Optical coherence tomography biomarkers as outcome predictors to guide dexamethasone implant use in patients with iERM: a randomized controlled trial.

BACKGROUND: We aimed to investigate the anatomical features of optical coherence tomography (OCT) and vitreous cytokine levels as predictors of outcomes of combined phacovitrectomy with intravitreal dexamethasone (DEX) implants for idiopathic epiretinal membrane (iERM) treatment. METHODS: A prospective, single-masked, randomized, controlled clinical trial included 48 eyes. They were randomly assigned in a 1:1 ratio to undergo the DEX group (combined phacovitrectomy with ERM peeling and Ozurdex implantation) and control group (phacovitrectomy only). Best-corrected visual acuity (BCVA) and central macular thickness (CMT) were assessed at 1 d, 1 week, 1 month, and 3 months. The structural features of OCT before surgery were analysed for stratified analysis. Baseline soluble CD14 (sCD14) and sCD163 levels in the vitreous fluid were measured using ELISA. RESULTS: BCVA and CMT were not significantly different in the DEX and control groups. Eyes with hyperreflective foci (HRF) at baseline achieved better BCVA (Ptime*group=0.746; Pgroup=0.043, Wald χ²=7.869) and lower CMT (Ptime*group = 0.079; Pgroup = 0.001, Wald χ²=6.774) responses to DEX during follow-up. In all patients, the mean vitreous level of sCD163 in eyes with HRF was significantly higher than that in eyes without HRF (P = 0.036, Z=-2.093) at baseline. In the DEX group, higher sCD163 predicted greater reduction in CMT from baseline to 1 month (r = 0.470, P = 0.049). CONCLUSIONS: We found that intraoperative DEX implantation did not have beneficial effects on BCVA and CMT over a 3-month period in all patients with iERM, implying that the use of DEX for all iERM is not recommended. In contrast, for those with HRF on OCT responded better to DEX implants at the 3-month follow-up and thier vitreous fluid expressed higher levels of sCD163 at baseline. These data support the hypothesis that DEX implants may be particularly effective in treating cases where ERM is secondary to inflammation. TRIAL REGISTRATION: The trail has been registered at Chinese Clinical Trail Registry( https://www.chictr.org.cn ) on 2021/03/12 (ChiCTR2100044228). And all patients in the article were enrolled after registration.

Comprehensive exploration of hub genes involved in oxidative stress in rhegmatogenous retinal detachment based on bioinformatics analysis.

Rhegmatogenous retinal detachment (RRD) is a type of ophthalmologic emergency, if left untreated, the blindness rate approaches 100 %. The RRD patient postoperative recovery of visual function is unsatisfactory, most notably due to photoreceptor death. We conducted to identify the key genes for oxidative stress (OS) in RRD through bioinformatics analysis and clinical validation, thus providing new ideas for the recovery of visual function in RRD patients after surgery. A gene database for RRD was obtained from the Gene Expression Omnibus (GEO) database (GSE28133). Then we screened differentially expressed OS genes (DEOSGs) from the database and assessed the critical pathways in RRD with Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Protein-protein interaction (PPI) networks and hub genes among the common DEOSGs were identified. In addition, we collected general information and vitreous fluid from 42 patients with RRD and 22 controls [11 each of epiretinal membrane (EM) and macular hole (MH)], examined the expression levels of proteins encoded by hub genes in vitreous fluid by enzyme-linked immunosorbent assay (ELISA) to further assess the relationship between the ELISA data and the clinical characteristics of patients with RRD. Ten hub genes (CCL2, ICAM1, STAT3, CD4, ITGAM, PTPRC, CCL5, IL18, TLR2, VCAM1) were finally screened out from the dataset. The ELISA results showed that, compared with the control group, patients with RRD: TLR2 and ICAM-1 were significantly elevated, and CCL2 had a tendency to be elevated, but no statistically significant; RRD patients and MH patients compared with EM patients: STAT3 and VCAM-1 were significantly elevated. We found affected eyes of RRD patients compared with healthy eyes: temporal and nasal retinal nerve fiber layer (RNFL) were significantly thickened. By correlation analysis, we found that: STAT3 was negatively correlated with ocular perfusion pressure (OPP); temporal RNFL was not only significantly positively correlated with CCL2, but also negatively correlated with Scotopic b-wave amplitude. These findings help us to further explore the mechanism of RRD development and provide new ideas for finding postoperative visual function recovery.

