GSH Levels Serve As a Biological Redox Switch Regulating Sulforaphane-Induced Cell Fate in Human Lens Cells.

Purpose: Sulforaphane (SFN) is a therapeutic phytochemical agent for many health conditions. SFN-induced cytotoxicity is shown to have promise in preventing posterior capsule opacification (PCO). In the current study, we aimed to elucidate key processes and mechanisms linking SFN treatment to lens cell death. Methods: The human lens epithelial cell line FHL124 and central anterior epithelium were used as experimental models. Cell death was assessed by microscopic observation and cell damage/viability assays. Gene or protein levels were assessed by TaqMan RT-PCR or immunoblotting. Mitochondrial networks and DNA damage were assessed by immunofluorescence. Mitochondrial membrane potential, activating transcription factor 6 (ATF6) activity, ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), and glutathione reductase (GR) activity were measured using different light reporter assays. SFN metabolites were analyzed by LC-MS/MS. Results: Treatment with N-acetylcysteine (NAC), a reactive oxygen species scavenger, prevented SFN-induced cell death in both models. NAC also significantly protected FHL124 cells from SFN-induced mitochondrial dysfunctions, endoplasmic reticulum stress (ERS), DNA damage and autophagy. SFN significantly depleted GSH, the major antioxidant in the eye, and reduced GR activity, despite doubling its protein levels. The most abundant SFN conjugate detected in lens cells following SFN application was SFN-GSH. The addition of GSH protected lens cells from all SFN-induced cellular events. Conclusions: SFN depletes GSH levels in lens cells through conjugation and inhibition of GR activity. This leads to increased reactive oxygen species and oxidative stress that trigger mitochondrial dysfunction, ERS, autophagy, and DNA damage, leading to cell death. In summary, the work presented provides a mechanistic understanding to support the therapeutic application of SFN for PCO and other disorders.

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells.

The present study aims to understand the mechanism of the lens epithelial cell's strong anti-apoptotic capacity and survival in the mature human lens that, on the one hand, maintains lens transparency over several decades, while on the other hand, increases the risk of posterior capsule opacification (PCO). Here we compared FHL124 cells and HeLa cells, spontaneously immortalized epithelial cell lines derived from the human lens and cervical cancer cells, respectively, of their resistance to TNFα-mediated cell death. TNFα plus cycloheximide (CHX) triggered almost all of HeLa cell death. FHL124 cells, however, were unaffected and able to block caspase-8 activation as well as prevent caspase-3 and PARP-1 cleavage. Interestingly, despite spontaneous NFκB and AP-1 activation and upregulation of multiple cell survival/anti-apoptotic genes in both cell types, only FHL124 cells were able to survive the TNFα challenge. After screening and comparing the cell survival genes, cFLIP was found to be highly expressed in FHL124 cells and substantially upregulated by TNFα stimulation. FHL124 cells with a mild cFLIP knockdown manifested a profound apoptotic response to TNFα stimulus similar to HeLa cells. Most importantly, we confirmed these findings in an ex vivo lens capsular bag culture system. In conclusion, our results show that cFLIP is a critical gene that is regulating lens epithelial cell survival.

Assessment of intraocular lens/capsular bag biomechanical interactions following cataract surgery in a human in vitro graded culture capsular bag model.

