May 2007
Volume 48, Issue 5
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Lens  |   May 2007
Activation of Src Kinases Signals Induction of Posterior Capsule Opacification
Author Affiliations
  • Janice L. Walker
    From the Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.
  • Iris M. Wolff
    From the Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.
  • Liping Zhang
    From the Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.
  • A. Sue Menko
    From the Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2214-2223. doi:10.1167/iovs.06-1059
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      Janice L. Walker, Iris M. Wolff, Liping Zhang, A. Sue Menko; Activation of Src Kinases Signals Induction of Posterior Capsule Opacification. Invest. Ophthalmol. Vis. Sci. 2007;48(5):2214-2223. doi: 10.1167/iovs.06-1059.

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

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Abstract

purpose. Posterior capsule opacification (PCO) is a complication of cataract surgery resulting from the proliferation, migration, and epithelial-to-mesenchymal transition (EMT) of lens epithelial cells that remain associated with the lens capsule. These changes cause a loss of vision. The authors developed a chick embryo lens capsular bag model to study mechanisms involved in the onset of PCO. Because Src family kinases (SFKs) signal cell proliferation, migration, and EMT, the authors examined whether the inhibition of SFKs can prevent PCO.

methods. After mock cataract surgery, chick lens capsular bags were pinned to a culture dish and grown in the presence or absence of the SFK inhibitor PP1. Cell movement was followed by photomicroscopy. Progression of proliferation and EMT in the PCO cultures was determined by Western blot analysis and immunofluorescence staining.

results. As occurs in PCO, lens cells in this model proliferated, migrated across the posterior capsule, and expressed EMT markers, α-smooth muscle actin (α-SMA), and fibronectin (FN). Lens cells treated with PP1 maintained an epithelial phenotype, accumulated cadherin junctions, and did not migrate to the posterior capsule, increase proliferation, or express EMT markers. Therefore, exposure to PP1 prevented PCO. Short-term inhibition of SFKs was sufficient to prevent EMT, but longer inhibition was necessary to prevent lens cell migration.

conclusions. Progression of PCO involved early activation of SFKs. Lens cell migration preceded EMT, and each of these two events required activation of an SFK signaling pathway. Suppression of SFK activation blocked PCO, suggesting SFKs as a therapeutic target for the prevention of PCO.

