TGF-ß2-induced alterations of m6A methylation in hTERT RPE-1 cells.

N6-methyladenosine (m6A) is a major type of RNA modification implicated in various pathophysiological processes. Transforming growth factor ß2 (TGF-ß2) induces epithelial-mesenchymal transition (EMT) in retinal pigmental epithelial (RPE) cells and promotes the progression of proliferative vitreoretinopathy (PVR). However, the role of m6A methylation in the EMT of human telomerase reverse transcriptase (hTERT) retinal pigmental epithelium (RPE)-1 cells has not been clarified. Here, we extracted RNA from RPE cells subjected to 0 or 20 ng/mL TGF-ß2 for 72 h and identified differentially methylated genes (DMGs) by m6A-Seq and differentially expressed genes (DEGs) by RNA-Seq. We selected the genes related to EMT by conjoint m6A-Seq/RNA-Seq analysis and verified them by qRT-PCR. We then confirmed the function of m6A methylation in the EMT of RPE cells by knocking down the methyltransferase METTL3 and the m6A reading protein YTHDF1. Sequencing yielded 5814 DMGs and 1607 DEGs. Conjoint analysis selected 467 genes altered at the m6A and RNA levels that are closely associated with the EMT-related TGF-ß, AGE-RAGE, PI3K-Akt, P53, and Wnt signaling pathways. We also identified ten core EMT genes ACTG2, BMP6, CDH2, LOXL2, SNAIL1, SPARC, BMP4, EMP3, FOXM1, and MYC. Their RNA levels were evaluated by qRT-PCR and were consistent with the sequencing results. We observed that METTL3 knockdown enhanced RPE cell migration and significantly upregulated the EMT markers N-cadherin (encoded by CDH2), fibronectin (FN), Snail family transcription repressor (SLUG), and vimentin. However, YTHDF1 knockdown had the opposite effects and decreased both cell migration and the N-cadherin, FN, and SLUG expression levels. The present study clarified TGF-ß2-induced m6A- and RNA-level differences in RPE cells, indicated that m6A methylation might regulate EMT marker expression, and showed that m6A could regulate TGF-ß2-induced EMT.

Autosomal dominant neovascular inflammatory vitreoretinopathy with CAPN5 c.731T > C gene mutation; clinical management of a family cohort and review of the literature.

BACKGROUND: To report a cohort of patients with clinically and genetically diagnosed autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) and showcase the spectrum of the disease utilizing multimodal imaging and genetic testing. Additionally, the utility of multimodal imaging in guiding treatment will also be illustrated. MATERIALS/METHODS: Five patients from a single-family pedigree in Ohio with clinical signs of ADNIV were evaluated. Medical history, family history, and complete ocular examinations were obtained during regular clinic visits. Multimodal imaging including ocular coherence tomography, fluorescein angiography, wide-field fundus photographs, and Humphrey visual field testing was obtained for all five patients. Additionally, genetic testing for the Calpain-5 (CAPN5) gene was conducted on all patients. RESULTS: All five patients were noted to have a CAPN5 c.731T > C (p.L244P) mutation on genetic testing. Using multimodal imaging to supplement the clinical examination, pathologic changes such as retinal vascular inflammation, macular edema, and tractional retinal membranes were well illustrated and monitored over time. This allowed for earlier intervention when appropriate such as with intraocular steroid or systemic anti-inflammatory treatments. CONCLUSION: Phenotypic presentation varied among patients in this series, but is consistent with the spectrum of pathologic changes previously described in patients with other CAPN5 gene mutations. Monitoring of patients with ADNIV utilizing multimodal imaging can help better assess progression of this disease and guide treatment decisions. Additionally, increased genetic testing in patients with inherited retinal diseases may reveal novel gene mutations that could serve as potential targets for future genetic treatment regimens.

Ligand-independent activation of platelet-derived growth factor receptor ß promotes vitreous-induced contraction of retinal pigment epithelial cells.

