Retinal pigment epithelial cells produce fibronectin

Antichick fibronectin antiserum, noncross-reactive to bovine fibronectin, was prepared to determine the production of fibronectin by cultured chick retinal pigment epithelial cells which were grown in the presence of fetal bovine serum. The typical fibrillary net pattern of fibronectin was observed by an indirect immunofluorescent technique when this specific antiserum reacted with cultured chick retinal pigment epithelial cells. Cultured chick retinal pigment epithelial cells were shown to produce fibronectin.     

Retinal pigment epithelial cells release inhibitors of neovascularization

Human retinal pigment epithelial (RPE) cells in culture were found to release a substance (or substances) that causes the regression of new blood vessels on the chick embryonic yolk sac and inhibits proliferation of fetal bovine aortic endothelial cells and human retinal microvessel endothelial cells in vitro. Neither astrocytes nor fibroblasts under identical test conditions released detectable inhibitors of neovascularization or endothelial cell growth. Subconfluent and superconfluent cultures of human RPE cells released higher levels of inhibitor than confluent cultures.     

Wnt3a诱导人晶状体上皮细胞细胞外基质合成和胶原凝胶收缩的机制研究

背景 晶状体上皮细胞(LECs)的细胞外基质(ECM)的产生、异常沉积和收缩是PCO发生的重要病理机制.Wnt3a蛋白参与机体组织上皮细胞ECM的产生和纤维化过程,但Wnt3a对晶状体组织中上皮间质转化(EMT)相伴行的ECM的产生和收缩的影响尚不清楚. 目的 研究Wnt3a对体外培养的人LECsECM合成的影响,并探讨其介导胶原凝胶收缩的相关分子机制. 方法 用脂质体介导转染技术将Wnt3acDNA表达载体瞬时转染人LECs系SRA01/04细胞作为Wnt3a转染组,对照组转染pcDNA3-HA表达载体.细胞转染后48 h,采用Western blot法测定各组细胞中Wnt3a、ECM主要成分Ⅰ型胶原纤维(Col-Ⅰ),Ⅳ型胶原纤维(Col-Ⅳ)和整合素β1(integrin β1)的表达;采用免疫荧光法检测α-SMA、细胞纤维状肌动蛋白(F-actin)在胞中的表达和分布.将SRA01/04细胞与Ⅰ型胶原凝胶混合,观察胶原收缩情况.结果 Wnt3a转染48 h,SRA01/04细胞内Wnt3a蛋白相对表达量(相对灰度值)明显升高,对照组细胞和Wnt3a转染组细胞中Wnt3a蛋白表达量分别为0.290±0.066和0.703±0.105,差异有统计学意义(t=5.782,P＜0.01).Western blot法检测显示,Wnt3a转染组SR A01/04细胞中的Col-Ⅰ和integrin β1蛋白的相对表达量分别为0.697±0.021和0.875±0.055,明显高于对照组的0.370±0.020和0.580±0.030,差异均有统计学意义(t=19.600、8.156,均P＜0.01),Wnt3a转染组Col-Ⅳ蛋白相对表达量为0.430±0.020,高于对照组的0.383±0.031,但差异无统计学意义(t=2.514,P＞0.05).免疫荧光检测显示,对照组细胞中F-actin和α-SMA蛋白主要表达于细胞膜,而Wnt3a转染组细胞中F-actin和α-SMA蛋白表达于细胞膜、细胞质和细胞核周围,阳性染色程度较对照组明显增强.Col-Ⅰ凝胶收缩试验结果显示,细胞与Col-Ⅰ胶原凝胶混合后24 h凝胶收缩面积比率明显低于混合后8h(64.1％±2.3％与98.9％±1.0％),Wnt3a转染组凝胶收缩面积比率明显低于对照组(64.1％±2.3％与93.9％±3.1％),差异均有统计学意义(均P＜0.01). 结论 Wnt3a过表达可诱导SRA01/04细胞ECM合成和细胞骨架重建,促进细胞收缩.     

