transforming-growth-factor-beta and Epiretinal-Membrane

Aging increases the risks for developing fibrocontractile membranes on the retina, which causes significant macular distortion, as in the idiopathic epiretinal membrane (iERM). Retinal Müller glial cells are components of these membranes and may play a key role in the iERM pathogenesis. The transforming growth factor-β (TGF-β) induces Müller cell transdifferentiation into myofibroblast, reducing glial cell markers (glutamine synthetase, GS, and glial fibrillary acidic protein, GFAP) and increasing α-smooth muscle actin (α-SMA). Our aim was to investigate the effect of the TGF-β inhibitor galunisertib (LY2157299) on the glial-mesenchymal transition and contraction of Müller cells. MIO-M1 human Müller cells were treated with TGF-β1 (10 ng/mL), galunisertib (5, 10 and 20 μM) and TGF-β1+galunisertib for 24h and 48h. Galunisertib cytotoxicity was analyzed by MTT and trypan blue, and TGF-β1 blockade by phospho-SMAD3 immunofluorescence. Caspase-3 (cell death indicator), GS, GFAP and α-SMA expression was examined by immunofluorescence, Western blotting, and qPCR analysis. Cell contractility was determined by collagen gel contraction assay with Müller cells incorporated. Galunisertib did not show cytotoxicity at the concentrations evaluated and maintained the Müller cells phenotype, ensuring the GS expression. Galunisertib inhibited the TGF-β1 pathway by decreasing phospho-SMAD3 immunoreactivity, attenuated the α-SMA expression, and prevented the contraction of Müller cells in collagen gel. Although more studies are needed, in vitro assays suggest that galunisertib may be a potential candidate to attenuate the formation of fibrocontractile membranes and prevent retinal detachment and consequent loss of vision.

Topics: Actins; Collagen; Ependymoglial Cells; Epiretinal Membrane; Humans; Neuroglia; Transforming Growth Factor beta; Transforming Growth Factor beta1

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

This work was aimed to further characterize cells of idiopathic epiretinal membranes (iERMs). We wanted to determine the contribution of 90-kDa heat shock protein (HSP90) to sustain the transforming growth factor-β (TGF-β)-mediated signal transduction pathway in iERM.. Immunofluorescence and confocal microscopy were carried out on deplasticized sections from 36 epiretinal membranes processed for electron microscopy and on frozen sections from five additional samples with antibodies against α-smooth muscle actin (αSMA), vimentin, glial fibrillary acidic protein (GFAP), SMAD2, HSP90α, type-II TGF-β1 receptor (TβRII), type-I collagen, and type-IV collagen. In addition, Müller MIO-M1 cells were transfected with HSP90 and challenged with TGF-β1.. Double and triple labeling experiments showed that a variable number of TβRII+ cells were present in 94.1% of tested iERMs and they were mostly GFAP-/αSMA+/vimentin+/HSP90α+. In almost half of the cases these cells contained type-I collagen, suggesting their involvement in matrix deposition. HSP90 overexpressing MIO-M1 cells challenged with TGF-β1 showed increased levels of TβRII, SMAD2, SMAD3, and phosphor-SMAD2. Nuclear SMAD2 staining could be observed in HSP90α+ cells on frozen sections of iERMs.. Cells in iERMs that express TβRII are also HSP90α+ and show the antigenic profile of myofibroblast-like cells as they are GFAP-/αSMA+/vimentin+. HSP90α-overexpressing MIO-M1 cells challenged with TGF-β1 showed an increased activation of the SMAD pathway implying that HSP90α might play a role in sustaining the TGF-β1-induced fibrotic response of iERM cells.

Topics: Ependymoglial Cells; Epiretinal Membrane; Fibrosis; HSP90 Heat-Shock Proteins; Humans; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

The epithelial-mesenchymal transition (EMT) is a key process in fibrogenic diseases where transdifferentiated myofibroblasts produce excessive amounts of extracellular matrix, resulting in organ dysfunction. Idiopathic epiretinal membrane (iERM) is a vision-threatening disorder characterized by fibrocellular proliferation and contraction on the central retina. Müller glial cells, which regulate retinal physiology and structure, are the major cellular components in the iERM tissue; however, the pathological role of this cell type remains incompletely understood. Here we revealed the involvement of Müller glial-mesenchymal transition (GMT), as an alternative to EMT, in the pathogenesis of iERM lacking epithelial contribution in nature. Of various pro-fibrotic cytokines, transforming growth factor (TGF)-β1 stimulation to human Müller glial cells exclusively increased mRNA and protein levels of several EMT-related molecular markers, together with the transcription factor SNAIL but not SLUG or TWIST. TGF-β1-stimulated Müller cells also exhibited EMT-related cell motility, while reducing the expression of glutamine synthetase (GS), a Müller glial marker. Notably, all of these TGF-β-induced EMT features were reversed by SNAI1 knockdown in Müller cells. iERM patient specimens demonstrated co-immunolocalization of SNAIL with TGF-β1, GS, and smooth muscle protein 22. Our data implicated a critical role of the TGF-β-SNAIL axis in Müller GMT to promote iERM formation.

