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

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nuclear contents become indistinguishable from the cytoplasm at the electron microscopy (EM) level (Kuwabara and Imaizumi, 1974). Immunofluorescence studies indicate that during this phase, cytoplasmic components, such as CP49, freely invade the nuclear volume (Sandilands et al., 1995a). A residual body, composed of condensed chromatin and putative nucleolar material, persists in the cytoplasm for an extended period after the loss of the nuclear envelope. In the chicken lens, final breakdown of the residual body occurs after a delay of 2–3 days and is accompanied by the sudden appearance of a large number of DNA strand scissions (Bassnett and Mataic, 1997). In the primate lens, a residual nuclear structure, perhaps consisting of nucleolar material, persists indefinitely (Bassnett, 1997). The condition of the nuclear DNA can be assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling) assay (Gavrieli et al., 1992), which labels 3 -OH ends of nicked DNA. The onset of fiber DNA degradation occurs quite suddenly at the time of or shortly after mitochondrial and ER breakdown. At this stage, the nuclear chromatin is in the process of condensing and is strongly labeled by the TUNEL assay (Bassnett, 1997; Bassnett and Mataic, 1997; Dahm et al., 1998a; Ishizaki et al., 1998; Wride and Sanders, 1998). The appearance of TUNEL-positive nuclear remnants is paralleled by the release of nucleosome-sized fragments of DNA into the fiber cell cytoplasm. When resolved by agarose gel electrophoresis, the cleaved fragments of DNA form an evenly spaced ladder (Appleby and Modak, 1977).

As organelles are degraded in cells bordering the OFZ, it is possible that other critical maturation events occur concomitantly. For example, many abundant membrane and cytoplasmic proteins of the lens (among them MIP, connexins, spectrin, and β-crystallins) are known to be truncated during differentiation. It is likely that at least some of these truncations occur at the border of the OFZ, perhaps as a result of bystander damage sustained during the proteolysis that is presumed to accompany organelle breakdown. For example, connexin 50 (connexin α8) is cleaved during differentiation into a 38-kD amino terminal fragment that remains embedded in the fiber plasma membrane and a 32-kD fragment that is derived from the carboxyl tail of the parent molecule and released into the cytoplasm. Proteolysis destroys a critical epitope, allowing the cells in which cleavage of connexin 50 has occurred to be identified by immunocytochemical techniques. This approach has demonstrated that proteolysis of connexin 50 occurs at the border of the OFZ, in cells undergoing organelle destruction (Lin et al. 1997).

9.7.2.2. Role of Caspases

Several authors have noted the similarity between organelle loss in the lens and classical apoptosis (Bassnett, 1997; Bassnett and Mataic, 1997; Dahm et al., 1998b; Dahm, 1999; Wride et al., 1999). Certainly, there are some striking similarities in the morphological features of the two processes, including the marginalization and condensation of chromatin and the cleavage of specific proteins (see below), although other features characteristic of apoptosis, such as membrane blebbing, are not observed during organelle loss. Recent findings also hint at conservation at the biochemical level.

A family of cysteine proteases has been shown to play a central role in apoptosis. These proteases, which are related to the interleukin-1 β-converting enzyme, are known as caspases (Alnemri et al., 1996). Caspases operate in a cascade, where proteolytic activation of upstream regulatory members of the family (e.g., caspase-8, -9, and -10) results in activation of effector caspases (e.g., caspase-3, -6, and -7). The effector caspases, in turn, cleave critical substrate molecules within the apoptotic cell, including poly(ADP-ribose) polymerase

(PARP), lamin B, DNA fragmentation factor (DFF45), and spectrin. The central role of caspases in apoptotic cell death is amply demonstrated by the observation that, in many instances, synthetic caspase inhibitors can prevent cells from dying in response to stimuli that would otherwise prove lethal.

