transforming-growth-factor-beta and Vitreoretinopathy--Proliferative

Proliferative vitreoretinopathy (PVR) is the major cause of failure in patients undergoing surgery for rhegmatogenous retinal detachment (RRD). Characterized by the formation of an abnormal contractile membrane within the eye, PVR can cause tractional retinal redetachment. Epithelial-to-mesenchymal transition (EMT), in which epithelial cells morphologically and phenotypically transdifferentiate into mesenchymal cells, is the major pathological process implicated in PVR. Among the various cell types involved in the process, retinal pigment epithelium cells are primary contributors although, after decades of research, the mechanisms underlying EMT have remained elusive. Recently, signaling pathways, some involving growth factors, have been demonstrated to contribute to EMT. In this article, we review research to date about the roles of such signaling, including including transforming growth factor-beta-, hepatocyte growth factor-, platelet-derived growth factor-, and Notch-, Wnt/β-catenin-, and Hippo-signaling pathways, in the EMT of PVR.

Topics: beta Catenin; Calcium-Binding Proteins; Epithelial-Mesenchymal Transition; Hepatocyte Growth Factor; Hippo Signaling Pathway; Humans; Intercellular Signaling Peptides and Proteins; Membrane Proteins; Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Receptors, Notch; Retinal Detachment; Serrate-Jagged Proteins; Signal Transduction; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative; Wnt Signaling Pathway

Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF-β) and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), can induce RPE-EMT; however, small molecule inhibitors of RPE-EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKβ) that selectively targets NF-κB signaling, can modulate TGF-β/TNF-α-induced RPE-EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKβ inhibition on RPE-EMT-associated factors using a second IKKβ inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE-EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.

Topics: Epithelial-Mesenchymal Transition; Humans; I-kappa B Kinase; Protein Serine-Threonine Kinases; Retinal Pigment Epithelium; Stem Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vitreoretinopathy, Proliferative

The purpose of this study was to investigate the effects of silibinin on epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) and proliferative vitreoretinopathy (PVR) formation, as well as its underlying molecular mechanism.. Cellular morphological change and EMT molecular markers were evaluated by using phase contrast imaging, qPCR, and Western blot (WB) to investigate the impact of silibinin on the EMT of ARPE-19 cells. Scratch assay and transwell assay were used to study the effect of silibinin on cell migration. An intravitreally injected RPE-induced rat PVR model was used to assess the effect of silibinin on PVR in vivo. RNA-seq was applied to study the molecular mechanism of silibinin-mediated PVR prevention.. Silibinin inhibited TGFβ1-induced EMT and migration of RPE in a dose-dependent manner in vitro. Moreover, silibinin prevented proliferative membrane formation in an intravitreal injected RPE-induced rat PVR model. In line with these findings, RNA-seq revealed a global suppression of TGFβ1-induced EMT and migration-related genes by silibinin in RPEs. Mechanistically, silibinin reduced TGFβ1-induced phosphorylation levels of Smad3 and Stat3, and Smad3 nuclear translocation in RPE.. Silibinin inhibits the EMT of RPE cells in vitro and prevents the formation of PVR membranes in vivo. Mechanistically, silibinin inhibits Smad3 phosphorylation and suppresses Smad3 nuclear translocation through the inhibition of Stat3 phosphorylation. These findings suggest that silibinin may serve as a potential treatment for PVR.

Topics: Animals; Epithelial-Mesenchymal Transition; Phosphorylation; Rats; Silybin; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

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)-β. 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-β2-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-β2-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.

Topics: Animals; Biomarkers; Cell Line; Cell Movement; Cell Transdifferentiation; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Humans; MicroRNAs; Models, Biological; Rabbits; Rats; Retinal Pigment Epithelium; Smad4 Protein; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

This 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.. Epiretinal 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.. PKA 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.. PKA 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.

Topics: Aged; Animals; Cells, Cultured; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Electroretinography; Epiretinal Membrane; Epithelial Cells; Female; Humans; Isoquinolines; Male; MAP Kinase Signaling System; Middle Aged; Retinal Pigment Epithelium; Smad Proteins; Sulfonamides; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

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.

Topics: Epithelial-Mesenchymal Transition; Gene Expression Regulation; Humans; Methyl-CpG-Binding Protein 2; Phosphorylation; Retinal Pigment Epithelium; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

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.

Topics: Actins; Cell Line; Cell Movement; Collagen Type I; Epithelial-Mesenchymal Transition; Ether; Humans; Pyrans; Retinal Pigment Epithelium; Signal Transduction; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

The aim of the present study was to investigate the effects of bradykinin (BK) on an epithelial-mesenchymal transition (EMT) model in retinal pigment epithelium (RPE) cells through exposure to transforming growth factor‑β1 (TGF‑β1). The aim was to improve the effect of BK on proliferative vitreoretinopathy (PVR) progression, and to find a novel method of clinical prevention and treatment for PVR. The morphology of ARPE‑19 cells was observed using an inverted phase‑contrast microscope. A Cell Counting Kit‑8 was used to assess the effects of TGF‑β1 on the proliferation of ARPE‑19 cells. Western blotting and immunofluorescence were used to detect the expression levels of the epithelial marker E‑cadherin, mesenchymal markers α‑smooth muscle actin (SMA) and vimentin, and phosphorylated (p) mothers against decapentaplegic homolog (Smad)3 and Smad7 of the TGF/Smad signaling pathway. Wound healing tests and Transwell assays were performed to detect cell migration ability. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis was performed to detect the expression levels of pSmad3 and Smad7 in the TGF/Smad signaling pathway. The results revealed that the addition of 10 ng/ml TGF‑β1 resulted in the expression of factors associated with EMT in ARPE‑19 cells. BK decreased the expression levels of the mesenchymal markers α‑SMA and vimentin, and increased the expression of the epithelial marker E‑cadherin. BK decreased cell migration in TGF‑β1‑induced EMT. These effects were reversed by HOE‑140, a specific BK 2 receptor antagonist. BK significantly downregulated the expression of pSmad3 and upregulated the expression of Smad7 in TGF‑β1‑treated ARPE‑19 cells, and the protective alterations produced by BK were inhibited by HOE‑140. In conclusion, 10 ng/ml TGF‑β1 resulted in EMT in ARPE‑19 cells and BK served a negative role in TGF‑β1‑induced EMT. BK had effects in TGF‑β1‑induced EMT by upregulating the expression of Smad7 and downregulating the expression of pSmad3 in TGF‑β/Smad signaling pathway, indicating that BK may be a novel and effective therapy for PVR.

