Vitreous Induces Components of the Prostaglandin E₂ Pathway in Human Retinal Pigment Epithelial Cells

Abstract

purpose. To investigate the alterations in gene expression when human retinal pigment epithelial (RPE) cells in culture are treated with vitreous as a model for the changes that occur in proliferative vitreoretinopathy.

methods. Human RPE cells were cultured with or without human vitreous or collagen. RNA was extracted and reverse transcribed. The RNAs expressed were compared by using DNA macroarrays. Messenger RNA levels were also measured using real-time reverse transcription polymerase chain reaction. Protein expression was examined by immunoblot analysis. Immunoassays were used to determine levels of prostaglandin E₂.

results. Vitreous treatment of RPE cells resulted in increased expression of two critical enzymes in the synthesis of prostaglandin E₂: membrane-associated prostaglandin E-synthase (mPGES) and cyclooxygenase (COX)-2. Increased levels of mPGES RNA and protein were still present at 48 hours of treatment, but the increase in COX-2 mRNA and protein was transient. The increase in the expression of mPGES was associated with an increase in the production of prostaglandin E₂ that was observed at 12 and 24 hours of treatment but not at 48 hours. Treatment with 100 μg collagen I per ml medium did not cause increased expression of mPGES and COX-2, even though both collagen- and vitreous-treatment caused a morphologic change in the RPE cells to a more fibroblast-like phenotype.

conclusions. Treatment of human RPE cells with vitreous induces changes in gene expression that are indicative of an inflammatory response.

Proliferative vitreoretinopathy (PVR) can be considered an aberrant wound-healing process characterized by the presence of epiretinal membranes (ERMs) within the vitreous or, in some cases, by subretinal membranes. Contraction of ERMs can lead to traction retinal detachment. ^{1 2} ERM cellular components include retinal pigment epithelial (RPE) cells, glial cells, fibroblasts, and inflammatory cells. ³ Many of the RPE cells in ERMs have undergone a morphologic transformation and show a fibroblastic phenotype. ^{3 4} RPE cells are thought to play a major role in the development and contraction of ERMs in PVR, ⁴ and a better understanding of the phenotypic changes in the RPE in this disease would help in the design of therapeutic approaches. 

Risk factors for development of PVR include breakdown of the blood–retinal barrier and/or inflammation and retinal tears. ⁵ In view of the importance of retinal breaks and the resultant contact between vitreous and RPE cells, it is interesting that exposure of RPE cells to vitreous in vitro in the presence of serum causes transformation from an epithelial to a fibroblast-like phenotype, similar to that seen in ERMs. ^{6 7 8} In this study, exposure of RPE cells to vitreous resulted in a sustained increase in the expression of membrane-associated prostaglandin E synthase (mPGES) and a transient increase in cyclooxygenase (COX)-2. Both of these enzymes participate in the synthesis of prostaglandins from arachidonic acid and both are induced by inflammatory mediators. ^{9 10 11} Arachidonic acid is metabolized to prostaglandin H₂ (PGH₂) by cyclooxygenase (COX)-1 or -2. COX-1 is constitutively expressed in many tissues, whereas COX-2 is usually induced by a variety of agents, including inflammatory mediators, although some cells express it constitutively. ¹² Prostaglandin E synthase (PGES) catalyzes the isomerization of PGH₂ to prostaglandin E₂ (PGE₂). Cytosolic PGES (cPGES) is constitutively expressed, ¹³ whereas mPGES is inducible. ¹⁰ cPGES is capable of converting COX-1- but not COX-2–derived PGH₂ to PGE₂ efficiently, ¹³ whereas the mPGES is preferentially coupled with COX-2 in producing PGE₂. ¹¹ It is possible that the inflammatory pathways associated with PGE₂ have a role in the processes that lead to the formation of PVR membranes. 