BACKGROUND: Epiretinal membranes in patients with proliferative vitreoretinopathy (PVR) consist of extracellular matrix and a number of cell types including retinal pigment epithelial (RPE) cells and fibroblasts, whose contraction causes retinal detachment. In RPE cells depletion of platelet-derived growth factor (PDGF) receptor (PDGFR)ß suppresses vitreous-induced Akt activation, whereas in fibroblasts Akt activation through indirect activation of PDGFRα by growth factors outside the PDGF family (non-PDGFs) plays an essential role in experimental PVR. Whether non-PDGFs in the vitreous, however, were also able to activate PDGFRß in RPE cells remained elusive. METHODS: The CRISPR/Cas9 technology was utilized to edit a genomic PDGFRB locus in RPE cells derived from an epiretinal membrane (RPEM) from a patient with PVR, and a retroviral vector was used to express a truncated PDGFRß short of a PDGF-binding domain in the RPEM cells lacking PDGFRß. Western blot was employed to analyze expression of PDGFRß and α-smooth muscle actin, and signaling events (p-PDGFRß and p-Akt). Cellular assays (proliferation, migration and contraction) were also applied in this study. RESULTS: Expression of a truncated PDGFRß lacking a PDGF-binding domain in the RPEM cells whose PDGFRB gene has been silent using the CRISPR/Cas9 technology restores vitreous-induced Akt activation as well as cell proliferation, epithelial-mesenchymal transition, migration and contraction. In addition, we show that scavenging reactive oxygen species (ROS) with N-acetyl-cysteine and inhibiting Src family kinases (SFKs) with their specific inhibitor SU6656 blunt the vitreous-induced activation of the truncated PDGFRß and Akt as well as the cellular events related to the PVR pathogenesis. These discoveries suggest that in RPE cells PDGFRß can be activated indirectly by non-PDGFs in the vitreous via an intracellular pathway of ROS/SFKs to facilitate the development of PVR, thereby providing novel opportunities for PVR therapeutics. CONCLUSION: The data shown here will improve our understanding of the mechanism by which PDGFRß can be activated by non-PDGFs in the vitreous via an intracellular route of ROS/SFKs and provide a conceptual foundation for preventing PVR by inhibiting PDGFRß transactivation (ligand-independent activation).

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

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

Impaired activity and membrane association of most calpain-5 mutants causal for neovascular inflammatory vitreoretinopathy.

Neovascular inflammatory vitreoretinopathy (NIV) is a rare eye disease that ultimately leads to complete blindness and is caused by mutations in the gene encoding calpain-5 (CAPN5), with six pathogenic mutations identified. In transfected SH-SY5Y cells, five of the mutations resulted in decreased membrane association, diminished S-acylation, and reduced calcium-induced autoproteolysis of CAPN5. CAPN5 proteolysis of the autoimmune regulator AIRE was impacted by several NIV mutations. R243, L244, K250 and the adjacent V249 are on ß-strands in the protease core 2 domain. Conformational changes induced by Ca2+binding result in these ß-strands forming a ß-sheet and a hydrophobic pocket which docks W286 side chain away from the catalytic cleft, enabling calpain activation based on comparison with the Ca2+-bound CAPN1 protease core. The pathologic variants R243L, L244P, K250N, and R289W are predicted to disrupt the ß-strands, ß-sheet, and hydrophobic pocket, impairing calpain activation. The mechanism by which these variants impair membrane association is unclear. G376S impacts a conserved residue in the CBSW domain and is predicted to disrupt a loop containing acidic residues which may contribute to membrane binding. G267S did not impair membrane association and resulted in a slight but significant increase in autoproteolytic and proteolytic activity. However, G267S is also identified in individuals without NIV. Combined with the autosomal dominant pattern of NIV inheritance and evidence that CAPN5 may dimerize, the results are consistent with a dominant negative mechanism for the five pathogenic variants which resulted in impaired CAPN5 activity and membrane association and a gain-of-function for the G267S variant.

Palmitic acid-induced microRNA-143-5p expression promotes the epithelial-mesenchymal transition of retinal pigment epithelium via negatively regulating JDP2.

