Pathogenesis Of Proliferative Vitreoretinopathy Research Articles

Polarity and epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy.

Under physiological conditions, retinal pigment epithelium (RPE) is a cellular monolayer composed of mitotically quiescent cells. Tight junctions and adherens junctions maintain the polarity of RPE cells, and are required for cellular functions. In proliferative vitreoretinopathy (PVR), upon retinal tear, RPE cells lose cell-cell contact, undergo epithelial-mesenchymal transition (EMT), and ultimately transform into myofibroblasts, leading to the formation of fibrocellular membranes on both surfaces of the detached retina and on the posterior hyaloids, which causes tractional retinal detachment. In PVR, RPE cells are crucial contributors, and multiple signaling pathways, including the SMAD-dependent pathway, Rho pathway, MAPK pathways, Jagged/Notch pathway, and the Wnt/β-catenin pathway are activated. These pathways mediate the EMT of RPE cells, which play a key role in the pathogenesis of PVR. This review summarizes the current body of knowledge on the polarized phenotype of RPE, the role of cell-cell contact, and the molecular mechanisms underlying the RPE EMT in PVR, emphasizing key insights into potential approaches to prevent PVR.

MeCP2-421-mediated RPE epithelial-mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial‐mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl‐CpG‐binding protein 2 (MeCP2), especially P‐MeCP2‐421 in the pathogenesis of PVR. The expressions of P‐MeCP2‐421, P‐MeCP2‐80, PPAR‐γ and the double labelling of P‐MeCP2‐421 with α‐SMA, cytokeratin, TGF‐β and PPAR‐γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α‐SMA, phospho‐Smad2/3, collagen I, fibronectin and PPAR‐γ; the expression of α‐SMA stimulated by recombinant MeCP2 in ARPE‐19; and the effect of TGF‐β and 5‐AZA treatment on PPAR‐γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF‐β. Our results showed that P‐MeCP2‐421 was highly expressed in PVR membranes and was double labelled with α‐SMA, cytokeratin and TGF‐β, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF‐β. TGF‐β inhibited the expression of PPAR‐γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5‐AZA) increased the expression of PPAR‐γ. α‐SMA was up‐regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF‐β as demonstrated by Chip assay. The results suggest that MeCP2 especially P‐MeCP2‐421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.

PDGFRβ 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 PDGFRβ was the predominant PDGFR isoform expressed in RPEMs. Indeed, CRISPR/Cas9-mediated depletion of PDGFRβ in RPEMs attenuated patient vitreous-induced Akt activation and cellular responses intrinsic to PVR including cell proliferation, migration, and contraction. We conclude that PDGFRβ appears to be the PVR relevant PDGFR isoform in RPEMs.

A Mouse Model of Proliferative Vitreoretinopathy Induced by Intravitreal Injection of Gas and RPE Cells.

PurposeDevelop a reproducible proliferative vitreoretinopathy (PVR) mouse model that mimics human PVR pathology.MethodsMice received intravitreal injections of SF6 gas, followed by retinal pigment epithelial cells 1 week later. PVR progression was monitored using fundus photography and optical coherence tomography imaging, and histologic analysis of the retina as an endpoint. We developed a PVR grading scheme tailored for this model.ResultsWe report that mice that received gas before retinal pigment epithelial injection developed more severe PVR. In the 1 × 104 retinal pigment epithelial cell group, after 1 week, 0 of 11 mice in the no gas group developed grade 4 or greater PVR compared with 5 of 13 mice in the SF6 gas group (P = 0.02); after 4 weeks, 3 of 11 mice in the no gas group developed grade 5 or greater PVR compared with 11 of 14 mice in the SF6 gas group (P = 0.01). We were able to visualize contractile membranes both on the retinal surface as well as within the vitreous of PVR eyes, and demonstrated through immunohistochemical staining that these membranes expressed fibrotic markers alpha smooth muscle actin, vimentin, and fibronectin, as well as other markers known to be found in human PVR membranes.ConclusionsThis mouse PVR model is reproducible and mimics aspects of PVR pathology reported in the rabbit PVR model and human PVR. The major advantage is the ability to study PVR development in different genetic backgrounds to further elucidate aspects of PVR pathogenesis. Additionally, large-scale experiments for testing pharmacologic agents to treat and prevent PVR progression is more feasible compared with other animal models.Translational RelevanceThis model will provide a platform for screening potential drug therapies to treat and prevent PVR, as well as elucidate different molecular pathways involved in PVR pathogenesis.

