Growth Of Lens Epithelial Cells Research Articles

Effects of Dexamethasone on DNA Synthesis in Lens Epithelial Cells Are Dependent on Cell Type and Growth Factor

Purpose To determine the factors that influence the ability of dexamethasone (dex) to inhibit or stimulate the growth of lens epithelial cells. Method Different growth factors with or without dex (10−6 M) were added to quiescent cultures of two clones of Nakano mouse lens epithelial cells (NK11) in serum-free medium. DNA synthesis was then measured after 8–12 hours by the incorporation of tritiated thymidine. Results Dex was found to both stimulate and inhibit mitogen-induced 3H-thymidine incorporation into the DNA of cultured mouse lens epithelial cells. Enhancement or repression by dex was found to depend on the growth factor used to stimulate the quiescent cell. EGF and insulin were consistently inhibited with dex. Basic fibroblast growth factor (bFGF) and retinoblastoma-derived growth factor (RbDGF) were both enhanced and inhibited by dex, depending on the growth factor concentration and the cell clone used for the experiment. Additionally, RbDGF protects against the dex inhibition of insulin stimulation, but not the inhibition of EGF stimulation. Progesterone, an inhibitor of the activation of the glucocorticoid receptor, blocks the dex inhibitory effect on the EGF and insulin stimulation of DNA synthesis. The ability of progesterone to affect the dex inhibition is consistent with the dex receptor modulating DNA synthesis. The dex effect on DNA synthesis, either stimulatory or inhibitory, was still seen if dex was added as late as 10 hours after the growth factor. Conclusions The study demonstrated that dex reduces the overall growth and activity of lens epithelial cells in vitro. This result provides insight into the risk of developing posterior subcapsular cataracts (PSC) in patients on oral glucocorticoid therapy. Understanding the basic mechanisms by which steroids mediate lens cell growth may provide the ability to more accurately predict who will develop PSC. The present studies show the difference in the effect of dex from lens cell to lens cell, but, more importantly, suggest a pattern of dependent variables that might prove useful in such predictions.

Amphiphilic silicones to mitigate lens epithelial cell growth on intraocular lenses

Silicone intraocular lenses (IOLs) that resist lens epithelial cell (LEC) growth would greatly improve patient outcomes. Herein, amphiphilic surface modifying additives (SMAs) were incorporated into an IOL-type diphenyl silicone to reduce LEC growth without compromising opto-mechanical properties. The SMAs were poly(ethylene oxide)-silane amphiphiles (PEO-SAs) [H-Si-ODMSm-block-PEO8-OCH3], comprised of a PEO segment and siloxane tether of varying lengths (m = 0, 13, and 30). These three SMAs were each blended into the addition cure diphenyl silicone at varying concentrations (5, 10, 15, 20, and 25 μmol g-1) wherein the wt% of PEO was maintained for all SMAs at a given molar concentration. The chemical crosslinking and subsequent retention of SMAs in modified silicones was confirmed. Key material properties were assessed following equilibration in both air and aqueous environments. Silicones modified with SMAs having longer tethers (m = 13 and 30) underwent rapid and substantial water-driven restructuring of PEO to the surface to form highly hydrophilic surfaces, especially as SMA concentration increased. The % transmittance was also maintained for silicones modified with these particular SMAs. The moduli of the modified silicones were largely unchanged by the SMA and remained in the typical range for silicone IOLs. When the three SMAs were introduced at the highest concentration, modified silicones remained non-cytotoxic and LEC count and associated alpha-smooth muscle actin (α-SMA) expression decreased with increasing tether length. These results demonstrate the potential of silicones modified with PEO-SA SMAs to produce LEC-resistant IOLs.

Identification of Differential Gene Expression Pattern in Lens Epithelial Cells Derived from Cataractous and Noncataractous Lenses of Shumiya Cataract Rat.

