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Lens  |   January 2013
Age-Related Differences in Signaling Efficiency of Human Lens Cells Underpin Differential Wound Healing Response Rates following Cataract Surgery
Author Affiliations & Notes
  • Lucy Jean Dawes
    From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; and The Save Sight Institute, University of Sydney, Sydney, Australia.
  • George Duncan
    From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; and The Save Sight Institute, University of Sydney, Sydney, Australia.
  • Ian Michael Wormstone
    From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; and The Save Sight Institute, University of Sydney, Sydney, Australia.
  • Footnotes
    3  Deceased January 17, 2007.
  • Corresponding author: Ian Michael Wormstone, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK NR4 7TJ; i.m.wormstone@uea.ac.uk
Investigative Ophthalmology & Visual Science January 2013, Vol.54, 333-342. doi:10.1167/iovs.12-10425
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      Lucy Jean Dawes, George Duncan, Ian Michael Wormstone; Age-Related Differences in Signaling Efficiency of Human Lens Cells Underpin Differential Wound Healing Response Rates following Cataract Surgery. Invest. Ophthalmol. Vis. Sci. 2013;54(1):333-342. doi: 10.1167/iovs.12-10425.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: Cataract surgery is blighted by posterior capsule opacification (PCO), which is more severe and frequent in the young than the elderly (>60 years). Our aim was to understand the biological basis for these age-related differences in PCO/wound healing rates.

Methods.: Human capsular bags were prepared by cataract surgery on donor lenses (young [<40 years] and elderly [>60 years] groups) and maintained in serum-free Eagle's minimum essential medium. Cell growth was determined using the MTS assay. Fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) levels were determined using ELISA. Protein synthesis rates were elucidated by 35S-methionine incorporation. U0126, SB203580, and SP600125 were used to disrupt ERK-, p38-, and JNK-mediated signaling, respectively. Level of total and phospho-ERK, -c-jun, -P38, and -JNK plus cytokines were detected using a BIOPLEX array system.

Results.: Following a 2-day culture period, significant decreases in IL-1β and IL-6, and increases in IL-10, IL-12, IL-13, and VEGF in the >60 years group were observed compared with their younger counterparts. Capsular bags (cells and capsule) from aged donors contained greater than or equal levels of HGF and FGF than younger counterparts and had greater rates of protein synthesis. Inhibition of ERK, p38, and JNK signaling significantly suppressed cell coverage on the posterior capsule. pERK, p-c-jun, p-p38, and pJNK were consistently lower in aged cell populations; total signaling protein expression was unaffected by age. Serum stimulation increased pERK, p-c-jun, and pJNK levels in cells of all ages; p-p38 was significantly increased in the >60 years group only.

Conclusions.: Ligand availability to cells is not a limiting factor as we age, but the ability to convert this resource into signaling activity is. We therefore propose that overall signaling efficiency is reduced as a function of age, which consequently limits wound-healing response rates after injury.

Introduction
Prevalence of cataracts increases dramatically as we age. 1 Currently, the only means to treat all forms of cataract is surgical intervention. 2 This procedure involves removal of a segment of the anterior capsule and extraction of the fibers. This produces a lens capsular bag comprising the entire cell-free posterior capsule and remaining anterior capsule with lens epithelial cells attached. This bag can house an artificial lens implant. Following the mechanical insult of surgery, cells grow and ultimately encroach on the visual axis. Light scattering changes are induced by the cells, giving rise to secondary visual loss termed posterior capsule opacification (PCO), which is also known as secondary cataract or after-cataract. 2 The rate of these changes is much greater in young patients (i.e., <40 years of age). 3,4 Therefore, we endeavored to understand the biological basis of this age-related difference in wound-healing response. 
To conduct the study, a human in vitro capsular bag model was employed, 5,6 which is based on a cataract operation and provides the same spatial organization as observed in vivo. This system also has the advantage of investigating a single human cell type and that the data can be related to physiological events in patients following cataract surgery. This capsular bag system not only serves as an excellent model to study PCO, but also provides a tool to investigate age-related wound healing. Using this in vitro experimental system, it has been shown that lens cells growing on capsular bags maintained in serum-free medium from a wide-age spectrum can effectively colonize the entire surface of the once cell-free posterior capsule. However, significant differences were observed, such that rate of growth of cells from the <40 years donors was greater than the >60 years donor counterparts (Fig. 1). 6 These findings also reflect the incidence of secondary blindness (PCO) following cataract surgery that is much greater in the young (i.e., <40 years). 3 However, stimulation with 10% fetal calf serum (FCS) can dramatically increase the rate of growth observed by the cells of aged capsular bags, but this supplement did not greatly enhance the growth rates of young donors (<40 years; Fig. 1). 6 Therefore, these data suggest that the capacity to divide is not lost with age, but the mechanisms driving the process are not driven maximally in serum-free (nonstimulated) conditions. One putative explanation for these age-related differences is replicative senescence and this notion is supported by work in rodents that demonstrate reduced proliferation with age. 7 The senescence model of aging commonly implicates shortening of the telomeres, which serve as a biological clock until replicative senescence results. 811 Telomere shortening can be countered by telomerase, 811 which is present in the lens epithelium. 12 However, it should be noted that telomere-independent senescence can also occur. 1315 Growth rate in aged lens cells on the capsular bag can be driven by serum addition to levels similar to the young. This would suggest that cell senescence, while likely to contribute, does not alone account for the observed differences in lens cell growth following surgery. Therefore, in the present study, we aimed to investigate differences in cell signaling efficiency during wound healing as a function of age. 
Figure 1. 
 
