Growth Of Lens Epithelial Cells Research Articles

Improved Performances of Intraocular Lenses by Poly(ethylene glycol) Chemical Coatings

Cataract surgery is a routine ophthalmologic intervention resulting in replacement of the opacified natural lens by a polymeric intraocular lens (IOL). A main postoperative complication, as a result of protein adsorption and lens epithelial cell (LEC) adhesion, growth, and proliferation, is the secondary cataract, referred to as posterior capsular opacification (PCO). To avoid PCO formation, a poly(ethylene glycol) (PEG) chemical coating was created on the surface of hydrogel IOLs. Attenuated total reflectance Fourier transform infrared spectroscopy, "captive bubble" and "water droplet" contact angle measurements, and atomic force microscopy analyses proved the covalent grafting of the PEG chains on the IOL surface while keeping unchanged the optical properties of the initial material. A strong decrease of protein adsorption and cell adhesion depending on the molar mass of the grafted PEG (1100, 2000, and 5000 g/mol) was observed by performing the relevant in vitro tests with green fluorescent protein and LECs, respectively. Thus, the study provides a facile method for developing materials with nonfouling properties, particularly IOLs.

061 Correction de la presbytie - l’enjeu de la prévention de l’opacification de la capsule postérieure

Posterior capsule opacification (PCO) still remains one of the major complications in cataract surgery. Problems especially emerge with the design of accommodative and multifocal intraocular lens (IOL). For proper function these IOLs require elastic lens capsules without any tendency of after cataract formation to avoid reduced mobility of the IOL and IOL decentration. Furthermore future concepts of presbyopia correction such as injectable IOL materials also highly depend on elastic lens capsules without any capsule opacification. The most accepted concept of PCO prevention today is the sharp edge design of an IOL. The sharp edge independently of IOL material can inhibit PCO development. Other current investigated strategies focus on the genetic and pharmacological manipulation of lens epithelial cells (LEC) or on the removal of these cells. A new in vitro PCO model for testing new strategies and substances which could influence LEC growth will be presented. This model is based on the stabilisation of the capsular bag by a capsular tension ring (CTR). It offers excellent stabilisation of the capsule and physiological conditions in organ culture. The influence of different CTRs on PCO was evaluated. We found, that CTRs do not completely prevent PCO development in vitro, however different CTR designs show different shapes of capsule expansion and different pattern of LEC growth. Cystamine, β-lactose and alkylphosphocholine, all antiproliferative but non toxic substrates were tested: Although none of the substances evaluated could fully prevent LEC migration in the in vitro PCO model, β-lactose and alkylphosphocholine were highly effective in delaying LEC migration on the capsule bag when compared to untreated LEC. In a third approach the laser photolysis system of A.R.C Laser® was adapted for capsule polishing by ablation of LEC. After cataract extraction multiple laser pulses were applied to mechanically remove LEC. The lowest efficient energy necessary for removal of LEC was evaluated in the in vitro model. The introduced in vitro model offers an easy and safe way to investigate the impact of innovative approaches on PCO prevention.

Evaluation of Lens Epithelial Cell Migration In Vivo at the Haptic-Optic Junction of a One-Piece Hydrophobic Acrylic Intraocular Lens

To evaluate the pattern of lens epithelial cell (LEC) ingrowth behind the intraocular lens (IOL) optic in patients implanted with the AcrySof SA60AT one-piece IOL. Retrospective case series. Patients implanted with the AcrySof SA60AT one-piece IOL with complete overlap of the capsulorrhexis and IOL optic and no ocular pathology were selected from the practices of six surgeons. High-resolution digital images of the distribution of LECs were captured. Circumferential location and extent of LEC growth behind the optic were measured. Mean +/- SD follow-up was 13.2 +/- 1.7 months for 40 patients (mean age 72 years). LEC migration beyond the edge of the IOL optic was displayed by 57.5% (23/40) of patients. LECs migrated into the visual axis in 22.5% (9/40) of patients, leading to a visual acuity of <6/9 (20/30) in 10% (4/40) of patients. Cell migration was located preferentially at the optic-haptic junction. Serial photographs were used to monitor LEC migration over time. In some cases, LECs extended from the opposing optic-haptic junctions to converge in the center of the IOL, creating a path across the optic. The optic-haptic junction of the AcrySof one-piece IOL is a point of weakness in the barrier effect of the square-edge IOL design that provides migrating LECs access to the posterior capsule.

