Use of Iris Pigment Epithelium to Replace Retinal Pigment Epithelium in Age-Related Macular Degeneration a Gene Expression Analysis

LABORATORY SCIENCES Use of Iris Pigment Epithelium to Replace Retinal Pigment Epithelium in Age-Related Macular Degeneration A Gene Expression Analysis

Hui Cai, MD, PhD; Min C. Shin, MD; Tongalp H. Tezel, MD; Henry J. Kaplan, MD; Lucian V. Del Priore, MD, PhD

Objective: To determine the gene expression profiles expressed only in RPE; 154 genes were expressed only in of primary retinal pigment epithelium (RPE) and iris pig- IPE. Twenty-two additional genes had greater than 3-fold ment epithelium (IPE) using microarrays. increased expression in RPE vs IPE, and 147 genes had greater than 3-fold decreased expression in RPE vs IPE. Methods: Primary RPE and IPE from 6 human donor eyes were collected, and total RNA was isolated. Differ- Conclusion: There are major differences in the gene ex- ences in gene expression were determined using a pression profiles of primary RPE vs IPE. human genechip (human U95Av2 [12 600 probes]; Affymetrix Inc, Santa Clara, Calif). Clinical Relevance: The different gene expression profiles of primary RPE vs IPE harvested from the same donor Results: Hierarchical cluster analysis differentiated the gene eyes infer that it may be difficult for IPE to replace all aspects expression profiles of RPE and IPE clusters into 2 distinct of damaged RPE function in transplantation studies. groups. A mean ± SD of 5308±416 gene probes were expressed in RPE vs 6130±205 in IPE. Sixty-eight genes were Arch Ophthalmol. 2006;124:1276-1285

EPLACEMENT OF DISEASED OR graphic atrophy, loss of RPE precedes cho- damaged retinal pigment riocapillaris and photoreceptor loss, and epithelium (RPE) has been RPE transplantation may prevent or re- an ongoing area of active in- verse these changes.25-28 In exudative AMD, vestigation for the last 3 de- the rationale for RPE transplantation is de- cades.R Efforts in this area are fueled by the ceptively simple, as the native RPE is ex- fact that RPE damage or dysfunction can cised unavoidably with the choroidal neo- lead to severe visual loss in many ocular dis- vascular complex during submacular orders; therefore, RPE replacement may surgery, and RPE removal leads to further prove beneficial to many patients with un- choriocapillaris atrophy and photorecep- treatable diseases. For example, RPE re- tor loss.29 Retinal pigment epithelium trans- placement may be a potential treatment for plantation may prevent subsequent cho- Leber congenital amaurosis, which devel- riocapillaris atrophy and improve the visual ops because of improper recycling of vi- prognosis.30-34 Initial clinical trials of RPE sual pigments by RPE from a defect in the transplantation in patients with exudative RPE65 gene.1 A defect in the MERTK gene or nonexudative AMD have resulted in in the Royal College of Surgeons rat model, nonsignificant improvement in vision; which leads to improper phagocytosis of however, graft survival is hampered by shed photoreceptor outer segments, was allograft rejection or by damage of Bruch’s treated successfully by RPE transplanta- membrane at the time of submacular tion, and homologous disease exists in hu- surgery.25-27,35-45 Author Affiliations: mans.2-23 Retinal pigment epithelium trans- Several major obstacles need to be over- Department of Ophthalmology, plantation may also have a role in the come before successful RPE replacement Harkness Eye Institute, management of age-related macular degen- in AMD, including the prevention of graft Columbia University, eration (AMD), which is the leading cause rejection by immune modulation or by tis- New York, NY (Drs Cai, Shin, and Del Priore); and Kentucky of blindness in patients older than 60 years sue matching. Retinal pigment epithe- 24 Lions Eye Center, University in the Western world. There are 2 ways lium is immunogenic, and the immune of Louisville (Drs Tezel in which RPE replacement may be a po- privilege of the subretinal space is rela- and Kaplan). tential treatment for advanced AMD. In geo- tive rather than absolute.46-56 The outer

