Thapsigargin-coated intraocular lenses inhibit human lens cell growth.

Cataract is responsible for rendering several million people blind throughout the world and is also by far the most common cause of low visual acuity. Although cataract surgery is common, routine and effective, posterior capsule opacification (PCO) occurs in 30-50% of patients following modern cataract surgery. This condition arises from stimulated cell growth within the capsular bag after surgery. The resulting decline in visual acuity requires expensive laser treatment, and PCO therefore prevents modern cataract surgery from being carried out routinely in underdeveloped countries. The present study, using a human lens capsular bag culture system, has confirmed that cells from a wide age range of donors proliferate in the absence of added serum protein and explains why PCO is such a common problem even in aged patients. This study also provides one possible solution for PCO by using polymethylmethacrylate (PMMA) implanted intraocular lenses as a drug delivery system. PMMA lenses coated with thapsigargin, a hydrophobic inhibitor of endoplasmic reticulum (ER) (Ca2+)-ATPase, greatly reduced cell growth in the capsular bag at relatively low coating concentrations (200 nM) but, more significantly, induced total cell death of the residual anterior epithelial cells at higher concentrations (>2 microM).

A human lens model of cortical cataract: Ca2+-induced protein loss, vimentin cleavage and opacification.

PURPOSE: Cortical cataract in humans is associated with Ca2+ overload and protein loss, and although animal models of cataract have implicated Ca2+-activated proteases in this process, it remains to be determined whether the human lens responds in this manner to conditions of Ca2+ overload. The purpose of these experiments was to investigate Ca2+-induced opacification and proteolysis in the organ-cultured human lens. METHODS: Donor human lenses were cultured in Eagle's minimum essential medium (EMEM) for up to 14 days. The Ca2+ ionophore ionomycin was used to induce a Ca2+ overload. Lenses were loaded with [3H]-amino acids for 48 hours. After a 24-hour control efflux period, lenses were cultured in control EMEM (Ca2+ 1.8 mM), EMEM + 5 microM ionomycin, or EMEM + 5 microM ionomycin + 5 mM EGTA (Ca2+ < 1 microM). Efflux of proteins and transparency were monitored daily. Protein distribution and cytoskeletal proteolysis were analyzed at the end of the experiment. Cytoskeletal proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were probed with anti-vimentin antibody (clone V9) and detected by enhanced chemiluminescence. RESULTS: Lenses cultured under control conditions remained transparent for 14 days in EMEM with no added supplements or serum. The lenses synthesized proteins and had a low rate of protein efflux throughout the experimental period. Ionomycin treatment resulted in cortical opacification, which was inhibited when external Ca2+ was chelated with EGTA. Exposure to ionomycin also led to an efflux of [3H]-labeled protein, amounting to 41% of the labeled protein over the 7-day experimental period, compared with 12% in ionomycin + EGTA-treated lenses. Efflux was accounted for by loss from the lens soluble protein (crystallin) fraction. Western blot analysis of the cytoskeletal protein vimentin (56 kDa) revealed a distinct breakdown product of 48 kDa in ionomycin-treated lenses that was not present when Ca2+ was chelated with EGTA. In addition, high-molecular-weight proteins (approximately 115 kDa and 235 kDa) that cross-reacted with the vimentin antibody were observed in ionomycin-treated lenses. The Ca2+-induced changes were not age dependent. CONCLUSIONS: Human lenses can be successfully maintained in vitro, remaining transparent for extended periods. Increased intracellular Ca2+ induces cortical opacification in the human lens. Ca2+-dependent cleavage and cross-linking of vimentin supports possible roles for calpain and transglutaminase in the opacification process. This human lens calcium-induced opacification (HLCO) model enables investigation of the molecular mechanisms of opacification, and the data help to explain the loss of protein observed in human cortical cataractous lenses in vivo.

Hepatocyte growth factor function and c-Met expression in human lens epithelial cells.

