B.J. Wagner

Posterior Capsular Opacification: A Problem Reduced but Not Yet Eradicated

Posterior capsular opacification (PCO) is the most frequent complication of cataract surgery. Advances in surgical techniques, intraocular lens materials, and designs have reduced the PCO rate, but it is still a significant problem. The only effective treatment for PCO, Nd:YAG laser capsulotomy carries vision-related complications and risks and puts a significant financial burden on the health care system. This review contains current knowledge about the mechanisms of PCO development. Posterior capsular opacification is caused mainly by remnant lens epithelial cell proliferation and migration, epithelial-mesenchymal transition, collagen deposition, and lens fiber generation. All of these processes are influenced by cytokines, growth factors, and extracellular matrix proteins. We also describe advances and improvements in surgical techniques, intraocular lens materials, and the designs and use of therapeutic agents leading to safe, effective, and less expensive strategies to eradicate PCO.

Characterization of lens proteins I. Identification of additional soluble fractions in rat lenses.

Abstract The soluble proteins of the rat lens have been fractionated on Sephadex G-200, superfine. Six distinct fractions have been obtained from the proteins of the normal lens. Fraction I is fastest moving and found to be α-crystallin. Fractions III–IV and V are found to be β- and γ-crystallins, respectively. Fraction II, which is not clearly defined, requires further investigation. Fraction VI is slowest moving and contains low molecular weight substances (protein and nucleotide) which diffuse through dialysis membranes. The resolution and reproducibility of the Sephadex G-200, superfine procedure are noteworthy. The Sephadex G-200, superfine, procedure has also been applied to the fractionation of calf soluble lens proteins. The elution profile of the calf lens is similar to that of the rat. The polyacrylamide electrophoretic gel patterns show certain similarities among the corresponding fractions of these mammalian proteins. Polyacrylamide disc gel electrophoresis (Tris-glycine buffer, Tris-glycine buffer with 7 m -urea and SDS) has also shown that the protein patterns of the Fractions I and V isolated by the present procedure are identical to the α- and μ-crystallins, respectively, isolated from DEAE-cellulose chromatography. Similar analyses of the galactose-induced cataract rat lens reveal a gradual decrease of Fraction V and an increase of Fraction I as the lens becomes increasingly opaque. A diminution of Fraction II has also been noticeable, even at the beginning of cataractogenesis. Fraction VI is found to be absent, or present in only trace amounts, in the cataract lenses. The qualitative and quantitative alterations of the protein composition of Fraction V from the normal to cataract lenses have been demonstrated by polyacrylamide gel electrophoresis.

Downregulation of MMP-2 and -9 by Proteasome Inhibition: A Possible Mechanism to Decrease LEC Migration and Prevent Posterior Capsular Opacification

PURPOSE The proliferation, epithelial-mesenchymal transition (EMT), and migration of residual lens epithelial cells (LECs) after cataract surgery leads to the development of posterior capsular opacification (PCO). The authors have shown that proteasome inhibition suppresses LEC proliferation and EMT. The present study investigates the prevention of LEC migration by proteasome inhibition through the suppression of matrix metalloproteinase (MMP) expression and activity. METHODS HLE B-3 and primary human LEC migration assays were performed using polycarbonate membrane inserts and 20% fetal bovine serum (FBS) as chemoattractant. Cultured cells were treated with 1 ng TGF-beta(2), with or without MG132 (proteasome inhibitor) or GM 6001 (MMP inhibitor). Capsular bags with intraocular lenses (IOLs) were prepared from human donor eyes and cultured in serum-free DMEM. The capsular bags were then treated with 1 or 10 ng/mL TGF-beta(2), with or without MG132 (2.5 or 10 muM, respectively). The medium was sampled and replaced every 2 days and analyzed for MMP-2 and -9 activities by SDS-PAGE zymography. Protein and RNA expression were analyzed by Western blotting and RT-PCR, respectively. RESULTS Proteasome inhibition blocks LEC migration in HLE B-3 and primary human LECs. To further evaluate the mechanism of decrease in LEC migration by proteasome inhibition, the authors measured MMP-2 mRNA and protein expression and MMP-2 and -9 activities. In HLE B-3 cells, TGF-beta(2) increased MMP-2 mRNA and protein levels; these increases were inhibited by MG132 cotreatment. Medium from HLE B-3 cultures showed MMP-2 and -9 activities, which were induced by TGF-beta(2) treatment and inhibited by MG132 co-treatment. TGF-beta(2) treatment also increased MMP-2 and -9 activities in IOL capsular bag cultures; these were progressively decreased by proteasome inhibition. CONCLUSIONS Proteasome inhibition decreases LEC migration. This inhibition is correlated with decreased MMP-2 and -9 activities, observed both with and without TGF-beta(2) treatment. These findings support proteasome inhibition as a therapeutic strategy to prevent PCO.

