Genome-wide association study of the common retinal disorder epiretinal membrane: Significant risk loci in each of three American populations.

Epiretinal membrane (ERM) is a common retinal condition characterized by the presence of fibrocellular tissue on the retinal surface, often with visual distortion and loss of visual acuity. We studied European American (EUR), African American (AFR), and Latino (admixed American, AMR) ERM participants in the Million Veteran Program (MVP) for genome-wide association analysis-a total of 38,232 case individuals and 557,988 control individuals. We completed a genome-wide association study (GWAS) in each population separately, and then results were meta-analyzed. Genome-wide significant (GWS) associations were observed in all three populations studied: 31 risk loci in EUR subjects, 3 in AFR, and 2 in AMR, with 48 in trans-ancestry meta-analysis. Many results replicated in the FinnGen sample. Several GWS variants associate to alterations in gene expression in the macula. ERM showed significant genetic correlation to multiple traits. Pathway enrichment analyses implicated collagen and collagen-adjacent mechanisms, among others. This well-powered ERM GWAS identified novel genetic associations that point to biological mechanisms for ERM.

Prevalence, Concordance, and Heritability of Vitreomacular Interface Abnormalities in a Twin Study.

Purpose: The relative importance of genetic factors in common vitreomacular interface (VMI) abnormalities is unknown. The aim of this classical twin study is to determine the prevalence case wise concordance between monozygotic and dizygotic twin pairs, and heritability of common VMI abnormalities, including epiretinal membrane (ERM), posterior vitreous detachment (PVD), vitreomacular adhesion (VMA), vitreomacular traction (VMT), lamellar macular holes (LMHs), and full-thickness macular holes (FTMHs). Methods: This is a single-center, cross-sectional classical twin study of 3406 TwinsUK participants over the age of 40 years who underwent spectral domain macular optical coherence tomography (SD-OCT) scans which were graded for signs of VMI abnormalities. Case wise concordance was calculated and the heritability of each VMI abnormality was estimated using OpenMx structural equation modeling. Results: In this population (mean age = 62.0 years [SD = 10.4 years], range = 40-89 years) the overall prevalence of ERM was 15.6% (95% confidence interval [CI] = 14.4-16.9) and increased with age, posterior vitreous detachment affected 21.3% (20.0-22.7), and VMA was diagnosed in 11.8% (10.8-13.0). Monozygotic twins were more concordant for all traits than dizygotic twins, and age, spherical equivalent refraction (SER), and lens status-adjusted heritability was estimated at 38.9% (95% CI = 33.6-52.8) for ERM, 53.2% (95% CI = 41.8-63.2) for PVD, and 48.1% (95% CI = 33.6-58) for VMA. Conclusions: Common VMI abnormalities are heritable and therefore have an underlying genetic component. Given the sight-threatening potential of VMI abnormalities, further genetic studies, such as genomewide association studies, would be useful to identify genes and pathways implicated in their pathogenesis.

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.

Gene expression analysis identifies two distinct molecular clusters of idiopatic epiretinal membranes.

Idiopathic epiretinal membranes (ERMs) are fibrocellular membranes containing extracellular matrix proteins and epiretinal cells of retinal and extraretinal origin. iERMs lead to decreased visual acuity and their pathogenesis has not been completely defined. Aim of this study was to provide a molecular characterization of iERMs by gene expression analysis. To this purpose, 56 iERMs obtained by pars plana vitrectomy were analyzed for the expression levels of genes encoding biomarkers of the cellular and molecular events occurring in iERMs. RT-qPCR analysis showed significant differences in the levels of cell population, extracellular matrix and cytokine/growth factor biomarkers among the iERMs investigated. Hierarchical clustering of RT-qPCR data identified two distinct iERM clusters, Cluster B samples representing transcriptionally "activated" iERMs when compared to transcriptionally "quiescent" Cluster A specimens. Further, Cluster B could be subdivided in two subgroups, Cluster B1 iERMs, characterized by a marked glial cell activation, and Cluster B2 samples characterized by a more pro-fibrotic phenotype. Preoperative decimal best-corrected visual acuity and post-surgery inner segment/outer grading values were higher in Cluster A patients, that showed a prevalence of fovea-attached type iERMs with near-normal inner retina, than in Cluster B patients, that presented more severe clinical and spectral domain optical coherence tomography (SD-OCT) features. In conclusion, this molecular characterization has identified two major clusters of iERM specimens with distinct transcriptional activities that reflect different clinical and SD-OCT features of iERM patients. This retrospective work paves the way to prospective whole-genome transcriptomic studies to allow a molecular classification of iERMs and for the identification of molecular signature(s) of prognostic and therapeutic significance.