Modification of the surface properties of a lens material to influence posterior capsular opacification

To study the effect of surface properties of materials on cellular behaviour and the formation of posterior capsular opacification (PCO). Polymethylmethacrylate, silicone and a hydrophobic acrylic were plasma treated and used in tissue culture. The changes in surface properties were quantified by dynamic contact angle measurements. Bovine lens epithelial cells (BLECs) were seeded onto these materials and cultured for 1 month. Serial photographs were taken. The cells were then fixed and stained to facilitate counting. Plasma treatment significantly increased the hydrophilicity of surfaces. BLECs grew on all surfaces but significantly more cells adhered to the treated than the untreated surfaces. On the untreated surfaces the BLECs had a fibroblastic morphology whereas on the treated surfaces the cells maintained their epithelial morphology. Posterior capsular opacification is a form of wound healing and the behaviour of lens epithelial cells is central to its progression. Emphasis has been on the elimination of residual lens epithelial cells to combat PCO. This study demonstrated that the phenotype of BLECs was influenced by the surface properties of the intraocular lens materials. Gas plasma treatment of the materials increased their hydrophilicity and allowed the adhered BLECs to maintain their normal epithelial morphology. We believe that controlled growth of lens epithelial cells may reduce the incidence of PCO.

Development of an accommodating intra-ocular lens—In vitro prevention of re-growth of pig and rabbit lens capsule epithelial cells

Cataract surgery is routinely performed to replace the clouded lens by a rigid polymeric intra-ocular lens unable to accommodate. By implanting a silicone gel into an intact capsular bag the accommodating properties of the natural lens can be maintained or enhanced. The implantation success of accommodating lenses is hampered by the occurrence of capsular opacification (PCO) due to lens epithelial cell (LEC) growth. In order to prevent LEC proliferation, a treatment regime using actinomycin D, cycloheximide and water was developed. The effectiveness of treatment was analyzed using an in vitro, MTT-based cell culture system and an ex vivo pig eye model in which the implanted lens-in-the-bag is cultured as a whole. LEC were exposed to treatment solutions for 5 min, then the cells were allowed to recover and to re-colonize the substratum. MTT conversion by cells was transiently inhibited by cycloheximide dissolved in water and by water alone. Exposure to actinomycin D resulted in a lasting inhibition of MTT conversion and consequently cell proliferation. These in vitro data could not be fully reproduced in the ex vivo pig eye model due to essential differences between both models. Treatment with actinomycin D containing solutions, however, resulted in a nearly complete absence of cells on the capsular wall. The pig eye model is a promising approach to further evaluate the effects of peri-surgical treatment during the accommodating intra-ocular lens implantation.

The study of posterior capsular opacification after cataract surgery Senile cataractous lens epithelial cells culture

目的 观察老年性白内障晶状体上皮细胞体外培养的生长特点,为研究老年性白内障及术后后囊浑浊的发生机制及防治奠定基础.方法应用改良组织块培养法对超声乳化术中老年性白内障晶状体前囊上皮细胞进行体外培养,在倒置显微镜下观察其生长和分化的规律.结果前囊接种3～5天,有新生上皮细胞自囊片的边缘长出并向四周延伸,第3～4周部分细胞内出现空泡和颗粒等结构改变,生长近于停止;传代培养细胞不能增生.结论老年性白内障晶状体前囊上皮细胞体外增生能力有限,改良组织块培养法培养晶状体上皮细胞简单有效。

Evaluating and defining the sharpness of intraocular lenses

To develop a new method for the evaluation of the optic edge design of intraocular lenses (IOLs). Charité Humboldt University and Berlin Eye Research Institute (Beri), Berlin, Germany. Poly(methyl methacrylate) IOLs with 11 edge designs were manufactured. Each lens design was evaluated using EPCO 2000. Also, edge's ability to stop cell growth was observed by placing each IOL into cell culture and observing cell growth over 18 days on average. Only 3 groups, those with the sharpest edge design, prevented the growth of lens epithelial cells onto the visual axis of the lens. The edge design that effectively stopped cell growth was characterized by an area above the edge of 13.5 microm2 at the most. Results shows that the edge design of IOLs plays an important role in the prevention of posterior capsule opacification. A specific optic edge design was most effective in this prevention.

