epidermal-growth-factor and Vitreoretinopathy--Proliferative

Abnormal proliferation and motility of retinal pigment epithelial cells leads to proliferative vitreoretinopathy (PVR). Melatonin is a known effective antitumour and anti-invasive agent, but whether it affects the formation and underlying mechanisms of PVR remains unclear. In this study, the results of the MTT assay, colony formation and propidium iodide (PI) staining with flow cytometry revealed that melatonin dose dependently inhibited epidermal growth factor (EGF)-induced proliferation of human ARPE-19 cells. Furthermore, melatonin reduced EGF-induced motility by suppressing cathepsin S (CTSS) expression. Pretreatment with ZFL (a CTSS inhibitor) or overexpression of CTSS (pCMV-CTSS) significantly inhibited EGF-induced cell motility when combined with melatonin. Epidermal growth factor induced the phosphorylation of AKT(S473)/mTOR (S2448) and transcription factor (c-Jun/Sp1) signaling pathways. Pretreatment of LY294002 (a PI3K inhibitor) or rapamycin (an mTOR inhibitor) markedly reduced EGF-induced motility and p-AKT/p-mTOR/c-Jun/Sp1 expression when combined with melatonin. Taken together, these data indicate that melatonin inhibited EGF-induced proliferation and motility of human ARPE-19 cells by activating the AKT/mTOR pathway, which is dependent on CTSS modulation of c-Jun/Sp1 signalling. Melatonin may be a promising therapeutic drug against PVR.

Topics: Cathepsins; Cell Line; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Gene Expression; Humans; Melatonin; Models, Biological; Protective Agents; Retinal Pigment Epithelium; Signal Transduction; Vitreoretinopathy, Proliferative

Proliferative vitreoretinopathy (PVR) is a common refractory eye disease that causes blindness and occurs after retinal detachment or retinal reattachment. Epidermal growth factor (EGF) has been shown to play an important role in the migration and proliferation of retinal pigment epithelium (RPE) cells, which promote PVR. Curcumin inhibits RPE cell proliferation, but it is not known whether it participates in the formation of PVR. Curcumin regulates the biological functions of EGF, which plays important roles in the development of PVR. This study aimed to evaluate the effect of curcumin on the regulation of EGF in PVR.. Rabbit RPE cells were cultured, and EGF expression was detected by immunocytochemistry. MTT assay was conducted to determine cell proliferation induced by different concentrations of EGF. Immunocytochemical staining was used to detect EGF expression after treatment with curcumin at varying concentrations. Real-time PCR (RT-PCR) and western blot analysis were used to detect the concentrations of EGF mRNA and protein after treatment with curcumin. After RPE cells and curcumin were injected into experimental rabbit eyes, the cornea, aqueous humor, lens, and vitreous opacity were observed and recorded simultaneously by indirect ophthalmoscopy, fundus color photography, and B-ultrasonography. The vitreous body was extracted, and the EGF content in the vitreous humor was measured by enzyme-linked immunosorbent assay (ELISA).. At each time point (24, 48, and 72 h), cell proliferation gradually increased with increasing EGF concentrations (0, 3, 6, and 9 ng/mL) in a dose-dependent manner. Cell proliferation between EGF concentrations of 9 and 12 ng/mL were no different, which suggested that 9 ng/mL EGF was the best concentration to use to stimulate RPE cell proliferation in vitro. Under all EGF concentrations (0, 3, 6, 9, and 12 ng/mL), RPE cell proliferation increased with time (from 24 to 72 h), suggesting a time-effect relationship. Curcumin downregulated EGF expression in RPE cells, which also indicated time-effect and dose-effect relationships. The best curcumin concentration for the inhibition of EGF expression was 15 µg/mL. RT-PCR and western blot analyses indicated that the EGF mRNA and expression of the protein in RPE cells treated with curcumin significantly decreased with time. Ocular examinations revealed that the vitreous opacity was lower and the proliferative membrane was thinner in the curcumin group compared with the control group. The PVR grade and the incidence of retinal detachment were significantly lower in the experimental group than in the control group. ELISA results showed that the EGF content in vitreous humor was higher in the control group than in the curcumin group. The curcumin and control groups were significantly different at each time point.. Curcumin inhibited RPE cell proliferation by downregulating EGF and thus effectively inhibited the initiation and development of PVR.