Topics: Bradykinin; Bradykinin B2 Receptor Antagonists; Cell Line; Cell Movement; Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Receptor, Bradykinin B2; Retinal Pigment Epithelium; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vitreoretinopathy, Proliferative

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 TGFβ-induced cell proliferation and migration. We further demonstrated that knockdown or inhibition of survivin attenuated the TGFβ signaling by showing reduced phospho-SMAD2 in BIRC5 knockdown or YM155-treated cells compared to controls. Inhibition of the TGFβ pathway using TGFβ receptor inhibitor also suppressed survivin expression in RPE cells. Our studies demonstrate that survivin contributes to EMT by cross-talking with the TGFβ pathway in RPE cells. Targeting survivin using small-molecule inhibitors may provide a novel approach to treat PVR disease.

Topics: Cell Line; Cell Proliferation; CRISPR-Cas Systems; Epithelial-Mesenchymal Transition; Humans; Inhibitor of Apoptosis Proteins; Retinal Pigment Epithelium; Signal Transduction; Survivin; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

Proteotoxic stress and transforming growth factor (TGFβ)- induced epithelial-mesenchymal transition (EMT) are two main contributors of intraocular fibrotic disorders, including proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). However, how these two factors communicate with each other is not well-characterized.. The aim was to investigate the regulatory role of proteotoxic stress on TGFβ signaling in retinal pigment epithelium.. ARPE-19 cells and primary human retinal pigment epithelial (RPE) cells were treated with proteasome inhibitor MG132 and TGFβ. Cell proliferation was analyzed by CCK-8 assay. The levels of mesenchymal markers α-SMA, fibronectin, and vimentin were analyzed by real-time polymerase chain reaction (PCR), western blot, and immunofluorescence. Cell migration was analyzed by scratch wound assay. The levels of p-Smad2, total Smad2, p-extracellular signal-regulated kinase 1/2 (ERK1/2), total ERK1/2, p-focal adhesion kinase (FAK), and total FAK were analyzed by western blot. The mRNA and protein levels of TGFβ receptor-II (TGFβR-II) were measured by realtime PCR and western blot, respectively.. MG132-induced proteotoxic stress resulted in reduced cell proliferation. MG132 significantly suppressed TGFβ-induced upregulation of α-SMA, fibronectin, and vimentin, as well as TGFβ-induced cell migration. The phosphorylation levels of Smad2, ERK1/2, and FAK were also suppressed by MG132. Additionally, the mRNA level and protein level of TGFβR-II decreased upon MG132 treatment.. Proteotoxic stress suppressed TGFβ-induced EMT through downregulation of TGFβR-II and subsequent blockade of Smad2, ERK1/2, and FAK activation.

Topics: Cell Movement; Cell Proliferation; Diabetic Retinopathy; Epithelial-Mesenchymal Transition; Focal Adhesion Kinase 1; Gene Expression Regulation; Humans; Leupeptins; MAP Kinase Signaling System; Primary Cell Culture; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Retinal Pigment Epithelium; Smad2 Protein; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

The transforming growth factor β (TGFβ) family plays an important role in the pathogenesis of many diseases, including fibrotic pathologies of the eyes. The difficulties of surgical procedures contribute to the search for new treatment strategies for proliferative vitreoretinopathy. Therefore, the aim of this study was to investigate the expression profile of TGFβ isoforms, their receptors, and TGFβ-related genes in human retinal pigment epithelial cells (RPE) after tacrolimus (FK-506) treatment in the presence or absence of lipopolysaccharide (LPS)-induced inflammation.. The expression profile was analyzed using oligonucleotide microarrays and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) techniques.. Analysis using oligonucleotide microarrays revealed 20 statistically significant differentially expressed TGFβ-related genes after LPS treatment in relation to control cells, and after tacrolimus and LPS treatment in relation to LPS-treated cells. Moreover, our results showed that mRNA levels for TGFβ2 and TGFβR3 after tacrolimus treatment, and for TGFβR3 after tacrolimus and LPS treatment in RPE cells were decreased. In turn, in the presence of LPS-induced inflammation, TGFβ2 mRNA level was increased.. These results can be important in regard to the treatment of proliferative vitreoretinopathy, pathogenesis of which is associated with processes regulated by TGFβ, such as inflammation, proliferation, epithelial-mesenchymal transition (EMT), and fibrosis.

Topics: Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Inflammation; Retinal Pigment Epithelium; RNA, Messenger; Signal Transduction; Tacrolimus; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR.

Topics: Activin Receptors, Type I; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type I; Cell Movement; Collagen; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Humans; Phosphorylation; Retinal Pigment Epithelium; Smad1 Protein; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

To investigate the effect of intravitreal tacrolimus on an animal model of proliferative vitreoretinopathy (PVR) and on growth factors implicated in its pathogenesis.. Twenty-one guinea pigs were randomly assigned to one of three groups of seven animals each: no-PVR/saline group (no PVR/intravitreal saline-injected group), PVR/saline group (dispase-induced PVR group, treated with control injections of intravitreal saline), and PVR/tacrolimus group (treatment group, dispase-induced PVR group treated with intravitreal tacrolimus injections). At the end of the experiment, eyes were enucleated and the identification of the stages of PVR was carried out. While a halves of the enucleated globes were evaluated histopathologically for PVR formation, the retinas of the other halves of globes were used for the preparation of retinal homogenates. The transforming growth factor-β, platelet-derived growth factor, and fibroblast growth factor levels in homogenized retina tissues were measured by the enzyme-linked immunosorbent assay method.. When assessing the average PVR stages in terms of severe PVR rates, the PVR/tacrolimus group had significantly improved when compared with the PVR/saline group. The PVR/tacrolimus group demonstrated significantly decreased levels of transforming growth factor-β, platelet-derived growth factor, and fibroblast growth factor when compared with the PVR/saline group and also demonstrated significant improvement in epiretinal membrane formation and retinal fold in the presence of histopathologic levels. The difference in degradation of photoreceptor cells between the PVR/tacrolimus and the PVR/saline groups was not statistically significant.. This study suggests that intravitreal tacrolimus application may suppress PVR development and that tacrolimus may merit investigation for the prophylaxis of PVR.