Development of the lens opacity known as cataract results in blurred vision and even blindness. This impairment of the visual process can be corrected by routine cataract surgery in which the lens fiber cell mass is removed. In most procedures, the lens capsule or capsular bag, the thick basement membrane that surrounds the lens, is left behind, and an artificial lens called an intraocular lens (IOL) is inserted within the capsular bag. The IOL allows lens function to be maintained. Not all lens cells are removed during cataract surgery; lens epithelial cells, tightly adherent to the lens capsule, remain attached to the basement membrane proteins that compose the capsule. 
From a few months to a few years after cataract surgery, posterior capsule opacification (PCO), also known as secondary cataract, develops in 20% to 40% of patients. The development of PCO is age related; older patients generally have a lower incidence of PCO than younger patients, particularly children and infants. 1 PCO occurs as the residual lens epithelial cells that line the inside surface of the equatorial lens capsule proliferate and migrate along the capsule until they reach its posterior aspects. These cells undergo an epithelial-to-mesenchymal transition (EMT) by which they become myofibroblastlike, express mesenchymal markers, and exhibit a contractile phenotype contributing to the wrinkling and fibrosis of the lens capsule. As a result, the lens capsule becomes clouded and vision is reduced. Although advances in cataract surgery and IOL design have reduced the frequency of PCO, it remains the major complication of cataract surgery. 
PCO cannot be predicted or prevented. When it leads to significant visual disturbance, it is treated with neodynium:YAG laser posterior capsulotomy, 1 a surgical procedure that restores vision by creating an opening in the opacified posterior capsule sufficient to let light pass to the retina. Although the procedure is quick and painless, it can result in a number of complications, including elevation of the intraocular pressure, damage to the IOL, dislocation of the IOL, or inflammation. 
Human capsular bag cultures developed by Liu et al. 2 have provided an ideal in vitro model for investigating the mechanism of PCO. Studies with this culture model have shown that TGF-β2 induces the EMT of lens epithelial cells and the contraction of the capsular bag that are characteristic of PCO. 3 We adapted this capsular bag model to the chick embryo lens. Because the incidence of PCO is highest in young patients and embryos are efficient at repairing damaged tissue, 4 it was predicted that the development of PCO in the embryonic lens would be rapid and that this model would provide a significant challenge to develop avenues for prevention. In addition, the ability to prepare large numbers of age-matched capsular bags makes it possible to analyze the signaling pathways that induce residual lens epithelial cells to proliferate, migrate along the lens capsule, and undergo EMT. With this PCO model, we believe we can uncover important therapeutic targets for PCO prevention to use for patients of all ages. 
In this study we investigated the role of Src family kinases (SFKs), a family of tyrosine kinases, in the signaling pathways that induce PCO. SFKs, activated downstream of integrins, play a central role in signaling pathways that direct cell proliferation, migration, and EMT. 5 6 7 Activation of Src kinases is required for normal wound-healing events, a process that shares many features with PCO. In the corneal epithelium, inhibitors of Src kinase activity interfere with wound healing by impeding cell migration. 8 Fibroblasts lacking SFK family members exhibit defects in migration. 9 10 Src kinases also play an essential role in signaling cell cycle progression. 11 12 SFK activity is required to maintain lens epithelial cells in a proliferative state. 13 Exposure of primary lens cell cultures to the SFK inhibitor PP1 blocks their proliferation and signals their withdrawal from the cell cycle, inducing the expression of cell cycle inhibitors p27 and p57. 13 In addition, activation of Src kinases plays a role in directing EMT. 5  
Although Src kinase may have a number of signaling roles in the induction of EMT, one of its important functions in this process is as a negative regulator of cadherin cell–cell adhesion junctions. Normal adherens junctions are thought to protect cells against the development of a mesenchymal phenotype, as occurs in many metastatic cancers. 14 SFK activity can disrupt cadherin-dependent cell–cell contacts in epithelial cells. 15 In lens epithelial cells, we have shown that the inhibition of SFK activity stabilizes cadherin-mediated cell–cell junctions, whereas constitutive activation of Src kinases causes induction of a mesenchymal phenotype and disassembly of cell–cell adhesion. 13  
Here we investigated whether the activation of Src kinases was responsible for the development of PCO and whether blocking the activation of Src kinases could prevent PCO. We found that the inhibition of SFK activity blocked lens epithelial cell proliferation and migration to the posterior capsule and EMT marker expression. Inhibition of PCO through the blocking of SFK activity was accompanied by an accumulation of β-catenin–containing cadherin junctions at cell–cell interfaces and maintenance of an epithelial cell phenotype. Blocking the activation of Src kinases for even a short time after mock cataract surgery was sufficient to block EMT, suggesting that Src kinases are among the earliest signals for the onset of PCO and are a potential therapeutic target for the prevention of PCO. 
Methods
Establishment of Chick Embryo Lens Capsular Bag Cultures
Chick embryo lens capsular bag PCO cultures were adapted from a methodology for human lenses developed by Liu et al. 2 that mimics cataract surgery. For our studies, lenses were removed from embryonic day (E)15 chick embryo eyes by dissection. Then an incision was made in the anterior lens capsule, from which the lens fiber cell mass was removed by hydroelution. Given that this is a new protocol, further details on preparation of the capsular bags to optimize for microscopic examination during inhibitor treatment are presented in Results. The resultant capsular bags were cultured in media (Media 199; ScienCell Research Laboratories, San Diego, CA) with 10% fetal calf serum, as indicated. Movement of the lens epithelial cells along the posterior aspects of the capsule was followed with a dissecting microscope (SMZ800; Nikon, Tokyo, Japan) and imaging software (Metamorph; Molecular Devices, Eugene, OR). Src kinase activity in the lens epithelial cells in the capsular bag cultures was suppressed using the Src family kinase inhibitor PP1 (4-Amino-1-tert-butyl-3-(1′-naphthyl)pyrazolo[3,4-d]pyrimidine; (Biomol, Plymouth Meeting, PA). PP1 was dissolved in DMSO and added to the culture medium at 1 to 10 μM, as noted. Typically, fresh inhibitor was added every 3 days; however, in some studies, PP1 or vehicle control (DMSO) was added once, at the time of cataract surgery, or only for short periods of times and then was washed away and replaced with media. 
Immunoblot Techniques
Samples were extracted in OGT buffer (44.4 mM n-Octyl β-D glucopyranoside, 1% Triton X-100, 100 mM NaCl, 1 mM MgCl2, 5 mM EDTA, 10 mM imidazole) containing 1 mM sodium vanadate, 0.2 mM H2O2, and a protease inhibitor cocktail (Sigma, St. Louis, MO). Protein concentrations were determined with an assay (BCA assay; Pierce, Rockford, IL). Proteins were separated on Tris-glycine gels (Novex, San Diego, CA), electrophoretically transferred to membrane (Immobilon-P; Millipore Corp., Bedford, MA), and immunoblotted as described previously. 16 For detection, reagent (ECL; Amersham Life Sciences, Arlington Heights, IL) was used. Immunoblots were scanned, and densitometry analysis was performed (1D software; Eastman Kodak, Rochester, NY). Values were normalized (± SEM) to those of day (D) 3 or D6, as indicated in the figure legends, and plotted on graphs. All gels were run under reducing conditions. Antibodies used included c-Src (Santa Cruz Biotechnology, Santa Cruz, CA; directed against a peptide in the Src C-terminus that is common to all SFK family members), phosphoSrc418 (Biosource, Camarillo, CA; the 418 site is common among SFK family members), β-actin (Sigma, St Louis, MO), α-smooth muscle actin (Sigma), β-catenin (BD Biosciences, Franklin Lakes, NJ), FAK Y397 (BD Biosciences), fibronectin (Developmental Studies Hybridoma Bank), and PCNA (Sigma). Fluorescence-conjugated phalloidin, which binds filamentous actin, was obtained from Molecular Probes (Eugene, OR). 
Immunofluorescence
Lens PCO cultures were fixed in 3.7% formaldehyde in PBS and permeabilized in 0.25% Triton X-100 in PBS before immunostaining. Cells were incubated with primary antiserum followed by rhodamine-conjugated secondary antibody (Jackson Laboratories, West Chester, PA). Samples immunostained with antibodies to α-smooth muscle actin and PCNA and counterstained with DAPI were examined with a digital imager (Eclipse 80i; Nikon) equipped with a camera (CoolSnap HQ; Photometrics, Tucson, AZ), and images were captured with image analysis software (Metamorph; Molecular Devices). Samples immunostained for β-catenin and counterstained with phalloidin were examined with a confocal microscope (LSM 510; Carl Zeiss, Oberkochen, Germany). 
Results
Chick Embryo Lens Capsular Bag Model
To study the pathways that signal the development of PCO, we developed a chick embryo lens capsular bag culture model that mimics the process of PCO as it occurs in vivo. Preparation of these cultures was based on the human model developed by Liu et al. 2 The technique involved mock cataract surgery, which leaves the lens epithelial cells of the equatorial zone attached to the lens capsule. Resultant capsular bags were pinned to the culture dish posterior capsule side down, and five equally spaced cuts were made in the anterior capsule that contained the lens epithelial cells (Fig. 1) . This modification to the technique made it possible to flatten the lens capsules on the culture substrate cell side up, providing a clear view of the cells in the equatorial zone and the posterior capsule, which at this point was devoid of cells (Fig. 1) . In these flattened capsular bag cultures, we could follow movement of the lens epithelial cells onto and across the posterior capsule, as occurs in PCO. 
Chick Embryo Lens Capsular Bag Model Mimics PCO
Proliferation of the lens epithelial cells, their migration from the equatorial zone across the posterior capsule, and their EMT are three essential elements in the development of PCO. Together, these events result in the wrinkling of the lens capsule and the scattering of light that causes loss of vision. 1 17 To determine whether our chick embryo lens capsular bag cultures recapitulated the normal progression of PCO, we monitored them for migration, proliferation, and EMT. Their migratory behavior was documented by photomicroscopy. Immediately after mock cataract surgery, the epithelial cells remained attached to their original position in the equatorial zone (Fig. 2A , arrow D0), and the posterior capsule remained devoid of cells. However, within 1 day, the equatorial epithelial cells began to migrate to the posterior capsule. While these cultures were observed through day 6, the lens cells typically migrated across the entire posterior capsule within 3 days (Fig. 2A , D3). The epithelial cells migrated as a sheet, as occurs in the human PCO culture model. 
