Download The Ocular Lens Epithelium

Bioscience Reports, Vol. 21, No. 4, August 2001 ( 2002)
MINI REVIEW
The Ocular Lens Epithelium
Suraj P. Bhat1
Receiûed April 12, 2001
An adult lens contains two easily discernible, morphologically distinct compartments, the
epithelium and the fiber-cell mass. The fiber-cell mass provides the lens with its functional
phenotype, transparency. Metabolically, in comparison to the fiber cells the epithelium is
the more active compartment of the ocular lens. For the purposes of this review we will
only discuss the surface epithelium that covers the anterior face of the adult ocular lens.
This single layer of cells, in addition to acting as a metabolic engine that sustains the
physiological health of this tissue, also works as a source of stem cells, providing precursor
cells, which through molecular and morphological differentiation give rise to fiber cells.
Morphological simplicity, defined developmental history and easy access to the experimenter make this epithelium a choice starting material for investigations that seek to
address universal questions of cell growth, development, epithelial function, cancer and
aging. There are two important aspects of the lens epithelium that make it highly relevant
to the modern biologist. Firstly, there are no known clinically recognizable cancers of the
ocular lens. Considering that most of the known malignancies are epithelial in origin this
observation is more than an academic curiosity. The lack of vasculature in the lens may
explain the absence of tumors in this tissue, but this provides only a teleological basis to a
very important question for which the answers must reside in the molecular make-up and
physiology of the lens epithelial cells. Secondly, lens epithelium as a morphological entity
in the human lens is first recognizable in the 5th–6th week of gestation. It stays in this
morphological state as the anterior epithelium of the lens for the rest of the life, making it
an attractive paradigm for the study of the effects of aging on epithelial function. What
follows is a brief overview of the present status and lacunae in our understanding of the
biology of the lens epithelium.
KEY WORDS: Eye lens; epithelial cells; fiber cells; programmed cell death; cataract.
THE EPITHELIUM AND ITS CELLS
Developmentally, all of the lens can be considered as an asymmetrically folded epithelium (Zamphigi et al., 2000) with region-specific morphological and physiological
specialization. A single layer of cells, the lens epithelium covers the anterior face of
the lens that faces the cornea and the outside world. The lens epithelium ends on
the rims of the anterior surface (oval or circular in shape, depending on the species)
1
Vision Molecular Biology Laboratory, Jules Stein Eye Institute, 100 Stein Plaza, BH 623, UCLA School
of Medicine, Los Angeles, CA 90095-0000; e-mail: [email protected]
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0144-8463兾01兾0800-0537兾0  2002 Plenum Publishing Corporation
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Fig. 1. Simplified drawing of the ocular lens. Note that the epithelium has three regions
of cells in non-dividing phase (central epithelium), in dividing phase (germinative) and
differentiating phase (equatorial). The anterior and posterior sutures are formed by the
meeting of the elongating fiber cells that make the bulk of the lens mass. The surrounding capsule (shaded area around the lens) indicates that the basal surface of the epithelium and the fiber cells is on the outside, while the apical surface faces the inside of
the lens. ‘‘Apical interface’’, an area of contact between the apical surfaces of the epithelial cells and the fiber cells is not indicated. It is the area just below the epithelial
layer.
of the lens (Fig. 1). It contains cells in the central region that do not divide and are
essentially ‘‘quiescent’’, surrounded by a germinative兾dividing zone of cells, followed
(at the equatorial fringe) by the dividing cells that differentiate into fiber cells. In
the newborn rat lens, as assessed by tritiated thymidine labeling in the intact animal,
the epithelium contains about 4.7% of dividing cells. This decreases to 1.7% by day
6 and then 0.8% by day 35 (Mikulicih and Young, 1963). A remarkable feature of
this epithelium is its capacity to divide and differentiate almost all through the life
of an individual. This feature of sustained growth is very much similar to its closest
embryological sibling, the cells in the skin. The difference between the two however
is noteworthy. The pattern of growth that results from the division and differentiation of the lens epithelial cells at the equatorial rim gives lens its unique physiognomy. The new (fiber) cells made from the lens epithelium, at its equatorial rim,
keep adding on top of the older fiber cells in such a way so that the oldest cells are
buried deep inside the lens. Thus, by peeling away, one fiber at a time one could
theoretically unveil a temporal catalog of the molecular changes starting from the
outside (the youngest or the most recent fibers) to the center (the oldest, the
‘‘nucleus’’ of the lens, which contains fibers laid in 5th兾6th week of gestation in
humans).
The cells in the lens epithelium represent typical epithelial morphology. They
are cuboidal, presenting a cobble-stone-like appearance in their native state and in
ûitro, if cultured without excessive passaging (Fig. 2). These cells in the epithelium
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Fig. 2. Photomicrographs of the native human lens epithelium (A) and cultured human fetal lens
epithelial cells (B). A, taken, with permission of the copyright holder, Association for Research in
Vision and Ophthalmology, from Mercantonio et al. (2000) Inûest. Ophthalmol. and Vis. Sci.
41:1130–1141. B, taken, with permission of the copyright holder, Academic Press, Orlandlo,
Florida, from Nagineni and Bhat (1988) Deûelopmental Biology 130:402–405.
are packed with very little intercellular space. Francois and Rabaey (Francois and
Rabaey, 1951) observed lens epithelium under a phase-contrast microscope. They
reported the presence of ‘‘pale polyhedral’’ and ‘‘dark star-shaped’’ cell types. A
recent in ûiûo study (Balaram et al., 2000) using the non-contact specular microscopy
recognized four morphological features of the live human lens epithelium. These
were categorized as ‘‘linear furrows’’, ‘‘columnar organization’’, ‘‘puffy clouds’’ and
‘‘black holes’’. These morphological features remain without a biochemical or molecular definition at this time. The diameter of the human lens epithelial cells ranges
from 9–17 mm (Brown and Bron, 1987). The cell size has been reported to increase
with age (Perry et al., 1979; Robinson et al., 1990), which suggests a change in the
cell density. Females have been reported to have higher cell density in the human
lens epithelium than in males (Konofsky et al., 1987; Guggenmoos-Holtzmann et
al., 1989). It must be recognized however, that both the cell size and cell density is
specific to a particular region or area of the lens epithelium. For example, in the
monkey lens, in the germinative zone, cells increase in size by about 30% from age
7 years to age 24.5 years, while the central zone epithelial cells (in the same period)
increase by 150%. Notably, within the same period the cells in the germinative zone
increase in number, while cells in the central epithelium seem to decrease in number
(by about 17%) (Kuszak, 1997).