Topics: Animals; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Guinea Pigs; Immunosuppressive Agents; Intercellular Signaling Peptides and Proteins; Intravitreal Injections; Platelet-Derived Growth Factor; Tacrolimus; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

Proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) are characterized by the development of epi-retinal membranes which may exert a tractional force on retina. A lot of inflammatory growth factors may disturb the local ocular cells such as retinal pigment epithelial (RPE) cells, causing them to migrate and proliferate in the vitreous cavity and ultimately forming the PVR membrane. In this study, the signal pathways mediating cell migration of RPE induced by growth factors were investigated. Hepatocyte growth factor (HGF), epidermal growth factor (EGF) or heparin-binding epidermal growth factor (HB-EGF) induced a greater extent of migration of RPE50 and ARPE19 cells, compared with other growth factors. According to inhibitor studies, migration of RPE cells induced by each growth factor was mediated by protein kinase C (PKC) and ERK (MAPK). Moreover, HGF coupled with EGF or HB-EGF had synergistic effects on cell migration and enhanced activation of PKC and ERK, which were attributed to cross activation of growth factor receptors by heterogeneous ligands. Furthermore, using the shRNA technique, PKCδ was found to be the most important PKC isozyme involved. Finally, vitreous fluids from PVR and PDR patients with high concentration of HGF may induce RPE cell migration in PKCδ- and ERK- dependent manner. In conclusion, migration of RPE cells can be synergistically induced by HGF coupled with HB-EGF or EGF, which were mediated by enhanced PKCδ activation and ERK phosphorylation.

Topics: Cell Line; Cell Movement; Drug Synergism; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Heparin-binding EGF-like Growth Factor; Hepatocyte Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Platelet-Derived Growth Factor; Protein Kinase C-delta; Retinal Pigment Epithelium; Signal Transduction; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

To explore the effect of glucosamine (GlcN) on transforming growth factor (TGF)-β signalling and several processes involved in proliferative vitreoretinopathy (PVR).. We evaluated the surface levels of TGF-β receptor and its binding of TGF-β in ARPE-19 cells. Release of cytokines and collagen, and expression of signalling intermediates were quantified. Migration was qualitatively and quantitatively examined. The morphology of cells undergoing PVR in vitro and in a mouse PVR model was observed.. Glucosamine reduced the surface levels of TGF-β receptor and the ability of ARPE-19 cells to bind TGF-β. In ARPE-19 cells, TGF-β1 plus epidermal growth factor (EGF) or TGF-β2 increased the expression of alpha-smooth muscle actin (α-SMA) and decreased the expression of zona occludens protein (ZO-1). Transforming growth factor-(β2) also caused the release of platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF) and type 1 collagen and increased the phosphorylation of SMAD2 and SMAD3. Platelet-derived growth factor and CTGF stimulated cell migration, and TGF-β2 stimulated wound closure, contraction of collagen and changes in cell morphology.. Treatment with GlcN counteracted all of these effects, and its administration in the mouse model reduced the morphologic appearance of PVR. Glucosamine could inhibit the TGF-β signalling pathway in retinal pigment epithelium cells and several of the downstream events associated with epithelial-mesenchymal transition and PVR.

Topics: Actins; Animals; Blotting, Western; Cell Movement; Cells, Cultured; Collagen; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Epithelial-Mesenchymal Transition; Glucosamine; Humans; Immunohistochemistry; Membrane Proteins; Mice; Phosphoproteins; Receptors, Transforming Growth Factor beta; Retinal Pigment Epithelium; Signal Transduction; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative; Zonula Occludens-1 Protein

The cytokine transforming growth factor-ß (TGF-ß) is a pivotal contributor to tissue fibrosis and a key cytokine in the pathogenesis of cellular transdifferentiation, epithelial-mesenchymal transition (EMT), and cell adhesion. This study evaluates the effect of decorin, a naturally occurring TGF-ß inhibitor, in an experimental rabbit model for proliferative vitreoretinopathy (PVR).. Traumatic PVR was induced in 50 rabbits divided into ten groups (n = 5). One group (GI) reveals a control with no treatment after trauma. Groups (GII-GIV) consisted of subgroups receiving phacovitrectomy at three different time points; (a) at the time of trauma, (b) 1 week following trauma, and (c) 2 weeks following trauma. GIII and GIV received 100 μg or 200 μg decorin, respectively. PVR severity was scored from 0 to 4. The amount of fibrosis was quantified using JMicroVision© software.. The control group GI developed severe PVR with tractional retinal detachment (TRD); (PVR score ≥2) in four rabbits out of five. Vitrectomy had a positive effect (p < 0.05) on PVR development when preformed immediately, however the developed fibrosis was high. The best results were obtained when surgery was used in conjunction with decorin that reduced both the PVR score and fibrosis development significantly (p < 0.05). Depending on dosage and time of vitrectomy, PVR could be completely avoided (PVR score = 0) in 16 rabbits out of 30. TRD was prevented in 13 rabbits out of 15 in GIII to 14 rabbits out of 15 in GIV. In decorin-treated eyes, vitrectomy outcome was best when preformed at 1 week after trauma. There were no drug-related toxic effects evident on clinical and histopathological examination.. In conclusion, in this rabbit model of PVR, adjuvant decorin application during vitrectomy effectively reduces fibrosis and TRD development. In conjunction with no obvious histopathological toxicity signs, decorin represents a promising substance to inhibit PVR reactions.

Topics: Animals; Decorin; Disease Models, Animal; Female; Fibrosis; Intravitreal Injections; Phacoemulsification; Rabbits; Retina; Retinal Detachment; Transforming Growth Factor beta; Vitrectomy; Vitreoretinopathy, Proliferative

Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment surgery or ocular trauma. Our previous study indicated that intravitreal co-injection of retinal pigmented epithelial (RPE)-J cells and platelet-rich plasma (PRP) (not RPE-J cells or PRP alone) in Wistar rat eyes can successfully induce a model of PVR. But which cells are involved in this process and why different induction methods, intravitreal injection of RPE-J cells or/and PRP, induced a different situation remain to be unknown. In this study, immunohistochemistry was performed to identify the main cell types involved in this process. The expression levels of transforming growth factor (TGF)-β2, platelet-derived growth factor (PDGF)-AA and PDGF-BB were tested using enzyme-linked immunosorbent assay (ELISA). The results showed that RPE cells, glial cells, fibroblasts and macrophages took part in the pathogenesis of this model. The expression levels and durations of TGF-β2 and PDGF-BB partially explained the different results induced by the different induction methods. This provides an experimental proof for attenuation of the experimental PVR by targeting at a specific cells or growth factor.