Materials and Methods

RPE Cell Culture

RPE cells were obtained from human donor eyes ¹⁴ (Lions’ Eye Bank, Columbia, SC, and Portland, OR). The protocol adhered to the tenets of the Declaration of Helsinki for research involving human tissue. The eyes were cut circumferentially above the equator, and the lens and iris tissue were removed. The vitreous was then taken out, cleared completely of any retinal tissue attached to it, and stored at −80°C. The choroid was separated from the sclera. Most of the RPE cells are attached to the choroid and were removed from it by incubation in 7.5% trypsin-EDTA solution (Life Technologies, Gaithersburg, MD). RPE cells were cultured in F-10 medium (Life Technologies) containing 10% fetal bovine serum (BD Biosciences-Clontech, Palo Alto, CA), 1% glutamine-penicillin-streptomycin (Glut-Pen-Strep; Irvine Scientific, Santa Ana, CA), 1% CaCl₂ and 1% culture supplement (ITS; BD Biosciences, San Diego, CA) and used at passages 2 to 6. The medium was removed from subconfluent RPE cells that were then treated with: 25% vitreous in complete medium, 100 μg collagen I per milliliter complete medium, or fresh complete medium for various times. The cells were subconfluent at the time of treatment and were still subconfluent at the time RNA was extracted. The vitreous gel was shredded with a syringe, diluted by adding three parts culture medium to one part vitreous, and filtered with a 0.22-μm filter bottle (PES; Corning Inc., Acton, MA). The vitreous treatment leads to a reproducible morphologic change after 48 hours (or less). ^{14 15} Collagen I (Cohesion Technologies Inc., Palo Alto, CA) was diluted in complete medium and filtered with a 0.22-μm filter bottle. 

Membrane Arrays

Cells were grown in 75-cm² bottles, and the RNA was isolated with a kit (ToTally RNA; Ambion, Inc., Austin, TX), according to the manufacturer’s directions, except that the cells were lysed in only 1.4 mL of denaturing solution and then the cell lysate was passed through a shredder (Qiashredder; Qiagen, Inc., Valencia, CA) to fragment the DNA. In addition, the acid-phenol extraction step in the manufacturer’s protocol was repeated twice. To remove genomic DNA, the purified RNA was dissolved in 180 μL of water to which 20 μL of 10× DNase buffer and 6 U DNase (Roche Molecular Biochemicals, Indianapolis, IN) were added and was incubated at 37°C for 60 minutes. The DNase was inactivated and removed by extraction with acidic phenol-chloroform (5:1, pH 4.5; Ambion, Inc.), followed by chloroform extraction and ethanol precipitation in the presence of 0.3 M sodium acetate. RNA was quantified and the quality checked from the absorbance at 260 and 280 nm. The integrity of the RNA was examined by nondenaturing gel electrophoresis, as described in the manufacturer’s instructions (Ambion, Inc.). ³²P-dATP-labeled cDNA was prepared and hybridized to human cancer gene arrays according to the manufacturer’s protocol (Atlas Arrays; BD Biosciences-Clontech). After washing, the blots were subjected to autoradiography (Biomax film; Eastman Kodak, Rochester, NY; Biomax MS intensifying screens; Eastman Kodak). 

