The primary functions of the ocular lens are to transmit incident light

and to focus it on the retina. This requires that the lens be transparent,
a condition dependent on the highly regular organization of the cells of
the lens and the high degree of short-range order of the proteins in the
lens cytoplasm.1 Protein concentration in lens fiber cells is extremely
high, resulting in an index of refraction significantly greater than that of
the surrounding fluids and enabling the lens to refract incident light.
Cataract occurs when the lens loses its transparency by either scattering
or absorbing light such that visual acuity is compromised. Cataracts can
result from genetic, metabolic, nutritional, or environmental insults or
may be secondary to other ocular or systemic diseases, such as diabetes
or retinal degenerative diseases.2 By far the most important risk factor is
age; aging-related cataract constitutes the great majority of all
cataracts. This type of cataract is a major public health problem in the
United States. In developing countries, where the availability of surgical
facilities is limited, aging-related cataract is the leading cause of
blindness. Because at present there is no efficacious nonsurgical therapy
for cataract, the problem is expected to increase in magnitude as the
world population becomes progressively older in coming decades.

To begin to understand the pathogenesis of cataract, it is necessary to

recognize that it is not a single disease. Even aging-related cataracts
vary greatly in the location of the opacity in the lens, the morphology
and appearance of the opaque region, and the rate of progression of
opacification. Because human aging-related cataract is caused by
multiple interacting factors, elucidation of the underlying molecular
mechanisms leading to opacification has been difficult. Most current
knowledge about the pathogenesis of cataract has come from the study
of animal models, because human cataract tissue has become very
difficult to obtain with the advent of extracapsular extraction and
phacoemulsification procedures. Although numerous animal models are
available and some have been investigated in detail, few of these
systems involve aging animals, raising questions as to how relevant the
results are to human aging-related cataracts.

In this chapter the emphasis will be on human aging-related cataract

and what is known about the mechanisms underlying the disease
process.

Back to Top

AGING-RELATED CATARACTS: GENERAL FEATURES

It would be a gross oversimplification to consider aging-related cataract
as a single disease. There appear to be three major types of aging-
related cataractscortical, nuclear, and posterior subcapsularwhich
differ in both the location in which the opacity initially appears and in the
pathology underlying the opacification. Many risk factors may be
common to all three types of aging-related cataracts, and although
cataracts often begin as a pure type, as they mature they typically
become mixed cataracts.

Cortical cataracts (Fig. 1) occur in the outer region (about 25%) of the
lens and are characterized by vacuoles, water clefts, and spokes. It is
generally believed that most cortical cataracts are osmotic in nature
that is, water accumulates in or between the lens cells, usually as a
result of ionic imbalances. The electrolyte imbalances likely arise as a
result of damage to the lens cell membranes, especially those of the lens
epithelial cells, which play the major role in maintaining the ionic and
metabolic homeostasis of the entire lens.3 Such damage could
compromise the normal permeability characteristics of the membrane or
damage specific membrane proteins responsible for ion transport. In
cortical cataracts, potassium levels decrease whereas sodium, chloride,
and calcium increase, with the resulting imbalance leading to an influx of
water.4 The vacuoles or lakes containing this water have a low
refractive index relative to the protein-rich cytoplasm in the fibers, and
the discontinuities create light scatter and cataract.

Fig. 1. A. Cortical cataract. Arrow points at a spoke opacity in

midperiphery of lens. B. Nuclear cataractslit-lamp photograph showing

opacification of the nuclear area.

Nuclear cataracts (see Fig. 1) occur in the central region of the lens and
appear to involve an acceleration of processes that occur during aging
even in the normal lens. The proteins accumulate postsynthetic
modifications, especially resulting from oxidation, leading to formation of
protein aggregates that scatter light.5 The proteins in the nucleus also
become progressively more pigmented with age; in some nuclear
cataracts the color can become dark brown or even black. In such cases
cataract may result from absorption of light rather than light scattering.
In contrast to the cortical cataract, nuclear cataracts tend to become
harder and less hydrated than normal, age-matched lens nuclei.

Posterior subcapsular cataracts occur at the posterior pole immediately

beneath the lens capsule (Fig. 2). These cataracts may result from
improper posterior suture formation or from abnormal differentiation of
lens fibers.6 In the latter instance lens epithelial cells may migrate to the
posterior pole. Posterior subcapsular cataracts are formed after radiation
with x-rays and long-term corticosteroid therapy and occur secondary to
retinal degeneration diseases; they also occur idiopathically.

Fig. 2. A. Posterior subcapsular cataract. Arrow points at cataract

located in the inner surface of the posterior capsule as seen by slit-lamp
biomicroscopy. B. Same eye, seen by retroillumination, showing the
irregular
surface of
the
cataract.

