The role of Bcl-xL in mouse RPE cell survival

PURPOSE. Retinal pigment epithelial (RPE) cell survival plays a critical role in normal physiology and in retinal diseases, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). We have previously demonstrated that Bcl-x(L) is an important cell survival protein in human RPE (hRPE) cells. Herein, we determined the role of Bcl-x(L) as a survival protein in mouse RPE (mRPE) cells. METHODS. Survival factor gene expression and Bcl-x(L) protein distribution were determined using qRT-PCR and immunohistochemistry, respectively. Cultured mRPE cells were transfected with two modified 2'-O-methoxyethoxy antisense oligonucleotides (ASOs): Bcl-x(L)-mismatched control and Bcl-x(L)-specific. Bcl-x(L) protein levels were analyzed using Western blot. To determine the effects of survival factor regulation in mRPE cells, cultured cells were treated for 24 hours with mouse TNF-α, human IL-1β, and human TNF-α. RESULTS. Bcl-x(L) was the most highly expressed survival factor in both mouse eyecup and cultured mRPE cells, whereas Bax was the most highly expressed antisurvival factor. Bcl-x(L) was expressed in the RPE layer, and the distribution among the retinal layers was similar to that observed in human eyecups. IL-1β and TNF-α had minimal effect on Bcl-x(L) and Bax expression and strongly upregulated Traf-1. Transfection with Bcl-x(L)-specific ASO resulted in markedly diminished Bcl-x(L) gene expression, Bcl-x(L) protein levels, and cell number. CONCLUSIONS. Bcl-x(L) is the most highly expressed survival gene in mRPE cells and is essential for mRPE cell survival. Our data suggest that mouse tissue is an appropriate model for investigations of RPE survival factor genes.     

RPE cell cultivation

Graefe's Archive for Clinical and Experimental Ophthalmology -     

胰蛋白酶对视网膜色素上皮细胞膜MHCⅡ分子表达的影响

目的 探讨不同浓度胰蛋白酶联合EDTA和机械刮擦等措施，分离培养的视网膜色素上皮(RPE)细胞并保持细胞膜HLA—DR分子活性的最佳条件。方法 常规培养人第3—6代RPE细胞，终浓度为500U／ml的IFN—γ刺激诱导培养4d，采取0．125%胰蛋白酶联合0．01％EDTA或机械刮擦等方法分离细胞；台盼蓝拒染活细胞计数；活细胞HLA—DR间接免疫荧光染色，流式细胞术分析；荧光显微镜观察。结果 0．125%胰蛋白酶 0．01％EDTA 机械刮擦组分离活细胞率最高；FCM检测HLA—DR免疫荧光染色细胞阳性率，0．125％胰蛋白酶 0．01％EDTA 机械刮擦组、机械刮擦组与对照组存在显著性差异；相对单细胞平均荧光强度，机械刮擦组与0．125％胰蛋白酶 0．01％，EDTA 机械刮擦组最高；荧光显微镜下可见机械刮擦组、0．125％胰蛋白酶 0．01％EDTA 机械刮擦组RPE细胞形态保持较好。结论 较低浓度胰蛋白酶结合EDTA联合机械刮擦方法能较好地保持RPE细胞膜结构及其糖蛋白的功能状态。     

生长因子对视网膜色素上皮的作用

RPE对多种生长因子敏感,本文主要介绍几种常见的生长因子对RPE细胞及其相关疾病所起的作用,以期从该角度研究治疗疾病的相关方法.     