BACKGROUND: The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is the most crucial step in the etiopathogenesis of proliferative vitreoretinopathy. This study aimed to investigate the role of miR-143-5p in the EMT of RPE cells induced by palmitic acid (PA). METHODS: ARPE-19 cells were treated with PA to induce EMT, followed by E-cadherin and α-smooth muscle actin (α-SMA) expression and the microRNA expression profile analyses. Subsequently, miR-143-5p mimics/inhibitors, and plasmids expressing its predicted target gene c-JUN-dimerization protein 2 (JDP2), were transfected in ARPE-19 cells using lipofectamine 3000, and followed by PA treatment. Their impacts on EMT were explored using wound healing and Western blot assays. Additionally, miR-143-5p mimics and JDP2-expressing plasmid were co-transfected into ARPE-19 cells and treated with PA to explore whether PA induced EMT of ARPE-19 cells via the miR-143-5p/JDP2 axis. RESULTS: PA decreased E-cadherin expression and increased those of α-SMA and miR-143-5p. Inhibiting miR-143-5p suppressed the migration of ARPE-19 cells and altered the expressions of E-cadherin and α-SMA. However, additional PA treatment attenuated these alterations. JDP2 was a target of miR-143-5p. Overexpression of JDP2 inhibited the EMT of ARPE-19 cells, resulting in α-SMA downregulation and E-cadherin upregulation, which were reversed by additional PA treatment via inhibiting JDP2 expression. Overexpression of miR-143-5p reversed the effect of JDP2 on the EMT of ARPE-19 cells and additional PA treatment markedly enhanced the effect of miR-143-5p mimics. CONCLUSION: PA promotes EMT of ARPE-19 cells via regulating the miR-143-5p/JDP2 axis, and these findings provide significant insights into the potential targeting of this axis to treat proliferative vitreoretinopathy.

Inflammation and Oxidative Stress Gene Variability in Retinal Detachment Patients with and without Proliferative Vitreoretinopathy.

This study investigated the association between certain genetic variations and the risk of developing proliferative vitreoretinopathy (PVR) after surgery. The study was conducted on 192 patients with primary rhegmatogenous retinal detachment (RRD) who underwent 3-port pars plana vitrectomy (PPV). The distribution of single nucleotide polymorphisms (SNPs) located in genes involved in inflammation and oxidative stress associated with PVR pathways were analyzed among patients with and without postoperative PVR grade C1 or higher. A total of 7 defined SNPs of 5 genes were selected for genotyping: rs4880 (SOD2); rs1001179 (CAT); rs1050450 (GPX1); rs1143623, rs16944, rs1071676 (IL1B); rs2910164 (MIR146A) using competitive allele-specific polymerase chain reaction. The association of SNPs with PVR risk was evaluated using logistic regression. Furthermore, the possible association of SNPs with postoperative clinical parameters was evaluated using non-parametric tests. The difference between two genotype frequencies between patients with or without PVR grade C1 or higher was found to be statistically significant: SOD2 rs4880 and IL1B rs1071676. Carriers of at least one polymorphic IL1B rs1071676 GG allele appeared to have better postoperative best-corrected visual acuity only in patients without PVR (p = 0.070). Our study suggests that certain genetic variations may play a role in the development of PVR after surgery. These findings may have important implications for identifying patients at higher risk for PVR and developing new treatments.

MicroRNA Expression in Subretinal Fluid in Eyes Affected by Rhegmatogenous Retinal Detachment.

Proliferative vitreoretinopathy (PVR) is an abnormal intraocular scarring process that can complicate cases of rhegmatogenous retinal detachment (RRD). Although previous studies have examined the relevance of microRNAs (miRNAs) in ophthalmic diseases, only a few studies have evaluated the expression profiles of microRNAs in subretinal fluid. We hypothesized that the expression profiles of specific miRNAs may change in response to RRD, in the subretinal fluid that is directly in contact with photoreceptors and the retinal pigment epithelium (RPE). We looked for a potential correlation between the expression of specific miRNAs in eyes with RRD and known clinical risk factors of PVR. A total of 24 patients (59 ± 11 years) who underwent scleral buckling procedure were enrolled in this prospective study. Twenty-four undiluted subretinal fluid samples were collected, RNA was isolated and qRT-PCR was performed to analyze the expression of 12 miRNAs. We found the existence of a positive association between the expression of miR-21 (p = 0.017, r = 0.515) and miR-34 (p = 0.030, r = 0.624) and the duration of symptoms related to retinal detachment. Moreover, the expression of miR-146a tended to decrease in patients who developed PVR. Subretinal fluid constitutes an intriguing biological matrix to evaluate the role of miRNAs leading to the development of PVR.

