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.

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.

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 colleagues^23,24 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.

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

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 (E_m) ionic data:

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

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.

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

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 pH_i is simply to saturate the perifusing medium with 100% carbon dioxide. This produces a rapid and reversible decrease in pH_i 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 (R_i) calculated from the increase in fast component in the voltage response to a step of current.⁸⁴ When pH_i and internal resistance are measured simultaneously, then a measure of the pH sensitivity of R_i 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

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 systems^92–94 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

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

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 glycoprotein^123,124 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 epithelium^117,130 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 lens^160,164 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.

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.

1. Duncan G: Brewster's contribution to the study of the lens of the eye. In Morrison-Low AD, Christie JRR (eds): Martyr of Science: Sir David Brewster 1781-1868. Edinburgh: Royal Scottish Museum, 1984:101

2. Brewster D: On the anatomical and optical structure of the crystalline lenses of animals, particularly that of the cod. Phil Trans Roy Soc Lond 123:323, 1833

3. Brewster D: On the cause and cure of cataract. Trans Roy Soc Edin 24: 11, 1865

4. Bassnett S, Missey H, Vucemilo I: Molecular architecture of the lens fiber-cell basal membrane complex. J Cell Sci 112:2155, 1999

5. Mathias RT, Rae JL, Balado GJ: Physiological properties of the normal lens. Physiol Revs 77:21, 1997

6. White TW, Goodenough DA, Paul DL: Targeted ablation of connexin 50 in mice results in microphthalmia and zonular pulverulent cataracts. J Cell Biol 143:815, 1998

7. Duncan G, Wormstone IM, Davies PD: The aging human lens: Structure, growth and physiological behaviour. Br J Ophthalmol 81:907, 1997

8. Reid TW: Growth control of cornea and lens epithelial cells. In Osborne N, Chader GJ (eds): Retinal and Eye Research. Vol 13. Oxford, UK: Elsevier, 1994:507

9. Liu CSC, Wormstone IM, Duncan G et al: A study of human lens cell growth in vitro: A model for posterior capsule opacification. Invest Ophthalmol Vis Sci 37:906, 1996

10. Wormstone IM, Liu CSC, Rakic JM et al: Human lens epithelial cell proliferation in a protein-free medium. Invest Ophthalmol Vis Sci 38:396, 1997

11. Kinsey VE, Reddy DVN: Studies of the crystalline lens xi. The relative role of the epithelium and capsule in transport. Invest Ophthalmol Vis Sci 4:104, 1965

12. Candia OA, Bentley PJ, Mills CD: Short-circuit current and active Na transport across isolated lens of the toad. Am J Physiol 220:539, 1971

13. Parmelee JT: Measurement of steady currents around the frog lens. Exp Eye Res 42:433, 1986

14. Patterson JW: Characterisation of the equatorial current of the lens. Ophthalmol Res 20:139, 1988

15. Duncan G: The site of the ion restricting membranes in the toad lens. Exp Eye Res 8:406, 1969

16. Duncan G, Stewart S, Prescott AR et al: Membrane and junctional properties of the isolated frog lens epithelium. J Membrane Biol 102:195, 1988

17. Prescott A, Duncan G, van Marle J et al: The correlated study of metabolic cell communication and gap junction distribution in adult frog lens. Exp Eye Res 58:737, 1994

18. Duncan G, Juett JR, Croghan PC: A simple chamber for measuring lens asymmetry potentials. Exp Eye Res 25:391, 1977

19. Musil LS, Beyer EC, Goodenough DA: Expression of the gap junction protein connexin 43 in embryonic chick lens: Molecular cloning, ultrastructural localization and post-translational phosphorylation. J Membrane Biol 116:163, 1990

20. Bassnett S, Kuszak JR, Reinisch HG et al: Intercellular communication between epithelial and fiber cells of the eye lens. J Cell Sci 107:799, 1994

21. Rae JL, Bartling J, Rae J et al: Dye transfer between cells of the lens. J Membrane Biol 150:89, 1996

22. Jacob TJC, Duncan G: Electrical coupling between fiber cells in amphibian and cephalopod lenses. Nature 290:704, 1981

23. Eisenberg RS, Rae JL: Current voltage relationships in the crystalline lens. J Physiol 262:285, 1976

24. Mathias RT, Rae JL, Eisenberg RS: The lens as a nonuniform syncytium. Biophys J 34:61, 1981

25. Thomas GR, Duncan G, Sanderson J: Acetylcholine-induced membrane potential oscillations in the intact lens. Invest Ophthalmol Vis Sci 39:111, 1998

