Quantification of retinal pigment epithelial phenotypic variation using laser scanning cytometry.

PURPOSE: Quantifying phenotypic variation at the level of protein expression (variegation) within populations of retinal pigment epithelium (RPE) cells may be important in the study of pathologies associated with this variation. The lack of quantitative methods for examining single cells, however, and the variable presence of pigment and/or lipofuscin complicate this experimental goal. We have applied the technique of laser scanning cytometry (LSC) to paraffin sections of mouse and human eyes to evaluate the utility of LSC for these measurements. METHODS: Mouse eyes were perfusion fixed in 4% paraformaldehyde and embedded in paraffin. Postmortem human eyes were fixed and dissected to obtain a 9-mm punch, which was then embedded in paraffin. A laser scanning cytometer equipped with violet, argon, and helium-neon lasers and the detectors for blue, green, and long red were used to record the fluorescence of each individual cell at all three wavelengths. Raw data were recorded and processed using the WinCyte software. Individual nuclei were identified by the fluorescence of the 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Next, RPE cells were uniquely identified in the green channel using an anti-retinal pigment epithelium-specific protein 65 kDa (anti-RPE65) monoclonal antibody with an Alexa Fluor 488-labeled secondary antibody. Mn-superoxide dismutase (MnSOD) was quantified in the long-red channel using an anti-MnSOD antibody and an Alexa Fluor 647-labeled secondary antibody. MnSOD(+) and RPE65(+) cells exhibited peaks in the plot of fluorescence intensity versus cell number, which could be characterized by the mean fluorescence intensity (MFI), the coefficient of variation (CV), and the percentage of total RPE cells that were also labeled for MnSOD. RESULTS: RPE cells can be uniquely identified in human and mouse paraffin sections by immunolabeling with anti-RPE65 antibody. A second antigen, such as MnSOD, can then be probed only within this set of RPE. Results are plotted primarily with the population frequency diagram, which can be subdivided into multiple regions. The data collected for each region include the MFI, the CV, and the number of cells that are immunolabeled in that region. Background interference from pigment or autofluorescent material can be successfully overcome by elevating the concentrations of fluorescent secondary antibodies. In the human and mouse eyes, age-related changes in MFI, CV, and percent RPE cells immunolabeled for MnSOD were observed. CONCLUSIONS: The extent of the variability of gene expression in RPE cells at the protein level can be quantified by LSC. Relative changes in the MFI, the CV, and/or percentage of RPE cells double labeled for a second antigen quantify the changes observed. The analysis of these data also suggest whether the effects observed are related to local changes in transcription (alterations of CV) or major changes of protein expression (MFI), which are likely to be due to changes in the chromatin structure. The changes of these variables with age suggest that the observed age-related variegation is primarily due to changes in the chromatin structure in individual cells.

The expression of native and cultured human retinal pigment epithelial cells grown in different culture conditions.

AIM: To determine the transcriptional proximity of retinal pigment epithelium (RPE) cells grown under different culture conditions and native RPE. METHODS: ARPE-19 cells were grown under five conditions in 10% CO(2): "subconfluent" in DMEM/F12+10% FBS, "confluent" in serum and serum withdrawn, and "differentiated" for 2.5 months in serum and serum withdrawn medium. Native RPE was laser microdissected. Total RNA was extracted, reverse transcribed, and radiolabelled probes were hybridised to an array containing 5,353 genes. Arrays were evaluated by hierarchical cluster analysis and significance analysis of microarrays. RESULTS: 78% of genes were expressed by native RPE while 45.3--47.7% were expressed by ARPE-19 cells, depending on culture condition. While the most abundant genes were expressed by native and cultured cells, significant differences in low abundance genes were seen. Hierarchical cluster analysis showed that confluent and differentiated, serum withdrawn cultures clustered closest to native RPE, and that serum segregated cultured cells from native RPE. The number of differentially expressed genes and their function, and profile of expressed and unexpressed genes, demonstrate differences between native and cultured cells. CONCLUSIONS: While ARPE-19 cells have significant value for studying RPE behaviour, investigators must be aware of how culture conditions can influence the mRNA phenotype of the cell.

Oxidative stress--induced single-strand breaks in chromosomal telomeres of human retinal pigment epithelial cells in vitro.

