduced in the tPA group in comparison with controls. Conclusion: Our results suggest that intravitreally injected tPA may show an inhibitory effect on PVR progression. Further exploration in clinical trials is desired.

MiRNA-124 regulates palmitic acid-induced epithelial-mesenchymal transition and cell migration in human retinal pigment epithelial cells by targeting LIN7C.

The present study revealed that palmitic acid (PA) treatment induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, which are involved in the progression of proliferative vitreoretinopathy (PVR). ARPE-19 cells were treated with PA followed by miRNA screening and EMT marker detection using qRT-PCR. Then, miR-124 mimic or inhibitor was transfected into ARPE-19 cells to explore the role of miR-124 on the EMT of ARPE-19 cells using transwell assay. The underlying mechanism of miRNA were predicted by bioinformatics method and confirmed by luciferase activity reporter assay. Furthermore, gain-of-function strategy was also used to explore the role of LIN7C in the EMT of ARPE-19 cells. The expression of miRNA or mRNA expression was determined by qRT-PCR and the protein expression was determined using western blot assay. The result presented that PA reduced the expression of E-cadherin/ZO-1 whilst increasing the expression of fibronectin/α-SMA. In addition, PA treatment enhanced the expression of microRNA (miR)-124 in ARPE-19 cells. Overexpression of miR-124 enhanced PA-induced upregulation of E-cadherin and ZO-1 expression and downregulation of fibronectin and α-SMA. Moreover, miR-124 mimic also enhanced the migration of ARPE-19 cells induced by PA treatment. Inversely, miR-124 inhibitor presented opposite effect on PA-induced EMT and cell migration in ARPE-19 cells. Luciferase activity reporter assay confirmed that Lin-7 homolog C (LIN7C) was a direct target of miR-124 in ARPE-19 cells. Overexpression of LIN7C was found to suppress the migration ability and expression of fibronectin and α-SMA, while increasing expression of E-cadherin and ZO-1; miR-124 mimic abrogated the inhibitive effect of LIN7C on the EMT of ARPE-19 cells and PA further enhanced this abolishment. Collectively, these findings suggest that miR-124/LIN7C can modulate EMT and cell migration in RPE cells, which may have therapeutic implications in the management of PVR diseases.

Predictors of Retinal Nerve Fiber Layer Parameters Following Scleral Buckling Surgery in Primary Rhegmatogenous Retinal Detachment.

Objective: This study aimed to examine predictors of retinal nerve fiber layer (RNFL) parameters following scleral buckling (SB) surgery for primary rhegmatogenous retinal detachment (RRD) and to determine the influence of the magnitude of change in qualitative and quantitative parameters on RNFL.Methods: In an observational prospective study, 40 subjects who underwent successful retinal reattachment with SB surgery done within one month of RRD were evaluated for the parameters of best-corrected visual acuity (BCVA), refractive error, intraocular pressure (IOP), axial length (AL), anterior chamber depth (ACD), angle opening distance (AOD 500 and AOD 750), trabecular iris surface area (TISA 500 and TISA 750), visual fields, and ganglion cell count (GCC) and RNFL before and three months after SB. We additionally noted qualitative factors like extent, location, and type of buckle; phakic status; and grade of proliferative vitreoretinopathy in the affected eye. The change in value of quantitative parameters was found. The influence of baseline values and magnitude of change of quantitative and qualitative parameters on average RNFL thickness and magnitude of change of RNFL thickness after SB was found.Results: Post-SB, average RNFL thickness reduced from 108.58±20.38 microns to 103.73±17.98 microns (p =0.042). The baseline temporal upper (TU), temporal lower (TL), and nasal lower (NL) RNFL thickness (p=0.01, p=0.02, p=0.01, respectively) and total deviation (TD) values of visual fields (p=0.01) correlated positively while baseline GCC gross loss of volume (p=0.01) correlated negatively with post-operative RNFL thickness. The TU, TL, and NL RNFL thickness (p=0.04, p=0.01, p=0.01, respectively) and average GCC (p=0.04) correlated negatively with the magnitude of change in RNFL. The magnitude of change in baseline parameters after surgery was correlated with the magnitude of change in average RNFL thickness. It was noticed that change in AL (p<0.01), TISA 500 (p=0.02), TISA 750 (p<0.01), GCC focal loss of volume (p=0.02), and temporal RNFL thickness (p<0.01) correlated positively while the change in refractive error correlated negatively (p=0.04). Except for the grade of proliferative vitreoretinopathy (PVR) (p=0.04), none of the qualitative parameters, including extent, type, and location of the buckle; and phakic status, had a significant association with post-operative average RNFL thickness or magnitude of its change.Conclusions: The predictors of average RNFL thickness following SB include AL; myopic shift; TISA; visual fields TD; average, TU, TL, and NL RNFL thickness; average GCC, gross and focal loss of volume; and grade of PVR. So an early surgery to prevent preoperative ganglion cell and RNFL loss and progression of PVR is recommended.

