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Posterior Capsule Opacification: A Cell Biological Perspective
Exp. Eye Res. (2002) 74, 337±347 doi:10.1006/exer.2001.1153, available online at http://www.idealibrary.com on
REVIEW Posterior Capsule Opaci®cation: A Cell Biological Perspective I . M I C H A E L WO R M S TO N E * School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, U.K. (Received Rochester 7 November 2001 and accepted in revised form 13 December 2001)
Key words: posterior capsule opaci®cation; cataract; lens; wound healing; growth.
Introduction Posterior capsule opaci®cation (PCO) is the most common complication of cataract surgery. At present the only means of treating cataract is by surgical intervention, and this initially restores high visual quality. Unfortunately, PCO develops in a signi®cant proportion of patients to such an extent that a secondary loss of vision occurs, which consequently requires further corrective laser surgery (Moisseiev et al., 1989 Sudhakar, Ravindran and Natchiar, 1989; Knight-Nanan, O'Keefe and Bowell, 1996; Schaumberg et al., 1998; Sundelin and Sjostrand, 1999) which is both expensive and not without risk (Ranta and Kivela, 1998). A modern cataract operation generates a capsular bag (Fig. 1(A)) which comprises a proportion of the anterior and the entire posterior capsule. The bag remains in situ, partitions the aqueous and vitreous humours, and in the majority of cases, houses an intraocular lens. The production of a capsular bag following surgery permits a free passage of light along the visual axis through the transparent intraocular lens and thin acellular posterior capsule. However, on the remaining anterior capsule, lens epithelial cells stubbornly reside despite enduring the rigours of surgical trauma. This resilient group of cells then begin to re-colonize the denuded regions of the anterior capsule, encroach onto the intraocular lens surface, occupy regions of the outer anterior capsule and most importantly of all begin to colonize the previously cell-free posterior capsule. Cells continue to divide, begin to cover the posterior capsule and can ultimately encroach on the visual axis (Fig. 1(B)). A thin cover of cells is insuf®cient to affect the light path, * Address correspondence to: I. M. Wormstone, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K. E-mail: [email protected]
0014-4835/02/03033711 $35.00/0
but subsequent changes to the matrix and cell organization can give rise to light scatter (Fig. 1(C)). If these changes are suf®ciently severe, vision is then seriously impaired and corrective surgery required. Prevalence of PCO The incidence of PCO reported is generally based on the requirement for follow-up treatment as a marker (Moisseiev et al., 1989; Sudhakar et al., 1989; KnightNanan et al., 1996; Schaumberg et al., 1998; Sundelin and Sjostrand, 1999). A relatively short-term study by Sudhakar et al. (1989) reported that 115 (11.5 %) of 1000 patients developed PCO 1 year following cataract surgery. Furthermore, it has been reported that the incidence of PCO requiring surgery increases with time after surgery (Schaumberg et al., 1998). Another useful study, carried out by Moisseiev et al. (1989), showed that the incidence of PCO 4 years after surgery is 41 %. This study also addressed the in¯uence of age on PCO incidence. The data revealed that the percentage of patients requiring secondary treatment was 37 % in a group of patients aged 4 60 years and 70 % when aged under 40 years. Paediatric patients are reported to have a rapid development of PCO (KnightNanan et al., 1996). This age-related phenomenon forms a rational basis for delaying the time at which cataract surgery is performed. A number of clinical studies have addressed the in¯uence of different IOLs on the incidence of PCO (Frezzotti and Caporossi, 1990; Nagata and Watanabe, 1996; Ursell et al., 1998; Hollick et al., 1999; Hayashi et al., 2001; Scaramuzza, Fernando and Crayford, 2001). The general theme is that an IOL can in¯uence the progression of PCO, and it is likely that this in¯uence is of a physical nature (Nagata and Watanabe, 1996; Nishi, Nishi and Sakanishi, 1998; Nishi, Nishi and Wickstrom, 2000). However, whilst the development of IOLs has helped reduce the incidence of PCO requiring corrective # 2002 Elsevier Science Ltd.
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F IG . 1. A schematic representation of (A) the post surgical capsular bag and (B) the extensive growth and modi®cation that gives rise to PCO. (C) A dark-®eld micrograph of a capsular bag removed from a donor eye that had undergone cataract surgery prior to death that exhibits light scattering regions beneath an intraocular lens.
surgery, perhaps by delaying the development of PCO, the problem is far from being eradicated. Furthermore, it should not be immediately assumed that a person who does not undergo corrective laser therapy has optimal clear vision. Laser therapy is employed when visual loss severely affects the life of an individual. Using laser capsulotomy rates as a barometer of PCO incidence is a useful tool, but a number of patients who do not undergo this secondary surgery are still likely to have a diminished visual quality, compared with the initial surgical result. Sundelin and Sjostrand (1999) examined this group of patients and found that a signi®cant proportion would bene®t from laser capsulotomy. In the vast majority of cases, if not all, lens epithelial cells will remain attached to the capsule following cataract surgery and therefore the sequence of events that give rise to PCO will commence. While the introduction of an IOL will play a part, the degree this will affect the patient is dependent on the regulation of
the cells themselves. The remainder of this review will therefore largely concentrate on the cell biological processes involved in PCO and the factors that in¯uence these events. 2. Methods Several approaches have been used to study PCO. These include cell culture studies (McDonnell, Krause and Glaser, 1988; McAvoy and Chamberlain, 1989; Ibaraki, Lin and Reddy, 1995; Kurosaka et al., 1995; Nishi et al., 1996a; Oharazawa et al., 1999; Wormstone et al., 2000); in vivo animal studies (Cobo et al., 1984; Behar-Cohen et al., 1995); capsular bag models (Nagamoto and Bissen-Miyajima, 1994; Liu et al., 1996; Wormstone et al., 1997; Saxby, Rosen and Boulton, 1998; Davidson et al., 2000b); in vivo observations (Ursell et al., 1998; Hollick et al., 1999); and analysis of post-mortem material (Kappelhof et al., 1987; Marcantonio et al., 2000; Saika et al., 2000;
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Wunderlich et al., 2000; Wormstone et al., 2000, 2001a). All these systems have strengths and weaknesses, but in concert they provide a powerful collection of tools to investigate PCO. Cell Culture Studies This is the simplest method of study and generally utilizes cell lines or primary cultures derived from native tissue to analyse growth characteristics. These experiments can therefore identify which factors can stimulate or inhibit growth. The drawbacks of using cell cultures are that the sub-structure of the growth matrix and the culture medium composition determine, to a large extent, not only the growth rate, but also the molecular characteristics of the cells. For example, native human lens epithelial cells express mainly the M1 muscarinic receptor subtype whereas the human lens epithelial cell line HLE-B3 expresses the M3 subtype (Collison et al., 2000). In addition, Krausz et al. (1996) have analysed the expression patterns of lens speci®c proteins or proteins expressed preferentially by lens cells using RT-PCR techniques. This work revealed that in some cases aA-crystallin was not expressed; however aB-crystallin and Pax6, the so-called eye master gene, were detected in the cell lines tested. Proteins associated with ®bre cells were not expressed under standard culture conditions. However, cell lines remain a valuable experimental tool. It would seem that their main uses are ®rst to provide indication of which molecules are potentially worthy of study, and moreover the concentrations at which they are effective. Secondly, once a system has been validated in a more complex experimental model or by circumstantial evidence obtained with postmortem material, then the more readily available cell lines are particularly good systems to investigate thoroughly speci®c regulatory pathways. In vivo Animal Studies The most commonly used model for this type of work is the rabbit. The main drawback with this approach, apart from ethical considerations, is that cell growth in animals such as a rabbit or cat may be different to that in primates. Furthermore, different species have been shown to react in different ways to primates following trauma to the eye (Bito, 1984). It is also dif®cult to study with any detail the progression of PCO in these systems with much of the information obtained from detailed end-point examinations. Capsular Bag Models This method of study has emerged in recent years and is based on a sham cataract operation. In effect a capsular bag is produced that is identical to that generated in vivo (Fig. 2). This system therefore has the
F IG . 2. Low power dark-®eld views of capsular bag preparations pinned out and immersed in culture medium. In the absence of an IOL (a) the disc shaped opening in the anterior capsule can clearly be seen, revealing the posterior beneath. When an IOL is present (b) the supporting loops (haptics) can distend the capsular bag and two creases (arrows) can be seen on the posterior capsule. The micrographs represent a ®eld of view of 1.8 1.25 cm. (Liu et al., 1996).
