Chapter 72A
Physiology of the Lens
GEORGE DUNCAN and I. MICHAEL WORMSTONE
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LENS STRUCTURE AND FUNCTION
INTERCELLULAR COMMUNICATION IN THE LENS
PERMEABILITY PROPERTIES OF THE LENS MEMBRANES
ACTIVE SODIUM AND POTASSIUM CONTROL MECHANISMS
SODIUM CONDUCTANCE
POTASSIUM CONDUCTANCE
CHLORIDE CONDUCTANCE
WATER MOVEMENT
pH REGULATION
CONTROL OF LENS CYTOPLASMIC CALCIUM
GROWTH FACTOR REGULATION
EXTRACELLULAR MATRIX
ACKNOWLEDGMENTS
REFERENCES

Cataract has been a recognized debilitating visual condition since Roman times and has been the subject of intense investigations by lens anatomists and physiologists for centuries.1 David Brewster2 was the first to recognize the importance of investigating cell structure in the lens. He used novel optical diffraction techniques, using a candle as a light source, to calculate very accurate values not only for fiber diameters in different regions of the lens but also pointed out the presence of ball-and-socket junction decorating the lateral faces of the fibers. Brewster also was the first to suggest that osmotic swelling played a role in cataract.3 Brewster's interests are still reflected today, although his candle has been replaced by the laser and electron microscope and his simple swelling manipulations by cataract-inducing transgenic technology. Great advances have been made in the past few years on how the cells within the lens are structured and organized, particularly in the role of the cytoskeleton and adhesion molecules in fiber cell movement4 and the role of cell-cell junctional communication in the maintenance of lens homeostasis5 and in cataract.6 Osmotic regulation of the lens also is of prime interest in cataract development, and calcium, pH, and second-messenger systems all play a role.7

It has long been appreciated that the lens continues to grow throughout life, and strenuous efforts also have been made to identify the various factors controlling this very well-regulated process (see growth factor section).8 It has become increasingly recognized in the past few years that lens cells continue to grow after modern extracapsular cataract extraction, and it is this growth that is critical to the development of posterior capsule opacification (PCO).9 Because a search for the factors modulating this type of growth can give insights into how lens growth in general might be controlled,10 this chapter deals with the congruent issues of normal lenshomeostasis, cataract, and PCO. Furthermore, because it is becoming increasingly obvious that many of the factors playing a critical role in human lens behavior do not always have a similar role in the animal lens, this chapter presents, as far as possible, data obtained from human lens investigations. Because this is not always possible, the species origin is given and the data evaluated in terms of known differences in behavior with the human lens.

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LENS STRUCTURE AND FUNCTION
The lens is an asymmetric structure bounded on the anterior surface by a single layer of epithelial cells and on the posterior surface by fiber cells (Fig. 1). This apparent asymmetry initiated a number of double-chamber experiments that appeared to show a translens (posterior to anterior) flux of sodium ions correlated with a translens short circuit current.11,12 Studies such as these have given rise to the “pump-leak model” of lens transport. However, more recent studies performed using a vibrating probe to measure radiating currents13,14 have shown asymmetries that are more marked between the equator and the two poles than there are between the two poles themselves, and this has given rise to the “internal flow model” for the lens.5

Fig. 1. Cross-sectional view of the mammalian lens. The lens is morphologically an asymmetric structure with a single layer of epithelial cells at the anterior surface. The anterior cells in Section A, although coupled to each other, do not appear to be well coupled to the fibers beneath nor does there appear to be many junctional complexes between fiber cells in this region. Cells in the equatorial section (E) do appear to be well coupled at the fiber-fiber and fiber-epithelium interface. Functionally, therefore, the lens appears to behave as two symmetric structures superimposed. The anterior (leaky) epithelium represents one system and the fiber cells represent the other. This model accounts for the radial distribution of currents around the lens and also explains why only a small asymmetry voltage is observed in double-chamber experiments. The posterior surface (P) has no epithelial cells, and the differentiated fibers in the inner cortex (IC) and nucleus (N) have lost their organelles. See text for further details.

There is little doubt that the membranes of the anterior epithelial cells have a very different array of channels and pumps than the fiber cell membranes. This fact, allied to an asymmetric distribution of internal gap junctions that seem to favor an internal flow of current toward the equator rather than from posterior to anterior, makes the two models difficult to reconcile.5 However, if an amalgam of the two asymmetry models is considered, then it is possible to merge the two separate ideas into one working hypothesis. The model is presented here and the evidence scrutinized later.

It is suggested that the lens consists of two symmetric and superimposed structures that are only intimately connected at the equator. In this model (see Fig. 1), the anterior epithelium and dynamic bow cells represent one system and the mature fiber cells another. The cells within each system are known to be in good electrical communication with other cells within the same system,15,16 but it is likely that good communication between the systems exists only at the bow region.17 If we also assume that the resting potential of the epithelial cells, on average, is higher than the mature fiber cell membrane potential (again on average), then current flows in at the fiber cell membranes and out through the epithelial cells. If the anterior epithelial layer is assumed to act as a leaky epithelium with little potential difference between the apical and basal surfaces, then the only net current that is observed is inward at the poles and outward at the equator. Furthermore, when the anterior and posterior surfaces are isolated, then the anterior epithelium lies in series with the rest of the lens and a small voltage difference (the net transepithelial voltage) wouldbe expected to be observed. In fact, using an oil to isolate the two surfaces of the bovine lens in a nondisruptive manner, Duncan and colleagues18 observed a maximum potential difference of + 6.5 mV (anterior positive). It should be noted that if the anterior epithelial cells were well-connected to the whole mass of underlying fibers, then a much greater asymmetry in polar currents would be expected than that actually observed as the high voltage of the anterior face membranes would give a strong outward current across the whole of the anterior surface.

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INTERCELLULAR COMMUNICATION IN THE LENS
It should be noted that there are two types of intercellular communication possible: electrical and molecular (or metabolic). It has been shown in several systems, including the lens, that metabolic communication can be severed while ionic communication remains intact.16 This important point should be borne in mind when picking one's way through the mine field of lens junction literature. It also should be remembered that there are possible species differences in the extent of communication between different regions of the lens and, furthermore, the pattern of communication can change as the lens develops.