Intraocular lenses (IOLs) are implanted during cataract surgery. For optimum results, stable positioning of the IOL in the capsular bag is important. Wound-healing events following cataract surgery lead to modification of the capsular bag and secondary visual loss due to posterior capsule opacification. At present, it is unclear how these biological events can affect stability of the IOL within the capsular bag. In the present study, a human in vitro graded culture capsular bag model was the experimental system. Capsulorhexis and lens extraction performed on human donor eyes generated suspended capsular bags (5 match-paired experiments). Preparations were secured by pinning the ciliary body to a silicone ring and maintained in 6 mL of medium for 84 days using a graded culture system: days 1-3, 5% human serum and 10 ng/mL transforming growth factor ß (TGFß2); days 4-7, 2% human serum and 1 ng/mL TGFß2; days 8-14, 1% human serum and 0.1 ng/mL TGFß2; days 15-84, serum-free Eagle's minimum essential medium (EMEM). A CT LUCIA 611PY IOL was implanted in all preparations. Quantitative measures were determined from whole bag images captured weekly. Images were registered using FIJI and analysed in ImageJ to determine capsular bag area; distortion; angle of contact; haptic stability; capsulorhexis area; and a fusion footprint associated with connection between the anterior and posterior capsules. Cell coverage and light scatter were quantified at end-point. The transdifferentiation marker, α-SMA was assessed by immunocytochemistry. Immediately following surgery, distortion of the capsular bag was evident, such that a long axis is generated between haptics relative to the non-haptic regions (short axis). The angle of contact between the haptics and the peripheral bag appeared inversely correlated to capsular bag area. Growth on the peripheral posterior capsule was observed 1 week after surgery and beneath the IOL within 1 month. As coverage of the posterior capsule progressed this was associated with matrix contraction/wrinkles of both the central posterior capsule and peripheral capsular bag. Cells on the central posterior capsule expressed αSMA. Fusion footprints formed in non-haptic regions of the peripheral bag and progressively increased over the culture period. Within and at the edge of the fusion footprint, refractive structures resembling lens fibre cells and Elschnig's pearls were observed. Cell attachment to the IOL was limited. An impression in the posterior capsule associated with the CT LUCIA 611PY optic edge was evident; cell density was much greater peripheral to this indent. Wound-healing events following surgery reduced capsular bag area. This was associated with the long/short axis ratio and angle of contact increasing with time. In summary, we have developed a human capsular bag model that exhibits features of fibrotic and regenerative PCO. The model permits biomechanical information to be obtained that enables better understanding of IOL characteristics in a clinically relevant biological system. Throughout culture the CT LUCIA 611PY appeared stable in its position and capsular bag modifications did not change this. We propose that the CT LUCIA 611PY optic edge shows an enhanced barrier function, which is likely to provide better PCO management in patients.

Aspirin inhibits TGFß2-induced epithelial to mesenchymal transition of lens epithelial cells: selective acetylation of K56 and K122 in histone H3.

Posterior capsule opacification (PCO) is a complication after cataract surgery that can disrupt vision. The epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) in response to transforming growth factor ß2 (TGFß2) has been considered an obligatory mechanism for PCO. In this study, we tested the efficacy of aspirin in inhibiting the TGFß2-mediated EMT of human LECs, LECs in human lens capsular bags, and lensectomized mice. In human LECs, the levels of the EMT markers α-smooth muscle actin (α-SMA) and fibronectin were drastically reduced by treatment with 2 mM aspirin. Aspirin also halted the EMT response of TGFß2 when introduced after EMT initiation. In human capsular bags, treatment with 2 mM aspirin significantly suppressed posterior capsule wrinkling and the expression α-SMA in capsule-adherent LECs. The inhibition of TGFß2-mediated EMT in human LECs was not dependent on Smad phosphorylation or MAPK and AKT-mediated signaling. We found that aspirin significantly increased the acetylation of K56 and K122 in histone H3 of human LECs. Chromatin immunoprecipitation assays using acetyl-H3K56 or acetyl-H3K122 antibody revealed that aspirin blocked the TGFß2-induced acetylation of H3K56 and H3K122 at the promoter regions of ACTA2 and COL1A1. After lensectomy in mice, we observed an increase in the proliferation and α-SMA expression of the capsule-adherent LECs, which was ameliorated by aspirin administration through drinking water. Taken together, our results showed that aspirin inhibits TGFß2-mediated EMT of LECs, possibly from epigenetic down-regulation of EMT-related genes.

The human capsular bag model of posterior capsule opacification.

