transforming-growth-factor-beta and Pterygium

Recently, it has been reported that miRNA is involved in pterygium, however the exact underlying mechanism in pterygium is unrevealed and require further investigation.. The differential expression of miRNA in pterygium was profiled using microarray and validated with quantitative real-time polymerase chain reaction (qRT-PCR). Human conjunctival epithelial cells (HCEs) were cultured and treated with transforming growth factor β (TGF-β) and epidermal growth factor (EGF) and transfected with miR-199a-3p/5p mimic and inhibitor. Markers of epithelial-mesenchymal transition (EMT) in HCEs were detected using western blot and immunohistochemistry. Cell migration ability was determined using wound healing and transwell assay, while apoptosis was determined by flow cytometry. The target genes of miR-199a were confirmed by the dual-luciferase reporter assay.. TGF-β and EGF could induced EMT in HCEs and increase miR-199a-3p/5p but suppress target genes, DUSP5 and MAP3K11. With the occurrence of EMT, cell migration ability was enhanced, and apoptosis was impeded. Promoting miR-199a-3p/5p expression could induce EMT in HCEs without TGF-β and EGF, while suppressing miR-199a-3p/5p could inhibit EMT in TGF-β and EGF induced HCEs. In a word, TGF-β and EGF induced EMT could be regulated with miR-199a-3p/5p-DUSP5/MAP3K11 axes. The validated results in tissues showed that, compared with control conjunctival tissues, miR-199a-3p/5p were more overexpressed in pterygium, while DUSP5/MAP3K11 were lower expressed. In addition, bioinformatics analysis indicated the miR-199a-3p/5p-DUSP5/MAP3K11 was belong to MAPK signalling pathway.. TGF-β and EGF induce EMT of HCEs through miR-199a-3p/5p-DUSP5/MAP3K11 axes, which explains the pathogenesis of EMT in pterygium and may provide new targets for pterygium prevention and therapy.

Topics: Dual-Specificity Phosphatases; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Humans; MicroRNAs; Pterygium; Transforming Growth Factor beta

To investigate the antifibrotic effects of sakuraso-saponin on a primary culture of human pterygium fibroblasts (HPFs) and normal human Tenon fibroblasts (HTFs) as compared to the effects of mitomycin C (MMC).. Samples of HPFs and HTFs were acquired during primary pterygium surgery. Cell toxicity, cell migration, and expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β (TGF-β) were evaluated in HPFs and HTFs after treatment with sakuraso-saponin and MMC. To determine the possible mechanisms underlying the antifibrotic effects of sakuraso-saponin, the expression of phosphorylated Smad2/3 was evaluated after treatment with sakuraso-saponin and MMC.. MMC (≥200 μg/mL) significantly reduced cell viability in both HPFs and HTFs, whereas sakuraso-saponin (1.0 μg/mL) decreased cell viability in HPFs only. Both sakuraso-saponin (1.0 μg/mL) and MMC (200 μg/mL) treatment significantly reduced the expression of α-SMA and TGF-β in HPFs (P < 0.05). It is interesting that the expression of α-SMA and TGF-β after treatment with sakuraso-saponin was significantly lower than that after treatment with MMC (P < 0.05). The expression of phosphorylated Smad2/3 protein was decreased by sakuraso-saponin and MMC in HPFs. Both sakuraso-saponin and MMC inhibited TGF-β1-induced cell migration as compared to the control in HPFs.. Sakuraso-saponin could be more effective than MMC for the reduction of fibrosis in HPFs. Our results might present the basis for its use as a promising candidate drug for adjuvant therapy to prevent recurrent pterygium after surgery.