Retinal pigment epithelial detachment

The vast majority of cases of AMD involve widespread disease with visible neovascularization, "occult" neovascularization, and serous as well as hemorrhagic detachments of the RPE. Accurate interpretation of the clinical and fluorescein angiographic findings in PEDs is difficult. The most noteworthy problem is the recognition of the presence and extent of associated neovascularization. Definitive guidelines for laser photocoagulation treatment of these PEDs have not yet been established. Large-scale, sophisticated clinical trials are needed to assess the efficacy and safety of laser treatment for this important manifestation of exudative AMD. Until these investigations are completed, certain cases of PEDs may be selected for laser treatment by one of three treatment techniques. A grid pattern photocoagulation to a serous PED may be carried out when there is no demonstrable or suspected underlying SRN and there is persistence and progression of the detachment with associated visual decline. Total photocoagulation of the PED which is suspected of having neovascularization can also be carried out if the fovea can be spared. Finally, photocoagulation of extrafoveal neovascularization beneath or at the margin of a PED can be performed in an attempt to obliterate the neovascularization, resolve the exudative manifestations, and stabilize or improve the vision. Only experienced retinal specialists who are well trained in the recognition of the complex clinical and fluorescein angiographic features of PEDs should attempt to treat these unusual and complicated cases.     

Retinal pigment epithelial detachment

Detachment of the retinal pigment epithelium is a prominent feature of many chorio-retinal disease processes, the most prevalent of which is age-related macular degeneration (AMD). Detachment of the retinal pigment epithelium may or may not be associated with choroidal neovascularization and may be caused by different types of pathogenesis, each associated with distinct angiographic features, natural course, visual prognosis, and response to treatment. The phrase "detachment of the retinal pigment epithelium" is used quite often, not always in the correct association and with no clear differentiation between its various types. It is important to identify the specific nature of detachment of the retinal pigment epithelium, and to establish an accurate diagnosis and treatment plan. Therefore, we present a review of the existing types of detachment of the retinal pigment epithelium with what we propose as being appropriate nomenclature and classification, and potential treatment recommendations.     

Retinal pigment epithelial cells are heterogeneous in their expression of MHC-II after stimulation with interferon-gamma

The pattern of interferon-gamma-induced major histocompatibility complex Class II antigen expression was evaluated on the retinal pigment epithelium. Experiments were performed in vitro using explant cultures of aged and fetal human eyes and in vivo in albino rabbits. The human explants were stimulated with 50 U ml-1 interferon-gamma for 3 days prior to immunostaining for Class II. The rabbit eyes were subretinally injected in vivo with 50 microl of interferon-gamma (500 U ml-1) and analyzed immunohistochemically 3 days later. A heterogeneous pattern of Class II expression was present in the interferon-gamma-stimulated retinal pigment epithelial cells, in both the in vivo and the in vitro experiments. In aged human eyes the percent of Class-II positive cells was higher in the periphery than in the posterior pole (macular region) after interferon-gamma stimulation (P<0.01). No such difference was found in the fetal eyes. These data demonstrate that retinal pigment epithelial cells are heterogeneous in their response to interferon-gamma. The results are supportive of previous studies demonstrating the structural and proliferative heterogeneity of the retinal pigment epithelium. Together, these studies provide support for the possibility of functional retinal pigment epithelial heterogeneity.     

Retinal pigment epithelial glycosaminoglycan metabolism: intracellular versus extracellular pathways. In vitro studies in normal and diseased cells