correct matrix i.e. the capsule and cellular distribution. A critical aspect of these models is the maintenance of the capsular bag shape. In order to achieve this, some groups have employed capsular rings (Nagamoto and Bissen-Miyajima, 1994; Saxby et al., 1998), while others have used ®ne pins to maintain the circular shape (Liu et al., 1996; Wormstone et al., 1997; Davidson et al., 2000b). Furthermore, some of these systems permit the insertion of an IOL and therefore the role it plays can also be investigated on a day to day
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basis. This model approach has been applied to human (Nagamoto and Bissen-Miyajima, 1994; Liu et al., 1996; Wormstone et al., 1997), canine (Davidson et al., 2000b) and bovine lenses (Saxby et al., 1998). In the case of humans and canines, which routinely undergo cataract surgery, the capsular bag culture system provides at present the most directly relevant in vitro data to the clinic; however, at least in the case of humans the rate limiting step is the availability of material.
In vivo Observations Due to improvements in technology, camera systems have been developed that can detail changes to the capsular bag within the visual axis (Ursell et al., 1998; Hollick et al., 1999). A patient can then be assessed at various time points and the changes observed. While this technique cannot at present provide information at the single cell level it does provide an excellent insight into the rates of PCO progression.
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Post-mortem Analysis This provides an important understanding of which molecules are present within the capsular bag as PCO progresses. Such molecules could be matrix components, cellular markers or growth factors. Techniques used to analyse the material include electron microscopy (Kappelhof et al., 1987; Marcantonio et al., 2000), immunochemistry (Marcantonio et al., 2000; Saika et al., 2000), RT±PCR (Wunderlich et al., 2000; Wormstone et al., 2001a) and ELISA (Wormstone et al., 2000, 2001a). This information can then be utilized when designing or comparing the other experimental systems described. While this type of investigation provides `scene of the crime' information it does not provide de®nitive proof that various molecular suspects are responsible for PCO development. Cell Regulation and Function The regulation of the processes involved in PCO is derived from the signals cells receive from the
F IG . 3. A schematic representation of autocrine and paracrine signalling control mechanisms of lens cells.
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environment. The source of these regulators can be from the lens cells themselves or from other ocular tissues and therefore form autocrine and paracrine signalling systems (Fig. 3). These two mechanisms are both likely to contribute to PCO development, as an adequate supply of signals is required to sustain viability and growth. If the supply of signals is insuf®cient this will lead to cell death, occurring presumably by apoptosis (Ishizaki et al., 1993). Paracrine Control Initially the focus was on increased levels of protein in the aqueous humour following surgery, generated largely by a disrupted blood±aqueous barrier and an in¯ammatory response (Pande, Spalton and Marshall, 1996). Interestingly, it is also reported that certain proteins are only detected in the aqueous humour following trauma to the eye (Namiki et al., 1992). The resulting cocktail of peptides and proteins therefore has great potential in regulating lens cell function. A considerable number of experiments have shown that addition of numerous factors can modulate lens cells from a variety of species. Transferrin, for example is believed to act as a survival factor and data reported using canine lens cells indicated that supplementing the culture medium with 200 mg ml 1 transferrin signi®cantly increases cell survival (Davidson et al., 1998). Furthermore, data show the ability of various agents to increase proliferation rates, migration and differentiation when added to cultures. The majority of these studies investigate cytokines, including growth factors. In the case of basic FGF, it has been shown that basic FGF could induce maximal rates of proliferation, migration and differentiation of rat lens explant epithelium at 0.15, 3 and 40 ng ml 1, respectively (McAvoy and Chamberlain, 1989). A similar trend was also found with acidic FGF, but the effective concentrations were considerably greater (Chamberlain and McAvoy, 1989). Differences in the sensitivity of human primary cells were also observed such that 10-fold higher concentrations of acidic FGF were required to achieve similar results to those observed with basic FGF (Ibaraki et al., 1995). Application of EGF to cell cultures has also been shown to induce proliferation (Hongo et al., 1993; Ibaraki et al., 1995; Majima, 1995). In one study (Hongo et al., 1993), using rabbit lens epitheial cells, concentrations of 0.1±100 ng ml 1 were found to stimulate growth, which was estimated by absorbance of methylene blue staining. However, the maximal response was achieved at 10 ng ml 1. This concentration of EGF also was found to be optimal for inducing proliferation of human lens epithelial cells in early sub-culture (Ibaraki et al., 1995). This study of human lens epithelial cells also examimed the potential to induce differentiation of epitheial cells to
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®bres. This was assessed by the capacity to form lentoid bodies that express gamma crystallin. Using this system EGF was a strong promoter of cell aggregation and lentoid body formation. This could have relevance to the formation of new ®bre cells in the periphery of the bag, commonly known as Soemerring's ring (Kappelhof et al., 1987; Marcantonio et al., 2000). TGF b is a protein that is becoming increasingly studied because of its association with ®brotic disorders. It therefore could have implications in the ®brotic changes that occur in PCO. TGF b is normally detected in the aqueous humour, but the majority is in the latent form (Cousins et al., 1991). However, following trauma, e.g. by surgery, active levels can be elevated (Ohta et al., 2000). The major isoform synthesized within the eye is TGF b2, however TGF b1 can also reach the aqueous humour from the blood. The proposed role of TGF b is to serve as an immune regulator in the eye (Streilein, 1996). However, these high levels could also in¯uence lens epithelial cells. Addition of TGF b to rat explants or bovine epithelial cells cultured on a collagen raft induced contraction of the underlying matrix (Liu et al., 1994; Kurosaka et al., 1995). Furthermore, addition of TGF b has been shown to induce transdifferentiation of rat lens epithelium that can be identi®ed by alpha smooth muscle actin expression (Gordon-Thomson et al., 1998). The induction of this event was isoform-dependent with TGF b isoforms 2 and 3 being 10-fold more potent than isoform 1. Recent studies have also shown that TGF b2 can stimulate greater wrinkle formation and increased expression of transdifferentiation markers in cultured human capsular bags (Wormstone et al., 2001b). Moreover, immunocytochemical studies have shown aSMA to be expressed within the capsular bag of postmortem material (Marcantonio et al., 2000). The greatest density found in these long-term cultures was on the outer surface of the anterior capsule. Saxby et al. (1998) have also shown that aSMA is expressed in serum cultured capsular bags. As the cells migrate across the posterior capsule the greatest levels are at the growing edge and following complete cover expression becomes ubiquitous. Alpha SMA expression was also observed in serum-free cultured canine capsular bags (Davidson et al., 2000b). More evidence to suggest that TGF b is important in PCO was reported by Wunderlich et al. (2000) who showed that the connective tissue growth factor was present in the post-mortem capsular bags. Expression of this particular growth factor is often associated with TGF b. Furthermore, lens cells are known to synthesize and secrete TGF b (Allen et al., 1998). Also, Saika et al. (2000) have identi®ed immunohistochemically the presence of TGF b isoforms and receptors.