Topics: Actins; Alkylating Agents; Blotting, Western; Cell Movement; Cell Survival; Cells, Cultured; Fibroblasts; Fibrosis; Humans; Mitomycin; Phosphorylation; Pterygium; Saponins; Smad2 Protein; Smad4 Protein; Tenon Capsule; Transforming Growth Factor beta

Vatalanib is a small-molecule tyrosine kinase inhibitor. We investigated the effects of vatalanib on the proliferation and migration of cultured human pterygial fibroblasts (HPFs).. Pterygium tissues were obtained after pterygium excision surgery and subjected to primary culture. HPFs were treated with vatalanib at various concentrations. Mitomycin C (MMC) was used as a positive control. Cell proliferation and migration assays were used to investigate the effects of vatalanib. Cell death was measured using flow cytometry analysis. Western blot analysis was performed to identify signaling molecules associated with the response to vatalanib.. Vatalanib inhibited both proliferation and migration of HPFs in a dose-dependent manner. Cell proliferation was significantly suppressed by vatalanib (10 and 100 μM) and MMC (0.004% and 0.04%) treatments. Migration assays revealed significant HPF delay when treated with vatalanib (1, 10, and 100 μM) and MMC (0.004% and 0.04%) compared with that in a negative control. Cell death analysis showed that high concentrations of vatalanib (100 μM) and MMC (0.004% and 0.04%) decreased cell numbers. Western blot analysis of vatalanib-treated cells showed vascular endothelial growth factor and transforming growth factor-β significantly reduced, but there was no alteration in p53 protein levels in HPFs.. These results indicate that vatalanib significantly suppressed the proliferation and migration of HPFs by decreasing vascular endothelial growth factor and transforming growth factor-β. Vatalanib showed less toxicity than that of MMC. Based on these results, vatalanib may potentially serve as a new adjuvant treatment after pterygium excision surgery.

Topics: Blotting, Western; Cell Movement; Cell Proliferation; Cells, Cultured; Fibroblasts; Humans; Phthalazines; Protein Kinase Inhibitors; Pterygium; Pyridines; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

To investigate the role of Tafazzin (TAZ) protein in regulating the proliferation of normal human conjunctiva epithelial cells and epithelial cells from pterygium tissue.. Conjunctiva epithelial cells were cultured in keratinocytes growth medium and treated with transformation growth factor β (TGFβ) to analyze the expression and translocation of TAZ protein by immunostaining and BrdU analysis. Immortalized conjunctiva epithelial cells (NHC) were treated with TGFβ, targeting siRNA, TGFβ receptor antibody or TGFβ receptor inhibitor, to study the involvement of TAZ and TGFβ signaling pathway in conjunctiva cell proliferation by cell adhesion assay. Conjunctiva tissues from a normal human eye and an eye with pterygium disease were collected for histological analyses and western blot to evaluate the TAZ protein expression in vivo.. TAZ expression was upregulated in mitotic conjunctiva epithelial cells, proliferating conjunctiva epithelial cells, TGFβ treated conjunctiva epithelial cells and human pterygium epithelium. TAZ siRNA induced less conjunctiva epithelial cell growth. Moreover, TGFβ receptor antibody and TGFβ receptor inhibitor rescued this anti-proliferative effect of TAZ siRNA.. TAZ is involved in human conjunctiva epithelial cells proliferation via regulating TGFβ signaling pathway.

Topics: Acyltransferases; Antibodies; Cell Adhesion; Cell Proliferation; Conjunctiva; Epithelial Cells; Gene Expression Regulation; Humans; Mitosis; Primary Cell Culture; Protein Transport; Pterygium; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