Pro-Fibrotic Role of Interleukin-4 in Influencing Idiopathic Epiretinal Membrane in Cataract Patients: Analysis From Clinical-Experimental Approaches.

Purpose: To evaluate the role of interleukin-4 in influencing idiopathic epiretinal membrane (iERM) formation and early progression post cataract surgery (PCS) from clinical and experimental perspectives. Methods: We quantified levels of IL-4 in aqueous humor (AH) samples from 22 iERM patients and 31 control subjects collected before and 20 hours after cataract surgeries using ELISA. After a 3-month follow-up, the association between IL-4 levels and iERM progression measurements was identified. In addition, in vitro studies were conducted to investigate the effects of IL-4 on primary rat retinal Müller glia proliferation, migration, and glial-mesenchymal transition (GMT). Results: Concentrations of IL-4 were significantly higher in preoperative AH samples from iERM patients versus controls (P = 0.006). Postoperatively, although IL-4 levels were elevated in both groups compared to their respective preoperative levels, they were even more obviously so in the iERM group (P < 0.001). Multivariate linear regression analyses revealed that, postoperatively, IL-4 level elevation was positively associated with macular volume and thickness increase (both P < 0.05) in iERM patients. However, no correlations were observed between IL-4 level (changes) and clinical characters in the controls. In vitro studies demonstrated that IL-4 promoted Müller glia proliferation and migration and increased the expression of GMT-related markers in a manner independent of transforming growth factor-ß1 (TGF-ß1). Conclusions: IL-4 plays a crucial pro-fibrotic role in iERM formation and early progression 3 months PCS possibly by stimulating Müller glia proliferation, migration, and GMT in a TGF-ß1-independent manner. Translational Relevance: The current study suggests the potential of IL-4 as a novel therapeutic target for iERM.

Co-Expression of Podoplanin and CD44 in Proliferative Vitreoretinopathy Epiretinal Membranes.

Epiretinal membranes (ERMs) are sheets of tissue that pathologically develop in the vitreoretinal interface leading to progressive vision loss. They are formed by different cell types and by an exuberant deposition of extracellular matrix proteins. Recently, we reviewed ERMs' extracellular matrix components to better understand molecular dysfunctions that trigger and fuel the onset and development of this disease. The bioinformatics approach we applied delineated a comprehensive overview on this fibrocellular tissue and on critical proteins that could really impact ERM physiopathology. Our interactomic analysis proposed the hyaluronic-acid-receptor cluster of differentiation 44 (CD44) as a central regulator of ERM aberrant dynamics and progression. Interestingly, the interaction between CD44 and podoplanin (PDPN) was shown to promote directional migration in epithelial cells. PDPN is a glycoprotein overexpressed in various cancers and a growing body of evidence indicates its relevant function in several fibrotic and inflammatory pathologies. The binding of PDPN to partner proteins and/or its ligand results in the modulation of signaling pathways regulating proliferation, contractility, migration, epithelial-mesenchymal transition, and extracellular matrix remodeling, all processes that are vital in ERM formation. In this context, the understanding of the PDPN role can help to modulate signaling during fibrosis, hence opening a new line of therapy.

Characterization of Different Types of Epiretinal Proliferations by Synchrotron Radiation-Based Fourier Transform Infrared Micro-Spectroscopy.

Pathological tissue on the surface of the retina that can be of different etiology and pathogenesis can cause changes in the retina that have a direct consequence on vision. Tissues of different etiology and pathogenesis have different morphological structures and also different macromolecule compositions usually characteristic of specific diseases. In this study, we evaluated and compared biochemical differences among samples of three different types of epiretinal proliferations: idiopathic epiretinal membrane (ERMi), membranes in proliferative vitreoretinopathy (PVRm), and proliferative diabetic retinopathy (PDRm). The membranes were analyzed by using synchrotron radiation-based Fourier transform infrared micro-spectroscopy (SR-FTIR). We used the SR-FTIR micro-spectroscopy setup, where measurements were set to achieve a high resolution that was capable of showing clear biochemical spectra in biological tissue. We were able to identify differences between PVRm, PDRm, and ERMi in protein and lipid structure; collagen content and collagen maturity; differences in proteoglycan presence; protein phosphorylation; and DNA expression. Collagen showed the strongest expression in PDRm, lower expression in ERMi, and very low expression in PVRm. We also demonstrated the presence of silicone oil (SO) or polydimethylsiloxane in the structure of PVRm after SO endotamponade. This finding suggests that SO, in addition to its many benefits as an important tool in vitreoretinal surgery, could be involved in PVRm formation.

