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Purpose To investigate the role of Gremlin-1, which is an endogenous antagonist of the bone morphogenetic protein (BMP) signaling pathway, in inducing epithelium-mesenchymal transition (EMT) in fetal RPE cells after repeated wounds. Methods Subconfluent repetitive passages in fetal RPE cells were regarded as a model of repeated wounds. A phase contrast microscope was used to observe the morphology and pigment formation in cells. The expression of GREM1 (Gene ID: 26585; OMIM 603054) and EMT- or RPE-related genes in cells was evaluated with quantitative PCR (qPCR). Recombinant human protein Gremlin-1 (0.1 μg/ml) was added every day to investigate the molecular effects of Gremlin-1 on fetal RPE cells. The cell migration rate was investigated using a cell wound scratch assay, and western blotting was used to analyze the representative proteins (P-cadherin, ZO-1, vimentin, Smad4, and phosphorylated-Smads). In addition, transfection of siRNA was used to explore the rescue effects on EMT cells through the downregulation of GREM1. Finally, LDN193189, which is a type of pan-inhibitor of BMP receptors, was used to verify whether complete blocking of the BMP pathway interferes with the redifferentiation in low-passage fetal cells, even if the cells were treated with transforming growth factor beta 1 (TGF-β) inhibitors. Results In fetal RPE cells, the expression of GREM1 were gradually upregulated with repetitive passages, and at the same time, the function-specific genes in fetal RPE cells (TJP1, PMEL, BEST1, RPE65, and MERTK) were downregulated while the EMT-specific genes were upregulated. In addition, GREM1 had a similar expression pattern as SNAI1, which is a key transcription factor to trigger EMT. Recombinant human Gremlin-1 promoted EMT with the upregulation of SNAI1 and elevated the cell migration rate in a cell scratch assay, as well as decreased the expression of two key transcription factors of RPE embryonic development (MITF and OTX2) and the RPE marker, RPE65. Furthermore, the EMT marker, vimentin, and the TGF-β pathway downstream transcription factor phosphorylated-Smad2 (p-Smad2) increased, but the epithelial marker, ZO-1, was reduced. Additionally, Smad4, which plays a role as a Snail1 cooperator by binding Smad3, was also increased. In contrast, GREM1 silencing increased the expression of MITF and OTX2, which means there was better redifferentiation in subconfluent fetal RPE cells, but it had little influence on p-Smad2 compared to the negative control group. Finally, by adding LDN193189, the BMP signaling pathway was blocked, and this block led to poor redifferentiation in low-passage cells, although the cells were treated with TGF-β inhibitors. In addition, as positive feedback to block the BMP pathway, GREM1 was subsequently upregulated. Conclusions In fetal RPE cells, Gremlin-1 induces EMT and inhibits redifferentiation by promoting the TGF-β pathway and inhibiting the BMP pathway. GREM1 silencing alleviates EMT and increases the redifferentiation of cells by relieving the blockade of the BMP pathway. However, GREM1 silencing has no effects on the TGF-β pathway. Thus, Gremlin-1 may serve as a novel target to treat proliferative vitreoretinopathy (PVR) and inhibit subretinal fibrosis, which is a risk factor for influencing the therapeutic effects of anti-vascular endothelial growth factor (anti-VEGF) on neovascular age-related macular degeneration (nAMD).

Weiwei Zhang,1,* Huan Hu,2,* Haixia Cheng,3,* Qinghuai Liu,1 Dongqing Yuan1 1Department of Ophthalmology, The First Affiliated Hospital with Nanjing Medical University, Nanjing, Jiangsu Province, People’s Republic of China; 2Department of Ophthalmology, Xishui People’s Hospital Affiliated to Hubei University of Science and Technology, Huanggang, Hubei Province, People’s Republic of China; 3Department of Ophthalmology, Children’s Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Dongqing YuanDepartment of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu Province 210029, People’s Republic of ChinaTel/Fax +86-25-68303160Email wuyuleng@126.comPurpose: To evaluate the changes in vessel density in patients with unilateral congenital cataract after cataract extraction.Materials and Methods: Children with unilateral congenital cataract were enrolled in our study. All of the patients underwent congenital cataract extraction and intraocular lens (IOL) implantation successfully. Optical coherence tomography angiography (OCTA) was performed to image the retinal vasculature in the macular and optic disc areas before and after surgery. The differences in vessel density and retinal thickness between groups were compared.Results: We found that the best corrected visual acuity (BCVA) was significantly improved one month after surgery compared with that before surgery (t=5.179, p< 0.001). The axial length was also changed one month after surgery (t=5.350, p< 0.001). The vessel density in the macular and optic disc areas of the affected eyes was significantly lower than that in the normal eyes, while the vessel density at the posterior pole was significantly improved one month after cataract extraction.Conclusion: The decrease in vessel density in the macular and optic disc areas might be a consequence of the congenital cataract. Cataract extraction can relieve the form deprivation of the affected eye and increase the vessel density at the posterior pole of the affected eye significantly.Keywords: unilateral congenital cataract, OCTA, cataract extraction, vessel density, form deprivation