Posterior capsule opacification (PCO) is the most common complication following cataract surgery and affects millions of patients. PCO is a consequence of surgical injury promoting a wound-healing response. Following surgery, residual lens epithelial cells grow on acellular regions of the lens capsule, including the central posterior capsule. These cells can undergo fibrotic changes, such that cell transdifferentiation to myofibroblasts, matrix deposition and matrix contraction can occur, which contribute to light scatter and the need for further corrective Nd:YAG laser capsulotomy in many patients. It is therefore of great importance to better understand how PCO develops and determine better approaches to manage the condition. To achieve this, experimental systems are required, and many are available to study PCO. While there may be a number of common features associated with PCO in different species, the mechanisms governing the condition can differ. Consequently, where possible, human systems should be employed. The human capsular bag model was established in a laboratory setting on donor eyes. A capsulorhexis is performed to create an opening in the anterior capsule followed by removal of the lens fibre mass. Residual fibre cells can be removed by irrigation/aspiration and if required, an intraocular lens can be implanted. The capsular bag is isolated from the eye and transferred to a dish for culture. The human capsular bag model has played an important role in understanding the biological processes driving PCO and enables evaluation of surgical approaches, IOLs and putative therapeutic agents to better manage PCO.

Resveratrol Inhibits Wound Healing and Lens Fibrosis: A Putative Candidate for Posterior Capsule Opacification Prevention.

Purpose: Posterior capsule opacification (PCO) is a common complication of cataract surgery. In addition to improved surgical methods and IOL designs, it is likely additional agents will be needed to improve patient outcomes. Presently no pharmacological agent is in clinical use to prevent PCO. Here we investigate the putative ability of resveratrol (RESV), a naturally occurring polyphenol, as a therapeutic agent. Methods: The human lens epithelial cell line FHL124, a human lens capsular bag model, and central anterior epithelium were used as experimental systems. Standard culture was in 5% fetal calf serum Eagle's minimum essential medium; 10 ng/mL transforming growth factor-ß2 (TGFß2) was used to induce fibrotic changes. A scratch wound assay was used to measure cell migration and the patch assay was used to assess matrix contraction by FHL124 cells. Protein expression was assessed by immunocytochemistry and Western blot and gene expression by quantitative RT-PCR. In capsular bags, cell growth across the posterior lens capsule, capsular wrinkling, and epithelial-to-mesenchymal transition were determined by image analysis. Results: In FHL124 cells, addition of 30 µM RESV significantly impeded cell migration in a wound-healing assay. RESV significantly inhibited TGFß2-induced expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) at both the message and protein levels, as well as significantly inhibiting matrix contraction induced by TGFß2. In human capsular bags, 30 µM RESV significantly inhibited cell growth. TGFß2-induced α-SMA expression and capsular wrinkling were also significantly inhibited by RESV treatment. RESV significantly suppressed expression of TGFß2-induced genes associated with fibrotic disease, including matrix metalloproteinase-2 in FHL124 cells, capsular bags, and central anterior epithelium. Conclusions: RESV can counter PCO-related physiological events in two human lens model systems. RESV therefore has the potential to be used as a candidate agent for the prevention of PCO, which in turn could benefit millions of cataract patients.

Development and Validation of a Deep Learning System to Detect Glaucomatous Optic Neuropathy Using Fundus Photographs.

Importance: A deep learning system (DLS) that could automatically detect glaucomatous optic neuropathy (GON) with high sensitivity and specificity could expedite screening for GON. Objective: To establish a DLS for detection of GON using retinal fundus images and glaucoma diagnosis with convoluted neural networks (GD-CNN) that has the ability to be generalized across populations. Design, Setting, and Participants: In this cross-sectional study, a DLS for the classification of GON was developed for automated classification of GON using retinal fundus images obtained from the Chinese Glaucoma Study Alliance, the Handan Eye Study, and online databases. The researchers selected 241 032 images were selected as the training data set. The images were entered into the databases on June 9, 2009, obtained on July 11, 2018, and analyses were performed on December 15, 2018. The generalization of the DLS was tested in several validation data sets, which allowed assessment of the DLS in a clinical setting without exclusions, testing against variable image quality based on fundus photographs obtained from websites, evaluation in a population-based study that reflects a natural distribution of patients with glaucoma within the cohort and an additive data set that has a diverse ethnic distribution. An online learning system was established to transfer the trained and validated DLS to generalize the results with fundus images from new sources. To better understand the DLS decision-making process, a prediction visualization test was performed that identified regions of the fundus images utilized by the DLS for diagnosis. Exposures: Use of a deep learning system. Main Outcomes and Measures: Area under the receiver operating characteristics curve (AUC), sensitivity and specificity for DLS with reference to professional graders. Results: From a total of 274 413 fundus images initially obtained from CGSA, 269 601 images passed initial image quality review and were graded for GON. A total of 241 032 images (definite GON 29 865 [12.4%], probable GON 11 046 [4.6%], unlikely GON 200 121 [83%]) from 68 013 patients were selected using random sampling to train the GD-CNN model. Validation and evaluation of the GD-CNN model was assessed using the remaining 28 569 images from CGSA. The AUC of the GD-CNN model in primary local validation data sets was 0.996 (95% CI, 0.995-0.998), with sensitivity of 96.2% and specificity of 97.7%. The most common reason for both false-negative and false-positive grading by GD-CNN (51 of 119 [46.3%] and 191 of 588 [32.3%]) and manual grading (50 of 113 [44.2%] and 183 of 538 [34.0%]) was pathologic or high myopia. Conclusions and Relevance: Application of GD-CNN to fundus images from different settings and varying image quality demonstrated a high sensitivity, specificity, and generalizability for detecting GON. These findings suggest that automated DLS could enhance current screening programs in a cost-effective and time-efficient manner.