PARP is a ubiquitous nuclear enzyme that binds to either doubleor single-stranded DNA breaks and catalyzes the transfer of an ADP-ribose moiety from NAD+ to itself and other nuclear acceptor proteins. The binding of PARP to broken DNA ends triggers a 500-fold stimulation in enzymatic activity. Following exposure of cells to environmental or endogenous genotoxic agents, PARP serves to trigger the base excision repair pathway. Caspase-mediated cleavage of PARP during apoptosis is thus thought to incapacitate the DNA damage surveillance network and eliminate fruitless attempts to repair the rapidly fragmenting DNA.

Like most eukaryotic cells, lens fibers possess extensive DNA repair machinery. Chicken fiber cells respond to x-radiation by an increase in nuclear ADP-ribosylation (Counis et al., 1985), presumably mediated by PARP. Interestingly, the capacity of lens fiber cells to repair damaged DNA decreases during fiber cell differentiation in vitro.

The catalytic and DNA-binding domains of PARP are separated by the caspase target sequence DEVD. Caspase-mediated cleavage of PARP generates 80-kDa and 20-kDa products corresponding to the catalytic and DNA-binding domains, respectively. The latter may play a direct role in the later stages of apoptosis by binding to DNA breaks and thereby preventing access by other repair enzymes.

Full-sized PARP ( 120 kDa) is observed in the developing rat lens but is progressively cleaved to an 80-kDa fragment (Ishizaki et al., 1998). In the outer fibers of the adult rat lens, the 80-kDa fragment is the predominant form. Similarly, in the chicken lens, cleaved PARP is only detected late in embryonic development, after the onset of organelle degradation (Wride et al., 1999).

Although there is no direct evidence that PARP is cleaved by caspase during fiber cell differentiation in vivo, in vitro experiments support this notion. Lens epithelial cells cultured under appropriate conditions undergo many of the morphological transformations observed during fiber differentiation in vivo, including progressive cleavage of PARP. The addition of the pan-caspase inhibitor zVAD-fmk to cultures of rat or chicken lens cells inhibited PARP cleavage (Ishizaki et al., 1998; Wride et al., 1999). Taken together, these observations suggest that PARP is degraded during fiber cell differentiation by the action of one or more caspases.

Immunofluorescence studies have indicated that the nuclear lamina is dismantled during organelle loss (Bassnett and Mataic, 1997; Dahm et al., 1998a) and that lamin B, a prominent component of the nuclear lamina, is ultimately degraded (Bassnett and Mataic, 1997). Lamin B is thought to be specifically susceptible to digestion by caspase-3 or caspase-7 (Slee et al., 2000).

Immuncytochemical studies indicate that DFF45 is expressed in the outer fiber cells and that cleavage to a 30-kD product is first observed at E12 in the chicken lens (Wride et al., 1999). This corresponds to the stage at which organelle loss is initiated.

Thus, at least three classic caspase substrates, PARP, lamin B, and DFF45, appear to be proteolytically cleaved at the border of the OFZ. All are well-characterized caspase-3 substrates but may be cleaved by other members of the caspase family. In contrast, the cleavage of alpha II-spectrin appears to be mediated specifically by caspase-3 (Janicke et al., 1998). Caspase-cleaved spectrin has recently been identified in the developing chicken lens at E12 (Lee et al., 2001), the stage at which organelle degradation commences in core fiber cells.

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Caspase-3 is itself cleaved, and thereby activated, in apoptotic cells. Caspase-3 has been localized in the embryonic chicken lens by immunocytochemistry, and a 17-kD fragment has been identified by Western blotting of lens lysates from >E12 embryos (Wride et al., 1999). Caspase-3 activity has not yet been measured directly in the developing lens. However, in lentoid cultures from rat or chicken, treatment with the pan-caspase inhibitors Z-VAD-FMK and Boc-D-FMK leads to a significant reduction in the number of TUNELlabeled nuclei (Ishizaki et al., 1998; Wride et al., 1999). Interestingly, in this in vitro setting, the caspase-3–specific inhibitor Z-DEVD-FMK is ineffective (Wride et al., 1999), perhaps suggesting the presence of other effector caspases that might substitute for caspase-3.