26. Alvarez LJ, Candia OA, Zamudio AC: Acetylcholine modulation of the short-circuit current across the rabbit lens. Exp Eye Res 61:129, 1995

27. Gandolfi SA, Duncan G, Tomlinson J et al: Mammalian lens inter-fiber resistance is modulated by calcium and calmodulin. Curr Eye Res 9:533, 1990

28. Mathias RT, Riquellme G, Rae JL: Cell to cell communication and pH in the frog lens. J Gen Physiol 98:1085, 1991

29. Bassnett S, Duncan G: The influence of pH on membrane conductance and intercellular resistance in the rat lens.J Physiol 398:507, 1988

30. Shestopalor VI, Bassnett S: Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core. J Cell Sci 113:1913, 2000

31. Duncan G: Role of membranes in controlling ion and water movements in the lens. In The Human Lens in Relation to Cataract. Ciba Symposium 19. London: Pitman, 1973:99

32. Broekhuyse RM: Biochemistry of membranes. In Duncan G (ed): Mechanisms of Cataract Formation in the Human Lens. London: Academic Press, 1981:151

33. Duncan G, Jacob TJC: Calcium and the physiology of cataract. In Human Cataract Formation. Ciba Foundation Symposium 106. London: Pitman, 1984:132

34. Rae JL, Kuszak JR: The electrical coupling of epithelium and fibers in the frog lens. Exp Eye Res 36:317, 1983

35. Menko AS, Klunks KA, Tai-Feng L et al: Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability and junctional protein expression. Dev Biol 123:307, 1987

36. Fitzgerald PG, Goodenough DA: Rat lens cultures: MIP expression and domains of intercellular coupling. Invest Ophthalmol Vis Sci 27:755, 1986

37. Miller TM, Goodenough DA: Evidence for two physiologically distinct gap junctions expressed by the chick lens epithelial cell. J Cell Biol 102:194, 1986

38. Vrensen G, van Marle J, van Veen H et al: Membrane architecture as a function of lens fiber maturation: A freeze fracture and scanning electron microscopic study of the human lens. Exp Eye Res 54:433, 1992

39. Rae JL, Shepard AR: Molecular biology electrophysiology of calcium-activated potassium channels from lens epithelium. Curr Eye Res 17:264, 1998

40. Shepard AR, Rae JL: Ion transporters and receptors in cDNA libraries from lens and cornea epithelia. Curr Eye Res 17:708, 1998

41. Stewart S, Duncan G, Marcantonio JM et al: Membrane and communication properties of tissue cultured human lens epithelial cells. Invest Ophthalmol Vis Sci 29:1713, 1988

42. Duncan G, Bushell AR: Ion analysis of human cataractous lenses. Exp Eye Res 20:223, 1975

43. Duncan G, Williams MR, Riach RA: Calcium, cell sig-nalling and cataract. In Osborne NN, Chader GJ (eds):Progress in Retina and Eye Research. Oxford, UK:Pergamon 1994:623

44. Goldman DE: Potential, impedance and rectification in membranes. J Gen Physiol 27:37, 1943

45. Duncan G: Relative permeability of the lens membranes to sodium and potassium. Exp Eye Res 8:315, 1969

46. Tomlinson J, Bannister SC, Croghan PC et al: Analysis of rat lens⁴⁵Ca²⁺ fluxes: Evidence for Na⁺ -Ca²⁺ exchange. Exp Eye Res 52:619, 1991

47. Delamere NA, Dean WL: Distribution of lens sodium-potassium-adenosine triphosphatase. Invest Ophthalmol Vis Sci 34:2159, 1993

48. Sweadner KJ: Isozymes of the Na⁺ /K⁺ ATPase. Biochim Biophys Acta 988:185, 1989

49. Moseley AE, Dean WL, Delamere NA: Isoforms of NaK ATPase in rat lens epithelium and fiber cells. Invest Ophthalmol Vis Sci 37:1502, 1996

50. Garner MH, Horwitz J: Catalytic subunit isoforms of mammalian lens NaK ATPase. Curr Eye Res 13:65, 1994

51. Delamere NA, Dean WL, Stidam JM et al: Differential expression of sodium pump catalytic subunits in the lens epithelium and fibers. Ophthalmic Res 28:73, 1996