Characterization of lens proteins. II. γ-Crystallin of normal and cataractous rat lenses

Abstract The γ-crystallin isolated from soluble normal rat lens cortex protein was fractionated into six subfractions (a-f), on SP-Sephadex C-25. Subfraction a, the first peak eluted, constituted 11% and subfractions (b-f) 33, 25, 13, 12 and 6%, respectively of the total protein. The amino acid compositions of the subfractions of the normal rat lens were determined. Arginine, glutamic acid, aspartic acid and tyrosine were the most abundant amino acids followed by glycine, serine and leucine. These data are similar to those previously reported on the subfractions of calf lens γ-crystallin. Glycine was found to be the only N-terminal amino acid in the rat γ-crystallin. The 5+ galactose-induced cataractous rat lens contained less than 10% of the γ-crystallin of the normal rat lens. Six corresponding subfractions were obtained from the γ-crystallin of the cataractous rat lens and the distribution was 5, 27, 41, 13, 12 and 2%. Preparations of γ-crystallin of the calf lens from Sephadex G-75 ( Bjork, 1961 ) and from Sephadex G-200, superfine ( Ocken et al., 1977 ) were fractionated on the SP-Sephadex columns. The separation of both preparations was identical. However, the profiles are different in that our procedure yielded 28% subfraction a while the other yielded 5–8%. The subfractions of γ-crystallin of both the rat and calf were subjected to polyacrylamide disc gel electrophoresis. The protein components shown in the gels were confirmed by the isoelectric focusing patterns. Instability of the subfractionated rat lens γ-crystallin was observed under our experimental conditions. This appears to be due to some form of equilibrium between the major and minor proteins in the subfractions.

Changes during aging in rat lens endopeptidase activity

Protein degradation has been implicated in lens aging and cataract development. We propose that proteolytic enzymes are important in these processes. In this study, lens neutral proteinase activity and thermal stability have been measured as a function of cell age and animal age in rat lens. Epithelial, cortical, and nuclear lens regions from animals between 1 and 25 months were analyzed. Specific activity and thermal stability were found to decrease with lens cell age, that is, from epithelium to nucleus. Specific activity in the cortex increased with animal age, while specific activity in the nuclear region remained constant with animal age. In the epithelium, specific activity showed no correlation with animal age. In 15 day old rats, thermal stability curves for the cortical enzyme were linear, indicating only one form of activity hydrolyzing synthetic substrate was present. At 1.25 months of age, thermal stability curves for the cortical enzyme were non-linear indicating that multiple forms of the activity were present.

Calf lens neutral proteinase activity using calf lens crystallin substrates

Activity of the calf lens neutral proteinase was measured with the calf lens crystallin fractions and purified α-crystallin A and B chains. For all substrates, rates of hydrolysis were linear for at least 1 hr. With α-crystallin, the rate of hydrolysis began to level off after 1 hr, but continued for at least 48 hr at a slower rate. The β L -crystallin fraction was hydrolyzed at the greatest rate, followed in decreasing order by α and β H -crystallin. ψ-Crystallin was not hydrolyzed. The purified B chains of α-crystallin were hydrolyzed at a greater rate than the A chains or unfractionated α 2 -crystallin. In unfractionated α 2 -crystallin a higher rate of degradation of B chain compared to A chain was shown by SDS gel was not extensive, polypeptide products of hydrolysis were identified

The bovine lens neutral proteinase comprises a family of cysteine-dependent proteolytic activities

Inhibitor studies with peptide substrates demonstrate that bovine lens neutral proteinase comprises three distinct activities. Diisopropylfluorophosphate distinguishes the activity hydrolyzing carbobenzoxy-Gly-Gly-Leu-p-nitroanilide (inhibited) from that hydrolyzing carbobenzoxy-Leu-Leu-Glu-2-naphthylamide (not inhibited). Leupeptin inhibits hydrolysis of the substrate carbobenzoxy-Leu-Leu-Arg-2-naphthylamide, but not hydrolysis of carbobenzoxy-Gly-Gly-Leu-p-nitroanilide or carbobenzoxy-Leu-Leu-Glu-2-naphthylamide, demonstrating the presence of the third activity. Inhibition of the three activities by thiol reagents suggests that each activity may be dependent on an active-site cysteine residue.