Topics: Animals; Becaplermin; Blood Platelets; Cell Transplantation; Cells, Cultured; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Glial Fibrillary Acidic Protein; Immunohistochemistry; Male; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rats; Rats, Wistar; Retina; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative; Vitreous Body

Cicatricial contraction of preretinal fibrous membrane is a cause of severe vision loss in proliferative vitreoretinal diseases such as proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). TGF-beta is overexpressed in the vitreous of patients with proliferative vitreoretinal diseases and is also detectable in the contractile membranes. Therefore, TGF-beta is presumed to contribute to the cicatricial contraction of the membranes, however, the underlying mechanisms and TGF-beta's importance among various other factors remain to be elucidated. Vitreous samples from PDR or PVR patients caused significantly larger contraction of hyalocyte-containing collagen gels, compared with nonproliferative controls. The contractile effect was strongly correlated with the vitreal concentration of activated TGF-beta2 (r = 0.82, P < 0.0001). PDR or PVR vitreous promoted expression of alpha-smooth muscle actin (alpha-SMA) and phosphorylation of myosin light chain (MLC), a downstream mediator of Rho-kinase (ROCK), both of which were dramatically but incompletely suppressed by TGF-beta blockade. In contrast, fasudil, a potent and selective ROCK inhibitor, almost completely blocked the vitreous-induced MLC phosphorylation and collagen gel contraction. Fasudil disrupted alpha-SMA organization, but it did not affect its vitreal expression. In vivo, fasudil significantly inhibited the progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells by electroretinographic and histological analyses. These results elucidate the critical role of TGF-beta in mediating cicatricial contraction in proliferative vitreoretinal diseases. ROCK, a key downstream mediator of TGF-beta and other factors might become a unique therapeutic target in the treatment of proliferative vitreoretinal diseases.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Animals; Blotting, Western; Immunohistochemistry; Muscle, Smooth; Phosphorylation; Protein Kinase Inhibitors; Rabbits; rho-Associated Kinases; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative; Wound Healing

To determine the effects of Smad7 gene transfer in the prevention of fibrogenic responses by the retinal pigment epithelium, a major cause of proliferative vitreoretinopathy after retinal detachment, in mice.. Retinal detachment-induced proliferative vitreoretinopathy in a mouse model. Forty-eight eyes received either an adenoviral gene transfer of Smad7 or Cre recombinase gene only. The eyes were histologically analyzed. A retinal pigment epithelial cell line, ARPE-19, was used to determine whether Smad7 gene transfection suppresses the fibrogenic response to transforming growth factor (TGF) beta2 exposure.. The Smad7 gene transfer inhibited TGF-beta2/Smad signaling in ARPE-19 cells and expression of collagen type I and TGF-beta1 but had no effect on their basal levels. In vivo Smad7 overexpression resulted in suppression of Smad2/3 signals and of the fibrogenic response to epithelial-mesenchymal transition by the retinal pigment epithelium.. Smad7 gene transfer suppresses fibrogenic responses to TGF-beta2 by retinal pigment epithelial cells in vitro and in vivo. Clinical Relevance Smad7 gene transfer might be a new strategy to prevent and treat proliferative vitreoretinopathy.

Topics: Adenoviridae; Animals; Blotting, Western; Cell Line; Collagen Type I; Disease Models, Animal; Fibronectins; Fibrosis; Fluorescent Antibody Technique, Indirect; Gene Expression; Genetic Vectors; Mice; Pigment Epithelium of Eye; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad7 Protein; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation; Vitreoretinopathy, Proliferative

Retinal pigment epithelial cells (RPEs) are thought to be one of the main components of fibrous membrane observed in eyes with proliferative vitreo-retinopathy. We investigated the signalling mechanisms of TGF-beta2-dependent collagen gel contraction by RPEs. An in vitro type I collagen gel contraction assay was performed to evaluate the effect of TGF-beta2 on gel contraction. The expression of alpha-smooth muscle actin (alpha-SMA) and the phosphorylation state of myosin light chain (MLC) were analyzed by Western blotting. The involvement of protein kinases such as p44/42 mitogen-activated protein kinase (MAPK), protein kinase C (PKC), p38 MAPK and phosphatidylinositol-3 kinase was investigated. The contribution of Rho-kinase and/or MLC-kinase was also evaluated using respective kinase inhibitors (Y27632, hydroxyfasudil and ML7). Additionally, RPEs were immunostained to examine whether the expression of alpha-SMA detected in our western blotting correlated to the stress fiber formation within the cells. TGF-beta2 caused time (0-5 days)-and dose (0 10 ng ml(-1))-dependent gel contraction associated with overexpression of alpha-SMA and phosphorylation of MLC (p < 0.01, respectively). PKC inhibitor (GF109203X, 5 microM) and p38 MAPK inhibitor (SB203580, 10 microM) significantly attenuated TGF-beta2-elicited gel contraction via partial downregulation of both alpha-SMA expression and MLC phosphorylation (p < 0.01, respectively). The gel contraction was prominently inhibited in the presence of Y27632 (10 microM) or hydroxyfasudil (10 microM) with strong suppression of MLC phosphorylation but had no significant effect on alpha-SMA expression. Treatment with ML7, in contrast, resulted in a marginal inhibition of MLC phosphorylation and gel contraction. Finally, pretreatment of the cells with Y27632 or hydroxyfasudil prevented the formation of stress fiber within the cells. These results indicate that TGF-beta2-dependent myofibroblastic transdifferentiation and MLC phosphorylation by RPEs involve both PKC and p38 MAPK pathways at least in part. Myofibroblastic transdifferentiation of RPEs appears to be independent of the Rho-kinase pathway, and the presence of alpha-SMA does not necessarily reflect the contractile potential of a cell. While Rho-kinase inhibitors are incapable of preventing myofibroblastic transdifferentiation itself, this pathway could be one of the critical targets of cell-mediated contraction of the tissue containing fibrillar collagens by trans

Topics: Actins; Animals; Cattle; Cells, Cultured; Collagen; Dose-Response Relationship, Drug; Intracellular Signaling Peptides and Proteins; Myosin Light Chains; Phosphorylation; Pigment Epithelium of Eye; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Recombinant Proteins; rho-Associated Kinases; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta2; Vitreoretinopathy, Proliferative

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.

Topics: Aged; Aged, 80 and over; Blotting, Western; Cell Culture Techniques; Cell Movement; Cell Proliferation; DNA; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Gene Expression Regulation; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Pigment Epithelium of Eye; Polymerase Chain Reaction; Proto-Oncogene Proteins c-akt; RNA, Messenger; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vitreoretinopathy, Proliferative

Proliferative vitreoretinopathy (PVR) is one of the major causes of the failure of retinal detachment surgery. Its pathogenesis includes a fibrotic reaction by the retinal pigment epithelium and other retina-derived non-neural cells, leading to fixation of the detached retina. We examined the role of p38 mitogen-activated protein kinase (MAPK) in transforming growth factor (TGF)-beta2-dependent enhancement of the fibrogenic reaction in a human retinal pigment epithelial cell line, ARPE-19, and also evaluated the therapeutic efficacy of inhibiting p38MAPK by adenoviral gene transfer of dominant-negative (DN) p38MAPK in a mouse model of PVR. Exogenous TGF-beta2 activates p38MAPK in ARPE-19 cells. It also suppresses cell proliferation, but this was unaffected by addition of the p38MAPK inhibitor, SB202190. SB202190 interfered with TGF-beta2-dependent cell migration and production of collagen type I and fibronectin, but had no effect on basal levels of these activities. While SB202190 did not affect phosphorylation of the C-terminus of Smads2/3, it did suppress the transcriptional activity of Smads3/4 as indicated by a reporter gene, CAGA12-Luc. Gene transfer of DN-p38MAPK attenuated the post-retinal detachment fibrotic reaction of the retinal pigment epithelium in vivo in mice, supporting its effectiveness in preventing/treating PVR.