Short-Term In Vitro ROS Detection and Oxidative Stress Regulators in Epiretinal Membranes and Vitreous from Idiopathic Vitreoretinal Diseases.

Background: A plethora of inflammatory, angiogenic, and tissue remodeling factors has been reported in idiopathic epiretinal membranes (ERMs). Herein we focused on the expression of a few mediators (oxidative, inflammatory, and angiogenic/vascular factors) by means of short-term vitreal cell cultures and biomolecular analysis. Methods: Thirty-nine (39) ERMs and vitreal samples were collected at the time of vitreoretinal surgery and biomolecular analyses were performed in clear vitreous, vitreal cell pellets, and ERMs. ROS products and iNOS were investigated in adherent vitreal cells and/or ERMs, and iNOS, VEGF, Ang-2, IFNγ, IL18, and IL22 were quantified in vitreous (ELISA/Ella, IF/WB); transcripts specific for iNOS, p65NFkB, KEAP1, NRF2, and NOX1/NOX4 were detected in ERMs (PCR). Biomolecular changes were analyzed and correlated with disease severity. Results: The higher ROS production was observed in vitreal cells at stage 4, and iNOS was found in ERMs and increased in the vitreous as early as at stage 3. Both iNOS and NOX4 were upregulated at all stages, while p65NFkB was increased at stage 3. iNOS and NOX1 were positively and inversely related with p65NFkB. While NOX4 transcripts were always upregulated, NRF2 was upregulated at stage 3 and inverted at stage 4. No significant changes occurred in the release of angiogenic (VEGF, Ang-2) and proinflammatory (IL18, IL22 and IFNγ) mediators between all stages investigated. Conclusions: ROS production was strictly associated with iNOS and NOX4 overexpression and increased depending on ERM stadiation. The higher iNOS expression occurred as early as stage 3, with respect to p65NFkB and NRF2. These last mediators might have potential prognostic values in ERMs as representative of an underneath retinal damage.

Proteomics and phosphoproteomics analysis of vitreous in idiopathic epiretinal membrane patients.

PURPOSE: The purpose of the present study was to characterize the idiopathic epiretinal membrane (iERM) through proteomics and phosphoproteomics analysis to facilitate the diagnosis and treatment of iERM. EXPERIMENTAL DESIGN: The vitreous of 25 patients with an iERM and 15 patients with an idiopathic macular hole were analyzed by proteomic and phosphoproteomic analysis based on tandem mass tag. PRM was used to verify the differential proteins. RESULTS: Proteomic analysis identified a total of 878 proteins, including 50 differential proteins. Tenascin-C, galectin-3-binding protein, glucose-6-phosphate isomerase, neuroserpin, collagen alpha-1(XI) chain, and collagen alpha-1(II) chain were verified to be upregulated in iERM by PRM. Phosphoproteomic analysis identified a total of 401 phosphorylation sites on 213 proteins, including 27 differential phosphorylation sites on 24 proteins. Mitogen-activated protein kinase-activated protein kinase (MAPKAPK)3 and MAPKAPK5 were predicted as the major kinases in the vitreous of iERM. Twenty-six of the differential proteins and phosphorylated proteins may be closely related to fibrosis in iERM. CONCLUSION AND CLINICAL RELEVANCE: Our results indicated the potential biomarkers or therapeutic targets for iERM, provided key kinases that may be involved in iERM. Fibrosis plays an essential role in iERM, and further exploration of related differential proteins has important clinical significance.

