Induction of adrenomedullin by hypoxia in cultured retinal pigment epithelial cells.

PURPOSE: To explore the effects of hypoxia on the production and secretion of adrenomedullin (ADM) and endothelin (ET)-1 in human retinal pigment epithelial (RPE) cells. METHODS: RPE cells were cultured under normoxic or hypoxic (1% O2) conditions. Expression of ADM and ET-1 was examined by Northern blot analysis and radioimmunoassay. Effects of ADM and ET-1 on the number of RPE cells were examined by modified 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: ADM mRNA expression levels and immunoreactive ADM levels in the medium were increased by hypoxia in all three human RPE cell lines (ARPE-19, D407, and F-0202). Immunoreactive ET was detected in the cultured media of D407 cells and ARPE-19 cells and identified as ET-1 by reversed-phase high performance liquid chromatography. Hypoxia treatment for 48 hours increased immunoreactive ET levels approximately 1.3-fold in the cultured media of D407, but not ARPE-19 cells. Hypoxia decreased the number of ARPE-19 cells and F-0202 cells, and the treatment with ADM ameliorated the hypoxia-induced decrease in the cell number. In contrast, exogenously added ET-1 had no significant effects on the number of ARPE-19 cells under normoxia and hypoxia. CONCLUSIONS: Hypoxia increased the expression of ADM in all three human RPE cell lines, whereas the induction of ET-1 by hypoxia was found only in D407 cells. ADM induced by hypoxia may have protective roles against hypoxic cell damage in RPE cells.

Short-term chlorambucil for refractory uveitis in Behcet's disease.

AIM: To evaluate the effects of short-term chlorambucil therapy in the management of uveitis due to Behcet's disease. METHODS: Forty-four patients with refractory uveitis associated with Behcet's disease who had had short-term chlorambucil therapy for about 23 weeks were included in the study. The frequency of attacks (number of attacks per year) and the longest period between the attacks were analyzed to evaluate the efficiency of the therapy. The therapy was judged to be effective if the patient had < or =1 attack a year and/or > or =1 year between the attacks. RESULTS: The mean follow-up time was 51.4 +/- 32.5 months (range: 13-122 months). Following the therapy, the mean frequency of attacks had decreased from 4.9 +/- 2.3 to 0.9 +/- 1.4 (p < 0.0001) and the mean longest period between the attacks was prolonged from 4.4 +/- 2.3 months to 25.7 +/- 23.1 months (p < 0.0001). The ratio of severe attacks had decreased from 74.1 +/- 34% to 51.3 +/- 36.6% (p = 0.0218). The best-corrected visual acuity was increased in 32.9%, decreased in 34.2%, and the same in 32.9% of the eyes. Phytsis bulbi developed in three (3.8%) eyes. New attacks were seen in 56.8% of patients and another immunosuppressive agent(s) was given to 40.9% of the patients 1-8 months after treatment. No serious side effects were observed during the chlorambucil therapy. CONCLUSION: Short-term chlorambucil therapy for refractory uveitis in Behcet's disease is effective in controlling the disease in two-thirds of patients.

Adrenomedullin in cultured human retinal pigment epithelial cells.

