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.     

TGF beta secretion modulates the density-dependent growth of pig retinal pigment epithelium in vitro

BACKGROUND: We aimed to identify the cytokine(s) responsible for the density-dependent growth regulation of pig retinal pigment epithelium (RPE) in vitro. METHODS: Confluent monolayers of primary pig RPE were established on bovine corneal endothelial extracellular matrix-coated tissue culture well inserts wrapped with dialysis membranes with different molecular weight cutoffs (0.5-50 kDa). These confluent RPE monolayers were then cocultured with first passage porcine RPE plated at a density of 1 cell/mm2, so that the newly plated RPE was bathed with different molecular weight fractions of the confluent cell media. Growth rates of the newly plated RPE were determined 72 h after plating and the molecular weight fraction of the confluent cell medium that inhibits the RPE proliferation was determined. First passage pig RPE (1 cell/mm2) were cocultured with confluent monolayers of primary pig RPE on inserts in the presence of different amounts of TGF-beta neutralizing antibody (0.1-100 microg/ml). Growth rates of the newly plated RPE were calculated 72 h after plating to determine the antibody concentration that would maximize the growth rate of the newly plated RPE in the presence of an adjacent confluent RPE monolayer. RESULTS: The growth rate of the newly plated RPE decreased when RPE were bathed with the 10- to 25-kDa fractions of medium from an adjacent confluent RPE monolayer. This growth inhibition reached statistical significance with the 25- to 50-kDa fractions (p < 0.05), and was abolished by adding pan-specific neutralizing antibody against TGF-beta (0.1-5 microg/ml). Blocking greater amounts of TGF-beta in the medium with higher doses of antibody (>10 microg/ml) also inhibited the growth of the newly plated RPE, in the presence or absence of a neighboring confluent cell layer. CONCLUSION: The TGF-beta family of cytokines mediates the density-dependent growth suppression of RPE in vitro. Neutralizing the effect of these cytokines by adding anti-TGF-beta antibodies can result in more rapid growth of the RPE in vivo.     

Inhibition of growth factor effects in retinal pigment epithelial cells

Several agents were examined for their effect on growth factor-stimulated processes in retinal pigment epithelial (RPE) cells. DNA synthesis was assessed by 3H-thymidine incorporation in density-arrested cells using previously determined maximally effective concentrations of various growth factors with and without test substances. Cell migration was assessed in Boyden chamber assays. For each test substance, trypan blue exclusion was used to determine noncytotoxic concentrations, and the effect of several concentrations were assessed on selected growth factors. The most effective, nontoxic concentration was then used for comparisons. Two cationic proteins, protamine and histone type II B, caused inhibition of RPE chemotaxis and 3H-thymidine incorporation induced by several growth factors, but a cationic polypeptide, polylysine, did not. Protamine and histone, were particularly effective inhibitors of acidic and basic fibroblast growth factors (FGF) but not if they were exposed to cells and then removed before growth factor addition. They had no effect on serum-stimulated chemotaxis or 3H-thymidine incorporation even when used in the presence of serum. Three anionic substances, heparin, pentosan polysulfate, and suramin, also inhibited RPE chemotaxis and 3H-thymidine incorporation induced by several different growth factors. They were less effective inhibitors of the FGFs than protamine and histone but were better inhibitors of serum-induced effects. Also unlike protamine and histone, the anionic substances maintained their inhibitory effect even when removed before growth factor addition. Since migration and proliferation of RPE cells are important processes in the pathogenesis of proliferative vitreoretinopathy, these agents and their mechanism of action deserve further study for potential therapeutic applications.     

Transforming growth factor-beta modulates effects of epidermal growth factor on corneal epithelial cells.

In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-beta caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-beta into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-beta itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-beta itself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-beta serves as a modulator of the effects of EGF.     

Interferon beta affects retinal pigment epithelial cell proliferation via protein kinase C pathways.

BACKGROUND: The aim of this study is to see the effect of interferon beta (IFN-beta) on cell proliferation and the protein kinase C (PKC) signaling pathway. METHODS: Proliferation of cultured human retinal pigment epithelium (RPE) cells, with various concentrations of IFN-beta, and with or without 3% fetal calf serum (FCS), was assessed by cell counting. Effects of short (3 h) or prolonged (48 h) exposure of RPE cells to natural human IFN-beta were assessed by (3)H-thymidine uptake. Cytosolic and membranous PKC activity over time in cells treated with IFN-beta and calphostin C was also measured. RESULTS: IFN-beta inhibited the increased proliferation by FCS in the prolonged-exposure assay. The PKC inhibitor calphostin C also showed an inhibitory effect on RPE cell growth and (3)H-thymidine uptake in the chronic exposure with FCS. Short treatment with IFN-beta had no inhibitory or stimulatory effect on (3)H-thymidine uptake. Cytosolic and membranous PKC activity was strongly upregulated after short IFN-beta exposure but returned to original levels after 1 h. PKC activity was downregulated both in the cytosol and membrane after 24 or 48 h. CONCLUSION: IFN-beta inhibited RPE proliferation in vitro and the effect is mediated by upregulation of the PKC pathway.     