Back to Top

OXIDATION
Oxidative damage to lens constituents, including nucleic acids, proteins,
and lipids, is believed to be a primary factor in aging-related
cataract.7 This belief is based on a large body of evidence of various
types. That oxidative stress can be cataractogenic is clear from abundant
data demonstrating, both in animals and in humans, that exposure of
the eye to x-rays or to high levels of other types of radiation, including
ultraviolet (UV) and microwaves, can cause cataract with definitive
oxidative effects in the lens. Likewise, exposure to hyperbaric oxygen,
either experimentally in animals or therapeutically in patients, can cause
cataracts.8 Further support for the oxidation hypothesis comes from
epidemiologic studies that have found an association between increased
exposure to sunlight, particularly its UV component, and aging-related
cataract.9,10 Biochemically, there is convincing evidence of oxidation
occurring as a function of aging, and more specifically of
cataractogenesis in the human lens. This is seen especially in the status
of the sulfur amino acid residues of the proteins of the lens. 11 In young
human lenses, the vast majority of these residues are in the reduced
state and are buried within the protein (i.e., are not accessible to
solvent). Analysis of normal lenses from persons ages 60 to 65 years
revealed that more than 50% of the thiols had become exposed as a
result of conformational changes secondary to structural modifications to
the long-lived proteins, yet little oxidation was detected. In contrast, in
human cataractous lenses, nearly all the thiols are exposed, and most
were found to be -oxidized. This oxidation was in the form of protein-
protein disulfides, mixed disulfides with low-molecular-weight thiol
compounds, or cysteic acid. Likewise, there was extensive oxidation of
the methionine thioether side chains to the sulfoxide or sulfone.
Glutathione, the major low-molecularweight thiol compound in the lens,
is markedly decreased in human cataracts and in almost all experimental
cataracts studied.12

SOURCES OF OXIDATIVE STRESS

To probe the role of oxidation in cataractogenesis, one must first

consider the environment of the lens in terms of both its particular
exposure to oxidative stresses and the array of mechanisms that are
present to combat or neutralize such stress. In the normal lens there is
clearly a balance between these two opposing forces; in many cataracts,
it appears that the balance has been lost and the antioxidative defenses
have been overwhelmed. The lens is an organ uniquely vulnerable to
oxidative damage because it is exposed to chronic oxidative stress from
multiple sources and has limited ability to renew or repair damaged
molecules. The major oxidants believed to be involved in creating
oxidative stress on the lens are the activated species of oxygen. These
include the one-electron reduction products of molecular oxygen (Fig.
3): superoxide anion (O-B32), hydrogen peroxide (H 2O2), and the
hydroxyl radical (OHB3), and an activated species of molecular oxygen
called singlet oxygen (1O2). The superoxide anion is a free radical formed
directly from molecular oxygen via one-electron reduction. It can be
formed in a variety of ways, including as an intermediate in numerous
metabolic reactions. It is not a strong oxidant but is quite reactive,
usually dismutating spontaneously, or more efficiently when catalyzed by
the enzyme superoxide dismutase (SOD), to produce H 2O2. H2O2 is a
stable product capable of diffusing into cells. Although it is not very
reactive itself, in the presence of reduced transition metal ions (e.g., iron
or copper), it can be converted into the extremely reactive and powerful
oxidant OHB3 via the Fenton reaction.

Fig. 3. Univalent reduction of

molecular oxygen.

Two potential sources of OHB3 or related, highly reactive species in the

lens have received much attention. The first of these is photo-oxidation,
because the lens is necessarily extensively exposed to sunlight, which
includes significant UV components. Although the cornea removes that
part of the spectrum below about 295 nm, some UV-B and most of the
UV-A reaches the lens. The fact that much of this UV radiation is
absorbed within the lens raises the possibility of either direct photo-
oxidation of the absorbing molecules or generation of activated oxygen
species. This latter process, termed photosensitized oxidation, usually
involves the triplet state of the sensitizer and can proceed via two
mechanisms. In the type I reaction, the sensitizer triplet reacts to
produce free radical species, including O-B32, which can then give rise
to H2O2 and OHB3. Alternatively, in a type II process the triplet sensitizer
transfers energy directly to molecular O 2 to produce singlet oxygen. In
either case, the activated species of oxygen produced are capable of
oxidizing certain protein side chains, nucleic acids, and lipids.

Epidemiologic studies have linked increased exposure to sunlight to a

higher risk of aging-related cataract. The action spectrum for this effect
is still controversial. The most authoritative epidemiologic study, done
with a group of Chesapeake Bay watermen, found a positive association
of UV-B exposure (290 to 320 nm) with cortical cataract, but no
association of UV-A exposure (320 to 400 nm) with cataract. 10 The
relation of UV-B exposure with cortical cataract is consistent with various
animal studies as well.

That UV-A may also play a role in cataract, despite epidemiologic

evidence to the contrary, remains an attractive theory to many
investigators.13 The human lens develops, as a function of age, yellow
pigments associated with the lens proteins, particularly in the lens
nucleus. It has been demonstrated that these pigments strongly absorb
UV-A and that in vitro exposure of pigmented proteins from human
lenses to UV-A can generate reactive oxygen species, including singlet
oxygen.14,15 Because oxidative damage to lens proteins is most
pronounced in the lens nucleus and because UV-B would be unlikely to
reach the lens nucleus, whereas UV-A is absorbed there in large
quantities, it is perhaps premature to ascribe all photo-oxidative damage
in the lens to UV-B.

A second potential source of oxidative stress on the lens that has

received much attention relates to the level of H 2O2 endogenously
present in the eyes of certain species, including humans. It has long
been believed that H2O2levels in aqueous humor are much higher than
those in plasma or in tissues outside the eye. Concentrations in the 20-
to 30-m range have been reported from several laboratories using
different analytic methods.11 Further, it has been reported that the
H2O2 level of aqueous from cataract patients may be substantially
increased.16 However, the findings of a recent study suggested that
levels of H2O2 were greatly overestimated in many earlier studies
because of interference with the assay by the high concentration of
ascorbate in aqueous.17 The absolute level of H2O2 in the eye remains a
controversial and important issue, but there is no doubt that this
compound can pose a significant hazard for the lens, as has been shown
in numerous studies using intact lenses and lens constituents in vitro.18,19