PDGFRß plays an essential role in patient vitreous-stimulated contraction of retinal pigment epithelial cells from epiretinal membranes.

Platelet-derived growth factor (PDGF) is associated with clinical proliferative vitreoretinopathy (PVR), which is characterized by formation of sub- or epi-retinal membranes that consist of cells including retinal pigment epithelial （RPE） cells and extracellular matrix. RPE cells play an important role in PVR pathogenesis. Previous findings indicated that PDGF receptor (PDGFR)α was essential in experimental PVR induced by fibroblasts. In RPE cells derived from epiretinal membranes from patients with PVR (RPEMs)， Akt was activated by PDGF-B but not PDGF-A, which suggested that PDGFRß was the predominant PDGFR isoform expressed in RPEMs. Indeed, CRISPR/Cas9-mediated depletion of PDGFRß in RPEMs attenuated patient vitreous-induced Akt activation and cellular responses intrinsic to PVR including cell proliferation, migration, and contraction. We conclude that PDGFRß appears to be the PVR relevant PDGFR isoform in RPEMs.

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.

Comprehensive circular RNA profiling of proliferative vitreoretinopathy and its clinical significance.

Proliferative vitreoretinopathy (PVR) is one of the major challenges in retinal surgery, which occurs in the patient with complex retinal surgery or penetrating eye injury. Circular RNAs (circRNAs) have emerged as important regulators in many biological processes and disease development. However, the characterization and function of circRNAs in PVR remains elusive. In this study, we identified 91 dysregulated circRNAs in the epiretinal membranes (ERMs) of PVR patients. We further investigated the expression pattern of circ_0043144. circ_0043144 was significantly up-regulated in the vitreous samples and the corresponding serum samples of the patients with PVR. circ_0043144 expression was significantly down-regulated after PVR operation. In vitro studies revealed that circ_0043144 was involved in the regulation of the proliferation, migration and secretion ability of ARPE-19 cells, which is critical for ERM formation. Collectively, this study indicates that circRNAs are potential regulators of the pathogenesis of PVR. circ_0043144 is a promising prognostic and diagnostic indicator for PVR diseases.

Pathologic Roles of Receptor-Associated Prorenin System in Idiopathic Epiretinal Membrane.

Receptor-associated prorenin system (RAPS) refers to the pathogenic mechanism whereby prorenin binding to (pro)renin receptor [(P)RR] dually activates tissue renin-angiotensin system (RAS) and RAS-independent signaling via (P)RR. The aim of this study is to determine the association of RAPS with idiopathic epiretinal membrane (iERM). Reverse transcription-PCR indicated the expression of RAPS components, including (P)RR and Ang II type 1 receptor (AT1R), in iERM tissues and human Müller glial cell line. Double-labeling analyses demonstrated that (P)RR and AT1R were detected in cells positive for glial fibrillary acidic protein, a marker for glial cells, and co-localized with prorenin and angiotensinogen, respectively. Administration of prorenin to Müller glial cells enhanced mRNA expression of fibroblast growth factor 2, while Ang II application stimulated the expression of glial cell line-derived neurotrophic factor, nerve growth factor, and transforming growth factor-ß1. These expression levels induced by prorenin or Ang II were reversed by (P)RR or AT1R blockade, respectively. Immunofluorescence revealed tissue co-localization of (P)RR and AT1R with the products of the upregulated genes in vitro. The present findings suggest the involvement of RAPS in the pathogenesis of iERM.

miRNAs in the vitreous humor of patients affected by idiopathic epiretinal membrane and macular hole.

