May 2001
Volume 42, Issue 6
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Lens  |   May 2001
FGF: An Autocrine Regulator of Human Lens Cell Growth Independent of Added Stimuli
Author Affiliations
  • I. Michael Wormstone
    From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; the
  • Katia Del Rio–Tsonis
    Department of Zoology, Miami University, Oxford, Ohio;
  • Gerald McMahon
    Sugen, Inc., South San Francisco, California; and the
  • Shigeo Tamiya
    From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; the
  • Peter D. Davies
    From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; the
    Department of Ophthalmology, West Norwich Hospital, United Kingdom.
  • Julia M. Marcantonio
    From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; the
  • George Duncan
    From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; the
Investigative Ophthalmology & Visual Science May 2001, Vol.42, 1305-1311. doi:https://doi.org/
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      I. Michael Wormstone, Katia Del Rio–Tsonis, Gerald McMahon, Shigeo Tamiya, Peter D. Davies, Julia M. Marcantonio, George Duncan; FGF: An Autocrine Regulator of Human Lens Cell Growth Independent of Added Stimuli. Invest. Ophthalmol. Vis. Sci. 2001;42(6):1305-1311. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Posterior capsule opacification (PCO) arises because of a persistent growth of lens epithelial cells. Cultured human lens cells residing on their native collagen capsule and maintained in serum-free medium actively grow and thus show an intrinsic capacity for regulation. In the present study, the authors investigated the role of the putative FGF autocrine system in human capsular bags.

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.