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Autocrine Control It is important to remember that the level of protein in the aqueous humour peaks shortly after surgery and slowly returns to basal levels i.e. low levels of protein within weeks or months (Kondo, Yamauchi and Nakatsu, 1995; Pande et al., 1996). However, PCO does not generally require secondary surgery until years later. This would suggest that the process of events seen in PCO has persisted some time after the initial exposure to high serum proteins has subsided. It would therefore seem that another regulatory system is involved and at this stage it would appear that autocrine control mechanisms become important. There is the possibility that the basal levels of growth factors in the aqueous humour, of which a high proportion of detectable protein is secreted by the lens, could slowly drive PCO. For example, McGahan et al. (1995) have reported that the lens cells are a major producer of the transferrin pool in the aqueous humour. Additional studies have also revealed autocrine pathways in lens cells (Majima, 1995; Lee and Joo, 1999; Wormstone et al., 2000, 2001a). In addition, there are a number of reports to indicate
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that an extensive production of cytokines and receptors occurs in the native lens (Majima, 1995; Weng et al., 1997; Lee and Joo, 1999; Wormstone et al., 2000, 2001a). However, following surgical trauma there is a modi®cation of the production levels of some proteins (Saika et al., 2000; Wormstone et al., 2000, 2001a). Ishizaki et al. (1993) in a study of rat lens cells found that the cells could survive in a protein-free medium. They also concluded that the ability to survive, and avoid undergoing cell death by apoptosis, was dependent on the underlying matrix and cell density. Furthermore, Wormstone et al. (1997) have shown using a human capsular bag model that lens cells maintained in serum-free medium without added proteins not only survive the surgical disruption but also actively proliferate and grow. The importance of this ®nding indicates autocrine regulation that is independent of added stimuli. A similar ability to grow actively in minimal conditions has also been reported in cultured bovine (Saxby et al., 1998) and canine (Davidson et al., 2000b) capsular bags. Wormstone et al. (2001a) also showed that cells residing on the posterior capsule can maintain phenotypic markers
F IG . 4. Dark-®eld (A and D) and phase contrast (B, C, E and F) images of cells within capsular bags cultured for 4 100 days in protein-free medium (A±C) and within a capsular bag removed from a donor eye that had previously undergone a cataract operation (D±F). (A) A modi®ed dark-®eld micrograph of a capsular bag cultured for 137 days. The circular outline of the anterior capsule edge (rhexis region) is clearly visible (white arrows). The anterior capsule (AC) has a consistent, smooth appearance and the homogeneous cell population is only visible using phase optics (see Fig. 1(B)). The cells on the posterior capsule are more apparent and there are also small cell-free areas (see Fig. 1(C)). Light scattering wrinkles are also apparent. (B) Phase image of cells that have re-colonized the anterior capsule of a capsular bag cultured for 465 days. Note that the cells still maintain a regular cobblestone appearance throughout this time. (C) Phase micrograph of cells growing on the central region of the posterior capsule from the same donor as 1(A). The cell distribution is heterogeneous with cells lying along wrinkles, whilst other areas are cell-free (stars). (D) The region in focus lies on the posterior capsule beneath the plastic intraocular lens (IOL) which was inserted at the time of surgery. Cell aggregations (*), cell-free areas (black arrows) and capsular wrinkling (white arrows) are all apparent. (E) In this phase image of the anterior capsule, large areas are out of focus due to the presence of the IOL. However, the regular cobblestone appearance of the anterior cells can be seen in areas within the focal plane (arrows). Additionally, there is a build-up of cellular material lying along the IOL support (*). (F) A higher power image of Fig. 4(E) that more clearly shows the cobblestone appearance of the epithelial cells on the anterior capsule. (Wormstone et al., 2001a).
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such as vimentin and alpha crystallin. However, on the posterior capsule cells maintained for 4 100 days showed some sites of regression. This pattern is similar to that seen on the posterior capsule of post-mortem capsular bags (Fig. 4). Interestingly, the cells remaining on the anterior capsule exhibited a regular cobblestone appearance and this was also the cell pattern observed on post-mortem bags (Fig. 4). Moreover, de novo protein synthesis was determined by 35S-methionine incorporation in both long-term capsular bag cultures and cultured post-mortem material. The ability of lens cells to synthesize the proteins required for survival and growth may be related to the restriction of nutrients to the surrounding humour by the blood±ocular barrier. As a consequence of the potentially de®cient environment the lens may have adapted by producing the necessary proteins to sustain itself rather than rely on scavenging the otherwise limited resources available. Other tissues residing behind barrier systems, e.g. the blood±brain barrier, have also been shown to display autocrine mechanisms to aid survival in a similar manner to the lens (Espinosa de los Monteros et al., 1990). Proliferation, in association with migration, is an important aspect of wound healing and PCO. Some interesting data have been reported using the BrdU labelling and detection technique to identify dividing cells (Rakic, Galand and Vrensen, 1997, 2000; Wormstone et al., 1997). In one study (Rakic et al., 1997), it was shown that simply performing a small capsulorhexis could up-regulate the rate of division in the lens relative to the intact lens. Moreover if the ®bres were removed, the rate of cell division was stimulated even more. Analysis of capsular bags following 3 days culture in serum-free medium showed increased numbers of positive cells (Wormstone et al., 1997). The distribution of these positive cells was predominately in the equatorial region, the natural site of division; however some cells were observed on the anterior capsule. Furthermore, if the capsular bags from aged donors (460 years) were established in the presence of serum, then the dividing population was increased, but the distribution pattern remained the same. It is notable 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, supplementing the medium with 10 % 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 in the young (540 years). These data also re¯ect the increased requirement for secondary surgery to treat PCO in young patients (Moisseiev et al., 1989; Knight-Nanan et al., 1996). Furthermore, the ®ndings suggest that powerful autocrine mechanisms in the young are active following surgical trauma. In addition, Rakic et al. (2000)
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examined post-mortem capsular bags, which were then cultured and ®xed following varying periods of culture. BrdU analysis showed that the divisional rate was similar to that previously observed in the whole lens; however, disruption to the capsular bag by removal of an IOL and residual material led to a 10fold increase in division. Other reports have also shown that in some cases YAG capsulotomy can induce a cellular reaction that can lead to coverage of the ablated region (Jones, McLeod and Boulton, 1995). Identi®cation of the autocrine signalling systems active within the capsular bag is beginning to emerge. Previous work on the native epithelium has shown that complete autocrine systems are in place. For example expression of EGF and EGFR message in addition to translated proteins in human lens epithelium have been reported (Majima, 1995; Lee and Joo, 1999). However, using capsular bag culture systems, which signalling components are present at various stages after surgery can be identi®ed and their relative roles in PCO progression can be tested. Basic FGF and FGF receptor 1, the elements of a potential autocrine pathway, were identi®ed using RT±PCR in cultured human lens capsular bags (Wormstone et al., 2001a). Basic FGF was also detected using ELISA. Interestingly, the degree of message for both the ligand and receptor appeared to be greater in cultured bags compared with native human lens epithelium. However, this study also reported that capsular bags removed from donors who had previously undergone cataract surgery also exhibited message for FGF and FGFR-1. The levels detected were similar to cultured capsular bags. The functional role of this autocrine system was tested, by blocking FGF receptor-1 using a speci®c inhibitor SU5402, which caused a retardation of growth. Hepatocyte growth factor and its receptor c-met have also been detected in capsular bags using ELISA and immunocytochemistry (Wormstone et al., 2000). HGF was also identi®ed by ELISA in capsular bags removed from donors who had previously undergone cataract surgery. Interestingly, actively growing cells on both the anterior and posterior capsules exhibited an elevated expression of receptor following surgical trauma compared to the expression in the native epithelium. Furthermore, high levels of HGF were detected in the medium in the ®rst few days of culture and declined with time. A similar pattern has also been observed with basic FGF (Wormstone et al., 2001a). Extracellular Matrix The importance of the lens capsule in the regulation of PCO is becoming more evident. The capsule is composed of a number of elements including collagen type IV and laminin, but as PCO progresses it is known that other matrix components, such as
