Ординатура / Офтальмология / Английские материалы / Development of the Ocular Lens_Lovicu, Lee Robinson_2004

11.2.2.3. Epithelial Differentiation and Maintenance

Besides growth regulation, there is evidence that growth and other regulatory factors play key roles in the differentiation and maintenance of the lens epithelial phenotype.

Retinoic Acid. As mentioned earlier in the discussion of lens induction, RA and its naturally occurring analogs appear to play important roles in regulating aspects of lens development. The importance of retinoid signaling has been established by applying exogenous RA (Hyatt et al., 1996a; Hyatt et al., 1996b), by deleting various combinations of RAR and RXR genes (Lohnes et al., 1993; Lohnes et al., 1994), and by overexpressing CRABP I (Perez-Castro et al., 1993). In lens cell cultures, RA has been shown to induce the expression of αB-, δ1- and γ F-crystallin genes (Gopal-Srivastava et al., 1998). In addition, RA has been shown to inhibit the epithelial-mesenchymal transition that lens cells undergo when cultured in type I collagen gels (Mattern et al., 1993). In this study, RA treatment induced the deposition of a basement membrane–like material, a result consistent with evidence that RA induces production of extracellular matrix components in lens cells, including type IV procollagen (Sawhney et al., 1997). Combined with experiments showing that RA is present in lens epithelial cells and the ocular media (Wakabayashi et al., 1994), these studies provide strong evidence that RA signaling influences many aspects of lens biology, including having an important role in maintaining the epithelial phenotype. This is supported by studies on the RA-inducible transcription factor AP-2α, which is expressed in the lens epithelium but not the fibers (Ohtaka-Maruyama et al., 1998a). AP-2α null mice develop an abnormal lens that adheres to the cornea and lacks a typical lens epithelium (West-Mays et al., 1999).

Wnt. Members of the Wnt growth factor family have been shown to be involved in the regulation of epithelial differentiation in a number of organ systems (see, e.g., Brisken et al., 2000; Itaranta et al., 2002; Miller and Sassoon, 1998; Tebar et al., 2001). Studies on the lens identified the expression of a number of Wnts and their Frizzled receptors in the chick and rodent lens epithelium (Jasoni et al., 1999; Stump et al., 2003). Wnt isoforms 5a, 5b, 7a, 7b, 8a, 8b and Frizzled receptors 1, 2, 3, 4 and 6 were detected in the postnatal rodent lens and showed similar patterns of expression (Stump et al., 2003). Expression was generally present throughout the lens epithelium and extended into the transitional zone, where fiber elongation occurs, but was lost in the more mature fibers of the lens cortex. Coreceptors, LDL-related proteins (LRP) 5 and 6, which are required for Wnt signaling through the ß-catenin pathway, were also expressed in the lens. In addition, expression of other molecules that are known to be involved in Wnt signaling and its regulation, including Dishevelleds, Dickkopfs, and secreted Frizzled-related proteins, were detected in the lens (Leimeister et al., 1998; Stump, 2003). Analysis of mice with a null mutation of Lrp6 (Pinson et al., 2000) provided more direct evidence of a role for Wnt/β-catenin signaling in differentiation of the lens (Stump et al., 2003). These mice had small eyes and aberrant lenses, characterized by an incompletely formed anterior epithelium, resulting in extrusion of the lens fibers into the overlying corneal stroma. Taken together, these results indicate a role for Wnt signaling in regulating the differentiation and formation of the epithelial sheet.

LEDGF. Lens epithelium–derived growth factor (LEDGF) has been recently identified and characterized (Shinohara et al., 2002). It was cloned from a lens epithelium

Figure 11.15. Diagram illustrating the epithelial-mesenchymal transition induced by TGFß. (Adapted from McAvoy et al., 2000.)

1998). As with the lenses cultured with TGFß, the cells in the transgenic plaques express markers of myofibroblastic/fibroblastic cells. These studies clearly show that TGFß disrupts the normal lens epithelial architecture and induces an epithelial-mesenchymal transition. In addition to induction of new patterns of gene expression, there is also evidence that TGFß down-regulates normal epithelial markers such as Pax-6, α-crystallin, and connexin 43 (Lovicu et al., 2002). Thus one of the effects of TGFß may be to inhibit the signaling pathways induced by growth factors involved in the maintenance of the lens epithelial phenotype. As a result, cells lose their normal cell-cell and cell-matrix associations. They also lose their normal polarity and monolayered arrangement as they undergo a marked phenotypic change (Fig. 11.15).

