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(Investigative Ophthalmology and Visual Science. 2004;45:2098-2106.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0863
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Changes in Retinal Pigment Epithelial Gene Expression Induced by Rod Outer Segment Uptake

Itay Chowers,1 Yoonhee Kim,2,3,4 Ronald H. Farkas,1 Tushara L. Gunatilaka,1 Abigail S. Hackam,1 Peter A. Campochiaro,1,5 Silvia C. Finnemann,2,3,4 and Donald J. Zack1,5,6,7

1From the Guerrieri Center for Genetic Engineering and Molecular Ophthalmology at the Wilmer Institute and the 5Departments of Neuroscience and 6Molecular Biology and Genetics, and the 7McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and 2The Margaret M. Dyson Vision Research Institute and the 3Departments of Ophthalmology and 4Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Rod outer segment (ROS) uptake, a crucial function of the retinal pigment epithelium (RPE), probably involve multiple proteins, yet only a small number have so far been identified. The goal of this study was to find additional genes involved in ROS uptake and degradation by identifying ROS-induced gene expression changes.

METHODS. Human RPE-derived ARPE-19 cells were harvested 3 and 12 hours after addition of bovine ROS. Gene expression profiles were compared with control cultures by using a custom human retina cDNA microarray and were validated by quantitative real-time RT-PCR (QPCR). ROS binding and internalization were quantitated with a fluorescence assay.

RESULTS. Alterations in the expression levels of multiple genes (especially ones involved in transcriptional regulation, signal transduction, or protein modification) were detected 3 hours after ROS challenge, whereas by 12 hours most had returned to baseline. QPCR results corroborated the microarray results for seven of the eight genes tested. Time-course QPCR experiments on an independent sample set demonstrated characteristic temporal expression changes for each gene. Protein levels of one of these, plasminogen activator inhibitor-1 (PAI-1), were tested and found to parallel the mRNA changes. In addition, exogenous PAI-1 inhibited ROS uptake by RPE cells in vitro, consistent with its putative function in integrin receptor regulation.

CONCLUSIONS. ROS uptake is associated with regulation of multiple RPE genes in a gene-specific temporal pattern. These genes are candidates for involvement in ROS uptake and degradation, particularly PAI-1, for which the study provided evidence suggesting that it may participate in the negative feedback of ROS uptake.

The importance of prompt daily clearance of shed rod outer segments (ROS) by the retinal pigment epithelium (RPE) in maintaining normal retina function is underscored by the association of photoreceptor death and retinal degeneration with perturbation in this process.1 2 3 4 Furthermore, histologic and experimental findings suggest that in age-related macular degeneration (AMD), the most common cause of irreversible blindness in the Western world, there may be alterations in ROS degradation by RPE cells.5 6 The relation between the disease process and these alterations is still unclear.

To date, four RPE plasma membrane receptor proteins have been described to be involved in ROS uptake (uptake including both ROS binding and internalization). These are a mannose receptor,7 the lipid scavenger receptor CD36,8 the integrin {alpha}vß5,9 10 and the receptor tyrosine kinase Mertk.11 In in vitro assays, {alpha}vß5 integrin mediates ROS binding to the RPE surface, whereas both CD36 and Mertk are involved in internalization of bound ROS.10 12 13 CD36 and Mertk probably activate signaling pathways to enable ROS internalization, but the pathway(s) involved have not yet been identified. After internalization, ROS are degraded in RPE lysosomes.14

Despite these exciting discoveries, much remains to be learned of ROS uptake by the RPE. For example, yet to be identified are additional genes involved in ROS uptake, related intracellular signal transduction, cellular preparation for subsequent cycles of ROS uptake, and modulating the RPE ROS uptake rhythm. It seems likely that the expression of at least some of the genes involved in ROS uptake are altered during the process. However, few studies have focused on RPE gene expression after ROS uptake. Ershov et al.15 16 reported that levels of early response genes including c-fos, zif-268, nur77, and cyclooxygenase-2 (COX2) peaked within an hour after ROS challenge to RPE cells in vitro, whereas levels of the peroxisome proliferator-activated receptor (PPAR)-{gamma} peaked after several hours. These investigators also described differential regulation of some of these genes, as well as increased levels of enzymes involved in prostaglandin synthesis and inhibition of ROS uptake on addition of prostaglandins to the culture medium.17 These findings suggest that ROS phagocytic challenge may regulate RPE phagocytic activity, at least in part, by altering RPE gene expression.