Real-Time Reverse Transcription–Polymerase Chain Reaction

Total RNA was extracted from RPE cells using a kit (RNeasy; Qiagen, Inc.) and treated with DNase (Qiagen, Inc.) while on the column according to the manufacturer’s protocol. The RNA’s quality and integrity were checked as just described, and the RNA was used to make cDNA. The 20-μL reverse transcription reaction consisted of 1× buffer (Omniscript; Qiagen, Inc.), 0.5 mM of each dNTP, 10 U RNasin (Promega, Madison, WI), 4 U reverse transcriptase, 1 μg total RNA, and either 500 ng oligo (dT)₁₅ primer (Promega; if PGES mRNA was to be measured) or 50 ng random hexamer primer (Promega; if COX-2 mRNA was to be measured). The reaction mix was covered with approximately 0.1 mL of mineral oil (Molecular Biology Grade, Sigma-Aldrich, St. Louis, MO), incubated at 37°C for 1 hour, diluted to 150 μL with water, and incubated in a boiling water bath for 10 minutes. Real-time PCR reactions were performed with a core reagent kit (SYBR Green PCR; Applied Biosystems, Foster City, CA). Messenger RNA levels for ribosomal protein, large, P0 (RPLP0) were measured as an internal standard. ^{16 17} Five microliters of the appropriate diluted reverse transcription mix (heated for 3 minutes in a boiling-water bath and quenched in ice water immediately before use) were added to a 96-well plate followed by 45 μL of PCR master mix. The final volume of the PCR reaction was 50 μL and consisted of 1× buffer (SYBR Green I; Applied Biosystems), 3 mM MgCl₂, 1 mM dNTP (0.2 mM each dNTP and 0.4 mM dUTP), 0.1 μM each of the appropriate forward primers (COX-2: 5′-GCC TGA TGA TTG CCC GAC T; mPGES: 5′-ACA TCT CAG GTC ACG GGT CTA; RPLP0: 5′-TTA AAC CCC CTC GTG GCA ATC) and reverse primer (COX-2: 5′-GCT GGC CCT CGC TTA TGA TCT, mPGES: 5′-TTC CTG GGC TTC GTC TAC TC; RPLP0: 5′-CCA CAT TCC CCC GGA TAT GA) and 1.25 U of Taq polymerase (AmpliTaq Gold; Applied Biosystems). The RPLP0 primers work with cDNA primed with oligo(dT)₁₅ or random hexamers. The concentration of RPLP0 cDNA was measured for all cDNAs at the same time the concentration of cDNA for the gene of interest was determined. The PCR products were detected with a real-time detection system (iCycler IQ; Bio-Rad Laboratories, Hercules, CA). Primers were designed using a computer program (Oligo; Molecular Biology Insights Inc., Cascade, CO) to cross an exon–exon boundary to minimize the chance that a signal was from contaminating DNA. To check that genomic DNA was not a problem, real-time PCR was performed, using mock reverse-transcription reactions in which the reverse transcriptase was omitted. All real-time PCR reactions included a melt curve to examine the specificity of PCR product and to ensure that primer-dimer artifacts did not interfere with the measurements. The real-time PCR assays were performed in triplicate for each sample, and the average cycle at which the PCR product crossed the threshold (Ct) was calculated. The ratio of target gene mRNA expression relative to that of the internal standard mRNA (RPLP0) was calculated according to the method of Pfaffl ¹⁸ (see the following equation, where E is the efficiency of real-time PCR amplification for the appropriate gene).  

\[\mathrm{ratio}\ {=}\ \frac{(\mathrm{E}_{\mathrm{target}})^{{\Delta}\mathrm{Ct}_{\mathrm{target\ (control-treated)}}}}{(\mathrm{E}_{\mathrm{RPLP0}})^{{\Delta}\mathrm{Ct}_{\mathrm{RPLP0\ (control-treated)}}}}\]

 

To calculate the efficiency, 10-fold serial dilutions of mPGES DNA, COX-2 DNA, or RPLP0 DNA were subjected to real-time PCR and the efficiency calculated from a plot of Ct versus the logarithm of the concentration of the DNA, using the equation E = 10^(−1/slope). The efficiency was 1.89 for mPGES, 1.94 for COX-2, and 1.87 for RPLP0, which implies that 89%, 94%, and 87% of the template was copied per PCR cycle for mPGES, COX-2, and RPLP0, respectively. Statistical analyses were performed with REST-XL version 2 software (http://www.wzw.tum.de/gene-quantification; developed by Michael Pfaffl). ¹⁹ This program may be conservative when determining significance with low numbers of samples. The REST-XL analysis showed that levels of RPLP0 gene expression did not vary significantly between control, vitreous-treated, and collagen-treated cells. 