Focal adhesion kinase (FAK) is strongly implicated in regulating migration because expression of dominant-negative forms of FAK in various cell types 18 19 and FAK null fibroblasts exhibit defects in migration. 20 21 FAK is a tyrosine kinase activated in response to integrins, resulting in the autophosphorylation of Y397, which creates a binding site for molecules such as Src kinases. 22 23 We measured FAK activation by Western blot analysis of PCO culture extracts with a phosphospecific FAK (Y397) antibody to determine whether lens cell migration to the posterior capsule could be correlated with FAK activation. FAK activation at Y397 increased after the capsular bags were placed in culture and remained high as the lens cells migrated across the posterior capsule, suggesting a role for FAK in lens cell migration during PCO (Fig. 2B)
The fact that lens cells moved across the posterior capsule as a sheet suggested that increased cell mass was necessary for a sufficient number of cells to populate the posterior capsule. We monitored cell proliferation in the chick lens capsular bag cultures by Western blot analysis and immunolocalization for PCNA expression, a cell cycle marker. The PCNA proliferation marker was expressed at D0 and as the cells populated the posterior capsule, but it slowed after the cells had completed their migration across the capsule (Fig. 2D) . On day 1, as the lens cells began migrating to the posterior capsule, nuclei in cells moving onto the capsule were PCNA positive (Fig. 2C) . However, by day 9, well after migration was completed, no nuclear staining for PCNA was detected in any region of the capsule (Fig. 2C) . These results suggest that cells only proliferate early in culture, when lens cells are migratory. 
To determine whether lens epithelial cells undergo EMT in this lens capsular bag model, we examined expression of the classical EMT markers αSMA and FN throughout the 6-day culture period. αSMA, a protein expressed by contractile mesenchymal cells, could not be detected in OGT extracts of chick embryo equatorial epithelial cells just after mock cataract surgery, and FN was expressed only at very low levels (Fig. 3A) . This result is consistent with the lack of expression of αSMA in any region of differentiation of E10 chick lenses (data not shown) and the low level of expression of FN in the anterior lens capsule. 24 25 αSMA is reported in lens epithelial cells of other species, 26 and, in all species examined, αSMA increases in lens cells on induction of EMT. 27 28 29 30 By day 6 in chick lens capsular bag cultures, lens epithelial cells exhibited typical characteristics of EMT, expressing high levels of αSMA and FN (Fig. 3A) , as occurs in clinical PCO and in anterior subcapsular cataract. Immunolocalization showed that the αSMA-expressing cells were present on the posterior capsule and within the original zone of the equatorial region (Fig. 3B) . Because migration to the posterior capsule typically occurs by day 3, migration of lens epithelial cells occurs before the onset of EMT. The migration and EMT of lens epithelial cells was accompanied by significant wrinkling and contraction of the lens capsule. Thus, the chick capsular bag model system recapitulates the PCO that occurs in vivo; lens epithelial cells proliferate, migrate along the posterior capsule, and undergo EMT, causing the wrinkling of the posterior capsule. Chick lens capsular bags cultured under serum-free media conditions migrated toward the posterior capsule, albeit more slowly than with the addition of serum, and induced the EMT markers αSMA and FN (data not shown). These results paralleled findings in the human capsular bag model, 31 suggesting that intrinsic qualities of lens cells lead to the progression of PCO. Consequently, these capsular bags provide an ideal model in which to determine the signaling pathways that are activated to induce PCO and to investigate potential pharmaceutical targets for the prevention of PCO. 
SFKs Role in PCO
SFKs are a family of tyrosine kinases that share common kinase and regulatory domains. Pharmaceutical inhibitors that block activation of the Src kinase domain effectively inactivate all SFK members. It is well known that SFKs have a pivotal role in the signaling pathways that regulate cell proliferation, migration, and EMT. The role of SFK signaling pathways in so many cell functions associated with the development of PCO led us to investigate the possibility that activation of these kinases was essential to the induction of PCO. To analyze the role of SFKs in PCO, chick embryo lens capsular bag cultures were incubated in the presence of the SFK inhibitor PP1. The efficacy of PP1 at inhibiting SFKs in the lens capsular bag cultures was demonstrated using the phospho-src418 antibody. This domain must be phosphorylated for SFKs to be active and is common among SFK family members. Exposure of the lens capsular bag cultures to PP1 from the beginning of the culture period effectively blocked activation of Src kinases (Y418 phosphorylation) early in culture (Fig. 4A) , and this activation block was maintained throughout the culture period (Fig. 4B) . We next examined whether the activation of SFKs was required for the induction of lens cell proliferation, the promotion of lens cell migration, and the transition of epithelial cells to a mesenchymal phenotype, all essential features of PCO. 
The effect of PP1 on lens epithelial cell migration was striking; continued exposure to PP1 throughout the culture period completely prevented lens epithelial cell migration onto the posterior capsule (Fig. 4B) . This was correlated directly with a decrease of FAK activation, a known Src target (data not shown). Src kinase signaling also was important for the proliferation of lens epithelial cells in the PCO cultures because blocking Src kinase activity suppressed PCNA expression (Fig. 5) . We examined whether Src kinase activity was required for the induction of EMT in the capsular bag cultures. Exposure to the Src kinase inhibitor PP1 suppressed the expression of FN and αSMA (Fig. 5) , both classical markers of EMT. The ability of the Src kinase inhibitor to block lens epithelial cell migration, proliferation and EMT in our chick lens capsular bag model was likely the reason this inhibitor was so efficacious in preventing PCO. 
SFK Inhibition Maintains Cadherin Junctions and an Epithelial Phenotype
Because SFK activation can signal the disassembly of cadherin cell–cell junctions 15 and the maintenance of cell–cell junctions can protect cells against the development of a mesenchymal phenotype, we determined whether the inhibition of PCO by PP1 involved changes in cadherin junctions. To examine the degree of cadherin junction assembly, chick lens capsular bags grown in the presence and absence of PP1 were immunostained for β-catenin, a major component of all cadherin junctions. The cells were costained for phalloidin, which detects the filamentous actin that links to cadherin junctions and provides a good marker of cell morphology. As lens cells migrated across the posterior capsule in the lens capsular bag cultures, significant changes occurred in cell morphology and in organization of their cell–cell junctions. Lens epithelial cells remaining on the flap (EQ region; Figs. 6D 6E 6F ) remained cuboidal, and β-catenin and F-actin localized to their cell–cell interfaces. That region contained significant overlap between β-catenin and actin filaments (Fig. 6F) . In contrast, cells that migrated to the posterior capsule appeared flattened and disorganized and had lost the typical epithelial morphology and tight cuboidal packing of normal lens equatorial cells (Figs. 6A 6B 6C) . Nonetheless, these cells retained some cell–cell interface staining for β-catenin that was not always coincident with actin fibers. In the presence of PP1, the cells maintained their epithelial phenotype and tight cuboidal packing and displayed a greatly increased level of cadherin junctions at their cell–cell interfaces, as indicated by staining for β-catenin (Fig. 6G) . In PP1-treated cultures, β-catenin colocalized with actin filaments at the cell–cell borders (Fig. 6I) . Inhibition of SFKs might have prevented PCO by maintaining the lens epithelial cell phenotype through the stabilization of cadherin junctions. 
SFKs Are Early Inducers of PCO
The development of PCO can be compared to the wound-healing process; not only is the induction of lens cell migration and proliferation similar to wound healing but cataract surgery can be viewed as an injury that inflicts insult to the lens. It is likely that lens cells may quickly respond to injury and that these early signals may give rise to PCO after cataract surgery. Because SFKs were active early in our capsular bag model, we determined whether short-term treatment of PP1 would be sufficient to block PCO from occurring. For this study, chick lens capsular bags were treated with PP1 or the vehicle control just after the mock surgery for only the first 5 minutes or the first day in culture. These short-term treatments with PP1 delayed migration but did not inhibit the eventual migration of lens cells onto the posterior capsule (Fig. 7A) . Although these short-term treatments with PP1 failed to keep lens cells from migrating onto the posterior capsule, they were sufficient to suppress induction of the EMT markers FN and αSMA (Fig. 7B) . These results demonstrate that early activation of SFKs is sufficient to signal the induction of EMT associated with the onset of PCO. Importantly, these studies show that the processes of migration and EMT in the development of PCO can be separated from one another because lens cells migrated to the posterior capsule but did not undergo EMT after short-term treatment with PP1, whereas continued suppression of SFKs blocked cell migration, proliferation, and EMT (Figs. 4 5) . These findings suggest that two mechanistically distinct SFK signaling pathways are involved in the induction of PCO. 
PP1 Dose Effectiveness
We examined the dose effectiveness of PP1 for the inhibition of migration and EMT in the PCO cultures. The typical dose for PP1 in culture studies is 10 μM. In this study, chick lens capsular bags were cultured in the presence of PP1 at concentrations ranging from 1.0 to 10 μM or were treated with vehicle control. Inhibitor was added at time 0, just after simulated cataract surgery and every 3 days thereafter. Lens capsular bag cultures were monitored for the migration of lens epithelial cells onto the posterior capsule and for the expression of the EMT markers FN and αSMA. The migration of lens epithelial cells onto the posterior capsule was blocked in a dose-dependent manner (Fig. 8A) . PP1 (3 μM) prevented lens cells from completely covering the posterior capsule, and 10 μM PP1 was most effective at preventing migration during the study (Fig. 8A) . Both doses blocked the expression of αSMA and FN. Even though migration to the posterior capsule was not blocked with a 1-μM dose of PP1, it was sufficient to suppress αSMA and FN expression (Fig. 8B) . These results suggested that low doses of PP1 are effective in slowing migration and preventing EMT associated with inducing PCO, and they provided additional evidence that two distinct Src pathways are involved in signaling migration and EMT in the development of PCO. 