PURPOSE: Hepatocyte growth factor (HGF) and its receptor c-met perform a multitude of functions. However, despite the significant degree of study of HGF and c-met in numerous tissues and cell types, relatively few investigations have been performed on the lens. In the current study, therefore, the role of HGF and the receptor c-met in human lens epithelial cells was investigated. METHODS: Anterior epithelium and capsular bags were prepared from human donor eyes and maintained in Eagle's minimum essential medium (EMEM) in a 5% CO(2) atmosphere at 35 degrees C. In addition, the human lens cell line FHL124, was routinely cultured and seeded onto glass coverslips (c-met immunodetection), 12-well plates (DNA and protein synthesis), and tissue culture dishes (migration). c-Met was detected by immunocytochemistry and fluorescence-activated cell scanning (FACS). HGF was measured using enzyme-linked immunosorbent assay (ELISA) techniques. Proliferation and protein synthesis were determined by [(3)H]thymidine and (35)S-methionine incorporation into DNA and proteins, respectively. Migration was assessed using a scratch-wound assay and time-lapse video microscopy. RESULTS: HGF was detected at all stages of culture of capsular bags in protein-free medium. Moreover, c-met was present on the native epithelium and after mechanical trauma was seen to be upregulated. Immunolocalization and FACS analysis demonstrated c-met expression on FHL124 cells throughout the whole population. Furthermore, FACS analysis showed that serum-maintained cells sustained a higher level of receptor expression relative to serum-deprived cells. Additionally, HGF was found to stimulate proliferation, protein synthesis, and migratory responses. CONCLUSIONS: c-Met receptors are expressed in native epithelium, capsular bag cultures, and FHL124 cells. Receptor is distributed across the entire cell population; however, this expression is environmentally and mechanically sensitive. HGF is also present in capsular bags at all stages of culture. In addition, HGF can stimulate migration, proliferation, and protein synthesis. It therefore appears that a multifunctional autocrine loop involving HGF and c-met is in place and could be important in the development of posterior capsule opacification.

Membrane and communication properties of tissue cultured human lens epithelial cells.

Explant cultures were established from capsule/epithelium preparations from both normal and cataractous lenses to investigate properties of human lens epithelial cells. The cultured cells were found to have similar membrane potentials to whole human lenses and isolated epithelia, and similar free ionic concentrations of potassium, sodium, and calcium (131 mM, 17 mM and 0.8 microM respectively) to whole human lenses. The free ionic concentrations were measured in both cases using neutral resin-filled electrodes. Cellular communication was investigated using electrical (two internal microelectrodes) and dye injection techniques. The electrical resistance of a confluent cell monolayer was approximately 4 M omega when the voltage measuring and current passing microelectrodes were in neighbouring cells or several cell diameters apart. Additionally, Lucifer Yellow dye injected into one cell spread rapidly over a wide area of cells. The cells thus appear to be extremely well coupled. Electrical communication could be disrupted by internal acidification (following exposure to 100% CO2), exposure to 1 mM octanol and by membrane depolarization. In the latter case the blockade was only partial. All uncoupling methods proved to be reversible. The diffusion of Lucifer Yellow dye was also inhibited by internal acidification and exposure to octanol. The sensitivity of both dye and electrical coupling to internal acidification and exposure to octanol is similar to that observed in hepatocytes and other tissues, whereas the effect on cell communication induced by changing the resting potential appears to occur only in a few cell types such as those of embryonic origin.

Lens cell populations studied in human donor capsular bags with implanted intraocular lenses.