The effects of age and serum on time to confluency of cells on the posterior capsule. Cell growth was observed using phase-contrast microscopy. Confluency was taken as the point when 100% of the area on the posterior capsule, within the confines of the capsulorhexis, was covered by epithelial cells. Serum data was taken from Liu et al. 5 The numbers of different capsules used to obtain the data are given on each bar of the graph. Reprinted with permission from Wormstone IM, Liu CS, Rakic JM, Marcantonio JM, Vrensen GF, Duncan G. Human lens epithelial cell proliferation in a protein-free medium. Invest Ophthalmol Vis Sci. 1997;38:396–404. Copyright 1997 Association for Research in Vision and Ophthalmology.
Figure 1. 
 
The effects of age and serum on time to confluency of cells on the posterior capsule. Cell growth was observed using phase-contrast microscopy. Confluency was taken as the point when 100% of the area on the posterior capsule, within the confines of the capsulorhexis, was covered by epithelial cells. Serum data was taken from Liu et al. 5 The numbers of different capsules used to obtain the data are given on each bar of the graph. Reprinted with permission from Wormstone IM, Liu CS, Rakic JM, Marcantonio JM, Vrensen GF, Duncan G. Human lens epithelial cell proliferation in a protein-free medium. Invest Ophthalmol Vis Sci. 1997;38:396–404. Copyright 1997 Association for Research in Vision and Ophthalmology.
Materials and Methods
Ethics Statement
All human donor eye tissue employed for this study was obtained from the East Anglian Eye Bank with written informed consent from next of kin. The research was approved by the UK National Research Ethics Committee (REC 04/Q0102/57) and followed the tenets of the Declaration of Helsinki regarding the use of human material. 
Capsular Bag Preparation
As previously described, a modern cataract surgery involves generation of a lens capsular bag, and it is this procedure that has been transferred from the operating theater to the laboratory. The model previously described by Liu et al. 5 was employed such that cataract surgery was performed in a laminar flow hood on donor eyes. The procedure involves washing the lens briefly with Eagle's minimum essential medium (EMEM) before creating a small rhexis in the anterior surface of the lens capsule and removing the central fibrous mass from donor globes. The product of this operation is a capsular bag. The capsular bag can then be removed from the eye following careful separation of the zonules and transferred to a tissue culture dish. The bag was subsequently secured to the dish using entomological pins (via penetration of the anterior and posterior capsule) and maintained in EMEM supplemented with 50 μg/mL gentamicin (Sigma-Aldrich, Poole, Dorset, UK) until appropriate end points. Coverage of the previously cell-free posterior capsule was captured using low-power phase microscopy. End point analysis of cell coverage on the previously cell-free posterior capsule was assessed with Java-based image analysis software (ImageJ; available in the public domain by National Institutes of Health, Bethesda, MD, http://rsbweb.nih.gov/ij/. The area of the posterior capsule within the capsulorhexis margin was determined and cell growth on this region was calculated as percent of coverage. 
Suspended Bead Array Analysis
Cytokines.
Culture medium sampled at day 2 was analyzed for levels of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1ra VEGF, G-CSF, basic fibroblast growth factor (FGF), MIP1α, MCP-1, IP-10, RANTES, GM-CSF, IFNγ, TNFα, MIP1β, eotaxin, and PDGF-BB using a commercially available 27-plex cytokine panel (Bio-Plex Suspended Multiplex Bead Array Assay kit; Bio-Rad, Hemmel Hempstead, UK) in accordance with manufacturer's instructions. Data from the reactions were acquired with a flow cytometry system (X Map-100; Luminex, Austin, TX) and accompanying software (Bio-Plex Manager software; Bio-Rad). The median fluorescence intensity was used as a measure of detection of protein. 
Signaling Proteins.
The cells within capsular bag preparations were lysed (Bio-Plex Cell Lysis Kit; Bio-Rad), according to the manufacturer's instructions. Lysates were precleared by centrifuging at 4500 rpm at 4°C for 20 minutes. Equal amounts of protein per sample were analyzed for phosphorylated and total levels of ERK1/2, P38, and JNK using a commercially available kit (Bio-Rad). Data from the reaction were acquired with a flow cytometry system (Luminex) and accompanying software (Bio-Rad). The median fluorescence intensity was used as a measure of detection of total and phosphorylated protein. Results are presented as the level of phosphorylated protein normalized to its corresponding total protein level. 
Cell Growth Assay
To test the effects of conditioned medium on lens cell growth a human lens epithelial cell line, FHL124, was employed. A total of 5000 cells were placed in each well and allowed to grow for 72 hours before a 24-hour serum-starvation period. At this point, cells were placed in experimental conditions and maintained for an additional 48 hours. FHL124 cell number was measured using a cell proliferation assay (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay; Promega, Fitchburg, WI). This is a colorimetric method for determining the number of viable cells based on the reduction of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. The plate was read at 490 nm using a plate reader (FLUOstar Omega; BMG LABTECH, Cary, NC). 
HGF, Basic FGF, and TGFβ2 Estimation Using ELISA
Capsular bags were placed in 0.5 mL of homogenizing medium (6 mM phosphate, 100 mM KCl, 225 mM NaCl, 1 mM EGTA, 1 mM EDTA, 10 mM mercaptoethanol, 10 mM N-ethylmaleimide, 50 μM phenylmethylsulfonyl fluoride [PMSF], and 5 μM E-64 and 1% v/v Tween 20 at pH 7.4). One hundred microliters of the homogenates were analyzed for human hepatocyte growth factor (HGF), human basic FGF (bFGF), or human active TGFβ2 using commercially available ELISA kits (R&D Systems, Minneapolis, MN). The optical density of each well was determined using a multiwell plate reader (Victor; EG&G Wallac, Cambridge, UK) 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 giving the estimated level of growth factor in picograms per milliliter. The limit of sensitivity of the system is 40 pg/mL for HGF, 3 pg/mL for basic bFGF, and 7 pg/mL in the case of TGFβ2; any sample below these limits was treated as being zero. Values were then used to determine total HGF or bFGF and TGFβ2 per bag. 
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 an additional two times. At the end of the wash period, the capsular bag was placed in 0.5 mL of homogenizing medium (6 mM phosphate; 100 mM KCl; 225 mM NaCl; 1 mM EGTA; 1 mM EDTA; 10 mM mercaptoethanol; 10 mM N-ethylmaleimide; 50 μM PMSF; and 5 μM E-64 and 1% v/v Tween 20 at 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 (1.5-mL Eppendorf tube; Eppendorf, Fremont, CA) and 950 μL of 5% trichloroacetic acid added. This was left for 30 minutes at 4°C, and 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 (PerkinElmer Life Sciences, Gaithersburg, MD) with appropriate backgrounds. Counts were corrected for decay. 
Statistical Analysis
A t-test analysis was performed using spreadsheet software (Excel; Microsoft, Redmond, WA) to determine any statistical difference between two groups. One-way ANOVA with Dunnett's post hoc analysis and two-way ANOVA were employed to assess multiple groups (SPSS 18; IBM, Armonk, NY). A 95% confidence interval was used to assess significance. 
Results
Cytokine Levels in the Culture Medium
Initially, we measured the level of cytokines in the culture medium of capsular bags (from donors aged 3–86 years) using a commercially available suspension array system (Bio-Plex System; Bio-Rad). Using this method, a significant decrease in IL-1β and IL-6 with age was detected (Fig. 2). However, a significant increase with respect to age was observed for IL-10, IL-12, IL-13, and VEGF (Fig. 2). No significant changes in IL-8, G-CSF, bFGF, IL-7, IL-9, MIP1α, MCP-1, IP-10, RANTES, and IL-15 were detected between age groups (Fig. 2). In addition, IL-2, IL-4, IL-5, IL-17, GM-CSF, IFNγ, TNFα, MIP1β, IL-1RA, eotaxin, and PDGF-BB was not detected in the culture medium of either group (Fig. 2). 
Figure 2. 
 