The relationship between cell density and age is interesting, although controversial. An earlier report (Guggenmoos-Holtzmann et al., 1989) that there is an agerelated decrease in the cell density in the lens epithelium has been recently confirmed
(Balaram et al., 2000). This recent study calculated loss of 675 cell兾mm2 in a 75-year
life span (that amounts to a loss of 14% of the cells). This estimate is based on the
unproven assumption that the rate of loss is linear with age, however it is not very
different from that reported in the aging monkey lens central epithelium (Kuszak,
1997). Others have found no such relationship (Fagerholm and Philipson, 1981;
Karin et al., 1987). Karim et al. (1987) reported a decrease in the mitotic index of
lens epithelial cells under normal as well as cataractous conditions. Harocopos et
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al. (1998) working with capsulotomy specimens from cataract surgeries and noncataractous lenses from eye banks concluded that there was no relationship between
cell density and the severity of cataract or between cell density and age. They however reported that in cataractous lenses the epithelium directly over the opaque (cataractous) had higher cell density when compared to that overlaying the transparent
regions. It is possible that a loss of a patch of cells overlying a cataractous fiber cell
area may lead to the activation of cell division and therefore, higher cell density.
This observation, however, needs to be confirmed.
The relationship between cell proliferation and cataracts is difficult to establish
particularly in a causal context. A recent study with sugar cataracts makes a good
example: A secondary complication of diabetes is cataractogenesis due to accumulation of sugar兾sugar alcohols in the lens causing pathological water uptake. Struck
et al. (2000) make an assessment of lens epithelial cell densities in type-II diabetics
(3691C346 cells兾mm2) vs. non-diabetes (4162C504 cells兾mm2). Among cataractous
lenses, the nuclear cataract had the highest cell density (4250C513 cell兾mm2). Interestingly, they found no significant differences in cell number between the females
(4036C525 cells兾mm2) and the males (3788C421 cell兾mm2) in disagreement with
earlier studies cited above. These authors also reported that the lowest mean cell
density in the lens epithelium was associated with posterior subcapsular cataracts
(3620C333 cells兾mm2). This is quite interesting because it suggests an aberration in
the mechanism that regulates (inhibits?) migration of cells to the posterior subscapsular area. Whether decreased cell density in the anterior epithelium is also related
to this aberration can only be speculated.
If it is accepted that there is a decrease in cell density either with age or with
cataracts we must ponder the question as to how does this happen? Do cells in the
lens epithelium die by apoptosis兾necrosis? If so what triggers these processes? It is
a common observation that cells when cultured at low densities do not grow well.
This is true of lens epithelial cells also. It has been shown that the medium from
high-density lens epithelial cell cultures promotes the survival of the cells in the lowdensity cultures suggesting that these cells secrete growth兾survival factors (Ishizaki
et al., 1993). At lower cell densities these factors are diluted and are, therefore, not
available to cells for their survival. This leads to the questions: Are there lens-specific
physiological factors that maintain a minimal cell density in the lens epithelium to
avoid intervention by apoptosis? How much decrease in cell density is tolerated
before it becomes pathological? Recently proteins兾factors that seem to enhance the
survival of lens epithelial cells in culture have been identified (Davidson et al., 1998;
Singh et al., 2000).
DEVELOPMENTAL HISTORY
Emergence of a transparent lens is the culmination of morphogenetic
remodeling, cell differentiation and development from one cell type, the surface
(head) ectoderm (Spemann, 1901; Lewis, 1904; Jacob and Sater, 1988; Saha et al.
1989; Grainger, 1992). Lens epithelium represents the region of the surface ectoderm
that is part of the lens ‘‘field’’ in the developing embryo. The lens morphogenesis
commences with the appearance of a thickening in the pre-determined area of the
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Fig. 3. Early stages in the formation of the lens vesicle (A–D) and the lens. Stage E represents
asymmetric morphogenesis in the lens vesicle. Posterior epithelial cells elongate (primary fiber cells)
and fill the lumen of lens vesicle (not completely filled in the drawing), resulting in the lens having
no epithelium on the posterior surface. Differentiating secondary fiber cells (shown in the illustration) from the anterior epithelium are the main contributor to the growth of the postnatal lens.
surface ectoderm (Spemann, 1962; Li et al., 1994), the future ‘‘lens placode’’, a point
where the lateral protuberance from the fore brain invaginates to form what becomes
the ‘‘optic vesicle’’ or ‘‘the optic cup’’, the surface ectoderm (lens placode) also
invaginates and pinches off as a lens vesicle. Studies done with excised surface ectoderm and the optic vesicle have shown that a physical contact between the two
surfaces is not essential for the formation of the lens vesicle (Jacob and Sater, 1988).
Molecular cues that emanate, possibly from both the interactive surfaces, are therefore diffusible. These inductive messengers have not yet been unequivocally identified. Gene products including growth factors and transcription factors associated
with early morphogenesis of the lens have been detailed (Ogini and Yasuda, 1998;
Jean et al., 1998) and will not be discussed here. It is however interesting to follow
the emergence of the incipient lens, complete with fiber cells so that a relationship
(albeit morphological兾embryological) between the adult lens epithelium and the rest
of the lens (fiber cells) can be envisioned. This process is polar (with respect to the
epithelium), in its progression and is briefly discussed below (Fig. 3).
The cells that line the lens vesicle have their apical surfaces pointing inwards
into the lumen of the vesicle and the basal surfaces outwards contacting the basement membrane that makes the ‘‘capsule’’ that surrounds the vesicle (and eventually
the lens). The transformation of this vesicle into a structure that is recognizable as
a lens starts with the differentiation of the posterior epithelial cells into fiber cells
(Fig. 3E). Differentiation involves elongation of these cells in an anterio-posterior
obliterating the lumen of the lens vesicle. These fiber cells are known as the ‘‘primary
fiber cells’’. This process brings the apical surface (ends) of the elongated fiber cells
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(posterior epithelial cells) in direct contact with the apical surfaces of the anterior
epithelial cells, this area of contact is known as the ‘‘apical interface’’ (Zamphigi et
al., 2000). The posterior surface of the lens thus lacks any epithelium having been
transformed into fiber cells. The anterior epithelium, which by dint of its distance
from the bottom of the optic cup presumably escapes the molecular influences of
the optic cup (future retina) remains undifferentiated. The proximity of embryonic
retina to the posterior lens vesicle has been suggested to promote lens fiber cell
differentiation and morphogenesis (McAvovy and Fernon, 1984). A large number
of growth factors have been implicated in this process but none unequivocally
(McAvovy and Chamberlain, 1990; Lang, 1999).
The asymmetric morphogenesis within the lens vesicle (Fig. 3) results in the
presence of an epithelial layer of cells on the anterior face of the lens covering a
mass of fiber cells. The future growth of the lens is dependent on this anterior epithelium. It is the cells at the equatorial zone of this epithelium that elongate in an
anterio-posterior fashion into ribbon-like long fiber cells that are responsible for the
growth of the lens beyond the 6-8th week of gestation. These fiber cells that are
produced from the edges of the anterior epithelium are known as ‘‘secondary fiber
cells’’ as opposed to ‘‘primary fiber cells’’ that were made from the posterior epithelial cells of the lens vesicle (Fig. 3).