PURPOSE: To determine whether adrenomedullin (ADM), a vasorelaxant peptide is produced and secreted by human retinal pigment epithelial (RPE) cells, whether ADM expression is regulated by inflammatory cytokines and a growth factor, and whether ADM has proliferative effects on these cells. METHODS: Production and secretion of ADM by cultured human RPE cells were examined by Northern blot analysis and radioimmunoassay. Regulation of the ADM expression by basic fibroblast growth factor, interferon (IFN)-gamma, tumor necrosis factor-alpha, interleukin (IL)1beta, or all-trans-retinoic acid was studied. In addition, proliferative effects of ADM on human RPE cells were examined by modified 3-(4,5-dimetylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: ADM mRNA was expressed constitutively in all three human RPE cell lines (F-0202, D407, and ARPE-19) examined. Immunoreactive ADM was detected in the cultured media by radioimmunoassay. Sephadex G-50 column chromatography of the cultured medium showed a single peak eluting in the position of ADM-(1-52). Treatment with IFN-gamma or IL-beta increased ADM mRNA levels and immunoreactive-ADM levels in the medium in dose- and time-dependent manners in ARPE-19 cells. Exogenously added ADM increased the number of F-0202 cells and ARPE-19 cells, and the treatment with ADM antibody or ADM-(22-52) (an ADM antagonist) decreased it. CONCLUSIONS: Human RPE cells produced and secreted ADM. IFN-gamma and IL-1beta induced ADM expression in ARPE-19 cells. Furthermore, ADM stimulated proliferation of RPE cells. These results raise the possibility that ADM is related to the pathophysiology of some inflammatory and proliferative ocular diseases.

Quantitative analysis of major histocompatibility complex class II-positive cells in posterior segment of Royal College of Surgeons rat eyes.

Potential antigen-presenting cells in the posterior segment of Royal College of Surgeons (RCS) rat eyes were analyzed quantitatively. Light microscopic immunohistochemistry was performed at postnatal days (P) 10, 20, 28, 42, 63, and 140 in the eyes of RCS rats and their congenic counterparts. Immunohistochemical studies were carried out using monoclonal antibodies against major histocompatibility complex (MHC) class II antigen (OX6), a cytoplasmic antigen in bone marrow-derived macrophages (ED1), a membrane antigen on resident tissue macrophages (ED2), and a microglia/macrophage marker (OX42). Some sections were stained by a double-labeling method using these antibodies. No MHC class II-positive cells were seen in dystrophic RCS rat retinas at P10. They were found, however, in the outer nuclear layer and debris of outer segments at P20. From P20 to P42 the number of cells increased, then decreased until P140. Congenic controls, however, showed no MHC class II-positive cells in the retina. Cells double-labeled with OX6 and ED1 were present in the outer nuclear layer at P42, but no OX6 or OX42 double-labeled cells were detected. Also, no ED2-positive cells were detected. Our results suggest that MHC class II-positive cells may play some role in retinal dystrophy.

Identification of a novel isoform of microphthalmia-associated transcription factor that is enriched in retinal pigment epithelium.

Mutations at the mouse locus encoding microphthalmia-associated transcription factor (Mitf) affect the development of many cell types, including retinal pigment epithelium (RPE), melanocytes, mast cells, and osteoclasts. Here we have identified a novel Mitf isoform, Mitf-a, and its human homologue MITF-A by cDNA cloning. MITF-A consists of 520 amino acid residues and differs in the amino-terminus from authentic melanocyte-type MITF (MITF-M). MITF-A mRNA is widely expressed and represents a predominant MITF isoform in cultured RPE cells, whereas MITF-M mRNA is exclusively expressed in melanocytes and melanoma cells. In situ hybridization analysis suggested that Mitf-a mRNA is enriched in the prospective RPE of mouse embryo. Moreover, transient cotransfection assays suggested that MITF-A activated transcription of the tyrosinase and tyrosinase-related protein 1 genes. MITF-A/Mitf-a therefore may play an important role in melanogenesis in RPE.

Transplantation of retinal pigment epithelium using viable cryopreserved cells.

