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Purpose: To investigate changes at a molecular level in the mouse corneal endothelium (CE) exposed to chronic cigarette smoke (CS)., Methods: Pregnant mice (gestation days 18-20) were placed in a whole-body exposure smoking chamber, and a few days later pups were born. After 3.5 months of CS exposure, a ConfoScan4 scanning microscope was used to examine the corneal endothelial cells (CECs) of CS-exposed and control (Ct) mice. The CE was peeled under a microscope and maintained as four biological replicates (two male and two female) for CS-exposed and Ct mice; each replicate consisted of 16 CEs. The proteome of the CE was investigated through mass spectrometry., Results: The CE images of CS-exposed and Ct mice revealed a difference in the shape of CECs accompanied by a nearly 10% decrease in CEC density (P < 0.00003) following CS exposure. Proteome profiling identified a total of 524 proteins exhibiting statistically significant changes in CE from CS-exposed mice. Importantly, proteins associated with Descemet's membrane (DM), including COL4α1, COL4α2, COL4α3, COL4α4, COL4α5, COL4α6, COL8α1, COL8α2, and FN1, among others, exhibited diminished protein levels in the CE of CS-exposed mice., Conclusions: Our data confirm that exposure to CS results in reduced CEC density accompanied by diminished levels of multiple collagen and extracellular matrix proteins associated with DM.

Purpose: The RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that βA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells., Methods: RPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of βA3/A1-crystallin., Results: βA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of βA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and vimentin, downregulation of E-cadherin, and increased cell migration. βA3/A1-crystallin binds to cortactin, and loss of βA3/A1-crystallin resulted in increased P-cortactinY421. The RPE from AMD samples had increased Snail and vimentin, and decreased E-cadherin, compared to age-matched controls., Conclusions: We introduced a novel concept of dry AMD initiation induced by lysosomal clearance defects in the RPE and subsequent attempts by RPE cells to avoid the resulting stress by undergoing EMT. We demonstrate that βA3/A1-crystallin is a potential therapeutic target for AMD through rejuvenation of lysosomal dysfunction and potentially, reversal of EMT.

Purpose: Mice typically produce apolipoprotein B (apoB)-48 and not apoB100. Apolipoprotein B100 accumulates in Bruch's membrane prior to basal deposit and drusen formation during the onset of AMD, raising the possibility that they are a trigger for these Bruch's membrane alterations. The purpose herein, was to determine whether mice that predominantly produce apoB100 develop features of AMD., Methods: The eyes of mice that produce apoB100 were examined for apoB100 synthesis, cholesteryl esterase/filipin labeling for cholesteryl esters, and transmission electron microscopy for lipid particles and phenotype., Results: Apolipoprotein B100 was abundant in the RPE-choroid of apoB100, but not wild-type mice by Western blot analysis. The apolipoprotein B100,(35)S-radiolabeled and immunoprecipitated from RPE explants, confirmed that apoB100 was synthesized by RPE. Apolipoprotein B100, but not control mice, had cholesteryl esters and lipid particles in Bruch's membrane. Immunoreactivity of ApoB100 was present in the RPE and Bruch's membrane, but not choroidal endothelium of apoB100 mice. Ultrastructural changes were consistent with aging, but not AMD when aged up to 18 months. The induction of advanced glycation end products to alter Bruch's membrane, did not promote basal linear deposit or drusen formation., Conclusions: Mice that produce apoB100 in the RPE and liver secrete lipoproteins into Bruch's membrane, but not to the extent that distinct features of AMD develop, which suggests that either additional lipoprotein accumulation or additional factors are necessary to initiate their formation., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Uveal melanoma is the most common primary intraocular malignancy in adults. Although local disease can be controlled with radiation therapy or enucleation, many cases are complicated by metastases, which account for the significant mortality from this disease. To date, no chemotherapeutic regimens effectively treat local or metastatic disease. Epigenetic silencing of tumor suppressor genes has been shown to be an important factor in the growth and metastasis of many cancers. One form of epigenetic alteration is DNA methylation, which often occurs at promoter elements resulting in the silencing of target gene transcription., Methods: We used 5-aza-2'-deoxycytidine (5-Aza), a well characterized demethylating agent that is US Food and Drug Administration approved to decrease DNA methylation in multiple uveal and cutaneous melanoma cell lines., Results: Demethylation of melanoma cell lines using 5-Aza causes significant decreases in growth, invasion, and clonogenicity. Treatment of melanoma cells with combined 5-Aza therapy and irradiation showed an even more pronounced effect on cell viability. In addition, treatment with 5-Aza decreased the number of metastases from the eye to the lung in a murine cutaneous melanoma xenograft model., Conclusions: We demonstrate in vitro and in vivo that demethylating agents such as 5-Aza may be promising chemotherapeutic agents for treating melanoma and decreasing progression to metastatic disease. These results provide proof of concept for an exciting potential therapy to reduce mortality from this disease. Future work will focus on identifying pathways that mediate these changes., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To determine the extent that auditory force feedback (AFF) substitution improves performance during a simulated ophthalmic peeling procedure., Methods: A 25-gauge force-sensing microforceps was linked to two AFF modes. The "alarm" AFF mode sounded when the force reached 9 mN. The "warning" AFF mode made beeps with a frequency proportional to the generated force. Participants with different surgical experience levels were asked to peel a series of bandage strips off a platform as quickly as possible without exceeding 9 mN of force. In study arm A, participants peeled with alarm and warning AFF modes, the order randomized within the experience level. In study arm B, participants first peeled without AFF, then alarm or warning AFF (order randomized within the experience level), and finally without AFF., Results: Of the 28 "surgeon" participants, AFF improved membrane peeling performance, reducing average force generated (P < 0.01), SD of forces (P < 0.05), and force × time above 9 mN (P < 0.01). Short training periods with AFF improved subsequent peeling performance when AFF was turned off, with reductions in average force, SD of force, maximum force, time spent above 9 mN, and force × time above 9 mN (all P < 0.001). Except for maximum force, peeling with AFF reduced all force parameters (P < 0.05) more than peeling without AFF after completing a training session., Conclusions: AFF enables the surgeon to reduce the forces generated with improved precision during phantom membrane peeling, regardless of surgical experience. New force-sensing surgical tools combined with AFF offer the potential to enhance surgical training and improve surgical performance.