An In Vitro Human Lens Capsular Bag Model Adopting a Graded Culture Regime to Assess Putative Impact of IOLs on PCO Formation.

Purpose: To develop a culture regime for the in vitro human lens capsular bag model that better reflects clinical events following cataract surgery and to use this refined model to evaluate the putative impact of IOLs on PCO formation. Methods: Capsulorhexis and lens extraction were performed on human donor eyes to generate capsular bags attached to the ciliary body by the zonules. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining in 6 mL serum-free EMEM for 28 days or in a graded culture system (days 1-3, 5% human serum and 10 ng/mL TGFß2; days 4-7, 2% human serum and 1 ng/mL TGFß2; days 8-14, 1% human serum and 0.1 ng/mL TGFß2; days 15-28, serum-free EMEM), which better mimics clinical changes. Preparations were monitored with phase-contrast and modified-dark-field microscopy. Cell coverage and light scatter were quantified using image analysis software. The transdifferentiation marker, α-SMA and matrix component, fibronectin were assessed by immunocytochemistry. To assess IOLs in the model, Alcon Acrysof or Hoya Vivinex IOLs were implanted in match-paired capsular bags. Results: Match-paired experiments showed that graded culture enhanced growth, facilitated matrix contraction, increased transdifferentiation, and promoted matrix deposition relative to serum-free culture. The graded culture protocol was applied to match-paired bags implanted with a Hoya Vivinex or an Alcon Acrysof IOL. The Vivinex demonstrated a lag in growth across the posterior capsule. However, by day 28, coverage was similar, but light-scatter was greater with Acrysof implanted. Cell growth on the Acrysof IOL anterior surface was significantly greater than Vivinex. Conclusions: The graded culture human capsular bag model serves as an excellent system to evaluate and develop intraocular lenses. The Hoya Vivinex IOL showed an overall better level of performance against postsurgical wound healing and PCO than the Alcon Acrysof using this model.

Growth factor restriction impedes progression of wound healing following cataract surgery: identification of VEGF as a putative therapeutic target.

Secondary visual loss occurs in millions of patients due to a wound-healing response, known as posterior capsule opacification (PCO), following cataract surgery. An intraocular lens (IOL) is implanted into residual lens tissue, known as the capsular bag, following cataract removal. Standard IOLs allow the anterior and posterior capsules to become physically connected. This places pressure on the IOL and improves contact with the underlying posterior capsule. New open bag IOL designs separate the anterior capsule and posterior capsules and further reduce PCO incidence. It is hypothesised that this results from reduced cytokine availability due to greater irrigation of the bag. We therefore explored the role of growth factor restriction on PCO using human lens cell and tissue culture models. We demonstrate that cytokine dilution, by increasing medium volume, significantly reduced cell coverage in both closed and open capsular bag models. This coincided with reduced cell density and myofibroblast formation. A screen of 27 cytokines identified nine candidates whose expression profile correlated with growth. In particular, VEGF was found to regulate cell survival, growth and myofibroblast formation. VEGF provides a therapeutic target to further manage PCO development and will yield best results when used in conjunction with open bag IOL designs.