When used as an alternative substrate following bare sclera removal of pterygium and other ocular surface diseases, amniotic membrane transplantation can reduce scarring on the reconstructed conjunctival surface. This study was carried out to determine if the amniotic membrane (AM) suppresses the expression of the TGFb signaling system in cultured normal conjunctival (HCF) and pterygial body fibroblasts (PBF).. HCF and PBF were cultured on AM and plastic wells in serum-containing and serum-free DMEM with or without TGF-beta1. Total RNA was extracted and subjected to Northern hybridization with probes of TGF-beta1, b2 and b3; TGF-beta receptors (TGF- beta R) type I, II and III; a-smooth muscle actin (alpha-SM), b1-integrin, CD44, fibroblast growth factor receptor 1 (FGF-R1/ flg) and platelet-derived growth factor receptor b (PDGFR-beta); and GAPDH as a loading control. MTT assay was used for cell proliferation.. Amniotic membrane markedly suppressed the transcript expression of TGF-beta2, b3 and all three types of TGF-beta receptors by both fibroblasts as compared to their cultures on plastic surface. In addition, expression of CD44 transcript was also markedly suppressed while that of b1 integrin, a-SM actin, and FGFR1/flg was mildly suppressed. In contrast, expression of TGF-beta1 and PDGFR-beta remained largely unchanged. The cell proliferation of HCF and PBF grown on AM was also significantly suppressed.. Amniotic membrane matrix uniquely suppresses TGF- beta signaling in both types of fibroblasts. It may also suppress signaling via CD44, b1 integrin and FGFR1/flg. As a result, the phenotype may become less mitogenic, contractile and fibrogenic. These data support in part why amniotic membrane transplantation has an anti-scarring effect for conjunctival surface reconstruction.

Topics: Actins; Amnion; Cell Division; Cells, Cultured; Conjunctiva; Fibroblasts; Filaggrin Proteins; Humans; Hyaluronan Receptors; Integrin beta1; Muscle, Smooth; Protein Isoforms; Pterygium; Receptors, Fibroblast Growth Factor; Receptors, Platelet-Derived Growth Factor; Receptors, Transforming Growth Factor beta; Reference Values; Signal Transduction; Transforming Growth Factor beta

Some fibroangiogenic factors have recently been shown to play potential roles in fibrovascular diseases. The aim of this study was to examine whether there is any relationship between growth factors and pterygium genesis. Twenty-three primary pterygia and 4 normal conjunctiva specimens were analyzed by indirect immunohistochemistry using specific antibodies against basic fibroblast growth factor (b-FGF), platelet derived growth factor (PDGF), transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha). Positive immunostaining of these growth factors was located in the epithelial cells, endothelial cells of vessels, basement membranes of vessels and epithelium, fibroblasts and infiltrating inflammatory cells in the pterygium. In the normal conjunctiva, positive immunolabeling for TGF-beta and PDGF was much weaker than in the pterygium. We conclude that growth factors may interact directly or indirectly in the pathogenesis of pterygium although proof of this awaits further studies.

Topics: Antibodies, Monoclonal; Conjunctiva; Fibroblast Growth Factor 2; Humans; Immunohistochemistry; Platelet-Derived Growth Factor; Pterygium; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Pterygium is a degenerative corneal limbal process and UV irradiation has been suggested as being a major environmental predisposing factor. The invasive nature of the fibroblasts associated with pterygia raises the question as to whether these cells are transformed. To test this hypothesis, we established fibroblast strains from autologous and heterologous pterygial and conjunctival specimens, respectively, from subjects between 40 to 50 yr of age, and compared their growth characteristics in culture. All pterygial fibroblast strains exhibited a reduced dependence on serum and exogenous growth factors for growth and reached a saturation population density that was threefold higher than conjunctival fibroblasts cultured under the same conditions. In addition, all pterygial fibroblast strains were able to form colonies in soft agar in 5% fetal bovine serum at a 6.0 to 7.5% efficiency. Under the same experimental conditions, none of the conjunctival fibroblast strains were able to grow. The results presented support the conclusion that pterygial fibroblasts have acquired many of the properties of the transformed phenotype.

Topics: Adult; Cell Adhesion; Cell Division; Cell Line, Transformed; Conjunctiva; Epidermal Growth Factor; Fibroblast Growth Factor 1; Fibroblasts; Humans; Male; Middle Aged; Phenotype; Pterygium; Time Factors; Transforming Growth Factor beta