The Shumiya cataract rat (SCR) is a model for hereditary cataract. Two-thirds of these rats develop lens opacity within 10-11 weeks. Onset of cataract is attributed to the synergetic effect of lanosterol synthase (Lss) and farnesyl-diphosphate farnesyltransferase 1 (Fdft1) mutant alleles that lead to cholesterol deficiency in the lenses, which in turn adversely affects lens biology including the growth and differentiation of lens epithelial cells (LECs). Nevertheless, the molecular events and changes in gene expression associated with the onset of lens opacity in SCR are poorly understood. In the present study, a microarray-based approach was employed to analyze comparative gene expression changes in LECs isolated from the precataractous and cataractous stages of lenses of 5-week-old SCRs. The changes in gene expression observed in microarray results in the LECs were further validated using real-time reverse transcribed quantitative PCR (RT-qPCR) in 5-, 8-, and 10-week-old SCRs. A mild posterior and cortical opacity was observed in 5-week-old rats. Expressions of approximately 100 genes, including the major intrinsic protein of the lens fiber (Mip and Aquaporin 0), deoxyribonuclease II beta (Dnase2B), heat shock protein B1 (HspB1), and crystallin γ (γCry) B, C, and F, were found to be significantly downregulated (0.07-0.5-fold) in rat LECs derived from cataract lenses compared to that in noncataractous lenses (control). Thus, our study was aimed at identifying the gene expression patterns during cataract formation in SCRs, which may be responsible for cataractogenesis in SCR. We proposed that cataracts in SCR are associated with reduced expression of these lens genes that have been reported to be related with lens fiber differentiation. Our findings may have wider implications in understanding the effect of cholesterol deficiency and the role of cholesterol-lowering therapeutics on cataractogenesis.

Evaluation of posterior capsule opacification of the Alcon Clareon IOL vs the Alcon Acrysof IOL using a human capsular bag model

BackgroundPosterior capsule opacification (PCO) after cataract surgery is influenced by intraocular lens (IOL) design and material. The following is an ex vivo comparison of PCO between the Clareon vs. the AcrySof IOL in human capsular bags.MethodsTwenty cadaver capsular bags from 10 human donors were used, with the novel hydrophobic IOL (Clareon, CNA0T0) being implanted in one eye and the other eye of the same donor receiving the AcrySof IOL (SN60WF) following phacoemulsification cataract surgery. Five capsular bags of 3 donors served as controls without IOL. Cellular growth of lens epithelial cells was photo-documented daily. The primary endpoint was the time until full coverage of the posterior capsule by cells. Furthermore, immunofluorescence staining of capsular bags for the fibrotic markers f-actin, fibronectin, alpha smooth muscle actin, and collagen type 1 were performed.ResultsThe new Clareon IOL did not show any disadvantages in terms of days until full cell coverage of the posterior capsule in comparison to the AcrySof (p > 0.99). Both, the Clareon (p = 0.01, 14.8 days) and the AcrySof IOL (p = 0.005, 15.7 days) showed a slower PCO development in comparison to the control (8.6 days). The fibrotic markers f-actin, fibronectin, alpha smooth muscle actin, and collagen type 1 were equally distributed between the two IOLs and differed from the control.ConclusionsA comparable performance has been found in the ex vivo formation of PCO between the two IOLs. Long-term clinical studies are necessary to reach final conclusions.

Ginsenosides induce extensive changes in gene expression and inhibit oxidative stress-induced apoptosis in human lens epithelial cells

BackgroundThe effect of ginsenosides on the growth and apoptosis of human lens epithelial (HLE) B3 cells exposed to H2O2 was investigated. In addition, the effect of ginsenosides on gene expression in HLE-B3 cells was analyzed using microarray assays to determine its molecular mechanism.MethodsHLE-B3 cells were treated with 1.75 M H2O2 in the presence or absence of 5, 10 or 20 μM ginsenosides. Cell viability and apoptosis were examined by MTT assays and flow cytometry, respectively, at 24 to 120 h after the treatment. Furthermore, HLE-B3 cells were treated with 20 μM ginsenosides for 8 days and total RNA was isolated and analyzed using the Affymetrix GeneChip Array. Principal component analysis was performed to visualize the microarray data.ResultsAddition of ginsenosides significantly alleviated the growth inhibitory effect of H2O2 on HLE-B3 cells and the percentage of viable cells was increased by more than 3 folds. Flow cytometric analysis showed that 6.16 ± 0.29% of H2O2-treated HLE-B3 cells were early apoptotic cells, and the percentage was reduced to 4.78 ± 0.16% (P < 0.05) in the presence of 20 μM ginsenosides. Principal component analysis revealed that ginsenoside caused extensive changes in gene expression in HLE-B3 cells. A total of 6219 genes showed significant differential expression in HLE-B3 cells treated with ginsenoside; among them, 2552 (41.0%) genes were significantly upregulated, whereas 3667 (59.0%) genes were significantly downregulated. FOXN2, APP and RAD23B were the top three upregulated genes while WSB1, PSME4 and DCAF7 were the top three downregulated genes in HLE-B3 cells treated with ginsenosides.ConclusionGinsenosides induce extensive changes in the expression of genes involved in multiple signaling pathways, including apoptotic signaling pathway and DNA damage response signaling pathway. Ginsenosides alleviate H2O2-induced suppression of the growth of HLB cells and inhibit H2O2-induced apoptosis of HLB cells.

