RGDRGD肽对人晶状体上皮细胞黏附与增生作用的影响

背景RGD肽是一类含有精氨酸-甘氨酸-天冬氨酸的小分子多肽,其在抑制肿瘤细胞黏附、转移和肿瘤新生血管的生成中起重要作用。研究证实RGD肽能够抑制晶状体上皮细胞（LECs）的黏附和增生,RGDRGD肽串联起来作用增强。目的研究RGDRGD肽对体外培养的人LECs黏附与增生的影响,并与RGD肽的作用进行比较。方法将在体外分离培养的LECs中分别加入250、500、1000mg／L的RGDRGD肽和RGD肽作为实验组,仅加入细胞培养液作为对照组。将LECs以2×10。／ml密度接种到含有纤维连接蛋白（FN）和I型胶原蛋白预孵化的96孔培养板中,培养1h后用MTT比色法检测RGDRGD肽与RGD肽对细胞黏附率的影响。将LECs接种于培养板,分别加入250、500、1000、2000mg／L的RGDRGD肽和RGD肽培养24、48、72h,检测RGDRGD肽和RGD肽对LECs增生的影响。结果RGDRGD肽对人LECs黏附率的抑制呈明显的剂量依赖性,随着其质量浓度的增加,对细胞黏附的抑制作用越强,500mg／L的RGDRGD肽比RGD肽抑制人LECs黏附的作用更强（P〈0．01）。RGDRGD肽对人LECs增生的抑制呈明显的时间剂量依赖性,1000mg／L的RGDRGD肽作用48h比RGD肽对人LECs增生的抑制作用更强（P〈0．01）。结论RGDRGD肽抑制LECs黏附与增生的作用强于RGD肽,为进一步临床应用提供了理论依据。     

Collagens XII and XIV (FACITs) in capsular opacification and in cultured lens epithelial cells

PURPOSE: We previously reported that extracellular matrix (ECM) accumulation in human capsular opacification included collagen types I, III, IV, V, and VI. To further characterize the ECM in capsular opacification we performed immunohistochemistry to localize collagen types XII and XIV (fibril-associated collagens with interrupted triple helices, or FACITs) in specimens of human capsular opacification and in cultures of bovine lens epithelial cells (LECs). METHODS: Cryosections and paraffin sections of human capsular opacification specimens or uninjured lens capsules, as well as cultured bovine LECs, were processed for immunohistochemistry using antibodies against collagen types I to VI, XII, and XIV. A rat crystalline lens was punctured through the central cornea and the eye was processed for immunohistochemistry for FACITs after healing intervals. RESULTS: In the absence of injury human LECs were unstained for FACITs, but as early as 10 days after operation, LECs in healing capsules were immunoreactive. Collagen types I, III, IV, V, and VI were also detected. ECM deposited in confluent LEC cultures stained for FACITs. Normal rat LECs were not stained for FACITs, but ECM accumulated in injured lens stained for them. CONCLUSIONS: LECs up-regulate FACITs during post-opera-tive healing. FACITs, as well as other collagen types, are deposited in ECM in healing injured rat lens, in human capsular opacification and in LEC cultures. ECM components may regulate LEC behavior during postoperative healing.     

Latent TGFβ binding protein-1 and fibrillin-1 in human capsular opacification and in cultured lens epithelial cells

BACKGROUND/AIM: It was previously reported that collagenous extracellular matrix (ECM) in human capsular opacification contained isoforms of transforming growth factor beta (TGFbeta). In the present study, the authors performed immunohistochemistry to examine whether ECM in human capsular opacification and in cultures of bovine lens epithelial cells (LECs) contained latent TGFbeta binding protein-1 (LTBP-1), TGFbeta1 latency associated peptide (beta1-LAP), and fibrillin-1, a suspected ligand of LTBP-1 as well as a component of the extracellular microfibrillar apparatus. The aim of the study was to further clarify the mechanism of TGFbeta1 deposition in ECM of capsular opacification. METHODS: Human capsular opacification specimens and uninjured lens capsules, as well as cultured bovine LECs, were processed for immunohistochemistry using antibodies against LTBP-1, beta1-LAP, fibrillin-1, and collagen type I. RESULTS: LTBP-1, beta1-LAP, and fibrillin-1 all were localised to the ECM in human capsular opacification. Uninjured lens epithelium stained for beta1-LAP, but not for LTBP-1 and fibrillin-1. ECM deposited in confluent LEC cultures stained for LTBP-1, beta1-LAP, and fibrillin-1, while cultures with only sparse cellularity were unstained for LTBP-1 or fibrillin-1. CONCLUSIONS: LECs upregulate LTBP-1 and fibrillin-1 during postoperative healing. LTBP-1, beta1-LAP, and fibrillin-1 colocalised to the ECM in capsular opacification and in confluent LEC cultures. TGFbeta1 is considered to deposit in ECM in the large latent form. ECM secreted by LEC may function as a scavenger or repository of TGFbeta.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases of fibrous humans lens capsules with intraocular lenses