The molecules (connexins) that form junctional complexes have been reviewed in detail recently,5,19 and only a brief summary is given here. Connexin 43 is the major junctional molecule in epithelial cells, whereas there is a much greater heterogeneity of connexins found in fiber cells, including connexins 46 and 50, although the latter is not found in mature fibers. Interestingly, connexin mutations can give rise to congenital cataracts,5 showing the importance of junction communication to the normal function of the lens. There is general agreement among lens physiologists that the fiber cells arecoupled electrically and that all epithelial cells are extremely well coupled, both electrically and metabolically.5,16,17,20 There is not the same degree of uniformity of opinion concerning the metabolic coupling between epithelial and fiber cells.17,20,21

FIBER-FIBER CELL COUPLING

Duncan15 first pointed out, from evidence obtained by inserting current-passing and voltage-measuring electrodes into the lens, that fiber cell membranes in the bulk of the lens did not present a great resistance to the passage of current within the lens. However, fiber cell membranes at the surface appeared to have a considerable resistance. This appears to be the case in every vertebrate lens so far investigated,5,15 although it should be pointed out that in the invertebrate lens, the equivalent fiber-like structures in the anterior portion of the lens are not coupled to the fiber plates in the posterior hemisphere. Interestingly, the fibers in both hemispheres are well coupled to their neighbors within the same hemisphere.22

The low internal resistance view was further refined by Eisenberg and colleagues23,24 who quantified the internal resistance and showed that it was not homogeneous, but was, in fact, lower in the equatorial regions in the cortex. This conclusion is satisfyingly in agreement with the distribution of gap-junctional complexes within the lens. They are found in much higher density in the equatorial regions.17,20

Electrical communication is so efficient within the lens that electrical signals arising from the activation of certain receptor systems can be coordinated so that the whole lens can be seen to oscillate. This pattern has been observed in the whole lens25 and in a lens clamped in a double-chamber system.26

Electrical communication also can be modulated or controlled, and lowering internal pH or increasing internal calcium has been shown to increase internal resistance.27–29 In the latter case, the uncoupling process is calmodulin-dependent.27 Mathias and coworkers28 pointed out that the nucleus of the amphibian lens does not uncouple even although the internal pH is reduced to very low values. This is interesting in view of the latest findings from the chick lens, in which it has been shown that the nuclear fibers are completely permeable to the passage of very large molecules.30 Duncan31 also has pointed out previously that the central regions of the lens contain relatively high levels of lysophospholipids32 and suggested that the nuclear fiber membranes were degenerate in some way. This, he argued, would explain why nuclear cataracts are homogeneous defects and involve always the whole nucleus whereas cortical cataracts that involve an increase in internal calcium can be localized and can involve only small, specific regions of the lens.33

Ionic coupling within the anterior epithelium is extremely efficient and although the calculated intercellular resistance of 25 Ωm17 is much higher than that of aqueous humor (1 Ωm), it is considerably lower than the value calculated for the fiber-fiber resistance (90 Ωm) in the bulk of the lens.24 It is interesting in this respect that ionic coupling in the bulk of the lens can be greatly reduced by conventional uncoupling agents such as octanol and low pH,27–29 whereas ionic coupling across the epithelium is relatively resistant to such treatment.16

There is only one report of ionic coupling between epithelial cells and fiber cells,34 but the model suggested in Figure 2 would allow for current injected into the fibers to flow into the epithelium, even if communication only existed between equatorial epithelia and underlying fibers. The space constant (λ) for the epithelium extends some 20 cell diameters,17 and so little decrement of voltage would be expected across the whole epithelium for currents injected into fiber cells anywhere in the lens.

Fig. 2. Human lens membrane voltage measured in eye bank and cataract lenses. Note that pure nuclear cataracts follow the pattern of normal lenses, while cortical cataracts have voltages considerably lower than normal lenses of a similar age.7,54

METABOLIC COUPLING IN THE LENS

The only noncontroversial statement in this area of lens research is that there is little agreement about the extent and nature of fiber-fiber or fiber-epithelium coupling. This lack of agreement may reflect species differences or the fact that some areas of the lens are better connected than others. Often, when the same experimental protocols are performed on lens cells from different species, different results are obtained. For example, when fluorescent dye is injected into a cultured chick lentoid body (expressing MIP26), then it spreads rapidly from the injected site into neighboring cells,35 while in a rat lentoid (similarly expressing MIP26), the dye remains within the injected cell.36 In the embryonic chick lens, dye injected into the posterior lens rapidly spreads throughout the lens and passes from fibers to epithelium.37 These communicating pathways are quite sensitive to low pH at early stages, but beyond stage 23, low pH could not prevent the flow of dye between fiber cells nor from fiber cells to epithelium.37 In amphibian and rat lenses, however, even ionic coupling between fiber cells is very sensitive to low pH.28,29 Furthermore, in these latter species, heroic efforts have to be made in terms of intrusive dissection of the anterior capsule and application of very-high-sensitivity imaging techniques to observe any flow between anterior epithelial and fiber cells.21 In the end, the authors of this latter study concluded that at most, one in four anterior epithelial cells are metabolically coupled to the underlying fibers. In fact, in humans, rats, and amphibians, there appear to be few junctional complexes between epithelial cells and underlying fibers, except, as has been mentioned above, in the equatorial region.17,20,38

Just as there are regional variations in electrical coupling in the bulk of the lens, there also are variations in the extent of metabolic coupling. Epithelial cells near the bow appear to be in communication with newly formed fiber cells,21 whereas short fiber cells that are actively elongating do not appear to pass dye efficiently to their neighbors and do not possess many gap-junctional plaques.17 However, longer, more mature, fibers with interdigitating junctions did pass injected dye efficiently to their neighbors.17,21 These fibers also possess abundant junctional complexes. Fibers that are more mature still and extend from the anterior to posterior pole do not appear to be metabolically coupled to their neighbors nor were they coupled efficiently to the anterior epithelial cells.17 Again, this behavior was in agreement with the lack of typical gap-junctional arrays in the membranes of these fibers. On pro-gressing to the nucleus, the fiber cell membranes appear to degenerate so that they do not impede the diffusion of very large molecules,30 at least in the chick, and neither can nuclear cells be uncoupled in the amphibian lens by low pH.28

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PERMEABILITY PROPERTIES OF THE LENS MEMBRANES
The permeability properties of the lens can be measured by a number of different methods including radiotracer techniques to assess directly the passage of ions and molecules and electrophysiologic techniques both to assess the overall electrical conductance of the lens but also to investigate the specific channel mechanisms involved. In the latter case, it is emerging from patch-clamp experiments that the lens possesses a remarkable variety of potassium, sodium, chloride, and calcium channels. The different methods used to identify the various channels have been reviewed recently in a number of publications.5,39,40 This review deals rather with the overall changes in lens conductance that occur with age and on exposure to oxidation, but it also deals with the changes that occur after the activation of certain receptors. It also should be remembered that ions may traverse membranes by a number of electrically silent systems, and they include the Na+ /H+ ,Na+ /Ca2+ HCO3-/Cl- exchange mechanisms as well as active transporters such as Na-K-ATPase. The movement of certain ions and even water itself also is restricted by interactions with other species within the lens, and this has to be taken into account in the overall picture of movement across the outer barriers and through the internal matrix of the lens. In the human lens, for example, although almost 100% of K+ ions are in free solution, ion-sensitive microelectrode measurements have shown that approximately 50% of internal sodium is complexed, whereas more than 99% of the total calcium is so tightly bound or sequestered that it does not contribute to the measured activity.33,41 Interestingly, a large fraction of the calcium that enters during cortical cataract also finally resides in the lens in a complexed form.42,43