PURPOSE: The aim of the present study was to assess the expression of miRNAs in the Vitreous Humor (VH) of patients with Macular Hole (MH) and Epiretinal Membrane (ERM) compared to a control group. METHODS: In this prospective, comparative study, 2-ml of VH was extracted from the core of the vitreous chamber in consecutive patients who underwent standard vitrectomy for ERM and MH. RNA was extracted and TaqMan® Low Density Arrays (TLDAs) were used to profile the transcriptome of 754 miRNAs. Results were validated by single TaqMan® assays. Finally, we created a biological network of differentially expressed miRNA targets and their nearest neighbors. RESULTS: Overall 10 eyes with MH, 16 eyes with idiopathic ERM and 6 controls were enrolled in the study. Profiling data identified 5 miRNAs differentially expressed in patients affected by MH and ERM with respect to controls. Four were downregulated (miR-19b, miR-24, miR-155, miR-451) and 1 was downregulated (miR-29a); TaqMan® assays of the VH of patients affected by MH and ERM, with respect to controls, showed that the most differentially expressed were miR-19b (FC -9.13, p:<0.00004), mir-24 (FC -7.52, p:<0.004) and miR-142-3p (FC -5.32, p:<0.011). Our network data showed that deregulation of differentially expressed miRNAs induces an alteration of several pathways associated with genes involved in both MH and ERM. CONCLUSION: The present study suggests that disregulation of miR-19b, miR-24 and miR-142-3p, might be related to the alterations that characterize patients affected by MH and ERM.

Gli1 Expression in Human Epiretinal Membranes.

Purpose: We evaluate the expression of Gli1 in human epiretinal membranes (ERM) and correlate this with clinical data. Methods: We prospectively recruited patients with ERM. A total of 33 human ERM specimens were immunolabeled with anti-Gli1 antibody and the number of total cells/hyperfield (HF), Gli1(+) cells/HF, and the percentage of Gli1(+) cells/total cells were calculated. We evaluated the interrelationship of cellular properties and clinical findings, such as presence of diabetic retinopathy (DR), retinal breaks, intraocular inflammation, central foveal thickness, maximal retinal thickness, retinal contraction, lamellar holes, pseudoholes, the attenuation or absence of an inner segment/outer segment (IS/OS) junction/external limiting membrane (ELM), cystic changes, and paravascular inner retinal defects. Results: Among 33 specimens, 25 specimens (75.8%) showed nuclear Gli1 expression. The mean Gli1(+) cells/total cells was 54.0 ± 36.7% (range, 0%-92.8%). There was significantly higher expression of Gli1(+) cells in ERM specimens from patients with DR (P = 0.014), and lower expression from patients with retinal breaks (P = 0.022). Epiretinal membrane specimens from patients with alteration of IS/OS junction/ELM or cystic changes on OCT showed higher percentage of Gli1(+) cells/total cells. Conclusions: Gli1 expression was detected in most ERM specimens. Patients who had DR or OCT findings indicating chronic retinal insults showed higher Gli1 expression. Gli1 may have a role in the pathogenesis of ERM after chronic retinal insults.

Th17 cell frequency and IL-17A concentrations in peripheral blood mononuclear cells and vitreous fluid from patients with diabetic retinopathy.

Objective To quantify T helper (Th)17 cells and determine interleukin (IL)-17A levels in peripheral blood mononuclear cell (PBMC) culture and vitreous fluid from patients with type 2 diabetes mellitus (T2DM) with diabetic retinopathy (DR). Methods Th17 cell frequency and IL-17A concentrations in PBMCs from 60 patients with T2DM with DR, 30 without DR and 30 sex- and age-matched healthy individuals were measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. IL-17A levels in vitreous fluid from 31 eyes with proliferative DR and diabetic macular oedema (DR group) and 32 eyes with an epiretinal membrane and macular hole (control group) that underwent vitrectomy were also examined by ELISA. Results Compared with the control group, the proportion of Th17 cells and IL-17A concentrations in PBMCs were significantly increased in patients without DR but decreased in those with DR. IL-17A concentrations and Th17 cell frequency in PBMCs tended to decrease with DR severity and were negatively correlated with body mass index, T2DM duration and glycated haemoglobin. Additionally, vitreous fluid IL-17A levels were significantly elevated in patients with DR compared with those of the control group. Conclusions We conclude that disturbances in Th17 cells and IL-17A levels are possibly associated with DR.