Effects of GSH,Pirenoxine and "Nei-Zhang-Qing" on the growth of bovine lens epithelial cells

目的 观察还原型谷胱甘肽(GSH)、卡林-U和内障清对牛晶状体上皮细胞(BLEC)生长的影响.方法通过噻唑蓝(MTT)法,观察不同浓度或质量浓度的GSH、卡林-U和内障清对BLEC在24、48和72 h生长的影响.结果GSH(≤400 μmol/L)、卡林-U(≤200 μg/L)、内障清(≤2 mg/L)对BLEC生长无明显影响;GSH(≥2 mmol/L)、卡林-U(≥1 mg/L)、内障清(≥10 mg/L)对BLEC生长有抑制作用.结论一定浓度或一定质量浓度的GSH、卡林-U、内障清对LEC生长有抑制作用。

Intraocular lens–capsular bag imaging with ultrahigh-resolution optical coherence tomography

To compare in vitro ultrahigh-resolution optical coherence tomography (UHR OCT) cross-sectional images of the pseudophakic human autopsy eye with histology to evaluate the potential of this imaging technique for enhanced visualization of the anterior segment, especially the capsular bag, intraocular lens (IOL), and posterior capsule opacification (PCO) formation after cataract surgery.Department of Medical Physics and Department of Ophthalmology, University of Vienna, Vienna, Austria, and Department of Oral and Maxillofacial Surgery Institute of Dentistry, University of Turku, Turku, Finland.Ultrahigh-resolution OCT images were acquired from 7 pseudophakic human autopsy eyes using 1.4 microm axial x 3.0 microm transverse resolution. The axial resolution with UHR OCT is 1.4 microm compared to 10.0 microm with the commercially available OCT. Plastic-embedded histologic sections were obtained in precise alignment with the OCT tomograms.Ultrahigh-resolution OCT cross-sectional tomograms corresponded to the histologic sections. With the wavelength used (800 nm), the anterior and posterior capsules, area of lens epithelial cell growth and extracellular matrix proliferation, and IOL could be clearly visualized. The extent of capsular bag adhesion to the IOL could be detected, as well as the amount of PCO formation.The improved resolution makes UHR OCT a powerful tool in anterior segment imaging and evaluation of the capacity of IOL materials and models to induce capsular bag adhesion. Ultrahigh-resolution OCT may also help in determining the area of origin of PCO after cataract surgery.

Electrical inhibition of lens epithelial cell proliferation: an additional factor in secondary cataract?

Cataract is the most common cause of blindness but is at least curable by surgery. Unfortunately, many patients gradually develop the complication of posterior capsule opacification (PCO) or secondary cataract. This arises from stimulated cell growth within the lens capsule and can greatly impair vision. It is not fully understood why residual lens epithelial cell growth occurs after surgery. We propose and show that cataract surgery might remove an important inhibitory factor for lens cell growth, namely electric fields. The lens generates a unique pattern of electric currents constantly flowing out from the equator and entering the anterior and posterior poles. We show here that cutting and removing part of the anterior capsule as in cataract surgery significantly decreases the equatorial outward electric currents. Application of electric fields in culture inhibits proliferation of human lens epithelial cells. This inhibitory effect is likely to be mediated through a cell cycle control mechanism that decreases entry of cells into S phase from G1 phase by decreasing the G1-specific cell cycle protein cyclin E and increasing the cyclin-Cdk complex inhibitor p27kip1. Capsulorrhexis in vivo, which reduced endogenous lens electric fields, significantly increased LEC growth. This, together with our previous findings that electric fields have significant effects on the direction of lens cell migration, points to a controlling mechanism for the aberrant cell growth in posterior capsule opacification. A novel approach to control growth of lens epithelial cells using electric fields combined with other controlling mechanisms may be more effective in the prevention and treatment of this common complication of cataract surgery.

Biological Compatibility of Polymethyl Methacrylate, Hydrophilic Acrylic and Hydrophobic Acrylic Intraocular Lenses