MicroRNAs in the Vitreous Humor of Patients with Retinal Detachment and a Different Grading of Proliferative Vitreoretinopathy: A Pilot Study.

PurposeAlthough the expression of microRNAs (miRNAs) in retinal pigment epithelial (RPE) cells undergoing epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of proliferative vitreoretinopathy (PVR), its expression in the vitreous of patients with primary retinal detachment (RD) and different PVR grading has not yet been investigated. We assessed the expression of miRNAs in the vitreous humor (VH) of patients diagnosed with RD and different grading of PVR.MethodsThe VH was extracted from the core of the vitreous chamber in patients who had undergone standard vitrectomy for primary RD. RNA was extracted and TaqMan Low-Density Arrays (TLDAs) were used for miRNA profiling that was performed by single TaqMan assays. A gene ontology (GO) analysis was performed on the differentially expressed miRNAs.ResultsA total of 15 eyes with RD, 3 eyes for each grade of PVR (A, B, C, and D) and 3 from unaffected individuals, were enrolled in this prospective comparative study. Twenty miRNAs were altered in the comparison among pathological groups. Interestingly, the expression of miR-143-3p, miR-224-5p, miR-361-5p, miR-452-5p, miR-486-3p, and miR-891a-5p increased with the worsening of PVR grading. We also identified 34 miRNAs showing differential expression in PVR compared to control vitreous samples. GO analysis showed that the deregulated miRNAs participate in processes previously associated with PVR pathogenesis.ConclusionsThe present pilot study suggested that dysregulated vitreal miRNAs may be considered as a biomarker of PVR and associated with the PVR-related complications in patients with RD.Translational RelevanceThe correlation between vitreal miRNAs and the pathological phenotypes are essential to identify the novel miRNA-based mechanisms underlying the PVR disease that would improve the diagnosis and treatment of the condition.

Epithelial-mesenchymal transition suppresses ERas to activate autophagy in retinal pigment epithelial cells in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a complex ocular disease that leads to detached retinas and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells plays a critical role in PVR occurrence. However, the core targets driven by the EMT process that lead to the pathogenesis of PVR remain unclear. In our study, the relationship between embryonic stem cell-expressed Ras (ERas) and EMT in RPE cells was investigated. The subretinal and epiretinal membrane specimens of human PVR were examined for ERas and hallmarks of autophagy and EMT using Western blotting and immunofluorescence. EMT was induced by transforming growth factor (TGF)-β1 or epidermal growth factor (EGF) in ARPE-19 cells. Autophagy was inhibited by U0126 or bafilomycin A1 in ARPE-19 cells. ERas was decreased and the classical autophagy biomarker microtubule associated protein 1 light chain 3 alpha (LC3) was upregulated in the subretinal and epiretinal membranes of PVR patients in vivo. Moreover, ERas was downregulated and autophagy was activated in RPE ARPE-19 cells in response to transforming growth factor (TGF)-β1 and epidermal growth factor (EGF) induction. Finally, overexpression of ERas in RPE cells inhibited autophagy via impaired formation of autophagosomes and lysosomes. Our study revealed the role of ERas in the pathogenesis of PVR through EMT and provided a novel therapeutic target for PVR prevention and treatment.

Protein Kinase A Inhibitor H89 Attenuates Experimental Proliferative Vitreoretinopathy.