PURPOSE: Posterior capsule opacification is an ongoing cellular redistribution process. The level of viable cell coverage was therefore determined in human donor capsular bags with implanted intraocular lenses, and cellular morphology and ultrastructure were investigated in relation to cell type and level of differentiation. METHODS: Donor capsular bags, retrieved at intervals of 4 months to 13 years after surgery, were investigated by phase optics before fixation. Postfixation techniques included scanning electron microscopy and transmission electron microscopy of sections and immunofluorescent staining of cytoskeletal proteins in wholemounts. RESULTS: All the capsular bags contained a large population of viable cells on the capsular surfaces. Cells on the anterior face of the anterior capsule and in the spaces around the intraocular lens had an elongated morphology and expressed alpha-smooth muscle actin. The cells formed light-scattering, multilayered aggregates and strands that were surrounded by layers of extracellular matrix. The regions between the intraocular lens and the equator of the bags were populated by monolayers of epithelial cells of normal morphology and ultrastructure, on both the anterior and posterior capsules. In some regions the apical surfaces of the two epithelial monolayers were in contact, and in some parts of the equatorial regions, differentiation of cells into well-organized fiberlike cells was evident. CONCLUSIONS: Human capsular bags contain a large population of viable cells for many years after cataract surgery. Cells in the regions around the intraocular lens undergo transition to a mesenchymal type. Cells peripheral to these regions can form a stable closed microenvironment in which both normal epithelial morphology and differentiation to fiberlike cells are maintained.

Calcium-induced opacification and proteolysis in the intact rat lens.

When intact rat lenses were incubated in artificial aqueous humor in the presence of 1 mM calcium and a sulfhydryl reagent p-chloromercuriphenyl sulfonate (pCMPS) a visible annular opacity developed within 4 hours. Combined photographic and ion-sensitive microelectrode investigations of the lenses demonstrated that the subsequent linear increase in opacification was accompanied by an increase in internal free calcium. Opacities were not observed in lenses incubated in the absence of either pCMPS or calcium. Gel electrophoresis of the soluble and urea-soluble fractions from lenses exposed to 1 mM calcium for periods of up to 14 hours showed no evidence for crystallin degradation and only minor proteolysis of cytoskeletal proteins. When lenses were incubated under identical conditions, but with 5 mM calcium, the degree of opacification increased up to approximately 8 hours and then remained constant. A progressive loss in cytoskeletal proteins was observed which correlated with a further increase in free calcium such that by 14 hours of incubation, when the internal calcium approached 1 mM, most of the spectrin and vimentin present in the cortex of the lens had disappeared. An unidentified 110-kilodalton protein also disappeared from lenses incubated in 5 mM calcium. These results indicate that proteolysis by calcium-dependent enzymes such as calpain may play a significant role in cytoskeletal regulation and metabolism in the lens. A role for cytoskeleton/membrane/crystallin interaction in calcium-induced opacification is discussed.

Human lens epithelial cell proliferation in a protein-free medium.

PURPOSE: The ocular humors are relatively low in protein, yet cell growth in the human capsular bag still occurs after extracapsular cataract extraction (ECCE) surgery. This resilient growth gives rise to posterior capsule opacification (PCO) in a significant proportion (30%) of patients. This study compared the ability of human lens cells to proliferate in serum-supplemented and protein-free medium. METHODS: Sham cataract operations were performed on human donor eyes. The capsular bag was dissected free, pinned flat on a petri dish, and incubated in Eagle's minimal essential medium (EMEM) alone or in EMEM supplemented with 10% fetal calf serum. Observations were made by phase-contrast microscopy. At the endpoint, capsules were studied by fluorescence or electron microscopy. Mitotic activity was identified using Bromo-2-deoxyuridine labeling and detection techniques. When required, an intraocular lens was implanted when surgery was performed. RESULTS: It was found that human lens cells from a wide age spectrum of donors proliferate and migrate on the lens capsule in the absence of added protein. The rate of growth was age-dependent, such that the posterior capsule was completely confluent after 8.0 +/- 0 days (n = 3) and 24.4 +/- 5.3 days (n = 3) for donor lenses aged < 40 years and > 60 years, respectively. The outgrowth of epithelial cells gave rise to capsular contraction, wrinkling, and increased light scatter. Growth on the anterior surface of the intraocular lens was less prolific than on the posterior capsule. CONCLUSION: The protein-free model replicates many features of clinically-observed PCO. The resilient cell growth on the natural collagen capsule explains the high prevalence of PCO, especially in younger patients, and suggests that inflammation and external growth factors are not necessary for PCO. Furthermore, the protein-free capsular bag system can be used to explore fundamental questions concerning the autocrine control of lens epithelial cell survival and growth.

