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1 From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; the 2 Department of Zoology, Miami University, Oxford, Ohio; 3 Sugen, Inc., South San Francisco, California; and the 4 Department of Ophthalmology, West Norwich Hospital, United Kingdom.
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Abstract |
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METHODS. Capsular bags were prepared from human donor eyes and maintained in a 5% CO2 atmosphere at 35°C. On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence cytochemistry. De novo protein synthesis was determined by the incorporation of 35S-methionine. Basic fibroblast growth factor (FGF) and FGF receptor (R)-1 were detected using enzyme-linked immunosorbent assay (ELISA) and reverse transcription–polymerase chain reaction (RT-PCR) techniques. FGFR-1 inhibition was achieved using the specific antagonist SU5402.
RESULTS. Human lens epithelial cells can maintain metabolic activity for more than 1 year in a protein-free medium. Basic FGF was shown to be present in capsular bags throughout culture and also in capsular bags removed from donor eyes that had previously undergone cataract surgery. Furthermore, FGFR-1 was identified. Inhibition of FGFR-1 caused a significant retardation of growth on the posterior capsule. On no occasion did any treated bag reach confluence, whereas all match-paired control samples did.
CONCLUSIONS. The results provide evidence that FGF plays an integral role in the long-term survival and growth of human lens epithelial cells, independent of external stimuli. Inhibition of FGFR-1 by specific synthetic molecules, such as SU5402, could provide a potential therapeutic approach to resolving PCO.
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Introduction |
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Many approaches have been used to investigate PCO, ranging from simple cell-line culture experiments to in vivo studies.4 5 6 These two extremes are both restricted, because cell-lines are not grown on their natural matrix, whereas in vivo experiments pose difficulty with on-going observation and, when animals are used, with potential species variation.7 To address many of the shortfalls, some groups, including this laboratory, have developed human in vitro capsular bag culture systems based on the cataract operation itself.8 9 Such models eliminate potential species differences and permit the cells to grow on their natural substrate, in a manner similar to that in vivo. Furthermore, cellular events are easily observed by standard microscopy. It was initially assumed that PCO develops as a result of external regulation, including inflammation generated at surgery.2 Although external influences may contribute to development of PCO, recent findings in this laboratory using a human capsular bag system, have clearly demonstrated that lens epithelial cells grown on their native collagen capsule are capable of migration and proliferation in a protein-free medium.10 11 This discovery suggests an intrinsic ability of lens cells to regulate cell function in an autocrine manner and also indicates why PCO is such a common and persistent problem.
In the present study we focused on the fibroblast growth factor (FGF) family of growth factors of which the most studied forms are acidic and basic FGF.12 FGF is capable of inducing several different biologic responses in a number of cell types, including lens epithelial cells. When lens epithelial explants are exposed to basic FGF the cells proliferate, migrate, and differentiate in a dose-dependent manner.13 Similar events arise in response to acidic FGF, but higher concentrations are required.14 Furthermore, Schweigerer et al.15 concluded from their studies of primary cultures of bovine lens cells that, although mRNAs for both acidic and basic forms were identified, most of the biologic activity present was due to basic FGF. Basic and acidic FGF have been detected in embryonic and mature lenses16 and are believed to play a fundamental role in development.17 18 19 20 21 22 23 24
A feature of FGF is its dependence on heparin or matrix-associated heparin sulfate proteoglycans for receptor interaction.25 The binding of heparin induces a conformational change that enables efficient binding of the ligand to the FGF receptor to occur. Acidic and basic FGF distribution in the rat lens parallels the distribution of heparin sulfate proteoglycans.16 The FGF receptors are all members of the tyrosine kinase receptor family. Although FGF receptors (R) 1, 2, and 3 appear to be expressed in lens cells of a variety of species,21 22 23 24 26 27 only FGFR-1 is universally expressed. Therefore, the evidence compiled from a range of species suggests that the necessary components are in place for a potential autocrine role for FGF in lens cell growth and, in particular, PCO. For the present study, it was important first to establish evidence for the presence of both FGF and its receptor in long-term serum-free capsular bags and in bags extracted from donor eyes that had previously undergone cataract surgery. Secondly, it was important to establish any effect of breaking the putative autocrine loop involving FGFR-1, and for this, the specific antagonist SU5402 was used.28
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Methods |
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Ex Vivo Specimens
Four donor eyes possessed capsular bags containing intraocular
lenses (IOLs) that had been generated by cataract surgery. The capsular
bag was dissected from the zonules and placed in homogenizing buffer
for protein synthesis and enzyme-linked immunosorbent assay (ELISA).