Thus, a strong circumstantial case has emerged that implicates caspases, particularly caspase-3, in organelle degradation: Caspase-3 activation coincides with the onset of organelle breakdown; four well-characterized caspase substrates (PARP, lamin B, DFF45, and spectrin) are cleaved during organelle degradation; and, at least in vitro, caspase inhibitors block certain aspects of organelle degradation. Given that effector caspases appear to be activated during organelle loss and that the expected apoptotic substrates are cleaved, it is pertinent to ask why lens fiber cells do not undergo classic apoptosis rather than organelle degradation. Interestingly, the nuclear events preceding organelle loss closely resemble those observed during apoptosis, but the membrane events do not. It is not currently understood how the nuclear and membrane elements of the apoptotic program have been dissociated from each other during lens organelle loss.

9.7.2.3. Role of Nucleases

The disintegration of the fiber cell nucleus is accompanied by the progressive cleavage of DNA. Strand scissions produce multiple 3 -OH ends in the DNA (Bassnett and Mataic, 1997) and ultimately release nucleosome-sized fragments into the cytoplasm (Appleby and Modak, 1977). The identity of the nuclease (or nucleases) responsible has not been determined, but several candidate enzymes have been detected.

The endonuclease responsible for DNA laddering during apoptosis is DNA fragmentation factor (DFF; Liu et al. 1997). In its inactive form, DFF is a heterodimer composed of a 45-kDa inhibitory chaperone subunit (DFF45) and a 40-kDa latent endonuclease subunit (DFF40). The DFF complex resides in the nucleus (Samejima and Earnshaw, 2000). Caspase-3 specifically cuts DFF45, resulting in the dissociation of the cleaved DFF45 from DFF40. The released endonuclease forms homo-oligomers that are the enzymatically active form of DFF40 (also known as CAD or CPAN). Studies on the cleavage preferences of DFF40 on naked DNA have demonstrated that this nuclease has a pH optimum of 7.5, requires Mg2+ (but not Ca2+), and is inhibited by Zn2+. DFF40 attacks the chromatin in the internucleosomal linker, producing blunt ends or ends with 1-base 5 -overhangs possessing 5 -phosphate and 3 -hydroxyl groups (Widlak et al., 2000). Prolonged incubation of DNA with DFF40 results in the generation of oligonucleosomal DNA ladders. Immunocytochemical studies on the lens have demonstrated the presence of DFF45, although expression is apparently restricted to the cytoplasm (rather than the nuclei) of epithelium and outer fiber cells (Wride et al., 1999). Significantly, immunoblotting experiments detected the 30-kDa cleaved form of DFF45 in the embryonic chicken lens only at >E12, suggesting that, in vivo, the activation of DFF may coincide with the initiation of organelle loss. It is tempting to speculate that DFF activation may result from proteolysis by caspase-3, especially as this enzyme is also known to be activated in the lens at E12 (Wride et al., 1999). However, incubation of lens cultures with a variety of caspase inhibitors failed to prevent cleavage

of DFF, indicating that, at least in vitro, DFF activation occurs via a caspase-independent pathway (Wride et al., 1999).

DNases may be grouped into three functional categories: Mg2+-dependent endonucleases (of which DFF40 is the best-studied example), Ca2+/Mg2+-dependent endonucleases, and cation-independent endonucleases (Counis and Torriglia, 2000). As a group, nucleases are poorly characterized at the molecular level. In only a few cases are primary sequences available, and rarely has a physiological role in chromatin breakdown been demonstrated unequivocally. The best-characterized Ca2+/Mg2+-dependent endonuclease is DNase I. This enzyme was first purified from bovine pancreas and has since been cloned and crystallized for structural studies. The cleavage of DNA by DNase I generates strand breaks with free 3 -OH ends, and this is the characteristic cleavage pattern observed during fiber cell maturation (Bassnett and Mataic, 1997; Ishizaki et al., 1998; Wride and Sanders, 1998). However, RT-PCR analysis of lens cDNA has failed to detect any transcripts for DNase I (Hess and FitzGerald, 1996). Therefore, if a Ca2+/Mg2+-dependent nuclease is involved in fiber denucleation, it is unlikely to be DNase I itself. A DNase I–like 30-kDa protein has been identified in the lens by DNase activity gels and immunoblots (Counis et al., 1991; Arruti et al., 1995; Torriglia et al., 1995). A 40-kDa DNase, inhibited by high concentrations of divalent ions but with an absolute requirement for Ca2+ and Mg2+, has also been demonstrated in both epithelial and fiber cells (Counis et al., 1991).