Herein, we describe use of a high-throughput approach to characterizing alterations in RPE gene expression specifically associated with ROS phagocytosis. A human RPE cell line (ARPE-19) that has been shown to phagocytize isolated ROS was studied.10 We used a custom human retina cDNA microarray that includes sequences representing more than 10,000 retina- and RPE-expressed genes.18 To validate and explore further the results of the microarray experiments, we followed the time course of mRNA level changes of several genes induced by ROS uptake using real-time quantitative PCR (QPCR). We also demonstrated for one of the genes, plasminogen activator inhibitor-1 (PAI-1), that its corresponding protein level correlated well with the mRNA changes. In addition, to begin exploration of the biological meaning of these findings, we tested the effect of exogenous PAI-1 on RPE-mediated ROS uptake.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Tissue Culture
The human retinal pigment epithelium cell line ARPE-19 was used in all experiments.19 Cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Invitrogen Corp., Carlsbad, CA), in 75-cm2 polystyrene flasks (Corning, Corning, NY) or 24-well polystyrene plates (Falcon, Franklin Lake, NJ) for the microarray and time-response experiments, respectively. Cultures were maintained for 6 weeks before ROS uptake experiments.

ROS were isolated from fresh bovine eyes in a dark room, as previously described,20 and stored in 2.5% sucrose in DMEM at –80°C. For uptake experiments, the culture medium was replaced with DMEM supplemented with 3% FCS and ROS (at a ratio of 10 ROS to 1 RPE cell) for 3 hours. Medium in control culture plates was also replaced with DMEM supplemented with 3% FCS and the same volume of 2.5% sucrose as that added to the study plates. For the longer time points (see the following sections), medium containing ROS was removed after 3 hours, cells were washed with PBS, and medium without ROS was added to the flasks.

Monitoring ROS Uptake by Immunostaining and Fluorescence Labeling
For validation of ROS phagocytosis by the ARPE-19 cells, cells were grown on chamber slides (Laboratory-Tek II; Nalge Nunc International, Naperville, IL). ROS were labeled with 1 mg/mL of fluorescein isothiocyanate (FITC; Molecular Probes, Eugene, OR), as previously described10 and added to the medium. After 3 hours, the incubation medium was removed, and cells were washed with PBS and were either incubated with the same medium without ROS for an additional 9 hours (total of 12 hours) or fixed with cold methanol for 20 minutes followed by a 1-hour incubation at room temperature with a mouse anti-bovine rhodopsin antibody.21 Secondary sheep anti-mouse IgG antibody conjugated to Cy3 (Sigma-Aldrich, St. Louis, MO) was then used to label the primary antibody. Images were recorded with a confocal system (Model TSP2; Leica, Bannockburn, IL).

Microarray Study Design
We studied gene expression changes in ARPE-19 cells 3 and 12 hours after ROS challenge using a retina and RPE custom cDNA microarray. The slide was constructed in our laboratory and contained sequences from more than 10,000 expressed sequence tags (ESTs) and genes that were selected to reflect the predicted human retina gene expression profile based on EST databases. A detailed description of array construction, probe labeling technique, and hybridization conditions, has recently been reported.18 Briefly, PCR products (fragments size average, roughly 0.9–1.0 kb) were spotted on coated slides (SuperAmine coating; TeleChem, Sunnyvale, CA) using an arrayer (MicroGrid II; BioRobotics, Cambridge, MA).

The 3- and 12-hour time points were selected in an attempt to identify genes that show changes in expression level during binding and internalization of ROS and during ROS degradation, respectively. Five replicates of control (only ROS medium with no ROS added) and study (ROS and medium added) plate pairs were studied at each time point (a total of 20 plates).