血管紧张素Ⅱ对体外培养的牛眼小梁细胞^3H胸腺核苷酸掺入率及胶原合成的影响

目的观测血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)对体外培养的牛眼小梁细胞(bovine trabecularmeshwork cells,TM cell)3H胸腺核苷酸(3H-thymidine,3H-TdR)掺入率及胶原合成的影响,探讨原发性青光眼的发病机制.方法(1)牛眼小梁细胞的体外培养应用免疫组化方法(neuronal specific enolas,NSE,Ⅷ因子相关抗原染色)、细胞鉴定、光学及电子透射显微镜对细胞进行形态学及生长特性的观察;(2)AngⅡ(1×10-7mol@L-1及1×10-8mol@L-1)以及其Ⅰ型受体(angiotensin receptor type I,AT1)拮抗剂孵育TM细胞,采用检测3H-TdR掺入率的方法了解细胞增殖状态,并用化学方法检测培养液中羟脯氨酸含量,间接推导胶原含量.结果牛眼小梁细胞培养成功,以上皮型为主.AngⅡ明显地增加了牛眼小梁细胞的3H-TdR摄入率,同时培养液中的羟脯氨酸含量亦相应增高.结论体外培养牛眼小梁细胞技术是研究小梁细胞特性的重要实验技术.AngⅡ可以诱导体外培养的牛眼小梁细胞的细胞增殖率升高,同时促使胶原合成增强.AT1受体拮抗剂可以部分阻断此促增殖效应.眼科学报2001;17209-212.     

Epitheliomesenchymal transdifferentiation of cultured RPE cells

Retinal pigment epithelium (RPE) cells of the proliferative vitreoretinopathy (PVR) membrane take on the shape of fibroblasts and participate in fibrosis, thus deviating from the character of epithelial cells. This study was undertaken to evaluate RPE cell transdifferentiation in vitro. During the culture of porcine RPE cells, primary and 10th-passaged RPE cells were investigated for cell growth in response to transforming growth factor (TGF) beta(2), change of phenotype and amount in collagen synthesis as well as expression of alpha-smooth-muscle actin (alpha-SMA). TGF-beta(2) inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, while the spindle-shaped 10th-passaged RPE cells were not inhibited by TGF-beta(2). The 10th-subcultured cells did not show much difference in the quality of collagen synthesis, other than type VIII collagen which was not produced. Collagen synthesis was dose-dependently stimulated by TGF-beta(2). The stimulation by TGF-beta(2) in the 10th-passaged RPE cells was much greater than in primary RPE cells. The 10th-subcultured RPE cells produced substantial alpha-SMA compared to alpha-SMA production by primary RPE cells. These results were also observed by confocal laser microscopy. These findings indicated that RPE metaplasia resulting in a change of biological cell behavior might be a necessary predisposing step in the development of PVR.     

维生素E对IL-6作用的视网膜色素上皮细胞增生及胶原合成的影响

目的 探讨白细胞介素-6(IL-6)对视网膜色素上皮(RPE)细胞增生及胶原合成的作用，以及维生素E对这种作用的影响。方法 采用放射自显影的方法测定RPE细胞增生和胶原合成。用含IL-6和不同浓度维生素E的EMDM培养基培养RPE细胞，加入氚标记的胸腺嘧啶核苷(^3H-TdR)和氚标记的脯氩酸(^3H-proline)，测定每分钟闪烁值cpm，反映^3H-TdR和^3H-proline的掺入量，即RPE细胞增殖和胶原合成情况。结果 IL-6与RPE细胞共同孵育后，^3H-TdR和^3H-proline的掺入量较单纯RPE细胞培养组明显升高，提示IL-6促进RPE细胞增牛明显，胶原合成显著增加：不同浓度的维生素E和IL-6共同培养的RPE细胞^3H-TdR和^3H-proline的掺入量较单纯IL-6培养的RPE细胞组明显降低。且维生素E浓度越高，^3H-TdR和^3H-proline的掺入量越低，提示维生素E抑制IL-6引起的RPE细胞的增生和胶原合成，且抑制作用随浓度的增加而增加。结论 IL-6对RPE细胞的增生和胶原合成有促进作用，维生素E抑制IL-6引起的RPE细胞的增生和胶原合成，抑制作用与维生素E浓度成正比。     