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 blood-retinal barrier, which is ordinarily formed by tight junctions between adjacent RPE, is compromised in AMD. Table 1. Donor Information on Study Eyes* The inner blood-retinal barrier may be compromised in some eyes as well, because neovascularization can origi- Donor No./ Time From Death nate in the retina in eyes with retinal angiomatous pro- Sex/Age, y to Cell Extraction, h Cause of Death liferation.57,58 Compromise of the blood-retinal barrier in- Microarray Data creases the probability that allogeneic RPE will undergo 1/F/63 18.6 Cardiovascular disease 2/M/71 26 Cardiopulmonary arrest immune rejection in the subretinal space. Several tech- 3/M/75 24 Lung cancer niques have been used to prevent immune rejection of 4/M/76 24 Myocardial infarction transplanted cells, including translocation of autolo- 5/M/85 15.5 Subarachnoid hemorrhage gous RPE suspensions or patches, transscleral biopsy tech- 6/F/86 28.2 Lung cancer niques to harvest autologous peripheral autologous RPE Polymerase Chain Reaction Data before transplantation, and transplantation of iris pig- 7/F/55 29 Chronic obstructive ment epithelium (IPE).59-71 pulmonary disease In principle, autologous IPE is an attractive candi- 8/F/65 32 Myocardial infarction date to replace diseased or damaged RPE because IPE and 9/F/71 25 Cardiac arrest 10/M/77 36 Cardiac arrest RPE share a common embryological origin. Iris pig- 11/M/81 10 Cardiac arrest ment epithelium can be obtained by performing a pe- 12/M/83 26 Myocardial infarction ripheral iridectomy before subretinal transplantation, and the donor cell population can be expanded in vitro. In *All donors were white. principle, iris biopsy can provide a ready source of do- nor cells and eliminate concerns about infectious disease transmission and immune rejection of the trans- given the expense and limited availability of human tissue, small sample sizes have been used in the past to generate important data planted cells. Iris pigment epithelium transplantation has 77-79 been performed in animal models of human disease and on gene expression within human tissue. in patients with AMD, without dramatic improvement in vision.35,59-74 Some aspects of IPE and RPE function have CYTOKERATIN LABELING been compared in vitro and in vivo, but, to our knowl- Cells from RPE and IPE were stained using a pancytokeratin edge, a detailed analysis of the gene expression profiles antibody to verify that all cells were of epithelial origin.39,45 For of IPE and RPE has not been performed. Data reported this purpose, RPE or IPE cells on a microscopic glass slide were herein should help answer the question of whether IPE fixed with 4% paraformaldehyde for 30 minutes and washed can replace human RPE effectively. with a phosphate-buffered saline solution. The cells were treated The development of microarray chips has made it tech- for 1 hour at room temperature with 3% bovine serum albu- nically feasible to compare the gene expression profiles of min (Sigma Chemical Co, St Louis, Mo) in phosphate- IPE and RPE harvested from the same human donor eyes. buffered saline solution to block nonspecific binding sites. The cells were then incubated at 37°C for 1 hour with a fluorescein The objectives of this study were to compare the gene ex- isothiocyanate–conjugated monoclonal antipancytokeratin an- pression profiles of primary RPE vs IPE harvested from the tibody to cytokeratins 5, 6, and 8 (Sigma Chemical Co). The same donor eyes and to determine the potential useful- cells were washed 3 times with a phosphate-buffered saline so- ness of replacing RPE with IPE in various disease states. lution and were examined under a fluorescence microscope. An irrelevant isotypic IgG primary antibody (antihuman von METHODS Willebrand antibody; Sigma Chemical Co) coupled with a fluorescein isothiocyanate–conjugated secondary antibody was also used and showed no background staining. All harvested cells PREPARATION OF ADULT HUMAN RPE AND IPE were positive for pancytokeratin, indicating that the cells were of epithelial origin. Primary RPE and IPE from the same human donors (aged 63, 71, 75, 76, 85, and 86 years) were obtained from the National Dis- ISOLATION OF TOTAL RNA FROM RPE AND IPE ease Research Interchange (Philadelphia, Pa), with cells harvested within 29 hours of death (Table 1). Because the study Cells from RPE or IPE (approximately 3ϫ105 to 5ϫ105) involved postmortem tissue without identification of individual were disrupted, and total RNA was isolated using a QIA patients, it was exempt from institutional review board ap- shredder and an RNeasy Mini Kit (QIAGEN Inc, Valencia, proval. Primary IPE and RPE cells were prepared from the ante- Calif). Briefly, 600 µL of lysing buffer (RLT) was added to rior and posterior poles of human cadaver eyes as previously de- cells in a 1.5-mL microfuge tube, and cell lysate was loaded scribed.75,76 On receipt in the laboratory, eyes were cleaned of onto a QIA shredder column and centrifuged for 2 minutes at extraocular tissue. Iris pigment epithelium was separated from 13 000 rpm. The homogenized lysate was then mixed with the stroma of the iris in the anterior segment of donor eye globes 600 µL of 70% ethanol and was applied to an RNeasy mini using forceps. Other anterior segment structures, vitreous, and spin column and was centrifuged for 15 seconds at 13 000 retina were removed, leaving an eyecup with native RPE on the rpm. Next, 700 µL of buffer RW1 and buffer RPE was added inner surface. For these studies, 500 000 primary RPE cells and and centrifuged sequentially for washing twice. Then, 60 µL 300 000 IPE cells were collected with trypsin from each pair of of ribonuclease-free water was used to elute total RNA from globes harvested from 6 human donors. The RPE and IPE cells the RNeasy mini spin column. All total RNA used in the were washed 3 times with cold Dulbecco phosphate-buffered sa- experiments was pure as determined by the ratio of absor- line and stored at –80°C before isolation of RNA in the DNA mi- bance (A) at 260 vs 280 nm (A260/A280 ratioϾ1.9). Total croarray study. Six pairs of eyes from human donors were used; RNA was stored at −80°C for later use.

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Figure 1. Hierarchical cluster analysis demonstrates that the gene expression profiles of adult retinal pigment epithelium (shaded boxes) and iris pigment epithelium (unshaded boxes) cluster into 2 distinct groups, with 1 discernable overlap indicated by the asterisk. The numerals indicate the age (in years) of the donors.

1.2 and Array Assist 3.01 (Stratagene, La Jolla, Calif) soft- 6400 ware. For the purpose of this study, gene expression was considered present if the gene was detected using the Affymetrix 5400 GCOS 1.2 statistical algorithm74 in at least 4 of 6 samples within a cell type (IPE or RPE) and the expression levels were at least 4400 50 on densitometry. Gene expression was considered absent 3400 within a cell type (IPE or RPE) if the gene was undetected using the Affymetrix GCOS 1.2 statistical algorithm in at least 4 180 of 6 samples within a given cell type. To minimize ambiguity, 120 we excluded any genes whose expression levels were margin- No. of Genes Detected ally present or absent. Genes were considered differentially ex- 60 pressed if they were present in both RPE and IPE, the expres- 0 sion levels were at least 50 on densitometry, and the difference RPEIPE Both RPE Only in Only in in expression level was greater than the 3-fold difference that and IPE RPE IPE was statistically significant (PϽ.01, t test).