All specimens showed signs of cell growth and of PCO development. No
exclusion criteria were adopted in this study, because these specimens
are extremely rare.
Anterior Lens Epithelium
After removal from the eye, the lens was transferred to a 35-mm
petri dish where it was placed anterior surface down. In some cases,
lenses had been mechanically damaged with the intention of disrupting
the epithelium. Using an insulin needle, the posterior capsule was
punctured and an incision made that separated the posterior capsule
into two halves. Two pins were then inserted at the ends of the
incision. A small cut was made near one of the pins before removing the
majority of the posterior capsule by curvilinear tear. This procedure
was then repeated on the other half. Six additional pins were inserted
through the periphery to maintain stability. The fiber mass was then
carefully removed and remaining fibers were cleared using surgical
forceps. The resultant preparations were then either prepared
immediately for immunocytochemical evaluation or cultured in 1.5 ml of
protein-free EMEM. Incubation was at 37°C in a 5%
CO2 atmosphere. The medium was sampled and
replaced every 3 to 4 days. All lenses used were from donors aged more
than 50 years.
35S-Methionine Incorporation into Newly Synthesized Proteins
35S-Methionine was added to the culture medium at 10 µCi/ml
for the final 2 days of incubation. Then the radioactive bathing medium
was sampled and replaced with fresh medium, and the preparations were
washed a further two times. At the end of the wash period, the capsular
bag was placed in 0.5 ml of homogenizing medium (composition in
millimolar: 6 phosphate, 100 KCl, 225 NaCl, 1 EGTA, 1 EDTA, 10
mercaptoethanol, 10 N-ethylmaliamide, 0.2
phenylmethylsulfonyl fluoride [PMSF], 0.005 E-64, and 1% vol/vol
Tween 20 [pH 7.4]), before storage in a -70°C freezer. The
preparations were thawed, homogenized, and centrifuged at 10,000 rpm
for 10 minutes. Fifty microliters of supernatant was transferred to
another container (Eppendorf, Fremont, CA) and 950 µl of 5%
trichloroacetic acid (TCA) added. This was left for 30 minutes at
4°C, then centrifuged at 10,000 rpm for 10 minutes, before removal of
the supernatant. One milliliter of 250 mM NaOH was added to the pellet
and left overnight. A 0.5-ml sample was taken from each container and
10 ml of scintillation fluid added (Hisafe Supermix) to each
before counting on a Wallac scintillation counter (Wallac; Perkin Elmer
Life Sciences, Gaithersburg, MD) with appropriate backgrounds. Counts
were corrected for decay.
Immunocytochemistry of the Capsular Bag
All reagents were from Sigma (Poole, UK) unless otherwise
stated. Three washes were performed, each for 15 minutes in
phosphate-buffered saline (PBS)/bovine serum albumin (BSA) and Nonidet
(0.02% and 0.05%, respectively). The pinned capsules were fixed for
30 minutes in 4% formaldehyde in PBS and permeabilized in PBS
containing 0.5% Triton X-100, also for 30 minutes. Nonspecific sites
were blocked with appropriate serum (1:50 in 1% BSA/PBS).
Anti-vimentin (Clone V9) and anti-human 
-crystallin (a gift from
Sam Zigler, National Eye Institute, Bethesda, MD) were diluted
1:100 and applied for 60 minutes at 35°C, followed by washing.
Vimentin and -crystallin were visualized with fluorescein
isothiocyanate (FITC)-conjugated secondary antibodies. The F-actin
cytoskeleton was stained with Texas red X-phalloidin (Molecular Probes,
Leiden, The Netherlands) for 30 minutes, and cell nuclei with
4[prime,6'-diamidino-2-phenylindole-2HCl (DAPI) at 1 mg/ml for 15
minutes, all at room temperature. The stained preparations were again
washed extensively, floated onto microscope slides, and mounted in
mounting medium (Vectashield; Vector, Peterborough, UK). Images were
viewed with either a fluorescence microscope (Eclipse E800; Nikon,
Tokyo, Japan) or a confocal microscope (Viewscan DVC-250; Bio-Rad,
Richmond, CA) with cooled CCD camera (Princeton Instruments, Marlow,
UK) and software (MetaMorph; Universal Imaging, West Chester, PA).
