Identification and localization of a beta-1 receptor from the integrin family in mammalian retinal pigment epithelial cells

The distribution of an adhesion receptor from the integrin family was mapped in cultured mammalian retinal pigment epithelial cells (RPE), and in primate RPE cells in vivo, using antibodies to the human fibronectin receptor (FnR) in conjunction with indirect immunofluorescence and immunoelectron microscopy. Protein homogenates from human RPE or MG-63 cells were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. On Western blots of proteins from both cell types, anti-FnR and a monoclonal antibody to the beta 1 subunit of human FnR each recognized a single band with a molecular mass of approximately 115 kDa. After 1-6 weeks in culture human, monkey and feline RPE cells gradually acquired a morphology and cytoskeletal arrangement typical of a mature cuboidal epithelium. The distribution of anti-FnR labeling changed dramatically in accordance with the cells' phenotype. In preconfluent cells, labeling consisted of streaks and flecks of fluorescence at the termini of stress fibers and at putative sites of cell substratum attachment. As the cells became confluent and acquired an epithelioid morphology, the bulk of anti-FnR labeling shifted to the peripheral cytoplasm and appeared as a cross-hatched meshwork. In fully differentiated RPE cells anti-FnR labeling consisted of a dense punctate pattern on or close to the apical cell surface that coincided with the distribution of f-actin as shown by phalloidin staining, and to the distribution of apical microvilli as identified by scanning electron microscopy. In addition, a compacted rim of fluorescence appeared at the cells' lateral margins that was also virtually identical to the phalloidin staining pattern. No basal surface labeling was apparent at this stage. At the ultrastructural level, FnR was localized to the apical surface of nonpermeabilized RPE cells and, in particular, to the plasma membrane of apical microvilli in vitro and in vivo using an indirect, pre-embedding method. The results strongly suggest that: (1) a membrane receptor/s containing the integrin beta 1 subunit is normally present on the plasma membrane of apical microvilli and on the lateral cell surfaces of cultured mammalian RPE cells; and (2) this receptor also is present on the plasmalemma of RPE apical microvilli in vivo.     

Exocytosis by retinal pigment epithelial cells

We observed cultured retinal pigment epithelial cells as they responded to the introduction of latex particles. The cells showed phagocytosis of particles 1 h after administration of latex and were filled with particles after 24 h. After 7 days, exocytosis of latex from basal plasma membrane was documented. Observation was repeated using a two-layer culture. 7 days after putting the retinal pigment epithelial layer containing latex particles on another layer without particles, we observed the appearance of latex particles in the lower layer that originally contained no particles. This demonstrated that cultured retinal pigment epithelial cells exocytose latex particles from basal cell membrane.     

生长因子与视网膜色素上皮细胞

目前 研究认为视网膜色素上皮(RPE)细胞是构成增生性视网膜疾病中增生膜的主要细胞成分。RPE细胞的增生和移行，使得增生组织长人玻璃体内，最终导致牵拉性视网膜脱离和失明。新近研究的某些生长因子在RPE细胞的增生和移行中发挥了重要作用。介绍几种主要的生长因子对RPE细胞的作用，为RPE细胞的实验研究和临床研究提供理论基础。     

Enhancement of membrane phospholipid breakdown and synthesis during phagocytosis in cultured chick retinal pigment epithelial cells

In chick cultured retinal pigment epithelial cells (RPE cells) phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are major membrane phospholipid constituents. The synthesis of these phospholipids occurred gradually, compared to rapid synthesis of phosphatidic acid (PA) and phosphatidylinositol (PI). Exposure of RPE cells to latex particles elicited active phagocytosis. In [3H] glycerol- or [3H] arachidonic acid-prelabeled cells, a transient decrease in PI was observed without increases in the level of either diacylglycerol (DG) or PA. However, the radioactivity of PC and PE significantly increased. The enhancement of de novo synthesis of phospholipids, measured by the incorporation of [3H] glycerol, was not stimulated within 2 hours after the addition of latex particles. The transient increase in the ratio of [14C] choline/[3H] glycerol in PC may indicate the accelerated synthesis of PC but not via a de novo synthetic pathway. The results obtained in the present study indicate that DG, produced by PI breakdown, may be utilized in PC synthesis for the replenishment of membrane lost during phagocytosis.     