PROTECTION FROM OXIDATIVE STRESS

To counteract this chronic oxidative stress, the lens has a variety of

defense mechanisms. First, the lens is nonvascular and receives all its
nutrients and other needs by means of diffusion from the surrounding
fluids. The lack of blood vessels eliminates potential light-scattering
elements in the lens and also reduces oxygen tension in and around the
lens, thereby lowering the potential for generation of reactive oxygen
species. The oxygen tension at the anterior surface of the lens is
estimated to be 10 mm, in contrast with the 160 mm at the external
surface of the cornea. Second, the lens has an unusually high
concentration of glutathione (GSH), a thiol-containing tripeptide that can
quench free radicals, detoxify H 2O2 and organic peroxides through its
role as a cofactor for glutathione peroxidase, and reduce disulfides. The
concentration of GSH in the lens is 5 mmol/L or greater on a whole-lens
basis and is much higher in the epithelium and outer cortical
region.20 Indicative of the highly reducing environment of the normal
lens, the amount of oxidized GSH is nearly undetectable, amounting to
only about 2% of total GSH. A second major antioxidant in the human
lens and in the lenses of other diurnal species is ascorbic acid. Ascorbate
is present at approximately 1 mmol/L in the aqueous humor of these
species, and there is a correlation between the relative amount of
ascorbate and H2O2 in the aqueous of different species, supporting the
earlier proposal that H2O2is formed in the eye through oxidation of
ascorbate.21 Although ascorbate in most situations functions as an
antioxidant, it also can act as a pro-oxidant and in some situations could
exacerbate oxidative stress.

The lens also has a full complement of antioxidant enzymes, including

catalase and glutathione peroxidase, which detoxify H2O2, and
superoxide dismutase, which eliminates the superoxide anion, albeit with
the generation of H2O2. The GSH redox cycle (Fig. 4) appears to be a
primary player in the antioxidant defenses of the lens. Under most
conditions, any GSH that is oxidized is rapidly rereduced by glutathione
reductase. Impairment of glutathione reductase prevents this process
and can lead to cataract. Further evidence for the importance of the GSH
redox cycle is the fact that under oxidative stress, the activity of the
hexose monophosphate shunt that provides the NADPH required for the
glutathione reductase reaction increases significantly.22 Several attempts
have been made to determine the relative importance of the antioxidant
-defense enzymes that detoxify H2O2. Using transfection methods in cell
cultures or transgenic and knockout technology in mice, the activities of
catalase or glutathione peroxidase have been selectively increased or
eliminated. These studies have not produced definitive answers.
Increasing catalase activity in a lens epithelial cell line did not
significantly increase the cell's ability to detoxify H 2O2 in the medium, yet
the cells did resist H2O2-induced stress better than the nontransfected
normal cells.23However, it has long been known that cataract is not
associated with acatalesemia. Likewise, knockout of the glutathione
peroxidase-1 gene in mice does not appear to cause cataract, although
one laboratory reported fiber cell membrane changes in the deep lens
nucleus, including blebbing, globularization, and vesicle formation. 24 A
second group did not observe such changes. 25 Lenses from the
knockouts do not seem to respond differently in vitro to H2O2-induced
stress than do wild-type lenses, although they are reported to be more
sensitive to lipid peroxide-induced oxidation. Based on the accumulated
data, it would seem that antioxidant protection is provided through
integration of several different systems with considerable redundancy.

Fig. 4. Glutathione redox cycle and its relation to the hexose

monophosphate (HMP) shunt and the detoxification of H 2O2 by
glutathione peroxidase.

The long-recognized association of GSH loss with cataract and the

marked increase in protein disulfide formation leading to aggregation
and insolubilization of proteins in many cataracts points to the thiol
redox balance as perhaps the most important factor in the ability of the
lens to protect itself from oxidative damage. Under conditions of
oxidative stress, GSH levels decrease but levels of the oxidized disulfide
GSSG and mixed disulfides of GSH with protein sulfhydryls increase.
These oxidations can be catalytically reversed by the activities of
glutathione reductase, an NADPH-dependent enzyme that converts
GSSG back to GSH, and thioltransferase, which reduces protein mixed
disulfides. Thioltrans-ferase, which has only recently been characterized
from lens,26 uses GSH as cofactor. Thus, GSH and the GSH redox cycle
appear to be the central elements in lens antioxidant defenses.

Back to Top

PHASE SEPARATION
As indicated above, a major source of light scatter and opacity in
cataracts is the formation of protein aggregates. It has been
demonstrated that such aggregates, which occur principally in the lens
nucleus, reach sizes that can cause considerable light scattering. The
aggregates form as a result of intermolecular crosslinks and noncovalent
attractions (e.g., hydrophobic interactions) between lens proteins.
Crosslinks include disulfides as well as nondisulfide covalent crosslinks
that may result from oxidative effects or from other types of protein
modifications, such as glycation. Hydrophobic interactions occur because
conformational changes, resulting from the accumulation of post-
translational modifications in the long-lived crystallins, expose
hydrophobic surfaces normally buried in the interior of the proteins. -
Crystallin, a major lens protein in all vertebrates and a member of the
small heat shock protein family, has been shown to act in a chaperone-
like manner to inhibit protein aggregation. 27 It appears likely that this
activity is a principal factor in the ability of the lens to remain
transparent for decades despite the chronic stresses to which it is
exposed and its very limited ability to synthesize and repair proteins.
Another mechanism leading to light scatter in the lens is phase
separation. Resulting from noncovalent, short-range attractive
interactions between proteins in concentrated solutions, phase
separation leads to reversible formation of protein-rich and protein-poor
regions.28 In the lens the existence of such regions in the cytoplasm of
lens fibers causes discontinuities in the index of refraction that create
light scatter. The reversible cold cataract (Fig. 5), which occurs in the
nuclear region of lenses from many young mammals on cooling, has
been shown to be a phase separation phenomenon. 29 The critical
temperature (Tc) is the temperature below which phase separation
occurs in a solution of proteins or other solutes. For certain proteins,
including some members of the -crystallin family, T c is only marginally
below body temperature.30 Because Tc is determined by the noncovalent
energy of attraction between proteins, modifications to proteins that
increase such attractive forces will raise T c. If Tc rises above body
temperature, phase separation may occur within the lens in vivo.