Figure 2. Number of genes expressed in retinal pigment epithelial (RPE) and iris pigment epithelial (IPE) cells, detected using Affymetrix human U95Av2 REAL-TIME POLYMERASE CHAIN REACTION chips. On average, a mean ± SD of 5308±416 genes were expressed in RPE cells and 6130±205 in IPE cells from 6 human donor eyes. Of these, 4895 genes were expressed in both cell types; 68 genes and 154 genes were Real-time polymerase chain reaction (PCR) was performed as expressed only in RPE cells and only in IPE cells, respectively. follows: RPE and IPE were harvested from 6 additional donors (aged 55, 65, 71, 77, 81, and 83 years), different from those DNA MICROARRAY EXPERIMENTS used to generate the microarray data. The RPE and IPE cells were harvested, and total RNA was prepared as already de- scribed. The LightCycler system (Roche Diagnostics, Welwyn A T7-(dT) oligomer, superscript reverse transcriptase II and 24 Garden City, United Kingdom) was used for real-time quan- DNA polymerase I (GIBCO BRL, Gaithersburg, Md) were used titative reverse transcriptase–PCR. An RNA Amplification Kit for first-strand and second-strand complementary DNA (cDNA) SYBR Green I (Roche Molecular Biochemicals, Mannheim, Ger- synthesis using total RNA as templates. Double-stranded cDNA many) was used to synthesize the first-strand cDNA and sub- was cleaned by Phase Lock Gels (Eppendorf AG, Hamburg, Ger- sequent amplification using gene-specific primers. The PCR re- many) phenol-chloroform extraction and ethanol precipita- action solution contained 0.5 µg of total RNA, 6-mM magnesium tion. Biotin-labeled antisense cRNA was produced by an in vitro chloride, and 0.5-µM of each primer (primer oligo sequences transcription reaction (ENZO BioArray High-Yield RNA Tran- are available online at http://www.columbia.edu/~hc2002 script Labeling Kit; Affymetrix Inc, Santa Clara, Calif) and in- /RPEvsIPE_data_onWeb/). Other components in the reverse cubated with fragmentation buffer (Tris-acetate, potassium ac- transcriptase–PCR master mix included buffer, enzyme, SYBR etate, and magnesium acetate [Sigma Chemical Co] at 94°C for Green I, and deoxyribonucleotide triphosphate. For reverse tran- 35 minutes). Target hybridization, washing, staining, and scan- scription, the 20 µL of reaction capillaries were incubated at ning probe arrays were performed according to an Affymetrix 55°C for 10 minutes, followed by incubation at 95°C for 30 sec- GeneChip Expression Analysis Manual.80 onds. Polymerase chain reaction was performed using an initial denaturation for 1 second at 95°C, followed by 45 cycles QUALITY CONTROL, DEFINITION of denaturation for 1 second at 95°C, annealing for 10 seconds OF GENE PRESENCE OR ABSENCE, at 55°C, and extension for 13 seconds at 72°C in a program- AND STATISTICAL ANALYSIS mable LightCycler. A melting curve analysis was performed by following the final cycle with incubation at 95°C for 1 second, For quality control, the Affymetrix human U95Av2 microar- at 65°C for 10 seconds, and then at a temperature transition ray chip included 20 housekeeping gene probes to measure the rate of 20°C per second to reach 95°C. Negative control samples consistency of the hybridization signals from the 3Ј, middle, for the reverse transcriptase–PCR analysis, which contained all and 5Ј fragments of messenger RNA (mRNA) coding re- reaction components except RNA, were performed simulta- gions.81 Gene expression analyses, including global normal- neously to determine when the nonspecific exponential am- ization and scaling, were performed using Affymetrix GCOS plification cycle number was reached.