It is reasonable to expect that the physiology and function of the lens epithelium
will be influenced by the aqueous humor in the front, by vitreous humor in the back
and by the ciliary body at the equatorial zone. The ciliary body, which is derived
from the anterior lip of the optic cup, interestingly is (at least morphologically)
reminiscent of the ‘‘undifferentiated’’ neuroretina, which has been suggested to
influence the differentiation of the posterior epithelial cells in the lens vesicle (see
Jacob and Sater, 1988; Saha et al., 1989; Grainger, 1992). Young and associates
(Mikulicih and Young, 1963) reasoned that the surrounding ciliary body must have
a regulatory influence on cell division and DNA synthesis in the equatorial region
of the lens epithelium. Whether the surrounding ciliary body has any molecular
influence on the growth and differentiation of the fiber cells in the equatorial region
of the lens has not been directly investigated. Nagineni and Bhat (1992), showed
that a co-culture of the ciliary body fibroblasts and the lens epithelial cells (both
isolated from the human fetal eyes) results in the appearance of differentiating fibers
at the interface of the fibroblasts and the lens epithelial cells. This investigation did
not address the question as to which cell type ûiz., the epithelial or the fibroblasts
differentiated to cells that expressed crystallins, although embryological history of
the two would suggest that it was the epithelial cells. This in ûitro culture system has
a promise of providing molecular identification of communicating influences
between these two cell types. A discussion on what growth factors and agents may
stimulate differentiation in the lens epithelial cells (Jean et al., 1998; Lang, 1999) is
beyond the scope of this review. However it is appropriate to comment on the positional integrity of the epithelium and the environment around it. This is elucidated
in two publications motivated by two different investigations about three decades
apart. Coulombre and Coulombre (1963) rotated the developing lens 180° thereby
exposing the epithelium to the vitreous humor and the retinal influences, this started
secondary fiber differentiation of the epithelial cells. Hightower and McReady (1991)
Ocular Lens Epithelium
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discovered that only when the anterior surface of the lens was exposed to selinite
did the cataract develop. Exposure of the posterior face that lacks the epithelial layer
had no effect. These observations directly point to a very important conclusion that
an alteration in the environment around the lens cannot only alter the developmental
status of the overlying epithelium but it can lead to physiological changes in the rest
of the fiber mass.
EPITHELIUM IS THE MAJOR SITE OF TRANSPORT/METABOLISM/
DETOXIFICATION
The overall metabolic status of the fiber cells in the absence of endoplasmic
reticulum, mitochondria and a nucleus is comparatively very low (Lieska et al.,
1992). There is no vascular system as we know it that would take nutrients to the
fiber cells and remove metabolic兾physiologic waste replenish the intra—and intercellular milieu of the lens. Mere diffusion as a process to sustain the slow but substantial physiology of the ocular lens will be insufficient to accomplish this
efficiently. A study of relative rates of transport across the anterior and posterior
surfaces of the lens had led to the model of the ‘‘pump-leak’’ system (Kinsey and
Reddy, 1965; Becker and Cotlier, 1962; Harris and Becker, 1965). The dynamics of
the active transport across the lens epithelium from the aqueous humor creates a
gradient of NaC and KC ions in the lens; high KC in the anterior and high NaC in
the posterior of the lens (Paterson, 1972). Epithelium does most of the work in
maintaining a low sodium concentration in the lens and an active resting potential,
pumping in KC and extruding NaC, which enters the lens from the posterior surface
by diffusion. There is however very high specific resistance (−1 MΩ cm2) that helps
reduce passive diffusion of ions into fiber cells. There are three isoforms of NaCKC
ATPase in the epithelium while there is only one in the fiber cells (Mosley et al.,
1996). Based on immunoblotting there are almost equal number of NaC兾KCATPase molecules in the epithelium and fiber cells per mg membrane protein, however for some unknown reasons the enzyme in the fiber cells is not highly active.
There are three carrier systems in the lens epithelium for the transport of basic,
neutral and acidic amino acids (see Reddy, 2000 for references). Interestingly lens
accumulates very high concentrations of taurine (2-aminoethanesulfonic acid). While
the significance of the presence of high concentrations of taurine in the lens epithelium remains speculative, changes in its concentrations are indicative of the
changes in the permeability characteristics of the epithelium, particularly in the cataractous lens when the concentration of taurine decreases (Reddy et al., 1976). Presence of carrier systems for the transport of taurine into the lens have been
demonstrated but not characterized at the molecular level. The taurine transporter
in retinal pigment epithelium seems to be regulated by calmodulin indicating that
Ca2C levels may thus determine the activity of the taurine transporter (Ramamoorthy et al., 1994). It is also possible that one of the functions of having taurine at
high concentrations may be regulation of the intracellular Ca2C levels (Huxtable,
1992). Ocular lens maintains a large gradient of Ca2+ and the lens epithelium plays
an important role in maintaining these levels (Jacob, 1983; Duncan and Jacob,
1984). Free Ca2C in the cells can either be increased by release from stored Ca2C
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complexes or in its absence, by transport of Ca2C from extracellular space. It is
believed that store-activated Ca2Centry is the major route of Ca2C influx into cells.
The presence of electrical current that flows across the plasma membrane following
this influx has been demonstrated and the existence of a ubiquitously distributed
store-operated Ca2C channel has been predicted (Parekh and Penner, 1997). These
have not yet been studied in the ocular lens epithelium. Interestingly, lens contains
high concentrations of myo-inositol (see Reddy, 2000). A NaC兾myo-inositol contransporter gene has been characterized in the bovine lens epithelium (Zhou and
Cammarata, 1997).
Human lens epithelial cells accumulate large amounts of polyol when exposed
to high galactose in culture (Lin et al., 1990). Aqueous humor contains glucose at
the same levels as in the blood (Chylack, 1971). It was observed very clearly that
glucose uptake occurs in the lenses even after the removal of the capsule and the
epithelium (Giles and Harris, 1959). Also, the capacity to transport glucose is comparable at both the anterior as well as posterior faces of the lens (Kern and Ho,
1973a). Oxidative phosphorylation consumed glucose transported via glucose transporters (GLUT) into the epithelium (Goodenough et al., 1980). In the rat lens
GLUT1 is predominantly expressed in the epithelium and GLUT3 in the cortical
fiber cells (Merriam-Smith et al., 1999). It is of interest to note that GLUT3, which
has a lower Km for glucose than GLUT1 is widely expressed in cells exposed to
easily accessible levels of glucose. Delineating the relationship of the expression of
different isoforms of GLUT transporters and sugar uptake by epithelium would be
important in any attempt of regulating the accumulation of glucose in the diabetic
lens.
The effect of glucocorticoids on the physiology of the lens epithelium is of
practical significance since administration of steroid hormones is part of the therapeutic schemes in treating glaucoma and other related inflammatory pathologies of
the eye. Cortisol and aldosterone can alter the physiological equilibrium within the
lens by altering the ionic composition of the lens and aqueous humor (Starka et al.,
1986). Appreciable expression of glucocorticoid and mineralocorticoid receptors and
their 11- β -hydroxysteroid dehydrogenases that regulate their activity at the prereceptor level in the lens epithelium was recently reported (Stokes et al., 2000).