Fig. 5. Cold cataract in a calf lens photographed in a special cooling

chamber. (Courtesy of Dr Rafat Ansari, NASA
Glenn Center)

Evidence for the involvement of phase separation phenomena in the

early stages of several experimental cataracts has been
presented.31 Further, agents that are believed to act as phase separation
inhibitors by reducing interprotein attraction, and thus T c, have been
shown to inhibit cataract development in some animal models. 32These
agents will be discussed later in this chapter.

Back to Top

LENS EPITHELIUM, DIFFERENTIATION, AND

DEVELOPMENT OF THE LENS
The epithelium, a single layer of cells on the anterior surface of the lens,
is crucial to the homeostasis of the entire organ. Virtually all metabolic
enzymes are at their highest levels in the epithelium. Because
mitochondria are lost from mature lens fibers, the epithelium, along with
the most peripheral, still elongating fiber cells, has the highest capacity
for energy production. Further, ion pumps such as the Na + ,K+ -ATPase
and transport systems that carry vital nutrients and metabolites into the
lens are most concentrated in the epithelium.3 The avascular lens
depends absolutely on the proper functioning of these systems. The
epithelium is also the location of the highest activity of enzyme systems
that protect the lens from toxic influences. For example, the antioxidant
enzyme systems such as catalase and the GSH redox cycle that detoxify
H2O2 are the most concentrated here. In some types of cataracts,
including sugar cataracts, the epithelium is the part of the lens that first
shows morphologic change.33 However, it is likely that in many cataracts
where opacification occurs in other parts of the lens that the process
leading to cataract actually originates with events occurring in the
epithelium.

With the development of extracapsular and phacoemulsification

procedures for cataract surgery, it has become increasingly difficult to
obtain intact cataractous lenses for use in research. The one portion of
the lens that can be obtained relatively intact from such procedures is
the central epithelium. Although the amount of tissue from a single lens
is very small, methods have been developed that can measure the
activities of various enzymes from single epithelia.34 Further, RNA
extracted from pooled epithelia has been used to generate cDNA
libraries, and it is also possible to study the expression of genes in
individual human epithelia.35 Further development of these technologies
should provide new approaches for probing the biology of this crucial
portion of the lens.

Another approach widely used in cataract research has been the study of
lens epithelial cell cultures. Because lens fibers are terminally
differentiated cells that no longer divide, the epithelial cells offer the only
opportunity for lens cell culture studies. Cultures from several species
have been established and used for a wide variety of studies, but only in
recent years has successful culture of human lens epithelial cells been
accomplished.36 The numbers of cells that can be obtained from human
primary cell cultures may not be adequate for many biochemical studies
because the number of passages is limited. Transformed cell lines from
human lens epithelial cells have also been produced; these grow better
and thus allow studies that are not feasible with the primary cell
cultures, but they also generally retain fewer lens-specific
characteristics.37

The processes whereby lens epithelial cells proliferate and differentiate

into lens fiber cells and the manner in which those fibers are arranged
into a functional, continuously growing lens are complex and highly
regulated but poorly understood. It is clear that any derangement in this
process leads to lens opacity. When differentiation of epithelial cells is
altered or incomplete, posterior subcapsular cataracts may occur
because of failure of fibers to form proper sutures at the posterior pole
of the lens or because undifferentiated epithelial cells migrate to the
posterior pole.6Such events are believed to be responsible for the
posterior subcapsular cataracts associated with x-irradiation or
corticosteroid therapy. Although the precise molecular defects
responsible for initiating these cataracts are unknown, it is becoming
clear that changes in effector molecules such as growth factors and
cytokines or altered signal transduction pathways can be potent initiators
of cataract.

One cataractogenic agent of this type that is currently receiving

considerable attention is the cyto-kine transforming growth factor
(TGF-). TGF- is believed to be involved in epithelial-mesenchymal
transformation, a process involved in the pathogenesis of various
diseases.38 It has been demonstrated that exposure of lens epithelial
explants to TGF- causes morphologic and molecular changes
resembling such transformation in other cell types. These changes are
also similar to changes occurring in human anterior subcapsular
cataracts and secondary cataract, including expression of smooth muscle
actin and type I collagen, proteins not normally present in lens epithelial
cells. Based on these findings, it was suggested that TGF- might be a
causative factor in the formation of anterior subcapsular
cataracts.39 Additional support for this hypothesis has come from the
demonstration that transgenic mice overexpressing active TGF- in the
lens develop anterior subcapsular cataracts40 and, more recently, that
injection of TGF- into the vitreous of rats causes similar anterior
subcapsular changes.41 In the latter study there were also morphologic
changes in the posterior subcapsular and cortical regions of the lens. Of
additional interest is the observation that estrogen may protect the lens
from the cataractogenic effect of TGF-.42

Back to Top

DIABETIC CATARACTS
Sugar cataracts have been the most thoroughly studied of all
experimental cataracts. Interest in this system was stimulated by the
observation of van Heyningen43 in 1959 of sorbitol accumulation in the
lenses of rats with sugar cataracts. Sorbitol is the polyol formed from
glucose by the enzyme aldose reductase, the first enzyme of the polyol
pathway (Fig. 6). This pathway was thought to be confined primarily to
the reproductive tissues such as the placenta and seminal vesicles.
However, since van Heyningen's findings, the polyol pathway has been
found to function not only in the lens but also in other tissues, including
the cornea, iris, retina, nerve, and kidney.44

Fig. 6. Polyol pathway.