Cytokine levels in the culture medium of capsular bag preparations from young and aged donors. Medium was sampled following a 2-day culture period and analyzed using a suspended bead array multiplex cytokine panel. Four donor lenses for the <40 years group and eight pairs for the >60 years group were analyzed. Data are expressed as mean ± SEM. *Indicates a significant difference between groups (Student's t-test, P ≤ 0.05).
Figure 2. 
 
Cytokine levels in the culture medium of capsular bag preparations from young and aged donors. Medium was sampled following a 2-day culture period and analyzed using a suspended bead array multiplex cytokine panel. Four donor lenses for the <40 years group and eight pairs for the >60 years group were analyzed. Data are expressed as mean ± SEM. *Indicates a significant difference between groups (Student's t-test, P ≤ 0.05).
Effect of Conditioned Medium on Lens Cell Growth
The addition of the day-2 culture medium, from aged donors (>60 years), to human lens cells (FHL124) significantly increased cell growth relative to serum-free EMEM control medium; exposure to medium from young donors did not significantly increase growth (Fig. 3). It should, however, be noted that the degree of stimulation by conditioned medium from old donors relative to 1% serum stimulation is weak (Fig. 3). This, therefore, suggests that the richest source of growth-promoting cytokines is not within the bathing medium, but is in association with the lens capsule. 
Figure 3. 
 
The effect of conditioned medium of capsular bag cultures from young (<40 years) and aged (>60 years) donors on growth of the human lens epithelial cell line FHL124 detected using the MTS assay. The data are expressed as mean ± SEM (n = 4). CM, conditioned medium; SF, serum free. *Represents a significant difference between untreated control and treatment group (P < 0.05; ANOVA with Dunnett's test).
Figure 3. 
 
