Retinal pigment epithelial cells produce a latent fibrinolytic inhibitor that is antigenically and biochemically related to type 1 plasminogen activator inhibitor produced by vascular endothelial cells

Conditioned media from retinal pigment epithelial (RPE) cells in culture contain active and latent plasminogen activator inhibitors (PAIs). Latent activity is unmasked by denaturants and accounts for the vast majority of total inhibitor activity. Activation by denaturants is an unusual characteristic previously described for PAI-1, the inhibitor produced by vascular endothelial cells. This property is not shared by PAI-2 or protease nexin. Reverse fibrin autography demonstrates that the PAI activity in RPE-conditioned media (RPE-CM) comigrates with purified endothelial cell-derived PAI-1 and has an apparent Mr of 50,000. Immunoblotting with a monospecific antiserum directed against endothelial cell-derived PAI-1 demonstrates a cross-reacting protein in RPE-CM at 50 kDa, and this same antiserum is able to immunoprecipitate a 50 kDa protein from [35S]methionine-labeled RPE-CM. These data suggest that RPE cells produce a PAI that is biochemically and immunologically related to PAI-1.     

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 pigment epithelial cells produce PDGF-like proteins and secrete them into their media

Human retinal pigment epithelial cells at confluence were used to condition serum-free Dulbecco's modified Eagle's medium. Conditioned media were exhaustively dialyzed against 0.5 N acetic acid, lyophilized, and subjected to Western blot analysis, using as primary antibody an IgG fraction prepared from goat antiserum directed against human platelet-derived growth factor. Native platelet-derived growth factor was resolved as a band with Mr of 30 kDa under non-reducing conditions, while bands with Mr of 36-38 kDa and 18.5 kDa were resolved from retinal pigment epithelial cell-conditioned media. Acid extracts of retinal pigment epithelial cells also contained bands at 36-38 kDa and media conditioned for 48 hr exhibited much denser bands than media conditioned for 24 hr. No bands were detected when non-immune goat IgG fractions were substituted for primary antibody and when conditioned media were prepared from several human fibroblast lines in the same manner as those prepared from retinal pigment epithelial cells, no detectable bands or only a faint shadow at 36 kDa were seen. Retinal pigment epithelial cell-conditioned media prepared in the presence of [35S]methionine were loaded on an anti-platelet-derived growth factor IgG affinity column, eluted, and subjected to SDS-polyacrylamide gel electrophoresis. Bands with Mr slightly less than 36 kDa and 18 kDa were visualized by autoradiography, demonstrating that the platelet-derived growth factor-like proteins in retinal pigment epithelial cell-conditioned media are newly synthesized. Two fractions eluted from the column also markedly stimulated fibroblast chemotaxis and incorporation of [3H]thymidine, both of which were neutralized by soluble anti-platelet-derived growth factor IgG. These data suggest that retinal pigment epithelial cells in culture produce platelet-derived growth factor-like proteins and secrete them into their media where they are capable of stimulating fibroblast chemotaxis and proliferation.     

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.     

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 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 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.     

Tissue plasminogen activator in cultured human trabecular meshwork cells. Predominance of enzyme over plasminogen activator inhibitor

Maintenance of patency of the trabecular meshwork, the major outflow channel of the anterior chamber of the eye, is necessary to prevent an excessive rise in intraocular pressure. Obstruction of flow due to clot formation results in severe glaucoma and damage to the optic nerve. We have found that human trabecular meshwork cells which have been passaged in tissue culture synthesize large amounts of tissue plasminogen activator (t-PA), based on functional, immunologic and molecular weight analysis. Trabecular cells express substantially more t-PA activity than vascular endothelium which produces t-PA for clot dissolution in the systemic circulation. Vascular cells produce excess t-PA inhibitor while trabecular cells make comparatively little. Trabecular meshwork cells are the first normal cell type reported in which the balance between t-PA and inhibitor is weighted towards the activator, indicating that fibrinolysis may be more important than clotting in the anterior chamber of the eye.     

Retinal pigment epithelium cells can influence endothelial cell plasminogen activators

Endothelial cells from both human retinal microvessels (HME) and fetal bovine aortic endothelium (FBAE) were grown in aggregate cultures alone, or with either retinal pigment epithelium (RPE) cells or fibroblasts. The levels of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in the conditioned media of the various aggregate types were measured. High PA levels were detected in the conditioned medium of pure endothelial cell aggregates (equal to 140% and 124% of urokinase control for HME and FBAE, respectively), and high PAI levels were associated with pure RPE aggregates (inhibiting 93% of the urokinase control). The conditioned medium of pure fibroblast aggregates had very low levels of either PA or PAI. When RPE cells were aggregated with FBAE or HME cells into mixed (heterogenous) aggregates, the PA measured in the conditioned medium was equal to 22% and 30% of the urokinase control, respectively. The PA level in the conditioned medium of mixed fibroblast-FBAE cell aggregates was higher, 104% of the control, and the difference was statistically significant (P less than 0.001). Co-incubation of pure RPE aggregates with pure FBAE aggregates or with pure HME aggregates resulted in PA activity in the conditioned medium that was equal to 110% and 96% of the control, respectively. The PA level found when pure FBAE cell aggregates were co-incubated with pure fibroblast aggregates was higher, 134% of the control, and the difference was statistically significant (P less than 0.001). Our results indicate that RPE cells can reduce endothelial cell PA, probably through both direct contact between the cells and PAI production. Fibroblasts did not have this influence on endothelial cell PA.     