The synthesis and turnover of glycosaminoglycans (GAGs) in different fractions of cultured feline retinal pigment epithelium (RPE) were characterized. In one method of fractionation, trypsin was used to separate the extracellular components (referred to as trypsin-soluble glycocalyx) from the intracellular components. As a second method, the basal extracellular matrix (basal ECM) was separated from the rest of the GAGs (cell-associated GAGs) by extracting the cell layer with NH4OH. The incorporation of 35SO4 into cetylpyridinium chloride-precipitable GAGs in the cell-associated and the intracellular fractions increased throughout the labeling period, while in the trypsin-soluble glycocalyx and the basal ECM incorporation approached a maximum. While heparan sulfate was the predominant GAG in all compartments, most was located extracellularly. The majority of dermatan sulfate was localized in the intracellular fraction. GAGs in the trypsin-soluble glycocalyx exhibited a rapid rate of turnover, while GAGs in the intracellular compartment and basal ECM turned over much more slowly. Ascorbic acid increased the incorporation of 35SO4 into ECM chondroitin sulfate/dermatan sulfate, but not heparan sulfate, on a per cell basis. Cycloheximide reduced incorporation of 35SO4-GAGs into both the cell-associated compartment and the basal ECM. In contrast, monensin caused a reduction in basal ECM GAGs while increasing the GAGs in the cell-associated compartment. The intracellular accumulation of GAGs and resultant pathology in alpha-L-iduronidase (alpha-L-id)-deficient RPE indicated that this pathway for the intracellular degradation of GAGs is important in normal RPE function. However, the turnover of GAGs in the trypsin-soluble glycocalyx was not affected by deficient alpha-L-id activity or by the subsequent intracellular accumulation of GAGs. Therefore, normal lysosomal activity in the RPE is not a prerequisite for maintaining the rate of extracellular GAG turnover within normal limits.     

The extracellular matrix of human retinal pigment epithelial cells in vivo and its synthesis in vitro

The production of extracellular matrix material by retinal pigment epithelium (RPE) may influence or mediate some of the many important functions of this tissue. Using immunohistochemical staining techniques, the extracellular matrix surrounding the RPE in vivo and the components produced by RPE in vitro have been investigated. Frozen sections of eye bank eyes showed antigen specific staining for collagen types I, III, and IV, and for fibronectin and laminin in Bruch's membrane and surrounding the RPE. Only very slight staining of the membrane was seen with antiserum against type II collagen, and there was no staining for type V collagen. Specific staining was demonstrated for the four collagens and glycoproteins in the extracellular matrix of RPE cells grown in culture. Once again, there was no staining for type V collagen. This study demonstrates that the RPE is capable of producing many of the components of the extracellular matrix found in Bruch's membrane and surrounding the RPE in vivo. This function may be important in the maintenance of a physical barrier to subretinal neovascularization, and may also play a role in such pathologic states as proliferative vitreoretinopathy.     

Retinal pigment epithelial cells in epiretinal membranes: an immunohistochemical study.

Immunohistochemical techniques were used to identify cells containing cytokeratins in sections or tissue-culture monolayers from ocular (reference) tissues and also from 22 epiretinal membranes obtained during closed microsurgery for macular pucker or massive preretinal retraction. Results of cytokeratin immunostaining in reference tissues indicated that this is a valuable means of determining the contribution and distribution of epithelial cells in epiretinal membranes, and that the epithelial cells in the membranes were probably derived from the retinal pigment epithelium. Epithelial cells were identified in 17 of the 22 epiretinal membranes, but they did not usually constitute the predominant cell type. We concluded that the fibroblasts or fibroblast-like cells thought to be responsible for the contraction of epiretinal membranes are seldom of retinal pigment epithelial origin. Biomicroscopic pigmentation of a membrane was shown to be a poor guide to its epithelial cell population.     

All-trans retinoic acid remodels extracellular matrix and suppresses laminin-enhanced contractility of cultured human retinal pigment epithelial cells