Characterization of the effects of retinal pigment epithelium-conditioned media on porcine and aged human retina

Background

Retinal pigment epithelium (RPE) cells produce neurotrophic factors that rescue photoreceptors from degeneration. Previously, we showed that conditioned medium (CM) from fetal vs adult RPE cells resulted in significantly better porcine retinal preservation, and possessed significantly higher levels of hepatocyte growth factor (HGF) and pigment epithelium-derived factor (PEDF). This study aimed to further describe the effects of human fetal RPE-CM on porcine and aged human retina, and to characterize its effects biochemically.

Methods

RPE-CM was harvested from passage-2 fetal RPE, 7 days after passage, 24-hours after exposure to basal medium. After culture in RPE-CM, porcine retinal morphology was assessed with confocal microscopy. The effects of RPE-CM on porcine and aged human retina survival were assessed by cytotoxicity and apoptosis biochemical assays. To characterize RPE-CM biochemically, effects of heating, digesting with proteinase-K, dilution, concentration, and fractionation were tested. Recombinant proteins and neutralizing antibodies were used to identify proteins that might contribute to the salutary effects of RPE-CM on porcine retina.

Results

Culturing porcine retina in RPE-CM significantly preserved outer nuclear layer width and the number of nuclei in cross-section, and significantly decreased photoreceptor axon retraction. RPE-CM decreased porcine retinal death by 17–34 % (p?<?0.05) compared to basal medium. Human retina from age-related macular degeneration (AMD) and non-AMD donors responded similarly after culture in RPE-CM. Heating, proteinase-K digestion, and dilution significantly diminished RPE-CM-mediated preservation of porcine retina, whereas concentrating RPE-CM significantly enhanced its preservation of porcine retina. Molecular cut filtration identified retina-preserving activity in the 3–100 kDa filtrate. PEDF or HGF at 90 % receptor occupancy significantly improved retinal preservation over 48 h of culture compared to basal medium. Neutralizing PEDF in RPE-CM decreased its ability to reduce retinal apoptosis by 23–27 % (p?<?0.05).

Conclusion

RPE-CM reduced biochemically and histologically measured degeneration in porcine retinae. This effect was concentration-dependent, and can be attributed to a protein component(s) in a 3–100 kDa molecular cut fraction. Human retina (including non-AMD and AMD Caucasian and non-AMD African-American) responds to culture in RPE-CM similarly to porcine retina. Receptor occupancy calculations and retinal viability data indicate that PEDF may be one of the components that contribute to retina preservation by RPE-CM.     

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。     

Serum-free Media for Culturing and Serial-Passaging of Adult Human Retinal Pigment Epithelium

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm⁻². Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9±2.7% and 47.1±4.1%, respectively) and DMEM with serum (87.2±5.6% and 52.9±10.5%, respectively) than DMEM without serum (59.2±5.6% and 33.1±6.9%, respectively;P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12±3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21±5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.     

Modulation of retinal pigment epithelial cell behavior by Agaricus bisporus lectin.

PURPOSE: To determine whether Agaricus bisporus lectin (ABL) binds retinal pigment epithelial cells (RPEs), to conduct a preliminary viability study of RPEs exposed to ABL, and to evaluate the effects of ABL on RPE proliferation and RPE-mediated matrix contraction in vitro. METHODS: Using cultured bovine RPEs, immunohistochemistry was used to study ABL binding. Morphologic and trypan blue exclusion techniques were used for toxicity studies. The effect of ABL on RPE proliferation was investigated by [methyl-3H]-thymidine incorporation. The effect of ABL on RPE-mediated matrix contraction was evaluated with RPE-populated three-dimensional collagen matrices. RESULTS: ABL bound to RPE cells. This binding was inhibited by asialomucin. No change in RPE morphology or trypan blue exclusion compared with controls was observed in RPEs incubated with 5 to 60 microg/ml ABL for 3 days. Twenty-four-hour incubations of RPEs with ABL significantly inhibited RPE proliferation in a dose-dependent way, 40 microg/ml ABL inhibited proliferation by 83% (SE 14, P<0.05). ABL showed a dose-dependent significant inhibition of RPE-mediated collagen matrix contraction over 3 days, with 93% inhibition compared with controls by 40 microg/ml lectin (P<0.05). The inhibitory effect of ABL on proliferation and gel contraction was partly reversible after eliminating ABL from the culture medium. CONCLUSIONS: Bovine RPE cells bind ABL, and preliminary evaluations suggest that levels of ABL that are nontoxic to the cells potently inhibit RPE proliferation and RPE-mediated matrix contraction. ABL deserves further investigation as a potential inhibitor of RPE proliferation and cell-mediated matrix contraction in anomalous reparative processes such as proliferative vitreoretinopathy and as a laboratory tool for RPE behavioral studies.     