Cataract is responsible for rendering millions of people blind throughout the world. 1 At present the only means of treatment is surgical intervention, and this initially restores high-quality vision. Unfortunately, within 2 years between 20% and 30% of patients experience a secondary loss of vision that requires further corrective surgery. 2 3 Modern cataract operations give rise to a lens capsular bag, wherein a proportion of the anterior and the entire posterior capsule remain in situ. Despite the various surgical manipulations, a significant proportion of lens epithelial cells remain on the anterior capsule. These cells subsequently grow across all available lenticular surfaces including, of particular importance, the previously cell-free posterior capsule surface. In doing so, the cells encroach on the visual axis, and the ensuing cellular and capsular changes that result from this growth induce light scatter, thus diminishing visual quality. This problem is commonly termed posterior capsule opacification (PCO), and its conventional treatment is to ablate the offending cells and posterior capsule using a Nd:YAG laser. 2 This surgical procedure is the second most common operation in the United States, with only cataract surgery itself being more frequent. 
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 lenses 16 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  
Methods
In Vitro Capsular Bag Model
The model previously described by Liu et al. 9 was used. After removal of corneoscleral discs for transplantation purposes, human donor eyes, or isolated lenses, obtained from the East Anglian or Bristol Eye Banks were used to perform a sham cataract operation. The resultant capsular bag was then dissected from the zonules and secured on a sterile 35-mm phenyl methylmethacrylate (PMMA) petri dish or a 24-well tissue culture plate. Eight entomologic pins (D1; Watkins and Doncaster, Kent, UK) were inserted through the edge of the capsule to retain its circular shape. At this point, approximately 50% of the remaining anterior capsule was covered with cells. Capsular bags were maintained in nonsupplemented Eagle’s minimum essential medium (EMEM). Incubation was at 35°C in a 5% CO2 atmosphere. The medium was sampled and replaced every 2 to 4 days. Ongoing observations were performed using phase-contrast microscopy. 
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 α-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 α-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. 
Results
Capsular Bag Preparations
Human lens cells can be successfully cultured for longer than 1 year in a protein-free medium, provided they remain on their natural substratum. The capsular bag illustrated in Figure 1A had been cultured for 137 days, and both the anterior and posterior capsular surfaces were covered with cells. In most cases, the anterior capsule was entirely covered with a monolayer of cells of typical cobblestone appearance (Fig. 1B) . However, in the case of cells growing on the posterior capsule, particularly in the center, regression occurred with time in some regions. Such regression took place between wrinkles in the areas highlighted in Figure 1C . It should be noted that although some regression occurred, most of the posterior capsule remained covered with cells. It is this area, lying on the visual axis, that influences visual quality. 
Detailed immunocytochemical evaluation of cytoskeletal elements and nuclear organization were performed on cells residing on the central posterior capsule. 30 Examination of the nuclei using DAPI indicated that the majority of cells possessed normal nuclei (Fig. 2A ). Immunofluorescent staining for vimentin, the intermediate filament protein expressed in lens epithelial cells, showed that this important protein was conserved in all cells on both the anterior and posterior capsules throughout culture (Fig. 2B) . All cells retained vimentin, which forms a filamentous network throughout the cytoplasm, and also F-actin, which is associated with submembranous regions (Figs. 2B 2C) . Moreover, the lens-specific protein α-crystallin was expressed in the cytoplasm of the majority of cells on the posterior capsule, but was not found in all cells (Fig. 2D)
Ex Vivo Preparations
On some occasions donor eyes were received that had previously undergone a cataract operation. The intact capsular bags were dissected from the eye by cutting the suspensory ligaments, and these preparations were cultured for a short period in protein-free medium so that protein synthesis studies could be performed. There were no apparent morphologic changes in the bags during this time. In all cases, ex vivo preparations contained an IOL, which would have been inserted at surgery some time before. It is clearly apparent from Figure 1D that there was considerable light scatter within the visual axis, underlying the IOL. The major sources of this light scatter were wrinkles and cell aggregations (Fig. 1D) . It is also notable that the posterior capsule possessed sites where regression had occurred. Closer inspection of the anterior capsule, however, revealed that a cell monolayer remained that also had a cobblestone-like appearance (Figs. 1E 1F) . This observation was further ratified by fluorescent cytochemical data that showed a regular distribution of a single layer of normal ovoid nuclei (Fig. 2F)
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. 
FGF and FGFR-1 Detection
FGF was detected using ELISA techniques in the culture medium of capsular bags in early stages of culture, but was below the detection limit in long-term cultures (Fig. 4A) . Data obtained from tissue homogenates are presented in Table 1 . Basic FGF was detected in capsular bags at all stages of culture. Furthermore, RT-PCR analysis showed that message for basic FGF was also present in the native lens epithelium, cultured capsular bags, and donor material that had previously undergone cataract surgery (Fig. 3A ). Furthermore, in all these preparations the message for FGFR-1 was present (Fig. 3B)
Effect of FGFR Inhibition
Inhibition of FGFR-1 by 10 μM SU5402 showed a marked attenuation of growth (Fig. 4B ). Of the five pairs of capsular bags used, retardation of growth was always seen in the treated preparation. The times taken to cover 50% of the posterior capsule within the region exposed by anterior capsule removal were 5.2 and 9.7 days for control and treated bags, respectively. Although cell growth was observed on the posterior capsule, with constant exposure to the inhibitor, the cells appeared to lose the dynamic profile typically associated with motility (Fig. 4C) . Instead, the growing edge became smooth, and in some cases vacuoles were observed (Fig. 4D) . No treated bags ever reached confluence, whereas their respective protein-free control cultures always did (Figs. 4B 4E 4F) . It is interesting to note that the level of bFGF secreted into the medium was significantly lower in the treated bags (Fig. 4A) . The cobblestone appearance of the anterior epithelium of treated bags did not, however, change over the 28-day culture period. Also of note, the treated cells still maintained metabolic activity, and FGF could also be detected (Table 1)
Discussion
The present data show that the cells within capsular bags continued to synthesize proteins after 1 year in culture and that cells on the posterior capsule contained vimentin and α-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 chondrocytes 31 and lens cells 10 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 agonist 15 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. 
 
Figure 1.
 