Immunological aspects of RPE cell transplantation

Retinal pigment epithelial (RPE) cells have several functions, including support of the neural retina and choroid in the eye and immunosuppression. Cultured human RPE cells directly suppress inflammatory immune cells. For instance, they directly suppress the activation of T cells in vitro. In contrast, transplanted allogeneic human RPE cells are rejected by bystander immune cells such as T cells in vivo. Recently, human embryonic stem cell-derived RPE cells have been used in several clinical trials, and human induced pluripotent stem cell (iPSC)-RPE cells have also been tested in our clinical study in patients with retinal degeneration. Major safety concerns after stem cell-based transplantation surgery include hyper-proliferation, tumorigenicity, or ectopic tissue formation, but these events have currently not been seen in any of these patients. However, if RPE cells are allogeneic, there are concerns about immune rejection issues that have been raised in previous clinical trials. We therefore performed a preclinical study of allogeneic iPSC-RPE cell transplantation in animal rejection models. We then conducted autogenic or allogeneic iPSC-RPE cell transplantation in clinical studies of patients with age-related macular degeneration. In this review, we focus on immunological studies of RPE cells, including iPSC-derived cells. iPSC-RPE cells have unique inflammatory (immunosuppressive and immunogenic) characteristics like primary cultured RPE cells. The purpose of this review is to summarize the current findings obtained from preclinical (basic research) and clinical studies in iPSC-RPE cell transplantation, especially the immunological aspects.     

RPE conditioned medium stimulates photoreceptor cell survival, neurite outgrowth and differentiation in vitro.

In the present study we have investigated retinal pigment epithelium-photoreceptor cell interactions in vitro, and their contributions to photoreceptor cell survival and differentiation. Preparations enriched for intact photoreceptor cells from neonatal rat retina were grown in either serum-free medium supplemented with RPE-conditioned medium (RPE-CM) or in serum-free medium alone. A variety of substrate conditions were tested for the best neurite outgrowth. Cultures were monitored for 7 days by light and electron microscopy, as well as by opsin, vimentin and carbonic anhydrase-C immunocytochemistry. RPE-CM was found to stimulate both proliferation of flat cells and photoreceptor differentiation. The number of photoreceptors bearing neurites and their neurite length measurements showed significant differences between the RPE-CM group and the control group within 20 hr in culture. Elimination of contaminating flat cells by the addition of an antimitotic drug prevented photoreceptor cell morphological maturation; however, these cells survived as round cell bodies without processes for at least 10 days in the presence of RPE-CM and expressed opsin during this period. Conditioned medium from the flat-cell monolayers did not support photoreceptor differentiation or their survival. However, the presence of flat cells was a requisite to achieve any neurite outgrowth even in the presence of RPE-CM. In the absence of RPE-CM, neither photoreceptors nor flat cells survived or proliferated. Heat and trypsin treatment of the RPE-CM abolished all its growth-supporting activities which indicates its proteinaceous nature. This represents the first time in vitro that an RPE-derived factor(s) has been shown to be responsible for photoreceptor cell survival and differentiation.     

PDGF和TGF-β1对兔RPE细胞α-SMA表达及收缩功能的影响

目的 研究血小板源性生长因子(PDGF-AB)和转化生长因子(TGF-β1)对兔视网膜色素上皮(RPE)细胞α-平滑肌肌动蛋白(α-SMA)的表达和RPE细胞诱导的胶原收缩的影响。方法免疫荧光染色法分析PDGF-AB和TGF-β1对α-SMA表达的影响。体外胶原凝胶收缩实验用于证实两种生长因子对兔RP细胞收缩的刺激作用。结果 PDGF-AB和TGF-β1均可显著诱导兔RPE细胞α-SMA的表达，TGF-β1的作用显著强于PDGF-AB；两种生长因子对RPE细胞诱导的胶原收缩有着相同显著的刺激作用。结论 PDGF-AB和TGF-β1均可增强RPE细胞的收缩力量．但二者可能是通过不同的途径达到这一效果，还需要更多的研究来分析α-S1MA在纤维增殖疾病中的作用。     

反义寡核苷酸对人视网膜色素上皮细胞EGFR表达和增生抑制作用的研究

目的 研究反义寡核苷酸(ODN)对人视网膜色素上皮(RPE)细胞，表皮生长因子受体(EGFR)表达和增生的影响。方法 采用人工合成反义ODN经阳性脂质体包裹后转染人RPE细胞。用MTT和细胞周期分析检测转染ODN后对细胞的生长抑制作用，采用半定量RT-PCR法与ELISA法检测EGFR mRNA及蛋白的表达水平。结果 显示转染反义ODN 24 h后，EGFR mRNA的表达抑制率为35．2％，EGFR蛋白抑制率为66．45％。48 h后．反义EGFR ODNS对EGFR蛋白的抑制作用消失。EGFR反义ODN对RPE细胞生长增生的抑制率约为40％。细胞周期分析EGFR反义ODN作用于细胞后，细胞G0／G1期细胞数明显增多，S期细胞数相应减少，而G2 M期细胞数亦明显减少。结论 EGFR反义SON可抑制人RPE细胞的生长和EGFR mRNA及其蛋白的表达。     

