Proliferation Of Human Retinal Pigment Epithelial Cells Research Articles

LYTAK1 attenuates proliferation of retinal pigment epithelial cells through TGF-β-mediated epithelial-mesenchymal transition via the ERK/AKT signaling pathway.

Retinal pigment epithelial (RPE) cells have crucial roles in the initiation and development of human ophthalmic diseases. Our previous study suggested that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a potential target in the progression and pathogenesis of human proliferative vitreoretinopathy disease. The present study further analyzed the role of TAK1 inhibitor, LYTAK1, in human RPE cells and explored the potential molecular mechanism of LYTAK1-mediated proliferation of human RPE cells. Proliferation of human RPE cells was investigated following treatment with LYTAK1 and knockdown of TGF-β. TGF-β-mediated epithelial-mesenchymal transition (EMT) through regulation of the extracellular signal-regulated kinase (ERK)/protein kinase B (AKT) signaling pathway was also explored to analyze the LYTAK1-mediated mechanism of proliferation in human RPE cells. The present results demonstrated that LYTAK1 administration suppressed TAK1 gene and protein expression in human RPE cells. LYTAK1 administration also inhibited proliferation and migration of human RPE cells in vitro. Outcomes indicated that LYTAK1 treatment downregulated expression levels of TGF-β1 and EMT markers, including cadherin, fibronectin and α-smooth muscle actin in human RPE cells. Notably, results demonstrated that the ERK/AKT signal pathway was blocked by LYTAK1 in human RPE cells. Knockdown of TGF-β markedly inhibited phosphorylation and activity of TAK1 and suppressed the LYTAK1-mediated ERK/AKT signaling pathway in RPE cells, which further canceled inhibition of RPE cell proliferation by LYTAK1. In conclusion, these findings indicated that LYTAK1 may inhibit RPE cell proliferation through the TGF-β-mediated EMT/ERK/AKT signaling pathway, suggesting that TAK1 may be a potential target for the treatment of RPE diseases.

Effect of chloride channel activity on retinal pigment cell proliferation and migration.

The present study aimed to investigate the effects of chloride channels (ClC) on the proliferation and migration of retinal pigment epithelial (RPE) cells, a primary component of proliferative vitreoretinopathy (PVR) membranes. An RPE cell model of phagocytosis was established using fibronectin‑coated latex beads. Cell proliferation was measured by live cell counting. The cell cycle and phagocytosis index was assessed by flow cytometry. Intracellular calcium concentration was quantified using Fura‑2‑acetoxymethyl ester. ClCs were blocked using 5‑nitro‑2‑(3‑phenylpropylamino) benzoic acid (NPPB) and tamoxifen (TAM). NPPB and TAM were identified to inhibit the proliferation of ARPE‑19 human adult RPE cells by arresting them in the G0/G1 phase, inhibit the phagocytosis of fibronectin, and decrease intracellular calcium levels, in a dose‑dependent manner. ClCs serve important roles in mediating human RPE cell proliferation and migration. The underlying mechanisms of action of ClCs are associated with the regulation of calcium. Targeting ClCs may provide a novel strategy to inhibit PVR formation.

Effect of Robo1 on Retinal Pigment Epithelial Cells and Experimental Proliferative Vitreoretinopathy

The Roundabout (Robo) family of proteins is related to the transmembrane receptors and plays a major role in neurogenesis. However, the role of the Robo proteins in proliferative retinopathy has not yet been defined. This study was conducted to determine whether Robo1 is expressed in the retina of patients with proliferative retinal disease and whether it has a pathobiological role in the disease. Immunohistochemistry was used to determine the presence and distribution of Robo1 in the pathologic membranes in proliferative retinopathy. Small interfering (si)RNA technology was used to knockdown Robo1 expression and to study its effects on retinal pigment epithelial (RPE) cells in vitro. The impact on PVR development of blocking Robo1 expression was determined by applying specific siRNA in a PVR rabbit model. The prevalences of PVR and retinal detachment were determined by indirect ophthalmoscope on days 1, 3, 7, 14, 21, and 28 after the injection of RPE cells into the vitreous. Immunohistochemistry showed that Robo1 expression was detected in GFAP-labeled glial cells and cytokeratin-labeled RPE cells in proliferative membranes. Robo1 expression was also detected in CD31-labeled vascular endothelial cells. Knockdown of Robo1 expression not only reduced human RPE cell proliferation in vitro but also effectively suppressed the development of PVR in a rabbit model. Robo1 is present in the extracellular matrix of proliferative membranes and may be derived from dedifferentiated RPE cells. Silencing the expression of Robo1 in RPE cells inhibited cell proliferation and suppressed the development of PVR in an animal model, indicating a potential therapeutic usefulness in treating PVR.