Topics: Animals; Cell Proliferation; Curcumin; Disease Models, Animal; Dose-Response Relationship, Drug; Epidermal Growth Factor; Rabbits; Retinal Pigment Epithelium; Vitreoretinopathy, Proliferative

Proliferative vitreoretinopathy (PVR) is mediated by proliferation and epithelial mesenchymal transition (EMT) of retinal pigment epithelium (RPE). Because heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) purified from human amniotic membrane exerts anti-inflammatory and anti-scarring actions, we hypothesized that HC-HA/PTX3 could inhibit these PVR-related processes in vitro. In this study, we first optimized an ARPE-19 cell culture model to mimic PVR by defining cell density, growth factors, and cultivation time. Using this low cell density culture model and HA as a control, we tested effects of HC-HA/PTX3 on the cell viability (cytotoxicity), proliferation (EGF + FGF-2) and EMT (TGF-β1). Furthermore, we determined effects of HC-HA/PTX3 on cell migration (EGF + FGF-2 + TGF-β1) and collagen gel contraction (TGF-β1). We found both HA and HC-HA/PTX3 were not toxic to unstimulated RPE cells. Only HC-HA/PTX3 dose-dependently inhibited proliferation and EMT of stimulated RPE cells by down-regulating Wnt (β-catenin, LEF1) and TGF-β (Smad2/3, collagen type I, α-SMA) signaling, respectively. Additionally, HA and HC-HA/PTX3 inhibited migration but only HC-HA/PTX3 inhibited collagen gel contraction. These results suggest HC-HA/PTX3 is a non-toxic, potent inhibitor of proliferation and EMT of RPE in vitro, and HC-HA/PTX3's ability to inhibit PVR formation warrants evaluation in an animal model.

Topics: Biomarkers; C-Reactive Protein; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Hyaluronic Acid; Protein Transport; Retinal Pigment Epithelium; Serum Amyloid P-Component; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1; Vitreoretinopathy, Proliferative

Proliferation and epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) are hallmarks of proliferative vitreoretinopathy. This study aims at clarifying the role of growth factors, such as epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), and transforming growth factor-β1 (TGF-β1), in controlling how RPE proliferates while undergoing EMT. When contact inhibition of post-confluent ARPE-19 cells was disrupted by EGTA, an increase of BrdU labeling was noted only in the presence of EGF and/or FGF-2, and was accompanied by EMT as evidenced by the loss of a normal RPE phenotype (altered cytolocalization of RPE65, N-cadherin, ZO-1, and Na,K-ATPase) and the gain of a mesenchymal phenotype (increased expression of vimentin, S100A4, and α-smooth muscle actin). EMT with proliferation by EGTA+EGF+FGF-2 was accompanied by activation of canonical Wnt signaling (judged by the TCF/LEF promoter activity, increased nuclear levels of and interaction between β-catenin and LEF1 proteins, and the replication by overexpression of β-catenin), abolished by concomitant addition of XAV939, a Wnt inhibitor, but not associated with suppression of Hippo signaling (negative expression of nuclear TAZ or YAP and cytoplasmic p-TAZ or p-YAP). The causative role of Wnt signaling on EMT with proliferation was confirmed by overexpression of stable S33Y β-catenin with EGTA treatment. In addition, contact inhibition disrupted by EGTA in the presence of TGF-β1 also led to EMT, but suppressed proliferation and Wnt signaling. The Wnt signaling triggered by EGF+FGF-2 was sufficient and synergized with TGF-β1 in activating the Smad/ZEB1/2 signaling responsible for EMT. These findings establish a framework for further dissecting how RPE might partake in a number of proliferative vitreoretinopathies characterized by EMT.

Topics: beta Catenin; Cell Proliferation; Cells, Cultured; Contact Inhibition; Egtazic Acid; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Fibroblast Growth Factor 2; Heterocyclic Compounds, 3-Ring; Homeodomain Proteins; Humans; Repressor Proteins; Retinal Pigment Epithelium; Signal Transduction; Smad Proteins; Transcription Factors; Transforming Growth Factor beta1; Vitreoretinopathy, Proliferative; Wnt Proteins; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

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

Proliferation and migration of retinal pigment epithelial (RPE) cells play a crucial role in proliferative vitreoretinopathy (PVR)-related pathology. Cytokines, including EGF, can result in RPE cell activation and cause PVR. In this study, integrin-alpha(5) expression was first studied in PVR membranes by immunofluorescence. Then the effect of EGF on integrin-alpha(5) expression was determined by flow cytometry, Western blot analysis and the reverse-transcription polymerase chain reaction (RT-PCR) in the ARPE-19 cell line. Proliferation and migration of ARPE-19 cells were measured by the methylthiazolyldiphenyl-tetrazolium bromide and Boyden chamber assays. We found that a higher level integrin-alpha(5) was present at the RPE cell surface in PVR compared with normal retina. EGF could dose dependently increase integrin-alpha(5) mRNA and protein levels in vitro. EGF promoted ARPE-19 cell proliferation and migration. Neutralizing integrin-alpha(5) by specific anti-integrin-alpha(5) antibody abolished most of the effects of EGF. The study provided evidence that EGF might influence PVR by promoting integrin-alpha(5) expression and subsequent proliferation and migration of RPE.