Morphology of live retinal pigment epithelial cells

We attempted to observe, by means of fluorescein angiography, the retinal pigment epithelial cells in pigmented rabbits. Fluorescein angiography was performed in 31 pigmented rabbits, after intravenous injection of 14mg/kg fluorescein sodium. The angiograms were evaluated as prints and as negative film under a light microscope. Animals were sacrificed and submitted to studies by scanning electron microscopy and fluorescein light microscopy. On fluorescein angiograms, we observed mosaic pattern which consisted of numerous polygonal spots overlying choroidal vasculature. Each polygonal spot showed central hypofluorescent area surrounded by hyperfluorescent rim. They were seen in all the eyes except 4 lightly pigmented eyes. They seemed to correspond, in size, to each retinal pigment epithelial cell. This pattern appeared from the early choroidal phase on, to become more distinct 5 to 15 minutes after dye injection. A hexagonal pattern was regularly seen away from the medullary rays by 3 or more disc diameters. The pattern became larger in the periphery than in the posterior pole. These angiographic findings closely matched those of retinal pigment epithelial cells as seen by scanning electron microscopy and fluorescein light microscopy in sizes and shapes. The findings indicate that it is possible to identify retinal pigment epithelial cells in pigmented rabbits by conventional fluorescein angiography.     

Phosphoinositide turnover and Ca2+ mobilization during phagocytosis in cultured chick retinal pigment epithelial cells

Ca2+ mobilization and phosphoinositide turnover were examined during phagocytosis of latex particles in cultured chick retinal pigment epithelial cells (RPE cells). Ca2+ influx into cells and Ca2+ efflux from the cells were enhanced by about 1.5-fold and 3-fold compared to control cells, respectively. A high content of Ca2+ in RPE cells has been reported. Therefore, the Ca2+ efflux observed here may be a reflection of Ca2+ release into cytosol from the intracellular storage site(s). In [3H] inositol-prelabeled RPE cells, addition of latex particles elicited decreases in phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol with the concomitant formation of inositol phosphates including inositol trisphosphate, indicating the hydrolysis of phosphoinositides by phospholipase C. These results suggest that phosphoinositide turnover may be closely coupled with Ca2+ mobilization during phagocytosis in RPE cells.     

Electrical estimulation of retinal pigment epithelial cells

We investigated and characterized the effect of externally applied electric fields (EF) on retinal pigment epithelial (RPE) cells by exposing primary cultures of human RPE cells (hRPE) and those from the ARPE19 immortalized cell line to various strengths of EF (EF-treated cells) or to no EF (control cells) under different conditions including presence or absence of serum and gelatin and following wounding. We evaluated changes in RPE cell behavior in response to EF by using a computer based image capture and analysis system (Metamorph). We found that RPE cells responded to externally applied EFs by preferential orientation perpendicular to the EF vector, directed migration towards the anode, and faster translocation rate than control, untreated cells. These responses were voltage-dependent. Responses were observed even at low voltages, of 50-300 mV. Furthermore, the migration of hRPE cell sheets generated by wounding of confluent monolayers of cells at early and late confluence could be manipulated by the application of EF, with directed migration towards the anode observed at both sides of the wounded hRPE. In conclusion, RPE cell behaviour can be controlled by an externally applied EF. The potential for externally applied EF to be used as a therapeutic strategy in the management of selected retinal diseases warrants further investigation.     

Mechanisms of apoptosis in retinal pigment epithelial cells

目的了解诱导视网膜色素上皮（ＲＰＥ）细胞在体外发生凋亡的因素，以推测该细胞凋亡在体内发生的可能机理。方法培养的人ＲＰＥ细胞分别给予低氧（３％），肿瘤坏死因子（ＴＮＦ），蛋白激酶Ｃ（ＰＫＣ）抑制剂及抗Ｆａｓ抗体处理２４～７２ｈ，同时也观察了纤维母细胞生长因子（ｂＦＧＦ）对体外凋亡诱导因子所致ＲＰＥ细胞凋亡的保护作用。以琼脂糖ＤＮＡ电泳及流式细胞计数法检测细胞凋亡，采用免疫组化染色及流式细胞计数法决定Ｆａｓ在ＲＰＥ细胞的表达。结果所用的细胞凋亡诱导剂均可使ＲＰＥ细胞发生典型的凋亡（ＤＮＡ片段形成），ｂＦＧＦ具有较强的抑制ＲＰＥ细胞凋亡发生的作用。３Ｈ胸腺嘧啶摄入表明，所有细胞凋亡诱导剂均能抑制ＲＰＥ细胞的生长。结论不同的诱导因子可诱导ＲＰＥ细胞在体外发生凋亡，其发生与多种凋亡信号系统调控和交互反应有关，因此，调节ＲＰＥ细胞凋亡可能会给某些视网膜变性类疾病提供新的治疗手段。     