Topics: Animals; Cell Line; Cell Movement; Cell Proliferation; Collagen Type I; Disease Models, Animal; Enzyme Inhibitors; Fibronectins; Fibrosis; Genetic Therapy; Humans; Imidazoles; Male; Mice; Mice, Inbred C57BL; p38 Mitogen-Activated Protein Kinases; Pigment Epithelium of Eye; Pyridines; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta2; Vitreoretinopathy, Proliferative

To study the involvement of apoptosis using different apoptosis markers in PVR pathogenesis.. The presence of mRNA coding for Fas, Fas ligand (FasL), and TNF-related apoptosis inducing ligand (TRAIL) was investigated in vitreous samples from 46 consecutive patients-25 with PVR, 11 with retinal detachment (RD) not complicated by PVR, and 10 with macular hole (MH)-using RT-PCR. From previously examined vitreous samples, 21 PVR, 9 RD, and 10 MH were examined for their levels of TGF-beta2 protein with sandwich ELISA kits. Five epiretinal membranes excised from five patients with PVR were also examined for apoptotic cell death using the terminal deoxytransferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) technique.. FAS mRNA was detected in 72% of patients with PVR, 55% of patients with RD and 20% of patients with MH. TRAIL mRNA was detected in 67% of patients with PVR, 89% of patients with RD, and 20% of patients with MH. FasL mRNA was detected in 20% of patients with PVR, 9% of patients with RD, and 10% of patients with MH. The median levels of Fas and TRAIL mRNA were significantly higher (P < 0.05) in patients with PVR than in those with MH hole but between patients with PVR and those with RD the difference was not significant (P > 0.05). A significant difference was detected between RD and MH for TRAIL mRNA levels (P = 0.008). For FasL, no significant difference between groups was found. TGF-beta2 was detected in all investigated vitreous samples. A significant difference was found between the PVR and MH groups (P = 0.001) and between the RD and MH groups (P = 0.004), but not between the PVR and RD groups (P < 0.05). The level of TGF-beta2 was significantly correlated to the level of TRAIL mRNA (r = 0.86), but no correlation was found between TGF-beta2 and Fas mRNA levels (r = 0.21). Four of five examined PVR epiretinal membranes showed positive staining for apoptotic cells using the TUNEL technique.. Apoptosis is one of the mechanisms that is involved in PVR pathogenesis. Different apoptosis markers suggest different pathways occur in PVR, including Fas/FasL, TRAIL, and TGF-beta2 mediated processes.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Apoptosis Regulatory Proteins; Biomarkers; Enzyme-Linked Immunosorbent Assay; Epiretinal Membrane; Fas Ligand Protein; fas Receptor; Female; Humans; In Situ Nick-End Labeling; Male; Membrane Glycoproteins; Middle Aged; Retinal Detachment; Retinal Perforations; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta; Transforming Growth Factor beta2; Tumor Necrosis Factor-alpha; Up-Regulation; Vitreoretinopathy, Proliferative; Vitreous Body

Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial-mesenchymal transition (EMT) following retinal detachment, playing a central role in formation of fibrous tissue on the detached retina and vitreous retraction (proliferative vitreoretinopathy (PVR)). We have developed a mouse model of subretinal fibrosis with implications for PVR in which retinal detachment is induced without direct damage to the RPE cells. Transforming growth factor-beta (TGF-beta) has long been implicated both in EMT of RPEs and the development of PVR. Using mice null for Smad3, a key signaling intermediate downstream of TGF-beta and activin receptors, we show that Smad3 is essential for EMT of RPE cells induced by retinal detachment. De novo accumulation of fibrous tissue derived from multilayered RPE cells was seen following experimental retinal detachment in eyes of wild type, but not Smad3-null mice. Expression of alpha-smooth muscle actin, a hallmark of EMT in this cell type, and extracellular matrix components, lumican and collagen VI, were also not observed in eyes of Smad3-null mice. Our data show that induction of PDGF-BB by Smad3-dependent TGF-beta signaling is likely an important secondary proliferative component of the disease process. The results suggest that blocking the Smad3 pathway might be beneficial in prevention/treatment of PVR.

Topics: Actins; Animals; Becaplermin; Biomarkers; Cell Differentiation; Cell Line; Cell Movement; Chondroitin Sulfate Proteoglycans; Collagen Type VI; Disease Models, Animal; DNA-Binding Proteins; Fluorescent Antibody Technique, Indirect; Humans; Keratan Sulfate; Lumican; Mice; Mice, Inbred Strains; Mice, Knockout; Pigment Epithelium of Eye; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Retinal Detachment; Signal Transduction; Smad3 Protein; Swine; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta2; Vitreoretinopathy, Proliferative

To determine whether silicone oil concentrates protein and growth factors in the retro-oil fluid.. A laboratory analysis of intraocular fluid and vitreous specimens obtained from patients undergoing removal of silicone oil, revision vitrectomy, or primary vitrectomy for macular hole, proliferative vitreoretinopathy (PVR), or retinal detachment. Patients were prospectively recruited from routine vitreoretinal operating lists. Vitreous cavity fluid and vitreous samples were analysed for the presence of transforming growth factor beta (TGF-beta2), basic fibroblast growth factor (bFGF), interleukin 6 (IL-6), and total protein using either commercially available enzyme linked immunosorbent assays (ELISA) or protein assay kits.. The median levels of bFGF, IL-6, and protein in the retro-oil fluid were raised (p<0.05) compared to all the other vitreous and vitreous cavity fluid samples. bFGF, IL-6, and protein levels were raised in PVR vitreous compared to non-PVR vitreous. TGF-beta2 levels were not significantly raised in retro-oil fluid or in PVR vitreous.. The concentration of fibrogenic (bFGF) and inflammatory (IL-6) growth factors and protein is raised in retro-silicone oil fluid. This may contribute to the process of retro-oil perisilicone proliferation and subsequent fibrocellular membrane formation.

