Differential chemokine regulation by Th2 cytokines during human RPE-monocyte coculture

PURPOSE: To determine the effects of the potent anti-inflammatory Th2 cytokines, interleukin (IL)-4, -10, and -13, on IL-8 and monocyte chemoattractant protein (MCP) 1 production by human retinal pigment epithelial (HRPE) cells, monocytes, and HRPE cell-monocyte cocultures. METHODS: Enzyme-linked immunosorbent assays were performed to determine IL-8 and MCP-1 secretion by HRPE cells, monocytes, and HRPE cell-monocyte cocultures stimulated with IL-1beta or TNF-alpha, either alone, or in combination with IL-4, -10, or -13, at various time points. RESULTS: IL-4 and -13, but not IL-10, enhanced constitutive and TNF-alpha-induced HRPE IL-8 and MCP-1 secretion. IL-4 also enhanced IL-1beta-induced HRPE IL-8. IL-4 and -13 reduced monocyte IL-8 and MCP-1, whereas IL-10 reduced monocyte IL-8 but enhanced MCP-1. Overlay of monocytes onto HRPE cell cultures resulted in increased IL-8 and MCP-1 secretion. IL-8 secretion by HRPE cell-monocyte cocultures was inhibited by IL-4, -10, and -13, whereas MCP-1 was inhibited only by IL-10. These cytokines also inhibited IL-1beta potentiation of IL-8, but not MCP-1 secretion by cocultures. IL-4 enhanced TNF-alpha potentiation of chemokine secretion by cocultures, whereas IL-10 had no effects. IL-13 potentiated TNF-alpha-induced MCP-1, but not IL-8 secretion by cocultures. CONCLUSIONS: IL-4, -10 and -13 have complex effects on chemokine secretion by HRPE cells, monocytes, and HRPE cell-monocyte cocultures. IL-10 appears to be the most consistently suppressive cytokine, suggesting potential therapeutic usefulness of IL-10 in the treatment of ocular inflammatory and proliferative diseases.     

Induction of interleukin-8 in human retinal pigment epithelial cells after denuding injury

AIM: To determine interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) expression in response to mechanical injury in human retinal pigment epithelial (HRPE) cells. METHODS: Enzyme linked immunosorbent assay (ELISA) was performed to determine IL-8 and MCP-1 secretion by HRPE cells after mechanical denudation. IL-8 and MCP-1 mRNA expression by HRPE cells was assessed using semiquantitative RT-PCR. The effects of immunosuppressive drugs, dexamethasone (DEX) and cyclosporin A (CSA), as well as immunosuppressive cytokines, interleukin 4 (IL-4), interleukin 10 (IL-10), and interleukin 13 (IL-13), on chemokine expression in HRPE cells after denuding injury were analysed. RESULTS: Mechanical injury induced HRPE IL-8 mRNA and IL-8 secretion. Although MCP-1 mRNA was enhanced slightly after denuding injury, MCP-1 secretion was not increased. DEX and CSA inhibited HRPE chemokine expression after injury. IL-4 and IL-13 enhanced IL-8 and MCP-1 production by HRPE cells after injury while IL-10 had no effect. CONCLUSIONS: These results suggest that IL-8 may be involved in retinal inflammatory responses to injury and that DEX and/or CSA treatment may help control the inflammatory components of retinal diseases such as proliferative vitreoretinopathy.     

Differential expression of chemokines by human retinal pigment epithelial cells infected with cytomegalovirus

PURPOSE: To evaluate the effects of human cytomegalovirus (HCMV) infection on chemokine gene expression and secretion by human retinal pigment epithelial (HRPE) cell cultures. METHODS: HRPE cells were infected with HCMV (strain AD169) at an MOI of 5. Culture supernatants, collected at various postinoculation days, were used for the analyses of chemokines by ELISA. The steady state levels of chemokine and chemokine receptor mRNA were analyzed by RT-PCR. Effects of interferon and MCP-1 on HCMV replication in HRPE cells were evaluated by plaque assays. RESULTS: HRPE cells infected with HCMV exhibited characteristic cytopathic effects. The reduction in the levels of monocyte chemotactic protein (MCP)-1 and -3 mRNA in HCMV-infected HRPE cells was observed in comparison to uninfected HRPE cells. In contrast, HCMV infection enhanced IL-8 mRNA levels, whereas regulated on activation normal T-cell expressed and secreted (RANTES) mRNA was not detectable in either control or infected HRPE cells. A significant decrease in MCP-1 (P < 0.01) and MCP-3 (P < 0.05), but a significant increase in IL-8 (P < 0.05), protein secretion was observed. Expression of the chemokine receptors CCR2, specific for MCP-1, and CXCR1 and CXCR2, specific for IL-8, were not altered by HCMV infection. Treatment of HRPE cultures with MCP-1 had no significant effect on HCMV replication in HRPE cells. CONCLUSIONS: HCMV infection in HRPE cells resulted in the modulation of MCP-1, MCP-3, and IL-8. Because chemokines facilitate the activation of leukocytes and their migration to the sites of inflammation, the modulation of chemokine production by the virus suggests a role for chemokines in immune evasion and/or immunopathogenesis of CMV retinitis.     