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 RESULTS pared the expression levels of genes that were associated with known RPE functions using a searchable database available at the Affymetrix Web site (http://www QUALITY CONTROL ASSESSMENT .affymetrix.com).85 Retinal pigment epithelium function– related genes detected in RPE but not in IPE included 2 Quality was assessed using the hybridization signals Ј Ј genes involved in phagocytosis (thrombospondin 1 and from the 3 , middle, and 5 fragments of mRNA of 20 ras-related C3 botulinum toxin substrate 2), 1 gene im- housekeeping genes coded in the Affymetrix DNA 81 portant for vitamin A metabolism (retinol dehydroge- chips. All 12 DNA chips passed quality control (data nase 5), 1 gene involved in angiogenesis (angiopoietin not shown). 1), and 15 genes involved in cell adhesion (Table 4). Using similar search techniques for analyzing function- HIERARCHICAL CLUSTER ANALYSIS related genes expressed only in IPE, we identified 32 genes that were not expressed in RPE; these were involved in Clustering analysis is a statistical technique to sort het- cell adhesion, extracellular matrix remodeling, phago- erogeneous samples into several distinct clusters so that cytosis, tight junction formation, and vitamin A metabo- samples within each cluster are more closely related to lism (Table 5). one another than samples from different clusters.82,83 Hi- erarchical cluster analysis yields a tree diagram, with the branches indicating the relationship of samples within COMMENT the cluster to other samples within and outside the clus- 84 ters. Hierarchical cluster analysis demonstrated that the In the last decade, investigators pioneered the use of gene expression profiles of RPE and IPE cluster into 2 IPE as a replacement for RPE in retinal degenerations, distinct groups with a discernable overlap in 1 sample including AMD.35 This use of IPE is based on the com- (Figure 1). mon embryological origin of these 2 cell lines, the ready availability of autologous IPE via iris biopsy, and the EXPRESSION PROFILES need to replace RPE in various disease states. Applica- OF ADULT RPE AND IPE CELLS tion of IPE transplantation for treatment of tapetoretinal degenerations due to a known gene defect, such as Among 12 600 gene probes on the Affymetrix human Leber congenital amaurosis and RPE-dependent forms U95Av2 microarray chip, a mean ± SD of 5308±416 gene of retinitis pigmentosa, is likely to be unfruitful because probes were expressed in 6 RPE samples, compared with autologous IPE and RPE would have the same genetic 6130±205 gene probes in 6 IPE samples. Of these, 4895 defect. The largest clinical application for autologous genes were expressed in all 6 samples of both cell types IPE transplantation may be in repair of age-related cell (Figure 2). Sixty-eight genes were expressed in RPE and tissue loss in AMD, in which transplanted IPE samples but absent from IPE samples based on the cri- could replace native RPE removed during submacular teria already defined (ie, present in 4 of 6 samples) surgery for exudative AMD or lost during the develop- (Table 2). Reverse transcriptase–PCR in a few genes con- ment of geographic atrophy in nonexudative AMD. firmed the microarray data. Of the 2500 most abun- To date, a few laboratory and clinical studies have been dantly expressed genes in adult RPE, 438 genes were ab- performed to determine the ability of IPE to survive af- sent from the 2500 most abundantly expressed genes in ter subretinal transplantation and to perform RPE func- IPE. Of the 2500 most abundantly expressed genes in IPE, tions, including outer segment phagocytosis, recycling 442 genes were absent from the 2500 most abundantly of visual pigment, and release of cytokines and other expressed genes in RPE. One hundred fifty-four genes growth factors.67 Previous authors have concluded that were detected in IPE that were undetected in RPE. Other IPE can survive at least 6 months after subretinal trans- details and the complete lists of genes expressed in RPE plantation, but proper interpretation of these results is and IPE are available at the URL provided in the “Real- confounded by difficulty in identifying transplanted cells Time Polymerase Chain Reaction” subsection of the unequivocally.65,66,73,86,87 Results of initial studies68,88 sug- “Methods” section. gested that subretinal or choroidal IPE transplantation may slow the rate of photoreceptor degeneration in the DIFFERENTIAL GENE EXPRESSION Royal College of Surgeons rat model for several months compared with untreated controls. However, the rescue After data normalization, there were 22 genes in RPE effect of transplanted IPE cells was no better than that samples whose expression levels were greater than 3-fold of sham surgery.69 higher compared with IPE samples, and 19 of these genes Iris pigment epithelium function in vitro and after were contained within the 2500 most abundantly ex- subretinal transplantation in vivo has also been previ- pressed genes in RPE (Table 3). There were 147 genes ously investigated. Iris pigment epithelium is capable of in IPE samples whose expression levels were greater than retinol metabolism,89 and transplanted IPE can ingest 3-fold lower compared with RPE samples, and 119 of these outer segments.69 The ability of cultured IPE to phago- genes were contained within the 2500 most abundantly cytose latex beads comprises 76% of the activity of expressed genes in IPE (details are available at the URL RPE.90 Cultured IPE maintains melanogenesis for up to provided in the “Real-Time Polymerase Chain Reac- 5 passages in tissue culture.91 Iris pigment epithelium tion” subsection of the “Methods” section). We com- and RPE form monolayers on Descemet membrane92,93

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Table 2. Genes Expressed in Adult Human Retinal Pigment Epithelium (RPE) but Not in Iris Pigment Epithelium (IPE)*