PURPOSE: To demonstrate that chronic hyperoxia induces single-stranded breaks in chromosomal telomeres as a measure of oxidative DNA damage in cultured RPE cells. METHODS: RPE340 cells were cultured in 40% and 20% (control) O(2). DNA damage was assessed by mean terminal restriction fragment (TRF) length, and the S1 nuclease assay was used to determine the frequency of single-strand breaks in telomeric DNA. The degree of oxidative stress in cells was estimated by flow cytometric analysis of reactive oxygen intermediate (ROI)-induced 2',7'-dichlorodihydrofluorescein diacetate fluorescence and Northern blot analysis of heme oxygenase-1 (HO-1) mRNA induction. RESULTS: The mean TRF length of cells grown in 40% O(2) shortened at a faster rate than those grown in 20% O(2). The S1 nuclease assay showed that the accelerated mean TRF length shortening was due to an increased accumulation of single-stranded breaks in telomeric DNA. The degree of ROI production and HO-1 mRNA induction was greater in cells treated with 40% than 20% O(2), an effect that was also larger in old than young passaged cells. CONCLUSIONS: RPE340 cells in vitro grown in chronic hyperoxia exhibited evidence of DNA damage with accelerated telomeric shortening via an increased accumulation of single-strand breaks in telomeric DNA. These changes could provide insight into aging of RPE cells by oxidative DNA damage.

Chemotaxis of rat retinal glia to growth factors found in repairing wounds.

Retinal glia migrate to sites in the vitreous body and on the inner and outer surfaces of the retina during a wide variety of pathological processes. Using in vitro cultures of rat retinal glia, we have evaluated an extensive list of homogeneous, well-characterized growth factors as chemoattractants for these cells. Humoral growth factors, fibroblast growth factors, extracellular matrix proteins and immune system cytokines all were without activity except for the platelet-derived growth factor (PDGF). Extracts of bovine ocular tissues including vitreous body and retina yielded significant activity.

Expression and splicing of FGF receptor mRNAs during APRE-19 cell differentiation in vitro.

PURPOSE: The expression and alternative splicing of the four FGF receptor (FGFR) mRNAs are regulated in a developmental- and tissue-specific fashion. Capability of differentiation in vitro of the retinal pigment epithelial cell line ARPE-19 has been previously demonstrated. In this study, the hypothesis that FGF receptor gene expression and the alternative splicing of the FGFR1 mRNA is regulated as a function of ARPE-19 differentiation in vitro was tested. METHODS: ARPE-19 cells were plated at sparse or confluent densities and maintained in culture up to 14 months. The expression of FGF receptors and the ratio of the FGFR1beta to FGFR1alpha splice variants of the FGFR1 transcript were quantified by a published PCR technique. Two in vivo samples of human RPE served as controls. RESULTS: Sparse cultures of ARPE-19 cells predominantly express FGFR1. When these cultures are allowed to differentiate, FGFR2 is also expressed. Samples of mRNA from RPE cells in vivo exhibit FGFR1 and FGFR2 expression as well as FGFR3 expression, a form that is minimally apparent in vitro. The ratio of the FGFR1beta to FGFR1alpha splice variant decreases as a function of cell differentiation in vitro and approaches the ratio observed in human RPE cells in vivo. Stimulation of cultures in vitro with FGF2 as a prototypical differentiation agent does not regulate the ratio of the FGFR1beta to FGFR1alpha splice variant. CONCLUSIONS: Differentiation of the ARPE-19 cell line in vitro recapitulates many but not all the in vivo patterns of FGFR expression and splicing. This in vitro system may be useful for selected studies on how cellular differentiation regulates FGF receptor gene expression and splicing.

Distribution of FGF-5 in the rhesus macaque retina.