Human RPE cell lysis of extracellular matrix: functional urokinase plasminogen activator receptor (uPAR), collagenase and elastase

Age-related macular degeneration (ARMD), proliferative vitreoretinopathy (PVR) and uveitis are characterized by RPE motility through the ECM of retinal lesions. The purpose of this study was to test the hypothesis that multiple proteolytic systems are functionally intact at the HRPE surface and peri-cellular region and that these activities are differentially modulated by IL-1beta. HRPE cells were evaluated: (1). as individual cells or cell extracts, (2). during migration across three-dimensional ECM-like layers and (3). in tissue sections. The urokirase plasminogen activator receptor (uPAR; CD87) was detected on HRPE cells as well as its functional activity. Although uPAR was associated with CD11b (CR3) on live resting cells, polarized migratory HRPE cells were found to dissociate uPAR from CR3; uPAR then translocated to anterior pole of the cell, where it enhanced PAI-1-inhibitable local proteolytic activity. The relative contribution of uPAR and collagenase in HRPE migration was evaluated using three-dimensional gelatin matrices. Interestingly, uPAR/uPA was found to play a key role in migration across these layers. IL-1 upregulated uPAR, collagenase, and elastase activities, suggesting that cytokines may affect the invasive program of HRPE cells in vivo. Immunohistochemistry for uPAR was performed in sections of human retina. Immunoreactive uPAR was present along the HRPE basolateral membrane in retinal sections and in sections of diseased retinal tissue at an enhanced level. Our results suggest that multiple proteolytic systems are present in association with HRPE and that the uPAR/uPA system may be particularly important.     

Gold nanoparticles downregulate VEGF-and IL-1β-induced cell proliferation through Src kinase in retinal pigment epithelial cells

Proliferative vitreo retinopathy (PVR) is one of the ocular complications, marked by the enhanced proliferation of various cells including retinal pigment epithelial cells (RPE). The aim of the present study is to analyze the effect of gold nanoparticles (Au-NP) on vascular endothelial growth factor (VEGF) and interleukin-1 beta (IL-1β)-induced cell spreading, migration and proliferation in RPE cells. Au-NP (300 nM) significantly blocked the VEGF-and IL-1β-induced cell spreading, migration and proliferation in bovine RPE cells (BRPEs). To elucidate the signaling mechanism of VEGF- and IL-1β-induced cell proliferation, BRPEs were treated with PP2, a Src inhibitor. Further, to clarify the possible involvement of the Src pathway on the inhibitory effect of Au-NPs, transient transfection assay was performed using dominant negative (DN) and constitutively active (CA) mutant plasmid of Src kinase. The results showed that VEGF and IL-1β exert their proliferative effects through the activation of Src kinase whereas CA Src rescued the inhibitory effect of Au-NP in presence or absence of VEGF and IL-1β in BRPEs. Further, an in vitro kinase assay was performed to identify the status of Src phosphorylation at Y419. We found that VEGF and IL-1β increased Src phosphorylation in BRPEs and Au-NP blocked the VEGF- and IL-1β-induced Src phosphorylation at Y419. Taken together, our result suggests that Au-NP could effectively inhibit the VEGF- and IL-1β-induced proliferation and migration by suppressing the Src kinase pathway in BRPEs and Au-NP might act as an effective therapeutic agent for the treatment of ocular diseases such as proliferative vitreo retinopathy.     