Western Blot Analysis

RPE cells were washed with ice-cold phosphate-buffered saline (PBS), and the proteins were extracted in lysis buffer (50 mM Tris-HCl [pH 8.0] containing 1 mM phenylmethylsulfonyl fluoride [PMSF], 50 mM NaF, 1% Nonidet P40 [Igepal; Sigma-Aldrich], 5 mM EDTA, and 1 μL protease inhibitor cocktail [Sigma-Aldrich]), per mL). An equal volume of 2× SDS gel-loading buffer (100 mM Tris-HCl [pH 6.8], 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 100 mM dithiothreitol) was added and the lysate was boiled for 10 minutes. For mPGES, proteins were separated on 12.5% acrylamide gels (Criterion gels; Bio-Rad Laboratories) and blotted onto membranes (Immobilon P^sq; Millipore Corp., Bedford, MA). For COX-2, proteins were separated on 4% to 15% acrylamide gels (Criterion; Bio-Rad Laboratories) and blotted onto membranes (Immobilon P; Millipore Corp.). Rabbit anti-human polyclonal antibodies against mPGES (Cayman Chemical Co., Ann Arbor, MI) and COX-2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used. Alkaline phosphatase–conjugated secondary antibodies were used and visualized with chemiluminescence reagent (Western Lightning CDP-Star; Perkin Elmer Life Sciences, Boston, MA). Images were then captured (Image Station 440CF; Eastman Kodak, Rochester, NY). 

Enzyme Immunoassay for PGE₂

Supernatant medium was taken from control RPE cells or RPE cells treated with vitreous for 12, 24, or 48 hours. The medium was frozen immediately in liquid nitrogen and then stored at −80°C until analyzed. PGE₂ was measured using an enzyme immunoassay kit (High Sensitivity PGE₂; Assay Designs, Inc., Ann Arbor, MI). 

Results

Array Analysis

To study changes in gene expression that accompany the exposure of RPE cells to vitreous, cells were grown for 48 hours in the presence of normal medium or in 25% vitreous, which resulted in a change in the morphology of the cells (Fig. 1) . RNA was extracted and ³²P-labeled cDNA was prepared. The latter was hybridized to gene arrays (Atlas Cancer Gene Arrays; Clontech), which were then washed and subjected to autoradiography. The regions of the arrays with cDNA spots corresponding to mPGES are shown in Figure 2 . A much higher expression of mPGES mRNA was reproducibly present in vitreous-treated cells (Fig. 2B , arrow) compared with cells grown in normal medium (Fig. 2A , arrow). Because of the expected variability between different RPE cell strains and between different vitreous donors, and also because of the possible artifacts associated with the use of array analysis for precise quantitation, the levels of mPGES mRNA after vitreous-treatment of different RPE cell strains were analyzed by real-time PCR rather than by array analysis. 

Effect of Vitreous on Expression of COX-2 and mPGES mRNA

To determine whether increased levels of mPGES mRNA were reproducibly present after vitreous treatment, regardless of RPE and vitreous donors, samples from multiple RPE cell and vitreous donors were examined. Real-time PCR was used to measure mRNA levels. An advantage of real-time PCR, in addition to its accuracy compared with other quantitative PCR methods, ²⁰ is that PCR primers can be designed to prime from unique regions of an mRNA and thus can be more gene specific than the longer probes used on membrane arrays. Details of the RPE cell strains and vitreous donors are given in Table 1 . In all cases and at all time points examined (6, 12, 24, and 48 hours) vitreous treatment resulted in an increase in the level of mPGES mRNA (Fig. 3A) . A significant increase in expression was observed by 6 hours of treatment, with higher levels at 24 hours that were sustained until at least 48 hours (Table 2 and Fig. 3A ). The average level of mPGES mRNA in vitreous-treated cells after 24 or 48 hours of treatment was 8.4 and 7.0 times that in control cells, respectively. 