Fold Probe Gene Name Symbol IPE† RPE† Gene Function Change 36346_at Retinal G protein–coupled receptor RGR 14.1 6915.7 Visual perception; phototransduction 492.2 35660_at S-antigen; retina and pineal gland (arrestin) SAG 12.1 3509.1 Rhodopsin-mediated signaling 289.6 34113_at Retinal pigment epithelium–specific protein 65 kDa RPE65 17.0 2468.9 Vitamin A metabolism; visual perception 145.4 39524_at Rhodopsin (opsin 2, rod pigment) (retinitis RHO 9.5 1248.1 Visual perception; phototransduction 131.3 pigmentosa 4) 32727_at Retinol dehydrogenase 5 (11-cis and 9-cis) RDH5 42.8 3866.5 Visual perception; metabolism 90.3 35887_at Retinaldehyde binding protein 1 RLBP1 43.2 3255.3 Vitamin A metabolism; transport; visual perception 75.3 40482_s_at Transcriptional activator of the c-Fos promoter CROC4 4.9 355.3 Cell development 73.1 35832_at Sulfatase 1 SULF1 15.6 965.2 Apoptosis; heparin sulfate proteoglycan 61.9 metabolism 39629_at Phospholipase A2, group V PLA2G5 28.6 1622.6 Phospholipid metabolism; lipid catabolism 56.8 614_at Phospholipase A2, group IIA (platelets, synovial PLA2G2A 49.6 2805.2 Lipid catabolism 56.6 fluid) 31408_at Retinal pigment epithelium–derived rhodopsin RRH 14.9 788.0 Receptor protein signaling pathway; visual 52.8 homologue perception 38291_at Proenkephalin PENK 8.8 411.8 Neuropeptide signaling pathway; cell-cell signaling 47.0 40035_at Kallikrein 11 KLK11 7.7 316.0 Proteolysis and peptidolysis 41.0 40041_at Kinetochore-associated 2 KNTC2 2.2 71.2 Mitotic sister chromatid segregation; mitosis 33.0 33630_s_at Spectrin, beta, nonerythrocytic 2 SPTBN2 13.3 407.3 Vesicle-mediated transport 30.6 32112_s_at Absent in melanoma 1 AIM1 17.7 525.3 Cell adhesion; sugar binding 29.6 41794_at Phosphodiesterase 6H, cGMP-specific, cone, PDE6H 5.1 131.5 Visual perception 25.9 gamma 40201_at Dopa decarboxylase (aromatic L-amino acid DDC 21.0 516.2 Amino acid metabolism; catecholamine 24.6 decarboxylase) biosynthesis 35730_at Alcohol dehydrogenase IB (class I), beta polypeptide ADH1B 9.0 202.6 Ethanol oxidation 22.5 41845_at Cone-rod homeobox CRX 49.1 963.0 Regulation of transcription; visual perception 19.6 36339_at Potassium channel, subfamily V, member 2 KCNV2 21.0 405.2 Cation transport; potassium ion transport 19.3 38112_g_at Chondroitin sulfate proteoglycan 2 (versican) CSPG2 16.9 319.6 Development; cell recognition 18.9 40897_at Phosphodiesterase 6A, cGMP-specific, rod, alpha PDE6A 21.9 400.1 Signal transduction; visual perception 18.2 38949_at Protein kinase C, theta PRKCQ 15.5 282.8 Regulation of cell growth; protein phosphorylation 18.2 41405_at Secreted frizzled-related protein 4 SFRP4 18.0 284.2 Signal transduction; embryo implantation 15.8 37857_at Protocadherin 21 PCDH21 38.2 601.2 Cell adhesion; homophilic cell adhesion 15.7 32817_at SEC14-like 2 (Saccharomyces cerevisiae) SEC14L2 20.4 280.5 Intracellular protein transport 13.7 36324_at Adenosine A2b receptor ADORA2B 14.6 199.1 Activation of MAPK; cellular defense response 13.7 39566_at Cholinergic receptor, nicotinic, alpha polypeptide 7 CHRNA7 11.4 148.1 Activation of MAPK; ion transport 13.0 34637_f_at Alcohol dehydrogenase 1A (class I), alpha ADH1A 15.0 181.3 Alcohol metabolism 12.1 polypeptide 266_s_at CD24 antigen (small cell lung carcinoma cluster 4 CD24 5.2 58.7 Humoral immune response 11.4 antigen) 39642_at Elongation of very long-chain fatty acids (FEN1/Elo2) ELOVL2 17.2 193.4 Fatty acid biosynthesis 11.2 36319_at Forkhead box F2 FOXF2 19.2 206.1 Transcription from Pol II promoter 10.7 41093_at Opioid-binding protein/cell adhesion molecule–like OPCML 11.3 119.0 Cell adhesion; neuronal cell recognition 10.5 35454_at Phospholipase C–like 4 PLCL4 17.6 173.7 Lipid metabolism; intracellular signaling cascade 9.9 31844_at Homogentisate 1,2-dioxygenase (homogentisate HGD 13.3 130.2 Tyrosine catabolism 9.8 oxidase) 38430_at Fatty acid binding protein 4, adipocyte FABP4 12.3 115.4 Transport 9.4 160031_at Cadherin 3, type 1, P-cadherin (placental) CDH3 26.2 200.4 Cell adhesion; visual perception 7.7 40328_at Twist homologue 1 (acrocephalosyndactyly 3) TWIST1 10.1 75.5 Morphogenesis; cell differentiation 7.4 33767_at Neurofilament, heavy polypeptide 200 kDa NEFH 21.6 155.8 Nucleosome assembly; neurogenesis 7.2 36867_at Neurofascin NFASC 25.2 179.3 Cell adhesion 7.1 40351_at Guanine nucleotide binding protein (G protein) GNB3 46.8 289.9 G protein–coupled receptor protein signaling 6.2 pathway 37749_at Mesoderm specific transcript homologue (mouse) MEST 34.6 210.5 Mesoderm development 6.1 37881_at Cluster including bone morphogenetic protein 11 . . . 22.6 136.9 Unknown 6.1 (BMP11) mRNA 40500_at National Disease Research Interchange family NDRG4 22.4 133.6 Response to stress; cell growth; cell differentiation 6.0 member 4 35965_at Heat shock 70 kDa protein 6 (HSP70BЈ) HSPA6 39.1 227.7 Protein folding; response to unfolded protein 5.8 35185_at Fatty acid–binding protein 7, brain FABP7 24.5 133.1 Fatty acid metabolism; regulation of cell 5.4 proliferation 37538_at Hypothetical protein LOC221362 LOC221362 42.3 229.2 Unknown 5.4 32398_s_at Low-density lipoprotein receptor–related protein 8 LRP8 23.5 122.8 Endocytosis; signal transduction 5.2 34043_at Chromosome 6 open reading frame 105 C6orf105 22.5 114.3 Unknown 5.1 41088_at Abhydrolase domain containing 2 ABHD2 25.3 123.4 Unknown 4.9 32110_at KIAA0523 protein KIAA0523 11.8 57.2 Transferase activity 4.8 34024_at Chloride channel 5 (nephrolithiasis 2, X-linked, Dent CLCN5 16.0 76.5 Ion and chloride transport 4.8 disease) 1599_at Cyclin-dependent kinase inhibitor 3 CDKN3 19.4 82.0 Regulation of cell cycle 4.2 33430_at Chromosome 18 open reading frame 10 C18orf10 35.0 146.4 Unknown 4.2

(continued)

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Fold Probe Gene Name Symbol IPE† RPE† Gene Function Change 39901_at Epidermal growth factor–like repeats and discoidin EDIL3 15.3 61.8 Cell adhesion; development 4.0 I–like domains 3 38201_at Branched chain aminotransferase 1, cytosolic BCAT1 20.8 79.5 Cell proliferation; amino acid metabolism 3.8 36376_at Solute carrier family 26, member 4 SLC26A4 29.6 110.8 Transport; perception of sound; sulfate transport 3.7 41069_at Cluster including chondromodulin I precursor mRNA . . . 15.6 57.0 Angiogenic inhibitory factor 3.7 38469_at Transmembrane 4 superfamily member 3 TM4SF3 37.3 133.0 Protein amino acid glycosylation 3.6 32671_at Tubulin tyrosine ligase–like family, member 4 TTLL4 46.8 163.8 Protein modification 3.5 39315_at Angiopoietin 1 ANGPT1 43.0 135.3 Angiogenesis; signal transduction 3.2 39109_at TPX2, microtubule-associated protein homologue TPX2 39.1 108.2 Mitosis; cell proliferation 2.8 38957_at Doublecortin and calmodulin kinase–like 1 DCAMKL1 20.7 56.2 Protein phosphorylation; endosome transport 2.7 38961_at KIAA1107 protein KIAA1107 27.6 69.3 Unknown 2.5 37053_at Adenosine triphosphatase, Ca2ϩ transporting, ATP2B2 25.8 53.2 Transport; cation transport; calcium ion transport 2.1 plasma membrane 2 36303_f_at Zinc finger protein 85 (HPF4, HTF1) ZNF85 31.6 56.8 Regulation of transcription, DNA dependent 1.8 36704_at Adaptor-related protein complex 4, sigma 1 subunit AP4S1 46.6 56.2 Transport; intracellular protein transport; 1.2 endocytosis