The effect of conditioned medium of capsular bag cultures from young (<40 years) and aged (>60 years) donors on growth of the human lens epithelial cell line FHL124 detected using the MTS assay. The data are expressed as mean ± SEM (n = 4). CM, conditioned medium; SF, serum free. *Represents a significant difference between untreated control and treatment group (P < 0.05; ANOVA with Dunnett's test).
Growth Factor Levels and Protein Synthesis Rates in the Capsular Bag
The level of growth factors within the capsular bag were assessed using commercial ELISA kits (R&D Systems) following a 2-day culture in serum-free medium (EMEM). At this stage of culture, cells are actively recolonizing the denuded regions of the anterior capsule and begin growing on the previously cell free posterior capsule. The loss of cells due to the mechanical stress of the cataract operation did not appear to show a bias to either age group. Therefore, the starting population of cells observed by coverage of the anterior capsule were comparable. Aged human donors (>60 years) contained significantly greater amounts of bFGF than younger donors (<40 years) with 858 ± 67 pg/mL for the former and 290 ± 71 pg/mL for the latter (Fig. 4A). The level of HGF present in capsular bags from aged human donors was not significantly different from their younger counterparts (Fig. 4B). In contrast, TGFβ2 was more abundant in capsular bags prepared from younger donors than from their older counterparts (Fig. 4C). In addition, detection of 35S-methionine incorporation into newly synthesized proteins revealed significantly greater rates of protein synthesis in lens cells from aged human donors compared with younger donors; following 2 days in serum-free culture, a 1.53-fold increase was observed (Fig. 4D). Therefore, the level of protein production and growth factors, in particular, does not correlate with the reduced growth rates observed in older donors relative to their younger counterparts previously reported. 6  
Figure 4. 
 
Basic fibroblast growth factor (A), hepatocyte growth factor (B), transforming growth factor beta 2 (C), and protein synthesis rates (D) detected in human lens capsular bag cultures from young (<40 years) and aged (>60 years) donors. Levels were obtained using commercially available ELISA kits and 35S-methionine incorporation. Six pairs of donor lenses were analyzed for the <40 years group and seven pairs for the >60 years group. Data are expressed as mean ± SEM. *Indicates a significant difference between groups (Student's t-test, P ≤ 0.05).
Figure 4. 
 
Basic fibroblast growth factor (A), hepatocyte growth factor (B), transforming growth factor beta 2 (C), and protein synthesis rates (D) detected in human lens capsular bag cultures from young (<40 years) and aged (>60 years) donors. Levels were obtained using commercially available ELISA kits and 35S-methionine incorporation. Six pairs of donor lenses were analyzed for the <40 years group and seven pairs for the >60 years group. Data are expressed as mean ± SEM. *Indicates a significant difference between groups (Student's t-test, P ≤ 0.05).
Inhibition of Signaling Pathways
Specific inhibitors U0126, SB203580, and SP600125 were used to disrupt ERK-, p38-, and JNK-mediated signaling, respectively (Fig. 5). In control preparations, cells could be seen growing on the anterior lens capsule and onto the previously cell-free peripheral posterior capsule. Ultimately, the cells grow beyond the capsulorhexis edge on to the central posterior capsule (arrowed edge, Fig. 5A). At end point, a significant proportion of this surface was covered with cells (Figs. 5B–G). Significant inhibition of cell growth onto the central posterior capsule was observed with application of each inhibitor (at 10 μM) relative to their match-paired nontreated (vehicle only) controls (Figs. 5E–G). It should be noted that in the U0126 bags, no cell cover of the posterior capsule was seen (Figs. 5B, 5E); however, cells were observed on the anterior capsule at end point (data not shown). 
Figure 5. 
 
The effects of signaling pathway inhibitors on cell coverage of the human lens capsular bag. (A) A top-view cartoon representation of the capsular bag showing the specific regions presented in (BD). Note that immediately following preparation of the capsular bags, the central posterior capsule (PC) is completely devoid of cells. (BD) Modified dark-field images of capsular bags showing the posterior capsule (central area) and outer anterior capsule captured after 14 days of culture. Experiments were carried out in a match-paired format, such that one bag was maintained in EMEM supplemented with 10 μM U0126, 10 μM SP600125, and 10 μM SB203580 to inhibit ERK-, JNK-, and p38-mediated signaling and the other exposed to vehicle only (control). (EG) Pooled data showing the level of cell coverage across the posterior capsule in the different treatment groups. Data are expressed as the mean ± SEM (n = 3). Donor ages for: U0126 match-paired experiments were 67.7 ± 6.5 (range 58–80); SP600125 match-paired experiments were 68.3 ± 6.7 (range 56–79); SB203580 match-paired experiments were 54.7 ± 6.8 (range 42–65). *Significant difference between treated and control groups (P ≤ 0.05, Student's t-test).
Figure 5. 
 