Topics: Enzyme-Linked Immunosorbent Assay; Eye Proteins; Fibroblast Growth Factor 2; Growth Substances; Humans; Immunosuppressive Agents; Interleukin-6; Prospective Studies; Retinal Detachment; Retinal Diseases; Retinal Perforations; Silicone Oils; Transforming Growth Factor beta; Transforming Growth Factor beta2; Vitrectomy; Vitreoretinopathy, Proliferative; Vitreous Body

Transforming growth factor (TGF)-beta2 and hepatocyte growth factor (HGF) have been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR) after retinal detachment surgery. The exact role of these factors in the early events, immediately after primary retinal detachment, is not yet known, and determining their roles was therefore the purpose of this study.. Subretinal fluids were collected prospectively from 144 patients during surgery for scleral buckling. TGF-beta2 and HGF were measured with commercially available ELISA kits. Thirty patients in whom a redetachment caused by postoperative PVR developed, were compared with 114 patients with an uncomplicated retinal detachment. The controls included 18 vitreous samples from patients with macular hole or pucker. Multivariate regression analysis was used to compare the relative roles of growth factors and clinical factors in the development of PVR.. The median amount of subretinal TGF-beta2 was approximately two times lower in patients with postoperative PVR (1.9 ng/mL) than in the uncomplicated detachment group (3.3 ng/mL; P=0.002). TGF-beta2 levels in the PVR-positive group were similar to control vitreous levels (1.8 ng/mL). Subretinal HGF concentrations were not significantly different between the two groups of patients (PVR positive: 8.8 ng/mL; PVR negative: 8.9 ng/mL), but were higher than control vitreous levels (4.6 ng/mL; P=0.01). Stepwise multivariate logistic regression analysis revealed that of all factors under study, decreased TGF-beta2 content was the exclusive predictor of postoperative PVR (P=0.01).. High TGF-beta2 levels in subretinal fluid at the time of primary retinal detachment may protect a patient against subsequent development of PVR.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Body Fluids; Enzyme-Linked Immunosorbent Assay; Exudates and Transudates; Female; Hepatocyte Growth Factor; Humans; Male; Middle Aged; Postoperative Complications; Prospective Studies; Recurrence; Retinal Detachment; Scleral Buckling; Transforming Growth Factor beta; Transforming Growth Factor beta2; Vitreoretinopathy, Proliferative

This study was conducted to investigate a method of gene therapy for proliferative vitreoretinopathy (PVR) by inhibiting type beta transforming growth factor (TGF-beta). PVR was induced in pigmented rabbits by intravitreal injection of 50 000 rabbit conjunctival fibroblasts after vitrectomy. Subsequently, the eyes received an intravitreal application of adenovirus vector encoding a soluble type II TGF-beta receptor (AdTbeta-ExR, n = 10) or adenoviral vector expressing beta-galactosidase (AdLacZ) (n = 10) or balanced salt solution (BSS) (n = 6). The eyes were examined ophthalmoscopically for 28 days after surgery, and the clinical stage of PVR was evaluated on a scale of zero to five. Histological examinations were performed on the treated eyes on day 28. All control eyes injected with AdLacZ or BSS developed PVR, characterized by retinal detachment and the formation of intravitreal membranes within 7 days. The eyes injected with AdTbeta-ExR also developed features of PVR, but the average severity from day 5 to day 28 was significant lower than in the control eyes (P < 0.05). TGF-beta plays an important role in PVR progression in a PVR model, and prevention of TGF-beta signaling could be therapeutically useful.

Topics: Adenoviridae; Animals; Enzyme-Linked Immunosorbent Assay; Fundus Oculi; Genetic Therapy; Genetic Vectors; Protein Serine-Threonine Kinases; Rabbits; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative; Vitreous Body

To determine whether hepatocyte growth factor (HGF) and connective tissue growth factor (CTGF) are expressed in human specimens of proliferative vitreoretinopathy (PVR) and to propose a model of PVR pathogenesis based upon the known activities of these growth factors. Methods Immunohistochemical methods (ABC Elite) were used to demonstrate the presence of HGF and CTGF in cryostat sections of five human PVR membranes.. In each of the five PVR membranes, stromal cells were immunohistochemically positive for both HGF and CTGF. Based upon this information and the known actions of these growth factors, a model of PVR pathogenesis was developed. In this model, injury of the retina induces an inflammatory response that upregulates HGF expression inducing the formation of multilayered groups of migratory retinal pigment epithelial cells (RPE). These RPE, present in a provisional extracellular matrix, come in contact with vitreous containing TGF-beta. The TGF-beta is activated, upregulating expression of CTGF. Under the influence of TGF-beta and CTGF, RPE become myofibroblastic and fibrosis ensues. Retinal traction induces further detachment continuing the cycle of retinal injury.. HGF and CTGF are expressed in PVR membranes and may play important roles in the pathogenesis of PVR. The expression and function of these growth factors should be critically examined in human PVR specimens, in in vitro cultures of RPE, and in animal models of PVR.

Topics: Connective Tissue Growth Factor; Growth Substances; Hepatocyte Growth Factor; Humans; Immediate-Early Proteins; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Models, Biological; Stromal Cells; Transforming Growth Factor beta; Up-Regulation; Vitreoretinopathy, Proliferative

The present study investigated the effects of transforming growth factor (TGF)-beta on retinal pigment epithelial (RPE) transformation in a simplified model and also whether or not TGF-beta exhibits similar proliferation effects on transformed RPE cells that it has on primary RPE cells. Furthermore, we examined the cell proliferation effects of RPE-conditioned medium (CM). A vertical wound measuring 2 mm in diameter was made on primary RPE monolayers. The expression of alpha-smooth muscle actin (SMA) by the cells located at the wound edges was observed using a confocal microscope under immunofluorescent staining. Cell proliferation was measured by incorporating 3H-thymidine into DNA. The presence of alpha-SMA was observed in the cells within the wound after treatment with TGF-beta2, while negative expression was observed in control cells. TGF-betas inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, but the spindle-shaped late-passaged RPE cells were not inhibited by these growth factors. The medium conditioned by RPE cells stimulated the proliferation of subconjunctival fibroblasts and inhibited the proliferation of primary RPE cells, in a manner similar to TGF-beta. These findings demonstrate that TGF-beta-stimulated RPE cells may evoke proliferative vitreoretinopathy through mesenchymal transformation and cell proliferation.