PURPOSE: To determine the distribution of mRNA transcribed from the FGF-5 gene and the distribution of FGF-5 protein in the normal adult retina of the rhesus macaque. METHODS: Freshly enucleated globes from rhesus macaque were prepared for Northern blot analysis, in situ hybridization, and immunohistochemical studies. A 350 base pair sequence from the human FGF-5 was subcloned and used to prepare 35S-labeled cRNAs for Northern and in situ experiments. Antipeptide antibodies were raised against an aminoterminal sequence as well as an internal sequence and were affinity purified on recombinant FGF-5 coupled to AffiGel-10. The specificity of antibody probes was verified by dot blot and Western blot analyses. RESULTS: Ganglion cells and photoreceptors express the highest levels of FGF-5 mRNA, although all neurons and the retinal pigment epithelium (RPE) were clearly labeled. Immunohistochemical staining similarly revealed a widespread distribution of the protein, with prominent labeling observed in photoreceptor inner segments, especially in cone cells. Plexiform layers and RPE cells were also clearly labeled. CONCLUSIONS: Clear evidence was established for the expression of FGF-5 in the retina, where it may play an important extracellular role as a secreted member of the FGF gene family.

Induction of an aging mRNA retinal pigment epithelial cell phenotype by matrix-containing advanced glycation end products in vitro.

PURPOSE: To determine an extensive mRNA phenotype of the established RPE cell line ARPE-19 when grown on a matrix modified by advanced glycation end products (AGEs). METHODS: Growth Factor Reduced Matrigel (Collaborative Biomedical Products, Bedford, MA) was nonenzymatically glycated with glycolaldehyde. ARPE-19 cells were seeded on both AGE-Matrigel and Matrigel and grown to confluence, and serum was withdrawn for 3 days. RNA was extracted, and microarray analysis was performed to characterize the genes, which are altered by a matrix modified by AGEs. Gene expression changes were confirmed by RT-PCR/Southern and Northern blot analysis. Apoptosis was measured by annexin V/propidium iodide labeling. RESULTS: Clusters of genes with altered expression were found related to cell differentiation, growth factors that regulate the RPE cell and basement membrane, and apoptosis. RT-PCR/Southern and Northern blot analysis confirmed the expression patterns of selected genes, and flow cytometry showed increased annexin V/propidium iodide-labeled cells when grown on AGE-Matrigel. CONCLUSIONS: Microarray analysis identified clusters of genes that could promote an aging RPE phenotype in vitro induced by a matrix modified with AGEs.

Ultrastructural and immunocytochemical changes in retinal pigment epithelium, retinal glia, and fibroblasts in vitreous culture.

Retinal pigment epithelial (RPE) cells, retinal glial, and fibroblasts, three cell types believed to play a role in the pathogenesis of epiretinal membrane formation, were maintained in vitreous culture to determine the influence of vitreous on their ultrastructure and expression of cytokeratin, glial fibrillary acidic protein (GFAP), vimentin, and glutamine synthetase (GS). Using a highly sensitive, preembedding technique for the immunolocalization of these antigens at the ultrastructural level, most RPE cells were found to lose cytokeratin and vimentin within 1 day after seeding on irradiated vitreous. The percentage of keratin-positive cells then increased with time in culture. If the vitreous was placed on RPE cells cultured in monolayer instead of placing the cells on the vitreous, keratin and vimentin were expressed these intermediate filament proteins diminished with time. Glutamine synthetase was found in RPE cells grown in monolayer with or without a vitreous overlay, but not in RPE cells grown on the surface of vitreous. Retinal glial grown on vitreous showed a time-dependent decrease in the number of cells expressing GFAP and a corresponding increase in cells expressing vimentin or GS. Some fibroblasts in vitreous culture expressed vimentin but not the other antigens evaluated. A substantial number of cells in each culture did not stain positively for cytokeratin, GFAP, vimentin, or GS. All three cell types showed phenotypic diversity at the ultrastructural level with each cell type being capable of assuming the same morphologic appearance under certain conditions. These results demonstrate the phenotypic plasticity of RPE cells, retinal glia, and fibroblasts when grown in contact with vitreous and provide further evidence that neither ultrastructure, intermediate filament protein expression, nor the presence of GS is sufficient to determine the cell type of origin of cells in epiretinal membranes.

Downregulation of differentiation specific gene expression by oxidative stress in ARPE-19 cells.