Dark-field (A, D) and phase-contrast (B, C, E, and F) images of cells within capsular bags cultured for more than 100 days in protein-free medium (A, B, and C) and within a capsular bag removed from a donor eye that had previously undergone a cataract operation (D, E, and F). (A) A modified dark-field micrograph of a capsular bag cultured for 137 days. The circular outline of the anterior capsule (AC) edge (rhexis region) is clearly visible (white arrows). The AC has a consistent, smooth appearance. The homogeneous cell population is visible only with phase optics (B). The cells on the posterior capsule are more apparent, and there are also small cell-free areas (C). Light-scattering wrinkles are also apparent. (B) Phase image of cells that have recolonized the anterior capsule of a capsular bag cultured for 465 days. The cells maintained a regular cobblestone appearance throughout this time. (C) Phase micrograph of cells growing on the central region of the posterior capsule from the donor in (A). The cell distribution is heterogeneous with cells lying along wrinkles, whereas other areas are cell free ( Image not available ). (D) The region in focus lies on the posterior capsule beneath the plastic IOL that was inserted at the time of surgery. Cell aggregations ( Image not available ), cell-free areas (black arrows), and capsular wrinkling (white arrows) are all apparent. (E) In this phase image of the anterior capsule, large areas are out of focus because of the presence of the IOL. However, the regular cobblestone appearance of the anterior cells can be seen in areas within the focal plane (arrows). In addition, there is a build-up of cellular material lying along the IOL support ( Image not available ). (F) A higher power image of (E) that more clearly shows the cobblestone appearance of the epithelial cells on the anterior capsule. Scale bars, (A, D) 500 μm; (B, C, and E) 200 μm; (F) 50 μm.
Figure 1.
 
Dark-field (A, D) and phase-contrast (B, C, E, and F) images of cells within capsular bags cultured for more than 100 days in protein-free medium (A, B, and C) and within a capsular bag removed from a donor eye that had previously undergone a cataract operation (D, E, and F). (A) A modified dark-field micrograph of a capsular bag cultured for 137 days. The circular outline of the anterior capsule (AC) edge (rhexis region) is clearly visible (white arrows). The AC has a consistent, smooth appearance. The homogeneous cell population is visible only with phase optics (B). The cells on the posterior capsule are more apparent, and there are also small cell-free areas (C). Light-scattering wrinkles are also apparent. (B) Phase image of cells that have recolonized the anterior capsule of a capsular bag cultured for 465 days. The cells maintained a regular cobblestone appearance throughout this time. (C) Phase micrograph of cells growing on the central region of the posterior capsule from the donor in (A). The cell distribution is heterogeneous with cells lying along wrinkles, whereas other areas are cell free ( Image not available ). (D) The region in focus lies on the posterior capsule beneath the plastic IOL that was inserted at the time of surgery. Cell aggregations ( Image not available ), cell-free areas (black arrows), and capsular wrinkling (white arrows) are all apparent. (E) In this phase image of the anterior capsule, large areas are out of focus because of the presence of the IOL. However, the regular cobblestone appearance of the anterior cells can be seen in areas within the focal plane (arrows). In addition, there is a build-up of cellular material lying along the IOL support ( Image not available ). (F) A higher power image of (E) that more clearly shows the cobblestone appearance of the epithelial cells on the anterior capsule. Scale bars, (A, D) 500 μm; (B, C, and E) 200 μm; (F) 50 μm.
Figure 2.
 
Immunocytochemical evaluation of cytoskeletal elements and nuclear organization of lens epithelial cells within cultured (A through E) and ex vivo (F) capsular bags. (A) Visualization of nuclear organization using DAPI shows cells residing on the posterior capsule after 117 days of culture. (B) Vimentin distribution in a group of cells on the posterior capsule cultured for 158 days. (C) F-actin distribution of cells growing on the posterior capsule, after 138 days of culture. (D) F-actin (red) and α-crystallin (green) distribution on the posterior capsule of the capsular bag shown in (B). (E) A micrograph showing the nuclear appearance of epithelial cells residing on the anterior capsule of a capsular bag cultured for 117 days. (F) A micrograph showing the nuclear appearance of epithelial cells residing on the anterior capsule of a capsular bag removed from a donor eye that had undergone a cataract operation 6 years earlier. Scale bars, (A, E, and F) 20 μm; (B, D) 33 μm; (C) 50 μm.
Figure 2.
 