PURPOSE: We located immunohistochemically the matrix metalloproteinases (MMP) -1, -2, -3 and -9 and the tissue inhibitors of matrix metalloproteinases (TIMP) -1 and -2 in the fibrous capsule of patients with intraocular lenses (IOLs). METHODS: During vitreoretinal surgery in 10 patients we obtained post-cataract surgery lens capsules with or without an IOL. The mean interval between the previous cataract operation and the extraction of the specimens was 35.2 months (range: 2-120 months). Circular sections of the anterior capsule with lens epithelial cells (LECs) were also obtained during cataract surgery. Specimens were processed for immunohistochemical identification of MMPs and TIMPs by light microscopy. RESULTS: While all the members of MMPs and TIMPs were not detected in the normal anterior capsules, they were detected in the ECM and/or LECs on the lens capsules extracted within 18 months after IOL implantations in all of the 4 patients, but were not observed in specimens obtained 18 months or longer postoperatively. In LECs of 1 capsule specimen 10 years postoperatively, MMP-1, but not other MMPs and TIMPs, was detected. CONCLUSIONS: MMPs and TIMPs were detected in the ECM and/or LECs on post-cataract surgery capsules. These proteins may be remodeling the newly deposited ECM and regulating LEC behavior on residual lens capsules in the early phase of healing after cataract surgery.     

Effect of minoxidil on rabbit lens epithelial cell behavior in vitro and in situ

BACKGROUND: Lens epithelial cell (LEC) proliferation and the associated production of extracellular matrix (ECM) are responsible for capsular opacification after cataract-IOL surgery. Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in procollagen hydroxylation. To evaluate the potential efficacy of minoxidil in inhibiting postoperative capsular opacification, we examined the effects of minoxidil on LEC behavior in cell and organ cultures. METHODS: We examined minoxidil effects on collagen production, migration and proliferation of cultured rabbit LECs as well as its ultrastructural effects, and also its effects on the cell population in organ-cultured capsular bag. RESULTS: No cytotoxicity was identified by MTT assay at the concentrations up to 3.0 mM of minoxidil, whereas it decreased the collagen production in LECs. Minoxidil also inhibited migration and proliferation of cells. Ultrastructural observation revealed the presence of dilated endoplasmic reticulum in LECs treated with minoxidil, indicating the accumulation of protein, probably underhydroxylated collagen precursors. The capsules cultured with minoxidil appeared less opaque than control specimens. On histological examination the numbers of cells on equatorial capsules were found to be significantly lower in minoxidil culture than in control culture. No lens cells were detected on the central posterior capsule of minoxidil culture, whereas they were seen in control. CONCLUSION: Minoxidil inhibited LEC migration and proliferation in vitro, as well as collagen secretion. Collagen secretion may be essential for LEC migration and proliferation. Minoxidil also attenuated repopulation of LECs on the inner surface of organ-cultured capsules. Minoxidil may be a potential inhibitor of postoperative capsular opacification.     

Selenium functionalized intraocular lenses inhibit posterior capsule opacification in an ex vivo canine lens capsular bag assay

The purpose of this study was to determine the inhibitory effect of selenocystamine coated intraocular lenses (IOLs) on the formation of posterior capsule opacification (PCO) in an ex vivo canine lens capsular bag assay. Selenocystamine was covalently bound to the surface of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) discs. Three groups of canine lens capsules (6 coated IOLs (SeIOLs), 7 non-coated control IOLs and 8 empty capsules) were cultured for 10 days. During the culture period PCO was scored based on visual inspection of the capsules using phase contrast microscopy. On day 10 all the capsules were prepared for light microscopic examination and lens epithelial cells (LECs) were quantified. Proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA) and cleaved caspase-3 were examined by immunohistochemistry. Additionally, cell viability assays were performed on LECs cultured in tissue culture medium pre-incubated with either a SeIOL or control IOL. The viability assays demonstrated that no detectable cytotoxic leachables were associated with the functionalized IOLs. The central posterior capsule was free of cells underneath all SeIOLs, although large numbers of LECs populated the capsular periphery. Apoptotic cells were observed underneath the periphery of some SeIOLs. Both the PCO scores and LEC counts of SeIOL containing capsules were significantly lower than those of control group capsules (p < 0.01 and p = 0.0004, respectively).The use of selenium functionalized IOLs resulted in a significant reduction of PCO in this ex vivo model. Binding of selenocystamine to a foldable IOL may provide an effective method to prevent population of the central posterior capsule with LECs.     