PURPOSE: To investigate how the differentiation of ARPE-19 cells affects the relative expression of the FGFR genes in response to oxidative stress. METHODS: After differentiation in vitro, APRE-19 cells were treated with t-butyl hydroperoxide (tBH) or hydrogen peroxide (H2O2) to induce oxidative stress. Viability and reactive oxygen intermediate (ROI) production were measured using standard assays. The mRNA expression of FGFR1, FGFR2, cellular retinaldehyde-binding protein (CRALBP), RPE65, and heme oxygenase-1 (HO-1) were measured by Northern blot analysis as a function of treatment with tBH and H2O2. RESULTS: ARPE-19 cells were viable at all tBH concentrations tested but showed progressive loss of viability at concentrations greater than 300 microM H2O2. Differentiated ARPE-19 cells treated with tBH or H2O2 resulted in upregulation of the HO-1 and FGFR1 transcripts. The expression of RPE-differentiated specific genes, including FGFR2, CRALBP, and RPE65 mRNAs, was downregulated with tBH or H2O2 treatment. CONCLUSIONS: Oxidative stress in differentiated ARPE-19 cells alters the expression of FGFR1, FGFR2, CRALBP, and RPE65 toward levels characteristic of the undifferentiated state. If similar changes take place in vivo, these events could alter the proliferative potential, viability, and even the function of the RPE.

Primary culture of human retinal glia.

Glial cells of the human retina participate in various pathologic processes characterized by cell migration, proliferation, and extracellular matrix production. To study these events in vitro, a procedure was developed to obtain primary cultures of human retinal glial cells. The cultures resulting from the processing of 130 globes contained cells with variable morphology including bipolar and multipolar or stellate cells. Most cells in the primary culture were labeled with antisera to the glial fibrillary acidic protein. The cultures were also examined with antibodies directed against factor VIII-related antigen and muscle-specific actin to determine the presence of endothelial cells and pericytes. A variable contamination of cells staining for the latter was found in these cultures (usually less than 10%). Together, these data indicated that the primary cultures arose principally from glial cells of the human retina but did not precisely identify the cell of origin.

Expression of fibroblast growth factor-5 by bovine choroidal endothelial cells in vitro.

PURPOSE: To demonstrate the expression of fibroblast growth factor-5 (FGF-5) by bovine choroidal microvascular endothelial (BCME) cells and to investigate its possible role as an autocrine mitogen in these cells. METHODS: Expression of FGF-5 by BCME cells was studied by a combination of Northern and Western blot analyses. Total RNA was isolated from BCME cultures at passages 5 through 8 and analyzed by Northern blot analysis for the presence of FGF-5 transcripts, using a 1-kb human complemetary DNA. Slot-blot analysis was performed to determine possible cross-reactivity between this probe and acidic and basic FGFs of human and bovine species. A previously characterized antibody directed against the aminoterminus of the human FGF-5 sequence was used in Western blot analyses to identify immunoreactive proteins released by BCME cells into the medium. Finally, the mitogenic activity of human recombinant FGF-5 on a variety of cell types was evaluated, using a cellular proliferation assay. RESULTS: Northern blot analysis provided evidence for the expression of two major FGF-5 transcripts at 4 kb and 3 kb and two minor transcripts at 2.2 kb and 1.7 kb. A single immunoreactive protein with a molecular weight of 34 kDa was identified by Western blot analysis of conditioned media. In cellular proliferation assays, human recombinant FGF-5 was not mitogenic in BCME cells but exhibited an approximate ED50 of 1.8 to 3.7 nM in BALB/c3T3 fibroblasts. This ED50 was within the range reported by the manufacturer, using a thymidine incorporation assay and a similar embryonic fibroblast cell line. Fibroblast growth factor-5 also stimulated proliferation of human retinal pigment epithelial cells. CONCLUSIONS: Bovine choroidal microvascular endothelial cells exhibit expression in vitro of FGF-5 at the messenger RNA and protein levels. Perivascular and endothelial cell staining for FGF-5 seen previously in choroidal neovascular membranes may therefore arise from expression by choroidal endothelial cells. Because nonglycosylated recombinant FGF-5 does not appear to be a mitogen in BCME cells in vitro, it is reasonable to question its role as an autocrine mitogen in vivo. Fibroblast growth factor-5 may instead be serving paracrine roles in the stimulation of fibroblasts and retinal pigment epithelial cells during the formation of choroidal neovascular membranes. Studies with fully glycosylated recombinant FGF-5 will be required, however, to assess the biologic activity of this member of the FGF gene family.

An experimental model of ectropion uveae and iris neovascularization in the cat.