Cyclooxygenase-2 gene expression and regulation in human retinal pigment epithelial cells

PURPOSE: Cyclooxygenases (COX) orchestrate a variety of homeostatic processes and participate in various pathophysiological conditions. The retinal pigment epithelium (RPE) cell performs a variety of regulatory functions within the retina. The conditions under which COX-1 and COX-2 are expressed and upregulated in human RPE (HRPE) cells were determined. METHODS: COX gene expression was examined using RT-PCR analysis of untreated HRPE cultures or cultures exposed to bacterial lipopolysaccharide or various cytokines. COX proteins were detected by immunohistochemistry and Western blot analysis. Prostaglandin (PG) production was analyzed by EIA. RESULTS: Examination of untreated RPE cells revealed the presence of COX-2 mRNA and the absence of COX-1 mRNA. Moreover, cytokine stimulation more readily enhanced COX-2 gene expression than COX-1 gene expression. IL-1 beta, the most potent inducer of COX-2, also resulted in detection of COX-2 protein by immunocytochemical staining and Western blot analysis. There was a direct relationship between both the appearance and amount of COX-2 mRNA and protein synthesis and the degree of PG synthesis by RPE cells. Furthermore, COX inhibitors significantly decreased PG production. Pretreatment of RPE cells with a NF-kappa B inhibitor, PDTC, resulted in dose-dependent decrease in IL-1 beta-induced COX-2 gene expression and PG production. CONCLUSIONS: COX-2 was the predominant isoform of cyclooxygenase in untreated HRPE cells. When HRPE cells were treated with proinflammatory cytokines, COX-2 gene expression and synthesis of PGs were enhanced. NF-kappa B mediated the induction of COX-2 gene expression in HRPE cells. These studies indicate that RPE cells may participate in normal and pathologic retinal conditions through the induction of COX-2.     

Macrophage modulation of retinal pigment epithelial cell migration and proliferation

Macrophages are fully differentiated cells that do not synthesize an extracellular matrix and do not contract; they do, however, produce substances that modify the behavior and functions of other cells, particularly those involved in the inflammatory and immune responses. Since macrophages are a ubiquitous component of periretinal membranes, we sought to determine whether they modulate proliferation and/or migration of retinal pigment epithelial (RPE) cells, functions that may be essential for the development of proliferative vitreoretinopathy (PVR). Using an in vitro assay, we found that macrophage supernatant contains factors that stimulate proliferation and migration of cultured human RPE cells. Since interleukin-1 (IL-1) is a product of activated macrophages that modulates a number of cellular functions, we also examined its effect on RPE proliferation and migration. We found that IL-1 increased migration but did not affect proliferation, and thus could not duplicate the effect of macrophage supernatant. Injection of activated macrophages into the vitreous of rabbits which had a retinal hole stimulated RPE cell proliferation in the area of the retinal hole, where the RPE cells were exposed. These findings suggest the ability of macrophages to modulate functions of RPE cells that are thought to be critical for the development of PVR. Macrophages may thus be an important part of the vitreous environment that favors the development of PVR.     

Effect of growth factors on bovine retinal pigment epithelial cell migration and proliferation

BACKGROUND: To examine the effects of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and transforming growth factor beta 2 (TGF(beta2)) on bovine retinal pigment epithelial (RPE) cell migration and proliferation. MATERIALS AND METHODS: Cultured bovine RPE cells were treated with 10 ng/ml PDGF, bFGF, aFGF, IGF-1, EGF, or TGF(beta2). RPE cell migration studies were performed in multiwell plates confluently covered with RPE cells. After inhibition of proliferation and denudation of half of each well, cells were incubated with various growth factors. Migration was measured as the number of cells that had entered the denuded area after 20 h. RPE cell proliferation was determined by [(3)H]thymidine incorporation after growth factor stimulation for 24 h. RESULTS: RPE cell migration was significantly enhanced after incubation with PDGF (stimulation of 213% compared to the negative control, p = 0.002), bFGF (206%, p = 0.003), aFGF (175%, p = 0.003), IGF-1 (150%, p = 0.003), and EGF (144%, p = 0.003). RPE cell proliferation was stimulated by bFGF (322% compared to the negative control, p < 0.005), PDGF (119%, p < 0.005), aFGF (121%, p < 0.005), and EGF (94%, p < 0.005). IGF-1 showed no significant effect on RPE cell proliferation; TGF(beta2) displayed no effect on RPE cell migration nor on proliferation. CONCLUSIONS: The peptide growth factors PDGF, bFGF, aFGF, IGF-1, and EGF play an important role in initiating RPE cell migration. Basic FGF, PDGF, aFGF, and EGF stimulate RPE cell DNA synthesis.     

