Release of extracellular vesicle miR-494-3p by ARPE-19 cells with impaired mitochondria.

BACKGROUND: Mitochondrial function in retinal pigmented epithelial (RPE) cells and extracellular vesicle (EV) formation/release are related through the lysosomal and exocytotic pathways that process and eliminate intracellular material, including mitochondrial fragments. We propose that RPE cells with impaired mitochondria will release EVs containing mitochondrial miRNAs that reflect the diminished capacity of mitochondria within these cells. METHODS: We screened ARPE-19 cells for miRNAs that localize to the mitochondria, exhibit biological activity, and are present in EVs released by both untreated cells and cells treated with rotenone to induce mitochondrial injury. EVs were characterized by vesicle size, size distribution, presence of EV biomarkers: CD81, CD63, and syntenin-1, miRNA cargo, and number concentration of EVs released per cell. RESULTS: We found that miR-494-3p was enriched in ARPE-19 mitochondria. Knockdown of miR-494-3p in ARPE-19 cells decreased ATP production and mitochondrial membrane potential in a dose-dependent manner, and decreased basal oxygen consumption rate and maximal respiratory capacity. Increased number of EVs released per cell and elevated levels of miR-494-3p in EVs released from ARPE-19 cells treated with rotenone were also measured. CONCLUSIONS: ARPE-19 mitochondrial function is regulated by miR-494-3p. Elevated levels of miR-494-3p in EVs released by ARPE-19 cells indicate diminished capacity of the mitochondria within these cells. GENERAL SIGNIFICANCE: EV miR-494-3p is a potential biomarker for RPE mitochondrial dysfunction, which plays a central role in non-neovascular age-related macular degeneration, and may be a diagnostic biomarker for monitoring the spread of degeneration to neighboring RPE cells in the retina.

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cells were grown under five conditions in 10% CO(2): "subconfluent" in DMEM/F12+10% FBS, "confluent" in serum and serum withdrawn, and "differentiated" for 2.5 months in serum and serum withdrawn medium.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.

A phase I trial of stereotactic external beam radiation for subfoveal choroidal neovascular membranes in age-related macular degeneration.

Effective treatment for neovascular age-related macular degeneration (AMD) is currently limited. Radiation therapy, a therapeutic approach with known antiangiogenic properties, has been investigated as a modality to prevent severe visual loss in AMD. Most of the studies using external beam radiation employed <25 Gy to the whole eye, which is below the dose of radiation that is toxic to the retina and optic nerve ( approximately 50 Gy and approximately 59 Gy, respectively). Stereotactic fractionated external beam radiation (St-EBR) is a method that allows radiation to be delivered to a small, defined area. We investigated the effects of St-EBR in incremental doses up to 40 Gy on neovascular AMD. Patients with clinical signs and fluorescein angiography demonstrating neovascular AMD, visual acuity (VA) better than 20/400 and ineligible for laser treatment (MPS criteria) or who refused to have laser photocoagulation were enrolled in the study. Each patient was treated with radiation at incremental dosages from 20 Gy to 40 Gy. After completion of the radiation course, all patients were followed-up at 3 and 7 weeks and 3, 6, and 12 months. Best-corrected VA (ETDRS), slit-lamp and fluorescein angiographic evaluations were performed at each visit. 94 eyes of 89 patients were treated from October 1997 to April 2000. The VA was 0.82+/-0.35 before treatment, 0.83+/-0.36 at 6 months, and 0.89+/-0.33 at 12 months. No patients suffered any significant acute side effects. No significant benefits in either VA or in membrane size were derived from increasing the doses of radiation. Our results are consistent with trends of a palliative benefit of radiotherapy in neovascular AMD and support further investigation of radiotherapy. Since there is no evidence that therapeutic effectiveness is dose dependent, our data provide no justification for potentially dangerous escalations in radiation dosage for treating neovascular AMD.

Clinical protocol. An open-label, phase I, single administration, dose-escalation study of ADGVPEDF.11D (ADPEDF) in neovascular age-related macular degeneration (AMD).