Topics: Adult; Cell Line; Cell Membrane; Cell Movement; Cell Proliferation; Cell Survival; Epidermal Growth Factor; Female; Fluorescent Antibody Technique; Gene Expression; Humans; Integrin alpha5; Male; Middle Aged; Retinal Pigment Epithelium; Vitreoretinopathy, Proliferative

Migration and proliferation of retinal pigment epithelial (RPE) cells play an important role in proliferative vitreoretinopathy. Epidermal growth factor receptor (EGFR) is a cell surface receptor with intrinsic tyrosine kinase activity. The engagement of the receptor by its ligand can induce intracellular mitogenic signal transduction pathways and stimulate proliferation, migration and differentiation of cells. This experiment aimed to investigate the activation and role of EGFR signal transduction pathway in proliferation of human RPE cells.. Cultured human RPE cells of the 3rd to 6th passages were studied by colorimetric assay for cellular growth and survival (MTT assay) to test the effects of EGF (0.1, 1, 10, 50, and 100 ng/ml) and fetal bovine serum (FBS) on proliferation of human RPE cells. An in vitro wound healing model was also set up, and the number of cells that had entered the denuded area was counted. The human RPE cells were cultured for 3 days with 0.1% FBS, 10% FBS, 10 ng/ml EGF + 0.1% FBS and a combination of EGF and 10% FBS, respectively. Immunohistochemical staining and in situ hybridization were used to observe the expressions of EGFR protein and mRNA, respectively. Activation of mitogen-activated protein kinase (MAPK) was detected by immunohistochemical method with specific antiphosphorylated extracellular signal-regulated kinase (ERK)1/2 antibody.. EGF stimulated proliferation and migration of cultured human RPE cells in a concentration-dependent manner. The maximum of the proliferation rate of RPE cells was 81.8% with EGF at a concentration of 10-100 ng/ml of EGF in serum-free Dulbecco's modified essential medium (DMEM) and 122.7% at a concentration of 1-10 ng/ml of EGF in 5% FBS DMEM (p < 0.001); there was a significant difference between serum-free DMEM groups and 5% FBS DMEM groups. The maximum of the migration rate of the cells was 438.9% at a concentration of 10-100 ng/ml of EGF in 10% FBS DMEM, 147% with 10% FBS, and only 36% with EGF in 0.1% FBS at the concentration of 10 ng/ml (p < 0.001). EGF promoted the expression of EGFR protein and mRNA in RPE cells. FBS cooperated with EGF in the stimulation of EGFR expression, and it had a stronger effect in the process than EGF alone. After 3 days of incubation with EGF, phosphorylated ERK1/2 was detectable in the nucleus of RPE cells, whereas cells presented immunostaining positive for phosphorylated ERK1/2 in the cytoplasm before stimulation, indicating that EGF could induce MAPK nuclear translocation.. EGF could induce EGF-EGFR-MAPK signal transduction pathway in human RPE cells in a concentration-dependent manner in vitro, which may play a key role in the activation of human RPE cell proliferation and migration.

Topics: Cell Movement; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoenzyme Techniques; In Situ Hybridization; Mitogen-Activated Protein Kinases; Phosphorylation; Pigment Epithelium of Eye; RNA, Messenger; Signal Transduction; Vitreoretinopathy, Proliferative

Proliferative vitreoretinopathy (PVR), a major reason for failure of retinal detachment surgery, is characterized by the formation of scarlike tissue that contains transdifferentiated retinal pigment epithelial (RPE) cells. The scar tissue occurs in response to growth factors such as transforming growth factor (TGF)-beta and epidermal growth factor (EGF). The authors postulate that transdifferentiation of RPE cells may arise via epithelial-to-mesenchymal transition (EMT). Bone morphogenetic proteins (BMPs) are expressed in the retina and have an antiproliferative role. Gremlin is expressed in the outer retina and is a BMP antagonist. The study was conducted to establish a model of PVR by inducing EMT in the human RPE cell line ARPE-19, using TGF-beta and EGF and to establish the contribution of gremlin to EMT.. ARPE-19 cells were cultured and stimulated with TGF-beta1, EGF, and gremlin. The expression of alpha-smooth muscle actin (alpha-SMA), vimentin, and zona occludens (ZO)-1 were examined via PCR, Western blot analysis, and immunofluorescence. Zymography was performed for matrix metalloproteinase (MMP) activity. Scratch assays were performed to assess migration.. A model of EMT was established in the ARPE-19 cell line. The characteristics of EMT include gain of alpha-SMA, loss of ZO-1, upregulation of MMP activity and enhanced migration. Gremlin plays an important role in this process, contributing to the gain of alpha-SMA, loss of ZO-1, and upregulation of MMP activity.. EMT occurs in vitro in the ARPE-19 cell line in response to the growth factors TGF-beta1 and EGF. EMT is also induced by Gremlin.