Because the substrate for mPGES is made by COX enzymes and because mPGES and COX-2 tend to be coinduced in some tissues, ^{21 22 23 24} real-time RT-PCR was used to explore the possibility of coordinate induction of the upstream enzyme, COX-2. At 3, 6, and 12 hours of vitreous-treatment, all RPE cell–vitreous donor combinations tested showed an increased level of COX-2 mRNA (Fig. 3B) . Only the 3-hour levels were significantly increased according to the results of the REST-XL statistical analysis (Table 2) , but the probability that the 6- and 12-hour vitreous-treated levels were the same as the control was fairly low (P ≤ 0.15). In contrast to the sustained increase seen for mPGES mRNA in vitreous-treated cells, the approximately threefold increase in COX-2 mRNA levels was transient, and, by 24 hours, expression of COX-2 mRNA in vitreous-treated cells was similar to that in control cells (Table 2) . 

One possible source of variation in these results is that RPE cell strains expressing a lower basal level of mPGES or COX-2 mRNA might exhibit a greater increase in mRNA on vitreous treatment than those that expressed higher basal levels. The correlation between initial levels of mRNA and the ratio of mRNA in vitreous-treated compared with control cells (Table 2) was examined at all time points at which the average ratio was more than 2.0. The correlation coefficients for mPGES were 0.67, −0.04, 0.60, and 0.32 at 6, 12, 24, and 48 hours, respectively, and for COX-2 the correlation coefficients were 0.67, 0.71, and 0.62 for 3, 6, and 12 hours, respectively. This suggests that some, but by no means all, of the variation in the response of the cells correlates with the levels of the mRNA in the untreated cells. 

Effect of Collagen on Expression of COX-2 and mPGES mRNA

Collagen is a major component of the vitreous, ²⁵ is a component of ERMs, ⁴ and has been shown to promote at least some of the changes that occur in human RPE cells in the presence of vitreous. ⁶ Therefore, the expression of COX-2 and mPGES mRNA was investigated after treatment of RPE cells for 6, 12, 24, or 48 hours with collagen I (100 μg/mL). However, in contrast to the results obtained after vitreous treatment, the levels of mPGES and COX-2 mRNA did not increase significantly in RPE cells treated with collagen (Fig. 4 ; Table 3 ), even though, in agreement with the data of Vidaurri-Leal et al., ⁶ there was a morphologic change in the cells comparable to that seen with vitreous (Fig. 1) . 

Effect of Vitreous on mPGES and COX-2 Protein Synthesis

Expression of mPGES and COX-2 protein was examined at various times after addition of vitreous. Control RPE cells exhibited a low level of mPGES protein expression. Vitreous treatment resulted in little change at 6 hours (Fig. 5B) but resulted in increased expression at both 24 and 48 hours (Fig. 5) with multiple RPE donors and vitreous donors (Fig. 5 and data not shown). COX-2 protein expression, which was barely detectable in control RPE cells, reached its peak at 6 hours after adding vitreous and then declined, with multiple RPE donors and vitreous donors (Fig. 6 , and data not shown). Thus, the time course of vitreous-induced protein expression paralleled the increase in levels of mRNA for each enzyme. 

PGE₂ Concentrations in Medium

Because there was an increase in both COX-2 and mPGES expression, it would be expected that the product of this metabolic pathway, PGE₂, would also increase. PGE₂ was measured in the medium of control and vitreous-treated cells by an enzyme immunoassay (Table 4) . Although there was considerable variation in the amount of PGE₂ released into the medium by RPE cells of different donors, vitreous treatment for 12 or 24 hours consistently caused an increase in the amount. On average, vitreous induced an 82% and 92% increase in the secretion of PGE₂ by RPE at 12 and 24 hours, respectively. At 48 hours, the levels of PGE₂ in the supernatant of vitreous-treated RPE had declined to levels close to that of the control (Table 4) . 