Influence of IL-1 beta and TNF-alpha on Fas expression of human retinal pigment epithelial cells in vitro.

PURPOSE: To observe Fas expression change of cultured human retinal pigment epithelial (RPE) cells by IL-1 beta and TNF-alpha. METHODS: With flow cytometry, immunohischemistry, and color imaging system, Fas expressions by exposure to IL-1 beta and/or TNF-alpha were measured. RESULTS: The gray degree values of Fas expression were 67.5 +/- 6.1 in IL-1 beta + TNF-alpha-treated group, 80.1 +/- 9.2 in IL-1 beta-treated group, and 70.4 +/- 6.4 in TNF-alpha-treated group, respectively. There were significant differences (P < 0.005) compared with control group (107.0 +/- 10.2). Flow cytometry showed that 15.0% cultured human RPE cells expressed Fas. Fas-positive in IL-1 beta, TNF-alpha, and IL-1 beta + TNF-alpha-treated groups expressed was 28.1%, 34.5%, and 65.2%, respectively. CONCLUSION: IL-1 beta, TNF-alpha, and combining both of them can up-regulate Fas protein expression, which may contribute to more Fas (+) cells in proliferative vitreoretinopathy (PVR). Minimizing this process by means of inducing apoptosis of Fas (+) proliferative cells of Fas/FasL pathway is a future preventive and therapeutic possibility for PVR.     

Cell-Associated Human Retinal Pigment Epithelium Interleukin-8 and Monocyte Chemotactic Protein-1: Immunochemical and In-situ Hybridization Analyses

Human retinal pigment epithelial (RPE) cells secrete chemokines, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in response to pro-inflammatory cytokines. In this study we (1) examined the efficiency of human RPE IL-8 and MCP-1 secretion, (2) determined the amount of neutrophil and monocyte chemotactic activity in human RPE cell conditioned media and cell extracts that is attributable to IL-8 and MCP-1, respectively, and (3) assessed the sensitivity of immunohistochemistry and in situ hybridization for detecting chemokine production by cytokine-stimulated human RPE cells. Conditioned media and extracts from human RPE cells stimulated with various physiologic concentrations of interleukin-1 beta (IL-1β) (0.2–20 ng ml⁻¹), tumor necrosis factor (TNF-α) (0.2–20 ng ml⁻¹) or interferon-gamma (IFN-γ) (10–1000 U ml⁻¹) were examined to compare secreted and cell associated levels of IL-8 and MCP-1 at various time points up to 24 hr.ELISA demonstrated that IL-8 and MCP-1 are both efficiently secreted by pro-inflammatory cytokine treated human RPE cells. Substantial dose- and time-dependent RPE secretion of IL-8 was observed following stimulation with IL-1β or TNF-α, but cell associated IL-8 was detectable only after high dose (20 ng ml⁻¹) IL-1β stimulation and comprised less than 1% of the total IL-8 induced. Dose- and time-dependent RPE cell MCP-1 secretion was also observed following IL-1β>TNF-α>IFN-γ stimulation, with an average of 4% of the total MCP-1 retained within RPE. Bioassays demonstrated neutrophil and monocyte chemotactic activity in conditioned media from stimulated RPE cells, but not in human RPE cell extracts. Inhibition of conditioned media-induced chemotaxis by specific anti-IL-8 or anti-MCP-1 antibodies demonstrated that IL-8 and MCP-1 were responsible for the majority of HRPE-derived neutrophil (>60%) and monocyte (53–57%) chemotactic activity, respectively.Using in situ hybridization IL-8 mRNA was readily detected within IL-1β>TNF-α stimulated RPE cells and MCP-1 mRNA easily visualized within IL-1β>TNF-α> or IFN-γstimulated cells. Immunohistochemistry to detect IL-8 was positive only in RPE cells exposed to high dose IL-1β(20 ng ml⁻¹) for 8 or 24 hr and was weak. Immunohistochemical staining for MCP-1 in RPE cells was more intense and was visualized within RPE cells stimulated with IL-β, TNF-α, or IFN-γ. This study demonstrates that: (1) RPE cells efficiently secrete IL-8 and MCP-1 upon stimulation with pro-inflammatory cytokines; (2) secreted IL-8 and MCP-1 account for the majority of human RPE neutrophil and monocyte chemotactic activity; (3) in situ hybridization readily detects IL-8 and MCP-1 mRNA in cytokine stimulated RPE cells; and (4) immunohistochemistry demonstrates cell-associated MCP-1 in cytokine stimulated RPE cells, but only minimal cell-associated IL-8.     