Early-onset Neovascular Inflammatory Vitreoretinopathy Due to Two de Novo CAPN5 Mutations in Chinese Patients: A Case Series.

PURPOSE: To explore the clinical and molecular characteristics, diagnosis, and treatment of early-onset autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) in Chinese patients. METHODS: A retrospective, interventional case series was assembled from three ADNIV patients. RESULTS: The three ADNIV cases harbored de novo CAPN5 mutations (p.Arg289Trp and p.Leu73Val). The ages of onset ranged from 11 months to 2 years. All the cases presented with vitreous opacity and subretinal inflammatory exudations. During the postoperative follow-up, all the patients manifested with exaggerated postoperative inflammatory responses. An intravitreal Ozurdex injection could not effectively control ocular inflammation in ADNIV. Laser spots after panretinal photocoagulation were partly visible. CONCLUSIONS: Two de novo CAPN5 mutations (p.Leu73Val and p.Arg289Trp) could cause early-onset ADNIV. Panretinal photocoagulation during vitrectomy and an intravitreal Ozurdex injection could not significantly stop the progression of subretinal exudations and ocular inflammation in early-onset ADNIV patients.

Vitreous humor proteome: unraveling the molecular mechanisms underlying proliferative and neovascular vitreoretinal diseases.

Proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and neovascular age-related macular degeneration (nAMD) are among the leading causes of blindness. Due to the multifactorial nature of these vitreoretinal diseases, omics approaches are essential for a deeper understanding of the pathophysiologic processes underlying the evolution to a proliferative or neovascular etiology, in which patients suffer from an abrupt loss of vision. For many years, it was thought that the function of the vitreous was merely structural, supporting and protecting the surrounding ocular tissues. Proteomics studies proved that vitreous is more complex and biologically active than initially thought, and its changes reflect the physiological and pathological state of the eye. The vitreous is the scenario of a complex interplay between inflammation, fibrosis, oxidative stress, neurodegeneration, and extracellular matrix remodeling. Vitreous proteome not only reflects the pathological events that occur in the retina, but the changes in the vitreous itself play a central role in the onset and progression of vitreoretinal diseases. Therefore, this review offers an overview of the studies on the vitreous proteome that could help to elucidate some of the pathological mechanisms underlying proliferative and/or neovascular vitreoretinal diseases and to find new potential pharmaceutical targets.

Development of Proliferative Vitreoretinopathy Is Attenuated by Chicken Ovalbumin Upstream Promoter Transcriptional Factor 1 Via Inhibiting Epithelial-Mesenchymal Transition.

Proliferative vitreoretinopathy (PVR) is an intractable condition after rhegmatogenous retinal detachment (RD), which is the primary cause of failure in retinal reattachment surgery. This study aimed to investigate the effects of chicken ovalbumin upstream promoter transcriptional factor 1 (COUP-TF1) in the development of proliferative vitreoretinopathy (PVR) both in vitro and in vivo. Adult retinal pigment epithelium cell line was used for in-vitro experiments. Immunocytochemistry assay, real-time quantitative polymerase chain reaction, and Western blot were used to measure the expression of COUP-TF1, alpha-smooth muscle actin (α-SMA), and E-cadherin. Epithelial-mesenchymal transition (EMT) was observed through cell counting kit-8 assay, wound healing tests, and the expression changes of related proteins. PVR rabbit models were established and evaluated by the images of fundus and vitreous cavity, pathological sections, and COUP-TF1 expression. As shown by our results, the proliferation and migration of the COUP-TF1 knockdown cells were reduced compared with the control cells with or without transforming growth factor-ß1 (TGF-ß1) treatment. After TGF-ß1 treatment, α-SMA expression was upregulated in ARPE-19 cells but kept the same in COUP-TF1 knockdown cells. E-cadherin expression was down-regulated in all the groups but the extent of the decrease in COUP-TF1 knockdown cells was smaller. EMT was attenuated in ARPE-19 cells after COUP-TF1 was knocked down. In the in-vivo experiment, PVR severity was attenuated and the retinal detachment rate decreased on the 14th and 28th day in COUP-TF1 knockdown group. In conclusion, COUP-TF1 is related to the development of PVR, and COUP-TF1 knockdown attenuates the progression of PVR. This suggests that COUP-TF1 can be a promising candidate for the treatment of PVR.