The Ca2+/Mg2+-independent nucleases include DNase II–like enzymes. DNase II is a ubiquitously expressed enzyme (Counis and Torriglia, 2000) and has been implicated in DNA fragmentation during apoptosis (Eastman, 1994; Belmokhtar et al., 2000). Four DNase II–like proteins have been identified in the lens by immunoblot analysis (Torriglia et al., 1995), and evidence for a role in fiber cell denucleation was provided by experiments in which antibodies to DNase II inhibited DNA degradation in isolated lens cell nuclei. Immunocytochemical localization of DNase II–like nucleases in fiber cells indicated that this enzyme is highly concentrated in the fiber cell nuclei (Torriglia et al., 1995; Counis et al., 1998). It should be noted, however, that DNase II digestion of DNA generates 3 -phosphate ends and that there is little evidence for accumulation of this kind of DNA damage during fiber differentiation in vivo (Bassnett, 1997; Bassnett and Mataic, 1997).

9.7.2.4. What Triggers Organelle Degradation?

Perhaps the most striking aspect of organelle degradation in the lens is that it appears to occur simultaneously in a thin shell of fiber cells. It is not known what serves to trigger organelle loss in this particular cell layer. In principal, the trigger could be spatial or temporal in nature (or both). The border of the OFZ is located approximately 800 µm beneath the lens surface in the embryonic chicken lens (Bassnett and Beebe, 1992) and 100–200 µm below the surface in the adult monkey lens (Bassnett, 1992).

Because the distance between the lens surface and the border of the OFZ remains relatively constant throughout chicken lens embryonic development (see Fig. 9.9), it has been suggested that organelle degradation may be triggered by a gradient of a diffusible substance (Bassnett and Mataic, 1997). As the volume of the lens increases, many standing gradients will be established because of the avascularity of the tissue and the relatively slow diffusion-limited exchange of metabolites with the surrounding humors of the eye. For example, glycolysis in the bulk of the tissue results in the establishment of a lactic acid gradient (Bassnett et al., 1987) and therefore a gradient of intracellular pH (Bassnett et al., 1987; Mathias et al., 1991). Any metabolite diffusing from the surface and consumed by the lens

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Figure 9.9. The relative sizes of the lens and the organelle-free zone (OFZ) during embryonic development. The images are drawn to scale from data presented in Bassnett and Beebe (1992). The arrows indicate the constant thickness of the layer of organelle-containing fiber cells.

will likely be found at lowest concentration in the lens core. Of particular relevance may be intracellular oxygen. Oxygen is a permeable species that will be consumed in mitochondria located in the outer layers. As the lens increases in volume, oxygen concentration in the lens core is expected to fall. If the oxygen reaches a sufficiently low concentration, the integrity of the mitochondria would be compromised, perhaps enough to trigger an apoptoticlike cascade, including release of cytochrome C and activation of the caspase pathway. This is an attractive hypothesis, but there is currently little experimental evidence to support it. Oxygen tension in the eye is low, and the noninvasive measurement of intracellular oxygen gradients in the lens is technically challenging.