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collagen types I, are actively synthesized (Wunderlich et al., 2000). Oharazawa et al. (1999) have investigated the role of ECM components in cell adhesion, proliferation and migration of a human lens cell line. Cell attachment was signi®cantly enhanced when cells were seeded onto matrix-coated dishes as opposed to non-coated dishes; however differences between the matrices tested were observed. Interestingly, maintenance on matrix-coated dishes did not enhance proliferation, but did increase rates of migration. However, it should be noted that a decrease in the overall cell number was observed when cells were cultured on ®bronectin or collagen. In contrast, when cultured on laminin, cell numbers increased. Parmigiani and McAvoy (1991) also showed that cells from all ages were capable of growing onto laminin, whereas growth onto ®bronectin was age-dependent. Lens epithelial cells are linked to the underlying matrix by cell adhesion molecules such as integrins. Immunocytochemical analysis of human anterior discs removed at the time of cataract surgery has identi®ed a2, a3, a5, b1 and b2 integrins (Nishi et al., 1997; Zhang et al., 2000). Furthermore, the importance of the b1 integrin has been validated by the addition of a monoclonal antibody. Using this tool, the number of viable cells remaining after 2 weeks of culture on collagen or laminin-coated dishes was reduced by the antibody in a dose-dependent manner (Nishi et al., 1997). Another interesting family of proteins that can regulate the matrix is the matrix metalloproteinases (MMPs). These enzymes are involved in normal physiological processes, such as morphogenesis, but also have been implicated in pathological conditions (Birkedal-Hansen, 1995). The most widely studied members of the MMP family in the eye are the gelatinases, and these are found in both the aqueous and vitreous humour (El-Shabrawi, Christen and Foster, 2000; Vaughan-Thomas, Gilbert and Duance, 2000). When examining the native human lens epithelium, Smine and Plantner (1997) could not detect MMP-2 (Gelatinase A), whereas MMP-9 (Gelatinase B) was not investigated. However, MT1MMP was identi®ed. This member of the family has been shown to convert pro-MMP-2 to its active form. Interestingly, Tamiya et al. (2000) have reported that neither MMP-2 nor -9 can be detected using gelatin zymography in the culture medium of clear cultured lenses; however following a sham cataract operation high levels were detected. Furthermore, Richiert and Ireland (1999) have shown that chick lens annular pad cells cultured on plastic, collagen type IV, ®bronectin or laminin do not express MMP-2 or -9 unless stimulated with TGF b or PDGF. Work carried out on the human capsular bag also shows that TGF b2 can sustain elevated levels of MMP-2 and -9 in the medium (Duncan et al., 2001). Furthermore, EGF was also found to stimulate expression of MMP-2, but not -9.
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Prevention of PCO A variety of different approaches have been applied to prevent PCO. At the surgical level attempts were made to polish the lens capsule during surgery and thus effectively eliminate the lens cell epithelial population. However, it now seems that total removal of the cells by this method is virtually impossible and even a small residual cell population can repopulate the posterior capsule (Davidson, Morgan and McGahan, 2000a). Therefore, the major area of development on the surgical front is in the design of intraocular lenses. These are constructed from many materials (Hollick et al., 1999; Apple et al., 2001; Hayashi et al., 2001; Scaramuzza et al., 2001). It would appear that the major bene®ts of an IOL in PCO prevention are its physical nature and it is reported that sharp edges at the optic edge or on capsular rings can create a barrier that will retard cell growth onto the posterior capsule (Nagata and Watanabe, 1996; Nishi et al., 1998). This improved design is undoubtedly bene®cial to the patient, but nevertheless YAG capsulotomy is still carried out on patients who have been implanted with these lenses, albeit at a lower incidence (Hayashi et al., 2001; Scaramuzza et al., 2001). A number of pharmacological antagonists have been proposed to inhibit lens cell growth leading to PCO, most of which have been tested in vitro (McDonnell et al., 1988; Behar-Cohen et al., 1995; Nishi et al., 1996b; Duncan et al., 1997). At the clinical level there are at present three major ways in which a pharmacological antagonist can be delivered to the target cells. These are by direct injection into the anterior chamber, modi®cation of the irrigating medium or by modi®cation of the IOL. The problem associated with any drug delivery is toxicity to other tissues, especially the corneal endothelium. At present the most likely mode of delivery is via the IOL. This could provide a more controlled means of drug delivery that can concentrate an agent at the site of the potentially proliferating cells more ef®ciently. In fact, modi®cation of the supporting loops of the IOL (known as haptics) to carry cytotoxic agents would allow direct delivery to the equatorial cells. The nature of drug attachment must be suf®ciently strong to prevent signi®cant leakage into surrounding tissues, yet suf®ciently weak to permit accumulation into contiguous cells. Behar-Cohen et al. (1995) have reported the effects of an FGF-saporin complex bound to a heparin surface modi®ed IOL in rabbits. When in contact with the cells the FGF binds to the appropriate receptors on the epithelial cells and internalizes the saporin, subsequently killing the cells. This particular system produced some side effects, including transient corneal oedema and iris depigmentation. Duncan et al. (1997) have also shown using a human capsular bag model that thapsigargin, a speci®c inhibitor of the endoplasmic reticulum CaATPase, when directly coated onto a PMMA IOL can effectively kill the
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entire cell population within the bag. However, this is a simple coating procedure and does not account for additional stresses associated with in vivo surgery, e.g. entry through the corneal/scleral tunnel. Therefore a drug release system that is both physically protective and provides appropriate drug delivery is necessary. Some studies have employed sustained drug delivery tools (Nishi et al., 1996b). However, the materials used are often opaque and therefore cannot be placed within the visual axis. Nevertheless such mechanisms could be used in the form of capsular rings or even in haptic production. 3. Conclusions PCO affects large numbers of cataract patients and is a condition that essentially arises from lens cell growth and gives rise to light scattering modi®cation of the posterior capsule. The development of PCO is dependent on both the increased levels of proteins in the aqueous humour shortly following surgery and the ability of lens cells to drive events persistently by autocrine mechanisms due to their association with the capsule. It is therefore essential to employ the methods available to identify both the paracrine and autocrine signals that underpin the various stages of PCO. This information could in turn provide appropriate targets for the development of future strategies for prevention. Acknowledgements The author would like to thank Professor George Duncan for his wise words and helpful discussions. He would also like to thank the members of the Norwich Eye Research Group and the East Anglian Eye Bank for their helpful support. Additional thanks go to Miss Yvette Reader for support and assistance in manuscript preparation. The author is also grateful to the Humane Research Trust, British Council for the Prevention of Blindness, BBSRC (SAGE initiative), the John and Pamela Salter Trust and Cambridge Antibody Technology for their ®nancial backing of his career to date.