Dose‐dependent density change of lens epithelial cells after in vivo UVR exposure

AbstractIntroductionOur research group focuses on the mechanism of ultraviolet radiation (UVR) induced lens damage. Posterior capsule opacification (PCO) is the most common complication for cataract surgery. PCO results from the growth and abnormal proliferation of lens epithelial cells (LECs) on the posterior capsule. Lens epithelium is the first target for toxic factors such as ultraviolet radiation. UVR‐300 nm (UVR‐B) is especially an important risk factor for cataract development. Our previous findings show that exposure to UVR‐B can damage the lens epithelium, affect the expression of proteins, induce apoptosis and lead to cataract formation. Therefore, it is possible to prevent PCO by sufficient dose exposure of ultraviolet radiation to residual lens epithelial cells after cataract surgery.PurposeTo determine the effect of different doses of UVR on the distribution of lens epithelial cells (LECs).MethodsAltogether, 40 Sprague Dawley rats were unilaterally exposed in vivo to 1, 3, 6 and 8 kJ/m2 UVR‐300 nm for 15 min. One week after UVR exposure, exposed and contralateral non‐exposed lenses were removed. One midsagittal section from each lens was stained with DAPI for fluorescence microscopy in order to determine the spatial distribution of LECs.ResultsThe LECs density difference between exposed and contralateral non‐exposed eyes at exposure dose of 1, 3, 6 and 8 kJ/m2 was estimated as a CI (95%), −0.1 ± 2.1, 0.1 ± 3.0, −4.1 ± 1.4 and −2.9 ± 2.7 Cell μm−1 10−2, respectively. Data was fit to a linear model considering the initial density difference at 0. The inclination coefficient was estimated as CI (95%), −0.4 ± 0.1 Cell μm kJ−1 1010.ConclusionUVR‐300 nm induces significant LECs density decrease at exposure dose ≥6 kJ/m2. With fitting to a linear model the evolution of dose‐cell density dependence can be determined.

Influence of super-hydrophobic silicone rubber substrate on the growth and differentiation of human lens epithelial cells.

Materials with low cell adhesion are advantageous for production of replacement intraocular lens (IOL) to prevent posterior capsular opacification (PCO). We evaluated the feasibility of compression molding for manufacture of silicone rubber with super-hydrophobic surface and low cell infiltrative characteristics compared to ordinary hydrophobic silicone rubber. Silicone specimens with complex surface topology (super-hydrophobic) or smooth surfaces (hydrophobic) were manufactured by vacuum deforming and molding. Contact angle, microscopic surface structure, and transparency were evaluated. Super-hydrophobic and smooth samples were compared for effects on proliferation, adhesion, and morphology of human lens epithelial cells (hLECs). Epithelial-mesenchymal transition (EMT) was examined by immunofluorescence expression of fibronectin (Fn), Alpha-smooth muscle actin (α-SMA), and vimentin. The surface contact angle of super-hydrophobic silicone was greater than that of smooth silicone (153.8° vs. 116°). The super-hydrophobic surface exhibited a micron-scale palisade structure under scanning electron microscopy (unit length, width, and height of 80, 25, and 25 μm, respectively). However, cell number per 50 × microscopic field on super-hydrophobic surfaces was markedly reduced 24 and 72 h post-seeding compared to smooth surfaces (p < 0.01). Cells were cuboidal or spherical after 72h on super-hydrophobic surfaces, and exhibited numerous surface microvilli with fluff-base polarity, while cells on smooth surfaces exhibited morphological characteristics of EMT. Expression levels of the α-SMA and vimentin were reduced on super-hydrophobic surfaces compared to smooth surfaces. Super-hydrophobic silicon inhibits proliferation, adhesion, and EMT of hLECs, properties that may prevent fibrosis following cataract surgery.

