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.

Genetic Linkage between CAPN5 and TYR Variants in the Context of Albinism and Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy Absence: A Case Report.

We present a case involving a patient whose clinical phenotype aligns with oculocutaneous albinism (OCA), yet exhibits a complex genotype primarily characterized by variants of unknown significance (VUS). An 11-year-old boy manifested iris hypopigmentation and translucency, pronounced photophobia, diminished visual acuity and stereopsis, nystagmus, reduced pigmentation of the retina, and foveal hypoplasia. Genetic testing was performed. A heterozygous missense VUS CAPN5 c.230A>G, p.(Gln77Arg), a heterozygous missense VUS TYR c.1307G>C, p.(Gly436Ala), and a heterozygous missense variant TYR c.1205G>A, p.(Arg402Gln) which was classified as a risk factor, were identified. We hypothesized that the TYR c.1307G>C, p.(Gly436Ala) variant is in genetic disequilibrium with the TYR c.1205G>A, p.(Arg402Gln) variant leading to deficient expression of melanogenic enzymes in retinal cells, resulting in the manifestation of mild OCA. Additionally, this study represents the case where we did not detect chiasmal misrouting in visual evoked potentials, nor did we observe a shift in the distribution of ganglion cell thickness from a temporal to a central position. Moreover, our patient's case supports the probable benign nature of the CAPN5 c.230A>G, p.(Gln77Arg) variant.

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.

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).

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.

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.

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.

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.

KRT8 phosphorylation regulates the epithelial-mesenchymal transition in retinal pigment epithelial cells through autophagy modulation.

Proliferative vitreoretinopathy (PVR) is a severe ocular disease which results in complex retinal detachment and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be critical in the pathogenesis of PVR. In this study, we focused on the potential impact of keratin 8 (KRT8) phosphorylation and autophagy on TGF-ß2-induced EMT of RPE cells and explored the relationship between them. Using immunofluorescence and Western blot analysis, the co-localization of KRT8 and autophagy marker, as well as the abundance of phosphorylated KRT8 (p-KRT8) expression, was observed within subretinal and epiretinal membranes from PVR patients. Moreover, during TGF-ß2-induced EMT process, we found that p-KRT8 was enhanced in RPE cells, which accompanied by an increase in autophagic flux. Inhibition of autophagy with pharmacological inhibitors or specific siRNAs was associated with a reduction in cell migration and the synthesis of several EMT markers. In the meantime, we demonstrated that p-KRT8 was correlated with the autophagy progression during the EMT of RPE cells. Knockdown the expression or mutagenesis of the critical phosphorylated site of KRT8 would induce autophagy impairment, through affecting the fusion of autophagosomes and lysosomes. Therefore, this study may provide a new insight into the pathogenesis of PVR and suggests the potential therapeutic value of p-KRT8 in the prevention and treatment of PVR.

Interleukin-6 promotes proliferative vitreoretinopathy by inducing epithelial-mesenchymal transition via the JAK1/STAT3 signaling pathway.

Purpose: Interleukin-6 (IL-6) is elevated in intraocular fluid from eyes with proliferative vitreoretinopathy (PVR), but the exact role of the cytokine is still unclear. We investigated the function and mechanism of IL-6 in retinal pigment epithelium (RPE) cell biology in vitro and in a mouse model in vivo. Methods: After treatment with various concentrations of IL-6, RPE cell proliferation was assessed with cell counting kit-8 (CCK-8) assay, and epithelial-mesenchymal transition (EMT) markers were evaluated using western blotting and immunofluorescent staining. The activation of JAK1/STAT3 signaling was determined with western blotting. Moreover, the effects of blockade of IL-6/JAK1/STAT3 signaling were investigated using pharmacological inhibitor S3I-201. For in vivo studies, the PVR model was induced with intravitreal injection of dispase/collagenase in wild-type and IL-6 knockout mice. The severity of PVR was evaluated with histological analysis. The expression of IL-6, gp130, and EMT markers was assessed with quantitative real-time PCR and western blotting. Results: IL-6 statistically significantly induced RPE cell proliferation and EMT in a dose-dependent manner in vitro, which was accompanied by rapid phosphorylation of JAK1 and STAT3. Blockade of the IL-6/JAK1/STAT3 pathway with S3I-201 apparently inhibited RPE proliferation and EMT. Furthermore, IL-6 and gp130 overexpression, and JAK1/STAT3 signaling hyperactivation were detected in the retinas of the wild-type mice at 1, 3, and 7 days after dispase/collagenase injection. Finally, we confirmed that IL-6 deficiency markedly alleviated mouse PVR development via inhibiting EMT. Conclusions: These findings indicate that IL-6 promotes PVR by inducing RPE proliferation and EMT via the JAK1/STAT3 signaling pathway. We provided new evidence that therapeutic strategies to block IL-6 may be beneficial for PVR.