Abbreviations: cGMP, cyclic guanosine 3Ј,5Ј-monophosphate; MAPK, mitogen-activated protein kinase; mRNA, messenger RNA. *Reverse transcriptase–polymerase chain reaction (PCR) performed on independent samples confirmed the microarray data in the following probes (number of positive PCR results/total number of PCR attempts): 36346_at (2/2), 35660_at (2/2), 34113_at (2/2), 39524_at (2/2), 32727_at (1/1), 35887_at (1/1), 40482_s_at (1/1), 35832_at (3/3), 31408_at (1/3), and 38949_at (2/3). †Densitometry readings from the DNA microarray chip. Gene expression was considered present within a cell type (IPE or RPE) if the gene was detected using the Affymetrix GCOS 1.2 statistical algorithm in at least 4 of 6 samples within a given cell type and the expression levels were at least 50 on densitometry. Gene expression was considered absent if the gene was detected in at least 4 of 6 samples within a given cell type and the expression levels were less than 50 on densitometry.

and exhibit similar growth on native and micropat- level but does not improve visual acuity.62,63 These poor terned human lens capsules.94 Iris pigment epithelium functional results are consistent with the suboptimal at- can form tight junctions, raising the possibility that tachment and survival of IPE and RPE on aged Bruch’s transplanted IPE could reestablish the blood-retinal membrane.99 Despite the lack of visual improvement, sub- barrier normally formed by RPE.67 retinal IPE transplantation in patients with AMD may pre- Cultured bovine IPE and RPE express retinol-binding vent recurrence of subretinal neovascularization.35,68 protein and cellular retinaldehyde–binding protein 1 We observed major differences in the gene expression (Table 2, probe set 35887_at), suggesting that both cell profiles of primary RPE vs IPE harvested from the same types have some capacity for transporting and metaboliz- donor eyes, including the lack of expression in IPE of genes ing retinol.95 Semiquantitative reverse transcriptase–PCR known to be critical for RPE function. For example, IPE demonstrates that bovine IPE and RPE express mRNA for does not express the gene for retinol dehydrogenase, whose cellular retinaldehyde–binding protein and 11-cis- gene product is necessary for recycling visual pigments. dehydrogenase at similar levels, but IPE expresses lower Recoverin is a visual cycle protein expressed in abun- levels of p63, the presumed RPE membrane receptor for dance in RPE but not in IPE, although its role in RPE func- retinoids.96 Iris pigment epithelium and RPE synthesize tion is unknown. Iris pigment epithelium does not ex- and release a similar but unidentical pattern of cytokines press other major functional RPE genes, including and their receptors, but major differences exist in the re- angiopoietin 1, S-antigen, and a transcriptional regulator lease of insulin and tumor necrosis factor ␣.97,98 Messen- of the c-Fos promoter. Numerous cell adhesion genes and ger RNA levels of vascular endothelial growth factor and additional genes related to RPE phagocytosis, tight junc- vascular endothelial growth factor receptor 2 (FLK-1) were tion formation, and vitamin A metabolism are missing from lower in IPE cells than in RPE cells, which may be an im- IPE, including thrombospondin 1 and ras-related C3 botu- portant functional difference because vascular endothe- linum toxin substrate 2 (Table 4). lial growth factor may maintain the choriocapillaris and For IPE to replace surgically excised or dysfunctional assist in the pathological development of choroidal neo- RPE, the transplanted IPE would probably need to de- vascularization in vivo.97,98 velop an expression profile that closely resembles that of Only a few clinical studies have been performed to date native RPE. Our results suggest that the native IPE gene on subretinal transplantation of IPE to replace surgi- expression profile may be a potential obstacle to success- cally excised RPE in patients with exudative AMD. Au- ful subretinal transplantation. Because the microenviron- tologous IPE transplantation has been performed in 35 ment of cells influences their behavior and gene expres- patients after removal of subfoveal choroidal neovascu- sion, we cannot exclude the possibility that the gene lar membranes, with no significant difference in visual expression profile of IPE may change after subretinal trans- acuity between patients who received transplants vs those plantation to more closely resemble that of native RPE. How- who underwent choroidal neovascularization removal ever, our data suggest that the expression levels of many alone. Autologous IPE translocation after submacular genes must change for IPE to resemble RPE. Some au- membranectomy can preserve foveal function at a low thors have suggested that transplanted IPE can serve as a

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Table 3. Genes Within Adult Human Retinal Pigment Epithelium (RPE) Up-regulated Greater Than 3-Fold Compared With Iris Pigment Epithelium (IPE)