Pharmacologic prevention of posterior capsule opacification: in vitro effects of preservative-free lidocaine 1% on lens epithelial cells

PURPOSE: To assess the in vitro effectiveness of preservative-free lidocaine 1% in removing lens epithelial cells (LECs) from the anterior capsule and to evaluate the effect of lidocaine on the LECs. SETTING: Center for Research on Ocular Therapeutics and Biodevices, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA. METHODS: Eight rabbits (16 eyes) were used in the study. After the rabbits were killed, the eyes were enucleated and divided into 2 groups. In Group 1 (n = 8 eyes), LECs were exposed to preservative-free lidocaine 1% or balanced salt solution (BSS) for 1, 2, or 5 minutes. The anterior capsules were then stained with trypan blue and alizarin red. Photomicrographs of each capsule were taken and analyzed for LEC damage. In Group 2 (n = 8 eyes), hydrodissection was performed with 1 of the agents, followed by phacoemulsification and cortical cleanup. The LEC attachment to the anterior capsule was evaluated by histopathology. RESULTS: Anterior capsule fragments irrigated with BSS showed no LEC nuclear staining; ie, no direct toxic effect. In those irrigated with preservative-free lidocaine 1%, the LECs showed mild toxicity; some cells showed blue nuclear staining. After hydrodissection with lidocaine, the capsules were almost free of LECs; after hydrodissection with BSS, the capsules showed a normal layer of LECs attached to the anterior capsule. CONCLUSIONS: Preservative-free lidocaine 1% may help diminish the amount of live LECs by facilitating cortical cleanup, by loosening the desmosomal area of cell-cell adhesion with decreased cellular adherence, or by a direct toxic effect. The use of this agent may help prevent posterior capsule opacification.     

Lens epithelial cell death after cataract surgery

PURPOSE: To determine whether lens epithelial cells (LECs) undergo apoptosis during healing after cataract surgery to further characterize the healing process of the postoperative lens capsule. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: Apoptotic cells were detected in human postoperative lens capsules using the terminal deoxynucleotidyl transferase deoxy-UTP nick-end labeling (TUNEL) method. The effect of exogenous transforming growth factor-beta2 (TGF-beta2) on mouse LEC apoptosis was also examined in an organ-culture system. RESULTS: Three of 17 postoperative specimens contained TUNEL-positive cells. In 2 lens capsules obtained earlier than 10 days, many TUNEL-positive cells, presumably apoptotic LECs, were observed beneath the residual anterior capsule. In cell multilayers in capsule opacification extracted later than 10 days, a few TUNEL-positive cells were seen in 1 specimen; most cells remained negative. In mouse lenses organ-cultured with 1.0 ng/mL TGF-beta2 for 48 hours, TUNEL-positive cells were detected beneath the lens capsule. CONCLUSIONS: Lens epithelial cells undergo apoptosis during healing after cataract surgery, especially in the early phase. Transforming growth factor-beta2 may be a factor inducing apoptosis in in vivo LECs.     

Failure of a discontinuous bend to prevent lens epithelial cell migration in vitro

PURPOSE: To assess the effect of substrate geometry (discontinuous bend) on lens epithelial cell (LEC) growth in vitro. SETTING: Department of Ophthalmology, St. Thomas' Hospital, London, United Kingdom. METHODS: Culture wells with central depths of 0.4 mm, 1.0 mm, or 3.0 mm and a sharp square-edged profile (discontinuous bend) or a round-edged profile (continuous bend) were produced from a block of poly(methyl methacrylate). Freshly harvested bovine LECs were attached to the center of each well and cultured using standard techniques. Observations were made of whether LECs grew out of the wells and of the time required to do so. RESULTS: Lens epithelial cells migrated out of all the wells. There was no significant difference in the rate at which they migrated out of round-edged and square-edged wells. CONCLUSIONS: In vitro, a sharp discontinuous bend did not appear to induce contact inhibition of migrating LECs nor did it significantly hinder the rate at which LECs migrated. Therefore, a discontinuous bend in the lens capsule in isolation is unlikely to be responsible for the observed reduction in posterior capsule opacification associated with the use of square-edged IOLs.     