PMD3 - METHODS FOR QUANTIFYING POSTERIOR CAPSULAR OPACIFICATION AND THE IMPACT OF INTRAOCULAR LENS MATERIAL ON SEVERITY SCORES - A SYSTEMATIC LITERATURE REVIEW

Posterior capsular opacification (PCO) results from the growth and abnormal proliferation of lens epithelial cells on the posterior capsule. Severity of PCO is assessed based on the presence or absence of opacification within the visual axis, subjective perception of reduced visual acuity and contrast sensitivity, and assessment of retroillumination images of the posterior capsule. The objective of this review was to identify the methods for quantifying PCO and assess the impact of intraocular lens (IOL) material and design on reported severity scores. A comprehensive literature search was performed for studies published in English between January 1996 and August 2017 in Embase®, MEDLINE®, MEDLINE®-In Process, and Cochrane library. Studies quantifying PCO using different scales were included and impact of lens material and design on the reported severity scores was assessed. A total of 63 RCTs were included in this review. Among the measurement systems used to assess PCO severity, Automated Quantification of After-cataract scale (AQUA; 20 studies), POCOman (13 studies) and Evaluation of Posterior Capsule Opacification scale (EPCO; 12 studies) were the most common. Other methods identified, included various subjective and objective scoring tools. Irrespective of the measurement system, severity of PCO increased with time and there is a trend in the literature to lower severity scores for eyes implanted with hydrophobic acrylic IOLs compared with IOLs of other materials. Furthermore, higher severity of PCO was observed with round optic edge IOLs compared to sharp optic edge IOLs of the same material. There is considerable heterogeneity in terms of the scoring systems used to quantify PCO. However, a trend exists in the available evidence suggesting that the lens material of the implanted IOL impacts on PCO severity. Moreover, sharp edge IOLs appear to provide a better barrier mechanism in preventing PCO than round edge IOLs.

Comparison of fibrotic response in the human lens capsular bag after femtosecond laser–assisted cataract surgery and conventional phacoemulsification

To compare the effect of different laser pulse energy settings in femtosecond laser-assisted cataract surgery with that of standard phacoemulsification and no energy at all used on posterior capsule opacification (PCO) invitro. Cell and Molecular Biology Research Laboratory, Department of Ophthalmology, Ludwig-Maximilians-University Munich, Real Eyes, Ophthalmology Center, Munich, and Institute for Clinical Pathology, Goethe University Frankfurt, Frankfurt, Germany. Experimental study. Fifteen cadaver capsular bags were cultivated from 8 human donors under standard cell culture conditions. For preparation of the capsular bag, 4 groups were established as follows: femtosecond laser-assisted cataract surgery standard energy (n=3), femtosecond laser-assisted cataract surgery high energy (n=3), phacoemulsification (n=6), and hydrodissection without energy (extracapsular cataract extraction) (n=3). Growth of lens epithelial cells was observed and photodocumented. The days until full cell coverage of the posterior capsule were documented. Capsular bags were stained for fibronectin, α-smooth muscle actin, and collagen type1. Cell growth patterns in all treatment groups were comparable, with no statistically significant differences detected at any timepoint measured (P=.81, Kruskal-Wallis). The markers for fibrosis were equally distributed in all groups, indicating an equal fibrotic reaction in all groups. Femtosecond laser-assisted cataract surgery did not increase different cellular responses in PCO formation comparison with phacoemulsification invitro, even when higher laser pulse energy levels were used.

Therapeutic potential of a dual mTORC1/2 inhibitor for the prevention of posterior capsule opacification: An in vitro study.

Mammalian target of rapamycin (mTOR) serves a central role in regulating cell growth and survival, and has been demonstrated to be involved in the pathological progression of posterior capsule opacification (PCO). In the present study, the potency of PP242, a novel dual inhibitor of mTOR complex 1/2 (mTORC1/2), in the suppression of the growth of human lens epithelial cells (HLECs) was investigated. Using a Cell Counting Kit-8 and a wound healing assay, it was demonstrated that PP242 inhibited the proliferation and migration of HLECs. In addition, western blot analysis indicated that PP242 completely inhibited mTORC1 and mTORC2 downstream signaling activities, whereas rapamycin only partially inhibited mTORC1 activity within LECs. Furthermore, PP242 treatment led to an upregulation of the expression levels of p53 and B cell lymphoma-2 (Bcl-2)-associated X and downregulation of Bcl-2. In addition, flow cytometric analysis demonstrated that PP242 induced the cell cycle arrest at the G0/G1 phase, which may have caused apoptosis and induced autophagy within the LECs. The results of the present study suggested that administration of PP242 may potentially offer a novel therapeutic approach for the prevention of PCO.