PARP-1 inhibition influences the oxidative stress response of the human lens.

Poly(ADP-ribose) polymerase-1 (PARP-1) is best characterised for its involvement in DNA repair. PARP-1 activity is also linked to cell fate, confounding its roles in maintaining genome integrity. The current study assessed the functional roles of PARP-1 within human lens cells in response to oxidative stress. The human lens epithelial cell line FHL124 and whole human lens cultures were used as experimental systems. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death was assessed by LDH release. The functional influence of PARP-1 was assessed using targeted siRNA and chemical inhibition (by AG14361). Immunocytochemistry and western blotting were used to assess PARP-1 expression and the alkaline comet assay determined the levels of DNA strand breaks. PARP-1 was generally observed in the cell nucleus in both the FHL124 cell line and whole human lenses. PARP-1 inhibition rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Interestingly, reduction of PARP-1 activity significantly inhibited H2O2-induced cell death relative to control cells. Inhibition of PARP-1 in whole human lenses resulted in a reduced level of lens opacity and cell death following exposure to H2O2 relative to matched pair controls. Thus, we show that PARP-1 could play a role in the fate of human lens cells, and these first observations in human lenses suggest that it could impact on lens opacity. Further studies are required to elucidate the regulatory processes that give rise to these effects.

Improving the drug development process by reducing the impact of adverse events: the case of cataracts considered.

Cataract was used as a model for the prevalence and economic impact of adverse events during the drug development process. Meta-analysis revealed a reported prevalence of cataract at 12.0% (1.0-43.3%), 3.8% (2.4-12.5%), 1.0% (0.0-8.1%), 1.7% (0.0-34.8%) and 3.8% (2.3-5.7%) of compounds in preclinical, Phase I, II, III and IV clinical trials, respectively. Utilising a human-based in vitro screening assay to predict cataractogenic potential in human could allow better selection of novel compounds at early-stage drug development. This could significantly reduce costs and ultimately increase the probability of a drug obtaining FDA approval for a clinical application.

An in vitro evaluation of the Anew Zephyr open-bag IOL in the prevention of posterior capsule opacification using a human capsular bag model.

PURPOSE: During cataract surgery an IOL is placed within the capsular bag. Clinical studies show that IOLs with a square edge profile and complete contact between the IOL and the anterior capsule (AC) are currently the best way to prevent posterior capsule opacification (PCO). This has been challenged by recent clinical and experimental observations, which suggest that if the capsular bag is kept open with separation of contact between the AC and posterior capsule (PC) by an "open-bag device" PCO is dramatically reduced. Therefore, the current study set out to evaluate the putative merits of an open-bag IOL (Anew Zephyr) in a human capsular bag model. METHODS: An in vitro organ culture model using the bag-zonular-ciliary body complex isolated from fellow human donor eyes was prepared. A capsulorhexis and lens extraction were performed, and an Alcon Acrysof IOL or Anew Zephyr IOL implanted. Preparations were secured by pinning the ciliary body to a silicone ring and maintained in 6 mL Eagle's minimum essential medium (EMEM) or EMEM supplemented with 2% vol/vol human serum (HS) and 10 ng/mL TGF-ß2 for 28 days. Cell growth and capsular modifications were monitored with phase-contrast and modified dark-field microscopy. RESULTS: In serum-free EMEM culture conditions, cells were observed growing onto the PC of preparations implanted with an Anew Zephyr IOL, but this was retarded relative to observations in match-paired capsular bags implanted with an Alcon Acrysof IOL. In the case of cultures maintained in 2% HS-EMEM plus TGF-ß2, the movement on to the PC was again delayed with the presence of an Anew Zephyr IOL. Differences in the degree of growth on the PC and matrix modifications were apparent with the different donors, but in each case the match-paired Alcon Acrysof implanted bag exhibited significantly greater coverage and modification of the capsule. CONCLUSIONS: The Anew Zephyr open-bag IOL performs consistently better than the Alcon Acrysof IOL in the human capsular bag model. We propose that the benefits observed with the Anew Zephyr result from a reduction in growth factor levels available within the capsular bag and a barrier function imposed by the ring haptic.

Effect of total lens epithelial cell destruction on intraocular lens fixation in the human capsular bag.