Immunocytochemical evaluation of cytoskeletal elements and nuclear organization of lens epithelial cells within cultured (A through E) and ex vivo (F) capsular bags. (A) Visualization of nuclear organization using DAPI shows cells residing on the posterior capsule after 117 days of culture. (B) Vimentin distribution in a group of cells on the posterior capsule cultured for 158 days. (C) F-actin distribution of cells growing on the posterior capsule, after 138 days of culture. (D) F-actin (red) and α-crystallin (green) distribution on the posterior capsule of the capsular bag shown in (B). (E) A micrograph showing the nuclear appearance of epithelial cells residing on the anterior capsule of a capsular bag cultured for 117 days. (F) A micrograph showing the nuclear appearance of epithelial cells residing on the anterior capsule of a capsular bag removed from a donor eye that had undergone a cataract operation 6 years earlier. Scale bars, (A, E, and F) 20 μm; (B, D) 33 μm; (C) 50 μm.
Table 1.
 
Basic FGF Content and Protein Synthesis Rate of Human Lens Capsular Bags
Table 1.
 
Basic FGF Content and Protein Synthesis Rate of Human Lens Capsular Bags
Preparation n 35S-Methionine Incorporation (dpm/day) FGF (pg/bag)
Capsular bag (t = 0) 5 560 ± 72
Capsular bag (t = 28 days) 5 58,599 ± 10,008 427 ± 98
Capsular bag (t = 28 days)-SU5402 treated 5 41,176 ± 3,477 360 ± 49
Capsular bag (t > 100 days) 5 43,164 ± 5,295 336 ± 65
Ex vivo capsular bag 4 30,401 ± 12,642 175 ± 88
Figure 3.
 
Expression of basic FGF and its receptor FGFR-1 using RT-PCR. Lanes 1 and 2: human native lens epithelium; lanes 3 and 4: human capsular bag cultured for 8 days; and lanes 5 and 6: donor lens that had undergone cataract surgery. Lanes 2, 4, and 6: RT-negative control samples. Lane M, base pair size markers. (A) Expression patterns of basic FGF (349 bp); (B) expression patterns of FGFR-1 (433 bp). The data presented represent the expression pattern observed in three separate specimens of each preparation studied.
Figure 3.
 
Expression of basic FGF and its receptor FGFR-1 using RT-PCR. Lanes 1 and 2: human native lens epithelium; lanes 3 and 4: human capsular bag cultured for 8 days; and lanes 5 and 6: donor lens that had undergone cataract surgery. Lanes 2, 4, and 6: RT-negative control samples. Lane M, base pair size markers. (A) Expression patterns of basic FGF (349 bp); (B) expression patterns of FGFR-1 (433 bp). The data presented represent the expression pattern observed in three separate specimens of each preparation studied.
Figure 4.
 
Influence of FGFR-1 inhibition on human lens cells in a protein-free medium. (A) ELISA detection of basic FGF in medium from cultured capsular bags. The medium was sampled and replaced at 2-day intervals (n = 5 in each case). Donor pairs were used for both control (black bars) and treated (white bars) groups. (B) Cell cover of the previously cell-free posterior capsule of capsular bags cultured in control protein-free medium (•) and exposed to 10 μM SU5402 (○). 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) 500μ m.
Figure 4.
 
Influence of FGFR-1 inhibition on human lens cells in a protein-free medium. (A) ELISA detection of basic FGF in medium from cultured capsular bags. The medium was sampled and replaced at 2-day intervals (n = 5 in each case). Donor pairs were used for both control (black bars) and treated (white bars) groups. (B) Cell cover of the previously cell-free posterior capsule of capsular bags cultured in control protein-free medium (•) and exposed to 10 μM SU5402 (○). 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) 500μ m.
The authors thank Pamela Keeley of the East Anglian Eye Bank, the staff members of the Bristol Eye Bank, and Diane Alden for technical assistance. 
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Figure 1.
 