Construction for Long Non-Coding RNA (lncRNA)-Associated Competing Endogenous RNA (ceRNA) Network in Human Retinal Detachment (RD) with Proliferative Vitreoretinopathy (PVR).

BACKGROUND The aim of this study was to analyze the long non-coding RNA (lncRNA)-associated competing endogenous RNA (ceRNA) network in human retinal tissues following detachment with proliferative vitreoretinopathy (PVR). MATERIAL AND METHODS Expression data of 19 human detached retinas with PVR and 19 normal retinas from postmortem donors were downloaded from Gene Expression Omnibust (GEO) database (GSE28133). The R package "limma" was utilized to discriminate the dysregulated lncRNA and mRNA profiles. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of differentially expressed mRNAs were performed using R packages "Clusterprofiler." The ceRNA network of dysregulated genes was constructed by using mircode, miRDB, miRTarBase and TargetScan databases, and was visualized by Cytoscape v3.6.1. RESULTS A total of 23 lncRNAs and 994 mRNAs were identified significantly expressed between the human detached retinas with PVR and the normal retina tissues, with thresholds of |log2FoldChange| >1.0 and adjusted P-value <0.05. The constructed ceRNA network (lncRNA-miRNA-mRNA regulatory axis) included 9 PVR-specific lncRNAs, as well as 27 miRNAs and 73 mRNAs. CONCLUSIONS We demonstrated the differential lncRNA expression profile and constructed a lncRNA-associated ceRNA network in human detached retinas with PVR. This may ferret out an unknown ceRNA regulatory network in human retinal detachment with PVR.

SINGLE NUCLEOTIDE POLYMORPHISMS IN RETINAL DETACHMENT PATIENTS WITH AND WITHOUT PROLIFERATIVE VITREORETINOPATHY.

PURPOSE: To investigate differences in genotype distributions of single nucleotide polymorphisms within genes, encoding inflammatory mediators, among patients with rhegmatogenous retinal detachment (RRD) and patients with proliferative vitreoretinopathy (PVR). METHODS: A genetic association study was performed on 191 Slovenian patients, divided into 2 groups: 113 RRD patients with PVR and 78 RRD patients without PVR. Genotype distributions were investigated within the following 13 single nucleotide polymorphisms: rs3760396 (CCL2), rs9990554 (FGF2), rs17561 (IL1A), rs2069763 (IL2), rs1800795 (IL6), rs1800871 (IL10), rs3008 (JAK3), rs2229094 (LTA), rs1042522 (TP53), rs7656613 (PDGFRA), rs7226855 (SMAD7), rs1800471 (TGFB1), and rs1800629 (TNF). RESULTS: Differences in genotype distributions between patients with RRD with or without PVR were detected in rs1800795 (IL6) (P = 0.04), rs1800871 (in the vicinity of the IL10) (P = 0.034), and rs1800471 (TGFB1) (P = 0.032). After adjustment none of the 13 analyzed single nucleotide polymorphisms showed statistically significant associations in single nucleotide polymorphism genotype distributions between patients with RRD with and without PVR. CONCLUSION: Further research is needed, particularly expanded multicentric population-based studies, to clarify the issue of genetic contribution to PVR from different genetic, clinical, and population-based aspects.

Proteomic evidence that ABCA4 is vital for traumatic proliferative vitreoretinopathy formation and development.

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment failure. The mechanism of PVR development is complex and still not completely elucidated. There are no proven methods for early prevention or clinical treatment. Retinal proteins are abnormally expressed during the entire PVR disease process. Due to the limitations of research methods and techniques, we do not fully understand the retinal protein changes in PVR. This proteomics study systemically analyzed and identified differential protein expression between retinas of PVR and non-PVR (normal) eyes. Retinal samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) coupled with mass spectrometry. Raw data were processed and analyzed by Maxquant software and then searched against the human UniProKB (201510) protein database. Differentially expressed proteins were selected and further validated in a human retinal pigment epithelial (RPE) cell line. The effects of dysregulated proteins on cell proliferation, apoptosis, and migration were studied. Systemic proteomics analysis identified several PVR-enriched proteins. The differentially expressed proteins were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation to find abnormal pathways involved in PVR. Retinal-specific ATP-binding cassette transporter (ABCA4) expression was one of the most increased proteins in PVR tissue. ABCA4 knockdown significantly reduced proliferation and affected the cell cycle in the human RPE cell line. ABCA4 knockdown also induced apoptosis and inhibited retinal cell migration. In conclusion, systemic proteomics analysis identified differentially expressed proteins in traumatic PVR, with ABCA4 being highly expressed. Disruption of ABCA4 expression induced apoptosis and inhibited cell proliferation and migration in a human RPE cell line.