PurposeThis study aimed to explore the role of the protein kinase A (PKA) pathway in proliferative vitreoretinopathy (PVR) and the effect of the PKA inhibitor H89 on experimental PVR.MethodsEpiretinal membranes (ERMs) were acquired from PVR patients and analyzed by frozen-section immunofluorescence. An in vivo model was developed by intravitreal injecting rat eyes with ARPE-19 cells and platelet-rich plasma, and changes in eye structures and vision function were observed. An in vitro epithelial-mesenchymal transition (EMT) cell model was established by stimulating ARPE-19 cells with transforming growth factor (TGF)-β. Alterations in EMT-related genes and cell function were detected. Mechanistically, PKA activation and activity were explored to assess the relationship between TGF-β1 stimulation and the PKA pathway. The effect of H89 on the TGF-β-Smad2/3 pathway was detected. RNA sequencing was used to analyze gene expression profile changes after H89 treatment.ResultsPKA was activated in human PVR membranes. In vivo, H89 treatment protected against structural changes in the retina and prevented decreases in electroretinogram b-wave amplitudes. In vitro, H89 treatment inhibited EMT-related gene alterations and partially reversed the functions of the cells. TGF-β-induced PKA activation was blocked by H89 pretreatment. H89 did not affect the phosphorylation or nuclear translocation of regulatory Smad2/3 but increased the expression of inhibitory Smad6.ConclusionsPKA pathway activation is involved in PVR pathogenesis, and the PKA inhibitor H89 can effectively inhibit PVR, both in vivo and in vitro. Furthermore, the protective effect of H89 is related to an increase in inhibitory Smad6.

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‐β2–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‐β2–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.

Gremlin mediates the TGF-β-induced induction of profibrogenic genes in human retinal pigment epithelial cells.

Proliferative vitreoretinopathy (PVR) is characterised by the contraction and growth of fibrotic membranes on the retina and within the vitreous body. Retinal pigment epithelial (RPE) cells, a major cellular component of the fibrotic membrane, is one of the cell types that have been previously reported to associate with PVR pathogenesis. During PVR, RPE cells undergo increased cell proliferation, migration and the secretion of extracellular matrix molecules, such as fibronectin and type I collagen. A variety of cytokines and growth factors are involved in the formation of the fibrotic membrane. Although gremlin has been reported to serve an important role in the regulation of epithelial-to-mesenchymal transition in PVR, the relationship between gremlin and the expression of profibrogenic factors in human RPE cells remains unclear. In the present study, gremlin promoted RPE cell proliferation and the expression of type I collagen and fibronectin. In addition, knocking down gremlin expression by siRNA significantly suppressed the transforming growth factor (TGF)-β1- and TGF-β2-induced expression of type I collagen and fibronectin in RPE cells. These findings suggest that gremlin may serve an important role in the development of PVR.

Vitreous levels of Lipocalin-2 on patients with primary rhegmatogenous retinal detachment.

PurposeTo measure vitreous levels of Lipocalin2 (LCN2) in patients with rhegmatogenous retinal detachment (RRD) and investigate potential association with presence of proliferative vitreoretinopathy (PVR).Materials and methodsCollection of undiluted vitreous samples from 24 patients suffering from RRD and 10 control patients undergoing vitrectomy for: vitreomacular traction (VMT) (n = 2), idiopathic epiretinal membrane (iERM) (n = 6) and full thickness macular hole (FTMH) (n = 2). Quantitative analysis of LCN2 has been made with flow cytometry. Lens status, duration of symptoms, quadrants of detachment, as well as level of PVR, were assessed. Statistical analysis included Mann-Whitney test, Kruskal-Wallis test, t-test, Spearman’s correlation coefficient and Fisher's exact test.ResultsMedian LCN2 was significantly higher in the RRD group as compared to control (p<0.001). Within the RRD group there was a positive correlation between LCN2 and PVR grade (rs = 0.94, p<0.001). Median LCN2 was 35,759 pg/ml (IR = 55,347) in grade C PVR, 9,387 pg/ml (IR = 3721) in grade B, 4,917 pg/ml (IR = non computable) in grade A and 3,921 pg/ml (2132) in the no PVR group. Median LCN2 was also significantly higher in pseudophakic patients as compared to phakic patients (p = 0.007). LCN2 also correlates with the extend of detachment (≤2 vs >2 quadrants, p<0.001) as well as with duration of symptoms (rs = 0.87, p<0.001). After multivariate linear regression analysis, only PVR was independently related with LCN2 concentration. In particular, increased PVR grading was associated with increased LCN2 concentration (coefficient b = 2.97, 95% confidence interval = 1.89 to 4.67, p<0.001).ConclusionA positive correlation between vitreous levels of LCN2 and PVR grading reveals a potential role in the pathogenesis and progression of PVR. Further studies could elucidate if LCN2 could be a therapeutic target.