Topics: Actins; Animals; Cell Division; Cells, Cultured; Culture Media, Conditioned; DNA; Mesoderm; Pigment Epithelium of Eye; Rabbits; Swine; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

Retinal pigment epithelial (RPE) cells are believed to play a pivotal role in the formation and contraction of epiretinal membranes in proliferative vitreoretinopathy (PVR). In the present study, an organ culture method was used that mimics the contractile stage of PVR, to investigate the contribution of a variety of growth factors in human RPE cell-mediated contraction of the retina.. Cultured human RPE cells were seeded onto bovine retinal explants. After attachment, cultures received one of the following exogenous growth factors: platelet-derived growth factor (PDGF)-AB, PDGF-BB, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-beta1, TGF-beta2, or interleukin (IL)-10; or a neutralizing antibody to PDGF and/or TGF-beta2. Control explants were either untreated or received a null antibody. Contraction was assessed by image analysis and expressed as percentage reduction in retinal area.. RPE cells produced a more than 50% contraction of the retina after 7 days in untreated samples. PDGF and TGF-beta2 stimulated RPE-mediated contraction by a further 20% at 100 ng/ml. IL-10 decreased contraction by 63%, whereas the other growth factors gave rise to similar contraction to untreated controls. Neutralizing antibodies against PDGF and TGF-beta2 reduced RPE-mediated contraction by up to 70% in comparison with untreated controls. The neutralizing antibodies also inhibited the effects of exogenous PDGF and TGF-beta2 on RPE-mediated contraction of the retina (P < 0.01).. These findings confirm a role for both PDGF and TGF-beta2 in RPE cell-mediated contraction of the retina. Such contraction can be inhibited by neutralizing antibodies against PDGF and TGF-beta2, which, together with IL-10, are putative candidates for therapeutic intervention in PVR.

Topics: Animals; Antibodies, Monoclonal; Cattle; Cell Adhesion; Dose-Response Relationship, Drug; Drug Combinations; Interleukin-10; Organ Culture Techniques; Pigment Epithelium of Eye; Platelet-Derived Growth Factor; Retina; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

Formation of scarlike epiretinal membranes (ERMs) constitutes potentially the end stage of evolution of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Among various cellular populations, ERMs contain cells with contractile features typical of myofibroblasts. The current study was conducted to investigate the presence of transforming growth factor (TGF)-beta1, TGF-beta receptor II (RII) and ED-A fibronectin (FN), the main inducers of myofibroblastic differentiation in ERMs in PDR and PVR.. Samples of ERM were obtained from 23 patients during microsurgery for PVR or PDR. Electron microscopy, immunohistochemistry, and confocal microscopy with antibodies recognizing beta-smooth muscle (SM) actin, desmin, TGF-beta1, TGF-beta receptors I and II, and ED-A FN were performed.. alpha-SM actin was detected in all ERMs, whereas desmin was present in 50% of the cases. ED-A FN was expressed in all ERMs in close relation with alpha-SM actin-positive myofibroblasts. In addition, TGF-beta1 and TGF-beta R II were always present, TGF-beta RII being expressed in both alpha-SM actin-positive and negative fibroblastic cells.. Myofibroblast accumulation is a key event in ERM-associated traction retinal detachment occurring during PVR and PDR. The current results suggest that the presence of alpha-SM actin-positive myofibroblasts is probably dependent on the concomitant neoexpression of TGF-beta1, TGF-beta RII, and ED-A FN. The results furnish new data on the mechanism of alpha-SM actin stimulation in fibroblasts in a human pathologic setting.

Topics: Actins; Adolescent; Adult; Aged; Aged, 80 and over; Diabetic Retinopathy; Epiretinal Membrane; Female; Fibroblasts; Fibronectins; Fluorescent Antibody Technique, Indirect; Humans; Immunoenzyme Techniques; Male; Microscopy, Confocal; Middle Aged; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

To investigate the ultrastructural distribution of platelet-derived growth factor (PDGF), transforming growth factor beta(1) (TGF-beta(1)) and their receptors in epiretinal membranes (ERM) and discuss the clinical significance of the distribution.. 25 membrane specimens were surgically removed by vitrectomy from 9 patients with rhegmatogenous retinal detachment. Double-staining techniques of immunoelectromicroscopy for eight antibodies (PDGF, TGF-beta(1), PDGF receptor subunits alpha and beta, TGF-beta subunits I and II, type I and III collagen) were used in these specimens as previously designed.. PDGF and TGF-beta(1) staining appeared to be stronger on early (< 2 months) and late (> 6 months) stage membranes than that of middle (2 - 6 months) stage of membranes, and the immunostaining intensity inverted with the degree of proliferative vitreoretinopathy (PVR). We found that the gold particles of PDGF and TGF-beta(1) tended to get together with that of type I and III collagen. In addition, the stainings of four growth factor receptors were frequently positive in a type of epithelial-like cells with rich cytoplasm components, and also the gold particles of PDGF and TGF-beta(1) were found around these cells.. A concentration change of PDGF and TGF-beta(1) exists in development of ERM throughout. The epithelial-like cells are of a type of active cells, and autocrine and paracrine activity may be involved in ERM pathogenesis. PDGF and TGF-beta(1) influence the contractile activity of ERM.

Topics: Epiretinal Membrane; Humans; Microscopy, Immunoelectron; Platelet-Derived Growth Factor; Receptors, Platelet-Derived Growth Factor; Receptors, Transforming Growth Factor beta; Retinal Detachment; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vitreoretinopathy, Proliferative

Proliferative vitreoretinopathy (PVR) is a major cause of failure of retinal detachment surgery. It is believed to be a wound-healing process in the retina. Many of the cellular functions are influenced by cytokines and growth factors such as interleukins (ILs). The present study was conducted to investigate the presence of transforming growth factor-beta 2 (TGF-beta2), basic fibroblast growth factor (bFGF), IL-1beta, IL-6, and protein in the vitreous of patients with retinal detachment and to determine the value of these mediators in predicting the future development of PVR.. A prospective study was conducted in 140 consecutive patients with rhegmatogenous retinal detachment in whom vitrectomy was considered necessary. Vitreous samples were analyzed for the presence of TGF-beta2, bFGF, IL-1beta, IL-6, and protein. Patients were then followed up for 3 months for the development of postoperative PVR.. The mean levels of TGF-beta2, bFGF, IL-1beta, and protein in the vitreous were significantly higher (P < 0.05) in patients with preoperative PVR compared with those without. The mean levels of TGF-beta2, bFGF, IL-6, and protein in the vitreous were significantly higher (P < 0.05) in patients who had postoperative PVR compared with those who did not. Multivariate logistic regression analysis showed IL-6 and protein to be significant (P < 0.05), independent, predictive risk factors for the development of PVR.. The various cytokines may play a role in the pathobiology of PVR. High vitreous levels of IL-6 and protein were identified as significant risk factors for PVR. A model was developed to predict the probability of development of postoperative PVR in these patients, and it may be used to indicate intravitreal pharmacologic treatment for those at risk.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Enzyme-Linked Immunosorbent Assay; Female; Fibroblast Growth Factor 2; Humans; Interleukin-1; Interleukin-6; Male; Middle Aged; Prospective Studies; Retinal Detachment; Transforming Growth Factor beta; Vitrectomy; Vitreoretinopathy, Proliferative; Vitreous Body