Lens fiber cells express TNFα and TNF receptors (TNFR1 and TNFR2), and it has been proposed that this proinflammatory cytokine may play a role in triggering lens fiber organelle degradation (Wride and Sanders, 1998). Of the 17 or more members of the TNF superfamily, TNF is probably the most potent inducer of apoptosis, acting largely through the type 1 receptor (Rath and Aggarwal, 1999). In vitro, the number of TUNEL-positive cells in differentiating lens cell cultures increases following application of TNFα or agonistic antibodies to TNFR1 and TNFR2. Conversely, neutralizing antibody to TNFα caused a significant reduction in TUNEL labeling (Wride and Sanders, 1998).

9.7.3. Cessation of Transcription and Translation

9.7.3.1. Transcription and Translation at the Border of the OFZ

Presumably, with the loss of the fiber cell nucleus, transcription stops and any residual translation must depend on the stability of extant mRNAs and the durability of the protein synthetic machinery. It is important, however, to determine precisely when transcription ceases. It is possible, for example, that transcription is terminated long before organelles are degraded. Morphological data suggest that transcription may stop in cells immediately adjacent to the OFZ. Coilin and fibrillarin are markers for distinct nuclear compartments (the coiled body and nucleolar compartments, respectively) that show characteristic distributions in transcriptionally active cells. In the lens, both of these markers disappear shortly before nuclear condensation. This has been interpreted as signifying transcriptional shutdown (Dahm et al., 1998a).

The transcriptional and translational competence of differentiating fiber cells has been assessed directly by testing the ability of a fiber cell to synthesize the autofluorescent protein GFP from a microinjected plasmid template (Shestopalov and Bassnett, 1999). Twenty-four hours after plasmid injections into superficial regions of the lens, strongly fluorescent fiber cells were observed. As injections were made at progressively greater depths, a point was reached where injections never resulted in GFP synthesis. The last GFP-synthesizing fiber was estimated to lie immediately adjacent to the OFZ. From these functional measures and correlative morphological analyses, it appears that fiber cells are transcriptionally and translationally competent until shortly before organelle degradation.

9.7.3.2. Stability of RNA in Mature Fiber Cells

Because they lack an ER, mature fiber cells presumably are no longer able to synthesize membrane proteins or secreted proteins. However, it is conceivable that residual synthesis of cytoplasmic proteins (e.g., crystallins) might continue in these cells. In this regard, it is interesting to note that certain mRNAs are unusually long lived in lens fibers. For example, Northern blot analysis has demonstrated the persistence of full-length δ-crystallin mRNA transcripts in the core region of lenses from three-month-old chickens (Treton et al., 1982). Thus, in comparison with most mRNAs (which have half-lives measured in minutes or hours), δ-crystallin mRNA in lens fibers is remarkably stable. The stability of this transcript may also reflect intrinsically low RNAse activity in the core of the lens or the presence of an endogenous ribonuclease inhibitor (Ortwerth and Byrnes, 1971, 1972).

Despite the persistence of specific mRNAs, it appears unlikely that significant protein synthesis occurs in cells within the OFZ. In the majority of these cells, ribosomes are completely absent (Kuwabara, 1975), and most attempts to metabolically label nascent proteins have proved unsuccessful (Wannemacher and Spector, 1968; Bagchi et al., 1981; Russell et al., 1996). Proteins that are highly expressed in differentiated cells are often translated from unusually stable mRNAs, and this is thought to account, in part, for their abundance. Thus, a half-life of >15 hours has been reported for myosin (Buckingham et al., 1974), immunoglobulin (Cowan and Milstein, 1974), and ovalbumin (Palmiter and Carey, 1974) mRNAs. By this argument, the stabilization of crystallin mRNA templates may be part of the mechanism by which fiber cells are able to synthesize these proteins in such abundance. The hypothesis that specific mRNAs are stabilized to allow their translation in anucleated core fiber cells appears to be less likely.