Purpose: Extensive clinical investigations of the biocompatibility of different intraocular lenses (IOLs) have been made in an effort to optimize the outcome of modern cataract surgery. The aim of this study was to add animal eye experimental implantation data regarding cellular reaction on the anterior surface of IOLs. Methods: Thirteen adult albino rabbits had phacoemulsification/aspiration of the crystalline lens followed by implantation of a posterior chamber IOL in each eye. Three types of IOLs were studied: Hydroview^® (Bausch & Lomb; n = 7), Acrysof^® (Alcon, USA; n = 7), and polymethyl methacrylate (PMMA; HOYA, Japan; n = 7). The animals were killed by intravenous pentobarbital 1, 4, or 8 weeks later. The IOLs were explanted and stained with hematoxylin and eosin, and observed under a light microscope. The shape of mouse ascites-induced macrophages on the anterior surface of the three different IOL types (Hydroview, PMMA, and Acrysof) was studied after 24 h of oven culture. Results: Hydrophilic acrylic IOLs showed the highest affinity for lens epithelial cell (LEC) outgrowth, and the lowest and slowest maturation rate reaction of macrophages. PMMA IOLs showed the lowest affinity for LEC outgrowth, and the highest reaction of macrophages. Hydrophobic acrylic IOLs showed intermediate results both regarding LECs and macrophages. Conclusions: Results suggest that IOL biomaterial properties are the key factor that influences the quantity of monocytes/macrophages as well as the process of their maturation/senescence. LEC outgrowth is influenced both by the biomaterial of IOLs and by the monocyte/macrophage reaction.

Inhibitory Effects of Arg-Gly-Asp (RGD) Peptide on Cell Attachment and Migration in a Human Lens Epithelial Cell Line

Posterior capsule opacification (PCO) after cataract surgery is caused by growth of residual human lens epithelial (HLE) cells on the posterior capsule. We have shown that extracellular matrix (ECM) is an essential factor for HLE cell attachment and migration. The purpose of this study was to examine the inhibitory effects of Arg-Gly-Asp (RGD) peptide on cell attachment and migration in an HLE cell line. HLE cell line cells (SRA 01/04) that were obtained by transfection of large T antigen of SV40 were cultured in the absence of serum. The culture dishes were coated with type IV collagen, laminin or fibronectin, and Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) RGD peptide (0.1, 0.3, 1.0, 2.0 mg/ml) was added to the medium. The number of attached cells was counted after 90 min of incubation, and the inhibitory effects of GRGDSP RGD peptide on cell attachment were calculated. Cell attachment on the fibronectin-coated dishes was inhibited by GRGDSP RGD peptide at concentrations higher than 0.3 mg/ml; the inhibitory rate was 80% at a concentration of 2.0 mg/ml. The inhibition of cell attachment by GRGDSP RGD peptide on laminin-coated dishes appeared only at a concentration of 2.0 mg/ml, whereas no effects were observed on the type IV collagen-coated dishes. The inhibitory effects of GRGDSP RGD peptide on cell migration were measured in medium containing 2.0 mg/ml of GRGDSP RGD peptide after 1, 3, 5 and 7 days of culture. Cell migration was inhibited by GRGDSP RGD peptide from 1 day of culture on the fibronectin-coated dishes and from 5 days of culture on the laminin-coated dishes, whereas no effects were observed on the type IV collagen-coated dishes. GRGDSP RGD peptide inhibited cell attachment and migration on laminin and fibronectin that have RGD sequences. These data suggested that RGD peptide may have the potential to prevent PCO.

Thioredoxin reductase may be essential for the normal growth of hyperbaric oxygen-treated human lens epithelial cells

We have shown previously with in vivo and in vitro animal models that the lens epithelium, in contrast to the nucleus, is remarkably resistant to hyperoxia. The main purpose of this study was to investigate the mRNA response of cultured human lens epithelial cells (LECs) to challenge by a high level of hyperbaric oxygen. Cells were treated for 3 hr with 50 atm of 99% O 2, and then cultured normally for various times up to 11 days. Although the cells appeared normal immediately after the O 2-treatment, they failed to grow and suffered 50% cell loss, as well as significant mitochondrial damage, during normal post-culture. Growth of the cells resumed after 3 days and by day 11, the number of O 2-treated cells was the same as the controls. Remarkably, the 3 hr O 2-treatment produced no immediate effects on either the cellular level of GSH, or on the activities of a number of antioxidant enzymes including glyceraldehyde-3-phosphate dehydrogenase, which is generally regarded as being highly sensitive to oxidation. In contrast, the activity of thioredoxin reductase (TrxR) was severely affected by the O 2, decreasing by 51% after the 3 hr exposure. O 2-induced death of the cells appeared to be caused by loss of ATP since a 31% decrease in ATP level occurred immediately after the O 2-treatment, in spite of a 46% increase in lactate production. Analysis with real-time PCR showed a maximum 3–6-fold increase in mRNA levels 9 hr after the 3 hr O 2-exposure for the enzymes heme oxygenase-1 (HO-1), MnSOD and TrxR1 (the cytoplasmic form of TrxR). These results were confirmed with the use of one-step RT-PCR and Northern blotting. Initial upregulation of message for HO-1 occurred a few hours before any upregulation of MnSOD could be detected, suggesting that release of free iron from the degradation of heme by HO-1 may have played a role in the upregulation of the dismutase. No significant changes in mRNA levels were observed for the antioxidant enzymes catalase, CuZnSOD, glutathione reductase and glutathione peroxidase, or for the antioxidant protein thioredoxin. Recovery of TrxR activity over a 4-day period appeared to parallel the return of the cells to a normal rate of growth. The results indicate that damaging effects of hyperoxia on cultured LECs occur primarily in the mitochondria, rather than in the cytoplasm. Cells avoid O 2-induced cell death, and return to a normal rate of proliferation by upregulating mRNA levels for HO-1, MnSOD and TrxR1. It appears that full activity of TrxR1, an enzyme required for the production of deoxyribonucletides for DNA synthesis, is essential for the normal growth of O 2-challenged LECs.