One PP1 Treatment Sufficient to Inhibit Migration and EMT
These studies demonstrated that SFK function is essential to the development of PCO in the chick lens capsular bags and that SFK inhibitors may be therapeutically useful for the prevention of PCO. We also determined whether a single treatment of PP1 at the time of mock cataract surgery would be sufficient to block PCO. PP1 at 3 μM and 10 μM or vehicle control were added once at time 0, just after the capsular bags were placed in culture. Cells in the capsular bags were monitored for cell migration onto the posterior capsule and for the expression of EMT markers. Although a single dose of PP1 could block migration and EMT, dose effectiveness was different: 10 μM was most effective at blocking migration, but 3 μM was sufficient to block EMT (Figs. 9A 9B) . These studies showed that a single dose of PP1 may be efficacious in preventing PCO, providing a possible therapeutic that could be applied at the time of cataract surgery. 
Discussion
Opacification of the posterior capsule is the major complication of cataract surgery, leading to loss of visual acuity. Several types of models are used to study PCO, including in vitro lens cell cultures, ex vivo capsular bag, and animal models. 17 Animal models are important but are difficult to use for studies examining the molecular details of pathways involved in inducing PCO. Functional studies are more easily performed in tissue culture models, and the ex vivo lens capsular bag model provides an excellent system to elucidate mechanisms of PCO because it closely mimics cataract surgery in vivo. In this study we developed a lens capsular bag model in the chick embryo based on the human capsular bag model developed by Liu et al. 2 The chick lens capsular bag model offers an advantage over the human model for studying PCO because tissue is unlimited and large numbers of age-matched capsular bags can be prepared for biochemical analysis. Given that the onset of PCO is age-related and younger patients are at a greater risk for PCO, our chick embryo model is likely more susceptible to PCO and more useful for identifying therapeutic targets to prevent PCO in patients of all ages. The chick capsular bag model also provides a valuable tool to screen and identify potential therapeutic targets for the prevention of PCO before testing in animals and in human ex vivo models. 
PCO represents a major socioeconomic problem, and the mechanisms underlying it remain to be elucidated. In this study we developed a chick lens capsular bag model that mimics PCO to identify mechanisms involved in the induction of PCO and the development of therapeutic targets for its prevention. The major features that lead to the onset of PCO in patients are similar in chick embryo and human capsular bag models. After mock cataract surgery, lens epithelial cells proliferate and migrate to the posterior capsule, a previously cell-free zone. During PCO in the lens capsular bag models, lens epithelial cells migrate as a sheet (also termed collective migration), maintaining cell–cell adhesions as they migrate toward the posterior capsule. This type of migration is similar to migration that occurs during epithelial wound healing and in certain cancer cell types. 32 Lens cells also undergo EMT, expressing classical EMT markers such as FN and αSMA. Consistent with findings by Wormstone et al. 31 for the human capsular bag model, our chick lens capsular bag model does not require exogenously added factors (serum-free conditions) to migrate and undergo EMT (data not shown). These processes lead to fibrosis—wrinkling of the lens capsule—which is responsible for the light scattering that contributes to loss of vision during PCO. 
Our studies have identified a role for SFKs in promoting PCO. Inhibition of SFKs was particularly efficacious in blocking PCO because their inhibition blocked all the major factors leading to PCO: proliferation, migration, and expression of EMT markers. In previous studies, a TGF-β pathway also was shown to signal the induction of lens cell EMT and the development of PCO. 3 33 34 The use of neutralizing antibodies to TGF-βII, 3 35 the inhibition of the proteasome, 36 or the addition of lithium 30 all block TGF-β–driven EMT and provide potential therapeutic approaches for PCO prevention. Interestingly, in mammary epithelial cells, SFKs are essential for TGF-βR-II–induced EMT. 37 It is possible that our results with SFK inhibition of PCO may be related to its impact on the TGF-β signaling pathway. 
Individual SFK family members may play discrete roles in PCO and are likely to differentially regulate the distinct processes that collectively cause this disease. Various SFKs are expressed in the lens, such as c-Src, Fyn, Lyn, Lck, and Yes (Ref. 38 and M. Leonard, unpublished data, 2006), and the SFK activity that we detect with the phosphoSrc418 antibody may reflect the activation of multiple SFK members that become specifically active at different times during the onset of PCO. Inhibition of SFKs with PP1 blocks all SFK family members (by competing with ATP binding). Although PP1 is commonly used as a specific inhibitor of SFK activity, a recent study using computational analysis and sequence alignment has identified some potential off-targets for PP1. 39 Although a major limitation of the computational studies is that conformational structures of potential off-targets were not considered when assessing selectivity to PP1, we performed studies to validate that PP1 functioned as a Src-specific inhibitor in blocking EMT. First, we demonstrated that PP1 effectively blocked EMT at concentrations as low as 1 μM, considerably below the concentration of 10 μM typically used in Src inhibition studies. Second, we examined whether PP1 affected the activity of an identified potential PP1 off-target, p38, when added to lens cell PCO cultures. Even at doses of 10 μM, p38 was not inhibited by PP1 in the lens capsular bag cultures (data not shown). This result is consistent with our earlier publication in which we show that p38 was upstream of Src signaling in cataract induction. In that study, PP1 had no effect on p38 activity, but p38 inhibitors suppressed Src activity. 40  
Although continued inhibition of SFKs blocked all the key features contributing to the onset of PCO, 5-minute treatment of PP1 at T0 (right after mock cataract surgery) was sufficient to suppress the expression of EMT markers but not of migration. Therefore, the migration of lens cells to the posterior capsule does not necessarily cause the cells to undergo EMT. These results allowed us to separate the processes of migration and EMT for the onset of PCO and identified two distinct SFK pathways, one controlling collective cell migration and the other regulating EMT. An SFK/FAK signaling pathway may regulate sheet migration (collective migration) of the lens cells onto the posterior capsule because FAK activation increased as lens cells migrated and SFK inhibition dampened FAK activation and blocked lens cell migration. FAK and SFKs regulate migration, 10 20 41 and recent reports describe a role for FAK in regulating cell–cell interactions for collective migration 42 and SFKs in collective migration of MDCK cells during wound healing. 43  
SFK-driven migration and EMT also could be regulated by the duration of the SFK signal because migration was inhibited only when SFK activation was continually suppressed, whereas EMT could be blocked by inhibiting SFK activity during the first 5 minutes in culture. Interruption of SFK activation for 5 minutes at later times in PCO culture, such as D1 or D2, did not suppress EMT markers (unpublished data, 2006), suggesting that the early Src kinase signal is responsible for signaling EMT. Although differential effects dependent on the duration of signal have not been previously described for SFKs, others report distinct consequences dependent on the duration of signaling for molecules such as PI3K 44 and ERK. 45 ERK signal duration in PC12 cells determines whether the cells will proliferate or differentiate. 46 47  
PCO is similar to the wound-healing process, and lens cells may activate SFKs quickly after injury (mock cataract surgery). In support of this hypothesis, we found that SFKs were active throughout PCO and that early activation of SFKs drives the initial signals that induce EMT and give rise to PCO. EMT is likely the underlying cause of PCO given that acquisition of the myofibroblast phenotype leads to contraction, wrinkling of the capsule, light scatter, and vision loss. SFKs may regulate EMT during PCO through changes in cell adhesion. In support of this hypothesis, blocking PCO in our chick lens capsular bag model through the inhibition of SFKs was accompanied by maintenance of an epithelial cell phenotype and increased β-catenin staining at cell–cell interfaces. Increased staining of β-catenin reflects an accumulation of cadherin junctions at cell–cell interfaces that can enhance junctional stability and preserve the epithelial phenotype. 
A central feature between cell–matrix and cell–cell adhesion systems is their common linkage to the cytoskeleton. SFKs are well-known regulators of the cytoskeleton and can regulate changes in cell adhesion. 5 48 One way in which SFKs can deregulate E-cadherin junctions is through the activation of FAK and the peripheral localization of myosin. 6 Therefore, SFK/FAK-mediated changes in cytoskeleton, such as through myosin contraction, may act as a control switch to regulate changes in cell–matrix and cell–cell adhesion for migration and EMT required for the development of PCO. 5 Cell–cell adhesion may play a role in coordinating waves of contraction that contribute to the myofibroblastlike phenotype, which causes fibrosis, wrinkling of the lens capsule, and light scattering and leading to loss of vision. Therefore, it is of great interest to our laboratory, but beyond the scope of these studies, to determine the role of FAK and SFK in regulating cell–cell junctions for collective migration and EMT during PCO. 
Our studies demonstrate that SFK function is essential to the development of PCO in the chick lens capsular bag model. Because SFKs regulate all the key processes leading to the onset of PCO, they may be especially efficacious in its prevention. Src inhibitors are being developed and tested for cancer clinical trials. 49 Therefore, it is particularly promising that SFK inhibitors can be used for the prevention of PCO; it may be possible to apply SFK inhibitors to the IOL or lens capsule at the time of cataract surgery. Further development of SFK inhibitors for the treatment of PCO will require testing in human capsular bags and animal models. Our model system represents an excellent model for PCO, and it facilitates the understanding of mechanisms of collective migration and EMT affiliated with tumor invasion and other disease processes and with wound healing. 
 