MicroRNA-4516 suppresses proliferative vitreoretinopathy development via negatively regulating OTX1.

Proliferative vitreoretinopathy (PVR) progression is associated with TGF-ß2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. In cancer cells, miR-4516 downregulates orthodenticle homeobox 1 (OTX1)-mediated cell invasion. Moreover, OTX1 is shown to be involved in invasion and EMT. The purpose of this study was to assess whether microRNA (miR-4516) suppresses EMT in RPE cells. EMT features were assessed using Western blotting, immunocytochemical staining, scratch-wound healing, modified Boyden chamber assay, and collagen gel contraction assay. For in vivo testing, a rabbit model was used, which involved induction of PVR by injection of transfected spontaneously arising RPE (ARPE) cells into the vitreous chamber. The putative target of miR-4516 was identified by luciferase reporter assay. Results showed that TGF-ß2-induced transdifferentiation and migration of RPE cells was inhibited by miR-4516 delivery. Overexpression of miR-4516 led to upregulation of zonula occludens-1, downregulation of α-smooth muscle actin and vimentin, and cell contractility-all EMT features-in the TGF-ß2-treated ARPE-19 cells. MiR-4516 regulated OTX1 expression negatively by binding to its 3'-UTR. TGF-ß2-induced phosphorylated ERK was inhibited in miR-4516-overexpressing ARPE-19 cells. MiR-4516 suppressed experimental PVR in vitro and in vivo. In conclusion, the overexpression of miR-4516 suppresses TGF-ß2-induced EMT in a PVR model, and its role in PVR depends on OTX1/ERK. Further research is needed to develop a feasible treatment method to prevent and treat PVR.

Exosomal miR-4488 and miR-1273g-5p inhibit the epithelial-mesenchymal transition of transforming growth factor ß2-mediated retinal pigment epithelial cells by targeting ATP-binding cassette A4.

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 ß2 (TGF-ß2)-induced ARPE-19 cells were used to stimulate the epithelial-mesenchymal transition (EMT) of cells. Exosomes derived from TGF-ß2-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-ß2-induced ARPE-19 cells. Overexpressed exosomal miR-4488 and miR-1273 g-5p could inhibit the EMT, migration, invasion, and promote apoptosis in TGF-ß2-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-ß2-induced ARPE-19 cells. In conclusion, our data showed that exosomal miR-4488 and miR-1273 g-5p could inhibit TGF-ß2-stimulated EMT in ARPE-19 cells through targeting ABCA4.

Repression of Smad4 by MicroRNA-1285 moderates TGF-ß-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)-ß. 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-ß2-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-ß2-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.

Targeting matrix stiffness-induced activation of retinal pigment epithelial cells through the RhoA/YAP pathway ameliorates proliferative vitreoretinopathy.

The purpose of this study was to investigate whether excessive extracellular matrix (ECM) deposition-induced mechanical matrix stiffness plays a key role in promoting retinal pigment epithelial (RPE) cell activation and the subsequent development of proliferative vitreoretinopathy (PVR). Human ARPE-19 cells were cultured on either 50 kappa (stiff) or 0.5 kappa (soft) gel-coated coverslips. Reverse and knockdown experiments were carried out to establish a model of matrix stiffness-induced activation in ARPE-19 cells in vitro. A PVR mouse model was established by the intravitreal injection of dispase. The effects of RhoA/YAP signalling blockade on matrix stiffness-induced ARPE-19 cell activation and PVR-induced retinal fibrosis were determined by using a combination of the Yes-associated protein (YAP) inhibitor verteporfin and the RhoA inhibitor C3 exoenzyme. Matrix stiffness stimulated YAP nuclear translocation and expression in ARPE-19 cells. The effect of YAP activation was dependent on F-actin cytoskeleton polymerization and RhoA activity, forming the RhoA/YAP signalling pathway. Upstream pharmacological blockade of RhoA by C3 exoenzyme or downstream blockade of YAP by verteporfin reduced the invasion, migration, and MMP expression of ARPE-19 cells and collagen gel contraction. Furthermore, blockade of RhoA/YAP signalling reduced PVR-induced retinal fibrogenesis and inhibited the TGF-ß/Smad pathway in vivo. RhoA/YAP signalling modulates matrix stiffness-induced activation of ARPE-19 cells. Targeting this signalling pathway could alleviate PVR-induced retinal fibrosis and suggests attractive novel therapeutic strategies for intervening in the progression of PVR.