A study of human lens cell growth in vitro. A model for posterior capsule opacification.

PURPOSE: After intraocular lens (IOL) implant surgery for cataract, cell growth on the posterior capsule is responsible for renewed visual impairment in approximately 30% of patients. The authors have, therefore, developed a human lens capsule system to study this growth in vitro. METHODS: Sham cataract surgery, including anterior capsulorhexis, nucleus hydroexpression, and aspiration of lens fibers, was performed on donor eyes. In some cases, a polymethylmethacrylate IOL implant was placed in the capsular bag. The capsular bag was dissected free, pinned flat on a plastic culture dish, covered with Eagle's minimum essential medium supplemented with 10% fetal calf serum and observed by phase-contrast and dark-field microscopy for as long as 100 days. At the end-point, capsules were examined by fluorescence microscopy for actin, vimentin, and chromatin. RESULTS: Within 24 hours, there was evidence of cell growth in the equatorial region. After 2 to 3 days, cells were normally observed growing from the rhexis onto the posterior capsule and across the anterior surface of the IOL, if present. Growth proceeded rapidly so that the posterior capsule, for example, was totally covered by a confluent monolayer of cells at 5.8 +/- 0.6 days and 7.2 +/- 0.7 days for capsules aged < 40 years and > 60 years, respectively. Total cover of the anterior IOL surface generally followed 4 to 5 days behind that of the capsule. Capsular wrinkles became increasingly apparent as time progressed, causing a marked rise in light scatter. An increase in capsular tension also occurred, and the actin filaments became more polarized near the wrinkles. CONCLUSIONS: The model presented here for posterior capsule opacification shows many of the changes seen in vivo, including rapid lens cell growth, wrinkling, tensioning, and light scatter in the posterior capsule. It will be possible to develop strategies for inhibiting cell growth with this system.

FGF: an autocrine regulator of human lens cell growth independent of added stimuli.

PURPOSE: Posterior capsule opacification (PCO) arises because of a persistent growth of lens epithelial cells. Cultured human lens cells residing on their native collagen capsule and maintained in serum-free medium actively grow and thus show an intrinsic capacity for regulation. In the present study, the authors investigated the role of the putative FGF autocrine system in human capsular bags. METHODS: Capsular bags were prepared from human donor eyes and maintained in a 5% CO(2) atmosphere at 35 degrees C. On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence cytochemistry. De novo protein synthesis was determined by the incorporation of 35S-methionine. Basic fibroblast growth factor (FGF) and FGF receptor (R)-1 were detected using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) techniques. FGFR-1 inhibition was achieved using the specific antagonist SU5402. RESULTS: Human lens epithelial cells can maintain metabolic activity for more than 1 year in a protein-free medium. Basic FGF was shown to be present in capsular bags throughout culture and also in capsular bags removed from donor eyes that had previously undergone cataract surgery. Furthermore, FGFR-1 was identified. Inhibition of FGFR-1 caused a significant retardation of growth on the posterior capsule. On no occasion did any treated bag reach confluence, whereas all match-paired control samples did. CONCLUSIONS: The results provide evidence that FGF plays an integral role in the long-term survival and growth of human lens epithelial cells, independent of external stimuli. Inhibition of FGFR-1 by specific synthetic molecules, such as SU5402, could provide a potential therapeutic approach to resolving PCO.

Susceptibility of the bovine lens 115kDa beaded filament protein to degradation by calcium and calpain.