PURPOSE: To evaluate the effect of complete destruction of lens epithelial cells (LECs) in the capsular bag on intraocular lens (IOL) stability. SETTING: School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. DESIGN: Comparative evaluation. METHODS: An in vitro organ culture model using the bag-zonule-ciliary body complex isolated from fellow human donor eyes was prepared. A capsulorhexis and fiber extraction were performed, and an Acrysof IOL was implanted. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining it in 6 mL Eagle minimum essential medium supplemented with 5% v/v fetal calf serum and 10 ng/mL transforming growth factor-ß2 for 3 weeks or more. One bag of each pair was treated with 1 µM thapsigargin to destroy all LECs. Observations of LEC growth were captured by phase-contrast microscopy, IOL stability by video microscopy, and endpoint analysis through scanning electron microscopy and immunocytochemistry. RESULTS: The LECs in control capsular bags migrated centrally, closing the bag and fixating the IOL between the anterior and posterior capsules, as seen clinically. These events were not observed in the thapsigargin-treated group. After a period of controlled orbital movement, the IOL in the control group stabilized quicker than in the treated bags. There was no IOL rotation in the bag; however, the IOLs in the treated group rocked with axial movement. CONCLUSIONS: The LECs appeared to aid stabilization of current IOL designs in the capsular bag. The results have clinical implications for IOL design and for strategies to prevent posterior capsule opacification. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Serum deprivation can suppress receptor-mediated calcium signaling in pterygial-derived fibroblasts.

PURPOSE: Pterygium is characterized as invasive, proliferative fibrovascular altered conjunctival tissue. The extensive vascular network is likely to significantly contribute to the progression of the disease. In the present study, we investigated the effects of reduced serum (to mimic a suppressed blood supply) on cell signaling events and the functional role of the calcium store in cultured, pterygial-derived fibroblasts. METHODS: Pure fibroblast cultures were established from cell outgrowths of pterygial tissue. Growth and migration of pterygial-derived fibroblast was evaluated using a patch growth assay, MTS assay, and a scratch wound assay. Intracellular calcium levels were determined using Fura-2 detection in response to ligand stimulation using a 96-well plate format. RESULTS: A progressive increase in serum concentration resulted in promotion of pterygial cell growth detected using the MTS assay. A significant increase in intracellular calcium level was observed in response to histamine (10 and 100 µM), ATP (10 and 100 µM), acetylcholine (10 and 100 µM) and epidermal growth factor (10 ng/mL) in serum-maintained cells. However, no significant changes were observed when cells were maintained in serum-free medium. Thapsigargin (1 µM), a Ca-ATPase inhibitor, induced a significantly greater increase in intracellular calcium level in the serum-maintained group relative to serum-starved cells. In both cases, elevation of intracellular calcium was reduced when calcium-free bathing medium was used. Preincubation of cells with 1 µM thapsigargin ablated ligand-induced calcium responses. Disruption of calcium signaling through thapsigargin treatment significantly perturbed cell growth and migration. CONCLUSIONS: Receptor-induced calcium signaling activity is suppressed in pterygial-derived fibroblasts in response to serum deprivation. This correlates with reduced growth rates and a depleted endoplasmic reticulum calcium store. The store plays a key role in cell growth and migration of pterygial-derived fibroblasts. Therefore, the strategic reduction of the vascular network in pterygium will affect the calcium store level and in turn affect functional responses associated with pterygia.

Sigma 1 receptor stimulation protects against oxidative damage through suppression of the ER stress responses in the human lens.

Stimulation of sigma-1 receptors is reported to protect against oxidative stress. The present study uses cells and tissue from the human lens to elucidate the relationship between the sigma 1 receptor, ER stress and oxidative stress-induced damage. Exposure of the human lens cell line FHL124 to increasing concentrations of H(2)O(2) led to reduced cell viability and increased apoptosis. In response to 30 µM H(2)O(2), levels of the ER stress proteins BiP, ATF6 and pEIF2α were significantly increased within 4h of exposure. Expression of the sigma 1 receptor was markedly increased in response to H(2)O(2). Application of 10 and 30 µM (+)-pentazocine, a sigma 1 receptor agonist, significantly inhibited the H(2)O(2) induced cell death. (+)-Pentazocine also suppressed the oxidative stress induced reduction of pro-caspase 12 and suppressed the induction of the ER stress proteins BiP and EIF2α. When applied to cultured human lenses, (+)-pentazocine protected against apoptotic cell death, LDH release and against H(2)O(2) induced opacification. These data demonstrate that stimulation of the sigma 1 receptor provides significant protection from oxidative damage and is, therefore, a putative therapeutic approach to delay the onset of diseases that may be triggered by oxidative damage, including cataract formation.