The effects of signaling pathway inhibitors on cell coverage of the human lens capsular bag. (A) A top-view cartoon representation of the capsular bag showing the specific regions presented in (BD). Note that immediately following preparation of the capsular bags, the central posterior capsule (PC) is completely devoid of cells. (BD) Modified dark-field images of capsular bags showing the posterior capsule (central area) and outer anterior capsule captured after 14 days of culture. Experiments were carried out in a match-paired format, such that one bag was maintained in EMEM supplemented with 10 μM U0126, 10 μM SP600125, and 10 μM SB203580 to inhibit ERK-, JNK-, and p38-mediated signaling and the other exposed to vehicle only (control). (EG) Pooled data showing the level of cell coverage across the posterior capsule in the different treatment groups. Data are expressed as the mean ± SEM (n = 3). Donor ages for: U0126 match-paired experiments were 67.7 ± 6.5 (range 58–80); SP600125 match-paired experiments were 68.3 ± 6.7 (range 56–79); SB203580 match-paired experiments were 54.7 ± 6.8 (range 42–65). *Significant difference between treated and control groups (P ≤ 0.05, Student's t-test).
Signaling Activity
Having confirmed the importance of the selected signaling pathways in cell coverage, through the inhibition studies described above, we have shown a common role for these different signaling pathways that unifies them. We then identified the level of total and phosphorylated (active) forms of signaling molecules in cells of capsular bag cultures (donors aged 3–86 years). To carry out these investigations, a suspended bead array (Bio-Plex System; Bio-Rad) was employed. One factor that could affect signaling activity is the level of total signaling proteins present in the cells. Evaluation of total ERK, p38, c-jun, and JNK (Fig. 6A) did not show any significant difference between age groups for each signaling protein studied (unpaired Student's t-test), nor was an overall effect of age on total signaling proteins observed using a two-way ANOVA approach with age and signaling component as the two factors (F = 0.542, P = 0.466). Lens cells growing on nonstimulated capsular bags generated from younger donors demonstrated enhanced activity of pERK, p-c-jun, p-p38, and p-JNK relative to their older counterparts (Fig. 6B). When separately comparing baseline level of each of the four signaling molecules studied in cells from young and aged capsular bags, no significant difference was determined (unpaired t-test). Although the difference between baseline levels in young and aged cells did not reach significance for any single signaling molecule assessed in isolation, a significant overall decline in signaling with age was observed for the experiment as a whole using two-way ANOVA with age and signaling component as the 2 factors (two-way ANOVA, F = 4.764; P = 0.035). 
Figure 6. 
 
Suspended bead protein array detection of baseline total and phospho-signaling molecules in cells within capsular bag cultures from young and aged donors. Cell lysates were obtained during log phase growth across the previously cell-free posterior capsule in different age groups maintained in nonstimulated (baseline) conditions. Four donor lenses for the <40 years group and eight pairs for the >60 years group were analyzed. (A) Total level of ERK, c-jun, p38 MAPK, and JNK in cells from young and aged donors. (B) Baseline levels of phosphorylated protein level of pERK, p-c-jun, p-p38 MAPK, and pJNK corrected for corresponding total protein level in cells from young and aged donors. Data presented are normalized to the mean <40 years nonstimulated (baseline) value for each signaling component and presented as mean ± SEM.
Figure 6. 
 
Suspended bead protein array detection of baseline total and phospho-signaling molecules in cells within capsular bag cultures from young and aged donors. Cell lysates were obtained during log phase growth across the previously cell-free posterior capsule in different age groups maintained in nonstimulated (baseline) conditions. Four donor lenses for the <40 years group and eight pairs for the >60 years group were analyzed. (A) Total level of ERK, c-jun, p38 MAPK, and JNK in cells from young and aged donors. (B) Baseline levels of phosphorylated protein level of pERK, p-c-jun, p-p38 MAPK, and pJNK corrected for corresponding total protein level in cells from young and aged donors. Data presented are normalized to the mean <40 years nonstimulated (baseline) value for each signaling component and presented as mean ± SEM.
Application of 10% serum to cultures promoted the level of p-ERK, p-JNK, and p-c-jun in cells from both young and aged donors; in the case of the p-p38 group, a significant increase in response to 10% serum was observed in cells from aged donors, but not in cells from <40 years group (P < 0.05, match-paired t-test; Fig. 7). It should also be noted that serum stimulation was found to elevate signaling activity in cells from the >60 years capsular bag group to levels that were equivalent or greater than in the nonstimulated <40 years group (Fig. 7). 
Figure 7. 
 
The effect of serum-stimulation on levels of phosphorylated signaling proteins in cells within capsular bag cultures from young and aged donors detected by suspended bead protein array. Cell lysates were obtained during log phase growth across the previously cell-free posterior capsule in different age groups following stimulation with 10% FCS for 30 minutes or maintained in control (baseline) conditions. These experiments were carried out using a match-paired format; four pairs of donor lenses were analyzed for the <40 years group and eight pairs for the >60 years group. The effect of serum-stimulation on levels of phosphorylated protein level of pERK, p-c-jun, p-p38, and pJNK corrected for corresponding total protein level in cells from young and aged donors. Data presented are normalized to the mean; <40 years nonstimulated (baseline) value for each signaling component and presented as mean ± SEM. *Represents a significant difference between age groups (P < 0.05, Student's t-test).
Figure 7. 
 