Gene Expression Analysis of the Irrigation Solution Samples Collected during Vitrectomy for Idiopathic Epiretinal Membrane.

PURPOSE: The analysis of gene expression in idiopathic epiretinal membranes (iERMs) may help elucidate ERM formation and its pathology. Here, we conducted a case-control study, in order to determine the expression levels of cytokines and other genes in eyes with macular hole (MH) or iERM. METHODS: Twenty eyes, obtained from seven male and 13 female patients, were included in the study. The average age of the study subjects was 69.1 ± 7.67 years, and 15 eyes had iERM, while five eyes had MH. Irrigation solution samples were collected during vitrectomy, centrifuged, and the levels of cytokine and other mRNAs in the sediment were assessed using real-time PCR. The expression level of 11 cytokine genes, four transcription factor genes, two cytoskeletal genes, and genes encoding two extracellular matrix proteins in eyes with MH or iERM were determined and compared. RESULTS: The expression levels of interleukin 6 (IL6), tumor growth factor B2 (TGFB2), vascular endothelial growth factor A (VEGFA), chemokine C-X-C motif ligand 1 (CXCL1), v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), glial fibrillary acidic protein (GFAP), and tenascin C (TNC) were significantly higher in eyes with iERM than in eyes with MH. The expression of these genes was not associated with the preoperative visual acuity of the investigated patients. CONCLUSIONS: The obtained results indicate that real-time PCR analysis of irrigation solution samples collected during vitrectomy can help assess the expression levels of several genes, and that iERM is associated with the expression of pro-inflammatory genes and the genes expressed during angiogenesis and wound healing process (IL6, TGFB2, VEGFA, CXCL1, RELA, GFAP, and TNC).

Epithelial Membrane Protein-2 in Human Proliferative Vitreoretinopathy and Epiretinal Membranes.

PURPOSE: To determine the level of epithelial membrane protein-2 (EMP2) expression in preretinal membranes from surgical patients with proliferative vitreoretinopathy (PVR) or epiretinal membranes (ERMs). EMP2, an integrin regulator, is expressed in the retinal pigment epithelium and understanding EMP2 expression in human retinal disease may help determine whether EMP2 is a potential therapeutic target. METHODS: Preretinal membranes were collected during surgical vitrectomies after obtaining consents. The membranes were fixed, processed, sectioned, and protein expression of EMP2 was evaluated by immunohistochemistry. The staining intensity (SI) and percentage of positive cells (PP) in membranes were compared by masked observers. Membranes were categorized by their cause and type including inflammatory and traumatic. RESULTS: All of the membranes stained positive for EMP2. Proliferative vitreoretinopathy-induced membranes (all causes) showed greater expression of EMP2 than ERMs with higher SI (1.81 vs. 1.38; P = 0.07) and PP (2.08 vs. 1.54; P = 0.09). However all the PVR subgroups had similar levels of EMP2 expression without statistically significant differences by Kruskal-Wallis test. Inflammatory PVR had higher expression of EMP2 than ERMs (SI of 2.58 vs. 1.38); however, this was not statistically significant. No correlation was found between duration of PVR membrane and EMP2 expression. EMP2 was detected by RT-PCR in all samples (n = 6) tested. CONCLUSIONS: All studied ERMs and PVR membranes express EMP2. Levels of EMP2 trended higher in all PVR subgroups than in ERMs, especially in inflammatory and traumatic PVR. Future studies are needed to determine the role of EMP2 in the pathogenesis and treatment of various retinal conditions including PVR.

Immunohistochemical Evaluation of Idiopathic Epiretinal Membranes and In Vitro Studies on the Effect of TGF-ß on Müller Cells.