Age-Related Macular Degeneration (AMD) is, together with Diabetic Retinopathy, the most common cause of vision loss among adults in the U.S. and other developed countries. In the U.S., at least 1.7 million people have impaired vision due to AMD. Every year, more than 165,000 people contract AMD and 16,000 go blind from it, predominantly from a rapidly progressing form of the disease called "wet" AMD. Wet AMD is characterized by serious or hemorrhagic detachment of the retinal pigment epithelium and choroidal neovascularization. The macula has the highest concentration of photoreceptors facilitating central vision and permitting high-resolution visual acuity. The damage caused by the leakage and fibrovascular scarring in wet AMD leads to profound loss of central vision and severe loss of visual acuity (usually 20/200 or worse). People with wet AMD have several limitations, including inability to read, inability to recognize faces or drive, and the disease often leads to blindness. The onset of severe visual changes in wet AMD can occur suddenly. More than 400,000 Americans are currently affected by this form of the disease, and the incidence is rising rapidly with the aging of the population. Therefore, the serious consequences of this disease along with the limited treatment options and their effectiveness make this a very good candidate for a gene transfer treatment approach. The investigational agent, Ad(GV)PEDF.11D, is an E1-, partial E3-, E4- deleted replication-deficient, adenovirus serotype 5, gene transfer vector. The transgene in this vector is the cDNA for human pigment epithelium-derived factor (PEDF). PEDF is one of the most potent known antiangiogenic proteins found in humans. While Ad(GV)PEDF.11D is able to transduce many somatic cell types, the natural barrier to other tissues created by the retina limits the ability of Ad(GV)PEDF.11D to affect tissues other than in the eye. Intravitreal administration of Ad(GV)PEDF.11D provides a convenient means of delivering PEDF to the relevant cells within the eye likely to result in a more prolonged duration of effect versus administration of the PEDF protein alone. In three murine disease models (the laser-induced choroidal neovascularization model, the VEGF transgenic model, and the retinopathy of prematurity model) significant inhibition of neovascularization (up to 85%) was demonstrated with doses of Ad(GV)PEDF vectors ranging from 1 x 10(8) to 1 x 10(9) pu. In toxicology studies performed in Cynomolgus monkeys, a dose-related inflammatory response was observed. A dose of 1 x 10(8) pu caused no adverse effects, while the inflammatory response observed at 1 x 10(9) pu was minimal and fully reversible. The observed inflammatory response at doses of 1 x 10(10) and 5 x 10(10) pu were increasingly severe. The proposed clinical trial is an open-label, dose-escalation, phase I study to investigate the safety, tolerability and potential activity of intravitreal injection of Ad(GV)PEDF.11D in patients with wet AMD. Ad(GV)PEDF.11D will be injected once via intravitreal injection into the eye with the most advanced AMD based on visual acuity. Subjects will be age 50 or over and have severe wet AMD in at least one eye defined by a best-corrected vision of 20/200 or worse. The primary objectives of this investigation are: (1) to assess the safety, tolerability and feasibility of intravitreal injection of Ad(GV)PEDF.11D in patients with severe, neovascular AMD, (2) to identify the maximum tolerated dose (MTD) of Ad(GV)PEDF.11D, and (3) to get some indication of potential activity of Ad(GV)PEDF.11D.

Inhibition of cultured human RPE cell proliferation and lysyl hydroxylase activity by hydroxy derivatives of minoxidil.

PURPOSE: To examine the antiproliferative and lysyl hydroxylase suppressing effects of 3'-hydroxyminoxidil and 4'-hydroxyminoxidil, derivatives of minoxidil devoid of the antihypertensive effect, on human retinal pigment epithelial (RPE) cells in culture. METHODS: Subconfluent and confluent cultures of RPE cells, exposed to 0.01 to 5 mM 3' or 4'-hydroxyminoxidil for 15 minutes to 7 days, were examined for proliferation, viability, and morphologic changes. Lysyl hydroxylase activity in confluent cultures exposed to 1 mM 3'- or 4'-hydroxyminoxidil was determined by measuring the amount of 3H2O released from L-(4,5-3H)lysine-labeled unhydroxylated procollagen substrate after vacuum distillation. RESULTS: Both compounds inhibited the proliferation of subconfluent cultures of RPE cells in a dose-dependent fashion. The 50% effect occurred at 0.25 mM for 3'-hydroxyminoxidil and 0.5 mM for 4'-hydroxyminoxidil. The antiproliferative effect was detectable within 24 hours, required a minimum 1-hour exposure, and persisted even after the drug was removed from the culture medium. Cell viability experiments provided no evidence for toxicity. In contrast, the number of cells at confluent density was not affected. Both 3'-hydroxyminoxidil and 4'-hydroxyminoxidil suppressed lysyl hydroxylase activity by 72%. CONCLUSIONS: The structure of minoxidil can be altered to reduce the antihypertensive activity while preserving the antiproliferative and lysyl hydroxylase suppressing effects. The hydroxy derivatives of minoxidil may be useful in the treatment of proliferative vitreoretinopathy, a disease with unwanted proliferation of RPE cells.