MMP2 activity is critical for TGFß2-induced matrix contraction--implications for fibrosis.

PURPOSE: The fibrotic lens disorder posterior capsule opacification (PCO) develops in millions of patients following cataract surgery. PCO characteristics are extensive extracellular matrix (ECM) production and contraction of the posterior lens capsule, resulting in light-scattering ECM modification (wrinkling). The pro-fibrotic cytokine transforming growth factor beta (TGFß) is central to PCO development. This study aimed to elucidate the role of the ECM modulators matrix metalloproteinases (MMPs) in TGFß-mediated PCO formation. METHODS: The human lens epithelial cell-line FHL-124 and human capsular bag models were employed. Gene expression of MMP family members was determined by oligonucleotide microarray and quantitative real-time RT-PCR. MMP2 and MT1-MMP protein levels were analyzed by ELISA and Western blotting, respectively. Matrix contraction was determined using an FHL-124 patch contraction assay; at end-point, cells were stained with Coomassie brilliant blue and area was determined using image analysis software. Cell coverage and wrinkle formation on the posterior capsule were also assessed using human capsular bag models. RESULTS: Active TGFß2 (10 ng/mL) increased gene and protein levels of MMP2 and MT1-MMP and induced matrix contraction in FHL-124 cells. Specific siRNA inhibition of MT1-MMP did not suppress TGFß2-induced matrix contraction. Active TGFß2-mediated contraction was prevented by broad-spectrum MMP inhibitor GM6001 (25 µM), MMP2 siRNA, and MMP2 neutralizing antibody (4 µg/mL). TGFß2-induced wrinkle formation was attenuated in human capsular bags treated with MMP2 neutralizing antibody (20 µg/mL). CONCLUSIONS: MMP2 plays a critical role in TGFß2-mediated matrix contraction, which appears to be independent of MT1-MMP. MMP2 inhibition provides a novel strategy for the treatment of PCO and potentially other fibrotic disorders.

A fully human in vitro capsular bag model to permit intraocular lens evaluation.

PURPOSE: To establish a fully human in vitro culture model with which to test the putative effects of intraocular lens (IOL) designs in preventing posterior capsule opacification (PCO) after cataract surgery. METHODS: A sham cataract operation was performed to prepare human capsular bags from donor lenses. In one capsular bag of a donor pair, an intraocular lens (PMMA round-edge IOL or acrylic IOL) was implanted while the other capsular bag remained aphakic. Bags were transferred to a Petri dish and secured anterior-face down using entomological pins. Capsular bags were maintained in Eagle's minimum essential medium supplemented with 2% human serum and 10 ng/mL TGF-ß to drive growth and matrix contraction. RESULTS: In the absence of an IOL, cells appeared within the central posterior capsule at 4.38 ± 0.26 days, whereas in the presence of a PMMA round-edge IOL or an acrylic IOL they appeared at 8 ± 0.41 days and 11 ± 0.7 days, respectively. Immunocytochemical analysis showed an accumulation of cells at the edge of the acrylic IOL and a less evident accumulation with the PMMA round-edge IOL. Moreover, matrix contraction was more prominent in the absence of an IOL but was still apparent, to a lesser degree, in the presence of a PMMA round-edge IOL. The acrylic IOL greatly suppressed matrix contraction. CONCLUSIONS: The authors have developed a fully human in vitro capsular bag system that relates well to clinical observations and permits the testing of novel intraocular lenses.

Activation of the innate immune response and interferon signalling in myotonic dystrophy type 1 and type 2 cataracts.