It is now generally accepted that the main ionic permeability barriers lie at the outer surface of the lens, whereas the inner fiber membranes present less of a barrier to the flow of ions because adjacentfibers are coupled through gap junctions or become fused or “degenerate.”5,15,31 The relative permeability of the different ionic species can be computed by fitting the Goldman equation44 to the membrane potential and (Em) ionic data:

where α = PNa/PK and β = PCl/PK. In most lensesso far studied, PNa <m25> PK, while PCl PK. In the amphibian lens, the relative permeabilities obtained in this way are in good agreement with the individual permeabilities calculated from radioisotope flux data obtained for the individual ions.45 The 42K (or 86Rb) and 36Cl efflux data are easiest to interpret as the major component follows single exponential kinetics.31,45 The 22Na and 45Ca2+ data kinetics are multiexponential in form, presumably because significant proportions of both of these ions are in a bound or complexed form.45,46

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ACTIVE SODIUM AND POTASSIUM CONTROL MECHANISMS
Sodium is distributed far from equilibrium in the normal lens and energy must be expended continuously to maintain the normal sodium, potassium, and water content of the lens.31 When the pump is inhibited by ouabain, then the sodium and water content increases and there is some loss of transparency due to localized refractive index changes. Several studies have shown that ouabain is only effective when applied to the anterior surface11,12,18 and, indeed, the largest fraction of Na-K-ATPase activity resides in the anterior epithelium. It should be noted, however, that ATPase activity is found associated with the membranes of all superficial lens cells.47 Interestingly, Western blot studies show that pump molecules are present on all lens membranes, even although activity is only found in those near the surface.47

The Na-K-ATPase pump consists of an α-catalytic subunit with an associated β subunit and is, in fact, a member of a multigene family of proteins.48 Three α subunits (α1, α2, α3) and three β subunits (β1, β2, β3) have been identified so far, and the expression of the three isoforms (the α form in particular) is quite tissue specific. Most kidney cell membranes express α1 and α3. Interestingly, the lens appears to express all three polypeptides in anterior epithelial cells but only the α1 form in fiber cells.49 It further appears that all three isoforms are not expressed homogeneously across the anterior epithelium, with α3 being located at the anterior pole and α1 predominating in the equatorial region.50

Sodium pump activity is under dynamic control itself and more α subunits are found in lenses stressed by exposure to amphotericin, for example. Interestingly, expression of the α2 subunit appears to be preferentially upregulated.51,52 More recently, it has been shown that Na-K-ATPase activity can be modulated by tyrosine kinase (TK) activation, which raises the very interesting possibility thatthis critical pump mechanism is under second-messenger control in the lens.53

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SODIUM CONDUCTANCE
The membrane potential of the human lens depolarizes with age (see Fig. 2) and the decline becomes most significant after the age of 50 years.54 Theion concentration and electrical data indicate thatPNa/PK progressively increases and as the measured electrical conductance and free Ca2+ activity also increases, Duncan and coworkers54 have suggested that all of these age-related changes could be explained by the progressive activation of a nonselective cation channel. Several studies have shown the presence of such a channel in a number of lens species.5,55,56 Interestingly, in the amphibian lens at least, this channel can be activated by pressure.56 After the age of 50, the lens becomes increasingly unable to accommodate. This is not because the ciliary muscles cease to function, but rather the lens properties became less elastic.57 The stretching and relaxation energy of the muscles would then be dissipated in the lens, leading to an increase in pressure on the lens membranes after the age of 50. The lens nonselective cation conductance also appears to be activated by oxidation58 and a progressive decline in glutathione content of the lens with age59 would also, therefore, tend to activate such a channel. It should be noted that there is a catastrophic decline in membrane potential in human cortical cataract with a concomitant increase in membrane permeability7,42,60 (see Fig. 2).

Although tetrodotoxin-sensitive sodium-specific conductance channels have been identified in tissue-cultured cells,5 there is as yet no evidence to suggest that neuronal-like sodium channels exist in native human lens cells.

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POTASSIUM CONDUCTANCE
Potassium ions are distributed near, or above, equilibrium levels in most lenses so far studied, and there is a huge diversity in the range of channelsthrough which K+ ions can diffuse. Patch-clamp and molecular cloning methods currently are in use to identify the channels that are responsible in thelens for K+ movement.5,39,40 The three types of channels identified by Mathias and his colleagues5 include inward rectifiers, large conductance calcium-activated channels (BK), and delayed rectifiers. The amphibian lens has been most extensively studied so far, and the overall conductance has beenmapped out by current-clamp techniques.61 The I-Vcurves show outward-rectifying properties and the I-V relationship in the normal lens is so steep, it cannot be predicted by Goldman theory. The overall conductance-voltage relationship is best explained by a combination of nonvoltage-sensitive and outwardly rectifying conductances. Removal of external calcium increases the former, largely by increasing Na+ conductance, while the latter is largely inactivated so that the conductance under Ca-free conditions is actually lower than the control conductance at voltages more depolarized than-70 mV. There are significant differences in the behavior of the overall potassium conductances of frog and rat lenses. In the former, the voltage-sensitive conductance is largely inactivated at the resting potential and the conductance is insensitive to blockade by TEA or quinine.62,63 However, on depolarizing by voltage or by adding external K+ , the voltage-sensitive conductance is activated and can be blocked by TEA. Interestingly, the voltage-sensitive conductance in the frog can be activated by low concentrations of the nonpermeant SH complexing agent PCMPS. Under these circumstances, the lens voltage hyperpolarizes to near -100 mV, and the conductance can now be blocked by TEA.63 In the rat lens, conversely, the resting conductance is very sensitive to blockage by TEA or quinine.64 Under these circumstances, the K+ conductance also can be blocked by the addition of external Ca2+ and the voltage depolarizes. Hence, although the K+ conductance in the rat lens can be modulated by Ca2+ , it is not strictly a Ca-activated conductance but rather an inactivated conductance. Significantly, 86Rb+ efflux from the human lens is reduced significantly by increasing external (so presumably internal) Ca2+ .60 At present, the only potassium channel identified by molecular cloning techniques from native human lens epithelial tissue is the inwardly rectifying channel 1RK1.65 This channel also has been identified in a number of animal lenses.5,39,40
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CHLORIDE CONDUCTANCE
Chloride ions are distributed near equilibrium across the lens membranes of a variety of species,31,45,66 and in the amphibian lens, the measured electrical conductance decreases when chloride ions are removed from the external solution.67 The isotopic chloride efflux rates from both amphibian and mammalian lenses are high. In addition, it should be noted that part of this efflux arises from exchange mechanisms and these would not be expectedto contribute to the electrical conductance.66,67Examples of such processes included the DIDS-sensitive HCO3-/Cl- and the Na+ /HCO3-/H+ /Cl- exchangers66,68 as well as the Na+ Cl-cotransporter.69 Undoubtedly, other exchange systems are likely to exist. Chloride channels have been identified by patch-clamp techniques in both epithelial and fiber membranes70 and are likely to play important roles in both volume70 and pH66 regulation.
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WATER MOVEMENT
The cytoplasm of the lens, in common with most animal cells, is isosmotic with the surrounding media.71 However, this is not an equilibrium situation, because of the large concentration of fixed negative charges with the membranes of the lens, and the lens has to expend energy continuously to maintain osmotic equilibrium.72 When the sodium pump is inhibited or the sodium permeability is increased, then the lens swells.31 The water permeability of lens membranes can either be determined by creating an osmotic gradient across the lens membranes and observing the rate of change of volume of the lens, or it can be determined by measuring the efflux of radioactive water. The first measurement determines the hydraulic permeability (Pos) and the second the diffusional permeability (Pd). Both permeabilities have been determined for the amphibian lens and the values are 100 μm-1 and 0.4 μm-1, respectively. These values are remarkably close to the two permeabilities measured in a wide range of cell types,71 and the large difference between the two indicates that much of the water movement can take place through water-filled pores. Water channels are known as aquaporins and lens epithelial cells contain an abundance of these.73 Interestingly, MIP26 is believed to be a very old (and not very efficient) member of the aquaporin family and is termed aquaporin O. When MIP26 is expressed in oocytes, it does, indeed, give rise to a small increase in water permeability and, significantly, in agreement with some other members of the same family, it also acts as a glycerol transporter.74 MIP26 could be termed the Swiss army knife of lens membrane proteins as it appears to have had a very large number of functions ascribed to it.74 The one role it seems to have escaped so far is of a “membrane chaperone” and its huge abundance in the lens might, indeed, indicate such a function!
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pH REGULATION
The lens must continually and actively extrude protons for two main reasons. The first reason, in common with most cells, is that the lens has a relatively high negative resting potential and thus there is an inwardly directed electrical gradient for mobile positive charge. For example, with a transmembrane potential of -60 mV and an external pH of the humors of approximately 7.3,29 the internal pH (pHi) at equilibrium would be predicted to be 6.2 from the Nernst equation. The measured pHi from a range of mammalian lens cells (including human) is close to 7,28,29,41 and so protons are not in equilibrium. The second important reason arises from the fact that because the lens is largely an anaerobic tissue, there is a continuous production of H+ from lactic acid.