Knockdown of survivin results in inhibition of epithelial to mesenchymal transition in retinal pigment epithelial cells by attenuating the TGFß pathway.

Proliferative vitreoretinopathy (PVR) is a common complication of open globe injury and the most common cause of failed retinal detachment surgery. The response by retinal pigment epithelial (RPE) cells liberated into the vitreous includes proliferation and migration; most importantly, epithelial to mesenchymal transition (EMT) of RPE plays a central role in the development and progress of PVR. For the first time, we show that knockdown of BIRC5, a member of the inhibitor of apoptosis protein family, using either lentiviral vector based CRISPR/Cas9 nickase gene editing or inhibition of survivin using the small-molecule inhibitor YM155, results in the suppression of EMT in RPE cells. Knockdown of survivin or inhibition of survivin significantly reduced TGFß-induced cell proliferation and migration. We further demonstrated that knockdown or inhibition of survivin attenuated the TGFß signaling by showing reduced phospho-SMAD2 in BIRC5 knockdown or YM155-treated cells compared to controls. Inhibition of the TGFß pathway using TGFß receptor inhibitor also suppressed survivin expression in RPE cells. Our studies demonstrate that survivin contributes to EMT by cross-talking with the TGFß pathway in RPE cells. Targeting survivin using small-molecule inhibitors may provide a novel approach to treat PVR disease.

Periostin in vitreoretinal diseases.

Proliferative vitreoretinal diseases such as diabetic retinopathy, proliferative vitreoretinopathy (PVR), and age-related macular degeneration are a leading cause of decreased vision and blindness in developed countries. In these diseases, retinal fibro(vascular) membrane (FVM) formation above and beneath the retina plays an important role. Gene expression profiling of human FVMs revealed significant upregulation of periostin. Subsequent analyses demonstrated increased periostin expression in the vitreous of patients with both proliferative diabetic retinopathy and PVR. Immunohistochemical analysis showed co-localization of periostin with α-SMA and M2 macrophage markers in FVMs. In vitro, periostin blockade inhibited migration and adhesion induced by PVR vitreous and transforming growth factor-ß2 (TGF-ß2). In vivo, a novel single-stranded RNAi agent targeting periostin showed the inhibitory effect on experimental retinal and choroidal FVM formation without affecting the viability of retinal cells. These results indicated that periostin is a pivotal molecule for FVM formation and a promising therapeutic target for these proliferative vitreoretinal diseases.

ASSOCIATION STUDY BETWEEN POLYMORPHISMS OF THE p53 AND LYMPHOTOXIN ALPHA (LTA) GENES AND THE RISK OF PROLIFERATIVE VITREORETINOPATHY/RETINAL DETACHMENT IN A MEXICAN POPULATION.

PURPOSE: To report the results of an association study between single-nucleotide polymorphisms of the p53 and LTA genes and the risk of proliferative vitreoretinopathy (PVR)/retinal detachment (RD) in a Mexican cohort. METHODS: A total of 380 unrelated subjects were studied, including 98 patients with primary rhegmatogenous RD without PVR, 82 patients with PVR after RD surgery, and 200 healthy, ethnically matched subjects. Genotyping of single-nucleotide polymorphisms rs1042522 (p53 gene) and rs2229094 (LTA gene) was performed by direct nucleotide sequencing. Allele frequencies, genotype frequencies, and Hardy-Weinberg equilibrium were assessed with HaploView software. RESULTS: No significant differences in the allelic distributions of the previously identified risk C allele for LTA rs2229094 were observed between RD subjects and controls (odds ratio [95% confidence interval] = 0.8 [0.5-1.2]; P = 0.3). Conversely, the C allele for rs1042522 in p53 was positively associated with an increased risk for RD (odds ratio [95% confidence interval] = 1.4 [1.01-1.9]; P = 0.04). No significant differences were observed when the subgroup of 82 RD + PVR subjects was compared with the subgroup of 98 patients with RD. CONCLUSION: The C allele for rs1042522 in p53 was genetically associated with a higher risk for RD but not for PVR in this cohort. This is the first association study attempting replication of PVR-associated risk alleles in a nonwhite population.