The 115kDa cytoskeletal beaded filament protein of bovine lens fibres is degraded during opacification induced by increased internal calcium. The monoclonal antibody R2D2 to this protein has been used in whole lenses and native homogenates to follow the process of degradation and the production of break-down products. In the opaque outer cortex of whole bovine lenses with an internal Ca2+ of 2.0mM, both the 115kDa parent protein and the main degradation product (57kDa) were reduced in amount by almost 60%. No additional products were detected by the antibody. When native homogenates were incubated overnight with 10mM Ca2+ the protein could no longer be detected in SDS gels, but faintly reactive bands were detected by the antibody. Since these changes were dependent on the presence of increased calcium they were compared with changes induced by incubating freshly isolated cytoskeletal proteins with Ca2+ and the Ca(2+)-activated protease, calpain. The 115kDa protein was shown to be susceptible to degradation by calpain, with the formation of a number of breakdown products. These results indicate that degradation of the beaded filament protein can be brought about by the activation of calpain. Since the enzyme is present in lens cortex it is likely to have a role in the protein degradation observed during Ca(2+)-induced opacification, and may also be involved in the changes occurring as the lens fibres mature.

Amino-acid transport and protein synthesis in the rabbit lens: absence of cryoprobe effect.

Recently developed organ-culture techniques have been used to investigate the effects of cryoprobe treatment on rabbit lenses. Uptake of 14C-tyrosine into cryoprobe treated and control lenses was followed for 96 h. Lens proteins were separated by gel filtration and incorporation of label measured in the individual crystallins. The cryoprobe treatment had no measurable effect on lens water, Na+, K+ or Ca++ content, tyrosine transport or the incorporation of tyrosine into the crystallins, during the period of the experiment.

Calcium-induced disruption of the lens cytoskeleton.

Ionic homeostasis is essential to lens clarity and the lens epithelium plays a large part in homeostasis, through vectorial transport. In most epithelia maintenance of vectorial function depends on the cytoskeleton. Capsule/epithelium preparations from human donor lenses have been used for immunohistochemical investigations of both normal epithelial cell cytoskeletal structure, and of structural changes induced by increasing cell calcium. A sustained increase in intracellular calcium, induced by incubation with A23187 and W7, led to a loss of cytoskeletal organisation. Changes induced depolymerisation of cytoplasmic actin filaments, disaggregation of microtubules and initial thickening, followed by breakdown, of vimentin intermediate filaments. In addition, spectrin staining, normally confined to the basal-lateral plasma membranes, became diffuse within the cytoplasm. Such disruptions of cytoskeletal structure would decrease ionic control, leading to increasing osmotic stress.

TGFbeta2 influences alpha5-beta1 integrin distribution in human lens cells.

TGFbeta plays a central role in posterior capsule opacification, in which cell proliferation and matrix deposition, accompanied by capsular wrinkling, are largely responsible for the increased light scatter involved. Human FHL124 cells were plated onto uncoated glass coverslips to form circular patches so that the central cells reached confluency while the peripheral cells grew outwards. Cell patches were exposed to serum free (SF) EMEM (control) or TGFbeta supplemented (10 ng ml(-1)) EMEM. Fibronectin (Fn), alpha5beta1 integrin and F-actin were localized by immunofluorescence techniques and analysed by confocal microscopy. In the confluent, central cells in SF medium alpha5beta1 showed a punctate distribution while Fn was present in strongly staining fibres. TGFbeta had no effect on integrin or Fn distribution in confluent cells. In the peripheral, motile cells of the patches in SF conditions alpha5beta1 was localized in well-defined focal adhesion plaques at the ends of actin stress fibres, while Fn was distributed in a punctate perinuclear pattern. TGFbeta had a profound dispersing effect on the integrin causing a widespread distribution of alpha5beta1 in the membrane with no apparent association with the actin filaments. The cells had a more fibroblastic morphology with increased deposition of Fn near the nucleus. All the TGFbeta-induced changes were inhibited by the TGFbeta antibody CAT152 (Cambridge Antibody Technology). Culture with a function-blocking alpha5 antibody or Fn antibody resulted in detachment of the peripheral cells from the patches, but the central cells remained intact. The patch culture method therefore provides a convenient means of investigating the differences between confluent and growing lens cells both in terms of the patterns of alpha5beta1 integrin and Fn and also in the response of the molecular arrangements of both to TGFbeta2.