The early studies on pH regulation were performed on amphibian75 and chick lenses76 and both showed that Na+ /H+ exchange was an important regulatory mechanism. However, the conclusions from the two studies differed on the role for HCO3-. In the chick, it appears that HCO3-/Cl- exchange is important, but in the amphibian system, HCO3- appears to exchange with Na+ rather than Cl-. More recent work performed on rat, bovine, and human lenses goes some way to reconcile these differences and has highlighted the importance of the com-plex Na+ -Cl--H+ -HCO3- quadruple exchanger. The most detailed studies have been performed on rat and bovine lenses,66,68,77 but the conclusions (Fig. 3) are directly applicable to the human lens77 (G. Duncan and M.R. Williams, unpublished data). The HCO3-/Cl- exchanger has been excluded since the inwardly directed gradient for chloride would ex-pel HCO3- from the lens and so tend to acidify fur-ther the internal contents. It is important to note that in the situation in which cells are bathed in a bicarbonate-free medium (see Fig. 3B), there is a lessening of pH regulatory powers for two reasons. First, the quadruple exchange is no longer transporting HCO3- into the lens but cycles Cl- instead. Second, the measured buffering capacity (β1) has been greatly reduced, presumably because of the loss of the HCO3-/CO2 buffer system. It is possible to separate the contributions from the different exchangers by manipulating the respective gradients and also by adding either the stilbene derivatives (SITS and DIDS), which inhibit the quadruple exchanger, or amiloride, which inhibits Na+ /H+ exchange.78,79 The very rapid acidification produced on inhibiting either of these systems has been observed in perifused bovine and human lens cells using the trapped-dye (BCECF) technique. Evidence for the lactate transporting system comes from driving the cotransporter in the reverse direction by adding lactate to the medium and in this case a pronounced acidification is observed, which is greatly augmented in the presence of amiloride.68

Fig. 3. Summary of acid-regulating systems present in bovine lens cells in the presence (A) and absence of bicarbonate (B). The original sources for the membrane potential and ironic data are given.68 Note the greater buffering capacity where HCO3- is present and that the quadruple ion exchanger can still operate in the absence of HCO3-. The lactate (L-) cotransporter is the only system that appears to be independent of other ionic gradients.

There is a considerable electrochemical gradient for Na+ to drive two of the coupled exchangers. However, there is no electrochemical gradient available to drive the outward movement of Cl- and, hence, the movements of the various charged species involved in the quadruple exchange system depend entirely on the sodium linkage. In the presence of external HCO3-, bicarbonate ions are distributed above equilibrium and, hence, are required to be actively imported. It appears that the quadruple exchanger represents the major mechanism by which this distribution is achieved. There is no evidence from the pH studies performed on mammalian lens cells and tissues that HCO3- moves across the membrane through channels80 and, indeed, such a movement would lend to drive HCO3- out of the cell down its electrochemical gradient.

INFLUENCE OF pH ON MEMBRANE CONDUCTANCE AND INTERCELLULAR COMMUNICATION

The internal pH of the lens is not greatly changed by an external acidification step of 0.5 unit, indicating both a lack of a proton conductance around normal pHs and also the power of lens pH regulatory mechanisms in general.29 However, the control mechanisms can be swamped by adding a weak-acid species to the medium, especially if this is accompanied by an external acidification. The most widely used technique to change pHi is simply to saturate the perifusing medium with 100% carbon dioxide. This produces a rapid and reversible decrease in pHi and intercellular coupling in a range of tissues,81,82 and the lens is no exception.28,29,83 Interestingly, interfiber communication in the inner core of the lens appears to be insensitive to acidifi-cation.28 In the frog lens, the total conductance changes in a complex manner on exposure to 100% carbon dioxide, but this is because there is both a large decrease in membrane conductance and a significant increase in internal resistance (Ri) calculated from the increase in fast component in the voltage response to a step of current.84 When pHi and internal resistance are measured simultaneously, then a measure of the pH sensitivity of Ri can be obtained. The resistance changes markedly within the range 6.6 to 7.2 and as the resting pH of the frog lens is around 7.2, then small perturbations from resting can produce significant changes in fiber coupling. In the frog lens, there also is a very significant decrease in membrane resistance at low pH, which is probably because of an increase in chloride conductance as the decrease is inhibited by replacing external chloride by the impermeant gluconate ion (Table 1).