Role of Gas6/Axl signaling in lens epithelial cell proliferation and survival

Axl is a receptor tyrosine kinase that is activated by Gas6, a growth factor that belongs to the vitamin K-dependent protein family. Although Gas6 binding to Axl has been shown to transmit mitogenic and/or antiapoptotic signals to a variety of cell types, the role of the Axl-Gas6 system in normal and pathological lens biology is not known. We demonstrate for the first time that Axl protein is expressed in normal rat and bovine lens and that its ligand, Gas6, is present in bovine aqueous humor. In addition, we have detected tyrosine-phosphorylated Axl in normal rat and bovine lens epithelial tissues. We further show that human recombinant Gas6 is able to act as a growth factor in cultured human lens epithelial cells by activating Axl and then the AKT signaling pathway. Gas6 mediates a survival and anti-apoptotic response in cultured human lens epithelial cells subjected to serum-starvation (48–72 hr), or treated with transforming growth factor β1 (5 ng ml −1, 48 hr) or tumor necrosis α (100 ng ml −1, 48 hr), as demonstrated by increased number of viable cells, and decreased DNA condensation or caspase-3 activity. In contrast, Gas6 is not able to block apoptosis induced by staurosporin (1 μ m, 5–24 hr) in human lens epithelial cells. Taken together, these data suggest that the Gas6/Axl signaling plays an important role in the control of lens epithelial cell growth and survival and hence in the maintenance of lens homeostasis.

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.

Results of hydrophilic acrylic, hydrophobic acrylic, and silicone intraocular lenses in uveitic eyes with cataract: Comparison to a control group

Purpose: To evaluate the uveal and capsular biocompatibility of hydrophilic acrylic, hydrophobic acrylic, and silicone intraocular lenses (IOLs) in eyes with uveitis. Setting: Department of Ophthalmology, University of Vienna, Vienna, Austria. Methods: This prospective study comprised 72 eyes with uveitis and 68 control eyes having phacoemulsification and IOL implantation by 1 surgeon. Patients received 1 of the following IOLs: foldable hydrophilic acrylic (Hydroview®, Bausch & Lomb), hydrophobic acrylic (AcrySof®, Alcon), or silicone (CeeOn® 911, Pharmacia). Postoperative evaluations were at 1, 3, and 7 days and 1, 3, and 6 months. Cell reaction was evaluated by specular microscopy of the anterior IOL surface and the anterior and posterior capsule reaction, by biomicroscopy. Results: Small round cell deposition was observed on all IOLs in the immediate postoperative period, especially in eyes with uveitis. This reaction decreased 3 to 6 months after surgery. Although the CeeOn 911 had a higher mean grade of small cells, there was no statistical difference between the 3 IOL types after 6 months in the uveitis and control groups. Foreign-body giant cells (FBGCs) increased after 1 week to 1 month. The AcrySof IOLs had the highest number of FBGCs; after 6 months, there was a statistically significant difference between the AcrySof and Hydroview uveitis groups ( P = .036) and the AcrySof and CeeOn 911 uveitis groups ( P = .003) but there was no difference among the 3 IOL types in the control group. Lens epithelial cell outgrowth persisted on the Hydroview IOLs in control eyes and regressed on all 3 IOL types in uveitic eyes and on the AcrySof and CeeOn 911 IOLs in control eyes ( P = .0001). Anterior capsule opacification (ACO) was more severe on all IOL types in uveitic eyes and on the CeeOn 911 IOL in control eyes. Posterior capsule opacification (PCO) was more severe in uveitic eyes. The Hydroview group had more severe PCO than the AcrySof and the CeeOn 911 groups in uveitis and control eyes. Six months postoperatively, the difference was significant ( P = .0001). There was no significant difference between the AcrySof and CeeOn 911 IOLs. Conclusions: Intraocular lens biocompatibility is inversely related to inflammation. Hydrophilic acrylic material had good uveal but worse capsular biocompatibility. Hydrophobic acrylic material had lower uveal but better capsular biocompatibility. Silicone showed a higher small cell count (mild) and more severe ACO but achieved PCO results comparable to FBGC results and better than those with the AcrySof lens 6 months after surgery. Despite the differences in IOL biocompatibility, all patients benefited from the surgery.