Lens epithelium is also the major site of detoxification and defense against oxidative insults (Reddy et al., 1980; Giblin, 2000). It contains active preteosome complexes (Andersson et al., 1998) and an active ubiquitin-dependent proteolysis
pathway (Shang et al., 1997) that degrades oxidized proteins such as α -crystallins
(Huang et al., 1995). The lens epithelium contains the highest concentration of
glutathione (GSH) and NADPH (Giblin et al., 1981). A concentration above 20 nM
has been reported. Glutathione is synthesized within the epithelium (Reddy et al.,
1966; Kern and Ho, 1973b). Presence of a sodium independent glutathione transporter has also been demonstrated in the lens (Kannan et al., 1995). The lens epithelium contains an active glutathione redox cycle (Glutathione reductase. NADPH,
hexose monophosphate shunt) that keeps GSSG reduced to GSH ensuring detoxification of oxidizing molecules. Epithelium is able to detoxify physiological concentrations of H2O2 enzymatically involving glutathione reductase, glutathione
peroxidase and the hexose monophosphate shunt (see review by Giblin, 2000). NaC兾
Ocular Lens Epithelium
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KC-ATPase, the main transport pump, is inhibited by peroxide thus inhibiting active
cation transport (Garner et al., 1983; Giblin et al., 1987). It was discovered very
early that peroxide is formed via the light-catalyzed oxidation of ascorbic acid in
the aqueous humor (Pirie, 1965). Since reactive oxygen species can lead to mutations
and carcinogenesis (Fridovich, 1986), it is to be expected that the lens epithelium
may have evolved an elaborate defense against oxidative (Hightower et al., 1994).
ELECTRICAL PROPERTIES
Direct electrical measurements have shown that the resting potential of the lens
(−69.6 mV for the rat lens) (Lucas et al., 1987), is mostly because of the KC gradient.
Cells control their volume by maintaining a negative resting potential. This is
achieved by keeping the concentration of intracellular NaC low and KC high. The
epithelial cells maintain their volume by restricting the inflow of the anions. Active
NaC兾KC transport, keeps the NaC兾KC ratio low. Both NaC兾KC ATPase and a
number of KC channels are synthesized in the epithelial cells. Because there is comparatively very little functional NaC兾KC ATPase and none or few KC channels in
the fiber cells the resting membrane potential of the lens must be considered to be
primarily due to activities in the epithelial layer and兾or superficial cortical layers of
fiber cells. NaC兾KC pump exchanges three NaC ions for two KC ions thus generating an outward current. Regional differences in the predominance of one or the
other isoform in the lens epithelium have been reported (Garner and Horwitz, 1994).
α 3 isoform is the predominant protein of the central epithelium, while α 1 isoform
is more predominant in cells nearer the equator. Functionally different isoforms
of NaC兾KC ATPase could have specific physiological consequences but regional
differences in the expression and activity of this pump have not been clearly correlated. A number of potassium channels have been cloned (Chandy and Gutman,
1995). In the lens presence of inward rectifiers (they conduct inward KC current
much faster than they do the outward current), outward rectifiers (delayed rectifiers)
and calcium-activated large-conductance KC channels have been found (Mathias et
al., 1997; Rae and Shephard, 1998; Rae and Shephard, 2000). A complete molecular
catalog of all the KC channels that exist in the lens epithelium will allow a regional
localization of specific types and their functional import.
Ocular lens has been called a syncytium (Goodenough, 1992; Mathias et al.,
1997) based on the fact that the entire lens is connected, the epithelial cells to epithelial cells, epithelial cells to fiber cells and fiber cells to fiber cells. The way lens is
placed, it is natural to think that the epithelium becomes the link that transports
nutrients between the aqueous and the vitreous humor compartments. However, it
was recognized very early that the electric current flux at the anterior as well as at
the posterior poles was directed inward into the lens. This current followed outward
direction only at the equatorial poles (Robinson and Patterson, 1983). This suggests
that regionally operated circulatory systems are followed by cellular fluids (see
review by Mathias et al., 1997). The inward transport being intercellular, and the
outward intracellular. Whether there exists a molecular兾biochemical link between
pumps, channels and gap junctions and these regional electrical conductances (circulatory systems) remains to be investigated. However, if the lens is treated as a single
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Fig. 4. A hypothetical schematic of the flow of current兾fluids in the ocular lens. Based on Mathias et al.
(1997) and Zamphigi et al. (2000). The thick dotted arrows from the anterior and the posterior follow
the orientation of the apical and the basal surfaces. It is presumed that the orientation of the cells兾fibers
at the equatorial region is such that facilitates the flow as shown (thick dotted arrows). The active NaC兾
KC transport is indicated by double headed open arrows on the anterior. Small dotted arrows in the
posterior indicate passive and facilitated diffusion. Thin arrows at the equator suggest some activity in
the direction of the arrow before the fiber cell differentiation is complete.
folded epithelium it is easier to relate the direction of the current (and therefore the
transport of the fluids) to the anatomy of the system (Fig. 4). Significantly then, the
direction of the inward current from the anterior and the posterior pole follows the
orientation of the apical and the basal surfaces of this epithelium (Zampighi, 2000).
Importantly, fluid transport in cultured lens epithelia and organ cultured rabbit lens
was recently shown to follow the path from basal to apical surfaces. This transport
was blocked by the inhibitors of KC channels and aquaporins (Fischbarg et al.,
1999). It is clear that transport within the fiber cells in the posterior of the lens and
in the epithelial cell layer in the anterior of the lens is dictated by different morphological and molecular parameters. Physically, the fiber cells present a much larger
surface than epithelial cells thus facilitating diffusion and molecularly, by the presence of gap junctions predominantly represented by MIP26 (major intrinsic membrane protein 26 kD, also known as aquaporin 0) (Gorin et al., 1984; Hamann et
al., 1998). Although existence of coupling between the epithelial cells and the underlying fibers has been questioned (Brown et al., 1990; Bassnett et al., 1994) there
seems to be no sound scientific evidence that suggests about 10% of the epithelial
cells and apical surfaces of the fibers are dye coupled. This coupling is as good as
between the epithelial cells themselves (Rae et al., 1996). The proximity of the apical
surfaces of the fiber cells and the epithelial cells (the apical interface) allows this
communication (Fig. 5). Using the uptake of the lipophilic cation as an indicator of
the resting potential in the rat lens, Cheng et al. (2000) suggested the presence of
two compartments, one that is fast and highly sensitive to the concentration of
external KC, while the other is less sensitive to external KC concentrations and slow.