 


 

The effects of internal acidification on membrane and communication properties of rat and frog lenses are listed in Table 1. Both respond with a significant depolarization and increase in internal resistance. However, the increase is much less in the rat, and the internal resistance of the human lens behaves in a similar manner to that of the rat when exposed to 100% carbon dioxide (G. Duncan, unpublished observations). Baldo and Mathias85 also concluded that the coupling resistance of the frog was greater than that of the rat, and it is interesting in this respect that although 30% of the lens fiber area in the equatorial cortex of the young rat lens is occupied by gap functions, only 5% of the corresponding area of the young frog lens is so specialized.86 Other factors that may limit the extent to which fibers may become uncoupled are the zones of membrane fusion occurring between fibers. These areas, although limited in extent, would permit an unregulated flow of ions and metabolites.87

The proteins of the lens appear to be stable when the cytoplasm is acidified as no light-scattering changes have been observed on exposing either rat or frog lenses to 100% carbon dioxide, at least for periods of up to 1 hour.29,84

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CONTROL OF LENS CYTOPLASMIC CALCIUM
In mature cortical cataract, although there is a general disruption of the normal internal ionic content,42 an increase in internal calcium appears to have a central role to play.43 Organ-culture studies of bovine and rat lenses previously had shown that increases in lens Na+ , Cl-, and H2O alone were insufficient to produce marked opacification and protein loss. An increase in lens calcium was required for proteolysis and opacification.43,88 These studies appeared to assign a critical role of calpain in the opacification process, but it has been pointed out that data from animal lenses cannot be immediately applied to the human lens as the ratio of calpain to its endogenous inhibitor calpastatin is much lower in the human lens than it is in the rat, for example.89 Very recent studies performed on the organ-cultured human lens indicate that calpain does have a critical role to play in human cortical cataract, since not only was an increase in internal calcium required for protein loss and opacification, but specific cleavage products of vimentin (a known substrate for calpain) were only detected in lenses in which calcium had increased.90

There also is evidence from studies performed on isolated fiber cells that calpain plays a role in the globulization process induced by calcium.91 It is interesting in this context that a localized increase in calcium can also give rise to localized changes in lens morphology.33 It is possible, therefore, that calcium has several roles to play in cataract. An increase in internal calcium can inhibit gap-junction communication and so helps seal off damaged cells.43 However, this would not explain why the spread of damage in the human lens also appears to be restricted in directions at right angles to the morphologic long axis of the fiber. It is possible that globulization or fusion of opposite facing fiber membranes may occur here, requiring a specific regional collapse of the cytoskeleton. In fact, it has been suggested previously that the lens cytoplasm acts as a stabilized gel until calcium enters to destroy this stability.88

Recently, it has become clear that calcium ions also have a more subtle role to play in lens physiology, and a number of calcium cell-signaling receptor systems have been identified in the human lens. These include the G-protein coupled muscarinic, purinergic, and histamine systems92–94 as well as the more familiar TK receptors stimulated by, for example, fibroblast growth factor (FGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF).43 The presence of the muscarinic system is particularly interesting as it has been shown that exposure to drugs that interfere with acetylcholine metabolism increases the risk of cataract formulation in both humans95 and fish.96 Activation of either class of receptors by their respective agonists leads to a release of inositol (1,4,5) trisphosphate (IP3) into the cytoplasm. IP3, in turn, binds with its membrane receptor in the endoplasmic reticulum (ER) and promotes the release of massive amounts of calcium from the ER through the receptor channel into the cell cytoplasm (Fig. 4). Typical calcium cell-signaling oscillations can be observed in human tissue culture cells92 exposed to low concentrations of acetylcholine, and calcium oscillations can then give rise to voltage oscillations that can be observed through the entire lens.25

Fig. 4. G-protein receptor signaling system. On activation by a ligand (such as acetylcholine), GTP is exchanged for GDP and the α subunit of GTP activates phosphophase C, which, in turn, hydrolyses phosphatidylinositol bisphosphate (PIP2) to form IP3. The IP3-activated calcium channel in the membrane releases stored calcium.92 The state of the store is relayed to a plasma membrane calcium channel by an unknown mechanism, and calcium can enter through this “capacitative” route.99 In the lens, this pathway is quite calcium specific.100 Calcium oscillations are possible as calcium is actively pumped into the emergency department from the cytoplasm by an active, thapsigargin-sensitive calcium pump.

The TK receptor system has long been known to play a critical role in lens growth and development.8 Because activation of either G-protein or TK receptors can lead to the release of IP3, there is the possibility of cross-talk between the two systems through the ER. In tissue-cultured rabbit cells, PDGF and ATP both release Ca2+ from ER stores and ATP modulates the growth response induced by PDGF.97 In fact, growth can be inhibited entirely by inactivating the ER system,98 either by thapsigargin, which prevents Ca2+ reuptake, or by caffeine, which inhibits Ca2+ efflux.97 Because PCO is fundamentally a problem of lens cell growth (see the following section), any agent that controls lens cell growth isa likely candidate to prevent the developmentof PCO.

The calcium store also has an important role in controlling calcium entry into the cell via the so-called capacitative entry pathway.99 Emptying the store, whether by activating the G-protein receptor directly or by inhibiting the reuptake mechanism, leads to an entry of calcium. In the lens, this pathway appears to be quite Ca2+ specific, and there is no concomitant Na+ entry, for example.100 Uncontrolled entry through this pathway might well explain why, in some localized human cataracts, there is a pronounced Ca2+ increase with no apparent change in Na+ content.43

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GROWTH FACTOR REGULATION
Cell regulation by growth factors has become a topic of great importance, and our understanding of the role of these factors in the lens has developed considerably over the past 3 decades. Growth factors are traditionally defined as “a large group of polypeptides that share the common property of inducing multiplication both in vivo and in vitro.”101 However, this definition is somewhat limited and perhaps does not encompass the full repertoire of growth factor function and regulation. An alternate definition is “secreted regulatory proteins that control the survival, growth, differentiation, and effector functions of tissue cells.”102 This latter definition seems more appropriate to the lens as the regulation of multiple cellular events is fundamental to normal lens function and disruption of the growth factors that regulate these processes can give rise to pathologic conditions such as cataract.

FIBROBLAST GROWTH FACTOR

The FGF family of growth factors comprises a number of members with molecular weights ranging from 15- to 32-kd heparin-binding, single-chain polypeptides.102,103 The most prominent forms are acidic and basic FGFs (also known as FGF-1 and FGF-2, respectively). A feature of FGF is dependence on heparin or matrix-associated heparin sulfate proteoglycans for receptor interaction, and FGF binds to two classes of sites: a low- and high-affinity form. The low-affinity form is associated with cell- or matrix-associated proteoglycans and is believed to be integral for the recruitment of FGF to the matrix or membrane. High-affinity binding is to the FGF receptor. The binding of heparin induces a conformational change, which enables efficient binding of the ligand to the FGF receptor to occur. There are five types of FGF receptor, and they are all members of the TK receptor family. However, their affinities to the different FGF family members are not equal.104 In addition, complexing with heparin can increase FGF longevity, as FGF is protected from proteolytic degradation.

Basic and acidic FGFs have been detected in embryonic and mature lenses105 and are believed to play a fundamental role in development.106–113 Moreover, basic and acidic FGF distribution in the rat lens parallels the distribution of heparin sulfate proteoglycans, which are integral for their function.105 Basic FGF also is detectable, using enzyme-linked immunosorbent assay (ELISA) techniques in capsular bags after 150 days of culture when maintained in nonsupplemented eagle's minimum essential medium.114 In addition, basic FGF also is secretedinto the culture medium at detectable levels duringearly stages of culture. More important, basic FGF also could be detected in ex vivo preparations.115 Although FGF receptors 1, 2, and 3 appear tobe expressed in lens cells of a variety of spe-cies,110–113,116,117 only FGFR-1 is universally expressed, whereas FGFR-2 appears to be absent from human lens cells.117

When rat lens epithelial explants were exposed to basic FGF, the cells were found to proliferate, migrate, and differentiate in a dose-dependent manner.118 Interestingly, similar events arise in response to acidic FGF, but higher concentrations are required.119 Differences in sensitivity of human primary cultures also were observed, such that 10-fold higher concentrations of acidic FGF were required to achieve similar results to those observed with basic fibroblast growth factor (bFGF).120 Furthermore, Schweigerer and coworkers121 concluded from their studies of primary cultures of bovine lens cells that although mRNAs for both acidic and basic forms were identified, most of the biologic activity present was because of basic FGF.

