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(Investigative Ophthalmology and Visual Science. 2002;43:2474-2480.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Vitreous Treatment of Cultured Human RPE Cells Results in Differential Expression of 10 New Genes

Norbert Kociok, Arno Hueber, Peter Esser, Ulrich Schraermeyer, Gabriele Thumann, Thomas T. Luther, Jens Jordan, Gerhard Welsandt and Bernd Kirchhof

From the Department of Vitreoretinal Surgery, Division of Ophthalmology, University of Cologne, Germany.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To analyze the differential gene expression in cultured human retinal pigment epithelial (RPE) cells after treatment with vitreous.

METHODS. Cultured human RPE cells were incubated for 48 hours with 25% human vitreous from donor eyes. Total RNA from treated and untreated cells was extracted. The gene expression was analyzed by differential expression analysis (DEmRNA-PCR). The differentially expressed genes were identified by gene bank searches. Differential expression was verified by a quantitative real-time RT-PCR fluorescent nucleic acid staining system. The in vivo mRNA expression of these genes in RPE cells was shown by gene-specific RT-PCR.

RESULTS. Vitreous treatment of human RPE cells resulted in the reduced expression of NFIB2, KE03 (NY-REN-25ag), PIG-B, DKFZp564BC462, LKHA, G3BP, PAM, UEV-1, and MAP1B calibrated to the expression of GAPDH when compared with their expression in untreated cells. The reduced expression after vitreous treatment was quantified by gene-specific quantitative real-time RT-PCR and varied from 0.69 to 0.17 compared with untreated cells. The mRNA expression of UDP-GalNac mRNA remained constant. The mRNA expression of eight of these genes was demonstrated in this study for the first time in human RPE cells.

CONCLUSIONS. Vitreous treatment of cultured RPE cells induces the differential expression of a variety of genes with functions in transcription, mediation of signal transduction and inflammation, glycosylation, ubiquitination and protein–protein interaction. Further examination of these genes may locate additional targets for treatment of diseases caused by contact of RPE cells with vitreous, typical in proliferative vitreoretinopathy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the adult eye, retinal pigment epithelial (RPE) cells form a differentiated and mitotically inactive monolayer between the sensory retina and choriocapillaris. Together with the underlying Bruch membrane, they form the outer blood–retina barrier and maintain the integrity of the photoreceptors, mainly through the phagocytosis and recycling of deteriorated photoreceptor outer segments. RPE cells that come into contact with vitreous humor under traumatic or pathologic situations may proliferate and initiate the formation of extracellular epiretinal membranes. The contraction of these membranes may develop into retinal detachment, resulting in diseases such as proliferative vitreoretinopathy (PVR) that can lead to blindness.1 2

One of the risk factors for PVR is the contact of RPE cells to human vitreous.3 4 When, during PVR, RPE cells proliferate into the vitreous, they change their normal cobblestone epithelial morphology into a fibroblast-like appearance, as has been found also in cultured RPE cells exposed to vitreous humor.5 6 Therefore, it is thought that vitreous contributes modulators that stimulate some functions of RPE cells that are believed to play a role in the pathogenesis of PVR.

This process, however, is not well understood at the molecular level. It has been shown that human vitreous stimulates migration but not proliferation of human RPE cells under serum-free conditions in vitro.5 Stimulation of proliferation of RPE cells and fibroblasts was observed, however, after admixture of albumin with the vitreous.5 Recently, it has been reported that treatment of human RPE cells with vitreous humor results in decreased expression of FGF-2 mRNA and protein.6 The knowledge of genes differentially regulated by vitreous treatment could help in further understanding of the molecular events within RPE cells that lead to PVR. We therefore analyzed the changes in gene expression of RPE cells treated with vitreous humor by a polymerase chain reaction (PCR)–based differential expression mRNA analysis (DEmRNA-PCR).7