Tranilast has been clinically used for various allergic diseases. Recently, it has also been found to inhibit excessive scarring in wound healing processes. In this study, we examined the effects of tranilast on the treatment for experimental proliferative vitreoretinopathy (PVR).. Cultured rabbit conjunctival fibroblasts were injected intravitreously (50000 cells/eye) into the rabbit vitreous to induce experimental PVR. Immediately after that, tranilast (0.5-5 mg/ml, 0.1 ml/eye) was injected into the vitreous. Injection of vehicle solution was used as a negative control. PVR was clinically evaluated by masked observers using ophthalmoscopy and graded into six stages: 0 (no PVR) to 5 (severe PVR). The amount of transforming growth factor beta1 (TGF-beta1) in the vitreous was measured by ELISA method. Functional and morphological changes induced by 5 mg/ml tranilast were sought by electroretinography, light microscopy, and electron microscopy on day 28.. The average stage of PVR in the eyes treated with tranilast (1 or 5 mg/ml) was significantly lower than that in the control group on days 14 and 28. There was no difference between the eyes treated with low-dose tranilast (0.5 mg/ml) and the control group. The amount of TGF-beta1 in the vitreous of tranilast-treated eyes was significantly lower than in the control group. The morphological and functional studies did not show any deleterious effect of tranilast on the retinal function and morphology.. Tranilast effectively inhibits the progression of PVR without showing apparent toxicity of the eye. This agent has therapeutic value for PVR.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Disease Progression; Electroretinography; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Histamine H1 Antagonists; ortho-Aminobenzoates; Rabbits; Retina; Transforming Growth Factor beta; Treatment Outcome; Vitreoretinopathy, Proliferative; Vitreous Body

Formation of vascularized membranes inside the vitreous leads to retinal detachment and blindness. In this paper it is shown that vitreal membranes are composed of newly formed vessels and myofibroblasts, immersed in a loose stroma with sparse histocytes. Vascular endothelial growth factor (VEGF) is clearly present in cellular constituents of the membranes and, therefore, represents a fundamental cytokine in their formation, while transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are not. Considering that the composition of vitreal membranes clearly resembles scar tissue, the absence of TGF-beta in the membranes could explain their peculiar histological appearance.

Topics: Adult; Aged; Endothelial Growth Factors; Humans; Immunohistochemistry; Lymphokines; Middle Aged; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vitreoretinopathy, Proliferative

Epiretinal tissue proliferations occurring during the evolution of ischemic microangiopathies or preretinal diseases are tough to cause retinal detachment by traction mechanisms. Cellular migration/proliferations and finally contraction are tough to be the pathogenic element. Myofibroblasts are contractile cells having features intermediate between those of the fibroblasts and smooth muscle. We conducted a study to explore whether such cells are present in preretinal membranes.. 8 membranes, preelevated during vitrectomy for proliferative vitreoretinopathy or diabetic proliferative vitreoretinopathy, were analysed with immunostaining technique searching for alpha-actine smooth muscle, desmine, which are specific markers for myofibroblasts and TGF-beta1, that is considered as the mean factor promoting the transformation of fibroblasts into myofibroblasts.. All the histological preparation showed abundant staining with antibody against alpha-actine smooth muscle, desmine and TGF-beta1.. Myofibroblasts are one of the major cellular element of preretinal membranes. They are scattered throughout the membrane and seem to account for their contractile properties.

Topics: Actins; Cell Division; Cell Movement; Desmin; Diabetic Retinopathy; Epiretinal Membrane; Fibroblasts; Humans; Myosins; Retinal Detachment; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative

To establish and quantify the presence of contraction-stimulating activity in pathologic vitreous and correlate this activity with clinical presentation and outcome, especially with proliferative vitreoretinopathy.. Contraction-stimulating activity of vitreous collected during surgery was quantified with a tissue culture assay using fibroblasts as target cells. The activity of each sample was correlated with patient history, clinical presentation, risk factors, proliferative disease, and postoperative proliferation.. Pathologic vitreous contained measurable quantities of contraction-stimulating activity and stimulated contraction in vitro, with elevated activities in samples from patients with proliferative vitreoretinopathy, epimacular proliferation, retinal detachment, retinal defects, pigmented cells in the vitreous, hemorrhage, or uveitis. Patients with postoperative proliferation had significantly elevated mean activities.. Levels of contraction-stimulating activity in pathologic vitreous correlate with some risk factors for the development of proliferative vitreoretinopathy and may ultimately be useful in the assessment of disease severity and the prediction of postoperative proliferation.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biological Factors; Cell Division; Cells, Cultured; Child; Collagen; Female; Fibroblasts; Humans; Male; Middle Aged; Retinal Diseases; Skin; Transforming Growth Factor beta; Vitrectomy; Vitreoretinopathy, Proliferative; Vitreous Body

Proliferative vitreoretinopathy (PVR) is characterized by cell proliferation and membrane formation on the vitreoretinal cavity of the eye. The membranes are composed of extracellular matrix, mainly collagen type I. To explore the possible mechanisms involved in PVR membrane formation, the authors analyzed the role of vitreous humor on collagen turnover.. The authors studied vitreous samples from ten patients with PVR and from five donor eyes (keratoplasty) as the control group. Human lung fibroblasts were used to study the influence of vitreous on collagen synthesis and cell proliferation. Neutralizing antibodies against transforming growth factor-beta 2 (TGF-beta 2) were used to inhibit the fibroblast collagen synthesis induced by the vitreous samples. Collagenolytic activity was analyzed in vitreous fluid using 3H-labeled collagen.. The authors found that samples obtained from patients with PVR significantly increased collagen synthesis (2979 +/- 963.26 versus 800 +/- 232 dpm of 3H-proline incorporated per milligram of vitreous-incubated protein; P < 0.00043), without affecting fibroblast replication. The collagen synthesis induced by the vitreous samples was inhibited by anti-TGF-beta 2 antibodies in both groups (0 and 481 +/- 59 dpm of 3H-proline incorporated per milligram of vitreous-incubated protein for control and PVR samples, respectively). Collagenolytic activity was considerably lower in vitreous derived from PVR samples compared with the control group (19.9 +/- 20.3 versus 234.1 +/- 19.1 micrograms of degraded collagen per milligram of vitreous-incubated protein; P < 0.0032).. These results suggest that a combined mechanism, including an increase of collagen synthesis mediated at least in part by TGF-beta 2 and a decrease of collagen degradation, may contribute to the exaggerated deposition of collagen observed in PVR membranes, and that vitreous should be considered as a part of the microenvironment that is participating actively in the pathogenesis of this vitreoretinal disorder.

Topics: Cell Division; Collagen; Collagenases; Fibroblasts; Humans; Immunoglobulin G; Lung; Retinal Detachment; Transforming Growth Factor beta; Vitreoretinopathy, Proliferative; Vitreous Body