Myotonic dystrophy (DM) is caused by a triplet repeat expansion in the non-coding region of either the DMPK (DM1) or CNBP (DM2) gene. Transcription of the expanded region causes accumulation of double-stranded RNA (dsRNA) in DM cells. We sought to determine how expression of triplet repeat RNA causes the varied phenotype typical of DM. Global transcription was measured in DM and non-DM cataract samples using Illumina Bead Arrays. DM samples were compared with non-DM samples and lists of differentially expressed genes (P≤ 0.05) were prepared. Gene set enrichment analysis and the Interferome database were used to search for significant patterns of gene expression in DM cells. Expression of individual genes was measured using quantitative real-time polymerase chain reaction. DMPK and CNBP expression was confirmed in native lens cells showing that a toxic RNA gain of function mechanism could exist in lens. A high proportion, 83% in DM1 and 75% in DM2, of the significantly disregulated genes were shared by both forms of the disease, suggesting a common mechanism. The upregulated genes in DM1 and DM2 were highly enriched in both interferon-regulated genes (IRGs) and genes associated with the response to dsRNA and the innate immune response. The characteristic fingerprint of IRGs and the signalling pathways identified in lens cells support a role for dsRNA activation of the innate immune response in the pathology of DM. This new evidence forms the basis for a novel hypothesis to explain the complex mechanism of DM.

The ocular lens: a classic model for development, physiology and disease.

Millions are rendered blind or exhibit visual impairment due to pathologies of the lens of the eye. Lens research therefore addresses the direct need to gain insights into the cellular and molecular basis of disease, but, moreover, serves as a valuable experimental system to answer fundamental biological questions. This themed issue showcases the scientific knowledge of the processes involved in the development, structure, ultrastructure, physiology and pathology of the lens and how this information has the potential to significantly further knowledge in various fields of research. The issue is divided into three main areas. Firstly, the lens is discussed as a developmental model for embryonic induction, as an elegant system for studying the role of growth factors in development, and for analysis of the molecular control and cellular basis of cellular differentiation. The genetic basis of disorders of lens development, including paediatric cataract (lens opacity), are also discussed in this section. Secondly, adult lens structure and ultrastructure are covered, as well as the lens as a model for homeostasis and solute exchange. Finally, the papers in the latter part of the special issue review lens pathology, including the lens as a model for normal and pathological ageing, vitreoretinal influences on lens function and cataract and the lens as a model for fibrotic disease. Overall, the articles highlight the lens as a continuing, very important and attractive model system for biologists working in many different research areas.

ADAM and ADAMTS gene expression in native and wound healing human lens epithelial cells.

PURPOSE: The ADAMs (a disintegrin and metalloproteinase) and the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin-like motifs) are extracellular proteases that mediate cellular interactions and cell signaling via the modulation of adhesion and the cleavage of cell surface protein ectodomains and extracellular matrix molecules. Gene expression profiling was undertaken to better understand the role of the ADAM and ADAMTS families in the clear native human lenses and following surgical injury with particular relevance to posterior capsule opacification. METHODS: To carry out profile analysis, the lens (t=0d) was dissected into three regions; anterior epithelia, equatorial region, and fiber cells. Capsular bag culture was undertaken as a means of assessing short-term changes (t=6d) and post-cataractous lens capsular bags (ex vivo) were used to predict long-term changes in ADAM/ADAMTS gene expression. RNA was isolated and quantitative real-time (TaqMan) reverse transcription-PCR (RT-PCR) performed. Data were analyzed in terms of cycle threshold number (C(T)) and also normalized relative to endogenous 18S rRNA. RESULTS: High expression of ADAM-9, -10, -15, and -17 was detected in all native lens regions. ADAM-15 expression was a characteristic of the native lens epithelia more than the fibers. Post-surgical injury, lens capsular bags showed a positive shift in ADAM/ADAMTS expression that was significant for ADAM-9, -15, and ADAMTS-3. Ex vivo capsular bags, with a long-term post surgical injury period, maintained high expression of ADAM-9 and -10 genes. CONCLUSIONS: The native human lens expresses ADAM and ADAMTS genes that are differentially regulated following surgical injury. Roles in maintaining cellular adhesion may be of particular importance to native lens tissue integrity and may be lost in the lens wound healing response following cataract surgery.