The effect of serum-stimulation on levels of phosphorylated signaling proteins in cells within capsular bag cultures from young and aged donors detected by suspended bead protein array. Cell lysates were obtained during log phase growth across the previously cell-free posterior capsule in different age groups following stimulation with 10% FCS for 30 minutes or maintained in control (baseline) conditions. These experiments were carried out using a match-paired format; four pairs of donor lenses were analyzed for the <40 years group and eight pairs for the >60 years group. The effect of serum-stimulation on levels of phosphorylated protein level of pERK, p-c-jun, p-p38, and pJNK corrected for corresponding total protein level in cells from young and aged donors. Data presented are normalized to the mean; <40 years nonstimulated (baseline) value for each signaling component and presented as mean ± SEM. *Represents a significant difference between age groups (P < 0.05, Student's t-test).
Discussion
The current body of data suggests that overall baseline signaling activity declines significantly with age. We would argue that this difference in signaling could account for the age-related differences in growth rate previously reported. 6 In serum-free conditions, activity of signaling components appears higher in the young relative to the old despite equivalent or greater levels of ligand availability. Total signaling protein expression is unaffected by age. Following serum stimulation, it is not the peak response per se that is critical, but whether or not serum stimulation can drive the level of signaling activity above that seen in young unstimulated cells. This provides a basis for serum-induced rejuvenation of growth in aged capsular bag cultures. Our findings suggest that multiple signaling pathways are responsible for wound-healing responses in the lens. It is therefore likely that disruption of multiple pathways may be needed to abolish or retard lens cell growth. Therapeutic agents to suppress activity of signaling molecules could be applied at the time of cataract surgery. This approach is likely to prevent or retard posterior capsule opacification in both young and old cataract patients. 
Of the six cytokines that showed a significant age-related difference, IL-6, IL-1β, IL-13, and VEGF are known to promote growth. 1619 IL-10, while not directly linked to proliferation, is reported to act largely as an immune suppressor; however, other studies suggest it can also possess a proinflammatory function. 20 IL-10 expression has also been correlated with VEGF levels and it is proposed that IL-10 mediates VEGF expression, 21 which is known to stimulate growth in many cells, 18 and is also implicated in lens growth. 22 While there are differences between the age groups in the makeup of cytokines, one can still anticipate that the putative overall growth potential within the medium is similar in each age group. This is largely borne out by exposure of a human lens epithelial cell line, FHL124, 23,24 to conditioned medium pooled from young and older donors. However, the degree of stimulation observed was weak relative to the addition of 1% FCS. This, therefore, suggests that the contents of the medium are not the driving force of growth in the capsular bag. As a result, attention was drawn to the lens capsule and growth factors that associate with it. For our investigations, we selected growth factors that are established promoters of lens cell growth 2527 and MAPK signaling. 25,28,29 For this purpose, we determined levels of bFGF 27,29 and HGF. 24,25 Moreover, bFGF and HGF require matrix association with heparan sulphate proteoglycans to facilitate receptor binding 3034 and, interestingly, these become more prominent as the lens capsule thickens with increasing age. 35 It is possible that this elevated rate of protein synthesis observed in older capsular bags results from an overproduction of growth factors, etcetera, in an attempt to drive cell growth to rates approaching the young. We subsequently asked the question: Does downstream activity of signaling components account for age-related growth rates? To address this notion, we focused on several signaling pathways known to be mediated by growth factors that are commonly associated with growth and migration of lens and nonlenticular cells—namely p-ERK, p-p38, p-c-jun, and p-JNK. 36,37 Through application of pathway inhibitors to the human capsular bag system, it was apparent that coverage of the posterior capsule can be regulated by these pathways and thus demonstrates a common regulatory role. The overall reduction in signaling activity of ERK, p38, c-jun, and JNK correlates well with age-related differences in vitro and in the clinic and thus provides a rationale to explain these phenomena. As near optimal cell growth is observed in nonstimulated <40 years capsular bag cultures, we hypothesized that the signaling motif observed in these cells was close to a threshold of activity required to grow at a maximum rate. We therefore added serum as a global stimulation to promote multiple receptor responses in order to determine whether signaling activity could be enhanced in cells of aged capsular bags. In all cases, signaling activity was enhanced and thus explains the basis for serum rejuvenated growth in aged lens capsular bags. One explanation for the differences in signaling activity in serum-free medium is the reduced level of receptors in the cells of older donors relative to the young. However, when provided with sufficient ligand, in the form of serum, the rate of growth observed by the cells of aged capsular bags was driven to rates observed in young donors. 6 Moreover, MAPK signaling can be dramatically increased in response to serum of young and aged donors. This, therefore, suggests that receptor availability is not the limiting factor. 
Another possibility that could explain both differences in cell coverage of the posterior capsule and signaling activity is the makeup of cell phenotype within the capsular bag. Clinically, fiber differentiation in PCO is seen as Soemmering's ring, and is more prevalent in the young. FGF is known to promote differentiation, but levels required to induce this are 10-fold greater than those needed to promote proliferation. 38 In our system, we do not observe fiber differentiation with serum-free culture. We have shown previously that transdifferentiation from an epithelial to a myofibroblast cell phenotype can occur in serum-free capsular bag cultures, to some degree. 39 TGF-β neutralizing antibody did not suppress this relatively low level transdifferentiation in capsular bags in serum-free medium. 39 Therefore, transdifferentiation can be stimulated by TGF-β independent mechanisms, presumably stress responses as a consequence of surgery. However, exposure to TGF-β dramatically increases myofibroblast marker expression. 39 We have assessed TGF-β levels within capsular bags of young and aged donors. Levels are relatively higher in the young than the old. This is interesting because TGF-β is actually a weak suppressor of growth. 23 Moreover, we do not observe any promotion of migration with TGF-β. 23,39 As TGF-βs promote myofibroblast expression in human lens cells, 23,3942 one can conclude that the myofibroblast phenotype does not exhibit an enhanced migratory function in the lens. Therefore, the cell population present within serum-free capsular bags will be made up of a majority, which retains some epithelial cell characteristics and a subgroup of myofibroblasts. Further investigations to discern the relative contributions of different cell phenotypes to signaling activity will be an interesting line of investigation in the future. 
We feel that our data has identified an interesting angle on age-related differences in wound healing. Elucidating the specific mechanisms regulating this reduced ability to convert ligand availability to signal pathway activation and growth will be a natural continuation of our work. 
Acknowledgments
The authors thank Lixin Wang for assistance in tissue preparations; the East Anglian and Bristol Eye Banks in providing donor material; Yvette Wormstone for research support and discussion; Jenny Gill for statistical advice; and John Reddan in providing the FHL124 cell line. 
References
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Footnotes
 Supported by the Biotechnology and Biological Sciences Research Council, Scientific Promotion of Ageing Research Capacity, and The Humane Research Trust.
Footnotes
 Disclosure: L.J. Dawes, None; G. Duncan, None; I.M. Wormstone, Alcon (C)
Figure 1. 
 