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aging human vitreous. Invest. Ophthalmol. Vis. Sci. 41, 3299±304. Weng, J., Liang, Q., Mohan, R. R., Li, Q. and Wilson, S. E. (1997). Hepatocyte growth factor, keratinocyte growth factor, and other growth factor-receptor systems in the lens. Invest. Ophthalmol. Vis. Sci. 38, 1543±54. Wormstone, I. M., Del Rio-Tsonis, K., McMahon, G., Tamiya, S., Davies, P. D., Marcantonio, J. M. and Duncan, G.2001aFGF: an autocrine regulator of human lens cell growth independent of added stimuli. Invest. Ophthalmol. Vis. Sci. 42, 1305±11. Wormstone, I. M., Liu, C. S., Rakic, J. M., Marcantonio, J. M., Vrensen, G. F. and Duncan, G. (1997). Human lens epithelial cell proliferation in a protein-free medium. Invest. Ophthalmol. Vis. Sci. 38, 396±404. Wormstone, I. M., Tamiya, S., Eldred, J. A., Reddan, J. R., Anderson, I. and Duncan, G. (2001b). Inhibition of TGF b2 mediated effects on human lens epithelial cells by the human monoclonal antibody CAT-152. Invest. Ophthalmol. Vis. Sci. 42, 2911 (Suppl.). Wormstone, I. M., Tamiya, S., Marcantonio, J. M. and Reddan, J. R. (2000). Hepatocyte growth factor function and c-Met expression in human lens epithelial cells. Invest. Ophthalmol. Vis. Sci. 41, 4216±22. Wunderlich, K., Pech, M., Eberle, A. N., Mihatsch, M., Flammer, J. and Meyer, P. (2000). Expression of connective tissue growth factor (CTGF) mRNA in plaques of human anterior subcapsular cataracts and membranes of posterior capsule opaci®cation. Curr. Eye Res. 21, 627±36. Zhang, X. H., Ji, J., Zhang, H., Tang, X., Sun, H. M. and Yuan, J. Q. (2000). Detection of integrins in cataract lens epithelial cells. J. Cataract Refract. Surg. 26, 287±91.
REVIEW Posterior Capsule Opaci®cation: A Cell Biological Perspective I . M I C H A E L WO R M S TO N E * School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, U.K. (Received Rochester 7 November 2001 and accepted in revised form 13 December 2001)
Key words: posterior capsule opaci®cation; cataract; lens; wound healing; growth.
Introduction Posterior capsule opaci®cation (PCO) is the most common complication of cataract surgery. At present the only means of treating cataract is by surgical intervention, and this initially restores high visual quality. Unfortunately, PCO develops in a signi®cant proportion of patients to such an extent that a secondary loss of vision occurs, which consequently requires further corrective laser surgery (Moisseiev et al., 1989 Sudhakar, Ravindran and Natchiar, 1989; Knight-Nanan, O'Keefe and Bowell, 1996; Schaumberg et al., 1998; Sundelin and Sjostrand, 1999) which is both expensive and not without risk (Ranta and Kivela, 1998). A modern cataract operation generates a capsular bag (Fig. 1(A)) which comprises a proportion of the anterior and the entire posterior capsule. The bag remains in situ, partitions the aqueous and vitreous humours, and in the majority of cases, houses an intraocular lens. The production of a capsular bag following surgery permits a free passage of light along the visual axis through the transparent intraocular lens and thin acellular posterior capsule. However, on the remaining anterior capsule, lens epithelial cells stubbornly reside despite enduring the rigours of surgical trauma. This resilient group of cells then begin to re-colonize the denuded regions of the anterior capsule, encroach onto the intraocular lens surface, occupy regions of the outer anterior capsule and most importantly of all begin to colonize the previously cell-free posterior capsule. Cells continue to divide, begin to cover the posterior capsule and can ultimately encroach on the visual axis (Fig. 1(B)). A thin cover of cells is insuf®cient to affect the light path, * Address correspondence to: I. M. Wormstone, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K. E-mail: [email protected]
0014-4835/02/03033711 $35.00/0
but subsequent changes to the matrix and cell organization can give rise to light scatter (Fig. 1(C)). If these changes are suf®ciently severe, vision is then seriously impaired and corrective surgery required. Prevalence of PCO The incidence of PCO reported is generally based on the requirement for follow-up treatment as a marker (Moisseiev et al., 1989; Sudhakar et al., 1989; KnightNanan et al., 1996; Schaumberg et al., 1998; Sundelin and Sjostrand, 1999). A relatively short-term study by Sudhakar et al. (1989) reported that 115 (11.5 %) of 1000 patients developed PCO 1 year following cataract surgery. Furthermore, it has been reported that the incidence of PCO requiring surgery increases with time after surgery (Schaumberg et al., 1998). Another useful study, carried out by Moisseiev et al. (1989), showed that the incidence of PCO 4 years after surgery is 41 %. This study also addressed the in¯uence of age on PCO incidence. The data revealed that the percentage of patients requiring secondary treatment was 37 % in a group of patients aged 4 60 years and 70 % when aged under 40 years. Paediatric patients are reported to have a rapid development of PCO (KnightNanan et al., 1996). This age-related phenomenon forms a rational basis for delaying the time at which cataract surgery is performed. A number of clinical studies have addressed the in¯uence of different IOLs on the incidence of PCO (Frezzotti and Caporossi, 1990; Nagata and Watanabe, 1996; Ursell et al., 1998; Hollick et al., 1999; Hayashi et al., 2001; Scaramuzza, Fernando and Crayford, 2001). The general theme is that an IOL can in¯uence the progression of PCO, and it is likely that this in¯uence is of a physical nature (Nagata and Watanabe, 1996; Nishi, Nishi and Sakanishi, 1998; Nishi, Nishi and Wickstrom, 2000). However, whilst the development of IOLs has helped reduce the incidence of PCO requiring corrective # 2002 Elsevier Science Ltd.
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F IG . 1. A schematic representation of (A) the post surgical capsular bag and (B) the extensive growth and modi®cation that gives rise to PCO. (C) A dark-®eld micrograph of a capsular bag removed from a donor eye that had undergone cataract surgery prior to death that exhibits light scattering regions beneath an intraocular lens.
surgery, perhaps by delaying the development of PCO, the problem is far from being eradicated. Furthermore, it should not be immediately assumed that a person who does not undergo corrective laser therapy has optimal clear vision. Laser therapy is employed when visual loss severely affects the life of an individual. Using laser capsulotomy rates as a barometer of PCO incidence is a useful tool, but a number of patients who do not undergo this secondary surgery are still likely to have a diminished visual quality, compared with the initial surgical result. Sundelin and Sjostrand (1999) examined this group of patients and found that a signi®cant proportion would bene®t from laser capsulotomy. In the vast majority of cases, if not all, lens epithelial cells will remain attached to the capsule following cataract surgery and therefore the sequence of events that give rise to PCO will commence. While the introduction of an IOL will play a part, the degree this will affect the patient is dependent on the regulation of
the cells themselves. The remainder of this review will therefore largely concentrate on the cell biological processes involved in PCO and the factors that in¯uence these events. 2. Methods Several approaches have been used to study PCO. These include cell culture studies (McDonnell, Krause and Glaser, 1988; McAvoy and Chamberlain, 1989; Ibaraki, Lin and Reddy, 1995; Kurosaka et al., 1995; Nishi et al., 1996a; Oharazawa et al., 1999; Wormstone et al., 2000); in vivo animal studies (Cobo et al., 1984; Behar-Cohen et al., 1995); capsular bag models (Nagamoto and Bissen-Miyajima, 1994; Liu et al., 1996; Wormstone et al., 1997; Saxby, Rosen and Boulton, 1998; Davidson et al., 2000b); in vivo observations (Ursell et al., 1998; Hollick et al., 1999); and analysis of post-mortem material (Kappelhof et al., 1987; Marcantonio et al., 2000; Saika et al., 2000;
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Wunderlich et al., 2000; Wormstone et al., 2000, 2001a). All these systems have strengths and weaknesses, but in concert they provide a powerful collection of tools to investigate PCO. Cell Culture Studies This is the simplest method of study and generally utilizes cell lines or primary cultures derived from native tissue to analyse growth characteristics. These experiments can therefore identify which factors can stimulate or inhibit growth. The drawbacks of using cell cultures are that the sub-structure of the growth matrix and the culture medium composition determine, to a large extent, not only the growth rate, but also the molecular characteristics of the cells. For example, native human lens epithelial cells express mainly the M1 muscarinic receptor subtype whereas the human lens epithelial cell line HLE-B3 expresses the M3 subtype (Collison et al., 2000). In addition, Krausz et al. (1996) have analysed the expression patterns of lens speci®c proteins or proteins expressed preferentially by lens cells using RT-PCR techniques. This work revealed that in some cases aA-crystallin was not expressed; however aB-crystallin and Pax6, the so-called eye master gene, were detected in the cell lines tested. Proteins associated with ®bre cells were not expressed under standard culture conditions. However, cell lines remain a valuable experimental tool. It would seem that their main uses are ®rst to provide indication of which molecules are potentially worthy of study, and moreover the concentrations at which they are effective. Secondly, once a system has been validated in a more complex experimental model or by circumstantial evidence obtained with postmortem material, then the more readily available cell lines are particularly good systems to investigate thoroughly speci®c regulatory pathways. In vivo Animal Studies The most commonly used model for this type of work is the rabbit. The main drawback with this approach, apart from ethical considerations, is that cell growth in animals such as a rabbit or cat may be different to that in primates. Furthermore, different species have been shown to react in different ways to primates following trauma to the eye (Bito, 1984). It is also dif®cult to study with any detail the progression of PCO in these systems with much of the information obtained from detailed end-point examinations. Capsular Bag Models This method of study has emerged in recent years and is based on a sham cataract operation. In effect a capsular bag is produced that is identical to that generated in vivo (Fig. 2). This system therefore has the
F IG . 2. Low power dark-®eld views of capsular bag preparations pinned out and immersed in culture medium. In the absence of an IOL (a) the disc shaped opening in the anterior capsule can clearly be seen, revealing the posterior beneath. When an IOL is present (b) the supporting loops (haptics) can distend the capsular bag and two creases (arrows) can be seen on the posterior capsule. The micrographs represent a ®eld of view of 1.8 1.25 cm. (Liu et al., 1996).