With this experimental design, RNA samples of differentially treated cells can be screened simultaneously and differentially expressed genes identified without preconceived assumptions. Herein, we report the detection of 10 differentially expressed genes in RPE cells due to treatment with vitreous humor. This is the first report of the demonstration of the expression of 8 of these 10 genes in RPE cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Vitreous Treatment
RPE cells were cultured from human donor eyes for keratoplasty within 10 hours after death. The cells were harvested by trypsinizing for 30 minutes with 0.2% trypsin containing 0.5 mM EDTA. The cells were centrifuged and seeded in culture flasks containing Dulbecco’s modified Eagle’s medium, supplemented with 15% fetal calf serum, 50 µg/mL gentamicin, and 2.5 µg/mL amphotericin. Phase-contrast examination of living cells was performed with a photomicroscope. RPE cell origin was confirmed by positive cytokeratin immunocytochemical analysis. The cells were kept in primary culture (P0) for approximately 3 weeks until confluence was reached. Subsequent passaging as a dissociated single-cell suspension was performed by trypsinization every 8 to 10 days. Cells were passaged or harvested for the experiments at the point of confluence. Human vitreous humor was dissected from donor eyes without any obvious ocular diseases within 10 hours after death and stored at -80°C after determination of the protein content by a Bradford assay (Bio-Rad Laboratories, Hercules, CA). For the experiments three different vitreous samples were used with a protein concentration varying from 0.8 to 1.1 mg/mL. The vitreous was diluted with serum-free medium to a final concentration of 25% vitreous and filtered through a 0.45-µm filter. Confluent cells of passage 6 were treated with 25% vitreous for 48 hours. The vitreous concentration and incubation time were chosen to allow comparison of our results with others.6 All control cultures were grown with the same cells at the same time and with the same serum until vitreous treatment, when control cells were incubated without serum for 48 hours.

Differentially Expressed mRNA PCR Analysis
RNA was isolated using an extraction reagent (TRIzol; Sigma, St. Louis, MO), according to the recommendations of the manufacturer, and dissolved in diethyl pyrocarbonate (DEPC)–treated water. DEmRNA-PCR analysis of the human RPE cells was performed as reported.7 Briefly, the complete RNA from RPE cells was reverse-transcribed with a system for DNA synthesis (Superscript Preamplification System for First-Strand cDNA Synthesis; Life Technologies, Rockville, MD) with oligo(dT) 12-18 primers. Diluted cDNA (40 ng) was used as a template for DEmRNA-PCR with the arbitrary primer P6 or P10 and primer T1, T5, or T7.7 Thermocycling was then performed (model PTC-100 thermocycler; MJ Research, Watertown, MA). Four microliters of the amplification reactions were run on a 0.4-mm nondenaturing 5% polyacrylamide gel in duplicates, and amplified products were visualized by silver staining. Bands with different levels of intensity between cDNA of vitreous-treated and untreated RPE cells were selected for subsequent reamplification. Reamplified PCR fragments were cloned in the pSTBlue1 vector (Novagen, Madison, WI). At least four insert-containing white bacterial colonies of each cloned reamplified PCR fragment were selected. The insert was amplified by colony PCR, as recommended by the manufacturer, and sequenced by terminator cycle sequencing.

Gene-Specific RT-PCR
With primer analysis software (Oligo, ver. 4.1; National Biosciences, Plymouth, MN), gene-specific primers were selected as shown in Table 1 . Aliquots of the diluted cDNAs of human RPE cells corresponding to 12.5 ng initially used total RNA, were mixed with PCR buffer (Qiagen, Hilden, Germany) containing Tris-HCl (pH 8.7 at 20°C), (NH4)2SO4, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 µM of each specific primer, and 1.25 U of polymerase (HotStarTaq; Qiagen) in a volume of 50 µL.


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  		Table 1. Gene-Specific Primers for RT-PCR

 
For gene-specific amplification, we used the following PCR cycle parameters: hot-start polymerase activation for 15 minutes at 95°C and 40 cycles of 95°C for 30 seconds, gene-specific annealing temperature as listed in Table 1 for 30 seconds, and 72°C for 1 minute, followed by a final extension at 72°C for 10 minutes. The amplified DNA fragments had the expected length listed in Table 1 . The correct sequences were verified by DNA sequencing. Genomic DNA contamination was excluded by control amplification reactions with nontranscribed RNA as templates. No products were found.