The effects of age and serum on time to confluency of cells on the posterior capsule. Cell growth was observed using phase-contrast microscopy. Confluency was taken as the point when 100% of the area on the posterior capsule, within the confines of the capsulorhexis, was covered by epithelial cells. Serum data was taken from Liu et al. 5 The numbers of different capsules used to obtain the data are given on each bar of the graph. Reprinted with permission from Wormstone IM, Liu CS, Rakic JM, Marcantonio JM, Vrensen GF, Duncan G. Human lens epithelial cell proliferation in a protein-free medium. Invest Ophthalmol Vis Sci. 1997;38:396–404. Copyright 1997 Association for Research in Vision and Ophthalmology.
Figure 1. 
 
The effects of age and serum on time to confluency of cells on the posterior capsule. Cell growth was observed using phase-contrast microscopy. Confluency was taken as the point when 100% of the area on the posterior capsule, within the confines of the capsulorhexis, was covered by epithelial cells. Serum data was taken from Liu et al. 5 The numbers of different capsules used to obtain the data are given on each bar of the graph. Reprinted with permission from Wormstone IM, Liu CS, Rakic JM, Marcantonio JM, Vrensen GF, Duncan G. Human lens epithelial cell proliferation in a protein-free medium. Invest Ophthalmol Vis Sci. 1997;38:396–404. Copyright 1997 Association for Research in Vision and Ophthalmology.
Figure 2. 
 
Cytokine levels in the culture medium of capsular bag preparations from young and aged donors. Medium was sampled following a 2-day culture period and analyzed using a suspended bead array multiplex cytokine panel. Four donor lenses for the <40 years group and eight pairs for the >60 years group were analyzed. Data are expressed as mean ± SEM. *Indicates a significant difference between groups (Student's t-test, P ≤ 0.05).
Figure 2. 
 
Cytokine levels in the culture medium of capsular bag preparations from young and aged donors. Medium was sampled following a 2-day culture period and analyzed using a suspended bead array multiplex cytokine panel. Four donor lenses for the <40 years group and eight pairs for the >60 years group were analyzed. Data are expressed as mean ± SEM. *Indicates a significant difference between groups (Student's t-test, P ≤ 0.05).
Figure 3. 
 
The effect of conditioned medium of capsular bag cultures from young (<40 years) and aged (>60 years) donors on growth of the human lens epithelial cell line FHL124 detected using the MTS assay. The data are expressed as mean ± SEM (n = 4). CM, conditioned medium; SF, serum free. *Represents a significant difference between untreated control and treatment group (P < 0.05; ANOVA with Dunnett's test).
Figure 3. 
 
The effect of conditioned medium of capsular bag cultures from young (<40 years) and aged (>60 years) donors on growth of the human lens epithelial cell line FHL124 detected using the MTS assay. The data are expressed as mean ± SEM (n = 4). CM, conditioned medium; SF, serum free. *Represents a significant difference between untreated control and treatment group (P < 0.05; ANOVA with Dunnett's test).
Figure 4. 
 
Basic fibroblast growth factor (A), hepatocyte growth factor (B), transforming growth factor beta 2 (C), and protein synthesis rates (D) detected in human lens capsular bag cultures from young (<40 years) and aged (>60 years) donors. Levels were obtained using commercially available ELISA kits and 35S-methionine incorporation. Six pairs of donor lenses were analyzed for the <40 years group and seven pairs for the >60 years group. Data are expressed as mean ± SEM. *Indicates a significant difference between groups (Student's t-test, P ≤ 0.05).
Figure 4. 
 
Basic fibroblast growth factor (A), hepatocyte growth factor (B), transforming growth factor beta 2 (C), and protein synthesis rates (D) detected in human lens capsular bag cultures from young (<40 years) and aged (>60 years) donors. Levels were obtained using commercially available ELISA kits and 35S-methionine incorporation. Six pairs of donor lenses were analyzed for the <40 years group and seven pairs for the >60 years group. Data are expressed as mean ± SEM. *Indicates a significant difference between groups (Student's t-test, P ≤ 0.05).
Figure 5. 
 