Collective Migration of Lens Epithelial Cell Induced by Differential Microscale Groove Patterns.

Herein, a micro-patterned cell adhesive surface is prepared for the future design of medical devices. One-dimensional polydimethylsiloxane (PDMS) micro patterns were prepared by a photolithography process. We investigated the effect of microscale topographical patterned surfaces on decreasing the collective cell migration rate. PDMS substrates were prepared through soft lithography using Si molds fabricated by photolithography. Afterwards, we observed the collective cell migration of human lens epithelial cells (B-3) on various groove/ridge patterns and evaluated the migration rate to determine the pattern most effective in slowing down the cell sheet spreading speed. Microgroove patterns were variable, with widths of 3, 5, and 10 µm. After the seeding, time-lapse images were taken under controlled cell culturing conditions. Cell sheet borders were drawn in order to assess collective migration rate. Our experiments revealed that the topographical patterned surfaces could be applied to intraocular lenses to prevent or slow the development of posterior capsular opacification (PCO) by delaying the growth and spread of human lens epithelial cells.

Vloga lečnih epitelnih celic pri razvoju zamotnitve zadnje lečne ovojnice in pri obnovi leče po operaciji prirojene sive mrene

Posterior capsule opacifcation–PCO is the most common complication after cataract surgery. Proliferation and migration of lens epithelial cells that remain in the capsular bag following cataract surgery can lead to the development of PCO, which is the main cause of deterioration of visual function. PCO shows the classic features of fbrosis, including hyperproliferation, migration, deposition of matrix and its shrinkage and transdifferentiation into myofbroblast. Astonishingly, the results of recent research show the importance of lens epithelium for lens regeneration following congenital cataract surgery. New minimally invasive cataract surgery removes only 1–1.5 mm of lens epithelium more laterally, so the major part of the epithelium remains in the capsular bag. Conceptually, the new method differs from the current practice, since it preserves the endogenous epithelial cells of the lens and achieves functional lens regeneration in rabbits and monkeys as well as in human infants with congenital cataracts. Pluripotency of lens epithelial cells and their stem cell nature are crucial for lens regeneration.Ex vivo cultures of the lens capsule explants can be used for testing the pharmacological agents for stimulating and inhibiting the growth of lens epithelial cells. Functionality of the cells and responses to pharmacological agents can be studied by analyzing the intra- and extra-cellular calcium (Ca2+) signaling. Stimulating the growth of lens epithelial cells is important in lens regeneration while inhibiting the growth of lens epithelial cells is important in preventing the development of PCO. In the article I described the methods for the analysis of lens epithelial cells after cataract surgeries, which are carried out in the laboratory of the Eye Hospital, University Medical Centre Ljubljana.

Inhibition effects of sulforaphane-induced autophagy flux to cell proliferation on ex vivo human capsular bags