52. Delamere NA, Manning RE, Liu Lx et al: Na,K-ATPase polypeptide upregulation responses in lens epithelium. Invest Ophthalmol Vis Sci 39:763, 1998

53. Okafor MC, Dean WL, Delamere NA: Thrombin inhibits active sodium-potassium transport in porcine lens. Invest Ophthalmol Vis Sci 40:2033, 1999

54. Duncan G, Hightower KR, Gandolfi SA et al: Human lens membrane cation permeability increases with age. Invest Ophthalmol Vis Sci 30:1855, 1989

55. Jacob TJC, Bangham JA, Duncan G: Characterization of a cation channel on the apical surface of the frog lens epithelium. Q J Exp Physiol 70:403, 1985

56. Rae JL, Mathias RT, Cooper K et al: Divalent cation effects on lens conductance and stretch-activated action channels. Exp Eye Res 55:135, 1992

57. Weale RA: Physical changes due to age and cataract. In Duncan G (ed): Mechanisms of Cataract Formation in the Human Lens. London: Academic Press, 1981:47

58. Sanderson J, Duncan G: pCMPS-induced changes in lens membrane permeability and transparency. Invest Ophthalmol Vis Sci 34:2518, 1993

59. Harding JJ, Crabbe MJC: The lens: Development, proteins, metabolism and cataract. In Davison H (ed): The Eye. Vol 1B. New York: Academic Press, 1984:207

60. Lucas VA, Duncan G, Davies PD: Membrane permeability characteristics of perifused human senile cataractous lenses. Exp Eye Res 42:151, 1986

61. Jacob TJC, Duncan G: Calcium controls both sodium and potassium permeability of lens membranes. Exp Eye Res 33:85, 1981

62. Duncan G, Patmore L, Pynsent PB: Impedance of the amphibian lens. J Physiol 312:17, 1981

63. Duncan G, Emptage NJ, Hightower KR: p-Chloro-mercuriphenylsulphonate activates a quinine-sensitive potassium conductance in frog lens. J Physiol 404:637, 1988

64. Lucas VA, Bassnett S, Duncan G et al: Membrane conductance and potassium permeability of the rat lens. Q J Exp Physiol 72:81, 1987

65. Ballabeni A, Trevisi A, Chiaponi C et al: Molecular characterization of the potassium channels of transparent and cataractous human lenses (submitted)

66. Duncan G, Dart C, Croghan PC et al: Evidence for a Na⁺ -Cl--H⁺ -HCO3- exchanger system in the mammalian lens. Exp Eye Res 54:941, 1992

67. Duncan G: Movement of sodium and chloride across amphibian lens membranes. Exp Eye Res 10:117, 1970

68. Williams MR, Duncan G, Croghan PC et al: pH regulation in tissue-cultured bovine lens epithelial cells. J Membrane Biol 129:179, 1992

69. Diecke FPJ, Beyer-Mears A: A mechanism for regulatory volume decrease in cultured lens epithelial cells. Curr Eye Res 16:279, 1997

70. Zhang JJ, Jacob TJC: ATP-activated chloride channel inhibited by an antibody to p-glycoprotein. Am J Physiol 36:C1095, 1994

71. Duncan G: Permeability of amphibian lens membranes to water. Exp Eye Res 9:188, 1970

72. Duncan G, Croghan PC: Mechanisms for the regulation of cell volume with particular reference to the lens. Exp Eye Res 8:421, 1969

73. Hasegawa H, Lian SC, Finkberner WE et al: Extrarenal tissue distribution of CHIP 28 water channels: In situhybridization and antibody staining. Am J Physiol 266:C893, 1994

74. Kushmerick C, Rice SJ, Baldo GJ et al: Ion, water and neutral solute transport in Xenopus oocytes expressing frog lens MIP. Exp Eye Res 61:351, 1995

75. Wolosin JM, Alvarez LJ, Candia OA: Cellular pH andNa⁺ -H⁺ exchange activity in lens epithelium of Bufo marinus toad. Am J Physiol 255:C595, 1988

76. Bassnett S: Intracellular pH regulation in the embryonic chicken lens epithelium. J Physiol 431:445, 1990

77. Williams MR: pH and Calcium Regulation in Lens Epithelial Cells: A Fluorimetric Dye Study. PhD Thesis, University of East Anglia, Norwich, UK, 1993