9.8. How Is the Process of Fiber Cell Differentiation Related to the Overall Shape of the Lens?

Lenses depend for their function on having a precise shape and refractive index. The shape of the lens is essentially the shape of the fiber cell mass, which is directly related to the shape of the fiber cells themselves. It is remarkable, therefore, that the shape of the lens may change dramatically during development and that the lenses of different species vary greatly in shape, from nearly spherical to distinctly oblate, with different anterior and posterior surface curvatures. To our knowledge, no studies have related the shape of the lens to the properties of individual fiber cells. What little is known about lens shape is that it is determined by the actions of factors outside the lens.

The first study to clearly demonstrate the effect of the extralenticular environment on lens morphogenesis was performed by Jane Coulombre and Chris Coulombre (1963). They

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showed that if the lens were rotated 180◦ so that the epithelium faced the retina, the lens repolarized, forming a new fiber mass from the epithelial cells and reforming an epithelium over what had previously been the basal ends of the fiber cells. In later experiments, these authors showed that when the original lens was removed and two lenses were implanted into the eye in its place, the two implanted lenses grew as if they were a single lens, irrespective of their orientation at the time of implantation (Coulombre and Coulombre, 1969). This “compound” lens eventually approximated the size and shape of the single lens from the contralateral, unoperated eye. This elegant experiment showed that the shape of the lens was not an intrinsic property of the tissue itself but was imposed by factors in the ocular environment. It would be interesting to extend this experiment to see whether lenses transplanted to the eye of a different species would assume a shape characteristic of the host or the donor.

9.9. Lens Pathology: Cataracts Caused by Abnormal Fiber Cell Differentiation

9.9.1. Posterior Subcapsular Cataracts

There are three major types of human age-related cataracts: nuclear, cortical, and posterior subcapsular. Nuclear and cortical cataracts occur in the fully differentiated cells of the lens nucleus and cortex, respectively. Typical posterior subcapsular cataracts (PSCs) are due to an accumulation of swollen cells at the posterior sutures, just beneath the posterior capsule. The morphology of PSCs suggests that they are caused by the abnormal differentiation of lens fiber cells (Streeten and Eshaghian, 1978; Eshaghian and Streeten, 1980).

Examination of the posterior regions of lenses with PSCs shows one or more “streams” of cells apparently migrating on the inner surface of the posterior capsule and extending from the lens equator to the plaque of swollen cells at the posterior pole. Examination of the cells in these streams shows that, as they migrate toward the posterior pole, they come to resemble swollen lens fiber cells (Eshaghian and Streeten, 1980). The swollen cells, along with cellular debris from the death of some of the migrating cells, accumulate at the posterior pole, causing the light scattering that is responsible for the cataract (Eshaghian and Streeten, 1980; Eshagian, 1982).

The causes of PSCs are not well understood. These cataracts occur with lower frequency than nuclear or cortical cataracts in most populations (approximately 10% of cataract surgeries). There is an increased risk for developing PSCs in patients on steroid therapy and in individuals with hereditary retinal degenerations, like retinitis pigmentosa. PSCs are one of the early signs of neurofibromatosis type 2 (NF2), a hereditary disease caused by mutations in the merlin tumor suppressor gene (Kaiser-Kupfer et al., 1989; Lim et al., 2000). However, there is little information to suggest why steroid therapy or retinal degeneration should lead to the formation of a PSC, and the mechanism of PSC formation in NF2 is not known. Research in this area has been hampered by the lack of an animal model that develops PSCs resembling those seen in human patients.

The morphology of the cells responsible for PSC formation suggests that this type of cataract is caused by a failure of some of the cells in the lens to differentiate properly. If a proportion of lens cells failed to elongate at the equator, they would remain attached to the posterior capsule and be carried to the posterior pole of the lens by the movement of the basal ends of adjacent fiber cells. Based on the mechanism of cell elongation proposed above,

one way that cells might fail to elongate would be if they lost their adhesive contacts with their neighbors. This would cause them to round up as they increased in volume rather than elongate. Such a view is consistent with the emerging role of the merlin tumor suppressor gene as a regulator of cell migration and, possibly, in connecting the actin cytoskeleton with the plasma membrane (Lim et al., 2000; Reed and Gutmann, 2001).