Quantification of mRNA in RPE Cells by Real-Time RT-PCR
The level of mRNA expression in vitreous-treated RPE cells compared with untreated cells was quantified by real-time RT-PCR, by using a nucleic acid fluorescent staining system (SYBR Green I reaction system; Eurogentec, Seraing, Belgium) on a thermal cycler (iCycler; Bio-Rad Laboratories). Using the primer analysis software (Oligo, ver. 4.1; National Biosciences) gene-specific primers suitable for real-time RT-PCR were selected as shown in Table 2 . The melting temperatures (Tm) of the primers chosen were between 58°C and 60°C wherever possible. The expected fragment length lies between 81 and 181 bp. With these primers, the mRNA expression of all genes together with GAPDH as calibrator were analyzed simultaneously in a single experiment in triplex reactions. The analysis was repeated twice. Aliquots of the diluted cDNAs of vitreous treated or untreated human RPE cells corresponding to 10 ng initially used total RNA, were mixed with 10x reaction buffer containing Tris-HCl and KCl, 3.5 mM MgCl2, 0.2 mM of each dNTP, 0.3 µM of each specific primer (0.15 µM for GAPDH primer), 0.5x to 1x fluorescent nucleic acid stain (Sybr Green I; Molecular Probes Europe, Leiden, Netherlands) and 1.25 U of DNA polymerase (HotGoldStar; Qiagen) in a volume of 50 µL. The following PCR cycle parameters were used: hot-start polymerase activation for 10 minutes at 95°C, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. Detection of the fluorescence product was performed during the last 10% of the cycles. To confirm amplification specificity, the PCR products from each primer pair were subjected to a melting curve analysis and subsequent agarose gel electrophoresis (data not shown). Genomic DNA contamination was excluded by control amplification reactions with nontranscribed RNA as templates. Only background fluorescence data were found. The quantification data were analyzed with the thermal cycler system software (iCycler iQ; Bio-Rad Laboratories), as described.8 After PCR, baseline subtraction was performed by the software, the log-linear portion of the fluorescence-versus-cycle plot was extended to determine a fractional cycle number at which a threshold fluorescence was obtained (threshold cycle, CT) for each analyzed gene and GAPDH as the reference. Because the efficiencies of target genes and the reference gene are approximately equal ({Delta}CT < 0.15), the comparative CT method was used for quantification of the target genes relative to GAPDH.


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  		Table 2. Primers for Real-Time RT-PCR

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differentially Expressed mRNA PCR Analysis
As expected, using the nonspecific primer combination P6 or P10 and T1, T3, T4, T5, T7, or T8 in the DEmRNA-PCR analysis of vitreous-treated human RPE cells resulted in the appearance of more than 50 bands for each primer combination elicited by silver staining. This primer-dependent pattern of the bands was reproducible. The increased amplification of the bands P6T1/4, P6T1/2, and P6T1/3 in cDNA derived from vitreous-treated RPE cells and the decreased amplification of the bands P10T1/1, P6T4/2, P6T5/2, P6T7/3, and P6T74 was consistently observed when comparing untreated with vitreous-treated RPE cells (Fig. 1 , top panel of each group) in all three investigated independent cell populations. Reamplification of the eluted cDNA from the excised bands (Fig. 1 , middle panel of each group) with the same primer combination, allowed the isolation of the specific PCR fragments for cloning and further analysis (Fig. 1 , bottom panel of each group).



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  		Figure 1. DEmRNA-PCR analysisusing P and T primers as indicated. Top and middle panels of each group: portions of silver-stained polyacrylamide gels showing differentially amplified bands in vitreous-treated (V) and untreated (nS) RPE cells (two lanes each), before (top) and after (middle) cutting off the band of interest. Bottom panel of each group: ethidium bromide-stained agarose gels showing PCR fragments in two lanes each, reamplified from the eluted bands cut from the polyacrylamide gel shown above, as shown in the middle panel. N, negative control; M, DNA marker (base pairs as indicated).