Figure 1.
 
Chick embryo lens capsular bag model. Lenses from E15 chicken embryos (A) are dissected, and a small cut is made in the anterior capsule where a needle is inserted to hydrostatically remove the fiber cell mass, leaving behind lens epithelial cells that are tightly attached to the capsule (B). This procedure mimics cataract surgery. Cuts are then made in the anterior region of the capsule (C), and individual flaps of the capsule containing lens epithelial cells are flattened and pinned onto the tissue culture dish capsule side down (D, E), allowing visualization of the lens epithelial cells as they migrate onto the posterior capsule (F). PC, posterior capsule.
Figure 1.
 
Chick embryo lens capsular bag model. Lenses from E15 chicken embryos (A) are dissected, and a small cut is made in the anterior capsule where a needle is inserted to hydrostatically remove the fiber cell mass, leaving behind lens epithelial cells that are tightly attached to the capsule (B). This procedure mimics cataract surgery. Cuts are then made in the anterior region of the capsule (C), and individual flaps of the capsule containing lens epithelial cells are flattened and pinned onto the tissue culture dish capsule side down (D, E), allowing visualization of the lens epithelial cells as they migrate onto the posterior capsule (F). PC, posterior capsule.
Figure 2.
 
Chick embryo lens capsular bag model mimics PCO: induction of migration and proliferation. (A) After mock cataract surgery, lens capsules were pinned down and lens epithelial migration onto the posterior capsule (PC) was examined from D0 to D6 by photomicroscopy with a digital camera and dissecting microscope. White arrow: lens capsule flap containing lens epithelial cells. Lens cells have already begun to migrate toward the posterior capsule as early as D1 and typically cover the posterior capsule by D3. (B) PCO cultures were extracted at 0 hour, 10 minutes, D1, and D6, and cell lysates were analyzed by immunoblotting for phospho-FAK Y397 and β-actin (loading control). An increase in FAK activation was observed as the cells migrated toward the posterior capsule. (C) PCO cultures were fixed on D1 and D9 and immunostained for PCNA and DAPI. Nuclear staining for PCNA was detected in lens cells migrating toward the posterior capsule on D1 but was not detected on D9. Scale bar, 100 μm. (D) PCO cultures were extracted on the days indicated, and the cell lysates were analyzed by immunoblotting for PCNA and β-actin (loading control). Similar to our localization studies for PCNA, PCNA was highly expressed early after mock surgery and decreased to very low levels as cells completed their migration to the posterior capsule. EQ, equatorial region of the capsule.
Figure 2.
 
Chick embryo lens capsular bag model mimics PCO: induction of migration and proliferation. (A) After mock cataract surgery, lens capsules were pinned down and lens epithelial migration onto the posterior capsule (PC) was examined from D0 to D6 by photomicroscopy with a digital camera and dissecting microscope. White arrow: lens capsule flap containing lens epithelial cells. Lens cells have already begun to migrate toward the posterior capsule as early as D1 and typically cover the posterior capsule by D3. (B) PCO cultures were extracted at 0 hour, 10 minutes, D1, and D6, and cell lysates were analyzed by immunoblotting for phospho-FAK Y397 and β-actin (loading control). An increase in FAK activation was observed as the cells migrated toward the posterior capsule. (C) PCO cultures were fixed on D1 and D9 and immunostained for PCNA and DAPI. Nuclear staining for PCNA was detected in lens cells migrating toward the posterior capsule on D1 but was not detected on D9. Scale bar, 100 μm. (D) PCO cultures were extracted on the days indicated, and the cell lysates were analyzed by immunoblotting for PCNA and β-actin (loading control). Similar to our localization studies for PCNA, PCNA was highly expressed early after mock surgery and decreased to very low levels as cells completed their migration to the posterior capsule. EQ, equatorial region of the capsule.
Figure 3.
 
EMT is induced in chick embryo capsular bag model. (A) Graphs depicting Western blot studies show relative expression of FN, αSMA, and β-actin in lens cell PCO cultures on D0, D3, and D6. Little change in β-actin was detected, whereas EMT markers FN and αSMA were highly induced by D6 in PCO cultures. Three experiments were quantified using 1D software (Eastman Kodak), and relative densities were plotted on graphs ± SEM. Data were normalized to D6 values for each graph. Error bars are present on D0 and D3 samples for the αSMA graph but are too small to be visualized in the figure. Representative Western blot is depicted below each graph. (B) D6 PCO cultures were fixed and immunostained for αSMA and DAPI. Cells were detected expressing αSMA on the posterior capsule and within the cells remaining on the flap. Scale bar, 100 μm.
Figure 3.
 
EMT is induced in chick embryo capsular bag model. (A) Graphs depicting Western blot studies show relative expression of FN, αSMA, and β-actin in lens cell PCO cultures on D0, D3, and D6. Little change in β-actin was detected, whereas EMT markers FN and αSMA were highly induced by D6 in PCO cultures. Three experiments were quantified using 1D software (Eastman Kodak), and relative densities were plotted on graphs ± SEM. Data were normalized to D6 values for each graph. Error bars are present on D0 and D3 samples for the αSMA graph but are too small to be visualized in the figure. Representative Western blot is depicted below each graph. (B) D6 PCO cultures were fixed and immunostained for αSMA and DAPI. Cells were detected expressing αSMA on the posterior capsule and within the cells remaining on the flap. Scale bar, 100 μm.
Figure 4.
 
Inhibition of Src family kinases blocks migration toward the posterior capsule. (A) Graph depicts the ratio of active SFKs (pSrc418) to Src in PCO cultures treated with DMSO (vehicle control) or 10 μM PP1 at 10 minutes and 30 minutes after preparation of the cultures. Data were normalized to 10 minutes DMSO treatment. An average of three experiments was quantified using 1D software (Eastman Kodak). PP1 effectively suppressed early activation of SFKs in PCO culture. (B) PCO cultures were treated with vehicle control DMSO (C) or PP1 throughout the duration of the culture period. Migration was followed by photomicroscopy, or cell lysates were collected and immunoblotted for pSrc (Src418) and Src. PP1 inhibited the migration of lens cells to the posterior capsule and blocked activation of Src kinases.
Figure 4.
 