Effect of tranilast on proliferation,collagen gel contraction,and transforming growth factor beta secretion of retinal pigment epithelial cells and fibroblasts

The efficiency of tranilast for the treatment of proliferative vitreoretinopathy (PVR) was investigated in vitro. A tetrazolium-based colorimetric assay showed that the 300-microM concentration of tranilast inhibited proliferation of bovine retinal pigment epithelial (RPE) cells and rabbit dermal fibroblasts with no toxicity. The contraction of collagen gels embedded with these cells was evaluated in the cultures. Compared with the gel incubated with minimal essential medium and 0.35% bovine serum albumin and/or fetal calf serum, tranilast inhibited gel contraction. Enzyme-linked immunosorbent assay revealed that a 300-microM concentration of tranilast inhibited transforming growth factor-beta(1) (TGF-beta(1)) secretion significantly (p < 0.01). These results suggest that tranilast may inhibit the proliferation of RPE cells and fibroblasts and contraction of intraocular fibrous membranes by suppressing TGF-beta(1) secretion from these cells with a potential to treat PVR.     

Differential regulation of key stages in early corneal wound healing by TGF-beta isoforms and their inhibitors

PURPOSE: Inhibition of TGF-beta reduces myofibroblast differentiation and fibrosis in the cornea. Determining the actions of distinct TGF-beta isoforms and their inhibitors during early corneal wound healing is an essential step in guiding therapeutic intervention. METHODS: Bovine serum-free corneal cell and wounded organ cultures were challenged with a range of concentrations of TGF-beta1, -beta2, and -beta3; IL-10; and neutralizing human monoclonal antibodies (mAbs) against TGF-beta1 (CAT-192) or -beta2, (CAT-152). Cultures were assessed for re-epithelialization, proliferation (cell counts and cresyl violet assay), morphology (histologic examination), repopulation of the area under the wound, and myofibroblast transformation (alpha-smooth muscle actin) between 0 and 5 days. RESULTS: TGF-beta1 delayed re-epithelialization, increased repopulation of the stroma, increased keratocyte proliferation and was the only isoform to promote myofibroblast differentiation. The anti-TGF-beta1 mAb, CAT-192 promoted re-epithelialization and reduced repopulation of the stroma. Exogenous TGF-beta3 had little effect on re-epithelialization but reduced repopulation of the stroma. IL-10 promoted corneal re-epithelialization at low doses but inhibited this response at high doses. Stromal repopulation was prevented by all doses of IL-10. TGF-beta2 or the anti-TGF-beta2 mAb, CAT-152 had little effect on any repair parameter. CONCLUSIONS: The results confirm TGF-beta1 as the principal isoform in corneal wound healing and suggest that inhibition of the action of TGF-beta1 can promote corneal wound healing. Treatment with the anti-TGF-beta1 mAb CAT-192 accelerates corneal re-epithelialization but reduces cell repopulation of the stroma. The cytokines TGF-beta3 and IL-10 have opposing actions to that of TGF-beta1.     

体外原代培养人视网膜色素上皮细胞

目的：探讨建立体外原代培养人类视网膜色素上皮细胞(RPE)技术。为研究溶血磷脂酸(lysophosphatidic acid，LPA)对培养的人类视网膜色素上皮细胞DNA合成与增殖的作用。方法：取自愿贡献的意外事故成年眼球，用2．5g／L胰酶消化获取人RPE细胞、150mL／L胎牛血清的DMEM培养液培养，细胞接近融合状态时进行传代培养。取自愿贡献的眼球并游离其视网膜色素上皮细胞，用DMEM加100mL／L血清及MEM氨基酸和庆大霉素，进行细胞传代培养。用溶血磷脂酸、转移因子β2(TGF—β2)及LPA TGF—β2进行视网膜色素上皮细胞增殖试验，用细胞染色法进行细胞计数，提取视网膜色素上皮细胞DNA，用Spectrophotometer进行DNA定量测定。结果：RPE原代细胞镜下为圆形，大小不一，内含较多的色素颗粒，胞核无法辨认。原代细胞培养贴壁后3d增殖速度明显加快，至4～5d即可基本融合，细胞浆内色素颗粒则随传代次数增多而逐渐减少。第3代培养的视网膜色素上皮细胞膜表面可见微绒毛，细胞质内细胞器丰富，线粒体量多，体积较小，内外膜分界清晰，嵴较短。色素颗粒散在分布于胞浆内，多数细胞质内数量较少呈高电子密度包含物。LPA对原代培养的视网膜色素上皮细胞增殖有促进作用。含有和／或缺乏10mL／L小牛血清的两种LPA(10μmol／L)均明显刺激视网膜色素上皮细胞增殖，但这种细胞的增殖被TGF-β2所抑制，LPA(10μmol／L)引起视网膜的色素上皮细胞DNA合成增加是正常对照组的两倍。     