Plumbagin induces RPE cell cycle arrest and apoptosis via p38 MARK and PI3K/AKT/mTOR signaling pathways in PVR.

BACKGROUND: This study aimed to explore the effects of plumbagin (PLB) on ARPE-19 cells and underlying mechanism. METHODS: Cultured ARPE-19 cells were treated with various concentrations (0, 5, 15, and 25 µM) of PLB for 24 h or with 15 µM PLB for 12, 24 and 48 h. Then cell viability was evaluated by MTT assay and DAPI staining, while apoptosis and cell cycle progression of ARPE cells were assessed by flow cytometric analysis. Furthermore, the level of main regulatory proteins was examinated by Western boltting and the expression of relative mRNA was tested by Real-Time PCR. RESULTS: PLB exhibited potent inducing effects on cell cycle arrest at G2/M phase and apoptosis of ARPE cells via the modulation of Bcl-2 family regulators in a concentration- and time-dependent manner. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways contributing to the anti-proliferative activities in ARPE cells. CONCLUSIONS: This is the first report to show that PLB could inhibit the proliferation of RPE cells through down-regulation of modulatory signaling pathways. The results open new avenues for the use of PLB in prevention and treatment of proliferative vitreoretinopathy.

MicroRNA-containing extracellular vesicles released from endothelial colony-forming cells modulate angiogenesis during ischaemic retinopathy.

Endothelial colony-forming cells (ECFCs) are a defined subtype of endothelial progenitors that modulate vascular repair and promote perfusion in ischaemic tissues. Their paracrine activity on resident vasculature is ill-defined, but mediated, at least in part, by the transfer of extracellular vesicles (EVs). To evaluate the potential of isolated EVs to provide an alternative to cell-based therapies, we first performed a physical and molecular characterization of those released by ECFCs. Their effects upon endothelial cells in vitro and angiogenesis in vivo in a model of proliferative retinopathy were assessed. The EVs expressed typical markers CD9 and CD63 and formed a heterogeneous population ranging in size from ~60 to 1500 nm by electron microscopy. ECFC EVs were taken up by endothelial cells and increased cell migration. This was reflected by microarray analyses which showed significant changes in expression of genes associated with angiogenesis. Sequencing of small RNAs in ECFCs and their EVs showed that multiple microRNAs are highly expressed and concentrated in EVs. The functional categories significantly enriched for the predicted target genes of these microRNAs included angiogenesis. Intravitreally delivered ECFC EVs were associated with the vasculature and significantly reduced the avascular area in a mouse oxygen-induced retinopathy model. Our findings confirm the potential of isolated EVs to influence endothelial cell function and act as a therapy to modulate angiogenesis. The functions associated with the specific microRNAs detected in ECFC EVs support a role for microRNA transfer in mediating the observed effects.

The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.

The effects of pleiotrophin in proliferative vitreoretinopathy.

PURPOSE: The purpose of our study was to investigate the effects of pleiotrophin (PTN) in proliferative vitreoretinopathy (PVR) both in vitro and in vivo. METHODS: Immunofluorescence was used to observe the PTN expression in periretinal membrane samples from patients with PVR and controls. ARPE-19 cells were exposed to TGF-ß1. The epithelial-to-mesenchymal transition (EMT) of the ARPE-19 cells was confirmed by observed morphological changes and the increased expression of α-SMA and fibronectin at both the mRNA and protein levels. We used specific small interfering (si)RNA to knock down the expression of PTN. The subsequent effects of PTN inhibition were assessed with regard to the EMT, migration, proliferation, cytoskeletal arrangement, TGF-ß signaling, PTN signaling, integral tight junction protein expression (e.g., claudin-1 and occludin), and p38 MAPK and p-p38 MAPK levels. Additionally, a PVR rat model was established by the intravitreal injection of ARPE-19 cells transfected with PTN-siRNA and was evaluated accordingly. RESULTS: PTN was highly expressed in PVR membranes compared to controls. PTN knockdown attenuated the TGF-ß1-induced migration, proliferation, cytoskeletal rearrangement, and expression of EMT markers such as α-SMA and fibronectin in the ARPE-19 cells, and these effects may have been mediated through p38 MAPK signaling pathway activation. PTN silencing inhibited the up-regulation of claudin-1 and occludin stimulated by TGF-ß1, and PTN knockdown inhibited the proliferative aspects of severe PVR in vivo. CONCLUSIONS: PTN is involved in the process of EMT induced by TGF-ß1 in human ARPE-19 cells in vitro, and PTN knockdown attenuated the progression of experimental PVR in vivo. These findings provide new insights into the pathogenesis of PVR.