Immunocytochemical localization of the channel proteins also suggests the existence
Ocular Lens Epithelium
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Fig. 5. Electron micrograph showing gap junctions between two anterior
epithelial cells and between epithelial and fiber cells at the ‘‘apical interface’’ in
rat lens. GJGGap junction. Magnification 75,000B. Courtesy Dr. G. Zampighi.
of two independent cell-to-cell networks (Zampighi et al., 2000). These are made of
different kinds of connexins (Goodenough et al., 1996); connexin 43 kD is at the
apical interface between the epithelial cells and fiber cells while connexin 43 kD is at
the apical interface between the epithelial cells and fiber cells while connexin 46兾
50 kD is on the lateral surfaces of the fiber cells. Molecular basis of the presence of
these two compartments remains to be understood.
LENS EPITHELIAL CELLS IN CULTURE
Natural course for an epithelial cell such as the lens epithelium is to divide and
differentiate to a terminal state that characterizes the final functional phenotype of
the tissue in question. It is for this reason that it is difficult to maintain epithelial
cells in culture indefinitely. Lens epithelial cells have been cultured in ûitro for a long
time with varied success (Okada et al., 1971; Beebe and Piatigorsky, 1977; Hamada
and Okada, 1978; Eguchi and Kodama, 1979; Ringens et al., 1982; Jacob, 1987;
Wormstone et al., 1997). The older the donor the harder it is to sustain a thriving
culture (Tassin et al., 1979; Reddan et al., 1981). Upon culture of the lens epithelial
cells, with time and passaging, there is usually a gradual loss of the crystallin synthesis (e.g., α B-crystallin), indicating that in ûitro these cells may have a tendency
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to stray from the pleuripotent state that must otherwise lead to fiber cell differentiation (van Venrooij et al., 1974; Vermorken et al., 1977; Hamada et al., 1979;
Ringens et al., 1982; Simonneau et al., 1983; Ibaraki et al., 1996). However, when
human fetal lens epithelial cells are cultured with deliberate slow passaging they can
be maintained fairly well in culture (see Fig. 2B). Not only do these cultures show
a sustained synthesis of α B-crystallin, but a progressive increase in the expression
of β B2-crystallin a marker indicative of fiber cell differentiation is also seen (Nagineni and Bhat, 1988; Nagineni and Bhat, 1989a). Arita et al. (1990) used nonprotein
binding membranes as substrates for the culture of human lens epithelial cells and
showed enhanced fiber cell differentiation as assessed by the appearance of ‘‘lentoid’’
bodies. This technique has allowed the culture of epithelial cells from older donor
lenses. Lens epithelial cells secrete their own capsular materials in culture (Grant et
al., 1972; Arita et al., 1993). When cultured on the lens capsule, lens epithelial cells
retain their ability to synthesize α B-crystallin (Vermorken et al., 1977).
In order to overcome the naturally restricted growth of native epithelial cells in
ûitro immortalized cell lines have been generated from primary lens epithelial cell
cultures, (Bloemendal et al., 1980). Two cell lines were generated recently from
human lens epithelial cells: One by transformation of the primary cultures with a
virus containing the simian virus-40 transforming gene (SV-40 T antigen) (Andley
et al., 1994) and the other by direct transfection with SV-40 large T-antigen gene
(within a plasmid) (Ibaraki et al., 1998). The later cell line seems to retain the
expression of α A-crystallin and β B2-crystallin. About fifty other lens epithelial cell
lines have been generated and investigated for the presence of different proteins
(markers?) (Sax et al., 1995; Krausz et al., 1996).
Do lens epithelial cells have a specific molecular marker? At this time we do
not know of any such markers, although different investigators have at times used
α -crystallins as lens cell markers, the fact is that a number of other non-lens cells in
culture express these proteins (Nagineni and Bhat, 1989b), albeit at very low levels.
This brings us to the unanswered questions: Does the loss of crystallin synthesis
indicate loss of lens-specific characteristics? How is a lens epithelial cell, which does
not express presumed markers (like α -crystallins) different from any other epithelial
cell? If α -crystallins are indicators of lens characteristics, how much and which α crystallin defines that characteristic? In the absence of any knowledge as to what
defines a lens-specific characteristic (morphologic or molecular), in an epithelial cell,
these are different questions to answer.
The next question is how representative are the cultured cells of the physiology
of the native epithelium? The answer to this question depends on the origin and
physiological characteristics of each cell line. However established transformed cells
(spontaneously or otherwise) must be used with discretion, particularly in the studies
of cell growth, differentiation, aging and apoptosis. The SV-40 transformed cells of
the hamster lens epithelial cells produce tumors in homologous hosts (Albert et al.,
1969). Long-term cultures of bovine epithelial cells produce tumors upon injection
into nude mice (Courtois et al., 1978). Transformed cell lines may present more than
one stable morphological and biochemical phenotype (Bloemendal et al., 1997).
Most of the work with lens epithelial cells thus far has been done with cell cultures
Ocular Lens Epithelium
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(lines) that have not been deliberately immortalized with the introduction of transforming (immortalizing) genes. A large number of experiments have utilized the
chicken embryonic lens epithelium system for studying cell-culture control and differentiation (Piatigorsky, 1981; Zelenka et al., 1997). Rat explant cultures have been
used for studying differentiation of fiber cells from epithelial cells in ûitro (McAvovy
and Fernon, 1984). Many investigations for metabolic studies have been done with
bovine and rabbit lens epithelial cells.
The study of lens-specific metabolism or molecular processes must always be
done with the knowledge that the very act of in ûitro culture may have changed the
molecular makeup of the cells. A few of these examples are discussed here. In the
primary cultures produced from the bovine lenses, the mitotically inactive cells show
calcium dependent spreading followed by DNA synthesis. These cells are very sensitive to C6-substituted purine derivatives that inhibit cell flattening as well as growth,
but this sensitivity to purine derivatives is lost as the cells are sub-cultured (Glaesser
et al., 1979). Culturing of the lens epithelial cells has been reported to result in
significant changes to energy metabolism of the cells (Piper et al., 1990). A calcium
dependent ATPase activity present on native bovine epithelial cells is lost upon culture. (Bergner and Glaesser, 1979) A recent comparative study of the muscarinic
receptors in the native lens epithelium vs. the transformed cell line, HLE-B3 (Andley
et al., 1994) is very informative. Both these cells in culture express muscarinic receptors, however the pattern of the subtypes that we expressed are very different. Native
lens epithelial cells almost exclusively express the MI subtype of the muscarinic
receptors. The transformed cell line HLE-B3 expresses M3 subtypes predominantly
(Collison et al., 2000). Recently, a cell line developed from the human fetal lens
epithelium (HFL124) with extended life span (without transformation, spontaneous
or deliberate) has been shown to maintain the expression of c-met receptor
expression similar to that in the native epithelium (Wormstone et al., 2000). Investigations on cultured cells have contributed extensively to our understanding of different biological processes and shall continue to be utilized for such defined purposes.