CRISPR-Based Genome Editing as a New Therapeutic Tool in Retinal Diseases.

Retinal diseases are the primary reasons for severe visual defects and irreversible blindness. Retinal diseases are also inherited and acquired. Both of them are caused by mutations in genes or disruptions in specific gene expression, which can be treated by gene-editing therapy. Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) system is a frontier of gene-editing tools with great potential for therapeutic applications in the ophthalmology field to modify abnormal genes and treat the genome or epigenome-related retinal diseases. The CRISPR system is able to edit and trim the gene include deletion, insertion, inhibition, activation, replacing, remodeling, epigenetic alteration, and modify the gene expression. CRISPR-based genome editing techniques have indicated the enormous potential to treat retinal diseases that previous treatment was not available for them. Also, recent CRISPR genome surgery experiments have shown the improvement of patient's vision who suffered from severe visual loss. In this article, we review the applications of the CRISPR-Cas9 system in human or animal models for treating retinal diseases such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR), then we survey limitations of CRISPR system for clinical therapy.

Connective tissue growth factor promotes retinal pigment epithelium mesenchymal transition via the PI3K/AKT signaling pathway.

Proliferative vitreoretinopathy (PVR) is a disease leading to the formation of contractile preretinal membranes (PRMs) and is one of the leading causes of blindness. Connective tissue growth factor (CTGF) has been identified as a possible key determinant of progressive tissue fibrosis and excessive scarring. Therefore, the present study investigated the role and mechanism of action of CTGF in PVR. Immunohistochemical staining was performed to detect the expression of CTGF, fibronectin and collagen type III in PRMs from patients with PVR. The effects and mechanisms of recombinant human CTGF and its upstream regulator, TGF­ß1, on epithelial­mesenchymal transition (EMT) and the synthesis of extracellular matrix (ECM) by retinal pigment epithelium (RPE) cells were investigated using reverse transcription­quantitative PCR, western blotting and a [3H]proline incorporation assay. The data indicated that CTGF, fibronectin and collagen type III were highly expressed in PRMs. In vitro, CTGF significantly decreased the expression of the epithelial markers ZO­1 and E­cadherin and increased that of the mesenchymal markers fibronectin, N­cadherin and α­smooth muscle actin in a concentration­dependent manner. Furthermore, the expression of the ECM protein collagen type III was upregulated by CTGF. However, the trends in expression for the above­mentioned markers were reversed after knocking down CTGF. The incorporation of [3H]proline into RPE cells was also increased by CTGF. In addition, 8­Bromoadenosine cAMP inhibited CTGF­stimulated collagen synthesis and transient transfection of RPE cells with a CTGF antisense oligonucleotide inhibited TGF­ß1­induced collagen synthesis. The phosphorylation of PI3K and AKT in RPE cells was promoted by CTGF and TGF­ß1 and the latter promoted the expression of CTGF. The results of the present study indicated that CTGF may promote EMT and ECM synthesis in PVR via the PI3K/AKT signaling pathway and suggested that targeting CTGF signaling may have a therapeutic or preventative effect on PVR.

Eupatilin attenuates TGF-ß2-induced proliferation and epithelial-mesenchymal transition of retinal pigment epithelial cells.