Single-Cell Protein and Transcriptional Characterization of Epiretinal Membranes From Patients With Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) remains an unresolved clinical challenge and can lead to frequent revision surgery and blindness vision loss. The aim of this study was to characterize the microenvironment of epiretinal PVR tissue, in order to shed more light on the complex pathophysiology and to unravel new treatment options. Methods: A total of 44 tissue samples were analyzed in this study, including 19 epiretinal PVRs, 13 epiretinal membranes (ERMs) from patients with macular pucker, as well as 12 internal limiting membranes (ILMs). The cellular and molecular microenvironment was assessed by cell type deconvolution analysis (xCell), RNA sequencing data and single-cell imaging mass cytometry. Candidate drugs for PVR treatment were identified in silico via a transcriptome-based drug-repurposing approach. Results: RNA sequencing of tissue samples demonstrated distinct transcriptional profiles of PVR, ERM, and ILM samples. Differential gene expression analysis revealed 3194 upregulated genes in PVR compared with ILM, including FN1 and SPARC, which contribute to biological processes, such as extracellular matrix (ECM) organization. The xCell and IMC analyses showed that PVR membranes were composed of macrophages, retinal pigment epithelium, and α-SMA-positive myofibroblasts, the latter predominantly characterized by the co-expression of immune cell signature markers. Finally, 13 drugs were identified as potential therapeutics for PVR, including aminocaproic acid and various topoisomerase-2A inhibitors. Conclusions: Epiretinal PVR membranes exhibit a unique and complex transcriptional and cellular profile dominated by immune cells and myofibroblasts, as well as a variety of ECM components. Our findings provide new insights into the pathophysiology of PVR and suggest potential targeted therapeutic options.

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

Purpose: To characterize vitreous microparticles (MPs) in patients with traumatic proliferative vitreoretinopathy (PVR) and investigate their role in PVR pathogenesis. Methods: Vitreous 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. Results: Vitreous 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. Conclusions: This 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.

Vitreous composition modification after transpalpebral electrical stimulation of the eye: Biochemical analysis.

Electrical stimulation (ES) of the eye represents a therapeutic approach in various clinical applications ranging from retinal dystrophies, age-related macular degeneration, retinal artery occlusion and nonarteritic ischemic optic neuropathy. In clinical practice, ES of the eye is mainly performed with a transcorneal or transpalpebral approach. These procedures are non-invasive and well-tolerated by the patients, reporting only minimal and transient adverse events, while serious adverse effects were not observed. Despite the growing literature on animal models, only clinical parameters have been investigated in humans and few data are available about biochemical changes induced by ES of the eye. The purpose of this study is to investigate the possible mechanism that regulates the beneficial effects of ES on retinal cells function and survival in humans. 28 patients undergoing pars plana vitrectomy (PPV) for idiopathic epiretinal membrane (iERM) were randomly divided in two groups: 13 patients were treated with transpalpebral ES before surgery and 15 underwent surgery with no prior treatment. Vitreous samples were collected for biochemical analysis during PPV. ES treatment leads to a reduction in the vitreous expression of both proinflammatory cytokines, namely IL-6 and IL-8, and proinflammatory lipid mediators, such as lysophosphatidylcholine. Indeed, we observed a 70% decrease of lysophosphatidylcholine 18:0, which has been proven to exert the greatest proinflammatory activities among the lysophosphatidylcholine class. The content of triglycerides is also affected and significantly decreased following ES application. The vitreous composition of patients undergoing PPV for iERM displays significant changes following ES treatment. Proinflammatory cytokines and bioactive lipid mediators expression decreases, suggesting an overall anti-inflammatory potential of ES. The investigation of the mechanism by which this treatment alters the retinal neurons leading to good outcomes is essential for supporting ES therapeutic application in various types of retinal diseases.

Role of Vital Dyes in Chromovitrectomy.

ABSTRACT: Chromovitrectomy, the intraocular application of dyes to assist visualization of preretinal tissues during vitreoretinal surgery, was introduced to avoid ocular complications related to internal limiting membrane peeling, inadequate removal of the vitreous, and incomplete removal of epiretinal membranes. Since 2000, chromovitrectomy has become a popular approach among vitreoretinal specialists. The first vital dye used in chromovitrectomy, indocyanine green, facilitated identification of the fine and transparent internal limiting membrane. Following indocyanine green, trypan blue was introduced to identify epiretinal membranes, and triamcinolone acetonide stained the vitreous well. Recently, additional natural dyes such as lutein and anthocyanin from the açaí fruit have been proposed for intraocular application during vitrectomy. The main goal of this review was to study the role of vital stains in chromovitrectomy and report the latest findings in the literature.

Osteopontin in vitreous and idiopathic epiretinal membranes.