Background Sulforaphane (SFN) is an effective chemopreventive agent and can regulate the biological molecular mechanisms to inhibit the overgrowth of cells.Autophagy is a biological process of maintaining cellular internal environment.Understanding the affection of SFN to biological behavior of human lens epithelial cells (LECs) and the association of SFN with autophagy is helpful for the prevention and target treatment of posterior capsule opacification (PCO). Objective This study was to investigate the eradication effeccts of SFN on residual lens cell population in vitro posterior capsule opacification (PCO) model and evaluate the mechanism of SFN-induced cell death. Methods In vitro human capsular bag models were generated from fresh donor eyes by phacoemulsification and were cultured in EMEM containing 2% fetal bovine serum (FBS). Different concentrations of SFN (0, 1, 10 and 100 μmol) were added in the medium for 30 days respectively according to grouping, and the growth of LECs was observed by optical microscope and immunofluorescence technique.FHL124, a human LEC line, was cultured with EMEM containing 5%FBS and divided into 0, 1, 10, 30 and 100 μmol SFN groups.Lactate dehydrogenase (LDH) release rate in the medium was detected to evaluate cell damage/death.The migration of the cells on capsular bags was assessed by scratch test.The ultrastructure and number of autophagosomes were examined under the transmission electron microscope.The expression of LC3 in the cells were detected using Western blot in the presence or absence of autophagy inhibitors. Results The cell coverage rates on the capsular bags were significantly lower in the 10 and 100 μmol/L SFN groups than those in the 0 and 1 μmol/L SFN groups, with a statistically significant difference among the groups (F=48.57, P<0.01). Immunofluorescence showed that the density of F-actin-and Vimentin-positive cells was evidently decreased in the 10 and 100 μmol/L SFN groups compared with 0 and 1 μmol/L SFN groups.The releasing levels of LDH (absorbancy) were 0.19±0.03, 0.39±0.06, 0.56±0.07, 0.68±0.08 and 0.89±0.09 in the 0, 1, 10, 30 and 100 μmol/L SFN groups, respectively, and the releasing level of LDH was gradually increased in the 10 and 100 μmol/L SFN groups in comparison with the 1 μmol/L SFN group (all at P<0.01). With the increase of SFN concentration, the reduction rate of scratched area decreased with the increase of SFN concentration, and the decrease of scratch area was significantly lower than that of adjacent low mass concentration group and the differences were statistically significant (P<0.05). The relative expressions of LC3-Ⅱ protein were 0.423±0.003, 14.543±0.024, 0.668±0.024 and 0.576±0.056 in the blank control group, SFN group, SFN+ 3-MA group and 3-MA group, respectively, and the relative expressions of LC3-Ⅱ protein were significantly lower in the SFN+ 3-MA group and 3-MA group than those in the SFN group (all at P<0.01). The number of autophagosomes was 4.07±0.32, 4.13±0.34, 9.21±0.53 and 21.02±1.34 in the blank control group, and 1, 10, 100 μmol/L SFN groups, and the number of autophagosomes in the 10 and 100 μmol/L SFN groups was significantly higher than that in the blank control group and 1 μmol/L SFN group (all at P<0.01). Conclusions SFN mediates LECs death by promoting autophagy in ex vivo capsular bags, and SFN may be a novel agent of potential chemopreventive and target treatment for PCO. Key words: Lens capsule, crystalline; Isothiocyanates; Thiocyanates/therapeutic use; Cell line; Lens epithelial cells; Models, biological; Autophagy/drug effects; Posterior capsule opacification

Sulforaphane promotes ER stress, autophagy, and cell death: implications for cataract surgery

Posterior capsule opacification (PCO) commonly develops following cataract surgery and is a wound-healing response that can ultimately lead to secondary visual loss. Improved management of this problem is required. The isothiocyanate, sulforaphane (SFN), is reported to exert cytoprotective and cytotoxic actions, and the latter may be exploited to treat/prevent PCO. SFN concentrations of 10 μM and above significantly impaired wound-healing in a human lens capsular bag model. A similar pattern of response was also seen with a human lens cell line, FHL124. SFN treatment promoted increased expression of endoplasmic reticulum (ER) stress genes, which also corresponded with protein expression. Evidence of autophagy was observed in response to SFN as determined by increased microtubule-associated protein 1A/1B-light chain 3 (LC3)-II levels and detection of autophagic vesicles. This response was disrupted by established autophagy inhibitors chloroquine and 3-MA. SFN was found to promote MAPK signaling, and inhibition of ERK activation using U0126 prevented SFN-induced LC3-II elevation and vesicle formation. SFN also significantly increased levels of reactive oxygen species. Taken together, our findings suggest that SFN is capable of reducing lens cell growth and viability and thus could serve as a putative therapeutic agent for PCO.Key messageSFN reduces lens epithelial cell growth, migration, and viability.SFN can promote ER stress and autophagy in lens cells.SFN promotes MAPK signaling, and inhibition of MEK can suppress SFN-induced autophagy.ER stress and autophagy in lens cells are likely promoted by ROS production.SFN may help prevent posterior capsule opacification after cataract surgery.

SiRNA Targeting mTOR Effectively Prevents the Proliferation and Migration of Human Lens Epithelial Cells.