 
DNA Sequencing and Gene Data Bank Homology Search
The reamplified PCR fragments were cloned into a pSTBlue1 vector, and the inserts were sequenced from both sides. Sequence alignment of the assembled bases of the PCR fragments with the gene bank sequences showed their identity with the coding sequences of the human genes referred to in this article. Using gene-specific primers for each gene (Table 1) , the gene’s basic mRNA expression was confirmed in human RPE cells freshly prepared from donor eyes (Fig. 2) . All the listed genes are expressed in human RPE cells and were detected using cDNA corresponding to the 12.5 ng total RNA used in the reverse-transcription reaction. The intensity of the bands varied, however. DKFZp564BC462, UDP-GalNac, NFIB2, and UEV-1 showed low expression; PIG-B and PAM showed moderate expression typical of GAPDH; and LKHA4, KE03, G3BP, and MAP1B showed very high expression in human RPE cells directly obtained from donor eyes.



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  		Figure 2. Gene specific RT-PCR. Ethidium bromide-stained 2% agarose gel with gene-specific amplicons (length listed in Table 1 ) of the genes indicated. M, DNA marker (base pairs as indicated).

 
Real-Time RT-PCR
Including the same amount of cDNA (10 ng per reaction) real-time RT-PCR was used to quantify the mRNA levels of the analyzed genes in human RPE cells due to vitreous treatment. After PCR baseline subtraction performed by the thermal cycler software, the log-linear portion of the fluorescence-versus-cycle plot was extended to determine a fractional cycle number at which a threshold (CT) fluorescence was obtained for each analyzed gene and GAPDH as the reference. This was performed on the RNA of vitreous-treated and untreated cells (Fig. 3 , for two examples). The threshold cycle for GAPDH was equal in vitreous-treated (19.93 ± 0.12) and untreated cells (19.93 ± 0.07). For each analyzed gene the {Delta}CT (gene minus GAPDH) was determined for treated and untreated cells. This ensures a calibration of each gene expression to the expression of the housekeeping gene GAPDH. The {Delta}{Delta} CT data of each gene, obtained by subtracting the {Delta}CT (gene minus GAPDH) data of vitreous-treated cells from the {Delta}CT (gene minus GAPDH) data of the untreated cells, resulted in GAPDH-calibrated gene expression in vitreous-treated cells compared with untreated cells (Fig. 4) . This analysis revealed a downregulation of 9 of the 10 analyzed genes from 0.17 ± 0.05 (DKFZp564BC462) to 0.69 ± 0.08 (NFIB2) compared with untreated cells. The relative expression of the other seven genes lies between those levels (UEV-1: 0.64 ± 0.09; G3BP: 0.62 ± 0.1; LKHA4: 0.51 ± 0.07; PAM: 0.5 ± 0.04; KE03: 0.47 ± 0.04; PIG-B: 0.37 ± 0.03; and MAP1B: 0.25 ± 0.04). One gene (UDP-GalNac) showed a constant expression (1.02 ± 0.36) in vitreous-treated RPE cells, compared with untreated cells (Fig. 4) .



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  		Figure 3. Real-time RT-PCR. Amplification plot of PIG-B (A) and LKHA4 (B) together with the housekeeping gene GAPDH as the internal calibrator of vitreous-treated (V) and serum-free medium (nS)–treated hRPE cells. The PCR baseline subtracted relative fluorescence units (RFU) of cycles 10 to 40 are shown. The line at 150 RFU represents the threshold used to determine the gene-specific CT. The curves below the threshold line or breaking through the line at cycle 36 to 38 (GAPDH) are the negative control with primers for the genes, but without cDNA.