Human telomerase accelerates growth of lens epithelial cells through regulation of the genes mediating RB/E2F pathway.

Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The catalytic core of human telomerase is composed of an RNA template known as hTER (human telomerase RNA) and a protein subunit named hTERT (human telomerase reverse transcriptase). We have been studying other functions of the telomerase besides its major role in telomere maintenance. In our previous work, we have demonstrated that the hTERT can functionally interact with a rabbit TER to regulate expression of other genes and also attenuate the induced apoptosis. Here we report that overexpression of hTERT in a human lens epithelial cell line accelerates growth of the transfected lens epithelial cells. Associated with the acceleration of cell growth, expression of p53, p21 and GCIP (Grap2 cyclin-D interacting protein) is downregulated in the hTERT-transfected cells. With the downregulation of p21 and GCIP, the retinoblastoma protein (RB) is completely hyperphosphorylated in the hTERT-transfected cells. As expected, in the presence of RB hyperphosphorylation, the E2F transactivity is upregulated. Inhibition of telomerase activity abolishes the observed growth acceleration and also the related molecular changes. Furthermore, expression of hTERT in telomerase-negative human lens epithelial cells derived from primary cultures also accelerates growth of the transfected cells. Taken together, our results suggest that hTERT, when overexpressed in human lens epithelial cells, accelerates cell growth rate through regulation of RB/E2F pathway and possibly other genes.

The R116C Mutation in αA-crystallin Diminishes Its Protective Ability against Stress-induced Lens Epithelial Cell Apoptosis

alphaA-crystallin is a small heat-shock protein expressed preferentially in the lens and is detected during the early stages of lens development. Recent work indicates that the expression of alphaA-crystallin enhances lens epithelial cell growth and resistance to stress conditions. Mutation of the arginine 116 residue to cysteine (R116C) in alphaA-crystallin has been associated with congenital cataracts in humans. However, the physiological consequences of this mutation have not been analyzed in lens epithelial cells. In the present study, we expressed wild type or R116C alphaA-crystallin in the human lens epithelial cell line HLE B-3. Immunofluorescence and confocal microscopy indicated that both wild type and R116C alphaA-crystallin were distributed mainly in the cytoplasm of lens epithelial cells. Size-exclusion chromatography indicated that the size of the alphaA-crystallin aggregate in lens epithelial cells increased from 500 to 600 kDa for the wild type protein to >2 MDa in the R116C mutant. When cells were exposed to physiological levels of UVA radiation, wild type alphaA-crystallin protected cells from apoptotic death as shown by annexin labeling and flow cytometric analysis, whereas the R116C mutant had a 4- to 10-fold lower protective ability. UVA-irradiated cells expressing the wild type protein had very low TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining, whereas cells expressing R116C mutant had a high level of TUNEL staining. F-actin was protected in UVA-treated cells expressing the wild type alphaA-crystallin but was either clumped around the apoptotic cells or was absent in apoptotic cells in cultures expressing the R116C mutant. Structural changes caused by the R116C mutation could be responsible for the reduced ability of the mutant to protect cells from stress. Our study shows that comparing the stress-induced apoptotic cell death is an effective way to compare the protective abilities of wild type and mutant alphaA-crystallin. We propose that the diminished protective ability of the R116C mutant in lens epithelial cells may contribute to the pathogenesis of cataract.