Modulation of matrix metalloproteinase and TIMP-1 expression by cytokines in human RPE cells

PURPOSE: The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is crucial for homeostasis of ocular extracellular matrices. To assess altered MMP activity as a determinant in the migration of human retinal pigment epithelial (RPE) cells, expression characteristics of several MMPs and TIMP-1 in RPE cell cultures were investigated. METHODS: Expression studies were performed with RT-PCR, ELISA, and immunofluorescence analysis. Secretion of MMP-2 was demonstrated by zymography. Migration of cytokine-stimulated RPE cells was evaluated with microporous membranes of permeable chambers. RESULTS: MMP-1, -2, -3, and -9; MT2-MMP; and TIMP-1 were expressed in cultured RPE cells. MMP-2 was detected on the cell surface and in secreted inactive and active forms. TGF-beta(2), IL-1beta, and TNF-alpha enhanced secretion of MMP-1, -2, and -3. TGF-beta(2) also stimulated MT2-MMP cell surface expression and release of TIMP-1. The mRNA levels of MMP-1, -2, and -3 and TIMP-1 were markedly increased by TNF-alpha and TGF-beta(2). MMP-2 mRNA levels were also upregulated by PDGF-BB. Migration of RPE cells stimulated by TGF-beta(2) or PDGF-BB was inhibited in presence of a synthetic MMP inhibitor. CONCLUSIONS: Proinflammatory cytokines and TGF-beta(2) play an important role in the upregulation of expression of MMP-1, -2, and -3 in RPE cells and account for a directional shift in the balance between MMPs and TIMPs. Facilitation of RPE cell migration stimulated by cytokines (i.e., TGF-beta(2) or PDGF-BB) in ocular diseases may be due to increased release of MMPs, in the presence of comparatively lower levels of their inhibitors.     

表皮生长因子、碱性成纤维细胞生长因子及5-氟尿嘧啶对培养的人视网膜色素上皮损伤愈合的体外研究

OBJECTIVE: To study the effects of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and 5-fluorouracil (5-Fu) on the cell migration and proliferation. METHODS: Phase-contrast microscopy, cell proliferation assay and wound -closure were used to study the process of cell proliferation and wound healing in a model of human retinal pigment epithelial (RPE) cell damage. RESULTS: It was found that EGF mainly stimulated proliferation and the migration of individual RPE cells from the wound edge, and induced cellular elongation, while bFGF mainly promoted proliferation and spread of the confluent monolayer into the wound defect, and induced enlargement and flattening of cells. But the effects of EGF, bFGF on wound closure were not very significant; 5-Fu inhibited the spread and proliferation of RPE cells, but had no effects on cell migration. CONCLUSIONS: EGF and bFGF have no significant effects on wound-healing, perhaps their effects on cell morphology are very important in cell transformation in proliferative vitreoretinopathy (PVR), and 5-Fu can only be a subsidiary drug to prevent PVR.     

Interleukin-6 (IL-6) gene expression and secretion by cytokine-stimulated human retinal pigment epithelial cells.

Retinal and choroidal inflammatory lesions are important causes of visual loss, but the mechanisms regulating intraocular inflammation remain poorly understood. By virtue of its position at the blood-retina barrier, the retinal pigment epithelium (RPE) cells may be critical to the initiation and propagation of ocular inflammation. Previously we showed that cytokine-stimulated RPE cells produce interleukin-8, a well-defined chemotactic factor for neutrophils and lymphocytes. In this study, we found that human RPE cells stimulated by human recombinant interleukin-1-beta (rIL-1 beta) or tumor necrosis factor-alpha (rTNF-alpha) produce interleukin-6 (IL-6). Using a plasmacytoma proliferation assay, significant levels of IL-6 were found in media of RPE cells stimulated with either rIL-1 beta or rTNF-alpha for 4 hr. Progressive accumulation of IL-6 in media overlying stimulated RPE cells occurred over the subsequent 20 hr. IL-1 beta was a significantly more potent stimulator of RPE IL-6 production than TNF-alpha, RPE IL-6 production in response to each of these cytokines was also dose-dependent over a range of 20 pg to 20 ng ml-1. Specific anti IL-6 antibody, but not control immunoglobulin, neutralized RPE-derived IL-6 activity in the plasmacytoma proliferation assays. RPE IL-6 mRNA levels were detectable 1 hr after cytokine stimulation, plateaued within 8 hr in 24-hr assays, and demonstrated dose-dependent kinetics in 6 hr assays. Lipopolysaccharide failed to induce RPE IL-6 mRNA expression or RPE IL-6 production.(ABSTRACT TRUNCATED AT 250 WORDS)     