The FGF family of growth factors obviously contributes greatly to the regulation of the lens, and disruption of this system is likely to increase the risk of cataract formation. Additionally, the multiple roles of FGF suggest it could also play a critical role in the development of PCO after cataract surgery.

EPIDERMAL GROWTH FACTOR

Epidermal growth factor is a low-molecular-weight polypeptide (6045 kd) that has been shown to enhance proliferation of a number of epithelial cell types.122 Initially, EGF exists in a precursor form and is a transmembrane glycoprotein123,124 in which the EGF sequence domain is external to the plasma membrane. However, little is known about the cleavage of the active EGF polypeptide.123,124 EGF is related in sequence and function to a number of other growth factors, including TGF-α, amphireg-ulin, and heparin-binding EGF. These related polypeptides comprise the EGF family.102 These factors mediate their stimulatory effects via the same receptor, which is a 170-kd glycoprotein and is characterized by its ligand-dependent TK.122

EGF has been identified in lens cells of a variety of species using many analytic procedures. Tripathi and coworkers125 examined homogenates of human lenses using a radioimmunoassay and found values to range from zero to 106 pg/mg water-soluble protein. They also suggested that the level of EGF detected showed a qualitative correlation with the degree of cataract formation. Additionally, EGF and EGF receptors have been found in human anterior capsule discs generated at surgery.126,127 Analysis of these preparations by reverse transcriptase polymerase chain reaction (RT-PCR) did not show any alteration in mRNA levels between samples from noncataractous, anterior polar cataract, and nuclear cataract patients. However, other reports suggest trauma to the epithelium can induce an upregulation of EGF receptor,128 and this may prove to be important after cataract surgery. Additional studies have shown EGF and EGFR to be present in rat129 and rabbit epithelium117,130 and in primary cultures of rabbit lens epithelial cells,117 primary human lens cells,117 and in a human lens cell line.131

Application of EGF to cell cultures has been shown to induce proliferation.120,126,130,132–134 In one study, using rabbit lens epithelial cells, concentrations of 0.1 to 100 ng/ml were found to stimulate growth, which was estimated by absorbance of methylene blue staining. However, the maximal response was achieved at 10 ng/ml. This concentration of EGF also was found to be optimal for inducing proliferation of human lens epithelial cells in early subculture.120 This study of human lens epithelial cells also examined the potential of EGF to induce differentiation of lens epithelial cells into fibers. This was assessed by the capacity to form lentoid bodies that express gamma crystallin. Using this system, EGF was a strong promoter of cell aggregation and lentoid body formation. Additionally, application to anterior discs removed at surgery suggested EGF could induce morphologic changes, such that exposure to 10 ng/ml EGF induced fibroblast-like cells to appear.135

HEPATOCYTE GROWTH FACTOR

Hepatocyte growth factor (HGF) is a heterodimeric glycoprotein secreted by cells largely of mesodermal origin.102 The heterodimer is a disulfide-linked heavy subunit A of 60 kd and a light subunit B of 32 kd.136–139 Activation of HGF takes place by a proteolytic cleavage of a single chain 92-kd precursor, and takes place in the extracellular environment.140,141 HGF binds to two classes of sites: low- and high-affinity forms. The low-affinity form is caused by binding to cellular or matrix-linked heparin sulfate proteoglycans and is linked with the loss of HGF/SF, presumably by degradation. The high-affinity binding site for HGF is the receptor met, the product of the proto-oncogene c-met. Met is a 190-kd heterodimer of two disulfide-linked subunits. The α subunit is extracellular, whereas the β subunit bears an extracellular portion involved in ligand binding. The receptor system elicits signaling responses by TK activity.

The first major study of HGF and met in thelens was performed by Weng and coworkers117 usingRT-PCR and western blot techniques. Their results showed that HGF and c-met mRNA were expressed in primary cultures of rabbit and human lens cells generated from young (younger than 2 years) do-nors. In addition, native rabbit epithelium was examined and also was found to express mRNA for both HGF and the receptor. In the case of rabbit cells, they also showed the mRNA was translated into protein. Following on from this work, Fleming and associates131 also identified expression ofmet RNA in the human lens epithelial cell line (HLE-B3), again using RT-PCR. Although RT-PCR and western blotting are powerful techniques for identifying the presence of RNA or protein in populations of cells, they do not provide information concerning distribution, particularly with respect to the receptor, within individual cells or a population. However, other techniques can be used to address these issues. Wormstone and coworkers,142 using immunocytochemistry, have reported the distribution of receptor to be mainly on the basolateral surface of HLE-B3 cells, and furthermore by use of fluorescence-activated cell scanning (FACS) analysis showed that the receptor was expressed in all cells within the population studied. Currently, no investigations have been performed on the lens fibers.

An early study143 into the functional role ofHGF suggested that concentrations below 20 ng/mlcould facilitate a wound-healing response; however, higher concentrations (up to 312 ng/ml) impaired the wound-healing process of the rabbit lens cell line NN1003a. Proliferation and migration play fun-damental roles in the wound-healing process, and HGF has been shown to induce both cell division and migration of lens cells.131,142 Interestingly, human lens cells cultured in unsupplemented EMEM, on their native collagen capsule, have been shown to secrete HGF into the bathing medium.114 Furthermore, HGF also has been detected, using ELISA techniques, in homogenates of cells cultured for 150 days under serum-free conditions.114 Therefore, this indicates a potential autocrine role of HGF in lens cell regulation and growth. Further roles for this growth factor will undoubtedly emerge, and its involvement in growth and wound-healing events could prove important in our understanding of both normal lens regulation and in pathologic conditions.

PLATELET-DERIVED GROWTH FACTOR

Platelet-derived growth factor is a 30-kd homodimer or heterodimer protein consisting of disulfide-bonded A and B polypeptide chains.102 It can therefore exist as AA, BB, or AB isoforms. The receptor system by which PDGF initiates its responses involves two binding proteins: an α subunit (Mr = 120 kd) and a β unit (121 kd). The α subunit of PDGF receptor can bind A and B chains and the β subunit only the B chain of PDGF. On ligand binding, two subunits of any combination dimerize to form the PDGF receptor. Again, like FGF, HGF, and EGF, PDGF also induces phosphorylation of tyrosine moieties on the receptor by activation of an internal TK domain.

