Julia M. Marcantonio

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 Soemmerrings ring formation and peripheral thickening of the capsular bag. Closer to the rhexis, cell swelling can result in globular Elschnigs 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.

Classification of human senile cataracts by nuclear colour and sodium content

Abstract Human cataractous lenses were photographed in vivo prior to surgery and then in vitro after extraction. They were also analysed for sodium concentration and protein content. The lenses were then classified according to a dual system taking into account the extent of nuclear involvement (colour groups I to V) and electrolyte changes (sodium bands A to E) in each lens. The protein data from lenses classified according to the above dual scheme confirmed that the colour of the lens is closely associated with the water-insoluble protein content of the lens but further demonstrated that an increase in the sodium concentration (osmotic involvement) is associated with a dramatic fall in the water-soluble and total protein content. As the osmotic and nuclear mechanisms lead to quite different changes in protein distribution and as most cataracts are mixed in nature, a dual classification system is essential in any study where it is intended to discriminate between the two mechanisms. It was found that both the in vivo and in vitro photographic methods gave a consistent measure of nuclear involvement. However, it was not always possible to relate the changes in lens sodium to the extent of light scattering seen either in the in vivo or in vitro photographs.

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.

Epithelial transdifferentiation and cataract in the human lens.

Anterior subcapsular cataracts cause a serious loss of vision and are normally associated with ocular trauma, inflammation or clinical skin conditions. They appear to be accompanied by epithelial cell growth and transdifferentiation where unscheduled production of a number of proteins, including alpha smooth muscle actin (alpha-sma), occurs. Clinical studies have also revealed an up-regulation of the TGFbeta signalling pathway in such cataracts. The present study, using phase contrast and immunofluorescent techniques, was undertaken to investigate the extent of alpha-sma expression in traumatic cataracts, in capsulorhexis specimens obtained during cataract surgery and in aged human lenses from donor eyes. The donor lenses were also exposed to trauma or TGFbeta in culture to observe their relative contribution to alpha-sma production. Dense anterior subcapsular cataracts were relatively rare (<1%), but all showed a pronounced up-regulation of alpha-sma, which was located both in anterior cells of normal appearance and in nucleated fibroblastic cells lying beneath the anterior epithelium. Surprisingly, more than 50% of capsulorhexis specimens from mature cataracts showed expression of alpha-sma, although to a limited extent. Alpha-sma was not expressed in any of the clear donor lenses and culture for 8 days in EMEM did not induce expression. Interestingly, unlike their young animal counterparts, human lenses failed to show the presence of alpha-sma when exposed to 10 ng ml(-1) TGFbeta. However, after culture, lenses with pre-existing cortical opacities did express alpha-sma, as did clear lenses subjected to injury or trauma. It appears that the greater the stress, the greater is the expression of alpha-sma. Cataract, and especially cortical cataract, should therefore be seen as associated with stress-induced signalling pathways in the lens that lead to the transdifferentiation of the anterior epithelial cells.

Interlenticular opacification in piggyback AcrySof intraocular lenses: explantation technique and laboratory investigations

BACKGROUND/AIMS Interlenticular opacification (ILO) is a recognised complication of piggyback intraocular lenses (IOLs). The aetiology, histopathology, and treatment are not clearly defined, however. METHODS Two pairs of AcrySof IOLs were explanted from a patient with bilateral ILO. The explantation technique and surgical challenges of IOL exchanges are described. The explanted IOL complexes and a sample of the anterior capsule were examined by phase, polarising, and immunofluorescence microscopy. RESULTS A 50 year old man developed ILO bilaterally after piggyback AcrySof IOL implantation. A central contact zone was surrounded by a homogeneous paracentral opacity possibly consisting of extracellular matrix previously laid down by proliferating lens epithelial cells (LECs). These opacities were in turn surrounded by interlenticular Elschnig pearl-type opacities contiguous with the same material filling the periphery of the capsular bag. The IOL complexes were very adherent to the capsular bag and they had to be separated with the help of high viscosity viscoelastic before a single one piece PMMA IOL implantation via large limbal incisions. The sample of anterior capsule showed a ridge configuration from the piling of LECs in the site of apposition with the anterior capsule and cells showing different characteristics on either side of the ridge. CONCLUSION Cellular proliferation, deposition of ECM from proliferating LECs, and capsular changes induced by cell metaplasia may lead to ILO formation in piggyback AcrySof IOLs. Careful separation of the AcrySof IOL complex from the capsule, meticulous clean up of the proliferating material, and implantation of single or dual in the bag PMMA IOLs through a large incision with capsulorrhexis enlargement may help in the prevention of recurrence of interface opacification.

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.

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.

Amino acid transport and protein synthesis in human normal and cataractous lenses

The ability of normal and cataractous human lenses to accumulate amino acids and synthesize proteins was studied under organ culture conditions. As expected, normal lenses regulate their internal ion levels, accumulate amino acids against a concentration gradient, and continue to synthesize proteins even in advanced age (greater than 60 yrs). The cataractous lenses fell basically into two groups. Those with a low internal sodium and calcium content behaved in a similar manner to normal lenses, but cataractous lenses with high sodium and calcium contents showed a markedly reduced ability to accumulate amino acid and synthesized less low molecular weight protein. They incorporated, however, a much higher proportion of labelled amino acid into high molecular weight protein. While sodium appears to be the ion involved in changes in amino acid accumulation, calcium seems to play a critical role in the disturbance of lens protein synthesis and also protein-protein interaction. Both loss of protein and accumulation of high molecular weight aggregates may be due to modifications induced by the calcium-activated protease, calpain. None of the changes appeared to be correlated with increasing nuclear brunescence.

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.

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.