Posterior capsule opacification (PCO) is the most common complication that causes visual decrease after extracapsular cataract surgery. The primary cause of PCO formation is the proliferation of the residual lens epithelial cells (LECs). The mammalian target of rapamycin (mTOR) plays an important role in the growth and migration of LECs. In the current study, we used small interfering RNA (siRNA) to specifically attenuate mTOR in human lens epithelial B3 cells (HLE B3). We aimed to examine the effect of mTOR-siRNA on the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of HLE B3 cells and explore the underlying mechanisms. The mTOR-siRNA was transfected into HLE B3 cells using lipofectamine 2000. The mRNA and protein levels of mTOR were examined to confirm the efficiency of mTOR-siRNA. The levels of mRNA and protein as well as the activity of mTOR down-stream effectors p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB, AKT) were examined using real-time PCR or Western blot, respectively. The cell proliferation was determined using cell counting kit (CCK) 8 and cell growth curve assay. The cell migration was examined using Transwell system and Scratch assay. MTOR-siRNA effectively eliminated mTOR mRNA and protein. The proliferation and migration were significantly suppressed by mTOR-siRNA transfection. mTOR-siRNA reduced the mRNA of p70S6K and AKT in a time-dependent manner. Furthermore, the phosphorylation of p70S6K and AKT was decreased by mTOR-siRNA. MTOR-siRNA also eliminated the formation of mTORC1 and mTORC2 protein complex and blocked the transforming growth factor (TGF)-β-induced EMT. Our results suggested that mTOR-siRNA could effectively inhibit the proliferation, migration and EMT of HLE B3 cells through the inhibition of p70S6K and AKT. These results indicated that mTOR-siRNA might be an effective agent inhibiting HLE cells growth and EMT following cataract surgery and provide an alternative therapy for preventing PCO.

Nox4 Plays a Role in TGF-β-Dependent Lens Epithelial to Mesenchymal Transition.

PurposeTransforming growth factor-β induces an epithelial to mesenchymal transition (EMT) in the lens, presented as an aberrant growth and differentiation of lens epithelial cells. Studies in other models of EMT have shown that TGF-β–driven EMT is dependent on the expression of the reactive oxygen species (ROS)–producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)–oxidase-4 (Nox4). We investigate the role of this enzyme in TGF-β–induced lens EMT and determine whether it is required for this pathologic process.MethodsRat lens epithelial explants were used to investigate the role of Nox4 in TGF-β–driven lens EMT. Nox1–4 expression and localization was determined by immunolabeling and/or RT-PCR. NADPH–oxidase–produced ROS were visualized microscopically using the fluorescent probe, dihydroethidium (DHE). VAS2870, a pan-NADPH oxidase inhibitor, was used to determine the specificity of Nox4 expression and its role in ROS production, and subsequently TGF-β–driven EMT.ResultsWe demonstrate, for the first time to our knowledge, in rat lens epithelial explants that TGF-β treatment induces Nox4 (but not Nox1–3) expression and activity. Increased Nox4 expression was first detected at 6 to 8 hours following TGF-β treatment and was maintained in explants up to 48 hours. At 8 hours after TGF-β treatment, Nox4 was observed in cell nuclei, while at later stages in the EMT process (at 48 hours), Nox4 was predominately colocalized with α-smooth muscle actin. The inhibition of Nox4 expression and activity using VAS2870 inhibited EMT progression.ConclusionsTransforming growth factor-β drives the expression of the ROS-producing enzyme Nox4 in rat lens epithelial cells and Nox4 inhibition can impede the EMT process.

Effect of femtosecond laser‐assisted lens surgery on posterior capsule opacification in the human capsular bag in vitro

To compare posterior capsule opacification (PCO) by observing lens epithelial cell growth in the human capsular bag invitro between conventional lens surgery using phacoemulsification (Phaco) technique and femtosecond laser-assisted lens surgery (FLACS). For the invitro human capsular bag model, 18 cadaver eyes from nine human donors underwent three types of lens surgery. Three groups consisting of six capsular bags were established, that is FLACS, Phaco and extracapsular lens extraction (ECCE). The capsular bag was transferred into equal cell culture conditions after using one of the defined surgical approaches. Cellular growth of lens epithelial cells was observed and photo-documented. The time until full cell-coverage of the capsular bag was measured. The human capsular bag model can be successfully prepared using FLACS. There was no statistically significant difference in time until cell-coverage of the human donor capsular bag invitro in all three surgical settings (ECCE versus Phaco p=0.6; ECCE versus FLACS p=1.0; Phaco versus FLACS p=1.0). In our invitro human capsular bag model, we could not observe a statistically significant difference in PCO formation using different surgical approaches of lens extraction. Therefore, PCO formation might not be attributed to the type of surgery. Furthermore, this study shows that FLACS can be used for the human capsular bag model preparation and validates the human capsular bag model for future research.

Sustained-release genistein from nanostructured lipid carrier suppresses human lens epithelial cell growth.