How aldose reductase initiates the sequence of events leading to diabetic

cataracts has been revealed through a series of observations in animal
models.45,46 The first change to occur in these diabetic rats is the
appearance of vacuoles at the lens periphery (Fig. 7); as the cataract
progresses, the vacuoles extend to the anterior cortex. A dense nuclear
opacity eventually develops. A clue to the role of aldose reductase in the
formation of diabetic cataracts was the histologic appearance of hydropic
lens fibers. Swollen lens fibers are found in the outermost regions of the
lens and are in striking contrast to those fibers in the deeper layers,
which are of normal size. Osmotic swelling of the affected lens fibers is
due to the accumulation of sorbitol. Polyols generally do not penetrate
biologic membranes very well; therefore, once formed in a cell they can
accumulate to unusually high levels if they are not rapidly metabolized.
This is what occurs in the lens fiber cells in diabetes because although
aldose reductase activity is high in rat lens, the activity of sorbitol
dehydrogenase is very low. The hypertonicity created by sorbitol
retention is corrected by an influx of water, which causes the lens fibers
to swell. The resulting osmotic swelling has deleterious effects in the
lens. For example, the membrane permeability is adversely affected;
consequently, substances such as potassium ions, amino acids, and
myoinositol, which are normally maintained in higher concentrations in
the lens than in the surrounding intraocular fluids, begin to leak out.
Sodium and chloride ions eventually build up within the lens as
potassium is lost, and these major electrolyte changes lead to the
nuclear opacity. Thus, the initial action of aldose reductase triggers a
series of events resulting in opacification of the lens.

Fig. 7. Changes leading to diabetic

cataract formation.

The development of the aldose reductase concept in diabetes was

greatly enhanced by taking advantage of an interesting property of the
two enzymes of the polyol pathway (see Fig. 6). Aldose reductase has
broad substrate specificity in that several sugars are attacked by this
enzyme. In fact, galactose is a better substrate than glucose. Second,
the polyol galactitol is not metabolized by sorbitol dehydrog-enase.
These two facts suggested that cataracts should form in galactosemic
rats as well as in diabetic rats and that they should form in the former
much earlier. This proved to be the case. A much greater accumulation
of polyol occurs and results in a much larger osmotic change in the lens
of a galactose-fed rat than in a diabetic rat. The use of a galactose rat
model has been extremely helpful in implicating aldose reductase not
only in cataracts but also in corneal keratopathy and retinopathy of
diabetic animals.46,47

The details of the role of aldose reductase in causing diabetic cataracts

were provided by lens culture studies in which cataracts were
produced in vitro by exposing lenses to incubating media with either
high glucose or galactose concentrations. The biochemical and the early
morphologic changes that occur in the lenses of diabetic or galactosemic
rats can be duplicated in the cultured lens. The biochemical changes
were shown to be caused by the osmotic consequences of polyol
accumulation. When these lenses were prevented from swelling by
appropriately increasing the tonicity of the medium, the biochemical
changes did not occur. Lens swelling was prevented by adding equivalent
amounts of polyol to the medium to offset the polyol formed in the lens.
In this osmotically compensated medium, the lens maintained in a
normal state of hydration was able to keep the biochemical parameters
at normal values despite the accumulation of polyol. Thus, the loss of
glutathione amino acids, potassium, and myoinositol or an increase in
sodium did not occur when the lens exposed to high-glucose medium
was kept from swelling. The ability of the lens to concentrate amino
acids is greatly impaired in the high-glucose or high-galactose medium.
However, when these media were made hyperosmolar to prevent lens
swelling, the lens retained its ability to concentrate amino acids and
myoinositol to normal levels.47 These findings indicated that the
biochemical changes observed in the early stages of diabetic cataracts
were caused by osmotic effects created by polyol accumulation.

Additional support for aldose reductase as the initiating mechanism in

diabetic cataract formation comes from the studies of two other animal
species. First are studies in two different mouse systems. Congenital
hyperglycemic mice are hyperglycemic throughout life but do not
develop cataracts, seemingly a conflict with the aldose reductase
hypothesis.48 Examination of the lens, however, revealed that the lenses
of these mice contained very little polyol despite the hyperglycemia. The
reason for this is that very little aldose reductase is present in these
lenses. As revealed later, a mouse's lens in general has little aldose
reductase, and so no cataracts are induced even when large amounts of
galactose are consumed. Recently, a transgenic mouse has been
engineered in which the aldose reductase gene coupled to a lens-specific
promoter is expressed in the lens.49 Transgenic lines with significant
expression of the enzyme in the lens were shown to be susceptible to
sugar cataract; wild-type littermates were not. Further, the severity of
the cataracts produced correlated positively with the level of aldose
reductase activity obtained from the lenses in different transgenic lines.
A second species of interest was the degu (Octodon degus).50This South
American rodent was brought into this country by immunologists for
study because it has a double thymus. When fed regular laboratory rat
chow, the degus developed cataracts. On investigation, it was revealed
that they had a blood glucose level of 150 mg/dl, hardly sufficient to
cause cataracts in rats but apparently high enough to cause a diabetic
cataract in the degu. Their lenses were found to have huge amounts of
sorbitol because of unusually high levels of aldose reductase, about five
times higher than in the rat lens. Thus, these two animals further
validate the concept that aldose reductase is the initiating factor in
diabetic cataracts. It is not the amount of glucose available by itself but
rather the amount of lens aldose reductase activity that determines
whether cataracts are formed when animals are subjected to a
hyperglycemic state.