All-trans retinoic acid (atRA) has been reported to inhibit the proliferation of retinal pigment epithelial (RPE) cells and used in treatment of proliferative vitreoretinopathy (PVR) in animal model. This study aimed at examining the effectiveness of atRA in inhibiting the extracellular matrix (ECM) biosynthesis by RPE cells and the RPE cell-mediated collagen gel contraction. Cultured RPE cells were treated with atRA and the expression of four ECM proteins (collagen types I, III, IV and laminin β1) was profiled. The results indicated that atRA treatment up-regulated de novo synthesis of collagen type I, but decreased that of laminin β1 in a dose-dependent manner. Moreover, the effect of atRA on RPE cell contraction was evaluated by measuring the area of collagen gel where RPE cells populated. Treatment with atRA significantly inhibited RPE cell-mediated collagen gel contraction. Addition of exogenous laminin nonapeptide into gels promoted RPE cell contraction, while atRA reversed the laminin-enhanced contractility. atRA treatment significantly suppressed the gene expression of integrin β3 but not αV subunit, and effectively inhibited the tyrosine phosphorylation of integrin β3 at residue 747 in RPE cells grown on laminin-coated dish, suggesting that atRA may suppress the RPE contractility through either inhibiting integrin β3 expression or abrogating the integrin β3-mediated signaling. In conclusion, atRA pharmacologically possesses a tissue-remodeling capacity and inhibits contractility of RPE cells. Therefore, atRA might be potentially a therapeutic agent for certain ocular disorders such as PVR.     

Retinal pigment epithelial detachment in sarcoidosis

PURPOSE: To report primary retinal pigment epithelial (RPE) detachments in sarcoidosis. DESIGN: Case report. METHODS: Clinical findings, fluorescence angiography and optical coherence tomography (OCT) results are presented and discussed. RESULTS: A 54-year-old Hispanic male with biopsy proved sarcoidosis presented with multiple RPE detachment in both eyes. Except for acute iritis, there were no other ocular manifestations of sarcoidosis. CONCLUSIONS: Detachments of the RPE may be a rare manifestation of ocular sarcoidosis.     

Retinal pigment epithelial hamartoma--unusual manifestations.

Hamartoma of the retinal pigment epithelium is an uncommon tumour of young adults. We have seen 2 patients with this clinical diagnosis, both with unusual manifestations. In one patient growth of the tumour was observed over a 5-year period. In the second patient arterial-arterial anastomoses were detected at a site distal to the tumour.     

Retinal pigment epithelial cells in culture secrete angiogenic inhibitors: in vitro study

Conditioned medium wherein bovine retinal pigment epithelial cells have been cultured (RPE-CM) inhibited proliferation of the capillary endothelial cells (CEC) of the bovine adrenal gland. The RPE-CM was fractionated into three fractions; molecular weight of more than 30 kilo Daltons (kDa) (30 kDa fraction), between 10 and 30 kDa (10 kDa-30 kDa fraction), and less than 10 kDa (10 kDa fraction). Each fraction was tested for its effect on the proliferation, morphology and movement of the CEC. The proliferation of CEC was inhibited in the more than 30 kDa fraction and less than 10 kDa fraction, but not in the 10 kDa-30 kDa fraction. The RPE-CM changed morphology of the CEC into slender shape. This morphological change was observed only in the more than 30 kDa fraction, and the CEC in the other fractions maintained normal morphology. When the CEC proliferation was arrested by hydroxyurea, RPE-CM and the more than 30 kDa fraction did not change morphology. Unfractionated RPE-CM and the more than 30 kDa fraction which changed the morphology of the CEC also inhibited movement of the CEC, such as the migration of cells from a confluent cell layer and single cell movement. These findings suggested that the RPE in culture secrete soluble anti-angiogenic factors into the medium.     

胶质细胞培养液对视网膜色素上皮细胞生长的影响

目的 观察体外培养的视网膜色素上皮细胞条件培养液对视网膜神经胶质细胞生长的影响。方法 用不同稀释度的视网膜条件培养液培养视网膜神经胶质细胞，细胞计数法观察细胞数量的变化；MTT法测定对细胞增生活性（吸光度A值）的影响；流式细胞仪观察细胞周期的变化。结果 随着视网膜条件培养液浓度的增加，视网膜胶质细胞的数量明显增加，吸光度A值升高，进入S期的细胞明显增加，显示体外培养视网膜色素上皮细胞促进视网膜神经胶质细胞增生。结论 视网膜色素上皮细胞和视网膜神经胶质细胞细胞间的相互作用对于PVR等的产生和发展是非常重要的。     