Minoxidil inhibits ocular cell proliferation and lysyl hydroxylase activity.

PURPOSE: To examine the antiproliferative and lysyl hydroxylase-suppressing effects of minoxidil on cultured proliferating and density-arrested human retinal pigment epithelial cells (hRPE) and Tenon's capsule fibroblasts (hTCF). METHODS: Proliferating and density-arrested hRPE and hTCF, exposed to minoxidil (0.1-5 mM) for 15 min to 7 days, were examined by proliferation assays, [3H]thymidine incorporation, trypan-blue exclusion, and phase-contrast microscopy. The lysyl hydroxylase-suppressing effects were examined in confluent hRPE exposed to minoxidil (0.01-1 mM) using L-[4,5-3H]-lysine-labeled procollagen substrate and measuring the amount of tritium released as 3H2O after vacuum distillation. RESULTS: Minoxidil (0.1-5 mM) inhibited the proliferation of subconfluent cultures of hRPE and hTCF in a dose-dependent manner with a half-maximal effect at 1.5 and 2.5 mM, respectively. The antiproliferative effect, detectable within 24 hr, occurred with a limited exposure period and persisted even after removal of minoxidil from the culture medium. In contrast, 1-5 mM minoxidil had minimal effect on density-arrested hRPE and hTCF. However, at doses above 3 mM, although minoxidil had no effect on the number of density-arrested hRPE, morphologic and viability experiments indicated signs of cytotoxicity. Minoxidil (0.1-1 mM) caused a maximum of 71% reduction in the activity of lysyl hydroxylase, an enzyme needed for stable cross-links in collagen. CONCLUSIONS: Minoxidil may be a useful drug for the treatment of conditions such as proliferative vitreoretinopathy and bleb scarring after trabeculectomy, disorders with unwanted cell proliferation and collagen production.

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.

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.

The antiproliferative effect of a transferrin-toxin on human retinal pigment epithelial cells and rabbit fibroblasts.

PURPOSE: To determine the effect of a rabbit transferrin conjugated to recombinant ricin A chain (Tfr-rRA) and the carboxylic ionophore monensin on proliferating and density-arrested human retinal pigment epithelial cells and rabbit dermal fibroblasts. METHODS: Cells were seeded on 24-well plates at 20,000 cells/cm2 and exposed to Tfr-rRA (0.1-10,000 ng/ml) with or without monensin (0.01 microM), and with or without human transferrin (65.7 mg/l) for 5 minutes to 7 days. Cells were studied morphologically and counted at 1, 2, 4, and 7 days. RESULTS: Tfr-rRA (10-10,000 ng/ml) killed proliferating human retinal pigment epithelial cells and rabbit dermal fibroblasts in a dose-dependent manner (p < or = 0.01) up to a maximum of 86% and 93%, respectively. In contrast, Tfr-rRA had minimal effect on density-arrested human retinal pigment epithelial cells and rabbit dermal fibroblasts. The cytotoxicity of Tfr-rRA was inhibited by the addition of human transferrin (65.7 mg/l), an effect that was partially overcome by longer treatment with Tfr-rRA. Monensin (0.01 microM) increased the cytotoxicity of Tfr-rRA by 4.8-fold over Tfr-rRA alone, shortened the onset of cell kill with Tfr-rRA from 48 to 24 hours (P = 0.04), and partially reversed the neutralizing effect of human transferrin. CONCLUSIONS: The results indicate that Tfr-rRA effectively inhibited the proliferation of human retinal pigment epithelial cells and rabbit dermal fibroblasts in vitro. The inhibitory effect could be modified by the addition of human transferrin or monensin. Thus, this ricin A chain conjugate may interrupt the proliferation of cells necessary in the pathogenesis of proliferative vitreoretinopathy.

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.

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.

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.

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.

The mRNA phenotype of a human RPE cell line at replicative senescence.