Purpose: Controlling the spread of uveal melanoma is key to improving survival of patients with this common intraocular malignancy. The Notch ligand Jag2 has been shown to be upregulated in primary tumors that metastasize, and we therefore investigated its role in promoting invasion and clonogenic growth of uveal melanoma cells., Methods: mRNA and protein expression of Notch pathway components were measured using qPCR and Western blot in uveal melanoma cell lines. Expression of Jag2 ligand was upregulated using Jag2-GFP-MSCV constructs or downregulated by sh-Jag2 in the uveal melanoma cell lines Mel285, Mel290, 92.1, and OMM1, and the effects on growth and invasion were assessed., Results: Jag2 was introduced into Mel285 and Mel290 cells, which have low baseline levels of both this ligand and Notch activity. Overall growth of the Jag2-expressing cultures increased somewhat, and a significant 3-fold increase in clonogenic growth in soft agar was also noted. Introduction of Jag2 increased motility in both wound-healing and transwell invasion assays. We also observed a significant increase in Jag2 and Hes1 mRNA in invasive OMM1 cells that had passed through a Matrigel-coated filter in the transwell assay when compared with noninvading cells. Loss-of-function studies performed in 92.1 and OMM1 lines using Jag2 shRNAs showed that downregulation of the ligand significantly suppressed cellular growth, invasion, and migration., Conclusions: Our data suggest that Jag2 may play an important role in promoting Notch activity, growth, and metastasis in uveal melanoma.

Purpose: Epidemiologic data indicate that people who consume low glycemic index (GI) diets are at reduced risk for the onset and progression of age-related macular degeneration (AMD). The authors sought corroboration of this observation in an animal model., Methods: Five- and 16-month-old C57BL/6 mice were fed high or low GI diets until they were 17 and 23.5 months of age, respectively. Retinal lesions were evaluated by transmission electron microscopy, and advanced glycation end products (AGEs) were evaluated by immunohistochemistry., Results: Retinal lesions including basal laminar deposits, loss of basal infoldings, and vacuoles in the retinal pigment epithelium were more prevalent in the 23.5- than in the 17-month-old mice. Within each age group, consumption of a high GI diet increased the risk for lesions and the risk for photoreceptor abnormalities and accumulation of AGEs., Conclusions: Consuming high GI diets accelerates the appearance of age-related retinal lesions that precede AMD in mice, perhaps by increasing the deposition of toxic AGEs in the retina. The data support the hypothesis that consuming lower GI diets, or simulation of their effects with nutraceuticals or drugs, may protect against AMD. The high GI-fed C57BL/6 mouse is a new model of age-related retinal lesions that precede AMD and mimic the early stages of disease and may be useful for drug discovery.