The most convincing evidence implicating aldose reductase in diabetic

cataract formation has evolved from the use of aldose reductase
inhibitors.51 In the beginning, inhibitors with marginal activity showed
that they were capable of delaying the onset of cataracts. With
development of more potent inhibitors, it is now possible to prevent
diabetic cataracts completely. About a dozen different aldose reductase
inhibitors can block polyol formation and prevent cataracts in diabetic or
galactosemic rats, dogs, or degus.46,50 Although questions have been
raised about the role of aldose reductase in sugar cataract development
because the catalytic efficiency of the enzyme in reducing aldoses is
low,52 the body of data outlined above overwhelmingly supports the
conclusion that it is the initiating factor leading to lens opacification in
the animal models of sugar cataract. The potential of aldose reductase
inhibitors as anticataract agents in humans will be discussed later in this
chapter.

Back to Top

ANIMAL MODELS OF CATARACT

Most of the information that has been learned about the underlying
mechanisms of lens opacification has been derived from the study of the
process in a variety of animal models. Some of these models have
resulted from spontaneous mutations; others are the result of exposing
normal animals to chemical or environmental toxicants. More recently,
genetically engineered animals (transgenic and knockout) have provided
additional cataract models. The following is a brief survey of some of the
better-studied models in each of these categories.

EXPERIMENTALLY INDUCED MODELS

Probably the earliest animal models of cataract to be systematically

studied were those in which cataract was induced experimentally. The
galactose cataract in rats, unquestionably the most studied and probably
the best understood of all cataract animal models (see above), dates
back to 1935. By the 1960s, studies were well underway on cataracts
induced in animals by radiation and by chemical agents such as
naphthalene, phenothiazines, and triparanol. More recent models have
provided insights into the process of cataractogenesis. One of the most
studied is the selenite cataract that is produced by injection of sodium
selenite into 10-day-old rat pups. 53Advantages of this model include the
reproducibility and rapidity of cataract formation. Severe nuclear
cataract occurs within 5 days, followed by cortical opacification several
weeks later. The hallmark of the selenite model is a marked increase in
calcium in the lens nucleus that precedes the formation of nuclear
cataract. The increased calcium activates the protease calpain II that
partially degrades various lens proteins, including - and -crystallins.
The selenite model has brought the role of proteolysis in
cataractogenesis more to the forefront. This model has also contributed
to knowledge of the role of oxidation in cataract
development.54 Oxidative stress, as evidenced by decreased GSH and
increased hexose monophosphate shunt activity, occurs within the first
day after selenite treatment, and it is believed that oxidative changes at
the cell membranes underlie the loss of normal calcium homeostasis.
The selenite model has been used to test possible anticataract agents,
including calpain inhibitors, antioxidants, and phase separation
inhibitors.

Two other animal model systems have been used to address aspects of
the role of oxidation in cataractogenesis. The first of these uses
buthionine sulfoximine, an inhibitor of GSH synthesis, to induce cataract
in neonatal rats or mice.55 Formation of cataract in this model requires
nearly total depletion of GSH in the lens and like the selenite model
occurs only in very young animals. After GSH depletion there is
progressive loss of cation homeostasis, osmotic swelling, increased
proteolysis of lens proteins, and lens fiber degeneration.56 This sequence
of events is similar to that found in the selenite cataract and other
osmotic-type cataracts.

A second model under study stems from the observation that subjects
repeatedly exposed to hyperbaric oxygen therapy are at risk of
developing nuclear cataracts. Guinea pigs exposed repeatedly to 2.5 atm
of pure oxygen develop changes specifically in the lens nucleus that are
similar to aging-related changes seen in the human lens.57These include
increased light scattering, disulfide formation, and membrane damage.
Unlike the previous systems, there is no increase in calcium in these
lenses, where specific losses of soluble protein seem to result from
disulfide formation and insolubilization rather than proteolysis.58

HEREDITARY MODELS

Spontaneously occurring hereditary cataracts have been studied in a

variety of species, including mice, rats, guinea pigs, and dogs. The
Nakano mouse was the first such model to be systematically
studied.59 Osmotic cataracts form about 20 days after birth and are
believed to be the result of impairment of Na + ,K+ -ATPase activity by a
polypeptide inhibitor that has not been fully characterized. 60 Two other
hereditary cataracts that have been extensively studied, in the Philly
mouse and the strain 13 guinea pig, have each been shown to result
from mutations in lens crystallin genes. The Philly mouse develops
visible cataracts at about 20 days61 of age that have been shown to be
associated with a deletion mutation in the B2-crystallin gene. This
mutation causes a four-residue deletion in the polypeptide that impairs
its stability.62 Interestingly, a human familial cataract has recently been
shown to result from a mutation in the B2-crystallin gene. 63 In the
strain 13 guinea pig model, nuclear cataracts are present at birth and
are believed to result from a splice site mutation in the gene for zeta-
crystallin, a major lens protein in guinea pigs that is also an
enzyme.64 Zeta-crystallin is an NADPH-dependent oxidoreductase, and
the mutant form of the protein has lost a complete 34-residue exon and
the ability to bind NADPH.65 Cataract may result from loss of a major
structural component of the lens (the crystallin) or from the markedly
decreased NADP(H) levels present in the lenses of the animals having
the mutant gene.