Topics: Actins; Biomarkers; Blotting, Western; Bone Morphogenetic Proteins; Cell Differentiation; Cell Movement; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; Fluorescent Antibody Technique, Indirect; Humans; Intercellular Signaling Peptides and Proteins; Membrane Proteins; Mesoderm; Models, Biological; Phosphoproteins; Pigment Epithelium of Eye; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta1; Vimentin; Vitreoretinopathy, Proliferative; Zonula Occludens-1 Protein

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

To compare the gene expression pattern of control postmortem retinas with retinas from patients with proliferative vitreoretinopathy (PVR), to determine the expression of the heparin binding epidermal growth factor-like growth factor (HB-EGF) by glial cells in fibroproliferative membranes, and to examine whether cells of the human Müller cell line, MIO-M1, respond to HB-EGF with proliferation, migration, and secretion of the vascular endothelial growth factor (VEGF).. To identify genes that were differently expressed in PVR and control retinas, the RNA from the neural retinas of seven postmortem donors and of two patients with PVR were analyzed for differential gene expression, by hybridization of labeled cRNA probes to an Affymetrix human genome microarray set. The results were validated by real time PCR experiments investigating RNA from 6 postmortem retinas and 4 PVR retinas. Epiretinal PVR membranes were immunohistochemically stained for colocalization of HB-EGF and the glial cell marker, glial fibrillary acidic protein (GFAP). The HB-EGF evoked proliferation of cultured Müller cells was investigated by a bromodeoxyuridine immunoassay, chemotaxis was assessed with a migration assay, and the release of VEGF was evaluated by ELISA.. Out of the 12,600 genes and expressed sequence tags investigated, the levels of 80 showed an increased expression, and 21 were expressed at decreased levels, in the retinas of PVR patients compared to the control retinas. The upregulated signals include genes for nuclear and cell cycle related proteins, extracellular secretory proteins, cytosolic signaling proteins, and proteins of the membrane and the extracellular matrix. The genes of the hepatocyte growth factor and of HB-EGF were found to be expressed in PVR retinas but not in control retinas. In epiretinal membranes of patients with PVR, HB-EGF immunoreactivity partially colocalized with GFAP. In cultured Müller cells, HB-EGF stimulated both proliferation and chemotaxis, and the secretion of VEGF, via activation of the extracellular signal regulated kinases 1 and 2 and of the phosphatidylinositol-3 kinase.. The development of PVR is accompanied by complex changes of the gene expression in the neural retina, with an upregulation of genes that support cell proliferation, cell signaling, cell motility, and extracellular matrix remodeling. HB-EGF is one of the factors that are significantly upregulated in PVR retinas. HB-EGF expression in fibroproliferative tissue and its stimulatory effect on glial cell proliferation, chemotaxis, and VEGF secretion suggest that HB-EGF may be a factor mediating glial cell responses during PVR.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Blotting, Western; Cell Culture Techniques; Cell Proliferation; Chemotaxis; DNA; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epiretinal Membrane; Expressed Sequence Tags; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Heparin-binding EGF-like Growth Factor; Hepatocyte Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Male; Microscopy, Confocal; Middle Aged; Neuroglia; Oligonucleotide Array Sequence Analysis; Retina; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation; Vascular Endothelial Growth Factor A; Vitreoretinopathy, Proliferative

To investigate the relationships between proliferative vitreoretinopathy (PVR) formation and the levels of human epidermal growth factor (h-EGF) and tumor necrosis factor (TNF) in serum and subretinal fluid (SRF) before and after surgery of retinal detachment with PVR.. Serum and SRF samples of 51 patients with retinal detachment and PVR were collected, and their h-EGF and TNF levels were measured by radioimmunoassays.. In comparison with the control group, the levels of h-EGF and TNF in serum and SRF in retinal detachment with PVR group were higher but not statistically significant (P > 0.05). Their levels in the serum were not related to the severity of the disease, while their levels in SRF increased significantly with the increase of the severity of the disease, and these two factors were positively correlated (P < 0.05). 5 days after the surgery of retinal detachment, the h-EGF and TNF levels in serum increased significantly (P < 0.01) and the levels decreased within 30 days but not to normal.. The h-EGF and TNF in SRF might have a synergic action and affect the formation and development of PVR. The h-EGF and TNF in serum take part in the repair of the operative trauma and inflammatory reaction. However, their high levels could interfere with the effect of operation.

Topics: Adult; Epidermal Growth Factor; Extracellular Space; Exudates and Transudates; Female; Humans; Intraoperative Period; Male; Middle Aged; Retinal Detachment; Tumor Necrosis Factor-alpha; Vitreoretinopathy, Proliferative