78. Thomas RC: Experimental displacement of intracellular pH and mechanisms of its subsequent recovery. J Physiol 354:3P, 1984

79. Thomas RC: Bicarbonate and pH_i response. Nature 337:601, 1989

80. Jentsch TK, von der Haar B, Keller SK et al: Response of the intracellular potentials of cultured bovine lens cells to ions and inhibitors. Exp Eye Res 41:131, 1985

81. Turin L, Warner AE: Intracellular pH in early Xenopus embryos: Its effect on current flow between blastomeres.J Physiol 300:489, 1980

82. Spray DC, Harris AL, Bennett MVL: Gap junctional conductance as a simple and sensitive function of intracellular pH. Science 211:712, 1981

83. Schutze SM, Goodenough DA: Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO₂ treatment with development. J Cell Biol 92:694, 1982

84. Emptage NJ, Duncan G, Croghan PC: Internal acidification modulates membrane and junctional resistance in the isolated lens of the frog Rana pipiens. Exp Eye Res 54:33, 1992

85. Baldo GJ, Mathias RT: Spatial variations in membrane properties in the intact rat lens. Biophys J 63:518, 1992

86. Kuszak JR, Shek YH, Carney KC et al: A correlative freeze-etch and electrophysiological study of communicating junctions in crystalline lenses. Curr Eye Res 4:1145, 1985

87. Kuszak JR, Macsai MS, Bloom KL et al: Cell to cell fusion of lens fiber cells in situ: Correlative light, scanning electron microscopic and freeze-fracture studies. J Ultrastruct Res 93:144, 1985

88. Marcantonio JM, Duncan G, Rink H: Calcium-induced opacification and loss of protein in the organ-cultured bovine lens. Exp Eye Res 42:617, 1986

89. David LL, Shearer TR: Role of proteolysis in lenses: A review. Lens Eye Toxic Res 6:725, 1989

90. Sanderson J, Marcantonio JM, Duncan G: A human lens model of cortical cataract: Calcium induces protein loss, vimentin cleavage and opacification in cultured human lens. Invest Ophthalmol Vis Sci 41:2251, 2000

91. Srivastava SK, Wang L-F, Ansari NH et al: Calcium homeostasis of isolated single cortical fibers of rat lens. Invest Ophthalmol Vis Sci 38:2300, 1997

92. Williams MR, Duncan G, Riach RA et al: Acetylcholine receptors are coupled to mobilization of intracellular calcium in cultured human lens cells. Exp Eye Res 57:381, 1993

93. Riach RA, Duncan G, Williams MR et al: Histamine and ATP mobilize calcium by activation of H₁ and P_2u receptors in human lens epithelial cells. J Physiol 486:273, 1995

94. Churchill GC, Louis CF: Roles of Ca²⁺ inositol trisphosphate and cyclic ADP-ribose in mediating intercellular Ca²⁺ signalling in sheep lens cells. J Cell Sci 111:1217, 1998

95. Shaffer RN, Hethrington J: Anticholinesterase drugs and cataract. Am J Ophthalmol 64:613, 1966

96. Fraser PJ, Duncan G, Tomlinson J: Effects of a cholinesterase inhibitor on salmon lens: A possible cause for the increased incidence of cataract in salmon Salmo solar. Exp Eye Res 49:293, 1989

97. Duncan G, Riach RA, Williams MR et al: Calcium mobilization modulates growth of lens cells. Cell Calcium 19:83, 1996

98. Duncan G, Wormstone IM, Liu CSC et al: Thapsigargin-coated intraocular lenses inhibit lens cell growth. Nat Med 3:1026, 1997

99. Berridge MJ: Elementary and global aspects of calcium signalling. J Physiol 499:291, 1997

100. Thomas GR, Sanderson J, Duncan G: Thapsigargin inhibits a potassium conductance and stimulates calcium influx in the intact rat lens. J Physiol 516:191, 1999

101. Heath JK: Growth Factors. Oxford, UK: Oxford University Press, 1993

102. Nicola NN: Cytokines and Their Receptors. Oxford, UK: Oxford University Press, 1994

103. Burgess WH, McCiag T: Heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem 58:575, 1989

104. Partanen J, Vainikka S, Alitalo K: Structural and functional specificity of FGF receptors. Phil Trans R Soc Lond 340:297, 1993