correct matrix i.e. the capsule and cellular distribution. A critical aspect of these models is the maintenance of the capsular bag shape. In order to achieve this, some groups have employed capsular rings (Nagamoto and Bissen-Miyajima, 1994; Saxby et al., 1998), while others have used ®ne pins to maintain the circular shape (Liu et al., 1996; Wormstone et al., 1997; Davidson et al., 2000b). Furthermore, some of these systems permit the insertion of an IOL and therefore the role it plays can also be investigated on a day to day
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basis. This model approach has been applied to human (Nagamoto and Bissen-Miyajima, 1994; Liu et al., 1996; Wormstone et al., 1997), canine (Davidson et al., 2000b) and bovine lenses (Saxby et al., 1998). In the case of humans and canines, which routinely undergo cataract surgery, the capsular bag culture system provides at present the most directly relevant in vitro data to the clinic; however, at least in the case of humans the rate limiting step is the availability of material.
In vivo Observations Due to improvements in technology, camera systems have been developed that can detail changes to the capsular bag within the visual axis (Ursell et al., 1998; Hollick et al., 1999). A patient can then be assessed at various time points and the changes observed. While this technique cannot at present provide information at the single cell level it does provide an excellent insight into the rates of PCO progression.
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Post-mortem Analysis This provides an important understanding of which molecules are present within the capsular bag as PCO progresses. Such molecules could be matrix components, cellular markers or growth factors. Techniques used to analyse the material include electron microscopy (Kappelhof et al., 1987; Marcantonio et al., 2000), immunochemistry (Marcantonio et al., 2000; Saika et al., 2000), RT±PCR (Wunderlich et al., 2000; Wormstone et al., 2001a) and ELISA (Wormstone et al., 2000, 2001a). This information can then be utilized when designing or comparing the other experimental systems described. While this type of investigation provides `scene of the crime' information it does not provide de®nitive proof that various molecular suspects are responsible for PCO development. Cell Regulation and Function The regulation of the processes involved in PCO is derived from the signals cells receive from the
F IG . 3. A schematic representation of autocrine and paracrine signalling control mechanisms of lens cells.
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environment. The source of these regulators can be from the lens cells themselves or from other ocular tissues and therefore form autocrine and paracrine signalling systems (Fig. 3). These two mechanisms are both likely to contribute to PCO development, as an adequate supply of signals is required to sustain viability and growth. If the supply of signals is insuf®cient this will lead to cell death, occurring presumably by apoptosis (Ishizaki et al., 1993). Paracrine Control Initially the focus was on increased levels of protein in the aqueous humour following surgery, generated largely by a disrupted blood±aqueous barrier and an in¯ammatory response (Pande, Spalton and Marshall, 1996). Interestingly, it is also reported that certain proteins are only detected in the aqueous humour following trauma to the eye (Namiki et al., 1992). The resulting cocktail of peptides and proteins therefore has great potential in regulating lens cell function. A considerable number of experiments have shown that addition of numerous factors can modulate lens cells from a variety of species. Transferrin, for example is believed to act as a survival factor and data reported using canine lens cells indicated that supplementing the culture medium with 200 mg ml 1 transferrin signi®cantly increases cell survival (Davidson et al., 1998). Furthermore, data show the ability of various agents to increase proliferation rates, migration and differentiation when added to cultures. The majority of these studies investigate cytokines, including growth factors. In the case of basic FGF, it has been shown that basic FGF could induce maximal rates of proliferation, migration and differentiation of rat lens explant epithelium at 0.15, 3 and 40 ng ml 1, respectively (McAvoy and Chamberlain, 1989). A similar trend was also found with acidic FGF, but the effective concentrations were considerably greater (Chamberlain and McAvoy, 1989). Differences in the sensitivity of human primary cells were also observed such that 10-fold higher concentrations of acidic FGF were required to achieve similar results to those observed with basic FGF (Ibaraki et al., 1995). Application of EGF to cell cultures has also been shown to induce proliferation (Hongo et al., 1993; Ibaraki et al., 1995; Majima, 1995). In one study (Hongo et al., 1993), using rabbit lens epitheial cells, concentrations of 0.1±100 ng ml 1 were found to stimulate growth, which was estimated by absorbance of methylene blue staining. However, the maximal response was achieved at 10 ng ml 1. This concentration of EGF also was found to be optimal for inducing proliferation of human lens epithelial cells in early sub-culture (Ibaraki et al., 1995). This study of human lens epithelial cells also examimed the potential to induce differentiation of epitheial cells to
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®bres. This was assessed by the capacity to form lentoid bodies that express gamma crystallin. Using this system EGF was a strong promoter of cell aggregation and lentoid body formation. This could have relevance to the formation of new ®bre cells in the periphery of the bag, commonly known as Soemerring's ring (Kappelhof et al., 1987; Marcantonio et al., 2000). TGF b is a protein that is becoming increasingly studied because of its association with ®brotic disorders. It therefore could have implications in the ®brotic changes that occur in PCO. TGF b is normally detected in the aqueous humour, but the majority is in the latent form (Cousins et al., 1991). However, following trauma, e.g. by surgery, active levels can be elevated (Ohta et al., 2000). The major isoform synthesized within the eye is TGF b2, however TGF b1 can also reach the aqueous humour from the blood. The proposed role of TGF b is to serve as an immune regulator in the eye (Streilein, 1996). However, these high levels could also in¯uence lens epithelial cells. Addition of TGF b to rat explants or bovine epithelial cells cultured on a collagen raft induced contraction of the underlying matrix (Liu et al., 1994; Kurosaka et al., 1995). Furthermore, addition of TGF b has been shown to induce transdifferentiation of rat lens epithelium that can be identi®ed by alpha smooth muscle actin expression (Gordon-Thomson et al., 1998). The induction of this event was isoform-dependent with TGF b isoforms 2 and 3 being 10-fold more potent than isoform 1. Recent studies have also shown that TGF b2 can stimulate greater wrinkle formation and increased expression of transdifferentiation markers in cultured human capsular bags (Wormstone et al., 2001b). Moreover, immunocytochemical studies have shown aSMA to be expressed within the capsular bag of postmortem material (Marcantonio et al., 2000). The greatest density found in these long-term cultures was on the outer surface of the anterior capsule. Saxby et al. (1998) have also shown that aSMA is expressed in serum cultured capsular bags. As the cells migrate across the posterior capsule the greatest levels are at the growing edge and following complete cover expression becomes ubiquitous. Alpha SMA expression was also observed in serum-free cultured canine capsular bags (Davidson et al., 2000b). More evidence to suggest that TGF b is important in PCO was reported by Wunderlich et al. (2000) who showed that the connective tissue growth factor was present in the post-mortem capsular bags. Expression of this particular growth factor is often associated with TGF b. Furthermore, lens cells are known to synthesize and secrete TGF b (Allen et al., 1998). Also, Saika et al. (2000) have identi®ed immunohistochemically the presence of TGF b isoforms and receptors.