Transplantation of retinal pigment epithelium (RPE) may have potential clinical application for the surgical treatment of RPE-specific retinal degeneration, including age-related macular degeneration. The feasibility of an RPE storage bank has been investigated by experimenting with transplantation using viable, cryopreserved RPE cells. Fresh and cultured fetal human and bovine RPE cells were cryopreserved in 90% fetal bovine serum containing 10% dimethyl sulfoxide. The viability of the cells before and after cryopreservation was evaluated by trypan blue dye exclusion test, microculture tetrazolium assay (MTA), tissue culture, and transplantation after cryopreservation. The origin of RPE cells before and after cryopreservation was assessed by immunocytochemistry, immunoblotting, and indirect ELISA of RPE-marker protein using cytokeratin for cultured fetal human RPE cells and by immunocytochemistry of cellular retinaldehyde-binding protein (CR-ALBP) for cultured bovine RPE cells. Freshly isolated and cryopreserved uncultured bovine RPE cells were transplanted by posterior transscleral approach into the subretinal spaces of adult albino rabbits and 23-day-old Royal College of Surgeons (RCS) rats with a 33 gauge Hamilton syringe. Following surgery, artificial retinal blebs were confirmed by fundus examination. Morphologic examination was performed postoperatively by light and electron microscopy in albino rabbits and by light microscopy in RCS rats up to 3 mo. Control subretinal injections using vehicle solution also were performed in RCS rats. Cultured fetal human and bovine RPE cells after cryopreservation were found to be viable, based on the results of trypan blue dye exclusion test, MTA, tissue culture, and transplantation. Expression and reexpression of cytokeratin intermediate filaments in cultured fetal human RPE were demonstrated by immunocytochemistry, immunoblotting, and indirect ELISA before and after cryopreservation. Immunocytochemistry of CRALBP before and after cryopreservation in uncultured bovine RPE cells disclosed expression and reexpression of RPE cell marker protein. No uncultured fetal human RPE cells showed proliferation in tissue culture after cryopreservation. In rabbits, light and electron microscopy disclosed xenografted RPE cells residing on Bruch's membrane of the host retina. No sign of graft vs. host reaction was observed. No morphologic difference was noted between the fresh and 10-day-old cryopreserved RPE cells in situ following transplantation at day 25. In RCS rats, subretinal injection of 3-wk-old cryopreserved bovine RPE cells partially rescued photoreceptor cells locally at the transplanted area observed at 3 mo postoperatively. The retinal photoreceptors at the inferior hemisphere of the transplanted eye and the eye injected with vehicle solution showed no rescue effect. We found that cryopreserved cultured fetal human RPE cells and uncultured and cultured bovine RPE cells can be used for RPE transplantation studies. The ability to create an RPE storage bank as a source of donor cells may result in several clinical advantages.

Associated ocular findings in pericentral pigmentary retinopathy.

Macular complications occurred in two isolated patients who had pericentral pigmentary retinopathy. One patient demonstrated bilateral bull's-eye maculopathy and a unilateral full-thickness macular hole. Later, she developed central retinal artery occlusion in the fellow eye. The second patient had a rhegmatogenous retinal detachment that was reattached by scleral buckling surgery, but a full-thickness macular hole was found 3 months postoperatively. In both patients, foveal ischemia may have played a role for the development of macular hole, resulting in poor visual prognosis in pericentral pigmentary retinopathy.

The properties of retinal pigment epithelial cells in proliferative vitreoretinopathy compared with cultured retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells, which proliferate and dedifferentiate under several pathological conditions, and cultured RPE cells have been considered a good model for comparison. In this investigation, we compared the properties of RPE cells in proliferative vitreoretinopathy with that of cultured human RPE cells. mRNAs of RPE cells from patients with proliferative vitreoretinopathy and from cultured human RPE cells were extracted, and reverse transcriptase-polymerase chain reaction was performed. We also examined cells that were aspirated from bare RPE surface from a patient with a giant retinal tear. We amplified the interleukin-6 gene in the proliferative membranes and cultured RPE cells. We also amplified the tyrosinase gene in seven of eight proliferative membranes, as well as tyrosinase-related proteins and cellular retinaldehyde-binding protein genes, but not the tyrosinase gene in cultured RPE cells. The cells aspirated from bare RPE surface showed reduced activity for expressing interleukin-6 and tyrosinase genes. The dedifferentiation characteristics of cultured RPE cells were different, in that they were less active than RPE cells in proliferative membranes for expressing the genes of melanogenesis, which are essential for pigment cells. Interleukin-6 and genes that were related to melanogenesis were expressed in the proliferative membranes and may play an important role in the generation of proliferative vitreoretinopathy.