105. Lovicu FJ, McAvoy JW: Localization of acidic fibroblast growth factor, basic fibroblast growth factor, and heparan sulphate proteoglycan in rat lens: Implications for polarity. Invest Ophthalmol Vis Sci 34:3355, 1993

106. Robinson ML, Overbeek PA, Verran DJ et al: Extracellular FGF-1 acts as a lens differentiation factor in transgenic mice. Development 121:505, 1995

107. Chow RL, Roux GD, Roghani M et al: FGF suppresses apoptosis and induces differentiation of fibre cells in the mouse lens. Development 121:4383, 1995

108. Stolen CM, Jackson MW, Griep AE: Overexpression of FGF-2 modulates fiber differentiation and survival in mouse lens. Development 124:4009, 1997

109. Lovicu FJ, Overbeek PA: Overlapping effects of different members of the FGF family on lens fiber differentiation in transgenic mice. Development 125:3365, 1998

110. De Iongh RU, Lovicu FJ, Chamberlain CG et al: Differential expression of fibroblast growth factor receptors during rat lens morphogenesis and growth. Invest Ophthalmol Vis Sci 38:1688, 1997

111. Del Rio-Tsonis K, Jung JC, Chiu I-M et al: Conservation of fibroblast growth factor function in lens regeneration. Proc Natl Acad Sci U S A 94:13701, 1997

112. Del Rio-Tsonis K, Trombley MT, McMahon G et al: Regulation of lens regeneration by fibroblast growth factor 1. Dev Dynamics 213:140, 1998

113. Ohuchi H, Koyama E, Myokai F et al: Expression pattern of two fibroblast growth factor receptor genes during chick eye development. Exp Eye Res 58:649, 1994

114. Wormstone IM, Duncan G, Liu CSC et al: Persistent lens cell activity throughout long-term culture of human capsular bags in protein-free medium. Invest Ophthalmol Vis Sci 39:S212, 1998

115. Wormstone IM, Del Rio-Tsonis K, McMahon G et al: Autocrine regulation and age-related expression of FGF in human lens cells. Invest Ophthalmol Vis Sci 41:S188, 2000

116. Marunouchi T, Hosoya H, Morioku T et al: Up-regulation of fibroblast growth factor receptor-1 in lens epithelial cells paralleled by growth stimulation. Exp Eye Res 67:611, 1998

117. Weng J, Liang Q, Mohan RR et al: Hepatocyte growth factor, Keratinocyte growth factor, and other growth factor-receptor systems in the lens. Invest Ophthalmol Vis Sci 38:1543, 1997

118. McAvoy JW, Chamberlain CG: Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration. Development 107:221, 1989

119. Chamberlain CG, McAvoy JW: Evidence that fibroblast growth factor promotes lens fibre differentiation. Curr Eye Res 6:1165, 1987

120. Ibaraki N, Lin LR, Reddy VN: Effects of growth-factors on proliferation and differentiation in human lens epithelial-cells in early subculture. Invest Ophthal Vis Sci 36:2304, 1995

121. Schweigerer L, Ferrara N, Haaparanta T et al: Basic fibroblast growth factor: Expression in cultured cells derived from corneal endothelium and lens epithelium. Exp Eye Res 46:71, 1988

122. Carpenter G, Wahl MI: The epidermal growth factor family. Handb Exp Pharmacol 95:69, 1990

123. Massague J, Pandiella A: Membrane-anchored growth factors. Annu Rev Biochem 62:515, 1993

124. Dernyck R: The physiology of transforming growth factor alpha. Adv Cancer Res 94:191, 1992

125. Tripathi RC, Borisuth NSC, Tripathi BJ et al: Radioimmunoassay of epidermal growth factor in lenses at various stages of development of cataract. Exp Eye Res 53:759, 1991

126. Majima K: Human lens epithelial cells proliferate in response to exogenous EGF and have EGF and EGF receptor. Ophthalmic Res 27:356, 1995

127. Lee EH, Joo C: Role of transforming growth factor-β in transdifferentiation and fibrosis of lens epithelial cells. Invest Ophthalmol Vis Sci 40:2025, 1999

128. Majima K, Kojima Y, Ouhashi F: Cell biological analysis with respect to cause of fibrous opacification of the anterior capsule after cataract extraction. Ophthalmologica 212:364, 1998

129. Fu SCJ, Zheng DR, Wang SL et al: Immunocytochemical localization of epidermal growth-factor (EGF) and its receptor (EGF-R) in rat lens epithelium. Invest Ophthalmol Vis Sci 34:1371, 1993