Lens epithelial cells have been studied to culture for a number of metabolic
and growth studies (Harding and Crabbe, 1984; McAvovy and Chamberlain, 1990;
Spector et al., 2000). Among the growth factors one that stands out is FGF. FGF
at concentrations of 0.15 ng兾ml supports proliferation while a concentration of
40 ng兾ml supports differentiation in rat lens explant culture system (McAvovy and
Chamberlain, 1989). Effect of FGF on the lens epithelial cells have also been demonstrated in transgenic animals where recombinant FGF1 controlled by fiber cellspecific α A-crystallin promoter, was synthesized and secreted from fiber cells. Adjacent epithelial cells were stimulated to form fiber cells (Robinson et al., 1995). At
this time no epithelial cell-specific promoter has been characterized that would direct
expression to lens epithelial cells only. It is interesting that lens epithelial cells retain
tissue-specificity when assayed for reporter gene assays with different crystallin promoters (Kondoh et al., 1983; Sax and Piatigorsky, 1994).
Another growth factor worthy of attention in the context of lens epithelial
biology is TGF-β . Exposure of intact lenses in ûitro to TGF-β leads to the appearance of spindle-shaped cells that contain α -smooth muscle actin (Hales et al., 1994).
However, in ûiûo, intravitreal injection of TGF-β , results in the phenotype of
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nucleated fiber cells in the cortex and the migration of the cells posteriorly (Hales et
al., 1999) indicating disruption in fiber cell differentiation. These data suggest that
TGF-β initiates an entirely different developmental program, namely, the transformation of the epithelial cell to the mesenchymal cell type. Mesenchymal transformation has been reported to be associated with anterior as well as posterior
subcapsular cataracts (see Marcantonio et al., 2000). Recently it was suggested that
expression of α -smooth muscle actin, an indicator of mesenchymal transformation,
is a common feature of lens epithelial cells in culture (Nagamoto et al., 2000). Epithelial兾mesenchymal transformation has developmental antecedents (Hay and Zuk,
1995) and points to the tenuous nature of the pleuripotency of the lens epithelium,
which when compromised may lead to secondary opacifications after cataract surgeries. This interesting aspect of lens epithelial biology deserves more attention.
PROGRAMMED CELL DEATH AND THE LENS EPITHELIUM
The interest in the study of programmed cell death in the lens epithelium was
generated recently by investigators probing the role of epithelium in cataractogenesis
(Li and Spector, 1996). These studies are based on the hypothesis that integrity of the
lens epithelium is essential for the normal functioning of the lens and that a decrease
in the cell number of the epithelium may lead to changes in homoeostasis that may in
turn lead to cataractogenesis. The role that apoptosis plays in the development and
tissue morphogenesis is well established (Steller, 1995). Apoptosis or cell death has
been morphologically documented in the very early stages of the lens vesicle formation
during development of the eye (Garcia-Porrero et al., 1984). This ‘‘morphogenetic’’
cell death (Gluksmann, 1951; Ishizaki et al., 1993) is possibly related to the loss of the
lens stalk, considering that at this time the lens vesicle is preparing to pinch off the
surface ectoderm. It is not clear whether apoptosis is essential to lens differentiation
and development after the formation of the lens vesicle? There is no experimental data
available to suggest that ‘‘apoptosis’’ is a major factor either in ‘‘primary fiber cell’’ or
‘‘secondary fiber cell’’ differentiation. This does not negate the role that apoptosis
may come to play in the final maturation of the differentiated fiber cell (Appleby
and Modak, 1977; Wride and Sanders, 1998). It is possible that during primary lens
fiber cell formation not all cells differentiate into fibers and a subset of posterior
epithelial cells may go through apoptosis. Another possible scenario would have an
important bearing on the phenotype of transparency is the migration of the differentiating cells to the posterior capsule. Under normal, healthy conditions cells that do
not stop at the equator may be triggered to die before they reach the posterior
capsule, which if they did, would nullify the phenotype of transparency by contributing to the development of the pathology known as ‘‘posterior subcapsular opacification’’. The fact that apoptosis may in some form attend fiber cell differentiation
is indicated by the reported involvement of caspases (cysteine-asparate proteases
known to initiate the cascade that leads to apoptosis) in the differentiation of the
fiber cells in the rat lens (Ishizaki et al., 1998).
It remains to be established if indeed there is a basal rate of apoptosis in the
normal adult lens epithelium. Ishizaki et al. (1993) found evidence of cell death
(phagocytosis) in the adult rat lens epithelium in the region of the ‘‘anterior suture’’
Ocular Lens Epithelium
551
(the area where the anterior ends of the lens fibers converge). The reasons as to why
this happens remain unclear. These investigators reported ∼5% pyknotic cells in the
young rats and about 1% in older rats. Cell division in the epithelium decreases with
age and that may have to do with the species-specific (Kuszak, 1995) and that there
is a (positional) relationship between sutures and the apoptotic cells in the epithelium then it must be assumed that contribution from apoptosis to the lens development兾physiology may be species-specific. As to why there should be an
association between the location of sutures and apoptosis remains unexplored.
Whether apoptosis has a definitive role in the maintenance of a specific number of
epithelial cells in the lens is debatable. Nonetheless, a role for apoptosis in regulating
the size of the lens by controlling the number of cells that reach terminal differentiation into fiber cells remains a possibility.
ULTRAVIOLET RADIATION (UVR) AND THE LENS EPITHELIUM
The single layer of epithelium is the first physical and cellular (biological)
defense against electromagnetic radiation in the ocular lens (Dillon, 1991). Some of
the direct effects of UVR exposure on cultured cells have been reviewed in detail
(Hightower, 1995). The consequences of UVR exposure on the epithelium must be
considered both in terms of mutagenic as well as cytotoxic effects (Worgul et al.,
1989, 1991). UVR exposure results in unscheduled DNA synthesis and repair
(Brenner and Grabner, 1982; Kleiman et al., 1990; Sidjanin et al., 1993). The action
spectrum for the rat lens in ûiûo shows that 305 nm, which reaches the lens much
more than the 290 nm band (part of UVB, 280–315兾320 nm), is harmful to the rat
lens. It is not possible to extrapolate directly from the rat lens to human condition,
a composite of location, intensity of exposure, genetics, diet and age. However it is
conceivable that the human lens epithelium accumulates insults due to UVR exposure in its genome over a period of time that are manifest in the aged lens (Merriam
et al., 2000). There is of course an age-dependence of UVR damage to different
molecular species including enzymes such as hexokinase, phosphofructokinase, isocitrate dehydrogenase and malate dehydrogenase (Reddy and Bhat, 1998). Loss of
hexokinase (Tung et al., 1988) would result in the inability of the lens to produce
NADPH and downstream antioxidants. It is conceivable that proteins (such as
NaC兾KC) ATPase, cytoskeletal elements, membrane proteins), which dependent
on –SH function will be damaged by exposure to increased oxidants.