Some or all of these TGFß-induced changes, as shown in the various animal models, are typically found in subcapsular cataracts in humans. Following ocular trauma or eye surgery or the onset of other conditions (e.g., atopic dermatitis and retinitis pigmentosa), anterior subcapsular cataracts (ASCs) can develop (Sasaki et al., 1998). These feature the development of subcapsular fibrotic plaques that obscure vision. Similar fibrotic plaques, as well as capsular wrinkles, also frequently arise in the lens bag after cataract surgery and lead to posterior capsular opacification (PCO; Wormstone, 2002). This condition, also known as “secondary cataract” and “aftercataract,” arises as a result of the aberrant growth and differentiation of epithelial cells that are invariably left behind after cataract surgery. In PCO and ASC, the opaque plaques are formed by abnormal accumulations of lens cells and extracellular matrix (Novotny and Pau, 1984; Pau et al., 1985; Wormstone, 2002). Immunolabeling studies of these cataracts have revealed the presence of cytoskeletal and extracellular matrix proteins not normally expressed by human lens cells. α-Smooth muscle actin and types I and III collagen have been immunolocalized in the plaques in ASC (Hatae et al., 1993; Schmitt-Graff et al., 1990; Wunderlich et al., 2000) and PCO (Frezzotti, 1990; Ishibashi et al., 1994; Saika et al., 1998; Wunderlich et al., 2000). Tenascin and fibronectin have also been localized in ASC and PCO (Saika et al., 1998; Wunderlich et al., 2000). In these cataracts, it also appears that an initial TGFß insult induces other factors, such as connective tissue growth factor and TGFß-inducible gene-H3, and other autocrine signaling events, including endogenous TGFß signaling, that may be central to the progressive fibrosis

HGF. This growth factor is frequently up-regulated during wound healing and has a major influence on cell spreading (it is also known as “scatter factor”) and invasive growth in metastasis (Trusolino and Comoglio, 2002). Consistent with this general role, HGF has been reported to be substantially up-regulated during the development of PCO (Wormstone, 2002). Its abundance in human capsular bag cultures and its ability to stimulate lens cell proliferation and migration indicate that it may contribute to the development of PCO (Wormstone et al., 2000).

11.3. Overview

The polarity of the lens is likely to depend on the influence of factors that inhibit or stimulate epithelial cell proliferation or differentiation. Growth factors that are known to be present in the anterior segment include FGF, TGFß, insulin/IGF, EGF/TGFα, PDGF, HGF, and Wnt. RA and its naturally occurring retinoid analogs make up another class of regulatory compounds that are present. Among these factors, FGF, insulin/IGF, EGF/TGFα, PDGF, and HGF have been shown to be mitogenic for cultured lens epithelial cells. This is consistent with their roles in other cellular systems, and it appears likely that at least some of these ligands function as lens mitogenic factors in vivo and that they probably work in concert. On the other hand, TGFß appears to be an inhibitor of the proliferation of lens cells and may have a role in maintenance of a mitotically quiescent central epithelium.

In addition to its role in normal lens biology, TGFß can induce pathological changes in the lens. TGFß has a major disruptive effect on the lens epithelial sheet and leads to the formation of plaques of aberrant cells that are characteristically found in subcapsular cataracts. Besides a direct effect, it is likely that TGFß destabilizes lens cells by down-regulating the molecules and mechanisms that are involved in the formation and maintenance of the normal epithelial phenotype. Currently little is known about the identity of such factors; however, emerging evidence indicates important roles for Wnt and RA. As with fiber differentiation, it is likely that the situation will be complex and that additional factors will be involved in activating complex signaling cascades that regulate gene expression and lens epithelial cell behavior (Fig. 11.16).

Clearly, there is much to do to develop a good understanding of the molecular interactions that determine the lens epithelial phenotype. Some momentum for this research may be gained by the growing realization that it is critical for understanding the molecular basis of cataracts involving aberrant epithelial growth and differentiation. These include subcapsular cataracts as well as posterior capsular opacification, which is a common sequel to cataract surgery and has become a major drain on health care budgets worldwide.