Age changes in bovine lens endopeptidase activity

Lens endopeptidase activity and thermal stability have been determined as a function of cell development, cell age, and animal age. Lenses from animals aged 3 months to 15 years (lens weights 1.15-2.80 g) were divided into epithelial (outermost), cortical (peripheral), and nuclear (central) regions. Changes accompanying cell development were determined by measuring specific activity in epithelial (undifferentiated), outer cortical (differentiating), inner cortical (mature) and nuclear (aged) regions of individual lenses. Thermal stability of the enzyme activity obtained from the outer cortical and nuclear regions of the same lenses was also determined. Specific activity and thermal stability were found to decrease as a function of lens cell development. Changes with cell development represent the effects of both differentiation and increasing cell age. To determine the effects of cell age alone, activity was determined in the same population of aged, fully differentiated cells in lenses of different ages. Specific activity decreased as a function of cell age alone. Changes with animal age were determined by comparing cells of the same developmental stage from animals of different ages (e.g., differentiating cells of the cortex in animals 3 months to 15 years old). Specific activity for the cortical region increased with animal age while specific activity in the nuclear region appeared to remain constant or decrease slightly with increasing animal age. Thermal stability of the enzyme activity from the cortex was different in young and adult lenses. The change in stability occurred early in the lifespan and was therefore more closely related to animal development than to aging.

A synthetic endopeptidase substrate hydrolyzed by the bovine lens neutral proteinase preparation

Lens neutral proteinase is thought to exhibit primarily endopeptidase activity. We have identified a synthetic endopeptidase substrate which is hydrolyzed by the bovine lens neutral proteinase preparation. Among 11 fluoro- and chromogenic endopeptidase substrates, only carbobenzoxy-glycylglycyl-L-leucyl-p-nitroanilide is effectively hydrolyzed. The activity hydrolyzing this substrate co-elutes with neutral proteinase activity upon gel filtration and specifically attacks the leucyl-p-nitroaniline bond. Optimal hydrolysis of the synthetic substrate is at neutral pH and high temperature (53 degrees C), analogous to the alpha-crystallin protein substrate obtained from lens. The rate of hydrolysis of the synthetic substrate increased proportionally with temperature between 20 and 60 degrees C, in contrast to alpha-crystallin. The rate of hydrolysis was linear for at least 1 h at 37 degrees C and there was no evidence of enzyme activation at high temperature.

Targeted disruption of specific steps of the ubiquitin-proteasome pathway by oxidation in lens epithelial cells

Several steps in the ubiquitin-proteasome pathway have been shown to be inhibited in models of oxidative stress and aging. We have designed similar models of aging and oxidation in the HLE B-3 human lens epithelial cell line. Following hydrogen peroxide (H2O2) treatment, B-3 cells exhibited an expected activation of c-fos. The effect of these same and similar treatments on the lens proteasome system was unexpected. The 2D gel pattern and the chymotrypsin-like activity of the 20S core were unaffected by this H2O2 treatment, contrary to previous experience in other culture systems. The critical role of proteolysis in the aging lens, and the strong tie between oxidation and proteasome changes, urged us to further model lens oxidation and investigate several steps of the ubiquitin-proteasome pathway with an alternative agent: the thiol-specific oxidant, diamide. The 20S core proteasome, de-ubiquitinating, and ATP-dependent 26S proteasome activities all showed decreases 10 min after diamide was applied, and recovered to near normal within 1h. The higher, 300 microM dose inhibited the 20S by 43%, the de-ubiquitinating activity by 17% and the 26S by 31%. The comparable susceptibility of the 20S activity and the 26S activity differs from several previously published models. Such differences may be the result of tissue or cell line-specific variants in either the components of the ubiquitin-proteasome pathway or in their modification by intracellular oxidants or reductants.