Ultraviolet radiation (300–320 nm) exposure leads to an alteration in the morphology of the epithelium. The morphological effects of UVR exposure encompass
general cell swelling including mitochondrial swelling, chromatin condensation and
fragmentation of the nuclear DNA. One of the more significant early effects is the loss
of positional identity of the epithelial cells. The epithelial cells from the bow region
migrate to the posterior capsule. A molecular or biochemical basis of the loss of this
positional integrity has not been investigated. It has been suggested that a disturbance
in calcium homeostasis interferes with the differentiation pathway of the lens epithelium into fiber cells, which leads to the migration of the bow region cells to the
posterior capsule. Many investigations suggest compromise of the calcium homeostasis possibly starts in the epithelium and propagates to the rest of the lens. Levels
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of Ca2C-increase have been shown to be dose-dependent in rabbit lenses exposed to
UVR. It is possible that this is because of the photo-oxidation of the Ca2C-ATPase
that contains approximately 30 thiol (SH) groups. In this context it is interesting to
note that Ca2C-ATPase activity increases with age (Brochman et al., 1989).
Different metabolites (Inoue et al., 2000) and drugs (Li et al., 1995b) have been
reported to initiate apoptosis in lens epithelial cells in culture. For the native lens
epithelium, however, environmental factors such as ultraviolet radiation (UVR)
become highly relevant in this context (Zigman, 2000) and Li and Spector (1996),
irradiated whole rat lenses in culture with 8 J兾s兾m2 of UVB (285–315 nm) for 60
min. As expected the cells in the equatorial zone were the first to show signs of
apoptosis followed by the central epithelial cells. They reported that more than 50%
of the cells were apoptotic by the 5th hour after irradiation and by 24 hr all the
epithelial cells in the irradiated lenses had died. This seems to be an extreme case of
exposure to UVR that leads to 50% cell death, but it underscores the fact that it is
the cells in the bow region that are first effected followed by those in the central
epithelium. As expected the dividing兾differentiating epithelial cells are more susceptible to UVR damage than the resting central epithelial cells. Based on morphological grounds, in the intact animal, the equatorial regions may not be exposed to as
much UV as the central epithelium. Recently, Michael et al. (1998) unilaterally
exposed Sprague–Dawley rat eyes to UVB (5 kJ兾m2, 300 nm) for 15 min and then
followed the fate of the lens epithelium at various time-points. TUNEL (terminal
deoxynucleotidyl transferase–deoxyuridine triphosphate nick end labeling)-positive
cells (indicative of cell-death) were detected only at the 24 hr time point and not at
1 hr or 6 hr or 1 week after exposure. Although the type of UVR used (long, 340–
400 nm vs. short 200–320 nm) may lead to temporal differences in the induction of
apoptosis, the fact that apoptotic cells and their phagocytosis by the neighboring
cells were only detectable at 24 hr (not before or after that) is interesting. That the
phagocytosis is completed quickly within hours is confirmed in an in ûitro study also
(Shui et al., 2000). These observations suggest that apoptosis may occur discretely
in the lens epithelium and only in isolated cells that become susceptible. Such a
thesis connotes two interesting corollaries: (1) Any isolated apoptotic cell may be
quickly eliminated by the surrounding cells(s) as a protective response against spread
of cell death. Importantly, therefore, it points to the existence of a potent mechanism
that strictly controls and inhibits cell death from spreading to the neighboring cells.
Thus in order to detect any apoptotic cells after an insult, in the normal lens epithelium a large number of cells and time points may have to be analyzed. (2) Presence or absence of apoptosis in the lens epithelium can be interpreted optimistically
as a process that eliminates the ‘‘dysfunctional’’ cells to keep the rest of the epithelium healthy. A pathological state may precipitate when this ability to remove
‘‘dysfunctional’’ cells is compromised, for example by aging or by exposure to harmful metabolites or environmental insults. The existence of a molecular mechanism
that keeps apoptosis discrete and rare in the lens epithelium remains to be identified.
APOPTOSIS AND CATARACT
It is clear that physical and兾or chemical insult may initiate apoptotic program
in lens cells, but whether apoptosis contributes to cataractogenesis remains
Ocular Lens Epithelium
553
unresolved. Li et al. (1995a) reported a significant number of apoptotic cells in capsular epithelia surgical samples from cataractous patients in comparison to those
obtained from normal donors. Haracopos et al. (1998) attributed presence of any
TUNEL-positive cells in casulotomy specimens most likely to be due to necrosis
caused by post-surgery handling of the specimen, thereby challenging the claim that
apoptosis may lead to cataractogenesis.
An interesting aspect of a relationship between cell density and apoptosis
deserves mention. Based on the ‘‘death by default’’ thesis (Raff et al., 1993; Ishizaki
et al., 1993), a decrease in cell density with age could lead to paucity of the presumed
survival factors that could in turn lead to further deterioration of the integrity of
the lens epithelium by initiating apoptosis. But before such conclusions are reached
it needs to be established firmly if (a) there is a decrease in cell density with age or
cataract and (b) this decrease in cell density is significant enough to trigger ‘‘cell
death’’. Based on the need for survival factors produced by neighboring cells in a
dense culture it will require large denuded areas to trigger ‘‘cell death’’ in the lens
epithelium. While such denuded ‘‘black holes’’ have been recently described (Balaram et al., 2000), their origin is unknown and their relationship to cataracts is unproven. Also, there remains the puzzle as to how the ‘‘black holes’’ are created in the
first place?
CATARACT AND CANCER
Cataractogenesis (loss of transparency) is the culmination of biochemical兾molecular events that lead to the compromise of the structural integrity of the fiber cells
and their proteins. The question of whether the initiating event for the development
of this pathology indeed lies in the molecular兾metabolic compromise of the epithelial
cell layer remains unsolved. Cataractous lenses and in particular the capsule兾epithelia have morphological features, typical of aging cells such as altered hexagonal
cellular arrays, changes in endoplasmic reticulum, Golgi apparatus and mitochondria (Straatsma et al., 1991). The lens epithelium兾lens is not known to become neoplastic except under experimental conditions when transforming genes are
introduced into the lens of transgenic animals (Mahon et al., 1987). The proliferative
status of epithelial cells in the cataractous lens is unknown. It is not clear if the cells
in the epithelium of the cataractous lens divide at the same rate as those in the
normal lens. The maintenance of the pleuripotent state of the lens epithelium during
cataractogenesis (posterior subcapsular opacification) is in question. Does epithelial
to mesenchymal transformation (discussed previously) represent an obligatory process attendant with cataractogenesis? Does increased length of telomeres in cataractous epithelia (see below) suggest a relationship to increased proliferation?
Harocopos et al. (1998) report that cataractous areas in the human lens are associated with areas of higher cell density in the lens epithelium. The phenotype of these
cells with respect to their oncogenic or benign is uninvestigated. Mitotically active
cells have been seen superimposed on the central epithelial cells in very advanced
cataracts (Vasavada et al., 1991). Colitz et al. (1999) found ‘‘fibrous metaplasia’’ in
canine hyper mature cataracts. Similarities between the causative agents in cataractogenesis and carcinogenesis have been pointed out previously (see Bloemendal,
554
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1991). However, the thesis that a molecular compromise of the genetic integrity of
the lens epithelium leads to a dysfunctional lens (cataract or otherwise) has actually
never been established or investigated directly. Cataract as a problem of uncontrolled proliferation has not been experimentally investigated. A general question as
to why the normal ocular lens兾lens epithelium remains non-cancerous has also
remained unexplored.