Extracellular release of acid hydrolases from cultured retinal pigmented epithelium

The intracellular and extracellular distribution of acid hydrolases in cultured retinal pigmented epithelium (RPE) was studied. Incubation of cultured RPE in medium containing 20 mM mannose-6-phosphate resulted in the extracellular release of approximately 15% of the cell-associated activity of several acid hydrolases. This represents an approximate 120% increase over control levels after 24 hr of culture with 20 mM mannose-6-phosphate. The extracellular release is not due to cell lysis, since no release of the cytoplasmic marker lactate dehydrogenase was seen. n-Acetyl-beta-glucosaminidase, alpha-mannosidase, and beta-glucuronidase were released into the extracellular medium, while acid phosphatase and beta-glucosidase were not. The release was specific for mannose-6-phosphate, and was dose-dependent. Inhibition of protein synthesis by treatment of RPE cells with cycloheximide (100 micrograms/ml) inhibited extracellular acid hydrolase release. RPE cells exhibited n-Acetyl-beta-glucosaminidase bound to the cell surface via a mannose-6-phosphate sensitive receptor. These results demonstrate a specific extracellular release of acid hydrolases by RPE and the presence of at least one acid hydrolase on the RPE cell surface. This may represent a mechanism for control of cell surface and extracellular levels of these enzymes in RPE via the mannose-6-phosphate receptor.     

Stimulation of DNA synthesis in human and bovine RPE by peptide growth factors: the response to TNF-alpha and EGF is dependent upon culture density

A range of concentrations of several peptide mitogens was tested for growth activity on bovine and human RPE cells under serum free conditions by analysis of 3H-thymidine incorporation within the first 24 hrs of exposure to the agents. For cultures which were subconfluent or in early confluence, TNF-alpha, a product of activated macrophages, was the most effective mitogen; little or no growth stimulation was observed for PDGF, EGF, NGF, IGF-1, IL-1B, bFGF or TGF-beta 1. For TNF-alpha and EGF the growth response was analyzed in cultures of varying density. TNF-alpha was more active in sparse RPE cultures whereas EGF stimulation was greater in dense cultures. The response to growth factors was similar in RPE cells from the two species sources, but the apparent magnitude of the response was greater for bovine cells because the growth rate in serum free medium, which was used as the basal reference, was lower for bovine RPE. It is concluded that culture conditions, especially the timing of the assay and the level of confluence of the cells, affect the detection of a growth response to peptide mitogens. Although several of the agents which were tested did not stimulate DNA synthesis in RPE in this study, they may nonetheless promote growth when assayed in combination with other agents or they may affect other biological functions of RPE cells.     

Retinoic acid promotes density-dependent growth arrest in human retinal pigment epithelial cells.

After retinal detachment the retinal pigment epithelium (RPE) undergoes a striking phenotypic change. It becomes dedifferentiated, proliferates to form multilayered colonies, and migrates into the subretinal space. These processes are important because they have been implicated in proliferative vitreoretinopathy and poor visual recovery after retinal reattachment; however the mechanisms by which they occur are unknown. In this study, the effect of retinoic acid on RPE cell morphology and growth in culture was examined. Cells grown in the presence of 1 microM retinoic acid do not exhibit cellular overgrowth and maintain characteristics associated with the morphologic appearance of mature RPE cells in vivo. Growth curves and 3H-thymidine incorporation suggest that retinoic acid inhibits RPE cell growth primarily after the cells have reached confluence. It may act by promoting density-dependent growth arrest. Dibutryl cyclic adenosine monophosphate also inhibits RPE cell growth and 3H-thymidine incorporation, but has little effect on cell morphology. However, in combination with retinoic acid it appears to have an additive effect on inhibition of cell growth and maintenance of a morphology like RPE in vivo. Retinoids have been demonstrated to modulate the growth and differentiation of several cell types. They are usually present in high levels in RPE cells. They become depleted in RPE in culture and such depletion may also occur in vivo after retinal detachment. This could play a role in the phenotypic alteration of RPE that occurs in association with retinal detachment.