Inhibitory effects of salmosin,a disintegrin,on posterior capsular opacification in vitro and in vivo

The proliferation, migration and transdifferentiation of the remaining lens epithelial cells (LECs) after cataract surgery are a major cause of posterior capsular opacification (PCO). It has previously been reported that salmosin, a novel disintegrin, significantly inhibits solid tumor growth in mice by perturbation of tumor-specific angiogenesis via blocking alpha v beta 3 integrin expressed on vascular endothelial cells. In this study, the inhibitory function of salmosin in PCO was investigated and was found that salmosin inhibits the attachment of bovine LECs and rabbit lens cells (N/N1003A) to extracellular matrix-coated plates. The anti-adhesive activity of salmosin was approximately 1000 times higher than that of synthetic Arg-Gly-Asp peptide. In addition, the cell proliferation and migration of bovine LECs and N/N1003A were strongly inhibited by salmosin, whereas the proliferation of corneal endothelial cells was less affected. LEC migration and proliferation were also decreased by salmosin treatment in rabbit eyes without any toxic effect in the cornea, iris and retina. In this study, salmosin was shown to specifically inhibit LEC migration and proliferation in an animal model. Therefore, the authors suggest that further investigation may show salmosin to be a good candidate for inhibiting PCO development.     

Prevention of posterior capsule opacification by intraoperative single-dose pharmacologic agents

PURPOSE: To determine whether an intraoperative single dose of dexamethasone, diclofenac, ethylenediaminetetraacetic acid (EDTA), a combination of EDTA and RGD peptide (arginine-glycin-aspartic acid sequence), or mitomycin-C (MMC) is a pharmacological means of preventing or reducing the development of posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, Celal Bayar University, School of Medicine, Manisa, and Department of Pathology, Dokur Eylül University, School of Medicine, Izmir, Turkey. METHODS: Fifty-four rabbits were randomly divided into 6 groups. Dexamethasone (4 mg/cc), diclofenac (2.5 mg/cc), EDTA (8 mg/cc), a combination of EDTA and RGD peptide (2.5 mg/cc), or MMC (0.04 mg/cc) was given, 0.1 cc by hydrodissection and 0.9 cc into the capsular bag after phacoemulsification. The sixth group served as a control group. After 3 months, the PCO was graded clinically and the proliferation of lens epithelial cells (LECs) was evaluated histologically. RESULTS: The drugs were significantly effective in preventing PCO compared with the control (P <.005). Dexamethasone had a weaker effect than the other drugs. In histological analysis, although monolayer LECs in the dexamethasone and diclofenac groups were observed, there was no proliferative activity on the posterior capsules in the EDTA, EDTA+RGD, and MMC groups in contrast to the multilayer cells in the control. CONCLUSIONS: Intraoperative single-dose application of EDTA, EDTA+RGD peptide combination, and MMC significantly prevented the development of PCO in rabbit eyes. Diclofenac was less effective but also reduced PCO. Although dexamethasone did not prevent the proliferation of LECs, it decreased PCO clinically.     

Detection of tumor necrosis factor alpha and interleukin 1 alpha gene expression in human lens epithelial cells

PURPOSE: To analyze the gene expression of interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF alpha) in human lens epithelial cells (LECs) by in situ RNA hybridization. SETTING: Department of Ophthalmology and Laboratory for Molecular Biology, Charité, Humboldt University, Berlin, Germany. METHODS: Anterior lens capsules with attached LECs were collected in RNase-free conditions from 10 consecutive patients during cataract surgery. Samples were then systematically analyzed by an in situ RNA-hybridization technique using specific gene probes for IL-1 alpha and TNF alpha, which were previously labeled with digoxigenin (DIG). RESULTS: The LECs tested positive for DIG-labeled gene probes in the described conditions. One (10%) patient showed a clearly detectable IL-1 alpha gene expression, and 7 (70%) showed a widely positive reaction for TNF alpha mRNA. CONCLUSION: The TNF alpha gene expression in LECs was more extended than that of IL-1 alpha in lens capsule samples from cataract surgery. Active synthesis of TNF alpha and IL-1 alpha may have consequences for postoperative inflammation and LEC proliferation.     

Use of a polylysine-saporin conjugate to prevent posterior capsule opacification.