Exosomal miR-4488 and miR-1273g-5p inhibit the epithelial-mesenchymal transition of transforming growth factor β2-mediated retinal pigment epithelial cells by targeting ATP-binding cassette A4

ABSTRACT Exosomal microRNAs (miRNAs) have been shown to be involved in the regulation of many disease progression, including proliferative vitreoretinopathy (PVR). However, the roles of exosomal miR-4488 and miR-1273 g-5p in PVR progression have not been demonstrated. Transforming growth factor β2 (TGF-β2)-induced ARPE-19 cells were used to stimulate the epithelial-mesenchymal transition (EMT) of cells. Exosomes derived from TGF-β2-induced ARPE-19 cells were identified by transmission electron microscopy and nanoparticle tracking analysis. The expression levels of miR-4488, miR-1273 g-5p and ATP-binding cassette A4 (ABCA4) were measured by quantitative real-time PCR. The promotion levels of exosomes markers, EMT markers, apoptosis markers and ABCA4 were determined by western blot analysis. The migration, invasion and apoptosis of cells were determined by transwell assay, wound healing assay and flow cytometry. Our data showed that miR-4488 and miR-1273 g-5p were lowly expressed in TGF-β2-induced ARPE-19 cells. Overexpressed exosomal miR-4488 and miR-1273 g-5p could inhibit the EMT, migration, invasion, and promote apoptosis in TGF-β2-induced ARPE-19 cells. In addition, ABCA4 was a target of miR-4488 and miR-1273 g-5p. Overexpressed ABCA4 also could reverse the negatively regulation of exosomal miR-4488 and miR-1273 g-5p on the EMT, migration, and invasion of TGF-β2-induced ARPE-19 cells. In conclusion, our data showed that exosomal miR-4488 and miR-1273 g-5p could inhibit TGF-β2-stimulated EMT in ARPE-19 cells through targeting ABCA4.

Upregulation of ASPP2 expression alleviates the development of proliferative vitreoretinopathy in a rat model

To investigate whether upregulation of apoptosis-stimulating p53 protein 2 (ASPP2) expression could alleviate the development of proliferative vitreoretinopathy (PVR) in a rat model. ASPP2-lentivirus or scrambled-lentivirus were transfected into ARPE-19 cells, followed with measurements of cell cytotoxicity by cell counting kit-8 assay. ASPP2 upregulation was confirmed by Western blotting and immunocytochemistry. Then ARPE-19 cells pretreated with ASPP2-lentivirus were intravitreally injected to Brown Norway rats to induce PVR models. PVR development and retinal function were evaluated by retinal photography and electroretinography, respectively. Finally, epithelial-mesenchymal transition as well as autophagy were investigated in rats' retinas via Western blotting. Protein expression of ASPP2 was significantly upregulated by ASPP2-lentivirus transfection in ARPE-19 cells. The development and progression of PVR were impeded significantly in rats with intravitreal injection of ARPE-19 cells pretreated with ASPP2-lentivirus. Accordingly, retinal functions were less affected and PVR grades were much lower in rats with ASPP2-lentivirus compared to scrambled-lentivirus treatment. Moreover, epithelial-mesenchymal transition and autophagy markers were decreased in the retinas of rats treated with ASPP2-lentivirus. ASPP2-lentivirus transfected to ARPE-19 cells mitigates the progression of PVR in rat models, which might be partly through reduced autophagy and attenuated epithelial-mesenchymal transition. ASPP2 might stand as a new approach for PVR treatment in the future.