In addition to these models, there are other mouse cataracts associated

with mutations in the major intrinsic protein, the major membrane
protein of the lens.66

Of particular interest are animal models of hereditary cataract in which

the lens opacifies later in life, because such models may be more
comparable to human aging-related cataracts. The Emory mouse 67 is the
best-studied model of this type. Two separate substrains have been
identified: one exhibits cataract onset at 5 to 6 months of age and the
other several months later.68 Morphologically, the cataracts are said to
resemble human cataracts more than do the cataracts in the various
early-onset cataract models. Although the extended time period needed
to reach opacity and the individual variability with respect to onset of
opacification make the use of the Emory mouse model more difficult,
there is no question that one of the greatest needs in cataract research
is a good animal model for human aging-related cataract. For that
reason, the Emory mouse model warrants further investigation.

A large collection of dominant cataract mutations were generated in mice

by paternal treatment of germ cells by x-ray or ethyl-nitrosourea. Many
of these have been studied, and in some specific mutations have been
identified. Graw69,70 has recently reviewed the data on these models.

GENETICALLY ENGINEERED MODELS

In the current age of genetic engineering, it is no longer necessary to

depend on nature to provide mutant strains of interest. Most of the
animal models now under study are engineered transgenics or
knockouts. Some have been produced specifically to study effects on the
lens; in other instances, cataracts have been found to occur in animals
engineered for other purposes. The lens has been a favorite subject for
transgenic studies because of the availability of lens-specific promoters
derived from lens crystallin genes.

One of the most studied transgenic mouse cataract systems is the HIV-1
protease transgenic mouse that develops cataracts at about postnatal
day 24. The HIV-1 protease is expressed specifically in the lens of these
animals under the control of the A-crystallin promoter.71 Formation of
the cataract is dependent on the activity of the HIV-1 protease because
specific inhibitors of the enzyme prevent cataract formation. The
biochemical hallmark of this cataract is proteolytic cleavage of certain
crystallins and of the major intrinsic membrane protein of lens fiber cells,
but it has been demonstrated that these cleavages are not the direct
result of the action of the HIV-1 protease. 72Rather, it appears that the
HIV-1 protease activates an endogenous cysteine protease that actually
cleaves the crystallins and major intrinsic membrane protein.

Two other transgenic models that have been particularly instructive were
mentioned above. The aldose reductase transgenic in which this enzyme
is expressed in the mouse lens at much higher than normal
levels49 provided the crucial confirmatory evidence that aldose reductase
initiates diabetic cataract in rodents. The TGF- transgenic
mouse40 demonstrated that anterior subcapsular cataracts would result
from excess exposure to this agent in vivo. Other transgenic mouse
models that develop cataract include the ectopic expression of -
interferon in the eye73 and the expression of various chimeric or mutated
type III intermediate filament genes in the lens. 74 Recently it has been
shown that overexpression of the bovine Na+ /myoinositol cotransporter
in lens fibers of transgenic mice causes lens swelling and cataract. 75 This
system is being studied as a model of osmotic cataract.

Models generated by gene knockout technology have also been used to

study the roles of specific proteins in the lens. The first lens crystallin to
be knocked out was A-crystallin. Complete elimination of A-crystallin
from the lens led to opacification starting in the lens nucleus and
spreading to involve the entire lens as the mouse ages. 76 A distinctive
feature of these lenses is dense inclusion bodies rich in B-crystallin that
are abundant in the central lens fibers. These findings demonstrate that
A is essential to maintaining a transparent lens, perhaps at least in part
by preventing the insolubilization of B. Interestingly, knockout of B-
crystallin does not appear to cause cataract, but the mice do ultimately
exhibit skeletal muscle degeneration, severe postural anomalies, and
premature death.77This phenotype probably reflects the fact that B is
expressed in muscle as well as in lens and other tissues, but the
mechanism responsible is not known. Among other knockout models
that have produced cataract are those in which the expression of
connexins, the proteins making up gap junction complexes, has been
disrupted.78 As noted earlier, the glutathione peroxidase knockout has
been used to study responses to oxidative stress by the lens.24,25

Back to Top

THERAPEUTIC APPROACHES TO CATARACT

As the leading cause of blindness worldwide, cataract presents a huge
potential market for pharmaceutical intervention. Although there is a
highly successful surgical cure for the disease, its availability is limited in
much of the world, and there are risks of complications to the surgery.
Thus, considerable effort has been made to develop effective
anticataract drugs. To date, no such drug has been licensed in the United
States. Several anticataract preparations have been available in various
parts of the world, but there is little or no evidence that any of them are
effective. The reader is referred to reviews of the subject for additional
information on these formulations and the rationales for their
development and use.79,80 Current efforts toward the development of
efficacious anticataract agents are summarized here.