Inhibition of Src family kinases blocks migration toward the posterior capsule. (A) Graph depicts the ratio of active SFKs (pSrc418) to Src in PCO cultures treated with DMSO (vehicle control) or 10 μM PP1 at 10 minutes and 30 minutes after preparation of the cultures. Data were normalized to 10 minutes DMSO treatment. An average of three experiments was quantified using 1D software (Eastman Kodak). PP1 effectively suppressed early activation of SFKs in PCO culture. (B) PCO cultures were treated with vehicle control DMSO (C) or PP1 throughout the duration of the culture period. Migration was followed by photomicroscopy, or cell lysates were collected and immunoblotted for pSrc (Src418) and Src. PP1 inhibited the migration of lens cells to the posterior capsule and blocked activation of Src kinases.
Figure 5.
 
Inhibition of Src family kinases inhibits proliferation and EMT in the chick embryo capsular bag model. Graphs depict relative expression of PCNA, FN, αSMA, and β-actin on D3 and D6 in the absence and presence of PP1. PP1 suppressed PCNA, FN, and αSMA expression. An average of 3 experiments was quantified with 1D software (Eastman Kodak), and relative densities were plotted in graphs ± SEM. Data were normalized to D6 vehicle control samples for FN and αSMA and to D3 vehicle control samples for PCNA and β-actin. Representative Western blot is depicted below each graph.
Figure 5.
 
Inhibition of Src family kinases inhibits proliferation and EMT in the chick embryo capsular bag model. Graphs depict relative expression of PCNA, FN, αSMA, and β-actin on D3 and D6 in the absence and presence of PP1. PP1 suppressed PCNA, FN, and αSMA expression. An average of 3 experiments was quantified with 1D software (Eastman Kodak), and relative densities were plotted in graphs ± SEM. Data were normalized to D6 vehicle control samples for FN and αSMA and to D3 vehicle control samples for PCNA and β-actin. Representative Western blot is depicted below each graph.
Figure 6.
 
Inhibition of SFKs causes an accumulation of β-catenin containing cadherin junctions at cell–cell interfaces and maintenance of an epithelial cell phenotype. PCO cultures were cultured from D0 in the presence or absence of 10 μM PP1. On D3, control and PP1-treated cultures were fixed and immunostained for the cadherin complex protein β-catenin (A, D, G) and were costained for F-actin with fluorescent phalloidin (B, E, H). Overlays of β-catenin and F-actin (C, F, I). In control treated capsular bags, epithelial cells located on the flap were cuboidal and maintained β-catenin at their cell–cell interfaces, whereas those lens cells that had migrated to the posterior capsule appeared more disorganized and lost their epithelial morphology and tight cuboidal packing but still labeled for β-catenin at cell–cell interfaces. PCO cultures treated with PP1 exhibited greater intensity of β-catenin staining at cell–cell interfaces than untreated cultures and maintained an epithelial cell phenotype. Smaller images depict the entire lens capsular bag treated with vehicle control (upper) or PP1 (lower). White boxes: regions of the lens capsular bag examined at higher magnification throughout the figure. Images are presented as projection images of 1.98 μm optical sections from z-stacks. Scale bar, 50 μm.
Figure 6.
 
Inhibition of SFKs causes an accumulation of β-catenin containing cadherin junctions at cell–cell interfaces and maintenance of an epithelial cell phenotype. PCO cultures were cultured from D0 in the presence or absence of 10 μM PP1. On D3, control and PP1-treated cultures were fixed and immunostained for the cadherin complex protein β-catenin (A, D, G) and were costained for F-actin with fluorescent phalloidin (B, E, H). Overlays of β-catenin and F-actin (C, F, I). In control treated capsular bags, epithelial cells located on the flap were cuboidal and maintained β-catenin at their cell–cell interfaces, whereas those lens cells that had migrated to the posterior capsule appeared more disorganized and lost their epithelial morphology and tight cuboidal packing but still labeled for β-catenin at cell–cell interfaces. PCO cultures treated with PP1 exhibited greater intensity of β-catenin staining at cell–cell interfaces than untreated cultures and maintained an epithelial cell phenotype. Smaller images depict the entire lens capsular bag treated with vehicle control (upper) or PP1 (lower). White boxes: regions of the lens capsular bag examined at higher magnification throughout the figure. Images are presented as projection images of 1.98 μm optical sections from z-stacks. Scale bar, 50 μm.
Figure 7.
 
Early SFK activation induces PCO. (A) PCO cultures were treated with vehicle control (DMSO) or 10 μM PP1 only during the first 0 to 5 minutes or the first day of the culture period. Lens cell migration was followed by photomicroscopy at D0, D2, and D9. Inhibition of SFKs for 0 to 5 minutes and for the first day of culture slowed migration onto the posterior capsule, which was evident on D2. (B) Cell lysates were collected on D9 and immunoblotted for expression of FN, αSMA, and β-actin (loading control). Short-term treatment with PP1 was sufficient to suppress the expression of FN and αSMA.
Figure 7.
 
Early SFK activation induces PCO. (A) PCO cultures were treated with vehicle control (DMSO) or 10 μM PP1 only during the first 0 to 5 minutes or the first day of the culture period. Lens cell migration was followed by photomicroscopy at D0, D2, and D9. Inhibition of SFKs for 0 to 5 minutes and for the first day of culture slowed migration onto the posterior capsule, which was evident on D2. (B) Cell lysates were collected on D9 and immunoblotted for expression of FN, αSMA, and β-actin (loading control). Short-term treatment with PP1 was sufficient to suppress the expression of FN and αSMA.
Figure 8.
 
Dose effectiveness of PP1. (A) PCO cultures were treated with vehicle control (DMSO) or 1, 3, or 10 μM PP1. Lens cell migration was followed by photomicroscopy and shown here at D1 and D3. PP1 at 3 μM and 10 μM, but not at 1 μM, prevented lens cells from covering the posterior capsule. (B) Cell lysates were collected on D6 and examined by immunoblotting for FN, αSMA, β-actin, pSrc, and Src. A dose-dependent decrease in active Src was observed with increasing amounts of PP1 treatment. Treatment of PCO cultures with 1, 3, and 10 μM PP1 suppressed the expression of FN and αSMA.
Figure 8.
 
Dose effectiveness of PP1. (A) PCO cultures were treated with vehicle control (DMSO) or 1, 3, or 10 μM PP1. Lens cell migration was followed by photomicroscopy and shown here at D1 and D3. PP1 at 3 μM and 10 μM, but not at 1 μM, prevented lens cells from covering the posterior capsule. (B) Cell lysates were collected on D6 and examined by immunoblotting for FN, αSMA, β-actin, pSrc, and Src. A dose-dependent decrease in active Src was observed with increasing amounts of PP1 treatment. Treatment of PCO cultures with 1, 3, and 10 μM PP1 suppressed the expression of FN and αSMA.
Figure 9.
 
A single dose of PP1 can prevent PCO. (A) PCO cultures were treated with a single dose of vehicle control (0, DMS0) or PP1 (3 or 10 μM). Lens cell migration was followed by photomicroscopy and documented here at D1 and D3. PP1 at 3 μM or 10 μM was sufficient to prevent the lens cells from covering the posterior capsule. (B) Cell lysates were collected on D6 and examined by immunoblotting for FN, αSMA, β-actin, pSrc, and Src. A single dose of PP1 at 3 or 10 μM lowered the activation level of Src kinases and effectively suppressed the expression of FN and αSMA.
Figure 9.
 