130. Hongo M, Itoi M, Yamamura Y et al: Distribution of epidermal growth-factor receptors in rabbit lens epithelial-cells. Invest Ophthalmol Vis Sci 34:401, 1993

131. Fleming TP, Song Z, Andley UP: Expression of growth control and differentiation genes in human lens epithelial cells with extended life span. Invest Ophthalmol Vis Sci 39:1387, 1998

132. Hollenberg MD: Receptors for insulin and epidermal growth factor: Relation to synthesis of DNA in cultured rabbit lens epithelium. Arch Biochem Biophys 171:371, 1975

133. Reddan JR, Wilsond-Ziedzic D: Insulin growth-factor and epidermal growth-factor trigger mitosis in lenses cultured in a serum-free medium. Invest Ophthalmol Vis Sci 24:409, 1983

134. Ohguro N, Fukuda M, Sasabe T et al: Concentration dependent effects of hydrogen peroxide on lens epithelial cells. Br J Ophthalmol 83:1064, 1999

135. Majima K, Kojima Y, Ouhashi F: Cell biological analysis with respect to cause of fibrous opacification of the anterior capsule after cataract extraction. Ophthalmologica 212:364, 1998

136. Miyazawa K, Tsubouchi H, Naka D et al: Molecular-cloning and sequence-analysis of cDNA for human hepatocyte growth-factor. Biochem Bioph Res Com 163:967, 1989

137. Nakamura T, Nishizawa T, Hagiya M et al: Molecular-cloning and expression of human hepatocyte growth-factor. Nature 342:440, 1989

138. Weidner KM, Arakaki N, Hartmann G et al: Evidence for the identity of human scatter factor and human hepatocyte growth-factor. Proc Natl Acad Sci USA 88:7001, 1991

139. Rubin JS, Chan AML, Bottaro DP et al: A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth-factor. Proc Natl Acad Sci USA 88:415, 1991

140. Naka D, Ishii T, Yoshiyama Y et al: Activation of hepatocyte growth-factor by proteolytic conversion of a single chain form to a heterodimer. J Biol Chem 267:20114, 1992

141. Naldini L, Tamagnone L, Vigna E et al: Extracellular proteolytic cleavage by urokinase is required for activationof hepatocyte growth-factor scatter factor. EMBO J 11:4825, 1992

142. Wormstone IM, Tamiya S, Marcantonio JM et al: C-met expression and hepatocyte growth factor function in human lens epithelial cells. Invest Ophthalmol Vis Sci S562, 1999

143. Smith DL, Reddan JR, Cooper KE: The effects of hepatocyte growth factor on lens epithelial wound healing. Invest Ophthalmol Vis Sci S201, 1997

144. Reneker LW, Overbeek PA: Lens-specific expression of PDGF-A alters lens growth and development. Dev Biol 180:554, 1996

145. Potts JD, Bassnett S, Kornacker S et al: Expression of platelet-derived growth-factor receptors in the developing chicken lens. Invest Ophthalmol Vis Sci 35:3413, 1994

146. Knorr M, Wunderlich K, Steuhl KP et al: Lens epithelial-cell response to isoforms of platelet-derived growth-factor. Graefes Arch Clin Exp Ophthalmol 231:424, 1993

147. Collison DJ, Coleman RA, James RS et al: Characterisation of muscarinic acetylcholine receptors in human lens cells. Invest Ophthalmol Vis Sci 41:2633, 2000

148. Brewitt B, Clark JI: growth and transparency in the lens, an epithelial tissue, stimulated by pulses of PDGF. Science 242:777, 1988

149. Brewitt B, Clark JI: A new method for study of normal lens development invitro using pulsatile delivery of PDGF or EGF in hl-1 serum-free medium. In Vitro Cell Dev Biol 26:305, 1990

150. Massague J: The transforming growth factor-beta family. Annu Rev Cell Biol 6:597, 1990

151. Roberts AB, Sporn MB: Physiological actions and clinical applications of transforming growth factor-beta (TGF-beta). Growth Factors 8:1, 1993

152. Roberts AB, Sporn MB: Differential expression of the TGF-beta isoforms in embryogenesis suggests specific roles in developing and adult tissues. Mol Reprod Dev 32:91, 1992

153. Yingling JM, Wang XF, Bassing CH: Signaling by the transforming growth-factor-beta receptors. BBA Rev Cancer 1242:115, 1995