 


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  		Figure 4. Comparison of gene expression. GAPDH-calibrated relative gene expression in vitreous treated human RPE cells compared with the untreated control. The gene names are listed below the columns. The relative GAPDH calibrated gene expression is listed inside the columns. GAPDH-calibrated gene expression in untreated cells was set as 1. Parallel lines: SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Exposure of RPE cells to vitreous results in changes in the morphology and biochemistry of RPE cells.1 2 9 In the current study, we used a DEmRNA-PCR analysis to investigate changes in the level of mRNA expression in RPE cells treated with vitreous. Recently, a decreased expression of FGF-2 mRNA and protein was shown in RPE cells treated with 25% vitreous.6 Using the same vitreous concentration and incubation time in our analysis, we additionally detected a variety of differentially expressed amplicons. By sequence alignment search in the gene data bank 10 of these were identified as part of the mRNA sequence of human genes. RT-PCR with gene-specific primers demonstrated their normal expression in ex vivo human RPE cells. This is the first report about mRNA expression of the genes NFIB2, KE03, PIG-B, DKFZp564BC462, LKHA4, PAM, UEV-1, and UDP-GalNac in RPE cells. The expression of MAP1B and G3BP in cultured RPE cells and in vivo has been shown previously.10 11

Using real-time RT-PCR as a quantification system, we quantified the downregulation of the mRNA of 9 of 10 analyzed genes in vitreous-treated RPE cells compared with the untreated control. Although increased expression for three of the amplicons later found to be parts of the genes PAM, UEV-1, and UDP-GalNac was detected initially in the DEmRNA-PCR analysis, the real-time RT-PCR analysis clearly showed decreased GAPDH-calibrated mRNA expression (PAM: 0.5; UEV-1: 0.6) and a constant expression for UDP-GalNac (Fig. 4) . This discrepancy could be due to the well-documented problems of differential display systems with the linearity of the PCR.12 Therefore, it is always necessary to use a second independent approach for quantification. Real-time PCR using green fluorescence (Sybr Green I; Molecular Probes Europe) is known as a versatile approach to quantitative PCR.13 This is demonstrated in our experiments by the constant GAPDH mRNA amount in differentially treated cells and by the confirmation of the decreased mRNA expression of the other seven genes found in the DEmRNA-PCR analysis by our real-time RT-PCR approach. Thus, we consider the decreased or constant expression found by real-time RT-PCR to be real.

Exclusive information has been published regarding the expression of MAP1B and G3BP in human RPE cells.10 11 By a semiquantitative approach, the investigators have shown an upregulation of the mRNAs of both genes in proliferating cells in culture and in epiretinal membranes surgically removed from patients with PVR. In the present study we demonstrated a downregulation of the mRNA expression of both genes in RPE cells cultured in serum-free medium due to vitreous treatment. The discrepancy of both results may be due to their different confluence status or different treatment of the cells (serum-free medium versus vitreous). It is known that the properties of cultured human RPE cells are affected by their confluence status, as was shown for the adherens junction.14 In addition, cells treated with serum-free, low-protein medium (with or without vitreous) would probably show altered behavior compared with cells grown in full medium. RPE cells in PVR membranes react differently to their microenvironments during a later stage of PVR in contrast to RPE cells that are freshly exposed to vitreous. Thus, it is not possible to directly compare the behavior of cultured RPE cells shown in this study and RPE cells in PVR membranes.

No information is available about the expression of the other genes in RPE cells, and only limited information exists about their expression and function in other tissues.

NFIB2 belongs to the family of nuclear factor (NF)- I or CCAAT box binding transcription factors (CTF).15 16 The CTF/NF-I proteins are individually capable of activating transcription and DNA replication.17 They are targets of gene expression regulatory pathways elicited by growth factors and interact with basal transcription factors and with histone H3.18

Pam has been shown to associate with c-Myc but not with N-Myc.19 Several motifs in Pam, especially the regulator of chromosome condensation (RCC)-1 motifs, give hints of possible roles for the Myc-Pam complex in chromatin modeling during the cell cycle or in RNA transportation.19 Recently, Pam has been additionally suggested as an integrator of transcriptional functions with other functions concerning cell-cycle transit, chromatin modeling, and apoptosis or the signaling of cell fate.20 Recently, it has been established that human RPE cells in culture express Myc mRNA and that antisense oligonucleotide inhibits human RPE cell proliferation.21

UEV-1 has been identified as a protein with expression that is regulated during in vitro differentiation of intestinal epithelial cells and which could play a role in modulating their mature phenotype and cell-cycle status.22 The protein was previously described as a transcriptional regulator, known as CROC-1.23 It has been demonstrated that UEV-1 protects cells from DNA-damaging agents24 and acts similarly to a DNA repair gene.25

The deduced amino acid sequence of KE03 protein contains ankyrin repeats. Ankyrin repeats are tandemly repeated modules of approximately 33 amino acids building an L-shaped structure consisting of a ß-hairpin and two {alpha}-helices.26 Many ankyrin repeat regions are known to function as protein–protein interaction domains.