FGF Estimation by ELISA
Commercially available ELISA kits for human basic FGF were
purchased from R&D Systems (Abingdon, UK). The optical density of each
well was determined using a multiwell plate reader (Victor; Wallac) at
a wavelength of 450 nm. The readings from the standard series were
plotted with logarithmic axes, and the data from the samples were then
applied to the graph, with the estimated level of growth factor
expressed in picograms per milliliter. The limit of sensitivity of the
system is 3 pg/ml. In the case of homogenate samples, values were used
to determine total FGF per bag.
Identification of Basic FGF and FGFR-1 by RT-PCR
RNA was isolated from human lens preparations using a kit
(RNeasy with the RNase-free DNase set; Qiagen, Valencia, CA).
Approximately 1 µg of RNA was used to make cDNA using a commercial
amplification system (Superscript Pre-amplification System for First
Strand cDNA Synthesis; Gibco, Grand Island, NY). reverse
transcription–polymerase chain reaction (RT-PCR) reactions were set
using standard methods. Briefly, the conditions were the following:
primers at 0.8 µM, 2.5 units of Taq (Promega, Madison, WI)
per 25-µl reaction, and 1.5 mM MgCl2 for FGF-2
and 1.7 mM for FGFR-1. The PCR was performed using the following
program: initial denaturation 95°C for 1
minute; denaturation 90°C for 30 seconds;
annealing: 54°C for FGF-2 and
51°C for FGFR-1 for 30 seconds; and extension
at 72°C for 45 seconds. Steps 2 through 4 were cycled 35 times with a
final extension at 72°C for 5 minutes. The oligonucleotide primers
(5'-3') used were designed according to Berger et al.29
For basic FGF forward: CTGTACTGCAAAAACGGG; reverse: AAAGTATAGCTTTCTGCC.
For FGFR-1, forward: CCTCTTCTGGGCTGTGCT; reverse: CGGGCATACGGTTTGGTT.
Forty percent of the product was run in a 1.7% agarose gel.
FGFR-1 Inhibition
SU5402 (Sugen, South San Francisco, CA), a specific inhibitor of
FGFR-1 was used. This is a 3-substituted inolin-2-one, which has
previously been shown to specifically inhibit autophosphorylation of
FGFR-1.28
Furthermore, to validate the compound in lens
cells, SU5402 was tested on a rabbit cell line NN1003A where it was
found to arrest basic FGF-induced proliferation and migration, but did
not inhibit similar responses to epidermal growth factor (EGF) or
hepatocyte growth factor (HGF). The latter two growth factors also use
tyrosine kinase receptors (data not shown).
All experiments involving SU5402 were performed on pairs of donor lenses, and a match-paired format was adopted. The control capsular bag was cultured in protein-free EMEM, whereas the experimental counterpart was incubated in the same medium containing 10 µM SU5402 for a 28-day duration.
Statistical Analysis
A t-test analysis was performed by computer (Excel;
Microsoft, Redmond, WA) to determine any statistical difference between
groups. When capsular bags of different origin and time points were
compared, a two-tailed t-test assuming equal variance was
used. In the case of FGF receptor inhibition experiments, in which both
lenses from a single donor were used, a match-paired t-test
was applied. The data are presented as mean ± SD, and the numbers
of individual preparations used are given in each case. A 95%
confidence interval was used to assess significance.
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Results |
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-crystallin was expressed in the
cytoplasm of the majority of cells on the posterior capsule, but was
not found in all cells (Fig. 2D)
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De Novo Protein Synthesis
To assess the general viability of the capsular bag system, the
capacity for de novo synthesis was investigated by quantifying
35S-methionine incorporation over the final 2 days of culture. The
results for capsular bags grown for 28 days and more than 100 days and
ex vivo preparations are presented in Table 1
. These data show that all preparations were capable of
synthesizing proteins.
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Discussion |
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-crystallin, both
markers of native lens epithelial cells. Furthermore, the cells on the
anterior capsule during this time maintained a homogeneous monolayer
formation, similar to that of native cells. It would therefore appear
that lens cells are capable not only of maintaining their phenotype in
the absence of external factors but that they actively proliferate
during this time. Most cell types require the presence of other cells
for their survival in culture,30
but
chondrocytes31
and lens cells10
32
have been
shown to be notable exceptions. Chondrocytes, although having the
ability to survive in protein-free medium, do not actively proliferate.