Purpose: To determine the effects of X-linked and autosomal recessive Alport syndrome on retinal basement membranes and how these result in the characteristic perimacular dot-and-fleck retinopathy, lozenge, and macular hole., Methods: The type IV collagen chains present in the normal retina were determined immunohistochemically. Ten patients with Alport syndrome underwent retinal photography and optical coherence tomography to determine the thickness of the internal limiting membrane (ILM) by segmentation analysis, the layers affected by the retinopathy, and any correlates of the lozenge and macular hole. Bruch's membrane was examined directly by electron microscopy in a donated Alport eye., Results: The alpha3alpha4alpha5 type IV collagen network was present in the normal ILM and in the retinal pigment epithelium basement membrane of Bruch's membrane. In Alport syndrome, the ILM/nerve fiber layer and Bruch's membrane were both thinned. The dot-and-fleck retinopathy corresponded to hyperreflectivity of the ILM/nerve fiber layer in the distribution of the nerve fiber layer. The lozenge and macular hole corresponded to temporal macular thinning. The thinning across the whole retina was principally due to thinning of the ILM/nerve fiber layer and inner nuclear layer., Conclusions: The Alport dot-and-fleck retinopathy results primarily from abnormalities in the ILM/nerve fiber layer rather than in Bruch's membrane. Thinning of the ILM/nerve fiber layer contributes to the retinopathy, lozenge, and macular hole, possibly through interfering with nutrition of the overlying retina or clearance of metabolic by-products.

Purpose: Matrix expansion is an early change in age-related maculopathy. The aim of this study was to determine whether connective tissue growth factor (CTGF) regulates the production of extracellular matrix components by retinal pigmented epithelial (RPE) cells., Methods: ARPE-19 cells were treated with CTGF and analyzed for fibronectin, laminin, and MMP-2 by RT-qPCR, Western blot analysis, or zymography. Cells were also pretreated with an MEK-1/2 inhibitor (PD98059) or a p38 inhibitor (SB203580) and an anti-CTGF antibody to analyze the signaling contributing to fibronectin, laminin, and MMP-2 production. Human maculas were analyzed for mRNA using laser capture microdissected RPE cells and by immunohistochemistry for the topographic distribution of CTGF., Results: CTGF induced fibronectin mRNA (P=0.006) and protein (P=0.006), and laminin mRNA (P=0.006) and protein (P=0.02) by ARPE-19 cells. CTGF also induced MMP-2 mRNA (P=0.002) and protein secretion (P=0.04). Using zymography, CTGF increased the latent and active forms of MMP-2 compared to controls (P=0.02). An anti-CTGF antibody inhibited fibronectin, laminin, and MMP-2 after CTGF stimulation. CTGF increased the phosphorylation of p38 and ERK1/2. Fibronectin and MMP-2 mRNA and protein were suppressed by a MEK-1/2 inhibitor, but not with a p38 inhibitor. Laminin expression was suppressed by both inhibitors. RT-qPCR analysis showed that macular RPE cells from human donors express CTGF. Immunohistochemistry of human maculas showed strong labeling of CTGF in Bruch membrane, including basal deposits and drusen., Conclusions: CTGF is increased in basal deposits and drusen of AMD specimens, and it induces matrix protein production in ARPE-19 cells through the ERK (p42/p44(mapk)) and p38(mapk) signaling pathways.

Purpose: Although light-induced oxidative stress in the retina has been extensively reported, little information regarding light-induced oxidative stress in choroidal endothelial cells (CECs) is available. In the current study, light-induced DNA oxidation and the activation of nuclear factor-kappaB (NF-kappaB), a major oxidative responsive transcription factor, were investigated in mouse CECs., Methods: Mice were exposed to green light. Light-induced DNA oxidation in CECs was detected by in situ 8-hydroxy-2-deoxyguanosine (8-oxo-dG) immunolabeling. CECs were isolated from the retinal pigment epithelium (RPE)/choroid by using immunomagnetic beads. The isolated CECs were immunochemically characterized by the expression of endothelial markers, CD31, and P1H12. The quality of total RNA from CECs was assessed by a bioanalyzer and RT-PCR. NF-kappaB activation in situ and in isolated CECs was investigated., Results: After a 3-hour exposure to light, the immunoreactivity to anti-8-oxo-dG antibody or anti-NF-kappaB p65 antibody in CECs in situ was significantly increased when compared with unexposed mice. Isolated CECs expressed CD31 and P1H12. The 28S/18S rRNA ratio of RNA isolated from CECs was 1.5:1. CD31 and von Willebrand Factor (vWF) transcripts were predominantly expressed in the RNA from isolated CECs. IkappaBalpha was more heavily phosphorylated in light-exposed than untreated CECs. IkappaBalpha expression levels were increased fivefold in isolated CECs after exposure to light compared to unexposed control subjects., Conclusions: Exposure to light induces oxidative stress in CECs in vivo. A method for CEC isolation from the mouse RPE/choroid with preservation of RNA quality has been developed. The results of this study may facilitate the ability to identify CEC-specific genes and gene products that respond to photo-oxidative stress.