Figure 12.1. Lens regeneration in the newt. Sequential steps from lens removal (1) to completion of lens regeneration (6). Frontal view of the eye at different stages of regeneration is indicated by F. The vertical lines indicate the dorsoventral plane of sectioning through the iris corresponding to the sections shown below and indicated by S. F, frontal; S, sagittal; m, macrophage. (From Eguchi, 1998.)

The major histological events of the lens regenerative process in the newt will be described first, followed by the cellular and molecular events that take place during the different stages.

12.2.1. Histology of the Regenerating Eye

Upon removal of the newt lens (Fig. 12.1, step 1), the pupillary margin of the dorsal iris swells, and the iris epithelium divides into inner and outer regions. A small vesicle soon forms at the tip of the dorsal iris due to the active proliferation of the PECs (Sato stages I and II, 4–8 days). The cells in this vesicle depigment as the cells divide (Sato stage III, 8–10 days; Fig. 12.1, step 2). The vesicle grows in size to form a vesicular lens rudiment corresponding to the lens vesicle in normal development (Sato stage IV, 9–15 days; Fig. 12.1, step 3). Then the inner part of the vesicle thickens as the cells in this region elongate to eventually differentiate into primary lens fibers (Sato stages V and VI, 12–16 days; Fig. 12.1, steps 4 and 5). Primary lens fibers continue to form on the inside of this lens vesicle, while secondary lens fibers form in the outer layers (Sato stage VIII and beyond). The growth of the lens is directed by the continuous differentiation of secondary lens fibers from lens epithelial cells. Finally, around 25 days postlentectomy (Sato stage X; Fig. 12.1, step 6) a fully regenerated lens is formed, with a lens epithelial layer surrounding a mass of lens fibers. At a later stage (Sato stage XI), the lens detaches from the dorsal iris and stands as an independent entity (Reyer, 1977).

12.2.2. Early Cellular and Molecular Events of Lens Regeneration

Among the first events to take place upon lentectomy are decondensation of chromatin and nucleolar activation of the dorsal iris PECs. These events are early signs that a cell is becoming ready for cell division and active transcription. Nucleolar activation, initiated with the enlargement of nucleoli and the increase in the amount of granule content (Eguchi, 1963, 1964; Karasaki, 1964; Dumont et al., 1970), is followed by active rRNA synthesis (Yamada and Karasaki, 1963; Reese et al., 1969). Along with RNA synthesis, cistrons for rRNA sequences are significantly amplified during the first days of this process (Collins, 1972).

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DNA synthesis follows the initial rRNA wave at about 4 days postlentectomy (Sato stage I; Eisenberg and Yamada, 1966; Yamada and Roesel, 1969; Eguchi and Shingai, 1971; Reyer, 1971), along with mitosis (Yamada and Roesel, 1971). Eguchi and Shingai (1971) reported that iris cell labeling peaks at 7 and 12 days, whereas Yamada and Roesel (1971) showed a peak at 7 and 15 days for mitotic figures. The first wave of actively proliferating PEC mostly participates in the replacement of the lens, whereas the second wave is believed to compensate for the loss of the iris cells used to regenerate the lens, and it includes cells from the dorsal as well as the ventral margin (Eguchi and Shingai, 1971). PECs undergo mitosis and start to depigment, reaching a peak of depigmentation at about day 13 (Sato stage IV). Sato (1940) initially described this depigmentation process histologically. Later, ultrastructural studies by others detailed the change in iris cell configuration, specifically the production of an intercellular space and the formation of cell projections where melanosomes await discharge, as well as the presence of phagocytotic cells (macrophages), which aid the process of cytoplasmic shedding (Eguchi, 1963; Karasaki, 1964; Dumont and Yamada, 1972; Yamada and Dumont, 1972; Reyer, 1990a , 1990b). For a detailed account of melanosome discharge and cytoplasmic shedding, consult Yamada’s (1977) review. Along with these changes, dedifferentiating PECs lose intercellular communication mediated via gap junctions, and their cell-cell adhesion decreases (Eguchi, 1976; Kodama and Eguchi, 1994). Also, the increased levels of alkaline phosphatase in the dorsal margin of the iris are reflective of the disorganization and dedifferentiation taking place there (Eguchi and Ishikawa, 1963). Interestingly, during this time, there is an increase in the number of cytoplasmic organelles (Eguchi, 1964; Karasaki, 1964; Yamada, 1967; Dumont and Yamada, 1972).

