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.

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.

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.

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-ÞB32), hydrogen peroxide (H₂O₂), and the hydroxyl radical (OH^ÞB3), 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 H₂O₂. 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 OH^ÞB3 via the Fenton reaction.

Two potential sources of OH^ÞB3 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-ÞB32, which can then give rise to H₂O₂ and OH^ÞB3. Alternatively, in a type II process the triplet sensitizer transfers energy directly to molecular O₂ 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 H₂O₂ endogenously present in the eyes of certain species, including humans. It has long been believed that H₂O₂ levels in aqueous humor are much higher than those in plasma or in tissues outside the eye. Concentrations in the 20- to 30-μm 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 H₂O₂ 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 H₂O₂ is 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 H₂O₂ in the medium, yet the cells did resist H₂O₂-induced stress better than the nontransfected normal cells.²³ However, 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.

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.

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 (T_c) is the temperature below which phase separation occurs in a solution of proteins or other solutes. For certain proteins, including some members of the γ-crystallin family, T_c is only marginally below body temperature.³⁰ Because T_c is determined by the noncovalent energy of attraction between proteins, modifications to proteins that increase such attractive forces will raise T_c. If T_c rises above body temperature, phase separation may occur within the lens in vivo.

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.³² These agents will be discussed later in this chapter.

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.⁶ Such 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 β (TGF-β). TGF-β 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-β 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-β 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-β in the lens develop anterior subcapsular cataracts⁴⁰ and, more recently, that injection of TGF-β 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-β.⁴²

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.

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).⁵⁰ This 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.

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.⁵³ Advantages 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 α- and β-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.⁵⁷ These 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 βB2-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 βB2-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 mouse⁶⁷ 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. Graw^69,70 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 αA-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.⁷² Rather, 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-β 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 γ-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 αA-crystallin. Complete elimination of αA-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 αB-crystallin that are abundant in the central lens fibers. These findings demonstrate that αA is essential to maintaining a transparent lens, perhaps at least in part by preventing the insolubilization of αB. Interestingly, knockout of αB-crystallin does not appear to cause cataract, but the mice do ultimately exhibit skeletal muscle degeneration, severe postural anomalies, and premature death.⁷⁷ This phenotype probably reflects the fact that αB 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

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.⁸⁴ It 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 α-tocopherol. Ascorbate has been reported to provide partial protection in various animal models of cataract, including selenite⁸⁹ and galactose.⁹⁰ Likewise, 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 β-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. Benedek²⁸ 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.^79,80

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