PURPOSE: To investigate osteopontin (OPN) expression in vitreous and in related idiopathic epiretinal membranes (ERMs), with respect to VEGF-A, IL8, MIP1α, IL6, and IL33, and correlate OPN expression with disease staging. METHODS: Fifteen (15) vitreous and allied ERMs were collected at the time of therapeutic vitreoretinal surgery. Additional 5 vitreous and 10 ERMs (historical collection) were used. Biochemical and molecular analysis of OPN was performed in clear vitreous, vitreal pelleted cells, and ERMs. Double-immunofluorescence analysis (OPN - GFAP and OPN - αSMA) was performed on paraffin and whole-mounted ERMs. Vitreal OPN levels were correlated to those of VEGF-A, IL8, MIP1α, IL6, and IL33. RESULTS: High OPN levels were observed in vitreal samples, and OPN transcripts were amplified in vitreal cells and related ERMs. OPN immunoreactivity was found in ERMs, mainly in GFAP-bearing (Muller cells) and to a less extend in αSMA-expressing (myofibroblasts) cells. OPN levels were highest at early stages of ERM formation and positively correlated to VEGF-A and MIP1α. CONCLUSIONS: High OPN levels in vitreous, OPN transcripts in vitreal cells/ERMs, OPN immunoreactivity in activated Müller cells and contractile myofibroblasts, as well as the correlation with VEGF-A and MIP1α fulfill the potential involvement of OPN in both inflammation and tissue remodeling that takes part in vitreoretinal interface disorders. The highest OPN levels at early stages of ERM formation would prospect OPN as a potential biomarker for disease severity.

Elevated M2 Macrophage Markers in Epiretinal Membranes With Ectopic Inner Foveal Layers.

Purpose: To analyze the differences in the vitreous cytokine profiles in epiretinal membrane eyes with and without an ectopic inner foveal layer (EIFL). Methods: Sixty eyes with epiretinal membrane (32 eyes without EIFL and 28 eyes with EIFL) were included. The vitreous samples were collected during surgery for epiretinal membrane. The cytokine levels of the vitreous were measured using a multiplex bead analysis. Results: The mean logMAR visual acuity was worse (0.42 vs. 0.37; P = 0.331) and the central foveal thickness was higher in the EIFL group (496.9 µm vs. 434.2 µm; P = 0.007) than they were in the group without EIFL. The mean EIFL thickness was 164.1 ± 67.7 µm in the EIFL group. On multiplex analysis of the vitreous cytokines, the levels of CD163 (21529 pg/dL vs. 10877 pg/dL; P = 0.002) and macrophage colony-stimulating factor (206 pg/dL vs. 159 pg/dL, P = 0.004) were significantly higher in the EIFL group than they were in the group without EIFL. Conclusions: Eyes with EIFL had increased vitreous levels of M2 macrophage markers. The activation of glial cell proliferation by M2 macrophages may contribute to EIFL formation.

Protein Kinase A Inhibitor H89 Attenuates Experimental Proliferative Vitreoretinopathy.

Purpose: This study aimed to explore the role of the protein kinase A (PKA) pathway in proliferative vitreoretinopathy (PVR) and the effect of the PKA inhibitor H89 on experimental PVR. Methods: Epiretinal membranes (ERMs) were acquired from PVR patients and analyzed by frozen-section immunofluorescence. An in vivo model was developed by intravitreal injecting rat eyes with ARPE-19 cells and platelet-rich plasma, and changes in eye structures and vision function were observed. An in vitro epithelial-mesenchymal transition (EMT) cell model was established by stimulating ARPE-19 cells with transforming growth factor (TGF)-ß. Alterations in EMT-related genes and cell function were detected. Mechanistically, PKA activation and activity were explored to assess the relationship between TGF-ß1 stimulation and the PKA pathway. The effect of H89 on the TGF-ß-Smad2/3 pathway was detected. RNA sequencing was used to analyze gene expression profile changes after H89 treatment. Results: PKA was activated in human PVR membranes. In vivo, H89 treatment protected against structural changes in the retina and prevented decreases in electroretinogram b-wave amplitudes. In vitro, H89 treatment inhibited EMT-related gene alterations and partially reversed the functions of the cells. TGF-ß-induced PKA activation was blocked by H89 pretreatment. H89 did not affect the phosphorylation or nuclear translocation of regulatory Smad2/3 but increased the expression of inhibitory Smad6. Conclusions: PKA pathway activation is involved in PVR pathogenesis, and the PKA inhibitor H89 can effectively inhibit PVR, both in vivo and in vitro. Furthermore, the protective effect of H89 is related to an increase in inhibitory Smad6.