Curiously, there appears to be few reports concerning the presence or absence of PDGF in the lens. However, Reneker and Overbeek144 have shown that low levels of PDGF mRNA are present in mouse lenses using in situ hybridization. However, with respect to the PDGF receptor isoforms, a greater body of literature is available. Potts and coworkers145 showed by several techniques that the α subunit was present in the developing chick lens. However, they could not detect the β subunit by RT-PCR or immunolocalization. Interestingly, the distribution of the α subunit alters in the chick throughout development, such that in the embryo, the receptor is expressed evenly across the entire epithelium, whereas in the neonatal, lens receptors are located more to the periphery.145 Furthermore, a similar pattern was observed in the developing mouse lens.144 In these receptor studies on noncultured material, the suggested receptor type is PDGFR-α. However, in cultured lens epithelial cells, the predominant isoform is PDGFR-β.146,131 This variation may be caused by species differences or alternatively because of the culture conditions, which have been shown to modify the expression of other receptor subtypes.147

PDGF has been reported to stimulate growth, measured by soluble protein content, in organ-cultured rat lenses, when applied in a pulsed manner. However, when PDGF was constantly exposed to the cultured lenses, a decreased growth was observed. Moreover, the pulsed application of PDGF aided the maintenance of lens clarity.148 In a mouse transgenic system, overexpression of PDGF-A increases the level of dividing cells, detected by BrdU labeling, throughout the epithelium.144 Interestingly, Knorr and associates146 did not observe any increase in proliferation of bovine lens epithelial cells after constant and pulsed exposure to all three isoforms of PDGF in a serum-free medium at concentrations of 5 to 50 ng/ml. However, Duncan and coworkers,97 using the rabbit lens cell line NN1003a cells, did observe PDGF-induced growth after 24 hours' exposure in a serum-free medium. However, this effect was only observed after applications of 100 and 500 ng/ml. Furthermore, Fleming and associates131 also showed 50 ng/ml PDGF could stimulate growth of HLE-B3 cells cultured in the presence of 1% serum. Additionally, PDGF has been shown to give a synergistic response when applied with insulin or EGF.149

Application of PDGF at 5 to 50 ng/ml to bovine lens epithelial cells invoked dose-dependent increases of the intracellular calcium level, with the maximum response being threefold higher than resting levels. Such effects have subsequently been observed in lens epithelial cells of other species.97,131 Additionally, Fleming and coworkers131 showed tyrosine phosphorylation after PDGF addition in HLE-B3 cells. Because PDGF induces an increase in cytosolic calcium in calcium-free medium, it appears that calcium is mobilized from internal calcium stores.146

PDGF alone is unlikely to initiate functional responses of lens cells in vivo; however, it is more likely to act as a competence factor, subsequently facilitating the actions of other agents on the lens. Alteration to PDGF levels or indeed any other agent that is influenced by PDGF in the eye may have potential pathogenic consequences to the lens.

TRANSFORMING GROWTH FACTOR β

The TGF-β family is comprised of homodimeric polypeptides that regulate many aspects of cellular function, including cell growth, differentiation, inflammation, and wound healing.150–152 Unlike many other growth factors that mediate their response via TK receptors, TGF-β initiates its response through cell surface serine/threonine kinase receptors, of which there are three major high-affinity forms.153 Although five members of the TGF-β family have currently been identified, only TGF-β1, -β2, and -β3 isoforms have been detected in mammals.152 TGF-β exists in both a latent and active form. ActiveTGF-β is likely to be released from the latent form via degradation of prosegments. This process may be performed by proteases such as plasmin and the cathepsins.154 It is believed that activation occursat the cell surface and is possibly caused by prosegments binding to the mannose 6-phosphate receptor, enabling proteolytic cleavage to ensue.155 In fact, mannose-6-phosphate has been used as an in vitro tool to negate TGF-β function.156 Additionally, the activity of TGF-β also can be regulated by proteins in surrounding fluids, such as the humors of the eye. In particular, α2-macroglobulin has a high affinity to TGF-β and may serve as a scavenger of free active TGF-β.157,158

TGF-β isoforms have been detected in mouse, rat, and human lens cells.117,127,159,160 In the embryonic mouse, TGF-β1, -β2, and -β3 were all identified in the lens fibers, but did not appear to be present in the epithelium.159 However, Gordon-Thomson and associates160 performed an investigation using rat lens tissue and probed for both protein and mRNA of TGF-β1, -β2, and -β3 using immunofluorescence and in situ hybridization, respectively. Interestingly, although all three isoforms of the protein were identified in the lens, only mRNA for TGF-β1 and -β2 was detectable. With respect to human lens cells, TGF-β1 and -β2 have been detected in primary cultures using RT-PCR techniques and in situ hybridization.117,127 In order to begin to understand theregulation and putative roles of TGF-β in lens cells, it is necessary to know the expression of receptors. Of the three major TGF-β receptor types, types I and II have been detected in chick lens cells using western blot techniques161 and, additionally, using RT-PCR techniques in a human lens cell line,131 in primary cells,117 and in native epithelium.127 This evidence infers that TGF-β receptors are expressed in lens cells; however, the techniques utilized to determine this fact do not permit the distribution to be identified. This information could be of great use in the future to understand the regulation and functioning capabilities of TGF-β in the lens.

Although TGF-β has been implicated in the regulation of lens cell proliferation, exposure of lens cells to TGF-β has been shown to generate inconsistent responses. Avian lens epithelial cells show increased rates of proliferation in the presence of TGF-β, and interestingly this response was enhanced when the cells were cultured on extracellular matrix (ECM) components.161 These TGF-β invoked responses contrast with those observed in lens cells of mammalian origin, which show reduced cell division after exposure to TGF-β.162,163 Indeed, some studies have suggested that active TGF-β in the aqueous humor may maintain the low mitotic activity of the lens160,164 and that a reduction of active TGF-β levels may contribute to increased rates of lens cell proliferation.164

Although TGF-β is likely to play an important role in the normal lens, it is becoming increasingly obvious that it can greatly influence transdifferentiation, which is proposed to be involved in certain pathologic conditions of the lens. This is perhaps best illustrated in the formation of subcapsular plaques. In one study,165 transgenic mice were generated by microinjection of a construct containing self-activating human TGF-β1 cDNA driven by thelens-specific alpha-crystallin promoter. Using this overexpression system, focal plaques formed that showed cells of spindle-shape morphology that exhibited expression of alpha smooth muscle actin, a transdifferentiation marker. These focal sites resembled plaques seen in human anterior subcapsular cataracts. Furthermore, TGF-β has been detected in anterior subcapsular cataracts of dogs using immunohistochemistry; however, the isoforms were not determined.166 Additionally, cultured rat lenses also have been found to generate capsular plaques in response to TGF-β exposure. In this study, the effects of TGF-β1, -β2, and -β3 were investigated, and the results showed that TGF-β2 and -β3 had a 10-fold greater potency to induce opacity.160 Interestingly, it also is reported that estrogen can protect against TGF-β-induced opacity167 and therefore may prove beneficial to women undergoing hormone replacement therapy. Further studies on rabbit and bovine lens culture models showed that after exposure to TGF-β1, an increased expression of fibronectin, type I collagen, and alpha smooth muscle actin was observed.127 The work performed by Gordon-Thomson and coworkers160 has now been advanced, such that TGF-β2 has been shown to induce plaques after intravitreous injection.168 Although there are TGF-β scavengers in the vitreous humor, most notably α2-macroglobulin,169 abnormal elevation of TGF-β in the vitreous humor could saturate these systems and therefore potentially give rise to subcapsular cataract formation. Such elevations could develop as a result of other ocular conditions such as retinopathies. At the human level, anterior epithelium generated by capsulorhexis has been studied and compared.127 In this study, the samples were separated into two groups based on whether the patient had a nuclear or anterior subcapsular cataract. These epithelia samples were subsequently analyzed using RT-PCR and mRNA levels of TGF-β1 and -β2 and TGF-β receptor II were higher in anterior epithelium removed from anterior subcapsular cataract patients than in those from nuclear cataract patients. The transdifferentiation of lens epithelial cells to fibroblast-like cells also is proposed to increase contractility,163,169 and this phenomenon in addition to plaque formation could be integral to the pathogenesis of PCO, which gives rise to visual impairment after cataract surgery.