Duplicate microarray experiments were performed comparing each experimental versus control flask pair (20 microarray slides). In the first microarray reaction for each pair, the study sample was labeled with Cy3 and the control with Cy5, and in the second labeling reaction, dye assignment was exchanged between the samples. This design controls for dye-specific differences in the labeling and hybridization reactions, as well as for slide-specific bias.

Microarray Procedure
At either the 3- or 12-hour time point, the media were was removed, cells were washed with PBS, and RNA was extracted (TRIzol; Invitrogen) according to the manufacturer’s protocol. Twenty micrograms of total RNA from each flask was used for each labeling reaction. RNA was treated with a DNase (DNAfree; Ambion, Austin, TX) followed by a purification step (RNesy kit; Qiagen, Valencia, CA) and indirect Cy3 or Cy5 (Amersham Biosciences, Inc., Piscataway, NJ) dye incorporation, according to an established protocol.22 Prehybridization and hybridization were preformed as previously described.18 Briefly, slides were incubated for 40 minutes in 1% bovine serum albumin (BSA), 5x SSC, and 0.1% SDS at 42°C; washed in water and isopropanol; and dried using compressed air. Cy3- and Cy5-labeled probes were combined in 40 µL hybridization buffer (50% formamide, 5x SSC, 0.1% SDS buffer, 10 µg poly(dA), and 10 µg Cot-1 DNA) and heated at 95°C for 3 minutes. Hybridization was performed under a coverslip (Fisher Scientific, Suwanee, GA) in a humidified hybridization chamber (GeneMachine, San Carlos, CA) for 18 hours in a water bath at 42°C. After hybridization, slides were washed in 1x SSC/0.2% SDS at 42°C, followed by washes in 0.1x SSC/0.2% SDS, and 0.1x SSC both at room temperature. Each wash lasted for 4 minutes, and then the slides were dried with compressed air. Slides were scanned using a confocal laser scanner (ScanArray 5000; Perkin Elmer, Wellesley, MA). Spot finding and quantification were performed on computer (Imagene; Biodiscovery Inc., Marina Del Rey, CA).

Image files generated from the highest laser power settings that did not include saturated spots were used for further analyses. Background subtraction, normalization, omission of data from noninformative spots (according to the Imagene software empty spot flag function), and combining data from duplicate experiments of each experimental/control flask pair were performed using another software package (Genesight; Biodiscovery). The normalized experimental/control sample ratios were then analyzed using the Significance Analyses of Microarray (SAM) software,23 and a one-class response analysis was applied. SAM calculates a score for each gene based on the multiple of the expression level change between the study and the control samples and the consistency of this change multiple (estimated by the SD of the expression ratios). SAM then assesses a false discovery rate (FDR), which is the median percentage of genes from a list of genes (sorted according to the SAM score) that are likely to be mistakenly identified as differentially expressed. This estimation is based on comparison of the SAM score distribution between the actual experimental data and the results of random permutations of the same data.23

Real-Time Quantitative RT-PCR
Array results were confirmed using real-time QPCR on both the same sample set used for array experiments and on a second independent set of samples. The independent sample set was also used to assess the time course of mRNA level changes after ROS challenge for selected genes. In addition, to validate that transcripts identified in RPE culture are also expressed by in vivo RPE, we performed QPCR using as a template cDNA from a human retinal pigment epithelium sample. The RPE was dissected from a cadaveric human eye (70-year-old white male) without known eye disease and with normal gross appearance of the retina and RPE. The eye was obtained through the National Disease Research Interchange (IRB exemption 4; NDRI, Philadelphia, PA), and was obtained in accordance with the provisions of the Declaration of Helsinki for research involving human tissue. The death to tissue preservation time interval was 3 hours and 15 minutes, and the death to RNA extraction time interval was 39 hours.