Phosphatidylinositol glycan of complementation class (PIG-B) encodes an endoplasmic reticulum transmembrane protein involved in the biosynthesis of mammalian glycosylphosphatidylinositol (GPI) anchors found in all eukaryotic cells.27 The PIG-B protein is essential for the transfer of the third mannose in an {alpha}-1,2 linkage to the core structure28 ; but, to date, no enzymatic activity has been shown.29 GPI-anchored proteins are assembled within membrane microdomains,30 which also accommodate cytoplasmic signaling molecules (e.g., G-proteins), suggesting mediation of signal transduction.31

UDP-GalNac(T2) belongs to the glycosyltransferase family 2.32 The enzyme catalyzes the initial reaction in O-linked oligosaccharide biosynthesis. The carbohydrate moieties of glycoproteins, glycolipids, and proteoglycans are thought to play important roles in intercellular recognition, which regulates differentiation, development, leukocyte trafficking, the immune response, and other tissue functions.33

LKHA4 is a member of the membrane alanyl dipeptidase (M1) family of metalloproteases. LKHA4 is known to hydrolyze the epoxide leukotriene-A4 to form an inflammatory mediator.34 This is the first report of the detection of this inflammatory mediator in human RPE cells. Recently, LKHA4 mRNA was detected in bovine and human corneal epithelium by RT-PCR and Northern blot analysis.35 For bovine retinal pericytes, it has been shown that the LKHA4-formed inflammatory mediator may play an important role in modulating fluid movement, vascular tone, and permeability in the microvasculature, particularly in inflammatory states.36 Preventing inflammation remains the first medical treatment of PVR.37 Therefore, it is important information that RPE cells are capable of expressing LKHA4 and that human vitreous downregulates the mRNA expression of this inflammatory mediator in RPE cells.

Presently, nothing is known about the human mRNA DKFZp564B1462. The sequence with the accession number AL080073 derived from the cDNA sequencing project of fetal brain of the German Cancer Research Center (DKFZ). A homology search of the human DNA bank revealed no homology to a known gene.

Analyzing the gene expression of cultured RPE cells treated with vitreous by DEmRNA-PCR analysis may result in new insight to the pathobiology of PVR. A complex network of gene action is involved in the transdifferentiation of RPE cells from a quiescent status to a proliferation status, ultimately leading to the development of proliferative retinopathy. The results of this article focus attention on the function of several new genes apparently participating in this process. This includes important cellular functions such as transcription (NFIB2, Pam with Myc), mediation of signal transduction (G3BP, PIG-B) and inflammation (LKHA4), glycosylation (UDP-GalNac[T2]), ubiquitination (UEV-1), and protein–protein interaction (MAP1B, KE03). Closer examination of these genes may lead to the definition of additional targets for treatment or prevention of diseases caused by contact of RPE cells to vitreous, such as PVR.


   Acknowledgements
 
The authors thank Beatrix Martiny and Claudia Gavranic for expert technical assistance.


   Footnotes
 
Supported by the Retinovit Foundation, the Köln Fortune Program, Faculty of Medicine, Cologne, and the German Research Foundation Grants He 840/6-3, Es 82/5-3, and He 840/5-3.

Submitted for publication July 24, 2001; revised November 13, 2001; accepted December 5, 2001.

Commercial relationships policy: N.

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: Norbert Kociok, Department of Vitreoretinal Surgery, Division of Ophthalmology, University of Cologne, Joseph-Stelzmann-Strasse 9, D-50931 Cologne, Germany; norbert.kociok{at}uni-koeln.de.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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R. Ganti, R. C. Hunt, S. K. Parapuram, and D. M. Hunt
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