Neovascularization of the iris (NVI) is one of the most frequently studied intraocular vascular proliferations in animal models. Ectropion uveae has not been a consistent finding in these studies. In this study, a surgical model of ectropion uveae and iris neovascularization was developed that involved lensectomy, vitrectomy, bipolar cautery and transection of all three principal branch veins in the cat eye. Twelve of 14 eyes that received this procedure developed postoperative retinal detachments with a clinical picture of hemorrhagic retinopathy. These eyes progressed to a clinical picture of NVI within 1-7 wk. Eight eyes developed ectropion uveae for as much as 300 degrees. At the light microscopic level, a fibrovascular membrane was apparent on the anterior iris stroma in 9 of 14 eyes and further involved the angle in six eyes.

Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5.

PURPOSE: To determine the polarity of fibroblast growth factor 5 (FGF5) secretions from retinal pigment epithelium (RPE) cells and to examine the viability and utility of the ARPE-19 cell line as a model for the study of RPE polarity. METHODS: Influenza infection and adenovirus-mediated gene transfer were used to deliver and express genes encoding influenza hemagglutinin (HA), p75-NTR (a neurotrophin receptor), low-density lipoprotein (LDL) receptor (LDLR), and FGF5 in confluent monolayers of ARPE-19 cells. The localization of HA, p75-NTR, and LDLR was determined by confocal microscopy. Domain selective biotinylation assays were used to quantitatively determine the polarities of p75-NTR and LDLR. The secretion of FGF5 into the apical and basal media of ARPE-19 cultures was examined by immunoblot analysis of conditioned media. RESULTS: Hemagglutinin and p75-NTR were found to be localized on the apical surface of infected and transduced ARPE-19 cells. In contrast, LDLR was associated preferentially with the basolateral membrane of ARPE-19 cells. Biotinylation studies indicated that 84% of p75-NTR was present on the apical surface, and 79% of LDLR was basolaterally polarized. Over the course of 6 hours, more than 90% of the total secreted FGF5 protein accumulated in the basolateral media. CONCLUSIONS: ARPE-19 cells exhibit a polarized distribution of cell surface markers when examined by either confocal microscopy or surface-labeling assays. This indicates that the ARPE-19 cell line is a valid model for studies of RPE cell polarity. FGF5, a secreted protein normally produced by RPE cells, is accumulated preferentially in the basal media after only 6 hours, suggesting that it is vectorially secreted from the basolateral surface of ARPE-19 cells.

Age and topographic variation of insulin-like growth factor-binding protein 2 in the human rpe.

PURPOSE: Previous studies have shown that insulin-like growth factor-binding protein (IGFBP)-2 is markedly upregulated in senescent RPE cells in vitro, and might therefore be a marker of senescent cells in vivo. This study was conducted to determine whether IGFBP-2 expression in human RPE cells from the macula and periphery varies with age in vivo. METHODS: Paraformaldehyde (4%)-fixed and optimal cutting temperature (OCT) compound-embedded human eyes from 17 patients were cryosectioned and subjected to high-sensitivity digoxigenin (DIG)-labeled cRNA in situ hybridization to determine the expression of IGFBP-2. Complementary immunohistochemistry experiments using a polyclonal anti-IGFBP-2 antibody were performed to confirm IGFBP-2 protein expression. Specimens were examined by light microscopy, and images were captured with a digital camera. The total numbers of RPE cells and IGFBP-2 mRNA expression-positive RPE cells were counted for each section, and the ratio of labeled RPE cells to total RPE cells counted was calculated for both macular and peripheral regions of each donor. RESULTS: IGFBP-2 mRNA expression was detected in the ganglion cell layer, inner and outer nuclear layers, and inner segments of photoreceptor cells in all 17 eyes. In 16 of 17 eyes, IGFBP-2 mRNA expression was detected in the RPE. In 11, the ratio of labeled cells to total RPE cells counted per section in the macula was 1.2 times greater than the ratio in the periphery (P = 0.008). The ratio of labeled RPE cells in the macula decreased with age (P = 0.0064). Immunohistochemistry studies for IGFBP-2 confirmed the expression pattern found by in situ hybridization. CONCLUSIONS: There is a topographical and age-related change in IGFBP-2 expression in RPE cells from human donor eyes. This distribution is likely not to represent senescent RPE cells in vivo.