Dark-field (A, D) and phase-contrast (B, C, E, and F) images of cells within capsular bags cultured for more than 100 days in protein-free medium (A, B, and C) and within a capsular bag removed from a donor eye that had previously undergone a cataract operation (D, E, and F). (A) A modified dark-field micrograph of a capsular bag cultured for 137 days. The circular outline of the anterior capsule (AC) edge (rhexis region) is clearly visible (white arrows). The AC has a consistent, smooth appearance. The homogeneous cell population is visible only with phase optics (B). The cells on the posterior capsule are more apparent, and there are also small cell-free areas (C). Light-scattering wrinkles are also apparent. (B) Phase image of cells that have recolonized the anterior capsule of a capsular bag cultured for 465 days. The cells maintained a regular cobblestone appearance throughout this time. (C) Phase micrograph of cells growing on the central region of the posterior capsule from the donor in (A). The cell distribution is heterogeneous with cells lying along wrinkles, whereas other areas are cell free ( Image not available ). (D) The region in focus lies on the posterior capsule beneath the plastic IOL that was inserted at the time of surgery. Cell aggregations ( Image not available ), cell-free areas (black arrows), and capsular wrinkling (white arrows) are all apparent. (E) In this phase image of the anterior capsule, large areas are out of focus because of the presence of the IOL. However, the regular cobblestone appearance of the anterior cells can be seen in areas within the focal plane (arrows). In addition, there is a build-up of cellular material lying along the IOL support ( Image not available ). (F) A higher power image of (E) that more clearly shows the cobblestone appearance of the epithelial cells on the anterior capsule. Scale bars, (A, D) 500 μm; (B, C, and E) 200 μm; (F) 50 μm.
Figure 1.
 
Dark-field (A, D) and phase-contrast (B, C, E, and F) images of cells within capsular bags cultured for more than 100 days in protein-free medium (A, B, and C) and within a capsular bag removed from a donor eye that had previously undergone a cataract operation (D, E, and F). (A) A modified dark-field micrograph of a capsular bag cultured for 137 days. The circular outline of the anterior capsule (AC) edge (rhexis region) is clearly visible (white arrows). The AC has a consistent, smooth appearance. The homogeneous cell population is visible only with phase optics (B). The cells on the posterior capsule are more apparent, and there are also small cell-free areas (C). Light-scattering wrinkles are also apparent. (B) Phase image of cells that have recolonized the anterior capsule of a capsular bag cultured for 465 days. The cells maintained a regular cobblestone appearance throughout this time. (C) Phase micrograph of cells growing on the central region of the posterior capsule from the donor in (A). The cell distribution is heterogeneous with cells lying along wrinkles, whereas other areas are cell free ( Image not available ). (D) The region in focus lies on the posterior capsule beneath the plastic IOL that was inserted at the time of surgery. Cell aggregations ( Image not available ), cell-free areas (black arrows), and capsular wrinkling (white arrows) are all apparent. (E) In this phase image of the anterior capsule, large areas are out of focus because of the presence of the IOL. However, the regular cobblestone appearance of the anterior cells can be seen in areas within the focal plane (arrows). In addition, there is a build-up of cellular material lying along the IOL support ( Image not available ). (F) A higher power image of (E) that more clearly shows the cobblestone appearance of the epithelial cells on the anterior capsule. Scale bars, (A, D) 500 μm; (B, C, and E) 200 μm; (F) 50 μm.
Figure 2.
 
Immunocytochemical evaluation of cytoskeletal elements and nuclear organization of lens epithelial cells within cultured (A through E) and ex vivo (F) capsular bags. (A) Visualization of nuclear organization using DAPI shows cells residing on the posterior capsule after 117 days of culture. (B) Vimentin distribution in a group of cells on the posterior capsule cultured for 158 days. (C) F-actin distribution of cells growing on the posterior capsule, after 138 days of culture. (D) F-actin (red) and α-crystallin (green) distribution on the posterior capsule of the capsular bag shown in (B). (E) A micrograph showing the nuclear appearance of epithelial cells residing on the anterior capsule of a capsular bag cultured for 117 days. (F) A micrograph showing the nuclear appearance of epithelial cells residing on the anterior capsule of a capsular bag removed from a donor eye that had undergone a cataract operation 6 years earlier. Scale bars, (A, E, and F) 20 μm; (B, D) 33 μm; (C) 50 μm.
Figure 2.
 