A single dose of PP1 can prevent PCO. (A) PCO cultures were treated with a single dose of vehicle control (0, DMS0) or PP1 (3 or 10 μM). Lens cell migration was followed by photomicroscopy and documented here at D1 and D3. PP1 at 3 μM or 10 μM was sufficient to prevent the lens cells from covering the posterior capsule. (B) Cell lysates were collected on D6 and examined by immunoblotting for FN, αSMA, β-actin, pSrc, and Src. A single dose of PP1 at 3 or 10 μM lowered the activation level of Src kinases and effectively suppressed the expression of FN and αSMA.
The authors thank Ni Zhai for her excellent technical assistance with the confocal imaging, and Michelle Leonard for critical reading of the manuscript. 
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Figure 1.
 
Chick embryo lens capsular bag model. Lenses from E15 chicken embryos (A) are dissected, and a small cut is made in the anterior capsule where a needle is inserted to hydrostatically remove the fiber cell mass, leaving behind lens epithelial cells that are tightly attached to the capsule (B). This procedure mimics cataract surgery. Cuts are then made in the anterior region of the capsule (C), and individual flaps of the capsule containing lens epithelial cells are flattened and pinned onto the tissue culture dish capsule side down (D, E), allowing visualization of the lens epithelial cells as they migrate onto the posterior capsule (F). PC, posterior capsule.
Figure 1.
 
Chick embryo lens capsular bag model. Lenses from E15 chicken embryos (A) are dissected, and a small cut is made in the anterior capsule where a needle is inserted to hydrostatically remove the fiber cell mass, leaving behind lens epithelial cells that are tightly attached to the capsule (B). This procedure mimics cataract surgery. Cuts are then made in the anterior region of the capsule (C), and individual flaps of the capsule containing lens epithelial cells are flattened and pinned onto the tissue culture dish capsule side down (D, E), allowing visualization of the lens epithelial cells as they migrate onto the posterior capsule (F). PC, posterior capsule.
Figure 2.
 
Chick embryo lens capsular bag model mimics PCO: induction of migration and proliferation. (A) After mock cataract surgery, lens capsules were pinned down and lens epithelial migration onto the posterior capsule (PC) was examined from D0 to D6 by photomicroscopy with a digital camera and dissecting microscope. White arrow: lens capsule flap containing lens epithelial cells. Lens cells have already begun to migrate toward the posterior capsule as early as D1 and typically cover the posterior capsule by D3. (B) PCO cultures were extracted at 0 hour, 10 minutes, D1, and D6, and cell lysates were analyzed by immunoblotting for phospho-FAK Y397 and β-actin (loading control). An increase in FAK activation was observed as the cells migrated toward the posterior capsule. (C) PCO cultures were fixed on D1 and D9 and immunostained for PCNA and DAPI. Nuclear staining for PCNA was detected in lens cells migrating toward the posterior capsule on D1 but was not detected on D9. Scale bar, 100 μm. (D) PCO cultures were extracted on the days indicated, and the cell lysates were analyzed by immunoblotting for PCNA and β-actin (loading control). Similar to our localization studies for PCNA, PCNA was highly expressed early after mock surgery and decreased to very low levels as cells completed their migration to the posterior capsule. EQ, equatorial region of the capsule.
Figure 2.
 
Chick embryo lens capsular bag model mimics PCO: induction of migration and proliferation. (A) After mock cataract surgery, lens capsules were pinned down and lens epithelial migration onto the posterior capsule (PC) was examined from D0 to D6 by photomicroscopy with a digital camera and dissecting microscope. White arrow: lens capsule flap containing lens epithelial cells. Lens cells have already begun to migrate toward the posterior capsule as early as D1 and typically cover the posterior capsule by D3. (B) PCO cultures were extracted at 0 hour, 10 minutes, D1, and D6, and cell lysates were analyzed by immunoblotting for phospho-FAK Y397 and β-actin (loading control). An increase in FAK activation was observed as the cells migrated toward the posterior capsule. (C) PCO cultures were fixed on D1 and D9 and immunostained for PCNA and DAPI. Nuclear staining for PCNA was detected in lens cells migrating toward the posterior capsule on D1 but was not detected on D9. Scale bar, 100 μm. (D) PCO cultures were extracted on the days indicated, and the cell lysates were analyzed by immunoblotting for PCNA and β-actin (loading control). Similar to our localization studies for PCNA, PCNA was highly expressed early after mock surgery and decreased to very low levels as cells completed their migration to the posterior capsule. EQ, equatorial region of the capsule.
Figure 3.
 
EMT is induced in chick embryo capsular bag model. (A) Graphs depicting Western blot studies show relative expression of FN, αSMA, and β-actin in lens cell PCO cultures on D0, D3, and D6. Little change in β-actin was detected, whereas EMT markers FN and αSMA were highly induced by D6 in PCO cultures. Three experiments were quantified using 1D software (Eastman Kodak), and relative densities were plotted on graphs ± SEM. Data were normalized to D6 values for each graph. Error bars are present on D0 and D3 samples for the αSMA graph but are too small to be visualized in the figure. Representative Western blot is depicted below each graph. (B) D6 PCO cultures were fixed and immunostained for αSMA and DAPI. Cells were detected expressing αSMA on the posterior capsule and within the cells remaining on the flap. Scale bar, 100 μm.
Figure 3.
 
EMT is induced in chick embryo capsular bag model. (A) Graphs depicting Western blot studies show relative expression of FN, αSMA, and β-actin in lens cell PCO cultures on D0, D3, and D6. Little change in β-actin was detected, whereas EMT markers FN and αSMA were highly induced by D6 in PCO cultures. Three experiments were quantified using 1D software (Eastman Kodak), and relative densities were plotted on graphs ± SEM. Data were normalized to D6 values for each graph. Error bars are present on D0 and D3 samples for the αSMA graph but are too small to be visualized in the figure. Representative Western blot is depicted below each graph. (B) D6 PCO cultures were fixed and immunostained for αSMA and DAPI. Cells were detected expressing αSMA on the posterior capsule and within the cells remaining on the flap. Scale bar, 100 μm.
Figure 4.
 
Inhibition of Src family kinases blocks migration toward the posterior capsule. (A) Graph depicts the ratio of active SFKs (pSrc418) to Src in PCO cultures treated with DMSO (vehicle control) or 10 μM PP1 at 10 minutes and 30 minutes after preparation of the cultures. Data were normalized to 10 minutes DMSO treatment. An average of three experiments was quantified using 1D software (Eastman Kodak). PP1 effectively suppressed early activation of SFKs in PCO culture. (B) PCO cultures were treated with vehicle control DMSO (C) or PP1 throughout the duration of the culture period. Migration was followed by photomicroscopy, or cell lysates were collected and immunoblotted for pSrc (Src418) and Src. PP1 inhibited the migration of lens cells to the posterior capsule and blocked activation of Src kinases.
Figure 4.
 
Inhibition of Src family kinases blocks migration toward the posterior capsule. (A) Graph depicts the ratio of active SFKs (pSrc418) to Src in PCO cultures treated with DMSO (vehicle control) or 10 μM PP1 at 10 minutes and 30 minutes after preparation of the cultures. Data were normalized to 10 minutes DMSO treatment. An average of three experiments was quantified using 1D software (Eastman Kodak). PP1 effectively suppressed early activation of SFKs in PCO culture. (B) PCO cultures were treated with vehicle control DMSO (C) or PP1 throughout the duration of the culture period. Migration was followed by photomicroscopy, or cell lysates were collected and immunoblotted for pSrc (Src418) and Src. PP1 inhibited the migration of lens cells to the posterior capsule and blocked activation of Src kinases.
Figure 5.
 