Yes-associated protein is essential for proliferative vitreoretinopathy development via the epithelial-mesenchymal transition in retinal pigment epithelial fibrosis.

This study was aim to investigate whether the progression of proliferative vitreoretinopathy (PVR) depended on the activation of Yes‐associated protein (YAP) and the subsequent epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cell. The effect of YAP activation on retinal fibrosis in a PVR mouse model and in human ARPE‐19 cells in vitro was studied. After treated with transforming growth factor‐β2(TGF‐β2), the expressions of fibrogenic molecules, YAP activation and the TGF‐β2‐Smad signalling pathway in ARPE‐19 cells were detected by Western blot and immunocytochemical analyses. The effect of YAP on change in fibrosis and EMT was tested by knockdown experiment using verteporfin (YAP inhibitor). YAP was upregulated in the PVR mouse model and during TGF‐β2–induced RPE cell EMT. In an in vivo study, verteporfin attenuated PVR progression in a mouse model. Additionally, YAP knockdown retained phenotype of RPE cells and ameliorated TGF‐β2–induced migration, gel contraction and EMT in vitro. YAP knockdown inhibited the TGF‐β2–induced upregulation of connective tissue growth factor (CTGF), smooth muscle actin (SMA‐α) and fibronectin. YAP was essential for the TGF‐β2–induced nuclear translocation and phosphorylation of Smad2/3. Our work provides direct evidence that YAP is an essential regulator of EMT and profibrotic responses in PVR and indicates that YAP inhibition could be a potential target in PVR therapeutic intervention.

Vitreous M2 Macrophage-Derived Microparticles Promote RPE Cell Proliferation and Migration in Traumatic Proliferative Vitreoretinopathy.

PurposeTo characterize vitreous microparticles (MPs) in patients with traumatic proliferative vitreoretinopathy (PVR) and investigate their role in PVR pathogenesis.MethodsVitreous MPs were characterized in patients with traumatic PVR, patients with rhegmatogenous retinal detachment (RRD) complicated with PVR, and control subjects by flow cytometry. The presence of M2 macrophages in epiretinal membranes was measured by immunostaining. Vitreous cytokines were quantified by ELISA assay. For in vitro studies, MPs isolated from THP-1 cell differentiated M1 and M2 macrophages, termed M1-MPs and M2-MPs, were used. The effects and mechanisms of M1-MPs and M2-MPs on RPE cell proliferation, migration, and epithelial to mesenchymal transition were analyzed.ResultsVitreous MPs derived from photoreceptors, microglia, and macrophages were significantly increased in patients with traumatic PVR in comparison with control and patients with RRD (PVR), whereas no significance was identified between the two control groups. M2 macrophages were present in epiretinal membranes, and their signature cytokines were markedly elevated in the vitreous of patients with traumatic PVR. Moreover, MPs from M2 macrophages were increased in the vitreous of patients with traumatic PVR. In vitro analyses showed that M2-MPs promoted the proliferation and migration of RPE cells via activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. However, M2-MPs did not induce the expression of fibrotic proteins, including fibronectin, α-smooth muscle actin, and N-cadherin in RPE cells.ConclusionsThis study demonstrated increased MP shedding in the vitreous of patients with traumatic PVR; specifically, MPs derived from M2 polarized macrophages may contribute to PVR progression by stimulating RPE cell proliferation and migration.

Repression of Smad4 by MicroRNA-1285 moderates TGF-β-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)-β. 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-β2-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-β2-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.