Anti-allergic Drugs Inhibit the Proliferation of Bovine Lens Epithelial Cells in Culture

To examine the inhibitory effects of tranilast, ketotifen fumarate, and disodium cromoglycate which are used clinically as anti-allergic agents, on the growth of bovine lens epithelial cells (LE) in culture. LE was grown in Dulbecco's modified Eagle's medium(DMEM) with 10% fetal calf serum and growth was measured with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide(MTT) and 5-bromo-2'deoxy-uridine(BrdU). Production of collagen, transforming growth factor-beta 1(TGF-beta 1), and basic-fibroblast growth factor(b-FGF) were measured with corresponding enzyme-linked immunosorbent assay(ELISA). Apoptotic cell death was detected by TdT-mediated dUTP-biotin nick end labelling method(TUNEL technique) and the DNA ladder method. Both ketotifen and tranilast inhibited the growth of LE, and half-inhibitory concentrations were 200 microM and 1,000 microM, respectively. Disodium cromoglycate did not inhibit LE proliferation significantly. Ketotifen and tranilast decreased the synthesis of collagen but had no obvious effect on TGF-beta 1 and b-FGF production. Apoptotic cell death was detected in LE treated with ketotifen or tranilast. Ketotifen and tranilast may be clinically useful for the prevention of aftercataract. Apoptotic cell death may be involved in the process.

Uveal and capsular biocompatibility of hydrophilic acrylic, hydrophobic acrylic, and silicone intraocular lenses

Purpose: To evaluate the long-term response of 6 types of 3-piece intraocular lenses (IOLs) by assessing the cellular reaction on the anterior IOL surface, the behavior of posterior and anterior capsule fibrosis, and flare. Setting: Department of Ophthalmology, Medical School, University of Vienna, Vienna, Austria. Methods: One hundred eighty eyes were prospectively randomized to receive 1 of 6 IOLs: hydrophilic acrylic Hydroview® (Bausch & Lomb) or MemoryLens® (ORC); hydrophobic acrylic AcrySof® MA60BM (Alcon) or AMO Sensar AR40® (Allergan); hydrophobic silicone CeeOn® 920 or CeeOn 911A (Pharmacia). The patients had standardized cataract surgery, postoperative medication, and follow-up. One year after surgery, 155 eyes were assessed. The cellular reaction was evaluated by specular microscopy of the anterior IOL surface. Anterior and posterior capsule opacification (PCO) was assessed semiquantitatively by biomicroscopy. Flare was measured with a Kowa FC-1000 laser flare-cell meter. Results: Regarding uveal biocompatibility, the hydrophobic acrylic IOLs showed the highest incidence of late foreign-body cell reaction (AcrySof, 30%; AR40, 17%) followed by the hydrophilic acrylic (MemoryLens, 8%; Hydroview, 4%) and silicone (CeeOn 920, 4%; CeeOn 911A, 0%) ( P = .0044). In all cases, the cellular reaction was low grade and clinically insignificant. Regarding capsular biocompatibility, some eyes developed lens epithelial cell (LEC) outgrowth on the anterior IOL surface. The highest incidence was in the hydrophilic acrylic group (Hydroview, 85%; MemoryLens, 27%) followed by the hydrophobic acrylic (AcrySof, 4%; AR40, 3%). No silicone IOL had LECs on the anterior surface. The difference among IOL groups was significant ( P = .0001). Anterior capsule opacification was more predominant in the hydrophobic IOL groups. Posterior capsule opacification of the central 3.0 mm area was lowest in the groups with a sharp-edged optic (CeeOn 911A, AcrySof) followed by the round-edged silicone (CeeOn 920), hydrophobic acrylic (AR40), and hydrophilic acrylic IOLs ( P = .0001). There was a significant difference in flare between the AR40 lens and the Hydroview, MemoryLens, CeeOn 911A, and CeeOn 920 ( P < .004). There was no statistically significant difference in the postoperative cell count at 1 year. The power calculation showed that the sample size was sufficient. Conclusions: The differences in cellular reaction, although clinically mild in normal eyes, indicate that there were more giant cells with hydrophobic acrylic IOLs and an increased tendency toward LEC outgrowth with hydrophilic lenses. The incidence of PCO was lowest in the hydrophobic IOL groups, especially in groups with a sharp-edged optic. Second-generation silicone IOLs with a sharp edge had good uveal and capsular biocompatibility 1 year after surgery.