Amino acid transport and crystallin synthesis in the bovine lens.

Bovine lenses were incubated in a defined, bicarbonate-free culture medium (EMEM) and the kinetics of amino acid uptake and protein synthesis investigated. The kinetics were interpreted in terms of a simple multi-compartment model. [14C]tyrosine was found to be totally exchangeable in the incubated lens and the rate constant for the exponential increase in activity was 0 . 0175 hr-1. The rate of influx was markedly reduced by incubating in the presence of ouabain (10(-5) M), which also caused a concomitant disturbance of the normal sodium and potassium distributions. The soluble proteins from the incubated lenses were fractionated on Sephadex G-200 and the rate of incorporation into the crystallins was shown to fall into two classes. The rate of synthesis of alpha and beta L crystallin was relatively rapid (rate constants approximately equal to 0 . 004 hr-1), while the synthesis rates of beta H and beta S/gamma were both much slower (0 . 001 hr-1). The efflux kinetics of [14C]tyrosine were determined and the rate of decrease of the free amino acid pool was identical to the rate of increase determined from an influx experiment. Hence the lenses are in a steady state with respect to free tyrosine throughout the incubation period (up to 160 hr). All classes of proteins continued to be synthesized during efflux experiments and there was no evidence for a breakdown of alpha or beta L crystallin during the time-course of these experiments. Ouabain slowed the rate of loss of tyrosine from the free amino acid pool, and this was interpreted in terms of an ouabain-induced decrease in synthesis rate rather than as a decrease in efflux rate from the lens. There was in fact a very marked decrease in the incorporation of [14C]tyrosine into the alpha and beta L crystallins on exposure to ouabain, and this decrease was apparent before any change in activity in the amino acid pool.

Changes in lens amino acid transport and protein metabolism during osmotic cataract produced by ouabain.

Osmotic (cortical) cataract was induced in bovine lenses during long-term organ culture by adding the cardiac glycoside, ouabain, to the incubation medium. This insult produces a much more rapid effect on protein synthesis than it does on amino acid influx, efflux or net protein loss. The kinetics of amino acid transport and protein synthesis were studied in detail using 14C-tyrosine in the incubation medium and a simple mathematical model was developed to help interpret the data. During efflux experiments, loss of radioactivity could be explained in terms of a single exponential process and ouabain was found to reduce the rate constant of loss after a delay of 50 hours. There was no delay, however, on the effect of ouabain on protein synthesis which was significantly reduced compared to control values. There was no effect of ouabain on net protein loss from the lens until after 120 hours, when significant amounts of 14-C labelled protein began to appear in the washing medium. This initiation of protein loss was accompanied by an increase in the amount of light scattered from the lens. Further studies are underway to determine which of the protein fractions are lost from the lens during osmotic insult.

Cell biology of posterior capsular opacification.

Posterior capsular opacification (PCO), a major complication of modern cataract surgery, necessitates further surgical intervention in 10-50% of patients. PCO results from the growth and transdifferentiation of lens epithelial cells left on the anterior capsule at the time of cataract surgery. These cells proliferate to form monolayers on the capsular surfaces, and such monolayers continue to line the anterior capsule leaflet many years after surgery. Some cells, however, differentiate or undergo a transition to another cell type, and these processes greatly contribute to PCO. Equatorial differentiation of cells to fibre-like structures leads to Soemmerring's ring formation and peripheral thickening of the capsular bag. Closer to the rhexis, cell swelling can result in globular Elschnig's pearls, which may occlude the visual axis. Cells at the rhexis edge and those in the space around the optic appear to undergo epithelial-mesenchymal transition. The resulting cells are fibroblastic in morphology, express the smooth muscle isoform of actin and secrete extracellular matrix containing proteins not normally present in the lens.

Calcium ionophore induced proteolysis and cataract: inhibition by cell permeable calpain antagonists.