The remarkable ability of the cells in the germinative zone of the lens epithelium
to keep dividing (Harding et al., 1971) beckons the question if this is related to the
presence of telomerase in these cells. Telomerase is an enzyme complex of ribonucleoprotein that replenishes the shortening 3′-ends of chromosomal DNA ends (containing 5′-TTAGGG-3′ repeats) after each replication, thereby helping maintain the
length of the chromosome ends (telomeres) in the dividing cells. Telomerase ensures
minimal telomere length in dividing cells thus preventing cell senescence or aging.
Telomerase is highly active in proliferating embryonic cells; stem cells and germ
cells, but is mostly absent in most normal adult somatic cells. The association of
telomerase with replicative ability is substantiated by the demonstration that cells in
culture attain unlimited proliferative potential upon transfection with telomerase
expressing vectors (Ferber, 1999; Bodnar et al., 1998; Vaziri et al., 1999).
The presence of telomerase in the lens epithelium raises interesting questions
about the relationship of telomerase to proliferation. Quite interesting is the finding
of increased telomere length in the cataractous epithelia as opposed to that in the
normal epithelium (Colitz et al., 1999). This study also reported that there was no
differences in telomere lengths in relation to age. These findings deserve attention
because telomere length would be expected to show a gradual decrease similar to
that seen in culture, as the cells get closer to senescence with each passage. Interestingly all regions of the lens epithelium, the quiescent, the germinative and the differentiating cells, all had same amount of telomerase activity as assayed by TRAPELISA method that measures the length of the telomeres with polymerase chain
reaction (Colitz et al., 1999). If the assay is quantitative it is a case of discordance
between the amount of telomerase activity and cell division. Consistent, although
not very convincing is the observation that the exposure of the lens epithelium to
radiation (including UVR) may physiologically predispose the cells to higher telomerase activity as a defense against UVR-induced damage to the cell DNA (Kleiman
et al., 1990). Lack of detectable telomerase activity, even in the limbal cells that
retain the proliferative potential in the cornea, a tissue also exposed to UVR (Egan
et al., 1998) weakens this explanation. At this time it is not possible to ascertain
if the increase of telomere length in the cataractous epithelial DNA is related to
hyperproliferation and if hyperproliferation is the result or the precipitating event
in the pathogenesis of the loss of transparency?
CONCLUSION
Table 1 is a summary of the alien features of the lens epithelium. We have not
considered a number of other interesting and important features of the lens
epithelium. These include but are not restricted to ultrastructural details (Ireland et
al., 1983; Rafferty et al., 1990; Kuszak, 1995) the basement membrane and its
Ocular Lens Epithelium
555
Table 1. Some Salient Features of the Lens Epithelium
Derived from surface ectoderm
Cell density is higher in females than males
Contains quiescent, non-dividing cells, dividing cells and differentiating cells
Produces cells that divide and differentiate
Contains cells that apoptosize
Contains epithelial cell :epithelial cell junctions
Contains epithelial cell :fiber cell junctions at the apical interface
Has ability for phagocytosis and endocytosis
Produces the basement membrane (collagen IV) that makes the lens capsule
Transports water, ions and nutrients into the lens
Pumps out NaC and transports in KC
Contains KC channels
Has a direct role in the maintenance of the resting potential of the ocular lens
Polarized, with apical surface facing the lumen of the lens and the basal surface facing the outside world
(aqueous humor)
Cells exposed to radiation (UVR)
Synthesised glutathione
Expresses glutathione transporters
Contains high concentration of taurine
components that make the lens capsule (Sawhney, 1995; Menko et al., 1988), the
process of endocytosis (Gorthy et al., 1971; Lo and Zhang, 1989) and its role in
transport of macromolecules and the aquaporins (Wintour, 1997). In addition there
are a large number of gene products that are constantly being discovered, which
have a role in the optimal functioning of this epithelium (Wen et al., 1995; Srivastava
et al., 1996; Padgaonkar et al., 1997; Andley et al., 1988; Dahm et al., 1999; Yan et
al., 2000). One of the best known promoters of the lens genes is that of α A-crystallin,
which directs expression specifically to the lens fiber cells (Sax and Piatigorsky,
1994). It has been used to study effects on lens epithelium (rather indirectly) by
targeting genes to the fiber cells. There is a need to search for lens epithelial cell
specific genes and a characterization of their promoters so that gene products may be
targeted to the lens epithelium specifically for functional studies and for therapeutic
possibilities.
A loss in the functional physiology in the lens epithelial cells with age may make
this epithelium susceptible to external (e.g., UVR) or internal physiological signals
(e.g., H2O2). In the theory of the oxidative stress as the initiating event for cataractogenesis of the lens, the initial and the primary target are the epithelial cells
(Spector, 1995; Sasaki et al., 1998). This can be investigated by examining the patterns of gene expression in the epithelium. One of the important questions that need
to be resolved is, does the lens accumulate DNA damage with age? Is it clear that
extensive analyses of the proliferative capabilities of the lens epithelium need to be
taken up in order to understand any special aspects of the genetic control of cell
proliferation in the lens epithelium. In this regard, lens epithelium, as a cell type that
seems to escape oncogenesis becomes a very relevant paradigm for understanding
replicative senescence as a tumor suppression strategy vs. replicative senescence as a
causative mechanism for tissue dysfunction and age-related pathologies (Campisi,
1999).
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It is only recently that investigators have started characterizing gene activity in
the epithelium under disease conditions (Kantrow et al., 1998; Sun et al., 2000). The
lens epithelium is exposed to a large number of physiological signals that come
through aqueous humor. These signals must create differential responses at the genetic level that define the physiology of the ocular lens in relation to the general
metabolism. One of the well-known signals is glucose. Glucose not only regulates
the glucose transporters but other glucose responsive genes either directly or
indirectly from metabolites derived from it (Vaulont et al., 2000). There may be a
plethora of genes that are modulated in an aging lens epithelium, those patterns of
gene activity remain to be discovered. With rapidly advancing technology of gene
expression profiling (Vishwanath et al., 1999) this ignorance may be very short-lived
and some of the peculiarities or lack thereof may be elaborated.
ACKNOWLEDGMENTS
I thank Dr. Guido Zampighi for providing the electron micrograph shown in
Fig. 5 and for his discussions about the anatomy of the lens. Thanks are due to
Sherry Yafai, Wendy Lam and Rick Gambino for their help with the manuscript
editing and illustrations. Thanks are due to Association for Research in Vision and
Ophthalmology, Rockville, MD and Academic Press, Orlando, FL for permission
to reproduce data presented in Fig. 2. Work in the laboratory of the author is
supported by grants from NIH, S.P.B. is a recipient of the Research to Prevent
Blindness Wassermann Merit Award.
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