8—氯腺苷对生长因子诱导的视网膜色素上皮细胞增殖抑制作用的研究

目的探讨８氯腺苷（８ｃｈｌｏｒｏａｄｅｎｏｓｉｎｅ,８ＣＬＡ）对生长因子诱导的视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ,ＲＰＥ）细胞增殖的抑制作用。方法通过ＲＰＥ细胞培养,采用３ＨＴｄＲ掺入测定８ＣＬＡ对肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ,ＴＮＦα）、白细胞介素１β（ｉｎｔｅｒｌｅｕｋｉｎ,ＩＬ１β）和碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ,ｂＦＧＦ）诱导的ＲＰＥ细胞ＤＮＡ合成的抑制作用。结果三种因子单独使用均可使ＲＰＥ细胞３ＨＴｄＲ掺入每分钟计数（ｃｏｕｎｔｐｅｒｍｉｎｕｔｅ,ＣＰＭ）值明显增高（Ｐ＜０．０５）,８ＣＬＡ在其终浓度为２～１６μｍｏｌ／Ｌ时可使三种因子刺激的ＲＰＥ细胞３ＨＴｄＲＣＰＭ值明显降低（Ｐ＜０．０５）。在ＴＮＦα、ＩＬ１β、ｂＦＧＦ和８ＣＬＡ组,分别于１６、８、２μｍｏｌ／Ｌ时,ＣＰＭ值与基础值相近（Ｐ＞０．０５）。结论８ＣＬＡ可抑制生长因子诱导的ＲＰＥ细胞增殖,为抗增殖药物提供了新的途径     

Hepatocyte growth factor stimulates proliferation and migration during wound healing of retinal pigment epithelial cells in vitro

PURPOSE: A defect in retinal pigment epithelial (RPE) cells may cause dysfunction of the neural retina, so rapid recovery of differentiated RPE cells is required after RPE injury. We investigated the effect of hepatocyte growth factor (HGF) on wound healing in RPE cells. METHODS: Confluent monolayers of bovine RPE cells were denuded, and the cells were allowed to recover in the presence or absence of HGF. The effect of HGF on RPE cell proliferation was evaluated by a 3-(4;5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetraz olium assay. In a migration assay, mitomycin C was used to inhibit proliferation, and the number of migrated cells was counted. The signaling pathways involved were examined using inhibitors of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 (PI3) kinase and protein kinase C pathways. RESULTS: At 80 ng/mL, HGF stimulated the wound closure of RPE monolayers and rendered the restituted cells more epithelioid in shape. HGF at 10 ng/mL stimulated RPE cell migration the most, whereas 80 ng/mL of HGF inhibited migration, but stimulated proliferation the most. In particular, PI3 kinase and MAPK inhibitor inhibited PRE cell migration and proliferation, respectively. CONCLUSIONS: HGF stimulated wound closure in cultured RPE cells, and rendered restituted cells epithelioid in shape. HGF may become a therapeutic candidate for RPE wound healing.     

PDGF-C and -D induced proliferation/migration of human RPE is abolished by inflammatory cytokines