To validate array results, equal amounts of RNA were pooled from the five replicates used for each study and control microarray experimental group. For the time-course experiments, duplicate samples were studied from each time point: RNA was extracted before addition of ROS and 15 and 30 minutes and 1, 3, 6, and 12 hours after addition of ROS, and prepared as described earlier. One-microgram aliquots of RNA from each pooled array experiment sample and time-course sample, and from human RPE, were then reverse transcribed (Superscript II transcriptase; Invitrogen) and used as template for QPCR reactions.

Reactions were performed using the thermocycler (Light-Cycler PCR machine; Roche, Nutley, NJ). Five serial twofold dilutions of one of the samples were defined as the standard for all the reactions. PCR products were quantified using the second derivate maximum values as calculated by the thermocycler analysis software. The second derivate maximum corresponds to the point at which the rate of change of fluorescence is the fastest, and it is a point at which the sample fluorescence can often be clearly distinguished from the background fluorescence. This value can be used to calculate the starting concentration of the cDNA of interest.

Expression levels were normalized to ribosomal phosphoprotein P0 mRNA levels.24 The primers used for real time PCR are listed in Table 1 . For each sample, each gene was tested in duplicate PCR reactions, and the mean of the two reactions was used to calculate expression levels. Reaction products were tested by agarose gel electrophoresis to confirm that there was only one PCR product and that it was of the expected size. As another quality control measure, we monitored the melting curves of PCR reactions to ensure that a reasonable curve was obtained and that the melting curve showed significant separation from that of primer dimers alone.


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  		TABLE 1. Primers Used for QPCR Experiments

 
Quantification of ROS Binding and Internalization
ARPE-19 cells were challenged in the presence or absence of 1 µM recombinant human PAI-1 (Molecular Innovations, Southfield, MI) with 10 FITC-ROS per cell in DMEM with 3% FCS for the duration of the experiment, chilled, washed three times with PBS containing 1 mM MgCl2 and 0.2 mM CaCl2 (PBS-CM) to remove excess ROS, and fixed in ice-cold methanol. In some cases, fluorescence derived from externally bound particles was quenched before fixation by using trypan blue to distinguish bound and internalized ROS from internalized ROS only.10 Fluorescence emission by fixed and mounted samples was quantified (Storm 860 Imager and ImageQuant 1.2; Molecular Dynamics, Sunnyvale, CA). The uptake index (representing bound and internalized particles) or the internalization index was calculated by dividing particle counts by nuclei counts of each area, thereby normalizing for phagocyte numbers. The binding index was calculated as the difference between uptake and internalization index. The correlation of phagocytic indices with phagosome numbers has been discussed in detail previously.25

Cell Lysis and Immunoblotting
After phagocytic challenge with OS, supernatants of ARPE-19 cells were collected, cleared by centrifugation, and denatured by boiling in reducing SDS-PAGE sample buffer for 3 minutes. Cells were rinsed with cold PBS-CM and solubilized by agitation for 30 minutes at 4°C in 50 mM HEPES (pH 7.4); 150 mM NaCl; 10% glycerol; 1.5 mM MgCl2; 1 mM EGTA; 1% Triton X-100; 1% sodium deoxycholate; 0.1% SDS; freshly supplemented with 2 mM each of aprotinin, leupeptin, and pepstatin; and 1 mM PMSF (all from Sigma-Aldrich). Whole-cell extracts representing equal numbers of cells (~30,000/lane) were separated on a 12% SDS-polyacrylamide gel under reducing conditions and transferred to nitrocellulose. Blots were incubated as indicated in the figure with primary polyclonal antibodies to actin (Sigma-Aldrich) or human PAI-1 (Molecular Innovations) and appropriate horseradish peroxidase–conjugated secondary antibodies followed by chemiluminescence detection (ECL; NEN, Boston, MA). X-ray films were scanned and signals quantified using NIH Image 1.61 (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD).