PURPOSE: The main characteristic of proliferative vitreoretinopathy (PVR) is migration, adhesion, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE). Eupatilin is a naturally occurring flavone that has the potential to inhibit cell proliferation and EMT. However, its efficacy on the PVR model induced by transforming growth factor-2 (TGF-ß2) is unknown. In this study, the potential effect of eupatilin on proliferation and EMT in the treatment of RPE was investigated. METHODS: Serum starved human RPE cells (ARPE-19) were treated with 10 ng/ml TGF-ß2 alone or co-treated with 25 µM eupatilin for 48 h. Quantitative real-time PCR and Western blot analysis were used to assess targets at the mRNA and protein expression level, respectively. Apoptosis and cell cycle progression was assessed by image-based cytometry. The effect of treatment on cell migration was evaluated by wound healing assay. RESULTS: Eupatilin inhibited TGF-ß2-induced RPE cell proliferation via regulating the cell cycle and inducing apoptosis. TGF-ß2 upregulated mRNA expression of mesenchymal markers fibronectin and vimentin was significantly downregulated by the treatment, while the epithelial markers E-cadherin and occludin expression was upregulated. The therapy significantly suppressed TGF-ß2 encouraged cell migration through downregulating the expression of transcription factors Twist, Snail, and ZEB1 induced by TGF-ß2. Furthermore, eupatilin significantly inhibited the expression of MMP-1, -7, and -9, and suppressed NF-κB signalling. CONCLUSION: These results suggest that eupatilin could inhibit the proliferation and transformation into fibroblast-like cells of RPE cells; thus the agent may be a potential therapeutic value in treating PVR.

Associations between Vascular Endothelial Growth Factor Gene Polymorphisms and Different Types of Diabetic Retinopathy Susceptibility: A Systematic Review and Meta-Analysis.

BACKGROUND: Vascular endothelial growth factor (VEGF) gene polymorphisms have been shown to be associated with the risk of diabetic retinopathy (DR), but the results were inconsistent. The aim of this study was to systematically assess the associations between VEGF gene polymorphisms and different types of DR (nonproliferative DR and proliferative DR). METHODS: Electronic databases PubMed, Embase, Web of Science, CNKI, and WANFANG DATA were searched for articles on the associations between VEGF gene polymorphisms and different types of DR up to November 6, 2019. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated, and subgroup analyses were conducted by ethnicity. Sensitivity analysis was conducted to assess the stability of the results. Publication bias was assessed by using the Egger regression asymmetry test and visualization of funnel plots. A systematic review was conducted for polymorphisms with a high degree of heterogeneity (I 2 > 75%) or studied in only one study. RESULTS: A total of 13 and 18 studies analyzed the associations between VEGF SNPs and nonproliferative DR (NPDR) as well as proliferative DR (PDR), respectively. There were significant associations between rs2010963 and NPDR in Asian (dominant model: OR = 1.29, 95%CI = 1.04 - 1.60); and rs2010963 is associated with PDR in total population (dominant model: OR = 1.20, 95%CI = 1.03 - 1.41), either Asian (recessive model: OR = 1.57, 95%CI = 1.04 - 2.35) or Caucasian (recessive model: OR = 1.83, 95%CI = 1.28 - 2.63). Rs833061 is associated with PDR in Asian (recessive model: OR = 1.58, 95%CI = 1.11 - 2.26). Rs699947 is associated with NPDR in the total population (dominant model: OR = 2.04, 95%CI = 1.30 - 3.21) and associated with PDR in Asian (dominant model: OR = 1.72, 95%CI = 1.05 - 2.84). CONCLUSIONS: Rs2010963, rs833061, and rs699947 are associated with NPDR or PDR, which may be involved in the occurrence and development of DR.

Normal vitreous promotes angiogenesis via activation of Axl.

Vitreous has been reported to prevent tumor angiogenesis, but our previous findings indicate that vitreous activate the signaling pathway of phosphoinositide 3-kinase (PI3K)/Akt, which plays a critical role in angiogenesis. The goal of this research is to determine which role of vitreous plays in angiogenesis-related cellular responses in vitro. We found that in human retinal microvascular endothelial cells (HRECs) vitreous activates a number of receptor tyrosine kinases including Anexelekto (Axl), which plays an important role in angiogenesis. Subsequently, we discovered that depletion of Axl using CRISPR/Cas9 and an Axl-specific inhibitor R428 suppress vitreous-induced Akt activation and cell proliferation, migration, and tuber formation of HRECs. Therefore, this line of research not only demonstrate that vitreous promotes angiogenesis in vitro, but also reveal that Axl is one of receptor tyrosine kinases to mediate vitreous-induced angiogenesis in vitro, thereby providing a molecular basis for removal of vitreous as cleanly as possible when vitrectomy is performed in treating patients with proliferative diabetic retinopathy.