12.2.3. Differentiation and Morphogenesis of the Regenerating Lens

As the dorsal iris cells divide and depigment, they form a lens vesicle that eventually thickens between 12 and 15 days postlentectomy (Sato stage V). An inner layer of cells is then formed, and these cells stop dividing and enter the differentiation mode, soon to become lens fiber cells (Eisenberg and Yamada, 1966; Yamada, 1966, 1967; Reyer, 1971). It is at this stage that β- and γ -crystallin proteins are first detected (McDevitt and Brahma, 1982). Just before this stage (Sato stage IV), the cells in the vesicle express α-, β-, and γ -crystallin mRNA (Mitashov et al., 1992; Mizuno et al., 2002). As these cells become primary fibers, they continue to synthesize β- and γ -crystallin proteins (Sato stages VI and VII), and α-crystallin protein is first detected (Takata et al., 1964, 1966; McDevitt and Brahma, 1982). The lens epithelial cells left in the outer layer continue to proliferate and begin to express α- and β-crystallin mRNA at Sato stage VI (Mizuno et al., 2002). Crystallin proteins are not evident until later stages in the epithelium, when α- and β- crystallin proteins are detected at Sato stage VIII (Takata et al., 1964, 1966; McDevitt and Brahma, 1982, 1990). As the epithelial cells reach the equatorial zone, they cease to divide and begin elongating to form secondary lens fibers that surround the primary lens fibers (Eguchi, 1964; Dumont and Yamada, 1972; Yamada and Dumont, 1972; Reyer, 1977, 1982; Yamada, 1977, 1982; Yamada and McDevitt, 1984). The secondary lens fibers express αA-, βB1-, and γ -crystallin genes at Sato stage VIII (Mizuno et al., 2002). No crystallin mRNAs are detected in terminally differentiated primary fibers (McDevitt and Brahma, 1982; Mizuno et al., 2002). By day 25 (Sato stage X), the lens has completely formed, and dividing cells are observed only in the lens epithelium. The differentiation of the lens vesicle to form a complete lens mimics the normal lens development process, where lens fibers differentiate from lens epithelial cells and even express crystallin genes similarly (McDevitt and Brahma, 1982, 1990; Mizuno et al.,

2002). In this sense, similar events might take place during development and regeneration of the lens even though inductive interactions such as those seen during lens development (between surface ectoderm and optic vesicle) are not necessary for lens regeneration.

12.2.4. Extracellular Matrix of the Regenerating Lens

A lens capsule is formed from the basal lamina of the iris epithelium. Before lentectomy, the basal lamina of the iris epithelium is composed of fibronectin, heparan sulfate proteoglycan (HSPG), and nidogen-entactin (Elgert and Zalik, 1989; Ortiz et al., 1992). Upon lentectomy and up to about day 15, the expression of fibronectin and nidogen-entactin decreases in the iris. During the differentiation phase of lens regeneration, the signal for these two extracellular matrix molecules in the iris epithelium and new capsule increases. HSPG was detected only after completion of regeneration in the iris and the lens capsule. On the other hand, laminin was found only in the new lens capsule during the differentiation stage of lens regeneration, at around 20 days (Ortiz et al., 1992). It has also been shown that the dorsal iris has high activity for enzymes such as hyaluronidase, which may promote the remodelisg of extracellular matrix components (Kulyk et al., 1987). It is clear from these studies that a significant change in the composition of the extracellular matrix is important for the cell type conversion that takes place during lens regeneration.

12.3. Problems Involved in the Study of Lens Regeneration

In the past one hundred years or so, there has been a significant effort to answer the basic questions about the process of lens regeneration. The main questions in the field of lens regeneration still stand: How can a terminally differentiated group of cells (iris PECs) transdiffrentiate and give rise to a completely functional lens? What is the mechanism? What molecules are involved or responsible? Can this ability be induced in other species?

The classic approaches to addressing these questions depended largely on the available technology and therefore concentrated on the cellular level, which was studied mostly by means of labeling and transplantation experiments. The early focus was on finding out, among other things, which cells are responsible for the regenerative capabilities, if there was a retinal factor involved in the process, and if there was a lens factor involved. The effort now is geared more toward discovering the molecular mechanism involved in the regenerative process by trying to identify the molecular masterminds of the process.