VITREOUS PROSTAGLANDIN E2 CHANGES AFTER TOPICAL ADMINISTRATION OF DICLOFENAC 0.1%, INDOMETHACIN 0.5%, NEPAFENAC 0.3%, AND BROMFENAC 0.09.

PURPOSE: To evaluate the vitreous concentration of different nonsteroidal anti-inflammatory drugs (NSAIDs) after topical administration and the related prostaglandin E2 (PGE2) levels in patients undergoing pars plana vitrectomy. METHODS: A prospective, randomized, investigator-masked study was performed. One hundred four patients scheduled for a pars plana vitrectomy for an epiretinal membrane or a macular hole were randomized to receive topical diclofenac 0.1%, indomethacin 0.5%, nepafenac 0.3%, bromfenac 0.09%, or placebo 3 days before surgery. At the beginning of surgery, a sample of undiluted vitreous was collected in each patient to assess NSAIDs concentration and PGE2 levels. RESULTS: The median vitreous concentrations were 203.35 (interquartile range 146.54-264.18) pg/mL for diclofenac, 243.45 (interquartile range 156.96-365.37) pg/mL for nepafenac, 438.21 pg/mL (interquartile range, 282.52-645.87) for its active metabolite amfenac, 350.14 (interquartile range, 290.88-481.95) pg/mL for indomethacin, and 274.59 (245.43-358.25) pg/mL for bromfenac. Vitreous PGE2 levels were significantly lower for all the NSAIDs groups compared with the control group (P < 0.001). A statistically significant higher vitreous PGE2 level was found in the diclofenac group compared with the other NSAIDs groups (P < 0.05). CONCLUSION: Topical NSAIDs achieve sufficient vitreous concentration to decrease vitreous PGE2 levels compared with the control group. The different efficacy in reducing PGE2 concentration may affect the management of posterior segment inflammation.

Histopathological Changes and Clinical Outcomes following Intervention for Sub-Internal Limiting Membrane Haemorrhage.

INTRODUCTION: Haemorrhage confined to the sub-internal limiting membrane (ILM) space can be associated with good visual recovery. There is controversy as to the best management of purely sub-ILM haemorrhage, which ranges from observation to immediate surgical intervention. METHODS: We studied a retrospective case series of patients with sub-ILM haemorrhage who underwent vitrectomy with subsequent histological analysis of the removed ILM. RESULTS: Sixteen patients underwent vitrectomy for sub-ILM haemorrhage. Five patients had underlying Terson syndrome, 6 had ruptured macro-aneurysms, and 5 had Valsalva retinopathy. Seven patients demonstrated cellular proliferation on the retinal surface of the ILM with staining for glial fibrillary acidic protein and cytokeratin 7, as well as CD68pg and Prussian blue. All but 1 of these cases were isolated from patients undergoing surgery >4 weeks following initial symptoms, the other presented at >2 weeks. Serial optical coherence tomography (OCT) was available in 8 patients; serial OCT in patients with delayed intervention demonstrated persistent inner retinal layer hyper-reflectance. Fourteen of 15 patients demonstrated symptomatic recovery and showed visual improvement with acuity ranging from -0.1 to 1.8 (mean 0.43) within 3 months of intervention (1 was lost to follow-up). The post-operative vision was 0.11 logMAR (mean; range -0.1 to 0.4) at 3 months in the group with intervention within 2 weeks of symptoms, and 0.9 logMAR (mean; range 0.0 to HM) in the group with delayed surgery. CONCLUSIONS: Early surgical intervention for sub-ILM haemorrhage resulted in good visual outcomes; delayed surgery may lead to proliferative vitreoretinopathy-like changes on the inner retinal surface of the ILM, and untreated cases may demonstrate persistent inner retinal changes potentially limiting visual prognosis despite subsequent surgical intervention.

Blockade of MDM2 with inactive Cas9 prevents epithelial to mesenchymal transition in retinal pigment epithelial cells.

Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-ß2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-ß2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-ß2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-ß2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-ß2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-ß2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-ß2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-ß2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.