RPE/ P Probe Gene Name Symbol IPE RPE IPE* Value† Gene Function 34853_at Fibronectin leucine–rich transmembrane FLRT2 30.5 1433.6 47.1 .002 Cell adhesion protein 2 33327_at Chromosome 11 open reading frame 9 C11 or f9 180.1 2648.8 14.7 .002 Unknown 36686_at Aldehyde dehydrogenase 1 family, ALDH1A3 82.8 1155.1 13.9 Ͻ.001 Alcohol and lipid metabolism member A3 33143_s_at Solute carrier family 16 SLC16A3 614.5 8328.9 13.6 Ͻ.001 Organic anion and monocarboxylic acid transport 38404_at Transglutaminase 2 TGM2 643.2 4602.3 7.2 Ͻ.001 Regulation of cell adhesion 718_at Protease, serine, 11 (insulinlike growth PRSS11 378.7 2050.4 5.4 Ͻ.001 Regulation of cell growth; proteolysis and factor binding) peptidolysis 1521_at Nonmetastatic cells 1, protein (NM23A) NME1 308.1 1622.2 5.3 Ͻ.001 Regulation of cell proliferation; nucleotide expressed in metabolism 36206_at Solute carrier family 16 SLC16A1 32.4 144.5 4.5 .001 Organic anion and mevalonate transport 32607_at Brain abundant, membrane-attached BASP1 1601.5 6818.9 4.3 .002 Unknown signal protein 1 239_at Cathepsin D (lysosomal aspartyl protease) CTSD 3047.9 12 547.0 4.1 .004 Proteolysis and peptidolysis 41585_at KIAA0746 protein KIAA0746 62.6 255.9 4.1 .01 Unknown 33203_s_at Forkhead box D1 FOXD1 91.3 373.2 4.1 .003 Regulation of transcription, DNA-dependent 39059_at 7-Dehydrocholesterol reductase DHCR7 546.9 2162.4 4.0 .003 Cholesterol biosynthesis 32174_at Solute carrier family 9 SLC9A3R1 787.9 2988.8 3.8 Ͻ.001 Protein complex assembly; receptor signaling pathway 33436_at Sex-determining region Y box 9 SOX9 124.7 433.0 3.5 .006 Skeletal development 34185_at Poly(rC) binding protein 4 PCBP4 470.6 1627.6 3.5 Ͻ.001 DNA damage response; induction of apoptosis 41207_at Chromosome 9 open reading frame 3 C9 or f3 119.8 396.0 3.3 Ͻ.001 Proteolysis and peptidolysis 40103_at Villin 2 (ezrin) VIL2 1739.4 5617.0 3.2 Ͻ.001 Cellular morphogenesis; cytoskeletal anchoring 39071_at Integrin, alpha V (vitronectin receptor, ITGAV 179.0 571.3 3.2 Ͻ.001 Cell-matrix adhesion; integrin-mediated antigen CD51) signaling pathway 33788_at Ectonucleoside triphosphate ENTPD4 122.6 381.8 3.1 .004 Uridine diphosphate catabolism diphosphohydrolase 4 35331_at Catenin (cadherin-associated protein), CTNNAL1 325.7 1013.7 3.1 .001 Cell adhesion alphalike 1 40960_at Uridine diphosphate–Gal:betaGlcNAc beta B4GALT1 739.2 2258.9 3.1 Ͻ.001 Carbohydrate metabolism; oligosaccharide 1,4-galactosyltransferase biosynthesis

*Fold-changes comparing RPE gene expression level with IPE. †t Test.

Table 4. Retinal Pigment Epithelium (RPE) Function–Related Genes Detected in RPE but Not in Iris Pigment Epithelium (IPE)

Mean IPE Mean RPE Expression Expression Probe Gene Name Symbol Functions Level* Level* 39315_at Angiopoietin 1 ANGPT1 Angiogenesis 43.0 135.3 1483_at Cadherin 4, type 1, R-cadherin (retinal) CDH4 Cell adhesion 90.0 220.0 160031_at Cadherin 3, type 1, P-cadherin (placental) CDH3 Cell adhesion 26.2 200.4 32058_at Carbohydrate sulfotransferase 10 CHST10 Cell adhesion 89.6 115.9 32112_s_at Absent in melanoma 1 AIM1 Cell adhesion 17.7 525.3 33410_at Integrin, alpha 6 ITGA6 Cell adhesion 27.0 56.3 35903_at Oligodendrocyte myelin glycoprotein OMG Cell adhesion 44.3 442.7 36148_at Amyloid beta (A4) precursor–like protein 1 APLP1 Cell adhesion 130.0 1302.5 37653_at Collagen, type XVIII, alpha 1 COL18A1 Cell adhesion 85.4 106.7 37857_at Protocadherin 21 PCDH21 Cell adhesion 38.2 601.2 38111_at Chondroitin sulfate proteoglycan 2 (versican) CSPG2 Cell adhesion 39.2 508.0 39901_at EGF-like repeats and discoidin I–like domains 3 EDIL3 Cell adhesion 15.3 61.8 40020_at Cadherin, EGF LAG 7-pass G-type receptor 3 CELSR3 Cell adhesion 74.8 101.5 41093_at Opioid-binding protein/cell adhesion OPCML Cell adhesion 11.3 119.0 molecule–like 41781_at Interacting protein (liprin), alpha 1 PPFIA1 Cell adhesion 33.8 47.0 32736_at ras-related C3 botulinum toxin substrate 2 RAC2 Phagocytosis 86.3 248.9 866_at Thrombospondin 1 THBS1 Phagocytosis 47.3 72.2 32727_at Retinol dehydrogenase 5 (11-cis and 9-cis) RDH5 Vitamin A metabolism 42.8 3866.5

Abbreviation: EGF, endothelin growth factor. *Mean values of DNA microarray chip densitometry scan signals; values in the IPE column are all below the detection level of the microarray for that particular gene.