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Validation of Outer Segment Uptake by ARPE-19 Cells
Previous studies showed that ARPE-19 cells phagocytose isolated ROS.10 19 In our study, after phagocytic challenge with isolated FITC-labeled ROS for 3 hours, most of the ARPE-19 cells had taken up ROS particles (data not shown). The identity of the FITC-labeled particles was also confirmed by rhodopsin immunostaining that showed a perinuclear colocalization with the FITC-labeled particles. No staining was evident when the primary antibody was omitted or when no ROS were added to the media (data not shown).

Microarray Studies
Initial evaluation of the data revealed that 6,620 and 7,130 of the 10,034 spots on the microarray were informative for the 3- and 12-hour time points, respectively. At a false-discovery rate (FDR) of 25% (25% of the genes in the list are predicted to be identified by chance alone and are not related to ROS uptake), based on SAM analysis, 41 genes with increased expression levels 3 hours after ROS uptake were identified (Table 2A) . The mean expression level increase of these genes was 1.4- to 4.1-fold compared with ARPE-19 cells challenged by media alone. Twenty-nine of these upregulated genes had been characterized, 1 gene was cloned but its function is still unknown, and 11 were expressed sequence tags (ESTs) from novel genes. Molecular function annotation showed that 10 of the genes with known function are involved in transcriptional regulation, 7 in signal transduction, and 6 in protein modification or interaction with other proteins; 2 are adhesion molecules, and the remaining 4 have a variety of other functions. Two of the genes, thioredoxin reductase 1, and dual specificity phosphatase 1, are involved in oxidative stress response (Fig. 1) . The expression levels of the four genes downregulated at the 3-hour time point were reduced 2.5- to 17.1-fold compared with the control group (Table 2A) . At a more stringent FDR of 10%, only the top six upregulated and two downregulated genes from Table 2A were predicted to be differentially expressed in ARPE cells 3 hours after ROS challenge.


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  		TABLE 2. Differential Gene Expression

 


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  		FIGURE 1. Molecular function breakdown of 29 genes with known function found to be upregulated 3 hours after ROS challenge. The number of genes in each functional class is in parentheses.

 
Twelve hours after ROS challenge, 10 genes were upregulated, whereas none was downregulated at 20% FDR. All 10 genes showed less than twofold increased expression. Six of these genes are characterized and four are ESTs of novel genes (Table 2B) .

QPCR Experiments
Eight genes that were identified by the array experiments as having an ROS uptake–associated expression pattern were also assessed by QPCR. These genes were selected based on the magnitude of expression level change associated with ROS uptake as well as on their known function, which make them potential candidates for involvement in the process. QPCR on the same samples used for microarray experiments confirmed the array results for seven of the eight genes tested. The amount of expression level change detected by the two methods correlated well in most cases, ranging from 1.9- to 4-fold according to the arrays and 1.3- to 4.2-fold, according to QPCR (Table 2A) . In one case (leukotriene A4 hydrolase) the array results indicated downregulation of the gene, whereas QPCR indicated no change in the expression level.

Expanded Time-Course Expression Level Analysis
We performed a more detailed time-course analysis of the expression levels of five of the genes that were indicated by the microarray analysis to be upregulated 3 hours after ROS challenge. The purpose of this experiment was both to validate the microarray results on an independent set of samples and to obtain more information about expression level changes associated with ROS challenge. We also evaluated two genes, early growth response 1 (EGR1) and Fos, that were reported by Ershov et al.15 to be upregulated after ROS uptake.

Although five of these seven genes showed expression level changes comparable to those identified by the microarrays or to those reported by Ershov et al., two genes, thioredoxin reductase 1 and activating transcription factor 4, showed fluctuating expression levels (Fig. 2) . These may result from large expression level variation of the two genes between different ARPE-19 cell populations that are not necessarily related to ROS uptake. Alternatively, these genes may show altered expression levels after ROS uptake that were missed by the time points that were tested. Indeed, QPCR experiments showed that the temporal pattern of transcription activation after ROS challenge is gene specific (Fig. 2) . Early growth response 1 levels peaked 15 minutes after ROS challenge, whereas DNA-damage–inducible transcript 3 mRNA levels increased gradually and peaked at 3 hours. FOS-like antigen 1 (Fosl1) mRNA levels also increased after 15 minutes, but the transcript level plateaued for several hours and declined after only 3 to 6 hours. By contrast, PAI-1 levels reached a plateau after 1 hour and maintained an increased level throughout the 12-hour experimental period.