Discussion

Prostaglandins have a major role in a variety of pathophysiological processes, including inflammation, fever, allergy and immunity, bone resorption and formation, gastric cytoprotection, transport of ions and water in the kidney, vascular homeostasis, reproduction, and pain sensation. ^{26 27} In the eye, prostaglandins regulate intraocular pressure, ²⁸ affect retinal blood flow, ²⁹ and have roles in ocular inflammation, ³⁰ corneal neovascularization, ³¹ and the disruption of the blood–retinal and blood–aqueous barriers. ^{32 33}  

The results reported herein show that vitreous-treatment of human RPE cells resulted in increased expression of two enzymes, COX-2 and mPGES, which are both involved in synthesis of PGE₂ and are often coinduced. ^{11 21 22 23 24 34} Although there was a coinduction of mPGES and COX-2 in the vitreous-treated RPE cells, the kinetics of the changes in mRNA and protein levels differed between the two enzymes, with the increase in COX-2 mRNA and protein declining by 12 hours, but the increase in mPGES mRNA and protein still observed after 48 hours of treatment. These kinetics for COX-2 and mPGES protein expression in RPE cells are similar to those reported in IL-1β–treated human synoviocytes. ^{11 34} The product of the COX-2-mPGES pathway is PGE₂, and an increase in PGE₂ synthesis occurred in vitreous-treated RPE cells. The increase in PGE₂ was not sustained at 48 hours, despite the continued increase in the level of mPGES protein, which may reflect the relatively early reduction in the expression of COX-2 that could have deprived mPGES of its substrate. In some systems, the induction of COX-2 and mPGES has been shown to be preferentially coupled to the inducible synthesis of PGE₂. ²¹ Thus, it is likely that mPGES and COX-2 are involved in the increased PGE₂ synthesis in vitreous-treated cells. However, proof of this necessitates further experimental validation. Of interest, Kahler et al., ³⁵ have reported that treatment of human fibroblasts with 10% vitreous isolated from patients with PVR causes an increase in synthesis of PGE₂. 

That COX-2 and mPGES mRNA were detectable in untreated cells indicates that mPGES and COX-2 may play a role in basal synthesis of PGE₂ in RPE cells, even though such synthesis is more usually associated with the constitutive COX-1/cPGES pathway. A recent report ³⁶ indicates that COX-2 may be the major isoform of COX in human RPE cells, although mRNAs for both COX-1 and -2 have been reported in rat RPE cells. ³⁷ Thus, RPE cells may express COX-2 constitutively, although at low levels, with increased synthesis during rod outer segment phagocytosis, ³⁷ under inflammatory conditions, ^{36 37} or after addition of vitreous. Our difficulty in detecting COX-2 protein in control RPE cells may have been due to the insensitivity of the immunoblot analysis method. Further experiments are needed to determine whether synthesis of PGE₂ in control RPE cells is associated with COX-2 and mPGES or with COX-1 and/or cPGES. 

The effect of vitreous on the prostaglandin pathway in RPE cells may be mediated by growth factors. It is known that growth factors such as IL-1, platelet-derived growth factor (PDGF), IFNγ, IL-6, TGF-β, and hepatocyte growth factor (HGF) are present in increased amounts in the vitreous of patients with PVR. ^{38 39 40 41 42 43} IL-1β and PDGF have been shown to increase the expression of COX-2 in RPE cells. ^{36 37} Production of PGE₂ is enhanced in IL-1β–treated RPE cells, and a combination of IL-1β with TNFα or IFNγ produced levels of PGE₂ that were greater than the levels produced by treatment with IL-1β alone. ³⁶  

Collagen II is the major collagen component of the vitreous, ²⁵ and collagens I and III are the major collagens in PVR ERMs. ⁴⁴ Both type I and type II collagen have been reported to cause a morphologic change in RPE cells similar to that in PVR or after treatment with vitreous. ⁶ Thus, the effect of collagen I on expression of mPGES and COX-2 in RPE cells was examined. Collagen I did not cause a significant induction of either COX-2 or mPGES mRNA in the RPE cells in the current experiments, although it caused a morphologic change. Vitreous is a complex mixture, and the criterion of morphologic change, although easy to observe, almost certainly does not reflect the true richness of the RPE response to vitreous. Although our data suggest that components of vitreous other than collagen are needed to induce mPGES and COX-2, the data do not show that collagen plays no role. It is possible that some other component is necessary along with collagen, or proteolysis of collagen by vitreous-induced proteases may be necessary to release active components, or collagen II may play a different role from collagen I. 