Purpose: To determine the expression profiles of morphologically normal human retinal pigment epithelial (RPE) cells that originate from the macula and periphery., Methods: Morphologically normal RPE cells from 15 human globes from donors aged 52 to 82 years old were laser capture microdissected. Total RNA from 5000 cells was SMART amplified, [33]P-labeled, and hybridized to a cDNA array containing 4325 known genes. Expression profiles were analyzed by hierarchical cluster analysis, Prediction Analysis of Microarrays (PAM), and Significance Analysis for Microarrays (SAM). Differentially expressed genes were evaluated further by real time RT-PCR., Results: The overall expression profiles of RPE cells from the macula and periphery were similar. Unsupervised and supervised hierarchical cluster analysis showed that patient genotype was a stronger separating factor than topographical location. SAM analysis identified 11 genes that were underexpressed by macular RPE cells. The expression patterns of these 11 genes were confirmed by real time RT-PCR, with 5 genes reaching statistical significance., Conclusions: Whereas the overall expression profiles were similar between cells from the macula and periphery, subtle differential expression of five genes could contribute to RPE phenotypic differences based on topographic location., (Copyright Association for Research in Vision and Ophthalmology)

Purpose: Low-dose D-galactose treatment in mice induces accelerated aging due to advanced glycation endproduct (AGEs) formation. The purpose of this study was to identify ultrastructural aging in the retinal pigment epithelium (RPE)-Bruch's membrane-choriocapillaris., Methods: Five-month-old C57Bl6 mice were injected daily with D-galactose or control buffer for 8 weeks. Eighteen-month-old mice were also treated with control buffer for 8 weeks. Eyes were prepared for electron microscopy and AGE-specific fluorescence at ex = 370 nm/em = 440 nm and ex = 330 nm/ex = 390 nm., Results: D-Galactose treatment induced AGE-specific fluorescence in lens and RPE/choroid compared to buffer-treated controls. In D-galactose-treated animals, the RPE had dilated and fewer basolateral infoldings. Bruch's membrane had alterations that included significant thickening, sub-RPE and prominent outer collagenous layer deposits, and choriocapillaris basement membrane duplication/splitting and thickening. The choriocapillaris endothelium displayed fenestration loss., Conclusions: Ultrastructural aging to the RPE-Bruch's membrane-choriocapillaris developed in mice treated with low-dose D-galactose. These changes could contribute to age-related changes that promote early age-related disease.

Purpose: To investigate the hypothesis that there are topographic and age-related changes in the expression of heme oxygenase (HO)-1 and catalase in the RPE., Methods: Cryosections of the macula and periphery of human eyes (n = 18; aged 27-87 years) were subjected to a high-sensitivity digoxigenin (DIG)-labeled cRNA in situ hybridization protocol to determine the expression of HO-1 and catalase. The immunoreactivity of HO-1 and catalase were also investigated in the same sample set. Specimens were examined by light microscopy, and images were captured with a digital camera. The total number of RPE cells and HO-1- and catalase-labeled RPE cells was counted in each section, and the ratio of labeled RPE cells to total RPE cells was calculated in both the macular and the peripheral regions of each donor eye., Results: There was a mosaic pattern of mRNA and protein expression of HO-1 and catalase in macular and peripheral RPE. Topographical differences in the expression of HO-1 at the mRNA level and catalase at both the mRNA and protein levels was also observed. The topographical differences between the expression of HO-1 in the macula and periphery protein were not statistically significant but showed similar trends. For HO-1, the only significant age-related decline in expression was observed in the macula and periphery. Expression of HO-1 at the protein level and that of catalase at both the mRNA and protein levels showed no significant decline with age., Conclusions: There is a possible age-related decline in HO-1 expression, whereas catalase expression remains unchanged with aging. Both exhibit mosaic patterns in the RPE monolayer.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.