The field of lens regeneration is in need of a revolution at the molecular level that will allow it to elucidate the molecular mechanisms by which iris cells destabilize, reenter the cell cycle, dedifferentiate, and finally redifferentiate into the final product, the lens. These cells undergo changes at many levels, including a phenotypic change, a change in cellcell communication, a change in cell–extracellular matrix interaction, and changes at the physiological and biochemical levels. All of these eventually must be carefully directed and orchestrated.

In the modern approaches, in vitro and in vivo models have been selected to study and understand cell conversion. In vitro models have been crucial for understanding cell type conversion. Several in vitro models used to study this phenomenon have been described (Eguchi, 1998). One of these includes the newt iris/retinal PEC model, in which iris/retinal PECs are isolated and grown under optimal conditions to transdifferentiate to lens cells (Eguchi, 1967, 1976, 1979; Zalik and Meza, 1968; Eguchi et al., 1974; Horstman and Zalik, 1974). Another in vitro model includes chick and human retinal PECs, which can actually

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be directed to transdifferentiate to either the lens phenotype or the retinal phenotype (Eguchi and Okada, 1973; Eguchi, 1976; Itoh, 1976; Yasuda, 1979; Masuda and Eguchi, 1982; Itoh and Eguchi, 1986a, 1986b; Kosaka et al., 1998). With these models, molecular switches for cell proliferation, dedifferentiation, and eventual differentiation can be carefully dissected. Some progress has been achieved, but there are still many pieces of the puzzle to be solved.

As for the in vivo model, the recent focus is on the elucidation of the factors that can induce or inhibit the process of lens regeneration in the newt eye. Approaches so far have concentrated on identifying important molecules involved in the process that are differentially expressed in the dorsal iris versus the ventral iris. Once a crucial molecule or factor is identified, functional studies to knock out or overexpress its function can be utilized. Using this approach, factors such as FGF and their receptors have been identified as essential for the process of lens regeneration (Del Rio-Tsonis et al., 1997; Del Rio-Tsonis et al., 1998). There are limitations as to the type of molecules (i.e., transcriptional factors) that can be manipulated with the existing technology.

Recently, Hayashi et al. devised an in vitro/in vivo system that recapitulates the in vivo conditions and is suitable for efficient genetic manipulation (Ito et al., 1999; Hayashi et al., 2001; Hayashi et al., 2002). Isolated PECs from the dorsal or ventral iris are cultured for about two weeks. Then the cells are aggregated and implanted into a lentectomized eye. The implanted ventral aggregates fail to form lenses, but the dorsal aggregates are successful in forming a lens regenerate. More importantly, transfection of genes has been established with an efficiency as high as 80% (Hayashi et al., 2001). Using this system, researchers can bypass the problem of in vivo transgenesis and create ventral transgenic aggregates with key genes that may control transdifferentiation of the dorsal iris. These transgenic aggregates can be implanted in host eyes to assay for their lens-forming potential. Similarly, transcription/translation of genes can be inhibited in vitro by the use of morpholino (Heasman, 2002) or RNAi (Zhou et al., 2002; Scherr et al., 2003) technology, creating thus a knockout condition in the system.

In the next sections, we discuss both the classic approaches and the modern approaches used to study the process of lens regeneration.

12.4. Classic Approaches to the Problems

Scientists in the field of lens regeneration have concentrated on trying to understand the intrinsic process of lens regeneration and transdifferentiation. Some of the basic questions pondered are discussed in this section.

12.4.1. How Does the Lens Regenerate? Which Cells Are Responsible for Its Regenerative Capabilities?

Following the histological description set up by Collucci (1891) and Wolff (1895), investigators accepted the idea that the lens regenerated from the dorsal iris PEC but still questioned whether these were the only cells involved. To address this issue, a series of experimental approaches were set forward in the first half of the 20th century, including microsurgical transplantation. With this approach it was shown that grafts of dorsal iris placed into the optic cavity of lentectomized eyes gave rise to lens (Wachs, 1914; Sato, 1930, 1935; Mikami, 1941). The most convincing experimental approach was the introduction of regeneration competent dorsal iris into the cavity of a lentectomized eye from a species of salamander that lacks the regenerating capacity (Ikeda, 1934; Amano and Sato, 1940; Reyer, 1956).