Enhanced expression of COX-2 and PGE₂ is associated with increased metastatic potential in cancer cells. ⁴⁵ Invasiveness probably results from increased expression of certain matrix metalloproteinases (MMPs), ⁴⁶ in that inhibitors of prostanoid synthesis also suppress the activation, or levels, of MMPs. ^{46 47} Treatment of some cancer cells with PGE₂ ⁴⁸ or its increased accumulation in cells cotransfected with COX-2 and mPGES ¹¹ results in increased motility and changes in cell morphology—phenomena that are similar to the changes in RPE cells in the presence of vitreous. ^{5 14 49} PGE₂ has been shown to cause morphologic changes in osteoblasts as a result of breakdown of actin filaments. ⁵⁰ Thus, the increased synthesis of PGE₂ in RPE cells treated with vitreous could have implications in conditions such as PVR, because PGE₂ may play a role in the morphologic and/or mobility changes in the RPE cells that facilitates their migration into the vitreous. 

The increased production of components of the prostaglandin pathway in RPE cells exposed to vitreous suggests a role for prostaglandins in the development of PVR. This is consistent with suggestions that an inflammatory response plays a role in PVR. ^{1 5} However, although PGE₂ may participate, or be necessary, it may well not be sufficient. In some systems in which changes have been shown to be PGE₂-dependent, PGE₂ alone has no effect; other comediators are also necessary. ^{47 51 52} Thus, PGE₂, through inflammation, could have the role of a facilitator or enhancer in the vitreous-induced changes in RPE cells. Suppressing the production of PGE₂ by inhibiting the synthesis of mPGES, thus reducing inflammation, could be part of a potential treatment in preventing the progression of PVR. 

 Supported by National Eye Institute Grants EY12711 (DMH) and EY10516 (RH) and by a University of South Carolina School of Medicine Dean’s Research Development Fund grant (DMH).

 Submitted for publication May 31, 2002; revised December 2, 2002; accepted December 31, 2002.

 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.

 

Figure 1.

 

Effect of vitreous or collagen treatment on morphology of low-passage human RPE cells. Cells were treated with normal medium (A) or medium containing 25% vitreous (B) or 100 μg collagen per mL (C) for 48 hours and examined by light microscopy.

Figure 2.

 

Part of a cDNA array showing the region for mPGES. Total RNA from control (A) or vitreous-treated (B) cells was copied into ³²P-labeled cDNA and hybridized to a human cancer cDNA array. A limited region of the autoradiograph is shown. Each cDNA spot on the array is spotted in duplicate. The two positive signals in the bottom row represent the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (overexposed) and α-tubulin (at right). The doublet indicated by the arrow represents mPGES.

Table 1.

 

Details of Cell Lines and Vitreous Donor Combinations Used

Time (h)	RPE Donor	Vitreous Donor	Collagen Treated	Passage Number	mRNA Assayed
										mPGES	COX-2
48	A	1	—	4	+	+
	B	2	—	6	+	+
	C	3	—	4	+	+
	D	4	—	4	+	+
	E	5	Yes	4	+	+
	F	6	Yes	2	+	+
	G	7	Yes	4	+	+
	H	8	—	3	+	+
24	A	9	Yes	3	+	+
	K	10	—	5	+	+
	K	11	Yes	5	+	+
	J	12	Yes	3	+	+
	J	13	Yes	4	+	+
12	A	9	Yes	3	+	+
	K	11	Yes	5	+	+
	J	13	Yes	4	+	+
	L	14	Yes	5	+	+
	M	15 + 16	Yes	4	+	+
	P	22	Yes	5	−	+
6	A	9	Yes	3	+	+
	K	11	Yes	5	+	+
	J	13	Yes	4	+	+
	N	17	Yes	4	+	+
	M	15 + 16	Yes	4	+	+
	Q	17	Yes	6	+	+
	R	23	Yes	4	+	+
3	L	18 + 19	—	6	−	+
	N	20	—	5	−	+
	O	21	—	5	−	+
	Q	17 + 24	—	6	−	+

Figure 3.