Inhibition of Src family kinases inhibits proliferation and EMT in the chick embryo capsular bag model. Graphs depict relative expression of PCNA, FN, αSMA, and β-actin on D3 and D6 in the absence and presence of PP1. PP1 suppressed PCNA, FN, and αSMA expression. An average of 3 experiments was quantified with 1D software (Eastman Kodak), and relative densities were plotted in graphs ± SEM. Data were normalized to D6 vehicle control samples for FN and αSMA and to D3 vehicle control samples for PCNA and β-actin. Representative Western blot is depicted below each graph.
Figure 5.
 
Inhibition of Src family kinases inhibits proliferation and EMT in the chick embryo capsular bag model. Graphs depict relative expression of PCNA, FN, αSMA, and β-actin on D3 and D6 in the absence and presence of PP1. PP1 suppressed PCNA, FN, and αSMA expression. An average of 3 experiments was quantified with 1D software (Eastman Kodak), and relative densities were plotted in graphs ± SEM. Data were normalized to D6 vehicle control samples for FN and αSMA and to D3 vehicle control samples for PCNA and β-actin. Representative Western blot is depicted below each graph.
Figure 6.
 
Inhibition of SFKs causes an accumulation of β-catenin containing cadherin junctions at cell–cell interfaces and maintenance of an epithelial cell phenotype. PCO cultures were cultured from D0 in the presence or absence of 10 μM PP1. On D3, control and PP1-treated cultures were fixed and immunostained for the cadherin complex protein β-catenin (A, D, G) and were costained for F-actin with fluorescent phalloidin (B, E, H). Overlays of β-catenin and F-actin (C, F, I). In control treated capsular bags, epithelial cells located on the flap were cuboidal and maintained β-catenin at their cell–cell interfaces, whereas those lens cells that had migrated to the posterior capsule appeared more disorganized and lost their epithelial morphology and tight cuboidal packing but still labeled for β-catenin at cell–cell interfaces. PCO cultures treated with PP1 exhibited greater intensity of β-catenin staining at cell–cell interfaces than untreated cultures and maintained an epithelial cell phenotype. Smaller images depict the entire lens capsular bag treated with vehicle control (upper) or PP1 (lower). White boxes: regions of the lens capsular bag examined at higher magnification throughout the figure. Images are presented as projection images of 1.98 μm optical sections from z-stacks. Scale bar, 50 μm.
Figure 6.
 
Inhibition of SFKs causes an accumulation of β-catenin containing cadherin junctions at cell–cell interfaces and maintenance of an epithelial cell phenotype. PCO cultures were cultured from D0 in the presence or absence of 10 μM PP1. On D3, control and PP1-treated cultures were fixed and immunostained for the cadherin complex protein β-catenin (A, D, G) and were costained for F-actin with fluorescent phalloidin (B, E, H). Overlays of β-catenin and F-actin (C, F, I). In control treated capsular bags, epithelial cells located on the flap were cuboidal and maintained β-catenin at their cell–cell interfaces, whereas those lens cells that had migrated to the posterior capsule appeared more disorganized and lost their epithelial morphology and tight cuboidal packing but still labeled for β-catenin at cell–cell interfaces. PCO cultures treated with PP1 exhibited greater intensity of β-catenin staining at cell–cell interfaces than untreated cultures and maintained an epithelial cell phenotype. Smaller images depict the entire lens capsular bag treated with vehicle control (upper) or PP1 (lower). White boxes: regions of the lens capsular bag examined at higher magnification throughout the figure. Images are presented as projection images of 1.98 μm optical sections from z-stacks. Scale bar, 50 μm.
Figure 7.
 
Early SFK activation induces PCO. (A) PCO cultures were treated with vehicle control (DMSO) or 10 μM PP1 only during the first 0 to 5 minutes or the first day of the culture period. Lens cell migration was followed by photomicroscopy at D0, D2, and D9. Inhibition of SFKs for 0 to 5 minutes and for the first day of culture slowed migration onto the posterior capsule, which was evident on D2. (B) Cell lysates were collected on D9 and immunoblotted for expression of FN, αSMA, and β-actin (loading control). Short-term treatment with PP1 was sufficient to suppress the expression of FN and αSMA.
Figure 7.
 
Early SFK activation induces PCO. (A) PCO cultures were treated with vehicle control (DMSO) or 10 μM PP1 only during the first 0 to 5 minutes or the first day of the culture period. Lens cell migration was followed by photomicroscopy at D0, D2, and D9. Inhibition of SFKs for 0 to 5 minutes and for the first day of culture slowed migration onto the posterior capsule, which was evident on D2. (B) Cell lysates were collected on D9 and immunoblotted for expression of FN, αSMA, and β-actin (loading control). Short-term treatment with PP1 was sufficient to suppress the expression of FN and αSMA.
Figure 8.
 
Dose effectiveness of PP1. (A) PCO cultures were treated with vehicle control (DMSO) or 1, 3, or 10 μM PP1. Lens cell migration was followed by photomicroscopy and shown here at D1 and D3. PP1 at 3 μM and 10 μM, but not at 1 μM, prevented lens cells from covering the posterior capsule. (B) Cell lysates were collected on D6 and examined by immunoblotting for FN, αSMA, β-actin, pSrc, and Src. A dose-dependent decrease in active Src was observed with increasing amounts of PP1 treatment. Treatment of PCO cultures with 1, 3, and 10 μM PP1 suppressed the expression of FN and αSMA.
Figure 8.
 
Dose effectiveness of PP1. (A) PCO cultures were treated with vehicle control (DMSO) or 1, 3, or 10 μM PP1. Lens cell migration was followed by photomicroscopy and shown here at D1 and D3. PP1 at 3 μM and 10 μM, but not at 1 μM, prevented lens cells from covering the posterior capsule. (B) Cell lysates were collected on D6 and examined by immunoblotting for FN, αSMA, β-actin, pSrc, and Src. A dose-dependent decrease in active Src was observed with increasing amounts of PP1 treatment. Treatment of PCO cultures with 1, 3, and 10 μM PP1 suppressed the expression of FN and αSMA.
Figure 9.
 
A single dose of PP1 can prevent PCO. (A) PCO cultures were treated with a single dose of vehicle control (0, DMS0) or PP1 (3 or 10 μM). Lens cell migration was followed by photomicroscopy and documented here at D1 and D3. PP1 at 3 μM or 10 μM was sufficient to prevent the lens cells from covering the posterior capsule. (B) Cell lysates were collected on D6 and examined by immunoblotting for FN, αSMA, β-actin, pSrc, and Src. A single dose of PP1 at 3 or 10 μM lowered the activation level of Src kinases and effectively suppressed the expression of FN and αSMA.
Figure 9.
 
A single dose of PP1 can prevent PCO. (A) PCO cultures were treated with a single dose of vehicle control (0, DMS0) or PP1 (3 or 10 μM). Lens cell migration was followed by photomicroscopy and documented here at D1 and D3. PP1 at 3 μM or 10 μM was sufficient to prevent the lens cells from covering the posterior capsule. (B) Cell lysates were collected on D6 and examined by immunoblotting for FN, αSMA, β-actin, pSrc, and Src. A single dose of PP1 at 3 or 10 μM lowered the activation level of Src kinases and effectively suppressed the expression of FN and αSMA.
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