154. Harpel JG, Metz CM, Kojima S et al: Control of transforming growth factor-beta activity: Latency vs. activation. Prog Growth Factor Res 4:321, 1992

155. Dennis PA, Rifkin DB: Cellular activation of latent transforming growth-factor-beta requires binding to the cation-independent mannose 6-phosphate insulin-like growth-factor type-II receptor. Proc Natl Acad Sci USA 88:580, 1991

156. Lee YS, Kay EP: TGF-beta stimulates synthesis of FGF-2 keratocytes and is inhibited by mannose 6-phosphate. Invest Ophthalmol Vis Sci 38:S2317, 1997

157. James K: Interactions between cytokines and alpha-2-macroglobulin. Immunol Today 11:16, 1990

158. Schulz MW, Chamberlain CG, McAvoy JW: Inhibition of transforming growth factor-beta-induced cataractous changes in lens explants by ocular media and alpha-2-macroglobulin. Invest Ophthalmol Vis Sci 37:1509, 1996

159. Pelton RW, Saxena B, Jones M et al: Immunohistochemical localization of TGF-beta-1, TGF-beta-2, and TGF-beta-3 in the mouse embryo: Expression patterns suggest multiple roles during embryonic-development. J Cell Biol 115:1091, 1991

160. Gordon-Thomson C, de Iongh RU, Hales AM et al: Differential cataractogenic potency of TGF-beta(1), -beta(2), and -beta(3) and their expression in the postnatal rat eye. Invest Ophthalmol Vis Sci 39:1399, 1998

161. Richiert DM, Ireland ME: TGF-beta elicits fibronectin secretion and proliferation in cultured chick lens epithelial cells. Curr Eye Res 18:62, 1999

162. Nishi O, Nishi K, Fujiwara T et al: Effects of the cytokines on the proliferation of and collagen synthesis by human cataract lens epithelial cells. Br J Ophthalmol 80:63, 1996

163. Kurosaka D, Nagamoto T: Inhibitory effect of TGF-beta-2in human aqueous-humor on bovine lens epithelial-cell proliferation. Invest Ophthalmol Vis Sci 35:3408, 1994

164. Wallentin N, Wickstrom K, Lundberg C: Effect of cataract surgery on aqueous TGF-beta and lens epithelial cell proliferation. Invest Ophthalmol Vis Sci 39:1410, 1998

165. Srinivasan Y, Lovicu FJ, Overbeek PA: Lens-specific expression of transforming growth factor beta 1 in transgenic mice causes anterior subcapsular cataracts. J Clin Invest 101:625, 1998

166. Colitz CMH, Malarkey D, Dykstra MJ et al: Histologic and immunohistochemical characterization of lens capsular plaques in dogs with cataracts. Am J Vet Res 61:139, 2000

167. Hales AM, Chamberlain CG, Murphy CR et al: Estrogen protects lenses against cataract induced by transforming growth factor-beta (TGF beta). J Exp Med 185:273, 1997

168. Hales AM, Chamberlain CG, Dreher B et al: Intravitreal injection of TGF beta induces cataract in rats. Invest Ophthalmol Vis Sci 40:3231, 1999

169. Liu J, Hales AM, Chamberlain CG et al: Induction of cataract-like changes in rat lens epithelial explants by transforming growth-factor-beta. Invest Ophthalmol Vis Sci 35:388, 1994

170. Reddan JR, Unaker N, Harding C et al: Induction of mitosis in the cultured rabbit lens initiated by the addition of insulin to medium KEI-4. Exp Eye Res 20:45, 1975

171. Beebe DC, Silver MH, Belcher KS et al: Lentropin, a protein that controls lens fiber formation, is related functionally and immunologically to the insulin-like growth-factors. Proc Natl Acad Sci USA 84:2327, 1987

172. Richardson NA, Chamberlain CG, McAvoy JW: IGF-1 enhancement of FGF-induced lens fiber differentiation in rats of different ages. Invest Ophthalmol Vis Sci 34:3303, 1993

173. Liu J, Chamberlain CG, McAvoy JW: IGF enhancement of FGF-induced fibre differentiation and DNA synthesis in lens explants. Exp Eye Res 63:621, 1996

174. Hutchings SE, Sato GH: Growth and maintenance of HeLa cells in serum-free medium supplemented with hormones. Proc Natl Acad Sci USA 75:901, 1975