Impaired RPE survival on aged submacular human Bruch's membrane

Resurfacing of diseased or iatrogenically damaged Bruch's membrane with healthy retinal pigment epithelium (RPE) has been proposed as adjunctive treatment for age-related macular degeneration (AMD). The purpose of this study was to determine whether cultured fetal human RPE cells can attach and differentiate on aged submacular human Bruch's membrane. Bruch's membrane was debrided to expose native RPE basement membrane, the superficial inner collagenous layer directly below the RPE basement membrane, or the deep inner collagenous layer. These are three surfaces that transplanted RPE cells will encounter in situ. Approximately 3146 cultured fetal RPE cells mm(-2) were seeded onto these three surfaces and grown in organ culture for 1, 7, or 14 days. Explants were bisected and examined histologically or analyzed with a scanning electron microscope. RPE nuclear density was measured on stained sections. Morphology and cell density were compared to cells seeded onto bovine corneal endothelial cell-extracellular matrix (BCE-ECM). In situ submacular RPE nuclear density was also measured in tissue sections of donor eyes ranging from 18 weeks gestation to 88 years of age to determine the effect of age on RPE density. Compared to cells seeded onto BCE-ECM at similar density, RPE cell coverage and cellular morphology on aged submacular human Bruch's membrane was poor at all time points. In contrast to cells on BCE-ECM, RPE cell density on Bruch's membrane decreased with time. In general, cell morphology on all three Bruch's membrane surfaces worsened by day-7 compared to day-1. Although some cells were more pigmented on RPE basement membrane and the deep inner collagenous layer at day-7, poor cellular morphology indicated the remaining cells were not well differentiated. At day-14, the cells were uniform and cuboidal on BCE-ECM, with cell density similar to that at day-7 and similar to in situ density of young donors (相似文献   

Embryonic stem cells that differentiate into RPE cell precursors in vitro develop into RPE cell monolayers in vivo

A culture system to generate eye-like structures consisting of lens, neural retina, and retinal pigmented epithelium (RPE) cells from undifferentiated embryonic stem cells has been established. Precursors of RPE cells that differentiated in the cultures were responsive to Wnt2b signaling and identified retrospectively to form secondary colonies consisting of only RPE-like cells in eye-like structures. These transplanted eye-like structures were capable of populating the developing chick eye as neuronal retina and RPE cells. The outgrowth of a single cell layer of mature RPE cells from the grafted eye-like structures confirmed the existence of precursors for RPE cells. These results suggest that the eye-like structures resulted from the normal developmental pathway responsible for generating eyes in vivo. If a functional effect of these cells can be established, such eye-like structures may be potentially used to establish therapy models for various eye diseases.     

Epidermal growth factor stimulation of RPE cell survival: contribution of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways

Epidermal growth factor (EGF) previously has been shown to stimulate short-term survival in vitro of cells derived from the native amphibian retinal pigment epithelium (RPE). In the present experiments, we have examined intracellular signaling pathways responsible for mediating these survival-specific growth factor effects, distinct from proliferative effects, using the human epithelial cell line RPE D407. When maintained as single cells in suspension culture in the absence of serum and exogenous survival factors, RPE D407 cell viability gradually declined over a 3-4 day period as a result of apoptotic cell death, a pattern similar to that seen for eye-derived RPE cells. Exposure to EGF (50 ng ml(-1)) enhanced cell survival by nearly 40% and caused a parallel increase in the tyrosine phosphate content of the EGF receptor (EGFR), as determined by immunoprecipitation and Western blotting. Both effects were completely blocked by 1 microm AG1478, an EGFR-selective tyrosine kinase inhibitor. EGF also stimulated phosphorylation of the phosphatidylinositol 3'-kinase (PI3K)-dependent effector kinase Akt, as well as that of the MEK-dependent mitogen-activated kinase (MAPK), extracellular signal-regulated kinase (ERK). Furthermore, EGF-induced protection was substantially reduced by either the PI3K inhibitor LY294002 (25 microm) or the MEK inhibitor U0126 (10 microm), under conditions in which phosphorylation of Akt and ERK1/2, respectively, was blocked. Our results indicate that EGF-stimulated survival of RPE D407 cells takes place as a result of signaling through both PI3K and ERK/MAPK pathways. Further, residual anti-apoptotic activity stimulated by EGF in the presence of both blockers suggests that additional as yet unidentified growth factor-dependent survival pathways exist.     