Chemotactic and Cytotoxic Effects of Minocycline on Human Retinal Pigment Epithelial Cells

To reveal the effects of minocycline, an anti-inflammatory and neuroprotective agent, on the viability and physiological properties of retinal pigment epithelial (RPE) cells and to compare the effects with those of triamcinolone acetonide. The proliferation of human RPE cells in vitro was investigated with a bromodeoxyuridine immunoassay; chemotaxis was examined with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. The gene expression of growth factors and MMP-9 was determined with real-time RT-PCR, and the secretion of VEGF was examined with ELISA. The phosphorylation of p38 MAPK and ERK1/2 proteins was determined with Western blot analysis. Minocycline at low concentrations (50 nM-20 microM) stimulated chemotaxis and decreased the proliferation of RPE cells. Minocycline at high concentrations (above 5 microM) decreased the viability of RPE cells through the induction of cell necrosis. The chemotactic effect of minocycline was mediated by the stimulation of autocrine PDGF signaling and the activation of p38 MAPK. Minocycline promoted the expression of PDGF-B, HGF, VEGF, and MMP-9 and increased the amounts of phosphorylated p38 and ERK1/2 proteins in RPE cells. Triamcinolone reduced PDGF-evoked chemotaxis and VEGF expression and secretion and had no significant effects on cell viability and proliferation. Triamcinolone did not reverse the effects of minocycline on cell proliferation, chemotaxis, or viability or the expression of VEGF. Low-dose minocycline induces the activation of RPE cells, as indicated by the activation of p38 and ERK1/2 and by enhanced chemotaxis mediated by autocrine PDGF signaling. High-dose minocycline induces RPE cell degeneration.

Epidermal Growth Factor Receptor in Cultured Human Retinal Pigment Epithelial Cells

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Migration of retinal pigment epithelial cells in vitro modulated by monocyte chemotactic protein-1: enhancement and inhibition.

The migration of retinal pigment epithelial (RPE) cells is an initial step in the development of proliferative vitreoretinopathy (PVR). This in vitro study was carried out to investigate the effects of monocyte chemotactic protein-1 (MCP-1) on the migration and proliferation of RPE cells. We used an in vitro wound healing model in which a small area of a confluent monolayer of human RPE (HRPE) cells was denuded with a razor blade. The cultures were subsequently incubated with MCP-1, IL-1beta, TNF-alpha, or combinations thereof. Neutralizing IgG1 of antihuman MCP-1, dexamethasone (DEX) or daunorubicin were also added to the cultures to test their inhibitory effects on migration of RPE cells. HRPE migration was measured as the number of cells that entered the denuded area. The effect of MCP-1 on proliferation of HRPE cells was examined by MTT assay. MCP-1 stimulated HRPE cell migration in a dose-dependent manner. IL-1beta or TNF-alpha slightly stimulated HRPE cell migration, but adding anti-MCP- IgG1 significantly reduced this effect. MCP-1-induced migration could be inhibited by DEX but not by daunorubicin. MCP-1 did not show a significant effect on HRPE cell proliferation. MCP-1 stimulates HRPE cell migration, suggesting that this chemokine regulates the development of PVR at the initial stage. The migration of HRPE cells induced by IL-1beta and TNF-alpha may be associated with the MCP-1 that HRPE cells secretes under the stimulation of these two cytokines. The knowledge that MCP-1-induced migration of HRPE cells is inhibited by DEX may be useful in devising an effective treatment for PVR.

Thalidomide and Prednisolone Inhibit Growth Factor-Induced Human Retinal Pigment Epithelium Cell Proliferation in vitro

Thalidomide and prednisolone were recently introduced as treatment modalities in age-related macular degeneration (AMD). Growth factor-induced activation of retinal pigment epithelial (RPE) cells is a crucial event in this disease. The purpose was to examine the effect of thalidomide and prednisolone on growth factor-preactivated RPE cells. Human RPE cells were stimulated with 10 ng/ml platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), or vascular endothelial growth factor (VEGF) for 24 h. Afterwards, thalidomide (50 µg/ml) or prednisolone (100 ng/ml) were added for 24 h. RPE cell proliferation was determined by [³H]-thymidine incorporation. PDGF and bFGF significantly stimulated human RPE cell proliferation (p < 0.005), the value for VEGF stimulation was not significant (p = 0.3). The effect of the growth factors was diminished after addition of thalidomide and prednisolone (p < 0.005). The current study shows that the inhibitory properties of thalidomide and prednisolone remain even after growth factor activation of the cells.

Growth factor combinations modulate human retinal pigment epithelial cell proliferation

Purpose. Retinal pigment epithelial (RPE) cell proliferation is an important step in the pathogenesis of ocular diseases such as proliferative retinopathy. Growth factors play a major role in modulating the development and progression of these diseases. The aim of this study was to examine the effects of single growth factors as compared to growth factor combinations on the stimulation of RPE cell proliferation. Methods. Human RPE cells were treated with 10 ng/ml IGF-1, PDGF, bFGF, EGF, VEGF, and TGFß 2, or with the combination of each of the growth factors. RPE cell proliferation was determined by [ 3 H]-thymidine incorporation after growth factor stimulation for 24 hr. Results. RPE cell proliferation was significantly increased when PDGF and bFGF were combined as compared to the single growth factor (p < 0.005). There was a decrease in DNA synthesis for combined growth factors as compared to the single growth factor for IGF-1 plus TGFß 2 (p < 0.05), IGF-1 plus VEGF (p < 0.005), PDGF plus TGFß 2 (p < 0.005), VEGF plus TGFß 2 (p < 0.005 as compared to VEGF), and bFGF plus TGFß 2 (p = 0.01 as compared to bFGF). The remaining growth factor combinations showed no significant synergistic effect on human RPE cell proliferation. Conclusions. These data lead to the implication that DNA synthesis in human RPE cell culture is synergistically affected by certain growth factor combinations, but not by all of them. This observation may be important for a better understanding of growth factor interactions occuring in proliferative diseases in the eye.