Developmental expression of bFGF in the bovine retina.

PURPOSE: To examine the expression of bFGF in the developing bovine retina. METHODS: Fetal bovine eyes at 90, 120, 150, and 180 days gestational age, as well as adult bovine eyes, were immunohistochemically stained for the presence of bFGF. Detailed characterization of the anti-bFGF antibodies by immunoblot and Western blot analysis against pure FGF gene family standards and crude extracts of bovine retina were also performed. RESULTS: Expression of bFGF occurs beginning at 150 days of gestation, a period when photoreceptor development and secondary capillary network development is in process. No bFGF expression was found at 90 days, but primary capillaries were already apparent at this stage of development. CONCLUSIONS: Expression of bFGF in the developing bovine retina may play a functional role in outer retinal development.

Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells.

PURPOSE: To investigate the relation of senescence-related beta-galactosidase activity and telomere shortening to replicative senescence in cultured human retinal pigment epithelial (RPE) cells. METHODS: A human RPE cell line was serially passaged until 80% of cells were nondividing in a 72-hour 5-bromo-2'-deoxyuridine (BrdU) labeling study. Early- and late-passage cells were double-stained for BrdU and senescence-related beta-galactosidase activity (pH 6). The average chromosomal telomere length at several population doublings was estimated by Southern blot analysis after double digestion of DNA with RsaI and HinfI and using a telomere-specific probe. RESULTS: BrdU-beta-galactosidase double-staining revealed an inverse correlation between the number of BrdU-labeled nuclei and beta-galactosidase-labeled cells as a function of population doubling level (PDL). At PDL 58, only 20% of all cells labeled for BrdU, whereas 57% stained for beta-galactosidase. The mean terminal restriction fragment length (TRF) was reduced from 10 kb in early (PDL 12) cultures to 4 kb in late (PDL 57) cultures. CONCLUSIONS: Senescence-related beta-galactosidase activity and mean TRF length may prove useful in studying the senescence of RPE cells in vitro. These techniques may be valuable in determining senescence of the retinal pigment epithelium in vivo, where senescent RPE cells could be involved in the development of age-related maculopathy and age-related macular degeneration.

Increase in the advanced glycation end product pentosidine in Bruch's membrane with age.

PURPOSE: To determine whether there is an age-related increase of pentosidine in human Bruch's membranes and to localize pentosidine and carboxymethyllysine (CML), two well-characterized, advanced glycation end products (AGEs) in aged human Bruch's membranes and choroid in vivo. METHODS: Human Bruch's membrane samples were isolated from the retinal pigment epithelium (RPE) and choroid and subjected to reversed-phase high-performance liquid chromatography to determine pentosidine content. A polyclonal anti-pentosidine antibody and a monoclonal antibody specific for carboxymethyllysine were used to localize AGEs in 20-month-old nondiabetic, 82-year-old nondiabetic, and 82-year-old diabetic globes. RESULTS: Human Bruch's membranes (n = 20) showed a linear age-dependent increase in pentosidine that reached approximately 0.17 millimoles pentosidine per mole hydroxyproline in late life (r = 0.896; P < 0.001). Immunohistochemical evaluation showed evidence of pentosidine in Bruch's membrane, choroidal extracellular matrix, and vessel walls in the 82-year-old nondiabetic and diabetic globes. A similar staining pattern was found with the anti-CML antibody. Basal laminar deposits and drusen stained with both antibodies in the elderly nondiabetic eye. In contrast, neither antibody stained the 20-month-old tissue. CONCLUSIONS: We provide biochemical and immunohistochemical evidence for the formation of pentosidine and CML structures in human Bruch's membrane and choroid with age. These changes could promote aging of the RPE-Bruch's membrane-choroid complex.

Senescence-associated beta-galactosidase histochemistry for the primate eye.