蛋白酪氨酸激酶抑制剂对RPE细胞趋化因子表达和Ca2+的影响

目的 研究蛋白酪氨酸激酶(protein tyrosine kinase,PTK)抑制剂染料木黄酮(Genistein)对白介素-1β(interleukin-1β,IL-1β)诱导的视网膜色素上皮(retinal pigment epithelium,RPE)细胞趋化因子表达和细胞内Ca2+的影响.方法 应用不同剂量IL-1β对RPE细胞进行刺激,RT-PCR法观察细胞IL-8和单核细胞趋化蛋白-1(monocyte chemotaetic protein-1,MCP-1)mRNA表达的变化.应用Genistein对RPE细胞培养进行干预,观察其对IL-1β诱导RPE细胞IL-8和MCP-1 mRNA表达的影响.应用Fura2/AM、荧光分光光度计检测IL-1β和Genistein对RPE细胞内Ca2+的影响.结果 RT-PCR结果显示,0.1μg·L-1、1μg·L-1、10μg·L-1IL-1β刺激组IL-8 mRNA相对表达值分别为0.446±0.133、0.727±0.054和0.523±0.106,对照组为0.272±0.088;1μg·L-1、10μg·L-1IL-1β刺激组与对照组比较,差异有统计学意义(P=0.001、0.015).10μg·L-1IL-1β诱导的RPE细胞MCP-1 mRNA相对表达值与对照组比较,差异有显著统计学意义(P=0.002).10 mg·L-1、50 mg·L-1 Genistein对IL-1β诱导的IL-8和MCP-1 mRNA表达具有明显的抑制作用,差异均有显著统计学意义(均为P<0.01).IL-1β引起细胞内ca2+浓度升高,Genistein对细胞内Ca2+作用与之相反.结论 Genistein对IL-1β诱导的1L-8和MCP-1 mRNA表达具有明显的抑制作用,其机制可能为抑制IL-1β诱导的细胞内Ca2+增加.     

Niosome-encapsulated auraptene reduced the mRNA expression of VEGF-A and PDGFs genes in human retina-derived RPE cell line

AIM: To evaluate the effect of auraptene (AUR) treatment in forms of free and encapsulated in niosome nanoparticles by investigating the mRNA expression level of vascular endothelium growth factor (VEGF)-A and platelet-derived growth factors (PDGFs) in human retinal pigment epithelium (RPE) cell line. METHODS: Niosome nanocarriers were produced using two surfactants Span 60 and Tween 80. RPE cell line was treated with both free AUR and niosome-encapsulated. Optimum dosage of treatments was calculated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of VEGF-A and PDGF-A, PDGF-B, PDGF-C, PDGF-D genes was measured after total RNA extraction and cDNA synthesis, using real-time polymerase chain reaction (RT-PCR). RESULTS: The highest entrapment efficiency (EE) was achieved by Span 60:cholesterol (1:1) with 64.3%. The half maximal inhibitory concentration (IC50) of free and niosome-encapsulated AUR were 38.5 and 27.78 µg/mL, respectively. Release study revealed that niosomal AUR had more gradual delivery to the cells. RT-PCR results showed reduced expression levels of VEGF-A, PDGF-A, PDGF-B, PDGF-C, and PDGF-D after treatment with both free and niosomal AUR. CONCLUSION: Niosomal formulation of Span 60: cholesterol (1:1) is an effective drug delivery approach to transfer AUR to RPE cells. VEGF-A, PDGF-A, PDGF-B, PDGF-C, and PDGF-D are four angiogenic factors, inhibiting which by niosomal AUR may be effective in age-related macular degeneration.     