Polyamine-dependent migration of retinal pigment epithelial cells

PURPOSE: Migration of retinal pigment epithelial (RPE) cells can be triggered by disruption of the RPE monolayer or injury to the neural retina. Migrating cells may re-establish a confluent monolayer, or they may invade the neural retina and disrupt visual function. The purpose of this study was to examine the role of endogenous polyamines in mechanisms of RPE migration. METHODS: Endogenous polyamine levels were determined in an immortalized RPE cell line, D407, using HPLC. Activities of the two rate-limiting enzymes for polyamine synthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (SAMdc), were measured by liberation of ((14)CO(2))(.) Migration was assessed in confluent cultures by determining the number of cells migrating into a mechanically denuded area. All measurements were obtained both in control cultures and in cultures treated with synthesis inhibitors that deplete endogenous polyamines. Subcellular localization of endogenous polyamines was determined using a polyamine antibody. RESULTS: The polyamines, spermidine and spermine, as well as their precursor, putrescine, were normal constituents of RPE cells. The two rate-limiting synthetic enzymes were also present, and their activities were stimulated dramatically by addition of serum to the culture medium. Cell migration was similarly stimulated by serum exposure. When endogenous polyamines were depleted, migration was blocked. When polyamines were replenished through uptake, migration was restored. Polyamine immunoreactivity was limited to membrane patches in quiescent cells. In actively migrating and dividing cells, immunoreactivity was enhanced throughout the cytoplasm. CONCLUSIONS: Polyamines are essential for RPE migration. Pharmacologic manipulation of the polyamine pathway could provide a therapeutic strategy for regulating anomalous migration.     

视网膜色素上皮细胞钙通道的研究现状

视网膜色素上皮(RPE)细胞对保护和维持视觉功能极为重要。RPE细胞的这些功能与钙通道及激活后的钙离子内流密切相关。RPE细胞钙通道的结构决定了其功能特征、蛋白质磷酸化、钙调蛋白及钙反馈调节的特性,由此形成的信号传递对许多生理活动进行精确调控。     

Tumor-associated retinal pigment epithelial proliferation simulating retinal pigment epithelial tear

A lesion with both the clinical and fluorescein angiographic appearance of the classical retinal pigment epithelial (RPE) tear was discovered over the dome of an actively growing metastatic choroidal tumor in a patient with a previous history of breast carcinoma. However, our patient exhibited no evidence of pigment epithelial detachment or age-related macular degeneration, the underlying cause of the RPE tear. Although we cannot be sure that a true RPE tear did not exist over our patient's tumor, more likely, we are observing a tumor induced zone of RPE dehiscence accompanied by RPE proliferation at its border, giving a very similar fundus appearance. Possible pathogenic mechanisms for the finding in our patient, and a comparison to those mechanisms responsible for the classical RPE tear, are discussed.     

巴曲酶对视网膜色素上皮细胞增生活性的影响

目的探讨巴曲酶对视网膜色素上皮细胞(retinal pigment epithelium,RPE)增生活性的影响。方法利用体外培养的RPE细胞系,应用噻唑兰比色法观察不同浓度的巴曲酶对RPE增生活性的影响。结果当巴曲酶浓度不大于1/10KU.mL-1时各浓度巴曲酶组(1/100KU.mL-1、1/32KU.mL-1、1/16KU.mL-1、1/10KU.mL-1)与对照组吸光度(optical density,OD)值差异无统计学意义(P=0.068、0.455、0.675、0.159);巴曲酶浓度为1/8KU.mL-1时OD值明显降低(P=0.000)。OD值与培养液渗透压呈明确的负相关(r=-0.951,P=0.000);巴曲酶组和等渗对照组OD值的差异没有统计学意义(P=0.749)。结论渗透压符合RPE生长要求的前提下,巴曲酶对体外培养的RPE增生无明显影响,其应用于玻璃体手术中控制眼内出血的可行性值得进一步研究。