The role of quantitative 99Tcm-glucoheptonate scintigraphy in the evaluation of acute uveitis in patients with Behçet's disease.

The purpose of this study was to evaluate the role of quantitative 99Tcm-glucoheptonate (99Tcm-GH) scintigraphy in the assessment of patients with Behçet's disease who suffered from ocular inflammation (uveitis). The study consisted of 13 patients with uveitis and five control subjects. There were a total of 25 eyes with chronic uveitis. Of these 25 eyes, 10 were in a state of remission, and the other 15 were in an acute phase of the illness. The study was performed by administering 370 MBq (10 mCi) 99Tcm-GH intravenously. Planar images were acquired 6 h later. Eye/scalp indices were quantified by drawing regions of interest (ROIs) around each eye and normalizing the mean counts per pixel by the mean counts in the scalp. The mean eye/scalp indices were 1.87 +/- 0.19 in controls and 1.98 +/- 0.19 in the affected eyes that were in remission (P = 0.23, nonsignificant). However, during the acute phase of the illness, the mean eye/scalp index was 2.18 +/- 0.28. The difference between controls and the eyes that were in the acute phase of the illness was significant (one way analysis of variance, P = 0.007). The mean value of the index for affected eyes in remission was not significantly different to that for eyes in the acute phase (P = 0.068, nonsignificant). These preliminary findings suggest that, despite previously published reports in animals with experimentally induced uveitis, 99Tcm-GH scintigraphy may not be a very sensitive method for evaluating human ocular inflammations.

The glucose-6-phosphatase system in the retina.

The retinal glucose-6-phosphatase system was studied in rabbits. Following subcellular fractionation of the retina by differential centrifugation, the microsomal fraction was used for spectrophotometrical assay of the phosphate release. Assays were performed with intact and Triton X-100-treated preparations. Intactness of the microsomal preparations was assessed by using 2 mM mannose-6-phosphate as a substrate. Latencies towards glucose-6-phosphate and mannose-6-phosphate were 13.1 +/- 4.1% (n = 5) and 92.5 +/- 2.8% (n = 5) respectively; the difference between them was significant (P < 0.001). Pyridoxal-5'-phosphate specifically inhibited the retinal glucose-6-phosphate translocase activity at concentrations of 5-10 mM. Postnatal development was studied at postnatal days 1, 10, 17, 25, 36, 46, 54 and 70. At the postnatal 17th day, the retinal glucose-6-phosphatase specific activity was 60.1 +/- 3.8 (n = 3) nmol/min/mg protein which was one-third of the adult level [173.6 +/- 26.2 (n = 6) nmol/min/mg protein] (P < 0.001). This finding suggested that, in the rabbit, development of this enzyme system might coincide with development of retinal glucose catabolism.

Response of Müller cells following experimental lensectomy-vitrectomy.

We used morphological, biochemical and immunohistochemical methods to assess the response of Müller cells after experimental lensectomy-vitrectomy in rabbits. We observed widened intercellular spaces between the Müller cells and nerve fibers of ganglion cells, and increased electron opacity in the Müller cells of eyes injected with silicone oil. No apparent morphological changes were detected in the Müller cells of air-injected eyes. The specific and total activities of Müller cell-marker enzymes (glucose 6-phosphatase and glutamine synthetase) showed an initial increase, followed by a decrease. Glial fibrillary acidic protein immunoreactivity was not found in the Müller cells of the normal rabbit retina but was exhibited after surgery. Our results showed that markers of Müller cells associated with glycogenolysis and/or gluconeogenesis, glutamate-glutamine cycle and cytoskeletal protein metabolism were affected by the experimental lensectomy-vitrectomy.