The role of mechanical stretch and TGF-β2 in epithelial-mesenchymal transition of retinal pigment epithelial cells.

To explore the effects and mechanisms of mechanical stress and transforming growth factor-beta2 (TGF-β2) on epithelial-mesenchymal transition (EMT) in cultured human retinal pigment epithelial (RPE) cells. Human RPE cells were inoculated on BioFex 6-well plates and RPE cells received 0, 1, 2, 3, or 4 mild stretch injuries delivered 3h apart after 24h of culture. The device of mechanical stress parameters were set to sine wave, frequency 1 Hz, stretch strength 20%. For treatment with TGF-β2, when the inoculated RPE cells in 6-well plates were around 60% confluent, serum was reduced to 0 for 12h and recombinant human TGF-β2 (0, 1, 5, 10 ng/mL) was added for 48h. α-SMA, Vimentin and N-Cadherin, fibronectin proteins expressions were detected by Western blotting, confocal cell immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). Then we detected the change of miRNA-29b and ascertained the changes of phosphatidylinositol 3-kinase-serine threonine protein kinase (PI3K/Akt) pathway after RPE cells were stretched by the device of mechanical stress and induced by TGF-β2 by Western blotting, confocal cell immunofluorescence and qRT-PCR. Mechanical stress induce EMT and activate the PI3K/Akt pathway in ways that lead to the EMT process. TGF-β2 induce RPE cells EMT and in a certain range and TGF-β2 decrease the miRNA-29b expression in RPE cells, and the inhibitory effect is more obvious with the increase of TGF-β2 concentration. Our findings are crucial steps in determining the critical roles of the PI3K/Akt signaling pathway and miRNA-29b in pathogenesis of proliferative vitreoretinopathy (PVR) which may be a potential target for preventing or treating PVR.

The effect of infliximab and octreotide on cytokine levels experimental proliferative vitreoretinopathy

Purpose: To investigate the efficiency of intravitreal octreotide, which has previously been shown to have benefits in the treatment of proliferative vitreoretinopathy (PVR), and intravitreal infliximab as a novel option in an experimental dispase-induced PVR model.Methods: A total of 28 pigmented guinea pigs were divided into four groups, and each group consisted of seven subjects. Group 1 (control) was treated with a 0.2 mL saline solution intravitreally from 1.5 mm behind the limbus. Group 2 (sham) was treated with 0.07 IU/0.1 mL dispase 0.1 mL saline solution using the same method. Group 3(infliximab) received 0.07 IU/0.1 mL dispase and 1 mg/0.1 mL infliximab, and group 4(octreotide) was treated with 0.07 IU/0.1 mL dispase and 1 mg/0.1 mL octreotide. An intravitreal injection of infliximab and octreotide was administered to groups 3 and 4 two times during the experiment. The subjects were held for a 10-week period to await for the formation of PVR. At the end of ten weeks, the eyes were enucleated, and tumour necrosis factor-alpha (TNF-α), interleukin 1(IL-1), interleukin 6 (IL-6), transforming growth factor (TGF-β), and platelet-derived growth factor (PDGF) and levels in homogenised retina tissue were measured using the enzyme linked-immuno-sorbent assay (ELISA) method.Results: Retinal TNF-α, IL-1, IL-6, and PDGF levels had significantly decreased in treatment groups compared to the sham group (p < 0.05). The decrease in the level of TGF-β was not statistically significant between the treatment and the sham groups (p > 0.05).Conclusions: Intravitreal infliximab can inhibit the development of PVR and reduce levels of cytokine, which plays an essential role in the pathogenesis of PVR. The results of our study suggest that it may be possible to identify the ideal adjuvant pharmacological drugs that are effective in preventing PVR.