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  		FIGURE 2. mRNA levels of five transcripts upregulated 3 hours after ROS challenge, according to our microarray experiments, and of two transcripts previously reported, as assessed by QPCR. Seven time points were evaluated. The y-axis shows the log2 ratio of the mRNA levels in the sample challenged with ROS to the control samples. The x-axis shows the time in minutes after ROS challenge. (A) DNA-damage–inducible transcript 3 (DDIT3); (B) fos-like antigen 1 (Fosl1); (C) plasminogen activator inhibitor type 1 (PAI-1); (D) early growth response 1 (EGR1); (E) Fos; (F) activating transcription factor 4 (ATF4); and (G) thioredoxin reductase 1 (TXNRD1).

 
All five transcripts that were identified by our array experiments and that were evaluated in these time-response experiments also showed detectable mRNA levels in RPE cells dissected from a donor human eye.

PAI-1 Production and Its Effect on ROS Uptake
We next evaluated the correlation between PAI-1 mRNA and protein level changes, as well as the functional significance of the increased PAI-1 expression level that is associated with ROS uptake. PAI-1 was selected for these experiments because of its known function as an integrin regulator.26 We found that PAI-1 is constitutively produced by ARPE-19 cells; furthermore, PAI-1 protein levels increased significantly 5 and 6 hours after ROS challenge (Fig. 3A) . To assess the possible function of PAI-1 in ROS uptake, we quantified ROS uptake in the presence of exogenous recombinant PAI-1 at a physiologic concentration. PAI-1 decreased ROS binding to ARPE-19 cells by 57% 2.5 hours after ROS challenge (Fig. 3B) , further suggesting that PAI-1 may play a role in RPE-mediated ROS uptake.



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  		FIGURE 3. ROS phagocytic challenge upregulates PAI-1 protein in ARPE-19 cells. Soluble PAI-1 inhibits ROS binding. (A) ARPE-19 cells received assay medium (medium) or ROS in medium (OS), for 3 to 6 hours as indicated before lysis. Immunoblot analysis was used to detect PAI-1 protein in samples containing an equal number of cells (PAI-1). Parallel gels blotted for actin confirmed equal loading of gels (actin). Quantification of band intensities revealed significant increases in PAI-1 protein in response to ROS by ARPE-19 cells ({blacksquare}) at 5 and 6 hours, compared with cells before challenge ({square}, / on x-axis) as determined by one-way analysis of variance followed by the Bonferroni post hoc test (P < 0.001). At earlier time points and at all time points after the addition of medium alone ( ), PAI-1 protein levels did not differ significantly from those in untreated control cells (P > 0.05). Data are the average results of three independent experiments ± standard deviation. (B) ARPE-19 cells were incubated with ROS for 2.5 and 5 hours in the presence of 2% PBS solvent ( ) or of 1 µM recombinant human PAI-1 ({blacksquare}). ROS total uptake and internalization were measured by fluorescence scanning of samples with and without trypan blue quenching of external OS, respectively. Binding indices and internalization indices were calculated. PAI-1 significantly reduced the OS binding index at the 2.5-hour time point by 57% ± 7% (t-test, P = 0.002) but had no effect on the internalization index, suggesting a primary effect on OS binding. Data are the average OS indices ± standard deviation (n = 3).

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
We found that ROS uptake is associated with expression level changes of multiple known and novel genes in ARPE-19 cells. We further demonstrated that these expression changes occur in a gene-specific manner in terms of timing and expression level changes. Expression level changes can occur within minutes after ROS challenge, with some genes returning to pre-ROS exposure levels within an hour, whereas other genes maintain high expression levels for several hours. Different challenges to RPE cells are likely to illicit not only distinct changes in patterns of gene expression, but also distinct kinetic patterns. Thus, the time points chosen for a particular experiment are likely to affect significantly the results that are obtained. In our experiments, genes from several functional classes showed differential expression—among them several that are involved in transcriptional regulation, signal transduction, and protein modification.