ALDOSE REDUCTASE INHIBITORS

The role of the enzyme aldose reductase as the initiator of cataract

formation in animal models of diabetes was discussed above. Many
agents have been developed that inhibit aldose reductase and that also
have been shown to block cataract formation in diabetic or galactose-fed
rats and dogs.51 There is no doubt that these diverse inhibitors prevent
cataract formation in these animals. However, it is not known whether
they would have efficacy in cataract prevention in humans with diabetes.
Aldose reductase is present in the human lens at much lower levels than
in the lenses of rats or dogs,81 whereas sorbitol dehydrogenase is
present at much higher levels. 82 Thus, accumulation of sorbitol in the
lenses of humans with diabetes is much less than in the animal
models.83 It has been established that persons with type II (adult-onset)
diabetes tend to develop cataract at an earlier age than the general
population.84It appears that in these persons, dia-betes is but one
additional risk factor in the constellation of stresses that may contribute
to aging-related cataract. In the case of the sudden cataracts that
sometimes occur in persons with type I diabetes, 85 rapid swelling of the
lens leads to opacities that morphologically resemble the cataract formed
in the diabetic and galactosemic animals. It has been suggested that this
latter type of cataract might be preventable by administration of an
aldose reductase inhibitor at the time of diagnosis of diabetes.85

Aldose reductase inhibitors have also been shown to prevent

naphthalene-induced cataract in rats,86 apparently because aldose
reductase activity is required for generation of the toxic metabolite of
naphthalene responsible for causing the cataract.87

ANTIOXIDANTS

Because oxidative processes are clearly involved in human aging-related

cataract formation11 and because epidemiologic evidence indicates a
reduced risk of cataract for persons with higher systemic levels of
antioxidant vitamins,88 there have been numerous studies of agents with
antioxidant activity as potential anticataract drugs. Initially these studies
focused on the antioxidant vitamins, ascorbic acid and -tocopherol.
Ascorbate has been reported to provide partial protection in various
animal models of cataract, including selenite89 and galactose.90Likewise,
vitamin E has been reported to retard cataract development in some
systems.91 With respect to human cataract, the evidence in support of
antioxidant vitamins comes from a variety of case-control and natural
history epidemiologic studies.88 It is hoped that more definitive data will
come from the Age-Related Eye Disease Study (AREDS), a 10-year
randomized trial sponsored by the National Eye Institute to test high-
dose supplementation with vitamin C, vitamin E, and -carotene in age-
related cataractogenesis.92

Another antioxidative approach to cataract therapy focuses on

maintenance of normal levels of glutathione in the lens. Because the
uptake of cys-teine has been shown to be limiting in the synthesis of
glutathione,93 cysteine prodrugs (i.e., compounds metabolized to
produce cysteine in the cell) have been used with positive effect in
several animal models of cataract, including naphthalene cataract. 94 The
nitroxide TEMPOL, a known radioprotective agent, has been shown to
protect against x-ray-induced cataract in rabbits, presumably by
inhibiting the formation of free radical species in the lens. 95 Other agents
with antioxidant capacity that have shown some efficacy in laboratory or
animal studies include lipoic acid,96 taurine,96 and py-ruvate.97 The
laboratory evidence on antioxidants is fragmentary and largely
preliminary, but taken with the obvious oxidative effects occurring during
cataractogenesis, this direction merits further investigation.

PHASE SEPARATION INHIBITORS

As discussed earlier, phase separation phenomena have also been

implicated in cataractogenesis. Because phase separation in the lens
would be potentiated by increased attraction between lens protein
molecules, it follows that decreasing such attractive forces would
decrease the likelihood of phase separation and the resultant light
scatter and opacification in the lens. Benedek 28 has discussed ways of
modifying lens crystallins to effect such changes. In vitro, the
modification of crystallins by the addition of hydrophilic groups such as
glutathione as mixed disulfides was found to decrease phase separation.
The use of several agents that exhibit such behavior in vitro has also
been attempted in animal models of cataract.32 Two agents that have
exhibited anti-cataract activity in several such models are pante-thine, a
disulfide derived from coenzyme A, and the radioprotective
phosphorothiate WR-77913. The delay in the time of onset and the
severity of cataract resulting from the systemic administration of these
agents in a variety of animal models is highly encouraging. The specific
mechanisms responsible for cataract inhibition by these agents remain
obscure.

Based on these findings, a small clinical trial was initiated to determine

whether pantethine could protect against the cataracts that commonly
occur 12 to 18 months after vitrectomy. Unfortunately, this trial was
stopped a few months after patient enrollment began because some
subjects suffered minor eye irritation, apparently resulting from the
pantethine eye drops (personal communication, John I. Clark, PhD). In
retrospect it appears that the vitrectomy model was not suitable for this
trial for several reasons. First, no animal model is available for
vitrectomy cataract, so preclinical tests could not be conducted. Second,
nearly all patients in the study had early-stage cataracts before
vitrectomy was performed. Because the weak noncovalent interactions
that pantethine is believed to influence are very early events in the
process of cataractogenesis, administration of pantethine or other phase
separation inhibitors probably would not prove effective in the
vitrectomy population. Finally, there is doubt about the ability of topical
pantethine to penetrate the human cornea and reach the lens. Although
this trial was interrupted and yielded no supporting evidence, the
potential use of inhibitors of protein aggregation as anticataract agents
remains attractive based on the animal and in vitro studies cited above.

OTHER AGENTS

Considerable controversy has surrounded the suggestion that aspirin

might have an anticataract effect. It was initially suggested that aging-
related cataract development might be inhibited in patients with
rheumatoid arthritis by aspirin taken for its anti-inflammatory
activity.98 The data regarding the potential efficacy of aspirin and other
analgesics as anticataract agents have recently been discussed. 52 Based
on several epidemiologic studies, it now appears that aspirin has little if
any protective effect against cataract formation.99 Many other agents
(e.g., Bendazac, Catalin, Phakan) are available outside the United States
and are given as anticataract eye drops, but none of them has
undergone definitive clinical trials or has been approved for such use in
the United States