Retinal pigment epithelial origin of bicarbonate response

The mechanisms of the bicarbonate-induced decrease in the ocular standing potential (bicarbonate response) were investigated in the cat. An intravenous infusion of 1.4% sodium bicarbonate solution caused a decrease in the standing potential. A high-bicarbonate solution decreased or increased the potential across the retinal pigment epithelium-choroid tissue of the excised cat eye when applied basally or apically, respectively, but did not affect the potential across the anterior portion of the excised eye or across the isolated neural retina. A high-bicarbonate solution principally depolarized the apical membrane of the retinal pigment epithelium (RPE) when applied basally, and hyperpolarized it when applied apically. These results suggest that the bicarbonate response in the cat is primarily due to the effects of an increase in bicarbonate concentration on the basal membrane of the RPE.     

Retinal pigment epithelial cells secrete substances that are chemotactic for monocytes

Monocyte/macrophages play a prominent role in several forms of retinal pathology including proliferative vitreoretinopathy, senile macular degeneration, and retinal wound healing. In each of these entities, the retinal pigment epithelium (RPE) is characteristically involved as well. Since RPE cells are known to secrete chemoattractants for astrocytes, we considered the possibility that they might secrete chemotactic factors for monocytes in addition. We have found in in vitro assays that a 5% concentration of medium from 6 different well-established RPE culture lines each consistently induced monocyte migration greater than that elicited by either buffer or unconditioned medium. "Checkerboard" analysis indicated that RPE culture supernatants induced optimal migration with a stimulus gradient (chemotaxis as opposed to chemokinesis alone). Chemotactic activity could be detected in eluates from ion exchange high performance liquid chromatography or gel filtration columns. Several peaks of activity suggested that more than one factor may be responsible for the ability to induce cell migration. The chemotactic activity was largely heat stable. The chemotactic factor induced only minimal migration of polymorphonuclear leukocytes. The secretion of chemotactic factors for monocytes could contribute significantly to the pathogenesis of several retinal diseases.     

Retinal microvessel extracellular matrix: an immunofluorescent study

The vasculature of the retina functions within a sheath of extracellular matrix (ECM). Unfortunately, little is known about the biochemical composition of this matrix. Abnormalities in the ECM of the retinal microvasculature are important in diabetic retinopathy as well as vasculopathies associated with connective tissue disorders. The ECM of unfixed frozen human retinal blood vessels was examined by indirect immunofluorescence using antibodies raised against collagen types I, II, III, IV, and V as well as the structural glycoproteins laminin and fibronectin. Antisera against collagen types I and IV as well as laminin and fibronectin stained a broad spectrum of retinal vessels, from large thick-walled vessels down to microvessels less than 10 micron in diameter. In contrast, antibodies against types III and V collagen were seen to stain primarily the walls of the larger vessels. Antibodies against type II collagen did not react with retinal vessels. Preincubation with the appropriate antigen or preimmune serum eliminated staining of the vessels by the antisera.     

Retinal pigment epithelial tears. Patterns and prognosis

Increasing experience with the diagnosis of retinal pigment epithelial (RPE) tears has led to expanded recognition and understanding of this clinical entity. The authors report 18 RPE tears followed for an average of 28 months; 16 were associated with age-related macular degeneration and 2 with presumed ocular histoplasmosis syndrome. Retinal pigment epithelial dehiscences fell into four categories: nine spontaneous tears associated with choroidal neovascularization, one tear associated with an RPE detachment without choroidal neovascularization, four iatrogenic tears occurring at krypton treatment of choroidal neovascularization, and four iatrogenic tears developing weeks to months after laser treatment of choroidal neovascularization. Eight patients had a final visual acuity of 20/100 or better, four were 20/200, and six were 20/400 or worse. Photocoagulation, particularly with the use of krypton red laser, may be modified on the basis of possible RPE tear formation. Heightened awareness of the possibility of inducing pigment epithelial rips should improve diagnosis and management of these cases.