Immunocytochemical evaluation of cytoskeletal elements and nuclear organization of lens epithelial cells within cultured (A through E) and ex vivo (F) capsular bags. (A) Visualization of nuclear organization using DAPI shows cells residing on the posterior capsule after 117 days of culture. (B) Vimentin distribution in a group of cells on the posterior capsule cultured for 158 days. (C) F-actin distribution of cells growing on the posterior capsule, after 138 days of culture. (D) F-actin (red) and α-crystallin (green) distribution on the posterior capsule of the capsular bag shown in (B). (E) A micrograph showing the nuclear appearance of epithelial cells residing on the anterior capsule of a capsular bag cultured for 117 days. (F) A micrograph showing the nuclear appearance of epithelial cells residing on the anterior capsule of a capsular bag removed from a donor eye that had undergone a cataract operation 6 years earlier. Scale bars, (A, E, and F) 20 μm; (B, D) 33 μm; (C) 50 μm.
Figure 3.
 
Expression of basic FGF and its receptor FGFR-1 using RT-PCR. Lanes 1 and 2: human native lens epithelium; lanes 3 and 4: human capsular bag cultured for 8 days; and lanes 5 and 6: donor lens that had undergone cataract surgery. Lanes 2, 4, and 6: RT-negative control samples. Lane M, base pair size markers. (A) Expression patterns of basic FGF (349 bp); (B) expression patterns of FGFR-1 (433 bp). The data presented represent the expression pattern observed in three separate specimens of each preparation studied.
Figure 3.
 
Expression of basic FGF and its receptor FGFR-1 using RT-PCR. Lanes 1 and 2: human native lens epithelium; lanes 3 and 4: human capsular bag cultured for 8 days; and lanes 5 and 6: donor lens that had undergone cataract surgery. Lanes 2, 4, and 6: RT-negative control samples. Lane M, base pair size markers. (A) Expression patterns of basic FGF (349 bp); (B) expression patterns of FGFR-1 (433 bp). The data presented represent the expression pattern observed in three separate specimens of each preparation studied.
Figure 4.
 
Influence of FGFR-1 inhibition on human lens cells in a protein-free medium. (A) ELISA detection of basic FGF in medium from cultured capsular bags. The medium was sampled and replaced at 2-day intervals (n = 5 in each case). Donor pairs were used for both control (black bars) and treated (white bars) groups. (B) Cell cover of the previously cell-free posterior capsule of capsular bags cultured in control protein-free medium (•) and exposed to 10 μM SU5402 (○). 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) 500μ m.
Figure 4.
 
Influence of FGFR-1 inhibition on human lens cells in a protein-free medium. (A) ELISA detection of basic FGF in medium from cultured capsular bags. The medium was sampled and replaced at 2-day intervals (n = 5 in each case). Donor pairs were used for both control (black bars) and treated (white bars) groups. (B) Cell cover of the previously cell-free posterior capsule of capsular bags cultured in control protein-free medium (•) and exposed to 10 μM SU5402 (○). 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) 500μ m.
Table 1.
 
Basic FGF Content and Protein Synthesis Rate of Human Lens Capsular Bags
Table 1.
 
Basic FGF Content and Protein Synthesis Rate of Human Lens Capsular Bags
Preparation n 35S-Methionine Incorporation (dpm/day) FGF (pg/bag)
Capsular bag (t = 0) 5 560 ± 72
Capsular bag (t = 28 days) 5 58,599 ± 10,008 427 ± 98
Capsular bag (t = 28 days)-SU5402 treated 5 41,176 ± 3,477 360 ± 49
Capsular bag (t > 100 days) 5 43,164 ± 5,295 336 ± 65
Ex vivo capsular bag 4 30,401 ± 12,642 175 ± 88
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