PURPOSE: To determine the feasibility of applying a polylysine-saporin (PLS) conjugate to the lens capsule at surgery to prevent lens epithelial cell (LEC) proliferation and posterior capsule opacification (PCO). SETTING: Department of Research & Development, Bausch & Lomb Surgical, and Department of Ophthalmology, Saint Louis University, St. Louis, Missouri, USA. METHODS: Fluorescein-labeled polylysine was applied to the lens capsule of rabbits after phacoemulsification and analyzed histologically to determine the extent of binding to the lens capsule and surrounding tissues. The cytotoxin saporin was conjugated to polylysine using bifunctional cross-linkers. This PLS conjugate was applied to LECs in culture and to the lens capsules of rabbits. These eyes were monitored for PCO. RESULTS: Polylysine primarily bound to the lens capsule membranes, with little or no binding to surrounding tissues. When PLS was added to LECs in culture, it was internalized and destroyed the cells. Of 9 rabbit eyes treated with PLS during surgery, 1 remained free of PCO for the life of the animal (40 weeks), while 6 showed a delay of cortical regrowth approximately 2 to 3 times that of control eyes. CONCLUSIONS: Polylysine bound selectively to the lens capsule membrane. The PLS conjugation resulted in a toxic agent that targeted the lens capsule and destroyed proliferating LECs. The application of a PLS conjugate during surgery may prevent PCO.     

Upregulation of alphavbeta6 integrin, a potent TGF-beta1 activator, and posterior capsule opacification

PURPOSE: To identify the predominant activation pathway of transforming growth factor (TGF)-beta1 in the lens capsule, studying the spatial and temporal expression pattern of alphavbeta6 and thrombospondin-1. Other PCO-related proteins were also studied. SETTING: Departments of Ophthalmology and Optometrics and Clinical Pathology, Medical School, University of Vienna, Vienna, Austria. METHODS: The lens capsules of 12 human donor eyes were cultivated in a protein-free medium for up to 28 days (cultivated lens capsules [CLCs]) after lens extraction. Ten intact lenses (ILs) served as the control group and were also cultured. During the culture period, cell dynamics were observed by phase-contrast microscopy. Proteins were detected by double immunofluorescence on frozen sections. RESULTS: In ILs, alphavbeta6 was absent but 91.6% of the CLCs showed extensive staining. Remnant lens epithelial cells (LECs) expressed alphavbeta6 immediately after lens extraction. The alphavbeta6 was detected throughout the culture period in all regions of the capsule. Thrombospondin-1 was absent in ILs and CLCs, suggesting that this protein is not significant in TGF-beta1 activation in the lens. Transforming growth factor-beta1 was abundantly expressed in all ILs and CLCs, slightly decreasing during intensive LEC proliferation and migration. The TGF-beta receptor II (RII) was expressed equally in all specimens, decreasing with culture time. Nonresident extracellular matrix proteins and alpha-smooth muscle actin were partially detected in CLCs but not in ILs. Latent TGF-beta binding protein 1 and collagen III were absent in all specimens. All cells found in the cultures expressed vimentin and alphaB-crystallin (LEC markers). CONCLUSION: Alphavbeta6 is the main activator of TGF-beta1 in the lens capsule and represents a new target for PCO prevention.     

Effect of posterior convexity of intraocular lenses on lens epithelial cell migration

PURPOSE:To investigate in vitro how the posterior convexity of an intraocular lens (IOL) affected the migration of lens epithelial cells (LECs) under its optic. METHODS: Porcine LECs were cultured for 9 days with polymethylmethacrylate (PMMA) IOLs in a cell culture chamber insert containing a collagen membrane on which the IOLs were implanted. The central sagittal optic depths of the implanted IOLs were 0, 0.158, 0.303, and 0.452 mm. The migration of LECs was observed with an inverted phase microscope. The cell-free area under the IOL optic, where LECs had not migrated, was measured. RESULTS: As time elapsed, LECs migrated onto the collagen membrane beneath the IOL optics from the periphery to the central area in a concentric fashion in all IOL configurations. At 5 days in culture, the greater central sagittal optic depths of the IOL optic were associated with wider cell-free areas (P=.0108). At 9 days in culture, LECs almost completely covered the collagen membrane under IOLs with 0-, 0.158- and 0.303-mm central sagittal optic depth whereas the cell-free area under the 0.452-mm IOL was 4.3+/-3.0% (P=.0029). CONCLUSIONS: The posterior convexity of an IOL optic has an inhibitory effect on LEC migration under the optic. However, this inhibition had little effect after 9 days in culture.     