Lens cells, on the other hand, show evidence of cell division, provided
they are maintained on their natural matrix.10
This
intrinsic ability of lens epithelial cells to maintain cellular
activity suggests that the cells themselves contribute greatly to PCO,
not only in the initial phases after operation, but also in later
stages when modification to the capsular bag organization contributes
to light scatter.2 It is important that the in vitro model replicate the pattern of in vivo growth as closely as possible. In this study, similar morphologies were observed with long-term capsular bags and with ex vivo PCO cultures. In both preparations, wrinkling of the capsule was evident, and this was associated with cell accumulation. Furthermore, in both cases regression of cells from regions of the posterior capsule had taken place. It was also observed that cells residing on the anterior capsule possessed a regular cobblestone appearance. It is important to note that ex vivo cultures also performed protein synthesis, verifying that they still possessed viable and metabolically active cells. These comparative observations provide further evidence to validate the appropriateness of the human capsular bag culture system for the study of PCO.
The evidence provided in this article clearly shows the persistent nature of the growth of lens epithelial cells. We further demonstrated that a known modulator of lens growth,13 14 15 basic FGF, was not only detectable in capsular bags throughout culture in protein-free medium, but by using RT-PCR we also showed that the cells within the capsular bag retained the ability to synthesize bFGF. RT-PCR analysis also shows that FGFR-1 can be actively synthesized by cells when cultured under protein-free conditions. In addition, basic FGF and corresponding mRNA for basic FGF and FGFR-1 were detected in ex vivo preparations. These results show that the necessary components of an autocrine signaling system are in place and can be actively maintained for prolonged culture periods in protein-free medium. Furthermore, the ability of cells within ex vivo specimens to synthesize basic FGF and FGFR-1 emphasizes the persistent nature of lens epithelial cells within the capsular bag.
The most direct way to study the functional role of a particular signaling system is to disrupt the pathway involved. This can be achieved either by chelating the agonist15 or disabling the receptor.23 In the present study we used the latter approach, which involved the addition of SU5402, a small synthetic compound that has been shown to exhibit specific inhibitory properties of the catalytic function of FGFR-1.28 The mechanism for this inhibition derives from the ability of this compound to penetrate cells and intercalate into the adenine-binding pocket of the catalytic core of the enzyme. The reagent is useful for our studies, because this specific inhibitory property does not affect extracellular ligand interactions or intracellular protein stoichiometry. The results obtained by this inhibition clearly demonstrate that the FGF activation of FGFR-1 was strongly involved in lens cell growth. It is significant that neither the overall protein synthesis rates nor internal levels of bFGF were obliterated (Table 1) . However, the secretion of bFGF into the medium was reduced by FGFR-1 blockade, at least at early time points. Therefore, these data indicate that the primary effect of FGFR-1 blockade was on the dynamic, actively growing cells, rather than on the static anterior epithelial cells.
In summary, we provide evidence of an autocrine system responsible for the long-term survival and growth of human epithelial cells independent of external stimuli. The FGF signaling system appears to play an important role in regulating cell growth in human lens cells, and therefore in PCO. Moreover, these data suggest that pharmacologic intervention with a synthetic agent may produce a therapeutic benefit in this disease.
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Acknowledgements |
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Footnotes |
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Submitted for publication October 31, 2000; accepted January 26, 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: I. Michael Wormstone, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. i.m.wormstone{at}uea.ac.uk
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).
(D) The region in focus lies on the posterior capsule
beneath the plastic IOL that was inserted at the time of surgery. Cell
aggregations (
). None of
the treated bags reached total confluence at any time
(n = 5, in each case). (C) Phase micrograph
of a control capsular bag cultured for 5 days. The anterior capsule
(AC) has a homogeneous appearance, and cells can be seen progressing
toward the center of the posterior capsule (PC). The cells have a
dynamic appearance at the leading edge. (D) Phase micrograph
of a SU5402-treated capsular bag cultured for 14 days. The cells did
not progress far across the posterior capsule in this time, and cells
at the leading edge lost their dynamic appearance, with some becoming
heavily vacuolated (arrows). (E) Dark-field
low-power micrograph of a control capsular bag cultured for 28 days.
The PC has a largely homogenous appearance, because it is completely
covered with cells. The light scatter in the center is due to the
presence of cell-induced wrinkles. (F) Capsular bag paired
to that shown in (E) but exposed to SU5402 for the 28-day
duration. The central region of the PC was devoid of cells, but some
wrinkling occurred in areas where cells were present. Scale bars,
(C, D) 200 µm; (E, F) 500
µm.