Vitreous Induces Components of the Prostaglandin E2
Pathway in Human Retinal Pigment Epithelial Cells
Sunil K. Parapuram, Ramapriya Ganti, Richard C. Hunt, and D. Margaret Hunt
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 ␮g 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. (Invest Ophthalmol Vis Sci. 2003;44:
1767–1774) DOI:10.1167/iovs.02-0528
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 – 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 –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-1but 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.
P
RPE Cell Culture
roliferative 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
From the Department of Pathology and Microbiology, University
of South Carolina School of Medicine, Columbia, South Carolina.
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.
Corresponding author: D. Margaret Hunt, Department of Pathology and Microbiology, University of South Carolina School of Medicine,
Columbia, SC 29208; mhunt@med.sc.edu.
Investigative Ophthalmology & Visual Science, April 2003, Vol. 44, No. 4
Copyright © Association for Research in Vision and Ophthalmology
MATERIALS
AND
METHODS
RPE cells were obtained from human donor eyes14 (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 ␮g 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-␮m 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-␮m
filter bottle.
1767
1768
Parapuram et al.
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 ␮L of water to which 20 ␮L 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-␮L 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 ␮g 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, SigmaAldrich, St. Louis, MO), incubated at 37°C for 1 hour, diluted to 150 ␮L
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 ␮L of PCR master mix. The final volume
of the PCR reaction was 50 ␮L 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 ␮M 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
IOVS, April 2003, Vol. 44, No. 4
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 Pfaffl18 (see the
following equation, where E is the efficiency of real-time PCR amplification for the appropriate gene).
ratio ⫽
(Etarget)⌬Cttarget (control-treated)
(ERPLP0)⌬CtRPLP0 (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 ␮L
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 Psq; 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 CDPStar; 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
IOVS, April 2003, Vol. 44, No. 4
RPE Cells, Prostaglandins, and PVR
1769
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 ␮g collagen per mL
(C) for 48 hours and examined by
light microscopy.
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
FIGURE 2. Part of a cDNA array
showing the region for mPGES. Total
RNA from control (A) or vitreoustreated (B) cells was copied into 32Plabeled 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 ␣-tubulin (at right). The doublet indicated by the arrow represents
mPGES.
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.
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Parapuram et al.
IOVS, April 2003, Vol. 44, No. 4
TABLE 1. Details of Cell Lines and Vitreous Donor
Combinations Used
mRNA
Assayed
Time
(h)
RPE
Donor
Vitreous
Donor
Collagen Passage
Treated Number mPGES
48
A
B
C
D
E
F
G
H
1
2
3
4
5
6
7
8
—
—
—
—
Yes
Yes
Yes
—
4
6
4
4
4
2
4
3
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
24
A
K
K
J
J
9
10
11
12
13
Yes
—
Yes
Yes
Yes
3
5
5
3
4
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
12
A
K
J
L
M
P
9
11
13
14
15 ⫹ 16
22
Yes
Yes
Yes
Yes
Yes
Yes
3
5
4
5
4
5
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
6
A
K
J
N
M
Q
R
9
11
13
17
15 ⫹ 16
17
23
Yes
Yes
Yes
Yes
Yes
Yes
Yes
3
5
4
4
4
6
4
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
3
L
N
O
Q
18 ⫹ 19
20
21
17 ⫹ 24
—
—
—
—
6
5
5
6
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫹
COX-2
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 ␮g/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).
RPE donors and vitreous donors were different, except that RPE
donor N is vitreous donor 19, RPE donor Q is vitreous donor 22, RPE
donor P is vitreous donor 17. A few RPE donors (A, J, L, and N) were
used at two different passages.
Because the substrate for mPGES is made by COX enzymes
and because mPGES and COX-2 tend to be coinduced in some
tissues,21–24 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 ⱕ 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 vitreoustreated 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,
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 ⱕ 0.05.
RPE Cells, Prostaglandins, and PVR
IOVS, April 2003, Vol. 44, No. 4
TABLE 2. Effect of Vitreous on mRNA Levels of mPGES and COX-2
Time
(h)
mPEGS mRNA
6
12
24
48
COX-2 mRNA
3
6
12
24
48
Average Ratio
Vitreous to Control
Experiments
(n)
P*
2.1
2.7
8.4
7.0
7
5
5
8
0.01
0.25
0.02
0.001
3.3
3.0
2.6
1.1
0.8
4
7
6
5
8
0.03
0.13
0.15
0.99
0.44
For each experiment, low-passage human RPE cells were treated
with complete medium or medium containing 25% vitreous for various
periods of time, and total RNA was extracted. Real-time PCR assays of
the target gene (mPGES or COX-2) and the standard gene (RPLP0) were
performed in triplicate, and the average values used to determine the
ratio of the target mRNA level in vitreous to control for that experiment. The average of multiple such experiments for each incubation
period is shown.
* The probability that the expression level of the target gene is the
same in the control group and the vitreous-treated group.
1771
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.11,21–24,34 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-1␤–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
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
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 ␮g 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.
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Parapuram et al.
IOVS, April 2003, Vol. 44, No. 4
TABLE 3. Effect of Collagen on mRNA Levels of mPGES and COX-2
Time
(h)
mPGES mRNA
6
12
24
48
COX-2 mRNA
6
12
24
48
Average Ratio
Collagen to Control
Experiments
(n)
P*
1.2
1.1
1.1
1.1
7
5
4
3
0.84
0.92
0.84
0.74
1.0
1.4
1.9
1.1
7
6
4
3
0.90
0.66
0.58
0.77
For each experiment, low-passage human RPE cells were treated
with complete medium or medium containing 100 ␮g collagen/mL for
various periods, and total RNA was extracted. Relative levels of mRNA
in collagen-treated and control cells were determined as described in
Table 2.
* The probability that the expression level of the target gene is the
same in the control group and the vitreous-treated group.
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 SDSpolyacrylamide gel electrophoresis and immunoblot analysis. Left: mobility of marker proteins (mass in kDa); arrow: position of the band
corresponding to COX-2.
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 COX1/cPGES pathway. A recent report36 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), IFN␥, IL-6, TGF-␤, and hepatocyte growth factor
(HGF) are present in increased amounts in the vitreous of
patients with PVR.38 – 43 IL-1␤ and PDGF have been shown to
increase the expression of COX-2 in RPE cells.36,37 Production
of PGE2 is enhanced in IL-1␤–treated RPE cells, and a combination of IL-1␤ with TNF␣ or IFN␥ produced levels of PGE2
that were greater than the levels produced by treatment with
IL-1␤ 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
RPE Cells, Prostaglandins, and PVR
IOVS, April 2003, Vol. 44, No. 4
TABLE 4. Effect of Vitreous on PGE2 Secretion
PGE2 (pg/mL)
Change in Presence
of Vitreous
Time
Control
Vitreoustreated
PGE2
(pg/mL)
% Change
12 h
70
172
174
140
380
220
70
208
46
100% increase
121% increase
26% increase
24 h
92
258
210
130
560
460
38
302
250
41% increase
117% increase
119% increase
48 h
400
245
450
225
50
⫺20
13% increase
8% decrease
RPE cells were treated with complete medium or medium containing 25% vitreous for 12, 24, or 48 hours. The medium was removed
and subjected to enzyme immunoassay for PGE2.
Data represent the average of duplicate determinations in each
sample. At each time point, multiple RPE donors and multiple vitreous
donors were used.
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 PGE248 or its increased
accumulation in cells cotransfected with COX-2 and mPGES11
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.
Acknowledgments
The authors thank Phil Fairey IV for his participation in obtaining some
of the preliminary data.
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