Local immunosuppression prolongs survival of RPE xenografts labeled by retroviral gene transfer

PURPOSE: To determine whether local immunosuppression with Cyclosporin A can influence the survival of human fetal retinal pigment epithelium (RPE) xenografts in the rabbit's subretinal space. METHODS: Cultured human fetal RPE cells were transduced with the gene for green fluorescent protein (GFP) using a lentiviral vector. The RPE was transplanted into the subretinal space of rabbits that received intravitreal cyclosporine either by weekly injections (0. 25-0.5 mg) or by slow release (approximately 2 microg/d) from a capsule sutured into the vitreal cavity after prior cryopexy. The transplanted RPE was followed by GFP fluorescence scanning laser ophthalmoscopy and by histology of the transplant site. RESULTS: RPE xenografts in eyes receiving intravitreal cyclosporine survived longer (several months) than they did in control eyes without cyclosporine. Survival was as long with slow release capsules as it was with weekly intravitreal injections at much higher concentrations of cyclosporine. CONCLUSIONS: Local immunosuppression of the eye with cyclosporine prolongs the survival of RPE xenografts in the subretinal space of rabbits, implying that rejection involves activated T lymphocytes. Local immunosuppression with slow release capsules is as effective as weekly injections at much higher concentrations.     

端粒酶反義寡核苷酸抑制視網膜色素上皮細胞增殖的實驗研究

目的使用脂質體介導的端粒酶反義寡核苷酸封閉端粒酶基因在視網膜色素上皮細胞的表達從而抑制視網膜色素上皮細胞的增殖,爲增殖性玻璃體視網膜病燮的治療探索一條基因治療途徑.方法使用端粒重復序列擴增法(Telomeric repeat amplification protocol,TRAP)定性、定量檢測濃度爲0umol/L、2umol/L、4umol/L、6umol/L、8umol/L脂質體介導的端粒酶反義寡核苷酸及濃度爲4umol/L的正義寡核苷酸在視網膜色素上皮細胞端粒酶活性表達.結果TRAP結果顯示随着端粒酶反義寡核苷酸濃度的增高,視網膜色素上皮細胞端粒酶活性逐漸降低,8umol/L幾乎没有表達,正義寡核苷酸很少抑制視網膜色素上皮細胞端粒酶活性.結論脂質體介導的端粒酶反義寡核苷酸能抑制視網膜色素上皮細胞端粒酶的活性從而抑制視網膜色素上皮細胞的增殖.     

视网膜色素上皮细胞的损伤因素与治疗进展

视网膜色素上皮（RPE）细胞在维持视网膜和脉络膜的正常功能和组织结构方面发挥重要作用,许多因素可导致RPE细胞的损伤,如缺氧、物理因素、化学物质和药物、免疫因素、氧化应激等。RPE细胞的损伤可导致多种眼底疾病,引起视功能的损害或丧失。深入研究引起RPE细胞损伤的因素对各种RPE细胞相关性眼病的预防和治疗具有十分重要的意义。近年来相关的研究日益受到关注,对RPE细胞损伤的影响因素及治疗方法进行综述。     

氧化损伤对视网膜色素上皮细胞中基质金属蛋白酶表达的影响

目的探讨氧化损伤对体外培养的人视网膜色素上皮(RPE)细胞金属蛋白酶(MMP-2,9)及其组织抑制剂(TIMP-1)表达的影响。方法将不同浓度的H2O2处理传代的细胞，检测活力后，采用明胶酶谱法和RT—PCR技术定量分析RPE细胞中MMP-2，9的蛋白水平和mRNA含量。采用RT—PCR和ELISA分别测定TIMP-1的mRNA表达和培养液中的浓度。结果不同浓度H2O2对RPE细胞增殖没有影响，MMP-2均呈高表达，但mRNA和蛋白均没有变化；MMP-9表达相对弱，且随着H2O2浓度的升高而增强，ELISA法检测不同浓度H2O2处理的RPE细胞培养液中TIMP-1浓度无显著差别。结论不同H2O2浓度下MMP-2、TIMP表达无明显差异，MMP-9随着H2O2浓度增加表达增强，表明糖尿病视网膜病变中存在的氧化损伤导致MMP-9表达升高。     

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.