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

Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-β2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-β2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-β2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-β2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-β2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-β2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-β2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-β2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.

MiR-194 suppresses epithelial-mesenchymal transition of retinal pigment epithelial cells by directly targeting ZEB1.

Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of proliferative vitreoretinopathy (PVR). Some microRNAs (miRNAs) participate in regulating RPE cell EMT as post-transcriptional regulators. However, the function of miR-194 in RPE cell EMT remains elusive. Here, the role of miR-194 in PVR was investigated. Retinal layers were obtained using laser capture microdissection (LCM). Gene expression at the mRNA and protein level in the tissues and cells was examined using quantitative reverse transcription (RT)-polymerase chain reaction and Western blotting, respectively. The related protein expression was observed by immunostaining. The effect of miR-194 on RPE cell EMT was examined by gel contraction, wound healing, and cell migration assays. RNAseq was performed in ARPE-19 with transfection of pSuper-scramble and pSuper-miR-194. The target gene of miR-194 was identified and confirmed via bioinformatics analysis and dual-luciferase reporter assay. ARPE-19 (adult retinal pigment epithelium-19) cells were treated with transforming growth factor (TGF)-β1 in the same fashion as the in vitro RPE cell EMT model. A PVR rat model was prepared by intravitreous injection of ARPE-19 cells with plasma-rich platelets. miR-194 was preferentially expressed in the RPE cell layer compared with the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer in rat retina. RNAseq analysis indicated that miR-194 overexpression was involved in RPE cell processes, including phagocytosis, ECM-receptor interaction, cell adhesion molecules, and focal adhesion. miR-194 overexpression significantly inhibited the TGF-β1-induced EMT phenotype of RPE cells in vitro. Zinc finger E-box binding homeobox 1 (ZEB1), a key transcription factor in EMT, was confirmed as the direct functional target of miR-194. Knockdown of ZEB1 attenuated TGF-β1-induced α-smooth muscle actin expression in ARPE-19 cells, and overexpression of miR-194 could significantly reduce the expression of some genes which were up-regulated by ZEB1. Exogenous miR-194 administration in vivo effectively suppressed PVR in the rat model, both functionally and structurally. Our findings demonstrate for the first time that miR-194 suppresses RPE cell EMT by functionally targeting ZEB1. The clinical application of miR-194 in patients with PVR merits further investigation.

Idelalisib inhibits vitreous-induced Akt activation and proliferation of retinal pigment epithelial cells from epiretinal membranes

Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8–10% of patients who undergo primary retinal detachment-reparative surgery and in 40–60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.

Proliferative vitreoretinopathy: revised concepts of pathogenesis and adjunctive treatment.

Proliferative vitreoretinopathy (PVR) remains a significant challenge for vitreoretinal surgeons. Its incidence, as a complication of retinal detachment, does not appear to have altered despite improvement in surgical techniques. Extensive laboratory research has been undertaken to investigate the pathogenesis of PVR and the use of adjunctive mediations to modify the disease process. To date these studies have not resulted in improved clinical management of PVR. Previous concepts of the pathogenesis of PVR, focussing on a central role for RPE cells, may be flawed and could potentially have contributed to the lack of clinical advances. Revised clinical classifications and novel approaches to adjunctive treatment may in the future result in improved surgical outcomes.