Topics: Aged; Biomarkers; Cell Movement; Cytokines; Cytoskeleton; Ependymoglial Cells; Epiretinal Membrane; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation; Humans; Male; Middle Aged; Nuclear Proteins; Snail Family Transcription Factors; Transforming Growth Factor beta; Twist-Related Protein 1

Retinal fibrosis, including epiretinal membrane (ERM) and proliferative vitreoretinopathy (PVR), is an ocular disease that can lead to blindness. An efficacious therapy remains an unmet medical challenge. In this study, we intended to explore the inhibitory effects of the nanoparticle-mediated delivery of plasminogen kringle 5 (K5-NPs) on laser-induced ERM and to identify the potential anti-fibrosis targets of K5-NPs. A rat model of laser-induced ERM was used. Control-NPs or K5-NPs were intravitreally injected. ERM formation was observed in vivo. The expression of fibrosis-associated factors including TGF-β, CTGF, and α-SMA was detected by qRT-PCR, Western blot, or immunohistochemistry. Our results showed that K5-NPs effectively inhibit laser-induced ERM formation. The expression of α-SMA, the marker of myofibroblast, apparently decreased in immunostained ERMs in the K5-NPs group. The expression of CTGF, a pro-fibrotic factor, in our ERM rat model was significantly downregulated by K5-NPs. Meanwhile, the expression of TGF-β, the upstream regulator of CTGF, was found to be suppressed by K5-NPs as well. The anti-fibrosis effect of K5-NPs might be achieved by interfering with TGF-β expression, thus abrogating the TGF-β-induced upregulation of CTGF and α-SMA. Our study has an important clinical relevance, demonstrating that K5-NPs might offer an additional efficacious clinical option for treating fibrosis-related diseases.

Topics: Actins; Animals; Blotting, Western; Connective Tissue Growth Factor; Epiretinal Membrane; Immunohistochemistry; Myofibroblasts; Nanoparticles; Peptide Fragments; Plasminogen; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

To investigate associations between expressions of advanced glycation end products (AGEs), transforming growth factor-beta (TGF-beta), tumour necrosis factor-alpha (TNF-alpha) and integrins and correlations between their expression and level of vascularization and proliferative activity in diabetic fibrovascular epiretinal membranes.. Membranes from eight patients with active proliferative diabetic retinopathy and nine patients with inactive proliferative diabetic retinopathy were studied by immunohistochemistry.. Blood vessels expressed AGEs, TGF-beta, TNF-alpha and alpha(v)beta(3) integrin in 5, 13, 8 and 8 membranes, respectively. Stromal cells expressed AGEs, TNF-alpha and alpha(v)beta(3) integrin in 15, 13 and 3 membranes, respectively. There was no immunoreactivity for alpha(v)beta(5), alpha(5)beta(1) and alpha(2)beta(1) integrins. There were significant correlations between number of blood vessels expressing CD34 and number of blood vessels expressing AGEs (r(s) = 0.496; P = 0.043), TGF-beta (r(s) = 0.777; P < 0.001) and TNF-alpha (r(s) = 0.699; P = 0.002). There were significant correlations between number of blood vessels expressing AGEs and number of blood vessels expressing TGF-beta (r(s) = 0.532; P = 0.028) and TNF-alpha (r(s) = 0.626; P = 0.007). The correlation between number of blood vessels expressing TNF-alpha and alpha(v)beta(3) integrin was significant (r(s) = 0.617; P = 0.008). Number of blood vessels expressing CD34 (P = 0.001), TGF-beta (P = 0.006) and TNF-alpha (P = 0.002) and stromal cells expressing AGEs (P = 0.001) and TNF-alpha (P = 0.004) were significantly higher in active membranes than in inactive membranes.. Interactions of AGEs, TGF-beta, TNF-alpha and alpha(v)beta(3) integrin might be involved in pathogenesis of proliferative diabetic retinopathy fibrovascular proliferation.