In Vitro Epiretinal Membrane Model and Antibody Permeability: Relationship With Anti-VEGF Resistance in Diabetic Macular Edema.

Purpose: Diabetic macular edema (DME) is characterized by an accumulation of fluid in the macula due to diabetic retinopathy. Currently, anti-VEGF drugs are the standard treatment worldwide for DME. This study aimed to assess whether the existence of epiretinal membrane (ERM) affects anti-VEGF efficacy, due to reduced permeability of the antibody through the ERM. Methods: We retrospectively examined clinical data of DME patients who underwent anti-VEGF treatment and evaluated whether clinical differences existed between DME eyes with ERM and those without ERM. We then created an in vitro ERM model using MIO-M1, ARPE-19, and NTI-4 cells on Transwell membranes and evaluated antibody permeability through this in vitro ERM model using fluorescently labeled antibodies. Results: Central retinal thickness (CRT) change between before and 1 month after first anti-VEGF treatment, as well as final CRT and final visual acuity 12 months after first anti-VEGF treatment, significantly differed between DME eyes with ERM and those without ERM. The in vitro ERM model led to production of collagen I in a manner similar to that of human ERM specimens. Fluorescence intensity of the lower chamber of the in vitro ERM model was significantly reduced in a dose-dependent manner. Conclusions: Clinical data analysis indicated that the existence of ERM in DME eyes lowered the efficacy of anti-VEGF treatment. Reduced antibody permeability through the in vitro ERM model suggested ERM presence was associated with resistance to anti-VEGF treatment in DME eyes with ERM.

P38 inhibition reverses TGFß1 and TNFα-induced contraction in a model of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a metaplasia in the vitreous of the eye manifested by the transformation of retinal pigment epithelial (RPE) cells and the development of contracting epiretinal membranes (ERM), which lead to retinal detachment and vision loss. While TGFß1 and TNFα have been associated with PVR, here we show that these cytokines act synergistically to induce an aggressive contraction phenotype on adult human (ah)RPE. Connected RPE detach upon contraction and form motile membranes that recruit more cells. TGFß1 and TNFα (TNT)-induced contracting membranes uniquely express muscle and extracellular rearrangement genes. Whole transcriptome RNA sequencing of patient-dissected PVR membranes showed activation of the p38-MAPK signaling pathway. Inhibition of p38 during TNT treatment blocks ahRPE transformation and membrane contraction. Furthermore, TNT-induced membrane contractility can be reversed by p38 inhibition after induction. Therefore, targeting the p38-MAPK pathway may have therapeutic benefits for patients with PVR even after the onset of contracting ERMs.

Macular vascular circulation and retinal oxygen saturation changes for idiopathic macular epiretinal membrane after vitrectomy.

PURPOSE: To investigate the postoperative changes in retinal-choroidal blood flow area and retinal vascular oxygen saturation among patients with idiopathic macular epiretinal membranes (iERMs). METHODS: This study prospectively included 24 eyes of 24 consecutive patients who underwent vitrectomy for iERMs. The flow area of choriocapillary layer, retinal superficial capillary plexuses (SCPs) and retinal deep capillary plexuses (DCPs) were performed using optical coherence tomography angiography with angiovue prototype software, and retinal vascular oxygen saturation was measured using retinal oximeter with the built-in software. The flow area of choriocapillary layers and mean retinal vessel oxygen saturation before and after surgery were compared. RESULTS: Three months after vitrectomy, the foveal flow area of choriocapillary layer increased clinically significantly from 1.5 ± 0.2 to 1.6 ± 0.2 mm2 (p = 0.02). The retinal vascular changes of SCPs and DCPs were not statistically significant. The mean retinal arterial oxygen saturation was 89.9 ± 11.3% preoperatively and increased to 94.5 ± 9.7% postoperatively (p = 0.04). Foveal retinal thickness revealed a clinically significant decrease from 547.8 ± 88.2 µm to 403.0 ± 47.5 µm after surgery (p < 0.05). Postoperative best corrected visual acuity had no statistically significant correlation with foveal flow area of the choriocapillary layer and retinal vascular oxygen saturation. CONCLUSIONS: There was a decrease of retinal thickness, an improved flow area of choriocapillary layer in macular region, and an increase of retinal arterial vascular oxygen among iERMs patients after vitrectomy.