To design and investigate the efficacy of a modified nanostructured lipid carrier loaded with genistein (Gen-NLC) to inhibit human lens epithelial cells (HLECs) proliferation. Gen-NLC was made by melt emulsification method. The morphology, particle size (PS), zeta potentials (ZP), encapsulation efficiency (EE) and in vitro release were characterized. The inhibition effect of nanostructured lipid carrier (NLC), genistein (Gen) and Gen-NLC on HLECs proliferation was evaluated by cell counting kit-8 (CCK-8) assay, gene and protein expression of the proliferation marker Ki67 were evaluated with real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence analyses. The mean PS of Gen-NLC was 80.12±1.55 nm with a mean polydispersity index of 0.11±0.02. The mean ZP was -7.14±0.38 mV and the EE of Gen in the nanoparticles was 92.3%±0.73%. Transmission electron microscopy showed that Gen-NLC displayed spherical-shaped particles covered by an outer-layer structure. In vitro release experiments demonstrated a prolonged drug release for 72h. The CCK-8 assay results showed the NLC had no inhibitory effect on HLECs and Gen-NLC displayed a much more prominent inhibitory effect on cellular growth compared to Gen of the same concentration. The mRNA and protein expression of Ki67 in LECs decreased significantly in Gen-NLC group. Sustained drug release by Gen-NLCs may impede HLEC growth.

Evaluation of laser capsule polishing for prevention of posterior capsule opacification in a human ex vivo model

To evaluate the efficacy of a laser photolysis (LP) system in preventing posterior capsule opacification (PCO) in a human ex vivo PCO model. Ars Ophthalmica Study Center, Department of Ophthalmology, General Hospital Linz, Medical Faculty of Johannes Kepler University, Linz, Austria, and the Department of Ophthalmology, Ludwig-Maximillians-University, Munich, Germany. Prospective randomized controlled laboratory trial. Open sky extracapsular cataract extraction following implantation of a capsular tension ring (CTR) into the capsular bag was performed in 28 human donor eyes. Donor eyes received LP treatment of the capsular bag fornix and the anterior capsule for 180 or 360 degrees, whereas the contralateral eyes served as a control group. Lens epithelial cell (LEC) growth onto the posterior capsule was determined objectively during 3 months of organ culture incubation. The mean interval until a complete monolayer of LECs on the posterior capsule had formed was 8.2 days ± 1.2 (SD) for control eyes and 9.4 days ± 1.1 for eyes with 180-degree LP treatment (P = .042). Eyes with 360-degree treatment showed no sign of LEC growth or migration onto the posterior capsule during the entire observation period. Transmission light microscopy revealed many residual LECs on the anterior lens capsule of untreated areas, whereas no evidence of remaining LEC in areas treated with LP was found. This study demonstrates complete and sustained PCO prevention by a prototype LP system in a capsular tension ring-based human ex vivo model. Laser capsule polishing has the potential to serve as a successful surgical strategy for PCO prevention. The authors have no proprietary or financial interest in any of the materials or equipment mentioned in this study.

Erufosine, a phosphoinositide-3-kinase inhibitor, to mitigate posterior capsule opacification in the human capsular bag model

To determine whether erufosine alone or erufosine-loaded intraocular lenses (IOLs) can inhibit growth of human lens epithelial cells after a single administration in the human capsular bag model. Laboratory for Cell Biology, Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Experimental study. Sixteen human cadaver eyes had sham cataract surgery. The capsular bag was transferred into cell culture. The tissue was exposed to the half maximum inhibitory concentrations of erufosine alone for 72 hours; solvent-only tissue served as a control. Erufosine is a potent inhibitor of phosphoinositide-3-kinase, a downstream kinase with major implications in posterior capsule opacification (PCO) pathogenesis. The IOLs were soaked with erufosine and implanted in the capsular bags; unsoaked IOLs served as controls. For both settings, the time until confluence of the capsular bag was measured. Cell growth was observed and photodocumented. Erufosine as a single therapeutic agent increased the time until confluence of the capsular bag, but not significantly compared with the control. When IOLs were soaked with erufosine, a long-term prophylactic effect was observed in this organ model for PCO, which is known to closely reflect the clinical situation. Erufosine-soaked IOLs effectively inhibited PCO formation as seen in long-term organ culture and might become of clinical relevance. Drs. Kampik and Eibl-Lindner are inventors of IOLs treated with alkylphosphocholines for pharmacological after-cataract prophylaxis, patent international application PCT/EP2010/051490. No other author has a financial or proprietary interest in any material or method mentioned.