 

Changes in levels of mRNA in vitreous-treated compared with control cells. The levels of mRNA for mPGES (A) or COX-2 (B) were measured after various periods of treatment with control medium or medium containing 25% vitreous using real-time RT-PCR. Real-time PCR assays for mPGES or COX-2 were performed in triplicate. Triplicate real-time PCR assays for RPLP0 were performed simultaneously so that this mRNA could be used as an internal standard to control for small differences in the amount of mRNA. The ratio of mRNA expression in vitreous-treated compared with control cells was calculated according to the method of Pfaffl. ¹⁸ Each point at a particular incubation time indicates a different RPE/vitreous donor pair (see Table 1 ). Ratios of more than one indicate an increase in mRNA. *P ≤ 0.05.

Table 2.

 

Effect of Vitreous on mRNA Levels of mPGES and COX-2

Time (h)	Average Ratio Vitreous to Control	Experiments (n)	P *
mPEGS mRNA
6	2.1	7	0.01
12	2.7	5	0.25
24	8.4	5	0.02
48	7.0	8	0.001
COX-2 mRNA
3	3.3	4	0.03
6	3.0	7	0.13
12	2.6	6	0.15
24	1.1	5	0.99
48	0.8	8	0.44

Figure 4.

 

Changes in levels of mRNA in collagen-treated compared with control cells. The levels of mRNA for mPGES (A) or COX-2 (B) were measured after various periods of treatment with control medium or medium containing 100 μg collagen/mL. Real-time PCR assays for RPLP0, mPGES, or COX-2 were performed in triplicate. Triplicate real-time PCR assays for RPLP0 were performed simultaneously so that this mRNA could be used as an internal standard to control for small differences in the amount of mRNA. The ratio of mRNA expression in collagen-treated compared with control cells was calculated according to the method of Pfaffl. ¹⁸ For details of the RPE donors used, see Table 1 . Values of more than one indicate an increase in mRNA.

Table 3.

 

Effect of Collagen on mRNA Levels of mPGES and COX-2

Time (h)	Average Ratio Collagen to Control	Experiments (n)	P *
mPGES mRNA
6	1.2	7	0.84
12	1.1	5	0.92
24	1.1	4	0.84
48	1.1	3	0.74
COX-2 mRNA
6	1.0	7	0.90
12	1.4	6	0.66
24	1.9	4	0.58
48	1.1	3	0.77

Figure 5.

 

Effect of vitreous on mPGES protein expression. RPE cells were treated with complete medium or medium containing 25% vitreous for various periods. Protein was then extracted and subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis. (A) Cells were treated with control or vitreous-containing medium for 48 hours. Lanes 1 and 2: RPE donor 1 and vitreous donor A; lanes 3 and 4: RPE donor 2 and vitreous donor B. (B) Cells from RPE donor 3 were treated for various periods with control or vitreous (donor C)-containing medium. (A, left) Mobility of marker proteins (mass given in kDa); arrow: position of the band corresponding to mPGES. (B) Results obtained after different times of treatment (6, 24, and 48 hours).

Figure 6.

 

Effect of vitreous on COX-2 protein expression. RPE cells (same donor for all lanes) were treated with complete medium (CON) or medium containing 25% vitreous (VIT; same donor for all lanes) for various periods. Protein was then extracted and subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis. Left: mobility of marker proteins (mass in kDa); arrow: position of the band corresponding to COX-2.

Table 4.

 

Effect of Vitreous on PGE₂ Secretion

Time	PGE2 (pg/mL)			Change in Presence of Vitreous
		Control	Vitreous- treated	PGE2 (pg/mL)	% Change
12 h	70	140	70	100% increase
	172	380	208	121% increase
	174	220	46	26% increase
24 h	92	130	38	41% increase
	258	560	302	117% increase
	210	460	250	119% increase
48 h	400	450	50	13% increase
	245	225	−20	8% decrease

The authors thank Phil Fairey IV for his participation in obtaining some of the preliminary data.