In vitro model for the study of human posterior capsule opacification

PURPOSE: To develop and evaluate a model for the organ culture of human lens capsules that reduces problems inherent in preexisting models for the study of in vitro posterior capsule opacification (PCO). METHODS: Human lenses (N = 110) were isolated from donor eyes and supported externally within a lens holder system by medical-grade cyanoacrylate glue, allowing visualization of the entire capsular bag. After capsulorhexis and lens extraction were performed, the capsule specimens were maintained at physiological conditions for up to 4 weeks. The area of lens epithelial cell (LEC) coverage over the posterior capsule surface was determined objectively on a daily basis using a graticule. Lens epithelial cell behavior was correlated with clinical data and other in vitro PCO models. RESULTS: Cyanoacrylate glue did not appear to be toxic to LECs at the concentration used. The amount of viable epithelium after nuclear extraction was dependent on the age and postmortem time of the specimen. Viable LEC cultures were obtained from eyes up to 9 days postmortem. The time from death to culture or from enucleation to culture did not influence LEC viability if it was fewer than 5 days. The LEC proliferation rates and confluence times were age dependent and correlated closely between pairs of eyes. CONCLUSIONS: Results show that the lens holder model is a more physiological method for supporting the capsule and is a robust, reproducible system for the study of LEC migration and proliferation. It allows visualization within the entire capsular bag. Intraocular lenses can be implanted in this system in a way that more closely resembles the in vivo scenario. This model can be used to evaluate therapeutic measures to prevent PCO.     

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.     

Lens epithelial cells in an in vitro capsular bag model: lens-in-the-bag versus bag-in-the-lens technique

PURPOSE: To evaluate the difference in lens epithelial cell (LEC) growth between lens-in-the-bag (LIB) implantation and bag-in-the-lens (BIL) implantation in an in vitro capsular bag model. SETTING: Antwerp University Hospital, Department of Ophthalmology and University of Antwerp, Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, Antwerp, Belgium. METHODS: Thirty capsular bags of human donor eyes were placed in culture for 4 weeks. They were divided into 3 groups: capsular bags without intraocular lens (IOL) implantation and capsular bags with LIB or BIL implantation. Each group was divided into 3 subgroups depending on the composition of the culture medium: calf serum only; calf serum enriched with basic fibroblast growth factor (b-FGF); calf serum enriched with transforming growth factor beta (TGF-beta(2)). All capsules were evaluated by confocal microscopy. A fourth group of noncultured capsules was kept as a control. RESULTS: Lens-in-the-bag implantation led to extensive proliferation and differentiation of LECs; BIL implantation showed little LEC proliferation and no differentiation. CONCLUSIONS: Bag-in-the-lens implantation reduced LEC proliferation and prohibited LEC differentiation in the remaining lens bag of human donor eyes, proving experimentally what has been observed clinically. This finding suggests that using this peripheral part of the capsular bag can promote postoperative accommodation.     

Water-mediated lysis of lens epithelial cells attached to lens capsule

PURPOSE: To investigate the effect of distilled deionized water (DDW) on lens epithelial cells (LECs) attached to the lens capsule. SETTING: Wound Healing Research Laboratory, Center for Vision Research, Westmead Hospital, Sydney, NSW, Australia. METHODS: Anterior capsulotomy specimens taken during routine cataract surgery were divided in half. One half was immersed in DDW and the other half in culture medium (control) for 1 to 5 minutes and photographed at intervals by phase-contrast microscopy. In further experiments, the capsules were exposed to DDW for 1 or 2 minutes and placed in culture for 1 week to determine whether LECs survive treatment and are capable of repopulating the lens capsule. RESULTS: Distilled-deionized water induced marked swelling of the cytoplasm within 60 seconds of treatment. At 120 seconds, there was disruption of the plasma membranes, with few intact cells remaining. In the control capsules, confluent monolayers of LECs covered the entire capsule surface with a halo of LECs growing on the surrounding plastic well. Viable LECs were observed in 1 of 3 capsules treated for 1 minute with DDW. These did not reach confluence or grow off the capsule onto the surrounding well. No viable LECs were seen on capsules exposed to DDW for 2 minutes. CONCLUSIONS: Short exposure of LECs to DDW induced extensive and rapid cell lysis. Distilled-deonized water may be a useful agent for instillation in the capsular bag during sealed-capsule irrigation to prevent posterior capsule opacification.