Dongqing Yuan, Weiwei Zhang, Songtao Yuan, Ping Xie, Qinghuai Liu Department of Ophthalmology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, People’s Republic of ChinaCorrespondence: Qinghuai LiuDepartment of Ophthalmology, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu Province 210029, People’s Republic of ChinaEmail liuqh@njmu.edu.cnPurpose: To evaluate the vision-related quality of life of vitrectomy combined with autologous internal limiting membrane (ILM) transplantation for refractory macular holes (MHs).Methods: There were 40 eyes with refractory MHs included, and all eyes received 23 G vitrectomy and ILM peeling with autologous ILM transplantation. Preoperative and postoperative basic conditions were recorded. The Chinese version of the vision-related quality-of-life scale was used to evaluate patients after operation. Quality of life, postoperative visual acuity, and size of MHs before operation were assessed with Spearman rank correlations.Results: All patients were followed up for 3 months after surgery. Mean postoperative best-corrected visual acuity had significantly improved after surgery. Vision-related quality of life of patients after surgery was closely related to the MH index, but negatively correlated with best-corrected visual acuity before and after surgery.Conclusion: The anatomical structure of refractory MHs with ILM peeling combined with autologous ILM transplantation was largely reduced, and the visual acuity of patients improved significantly.Keywords: refractory macular hole, internal limiting membrane transplantation, vision-related quality of life

Purpose The aim of this study was to assess the roles of plasma cytokines in diabetic retinopathy (DR) and their relationship with the severity of DR. Methods This study included 59 diabetic patients and 19 non-diabetic controls. The plasma concentrations of endothelial growth factor (EGF), eotaxin, fibroblast growth factor 2 (FGF-2), Flt-3 ligand (Flt-3L), fractalkine, granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), growth-related oncogene (GRO), interferon (IFN)-α2, IFN-γ, interleukin (IL)-1α, IL-1β, IL-1Ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IFN-inducible protein-10 (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage-derived cytokine (MDC), macrophage inflammatory protein (MIP)-1α, MIP-1β, sCD40L, sIL-2Rα, transforming growth factor (TGF)-α, tumor necrosis factor (TNF)- α, TNF-β, and VEGF were measured with Luminex multiplex bead immunoassay. The levels of these cytokines were investigated according to the DR stage. Results The plasma level of ten cytokines—MCP-1, IL-6, IL-7, IL-9, IL-13, IL-15, IL-17, sCD40L, sIL-2Rα and TNF-β—increased significantly in the diabetic group compared to the controls. The Flt-3L, IL-1Ra, IL-3, IL-5, and IL-12 (p40) levels were lower in the diabetic group than in the control group. The TNF-α plasma level was significantly elevated in patients with proliferative diabetic retinopathy (PDR) compared with the levels in patients with non-proliferative diabetic retinopathy (NPDR) and patients with no apparent diabetic retinopathy (NDR). Conclusions TNF-α might be involved in the progression of DR, especially in the pathogenesis of PDR. TNF-α is a potential cytokine for the prognosis of DR and might act as a therapeutic target in DR.

Purpose To evaluate the precise association of complement factor H (CFH) Val62Ile polymorphism with age-related macular degeneration (AMD) susceptibility. Methods We performed a meta-analysis using databases including PubMed, EMBASE, and Web of Science to find relevant studies. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using fixed-effect and random-effects models. The inconsistency index (I2) was used to assess heterogeneity. Funnel plots and Egger’s test were used to evaluate publication bias. Sensitivity analysis was also performed. Results Fourteen studies including 4,438 patients with AMD and 6,099 controls based on the search criteria were involved in the meta-analysis. In overall populations, the pooled OR1 for genotype GA+GG versus homozygous genotype AA was 2.28 (95% confidence interval (CI): 1.48–3.52), the OR2 of heterozygous genotype GA versus AA was 1.58 (95% CI: 1.13–2.19), the OR3 of homozygous genotype GG versus AA was 2.90 (95% CI: 1.95–4.30), and the OR4 of allele G versus A was 1.77 (95% CI: 1.43–2.21). In Asian populations, our results provided substantial evidence that the Val62Ile variant was significantly associated with AMD (OR4=1.85, 95% CI: 1.63–2.09). However, in Caucasian populations, no significant association of Val62Ile with AMD was established in all circumstances. Conclusions Our analysis provides substantial evidence that the Val62Ile variant is significantly associated with AMD in Asian populations. However, our results have demonstrated no link between the Val62Ile polymorphism and AMD in Caucasian populations.