Two dipeptide aldehyde cell permeable calpain inhibitors, cBz-Val-Phe and calpeptin, have been assessed for their ability to prevent cytoskeletal proteolysis and loss of transparency in whole rat lenses. Calcium overload, induced by ionomycin in artificial aqueous humor with 1mM calcium, resulted in lens opacification and degradation of cytoskeletal proteins including spectrin, filensin, and vimentin. No such changes resulted from incubation in ionomycin in the absence of calcium. In calcium overload lenses both inhibitors gave some protection against cytoskeletal protein degradation and loss of transparency. These experiments indicate that calpain has a role in cortical opacification in high calcium lenses and that cell penetrating calpain inhibitors do indeed enter lens cells and reduce both proteolysis and opacification.

Calcium-induced opacification and loss of protein in the organ-cultured bovine lens.

A long-term system of organ culture for bovine lenses was used to investigate the effect of osmotic stress on lens opacification and crystallin loss. Lenses were pre-incubated in control medium containing L-[U-14C]tyrosine so that labelled crystallins were produced. The fate of these crystallins was studied in relation to two forms of osmotic stress. The addition of either ouabain or EGTA to the medium induced severe osmotic swelling and disturbance of the lens monovalent cation balance, but only the former treatment was followed by an increase in lens calcium. The changes due to osmotic stress were accompanied by loss of transparency and protein only in the lenses with increased calcium. Both opacification and increased calcium were found largely to be confined to the outer cortical fibres. Protein loss increased with time as lens calcium continued to increase. The protein recovered from the incubation medium was characterized by gel filtration and immunological techniques. The first protein detected was beta L-crystallin, and this formed the major part of the lost protein throughout, although alpha- and gamma-crystallins were detected at a later stage. Increased calcium also resulted in a change in the susceptibility of the crystallins to aggregation, since there was an increase in [14C]tyrosine incorporated into the lens high-molecular-weight (HM) fraction after exposure to ouabain, but not after exposure to EGTA. The relevance of these findings to human cataract is discussed.

Induction of matrix metalloproteinases 2 and 9 following stress to the lens.

Matrix metalloproteinase 2 and 9 (MMP-2 and 9, also known as gelatinase A and B) have been implicated in a number of eye diseases, but their possible involvement in lens pathology is yet to be determined. In the present study, we therefore investigated a possible role of matrix metalloproteinases in cataract and posterior capsule opacification. Whole porcine lenses were removed from the eye and cultured in either Eagles Minimum Essential Medium (EMEM) or EMEM supplemented with 1 m M hydrogen peroxide. The medium was sampled and changed every 2 days. On some occasions a sham cataract operation was performed on cultured lenses. The resulting capsular bag was secured to a Petri dish and cultured in EMEM. Culture media from all preparations were analysed for MMP-2 and 9 activity by gelatin zymography. Media samples from lenses which maintained clarity over the 6 day culture period did not display any detectable gelatinolytic activity. However, media from cataractous lenses demonstrated a gelatinolytic band, which had similar molecular weights to the pro-form of MMP-2. In addition to this band, bands with a similar molecular weight to pro-MMP-9 and its dimeric form were also detected in samples obtained from capsular bag preparations within 24 hr. The data presented indicate that normal lenses have undetectable gelatinase activity. However, there is an associated expression of gelatinases with pathological states of the lens, and therefore gelatinase expression could play an important role in cataractogenesis and posterior capsule opacification.

Calcium-induced cleavage and breakdown of spectrin in the rat lens.

Incubation of intact rat lenses under conditions that stimulated a net influx of calcium resulted in a pronounced loss of transparency and a major decrease in the levels of spectrin. The progressive loss of this cytoskeletal component coincided with the appearance of polypeptides of approximately 150 kDa which showed immunoreactivity with an antibody raised to spectrin. These bands disappeared on further incubation. It is, therefore, suggested that a calcium-activated protease is present in the lens which is capable of degrading spectrin by the initial removal of approximately 90 kDa fragments. This process calcium-induced proteolysis may be the basis for the cytoskeletal reorganisation observed during the differentiation of lens fibre cells and may be involved in cataract development.