PURPOSE: To develop a senescence-associated beta-galactosidase histochemistry and bleaching protocol for the primate posterior pole. METHODS: Rhesus monkey eyes of different ages were enucleated after death, fixed in 4% paraformaldehyde for up to 16 hours, and cryoprotected using a graded sucrose infiltration technique. Ten-micrometer tissue sections were treated with beta-galactosidase, pH 4 (lysosomal) or pH 6 (senescence-associated) activity, for various times. Bleaching of retinal pigment epithelial (RPE) cell and choroidal melanocyte pigment was performed after beta-galactosidase histochemistry using 0.1% to 1% potassium permanganate incubation for 1 minute to 2 hours followed by 0.5% oxalic acid immersion. RESULTS: A 6-hour incubation with beta-galactosidase, pH 4 or 6, demonstrated optimal staining of the RPE. Uniform staining of the RPE for pH 4 beta-galactosidase was seen in both young and old eyes. In contrast, senescence-associated beta-galactosidase (pH 6) staining was seen in the RPE of 16 and 29-year-old, but not 1- and 2-year-old eyes. Senescence-associated beta-galactosidase staining was evident in RPE cells adjacent to cuticular drusen. Optimal bleaching without loss of beta-galactosidase staining was obtained using a 25-minute incubation with 0.05% permanganate. CONCLUSIONS: The senescence-associated beta-galactosidase histochemistry assay, adapted for use in the primate posterior pole, showed staining of RPE cells in older eyes. Visualization of beta-galactosidase activity in the RPE was enhanced by permanganate bleaching of melanin pigment. This technique could be valuable for identifying senescent RPE cells in human eyes.

Induction of microsomal dimethylnitrosamine demethylase by pyrazole.

Pyrazole, a potent inhibitor of alcohol dehydrogenase, was found to be a potent inducer of the activity of low Km dimethylnitrosamine demethylase (DMN-d). One injection of pyrazole (200 mg/kg body wt) to weanling Wistar rats changed the microsomal DMN demethylase activity by 1.7, 1.9 and 2.5 times the control values at 6, 12 and 24 hr after the injection respectively. Pyrazole administration reduced arylhydrocarbon hydroxylase (AHH) activity. When animals were injected with pyrazole (200 mg/kg body wt) for 1, 2, 3 or 4 consecutive days, the values for DMN-d activity were 277, 297, 306 and 319% of the control values. The corresponding values for AHH were 91, 67, 57 and 45% for 1, 2, 3 and 4 injections respectively. pyrazole-induced DMN-d activity was NADPH dependent and was inhibited by CO; n-butanol gave a 50% inhibition at a concentration of 2 X 10(-3) M. The corresponding value for metyrapone was 1 X 10(-2) M. Cytochrome P-450 was slightly increased by pyrazole and its CO-complex gave an absorption maximum around 451 nm. When the microsomal proteins were separated using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, a large increase in a band at about 51,000 daltons was found in the liver microsomes of pyrazole-treated animals.

The use of hyperoxia to induce chronic mild oxidative stress in RPE cells in vitro.

PURPOSE: To establish a model of mild and chronic oxidative stress using hyperoxia for retinal pigment epithelial (RPE) cells in vitro. METHODS: RPE340 cells and WI38 lung fibroblasts were grown in normal oxygen (20% O2) and hyperoxia (40% O2 or 60% O2). After cell viability was examined, the levels of reactive oxygen intermediates (ROI) by flow cytometry and heme oxygenase-1 (HO-1) mRNA by northern analysis were measured as markers of oxidative stress in both cell types. Proliferative ability and gene expression pattern of growth factors were studied to demonstrate the phenotypic changes induced by mild oxidative stress upon these cells. RESULTS: While decreased by 60% O2, 40% O2 did not affect viability in both cell types, ROI production and HO-1 mRNA expression were elevated in hyperoxia compared to controls, but were inhibited with the antioxidant dehydro-ascorbic acid (DHA). The proliferation of cells by hyperoxia was inhibited in both cell types. The expression of growth factors induced by hyperoxia was cell type dependent. Fibroblast growth factor-2 mRNA was unchanged in RPE cells, but was increased in fibroblasts. Transforming growth factor-beta2 was decreased in RPE cells, but unchanged in fibroblasts. Vascular endothelial growth factor was downregulated in RPE cells, while upregulated in fibroblasts. Connective tissue growth factor was decreased in RPE cells, but was unchanged in fibroblasts. CONCLUSIONS: The results demonstrate that hyperoxia induces mild oxidative stress which alters the phenotype of cells in a cell type specific manner.