PURPOSE: The role of growth factors and inflammation in regulating retinal pigment epithelial (RPE) function is complex and still poorly understood. The present study investigated human RPE cell proliferation and migration mediated by platelet-derived growth factor (PDGF) and inflammatory cytokines. METHODS: Human fetal RPE (hfRPE) cells were obtained as previously described. Gene expressions of PDGF isoforms and their receptors were detected using real-time PCR. Protein expression, activity, and localization of PDGFR-alpha and -beta were analyzed by Western blot and immunohistochemistry. BrdU incorporation and wound healing assays were used to test the effects of different PDGF isoforms and inflammatory cytokines on hfRPE proliferation and migration. Annexin-V and phalloidin staining were used to detect apoptosis and the actin cytoskeleton, respectively. RESULTS: PDGF-C and PDGF-D proteins are expressed in native human adult RPE, and mRNA levels are up to 100-fold higher than PDGF-A and -B. PDGFR-alpha and -beta proteins are expressed in native adult RPE and hfRPE (mainly localized to the apical membrane). In hfRPE, these receptors can be activated by PDGF-CC and -DD. PDGF-CC, -DD, and -BB significantly increased hfRPE proliferation, whereas PDGF-DD, -BB, and -AB significantly increased cell migration. An inflammatory cytokine mixture (TNF-alpha/IL-1beta/IFN-gamma) completely inhibited the stimulatory effect of PDGF-BB, -CC, and -DD; in contrast, this mixture stimulated the proliferation of choroidal cells. This inflammatory cytokine mixture also induced apoptosis, significant disruption of actin filaments and zonula occludens (ZO-1), and a decrease in transepithelial resistance. CONCLUSIONS: These results suggest that proinflammatory cytokines in vivo can inhibit the proliferative effect of PDGF on human RPE and, at the same time, stimulate the proliferation of choroidal cells. They also suggest an important role of proinflammatory cytokines in overcoming local proliferative/wound-healing responses, thereby controlling the development of disease processes at the retina/RPE/choroid interface.     

姜黄素、丹参单体和苦参碱抑制IL-1β诱导下兔RPE细细胞增生的体外实验研究

背景 增生性玻璃体视网膜病变(PVR)是临床上常见的致盲眼病.视网膜色素上皮(RPE)细胞是PVR发生和发展中的关键细胞,研究中药对体外培养RPE细胞的作用及原理对于防治PVR并揭示其发病机制具有重要意义. 目的 研究姜黄素、丹参单体、苦参碱对白细胞介素-1β(IL-1β)诱导的兔RPE细胞增生的抑制效果.方法 取第3～4代体外培养的有色兔RPE细胞,采用透射电子显微镜鉴定细胞结构.采用MTT法检测2.5、5.0、10.0、20.0 μg/L IL-1β培养后24、48和72 h RPE细胞的增生率;并检测5、10、20 μg/ml姜黄素和丹参单体IH763-4以及100、200、400 μg/ml苦参碱对IL-1β诱导RPE细胞增生的抑制率.采用线性回归分析计算各药物的半数抑制率(IC50)剂量.结果 初分离的兔RPE细胞呈球形,细胞中可见大量黑色素颗粒;第4代细胞色素颗粒明显减少,形态更加狭长.免疫细胞化学染色结果显示,细胞角蛋白(AE1/AE3)在细胞质表达呈阳性.透射电子显微镜下可见细胞顶端有大量的微绒毛,细胞间可见连接复合体.不同质量浓度IL-1β组培养后24、48、72 h细胞增生率总体比较差异均有统计学意义(F时间=30.33,P=0.00;F浓度=9.37,P=0.00);组内相邻培养时间点间细胞增生率比较差异均有统计学意义(均P＜0.05),同一培养时间点相邻质量浓度IL-1β组间细胞增生率比较,差异均有统计学意义(均P＜0.05).10.0μ,g/LIL-1β培养72 h后细胞增生率达峰值.不同质量浓度姜黄素、丹参单体和苦参碱不同培养时间点细胞抑制率总体比较差异均有统计学意义(姜黄素:F时间=128.75,P=0.00;F质量浓度=334.05,P=0.00.丹参单体:F时间=39.32,P=0.00;F质量浓度=165.57,P=0.00.苦参碱:F时间=267.76,P=0.00;F质量浓度=912.34,P=0.00).3种药物对RPE细胞的抑制作用均呈明显的剂量和时间依赖性,组内相邻培养时间点间细胞抑制率比较差异均有统计学意义(均P＜0.05),相邻质量浓度药物组间细胞抑制率的比较差异均有统计学意义(均P＜0.05).培养后24、48及72 h,姜黄素的IC50分别为26.77、19.01和9.45 μg/ml,丹参单体的IC50分别为33.72、23.47和12.56μg/ml,苦参碱的IC50分别为570.96、352.25和97.50 μg/ml. 结论 IL-1β可促进RPE细胞增生,10.0 μg/L IL-1β促增生作用最显著;姜黄素、丹参单体、苦参碱均可抑制IL-1β诱导的RPE细胞增生,其抑制作用均呈时间和剂量依赖性,其中姜黄素抗增生作用最强.