Topics: Antigens, CD34; Diabetic Retinopathy; Endothelium, Vascular; Epiretinal Membrane; Glycation End Products, Advanced; Humans; Immunoenzyme Techniques; Integrins; Ki-67 Antigen; Retinal Vessels; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

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

We studied the cellular constituents and the expression of vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta2) in idiopathic epiretinal membranes (ERMs), attempting to correlate the presence of these growth factors with fluorescein leakage during angiography and with the amount of macular scar tissue.. Idiopathic ERMs were excised at vitrectomy in 41 consecutive cases and stained for glial fibrillary acidic protein (GFAP), vimentin, cytokeratin, desmin and actin. A subset of 13 cases for which fluorescein angiograms and colour retinal photographs were available was further studied for the presence of VEGF and TGF-beta2 in the ERMs, fluorescein leakage and amount of macular scar tissue.. Of the 41 ERMs, 31 (76%) were found to be fibroglial by light microscopy; 40 (98%) stained for GFAP, 39 (95%) for vimentin, 10 (24%) for cytokeratin, 3 (7%) for desmin and 11 (27%) for actin. Of the 13 ERMs in the subset, staining was positive for VEGF in 11 (85%) and for TGF-beta2 in 11 (85%). There was no statistically significant relationship between the presence of VEGF and leakage (p = 0.68) or between the presence of TGF-beta2 and scar size (p = 0.90). When both VEGF and TGF-beta2 were present, there was likely to be leakage or a large scar, or both, which suggested that an interaction exists between the two growth factors (p = 0.057). When leakage occurred, large scars were 2.5 times less likely to be present; when no leakage occurred, large scars were 2.5 times more likely to be present (odds ratio 0.4; Yules association coefficient -0.43).. The cells constituting idiopathic ERMs were primarily fibroglial with minimal staining evidence for the presence of contractile proteins in their cytoplasm. VEGF and TGF-beta2 were present in 85% of specimens. Although there was no direct correlation between the presence of these growth factors and either fluorescein leakage or the abundance of scar tissue respectively, there was some evidence for the interaction of these growth factors in producing either leakage or abundance of scar tissue.

Topics: Capillary Permeability; Cytoskeletal Proteins; Epiretinal Membrane; Fluorescein Angiography; Humans; Immunoenzyme Techniques; Photography; Retinal Vessels; Transforming Growth Factor beta; Transforming Growth Factor beta2; Vascular Endothelial Growth Factor A; Vitrectomy

To evaluate the results of a third macular hole surgery in eyes with recurrent macular holes and two prior macular hole surgeries.. Retrospective consecutive noncomparative case series.. Sixteen eyes of sixteen patients with two prior macular hole surgeries with recurrent macular hole.. A third vitreous surgery was performed in each eye using a long-acting gas bubble.. Closure of the macular hole and change in visual acuity.. The macular hole was closed in 12 of 16 eyes (75%) at 3 months after the third surgery. Visual acuity improved 2 or more Snellen lines in 9 of 16 eyes (56%), and 5 of 16 eyes (31%) achieved 20/40 or better vision. Six eyes (37.5%) had cataract surgery after the third macular hole surgery, and visual acuity results were similar in eyes with or without cataract surgery. Successful closure of the macular hole improved the visual acuity from 20/80 -1 to 20/50 +1 (P < 0.001). Eyes in which one of the previous surgeries had been temporarily successful in closing the macular hole improved from a mean of 20/80 to 20/40 (P = 0.003). Eyes in which both prior macular hole surgeries had been primary failures had minimal benefit with a preoperative visual acuity of 20/100 +1 and a postoperative visual acuity of 20/100 +2 (P = 0.67).. Repeat macular hole surgery should be considered in eyes with recurrent macular holes and two prior surgeries when the macular hole was temporarily closed by at least one of the two previous surgeries. Successful closure of a macular hole in such cases usually results in significant visual acuity improvement.

Topics: Adult; Aged; Chemotherapy, Adjuvant; Epiretinal Membrane; Female; Humans; Intraoperative Complications; Male; Middle Aged; Platelet-Derived Growth Factor; Recombinant Proteins; Recurrence; Reoperation; Retinal Perforations; Retrospective Studies; Transforming Growth Factor beta; Treatment Outcome; Visual Acuity; Vitrectomy

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

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