Based on their known function, some of the differentially expressed genes associated with ROS uptake are of particular interest. We selected one of these genes for study in further detail. PAI-1 has been shown to be produced by human RPE,27 28 29 30 is implicated in choroidal neovascularization,31 and has been reported to be downregulated in an in vitro model of RPE replication senescence.32 It has been shown to regulate adipocyte motility through the interaction of vitronectin with its receptor, the integrin {alpha}vß3.26 As integrin {alpha}vß5, another vitronectin receptor, plays a role in ROS binding by RPE cells,9 10 we speculated that PAI-1 may regulate the interaction of RPE and ROS.

We first confirmed previous reports demonstrating that RPE cells in culture constitutively produce PAI-1. We then showed that PAI-1 mRNA and protein levels increase after ROS challenge. Further experiments showed that addition of recombinant PAI-1 to the culture medium results in decreased ROS uptake by ARPE-19 cells. These observations support our hypothesis that PAI-1 is involved in ROS uptake regulation. Conceivably, PAI-1 serves in a negative feedback pathway decreasing continuous ROS uptake by RPE cells after initial phagocytosis. Tripathi et al.33 have described increased secretion of tissue plasminogen activator (tPA) from cultured porcine RPE cells in response to outer segment challenge; however, the functional significance of this finding is still unclear.

CD81 antigen, another gene of potential functional significance in ROS uptake, also showed increased expression levels after ROS challenge. This transmembrane protein belongs to the tetraspanin family, which is known to be associated with receptors and in particular, integrins.34 CD81 was shown to be involved in glial cell phagocytosis35 but its potential role in ROS uptake by the RPE has not been suggested previously.

We also found that coagulation factor III (F3) expression is upregulated in association with ROS uptake. F3 is also known to be upregulated in macrophages after phagocytosis,36 but this has not been described in RPE cells. This finding adds to published data indicating similarities in the phagocytosis process between RPE and macrophages. In particular, all known phagocytic receptors of RPE cells also mediate phagocytosis in macrophages.7 10

Our findings demonstrate the feasibility of using expression data to study the mechanisms of RPE-mediated ROS uptake and degradation. The findings indicate that multiple genes, in addition to the ones previously identified, are regulated by and potentially involved in ROS uptake. These genes should be interesting targets for additional studies designed to obtain a more comprehensive view of the mechanisms regulating ROS metabolism, and such increased understanding should provide insights into both normal retinal/RPE physiology and how it is altered in disease.


   Footnotes
 
Supported in part by grants from the National Eye Institute, Foundation Fighting Blindness, Macula Vision Foundation, and Santen Pharmaceutical and generous gifts from Marshall and Stevie Wishnack and Robert and Clarice Smith. PAC is the George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience and DJZ is the Guerrieri Professor of Genetic Engineering and Molecular Ophthalmology, the Johns Hopkins University School of Medicine. DZJ is a recipient of the Senior Investigator Award and SCF is a recipient of the Career Development Award from Research to Prevent Blindness.

Funding for the study described in this article was partially provided by Santen Pharmaceuticals, Inc. Under a licensing agreement between Santen Pharmaceuticals, Inc. and the Johns Hopkins University, IC and DJZ are entitled to a share of any royalties received by the University on sales of products described in this article. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

Submitted for publication August 10, 2003; revised January 22, 2004; accepted February 24, 2004.

Disclosure: I. Chowers, (P); Y. Kim, None; R.H. Farkas, T.L. Gunatilaka, None; A.S. Hackam, None; P.A. Campochiaro, None; S.C. Finnemann, None; D.J. Zack, Santen Pharmaceutical (F), (P)

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Donald J. Zack, 809 Maumenee Building, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287; dzack{at}bs.jhmi.edu.


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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