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Autocrine Control It is important to remember that the level of protein in the aqueous humour peaks shortly after surgery and slowly returns to basal levels i.e. low levels of protein within weeks or months (Kondo, Yamauchi and Nakatsu, 1995; Pande et al., 1996). However, PCO does not generally require secondary surgery until years later. This would suggest that the process of events seen in PCO has persisted some time after the initial exposure to high serum proteins has subsided. It would therefore seem that another regulatory system is involved and at this stage it would appear that autocrine control mechanisms become important. There is the possibility that the basal levels of growth factors in the aqueous humour, of which a high proportion of detectable protein is secreted by the lens, could slowly drive PCO. For example, McGahan et al. (1995) have reported that the lens cells are a major producer of the transferrin pool in the aqueous humour. Additional studies have also revealed autocrine pathways in lens cells (Majima, 1995; Lee and Joo, 1999; Wormstone et al., 2000, 2001a). In addition, there are a number of reports to indicate
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that an extensive production of cytokines and receptors occurs in the native lens (Majima, 1995; Weng et al., 1997; Lee and Joo, 1999; Wormstone et al., 2000, 2001a). However, following surgical trauma there is a modi®cation of the production levels of some proteins (Saika et al., 2000; Wormstone et al., 2000, 2001a). Ishizaki et al. (1993) in a study of rat lens cells found that the cells could survive in a protein-free medium. They also concluded that the ability to survive, and avoid undergoing cell death by apoptosis, was dependent on the underlying matrix and cell density. Furthermore, Wormstone et al. (1997) have shown using a human capsular bag model that lens cells maintained in serum-free medium without added proteins not only survive the surgical disruption but also actively proliferate and grow. The importance of this ®nding indicates autocrine regulation that is independent of added stimuli. A similar ability to grow actively in minimal conditions has also been reported in cultured bovine (Saxby et al., 1998) and canine (Davidson et al., 2000b) capsular bags. Wormstone et al. (2001a) also showed that cells residing on the posterior capsule can maintain phenotypic markers
F IG . 4. Dark-®eld (A and D) and phase contrast (B, C, E and F) images of cells within capsular bags cultured for 4 100 days in protein-free medium (A±C) and within a capsular bag removed from a donor eye that had previously undergone a cataract operation (D±F). (A) A modi®ed dark-®eld micrograph of a capsular bag cultured for 137 days. The circular outline of the anterior capsule edge (rhexis region) is clearly visible (white arrows). The anterior capsule (AC) has a consistent, smooth appearance and the homogeneous cell population is only visible using phase optics (see Fig. 1(B)). The cells on the posterior capsule are more apparent and there are also small cell-free areas (see Fig. 1(C)). Light scattering wrinkles are also apparent. (B) Phase image of cells that have re-colonized the anterior capsule of a capsular bag cultured for 465 days. Note that the cells still maintain a regular cobblestone appearance throughout this time. (C) Phase micrograph of cells growing on the central region of the posterior capsule from the same donor as 1(A). The cell distribution is heterogeneous with cells lying along wrinkles, whilst other areas are cell-free (stars). (D) The region in focus lies on the posterior capsule beneath the plastic intraocular lens (IOL) which was inserted at the time of surgery. Cell aggregations (*), cell-free areas (black arrows) and capsular wrinkling (white arrows) are all apparent. (E) In this phase image of the anterior capsule, large areas are out of focus due to the presence of the IOL. However, the regular cobblestone appearance of the anterior cells can be seen in areas within the focal plane (arrows). Additionally, there is a build-up of cellular material lying along the IOL support (*). (F) A higher power image of Fig. 4(E) that more clearly shows the cobblestone appearance of the epithelial cells on the anterior capsule. (Wormstone et al., 2001a).
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such as vimentin and alpha crystallin. However, on the posterior capsule cells maintained for 4 100 days showed some sites of regression. This pattern is similar to that seen on the posterior capsule of post-mortem capsular bags (Fig. 4). Interestingly, the cells remaining on the anterior capsule exhibited a regular cobblestone appearance and this was also the cell pattern observed on post-mortem bags (Fig. 4). Moreover, de novo protein synthesis was determined by 35S-methionine incorporation in both long-term capsular bag cultures and cultured post-mortem material. The ability of lens cells to synthesize the proteins required for survival and growth may be related to the restriction of nutrients to the surrounding humour by the blood±ocular barrier. As a consequence of the potentially de®cient environment the lens may have adapted by producing the necessary proteins to sustain itself rather than rely on scavenging the otherwise limited resources available. Other tissues residing behind barrier systems, e.g. the blood±brain barrier, have also been shown to display autocrine mechanisms to aid survival in a similar manner to the lens (Espinosa de los Monteros et al., 1990). Proliferation, in association with migration, is an important aspect of wound healing and PCO. Some interesting data have been reported using the BrdU labelling and detection technique to identify dividing cells (Rakic, Galand and Vrensen, 1997, 2000; Wormstone et al., 1997). In one study (Rakic et al., 1997), it was shown that simply performing a small capsulorhexis could up-regulate the rate of division in the lens relative to the intact lens. Moreover if the ®bres were removed, the rate of cell division was stimulated even more. Analysis of capsular bags following 3 days culture in serum-free medium showed increased numbers of positive cells (Wormstone et al., 1997). The distribution of these positive cells was predominately in the equatorial region, the natural site of division; however some cells were observed on the anterior capsule. Furthermore, if the capsular bags from aged donors (460 years) were established in the presence of serum, then the dividing population was increased, but the distribution pattern remained the same. It is notable 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, supplementing the medium with 10 % 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 in the young (540 years). These data also re¯ect the increased requirement for secondary surgery to treat PCO in young patients (Moisseiev et al., 1989; Knight-Nanan et al., 1996). Furthermore, the ®ndings suggest that powerful autocrine mechanisms in the young are active following surgical trauma. In addition, Rakic et al. (2000)
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examined post-mortem capsular bags, which were then cultured and ®xed following varying periods of culture. BrdU analysis showed that the divisional rate was similar to that previously observed in the whole lens; however, disruption to the capsular bag by removal of an IOL and residual material led to a 10fold increase in division. Other reports have also shown that in some cases YAG capsulotomy can induce a cellular reaction that can lead to coverage of the ablated region (Jones, McLeod and Boulton, 1995). Identi®cation of the autocrine signalling systems active within the capsular bag is beginning to emerge. Previous work on the native epithelium has shown that complete autocrine systems are in place. For example expression of EGF and EGFR message in addition to translated proteins in human lens epithelium have been reported (Majima, 1995; Lee and Joo, 1999). However, using capsular bag culture systems, which signalling components are present at various stages after surgery can be identi®ed and their relative roles in PCO progression can be tested. Basic FGF and FGF receptor 1, the elements of a potential autocrine pathway, were identi®ed using RT±PCR in cultured human lens capsular bags (Wormstone et al., 2001a). Basic FGF was also detected using ELISA. Interestingly, the degree of message for both the ligand and receptor appeared to be greater in cultured bags compared with native human lens epithelium. However, this study also reported that capsular bags removed from donors who had previously undergone cataract surgery also exhibited message for FGF and FGFR-1. The levels detected were similar to cultured capsular bags. The functional role of this autocrine system was tested, by blocking FGF receptor-1 using a speci®c inhibitor SU5402, which caused a retardation of growth. Hepatocyte growth factor and its receptor c-met have also been detected in capsular bags using ELISA and immunocytochemistry (Wormstone et al., 2000). HGF was also identi®ed by ELISA in capsular bags removed from donors who had previously undergone cataract surgery. Interestingly, actively growing cells on both the anterior and posterior capsules exhibited an elevated expression of receptor following surgical trauma compared to the expression in the native epithelium. Furthermore, high levels of HGF were detected in the medium in the ®rst few days of culture and declined with time. A similar pattern has also been observed with basic FGF (Wormstone et al., 2001a). Extracellular Matrix The importance of the lens capsule in the regulation of PCO is becoming more evident. The capsule is composed of a number of elements including collagen type IV and laminin, but as PCO progresses it is known that other matrix components, such as