175. McGahan MC, Harned J, Goralska M et al: Transferrin secretion by lens epithelial cells in culture. Exp Eye Res 60:667, 1995

176. Davidson MG, Harned J, Grimes AM et al: Transferrin in after-cataract and as a survival factor for lens epithelium. Exp Eye Res 66:207, 1998

177. Tripathi RC, Borisuth NSC, Tripathi BJ: Detection, quantification, and significance of basic fibroblast growth-factor in the aqueous-humor of man, cat, dog and pig. Exp Eye Res 54:447, 1992

178. ArakiSasaki K, Danjo S, Kawaguchi S et al: Human hepatocyte growth factor (HGF) in the aqueous humor. Jpn J Ophthalmol 41:409, 1997

179. Ohashi Y, Motokura M, Kinoshita Y et al: Presence of epidermal growth-factor in human tears. Invest Ophthalmol Vis Sci 30:1879, 1989

180. Namiki M, Tagami Y, Yamamoto M et al: Presence of epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF) in human aqueous. Acta Soc Ophthalmol Jap 96:652, 1992

181. Cousins SW, McCabe MM, Danielpour D et al: Identification of transforming growth-factor-beta as an immunosuppressive factor in aqueous-humor. Invest Ophthalmol Vis Sci 32:2201, 1991

182. Dernouchamps JP, Vaerman JP, Michiels J et al: La transferrine des liquides endoculaires chez le lapin. Ophthalmologica 170:72, 1975

183. Li J, Tripathi RC, Chalam KV et al: Expression of PDGF-Aand PDGF-B mRNAs by trabecular cells and lack of detectable level of PDGF-BB in aqueous humor. Invest Ophthalmol Vis Sci 37:3792, 1996

184. Ishizaki Y, Voyvodic JT, Burne JF et al: Control of lens epithelial cell survival. J Cell Biol 121:899, 1993

185. Kohno T, Sorgente N, Ishibashi T et al: Immunofluorescent studies of fibronectin and laminin in the human eye. Invest Ophthalmol Vis Sci 28:506, 1987

186. Iwata S: Physiological and biological characteristics of ocular lens capsule. IOL 2:13, 1988

187. Parmigiani CM, McAvoy JW: The roles of laminin and fibronectin in the development of the lens capsule. Curr Eye Res 10:501, 1991

188. Olivero DK, Furcht LT: Type IV collagen, laminin and fibronectin promote the adhesion and migration of rabbit lens epithelial cells in vitro. Invest Ophthalmol Vis Sci 34:2825, 1993

189. Oharazawa H, Ibaraki N, Lin LR et al: The effects of extracellular matrix on cell attachment, proliferation and migration in a human lens epithelial cell line. Exp Eye Res 69:603, 1999

190. Menko AS, Philip NJ: Beta(1) integrins in epithelial tissues: A unique distribution in the lens. Exp Cell Res 218:516, 1995

191. Nishi O, Nishi K, Akaishi T et al: Detection of cell adhesion molecules in lens epithelial cells of human cataracts. Invest Ophthalmol Vis Sci 38:579, 1997

192. Walker JL, Menko AS: Alpha 6 integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching. Dev Biol 210:497, 1999

193. Zhang XH, Ji J, Zhang H et al: Detection of integrins in cataract lens epithelial cells. J Cataract Refract Surg 26:287, 2000

194. Birkedal-Hansen H: Proteolytic remodeling of extracellular matrix. Curr Opin Cell Biol 7:728, 1995

195. Basbaum CB, Werb Z: Focalized proteolysis: Spatial and temporal regulation of extracellular matrix degradation at the cell surface. Curr Opin Cell Biol 8:731, 1996

196. Smine A, Plantner JJ: Membrane type 1 metalloproteinase in human ocular tissues. Curr Eye Res 16:925, 1997

197. Butler GS, Butler MJ, Atkinson SJ et al: Membrane type 1 metalloproteinase “receptor” regulates the concentration and efficient activation of progelatinase A: A kinetic study. J Biol Chem 273:1998

198. Belien AT, Paganetti PA, Schwab ME: Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter. J Cell Biol 144:373, 1999

199. d'Ortho MP, Will H et al: Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases. Eur J Biochem 250:751, 1997

200. Richiert DM, Ireland ME: Matrix metalloproteinase secretion is stimulated by TGF-β in cultured lens epithelial cells. Curr Eye Res 19:269, 1999