9.9.2. Secondary Cataracts

Modern cataract surgery is performed by peeling off the central portion of the lens epithelium, breaking up and removing the lens fiber mass, and implanting a clear plastic lens into the remaining “capsular bag.” Epithelial cells from the lens equator sometimes migrate on the denuded posterior capsule beneath the lens implant. There they may differentiate into small clusters of lens fiber cells (Elschnig’s pearls) that resemble the lentoid bodies that often form in primary cultures of lens epithelial cells. They may also form plaques of fibrous tissue. These plaques contain myofibroblast-like cells embedded in an abundant extracellular matrix. Both Elschnig’s pearls and fibrous plaques scatter light and degrade the visual image, forming secondary cataracts. Secondary cataracts are also often referred to as “posterior capsule opacification” (PCO) or “after cataracts.”

The cells in Elschnig’s pearls closely resemble lens fiber cells (Kappelhof et al., 1986; Sveinsson, 1993; Marcantonio and Vrensen, 1999). It is probable that these fiberlike cells are derived from equatorial epithelial cells that have migrated onto the posterior capsule, responded to the normal environment there, and begun the process of fiber cell differentiation. The abnormal size and shape of these lentoids are likely to be due to the abnormal physical environment in which they are differentiating. They do not have a “template” of preexisting fiber cells on which to elongate, possibly explaining their swollen shape, and the size of the tiny lenslike structures that are formed are disproportionately small for the adult eye. We know of no studies that indicate the degree to which the biochemical characteristics of these fiberlike cells resemble authentic lens fiber cells.

In additions to forming Elschnig’s pearls on the posterior capsule, lens fiber cells frequently differentiate near the lens equator. Here they often surround the haptics that position the IOL in the capsular bag. The aggregate of fiberlike cells that forms in this region is called Soemmering’s ring. Because it is positioned outside of the optic axis, Soemmering’s ring does not degrade the visual image. In young animals that have had experimental cataract surgery, the differentiation of fiber cells at the lens equator can re-form a fiber mass that has optical qualities comparable to the original (Gwon et al., 1989; Gwon et al., 1990; Gwon et al., 1992).

In contrast to the similarity of the cells in Elschnig’s pearls to lens fiber cells, the fibrous plaques found in secondary cataracts result from the transdifferentiation of epithelial cells into myofibroblast-like cells (Novotny and Pau, 1984; Schmitt-Graff et al., 1990; Hales et al., 1994; Marcantonio and Vrensen, 1999; Nagamoto et al., 2000). These cells are contractile and express α-smooth muscle actin, collagen type I, and a variety of other markers of fibrotic connective tissues (Hales et al., 1994; Hales et al., 1995; Kurosaka et al., 1995; Lee and Joo, 1999; Marcantonio and Vrensen, 1999; Nagamoto et al., 2000; Wunderlich et al., 2000). In experimental studies, lens epithelial cells can be stimulated to acquire many of the properties of myofibroblasts by treatment with the cytokine TGFβ (Hales et al., 1994; Kurosaka et al., 1995; Lee and Joo, 1999). However, it has not yet been demonstrated that signaling through this pathway is responsible for myofibroblast differentiation during secondary cataract formation in vivo.

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9.10. Concluding Remarks

The transparency and refractive properties of the lens depend directly on cellular features that arise during fiber cell differentiation. Therefore, a detailed knowledge of the differentiation process is central to an understanding of the fundamental optical properties of the lens. It also provides the basic information necessary to understand the etiology of certain types of cataract.

In this chapter we divided fiber cell differentiation into four stages that are identified by major physical landmarks common to all lenses. We hope that these stages provide a useful conceptual framework for the design and interpretation of future studies.

The formation of lens fiber cells is one of the most extreme examples of cell differentiation in nature, culminating in a degree of specialization scarcely equaled by any other cell type. However, as this chapter illustrates, there are many areas that remain to be explored before we can claim to have a full understanding of the events that underlie the differentiation process.