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.     

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.     

Insulin-like growth factor I stimulates proliferation, migration, and plasminogen activator release by human retinal pigment epithelial cells

The migration of retinal pigment epithelial (RPE) cells from their normal anatomic position to a new position in the vitreous cavity is a critical feature of proliferative vitreous retinopathy. To determine if insulin-like growth factor I (IGF I), which is present in the vitreous fluid of diabetics, stimulates RPE cells, we examined the effects of IGF I on the proliferation, chemotaxis, and release of plasminogen activator by these cells. At the concentrations of IGF I tested, significant proliferation of RPE cells is seen. Significant chemotaxis of the RPE cells also is seen at all the concentrations of IGF I tested. The mean number of migrating cells per high-powered field in control studies was 43 +/- 13 (x +/- SEM), and for IGF I at 2.5 ng and 50 ng/ml the mean numbers of migrating cells were 96 +/- 17 and 483 +/- 62, respectively (P less than 0.001 for each comparison). The IGF I response was noted to be dose-dependent. The chemotactic response noted at 50 ng/ml of IGF I was greater than the positive chemotactic control of 10% fetal calf serum. Addition of alpha IR-3, an IGF I receptor antibody, eliminated the IGF I chemotactic response. The effect of IGF I on the secretion of plasminogen activators was assessed using an immunological assay for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). Media conditioned by RPE cells have measureable levels of PAI and t-PA antigen. IGF I supplementation resulted in an increase of t-PA secretion and PAI secretion over basal levels. These studies support a role for IGF I in modulating RPE cell function.     

Retinal pigment epithelial cells in post mortem HLA typing of corneal donors.

Corneas used for transplantation are typically obtained from donors up to 48 hr post mortem. By this time, standard HLA typing usually is impossible because of the lack of viable lymphocytes in spleen and peripheral blood. To increase the number of HLA-typed corneas, we developed a method in which the retinal pigment epithelial (RPE) cells of the donor eye are isolated, cultured in the presence of 1000 IU/ml interferon-gamma (IFN-gamma), and, after 4 d, are typed for HLA Class I and Class II antigens in the standard NIH cytotoxicity assay. Sixty five donors were typed simultaneously using peripheral blood lymphocytes and RPE. One hundred and sixteen out of 120 HLA-A antigens, 125/127 HLA-B, 106/108 HLA-C, and 92/100 HLA-DR antigens were identical using the same technique. Donor age, sex, cause of death, time of enucleation post mortem, and time of RPE preparation post mortem, as well as duration of culture period prior to stimulation with IFN-gamma did not correlate with the results of HLA typing. These data show that RPE cells can substitute for lymphocytes in post mortem HLA typing. Consequently, every donor with corneas suitable for transplantation can be prepared for matched transplantation.     

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。     

Roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy

AIM:To investigate the role of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) in proliferative diabetic retinopathy (PDR) and to discuss the correlations among t-PA, PAI and vascular endothelial growth factor (VEGF) expressions.METHODS: A total of 36 vitreous samples were collected from 36 patients with PDR (PDR group), and 17 vitreous samples from 17 patients with idiopathic macular hole were used as control. The concentrations of t-PA, PAI and VEGF in samples were determined by ELISA method. The correlations among t-PA, PAI and VEGF expressions were discussed.RESULTS: The concentrations of t-PA, PAI and VEGF in the PDR group were significantly higher than those in the control group (P<0.001). The t-PA and PAI expressions were highly correlated with the VEGF expression (P<0.001).CONCLUSION:In addition to VEGF, a variety of bioactive substances, such as t-PA and PAI, are involved in the pathogenesis involved in the angiogenesis of PDR. VEGF can activate t-PA expression, resulting in collagen tissue degradation and angiogenesis. VEGF may also activate the mechanism for endogenous anti-neovascularization.     

Retinal pigment epithelial cells: autocrine and paracrine stimulation of extracellular matrix contraction

Background: This study was carried out to examine the biological activity of contraction promoters produced by dedifferentiating retinal pigment epithelial cells (RPE) and to evaluate the importance of autocrine and paracrine effects within a semi-closed environment like the vitreal cavity. Methods: RPE at different stages of dedifferentiation in culture were examined for their ability (a) to generate tractional forces in vitro, with and without serum stimulation, and (b) to produce and release contraction-stimulating proteins. Autocrine versus paracrine effects of cell-secreted promoters were tested by using RPE or human dermal fibroblasts (HDF) as target cells. The contraction-stimulating activity of the cell-secreted promoters was partially characterized and compared to the activity of defined promoters. Results: Our study confirmed that RPE can synthesize and secrete cell-contraction-promoting factor(s) active in stimulating the development of tractional forces by RPE as well as HDF. The quantity of biological activity secreted per cell decreases with progressive dedifferentiation, yet the responsiveness of the cell to contraction promoters increases. The contraction promoter(s) synthesized by RPE is partially distinct from the promote rs in serum, TGF-1 and 2, IGF-1, ET-1 and PDGF. The contraction-promoting effects of the RPE product(s) can be completely blocked by staurosporine. Conclusion: Dedifferentiation of RPE is characterized by increasing capacity to generate tractional forces and decreasing synthetic capacity. RPE within a semi-closed system like the vitreal cavity can, theoretically, act both as promoting and active component of traction-related events (tractional retinal detachment).