PURPOSE: The purpose of this study was to investigate the presence of type VI collagen and glial cells in idiopathic epiretinal membrane (iERM) and the role of TGF-ß in the expression of collagens and α-smooth muscle actin (α-SMA) in retinal Müller cells. METHODS: Idiopathic ERM samples from vitrectomy were analyzed for glial acidic fibrillary protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), α-SMA, and type VI collagen using flat-mount immunohistochemistry. To study intracellular collagen expression in relation to cellular phenotype, spontaneously immortalized human Müller cells (MIO-M1) were treated with TGF-ß1 for 48 hours, and the expression of α-SMA and intracellular type I, II, IV, and VI collagens was studied by using immunocytology. Findings in Müller cells were compared with those in fetal lung fibroblasts and newborn skin fibroblasts. RESULTS: A colocalization of GFAP/CRALBP and GFAP/α-SMA was found in iERM, indicating a dynamic process of activation of retinal Müller cells in vivo. Transforming growth factor-ß1 induced up-regulation of α-SMA stress fibers in retinal Müller cells and both types of fibroblasts in vitro. The intracellular staining intensity of type I, II, and VI collagens was decreased in retinal Müller cells containing α-SMA stress fibers, whereas the intracellular staining intensity of type I and VI collagens in both types of fibroblasts was not affected. CONCLUSIONS: Type VI collagen and activated retinal Müller cells are present in iERM. Transforming growth factor-ß1 induces an up-regulation of α-SMA stress fibers in retinal Müller cells and fibroblasts and appears to have a cell-specific effect on intracellular collagen expression.

Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy.

PURPOSE: Epiretinal fibrovascular membranes (FVMs) are a hallmark of proliferative diabetic retinopathy (PDR). Surgical removal of FVMs is often indicated to treat tractional retinal detachment. This potentially informative pathological tissue is usually disposed of after surgery without further examination. We developed a method for isolating and characterizing cells derived from FVMs and correlated their expression of specific markers in culture with that in tissue. METHODS: FVMs were obtained from 11 patients with PDR during diabetic vitrectomy surgery and were analyzed with electron microscopy (EM), comparative genomic hybridization (CGH), immunohistochemistry, and/or digested with collagenase II for cell isolation and culture. Antibody arrays and enzyme-linked immunosorbent assay (ELISA) were used to profile secreted angiogenesis-related proteins in cell culture supernatants. RESULTS: EM analysis of the FVMs showed abnormal vessels composed of endothelial cells with large nuclei and plasma membrane infoldings, loosely attached perivascular cells, and stromal cells. The cellular constituents of the FVMs lacked major chromosomal aberrations as shown with CGH. Cells derived from FVMs (C-FVMs) could be isolated and maintained in culture. The C-FVMs retained the expression of markers of cell identity in primary culture, which define specific cell populations including CD31-positive, alpha-smooth muscle actin-positive (SMA), and glial fibrillary acidic protein-positive (GFAP) cells. In primary culture, secretion of angiopoietin-1 and thrombospondin-1 was significantly decreased in culture conditions that resemble a diabetic environment in SMA-positive C-FVMs compared to human retinal pericytes derived from a non-diabetic donor. CONCLUSIONS: C-FVMs obtained from individuals with PDR can be isolated, cultured, and profiled in vitro and may constitute a unique resource for the discovery of cell signaling mechanisms underlying PDR that extends beyond current animal and cell culture models.

Aquaporin-1 expression in proliferative vitreoretinopathy and in epiretinal membranes.

PURPOSE: Aquaporin-1 (AQP1) is involved in cell migration and proliferation; therefore, the purpose of the study was to investigate its expression in proliferative vitreoretinopathy (PVR) and epiretinal membranes (ERM). METHODS: 19 membranes from PVR and ERM were collected following eye surgery. AQP1 mRNA and protein expressions were determined by RT-qPCR and immunofluorescence in the membranes from PVR and ERM. RESULTS: AQP1 mRNA and protein were expressed in both PVR and ERM as shown by RT-qPCR and immunofluorescence. AQP1 protein expression was heterogeneous among and between PVR and ERM and colocalized with alpha-smooth muscle actin ( α SMA) and with glial fibrillary acidic protein (GFAP). There were a higher percentage of cells coexpressing AQP1 and α SMA than AQP1 and GFAP. GFAP and α SMA did not colocalize. CONCLUSION: Our data show for the first time AQP1 expression in both PVR and ERM. AQP1 is expressed mostly by the α SMA-positive cells, presumably myofibroblasts, but also by GFAP-positive cells, assumed to be glial cells. These original findings warrant further functional investigations aiming at studying the potential role of AQP1 in cell migration and proliferation occurring during the development of PVR and ERM.