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Table 5. Retinal Pigment Epithelium (RPE) Function–Related Genes Detected in Iris Pigment Epithelium (IPE) but Not in RPE

Mean IPE Mean RPE Expression Expression Probe Gene Name Symbol Function Level Level 208_at Catenin (cadherin-associated protein), alpha 2 CTNNA2 Cell adhesion 61.4 21.8 245_at Selectin L (lymphocyte adhesion molecule 1) SELL Cell adhesion 79.3 34.1 33302_at Sarcospan (K-ras oncogene–associated gene) SSPN Cell adhesion 104.4 40.4 33756_at Amine oxidase, copper-containing 3 AOC3 Cell adhesion 237.8 39.9 34193_at Cell adhesion molecule with homology to L1CAM CHL1 Cell adhesion 172.5 29.7 34619_at Desmocollin 1 DSC1 Cell adhesion 102.7 10.2 35245_at Coagulation factor V (proaccelerin, labile factor) F5 Cell adhesion 152.9 11.4 35678_at BH-protocadherin (brain-heart) PCDH7 Cell adhesion 177.9 27.5 36256_at Limbic system–associated membrane protein LSAMP Cell adhesion 59.5 35.4 36824_at Astrotactin ASTN Cell adhesion 79.5 11.9 37286_at Neuronal cell adhesion molecule NRCAM Cell adhesion 52.9 32.7 38160_at Lymphocyte antigen 75 LY75 Cell adhesion 63.6 15.9 40676_at Integrin beta 3 binding protein (beta3-endonexin) ITGB3BP Cell adhesion 120.4 44.5 33468_at Desmoglein 2 DSG2 Cell adhesion 69.7 42.7 41826_at Serotonin-7 receptor pseudogene KIAA1467 Cell adhesion 87.0 34.7 34531_at Fibronectin leucine–rich transmembrane protein 1 FLRT1 Cell adhesion, ECM 263.8 49.0 38427_at Collagen, type XV, alpha 1 COL15A1 Cell adhesion, ECM 53.8 34.7 658_at Thrombospondin 2 THBS2 Cell adhesion, ECM 64.0 29.2 36917_at Laminin, alpha 2 (merosin, congenital muscular dystrophy) LAMA2 Cell adhesion, ECM 172.2 48.1 34775_at Tetraspanin 1 TSPAN1 Cell adhesion, proliferation 335.0 49.8 32305_at Collagen, type I, alpha 2 COL1A2 ECM 92.7 24.5 33596_at Sparc/osteonectin, cwcv- and kazal-like domains proteoglycan 3 SPOCK3 ECM 56.2 6.6 36470_s_at Dystrobrevin, alpha DTNA Phagocytosis 68.6 19.2 435_g_at Histamine 1 histone family, member 0 H1F0 Phagocytosis 106.8 39.7 1501_at Insulinlike growth factor 1 (somatomedin C) IGF1 Proliferation 117.4 21.8 32027_at PDZ domain–containing 1 PDZK1 Proliferation 82.0 13.4 33864_at Zinc finger, MYND domain–containing 11 ZMYND11 Proliferation 52.8 35.3 748_s_at MAX interactor 1 MXI1 Proliferation 142.3 47.6 37327_at Epidermal growth factor receptor EGFR Proliferation, vitamin A 66.4 39.6 32656_at Multiple PDZ domain protein MPDZ Tight junction 55.8 23.9 35416_at Cubilin (intrinsic factor–cobalamin receptor) CUBN Vitamin A 67.0 13.4 821_s_at Folate receptor 1 (adult) FOLR1 Vitamin A 84.4 41.7

Abbreviation: ECM, extracellular matrix.

potential reservoir for a single growth factor or cytokine Financial Disclosure: None reported. and thereby rescue adjacent cells from the effects of pro- Funding/Support: This study was supported by the Rob- gressive tapetoretinal degeneration.100 For example, IPE that ert L. Burch III Fund, the Macula Society, the Founda- is induced to transcribe the BNDF gene protects against reti- tion Fighting Blindness, and the Commonwealth of Ken- nal damage due to N-methyl-D-aspartate–induced neuro- tucky Research Challenge Trust Fund (Dr Kaplan) and nal death and light toxicity.101,102 Iris pigment epithelium by unrestricted funds from Research to Prevent Blind- that is genetically modified to express pigment epithelial– ness. Dr Tezel is the recipient of a Research to Prevent derived factor inhibits choroidal neovascularization in a rat Blindness Career Development Award. model of laser-induced choroidal neovascularization and increases the survival of and preserves the rhodopsin ex- REFERENCES pression of photoreceptor cells in the Royal College of Sur- 70 geons rat model. For such applications, the striking dif- 1. Marlhens F, Bareil C, Griffoin JM, et al. Mutations in RPE65 cause Leber’s con- ference in the gene expression profiles between RPE and genital amaurosis. Nat Genet. 1997;17:139-141. IPE may be less of an obstacle to successful cell-based 2. Vollrath D, Feng W, Duncan JL, et al. Correction of the retinal dystrophy phe- therapy. Additional studies, including determination of the notype of the RCS rat by viral gene transfer of Mertk. Proc Natl Acad SciUSA. gene expression profiles of IPE and RPE after subretinal 2001;98:12 584-12 589. 3. Gal A, Thompson DA, Weir J, et al. Mutations in MERTK, the human ortho- transplantation, are needed to determine if the microen- logue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa. Nat Genet. vironment of the subretinal space will have a marked effect 2000;26:270-271. on the gene expression profile of IPE. 4. Coffey PJ, Girman S, Wang SM, et al. Long-term preservation of cortically dependent visual function in RCS rats by transplantation. Nat Neurosci. 2002; 5:53-56. Submitted for Publication: August 8, 2005; final revi- 5. Castillo BV Jr, del Cerro M, White RM, et al. Efficacy of nonfetal human RPE for sion received November 23, 2005; accepted December photoreceptor rescue: a study in dystrophic RCS rats. Exp Neurol. 1997; 12, 2005. 146:1-9. Correspondence: Lucian V. Del Priore, MD, PhD, De- 6. Gaur V, Agarwal N, Li L, Turner JE. Maintenance of opsin and S-antigen gene expression in RCS dystrophic rats following RPE transplantation. Exp Eye Res. partment of Ophthalmology, Harkness Eye Institute, Co- 1992;54:91-101. lumbia University, 635 W 165th St, New York, NY 10032 7. Girman SV, Wang S, Lund RD. Cortical visual functions can be preserved by ([email protected]). subretinal RPE cell grafting in RCS rats. Vision Res. 2003;43:1817-1827.

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