PURPOSE: To explore the changes in expression of a set of genes in a single retinal pigment epithelial (RPE) cell line and two fibroblast cell lines as controls under culture conditions previously used for the analysis of senescent gene expression. METHODS: A single human RPE cell line, which had previously been characterized using known markers of senescence, and two fibroblast cell lines were grown to replicative exhaustion. The mRNA phenotype of genes known to be altered by senescence were studied by quantitative Northern analysis. RESULTS: The mRNA phenotype of cells changes at replicative senescence yielding a synthetic phenotype which is similar to cells found in repairing wounds. Of the genes studied, urokinase-type plasminogen activator and plasminogen activator inhibitor-1 were regulated in RPE cells similar to fibroblasts at senescence. The largest changes noted for any single gene were the upregulation of insulin growth factor binding protein 2, and the downregulation of collagen I alpha 2, basic fibroblast growth factor, and fibroblast growth factor-5. CONCLUSIONS: This study demonstrates an altered mRNA phenotype of a human RPE cell line grown to replicative exhaustion. This analysis of a single cell line emphasizes the variability of results based on a single cell line or tissue specimen and indicates the need for additional study.

Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye.

PURPOSE: To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes. METHODS: Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis. RESULTS: HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. CONCLUSIONS: A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.

Treatment with intravitreal steroid reduces blood-retinal barrier breakdown due to retinal photocoagulation.

The effect of corticosteroid treatment on blood-retinal barrier breakdown caused by argon-laser panretinal photocoagulation was evaluated in the rabbit eye. One day before photocoagulation, eyes were given either a sub-Tenon (20-mg) or intravitreal (2-mg) injection of triamcinolone acetonide. The severity of blood-retinal barrier breakdown was measured after photocoagulation using rapid sequential magnetic resonance imaging following intravenous administration of gadolinium diethylenetriaminepentaacetic acid. Leakage of gadolinium diethylenetriaminepentaacetic acid into the vitreous space was significantly lower in eyes that received intravitreal triamcinolone acetonide than in control eyes (P = .007); however, sub-Tenon triamcinolone acetonide produced no significant reduction in leakage (P = .65) compared with controls. Fluorescein angiography supported the magnetic resonance imaging findings. We conclude that retinal photocoagulation in the rabbit eye produces blood-retinal barrier breakdown that is partially amenable to corticosteroid treatment.

Distribution of the collagen IV isoforms in human Bruch's membrane.

AIMS: To determine the distribution of the alpha1 to alpha6 chains of type IV collagen in Bruch's membrane of the human posterior pole. METHODS: Cryosections (10 micro m) from 18 human eyes (20 months to 83 years old) were acid treated, blocked with 10% normal goat serum, incubated for 1 hour with monoclonal antibodies against type IV collagen isoform specific peptides at 1:75 dilution, and visualised with an ABC staining kit. RESULTS: In Bruch's membrane, the alpha1(IV) and alpha2(IV) chains were identified in retinal pigment epithelial (10/18 = 55%) and choriocapillaris basement membranes (18/18 = 100%); the alpha3(IV), alpha4(IV), and alpha5(IV) chains were also found in the retinal pigment epithelial basement membrane (13/18 = 72%). In the choroid, the alpha1(IV) and alpha2(IV) chains were detected in the blood vessels (18/18=100%). The alpha6(IV) chain was not identified in any sections. CONCLUSION: The heterogeneous distribution of alpha1-2(IV) and alpha3-5(IV) in Bruch's membrane could give insights into the function of this structure in health, ageing, and diseases such as age related macular degeneration.

Inhibition of proliferating lens epithelium with antitransferrin receptor immunotoxin.

We investigated the effect of an antitransferrin receptor immunotoxin (454A12-rRA) on proliferating human and baboon lens epithelium in vitro. Human and baboon lens epithelial cells grown in modified TC-199 medium at 35 degrees Celsius in 7% CO2 were seeded in 24 well plates at a density of 17,500 cells/ml to 40,000 cells/ml. The cells were exposed to various concentrations of 454A12-rRA for seven days. The sensitivity of proliferating human lens epithelium to 454A12-rRA was dependent on the dose, with a 60% to 70% reduction in cell counts at immunotoxin concentrations of 100 ng/ml and above. The immunotoxin had no significant effect on baboon lens epithelium in vitro, which suggests that it is specific for human tissue. By preventing the proliferation of human lens epithelial cells, immunotoxin 454A12-rRA may be useful in the management of posterior capsule opacification after planned extracapsular cataract surgery.