Elucidation of the regulatory mechanisms of TGF-β, both in and around the lens, would greatly enhance our knowledge of the normal lens and the development of clinically important pathologies.

OTHER GROWTH FACTORS

In this section, we have earlier highlighted a handful of growth factors that seem to play fundamental roles in the regulation of lens cells. However, it should not be forgotten that this field is extensive, and other factors that we cannot discuss in such detail also may contribute to physiologic regulation of the lens.

Insulin and insulin-like growth factor (IGF) have been reported to stimulate proliferation of mam-malian lens cells.120,170 Additionally, application of either of these factors can promote lens fiber formation in the chick, rat, and human.120,171–173 Furthermore, the effects of insulin and IGF can act synergistically with other growth factors such as PDGF, EGF, and FGF.149,171

Transferrin is an iron-binding protein that has been identified as one of the serum factors essential for growth in tissue culture.174 Transferrin makes up a large component of the soluble protein in the ocular fluids, and it has been shown that transferrin is synthesized and secreted by both rabbit and human lens cells.175,176 Furthermore, transferrin has been shown to act as a survival factor and is upregulated after cataract surgery.176

FACTORS IN THE AQUEOUS HUMOR

Apart from producing their own growth factors,114,176 lens cells are subject to the influence of paracrine factors from the surrounding humors. Not surprisingly, growth factors have been detected in the aqueous humor in a variety of species. Growth factors detected in the aqueous include FGF,177 HGF,178 EGF,179,180 TGF-β,181 and transferrin.182,175 However, some reports suggest that certain factors are not naturally abundant in the aqueous humor and can only be observed under pathologic conditions or trauma.179,180,183 The relationship between the paracrine and autocrine factors in the regulation of lens physiology is likely to be critical in the maintenance of lens clarity.

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EXTRACELLULAR MATRIX
The importance of the lens capsule is now becoming more evident. Recently, it has been shown that lens epithelial cells residing on their natural matrix cannot only survive but also proliferate and migrate when cultured in protein-free media.10,184 Human lens epithelial cells maintain the capacity to undergo protein synthesis and still express lens cell phenotypic markers after long-term culture.115 It would therefore appear that lens cells growing on their native substratum synthesize the necessary factors required to maintain cell survival and growth functions.114,176

The lens capsule is composed of a number of elements including collagen type IV, laminin, and fibronectin.185–188 Oharazawa and coworkers189 investigated the role of these particular ECM components in cell adhesion, proliferation, and migration of a human lens cell line. Cell attachment was found to be enhanced significantly when cells were seeded onto matrix-coated dishes as opposed to noncoated dishes; however, no difference among the three ECM components tested was observed. Additionally, the matrix components did not enhance proliferation but did stimulate migration. However, it should be noted that a decrease in the overall cell number was observed when cultured on collagen and fibronectin matrices. In contrast, when cultured on laminin, cell numbers increased. This result agrees with those results of previous reports that suggest laminin is critical for cell growth. Previously, it has been reported that EGF could not initi-ate growth of bovine lens epithelial cells ontononcoated culture dishes, but could promotegrowth onto laminin. Furthermore, Parmigiani andMcAvoy187 showed that cells from all ages of rat would grow onto laminin, whereas growth onto fibronectin was age-dependent.

Lens epithelial cells are linked to the underlying matrix by cell adhesion molecules such as integrins. Reports have shown the presence of α integrin subunits in both the embryonic and mature lens.190–193 In the embryonic lens, α3 and α6 integrins were detected using immunoprecipitation followed by western blot analysis. However, the presence of α1, α5, and αV integrins was not detected. In the embryonic system, α3 is strongly associated with the epithelium and α6 with the fibers. The association of α6 with the fibers was along their lateral borders, the site of attachment to the capsule, and at their interface with epithelial cells. Interestingly, immunohistochemical examination of human anterior disc specimens removed at surgery indicated α2, α3, and α5 integrins were present.193 With respect to β integrins, the β1 and β4 subunits have been identified in the embryonic lens,190,192 whereas Nishi and coworkers191 also have confirmed the presence of β1 integrin in the adult human; however, they did not identify β2 and β3 integrins. In contrast, another group has reported that integrin β2 is present in similar specimens.193 Furthermore, the importance of β1 integrin has been validated by the addition of a monoclonal antibody. Using this tool, the number of viable cells remaining after 2 weeks of culture on collagen or laminin-coated dishes was reduced by the antibody in a dose-dependent manner.191

Another interesting family of molecules that could regulate the lens capsule is the matrixmetalloproteinases (MMPs), a family of zinc-endopeptidases capable of degrading ECM components. These enzymes are involved in normal physiologic processes, such as morphogenesis, but also have been implicated in pathologic conditions such as tumor metastasis.196,197 The most widely studied members of the MMP family in the eye are the gelatinases, and these are found in both the aqueous and vitreous humors.

Despite the growing evidence to suggest gelatinases play an important role in normal and pathologic conditions of ocular tissues, little is known of their role within the lens. Smine and Plantner196 could not detect MMP-2 (gelatinase A) in the lens from postmortem human eyes, whereas MMP-9 was not investigated. However, MT1-MMP was identified. This member of the MMP family has been shown to convert pro-MMP-2 to its metabolically active form.197 However, MT1-MMP also can perform other functions independent of gelatinase A.198,199 Richiert and Ireland200 have shown that chicken lens anular pad cells cultured on plastic, collagen type IV, fibronectin, or laminin do not express MMP-2 or MMP-9 unless TGF-β or PDGF is added to the culture medium. It would therefore appear that lens cells are capable of synthesizing MMP-2 and MMP-9, but seemingly require stimuli to induce expression. Such factors could be generated on stress or injury to the lens.

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ACKNOWLEDGMENTS
We thank BBSRC, the Humane Research Trust, and NEI (EY 10558) for support during the preparation of this chapter. We also thank Drs. Ken Hightower, Mark Williams, and Julia Marcantonio for many stimulating discussions.
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