The effects of signaling pathway inhibitors on cell coverage of the human lens capsular bag. (A) A top-view cartoon representation of the capsular bag showing the specific regions presented in (BD). Note that immediately following preparation of the capsular bags, the central posterior capsule (PC) is completely devoid of cells. (BD) Modified dark-field images of capsular bags showing the posterior capsule (central area) and outer anterior capsule captured after 14 days of culture. Experiments were carried out in a match-paired format, such that one bag was maintained in EMEM supplemented with 10 μM U0126, 10 μM SP600125, and 10 μM SB203580 to inhibit ERK-, JNK-, and p38-mediated signaling and the other exposed to vehicle only (control). (EG) Pooled data showing the level of cell coverage across the posterior capsule in the different treatment groups. Data are expressed as the mean ± SEM (n = 3). Donor ages for: U0126 match-paired experiments were 67.7 ± 6.5 (range 58–80); SP600125 match-paired experiments were 68.3 ± 6.7 (range 56–79); SB203580 match-paired experiments were 54.7 ± 6.8 (range 42–65). *Significant difference between treated and control groups (P ≤ 0.05, Student's t-test).
Figure 5. 
 
The effects of signaling pathway inhibitors on cell coverage of the human lens capsular bag. (A) A top-view cartoon representation of the capsular bag showing the specific regions presented in (BD). Note that immediately following preparation of the capsular bags, the central posterior capsule (PC) is completely devoid of cells. (BD) Modified dark-field images of capsular bags showing the posterior capsule (central area) and outer anterior capsule captured after 14 days of culture. Experiments were carried out in a match-paired format, such that one bag was maintained in EMEM supplemented with 10 μM U0126, 10 μM SP600125, and 10 μM SB203580 to inhibit ERK-, JNK-, and p38-mediated signaling and the other exposed to vehicle only (control). (EG) Pooled data showing the level of cell coverage across the posterior capsule in the different treatment groups. Data are expressed as the mean ± SEM (n = 3). Donor ages for: U0126 match-paired experiments were 67.7 ± 6.5 (range 58–80); SP600125 match-paired experiments were 68.3 ± 6.7 (range 56–79); SB203580 match-paired experiments were 54.7 ± 6.8 (range 42–65). *Significant difference between treated and control groups (P ≤ 0.05, Student's t-test).
Figure 6. 
 
Suspended bead protein array detection of baseline total and phospho-signaling molecules in cells within capsular bag cultures from young and aged donors. Cell lysates were obtained during log phase growth across the previously cell-free posterior capsule in different age groups maintained in nonstimulated (baseline) conditions. Four donor lenses for the <40 years group and eight pairs for the >60 years group were analyzed. (A) Total level of ERK, c-jun, p38 MAPK, and JNK in cells from young and aged donors. (B) Baseline levels of phosphorylated protein level of pERK, p-c-jun, p-p38 MAPK, and pJNK corrected for corresponding total protein level in cells from young and aged donors. Data presented are normalized to the mean <40 years nonstimulated (baseline) value for each signaling component and presented as mean ± SEM.
Figure 6. 
 
Suspended bead protein array detection of baseline total and phospho-signaling molecules in cells within capsular bag cultures from young and aged donors. Cell lysates were obtained during log phase growth across the previously cell-free posterior capsule in different age groups maintained in nonstimulated (baseline) conditions. Four donor lenses for the <40 years group and eight pairs for the >60 years group were analyzed. (A) Total level of ERK, c-jun, p38 MAPK, and JNK in cells from young and aged donors. (B) Baseline levels of phosphorylated protein level of pERK, p-c-jun, p-p38 MAPK, and pJNK corrected for corresponding total protein level in cells from young and aged donors. Data presented are normalized to the mean <40 years nonstimulated (baseline) value for each signaling component and presented as mean ± SEM.
Figure 7. 
 
The effect of serum-stimulation on levels of phosphorylated signaling proteins in cells within capsular bag cultures from young and aged donors detected by suspended bead protein array. Cell lysates were obtained during log phase growth across the previously cell-free posterior capsule in different age groups following stimulation with 10% FCS for 30 minutes or maintained in control (baseline) conditions. These experiments were carried out using a match-paired format; four pairs of donor lenses were analyzed for the <40 years group and eight pairs for the >60 years group. The effect of serum-stimulation on levels of phosphorylated protein level of pERK, p-c-jun, p-p38, and pJNK corrected for corresponding total protein level in cells from young and aged donors. Data presented are normalized to the mean; <40 years nonstimulated (baseline) value for each signaling component and presented as mean ± SEM. *Represents a significant difference between age groups (P < 0.05, Student's t-test).
Figure 7. 
 
The effect of serum-stimulation on levels of phosphorylated signaling proteins in cells within capsular bag cultures from young and aged donors detected by suspended bead protein array. Cell lysates were obtained during log phase growth across the previously cell-free posterior capsule in different age groups following stimulation with 10% FCS for 30 minutes or maintained in control (baseline) conditions. These experiments were carried out using a match-paired format; four pairs of donor lenses were analyzed for the <40 years group and eight pairs for the >60 years group. The effect of serum-stimulation on levels of phosphorylated protein level of pERK, p-c-jun, p-p38, and pJNK corrected for corresponding total protein level in cells from young and aged donors. Data presented are normalized to the mean; <40 years nonstimulated (baseline) value for each signaling component and presented as mean ± SEM. *Represents a significant difference between age groups (P < 0.05, Student's t-test).
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