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collagen types I, are actively synthesized (Wunderlich et al., 2000). Oharazawa et al. (1999) have investigated the role of ECM components in cell adhesion, proliferation and migration of a human lens cell line. Cell attachment was signi®cantly enhanced when cells were seeded onto matrix-coated dishes as opposed to non-coated dishes; however differences between the matrices tested were observed. Interestingly, maintenance on matrix-coated dishes did not enhance proliferation, but did increase rates of migration. However, it should be noted that a decrease in the overall cell number was observed when cells were cultured on ®bronectin or collagen. In contrast, when cultured on laminin, cell numbers increased. Parmigiani and McAvoy (1991) also showed that cells from all ages were capable of growing onto laminin, whereas growth onto ®bronectin was age-dependent. Lens epithelial cells are linked to the underlying matrix by cell adhesion molecules such as integrins. Immunocytochemical analysis of human anterior discs removed at the time of cataract surgery has identi®ed a2, a3, a5, b1 and b2 integrins (Nishi et al., 1997; Zhang et al., 2000). Furthermore, the importance of the b1 integrin has been validated by the addition of a monoclonal antibody. Using this tool, the number of viable cells remaining after 2 weeks of culture on collagen or laminin-coated dishes was reduced by the antibody in a dose-dependent manner (Nishi et al., 1997). Another interesting family of proteins that can regulate the matrix is the matrix metalloproteinases (MMPs). These enzymes are involved in normal physiological processes, such as morphogenesis, but also have been implicated in pathological conditions (Birkedal-Hansen, 1995). The most widely studied members of the MMP family in the eye are the gelatinases, and these are found in both the aqueous and vitreous humour (El-Shabrawi, Christen and Foster, 2000; Vaughan-Thomas, Gilbert and Duance, 2000). When examining the native human lens epithelium, Smine and Plantner (1997) could not detect MMP-2 (Gelatinase A), whereas MMP-9 (Gelatinase B) was not investigated. However, MT1MMP was identi®ed. This member of the family has been shown to convert pro-MMP-2 to its active form. Interestingly, Tamiya et al. (2000) have reported that neither MMP-2 nor -9 can be detected using gelatin zymography in the culture medium of clear cultured lenses; however following a sham cataract operation high levels were detected. Furthermore, Richiert and Ireland (1999) have shown that chick lens annular pad cells cultured on plastic, collagen type IV, ®bronectin or laminin do not express MMP-2 or -9 unless stimulated with TGF b or PDGF. Work carried out on the human capsular bag also shows that TGF b2 can sustain elevated levels of MMP-2 and -9 in the medium (Duncan et al., 2001). Furthermore, EGF was also found to stimulate expression of MMP-2, but not -9.
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Prevention of PCO A variety of different approaches have been applied to prevent PCO. At the surgical level attempts were made to polish the lens capsule during surgery and thus effectively eliminate the lens cell epithelial population. However, it now seems that total removal of the cells by this method is virtually impossible and even a small residual cell population can repopulate the posterior capsule (Davidson, Morgan and McGahan, 2000a). Therefore, the major area of development on the surgical front is in the design of intraocular lenses. These are constructed from many materials (Hollick et al., 1999; Apple et al., 2001; Hayashi et al., 2001; Scaramuzza et al., 2001). It would appear that the major bene®ts of an IOL in PCO prevention are its physical nature and it is reported that sharp edges at the optic edge or on capsular rings can create a barrier that will retard cell growth onto the posterior capsule (Nagata and Watanabe, 1996; Nishi et al., 1998). This improved design is undoubtedly bene®cial to the patient, but nevertheless YAG capsulotomy is still carried out on patients who have been implanted with these lenses, albeit at a lower incidence (Hayashi et al., 2001; Scaramuzza et al., 2001). A number of pharmacological antagonists have been proposed to inhibit lens cell growth leading to PCO, most of which have been tested in vitro (McDonnell et al., 1988; Behar-Cohen et al., 1995; Nishi et al., 1996b; Duncan et al., 1997). At the clinical level there are at present three major ways in which a pharmacological antagonist can be delivered to the target cells. These are by direct injection into the anterior chamber, modi®cation of the irrigating medium or by modi®cation of the IOL. The problem associated with any drug delivery is toxicity to other tissues, especially the corneal endothelium. At present the most likely mode of delivery is via the IOL. This could provide a more controlled means of drug delivery that can concentrate an agent at the site of the potentially proliferating cells more ef®ciently. In fact, modi®cation of the supporting loops of the IOL (known as haptics) to carry cytotoxic agents would allow direct delivery to the equatorial cells. The nature of drug attachment must be suf®ciently strong to prevent signi®cant leakage into surrounding tissues, yet suf®ciently weak to permit accumulation into contiguous cells. Behar-Cohen et al. (1995) have reported the effects of an FGF-saporin complex bound to a heparin surface modi®ed IOL in rabbits. When in contact with the cells the FGF binds to the appropriate receptors on the epithelial cells and internalizes the saporin, subsequently killing the cells. This particular system produced some side effects, including transient corneal oedema and iris depigmentation. Duncan et al. (1997) have also shown using a human capsular bag model that thapsigargin, a speci®c inhibitor of the endoplasmic reticulum CaATPase, when directly coated onto a PMMA IOL can effectively kill the
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entire cell population within the bag. However, this is a simple coating procedure and does not account for additional stresses associated with in vivo surgery, e.g. entry through the corneal/scleral tunnel. Therefore a drug release system that is both physically protective and provides appropriate drug delivery is necessary. Some studies have employed sustained drug delivery tools (Nishi et al., 1996b). However, the materials used are often opaque and therefore cannot be placed within the visual axis. Nevertheless such mechanisms could be used in the form of capsular rings or even in haptic production. 3. Conclusions PCO affects large numbers of cataract patients and is a condition that essentially arises from lens cell growth and gives rise to light scattering modi®cation of the posterior capsule. The development of PCO is dependent on both the increased levels of proteins in the aqueous humour shortly following surgery and the ability of lens cells to drive events persistently by autocrine mechanisms due to their association with the capsule. It is therefore essential to employ the methods available to identify both the paracrine and autocrine signals that underpin the various stages of PCO. This information could in turn provide appropriate targets for the development of future strategies for prevention. Acknowledgements The author would like to thank Professor George Duncan for his wise words and helpful discussions. He would also like to thank the members of the Norwich Eye Research Group and the East Anglian Eye Bank for their helpful support. Additional thanks go to Miss Yvette Reader for support and assistance in manuscript preparation. The author is also grateful to the Humane Research Trust, British Council for the Prevention of Blindness, BBSRC (SAGE initiative), the John and Pamela Salter Trust and Cambridge Antibody Technology for their ®nancial backing of his career to date.
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