Phosphoinositide 3-kinase δ inactivation prevents vitreous-induced activation of AKT/MDM2/p53 and migration of retinal pigment epithelial cells

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play a critical role in transmitting signals from cell-surface molecules to intracellular protein effectors. Key PI3Ks include PI3Kα, PI3Kβ, and PI3Kδ, which are regulated by receptors. The signaling pathway comprising the PI3Ks, along with a Ser/Thr kinase (AKT), a proto-oncogene product (mouse double minute (MDM)2), and a tumor suppressor protein (p53), plays an essential role in experimental proliferative vitreoretinopathy (PVR), which is a fibrotic blinding eye disorder. However, which PI3K isoforms are involved in PVR is unknown. A major characteristic of PVR is the formation of epi (or sub)-retinal membranes that consist of extracellular matrix and cells, including retinal pigment epithelium (RPE) cells, glial cells, and macrophages. RPE cells are considered key players in PVR pathogenesis. Using immunoblotting and immunofluorescence analyses, we herein provide the evidence that PI3Kδ is highly expressed in human RPEs when it is primarily expressed in leukocytes. We also found that PI3Kδ inactivation through two approaches, CRISPR/Cas9-mediated depletion and a PI3Kδ-specific inhibitor (idelalisib), not only blocks vitreous-induced activation of AKT and MDM2 but also abrogates a vitreous-stimulated decrease in p53. Furthermore, we demonstrate that PI3Kδ inactivation prevents vitreous-induced proliferation, migration, and contraction of human RPEs. These results suggest that PI3Kδ may represent a potential therapeutic target for RPE-related eye diseases, including PVR.

The polyether ionophore salinomycin targets multiple cellular pathways to block proliferative vitreoretinopathy pathology.

Proliferative vitreoretinopathy (PVR) is characterized by membranes that form in the vitreous cavity and on both surfaces of the retina, which results in the formation of tractional membranes that can cause retinal detachment and intrinsic fibrosis of the retina, leading to retina foreshortening. Currently, there are no pharmacologic therapies that are effective in inhibiting or preventing PVR formation. One of the key aspects of PVR pathogenesis is retinal pigment epithelial (RPE) cell epithelial mesenchymal transition (EMT). Here we show that the polyether ionophore compound salinomycin (SNC) effectively inhibits TGFβ-induced EMT of RPE cells. SNC blocks the activation of TGFβ-induced downstream targets alpha smooth muscle actin (αSMA) and collagen 1 (Col1A1). Additionally, SNC inhibits TGFβ-induced RPE cell migration and contraction. We show that SNC functions to inhibit RPE EMT by targeting both the pTAK1/p38 and Smad2 signaling pathways upon TGFβ stimulation. Additionally, SNC is able to inhibit αSMA and Col1A1 expression in RPE cells that have already undergone TGFβ-induced EMT. Together, these results suggest that SNC could be an effective therapeutic compound in both the prevention and treatment of PVR.

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.

Protective Effects of Fucoidan on Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells and Progression of Proliferative Vitreoretinopathy

Background/Aims: Proliferative vitreoretinopathy (PVR) is a severe blinding complication of rhegmatogenous retinal detachment. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is thought to play a pivotal role in the pathogenesis of PVR. Fucoidan, a marine extract, reportedly has many benefits effects in a variety of tissues and organs such as anti-inflammation, anti-oxidative stress, and anti-carcinogenesis. In this study, we investigated the potential role of fucoidan on EMT in RPE cells and its effect on the development of PVR. Methods: MTS, Transwell, and collagen gel contraction assays were employed to measure the viability, migration, and contraction of RPE cells, respectively. mRNA and protein expression were evaluated via real-time quantitative PCR and western blot analysis, respectively. In vivo, a pigmented rabbit model of PVR was established to examine the anti-PVR effect of fucoidan. Results: Fucoidan reversed the transforming growth factor (TGF)-β1-induced EMT of RPE cells, including the increased expression of α-smooth muscle actin (α-SMA) and fibronectin and down-regulation of E-cadherin in human primary RPE cells. Moreover, the upregulation of phosphorylated Smad2/3 induced by TGF-β1 was suppressed by fucoidan. Fucoidan also inhibited the migration and contraction of RPE cells induced by TGF-β1. In vivo, fucoidan inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that fucoidan suppressed the formation of α-SMA-positive epiretinal membranes. Conclusion: Our findings regarding the protective effects of fucoidan on the EMT of RPE cells and experimental PVR suggest the potential clinical application of fucoidan as an anti-PVR agent.